The fatty acid composition of the moss Rhytididelphus squarrosus (Hedw.) Warnst. (Hylocomiaceae) collected in Germany during winter time was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Nine fatty acids were identifi ed in its chloroform:methanol extract 1:1: arachidonic acid (30.7%), α-linolenic acid (19.1%), linoleic acid (15.1%), cis-5,8,11,14,17-eicosapentaenoic acid (14.4%), palmitic acid (11.9%), cis-8,11,14- eicosatrienoic acid (4.1%), oleic acid (2.3%), γ-linolenic acid (1.4%) and stearic acid (1.0%). Th e results indicate that this plant species can be a good source of arachidonic acid collected during the winter.Sastav viših masnih kiselina mahovine Rhytididelphus squarrosus (Hedw.) Warnst. (Hylocomiaceae), sakupljene u Nemačkoj za vreme zimskog doba, preliminarno je ispitivan GC i GC-MS analizom. U njenom ekstraktu hloroform: metanol 1:1 identifikovano je devet viših masnih kiselina: arahidonska kiselina (30.7%), α-linolenska kiselina (19.1%), linolna kiselina (15.1%), cis-5,8,11,14,17-eikosapentaenska kiselina (14.4%), palmitinska kiselina (11.9%), cis-8,11,14-eikosatrienska kiselina (4.1%), oleinska kiselina (2.3%), γ-linolna kiselina (1.4%) i stearinska kiselina (1.0%). Dobijeni rezultati ukazuju da je ova biljna vrsta dobar izvor arahidonske kiseline u ispitivanom vremenskom periodu

Pejin, Boris

Sabovljević, Aneta

Sabovljević, Marko

Tešević, Vele

Vajs, Vlatka

Vujisić, Ljubodrag V.

Botanica Serbica

English

 CER - Central Repository of the Institute of Chemistry;  Technology and Metallurgy

35 (2): (2011) 99-101
UDK 582.273:581.42(498) 
Original Scientifi c Paper
Received 11 November 2010   Revision accepted 17 March 2011
© 2011 Institute of Botany and Botanical Garden Jevremovac, Belgrade✳correspondence: borispejin@yahoo.com
An insight into fatty acid chemistry of Rhytididelphus 
squarrosus (Hedw.) Warnst.
Boris Pejin1*, Ljubodrag Vujisić2, Aneta Sabovljević3, Marko Sabovljević3, Vele 
Tešević1 and Vlatka Vajs2 
1 Department of Organic Chemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 16, 11000 Belgrade, 
Serbia 
2 Center of Chemistry, Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Njegoševa 12, 
11000 Belgrade, Serbia 
3 Institute of Botany and Garden, Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, Serbia
ABSTRACT: Th e fatty acid composition of the moss Rhytididelphus squarrosus (Hedw.) Warnst. (Hylocomiaceae) 
collected in Germany during winter time was analyzed by gas chromatography (GC) and 
gas chromatography-mass spectrometry (GC-MS). Nine fatty acids were identifi ed in its 
chloroform:methanol extract 1:1: arachidonic acid (30.7%), α-linolenic acid (19.1%), linoleic 
acid (15.1%), cis-5,8,11,14,17-eicosapentaenoic acid (14.4%), palmitic acid (11.9%), cis-8,11,14-
eicosatrienoic acid (4.1%), oleic acid (2.3%), γ-linolenic acid (1.4%) and stearic acid (1.0%). Th e 
results indicate that this plant species can be a good source of arachidonic acid collected during the 
winter. 
   Key words: moss; Rhytididelphus squarrosus; fatty acids; GC FID; GC-MS. 
INTRODUCTION
Bryophytes (mosses, liverworts and hornworts) are 
the second biggest group of land plants aft er the 
fl owering plants with approximately 28.000 species 
spread worldwide. Although the chemical structure 
and occurrence of fatty acids and their derivatives in 
bryophytes have been reviewed by Huneck (1969), their 
chemistry is still poorly known with information on this 
being very scattered (Asakawa 2007). Partly, this is due 
to diffi  culties in collecting large quantities of pure material 
of a certain species. Th e most abundant fatty acids of 
these plants are also common to most of other organisms. 
However, typical for many of them is a high content 
of long-chain polyunsaturated fatty acids, particularly 
arachidonic acid and cis-5,8,11,14,17-eicosapentaenoic 
acid, compounds which are not found very abundantly in 
the rest of the plant kingdom (Gellerman et al. 1975). 
Indeed, the occurrence of these polyunsaturated fatty 
acids constitutes an outstanding compositional diff erence 
between mosses and other higher plants. On the other 
hand, it is known that these fatty acids are constituents of 
membranes in animal tissues which are essential for many 
physiological processes. Arachidonic acid is a precursor 
of the biologically active prostaglandins and leukotrienes 
(Marx 1982). 
As part of our ongoing research on moss chemistry 
(Pejin et al. 2010, 2011a, 2011b, 2011c), a phytochemical 
investigation was conducted on the pleurocarp 
moss Rhytididelphus squarrosus (Hedw.) Warnst. 
(Hylocomiaceae). Two reports on fatty acids from the moss 
R. squarrosus have been published to date (Guschina & 
Harwood 2000, 2002). Th e most abundant fatty acid of 
this species collected during the summer near Cardiff , UK 
100 vol. 35 (2)
(July and September, 1998) from both lead-contaminated 
and non-contaminated areas was arachidonic acid (28.6% 
and 24.8%, respectively) (Guschina & Harwood 2002). 
Th e objective of this study was to identify the pattern of 
fatty acids of the same bryophyte species during the winter 
time as a contribution to its chemistry. 
MATERIAL AND METHODS
R. squarrosus was collected in Königforst near Köln 
(Germany) in December 2007. A voucher specimen was 
deposited in the Herbarium of the Institute of Botany, 
University of Belgrade, Serbia (bryophyte collection - 
BEOU No. 4707).
Th e moss was carefully selected and cleaned from soil 
and other contaminants. Th e gametophyte tips were used 
for the extraction. Air-dried parts of the sample were ground 
(1g) and extracted three times with chloroform:methanol 
1:1 for 1h at room temperature (9.88%, extract yield). Th e 
extract was evaporated to dryness and trans-esterifi ed with 
5% H2SO4 in MeOH (v/v) for 4h at 80°C. Th e resulting 
methyl esters of fatty acids were analysed by comparing 
their GC FID chromatograms with that of a standard 
mixture (Supelco 37) obtained under the same conditions, 
and/or by analysis of GC-MS data using NIST 5 and Wiley 
7 libraries.
GC analysis was performed on an Agilent 7890A GC 
system equipped with 5975C MSD and FID detectors, 
using a DB-23 column (30 m × 0.25 mm × 0.25 μm). 
Injection volume was 1 μL and injector temperature was 
220°C with a 10:1 split ratio. Carrier gas (He) fl ow rate was 
0.9 ml/min while the column temperature was linearly 
programmed in a range of 150-240°C at a rate of 4°C/min 
and then held at 240°C for 10 min. Th e transfer line was 
heated at 240°C. Th ree assay replicates and three biological 
replicates were analysed. Th e FID detector temperature 
was 300°C. EI mass spectra (70 eV) were acquired in the 
m/z range 40-500.
RESULTS
Nine fatty acids were identifi ed in the chloroform:methanol 
1:1 extract: arachidonic acid (30.7%), α-linolenic 
acid (19.1%), linoleic acid (15.1%), cis-5,8,11,14,17-
eicosapentaenoic acid (14.4%), palmitic acid (11.9%), 
cis-8,11,14-eicosatrienoic acid (4.1%), oleic acid (2.3%), 
γ-linolenic acid (1.4%) and stearic acid (1.0%) (Fig.1).
Th ese results for the composition and proportion of 
fatty acids are in good agreement with the previous report 
(Guschina & Harwood 2002), particularly regarding the 
content of arachidonic acid (Fig.2); the main diff erence 
is in presence of cis-13,16,19-docosatrienoic acid which 
was detected only in the earlier study (2.1%). Seasonal- 
and habitat- related changes in the fatty acid composition 
of various bryophytes exist and these vary from species 
to species (Karunen 1990). Th e major fatty acid of R. 
squarrosus seems to be arachidonic acid regardless of 
place and time of its collection. However, our sample 
collected during the winter had slightly more arachidonic 
acid (30.7%) in comparison with those collected in July 
(28.6%) and September (24.8%) as reported by Guschina 
& Harwood (2002). Since, the plants were not from the 
same habitats, in addition to time of the year, this variation 
could also be due to habitat diff erences.
Fig. 2. Arachidonic acid 
Fig. 1. Fatty acid methyl ester 
chromatogram for R. squarrosus; 
palmitic acid (RT 13.156 min); stearic 
acid (RT 16.205 min); oleic acid (RT 
16.673 min); linoleic acid (RT 17.526 
min); γ-linolenic acid (RT 18.067 
min); α-linolenic acid (RT 18.573 
min); cis-8,11,14-eicosatrienoic acid 
(RT 21.154 min); arachidonic acid 
(RT 21.502 min); cis-5,8,11,14,17-
eicosapentaenoic acid (RT 22.545 
min).
101B. Pejin et al.: An insight into fatty acid chemistry of Rhytididelphus squarrosus (Hedw.) Warnst.
Guschina & Harwood (2002) identifi ed 17 fatty acids, 
7 of which were in trace amounts which is in accordance 
with the 9 fatty acids recorded in this study. 
For a general consideration of the seasonal-related 
changes in the fatty acid composition of this moss species, 
more replicate samples would need to be included in a 
follow-up study, including in vitro establishment of this 
moss.
Acknowledgments – Th is work was supported by the 
Ministry of Science and Technological development of 
the Republic of Serbia (Research grants No. 142053 and 
143015). 
REFERENCES 
Asakawa Y. 2007. Biologically active compounds from 
bryophytes. Pure Appl. Chem. 79: 557-580.
Huneck S. 1969. Moosinhaltstoff e, eine Uebersicht. J. Hattori 
Bot. Lab. 32: 1-16. 
Gellerman JL, Anderson WH, Richardson DG & 
Schlenk H. 1975. Distribution of arachidonic and 
eicosapentaenoic acids in the lipids of mosses. Biochim. 
Biophys. Acta 338: 277-290.
Marx JL. 1982. Th e leukotriens in allergy and infl ammation. 
Science 215: 1380-1383.
Pejin B, Vujisic Lj, Sabovljevic M, Sabovljevic A, 
Tešević V & Vajs V. 2010. Preliminary 
analysis of fatty acid chemistry of Kinbergia praelonga and 
Kindbergia stokesii (Brachytheciaceae). J. Serb. Chem. Soc. 
75: 1637-1640.
Pejin B, Bianco A, Newmaster S, Sabovljevic M, Vujisic 
Lj, Tesevic V, Vajs V & De Rosa S. 2011a. Fatty acids of 
Rhodobryum ontariense (Bryaceae). Nat. Prod. Res. (in 
press)
Pejin B, Vujisic Lj, Sabovljevic M, Tesevic V & Vajs V. 
2011b. Th e moss Mnium hornum, a promising resource of 
arachidonic acid. Chem. Nat. Compd.(in press)
Pejin B, Vujisic Lj, Sabovljevic M, Tesevic V & Vajs 
V. 2011c. An insight into fatty acid composition of 
Calliergonella cuspidata. Asian J. Chem. (in press)
Guschina IA & Harwood JL. 2000. Eff ect of copper and lead 
on lipid metabolism in bryophytes and lichens. Biochem. 
Soc. Trans. 28: 910-912.
Guschina IA & Harwood JL. 2002. Lipid metabolism of 
the moss Rhytididelphus squarrosus (Hedw.) Warnst. from 
lead-contaminated and non-contaminated populations. J. 
Exp. Bot. 53: 455-463. 
Karunen P. 1990. Th e acyl lipids of bryophytes. In: 
Zinsmeister HD & Mues R (eds.), Bryophytes, their 
chemistry and chemical taxonomy, pp. 121-141, Clarendon 
Press, Oxford. 
Sastav viših masnih kiselina mahovine Rhytididelphus squarrosus (Hedw.) Warnst. (Hylocomiaceae), sakupljene u Nemačkoj za vreme zimskog doba, preliminarno je ispitivan GC i GC-MS analizom. U njenom ekstraktu 
hloroform:metanol 1:1 identifi kovano je devet viših masnih kiselina: arahidonska kiselina (30.7%), α-linolenska 
kiselina (19.1%), linolna kiselina (15.1%), cis-5,8,11,14,17-eikosapentaenska kiselina (14.4%), palmitinska kiselina 
(11.9%), cis-8,11,14-eikosatrienska kiselina (4.1%), oleinska kiselina (2.3%), γ-linolna kiselina (1.4%) i stearinska 
kiselina (1.0%). Dobijeni rezultati ukazuju da je ova biljna vrsta dobar izvor arahidonske kiseline u ispitivanom 
vremenskom periodu.  
Ključne reči: mahovina; Rhytididelphus squarrosus (Hedw.) Warnst; masne kiseline; GC FID; GC-MS.
Osvrt na hemiju masnih kiselina Rhytididelphus 
squarrosus (Hedw.) Warnst.
Boris Pejin, Ljubodrag Vujisić, Aneta Sabovljević, Marko Sabovljević, Vele 
Tešević, Vlatka Vajs
REZIME


Osvrt na hemiju masnih kiselina Rhytididelphus squarrosus (Hedw.) Warnst

Faculty of Chemistry Repository - Cherry

35 (2): (2011) 99-101UDK 582.273:581.42(498) Original Scientifi c PaperReceived 11 November 2010   Revision accepted 17 March 2011© 2011 Institute of Botany and Botanical Garden Jevremovac, Belgrade✳correspondence: borispejin@yahoo.comAn insight into fatty acid chemistry of Rhytididelphus squarrosus (Hedw.) Warnst.Boris Pejin1*, Ljubodrag Vujisić2, Aneta Sabovljević3, Marko Sabovljević3, Vele Tešević1 and Vlatka Vajs2 1 Department of Organic Chemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia 2 Center of Chemistry, Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Njegoševa 12, 11000 Belgrade, Serbia 3 Institute of Botany and Garden, Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, SerbiaABSTRACT: Th e fatty acid composition of the moss Rhytididelphus squarrosus (Hedw.) Warnst. (Hylocomiaceae) collected in Germany during winter time was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Nine fatty acids were identifi ed in its chloroform:methanol extract 1:1: arachidonic acid (30.7%), α-linolenic acid (19.1%), linoleic acid (15.1%), cis-5,8,11,14,17-eicosapentaenoic acid (14.4%), palmitic acid (11.9%), cis-8,11,14-eicosatrienoic acid (4.1%), oleic acid (2.3%), γ-linolenic acid (1.4%) and stearic acid (1.0%). Th e results indicate that this plant species can be a good source of arachidonic acid collected during the winter.    Key words: moss; Rhytididelphus squarrosus; fatty acids; GC FID; GC-MS. INTRODUCTIONBryophytes (mosses, liverworts and hornworts) are the second biggest group of land plants aft er the fl owering plants with approximately 28.000 species spread worldwide. Although the chemical structure and occurrence of fatty acids and their derivatives in bryophytes have been reviewed by Huneck (1969), their chemistry is still poorly known with information on this being very scattered (Asakawa 2007). Partly, this is due to diffi  culties in collecting large quantities of pure material of a certain species. Th e most abundant fatty acids of these plants are also common to most of other organisms. However, typical for many of them is a high content of long-chain polyunsaturated fatty acids, particularly arachidonic acid and cis-5,8,11,14,17-eicosapentaenoic acid, compounds which are not found very abundantly in the rest of the plant kingdom (Gellerman et al. 1975). Indeed, the occurrence of these polyunsaturated fatty acids constitutes an outstanding compositional diff erence between mosses and other higher plants. On the other hand, it is known that these fatty acids are constituents of membranes in animal tissues which are essential for many physiological processes. Arachidonic acid is a precursor of the biologically active prostaglandins and leukotrienes (Marx 1982). As part of our ongoing research on moss chemistry (Pejin et al. 2010, 2011a, 2011b, 2011c), a phytochemical investigation was conducted on the pleurocarp moss Rhytididelphus squarrosus (Hedw.) Warnst. (Hylocomiaceae). Two reports on fatty acids from the moss R. squarrosus have been published to date (Guschina & Harwood 2000, 2002). Th e most abundant fatty acid of this species collected during the summer near Cardiff , UK 100 vol. 35 (2)(July and September, 1998) from both lead-contaminated and non-contaminated areas was arachidonic acid (28.6% and 24.8%, respectively) (Guschina & Harwood 2002). Th e objective of this study was to identify the pattern of fatty acids of the same bryophyte species during the winter time as a contribution to its chemistry. MATERIAL AND METHODSR. squarrosus was collected in Königforst near Köln (Germany) in December 2007. A voucher specimen was deposited in the Herbarium of the Institute of Botany, University of Belgrade, Serbia (bryophyte collection - BEOU No. 4707).Th e moss was carefully selected and cleaned from soil and other contaminants. Th e gametophyte tips were used for the extraction. Air-dried parts of the sample were ground (1g) and extracted three times with chloroform:methanol 1:1 for 1h at room temperature (9.88%, extract yield). Th e extract was evaporated to dryness and trans-esterifi ed with 5% H2SO4 in MeOH (v/v) for 4h at 80°C. Th e resulting methyl esters of fatty acids were analysed by comparing their GC FID chromatograms with that of a standard mixture (Supelco 37) obtained under the same conditions, and/or by analysis of GC-MS data using NIST 5 and Wiley 7 libraries.GC analysis was performed on an Agilent 7890A GC system equipped with 5975C MSD and FID detectors, using a DB-23 column (30 m × 0.25 mm × 0.25 μm). Injection volume was 1 μL and injector temperature was 220°C with a 10:1 split ratio. Carrier gas (He) fl ow rate was 0.9 ml/min while the column temperature was linearly programmed in a range of 150-240°C at a rate of 4°C/min and then held at 240°C for 10 min. Th e transfer line was heated at 240°C. Th ree assay replicates and three biological replicates were analysed. Th e FID detector temperature was 300°C. EI mass spectra (70 eV) were acquired in the m/z range 40-500.RESULTSNine fatty acids were identifi ed in the chloroform:methanol 1:1 extract: arachidonic acid (30.7%), α-linolenic acid (19.1%), linoleic acid (15.1%), cis-5,8,11,14,17-eicosapentaenoic acid (14.4%), palmitic acid (11.9%), cis-8,11,14-eicosatrienoic acid (4.1%), oleic acid (2.3%), γ-linolenic acid (1.4%) and stearic acid (1.0%) (Fig.1).Th ese results for the composition and proportion of fatty acids are in good agreement with the previous report (Guschina & Harwood 2002), particularly regarding the content of arachidonic acid (Fig.2); the main diff erence is in presence of cis-13,16,19-docosatrienoic acid which was detected only in the earlier study (2.1%). Seasonal- and habitat- related changes in the fatty acid composition of various bryophytes exist and these vary from species to species (Karunen 1990). Th e major fatty acid of R. squarrosus seems to be arachidonic acid regardless of place and time of its collection. However, our sample collected during the winter had slightly more arachidonic acid (30.7%) in comparison with those collected in July (28.6%) and September (24.8%) as reported by Guschina & Harwood (2002). Since, the plants were not from the same habitats, in addition to time of the year, this variation could also be due to habitat diff erences.Fig. 2. Arachidonic acid Fig. 1. Fatty acid methyl ester chromatogram for R. squarrosus; palmitic acid (RT 13.156 min); stearic acid (RT 16.205 min); oleic acid (RT 16.673 min); linoleic acid (RT 17.526 min); γ-linolenic acid (RT 18.067 min); α-linolenic acid (RT 18.573 min); cis-8,11,14-eicosatrienoic acid (RT 21.154 min); arachidonic acid (RT 21.502 min); cis-5,8,11,14,17-eicosapentaenoic acid (RT 22.545 min).101B. Pejin et al.: An insight into fatty acid chemistry of Rhytididelphus squarrosus (Hedw.) Warnst.Guschina & Harwood (2002) identifi ed 17 fatty acids, 7 of which were in trace amounts which is in accordance with the 9 fatty acids recorded in this study. For a general consideration of the seasonal-related changes in the fatty acid composition of this moss species, more replicate samples would need to be included in a follow-up study, including in vitro establishment of this moss.Acknowledgments – Th is work was supported by the Ministry of Science and Technological development of the Republic of Serbia (Research grants No. 142053 and 143015). REFERENCES Asakawa Y. 2007. Biologically active compounds from bryophytes. Pure Appl. Chem. 79: 557-580.Huneck S. 1969. Moosinhaltstoff e, eine Uebersicht. J. Hattori Bot. Lab. 32: 1-16. Gellerman JL, Anderson WH, Richardson DG & Schlenk H. 1975. Distribution of arachidonic and eicosapentaenoic acids in the lipids of mosses. Biochim. Biophys. Acta 338: 277-290.Marx JL. 1982. Th e leukotriens in allergy and infl ammation. Science 215: 1380-1383.Pejin B, Vujisic Lj, Sabovljevic M, Sabovljevic A, Tešević V & Vajs V. 2010. Preliminary analysis of fatty acid chemistry of Kinbergia praelonga and Kindbergia stokesii (Brachytheciaceae). J. Serb. Chem. Soc. 75: 1637-1640.Pejin B, Bianco A, Newmaster S, Sabovljevic M, Vujisic Lj, Tesevic V, Vajs V & De Rosa S. 2011a. Fatty acids of Rhodobryum ontariense (Bryaceae). Nat. Prod. Res. (in press)Pejin B, Vujisic Lj, Sabovljevic M, Tesevic V & Vajs V. 2011b. Th e moss Mnium hornum, a promising resource of arachidonic acid. Chem. Nat. Compd.(in press)Pejin B, Vujisic Lj, Sabovljevic M, Tesevic V & Vajs V. 2011c. An insight into fatty acid composition of Calliergonella cuspidata. Asian J. Chem. (in press)Guschina IA & Harwood JL. 2000. Eff ect of copper and lead on lipid metabolism in bryophytes and lichens. Biochem. Soc. Trans. 28: 910-912.Guschina IA & Harwood JL. 2002. Lipid metabolism of the moss Rhytididelphus squarrosus (Hedw.) Warnst. from lead-contaminated and non-contaminated populations. J. Exp. Bot. 53: 455-463. Karunen P. 1990. Th e acyl lipids of bryophytes. In: Zinsmeister HD & Mues R (eds.), Bryophytes, their chemistry and chemical taxonomy, pp. 121-141, Clarendon Press, Oxford. Sastav viših masnih kiselina mahovine Rhytididelphus squarrosus (Hedw.) Warnst. (Hylocomiaceae), sakupljene u Nemačkoj za vreme zimskog doba, preliminarno je ispitivan GC i GC-MS analizom. U njenom ekstraktu hloroform:metanol 1:1 identifi kovano je devet viših masnih kiselina: arahidonska kiselina (30.7%), α-linolenska kiselina (19.1%), linolna kiselina (15.1%), cis-5,8,11,14,17-eikosapentaenska kiselina (14.4%), palmitinska kiselina (11.9%), cis-8,11,14-eikosatrienska kiselina (4.1%), oleinska kiselina (2.3%), γ-linolna kiselina (1.4%) i stearinska kiselina (1.0%). Dobijeni rezultati ukazuju da je ova biljna vrsta dobar izvor arahidonske kiseline u ispitivanom vremenskom periodu.  Ključne reči: mahovina; Rhytididelphus squarrosus (Hedw.) Warnst; masne kiseline; GC FID; GC-MS.Osvrt na hemiju masnih kiselina Rhytididelphus squarrosus (Hedw.) Warnst.Boris Pejin, Ljubodrag Vujisić, Aneta Sabovljević, Marko Sabovljević, Vele Tešević, Vlatka VajsREZIME

Central Repository of the Institute of Chemistry, Technology and Metallurgy (CER)

Cherry - Repository of the Faculty of Chemistry;  University of Belgrade

35 (2): (2011) 99-101
UDK 582.273:581.42(498) 
Original Scientifi c Paper
Received 11 November 2010   Revision accepted 17 March 2011
© 2011 Institute of Botany and Botanical Garden Jevremovac, Belgrade✳correspondence: borispejin@yahoo.com
An insight into fatty acid chemistry of Rhytididelphus 
squarrosus (Hedw.) Warnst.
Boris Pejin1*, Ljubodrag Vujisić2, Aneta Sabovljević3, Marko Sabovljević3, Vele 
Tešević1 and Vlatka Vajs2 
1 Department of Organic Chemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 16, 11000 Belgrade, 
Serbia 
2 Center of Chemistry, Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Njegoševa 12, 
11000 Belgrade, Serbia 
3 Institute of Botany and Garden, Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, Serbia
ABSTRACT: Th e fatty acid composition of the moss Rhytididelphus squarrosus (Hedw.) Warnst. (Hylocomiaceae) 
collected in Germany during winter time was analyzed by gas chromatography (GC) and 
gas chromatography-mass spectrometry (GC-MS). Nine fatty acids were identifi ed in its 
chloroform:methanol extract 1:1: arachidonic acid (30.7%), α-linolenic acid (19.1%), linoleic 
acid (15.1%), cis-5,8,11,14,17-eicosapentaenoic acid (14.4%), palmitic acid (11.9%), cis-8,11,14-
eicosatrienoic acid (4.1%), oleic acid (2.3%), γ-linolenic acid (1.4%) and stearic acid (1.0%). Th e 
results indicate that this plant species can be a good source of arachidonic acid collected during the 
winter. 
   Key words: moss; Rhytididelphus squarrosus; fatty acids; GC FID; GC-MS. 
INTRODUCTION
Bryophytes (mosses, liverworts and hornworts) are 
the second biggest group of land plants aft er the 
fl owering plants with approximately 28.000 species 
spread worldwide. Although the chemical structure 
and occurrence of fatty acids and their derivatives in 
bryophytes have been reviewed by Huneck (1969), their 
chemistry is still poorly known with information on this 
being very scattered (Asakawa 2007). Partly, this is due 
to diffi  culties in collecting large quantities of pure material 
of a certain species. Th e most abundant fatty acids of 
these plants are also common to most of other organisms. 
However, typical for many of them is a high content 
of long-chain polyunsaturated fatty acids, particularly 
arachidonic acid and cis-5,8,11,14,17-eicosapentaenoic 
acid, compounds which are not found very abundantly in 
the rest of the plant kingdom (Gellerman et al. 1975). 
Indeed, the occurrence of these polyunsaturated fatty 
acids constitutes an outstanding compositional diff erence 
between mosses and other higher plants. On the other 
hand, it is known that these fatty acids are constituents of 
membranes in animal tissues which are essential for many 
physiological processes. Arachidonic acid is a precursor 
of the biologically active prostaglandins and leukotrienes 
(Marx 1982). 
As part of our ongoing research on moss chemistry 
(Pejin et al. 2010, 2011a, 2011b, 2011c), a phytochemical 
investigation was conducted on the pleurocarp 
moss Rhytididelphus squarrosus (Hedw.) Warnst. 
(Hylocomiaceae). Two reports on fatty acids from the moss 
R. squarrosus have been published to date (Guschina & 
Harwood 2000, 2002). Th e most abundant fatty acid of 
this species collected during the summer near Cardiff , UK 
100 vol. 35 (2)
(July and September, 1998) from both lead-contaminated 
and non-contaminated areas was arachidonic acid (28.6% 
and 24.8%, respectively) (Guschina & Harwood 2002). 
Th e objective of this study was to identify the pattern of 
fatty acids of the same bryophyte species during the winter 
time as a contribution to its chemistry. 
MATERIAL AND METHODS
R. squarrosus was collected in Königforst near Köln 
(Germany) in December 2007. A voucher specimen was 
deposited in the Herbarium of the Institute of Botany, 
University of Belgrade, Serbia (bryophyte collection - 
BEOU No. 4707).
Th e moss was carefully selected and cleaned from soil 
and other contaminants. Th e gametophyte tips were used 
for the extraction. Air-dried parts of the sample were ground 
(1g) and extracted three times with chloroform:methanol 
1:1 for 1h at room temperature (9.88%, extract yield). Th e 
extract was evaporated to dryness and trans-esterifi ed with 
5% H2SO4 in MeOH (v/v) for 4h at 80°C. Th e resulting 
methyl esters of fatty acids were analysed by comparing 
their GC FID chromatograms with that of a standard 
mixture (Supelco 37) obtained under the same conditions, 
and/or by analysis of GC-MS data using NIST 5 and Wiley 
7 libraries.
GC analysis was performed on an Agilent 7890A GC 
system equipped with 5975C MSD and FID detectors, 
using a DB-23 column (30 m × 0.25 mm × 0.25 μm). 
Injection volume was 1 μL and injector temperature was 
220°C with a 10:1 split ratio. Carrier gas (He) fl ow rate was 
0.9 ml/min while the column temperature was linearly 
programmed in a range of 150-240°C at a rate of 4°C/min 
and then held at 240°C for 10 min. Th e transfer line was 
heated at 240°C. Th ree assay replicates and three biological 
replicates were analysed. Th e FID detector temperature 
was 300°C. EI mass spectra (70 eV) were acquired in the 
m/z range 40-500.
RESULTS
Nine fatty acids were identifi ed in the chloroform:methanol 
1:1 extract: arachidonic acid (30.7%), α-linolenic 
acid (19.1%), linoleic acid (15.1%), cis-5,8,11,14,17-
eicosapentaenoic acid (14.4%), palmitic acid (11.9%), 
cis-8,11,14-eicosatrienoic acid (4.1%), oleic acid (2.3%), 
γ-linolenic acid (1.4%) and stearic acid (1.0%) (Fig.1).
Th ese results for the composition and proportion of 
fatty acids are in good agreement with the previous report 
(Guschina & Harwood 2002), particularly regarding the 
content of arachidonic acid (Fig.2); the main diff erence 
is in presence of cis-13,16,19-docosatrienoic acid which 
was detected only in the earlier study (2.1%). Seasonal- 
and habitat- related changes in the fatty acid composition 
of various bryophytes exist and these vary from species 
to species (Karunen 1990). Th e major fatty acid of R. 
squarrosus seems to be arachidonic acid regardless of 
place and time of its collection. However, our sample 
collected during the winter had slightly more arachidonic 
acid (30.7%) in comparison with those collected in July 
(28.6%) and September (24.8%) as reported by Guschina 
& Harwood (2002). Since, the plants were not from the 
same habitats, in addition to time of the year, this variation 
could also be due to habitat diff erences.
Fig. 2. Arachidonic acid 
Fig. 1. Fatty acid methyl ester 
chromatogram for R. squarrosus; 
palmitic acid (RT 13.156 min); stearic 
acid (RT 16.205 min); oleic acid (RT 
16.673 min); linoleic acid (RT 17.526 
min); γ-linolenic acid (RT 18.067 
min); α-linolenic acid (RT 18.573 
min); cis-8,11,14-eicosatrienoic acid 
(RT 21.154 min); arachidonic acid 
(RT 21.502 min); cis-5,8,11,14,17-
eicosapentaenoic acid (RT 22.545 
min).
101B. Pejin et al.: An insight into fatty acid chemistry of Rhytididelphus squarrosus (Hedw.) Warnst.
Guschina & Harwood (2002) identifi ed 17 fatty acids, 
7 of which were in trace amounts which is in accordance 
with the 9 fatty acids recorded in this study. 
For a general consideration of the seasonal-related 
changes in the fatty acid composition of this moss species, 
more replicate samples would need to be included in a 
follow-up study, including in vitro establishment of this 
moss.
Acknowledgments – Th is work was supported by the 
Ministry of Science and Technological development of 
the Republic of Serbia (Research grants No. 142053 and 
143015). 
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Sastav viših masnih kiselina mahovine Rhytididelphus squarrosus (Hedw.) Warnst. (Hylocomiaceae), sakupljene u Nemačkoj za vreme zimskog doba, preliminarno je ispitivan GC i GC-MS analizom. U njenom ekstraktu 
hloroform:metanol 1:1 identifi kovano je devet viših masnih kiselina: arahidonska kiselina (30.7%), α-linolenska 
kiselina (19.1%), linolna kiselina (15.1%), cis-5,8,11,14,17-eikosapentaenska kiselina (14.4%), palmitinska kiselina 
(11.9%), cis-8,11,14-eikosatrienska kiselina (4.1%), oleinska kiselina (2.3%), γ-linolna kiselina (1.4%) i stearinska 
kiselina (1.0%). Dobijeni rezultati ukazuju da je ova biljna vrsta dobar izvor arahidonske kiseline u ispitivanom 
vremenskom periodu.  
Ključne reči: mahovina; Rhytididelphus squarrosus (Hedw.) Warnst; masne kiseline; GC FID; GC-MS.
Osvrt na hemiju masnih kiselina Rhytididelphus 
squarrosus (Hedw.) Warnst.
Boris Pejin, Ljubodrag Vujisić, Aneta Sabovljević, Marko Sabovljević, Vele 
Tešević, Vlatka Vajs
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Osvrt na hemiju masnih kiselina Rhytididelphus squarrosus (Hedw.) Warnst

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