Bioactive Volatile Compounds from Plants - ACS Publications

Chapter 13

Volatile Compounds from Flowers Analytical and Olfactory Aspects H. Surburg, M. Guentert, and H. Harder

Downloaded by STANFORD UNIV GREEN LIBR on June 29, 2012 | http://pubs.acs.org Publication Date: April 6, 1993 | doi: 10.1021/bk-1993-0525.ch013

Haarmann and Reimer GmbH, D-3450 Holzminden, Germany

A short survey of the different procedures for the isolation of volatile constituents from fragrant flowers is presented. It is shown, that the three most common methods (dynamic and vacuum headspace sampling, steam distillation) give concentrates whose composition differs both quantitatively and, in many cases, qualitatively. Various results are used by way of example in an attempt to explain how and in what way the different processes lead to different products. The range of usefulness of each method is discussed. For perfumery applications we prefer to use the vacuum headspace technique, which produces odor concentrates with the best sensory properties. Some of the results of these investigations are presented in detail. In recent years much of the research carried out in the fragrance industry has been directed towards investigation of naturalflowerfragrances.The aim was to develop a new generation offragrancecompositions more closly related to nature and, thus, to meet the increasing demands of the consumer for more natural products. Consequently, research results gained importance as marketing instruments. But the search for newfragrancesubstances and new ideas for creating perfume compositions also played an important role in investigations of natural scents. These research activities were essentially stimulated by the rapid development of modern analytical equipment, which enables us today to analyze complex mixtures in the submicrogram region and to elucidate the constitution of an unknown compound, even if isolated in microgram quantities. Especially in the case of flowers it appeared that isolation of volatile constituents for analytical and perfumery purposes, using classical methods like extraction and distillation, yielded in most cases, products which did not reproduce the organoleptic properties of the natural material. Consequently, attempts were made to trap fragrant volatiles of flowers direcdy by so-called "headspace" procedures. However, these methods produced such small amounts of odor concentrates that analysis of the constituents could be accomplished only with the aid of modern analytical techniques. As a result of this research several papers have been published in recent years dealing with the investigation of flower volatiles. The authors describe the use of very different headspace methods, leading to different, sometimes inconsistent results (7-77).

0097-6156/93A)525-O168$06.00A) © 1993 American Chemical Society

In Bioactive Volatile Compounds from Plants; Teranishi, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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Until very recently hardly any comparative studies existed (10,13), which attempted to clarify these contradictions; for example, through the application of the different headspace methods to one and the same land of flower. Some years ago, we began to perform detailed studies in this field. In the present paper we will describe the experience that we have gained using the different methods for the isolation of volatile constituents of flowers. We will present various results in an attempt to clarify how and in what way the different headspace processes lead to different products, what their advantages and disadvantages are, what risks they involve and how they have to be assessed for perfumery applications.


Different Headspace Methods for Isolation of Volatile Constituents of Flowers First, a short survey will summarize the methods which are generally used to isolate the volatile constituents of flowers. Since two very detailed reviews of the subject have just been published (77,72), only the principles will be explained briefly. "Dynamic Headspace". Following dynamic headspace accumulation, the volatiles released by the plant are purged by a gas stream and trapped by adsorption. The adsorption materials used may be charcoal, Tenax or similar macroporous resins. In a second step a concentrate of the volatile compounds is obtained by desorption with a solvent or by heating (3,5,10,13,14). Very sophisticated variations and modifications of this method have been reported for trapping the fragrant volatiles of living flowers (5,10,14-18). For example, devices have been constructed to enclose a flower which is still connected to the living plant, or the emitted volatiles are sucked off instead of being purged (79). "Vacuum Headspace". However, since in the products of dynamic processes fewer volatile compounds are found only in reduced quantities or sometimes not at all, a further method was developed: the vacuum headspace method. This method is basically a form of vacuum steam distillation. The flowers are subjected to a vacuum; the volatile components distill off together with the water contained in the plant and are condensed at low temperatures, thus providing the fragrance concentrate immediately (8,9).

Results and Discussion Comparison of Dynamic and Vacuum Headspace Methods. In recent years we have applied both the dynamic and the vacuum headspace methods for the enrichment of volatile constituents of flowers. To allow comparison we also used in some cases steam distillation at normal pressure, in the modification recommended by Likens and Nickerson. These investigations have demonstrated clearly that not only the quantitative but also the qualitative composition of the respective products differs and depends heavily on the method used. This is shown by the following examples. Lily of the Valley. The volatile constituents of lily of the valley flowers were enriched in two ways. First we used dynamic headspace sampling during a period of 24 h trapping the volatiles on Tenax and following this by desorption with ether. The second method was the vacuum headspace procedure (8). The composition of the products obtained is shown in Table I which presents some characteristic compounds. It is evident, that the vacuum headspace concentrate contains higher proportions of higher-boiling, more polar compounds, whereas the low-boiling, less polar components are better represented in the concentrate of the dynamic process. When tested on a smelling blotter, the vacuum product displayed a more typical lily of the valley odor. This may be due to the higher content of compounds like dihydrofarnesol and farnesol.



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Hoya carnosa (Wax-Plant). The flowers of Hoya carnosa emit their typical odor only at night To collect the fragrant volatiles, we placed an entire potted plant in a 200 1 plastic bag. During the period from 9 p.m. to 6 a.m. a stream of air was passed through this system and led through a Tenaxfilterat the outlet. In another experiment a flower was cut at midnight and submitted to the vacuum headspace procedure. The composition of the two products obtained is depicted in Table Π. It can be seen that the dynamic process led to higher concentrations of low-boiling compounds like sabinene and linalool. In contrast, the vacuum headspace concentrate contained higher proportions of high-boiling and more polar constituents, e.g. 2phenylethanol, benzyl alcohol, methyl salicylate, eugenol. The higher content of benzaldehyde in the dynamically obtained product is probably due to an oxidation re action during the sampling period, because the vacuum headspace concentrate con tains smaller amounts of benzaldehyde but larger quantities of benzylalcohol. Again, the product of the vacuum process had superior organoleptic properties. Phlox. The volatiles of pink Phlox flowers were isolated under the same condi tions as described above for lily of the valley flowers. Again, as Table ΙΠ shows, the dynamic process yields higher proportions of the aldehydic components, whereas the concentrate obtained by vacuum contained higher quantities of the corresponding al cohols. The reason might be an oxidation reaction and/or the higher volatility of the aldehydes in comparison to the alcohols. Cinnamic alcohol could not be detected by the dynamic method; in contrast, the vacuum headspace method produced it in sut> stantial amounts. Curiously, the flowers of white Phlox did not contain this substance at all, although the composition is otherwise very similar. The two isomeric hydroxyketones and the corresponding diol are present only in the vacuum headspace concentrate. In our experience, they are frequently found when this method is used for isolating flower volatiles. As the origin of these com pounds is not yet clear, they may be formed as a result of the destruction of the plant material (see below). For the sake of completeness it should be mentioned that lilac alcohols, eugenol methyl ether, isoeugenol methyl ether and germacrene-D are addi tional main constituents of the volatiles of Phlox flowers. Lilac (Syringa vulgaris hybrids). Investigation of the fragrance of purple lilac under the conditions described above produced analogous results, see Table IV. The concentrate obtained by the dynamic process consists mainly of low-boiling com pounds like (E)-ocimene and benzyl methyl ether. In contrast, the quantity of these compounds in the vacuum headspace concentrate is much lower. There, the emphasis is on the higher-boiling compounds like lilac aldehydes and alcohols, anisaldehyde etc. Again, the dynamic method produces no cinnamic alcohol; indole and 8-oxolinalool are also missing. The qualitative composition of the vacuum headspace concentrates from lilac flowers of different colors are generally comparable. Quantitatively, few exceptions can be noted. Red lilac appears to contain only small amounts of ocimene and white lilac produces higher proportions of indole. Accumulation of Low-Boiling Compounds During Dynamic Processes; Repro ducibility of Headspace Methods. The aforementioned results indicate that one of the main differences between the two methods is the higher content of low-boiling compounds in the dynamically obtained headspace concentrate. In order to examine whether and in what way low boiling compounds accumulate during dynamic proces ses, we investigated the behavior of test mixtures. The test mixture shown in Table V consists of nearly equal amounts (by weight) of compounds with different boiling points and polarities, as they typically occur in na tural flowerfragrances.For the experiments a small amount was dabbed on a filter paper. The evaporating substances were purged by a constant stream of air


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Table L Composition of Headspace Concentrates Obtained by Different Methods from Lily of the Valley Flowers (ConwaUaria majalis L.), [%]


Compound

dynamic

vacuum

2 18 3 5 10 0.02 snowball* (Viburnum χ burkwoodii Burkw. et Skipw.); 3,5-dimethoxytoluene in yellow roses and related sorts (730), narcissus (6), sour cherry blossoms* {Prunus cerasus L.) and mimosa*; 1,3,5-trimethoxybenzene in olive tree blossoms (8) and some red roses* (*results of these authors, unpublished work). Grape hyacinth (Muscari armeniacum Leichtl. ex Bak., Liliaceae) is a small plant which flowers in spring. Its flowers give off a fruity, floral, slightly peach-like odor. 2-Phenylethanol and 2-(4-methoxyphenyl)-ethanol are the main constituents of the corresponding odor concentrate (see Table ΧΠ). Acetophenone and 1-phenylethanol are responsible for the typical topnote. In addition to the well-known 2-phenylethyl benzoate we also identified 1-phenylethyl benzoate, a compound hitherto found only once in nature, namely as a constituent of Piper hookeri (37). Skimmia japonica Thunb. is a evergreen shrub native to Eastern Asia. The little white flowers emit a characteristic floral, lily of the valley-like odor. In keeping with this impression we found farnesol and 2,3-dihydrofarnesol (see Table ΧΙΠ) exactly as in lily of the valley flowers. Together with nerolidol and 2-phenylethanol these com pounds create the typical note. Other main constituents like phenylacetaldoxime and phenylacetonitrile constitute an additional similarity to lily of the valley. 5-Methyl-2heptanone and the related alcohol are important for the topnote, displaying a fresh, fatty and slightly citrus-like odor. It is remarkable that these compounds have not previously been identified as flower constituents. The first was identified as a trace component in a basil oil (32), the latter as a volatile constituent of roasted beef flavor (33).

The typical odor of German chamomile [Chamomilla recutita (L.) Rauschert] flowers is attributed to a number of low-boiling components, which occur in the es sential oil in only very small amounts, e.g. ethyl and propyl 2-methylbutyrate. These compounds are found in much higher concentrations in the vacuum headspace con centrate of fresh flowers (see Table XIV). But methyl salicylate, 2-methoxy-4methylphenol and indole also contribute essentially to the characteristic chamomile note, even though they are present in very low concentrations. While monoterpene hydrocarbons such as the ocimenes and p-cymene appear at comparatively high


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Table XL Composition of a Vacuum Headspace Concentrate Obtained at 2.00 p.m. from a Non-Fragrant Hoya carnosa Flower Compound

%

Sabinene Benzaldehyde Benzyl alcohol Isoamyl alcohol Linalool 2-Phenylethanol Methyl salicylate endo-2-Hydroxycineole Eugenol

0.1 0.1 29 35 4 4 1 18 0.5

4-11%

4-6%

Figure 3. Theaspiranones identified in the vacuum headspace concentrate of Reseda odorata flowers.

Table ΧΠ. Compounds Identified in the Vacuum Headspace Concentrate of the Flowers of Grape Hyacinth (Muscari armeniacum) Compound Acetophenone 2-Phenylethanol 1-Phenylethanol 4-Hydroxylinalool 2- (4-Methoxyphenyl)-ethanol (E^)-a-Famesene 1- Phenylethyl benzoate 2-Phenylethyl benzoate

% 2 20 1 3 31 2 3 2


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BIOACTIVE VOLATILE COMPOUNDS F R O M PLANTS

Table XEDL Compounds Identified in the Vacuum Headspace Concentrate of Flowers of Skimmia japonica


Compound Farnesol 23-Dihydrofarncsol Nerolidol Pbenylacetaldehydoxime Phenylacetonitrile 2-Phenylethanol Gennacrene-D S-Methyl-2-heptanone 5-Methyl-2-heptanol

%

19 1 12 13 7 22 2 2 1

concentrations in the headspace concentrate, the content of the higher-boiling main constituents generally corresponds to that of the essential oil, with the exception of chamazulene, a known artifact produced only under steam distillation conditions. Iceland poppy (Papaver nudicaule L.) is a poppy originating from Arctic regions and is now a popular garden plant due to its beautifully colored flowers. Their odor is strong, very chemical, smoky, and clove-like. This sensory impression is reflected by the constituents identified in the odor concentrate (see Table XV), mainly by 2methoxy- and 4-methoxyphenol, their corresponding methyl ethers and eugenol, which is by far the main component The topnote is influenced by smaller amounts of acetophenone and 1-phenylethanol. Dames-violet or sweet rocket (Hesperis matronalis L.) belongs to the family of Brassicaceae. It is found frequently in old gardens. The color of the flowers which appear in the early summer varies from light purple to lilac. In the evening, the flow ers develop a pleasant green-floral, clove-like odor. Accordingly, the following com pounds were found in the odor concentrate: benzyl alcohol, 1,8-cineole, benzyl ace tate, linalool, oc-terpineol, cinnamic alcohol, eugenol, isoeugenol, isoeugenol methyl ether and benzyl benzoate. It should be noted that the concentration of the cis-isomers of isoeugenol and its methyl ether is about 3-5 times higher than that of the transisomers (0.5 to 0.1% for the former and 0.2 to to 0.07% for the latter). The content of long-chained aliphatic compounds such as hexadecanal (0.2%), hexadecanol (0.2%) and hexadecyl acetate (0.5%) demonstrates again, how well even high-boiling con stituents are recovered by the vacuum headspace method. The flowers of the common cherry laurel (Prunus laurocerasus L.) emit a typical cherry odor combined with a pronounced anthranilate note. By far the main constitu ent of the odor concentrate is benzaldehyde. The impression of anthranilate-like com pounds is caused by 2-aminoacetophenone, which we previously detected in the flow ers of Prunus padus L. (bird cherry) too (34). Large amounts of lilac aldehydes and alcohols and linalool derivatives oxidized at C-8 (8-Oxolinalool, (E>- and (Z)-8hydroxylinalool, 6,7-dihydro-8-hydroxylinalool) are also found. These compounds have also been identified as constituents of the flowers of bird cherry, a related plant The occurrence of various 4-oxoisophorone derivatives (see Figure 4) proved to be a characteristic property of cherry laurel blossoms. All these compounds are found in a range of 0.1 to 1%. Erysimum χ allionii (Brassicaceae) is a plant closely related to the wallflower. The deep orange-colored flowers display a heavy floral odor, but without the anisic violet note of wallflower. The quality of the identified constituents confirmed the organo leptic impression (see Table XVI). It should be mentioned that, in contrast to wall flower, no mustard oil compounds could be detected in the odor concentrate.


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Table XIV. Comparison of the Constituents of the Essential Oil and a Vadium Headspace Concentrate of German Chamomile Flowers [%]


Compound Ethyl 2-mcthylbutyratc Propyl 2-mcthylbutyratc 2-Mcthylbutyl propionate (Z)-3-Hcxcnyl acetate (Z)-3-Hcxenyl propionate Artemisiaketone Arternisiaalkohol 2-Methylbutyl 2-mcthylbutyrate Methyl salicylate 2-Methoxy-4-methylphenol Indole p-Cymcne (Z)-Ocimene (E)-Ocimcne (E)-beta-Farnescne Germacrenc-D Bicyclogermacrene Bisabolol oxide Β Bisabolone oxide Bisabolol Bisabolol oxide A Chamazulene En-In dicycloether

vacuum

oil

1

0.1 0.06 tr. tr. tr. 0.44 tr. tr.

1.0 0.5 0.2 0.8 0.3 10 5 0.7 0.2 0.4 0.04 0.7 0.9 4.3 23.0 2.2 2.7 4.0 3.1 0.9 28.2

-

0.1 0.2 0.8 26.2 0.3 1.1 4.2 5.1 2.0 6.8 1.2 3.0

.

0.6

1. Commercial grade

Table XV. Compounds Identified in the Vacuum Headspace Concentrate of Ice land Poppy (Papaver nudicaule) Flowers Compound

%

Acetophenone 1- Phenylethanol 2-Methoxyphcnol 1,2-Dimcthoxybenzcne 1,4-Dimethoxybenzene 4-Methoxyphenol Eugenol 2-(4-Methoxyphenyl)-ethanol Methyl eugenol 2-Heptadecanone

0.4 0.1 2 1 6 8 45 11 2 12


BIOACTTVE VOLATILE COMPOUNDS F R O M PLANTS

184

Table XVL Compounds Identified in the Vacuum Headspace Concentrate of Erysimum χ allionii Flowers %


Compound

34 39 1 2 0.4 1 3 2 2 2

Benzyl alcohol 2-Phenylethanol Linalool Methyl salicylate Anthranilaldehyde 5-Hydroxylinalool Indole Methyl 2-methoxybenzoate Caryophyllene Benzyl benzoate

HO

HO

HO

HO

HO

Figure 4. Isophorone derivatives found in the vacuum headspace concentrate of flowers of common cherry laurel The southern Meditearranean coast of Turkey is home to a narcissus species named "Nergiz" by the locals. It is a small tazetta-like plant with several doubled flowers on each stem. The odor concentrate contained mainly trivial compounds such as benzyl alcohol, (E)-ocimene, benzyl acetate, indole, methyl cinnamate etc. Some uncommon minor components are noteworthy: 2-methoxybenzylalcohol and the corresponding acetate have previously been found only in hyacinth (7) and narcissus (35) flowers. The homologous 2-(2-methoxy)phenylethyl alcohol is a new natural product. Its ace tate is already known, but only as a narcissus constituent (55). Acknowledgement We would like to thank the management of Haarmann & Reimer for permission to publish this paper, the staff of the H&R research department for their valuable coop eration, especially Miss S. Allerkamp and Mrs. Chr. Voessing for their skilful techni cal assistance and Dr. P. Werkhoff for helpful discussions. We are grateful to In Bioactive Volatile Compounds from Plants; Teranishi, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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Dr. P. Calzavara, H&R Italy, for carrying out headspace experiments in the Mediter ranean region and Dr. H. Harder and A. Landi for sensory evaluations.


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Bioactive Volatile Compounds from Plants - ACS Publications

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