New insights into plant volatiles and es

() 1 New Insights into Plant Volatiles and Essential Oils K Hüs ü Ca BAŞER1,2,3,4* & Fatih DEMIRCI2,3 1Bahçeşehir U iversity, Te h ology Tra sfer Offi e, Beşiktaş, Ista ul, Turkey 2Bade io Biote h olo[.]

New Insights into Plant Volatiles and Essential Oils

K Hüs ü Ca BAŞER 1,2,3,4 * & Fatih DEMIRCI 2,3

1 Bahçeşehir U iversity, Te h ology Tra sfer Offi e, Beşiktaş, Ista ul, Turkey

2 Bade io Biote h ology Ltd, Eskişehir, Turkey

3 Anadolu University, Faculty of Phar a y, Depart e t of Phar a og osy, Eskişehir, Turkey

4 King Saud University, Faculty of Science, Department of Botany and Microbiology, Riyadh, Saudi Arabia

www.khcbaser.com , www.badebio.com

Abstract

Plant volatiles and essential oils comprise thousands of secondary metabolites in a wealth of biochemical, biological and pharmacological diversity Throughout our research over three decades

we have employed many innovative techniques such as pilot scale distillation to molecular

distillation, microdistillation, absorptive and headspace etc techniques for the isolation; and several

chromatospectral techniques for the chemical characterization Beside semisyntheses, we have employed biotransformation techniques to modify the chemical composition of essential oils and volatiles for further investigations and applications Biological and pharmacological evalutions of the

essential oils, volatiles and their derivatives were in a broad spectrum both in vitro and in vivo

Antioxidant, antimicrobial, antimalarial, antiprotozoal, cytotoxicity, genotoxicity assays; anticancer assays including cancer cell lines, apoptotic and (anti)angiogenic activities; antiinflammatory,

choleretic, hepatoprotective, analgesic, woundhealing activities, etc However, in recent years we

use the combination of essential oil components such as the nature does for the enhancement or decrease of the biological/pharmacological activity Due to their highly volatile nature and solubility problems it is difficult to work with essential oils and volatiles, however, current research suggest that the bright future potential on this topic is still promising for diverse interdisciplinary collaborative work

Plan of presentation

- Flora of Turkey

- Our research group

- Infrastructure, Capabilities and Research Staff

- Essential oil and volatiles isolation

- - Headspace (HS) Techniques

- - Static and Dynamic HS Trapping

- - Microdistillation

- - Molecular distillation

- - Microwave distillation

- - Spinning band fractional distillation system

- - SFE

- Chromatospectral Techniques for authentication and analysis

- - Preparative GC

- - Multidimensional GC/MS (Enantiomeric separations)

- - GC/MS, GC/FT-IR, GC-AED

- - HPTLC – prepTLC

- Applications

- - Microbial transformations

- - Antimicrobial activity testing

- - Bioactivities: Antioxidant, antimicrobial, antimalarial, antiprotozoal, cytotoxicity, genotoxicity assays; anticancer assays including cancer cell lines, apoptotic, angiogenic and antiangiogenic activities; antiinflammatory, choleretic, hepatoprotective, analgesic,

woundhealing activities, etc

- Commercialization of research activities

Flora of Turkey

Turkey is situated at the junction of two continents and has land in Asia and Europe between 42oN and 36oN latitudes Its land mass is about 0.8 million square kilometer supporting a population of 75 million Turkey has a transect between the sea level and Mount Ararat (5137 m) It is under the influence of three different climates: Mediterranean, Continental and Oceanic, and is at the junction of three important phytogeographic regions: Irano-Turanian, Euro-Siberian and Mediterranean

This is the reason why the flora of Turkey is rich and diverse It comprises 12.068 flowering plant taxa belonging to 10.279 species This is ca 2000 less than the number of flowering plant species growing in Europe 1/3 of the flora comprises aromatic plants and >33% of the flora consists of endemic species 1000 plant taxa are used in folk medicine variously

Flora of Turkey is well documented in the Flora of Turkey and the East Aegean Islands, edited by the late Prof Dr P H Davis and published by Edinburgh University Press in 10 volumes (9+1 supplement) between 1965-1988 Vol 11, the second supplement, edited by Prof Dr Tu a Eki , Prof Dr Adil Gü er, Prof Dr Neri a Özhatay a d Prof Dr K Hüs ü

Ca Başer was published in 2000 by the same publisher

Our Research Group

Our research group has been screening especially the aromatic the flora of Turkey in the last

33 years systematically for their essential oil yield and compositions as well as their biological activities 33 genera out of 46 in the family Lamiaceae have been investigated by our group The other important families investigated are Apiaceae and Asteraceae, etc

Pharmacognosy Department at the Faculty of Phar a y of A adolu U i ersity i Eskişehir, Turkey was founded in 1968 Pharmacognosy facilities were strengthened in 1980s with the advent of TBAM ( Medicinal and Aromatic Plant and Drug Research Centre) and with its staff strength of 25 pharmacists, chemists, biologists and chemical engineers under the direction

of Prof Dr K Hüs ü Ca Başer

The group published its first research paper in 1982 (1) and made 11 publications until 1991 The first paper on essential oils appeared in 1991 (2) and since then >1000 research papers have been published by our group, over 90% of them being on essential oils

Research interests of the department include botany and ethnobotany, chemistry, analysis, biological activity and pharmacology, enzyme inhibitory activities, technology;

biotransformations, biopolymerizations etc of essential oils and volatile compounds;

phytochemical and biological screening programmes on the analysis f the aromatic plants of Turkey; collaborative work with Europe, USA, South Africa, Uzbekistan, Kazakhstan, Japan, Pakistan, India, Mongolia, Brunei

We have contributed chapters on essential oils in the following important books:

Analysis and Quality Assessment of Essential Oils, K.H.C Baser, In: K Tuley De Silva (Ed.) A Manual on the Essential Oil Industry, UNIDO, Vienna , pp 155-177 (1995)

Chemistry of Essential Oils, K.H.C Baser and F Demirci In: Flavours and Fragrances:

Chemistry, Bioprocessing and Sustainability, R.G Berger (Ed.), Springer, Berlin, pp 43-86 (2007)

Handbook of Essential Oils: Science, Technology and Applications, K.H.C Baser and G

Buchbauer (Eds.), CRC Press, Boca Raton, London, New York (2010)

Essential Oils, K Husnu Can Baser and F Demirci, Kirk-Othmer Encyclopedia of Chemical Technology, 4th Edition, Wiley, 1 –37 (2011)

Analysis of Essential Oils and Fragrances by Gas Chromatography, K.H.C Baser and T Ozek, In: Gas Chromatography, Ed C Poole, Elsevier, Oxford, pp 519-527 (2012)

The department is well equipped with basic infrastructure and laboratories as well as

sophisticated chromatospectral instrumentation such as Gas Chromatography/Mass

Spectrometry (GC/MS) systems, Multidimensional GC/MS system, Preparative GC, Liquid Chromatography/Mass Spectrometry (LC/MSMS) system, fully fledged High Performance Thin Layer Chromatography (HPTLC) system, High Pressure Liquid Chromatography (HPLC) systems, Medium Pressure Liquid Chromatography (MPLC) system, Flash Chromatography and has access to 500 MHz NMR, GC-FTIR, GC-AED (Atomic Emission Detector), TLC scanner, Chromatotron and pilot plants and semi-industrial plants for large scale extraction and distillation, chemical reactor

The laboratories possess all necessary facilities for essential oil research and development such as numerous sets of small and medium scale Clevenger apparata, Likens-Nickerson Simultaneous Distillation-Extraction (SDE) apparata, microdistillation, microwave distillation, Short Path Distillation (Molecular Distillation) systems, dynamic headspace trapping set up, Headspace Solid Phase Microextraction (HS-SPME) set up, Supercritical Fluid Extraction (SFE) system, Extraction with Fluidized Gasses (Phytosol) equipment, Spinning Band Fractional Distillation system, etc

Applications

Due to the paucity of time, I shall not elaborate on the phytochemical research carried out by our group, but focus mainly on the more recent innovative activities concerning microbial transformations and biological activity studies References given in this text are restricted to our papers published in the last 5 years

Microbial Transformations (Biotransformations)

In 1929, Bio J.B.S Haldane stated that microorganisms can be your friend if you know how

to o u i ate ith the sayi g Why trou le to ake o pou ds yourself he a ug ill do it for you?

Biotransformation is the use of microorganisms or enzymes as biocatalysts to produce novel

or known metabolites from particular substrates When a substrate is added to a microbial medium, the microorganism metabolizes the substrate by first making it more soluble in its body fluid During this stage, functional groups are added to the molecule Such compounds are extracted from the medium before they are completely metabolized

Biotransformations are useful techniques employed in biotechnology, nanotechnology and green chemistry One of the major advantages of biotransformation is the production of natural metabolites from natural starting materials, generally resulting in more or less bioactive compounds from micro to ton scale Biotransformation processes are generally considered as environmental friendly due to mild conditions in aqueous medium where fermention conditions are relatively simple and safe resulting in highly regio- and/or

stereoselective one pot reactions in a relatively short time ranging from hours to weeks Such reactions generally mimic in vivo biosyntheses of compounds, therefore, are useful in

elucidating biosynthetic pathways under in vitro conditions Biotransformations are

considered low energy consuming, hence more economical compared to conventional chemical syntheses

Our research group possesses more than 150 microorganisms for use in microbial

transformations as well as antimicrobial screening tests We, either use volatile compounds isolated from essential oils or essential oils as such for microbial transformations Example of some substrated studies include terpenes such as p-cymene, terpinolene, a-pinene,

b-pinene, carvone enantiomers, carvacrol and thymol and their methyl ethers, and several sesquiterpenes

The metabolites are either extracted from the medium by immiscible solvent extraction or by headspace solid phase microextraction (HS-SPME) Analyses are mainly performed by Thin Layer Chromatography (TLC) and GC/MS

Some of our results are summarized in the following figures and some representative

publications are listed in the references (3-10)

Bioassays

In vitro and in vivo microtechniques

1 Antimicrobial (Agar diffusion, microdilution, TLC-bioautography)

- Antibacterial (MRSA, Serratia, E coli, H pylori )

- Antifungal (human and plant pathogens)

- Anticandidal

2 Antioxidant

- 96 well format – spectrophotometrically

- TLC bioautography

3 Enzyme inhibition assays (UV spectrophotometry – 96 well format, TLC bioautography)

- Acetylcholine esterase (AchE)

- Butyrylcholine esterase (BuChE)

- Lipoxygenase (LOX)

- Cycloxygenase (COX)

- Neuraminidase (NEUR)

- -Glycosidase (-Gly)

- Lipase

4 Bioluminex – Vibrio fischeri toxicity assay (TLC-bioautography)

5 Chorioallantoic membrane assay (CAM) – Angiogenic/antiangiogenic activity

Antimicrobial assays

Due to the fact that pathogenic microorganisms are gaining resistance to antibiotics,

research into discovering new antimicrobial agents is a hot area Discovering new

microbiostatic and microbicidal agents has been our aim in performing antimicrobial assays

We use various in vitro techniques such as agar diffusion, microdilusion and

TLC-bioautographic methods against Gram + and Gram - bacteria such as E coli, MRSA

(Methicillin-resistant Staphylococcus aureus), Serratia, Helicobacter pylori, etc., human and plant pathogenic fungi (Aspergillus, Penicillium, Fusarium, Cladosporium, etc.), yeasts such as Candida spp

Some representative publications can be found in references 11-47

Antioxidant assays

Oxidation reactions are vital for biological systems However, oxidations may also cause damage to their normal functioning through oxidative stress caused by free radicals such as ROS (reactive oxygen species) Antioxidants inhibit the oxidation of molecules at cellular level They are also used for preservation of foods, cosmetics, and other products prone to oxidation

Essential oils and their components are valuable and promising sources of natural

antioxidants We test antioxidant activity by the following assays: Total phenol content, total flavonoid content, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH●

) scavenging activity, TLC bioautography, iron(III) to iron (II) reduction, determination of inhibition of linoleic acid peroxidation, determination of inhibition of -carotene/linoleic acid co-oxidation, ABTS radical scavenging activity, estimation of Trolox Equivalent Antioxidant Capacity (TEAC) using ABTS radical, online HPLC-ABTS●

and HPLC-DPPH ●

screening tests

A list of our representative publications are given in references 48-56

Enzyme inhibitory activity assays

Enzymatic reactions play a major role in many pathogeneses Therefore, inhibition of

responsible enzymes for pathogenies is important to stop progression of a disease Many drugs are enzyme inhibitors Therefore, finding new enzyme inhibitors for various diseases is

a promising and rewarding research area

We perform the following enzyme inhibitory assays:

Acetylcholine esterase (AchE) and Butyrylcholine esterase (BuChE) assays for the discovery of anti-Alzheimer agents and insecticides

Lipoxigenase (LOX) and Cycloxygenase (COX) inhibitory activities are for antiinflammatory agents

Neuraminidase (NEUR) inhibition is for the treatment of viral diseases such as influenza

-Glycosidase and Lipase inhibitions are important for diabetes and obesity type metabolic disorders

Related references are 57-65

Vibrio fisheri toxicity assay is for the in vitro determination of acute and chronic toxicity It is

a practical, fast and sensitive assay suitable for microdilution and TLC-bioautography of

indvidual compounds and mixtures Vibrio fischeri is a luminescent Gram (-) marine

bacterium The Latin name of this organism has recently been changed to Aliivibrio fischeri

This bacterium emits luminescence while alive When it is exposed to toxic chemicals it loses its luminesces and dies Bioluminex method measures the acute and chronic LD50 values of the tested samples

Related references 14, 15, 25, 26, 28

Chorioallantoic membrane assay (CAM)

Angiogenesis and anti-angiogenesis have been associated with tumour pathogenesis CAM

of the chick embryo serves as an excellent vascularization (angiogenesis) environment The test is conducted on fertilized chicken eggs at early embryo stage Test sample is introduced onto the chorioallantoic membrane in pellet form for 24 hours Stereomicroscopic

examination allows to distinguish membrane toxicity, angiogenic effect, antiangiogenic effect

as well as antiinflammatory effect In anti-angiogenic effect, capillaries around the pellet diminish, however, in angiogenesis development of new capillaries are evident and

measurable Using an inflammatory agent like SDS (Sodium dodecylsulfate) causes

membrane irritation seen as extrene vascularization in a star like form An antiinflammatory agent reverses this effect Related references are 66-72

Insecticidal and antiparasitic activities

Mosquito repellent, biting deterrence, larvicidal, acaricidal, anti-tick and insecticidal assays are conducted in collaboration with Mississippi University (OleMiss), Oxford, MS (USA) Antiprotozoal and antileishmanial actvities were also conducted in collaboration with UK and Pakistani collaborators (73-80)

Pharmacological and clinical studies

The only clinical study we have conducted is on oregano water which is the hydrosol of Origanum onites oil distillation The study was conducted on 48 patients having mild

hyperlipidaemia 32 of the patients were in the study group Patients in the study group were given 25 ml of the hydrosol daily for three months Beneficial effects of the hydrosol on lipid profiles, antioxidant status, endothelial functions of mild hyperlipidaemic functions (81) Pharmacological studies conducted include hepatoprotective and ischemia reperfusion defects inhibition of carvacrol in liver, antiinflammatory, antinociceptive, anticolitis,

anticonvulsant, anticancer activities of essential oils and natural aromachemicals (82-86)

Commercialization of Research Activities

Research for the sake of research is good for the generation new knowledge However, if it only helps publication of new articles, innovation is buried in libraries Therefore, it is the duty of a responsible researcher to find ways of application of research results This is best achieved by collaboration with commercial companies interested in research and

development, or by establishing a new company by the researcher himself There are ample opportunities for innovative researchers nowadays in many countries including Turkey, Europe and the world In Turkey, university researchers can set up their spin off companies in technoparks with high incentives We have taken up this opportunity to set up our company

BadeBio Biotechnology Ltd (www.badebio.com) at Anadolu University Technopark in

Eskisehir, Turkey in order to implement our research ideas on commercial level

We have our own laboratories and have access to all the research facilities of the university

at a nominal fee We are currently implementing R&D projects for a major American flavours and fragrances supplier company, and for other foreign and Turkish companies for the development of novel products

We have conducted R&D projects and taken part in European project consortia as a micro-SME

We have organized 28th and 42nd International Symposium on Essential Oils (ISEO) in 1997 and 2011 respectively in Eskişehir a d Antalya, Turkey and International PSE Symposium on Terpenes in 2010 in Istanbul 45th ISEO (www.iseo2014.org) is organized by us on 7-10 September 2014 in Istanbul, Turkey

REFERENCES

1) H.Guinaudeau, A.J.Freyer, R.D.Minard, M.Shamma, K.H.C.Baser, Structural Variations Among the

Aporphine-Benzylisoquinoline Dimers,Tetrahedron Letters, 23(25) 2523-2526 (1982)

2) K.H.C.Baser, E Sezik, G Tumen, Composition of the Essential Oil of Ziziphora clinopodioides Lam.,

J Essent Oil Res., 3(4) 237-239 (1991)

3) Kira , Ö Özşe , T Çelik, “ İlha , B Ye i e Gürsu, F Demirci, Microbial Transformations of

Isophorone by Alternaria alternata and Neurospora crassa, Nat Prod Comm 8 (1) 59-61, 2013

4) G Iscan, N Kirimer, F Demirci, B.Demirci , Y Noma, K.H.C Baser, Biotransformation of (R)-(-)-alpha-phellandrene: Antimicrobial activity of its major metabolite, Chemistry and Biodiversity, 9, 1525-1532, 2012

5) İ Kira , Z Dur eyla , N Kiri er, K H.C Başer, Y No a, F De ir i, Biotra sfor atio of α-Cedrol

and Caryophyllene Oxide by the Fungus Neurospora crassa, Nat Prod Commun 5 (4) 515-518, 2010

6) F Demirci, T Akar, TA Demir, I Kiran, N Kirimer, K.H.C Baser, Hydroxylation of selected

sesquiterpenes by the fungus Neurospora crassa, Planta Medica, 72, 981, 2006

7) F Demirci, N Kirimer, Y Noma, K.H.C Baser, Biotransformation of (-)-Carvone, Z Naturforsch C

59 389-392, 2004

8) A Farooq, M.I Choudhary, Atta-ur-Rahman, S Tahara, K.H.C Baser, F Demirci, Detoxification of Terpinolene by Plant Pathogenic Fungus Botrytis cinerea, Z Naturforsch C 57 (9/10), 863-866, 2002 9) A Farooq, S.Tahara, M I Choudhary, A Rahman, K H C Baser, F Demirci, The Microbiological Oxidation of (-)-alpha-Pinene by Botrytis cinerea, Z Naturforsch C 57 (7/8), 686-690, 2002

10) F Demirci, N Kirimer, B Demirci, Y Noma, K H C Baser, Screening of Biotransformation

Products of Carvone Enantiomers by Headspace-SPME/GC-MS, Z Naturforsch C 56 (1/2), 58-64,

2001

11) S Celen, A.D Azaz, M Kurkcuoglu, K.H.C Baser, Chemical Composition of Endemic Thymus spathulifolius Hausskn and Velen Essential Oil and its Antimicrobial and Antioxidant Activity from

Turkey, J Essent Oil Bear Plants (JEOBP) 15(4) 628-636 (2012)

12) G Iscan, B Demirci, F Demirci, F Goger, N Kirimer, Y.B Kose, K.H.C Baser, Antimicrobial and

Antioxidant Activities of Stachys lavandulifolia subsp lavandulifolia Essential Oil and its Infusion, Nat

Prod Commun 7(9) 1241-1244 (2012)

13) G Iscan, N Kirimer, F Demirci , B.Demirci , Y Noma and K.H.C Baser, Biotransformation of ( R

)-(-)-alpha-phellandrene: Antimicrobial activity of its major metabolite, Chemistry and Biodiversity, 9, 1525-1532 (2012)

14) K Polatoglu, F Demirci, B Demirci, N Goren, K.H.C Baser, Essential Oil Composition and

Antimicrobial Activities of Tanacetum chiliophyllum (Fisch & Mey.) Schultz Bip

var monocephalum Grierson from Turkey, Rec Nat Prod 6(2) 184-188 (2012)

15) K Polatoglu, F Demirci, B Demirci, K.H.C Baser, N Goren, Biological activity and the essential

composition of Tanacetum chiliophyllum var chiliophyllum chemotypes from Turkey, Ind Crops

Prods., 39, 97-105 (2012)

16) B Kunduhoglu, M Kurkcuoglu, M E Duru, K.H.C Baser, 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., J Med Plants Res 5(29)

6484-6490 (2011)

17) G Iscan, Y B Kose, B Demirci, K.H.C Baser, The Anticandidal Essential Oil of Nepeta

transcaucasica Grossh., Chemistry and Biodiversity,8, 2144-2148 (2011)

18) N Tabanca, B Demirci, I Gurbuz, F Demirci, J.J Becnel, D.E Wedge, K.H.C Baser, Essential Oil

Composition of Five collections of Achillea biebersteinii from Central Turkey and Their Antifungal and

Insecticidal Activity, Nat Prod Commun 6(5) 701-706 (2011)

19) A Abdel-Megeed, A.N Al-Rahma, A.A Mostafa, K.H.C Baser, Biochemical characterization of

antimicrobial activity of glycolipids produced by Rhodococcus erythropolis, Pakistan J Bot., 43(2)

1323-1334 (2011)

20) M Kurkcuoglu, G Iscan, F Demirci, H Malyer, E Erdogan, K.H.C Baser, Composition and

Antibacterial Activity of The Essential Oil of Ferulago confusa Velen, J Essent Oil Res., 22(6) 490-492

(2010)

21) T Ozek, N Tabanca, F Demirci, D E Wedge, K.H.C Baser, Enantiomeric distribution of some linalool containing essential oils and their biological activities, Rec Nat Prod., 4(4) 180-192 (2010) 22) N Bilge Oral, L Vatansever, B Duman Aydin, C Sezer, A Guven, M Gulmez, K.H.C Baser, M

Kurkcuoglu, Effect of Oregano Essential Oil on Biofilms Formed by Staphylococi and Escherichia coli,

Kafkas Universitesi Veteriner Fakultesi Dergisi, 16 (Suppl A), 23-29 (2010)

23) N Tabanca, U.R Bernier, M Tsikola, J.J.Becnel, B Sampson, C Werle, B Demirci, K.H.C Baser,

E.K Blythe, C Pounders, D.E Wedge, Eupatorium capillifolium Essential Oil: Chemical Composition,

Antifungal Activity, and Insecticidal Activity, Nat Prod Commun 5(9) 1409-1416 (2010)

24) T Serbetci, B Demirci, C Bozkurt Guzel, S Kultur, M Ergü e , K.H.C Baser, Essential Oil

Composition, Antimicrobial and Cytotoxic Activities of Two Endemic Stachys cretica Subspecies

(Lamiaceae) from Turkey, Nat Prod Commun 5(9) 1369-1374 (2010)

25) K Polatoglu, F Demirci, B Demirci, N Goren, K.H.C Baser, Essential oil composition and

antibacterial activity of Tanacetum argenteum (Lam.) Willd ssp argenteum and T densum (Lab.) Schultz Bip ssp amani Heywood from Turkey, J Oleo Sci 59 (7) 361-367 (2010)