nveo 2015, volume 2, issue 1 · 2019. 6. 17. · nat. volatiles & essent. oils, 2015;2(1):1-10 jan...

NVEO 2015, Volume 2, Issue 1

CONTENTS

1. The European Flavouring regulation and how to deal with“Restricted Substances”. / Pages:

1-10

Jan Demyttenaere

2. Composition of the essential oil of Pink Chablis™ bluebeard (Caryopteris ×clandonensis

‘Durio’) and its biological activity against the yellow fever mosquito Aedes aegypti / Pages:

11-21

Eugene K. Blythe, Nurhayat Tabanca, Betul Demirci, Ulrich R. Bernier, Natasha M.

Agramonte, Abbas Ali, K. Hüsnü Can Başer and Ikhlas A. Khan.

3. Evaluation of transdermal administration of -cyperone (4,11- selinadien-3-one) isolated from

purple nutsedge (Cyperus rotundus) essential oils as a new drug delivery treatment method

for lowering cholesterol. / Pages: 22-31

Hayfaa Al-Shammary, Sahar Al-Saadi, Fatima Anad

4. Geographical Variation of Ajuga laxmannii (L.) Bentham Essential Oil / Pages: 32-36

Yavuz Bülent Köse, Sevim Alan, Betül Demirci, K. Hüsnü Can Başer.

5. Essential oil content of cultivated Satureja spp. in Northern Greece / Pages: 37-48

Marilena Papadatou, Catherine Argyropoulou, Catherine Grigoriadou, Eleni Maloupa, Helen Skaltsa

Nat. Volatiles & Essent. Oils, 2015;2(1):1-10 Jan C.R. Demyttenaere

1

REVIEW

The European Flavouring regulation and how to deal with

“Restricted Substances”

Jan C.R. Demyttenaere*

EFFA (European Flavour Association), Kunstlaan 6, 1210 Brussels, Belgium

*Email: [email protected]

Abstract

The European Flavouring Regulation contains a list of so-called “restricted substances” (RS), i.e. substances that occur naturally in

source materials for flavourings and food ingredients with flavouring properties, but whose presence in certain foods is restricted

and/or for which maximum levels are set. This list is the Annex III to the Flavouring Regulation, the most important part of which is

Part B, listing 11 substances which are naturally present in flavourings and food ingredients with flavouring properties and to which

“maximum levels” apply in specific food categories. The current paper provides some legal aspects with regard to those “restricted

substances” and reports on a new method which has been developed by the Working Group on Methods of Analysis of the

International Organization of the Flavor Industry (IOFI) for the rapid routine determination of β-asarone, coumarin, menthofuran,

methylchavicol, methyleugenol, pulegone, safrole, and α- and β-thujones in flavourings and their raw materials by gas

chromatography-mass spectrometry (GC-MS), using selected-ion monitoring and internal standards. The paper will further focus on

Business-to-Business requirements when flavourings are sold to food producers (customers) and provide some elements from EFFA’s

Guidance Document.

Keywords: Volatile Restricted Substances, European Flavouring Regulation, Annex III, Methods of Analysis, routine determination,

GC-MS

Introduction

The European Flavouring Regulation (EC) No 1334/2008 (Official Journal, 2008) entered into force on 20

January 2009 and applies since 20 January 2011. According to this Regulation certain substances, most of

which are common constituents of natural (food) ingredients, are restricted. These “restricted substances”

(RS) as they are called, are substances that occur naturally in source materials for flavourings and food

ingredients with flavour properties, but whose presence in certain foods is restricted and/or for which

maximum levels are set (Demyttenaere, 2012).

Thus, two types of restrictions are foreseen: Annex III Part A of the Flavouring Regulation lists 15 substances

“which shall not be added as such to food”, whereas Part B of Annex III lists 11 substances which are naturally

present in flavourings and food ingredients with flavouring properties and to which “maximum levels” apply

in specific food categories. Also elsewhere in the world flavour regulations contain such lists of so-called

“Restricted substances”. For example a list of substances to be controlled can be found in the Mercosur

Technical Regulation Concerning Flavourings (Mercusor, 2006), the new Russian Federation Customs Union

Technical Regulation on Food Additives (Russian Federation Customs Union, 2012), and many flavour

regulations of South-East Asian countries, such as Malaysia, Indonesia, Singapore, etc.

Only a few publications refer to the determination of RS in compound flavourings or their raw materials, and

the latter only concern the analysis of one or two individual RS in single essential oils (Royal Society of

Chemistry, 2002; Archer, 1988; De Jager, Perfetti, & Diachenko, 2008; Otto, Wohlschlager, Grüner,

Weinreich, & Parlar, 2001). This paper reports on a method for the determination of some of these RS in


2

flavourings and their raw materials by gas chromatography-mass spectrometry using selected-ion monitoring

(GC-MS-SIM) and internal standards. The method is intended for flavour-industry laboratories in order to

enable them to inform their customers (food industry) of the amounts of these substances in commercial

flavourings, but is not intended for their analysis in finished foods.

Legal background

Current Flavouring Regulation in EU

On 31 December 2008 the new Flavouring Regulation was published in the Official Journal of the EU, which

entered into force on 20 January 2009 and which officially applies since 20 January 2011.

The full title of this Regulation is: Regulation (EC) No 1334/2008 of the European Parliament and of the Council

on flavourings and certain food ingredients with flavouring properties for use in and on foods and amending

Council Regulation (EEC) No 1601/91, Regulations (EC) No 2232/96 and (EC) No 110/2008 and Directive

2000/13/EC.

Since the application of this new Regulation, the former Council Directive 88/388/EEC of 22 June 1988

(Official Journal, 1988a) as well as its amendment Directive 91/71/EEC (Official Journal, 1991) and the

Commission Decision 88/389/EEC (Official Journal, 1988b) have been repealed. As many essential oils and

extracts either contain flavouring substances, or are regarded as “food ingredients with flavouring properties”

this new Flavouring regulation will have an impact on essential oils and their use as flavouring ingredients for

food products. Extracts and essential oils contain certain constituents (substances) that according to this

regulation “should not be added as such to food” or to which maximum levels apply. In particular the

application of maximum levels of these substances will have an impact on how and when extracts, essential

oils but also herbs and spices may or can be applied to food.

Maximum levels of Restricted Substances

Apart from the fact that the former Flavouring Directive 88/388/EC now has been replaced by a Regulation,

there are many changes that will have an impact on how essential oils and extracts will be used as source of

flavours. The most important issue is how the “Restricted Substances” are addressed. This is addressed by

Art. 6 of the Flavouring Regulation: “Presence of certain substances” which refers to Annex III with the same

title. This article clearly states in the first paragraph that “Substances listed in Part A of Annex III shall not be

added as such to food.”

However, when it concerns the levels of these substances coming from the use of flavourings and food

ingredients with flavouring properties (such as extracts, essential oils, herbs and spices) the Regulation

further specifies (Art. 6.2):

2. Without prejudice to Regulation No 110/2008 maximum levels of certain substances, naturally

present in flavourings and/or food ingredients with flavouring properties, in the compound foods

listed in Part B of Annex III shall not be exceeded as a result of the use of flavourings and/or food

ingredients with flavouring properties in and on those foods. The maximum levels of the substances

set out in Annex III apply to foods as marketed, unless otherwise stated. By way of derogation from

this principle, for dried and/or concentrated foods which need to be reconstituted the maximum levels

apply to the food as reconstituted according to the instructions on the label, taking into account the

minimum dilution factor.


3

This means that maximum levels of these substances also apply when the substances come from any type of

food ingredients with flavouring properties; the only exception is given to dried and/or concentrated foods

which can have higher levels before they are diluted, and/or reconstituted. Upon dilution, and/or

reconstitution, the normal maximum levels apply again.

The main difference between the former Flavouring Directive 88/388 and the new Flavouring Regulation is

that in the Directive 88/388 there was only one list (Annex II) of substances to which the maximum levels

apply – all those substances may not be added as such to food. In contrast, in the new Flavouring Regulation,

the Annex III is split in two parts: Part A with “Substances which shall not be added as such to food” and Part

B establishing: “Maximum levels of certain substances, naturally present in flavourings and food ingredients

with flavouring properties, in certain compound food as consumed to which flavourings and/or food

ingredients with flavouring properties have been added.”

Part A of Annex III contains 15 substances, whereas Part B contains 11 substances.

Table 1 lists the 15 Substances of Part A of Annex III “which shall not be added as such to food” and Table 2

lists the 11 substances of Part B with their respective maximum levels in the various compound foods

according to the current Flavouring Regulation.

Table 1. Annex III, Part A of Regulation (EC) No 1334/2008: Substances which shall not be added as such to food

Agaric acid Aloin Capsaicin

1,2-Benzopyrone, coumarin Hypericine Beta-asarone

1-Allyl-4-methoxybenzene, estragolea Hydrocyanic acid Menthofuran

4-Allyl-1,2-dimethoxybenzene, methyleugenol Pulegone Quassin

1-Allyl-3,4-methylene dioxy benzene, safrole Teucrin A Thujone (alpha and beta)

aSubstances in bold are “new” (i.e. not in the former Directive 88/388/EC Annex II)

Table 2. Maximum levels of certain substances, naturally present in flavourings and food ingredients with flavouring

properties, in certain compound food as consumed to which flavourings and/or food ingredients with flavouring

properties have been added (Annex III, Part B to Flavouring Regulation (EC) No 1334/2008).

Name of the substance

Compound food in which the presence of the substance is restricted

Maximum level mg/kg

Beta-asarone Alcoholic beverages 1.0

1-Allyl-4-methoxybenzene,

Estragol(*)

Dairy products

Processed fruits, vegetables (incl. mushrooms, fungi, roots, tubers, pulses and legumes), nuts and seeds

Fish products

Non-alcoholic beverages

50

50 50

10

Hydrocyanic acid Nougat, marzipan or its substitutes or similar products

Canned stone fruits

Alcoholic beverages

50 5

35

Menthofuran Mint/peppermint containing confectionery, except micro breath freshening confectionery

Micro breath freshening confectionery

Chewing gum

Mint/peppermint containing alcoholic beverages

500

3000

1000

200


4

4-Allyl-1,2-dimethoxy-benzene,

Methyleugenol (*)

Dairy products

Meat preparations and meat products, including poultry and game

Fish preparations and fish products

Soups and sauces

Ready-to-eat savouries


20

15

10 60

20

1

Pulegone Mint/peppermint containing confectionery, except micro breath freshening confectionery

Micro breath freshening confectionery

Chewing gum

Mint/peppermint containing non-alcoholic beverages

Mint/peppermint containing alcoholic beverages

250 2000

350

20 100

Quassin Non-alcoholic beverages

Bakery wares

Alcoholic beverages

0.5

1

1.5

1-Allyl-3,4-methylene dioxy benzene, safrole (*)

Meat preparations and meat products, including poultry and game

Fish preparations and fish products

Soups and sauces


15 15

25

1

Teucrin A Bitter-tasting spirit drinks or bitter1

Liqueurs2 with a bitter taste

Other alcoholic beverages

5

5

2

Thujone (alpha and beta) Alcoholic beverages, except those produced from Artemisia species

Alcoholic beverages produced from Artemisia species

Non-alcoholic beverages produced from Artemisia species

10 35

0.5

Coumarin Traditional and/or seasonal bakery ware containing cinnamon in the labelling

Breakfast cereals including muesli

Fine bakery ware with exception of traditional and/or seasonal bakery ware containing cinnamon in the labelling

Desserts

50

20

15

5

(*) The maximum levels shall not apply where a compound food contains no added flavourings and the only food ingredients with flavouring properties which have been added are fresh, dried or frozen herbs and spices. After consultation with the Member States and the Authority, based on data made available by the Member States and on the newest scientific information, and taking into account the use of herbs and spices and natural flavouring preparations, the Commission, if appropriate, proposes amendments to this derogation. (1) As defined in Annex II, paragraph 30 of Regulation (EC) No 110/2008. (2) As defined in Annex II, paragraph 32 of Regulation (EC) No 110/2008.

Another significant change is that under the former Directive 88/388 the limitations applied to all food or

beverage categories mentioned under Annex II to this Directive, i.e. all categories covered by the term

“Foodstuffs” were limited to a certain (same) maximum level and the same applied to all “Beverages”; apart

from the general limitations/restrictions some exceptions (for certain more particular food categories)

applied (i.e. “special restrictions”).

Under the current Flavouring Regulation, the maximum levels only apply to the specific food categories

(referred to as “Compound food in which the presence of the substance is restricted”) mentioned in the

second column of Annex III, with a specific maximum level, mentioned in the third column. Thus, only those

categories contributing most to the consumers’ exposure are restricted. This is also referred to as the “major

contributor approach” and is outlined in Recital (10): “Maximum levels for certain naturally occurring

undesirable substances should focus on the food or food categories which contribute most to dietary intake.”


5

It is a pragmatic solution which was adopted to make the controls by the Member States more efficient as

they will focus on foodstuffs who contribute the most to the intake of substances of toxicological concern

and no longer to foodstuffs and beverages in general.

This means that “compound foods” (i.e. certain foodstuffs/food categories or beverages) that are not

mentioned in Annex III are not restricted. For example thujone used to be restricted to all foodstuffs (0.5

mg/kg) and all beverages (0.5 mg/kg) with the exception of certain alcoholic beverages and foodstuffs to

which higher levels applied (old Annex II); today thujone is only limited to alcoholic beverages (with two

different levels depending on whether or not they have been produced from Artemisia species: respectively

35 and 10 mg/kg) and to non-alcoholic beverages produced from Artemisia species (maximum level 0.5

mg/kg),but not to any other non-alcoholic beverages, nor to foodstuffs.

“New” restricted substances

Further compared to the former Flavouring Directive 88/388, some “restricted substances” are new, e.g.

methyleugenol, estragole, menthofuran, etc. This is because since the publication (and amendment) of the

former Directive some new scientific evidence has become available that suggested that there would be

some toxicological concern for these substances. As explained in Recital (8): “Since 1999, the Scientific

Committee on Food and subsequently the European Food Safety Authority [EFSA] […] have expressed opinions

on a number of substances occurring naturally in source materials for flavourings and food ingredients with

flavouring properties which, according to the Committee of Experts on Flavouring Substances of the Council

of Europe, raise toxicological concern. Substances for which the toxicological concern was confirmed by the

Scientific Committee on Food should be regarded as undesirable substances which should not be added as

such to food.”

It should be noted that for the same reason, as stipulated in Art. 22 of the Flavouring Regulation, the Annex

III can be amended “to reflect scientific and technical progress” […] “following the opinion of the Authority”

(i.e. European Food Safety Authority, EFSA).

An important example is methyleugenol (4-allyl-1,2-dimethoxybenzene). In 1999 methyleugenol was

evaluated by the Committee of Experts on Flavouring Substances (CEFS) of the Council of Europe. The

conclusions of this Committee were:

"Available data show that methyleugenol is a naturally-occurring genotoxic carcinogen compound with a

DNA-binding potency similar to that of safrole. Human exposure to methyleugenol may occur through the

consumption of foodstuffs flavoured with aromatic plants and/or their essential oil fractions which contain

methyleugenol. In view of the carcinogenic potential of methyleugenol, it is recommended that absence of

methyleugenol in food products be ensured and checked with the most effective available analytical method"

(Council of Europe, 1999).

Methyleugenol was subsequently evaluated by the Scientific Committee on Food (SCF) and an opinion on its

safety was published in 2001 (Scientific Committee on Food, 2001).

The conclusion of the SCF was:

“Methyleugenol has been demonstrated to be genotoxic and carcinogenic. Therefore the existence of a

threshold cannot be assumed and the Committee could not establish a safe exposure limit. Consequently,

reductions in exposure and restrictions in use levels are indicated.”


6

An equally important but similar example is estragole (1-allyl-4-methoxybenzene) also known as

methylchavicol. In 2000 the Committee of Experts on Flavouring Substances (CEFS) of the Council of Europe

evaluated estragole and based on their findings (it was found to be a naturally occurring genotoxic carcinogen

in experimental animals), a limit of 0.05 mg/kg (detection limit) was recommended (Council of Europe, 2000).

Estragole was subsequently evaluated by the Scientific Committee on Food (SCF) and an opinion on its safety

was published in 2001 (Scientific Committee on Food, 2001).

The conclusion of the SCF was:

“Estragole has been demonstrated to be genotoxic and carcinogenic. Therefore the existence of a threshold

cannot be assumed and the Committee could not establish a safe exposure limit. Consequently, reductions in

exposure and restrictions in use levels are indicated.”

As a consequence, both methyleugenol and estragole have been added to Annex III of the new Flavouring

Regulation as “restricted substances”.

Analysis of volatile restricted substances

Many published methods exist for the determination of RS in finished foods and beverages, however only a

few refer to their determination in compound flavourings or their raw materials – the latter mainly concern

the analysis in single essential oils of one or two individual RS, and are often based on a direct GC or LC

analysis (Royal Society of Chemistry, 2002; Archer, 1988; De Jager et al., 2008; Otto et al., 2001).

Sample preparation for compound flavourings is generally simpler than for finished foodstuffs, but their

complexity necessitates the use of GC-MS with selected-ion monitoring in order to deal with the

interferences arising from the wide variety of constituents present in a typical compound flavouring.

Reporting obligations by flavour industry

Flavour companies need a rapid routine method for the simultaneous determination of multiple RS in

compound flavourings, since it is in their interest to inform their customers of the levels of any of these in

their flavourings, and the guidelines of EFFA commit the industry to provide such information. According to

the first principle of HACCP (Hazard Analysis and Critical Control Point) as laid down in Regulation

852/2004/EC (Official Journal, 2004), the flavour manufacturers shall put in place suitable procedures in

order to identify any hazard that must be prevented, eliminated or reduced to acceptable levels.

The following recommendations are stated in the EFFA Guidance Document (EFFA, 2013) in relation to the

reporting of the presence of Restricted Substances in flavourings to customers (food business operators):

Recommendation 1)

Flavour producers commit themselves to control the potential presence of ANNEX III A substances in

flavourings in case these substances are also listed in Annex III part B and hence subject to maximum

limits in specified applications. In the Quality Assurance System of flavour producers, these substances

should be identified.

Recommendation 2)

Flavour producers commit themselves to communicate to the customer any relevant “RS” levels in

flavourings irrespective of the intended use of the flavourings, even if the flavoured food is not

covered by any food category mentioned in Annex III B.


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Development of a method of analysis of Restricted Substances

The Working Group on Methods of Analysis (WGMA) of the International Organization of the Flavour Industry

(IOFI) has previously already published guidelines for the quantitative analysis of volatile flavouring

substances by GC (IOFI, 2011) and by GC/MS using SIM (IOFI, 2012). The IOFI WGMA has now developed a

method suitable for this purpose (IOFI, 2015). It should be noted that the method is not intended for the

analysis of RS in finished foods and beverages.

The method is based on the analysis of flavourings and their volatile raw materials by gas chromatography-

mass spectrometry (GC-MS), using selected-ion monitoring (SIM) and internal standards. It has been

evaluated by 9 flavour-industry laboratories using a complex surrogate flavouring. This surrogate flavouring

contained the following added volatile restricted substances (as standards): β-asarone, coumarin,

menthofuran, methylchavicol, methyleugenol, pulegone, safrole, and the thujones (α- and β-isomers) (See

Figure 1).

Figure 1. Volatile restricted substances covered by the method of analysis


8

For more details about the developed method of analysis we refer to the paper of the IOFI WGMA (IOFI,

2015).

The main principles of the method are summarised below:

dilution of flavourings or their volatile raw materials in a suitable solvent of low volatility, e.g. iso-

octane, ethanol;

addition of 1 or more internal standards;

direct split injection in a GC/MS system with a quadrupole or a magnetic-sector analyser;

suggested internal standards are 1,4-dibromobenzene and 4,4’-dibromodiphenyl which provide very

distinctive isotopic patterns, always in a ratio close to 1:2:1, making it possible to use the central

molecular ion as quantifier and the two others as qualifiers;

non- or semi-polar columns are recommended; columns with a polar phase can be used, but long-

term stability should be monitored.

Some general considerations with regard to the instrumentation of choice are summarised below:

Gas Chromatograph coupled with Mass Spectrometer: quadrupole or magnetic-sector analyser;

ion-trap detectors for quantification are unsuitable – inter-laboratory testing has shown that results

from these are unreliable for the quantitation of volatile flavouring substances;

Capillary columns with non-polar, polar or semi-polar column (all types were used during the method

development & validation) can be used;

Carrier gas: Helium;

Injection volume: varying from 0,2 to 1 µL;

Split ratio: varying from 1/10 to 1/100;

Injector Temperature: 250°to 300°C;

Typical temperature program: from 60°C to MAOT @ 5°C/min.

Details about the MS-parameters (recommended selected ions for SIM mode) can be found in the paper of

the IOFI WGMA.

Conclusions on method performance

The method was evaluated with a surrogate flavouring purposely designed to provide a “worst-case” product

in terms of complexity and which contained all of the analytes at concentrations that would be likely to

produce levels in finished foods at around those fixed as maximum under EU legislation (flavourings are used

at a dilution of at least 200-fold, usually more). In this validation test, 9 laboratories took part, using four

different column types and two different flavour carriers (ethanol and 1,2-propanediol).

Overall, the reproducibility was very high, resulting in relative standard deviations of less than about 20%,

and recoveries of 80-120 %.

ACKNOWLEDGMENT

The author wishes to acknowledge the members of the IOFI Working Group Methods of Analysis for their input and contribution.

DISCLAIMER

The views expressed are purely those of the author and may not in any circumstances be regarded as stating an official

position of the European Flavour Association


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REFERENCES

Archer, W. (1988) Determination of safrole and myristicin in nutmeg and mace by high-performance liquid

chromatography. J. Chromatogr., 438, 117.

Council of Europe (1999) Committee of Experts on Flavouring Substances, 1999. Publication datasheet on

Methyleugenol. Document RD 4.14/2-45 submitted by the delegation of Italy for the 45th meeting in Zurich, October

1999.

Council of Europe (2000) Committee of Experts on Flavouring Substances, 2000. Final version of the publication

datasheet on estragole. Document RD 4.5/1-47 submitted by Italy for the 47th meeting in Strasbourg, 16 – 20 October

2000.

De Jager, L.S., Perfetti, G.A., & Diachenko, G.W. (2008) Comparison of headspace-SPME-GC-MS and LC-MS for the

detection and quantification of coumarin, vanillin, and ethyl vanillin in vanilla extract products. Food Chem., 107, 1701.

Demyttenaere, J. C. R (2012) The new European Union Flavouring Regulation and its impact on essential oils: production

of natural flavouring ingredients and maximum levels of restricted substances. Flavour and Fragrance Journal, 27, 3–12.

EFFA (2013) EFFA Guidance Document on the EC Regulation on Flavourings as amended, 2013. The EFFA-website:

www.effa.eu Publications Guidance Documents

IOFI (2011) Guidelines for the quantitative gas chromatography of volatile flavouring substances. Flavour Fragr. J., 26,

297; open access at http://onlinelibrary.wiley.com/doi/10.1002/ffj.2061/pdf

IOFI (2012) Guidelines for the quantitative analysis of volatile flavouring substances by GC/MS using SIM. Flavour Fragr.

J., 27, 224; open access at http://onlinelibrary.wiley.com/doi/10.1002/ffj.3092/pdf

IOFI (2015) Determination of Volatile "Restricted Substances" in Flavourings and their Volatile Raw Materials by GC-MS.

In press, Flavour Fragr. J., 30, 160-164.

Mercosur (2006) Technical Regulation Concerning Flavourings, Mercosur GMC/RES N°10/06, 2006,

http://archivo.presidencia.gub.uy/sci/decretos/2012/07/msp_33.pdf

Offical Journal (1988a) Council Directive 88/388/EC of 22 June 1988 on the approximation of the laws of the Member

States relating to flavourings for use in foodstuffs and to source materials for their production (OJ L 184, 15.7.1988, p.

61).

Official Journal (1988b) Council Decision 88/389/EEC of 22 June 1988 on the establishment, by the Commission, of an

inventory of the source materials and substances used in the preparation of flavourings (OJ L184, 15.7.1988, p. 67).

Official Journal (1991) Commission Directive 91/71/EEC of 16 January 1991 completing Council Directive 88/388/EEC on

the approximation of the laws of the Member States relating to flavourings for use in foodstuffs and to source materials

for their production (OJ L42, 15.2.1991, p. 25).

Official Journal (2008) Regulation (EC) No 1334/2008 of the European Parliament and of the Council on flavourings and

certain food ingredients with flavouring properties for use in and on foods and amending Council Regulation (EEC) No

1601/91, Regulations (EC) No 2232/96 and (EC) No 110/2008 and Directive 2000/13/EC (OJ L354, p. 34).

Otto, F., Wohlschlager, H., Grüner, S., Weinreich, B., & Parlar, H. (2001) A new GC-MS method for determining safrole

and coumarin in spices, flavourings and extracts (in German). In Advances in Food Sciences, 23(1), 31.

Royal Society of Chemistry (2002) Analytical Methods Committee, Essential Oils Subcommittee, Application of gas-liquid

chromatography to the analysis of essential oils. Part XVIII. Determination of safrole in oils of cinnamon leaf, Litsea

cubeba, and nutmeg. Analyst, 127(3), 428.

http://www.effa.eu/http://onlinelibrary.wiley.com/doi/10.1002/ffj.2061/pdfhttp://onlinelibrary.wiley.com/doi/10.1002/ffj.3092/pdfhttp://archivo.presidencia.gub.uy/sci/decretos/2012/07/msp_33.pdf


10

Russian Federation Customs Union (2012) Technical Regulation on Food Additives, Annex 20 of TR TS 029/2012,

http://gain.fas.usda.gov/Recent%20GAIN%20Publications/Customs%20Union%20Technical%20Regulation%20on%20F

ood%20Additives_Moscow_Russian%20Federation_6-25-2013.pdf

Scientific Committee on Food (2001) Opinion of the Scientific Committee on Food on Methyleugenol (4-Allyl-1,2-

dimethoxybenzene). SCF/CS/FLAV/FLAVOUR/4 ADD1 FINAL. European Commission, Health & Consumer Protection

Directorate-General.

Scientific Committee on Food (2001) Opinion of the Scientific Committee on Food on Estragole (1-Allyl-4-

methoxybenzene). SCF/CS/FLAV/FLAVOUR/6 ADD2 FINAL. European Commission, Health & Consumer Protection

Directorate-General.

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Nat. Volatiles & Essent. Oils, 2015;2(1):11-21 Blythe et al.

11

RESEARCH ARTICLE

Composition of the essential oil of Pink Chablis™ bluebeard (Caryopteris ×clandonensis 'Durio') and its biological activity against the yellow fever mosquito Aedes aegypti

Eugene K. Blythe1,*, Nurhayat Tabanca2, Betul Demirci3, Ulrich R. Bernier4, Natasha M. Agramonte4,

Abbas Ali2, K. Hüsnü Can Başer3,5 and Ikhlas A. Khan2,6,7

1 Coastal Research and Extension Center, Mississippi State University, South Mississippi Branch Experiment Station,

Poplarville, MS 39470, USA 2 National Center for Natural Products Research, The University of Mississippi, University, MS 38677, USA 3 Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, Eskişehir, 26470, TURKEY 4 Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, Gainesville, FL 32608, USA 5 Botany and Microbiology Department, College of Science, King Saud University, Riyadh 11451, SAUDI ARABIA 6 Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University, MS 38677, USA 7 Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11451, SAUDI ARABIA

*Corresponding author. Email: [email protected]

Abstract

Caryopteris ×clandonensis A. Simmonds ex C. H. Curtis 'Durio' Pink Chablis™, (Lamiaceae) a pink-flowered cultivar distinctive among

the typically blue-flowered cultivars of bluebeard, is valued as a small, deciduous shrub in the landscape for its mounded growth

habit, showy flower display in summer, and attractiveness to insect pollinators. As part of a broader research program examining

aromatic compounds from ornamental species as natural alternatives to synthetic chemicals for control of insect pests, the essential

oil of Pink Chablis™ bluebeard was investigated for its chemical composition and bioactivity as a repellent and larvicide against the

yellow fever mosquito [Aedes aegypti (L.) (Diptera: Culicidae)]. Essential oil from the aerial parts of this mildly aromatic ornamental

species was extracted by water distillation and analyzed by gas chromatography and gas chromatography mass spectrometry. The

primary compounds in the essential oil were α-copaene (8.3%), limonene (7.2%), and δ-cadinene (6.3%), followed by trans-p-mentha-

2,8-dien-1-ol (4.6%), trans-p-mentha-1(7),8-dien-2-ol (4.5%), cis-p-mentha-2,8-dien-1-ol (4.0%), and hotrienol (3.8%). Against the

yellow fever mosquito, the essential oil exhibited mild repellency compared to DEET (N,N-diethyl-3-methylbenzamide) as a reference

standard. It exhibited weak activity as a mosquito larvicide.

Keywords: Caryopteris ×clandonensis, Aedes aegypti, mosquito control, mosquito larvicide, mosquito repellent

Introduction

Aedes aegypti L. (Diptera: Culicidae), the yellow fever mosquito, transmits viral pathogens, including yellow

fever, dengue fever, and chikungunya, which can cause serious human illness and death (World Health

Organization, 2014a, 2014b, 2014c). Insecticides have been the primary control measure for mosquito

management, as well as control of a wide range of other insect pests in agriculture and public health

situations. Frequent use of any single insecticide class, such as pyrethroids, can lead to non-target effects and

the development of insecticide resistance (Liu, Xu, Zhu, & Zhang, 2006; Maharaj, 2011). Consequently, there

exists an urgent need to develop alternative insecticides to supplement pyrethroids for control of a wide

variety of insect-vectored diseases (Maharaj, 2011; Pridgeon, Becnel, Clark, & Linthicum, 2009b; Pridgeon et

al., 2008).


12

An alternative to conventional insecticides is the use of natural products from plants that produce

phytochemicals as defense mechanisms against microorganisms and predators. Such chemicals may serve as

candidate products for controlling a wide variety of insect vectors. Recent efforts have focused on

identification and utilization of plant extracts or phytochemicals as potential sources of commercial mosquito

control agents or bioactive chemical compounds (Quinn, Bernier, & Booth, 2007; Yang et al., 2002). Members

of the Lamiaceae (mint family), in particular, have been shown to be sources of essential oils having

insecticidal and insect repellent properties (Ayvaz, Sagdic, Karaborklu, & Ozturk, 2010; Çalmaşur, Aslan, &

Şahin, 2006; Conti, Canale, Cioni, & Flamini, 2010; Odeyemi, Masika, & Afolayan, 2008; Tabanca et al., 2013;

Yildirim, Kordali, & Yazici, 2011).

The genus Caryopteris Bunge (Lamiaceae) consists of 16 species native to China and East Asia (Abu-Asab,

Cantino, Nowicke, & Sang, 1993; Flora of China Editorial Committee, 1994). The composition of essential oils

has previously been investigated for several species: C. forrestii Diels (Pu, Shi, Yang, Zhang, & Lü, 1984); C.

incana (Thunberg ex Houttuyn) Miquel (Chu, Liu, Zhou, Du, & Liu, 2011; Kim, 2008; Pu et al., 1984), C.

mongholica Bunge (Shatar & Adams, 1999), C. tangutica Maximowicz (Dai, Zhang, & Liao, 2012; Yan & Wang,

2009), and C. trichosphaera W. Smith (Pu et al., 1984). Caryopteris incana has been identified as a source of

new glycosides (Park et al., 2014) and C. mongholica has yielded new alkaloids (Dumaa et al., 2012). Essential

oil of C. incana has demonstrated strong insecticidal activities against the maize weevil, Sitophilus zeamais

Mots. (Coleoptera, Dryophthoridae) (Chu et al., 2011).

Caryopteris ×clandonensis A. Simmonds ex C. H. Curtis (bluebeard, blue mist shrub, false spirea) is a hybrid

between C. incana and C. mongholica, originating as a chance seedling in the garden of Arthur Simmonds in

Surrey, England, in 1933. Since then, additional ornamental cultivars have been selected by horticulturists

into the nursery trade (Chicago Botanic Garden, 2014). C. ×clandonensis is valued in the landscape for its

mounded growth habit, showy display of blue flowers in summer, and attractiveness to insect pollinators. C.

×clandonensis 'Durio' Pink Chablis™, unique among the bluebeards in having pink flowers (Figures 1-3), was

discovered as a chance seedling in 1998 by Dalton Durio of Louisiana Nursery, Opelousas, LA, USA [U.S. Plant

Patent No. PP16,913 (Durio, 2006)].

Previous research has examined chemical constituents of C. ×clandonensis. Caryopteris ×clandonensis was

found to be a source of two new keto-glycosides, clandonoside and 8-O-acetylclandonoside (Hannedouche,

Jacquemond-Collet, Fabre, Stanislas, & Moulis, 1999), the pyranojuglone pigment α-caryopterone

(Matsumoto, Mayer, & Eugster, 1969), and quinones with strong molluscicidal activity (Hannedouche,

Souchard, Jacquemond-Collet, & Moulis, 2002). Essential oil of Caryopteris ×clandonensis was found to be

less effective than oils from other aromatic plants when tested in vapor phase against foodborne bacteria

(Nedorostova, Kloucek, Kokoska, Stolcova, & Pulkrabek, 2009).

In a cooperative effort involving multiple institutions, we are evaluating new plant extracts and pure

compounds for mosquito repellent and larvicidal activity as part of the Department of Defense Deployed

War-Fighter Protection (DWFP) research program (Cope, Strickman, & White, 2008; Linthicum et al., 2007).

The DWFP program emphasizes identification and testing of new classes of chemistry for control of insect

vectors and new tools for chemical application suited to the protection of troops and human populations

after natural disasters. Taking into account the necessity of developing new mosquito repellents with more

favorable environmental properties, the objectives of the current study were to determine the composition

of essential oil obtained from the ornamental shrub Caryopteris ×clandonensis 'Durio' Pink Chablis™

(Lamiaceae) and to examine the repellent and larvicidal activities of the essential oil against the yellow fever

mosquito, Aedes aegypti.


13

Materials and Methods

Plant Material and Essential Oil Distillation

Plants of C. ×clandonensis 'Durio' Pink Chablis™ (Spring Meadow Nursery Inc., Grand Haven, MI, USA), a

vegetatively propagated clone of bluebeard, were used in this study. Plants were grown outdoors in 11.4-L

containers in a peatmoss and pine bark-based substrate at the South Mississippi Branch Experiment Station

(SMBES) in Poplarville, MS (30°50'26"N, long. 89°32'46"W; USDA hardiness zone 8b). Voucher specimen #9

was deposited at the SMBES for future reference. Aboveground parts were harvested from 9-month-old

plants in June 2009 and air-dried for three weeks inside an air-conditioned building (25°C max.). Dried plant

material was packed loosely into cardboard boxes to avoid crushing and stored in the same building until

shipment to the National Center for Natural Products Research in Oxford, MS for distillation of essential oils.

The air-dried aerial parts of C. ×clandonensis were subjected to water distillation using a Clevenger-type

apparatus to obtain the oil (Figure 4). Light olive-green oil was obtained with a yield of 0.05% (v/w).

Gas Chromatography and Gas Chromatography–Mass Spectrometry Analysis of Essential Oil

The essential oil was analyzed by gas chromatography (GC) with a flame ionization detector (FID) and gas

chromatography–mass spectrometry (GC-MS) using an Agilent 5975 GC-mass selective detector (MSD)

system. For the GC-MSD analysis, an Innowax fused silica capillary (FSC) column (60 m × 0.25 mm, 0.25 µm

film thickness) was used with helium as the carrier gas (0.8 mL/min). The oven temperature was kept at 60

°C for 10 min, then programmed to 220 °C at a rate of 4 °C/min, then maintained constant at 220 °C for 10

min, and finally programmed to 240 °C at a rate of 1 °C/min. The injector temperature was set at 250 °C. The

split flow was adjusted at 50:1. Mass spectra were recorded at 70 eV with the mass range m/z 35 to 450. The

GC analysis was performed using an Agilent 6890N GC system. FID detector temperature was set to 300 °C

and the same operational conditions were applied to a duplicate of the same column used in GC-MS analysis.

Simultaneous auto injection was done to obtain equivalent retention times. Relative percentages of the

separated compounds (Table 1) were calculated from integration of the peak areas in the GC-FID

chromatogram.

Individual components were identified by comparison of retention times with authentic samples or by

comparison of their relative retention index (RRI) to a series of n-alkanes (Curvers , Rijks, Cramers, Knauss, &

Larson, 1985) and by computer matching with commercial mass spectral libraries (Wiley GC/MS Library,

MassFinder 3 Library) and in-house “Baser Library of Essential Oil Constituents” built up from the authentic

samples, known oils, and mass literature data (ESO, 2000; Joulain & König, 1998; König, Joulain, & Hochmuth,

2004; McLafferty and Stauffer, 1989).

Mosquito Bioassays

Mosquitoes

Aedes aegypti (1952 Orlando strain) larvae and adults used in these studies were from a laboratory colony

maintained at the Mosquito and Fly Research Unit at the Center for Medical, Agricultural, and Veterinary

Entomology, USDA-ARS, Gainesville, FL, USA. For larval bioassays, the eggs were hatched and the larvae

were maintained at an ambient temperature of 78 ± 3 °C.

Mosquito repellent assay (Cloth patch assay)

Repellency was determined as the Minimum Effective Dosage (MED), which is the minimum threshold

surface concentration necessary to prevent mosquitoes from biting through the treated surface (Schreck,

Posey, & Smith, 1977) Approximately 500 (± 10%) mosquitoes were collected and loaded into a test cage


14

(size of 45 cm x 37.5 cm x 35 cm) and held in the cage for 25 (± 2.5) min before initiating repellency assays.

Serial dilutions were then made such that the concentrations on the cloth for the remaining 1 mL solution

were: 0.375, 0.094, 0.047, 0.023, and 0.011 mg/cm2. Each concentration was tested to determine the point

where the repellent failed for each of the volunteers in the study; this concentration was averaged and

reported. Each test was conducted by having a volunteer affix the treated cloth onto a plastic sleeve to cover

a 32 cm2 window previously cut into the sleeve. Each of the volunteers wore this sleeve/cloth assembly above

a nylon stocking covering their arm, with their hands protected by a glove (Katritzky et al., 2010). The arm

with the sleeve/cloth assembly was inserted into a cage, where approximately 500 female Ae. aegypti

mosquitoes (aged 6-10 days) had been preselected as host-seeking using a draw box (Posey & Schreck, 1981).

Failure of the repellent treatment is 1% bite through, i.e. the volunteer receives 5 bites through the cloth

over the sleeve window in the 1 minute assay. There were three human volunteers in this study and all three

provided written informed consent to participate in this study as part of a protocol (636-2005) approved by

the University of Florida Human Use Institutional Review Board (IRB-01).

Mosquito larvicidal assay

Bioassays were conducted using the system described by Pridgeon et al. (2009a) to determine the larvicidal

activity of the essential oils against Ae. aegypti. Five 1-d-old larvae were transferred to individual wells of a

24-well tissue culture plates in a 30-40 µL droplet of water. Fifty µL of larval diet of 2% slurry of 3:2 beef liver

powder (Now Foods, Bloomingdale, Illinois) and Brewer’s yeast (Lewis Laboratories Ltd., Westport, CT) and

1 mL of deionized water were added to each well by using a Finnpipette® stepper pipetter (Thermo Fisher,

Vantaa, Finland). C. ×clandonensis 'Durio' essential oil was diluted in DMSO. Eleven microliters of the test

chemical was added to the wells, while 11 µL of DMSO was added to the control treatments. After treatment

application, the plates were swirled in clockwise and counterclockwise motions and front to back and side to

side five times to ensure even mixing of the tested compounds. Permethrin (46.1% cis – 53.2% trans;

Chemical Service, West Chester, PA) at 0.025 ppm was used as positive control. Larval mortality was recorded

24 h post treatment.

Results and Discussion

A total of 50 compounds were identified in the essential oil of C. ×clandonensis 'Durio' Pink Chablis™ (Table

1). The main components were characterized as α-copaene (8.3%), limonene (7.2%) and δ-cadinene (6.3%),

followed by trans-p-mentha-2,8-dien-1-ol (4.6%), trans-p-mentha-1(7),8-dien-2-ol (4.5%), cis-p-mentha-2,8-

dien-1-ol (4.0%), and hotrienol (3.8%). Among the main compounds in essential oil of C. ×clandonensis 'Durio',

limonene and δ-cadinene have also been previously reported as major compounds in essential oil of C.

mongholica from Mongolia (Shatar & Adams, 1999), with limonene also reported as a major compound in

essential oil of C. incana from Jiangxi, China (Sun, Ye, & Chen, 2004). Otherwise, main components in C.

×clandonensis 'Durio' essential oil mostly differed from those previously reported for C. incana and C.

mongholica, the parent species of C. ×clandonensis. Shatar & Adams (1999) reported main constituents of

essential oil from leaves and flowers of C. mongholica from Mongolia were α-thujene (18.7%); (E)-β-ocimene

(11.0%); limonene (8.8%); β-pinene (8.0%) terpinene-4-ol (7.2%); α-pinene (6.3%); sabinene (5.6%);

sylvestrene (2.4%); γ-terpinene (2.3%); germacrene-D (2.3%) and δ-cadinene (2.1%). Composition of essential

oils from aerial parts of C. incana from China and Korea differed by source of the plant material (Chu et al.,

2011; Sun, Ye, & Chen, 2004; Pu et al., 1984; Kim, 2008). Main components produced by plants were estragole

(24.8%), linalool (14.0%), 1,8-cineol (5.2%), and δ-guaiene (4.1%) using plants from Guangdong, China (Chu

et al., 2011).; linalool (16.3%), perillalcohol (15.3%), carvone (14.7%), and orthodene (9.7%) using plants from

Jiangxi, China (Sun, Ye, & Chen, 2004); limonene (38.5%), α-terpenene (17.3%), β-pinene (12.9%) and ρ-


15

cymene (12.6%) using plants from Sichuan, China (Pu et al., 1984); and 4,6,6-tri-methyl [1S-(1α,2β,5α)]-

bicyclo[3.1.1]hept-3-en-2-ol (11.8%), τ-cadinol (9.4%), myrtenyl acetate (9.2%), pinocarvone (7.0%), 1-

hydroxy-1,7-dimethyl-4-isopropyl-2,7-cyclodecadiene (6.3%), and δ-3-carene (6.2%) using plants from Korea

(Kim, 2008).

The mosquito repellent assay using Ae. aegypti mosquitoes revealed the essential oil of C. ×clandonensis

'Durio' to have a MED for repellency of 0.250 ± 0.109 mg/cm2; however, this indicated a mild ability to repel

Ae. aegypti compared with the reference standard, DEET (MED=0.039 ± 0.014 mg/cm2). In the mosquito

larvicidal screening assay, C. ×clandonensis 'Durio' essential oil gave 90%, 20% and 0% mortality of the 1-d-

old Ae. aegypti larvae at the concentrations of 125, 62.5 and 31.25 ppm, respectively.

This study provides the first report on the composition of the essential oil of the interspecific ornamental C.

×clandonensis 'Durio' Pink Chablis™ and its assessment as a mosquito repellent and larvicide. Although the

essential oil exhibited mild repellency and weak larvicidal activity against Ae. aegypti, further investigation

for unique chemical constituents may be warranted based on previous findings with C. ×clandonensis

(Hannedouche et al., 1999; Hannedouche et al., 2002) and other species of Caryopteris (including C.

mongholica, a parent of C. ×clandonensis) (Dai et al., 2012; Dumaa et al., 2012; Park et al., 2014). Being a

vegetatively propagated clone, chemical constituents of C. ×clandonensis 'Durio' are likely to remain more

consistent from one harvest to another than would wild-collected forms of Caryopteris.

Table 1. Composition of the essential oil of Caryopteris ×clandonensis 'Durio' Pink Chablis™.

RRI Compound % Identification method

1032 α-Pinene 0.3 tR, MS

1076 Camphene 0.1 tR, MS

1118 β-Pinene 0.4 tR, MS

1203 Limonene 7.2 tR, MS

1220 cis-Anhydrolinalool oxide 0.5 MS

1224 o-Mentha-1(7),5,8-triene 2.5 MS

1253 trans-Anhydrolinalool oxide 0.4 MS

1261 menthatriene isomer* 6.6 MS

1280 p-Cymene 0.3 tR, MS

1319 Dihydrotagetone 0.1 MS

1408 1,3,8-p-Menthatriene 0.3 MS

1452 α,p-Dimethylstyrene 2.4 MS

1452 1-Octen-3-ol 0.9 MS

1478 cis-Linalool oxide 0.2 MS

1492 Cyclosativene 0.5 MS

1497 α-Copaene 8.3 tR, MS

1553 Linalool 2.7 tR, MS

1612 β-Caryophyllene 0.5 tR, MS

1616 Hotrienol 3.8 MS

1639 trans-p-Mentha-2,8-dien-1-ol 4.6 MS

1661 Alloaromadendrene 0.3 MS

1678 cis-p-Mentha-2,8-dien-1-ol 4.0 MS

1700 p-Mentha-1,8-dien-4-ol 0.1 MS

1704 Myrtenyl acetate 0.4 MS

1706 α-Terpineol 0.4 tR, MS

1708 Ledene 0.6 MS

1740 α -Muurolene 0.3 MS

1751 Carvone 2.7 tR, MS

1773 δ-Cadinene 6.3 MS


16

1797 p-Methyl acetophenone 0.2 MS

1807 Perilla aldehyde 0.2 tR, MS

1811 trans-p-Mentha-1(7),8-dien-2-ol 4.5 MS

1845 trans-Carveol 2.3 tR, MS

1849 Calamenene 1.0 MS

1864 p-Cymen-8-ol 0.4 tR, MS

1896 cis-p-Mentha-1(7),8-diene-2-ol 2.0 MS

1941 α-Calacorene 3.1 MS

1984 γ-Calacorene 0.9 MS

2008 Caryophyllene oxide 0.4 tR, MS

2057 Ledol 1.2 MS

2080 Cubenol 0.2 MS

2088 1-epi-Cubenol 0.3 MS

2089 6-Methyl-5(3-methylphenyl)-2-heptanone 0.5 MS

2104 Viridiflorol 0.5 MS

2161 Muurola-4,10(14)-dien-1-ol 1.3 MS

2198 Thymol 2.7 tR, MS

2239 Carvacrol 0.6 tR, MS

2256 Cadalene 2.2 MS

2289 Oxo--Ylangene 2.0 MS

2411 4-Isopropyl-6-methyl-1-tetralone 0.4 MS

Total 84.6

*: Correct isomer could not identified; RRI;Relative retention indices calculated against n-alkanes; % calculated from FID data;

Identification method, tR, identification based on the retention times of genuine compounds on the HP Innowax column; MS,

identified on the basis of computer matching of the mass spectra with those of the Wiley and MassFinder libraries and comparison

with literature data.

Figure 1. C. ×clandonensis 'Durio' Pink Chablis™ growing in a landscape setting. (Photo by Spring Meadow Nursery, Inc.)


17

Figure 2. Inflorescences of C. ×clandonensis 'Durio' Pink Chablis™. (Photo by Spring Meadow Nursery, Inc.)

Figure 3. Close-up of the flowers of C. ×clandonensis 'Durio' Pink Chablis™. (Photo by Spring Meadow Nursery, Inc.)


18

Figure 4. Essential oil of Caryopteris ×clandonensis 'Durio' Pink Chablis™ was obtained from aerial parts by water

distillation using a Clevenger-type apparatus and the oil was subjected to mosquito repellent and larvicidal bioassays.

ACKNOWLEDGMENTS

This study was supported in part by USDA/ARS grant No. 56-6402-1-612, Deployed War-Fighter Protection Research Program Grant funded by the U.S. Department of Defense through the Armed Forces Pest Management Board, and a Special Research Initiative grant from the Mississippi Agricultural and Forestry Experiment Station. We thank Nathan Newlon, Greg Allen, Dr. Maia Tsikolia, and Dr. James J. Becnel, USDA-ARS, Gainesville, FL for supplying mosquito eggs. We also thank Cecil Pounders, Eric Stafne, and Stephen Stringer for reviewing an early draft of the manuscript. This paper was approved for publication as Journal Article No. J-00000 of the Mississippi Agricultural and Forestry Experiment Station, Mississippi State University.

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Nat. Volatiles & Essent. Oils, 2015;2(1):22-31 Al-Shammary et al.

22

RESEARCH ARTICLE

Evaluation of transdermal administration of -cyperone (4,11-selinadien-3-one) isolated from purple nutsedge (Cyperus rotundus) essential oils as a new drug delivery treatment method for lowering cholesterol

Hayfaa A. Al-Shammary1,*, Sahar Malik Al-Saadi2, Fatima J. Anad3

1,3 Department of Medical Analysis, Thi-Qar University, Thi-Qar, IRAQ

2 Department of Biology, University of Basrah, Basrah , IRAQ

*Corresponding author. Email: [email protected]; [email protected]

Abstract

The compound -cyperone (4, 11-selinadien-3-one) isolated from purple nutsedge (Cyperus rotundus) essential oil was investigated

for cholesterol lowering effect. In this study, we removed the hair from the back area of the rats, this compound gave us a good

result as an alternative drug for lowering serum cholesterol levels via the transdermal route of administration. This compound

significantly decreased total serum cholesterol level in rats at p ≤ 0.01.

Keywords: Transdermal administration, cholesterol-lowering drug, Cyperus rotundus, liver

Introduction

Cholesterol

Cholesterol plays a unique role among the many lipids in mammalian cells. This is based partly on its

biophysical properties, which allow it to be inserted into or extracted from membranes relatively easily.

Additionally, it plays an important role in organizing other lipids in a bilayer. Because of the importance of

sterols, cells have evolved complex mechanisms to tightly regulate their abundance and distribution.

Since cholesterol homeostasis is critical at the whole body level, cells have various dedicated pathways for

the uptake of cholesterol from low-density lipoproteins (LDL) and export to high-density lipoproteins (HDL).

Most mammalian cells synthesize cholesterol endogenously, but it can also be delivered by lipoprotein

carriers (Maxfield and van der Meer, 2010). Common cholesterol-lowering drugs include statins, fibrates,

niacin, resins, and cholesterol absorption inhibitors. All of these drugs have side effects such as diarrhoea,

constipation, stomach pain, cramps, bloating, nausea, vomiting, headaches, drowsiness, dizziness, muscle

aches or weakness, flushing, and sleep problems (American Academy of Family Physicians, 2011).

Cyperus rotundus L. (Cyperaceae)

Cyperus rotundus (Cyperaceae) is a multipurpose plant, A number of pharmacological and biological activities

including anti-candida, anti-inflammatory, anti-diabetic, anti-diarrhoeal, cytoprotective, anti-mutagenic,

antimicrobial, antibacterial, antioxidant, cytotoxic and apoptotic, anti-pyretic and analgesic activities have

been reported for this plant (Durate et al.,2005; Sundaram et al.,2008; Raut and Gaikwad, 2006; Uddin et al.,

mailto:[email protected]


23

2006; Kilani et al. ,2005 ; Zhu et al., 1997; Kilani et al., 2007; Kilani et al., 2008; Dhillon et al., 1993; Pal and

Dutta, 2006; Kilani et al., 2008; Sandeep et al., 2010; Sawanee et al., 2008 ;). It is a source of natural

antioxidant (Nagulendran et al., 2007) and protective measure against mosquito bites by using the hexane

extract of its tuber (Singh et al., 2009), and it has antinociceptive effect, but Poonam et al., 2004 had shown

it has limited activity against different forms of infectious diarrhoea .

The skin and transdermal drug delivery (TDD)

The skin, also known as the integument or cutaneous layer, is the largest single organ of the body. It accounts

for 15-20% of the total body weight and has a surface area of 1.5-2 m2 in adults. The skin is composed of the

epidermis, an epithelial layer of ectodermal origin consisting of keratinized squamous epithelium, and a

dermal layer comprising mesodermal connective tissue. The highly vascular dermis nourishes and supports

the epidermis and consists of a thick layer of dense, fibroelastic connective tissue, which contains many

sensory receptors. Beneath the dermis lies the subcutaneous tissue or hypodermis, which is loose connective

tissue that may contain pads of adipocytes (Mesher, 2010). TDD systems are used to topically administer

medication in the form of patches that deliver drugs at a pre-determined and controlled rate for a systemic

effect. Currently, about 74% of drugs are taken orally, but this route of administration is not always effective.

TDD systems have emerged to improve drug delivery characteristics (Kumar et al., 2012).

The skin has been investigated as a route to deliver drugs topically, regionally, or systemically, but

unfortunately dermis and TDD are often limited by poor drug permeability (Fang et al., 2003). Low

permeability can be attributed mainly to the outermost layer of the skin (the stratum corneum), which serves

as a rate-limiting lipophilic barrier against the uptake of chemical and biological toxins and loss of water

(Shah, 1994; Hadgraft, 2004). The epidermal cell membranes in the stratum corneum are so tightly joined

that there is hardly any intercellular space through which polar non-electrolyte molecules and ions can

diffuse (Hsieh, 1994). The proteins and lipids of the stratum corneum form a complex interlocking structure,

resembling bricks and lipid mortar (Shah, 1994). The major lipids found in the stratum corneum include

cholesterol and fatty acids (Law et al., 1995). Ceramides, in particular ceramide 2 and ceramide 5, play an

important role in the overall lipid matrix organization of the stratum corneum and in skin barrier function

(Chen et al., 2000). Ceramides are tightly packed in lipid layers owing to the strong hydrogen bonding

between opposing amide headgroups. This specifies a transverse organization in addition to the lateral

orthorhombic chain organization of ceramide molecules. Different routes through which molecules can cross

the stratum corneum include the transcellular, intercellular, and appendageal (i.e., through the

eccrine/sweat glands or hair follicles) routes (Figure 1. by Barry, 2001).


24

Figure 1. Drug permission through the skin and Mechanism of skin permission (Barry, 2001)

Many studies have shown that the skin can absorb chemical compounds like coumarin. A study by Yourick

and Bronaugh (1997) indicated that coumarin absorption is significant in the skin. Systemic coumarin

absorption must be expected after dermal contact with coumarin-containing products. The use of coumarin

in food was banned by the Food and Drug Administration because of reports that it induced hepatotoxicity

in rodents. Yokoi et al. (2008) showed that rats topically treated with 0.42 mg GeO2/g ointment had

significantly higher germanium concentrations in the plasma, liver, and kidney than the corresponding

concentrations in control rats. This finding indicates that the skin is permeable to inorganic germanium ions

or germanate. Another study on the short-term dermal absorption and penetration of an organic chemical

(dibromomethane) in aqueous solutions found that the amount of chemical in the skin and its fate during

short exposures is important. The square-root-of-time approach predicted the total amount of chemical,

which penetrated the skin and was absorbed, better than the steady-state approach (McDougal and Jurgens-

Whitehead, 2001).

Chemical enhancers can be divided into two broad categories: those that change partitioning into the stratum

corneum and those that influence diffusion across the stratum corneum (Thomas and Finnin, 2004).

Examples of chemical penetration enhancers include sulfoxides (dimethylsulfoxide), alcohols (ethanol),

polyols (propylene glycol), alkanes, fatty acids (oleic acid), esters, amines and amides (urea,

dimethylacetamide, dimethylformamide and pyrrolidones), terpenes, cyclodextrins, surfactants (non-ionic,

cationic, and anionic), and ozone (Walker and Smith, 1996; Foldvari, 2000).

TDD offers many advantages, such as reduced side effects, improved patient compliance, elimination of the

first-pass metabolism, and sustained drug delivery (Park et al., 2014; Singh and Morris, 2011). Terpenes are

constituents of essential oils that are well-recognized penetration enhancers of drugs across the human skin,

and have been receiving considerable interest in the pharmaceutical industry for this application (Cornwell

and Barry, 1993). In general, they have low systemic toxicity and do not cause skin irritation, in addition to

having good penetration-enhancing abilities (Cornwell et al., 1996). They are clinically acceptable and

relatively safe skin penetration enhancers for both lipophilic and hydrophilic drugs (Gao and Singh, 1998).

Terpenes are arguably the most advanced and established category of penetration enhancers and are

classified as generally regarded as safe by the Food and Drug Administration (Aqil et al., 2007).

The aim of this study was to evaluate transdermal administration of -cyperone (4,11-selinadien-3-one),

which was isolated and identified from purple nutsedge (Cyperus rotundus) essential oil, as a new treatment

method for lowering cholesterol.

http://libhub.sempertool.dk.tiger.sempertool.dk/gmt/ivsl/synergy/02724332_2001_21_4_719-726/10.1111/0272-4332.214145http://libhub.sempertool.dk.tiger.sempertool.dk/gmt/ivsl/synergy/02724332_2001_21_4_719-726/10.1111/0272-4332.214145http://libhub.sempertool.dk.tiger.sempertool.dk/gmt/ivsl/synergy/02724332_2001_21_4_719-726/10.1111/0272-4332.214145http://libhub.sempertool.dk.tiger.sempertool.dk/gmt/ivsl/synergy/02724332_2001_21_4_719-726/10.1111/0272-4332.214145http://libhub.sempertool.dk.tiger.sempertool.dk/libhub?func=search&query=au:%22Julia%20L.%20Jurgens-Whitehead%22&language=enhttp://libhub.sempertool.dk.tiger.sempertool.dk/libhub?func=search&query=au:%22Julia%20L.%20Jurgens-Whitehead%22&language=enhttp://www.ncbi.nlm.nih.gov/pubmed?term=Singh%20I%5BAuthor%5D&cauthor=true&cauthor_uid=23071913http://www.ncbi.nlm.nih.gov/pubmed?term=Morris%20AP%5BAuthor%5D&cauthor=true&cauthor_uid=23071913


25

Materials and Methods

Laboratory animals

White laboratory rats (Rattus norvegicus; n= 32) were housed under standard laboratory temperature (22-

25 °C) and light conditions. The animals were divided into 2 groups (control group and treatment group).

Each group consisted of 16 rats (8 male and 8 female). Hair on the dorsal area of each rat (approximately 2.5

cm2) was removed by using sugar syrup (Figure 3). Animals in the control and treatment groups were treated

topically with 0.1 mL of distilled water and 0.1 mL of 0.25 mg concentration -cyperone (4,11-selinadien-3-

one), respectively, twice daily for 30 days. At the end of the experiment, the rats were generally anesthetized

by chloroform inhalation and then sacrificed. Blood samples were collected directly from the heart by using

disposable 10-mL syringes. Then, blood samples were transferred into anticoagulant-free test tubes and

allowed to clot at room temperature. Next, the samples were centrifuged at 3000 rpm for 15 min and the

serum was collected and frozen at -20 C until analysis. The animals were treated in accordance with the

Ethical Guide for the Care and Use of Laboratory Animals (National Research Council, 2002).

Figure 2. Rat with removal area

Histological study

Tissue sections were prepared from the liver according to the method described by Luna (1968). Liver

sections were isolated from the sacrificed animal, cut into small pieces, and prepared as follows: fixationin

10%, dehydration, clearing, impregnation, embedding, trimming and cutting, sectioning, mounting, and

staining.

Statistical analysis

Data are expressed as the mean ± standard error of the mean (SEM) and were analyzed by two-way analysis

of variance. Differences between means were considered significant at P ≤ 0.01 using the Fisher’s least

significant difference (LSD) test.


26

Results and Discussion

The effect of -cyperone (4,11-selinadien-3-one) is shown in Table 1. Compared to the control group, both

male and female animals in the treatment groups showed significantly decreased serum cholesterol levels (p

≤ 0.01). The decrease in serum cholesterol levels in males treated with -cyperone (4,11-selinadien-3-one)

was significantly higher than that in females, indicating that gender affected by the treatment.

Table 1. Effect of -cyperone (4, 11-selinadien-3-one) on blood serum cholesterol levels in rats

Treatment Mean ± SEM mmol\liter

Cholesterol, mmol\liter Treatment

Males Females 87.70 ± 4.76 A 88.40 ± 8.18 a 87.00 ± 5.49 a Control 75.00 ± 6.42 B 68.60 ± 5.48 c 81.30 ± 11.62 b -Cyperone 78.50 ± 15.00 B 84.15 ± 4.03 A Mean of sex 0.41

sex 2.21

interaction 0.75

LSD

* Capital letters indicate significantly different mean values (P < 0.01) for either treatment or sex. Small letters indicate interactions.

The results of our study showed that transdermal treatment with -cyperone (4,11-selinadien-3-one)

isolated from C. rotundus significantly decreased serum cholesterol levels. This compound dissolved in

distilled water. The transdermal drug delivery (TDD) system provided an appealing alternative, minimizing

the limitations associated with oral and parenteral drug administration (Alexander et al., 2012). The

molecular mass of -cyperone (218.34 Da) is within the range suggested by Barry, 2001, who showed that

low molecular mass (


27

continuous stratum corneum between these appendages (Barry, 2001). This oil may penetrate the skin

through intercellular or intracellular (transcellular) mechanisms (Barry and William, 1995). The mechanisms

by which terpenes act on the lipid bilayer of the stratum corneum have been reviewed by (Jain et al., 2002).

Many basic aspects of cholesterol homeostasis are not well understood. Our results may be due to absorption

of -cyperone by the skin and its subsequent effect on proprotein convertase subtilisin/kexin type 9 (PCSK9).

This protease has been identified from recent human genetic screens (Mousavi et al., 2009; Horton et al.,

2009) and undergoes autocatalytic processing in the secretory pathway. The mature form is found in plasma,

and it binds to the EGF AB domains of the LDL-receptor (LDLR), leading to its lysosomal degradation (Mousavi

et al., 2009; Bottomley et al., 2009; Zhang et al., 2008 and Zhang et al., 2007). Introduction of PCSK9 into the

circulation of mice through parabiosis reduced hepatic LDLR levels, which is consistent with PCSK9 interacting

with LDLR on the cell surface (Lagace et al., 2006). Studies have shown that organic or inorganic compounds

absorbed by the skin (Yokoi et al., 2008) can reach the liver through the systemic circulation and can affect

liver metabolism. PCSK9 is expressed predominantly in the liver, small intestine, kidney, and brain (Seidah et

al., 2003), and it is also present in human plasma (Lagace et al., 2006). Tavori et al. (2013) proposed that LDLR

represented the main route of elimination of PCSK9 and that a reciprocal regulation between these two

proteins controlled serum PCSK9 levels, hepatic LDLR expression, and serum LDL levels. Our data were

consistent with this scenario, as the transdermal administration of -cyperone may have decreased total

blood serum cholesterol levels by inhibiting PCSK9-mediated LDLR degradation.

The binding of LDL to its receptor and its uptake by receptor-mediated endocytosis have been summarized

in a recent review by (Goldstein and Brown, 2009). Alternatively, -cyperone could affect PCSK9 mRNA

expression. Cameron et al. (2008) investigated the effect of berberine, a natural plant extract, on PCSK9

expression in HepG2 cells. Berberine decreased PCSK9 mRNA and protein levels and increased LDLR mRNA

expression in a time- and dose-dependent manner. The two possible mechanisms underlying this could be

increased degradation of PCSK9 mRNA, or decreased transcription of the PCSK9 gene.

PCSK9 plays a critical role in cholesterol metabolism by enhancing degradation of the LDLR protein in the

liver (Bedi et al., 2008); therefore, it is possible that -cyperone could affect cholesterol metabolism. Another

explanation is that -cyperone may affect the cholesterol biosynthesis pathway. Zaragozic acid (ZA), a

secondary metabolite produced by fungi, has good therapeutic potential because of its ability to decrease

cholesterol biosynthesis. Bedi et al. (2008) showed that ZA administration could regulate hepatic expression

of the PCSK9 gene in rats. Administration of ZA resulted in increased PCSK9 mRNA and protein levels in rat

liver, and a concomitant increase in hepatic LDLR mRNA levels, LDLR protein turnover, and decreased serum

cholesterol levels. Both the binding of PCSK9 to LDLR and its self-association are enhanced under the low pH

conditions that are present in endosomes (Zhang et al., 2007; Fan et al., 2008). For many recycling receptors,

occupancy by multivalent ligands can cause retention in the cell or delivery to late endosomes (Marsh et al.,

1995). The mechanism of PCSK9-mediated regulation of LDLR is now a major target of therapeutic strategies

for reducing circulating LDL.

ACKNOWLEDGMENT

The authors thank University of Thi- Qar \College of science to \Department of medical analysis and our colleague Prof.

Dr. Assad Y. Ayied for his excellent statistical analysis of our data, also we would like to thank our families for their

support.


28

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