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NVEO 2019, Volume 6, Issue 1

CONTENTS

1. The history of the ISEO: 1969-2019 / Pages: 1-17 Jan Karlsen

2. Essential oil composition of two endemic Centaurea species from Turkey / Pages: 18-24 Hüseyin Servi, Sezgin Çelik and Ramazan Süleyman Göktürk

3. Antiglycation and antiaggregation potential of thymoquinone / Pages: 25-33 Dinesh Kumar and Ahmad Ali

4. Volatile compositions of three critically endangered and endemic species of the genus Crocus L. (Iridiaceae) and comparison with C. sativus L. (Saffron) / Pages: 34-39 Sevim Küçük, Melike Sayarer and Betül Demirci

5. Characterization of Salvia verticillata L. subsp. amasiaca (Freyn & Bornm.) Bornm. essential oil from Turkey / Pages: 40-46 Nilüfer VURAL, İsmihan GÖZE and Nazlı ERCAN

Nat. Volatiles & Essent. Oils, 2019; 6 (1): 1-17 Karlsen J.

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REVIEW

The history of the ISEO: 1969-2019 Jan Karlsen

Department of Pharmaceutics, University of Oslo, Oslo, NORWAY

Jan Karlsen, PhD, Pharm., Professor of Galenic Pharmacy, passed away 21st of March 2019.

Abstract

Half of a decade of essential oil research was reviewed in a historical aspect starting from the first meeting in 1969, which was

transformed to an international high scientific level named; Inteional Symposium on Essential Oils (ISEO).

Keywords: Essential oils, historical, meeting

Introduction

The International Symposium on Essential Oils (ISEO) was initiated by a few enthusiastic scientists in the heart

of Europe a half Century ago. Our 50 ISEO meetings have been a great inspiration to scientists working in the

field of essential oils and related areas. We have moved from the first applications of gas chromatography

(GC) on the volatiles of plants separating an essential oil into a limited number of constituents to the fantastic

separations described in publications during the last 5 years. With the much improved separations of the

constituents during the 1970s new research areas within the study of essential oils opened up. Single

constituents could be isolated, identified by structure elucidation, and by retention indices. The ISEO

meetings could benefit from this by changing or better enlarging the research topics to attract more scientists

to the meetings. The ISEO symposia became a meeting place where contact across scientific areas was made.

Having essential oils as a main topic, but allowing the meetings to be held in places where the interest of

essential oils had different goals and aims, allowed the meetings to become varied in the outlook of terpene

and volatiles research.

Timelines and venues

Over the years, ISEOs were organized:

Table 1. Meetings to Symposia, Venue and Organizers

Year Venue Host

initiation 1969 Leiden, The Netherlands A. Baerheim-Svendsen

1 1970 Leiden, The Netherlands A. Baerheim-Svendsen

2 1971 Freiburg, Germany F.-W. Hefendehl

3 1972 Helsinki, Finland M. von Schantz



6 1975 Leiden, The Netherlands A. Baerheim-Svendsen

7 1976 Würzburg, Germany K.-H. Kubeczka


9 1978 Münster, Germany H. Hörster



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11 1980 Groningen, The Netherlands M.H. Boelens, H. Hendriks

12 1981 Marburg, Germany K.-H. Kubezka


14 1983 Freising-Weihenstephan, Germany Ch. Franz

15 1984 Leiden The Netherlands A. Baerheim-Svendsen

16 1985 Holzminden/Neuhaus, Germany E.-J. Brunke

17 1986 Bad Bevensen, Germany E. Stahl-Biskup

18 1987 Nordwijkerhout, The Netherlands J.J.C. Scheffer

19 1988 Zürich-Greifensee, Switzerland D. Lamparsky, R. Kaiser


21 1990 Lahti, Finland R. Hiltunen

22 1991 St.Vincent, Italy C. Bicchi

23 1992 Auchincruive, UK S. Deans, K. Svoboda

24 1993 Berlin, Germany P. Weyerstahl, H. Schilcher

25 1994 Grasse, France D. Joulain

26 1995 Hamburg, Germany K.-H. Kubeczka

27 1996 Wien/Vienna, Austria Ch. Franz, G. Buchbauer

28 1997 Eskisehir, Turkey K.H.C. Baser, N. Kirimer

29 1998 Frankfurt, Germany W. König, A. Mosandl

30 1999 Leipzig/Miltitz, Germany Bell Flavours & Fragrances

31 2000 Hamburg, Germany K.-H. Kubeczka, W. König

32 2001 Wroclaw, Poland S. Lochynski,

33 2002 Lisbon, Portugal A. Figueiredo


35 2004 Giardini Naxos, Italy L. Mondello, P. Dugo

36 2005 Budapest, Hungary E. Nemeth

37 2006 Grasse, France D. Joulain

38 2007 Graz, Austria Ch. Franz, J. Novak, R. Bauer

39 2008 Quedlinburg, Germany J. Schulz

40 2009 Savigliano, Italy C. Bicchi, P. Rubiolo

41 2010 Wroclaw, Poland S. Lochynski

42 2011 Antalya, Turkey K.H.C. Baser, F. Demirci

43 2012 Lisboa, Portugal A. Figueiredo

44 2013 Budapest, Hungary E. Nemeth

45 2014 Istanbul, Turkey K.H.C. Baser, F. Demirci

46 2015 Lublin, Poland A. Ludwiczuk

47 2016 Nice, France N. Baldovini

48 2017 Pecs, Hungary G. Horvath

49 2018 Nis, Serbia N. Radulovic

50 2019 (to be organized) Vienna, Austria J. Novak, I. Stappen, Ch. Franz

In 1965 two Norwegians came to De Rijksuniversiteit te Leiden, Leiden, The Netherlands: Professor Anders

Baerheim Svendsen and me as this assistant started to reorganize the Department of Pharmacognosy from

a descriptive, botanical research oriented department into a department for the study of interesting medical

natural products with emphasis on chromatographic techniques, isolation of pure compounds and

spectroscopic structure elucidation. Anders Baerheim Svendsen had already a group of friends as professors


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of Pharmacognosy in Germany, Switzerland and Brazil who wanted to change the emphasis of this university

subject of pharmacognosy from botany into a study of natural products with interesting biological/medical

effects following the ideas of Max von Schantz in Helsinki, Iconomou in Athens, Richard Wasicky in Sao Paolo.

This was the time when the chromatographic techniques were in very active development, and especially gas

chromatography. We have decided to choose essential oils as research topic as this was obviously the most

complex, demanding and difficult natural occurring samples to study at that time using the available

separation techniques. We were both fascinated by the separation power of the new gas chromatographic

equipment as this obviously would lead to a better understanding of the complexity of volatiles in plant

material. We were given a very nice budget by the director of the institute (Pharmaceutisch Laboratorium)

and were able to purchase the first gas chromatographs within few weeks after arriving in Leiden in 1965).

Figure 1. Prof. Dr. Anders Baerheim-Svendsen walking on the streets of Leiden taking photos of our “new” city.

These were the then excellent Varian GC’s. However, very soon we discovered that the GC instrumentation

available needed improvements for very volatile compounds analysis. We also had to develop preparative

GC equipment to isolate pure compounds for the NMR, MS or FT-IR. An active collaboration with the

company Becker Delft BV, Delft was very practical for this project. We had a lot of fun modifying a water bath

to be used as the GC-oven allowing us to do GC-analyses down to and below zero degrees. The spectroscopic

instrumentations were also in a very active industrial development (improving the sensitivity) following the

possibility to isolate pure natural products from plants by the new chromatographic instrumentation

available. It was period of hectic instrumentation development and something of a paradise for them in

Leiden as we had a modern instrument workshop at their disposal who could help them in the improvement

of the instrumentation and by making it possible to build new equipment. Parallel with the development of


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gas chromatography we were building equipment in 1966 for column chromatography under pressure, an

activity which started the instrumentation development of HPLC!

Figure 2. A very satisfied Prof. A. Baerheim Svendsen, when he and myself managed to run the first terpene analysis from Juniperus communis L. in August 1965 in the new laboratory

During the next years, we were occupied developing and improving the chromatographic instruments and to

establish the Department of Pharmacognosy as a centre for natural products research. Working in a very

active area of natural products it was natural to seek other young scientists interested in essential oil analysis

for regular discussions. This was thoroughly discussed between Anders and me planning for the future as we

wanted to make a discussion group, which would hopefully last for many years. This led them to approach

K.-H. Kubeczka who was using GC instrumentation and interested in method development and F.W.

Hefendehl, who had just published some interesting papers on the essential oil in Mentha leaves by analysing

the essential oil in single oil glands /cells in leaves. Another interested young scientist was Simo Juvonen from

Helsinki, who did some outstanding research on the needle of Pinus-species in Finland.


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Figure 3. Anders and myself were testing the newly made brandy of cloudberries to observe if we had managed to keep the taste and smell during the production. Many of the flavouring agents naturally occurring in the plant material disappeared without a trace during the production procedures and posed a big problem for the product development. This was often the driving force behind our interest in the natural volatiles and initiated also contact with flavour and fragrance industry in The Netherlands and abroad.

Anders and myself approached Karl-Heinz Kubeczka and Friedrich Wilhelm Hefendehl at the GA meeting in

Würzburg, in 1968 with a proposal to make a discussion group for all aspects of essential oil research and we

agreed to participate. One could say that our meeting in Bürgerspital in Würzburg with many bottles of

“Eschendorfer Lump” started the ISEO idea.

In The Netherlands, I was a member of a chromatography discussion group since 1966, and found this kind

of arrangement very suitable for a discussion group in pharmacognosy and specifically on the applications of

chromatography of the terpenes. The driving force behind the development was the change of research in

pharmacognosy from emphasis on botany to the chemical study of natural occurring compounds. To make

this practical we decided to meet a day after the annual GA meetings to start the discussion group with any

questions regarding the essential oils (separation of terpenes, chemistry, chemotaxonomy or other relevant

topics regarding the constituents of essential oils). Even then we discussed to enlarge the topics to other

volatiles in nature in general but left this question to future meetings. It was a very active time in

instrumentation development (end of 1960 and far into the 1970) where we introduced standard capillary

GC, enantiomeric stationary phases, micromanipulators used in medicine to pick out single oil cells for

analysis, solid sampling GC to improve the sensitivity, and micro cells for IR analyses. It was the time also in

ISEO for discussing instrument improvements as main topic for the meetings. This was the start of the ISEO

meetings.


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In 1969, we had for disposal capillary GC instrumentation, and the first meeting with invited speakers took

place in Leiden with two main speakers on capillary GC applications. The heads of analytical research from

British Petroleum (BP) and from Shell gave their experiences from the petrochemical analysis by capillary GC.

We also had nice collaboration with a company in Delft (Becker Delft BV), on the development of preparative

GC equipment and a representative from this company was giving a lecture, too. It was a time when the

instrumental development fitted perfectly with the complexity of the essential oil composition. I also

especially remember Dr. Herout from Prague, who was one of the authors of a large number of publications

on the structure of sesquiterpenes from essential oils together with Sorm and co-workers. Herout visited him

several times in Leiden to discuss instrumental developments. I can never forget how he described the

continuously distillation to isolate 50 Litres of Mentha-oil and the fractionated distillation to isolate enough

of single sesquiterpenes to be able to do NMR analysis and structure elucidation. Whether the compounds

isolated were artefacts formed during hours of distillation or not, was not a topic of discussion! The additional

knowledge gathered on the composition of the essential oil compounds, developing NMR as well as

hyphenated GC-MS techniques matched very well anyone interested in essential oils. Gradually the

instrumental techniques became better and much more sensitive. For many years, the discussion on

terpenes in plants focussed upon why the plants accumulated these lipophilic constituents in special

“containers” in the living plant. What was the biochemical, and what was the biological reason of these

compounds?

We focussed on the “essential oils” because this was a well-known product of pharmaceutical interest and

because the analytical techniques at that time (around 1970) was very suitable for essential oil

characterization. However, the scientific community consisted of a conservative group of scientists, and it

took many years before a more thorough discussion on the biological aspects of terpenes in plants,

enzymology of terpene formation and biological effects of volatiles took place. It was said that we have spent

the best part of the last 20 years for these discussions. Separations techniques are no longer a big problem,

structure elucidation of isolated volatiles likewise relatively easy and solvable.

Very soon the one-day meeting in connection with the GA meetings grew in popularity and we decided to

set up a separate meeting for anyone interested in essential oils /terpenes, the Symposium Ätherische Öle,

which was changed around 1980 into International Symposium on Essential Oils (ISEO). Young scientists from

many areas found the ISEO meetings very instructive and we became like a family of scientists with different

backgrounds but with the same goal to gain better knowledge about the terpenes and the essential oils. We

had discussed how to promote the meetings and decided upon inviting young scientists from every area

interested in essential oils and not to limit the participation to specific groups. Some of these former young

scientists are still members of the scientific committee of the ISEO.

From the start, we did not want to make a new society of scientists with rules and regulations. We wanted

to keep it at the informal level without any other guiding group than a permanent scientific committee (also

known as 'Monday Evening Group'). We also discussed the issue of reaching a broader variety of scientists

such as those interested in synthetic chemistry, separation techniques, structure elucidation, traditional

pharmacognosy, biodiversity, agronomy and biological effects of terpenes, all could attend these future

meetings. The main issue for the ISEO Scientific Committee was to decide upon the next venue so naturally

we had a discussion on the suitable places. The person in charge of the next meeting should be or become

member of the scientific committee. This system has worked now for 50 years without any trouble.


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With the passing of the years, we moved on in their careers and changed positions. Some of them stayed on

at the university and some joined other governmental laboratories or industries and this was very healthy

for new recruitment of scientists for the ISEO.

Friedrich Wilhelm Hefendehl joined the governmental laboratory in Berlin and disappeared from the ISEO

meetings. Karl-Heinz Kubeczka wanted to enlarge the ISEO meetings and the number of participants and

proposed more contact with industry. He did a lot of promotion of the ISEO and attracted the interest (and

participants) from industry. I went back to Norway and joined the Department of Drug formulation at the

University of Oslo. Simo Juvonen bought a pharmacy in Finland and became the traditional retail pharmacy

owner/apotheker. Karlsen kept his subscription of the journal “Perfumer and Flavorist” to follow the

development of flavour and fragrance chemistry and participated in the world-wide meetings of the Flavour

and Fragrance industry (International Congresses on Essential Oils, Fragrances and Flavours). In this way, he

kept contact with the research area of terpenes and essential oils although his research area now had focus

upon drug formulation, encapsulation and stability problems of natural products.

Many new members of the ISEO committee joined in the study of the terpenes and volatiles. After a spell of

5-6 years, working with pharmaceutical industry and drug formulation I joined again the ISEO meetings, since

he missed his old scientist friends from the terpene research area.

The topics of the ISEO has changed according to discussion of the scientific committee. The flavour and

fragrance industry with their representatives joined in the years 70, hosted meetings, supporting the annual

venue of the ISEO, and made it possible to keep this annual meeting running for many years. Emphasis of the

meeting changed from purely analytical meetings via agricultural problems of the essential oil plants

standardization up to microbiological applications - then back again to hyphenated techniques and their

application to essential oils. I was particularly happy that we could invite Rodney Croteau and some of his

collaborators to discuss the biosynthesis of the terpenes, a topic that we should again focus upon, as well as

biotransformation of the terpenes (which terpenes are really present in the living plant). In the 1980s more

investigations on the biological effects of the volatile terpenes started to emerge from the poster sessions.

We even had an aromatherapist (Maria Lis-Balchin) giving lectures some years at the ISEO.

Katerina Svoboda published some fantastic pictures of essential oil cells in the living plant as a book and

Alexander Pauli collected an enormous data base on the antimicrobial effect of specific essential oils. We had

Roman Kaiser showing how plants (especially Orchids!) emitted complex scents at different times of the

day/night to attract specific insects, and his transportable laboratory with a hot-air balloon with head-space

collections of emitted fragrance mixtures will not easily be forgotten. We still see the pictures of the

laboratory moving between the treetops in the jungle of the Amazons by the help of a hot-air balloon. His

book “The Scent of Orchids” has always been one of the favourite books of scents and perfumery.

During these years all aspects of essential oil research has been covered. It was quite natural that Gerhard

Buchbauer and K. Hüsnü Can Başer invited authors to join them in making a “Bible” for essential oil research

resulting in “The Handbook of Essential Oils “, which has become a great success. Not to forget the books

edited by and also written/collected by Karl-Heinz Kubeczka and other colleagues on the analysis of essential

oils (see Figures 4-11).

There are many scientists who have contributed to the success of the ISEOs: Anders Baerheim Svendsen,

Karl-Heinz Kubeczka, Friedrich Wilhelm Hefendehl, Chlodwig Franz, Heinz Schilcher, Peter Weyerstahl, Carlo

Bicchi, Roman Kaiser, Wilfried König, Brian Lawrence, Daniel Joulain, Eva Nemeth, Gerhard Buchbauer,

Stanislaw Lochynski, K. Hüsnü Can Baser, Fatih Demirci, Hans Scheffer, Luigi Mondello, Yoshinori Asakawa,


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Yoshiaki Noma, Nicolas Baldovini, Armin Mosandl, Elisabeth Stahl, Katerina P. Svoboda, Patricia Rubiolo,

Agnieszka Ludwiczuk, Heinz Hörster, Danuta Kalemba, Otto Sticher, Ana Cristina Figueiredo, Niko

Radulovic…..and many others.

Gradually, more emphasis on the biological effects of volatile terpenes became the main issue of many of

the later meetings on the ISEO. Even though there were more development in automation of the

chromatographic equipment, new two-dimensional methods of GC and HPLC were applied to terpene

analyses, terpene encapsulation became an interesting research area and the use of terpene-containing

plants as additives to animal feed could become an interesting field for the future etc. In this way, the topics

of the ISEO changed according to the interest from the applied field of volatile oils.

Changes of topics/Topic development

We started out with long discussions on the separation techniques for the terpenes and were trying to

improve the methods during the first 10 years of the ISEO. Many of the ISEO participants were also interested

in the biochemistry of the lower terpenes, and in the biosynthesis of these compounds. It was a great

pleasure that Rodney Croteau agreed to come and give an overview and the latest results of his work on this

very interesting group of natural products. He clearly enabled to understand the complexity of the

biosynthesis of the terpenes, the complexity of enzymes active in the plant and the difficulties met when

trying to manipulate the enzymatic system. His talk gave many of them ideas for future research. However,

one should not forget another very important development in the chromatographic separation of the

terpenes, the development of the enantiomeric column, allowing to differentiate between (+) and (-)-

enantiomers of the monoterpenes. Instrumental in this area were Armin Mosandl and Wilfried König, who

both gave several and interesting talks on the importance of enantiomeric forms of the volatiles. This could

easily be applied to the verification of the oils in the market as well as giving them new ideas (and problems)

in the field of the formation of the terpenes. Meanwhile, the separation of the terpenes had been brought

so far that the publication of lists of constituents in an essential oil made little sense since many of the

compounds detected could not possibly be present and the components identified on the basis of retention

time on a single column and mass spectrum/library search became dubious. What was really present in the

plant and what was purely artefacts originating in the isolation process of the essential oil was an important

topic of discussion in the papers. This topic still needs further evaluation Two-dimensional GC efficiently

reported on by Carlo Bicchi and Luigi Mondello clearly showed that many of the so-called baseline separated

single peaks contained two or often more compounds. A proposal to enlarge the topics of the ISEO also to

include other natural volatiles seemed “natural” as both analytical techniques, biosynthesis and biological

effect studies will have to cover both groups. Essential oils are, after all, an artificial name of a group of

natural products originally isolated by distillation and being lipophilic. In his opinion, this is a too narrow

definition for the interest of the ISEO, and we needed to cover all natural occurring volatiles in the future!

The group “essential oils” is a commercial product well known to pharmacists for several hundreds of years

and has been kept by the ISEO maybe with a kind of respect to history. Volatiles in general requires special

isolation techniques as well as typical formulation like encapsulation, which will enable us to study their

biological effect without the compounds disappearing before the experimental endpoint! In our opinion, a

natural development would be to enlarge our interest to volatiles in general as the industrial interest in

volatiles is growing.

One must not forget Chlodwig Franz who generously hosted special scientific committee meetings at his

house in Austria, to discuss the future of the ISEO meetings, namely “the Bach Meetings”. A very laudable

initiative for important discussions between the most active members of our “essential oil family/ scientific


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committee”. These meetings have resulted in recommendations for the scientists studying terpenes and

essential oils. These recommendations are guidelines for younger scientists to ensure repeatability of the

experiments carried out on volatiles (Franz, 2002; .

Meanwhile, the perfumery industry partly moved on to synthetic compounds and away from the essential

oils as raw material for their products. Interest in the essential oils seemed to diminish. However,

biotransformation of terpenes, terpenes as antibacterial, importance of the terpenes for the functioning of

the plant and not to forget the renewed interest in the essential oils from other parts of the world seemed

to give the ISEO new energy. The biological effects of the terpenes/essential oils were taking over the

chemistry and separation techniques as main interest for the participants, as e.g. the use of essential oils in

animal health and feeding. This was regarded by me to be an important direction of the essential oil research.

Instead of only investigating the essential oils for the benefit of perfumery, now we could really study the

essentials of the terpene formation and functioning. ISEO had moved from the area of academic interest into

separation techniques, to industrial products like perfumery, and then back to the biological effect in the

plant and in medical applications. For the biological effects of the volatiles in the plants we would like to

emphasize the work of Roman Kaiser on the scent of orchids and their diurnal variation. Interestingly, if one

look at the research speciality of the participants, it covers a very broad aspect of essential oil research from

agricultural problems, governmental regulations and separation techniques to biological effects, formulation

of terpene products, synthesis of olfactory important copies of natural occurring compounds and stability of

the volatile terpenes. In our opinion this is the key to the success we have had with the ISEO meetings and

kept it alive for 50 years.

Figure 4. Publication of the workshops edited by K.-H. Kubeczka (1976-1978).


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Figure 5. Proceedings of the 15th ISEO, 1984, released by M. Nijhoff & Dr. W. Junk Publishers (1985).

Figure 6. Special issue published in FFJ, 1987 by K.-H. Kubeczka.


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Figure 7. Proceedings of the 27th ISEO, edited by Ch. Franz, A. Mathe, G. Buchbauer (1997).

Figure 8. Proceedings of the 28th ISEO, edited by K. H. C. Başer & N. Kirimer (1998).


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The participants

The most important task of an informal organization is the attraction of younger scientists as new regular

participants. There is a permanent slow change in the Scientific Committee due to the fact that no official

‘elections of the board’ are necessary but elder members of the committee retire and by that way younger

scientists are able to influence and define ISEO anew. So far, it seems that this gradual change is continuous.

The future and status 2019:

Actually, we have at our disposal

- an annual meeting (ISEO)

- a web site: http://iseo-pc.org

- journals (JEOR, FFJ and recently NVEO)

- a regular number of participants

- a scientific committee that works

- and after 50 years a definite place in the scientific community with a number of younger scientists

participating in the organization and the meetings.

Figure 9: Number of participants at ISEO Meetings from 1974 (Freiburg) until 2009 (Savigliano).


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It is first and foremost this last point that has kept ISEO alive and active all these years. The running of the

ISEO does not depend upon only “older, experienced” scientists but equally well upon new members of the

ISEO family. The open organization of the ISEO is very special but it works with special arrangements for

young scientists. Let us celebrate the 50th anniversary of the ISEO as it is!

Figure 10. Two abstracts by the lectures made by Karl-Heinz Kubeczka at the 25th and the 40th ISEO meeting

covering the historical developments

Figure 11. Progress in Essential Oil Research, edited by E.-J. Brunke (1986)


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Important guidelines

“for good practices of carrying out the study of essential oils and natural volatiles”

These guidelines are intended to be of assistance to researchers working in the field of essential oils (EO) and

natural volatiles, and set the basis of good practice in experimental research and communication of results.

These guidelines are not all mandatory, as depending on the specific goals of the research considered, i.e.

some recommendations may not be essential if we are not directly related to the main goal of the study.

There are still many scientific papers, poster presentations and lectures presented during symposia in which

the term of EO is used incorrectly. Because of this, at the beginning of these guidelines, it is necessary to

define what is the ‘Essential Oil’. EO are complex mixtures of volatile compounds produced by living

organisms and isolated by physical means only (pressing and distillation) from a whole plant or plant part of

known taxonomic origin. In contrast, extracts obtained by solvent extraction with different organic solvents

or by supercritical fluid extraction (SFE) may not be considered as essential oils.

These guidelines are formally divided into those dealing with chemical and biological issues, but the biological

part cannot be considered separately from the chemical one, as both are generally dependent one on

another.

1. The chemical issues span from sampling of the material to identification and quantification of

the volatile constituents.

The sampling of the material has to be representative and in accordance with the goal of the study.

Researchers should define if characterization of a species, catching of intraspecific variability, comparison of

different populations, accessions or detecting the role of special factors on the EO variability is the focus of

the study. And, for example, a study demonstrating an infraspecific variability of EO composition should be

based on irreproachable and statistically significant sampling procedures, while a description of the volatiles

of a rare and endangered new species may rely on the analysis of a single sample. In parallel with this, care

should be taken to sampling the appropriate plant part, at the appropriate time and developmental phase

each of which should be defined. Besides, geographical coordinates of the collection place are mandatory.

The valid botanical identification of plant material has to be backed up by a deposition of voucher specimens

in a herbarium or a collection, freely available to other interested researchers. Good quality photographs or

scans of the sample specimen should be provided as supplementary material, to facilitate the confirmation

of the correct botanical identification.

Repeatable and clearly described sample preparation methods should be used, including the biological and

laboratory replicates. Contamination and artefact formation should be avoided/kept to a minimum as much

as possible in the context of the main goal of the study. The yield of the volatiles should be expressed as a

percentage based on dry material content of the plant sample.

The GC-MS-based identification of constituents has to rely upon repeatable and accurate analytical

procedures. It should be based on the comparison with authentic mass spectra and retention index data. The

use of columns of different polarity to provide a set of different retention indices is encouraged. Co-injection

experiments of reference compounds are highly recommended. Other means of identification, such as

chemical transformations, fractionations leading to structural analysis etc., significantly improve the quality

of the work and are much appreciated. Determination of the enantiomeric ratio of the constituents by chiral

GC is another highly welcomed addition. The raw GC-MS electronic files ( .D, .MS, .MSF, .R##, .SMS etc. type

files) of the main analyses described in the work should be provided as supplementary data.


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Quantification of constituents has to be performed by internal standardization using GC-FID or GC-MS as

recommended by IOFI and reliable recent publications (Cachet et al., 2016; Begnaud & Chaintreau, 2016).

2. The biological properties of EO samples and/or their components constitute the second main

part of EO research.

Testing of biological activities can be done in vitro or in vivo, on 3 different forms of EO: on the entire oil as

such, on the oil in a soluble form (to improve its bioavailability), or on encapsulated oil (to enhance its contact

time with the targeted biological system). Here again, the experimental protocols should be validated and

repeatable, in direct link with the goal of the study, and described in sufficient detail to be reproduced by

other researchers.

Many types of biological activities can be explored to characterize the potential valorization of EOs for

human, veterinary and agricultural applications (antimicrobial, antioxidant, various systems including central

nervous system, upper respiratory tract, cardiovascular system, dermatological applications; insect

repellent/attractant, pest management etc.). Many other types of biological activities deserve attention and

should be studied to expand this non-exhaustive list of potential EO research axes. However, it should be

considered that the effect of complex mixtures such as EOs on even more complex systems (living organisms)

is far from simple and should not be restricted to the effect of a single substance on a specific target, but

rather an intricate combination of synergistic/antagonistic interactions requiring advanced testing

procedures. In this context, it must be stressed that studies without a sound chemical characterization of the

EO sample tested are completely meaningless and even in some instances hazardous. In biological activity

tests, both in vitro and in vivo, safety issues need to be followed and non-toxic concentrations need to be

applied/considered according to current literature.

There are several methods to perform antimicrobial activity of different extracts, compounds. A review

published in 2016 summarized the in vitro assays (Balouiri et al., 2016). The examination of antimicrobial

activity of essential oils or components is recommended according to standards published by the Clinical and

Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing

(EUCAST). The following data are recommended to indicate in the article:

- Source of essential oil and/or components (e.g. distilled or obtained from market)

- Solvent should be indicated

- Positive and negative controls are also recommended

- Type of strains (e.g. ATCC or from other culture collection)

- In case of strains isolated from clinical samples, ethical approval or certificate should be recommended

- Growth conditions of microorganisms

- If new method is applied, detailed description should be recommended

- In case of interaction/combination tests, intervals of FICI should be indicated

- Calculation of values of activity should be described (e.g. MIC, MBC)

- Statistical analysis should be recommended (e.g. in case of diameter of inhibition zones)

To study the antimicrobial activity of EOs the following methods are recommended (due to the volatility and

hydrophobic character of EOs):

- broth dilution (macro and/or micro, anti-biofilm)

- direct bioautography

- vapour-phase-mediated antimicrobial (VMAA) (Feyaerts et al. 2017 and 2018)


16

Bach resolutions 2018

The 2nd respective “Brainstorming Workshop” was organized at Bach Castle, Carinthia, Austria on 2-4 March

2018. In 2001 the initial idea of the Workshop was raised by Prof. Chlodwig Franz, who invited 15 colleagues

to Austria. In this year, 12 colleagues, who are the members of the Permanent Scientific Committee of ISEO

(International Symposium on Essential Oils) met again to take part in the Workshop. After two days of open-

space and fruitful discussion concerning the present state and future trends in essential oil (EO) research, the

group elaborated and adopted the following résumé:

In the scientific literature and research papers (manuscripts) quite often insufficient data concerning, e.g.

description of plant samples, the parameters of GC experiments, as well as qualitative and quantitative

analyses of EOs and/or natural volatiles can be found making the results doubtful. Therefore, there is a

need to prepare guidelines, which are intended to be of assistance to researchers working in the field of

EO and natural volatiles, and set the basis of good practice in experimental research and communication

of results. It should be clearly defined as to what the EO and what the volatiles are.

These guidelines consist of mandatory and recommended points depending upon the specific goals of the

research considered, i.e. some recommendations may not be essential if we are not directly related to

the main goal of the study.

These guidelines are formally divided into two main parts: chemical part and biological testing. The

biological part cannot be considered separately from the chemical one, as both are generally dependent

on each other. The chemical part includes sampling, sample preparation, identification of constituents

and quantification of constituents. Every step related to chemical or biological parts should be statistically

appropriate. Repeatability, accuracy and validation are highly important characters in the studies of EOs.

During ISEO Symposia, workshops are planned to be organized with the topic “How to conduct research

into EOs and natural volatiles?” The above mentioned guidelines (standards) will be introduced to help

researchers in their laboratory work. On the other hand, these workshops may give answer to the

researchers’ question “How to set up a biological activity test?”

If commercial essential oils are used for safety, pharmacological or clinical studies, their identity and

chemical composition (including chiral analysis) has to be ascertained.

There are several scientific hot-topics associated with EOs and volatiles, e.g. their role in animal feeding,

in nutrition and in food industry. Therefore, the issue of safety and toxicology of these natural chemicals

should not be neglected.

There are only a few numbers of human clinical studies involving EOs. More clinical trials and safety

considerations are required.

Synergism between Western and Eastern Medicine and the discovery of traditional use of EOs should be

explored.

Endophytes live in symbiosis with a plant, but most of the endophyte-plant relationships are not well

understood. However, endophytes may often produce metabolites with diverse biological activities. The

role of endophytes in aromatic plants should, therefore, be studied.

Genetics, metabolomic approaches and bioinformatics are worth involving in EO research. In 2001, the

1st Bach Resolution concluded that “Biodiversity of the chemical composition of aromatic plants, both

genetically and environmentally induced, will provide a valuable background for the identification of a

gene coding for individual terpene synthesis. The better understanding of the biosynthesis of terpenes

leads the way to molecular tailoring of terpene synthesis.” This statement is still valid.


17

In the future, the ISEO Permanent Scientific Committee would like to inform researchers working in the field

of EO and natural volatiles about the event of ISEO Symposia on the following official website: http://iseo-

pc.org/.

The members of Permanent Scientific Committee of ISEO present at the 2nd “Brainstorming Workshop” at

Bach Castle, Carinthia, Austria:

Nicolas Baldovini, K. Husnu Can Baser, Carlo Bicchi, Fatih Demirci, Chlodwig Franz, Gyorgyi Horvath, Jan

Karlsen, Stanislaw Lochynski, Agnieszka Ludwiczuk, Eva Nemeth-Zamborinè, Niko Radulovic, Patrizia Rubiolo.

ACKNOWLEDGMENT

The manuscript submitted was not completed, due to the sudden demise of Jan. With the help of NVEO Editorial the

work was prepared in the present form. Special thanks need to be addressed to Prof. Dr. Elisabeth Stahl-Biskup and Prof.

Dr. Chlodwig Franz for their invaluable contributions.

REFERENCES

Balouiri, M., Sadiki, M., & Ibnsouda, S. K. (2016). Methods for in vitro evaluating antimicrobial activity: A review. Journal

of Pharmaceutical Analysis, 6(2), 71-79.

Begnaud, F. & Chaintreau, A. (2016). Good quantification practices of flavours and fragrances by mass spectrometry.

Philosophical Transactions of the Royal Society A, 374(2079), 20150365.

Cachet, T., Brevard, H., Chaintreau, A., Demyttenaere, J., French, L., Gassenmeier, K., Joulain, D., Koenig, T., Loesing, G.,

Marchant, M., Merle, Ph., Saito, K., Schippa, C., Sekiya, F., Smith, T. (2016). IOFI recommended practice for the use of

predicted relative-response factors for the rapid quantification of volatile flavouring compounds by GC-FID. Flavour and

Fragrance Journal, 31(3), 191-194.

E.-J. Brunke (ed.) (1986). Progress in Essential Oil Research Walter de Gruyter & Co., Berlin.

Fanz, Ch. (2002). Bach Resolution on Essential Oils and Plant Phenolics (Antioxidants). Journal of Essential Oil Research,

14(2), 79.

Feyaerts, A. F., Mathé, L., Luyten, W., De Graeve, S., Van Dyck, K., Broekx, L., & Van Dijck, P. (2018). Essential oils and

their components are a class of antifungals with potent vapour-phase-mediated anti-Candida activity. Scientific Reports,

8(1), 3958.

Feyaerts, A. F., Mathé, L., Luyten, W., Tournu, H., Van Dyck, K., Broekx, L., & Van Dijck, P. (2017). Assay and

recommendations for the detection of vapour-phase-mediated antimicrobial activities. Flavour and Fragrance Journal,

32(5), 347-353.

Received : 19.02.2019

Accepted: 13.03.2019

Nat. Volatiles & Essent. Oils, 2019; 6(1): 18-24 Servi et al.

18

RESEARCH ARTICLE

Essential oil composition of two endemic Centaurea species from Turkey

Hüseyin Servi1,*, Sezgin Çelik2 and Ramazan Süleyman Göktürk3

1 Department of Pharmaceutical Botany, Faculty of Pharmacy, Altinbas University, 34144, Istanbul, Turkey 2 Department of Molecular Biology and Genetic, Faculty of Arts & Science, Yıldız Technical University, 34220, Istanbul,

Turkey 3 Department of Biology, Faculty of Science, Akdeniz University, 07058, Antalya, Turkey

*Corresponding author. Email: [email protected]

Abstract

Essential oil composition of Centaurea austroanatolica Hub.-Mor. and Centaurea kizildaghensis Uzunh., E. Doğan & H. Duman were

analyzed by means of gas chromatography-mass spectrometry (GC-MS). Sixty and fourty six compounds were identified in the

essential oils of C. austroanatolica and C. kizildaghensis, respectively. The major components were determined hexadecanoic acid

(21.3%), caryophyllene oxide (4.8%), dodecanoic acid (4.2%) and heptacosane (3.5%) in C. austroanatolica oil, hexadecanoic acid

(24.4%), phytol (9.3%), caryophyllene oxide (3.8%) and salvial-4(14)-en-1-one (3.1%) in C. kizildaghensis oil.

Keywords: Centaurea austroanatolica, Centaurea kizildaghensis, essential oil, GC-MS

Introduction

Centaurea L. species are member of Asteraceae family and distributed mainly in Southwest and Central and

Eastern regions in Turkey. Centaurea has 226 species in 34 sections, of which 66% are endemic of the country.

Turkey is the main center of diversity for Centaurea (Kültür et al., 2016). The aerial parts of several species of

Centaurea are used in the traditional medicine for the treatment of diabetes, diarrhea, hypertension, malaria,

microbial infections, rheumatism and ulcers (Baytop, 1999; Sarker et al., 1997; Uğur et al., 2009). But there

is no report any medicinal uses of C. austroanatolica and C. kizildaghensis in traditional medicine in Turkey.

Even though Centaurea L. is one of the largest genera of Asteraceae family, reports on the analysis of the

essential oils of this genus are limited. There is only one report chemical composition and antimicrobial

activity of endemic Centaurea austroanatolica. The main components of the chloroform extract of Centaurea

austroanatolica were caryophyllene oxide (21.32%), spathulenol (10.86%), n-tricosanol (9.58%) and geranyl

isovalerate (8.71%). The chloroform extract showed significant antibacterial activity toward all bacteria test

(Uğur et al., 2009). Additionally, anthocyanin content was detected in C. kizildaghensis (Gokbel et al., 2015).

Flavonoids from Turkish C. austroanatolica and C. kizildaghensis were investigated. Apigenin was identified

from both species, genkwanin and quercetin from C. austroanatolica and genkwanin-4'-methyl ether from C.

kizildaghensis (Uddin et al., 2017).

According to our literature survey there is only one report on the essential oil composition of any C.

austroanatolica. This prompted us to investigate the essential oil composition of these species. To the best

of our knowledge this is the first report on the essential oil composition of C. austroanatolica and C.

kizildaghensis.


19

Materials and Methods

Plant materials

Plant materials were collected during the flowering period; C. austroanatolica on 19.06.2015 from Antalya,

Kumluca District (250 m) and C. kizildaghensis on 11.07.2015 from Konya, Derebucak District (1960 m) in

Turkey. Voucher specimens have been deposited in the Herbarium of Akdeniz University (Voucher no. AKDU

4139 and AKDU 4140 for C. kizildaghensis and C. austroanatolica respectively), Turkey.

Isolation of the essential oils

Aerial parts of the air dried plants subjected to hydrodistillation for 3 h, using a Clevenger-type apparatus to

produce essential oils. Condenser of the Clevenger was attached to a microchiller that set to 4°C. C.

kizildaghensis and C. austroanatolica afforded oils from the aerial parts with 0.01 and 0.02% (v/w) yields,

respectively. The oils were recovered with 1 mL n-hexane and preserved in amber vials under -20°C until the

day they were analyzed.

Gas chromatography/mass spectrometry analysis

The GC-MS analysis was performed with an Agilent 5975C Inert XL EI/CI MSD system operating in EI mode.

Essential oil of C. kizildaghensis and C. austroanatolica were diluted 1/100 and 1/65 (v/v) with n-hexane,

respectively. Injector and MS transfer line temperatures were set at 250˚C. Innowax FSC column (60 m (×)

0.25 mm, 0.25 µm film thickness) and helium as carrier gas (1 mL/min) were used in both GC/MS analyses.

Splitless injection was employed. Oven temperature was programmed to 60˚C for 10 min. and raised to 220˚C

at rate of 4˚C/min. Temperature kept constant at 220˚C for 10 min. and then raised to 240˚C at a rate of

1˚C/min. Mass spectra were recorded at 70 eV with the mass range m/z 35 to 425. Relative percentage

amounts of the separated compounds were calculated from integration of the peaks in MS chromatograms.

Identification of essential oil components were carried out by comparison of their relative retention indices

(RRI) obtained by series of n-alkanes (C5 to C30) to the literature and with mass spectra comparison (Baser

et al., 2006a, 2006b, 2008; Demirci et al., 2013; Maggio et al., 2012; Moronkola et al., 2009a, 2009b;

Noorizadeh and Farmany, 2010; Özcan et al., 2001; Polatoğlu et al., 2010, 2011, 2013, 2014, 2015, 2016,

2017; Saidana et al., 2008; Schepetkin et al., 2016; Tabanca et al., 2001, 2006; Viegas and Bassoli, 2007). Mass

spectra comparison was done by computer matching with commercial Wiley 8th Ed./NIST 05 Mass Spectra

library, Adams Essential Oil Mass Spectral Library and Pallisade 600K Complete Mass Spectra Library. The

analysis was carried out in triplicate and the results were given as the mean ± standard deviation.

Results and Discussion

Essential oil composition of Centaurea austroanatolica Hub.-Mor. and Centaurea kizildaghensis UzunH., E.

Doğan & H. Duman were analyzed by means of gas chromatography-mass spectrometry (GC-MS). Sixty

compounds were identified in the essential oil of C. austroanatolica that represent 76.1 ± 1.8% (n=3) of the

oil. The main components of the essential oil were hexadecanoic acid (21.3 ± 0.9%), caryophyllene oxide (4.8

± 0.1%), dodecanoic acid (4.2 ± 0.1%) and heptacosane (3.5 ± 0.1%). Forty six compounds were identified in

the essential oil of C. kizildaghensis that represent 76.1 ± 0.5% (n=3) of the oil. The main components of the

essential oil were hexadecanoic acid (24.4 ± 0.2%), phytol (9.4 ± 0.3%), caryophyllene oxide (3.8 ± 0.1%) and

salvial-4(14)-en-1-one (3.1 ± 0.1%). The essential oil composition of C. austroanatolica and C. kizildaghensis

are given in Table 1.


20

Hexadecanoic acid is the main compound of the both species. Hexadecanoic acid was previously found as the

major compound of essential oils of C. wagenitzii, C. tossiensis, C. luschaniana, C. iberica, C. hyalolepis, C.

polyclada, C. aladagensis, C. hierapolitana, C. cadmea, C. calolepis, C. reuterana var. reuterana, C. depressa,

C. urvillei ssp. urvillei, C. solstitalis ssp. solstitalis, C. balsamita, C. behen, C. aggregata subsp. aggregata and

C. pichleri ssp. pichleri from Turkey (Erdoğan et al., 2017; Erel et al., 2013; Karamenderes 2008; Köse et al.,

2007, 2008; Tastan et al., 2017; Tuzun et al., 2017). Hexadecanoic acid was shown to increase the possibility

of coronary heart diseases. People should be worried to use of C. austroanatolica and C. kizildaghensis

essential oil might cause serious cardiac problems (Connor, 1999). The essential oil obtained from C.

austroanatolica have considerable differences than the chloroform extract of C. austroanatolica. These

differences in the previous literature and present data could be related to different collection times, climatic

and soil conditions, ecological factors, methods and instruments employed in analysis or different genotypes.

We believe the results obtained from this research will stimulate further research on the chemistry of

Centaurea species.

Table 1. The essential oil composition of C. kizildaghensis and C. austroanatolica

C. kizildaghensis C. austroanatolica

No RRI1 RRI

Lit.2

Compound I3 II III Mean4 SD5 I II III Mean SD Id. Met.6

1 1236 1239 2-pentyl furan 0.8 0.8 0.7 0.8 0.1 0.3 0.3 0.3 0.3 0.0 RI, MS

2 1398 1399 Nonanal 0.7 0.7 0.6 0.7 0.1 0.2 0.2 0.2 0.2 0.0 RI, MS

3 1401 1400 Tetradecane 0.1 0.1 0.1 0.1 0.0 - - - - - RI, MS, Ac

4 1489 1497 α-Copaene - - - - - 0.5 0.5 0.5 0.5 0.0 RI, MS

5 1499 1505 Dihydroedulan II 0.5 0.5 0.4 0.5 0.1 0.2 0.2 0.2 0.2 0.0 RI, MS

6 1504 1505 Decanal - - - - - 0.4 0.4 0.4 0.4 0.0 RI, MS

7 1530 1535 Dihydroedulan I - - - - - 0.1 0.1 0.2 0.1 0.1 RI, MS

8 1549 1553 (Z)-Theaspirane - - - - - 0.2 0.2 0.3 0.2 0.1 RI, MS

9 1549 1553 β-linalool 0.4 0.4 0.4 0.4 0.0 - - - - - RI, MS

10 1599 1600 β-Elemene - - - - - 0.1 0.1 0.1 0.1 0.0 RI, MS

11 1602 1600 Hexadecane - - - - - 0.2 0.2 0.2 0.2 0.0 RI, MS, Ac

12 1608 1608 β-caryophyllene 0.6 0.6 0.7 0.6 0.1 0.9 1.0 1.0 1.0 0.1 RI, MS

13 1633 1638 β-cyclocitral 0.4 0.4 0.4 0.4 0.0 - - - - - RI, MS

14 1659 1661 Safranal 0.4 0.4 0.4 0.4 0.0 - - - - - RI, MS

15 1661 1664 1-nonanol 0.4 0.4 0.3 0.4 0.1 0.2 0.2 0.2 0.2 0.0 RI, MS

16 1683 1687 α-humulene 0.1 0.1 0.1 0.1 0.0 - - - - - RI, MS

17 1723 1723 Germacrene D 0.4 0.4 0.3 0.4 0.1 0.4 0.4 0.4 0.4 0.0 RI, MS

18 1735 1742 β-Selinene - - - - - 0.3 0.3 0.3 0.3 0.0 RI, MS

19 1762 1763 Naphthalane 0.9 0.9 0.9 0.9 0.0 - - - - - RI, MS

20 1764 1766 Decanol - - - - - 0.1 0.1 0.1 0.1 0.0 RI, MS

21 1770 1773 δ-Cadinene - - - - - 0.2 0.2 0.2 0.2 0.0 RI, MS

22 1776 1779 (E,Z)-2,4-Decadienal - - - - - 0.2 0.2 0.2 0.2 0.0 RI, MS

23 1823 1827 (E,E)-2.4-Decadienal - - - - - 0.6 0.6 0.5 0.6 0.1 RI, MS

24 1825 1830 Tridecanal - - - - - 0.3 0.3 0.3 0.3 0.0 RI, MS

25 1835 1838 β-damascenone 1.5 1.5 1.4 1.5 0.1 0.6 0.7 0.7 0.7 0.1 RI, MS

26 1849 1849 Dihydro-β-ionone 0.7 0.7 0.7 0.7 0.0 - - - - - RI, MS


21

27 1863 1864 (E)-Geranyl acetone 1.0 1.1 0.9 1.0 0.1 0.5 0.5 0.5 0.5 0.0 RI, MS

28 1901 1900 Nonadecane 0.3 0.3 0.3 0.3 0.0 0.1 0.1 0.1 0.1 0.0 RI, MS, Ac

29 1929 1932 Neophytadiene isomer 0.3 0.3 0.4 0.3 0.1 - - - - - RI, MS

30 1936 1941 α-Calocarene - - - - - 0.3 0.3 0.3 0.3 0.0 RI, MS

31 1953 1958 (E)-β-ionone 1.8 1.8 1.8 1.8 0.0 0.8 0.8 0.9 0.8 0.1 RI, MS

32 2001 2000 Eicosane 1.3 1.4 1.3 1.3 0.1 - - - - - RI, MS, Ac

33 2005 2007 Caryophyllene oxide 3.8 3.9 3.8 3.8 0.1 4.6 4.9 4.8 4.8 0.2 RI, MS

34 2029 2031 Salvial-4(14)-en-1-one 3.0 3.1 3.0 3.0 0.1 0.5 0.5 0.5 0.5 0.0 RI, MS

35 2037 2037 Pentadecanal 0.5 0.4 0.5 0.5 0.1 - - - - - RI, MS

36 2039 2036 Hexadecanal - - - - - 0.9 0.9 0.9 0.9 0.0 RI, MS

37 2044 2050 (E)-Nerolidol 0.3 0.3 0.3 0.3 0.0 0.3 0.3 0.3 0.3 0.0 RI, MS

38 2064 2063 Humulene epoxide II - - - - - 0.8 0.9 0.9 0.9 0.1 RI, MS

39 2098 2092 β-Oplopenone - - - - - 0.3 0.3 0.3 0.3 0.0 RI, MS

40 2104 2100 Heneicosane 2.3 2.3 2.3 2.3 0.0 1.0 1.0 1.0 1.0 0.0 RI, MS, Ac

41 2133 2131 Hexahydro farnesyl acetone 1.9 1.9 1.9 1.9 0.0 1.8 1.8 1.8 1.8 0.0 RI, MS

42 2140 2142 Spathulenol 1.4 1.5 1.4 1.4 0.1 2.4 2.5 2.4 2.4 0.1 RI, MS

43 2171 2192 Nonanoic acid 0.1 0.1 0.1 0.1 0.0 2.0 2.1 2.2 2.1 0.1 RI, MS

44 2173 2150 Nor-copaanone 1.5 2.0 2.0 1.8 0.3 - - - - - RI, MS

45 2187 2187 t-Cadinol - - - - - 1.8 1.7 1.6 1.7 0.1 RI, MS

46 2195 2198 1-Docosene - - - - - 0.9 0.9 0.9 0.9 0.0 RI, MS

47 2203 2200 Docosane 0.7 0.8 0.8 0.8 0.1 1.3 1.3 1.3 1.3 0.0 RI, MS, Ac

48 2226 2239 Carvacrol - - - - - 0.9 0.8 0.8 0.8 0.1 RI, MS

49 2241 2228 Isospathulenol 0.1 0.1 0.1 0.1 0.0 0.4 0.4 0.4 0.4 0.0 RI, MS

50 2251 2253 β-Eudesmol - - - - - 1.1 1.2 1.2 1.2 0.1 RI, MS

51 2277 2282 Decanoic acid 0.7 0.7 0.7 0.7 0.0 1.0 1.0 1.0 1.0 0.0 RI, MS

52 2282 2289 Oxo-α-ylangene 0.5 0.5 0.5 0.5 0.0 0.5 0.5 0.5 0.5 0.0 RI, MS

53 2295 2296 Isophytol - - - - - 0.3 0.3 0.3 0.3 0.0 RI, MS

54 2303 2300 Tricosane 2.0 2.0 2.0 2.0 0.0 1.9 1.9 1.9 1.9 0.0 RI, MS, Ac

55 2311 2316 Caryophylla-2(12),6(13)dien-5-α-ol - - - - - 0.4 0.4 0.4 0.4 0.0 RI, MS

56 2315 2315 2,4-bis(tert-butyl)phenol 0.6 0.6 0.6 0.6 0.0 - - - - - RI, MS

57 2352 2324 Caryophylladienol-II - - - - - 1.5 1.6 1.6 1.6 0.1 RI, MS

58 2353 2389 Caryophyllenol-I - - - - - 0.7 0.7 0.7 0.7 0.0 RI, MS

59 2383 2381 Farnesyl acetone 0.9 0.9 0.8 0.9 0.1 0.2 0.2 0.3 0.2 0.1 RI, MS

60 2393 2399 Aromadendrene oxide 0.5 0.5 0.4 0.5 0.1 - - - - - RI, MS

61 2394 2392 Caryophyllenol-II - - - - - 1.4 1.4 1.4 1.4 0.0 RI, MS

62 2399 2423 γ-Cadinene-15-al - - - - - 0.3 0.3 0.3 0.3 0.0 RI, MS

63 2402 2400 Tetracosane 0.3 0.3 0.3 0.3 0.0 0.7 0.7 0.7 0.7 0.0 RI, MS, Ac

64 2488 2492 Dodecanoic acid 1.3 1.3 1.3 1.3 0.0 4.2 4.2 4.3 4.2 0.1 RI, MS

65 2504 2500 Pentacosane 0.7 0.7 0. 0.7 0.0 1.6 1.6 1.9 1.7 0.2 RI, MS, Ac

66 2542 2551 Geranyl linalool - - - - 0.2 0.4 0.4 0.3 0.1 RI, MS

67 2608 2600 Hexacosane - - - - - 0.6 0.6 0.5 0.6 0.1 RI, MS, Ac


22

68 2618 2614 Phytol 9.6 9.6 9.1 9.4 0.3 1.8 1.9 1.8 1.8 0.1 RI, MS

69 2700 2713 Tetradecanoic acid 1.1 1.2 1.2 1.2 0.1 2.1 2.2 2.1 2.1 0.1 RI, MS

70 2705 2700 Heptacosane 1.7 1.7 1.7 1.7 0.0 3.6 3.5 3.4 3.5 0.1 RI, MS, Ac

71 2776 2783 1-Docosanol 1.5 1.5 1.5 1.5 0.0 - - - - - RI, MS

72 2806 2809 Pentadecanoic acid 1.0 1.0 1.0 1.0 0.0 1.3 1.4 1.3 1.3 0.1 RI, MS

73 2916 2931 Hexadecanoic acid 24.3 24.3 24.7 24.4 0.2 20.2 22.0 21.6 21.3 0.9 RI, MS

74 2985 2990 Docosanal 1.5 1.5 1.5 1.5 0.0 - - - - - RI, MS

Total 75.8 76.7 75.8 76.1 0.5 74.1 77.3 77.0 76.1 1.8

In addition to the above data, diisobutyl phthalate is a common plasticizer contaminant and it was detected as a considerable component as 1.0 percentage for C. austroanatolica. 1RRI: Relative retention time indices calculated against n-alkanes (C5-C30). 2RRI Lit.: Relative retention time given in the literature for the compound in similar columns and analysis conditions. 3The results of the analysis in each replicate. 4,5The analysis were carried out in triplicate results are given as mean % area ± standard deviation (SD), calculated from MS data. 6Identification method: RI: identification based on the relative retention times (RRI) of genuine compounds on the HP Innowax column and the literature data; MS: identification based on MS comparison with the database or the literature data, Ac: Identification is done according to RRI and MS values of the authentic compounds.

REFERENCES

Başer, K. H. C., Özek, G., Özek, T., & Duran, A. (2006a). Composition of the essential oil of Centaurea huber‐morathii

Wagenitz isolated from seeds by microdistillation. Flavour and fragrance journal, 21(3), 568-570.

Başer, K. H. C., Özek, G., Özek, T., Duran, A., & Duman, H. (2006b). Composition of the essential oils of Rhabdosciadium

oligocarpum (Post ex Boiss.) Hedge et Lamond and Rhabdosciadium microcalycinum Hand.‐Mazz. Flavour and fragrance

journal, 21(4), 650-655.

Başer, K. H. C., Demirci, B., & Dadandi, M. Y. (2008). Comparative essential oil composition of the natural hybrid Phlomis

x vuralii Dadandi (Lamiaceae) and its parents. Journal of Essential Oil Research, 20(1), 57-62.

Baytop, T. (1999). Geçmişte ve bugün Türkiye’de bitkiler ile tedavi. Nobel Tıp Kitabevi.

Connor, W. E. (1999). Harbingers of coronary heart disease: dietary saturated fatty acids and cholesterol. Is chocolate

benign because of its stearic acid content?. The American Journal of Clinical Nutrition, 70(6), 951-952.

Demirci, B., Yasdıkcıoglu, G.K. & Baser, K.H.C. (2013). Sesquiterpene hydrocarbons of the essential oil of Actinolema

macrolema Boiss. Turkish Journal of Chemistry, 37(6), 917-926.

Erel, S. B., Demirci, B., Demir, S., Karaalp, C., & Hüsnü Can Baser, K. (2013). Composition of the essential oils of Centaurea

aphrodisea, C. polyclada, C. athoa, C. hyalolepis and C. iberica. Journal of Essential Oil Research, 25(2), 79-84.

Erdoğan, T., Sümer, B., Özçınar, Ö., Çakılcıoğlu, U., Demirci, B., Başer, K. H. C., & Kıvçak, B. (2017). Essential Oil

Composition of Three Centaurea Species from Turkey: Centaurea aggregata Fisch. & Mey. ex. DC. subsp. aggregata, C.

balsamita Lam. and C. behen L. Records of Natural Products, 11(1), 69-73.

Gökbel, H., Özcan, M. M., Duran, A., Hamurcu, M., Çelik, M., Uslu, N., & Demiral, T. (2015). Mineral Contents and

Bioactive Properties of Centaurea spp. Leave and Seeds. Iranian Journal of Chemistry and Chemical Engineering (IJCCE),

34(4), 79-88.

Karamenderes, C., Demirci, B., & Baser, K. H. C. (2008). Composition of essential oils of ten Centaurea L. taxa from

Turkey. Journal of Essential Oil Research, 20(4), 342-349.

Köse, Y. B., İşcan, G., Demirci, B., Başer, K. H. C., & Celik, S. (2007). Antimicrobial activity of the essential oil of Centaurea

aladagensis. Fitoterapia, 78(3), 253-254.

Köse, Y. B., Demirci, B., Başer, K. H. C., & Yücel, E. (2008). Composition of the essential oil of three endemic Centaurea

species from Turkey. Journal of Essential Oil Research, 20(4), 335-338.


23

Kültür, Ş., Bona, M., Nath, E.Ö. (2016). A new species of Centaurea (Asteraceae) from East Anatolia, Turkey. Phytotaxa,

247(1), 85-91.

Maggio, A., Rosselli, S., Bruno, M., Spadaro, V., Raimondo, F. M., & Senatore, F. (2012). Chemical composition of

essential oil from Italian populations of Artemisia alba Turra (Asteraceae). Molecules, 17(9), 10232-10241.

Moronkola, D.O., Ogunwande, I.A., Başer, K.H.C., Ozek, T., & Ozek, G. (2009a). Essential oil composition of Gmelina

arborea Roxb., Verbenaceae, from Nigeria. Journal of Essential Oil Research, 21(3), 264-266.

Moronkola, D. O., Ogunwande, I. A., Oyewole, I. O., Başer, K. H. C., Ozek, T., & Ozek, G. (2009b). Studies on the volatile

oils of Momordica charantia L.(Cucurbitaceae) and Phyllanthus amarus Sch. et Thonn (Euphorbiaceae). Journal of

Essential Oil Research, 21(5), 393-399.

Noorizadeh, H., & Farmany, A. (2010). Exploration of linear and nonlinear modeling techniques to predict of retention

index of essential oils. Journal of the Chinese Chemical Society, 57(6), 1268-1277.

Özcan, M., Akgül, A., Başcr, K. H. C., Özck, T., & Tabanca, N. (2001). Essential oil composition of sea fennel (Crithmum

maritimum) form Turkey. Food/Nahrung, 45(5), 353-356.

Polatoglu, K., Demirci, F., Demirci, B., Gören, N., & Baser, K. H. C. (2010). Antimicrobial activity and essential oil

composition of a new T. argyrophyllum (C. Koch) Tvzel var. argyrophyllum chemotype. Journal of Oleo Science, 59(6),

307-313.

Polatoglu, K., Demirci, B., Goren, N., & Baser, K.H.C. (2011). Essential oil composition of Tanacetum kotschyi from Turkey.

Chemistry of Natural Compounds, 47(2), 297-299.

Polatoğlu, K., Karakoç, Ö. C., & Gören, N. (2013). Phytotoxic, DPPH scavenging, insecticidal activities and essential oil

composition of Achillea vermicularis, A. teretifolia and proposed chemotypes of A. biebersteinii (Asteraceae). Industrial

crops and products, 51, 35-45.

Polatoglu, K., Sen, A., Bulut, G., Bitis, L., & Gören, N. (2014). Essential Oil Composition of Centaurea stenolepis Kerner.

from Turkey. Journal of Essential Oil Bearing Plants, 17(6), 1268-1278.

Polatoğlu, K., Arsal, S., Demirci, B., & Başer, K. H. C. (2015). DPPH scavenging, PRAP activities and essential oil

composition of edible Lathyrus ochrus L. (Cyprus Vetch, Luvana) from Cyprus. Journal of Oleo Science, 64(3), 309-314.

Polatoğlu, K., Arsal, S., Demirci, B., & Başer, K. H. C. (2016). Unexpected Irregular Monoterpene “Yomogi Alcohol” in the

Volatiles of the Lathyrus L. species (Leguminosae) of Cyprus. Journal of Oleo Science, 65(3), 241-249.

Polatoğlu, K., Servi, H., Özçınar, Ö., Nalbantsoy, A., & Gücel, S. (2017). Essential Oil Composition of Endemic Arabis

purpurea Sm. & Arabis cypria Holmboe (Brassicaceae) from Cyprus. Journal of Oleo Science, 66(1), 65-70.

Saidana, D., Mahjoub, M. A., Boussaada, O., Chriaa, J., Chéraif, I., Daami, M., & Helal, A.N., et al. (2008). Chemical

composition and antimicrobial activity of volatile compounds of Tamarix boveana (Tamaricaceae). Microbiological

Research, 163(4), 445-455.

Sarker, S. D., Savchenko, T., Whiting, P., Šik, V., & Dinan, L. N. (1997). Moschamine, cis-moschamine, moschamindole

and moschamindolol: four novel indole alkaloids from Centaurea moschata. Natural Product Letters, 9(3), 189-199.

Schepetkin, I. A., Kushnarenko, S. V., Özek, G., Kirpotina, L. N., Sinharoy, P., Utegenova, G. A., & Khlebnikov, A. I. et al.

(2016). Modulation of human neutrophil responses by the essential oils from Ferula akitschkensis and their constituents.

Journal of Agricultural and Food Chemistry, 64(38), 7156-7170.

Tabanca, N., Kırımer, N., Demirci, B., Demirci, F., & Başer, K. H. C. (2001). Composition and antimicrobial activity of the

essential oils of Micromeria cristata subsp. phrygia and the enantiomeric distribution of borneol. Journal of Agricultural

and Food Chemistry, 49(9), 4300-4303.


24

Tabanca, N., Demirci, B., Ozek, T., Kirimer, N., Baser, K.H.C., Bedir, E., & Wedge, D.E., et al. (2006). Gas chromatographic–

mass spectrometric analysis of essential oils from Pimpinella species gathered from Central and Northern Turkey.

Journal of Chromatography A, 1117(2), 194-205.

Tastan, P., Fafal, T., Tüzün, B. S., Gönenç, T., Demirci, B., & Kivcak, B. (2017). Composition of essential oil and fatty acids

of Centaurea pichleri ssp. pichleri. International Journal of Secondary Metabolite, 4(3, Special Issue 1), 37-42.

Tuzun, B. S., Gonenc, T., Tastan, P., Fafal, T., & Demirci, B. (2017). Essential Oil and Fatty Acid Composition of Centaurea

solstitialis ssp. solstitialis. International Journal of Secondary Metabolite, 4(3, Special Issue 1), 58-62.

Uddin, S., Alnsour, L., Segun, P., Servi, H., Celik, S., Göktürk, R. S., Al-Groshi, A., Al-Majmaie, S., Guetchueng, S. T., Nahar,

L., et al. (2017). Flavonoids from two Turkish Centaurea species and their chemotaxonomic implications. Trends in

Phytochemical Research, 1, 243-248.

Ugur, A., Sarac, N., Ceylan, O., & Duru, M. (2009). Chemical composition of endemic Centaurea austro-anatolica and

studies of its antimicrobial activity against multi-resistant bacteria. Acta Pharmaceutica, 59(4), 463-472.

Viegas, M. C., & Bassoli, D. G. (2007). Utilização do índice de retenção linear para caracterização de compostos voláteis

em café solúvel utilizando GC-MS e coluna HP-Innowax. Química nova, 30, 2031-2034.

Received : 21.01.2019


Nat. Volatiles & Essent. Oils, 2019; 6(1): 25-33 Kumar & Ali

25

RESEARCH ARTICLE

Antiglycation and antiaggregation potential of thymoquinone

Dinesh Kumar and Ahmad Ali*

Department of Life Sciences, University of Mumbai, Vidyanagari, Santacruz (East), Mumbai, Maharashtra, INDIA


Abstract

The consequences of Diabetes are manifested due to the accumulation of glucose. The carbonyl group of sugars reacts with the

amino group of proteins leading to generation of harmful products collectively known as advanced glycation end products (AGEs).

These products have been shown to be involved in the various secondary complications of Diabetes and neurodegenerative disorders.

The present study involves the assessment of role of Thymoquinone in the process of glycation.

The in vitro glycation system consisted of BSA and glucose and incubated in the presence and absence of thymoquinone for four

weeks at 37°C. The amount of glycation products was measured by standard methods like browning, total AGEs by

spectrofluorimetry. The aggregation of protein was checked by aggregation index and Congo red assays. The effect of thymoquinone

was also checked on the glycation of DNA and the sample was analysed by agarose gel electrophoresis. The presence of

thymoquinone resulted in the decrease in browning and amount of total AGEs significantly. There was also a drastic decrease in the

glycation-induced aggregation of BSA and reversal of glycoxidative damage of DNA in the presence of thymoquinone. It can be

concluded from these results that thymoquinone is potential antiglycating agent and it can be used to prevent the glycation-induced

damage of biomolecules.

Keywords: Advanced glycation end-products (AGEs), antiglycation, aggregation, DNA damage, thymoquinone

Introduction

Diabetes has affected millions of people worldwide and has become a major socioeconomic issue. The

condition begins with a slow accumulation of glucose in the body and reaches a toxic level after sometime.

The reactive group of sugars, carbonyl, interacts covalently with the amino group of other biomolecules like

proteins by a process known as glycation or Maillard reaction after its discover Louise Camille Maillard

(Kikuchi et al., 2003). This reaction leads to generation of Schiff’s bases which get converted to Amadori

products. The rearrangement of these metabolites lead to generation of a group of harmful compounds

commonly known as advanced glycation end products (AGEs) (Ahmad, 2005). All these intermediates of

glycation process are also known to generate free radicals and cause weakening of the antioxidant defense

mechanisms which damages cellular organelles and enzymes (Ahmad et al., 2014). The rate of formation of

AGEs increases during the oxidative and carbonyl stress caused due to accumulation of reactive oxygen

species (ROS) and dicarbonyl compounds (Sadowska-Bartosz & Bartosz, 2015). The biological reactions

leading to AGE formation and free radical generation are closely related and are often called as glycoxidation

process. They have been shown to be implicated in various pathophysiological conditions like diabetes,

neurodegenerative disorders and cancer (Ahmad et al., 2014).

Glycation has received a lot of attention in the last two decades because of its involvement in the secondary

complications of diabetes and many neurodegenerative disorders. There has been effort to develop a drug

which can inhibit the generation of AGEs in the body or treat the conditions due to accumulation of AGEs.

Although some synthetic drugs have been approved for the treatment but they have been found to exert

harmful effects on the body for example, aminoguanidine (Thornalley, 2003). Recent developments in the

https://mail.google.com/mail/u/0/h/7safal5hrmxj/?&cs=wh&v=b&[email protected]


26

field of photochemistry and analytical methods have led to discovery of natural products from plants or

derivatives of the natural products synthesized and tested in the laboratories. Some of these natural

compounds such as curcumin, eugenol, rutin, garcinol etc. have shown to possess significant antioxidant and

antiglycating potentials (Khan & Gothalwal, 2018).

Nigella sativa (black cumin) seeds have been used as traditional medicine since centuries (Ali & Blunden,

2003). Thymoquinone, the main phytoconstituent of black cumin seed, has been reported to have

hepatoprotective, anti-inflammatory, anti-oxidant, cytotoxic and anti-cancer chemical properties

(Khader & Eckl, 2014). There are a very few reports on understanding the mechanism of antiglycating and

antiaggregation potentials of thymoquinone (Anwar et al., 2014). Therefore, thymoquinone was used in the

present study to check its role in the prevention of glycation and glycation-induced processes like protein

aggregation and glycoxidation. The results indicate significant role of thymoquinone in the prevention of

accumulation of early as well as advanced glycation end products. Glycation-induced aggregation and DNA

damage were also prevented in the presence of thymoquinone. Antioxidant potential of thymoquinone was

also measured using DPPH assay. This study is an important step towards understanding the possible

mechanism of thymoquinone in the prevention of glycation in the secondary complications of diabetes.


Materials

Bovine serum albumin, Agarose, methylglyoxal and thymoquinone were purchased from Sigma-Aldrich.

Lysine and DPPH (2,2-diphenyl-1-picrylhydrazyl) were procured from HiMedia. pBR322 was purchased from

Thermo Fisher. All other chemicals used were of high analytical grade.

Methods

Incubation of thymoquinone in vitro glycation system

10 mg/mL aqueous solution of BSA was incubated with glucose (100 mg/mL) with or without thymoquinone

(10 µM and 20 µM) in 100 mM phosphate buffer (pH 7.4) at 37 °C for 28 days. The bacterial contamination

during the prolonged incubation was prevented by adding 3 mM sodium azide.

Measurement of browning

The extent of browning was measured at 420 nm using Shimadzu UV 1800 spectrophotometer (Rondeau et

al., 2007) and relative percentage of absorbance was used to plot the graph.

Estimation of fluorescent AGEs

The measurement of fluorescent intensity was carried out at excitation (370 nm) and emission (438 nm)

wavelengths (Ali et al., 2017). Cary Eclipse Fluorescence spectrophotometer was used for the measurement

of fluorescent AGEs.

Determination of protein aggregation index

The effect of thymoquinone was also checked on the protein aggregation by measuring the absorbance of

glycated samples in the presence/absence of thymoquinone. The aggregation index was calculated by the

following formula

Aggregation index = [A340 / (A280 – A340)] * 100

A280 and A340 - absorbance at 280 nm and 340 nm respectively (Pandey et al., 2018).


27

Congo red assay

Congo red dye bound to amyloid cross β-structure was checked by recording absorbance at 530 nm (Ali et

al., 2017). The glycated sample (50 μL) was mixed with 50 μL of 100 μM Congo red dye and kept at 25 °C for

20 minutes. The volume was made sufficient with distilled water (1 mL) for spectrophotometric analysis at

530 nm.

In vitro glycation of plasmid DNA in the presence of thymoquinone

The effect of thymoquinone on the glycation-mediated DNA strand breakage was performed according to a

previous method with minor modifications (Ali et al., 2014). The pBR322 plasmid (0.25 μg) in 100 mM

potassium phosphate buffer (pH 7.4) was incubated with lysine (20 mM), MG (20 mM) and FeCl3 (100 μM) in

presence and absence of thymoquinone (10 µM and 20 µM). The reaction mixture of samples was incubated

at 37 °C for two hours. pBR322 plasmid DNA (0.25 μg) in 100 mM potassium phosphate buffer (pH 7.4)

without glycation system was used as control. The reaction was stopped by freezing the samples at -20 °C.

Agarose gel electrophoresis of glycated plasmid DNA sample

Ten microliters of samples were mixed with 2 μL of 6X gel loading dye and loaded on to 1% agarose gel.

Electrophoresis was carried out initially at 90 V and once the samples left the well, voltage was decreased to

85 V. As soon as the dye band reached two-thirds of gel length, electrophoresis was terminated and gel was

stained using ethidium bromide solution (final concentration 5 μg/mL) for 20 min in dark. Subsequently the

gel was visualized under Gel-Doc and bands analysed with the help of control.

DPPH• assay

The thymoquinone was screened for its antioxidant potential by DPPH radical scavenging assay with some

minor modification (Lutterodt et al., 2010). 0-25 µM concentrations of thymoquinone (1 mM stock) and 100

µL DPPH• (1 mM) were mixed and volume made up to 1 mL with methanol, then incubated at 37 °C for 30

minutes and absorbance was recorded at 517 nm.


Results

Effect of Nigella sativa seed extracts on browning

BSA was glycated with glucose at 37 °C for 28 days in the presence and absence of thymoquinone. Initial

indicator for glycation, browning, was measured spectrophotometrically at 420 nm. Thymoquinone caused

inhibition of glycation at 10 µM (21.67%) and 20 µM (28.04%) of glycation as compared to glycated BSA

(100%) (Figure 1). These results indicate that the decrease in browning in the presence of thymoquinone can

be correlated with less formation of glycated products.


28

Figure 1. Measurement of Browning.

Estimation of fluorescent total AGEs in presence of thymoquinone

The extent of fluorescent AGE formation was checked by measuring the fluorescent intensity in glycated BSA

with glucose as shown in Figure 2. The results showed that the addition of thymoquinone into the solution

greatly reduced the formation of fluorescent AGEs by 31.13% (10 µM) and 78.64% (20 µM).

Figure 2. Measurement of Total Flourescent AGEs.

Effect of Nigella sativa seed extracts on protein aggregation Index

Aggregation of protein is the late stage of non- enzymatic glycation process in which there is formation of

cluster when the carbonyl group is bound to protein. The aggregation index showed very significant reduction

of amyloid cross-β structure in presence of thymoquinone in comparison of glycated protein (Figure 3).


29

Figure 3. Measurement of Aggregation index.

Measurement of amyloid cross-β structure in glycated BSA in presence of thymoquinone

The measurement of amyloid cross-β structure is one of the vital quantitative estimation of glycated protein

and done by performing Congo red assay. Formation of amyloid cross-β structure in the glycated BSA

approximately decreased by 19.98% and 41.94% at 10 µM and 20µM respectively (Figure 4).

Figure 4. Congo Red Assay for the measurement of protein aggregation.


30

Figure 5. Glycoxidative damage of DNA.

LANE DESCRIPTION:

Lane 1- DNA alone

Lane 2- DNA + TQ (10 µM)

Lane 3- DNA + Lysine (20 mM) + MG (20 mM) + FeCl3 (100 µΜ)

Lane 4- Lane 2 + TQ (10 µM)

Effect of thymoquinone on the glycation of DNA

Strand breakage was observed for DNA incubated with methyl glyoxal, lysine and ferric chloride for 2 hours

at 37 °C (Figure 5, Lane 3) as compared to control (Figure 5, Lane 1). The thymoquinone (10 µM) caused the

reversal of the strand breakage (Figure 5, Lane 4) and had no effect on its own (Figure 5, Lane 2).

DPPH• assay

It was found that thymoquinone caused reduction in free radical generation and inhibition increased from 8

% to 20 % in a concentration dependent manner (Table 1).

Table 1. DPPH• Assay.

Samples Scavenging Activity (%) ± S.E.

5 µM TQ 8.27 ± 0.44

10 µM TQ 12.10 ± 0.91

15 µM TQ 15.71 ± 0.29

20 µM TQ 19.01 ± 0.95

25 µM TQ 20.99 ± 0.41


31

Conclusion

Advanced glycation end products have been implicated in many health complications. The major challenges

scientists have faced as far as treatment of glycation related disorders is concerned is their detection and

prevention. Many qualitative and quantitative methods have been developed from last few decades for the

detection of the formation of glycation products and glycation induced processes (Banan & Ali, 2016). These

methods include browning, total AGEs estimation, Congo red assay for amyloid cross β-structure, aggregation

index and electrophoretic mobility of biomolecules. Traditionally glycated samples have been analyzed by

different methods including spectrophotometric and electrophoretic techniques (Ali & Sharma, 2015).

Recently advanced techniques like spectroflourimetry, HPLC and LC-MS have found application in the

quantification of AGEs (Poulsen et al., 2013).

In last few decades focus has been shifted to utilize natural compounds for the prevention and cure of

diseases. Black cumin seeds are sources of several natural compounds which cure many diseases (Najmi et

al., 2012). Although there are many reports in literature on antidiabetic properties of black cumin seeds there

are a very few reports on understanding the mechanism of antiglycating and antiaggregation potentials of

black cumin seed extracts (Pandey et al., 2018; Zafer et al., 2013). In the present study thymoquinone was

used to analyze its preventive role in the process of glycation and consequences of glycation induced

processes like aggregation and glycoxidation. Measurement of extent of browning has been used as a

conventional indicator of the process of glycation. It can be seen from the results obtained that

thymoquinone reduced the extent of browning significantly. The amount of total fluorescent AGEs were

checked using spectrofluorometer and thymoquinone was found to be very potent in inhibiting the formation

of glycation products. Previously Khan et al. (2014) have also reported the antiglycating properties of

thymoquinone.

Glycation also leads to formation of protein cross-linking and aggregates which have been implicated in

neurodegenerative disorders (Ali et al., 2014). In the present study protein aggregation index and Congo red

methods were used to measure the glycation-induced aggregation of the glycated BSA. The most significant

reduction in the amyloid cross β-structure was observed for BSA samples glycated with glucose in the

presence of thymoquinone. Spectroscopic analysis of glycated sample at the different wavelengths gives an

idea of the extent of protein aggregation. A ratio of absorbance taken at 280 and 340 nm is used to calculate

the aggregation index. The presence of thymoquinone reduced the aggregation index significantly. These

results indicate the antiaggregation potential of thymoquinone.

The accumulation of AGEs leads to generation of reactive oxygen species in vitro and in vivo. Macromolecular

structures like DNA and proteins are very prone to these free radicals. In the present study the effect of

glycation-induced DNA damage was studied in the presence and absence of thymoquinone. Addition of metal

ions catalysed the generation of free radicals and led to enhanced strand breakage of DNA. There was

significant inhibition/reversal of DNA damaged by glycation in the presence of thymoquinone. Glycoxidative

damage of DNA was prevented by the seed extract of Nigella sativa (Pandey et al., 2018). In another study

Losso et al. (2011) have reported the application of thymoquinone in the prevention of AGE formation.

Thymoquinone was also found to be good scavenger of oxidants (Solati et al. 2014). Earlier reports have

shown the antiglycating potential of thymoquinone (Anwar et al., 2014; Khan et al., 2014) but there was a

lack of information on the preventive role of this active constituent of Nigella sativa on glycation-induced

processes.


32

It can be concluded from this study that the thymoquinone possess antiglycating, anti-aggregation and

antioxidant potential. The thymoquinone can be used for preventing glycation-mediated secondary

complications of diabetes, glycoxidative damage of DNA as well as protein aggregation mediated neurological

disorders. Further studies need to be carried out to understand the mechanism of inhibition of glycation by

thymoquinone.

ACKNOWLEDGEMENTS

The study was funded by Research Society for the Study of Diabetes in India (RSSDI/HQ/Grants/2017/342).

REFERENCES

Ahmed, N. (2005). Advanced glycation end products-role in pathology of diabetic complications. Diabetes Res Clin Pract,

67, 3-21.

Ahmad, S., Khan, M.S., Akhter, F., Khan, M.S., Khan, A., Ashraf, J.M. & Shahab. (2014). Glycoxidation of biological

macromolecules: a critical approach to halt the menace of glycation. Glycobiology, 24, 979-990.

Ahmad, S., Moinuddin, S.U., Khan, M.S., Habeeb, S., Alam, K. & Ali, A. (2014). Glyco-oxidative damage to human DNA –

Neo-antigenic epitopes on DNA molecule could be a possible reason for autoimmune response in type 1 diabetes.

Glycobiology, 24, 281-291.

Ali, A., More, T. A., Hoonjan, A. K., & Sivakami, S. (2017). Antiglycating potential of acesulfame potassium: an artificial

sweetener. Applied Physiology, Nutrition, and Metabolism, 42(10), 1054-1063.

Ali, B.H., & Blunden, G. (2003). Pharmacological and toxicological properties of Nigella sativa. Phytotherapy

Research, 17, 299–305.

Ali, A., Sharma, R. & Sivakami, S. (2014). Role of natural compounds in the prevention of DNA and proteins damage by

glycation. Bionano Front, 7, 25-30.

Ali, A. & Sharma, R. (2015). A comparative study on the role of lysine and BSA in glycation-induced damage to

DNA. Bioscience and Bioengineering Communications, 1, 38-43.

Anwar, S., Khan, M. A., Sadaf, A., & Younus, H. (2014). A structural study on the protection of glycation of superoxide

dismutase by thymoquinone. International Journal of Biological Macromolecules, 69, 476-481.

Banan, P. & Ali, A. (2016). Preventive effect of phenolic acids on in vitro glycation. Annals of Phytomedicine, 5, 97-102.

Khan, M. A., Anwar, S., Aljarbou, A. N., Al-Orainy, M., Aldebasi, Y. H., Islam, S., & Younus, H. (2014). Protective effect of

thymoquinone on glucose or methylglyoxal-induced glycation of superoxide dismutase. International Journal of

Biological Macromolecules, 65, 16-20.

Khan, M.N. & Gothalwal, R. (2018). Herbal origins provision for non-enzymatic Glycation (NEGs) inhibition. Frontiers in

Medicinal Chemistry and Drug Discovery, 2(1), 10-015.

Khader, M., & Eckl, P. M. (2014). Thymoquinone: an emerging natural drug with a wide range of medical applications.

Iranian Journal of Basic Medical Sciences, 17(12), 950.

Kikuchi, S., Shinpo, K., Takeuchi, M., Yamagishi, S., Makita, Z., Sasaki, N., & Tashiro, K. (2003). Glycation - a sweet tempter

for neuronal death. Brain Research Reviews, 41, 306-323.

Losso, J.N., Bawadi, H.A. & Chintalapati, M. (2011). Inhibition of the formation of advanced glycation end products by

thymoquinone. Food Chemistry, 128, 55-61.

Lutterodt, H., Luther, M., Slavin, M., Yin, J.J., Parry, J., Gao, J.M., & Yu, L. (2010). Fatty acid profile, thymoquinone

content, oxidative stability, and antioxidant properties of cold-pressed black cumin seed oils. LWT - Food Science and

Technology, 43, 1409–1413.


33

Najmi, A., Nasiruddin, M., Khan, R.A. & Haque, S.F. (2012). Therapeutic effect of Nigella sativa in patients of poor

glycemic control. Asian Journal of Phytomedicine and Clinical Research, 5, 224-228.

Pandey, R., Kumar, D. & Ali, A. (2018). Nigella sativa seed extracts prevent the glycation of protein and DNA. Current

Perspectives on Medicinal and Aromatic Plants, 1, 1-7.

Poulsen, M.W., Hedegaard, R.V., Andersen, J.M., de Courten, B., Bügel, S., Nielsen, J. & Dragsted, L.O. (2013). Advanced

glycation end products in food and their effects on health. Food and Chemical Toxicology, 60, 10-37.

Rondeau, P., Armenta, S., Caillens, H., Chesna, H. & Bourdon, E. (2007). Assessment of temperature effects on b-

aggregation of native and glycated albumin by FTIR spectroscopy and PAGE: Relations between structural changes and

antioxidant properties. Archives of Biochemistry and Biophysics, 460, 141-50.

Sadowska-Bartosz, I. & Bartosz, G. (2015). Prevention of protein glycation by natural compounds. Molecules, 20, 3309-

3334.

Solati, Z., Baharin, B. S., & Bagheri, H. (2014). Antioxidant property, thymoquinone content and chemical characteristics

of different extracts from Nigella sativa L. seeds. Journal of the American Oil Chemists' Society, 91(2), 295-300.

Thornalley, P. J. (2003). Use of aminoguanidine (Pimagedine) to prevent the formation of advanced glycation

endproducts. Archives of Biochemistry and Biophysics, 419(1), 31-40.

Zafar, H., Hussain, F., Zafar, S. & Yasmin, R. (2013). Glycation inhibition by Nigella sativa (Linn) – an in vitro model. Asian

Journal of Agricultural Biology, 1, 187-189.

Received : 02.18.2019


Nat. Volatiles & Essent. Oils, 2019; 6(1): 34-39 Küçük et al.

34

RESEARCH ARTICLE

Volatile compositions of three critically endangered and endemic species of the genus Crocus L. (Iridiaceae) and comparison with C. sativus L. (Saffron)

Sevim Küçük1,*, Melike Sayarer1 and Betül Demirci2

1 Department of Pharmaceutical Botany, Anadolu University, Faculty of Pharmacy, 26470 Eskisehir, TURKEY 2 Department of Pharmacognosy, Anadolu University, Faculty of Pharmacy, 26470 Eskisehir, TURKEY


Abstract

This study was carried out upon chemical researches of three species Crocus L. that distribute in Central Anatolia (Eskişehir), Turkey.

The volatile compounds were obtained by microdistillation of the stylus, stigma-tepal parts of three critically endangered and

endemic species of the genus Crocus L., viz. C. chrysanthus (Herb.) Herb., C. antalyensis B. Mathew and C. ancyrensis (Herb.) Maw,

and the stylus, stigma parts of C. sativus L. and the volatiles were analyzed by GC-FID and GC-MS, simultaneously. Ethyl cinnamate

(14.8%), heptanal (14%), and hexahydrofarnesyl acetone (12.8%) of C. chrysanthus. Hexanal (17.7%), nonanal (17.1%), and undecanal

(14.7%) of C. antalyensis. β-Isophorone (14.4%), heptanal (11.5%), and heneicosane (8.5%) of C. ancyrensis. Safranal (77.9%), α-

isophorone (13.5%), and β-isophorone (2.2%) were detected as main constituents in sample of C. sativus. In addition, chemical

structures of C. ancyrensis, C. chrysanthus and C. antalyensis are given in this study for the first time.

Keywords: Iridaceae, Crocus, Saffron, Endemic, GC-FID, GC-MS, Microdistillation

Introduction

Turkey is a rich country regarding the species occurrence of Crocus L. (Iridaceae). Crocus is distributed mainly

in the Mediterranean region and includes 80 species worldwide (Mathew, 1984). There are 70 taxa (including

subsp. and var.) of Crocus in Turkey (Güner et al 2000). Thirty-one of these are endemics for Turkey (Erol,

2011). Many species of the family Iridaceae are grown in parks and gardens as ornamental plants due to their

beautiful flowers (Baytop, 1999).

The known chemical components of C. sativus can be listed as follows. Crosetins: crosin-1, crosin-2, crosin-3,

crosin-4, crocetin, protocrosin, picrosin, safranal Flavonoids: camphorol, astragalin, helicrisoside, crosatoside

A, camphorol 3-O-β-D-glucopyranosyl (1→2) β-D-glucopyranoside, camphorol-3-O-β-D-glucopyranosyl

(1→2) β-D-6-acetylglucopyranoside, quercetin-3-O-β-D-glucopyranoside, isorhamnetin-3-O-β-D-

glucopyranoside Pigments: carthamin, precaharthamine, safflor yellow A, B, β-carotene, zeaxanthin,

lycopene Phenolic compounds: chlorogenic acid, caffeic acid, catechol, crosatoside B, 3,8-dihydroxy-1-

methylantraquinone-2-carboxylic acid Triterpenes: ursolic acid, oleanolic acid, β-sitosterol, campesterol,

stigmasterol Amino acids: 3,4-dihydroxyphenylalanine, proline, asparagine, arginine, glutamine, glutamic

acid Organic acids: palmitic acid, palmitoleic acid, oleic acid, linoleic acid, linolenic acid Other components:

mangicrosine, nonacosane ( Bensky et al. 2004). Saffron's yellow-orange color crosin, sharp taste picrocrosin,

aroma comes from safranal (Gruenwald et al. 2007).

Some Crocus species were used for medicaments making dye and perfume ((Abdullaev, 2003). The saffron

(Crocus sativus L.) was the first to be cultivated and has been grown for economic purposes since ancient


35

times (Abdullaev, 2003) pointed out that the saffron could be useful in cancer chemoprevention in the

immediate future. (Özdemir et al. 2006).

The Crocus is regarded as an ornamental plant as they grow like tulipa and flower in different colours.

Because of these properties the Crocus species can sustain their life forms when they are cultivated in parks

and gardens. It is estimated that the Crocus which affects people positively with their lovely flowers is going

to be of an important economic value in the near future. Some studies reported that the Crocus species have

antitumor, antimutagenic, cytotoxic activities and inhibits nucleic acid synthesis in human (Kravkaz et al.

2006; Fatehi et al. 2003).

The species known as endemic C. ancyrensis (Herb.) Maw “Ankara Çiğdemi”, C. chrysanthus (Herb.) Herb.,

“Sarı Çiğdem”, endemic C. antalyensis B. Mathew “Antalya Çiğdemi” and C. sativus L. “Safran, Çiğdem” in

Anatolia (Guner et al. 2012).

The aim of this study to investigate chemical characteristics of C. ancyrensis, C. chrysanthus, C. antalyensis

and C. sativus. In addition, volatile compositions of C. ancyrensis, C. chrysanthus and C. antalyensis are given

in this study for the first time.


Plant material

Plant materials were collected from different localities in Eskişehir, Turkey and they were identified as

herbarium materials (Table 1). Voucher specimens are kept at the Herbarium of the Faculty of Pharmacy,

Anadolu University (ESSE) in Eskişehir, Turkey.

Table 1. Information on the plant materials.

Species Collection date

Collection site Voucher specimens no (ESSE)

C. ancyrensis 10.03.2013 B3:Eskişehir: Bozdağ, 39° 56’ 32’’ K - 030° 30’ 54’’ D, 1079 m 14623

C. chrysanthus 10.03.2013 B3:Eskişehir: Hekimdağ,39°54’34’’K-030° 33’13’’D, 1289 m 14625

C. antalyensis 10.03.2013 B3:Eskişehir: Bozdağ,39°56’32’’K-030° 30’54’’D, 1079 m 14624

C. sativus 20.042013 Eskişehir Geçit Kuşağı Tarımsal Araştırma Enstitüsü (TAGEM) 15406

Isolation of volatile components

Microdistillation

The volatiles were obtained after microdistillation of the plant material (650 mg) using an Eppendorf

MicroDistiller® containing 10 mL of distilled water per sample vial. The sample vial was heated to 108 °C at a

rate of 20 °C/min for 90 min followed by heating at 112 °C at the rate of 20 °C/min for 30 min. The sample

was subjected to a final post-run for 2 min under the same conditions. The collecting vial, containing a

solution of NaCl (2.5 g) and water (0.5 mL) n-hexane (350 μL) to trap volatile components, which were cooled

to -5°C during distillation. Thereafter, the organic layer in the collection vial was separated and analyzed by

gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) simultaneously.


36

GC-FID and GC-MS analyses

Gas Chromatography-Flame Ionization Detection (GC-FID) and Gas Chromatography-Mass Spectrometry (GC-

MS) analyses processes were performed with reference to Demirci et al., (2017).


In this study, the stylus-tepal parts of C. chrysanthus, C. antalyensis, C. ancyrensis and the stigma parts of C.

sativus were distilled using Eppendorf Microdistiller® and analyzed by GC-FID and GC-MS. Ethyl cinnamate

(14.8%), heptanal (14%), hexahydrofarnesyl acetone (12.8%) were detected as main constituents in sample

of C. chrysanthus, hexanal (17.7%), nonanal (17.1%), undecanal (14.7%) of C. antalyensis, β-Isophorone

(14.4%), heptanal (11.5%), heneicosane (8.5%) of C. ancyrensis. In the oil of C. sativus safranal (77.9%) α-

isophorone (13.5%), and β-isophorone (2.2%) were detected as main constituents (Table 2).

There is a lot of works available in the literature related volatile and non-volatile components of the Crocus

sativus. In previous work, volatile and colour compounds of Crocus sativus stigmas were obtained by

microdistillation and extraction techniques, respectively. The samples were analyzed by GC-FID, GC-MS, HPLC

systems (Başer et al. 2007). Previously, safranal, α-isophorone and β-isophorone were reported as the main

components for C. sativus flowers by microdistillation method (Başer et al. 2007). Caballero-Ortega et al.

have investigated and compared active compounds with the HPLC technique on 11 C. sativus samples

obtained from different sites and sources (Caballero-Ortega et al. 2007). Zheng et al. have reported on

chemical composition determination studies using the GC-MS method in their study of different parts of C.

sativus such as stigma, stamen, and periant (Zheng et al. 2011). Maggi et al. conducted chemical composition

studies on 418 C. sativus samples obtained from various sources around the world and found that saffron-

specific aromatic properties originate from the safranal named compound in their research and comparison

with the volatile compounds they obtained with the GC-MS technique (Maggi et al. 2009). Zhu et al. have

comparatively examined the specimens in their study of the volatile oil composition they made on the C.

sativus corm extract and stigma sections (Zhu et al.2008). Esmaeili et al. have identified phenolic compounds

of C. sativus by GC-MS method (Esmaeili et al 2011). In a study by Campo et al. the content of picrocrosin on

345 C. sativus samples obtained from various sources was comparatively determined (Campo et al. 2010).

Masuda et al. performed comparative volatile compound analysis on the corms in their study with C. sativus

and C. vernus (Masuda et al.2012). Esmaeilian et al. have reported on chemical composition determination

with GC-MS on C. sativus stigmas obtained at different harvest times. Esmaeili et al. conducted studies on

antioxidant activity and identification of phenolic compounds on C. sativus (Esmaeilian et al. 2012). Goupy et

al. have done studies to identify flavonol, anthocyanin, luteindiesters on C. sativus tepals (Goupy et al. 2013).

Norbaek et al. were found in C. chrysanthus flowers in the anthocyanidins by HPLC method. Obtained

anthocyanins are: 3-O- (6-O-malonyl-β-D-glucosyl) -7-O- (6-O-malonyl-O-malonyl- β-D-glucoside) -7-O- (6-O-

malonyl-β-D-glucoside). Obtained camphorol, quercetin and myricetin flavonoids from C. chrysanthus

periant segments and nine flavonol glycosides in their study on C. antalyensis flowers (Norbaek et al. 1998-

1999). Norbaek et al. anthocyanins such as delfinidine, petudine and petunidine in the C. antalyensis periant

segments (Norbaek et al. 1999).

To the best of our knowledge, this is the first report describing the volatile compositions of C. ancyrensis, C.

chrysanthus and C. antalyensis. In this study these compounds are encountered in the samples and it is

thought that the samples may be alternative sources to C. sativus.


37

Table 2. The volatile composition of the Crocus species.

No RRI Compounds C.ancyrensis

%

C. chrysanthus

%

C. antalyensis

%

C. sativus

%

1 1093 Hexanal -- -- 17.7 --

2 1194 Heptanal 11.5 14.0 -- --

3 1400 Nonanal 8.2 -- 17.1 --

4 1400 Tetradecane -- 5.6 -- --

5 1415 β-Isophorone 14.4 4.8 -- 2.2

6 1503 2,6,6-Trimethyl-1,4-Cyclohexadiene-1-Carboxaldehyde

--

--

--

1.9

7 1548 (E)-2-Nonenal 5.2 -- 7.1 --

8 1600 α-Isophorone 5.8 4.1 -- 13.5

9 1617 Undecanal -- -- 14.7 --

10 1661 Safranal 2.3 5.3 -- 77.9

11 1700 Heptadecane -- 1.5 -- --

12 1714 6-Oxoisophorone -- 2.5 -- 1.2

13 1719 Borneol -- tr -- --

14 1793 Dihidro-4- Oxoisophorone -- -- -- 0.3

15 1800 Octadecane 2.4 2.8 1.8 --

16 1900 Nonadecane 4.2 3.0 5.7 --

17 1958 (E)-β- Ionone -- -- -- 0.5

18 1960 4-(2,2,6-trimethylcyclohexan -1-yl)-3-buten-2-one

--

--

--

0.9

19 2000 Eicosane -- -- 9.2 --

20 2100 Heneicosane 8.5 6.4 -- --

21 2131 Hexahydrofarnesyl acetone -- 12.8 9.9 --

22 2157 (E)-Ethyl cinnamate -- 14.8 -- --

23 2174 Fokienol -- 1.4 -- --

24 2300 Tricosane 3.4 3.1 -- --

25 2400 Tetracosane -- 2.8 -- --

26 2500 Pentacosane -- 4.2 -- --

27 2700 Hexacosane -- 6.8 -- --

Total 65.9 95.9 83.2 98.4

RRI: Relative retention indices calculated against n-alkanes. % Calculated from FID data. Tr Trace (|<0.1 %).

ACKNOWLEDGMENT

The authors are grateful to İsmail Kara for collecting the C. sativus. Part of this work was the MSc thesis project of Melike

Sayarer (2015). Anatomical, morphological and chemical research on some Crocus L. species in Eskişehir.

REFERENCES

Abdullaev, F.I. (2003). Crocus sativus against cancer. Archives of Medical Research, 34(4), 354.

Başer, K.H.C., Koşar, M., Demirci, B. & Kara, İ. (2007). Chemical composition and free radical scavenging activity of

saffron, Planta Medica, 73, 7.

Baytop, T. (1999). Türkiye’de bitkiler ile tedavi: geçmişte ve bugün, Nobel Tıp Kitabevleri, İstanbul, Türkiye, p.360.


38

Bensky, D., Clavey, S., Stöger, E. (2004). Chinese herbal medicine, Materia Medica. Third edition, Eastland Press, Seattle,

USA. Gruenwald J, Brendler T, Jaenicke C. PDR for Herbal Medicines. 4th ed., Montvale, N.J. 2007, 653-654.

Caballero-Ortega, H., Pereda-Miranda, R., Abdullaev, F.I. (2007). HPLC quantification of major active components from

11 different saffron (Crocus sativus L.) sources. Food Chemistry, 100, 1126–1131.

Campo, C., Carmona, M., Maggi, L., Kanakis, C.D., Anastasaki, E.G., Tarantilis, P.A., Polissiou, M.G., Alonso, G.L. (2010).

Picrocrocin content and quality categories in different (345) worldwide samples of saffron (Crocus sativus L.). Journal of

Agricultural and Food Chemistry, 58(2), 1305-1312.

Davis, P.H. (1984). Flora of Turkey and the East Aegean Islands. Vol. 8. Univesity Press. Edinburgh.

Demirci, B., Yusufoglu, H. S., Tabanca, N., Temel, H. E., Bernier, U. R., Agramonte, N. M., Alqasoumi, S. I., Al-Rehaily, A.

J., Başer, K.H.C., Demirci, F. (2017). Rhanterium epapposum Oliv. essential oil: Chemical composition and antimicrobial,

insect-repellent and anticholinesterase activities, Saudi Pharmaceutical Journal, 25, 703-708.

Erol, O., Şık, L., Kaya, H.B., Tanyolaç, B., & Küçüker, O. (2011). Plant Systematic Evolution, 294, 281-287.

Esmaeili, N., Ebrahimzadeh, H., Abdi, K., Safarian, S. (2011). Determination of some phenolic compounds in Crocus

sativus L. corms and its antioxidant activities study. Pharmacognosy Magazine, 7(25), 74-80.

Esmaeili, N., Ebrahimzadeh, H., Niknam, V., Mirmasoumi, M., Abdi, K., Safarian, S. (2013). The study on phenolic

compounds in Iranian Crocus sativus L. corms by GC-MS analysis. ISHS Acta Horticulture 850: III International Symposium

on Saffron: Forthcoming Challenges in Cultivation. Research and Economics.

Esmaeilian, Y., Galavi, M., Ramroudi, M., Mashhadi, M., Boojar, A. (2012). Diurnal variability of stigma compounds of

saffron. Annals of Biological Research, 3(3), 1562-1568.

Güner, A., Özhatay, N., Ekim, T., & Başer, K.H.C. (2000). Flora of Turkey and The East Aegean Islands; Univ. Press:

Edinburgh. 364-367, 514.

Guner, A., Aslan, S., Ekim, T., Vural, M., & Babac, M.T. (2012). List of Turkish Flora (Vascular Plants). Publication of

Nezahat Gokyigit Botanical Garden and Flora Research Founda-tion; 888.

Fatehi, M., Rashidabady, T., & Hassanabad, F. (2003). Effects of Crocus sativus extract on rat blood pressure and on

response induced by electrical field stimulation in rat isolated vas defens and guinea-pigileu, Journal of

Ethnopharmacology. 84, 199-203.

Goupy, P., Vian, M.A., Chemat, F., Veyrat, C. (2013). Identification and quantification of flavonols, anthocyanins and

lutein diesters in tepals of Crocus sativus by ultra-performance liquid chromatography coupled to diode array and ion

trap mass spectrometry detections. Industrial Crops and Products, 44, 496-510.

Joulain, D., & Koenig, W. A. (1998). The Atlas of Spectra Data of Sesquiterpene Hydrocarbons, EB-Verlag, Hamburg.

Kravkaz, I. S., Vurdu, H., & Türkyılmaz, E. (2006). Colchicums (Crocus ssp.) as a potential or ornamental plant, Gazi Univ.

Kastamonu Journal of Forest Faculty. 6,135-140.

Mathew, B. (1984). Crocus L In: Davis PH (eds), Flora of Turkey and The East Aegean Island, Edinburg at the University

Press, Edinburg, 8, 413-438.

Maggi, L., Carmona, M., Campo, C.P., Kanakis, C.D., Anastasaki, E., Tarantilis, P.A., Polissiou, M.G., Alonso, G.L. (2009).

Worldwide market screening of saffron volatile composition. Journal of the Science of Food and Agriculture, 89(11),

1950-1954.

Masuda, A., Mori, K., Miyazawa, M. (2012). Comparative analysis of volatile compounds from corms of Crocus sativus

and Crocus vernus. Chemistry of Natural Compounds, 48(2), 319-321.

Norbaek, R., Kondo, T. (1998). Anthocyanins from flowers of Crocus (Iridaceae). Phytochemistry, 47(5), 861-864.


39

Norbek, R., Kondo, T. (1999). Flavonol glycosides from flowers of Crocus speciosus and C. antalyensis, Phytochemistry.

51, 1113-1119.

Norbek, R., Kondo, T. (1999). Further anthocyanins from flowers of Crocus antalyensis (Iridaceae). Phytochemistry, 50,

325-328.

Norbaek, R. Nielsen, J.K., Kondo, T. (1999). Flavonoids from flowers of two Crocus chrysanthus-biflorus cultivars: “Eye-

catcher” and “Spring Pearl” (Iridaceae). Phytochemistry, 51, 1139-1146.

Özdemir, C., Barana, P., & Akyol, Y. (2006). The Morphology and Anatomy of Crocus flavus Weston subsp. flavus

(Iridaceae). Turkish Journal of Botany, 30, 175-180.

Zheng, C.J., Li, L., Ma, W.H., Han, T., Qin, L.P. (2011). Chemical constituents and bioactivities of the liposoluble fraction

from different medicinal parts of Crocus sativus. Pharmaceutical Biology, 49(7), 756-763.

Zhu, Y., Han, T., Hou, T.T., Hu, Y., Zhang, Q.Y., Rahman, K., Qin, L.P. (2008). Comparative study of composition of essential

oil from stigmas and of extract from corms of Crocus sativus. Chemistry of Natural Compounds, 44(5), 666-667.

Received : 08.04.2019


Nat. Volatiles & Essent. Oils, 2019; 6(1): 40-46 Vural et al.

40

RESEARCH ARTICLE

Characterization of Salvia verticillata L. subsp. amasiaca (Freyn & Bornm.) Bornm. essential oil from Turkey

Nilüfer VURAL1*, İsmihan GÖZE2 and Nazlı ERCAN3

1 Department of Chemical Engineering, Faculty of Engineering, Ankara University, Ankara, TURKEY 2 Göze Pharmacy, Çarşıbaşı Street, No:7, 58000, Sivas, TURKEY 3 Department of Biochemistry, Faculty of Veterinary Medicine, Cumhuriyet University, Sivas, TURKEY


Abstract

The essential oil obtained by hydrodistillation from the aerial parts of Salvia verticillata L. subsp. amasiaca (Freyn&Bornm.) Bornm.

from Turkey was analyzed by GC-MS. Overall, 21 compounds were identified representing 97.27% of the total oil. 1,8-Cineole 15.9%,

trans-caryophyllene 13.3%, spathulenol 8.3%, germacrene-D 7.5%, carvacrol 6.3% and β-pinene 4.9 % as main constituents in the oil.

Keywords: Salvia verticillata, Lamiaceae, essential oil, GC-MS

Introduction

Lamiaceae is the third largest family based on the taxon number and fourth largest family based on the

species number in Turkey (Celep and Dirmenci, 2017). Salvia, the largest genus of Lamiaceae, includes about

945 species, widespread throughout the world. This genus is represented, in the flora of Turkey by 100

species and 107 taxa, 54 % of which are endemic (Davis,1982; Güner et al., 2000; Chalchat et al., 2001; Celep

and Dirmenci, 2017; Başer and Kırımer, 2018). The second largest geographical distribution of Lamiaceae taxa

occurs in Central Anatolia (rate of endemism 36%), after the Mediterranean region (Celep and Dirmenci,

2017).

The interest in Salvia has increased remarkably over the last 15 years, due to the diversity of species, world

distribution and high ecological, structural and functional diversity (Claßen-Bockhoff, 2017). The genus Salvia

has recently attracted great attention due to its notable biological activities. In Turkish folk medicine, Salvia

species, also known as “adaçayı, elmaçayı, karabaşotu, dadırak, hart şalbası and yağlıkara” are used as

diuretic, carminative, antiseptic, against colds, stomachache, sore throat, inflammations in the mouth and

infections. They are also consumed as an herbal tea and used as food flavor (Adams, 2001; Aşkun et al., 2010;

Tabanca et al.,2017). Two subspecies are currently recognized, based on the colour of the inflorescence axis:

S. verticillata subsp. verticillata, with a wide distribution range in Europe and S. verticillata subsp. amasiaca

(Freyn & Bornm.) Bornm, limited to Turkey and Western Asia (Giuliani et al., 2018). Especially in the form of

infusion and decoction of Salvia subsp. amasiaca is used in the treatment of diseases such as abdominal pain,

stomachache (Sezik et al. 2001) cardiovascular diseases (Kultur, 2007), laxative, colds, nausea (Altundağ and

Ozturk, 2011). Previous studies showed that the essential oils and extracts Salvia verticillata subsp. amasiaca

have biological activity such as antioxidant, antimicrobial, anticholinesterase, antidiabetic (Tepe at al.,2007;

Eidi et al., 2011; Kunduhoglu et al.,2011; Erbil and Dığrak, 2015) and antimycobacterial activities (Aşkun et

al., 2010).


41

Although Turkey is a country rich in endemic Salvia species, only a few studies have been conducted on the

essential oil components of Salvia verticillata subsp. amasiaca. In the present study, S. verticillata subsp.

amasiaca essential oil, one of the two subspecies of Salvia verticillata, which is thought to be especially

important due to chemical variation, was collected and investigated from the Sivas region.

Plant material

Aerial parts of Salvia verticillata subsp. amasiaca were collected from Gürün, Yazyurdu village (August/1550

m), Sivas, Turkey. The voucher specimen was identified by Dr. Erol Dönmez at the Department of Biology,

Cumhuriyet University, Sivas-Turkey and deposited at the Herbarium (CUFH-Voucher No: ED 11019).

Isolation of the essential oil

The air-dried and finely ground aerial parts were distilled for 3h using a Clevenger-type apparatus. The oil

(yielded 0.02% v/w) was dried over anhydrous sodium sulphate and stored at +4 °C.

Gas chromatography- mass spectrometry (GS/MS) analysis

The chemical composition of the Salvia verticillata subsp. amasiaca essential oil was analysed using a

Shimadzu QP-5000 gas chromatograph-mass spectrometer equipped with a GL Science capillary column TC-

5 (30 m × 0.25 mm i.d.,0.25 mm) and a 70 eV EI Quadrupole detector.

Helium was the carrier gas, at a flow rate of 1.9 mL/min. Injector and MS transfer line temperatures were set

at 250 and 280°C, respectively. The column temperature was initially at 40°C held for 2 min, then gradually

increased to 125°C at a 2°C/min rate, held for 2 min, and finally increased to 250°C at 5°C/ min held for 2 min.

Diluted samples (1:100 v/v, in acetone) of 1.0 µL were injected manually and splitless.

Identification

The components were identified by comparison with their relative retention indices and MS (NBS75K library

data of the GC–MS system) as well as the literature (Adams, 2001).


In this present study, the composition of Salvia verticillata subsp. amasiaca oil was analysed by GC-MS,

compounds representing 97.27% of the oil were identified, with 1.8-cineole 15.9%, trans- caryophyllene

13.3%, spathulenol 8.3%, germacrene D 7.5 %, carvacrol 6.3 % and β-pinene 4.9 % as main constituents as

seen at Table 1.

Previous reports indicate that β-Pinene, α-pinene ,1,8-cineole caryophyllene, carvacrol, spathulenol and

germacrene-D are the main and/or characteristic constituents of Salvia verticillata subsp. amasiaca essential

oil (Başer,1993; Başer,2002; Başer and Kırımer, 2006; Altun et al., 2007; Aşkun et al.,2010; Kunduhoglu et

al.,2011; Başer and Kırımer, 2018).


42

Table 1. Components of S. verticillata subsp. amasiaca essential oil

RRI Retention time (Rt)a

Compounds %

954 13.555 campheneb 1.85

975 15.067 sabineneb 2.45

977 15.183 β-pinenec 4.89

990 16.418 β-myrceneb 2.05

1024 18.268 p-cymene 3.45

1030 18.531 1,8-cineoleb 15.89

1044 19.820 cis-β-ocimene 3.15

1096 23.808 linalool 3.97

1102 23.900 thujyl alcoholc 3.97

1126 24.173 pulegone 2.76

1138 25.800 cis-sabinol 3.81

1165 27.712 borneol 3.54

1194 36.580 myrtenol 1.70

1295 38.625 carvacrolb 6.28

1375 41.486 α-copaenec 2.98

1406 45.983 trans-caryophyllene 13.25

1435 47.202 aromadendreneb 2.98

1480 49.255 germacrene Db

7.47

1486 52.125 β-selinenec 0.57

1620 53.543 spathulenol 8.28

1719 56.125 farnesol 1.95

Ʃ Monoterpene compounds

Ʃ Sesquiterpene compounds

Total identified

59.79

37.48

97.27%

RRI Relative retention indices calculated against n-alkanes. % calculated from FID data. aRetention time (as min). bCompounds listed in order of elution from a OV-5 column. cIdentification of components based on standard compounds.

In order to assess the variability in the essential oil composition of our sample, a qualitative comparison was

performed with respect to the profiles known in the literature, referring to 23 populations from a broad

geographical range, from the Yugoslavia, Serbia, Romania, Greece, Italy, Turkey region to the Iran (Table 2).

Essential oil data of S. verticillata subsp. amasiaca in the literature is limited to a few studies in Turkey (Başer

et al., 1993; Başer, 2002; Başer and Kırımer,2006; Altun et al.,2007; Aşkun et al., 2010; Kunduhoglu et al.,

2011; Hatipoglu et al., 2016; Başer and Kırımer,2018).

The essential oil yields obtained in the present and the other previous studies in literature were extremely

variable from levels below the limit of quantification up to 1.3% (Altun et al., 2007) in Turkish populations.

At the same time, the total number of the identified compounds was very different.

According to the present literature, S. verticillata volatile chemicals appeared to be characterized by a high

level of complexity. A general chemical polymorphism exists due to the geographical provenance, the

processed plant parts, the employed techniques and the investigated subspecies.

However, monoterpene hydrocarbons constituted the most represented class in a few studies taken into

consideration in accordance to our data (Altun et al. ,2007; Aşkun et al., 2010) in S. verticillata subsp.

amasiaca.


43

In Turkey, Başer et al., (1993) reported that carvacrol 27.5% and spathulenol 17% were the main components

in S. verticillata subsp. amasiaca. At the same time, β -caryophyllene 17% (Başer, 2002; Başer and Kırımer,

2006) and germacrene-D 37% (Başer and Kırımer, 2018) were also determined. Altun et al., 2007 reported,

β-pinene 23.0%, α-pinene 21.6%, ß-phellandrene 13%, limonene 11%, 1,8-cineole 10.9% in a Bitlis sample.

Askun et al., (2010) determined different main constituents β-pinene 21.4%, 1,8-cineole 16.1%, α-copaene

5.4%, alloaromadendrene 5.1%, α-gurjunene 4.6% in S. verticillata subsp. amasiaca collected from the Bitlis-

Tatvan geographical region. Kunduhoğlu et al., (2001) reported that S. verticillata subsp. amasiaca collected

from Bilecik, Bozüyük, Kozpınar had high levels of germacrene D 36.6%, β-caryophyllene 7.6%, hexadecanoic

acid 6.7% and β-copene 5.7%. The contents of S. verticillata subsp. amasiaca essential oil have been

reported mainly hydrocarbons and their derivatives by Hatipoglu et al., 2016. According to our survey of the

available literature on the composition of S. verticillata subsp. amasiaca our data partially agrees with the

previous studies (Table 2).

Table 2. Previous reports on S. verticillata and S. verticillata subsp. amasiaca* essential oils

Main compounds (%) Origin References

β-caryophyllene 24.7%, γ-muurolene 28.8%, limonene 8.9%, α-humulene 7.8%

Iran Şefidkon et al., 1999

β-caryophyllene 13.3%, γ-muurolene 10.3%, trans-chrysanthenol 6.1% Yugoslavia Chalcat et al., 2001

Germacrene D 48%, (E)-caryophyllene 13.4%, α-humulene 7.2%, α -cadinol 10.4%

Serbia

North Serbia

Krstic et al., 2006

α-Pinene 30.7%, p-cymene 23%, β-pinene 7.6%, lauric acid isopropyl ester 16.8%

Greece Pıtarokili et al., 2006

β-Caryophyllene 31.5%, germacrene D 16.2%, limonene 15.5%, α-pinene 10.4%, α-humulene 9.4%

Iran Yousefzadi et al., 2007

β-Caryophyllene 13.5%, germacrene-D 22.8%,valeronene 8.9%, γ-elemene 4.6%

Romania Pădure et al., 2008

β-Caryophyllene 16%, α-caryophyllene 14.5%, spathulenol 8.6% Eastern Romania Coisin et.al, 2012

E-Caryophyllene 14.7-17.8%, a-gurjunene 12.8- 3.4%, β- phellandrene 6.6- 14.2%, α-humulene 7.6- 10.11 %, germacrene D 5.1 %, sabinene 4.5 %

Iran Nasermoadeli et al., 2013

E-Caryophyllene 16.9–40.9%, spathulenol 0–17.5%, α-humulene 5.4–14.3%, bicyclo-germacrene 3.4–6.4%

Iran Rajabi et al., 2014

trans-Caryophyllene 24.4%, β-phellandrene 9.0%, α-humulene 8.6%, bicyclogermacrene 6.3%, spathulenol 5.9%, β-pinene 5%

Iran Khosravi Dehaghi et al., 2014

1,8-Cineole 38.3 %, camphor 22.9% Iran and West Azerbaijan)

Forouzin et al., 2015

Germacrene D 13.8%, spathulenol 10%, limonene 4.5%, 1,8- cineole 4.5% Turkey (Elazıg, Baskil) Doğan et al., 2015

trans-Caryophyllene 18.8%, Germacrene-D 9.49%, spathulenol 7.5%, sabinene 6.5%, α-caryophyllene 5.8%

Iran (the central Alborz) Mahdavi et al., 2015

Spathulenol 31.0%, α-pinene 8.2%, limonene 4.1%) and hexahydrofarnesyl acetone 3.8%

Turkey (Şırnak, Hakkari)

Tabanca et al., 2017

Germacrene-D 39.5-40.7 %, bicyclogermacrene 11.5-14.8%, β-caryophyllene 7.5-11.9%, spathulenol 3.1-6.6%, α-humulene 2.7-5.6%,

Milan, Italy Giuliani et al.,2018

Carvacrol 27. 5 %, spathulenol 17% Turkey Başer et al., 1993

β -Caryophyllene 17% Turkey Başer, 2002

β -Caryophyllene 17%, Carvacrol 27 %,spathulenol 17% Turkey Başer and Kırımer, 2006

β -Pinene 23.0%, α-pinene 21.6%, ß-phellandrene 13%, limonene 11%, 1,8-cineole 10.9%

Turkey (Bitlis Tatvan) Altun et al., 2007

http://www.sciencedirect.com/science/article/pii/S0926669014003951


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β-Pinene 21.4%, 1,8-cineole 16.1%, α-copaene 5.4%, alloaromadendrene 5.1%, α-gurjunene 4.6%

Turkey (Bitlis Tatvan) Aşkun et al., 2010

Germacrene-D 36.6%, β-caryophyllene 7.6%, hexadecenoic acid 6.7%, β- copaene 5.7%, spathulenol 4.5%

Turkey (Bilecik, Bozüyük Kozpınar)

Kunduhoglu et al., 2011

β-Caryophyllene 17%, germacrene-D 37% Turkey Başer and Kırımer,2018

Contrary to our study, the investigations on the spontaneous populations of S. verticillata subsp. amasiaca

from Turkey (Başer et al., 1993; Başer and Kırımer, 2006; Kunduhoğlu et al., 2011; Hatipoglu et al., 2016;

Başer and Kırımer, 2018) documented a dominance of sesquiterpenes in the essential oils. At the same time,

the investigations on the populations of S. verticillata subsp. verticillata in the literature give similar results

(Table 2).

The only ubiquitous compound is beta-caryophyllene, even if detected in variable relative amounts. Among

the most frequent compounds, that are also the most abundant ones, germacrene-D, spathulenol and

carvacrol must be cited.

Conclusion

In conclusion, dominance of sesquiterpenes may be specific to both S. verticillata subsp. verticillata and S.

verticillata subsp. amasiaca. In order to find an answer to this problem, it is necessary to characterize

additional samples from two sub-species.

REFERENCES

Adams, R.P. (2001). Identification Of Essential Oil Components by GC-MS. Carol Stream IL: Allured Publishing

Corporation.

Altun, M., Ünal, M., Kocagöz, T., Gören, A.C. (2007). Essential oil compositions and antimicrobial activity of Salvia

species. Journal of Essential Oil Bearing Plants, 10, 3, 251-258.

Altundag, E., Ozturk, M. (2011). Ethnomedicinal studies on the plant resources of east Anatolia, Turkey. Procedia Social

and Behavioral Sciences, 19, 756-777.

Aşkun, T., Başer, K.H.C., Tumen, G., Kurkcuoğlu, M. (2010). Characterization of essential oils of some Salvia species and

their antimycobacterial activities. Turkish Journal of Biology, 34, 89-95.

Başer, K.H.C. (1993). Essential oils of Anatolian Labiatae. A Profile. Acta Horticulturae, 333, 217-237.

Başer, K.H.C. (2002). Aromatic biodiversity among the flowering plant taxa of Turkey. Pure and Applied Chemistry, 74,

527-545.

Başer, K.H.C. & Kırımer, N. (2006). Essential Oils of Lamiaceae Plants of Turkey, Acta Horticulturae, 723, 163-171.

Başer, K.H. C., Kırımer, N.(2018) Essential oils of Anatolian Lamiaceae - An update. Natural Volatiles and Essential Oils,

5(4):1-28

Celep, F., Dirmenci, T. (2017). Systematic and Biogeographic overview of Lamiaceae in Turkey. Natural Volatiles and

Essential Oils, 4(4): 14-27.

Chalchat, J.C., Gorunovic, M.S., Petrovic, S.D., Maksimovic, Z.A. (2001). Chemical composition of two wild speciesof the

genus Salvia L. From Yugoslavia: Salvia aethiopis and Salvia verticillata. Journal of Essential Oil Research, 13, 416-418.

Claßen-Bockhoff, R. (2017). Stamen construction, development and evolution in Salvia s.l. Natural Volatiles and

Essential Oils, 4(4): 28-48.

Coisin, M., Burzo, I., Ştefan, M., Rosenhech, E., Zamfirachem, M. (2012). Chemical composition and antibacterial activity


45

of essential oil of three Salvia species, wide spread in Eastern Romania. Analele Ştiinţifice ale Universităţii „Al. I. Cuza”

Iaşis. II a. Biologie vegetală., 58(1), 51-58.

Davis, P.H. (1982). Flora of Turkey and the East Aegean Islands. University Press, Edinburgh Vol. 7. pp. 102-126.

Dogan, G., Hayta, S., Yuce, E., Bagci, E. (2015). Composition of the essential oil of two Salvia taxa (Salvia sclarea and

Salvia verticillata subsp. verticillata) from Turkey. Natural Science and Discovery, 1(3),62-67

Eidi, A., Eidi, M., Shahmohammadi, P., Mozaffarian V., Rustaiyan, A. ve Mazooji, A. (2011). Antidiabetic effect of Salvia

verticillata L. aerial parts in normal and streptozotocin-induced diabetic rats. International Journal of Pharmacology, 7,

66-73.

Erbil, N. and Dığrak, M. (2015). Total phenolic and flavonoid contents, antimicrobial and antioxidant properties of Salvia

verticillata L. var. amasiaca and Salvia microstegia Boiss & Bal from Turkish Flora. Journal of Microbiology and

Antimicrobial Agents, 1, 23-29.

Forouzin, F., Jamei, R., Heidaril, R. (2015). Compositional Analysis and Antioxidant Activity of volatile components of two

Salvia spp. Tropical Journal of Pharmaceutical Research, 14(11), 2009-2013.

Giuliani, C., Ascrizzi, R., Lupi, D., Tassera, G., Santagostini, L., Giovanetti, M., Flamini, G., Fico, G. (2018). Salvia verticillata:

Linking glandular trichomes, volatiles and pollinators. Phytochemistry, 155, 53-60.

Güner, A., Özhatay, N., Ekim, T., Başer, K.H.C. (2000). Flora of Turkey and The East Aegean Islands, Flora of Turkey and

the East Eagean Islands, (Vol II) Edinburg: Edinburg Universty Press.

Khosravi Dehaghi, N., Ostad, S.N, Maafi, N., Pedram, S., Ajani, Y., Hadjiakhoondi A., Kanavi, M. (2014). Cytotoxic activity

of the essential oil of Salvia verticillata L. Research Journal of Pharmacognosy 1(3),27-33.

Kultur, S. (2007). Medicinal plants used in Kırklareli Province (Turkey). Journal of Ethnopharmacology, 111, 341-364.

Kunduhoglu, B., Kurkcuoglu, M., Duru, M.E., Baser, K.H.C. (2011). Antimicrobial and anticholinesterase activities of the

essential oils isolated from Salvia dicroantha Stapf., Salvia verticillata L. subsp. amasiaca (Freyn and Bornm.) Bornm.

and Salvia wiedemannii Boiss. Journal of Medicinal Plants Research, 5(29), 6484-6490.

Krstic, L., D Malencic, G Anackov (2006). Structural investigations of trichomes and essential oil composition of Salvia

verticillata. Botanica Helvetica, 116(2), 159-168.

Mahdavi, M., Jouri, M.H., Mahzooni-Kachapi, S., Halimi’Jelodar, S. (2015). Study of chemical composition and

antibacterial effects of essential oils of Stachys lavandulifolia Vahl., Salvia verticillata L., and Tanacetum polycephalum

Schultz-Bip. on some microbial lineages. International Journal of Farming and Allied Sciences, 4(3),197-206.

Nasermoadeli, S., Rowshan, V., Abotalebi, A., Nasermoadeli, L., Charkhchian, M (2013). Comparison of Salvia verticillata

essential oil components in wild and cultivated population. Annals of Biological Research, 4(5),252-255.

Pădure-Ioana, M., Burzo I., Mihăiescu D., Bădulescu L., Dobrescu A., Delian E. (2008). Chemıcal constıtuents of the

essentıal oıls of eıght specıes of Salvia L. (Lamiaceae) from Romania. Acta Horti Botanici Bucurestiensis, 35,1-7.

Pitarokili, D., Tzakou, O., Loukis A. (2006). Essential oil composition of Salvia verticillata, S. verbenaca, S. glutinosa and

S. candidissima growing wild in Greece. Flavour and Fragrance Journal, 21(4), 670-673.

Rajabi, Z., Ebrahimi, M., Farajpour, M., Mirza, M., Ramshini, H. (2014). Compositions and yield variation of essential oils

among and within nine Salvia species from various areas of Iran. Industrial Crops and Products, 61, 233–239.

Rzepa, J., Wojtal, L., Staszek, D., Grygierczyk, G., Labe, K., Hajnos, M., Kowalska, T., Waksmundzka-Hajnos, M. (2009).

Fingerprint of selected Salvia species by HS-GC-MS analysis of their volatile fraction, Journal of Chromatographic

Science, 47, 575-580.

Sezik, A., Yesilada, E., Honda, G., Takaishi, Y., Takeda, Y., Tanaka, T. (2001). Traditional medicine in Turkey X. Folk

medicine in Central Anatolia. Journal of Ethnopharmacology, 75, 95-115.


46

Şefidkon, F., Khajavi, M.S. (1999). Chemical composition of the essential oils of two Salvia species from Iran: Salvia

verticillata L. and Salvia santolinifolia Boiss. Flavour and Fragrance Journal, 14(2), 77-78.

Tabanca, N., Demirci, B., Aytaç, Z., Başer, K.H.C. (2017). The chemical composition of Salvia verticillata L. subsp.

verticillata from Turkey. Naural Volatiles and Essential Oils, 4(1), 18-28.

Tepe, B., Eminagaoglu, O., Akpulat, A., Aydin, E (2007). Antioxidant potentials and rosmarinic acid levels of the

methanolic extracts of Salvia verticillata (L.) subsp. verticillata and S. verticillata (L.) subsp. amasiaca (Freyn & Bornm.)

Bornm. Food Chemistry, 100(3), 985-989.

Yousefzadi, M., Sonboli, A., Karimi, F., Ebrahimi, S.N., Asghari, B., Zeinalia, A. (2007). Antimicrobial activity of some Salvia

species essential oil. Zeitschrift für Naturforschung C, 62, 514–518.

Received : 09.08.2017


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