FLAVOUR AND FRAGRANCE JOURNAL, VOL. 6,69-73 (1991)

Essential Oils and Glycosidic Bound Volatiles from Leaves, Stems, Flowers and Roots of Hyssopus oficinalis L. (Lamiaceae)

Gudrun Schulz and Elisabeth Stahl-Biskup Lehrstuh1,fur Pharmakognosie der Universirai Hamburg, Bundesstrasse 43, 0-2000 Hamburg 13, FRG

The essential oils from different parts of hyssop (Hyssopus ojicinalis L.) were investigated by means of GC and GC-MS at three stages of development of the plant. Besides the main components pinocamphone, camphor and fi- pinene, 15 other terpenes were detected, among which were isopinocamphone, a- and b-phellandrene, germacrene D, and some derivatives of myrtenol. The sesquiterpene alcohol hedycaryol was found to be converted to elemol during G C and MS analysis.

Compared with the essential oil content (0.03-0.16 % of the fresh plant material), the glycosidic bound volatiles were present in lower concentrations (0.01-0.06 %). The glycosidic fraction was hydrolysed by means of Pectinol C and fi-glucosidase yielding among others octan-3-01, linalol, cis-nerolidol, benzyl alcohol, phenylethanol, eugenol and o-vanillin. The bicyclic terpenes myrtenol and verbenol could only be detected in small amounts as glycosides of the leaves. This fact gives reason for doubt about a direct connection between the glycosidic bound volatiles and the biogenesis of the essential oil components in hyssop.

K E Y WORDS H y s s o p s oj’icinalis L. Lamiaceae Essential oil GC-MS Pinocamphone Glycosidic bound volatiles

INTRODUCTION

Glycosidic bound volatiles which are considered to be the precursors of the aroma components of plants, are of increasing technological interest. They are also thought to play a role in the biogene- sis of the essential oil components as transport forms, or as accumulation forms in undifferentiated cell cultures. Two review papers’.’ have been pub- lished on these matters.

The most comprehensive analysis of the essential oil of hyssop, H y s s o p s oficinalis L., so far has been performed by Joulain in 1976.3 He found the bi- cyclic monoterpenes pinocamphone and isopino- camphone to be the main compounds among the oxygen-containing monoterpenes. The question arises as to whether the bicyclic skeleton is also present in glycosidic form. If not, the glycosides might be considered as independent entities in the biogenesis of the essential oil components in hys- sop. In order to get more insight in the role of glycosidic bound volatiles in hyssop, we carried out an investigation of their occurrence in different parts of this plant.

EXPERIMENTAL

Plant Material

H . oficinalis was grown on the test fields of our institute near Hamburg in 1987. The plants were harvested at three stages of devel- opment: (1) in May, before the flower buds appeared; (2) at the beginning of July, when the flowering started; (3) at the beginning of September, when the flowering had ended.

Essential Oil

Fresh plant material (leaves, stems, roots 25 g of each, flowers 5 g) was hydrodistilled for 2.5 h ac- cording to the method of S p r e ~ h e r , ~ using n-hexane (1 ml) as the solvent.

Isolation of the Glycosidic Bound Volatiles

Fresh plant material (leaves, stems, roots, 250 g of each, flowers 50 g) was ground in liquid nitrogen and extracted twice with methanol (250 ml) at

0882-5734/91/01~69-05$05.00 ,J: 1991 by John Wiley & Sons, Ltd.

Received 6 March 1990 Accepted 6 June I990

70 G. SCHULZ A N D E. STAHL-BISKUP

room temperature for 24 h. The extract was filtered and concentrated to 5 ml. The methanolic extract was mixed with silica gel (for column chromatogra- phy) to obtain a dry mixture, which was applied to the top of a glass column (1.5 cm i.d.) packed with a 10 cm high layer of silica gel (Silica gel 60, 0.040-0.063 mm, Merck, Darmstadt, FRG). The essential oil was removed by elution with 100 ml n- hexane-ethyl acetate (1 :1) and dichloromethane. Subsequently, the polar glycosides were eluted with 100ml ethyl acetate and 100ml methanol. Both fractions were separately concentrated to 1 ml. After separate hydrolysis and G C analysis, the results were combined to draw up Table 2.

Enzymatic Hydrolysis

The glycosidic fractions were further concen- trated to dryness and the residues dissolved in a citrate buffer (pH 5.0,5 ml). /3-Glucosidase (15 mg 'Emulsin from Amygdalus communis', Serva, Hei- delberg, FRG) was added and after incubation at 37°C for 24 h, the hydrolysed volatiles were ex- tracted three times with n-hexane (5 ml). The com- bined hexane extracts were dried over anhydrous sodium sulphate and concentrated to 1 ml. The hydrolysis by means of Pectinol C (1.0 g, Rohm- Pharma, Darmstadt, FRG) was accomplished in the same way, but the incubation period lasted 16 h. The concentration of the glycosides in the fresh plant material was found to be 0.01 -0.06 %, as calculated from the GC peak areas of the corre- sponding aglycones including a stoichiometric amount of glucose.

Gas Chromatography

A Varian 3700 gas chromatograph equipped with FID, an on-column-injection system and a fused-silica capillary column, Supelcowax 10, 30 m x 0.32 mm i.d., film thickness 0.25 pm, was used. Carrier gas: helium, 1.5 ml/min. Oven tem- perature programme: 60°C for 5 min, then 3"C/min to 220°C. On-column-injector temperature pro- gramme: 60-25ODC, at 100"C/min. Detector tem- perature: 250°C.

Quantitative Estimation

Quantitative GC peak estimation was achieved by means of a Varian 270 Integrator. For the calculation of the absolute amounts (mg/kg), trans-

hex-2-en-1-01, linalol, or o-vanillin were used as external standards.

GC-MS

A Hewlett Packard 590085A mass spectrometer (ion source 2 W C , EI 70eV) was coupled to a Hewlett Packard 5840A gas chromatograph, equipped with a fused-silica capillary column, OV- 101, 25 m x 0.32 mm i.d.

RESULTS AND DISCUSSION

Essential Oil

The essential oil content of the fresh roots, stems, leaves, and flowers from hyssop varied from 0.03% to 0.16%. By means of GC, 60 compounds could be detected, 29 of which were identified by co-chromatography with au- thentic compounds as well as by GC-MS (Table 1).

the bi- cyclic monoterpenes pinocamphone and P-pin- ene were found to be the main components, not only in the oils from the flowers and leaves of the plant but also in the root oils. Additionally camphor was detected in a high concentration in the oils from the flowers and from the leaves. In another study, using a headspace method, isopinocamphone has been found as main component of hyssop oiL6 In the oils we investigated, this compound was found only in low concentrations. Pinocam- phone and isopinocamphone are responsible for the fresh and spicy flavour of hyssop herb. Among the sesquiterpene alcohols, in the higher retention times area, an unusually broad peak was observed in the chromatograms of the essential oils of the aerial parts of the plant. This was caused by hedycaryol, a cyclo- deca- 1,5-diene sesquiterpene, rearranging to ele- mol by a Cope rearrangement during GC. Elemol itself seems to be an artefact.

In agreement with previous

Glycosidic Bound Voiatiles

In comparison to the essential oil content of hyssop, the glycosidic bound volatiles occurred in considerably lower amounts: 0.01 -0.06%. The same has been observed during previous investigations of other Lamiaceae species and only marjoram (Ori-

VOLATILES FROM HYSSOPUS OFFICINALIS 71

Table 1. Composition of the essential oils from flowers, leaves, stems and roots of Hyssopus oficinalis L. at three stages of development ( 1 , 2, 3); tr = < 0.05 %

Constituents

Flowers Leaves Stems Roots (%) (%I (7% (%I 2 1 2 3 1 2 3 1 2 3

a-Pinene Camphene b-Pinene Sabinene a-Phellandrene a-Terpinene Limonene 8-Phellandrene cis-b-Ocimene y-Terpinene trans-j-Ocimene Terpinolene Unknown Myrtenyl methyl ether Unknown trans-Sabinene hydrate Unknown Camphor Pinocamphone Isopinocamphone j-Car yophy Ilene Unknown Methyl myrtenate Unknown Estragole Unknown a-Humulene Unknown Germacrene D M yrtenol Unknown Unknown p-Caryophyllene epoxide trans-Nerolidol Sesquiterpene alcohol Sesquiterpene alcohol Hedycaryol Spathulenol T-Cadinol a-Cadinol

Oil content (g/IOO g fresh material)

0.5 tr

10.0 1.7 2.0 tr 0.9 5.8 0.1 tr

1 .1 tr

2.0

0.1 0.2

20.6 34.3 0.9 6.9

0.9

0.3

0.2 0.1 3.4 2.7 1.3

-

-

-

-

-

-

- tr 0.1

1.8 tr

tr

0.06

-

-

0.4 0.3

13.9 1.5 1.2 0.1 0.8 0.3 0.1 0.1 0.2 0.1

1.9

0.1 0.4

15.4 31.8 2.0 2.9

2.2

0.4 0.1 0.5 0.1 2.3 1.7 0.8

-

-

-

-

- -

0.1 0.1 0.1 8.5 tr

tr

0.13

-

0.5 0.1

11.6 1.4 1.6 0.1 0.7 4.0 0.1 0.3 0.7 0.1

1.1

0.2 2.6

16.4 35.3

1.8 1 .o 1.6

0.4 0.1 0.7 0.2 3.7 2.6 0.7

-

-

-

-

-

-

0.2 0.1 0.1 6.4 0.1

tr

0.16

-

0.9 0.1

12.7 1.6 1.3 tr

0.8 2.9 0.3 tr tr tr

0.5

0.1 0.2 3.4

60.2 1 .1 1.2

1.5

0.2 0.2 0.5 0.2 1.3 1.3 1.5

-

-

-

-

- -

0.3 tr tr 2.6 0.4

tr

0.10

-

5.7 2.7

0.5 0.4 - -

- -

- -

- -

0.6 0.6 tr 0.2 - - - -

- -

- - - -

1.2 0.9 0.5 0.4

1.0 0.6 2.9 2.3

29.3 34.6 1.2 0.9 2.4 2.0 2.6 2.2 2.4 1.6

2.1 2.3

0.7 1 .o 2.7 2.2 2.1 1.7 1.8 1.1 3.2 2.7 4.1 3.5 1.3 1.1

- -

- -

- -

- -

- -

- __ 12.1 16.2 3.6 3.1 2.2 1.6 1.2 2.0

0.07 0.09

0.8 - - -

-

-

-

0.1 - -

-

-

-

1.2 -

-

-

2.6 34.8

1 .o 2.9 2.7 2.5

1.6

0.9

3.0 2.0 1.9 3.4 4.9 1.2

-

-

__

- -

11.4 2.2 1.2 0.8

0.05

11.7 0.3 7.6 0.5 -

-

1 .o 0.3 - -

-

-

4.9 0.4 -

-

tr 2.7

30.7 1.1 3.9

2.2 1.8 tr

0.2 5.4 1 . 1 0.9

15.6 1.6 tr

0.6

1.2

-

-

__

-

-

-

-

0.03

5.0 1.1 6.2 0.8 -

-

1.8 0.9 -

- - -

4. I 0.5 -

-

tr 3.1

21.7 tr 5.4

3.9 2.3 tr

0.4 6.3 2.2 1.4

13.1 3.1 tr

tr

0.5

-

-

-.

-

-

-

-

0.03

5.6 0.5 6.6 0.5 -

-

1.5 0.7 - -

- -

3.2 0.6 -

-

tr 2.9

23.1 0.6 4.7

3.2 2.1 0.8

tr 7.5 1.9 1.1

13.9 3.8 tr

1 .o 0.9

-

-

-

-

-

-

-

0.04

ganum majorana, syn. Majorana hurtensis) ap- peared to be an e~cept ion .~

The glycosides were extracted with methanol, cleaned up on a silica gel column and then treated alternatively with Pectinol C or P-glucosidase to obtain the free volatiles. The two enzymes yielded different hydrolysis products. P-Glucosidase, a more specific enzyme than Pectinol C, produced mainly myrtenol, eugenol, and o-vanillin, whereas Pectinol C, an unspecific enzyme mixture, liberated

predominantly octan-3-01, linalol, benzyl alcohol, phenylethanol, j?-ionone, and cis-nerolidol. This may mean that a P-linkage between myrtenol, eugenol and o-vanillin, respectively, occurs with glucose and/or that P-glucosidase has a higher affinity to the phenolic rather than to the non- phenolic glycosides.

Table 2 shows the combined composition of the fractions obtained by hydrolysis. In total, 32 com- pounds were detected, 20 of which were identified

72 G. SCHULZ AND E. STAHL-BISKUP

Table 2. Volatiles obtained by enzymatic hydrolysis of the glycosidic fraction from flowers, leaves, stems and roots of Hyssopus officinalis L. at three stages of development (1, 2, 3); tr = < 0.05%

Flowers Leaves (mg/kg) (mg/kg)

Constituents 2 1 2 3

Unknown Unknown Hexan- 1-01 Octan-3-01 Oct-1-en-3-01 Unknown Unknown Linalol Monoterpene alcohol Terpinen-4-01 Verbenol a-Terpineol Unknown Myrtenol Monoterpene alcohol Nerol G e r a n i o 1 Benzyl alcohol Phenylethanol 8-Ionone cis-Nerolidol Sesquiterpene alcohol Elemol Spathulenol Eugenol T-Cadinol Sesquiterpene alcohol Sesquiterpene alcohol Unknown Sesquiterpene alcohol p-Vanillin o - V a n i 11 in

Total (mg/kg fresh material)

1

-

0.50 1.05

0.10 0.97

0.12

2.85

0.34

-

-

-

-

- -

1.60 0.79 0.56 3.17 2.16 0.61 -

-

21.53

62.44 tr tr 3.04 0.85

tr

-

-

-

113.16

tr

0.99 0.10 1.56 1.45 0.80 1.61 tr

79.00 1.61 0.68

2.80 1.47 0.90 tr 3.04 3.98 0.31 1.96

60.85 1.10

44.32 0.60 3.19

82.38 tr 1.50 tr 1.01

297.22

-

-

-

0.70

0.94 tr

4.79 tr

0.24 0.65 1.26 5.75 0.10

0.50 1.35 tr

0.14 0.09 0.21 0.28

-

-

-

-

-

0.45 3.45

39.81 0.54 0.60 tr 1.47

3.80 2.14

69.97

-

1.01

1.50 tr 0.37 tr

0.25 0.60 0.90 7.83 0.20

0.63 1.92 tr

0.23 0.24 0.22 0.40

-

-

-

- - 0.56 1.97

28.00 0.57 0.88 tr 1.28

1.25 1.20

52.80

-

-

-

-

- -

- -

- -

0.22 -

-

- -

0.05 0.16 - -

0.68 0.15

0.10

tr 6.57 0.20

0.62 0.95

-

-

-

- -

1.20

10.90

3 1

- - - - -

- -

- - 0.37 -

- - - 0.23 0.35 -

- 0.74 0.47

0.28

0.40 15.35 0.45

0.55 1.08

-

-

-

- - 2.00

2 1.45

-

-

__ 1.55 -

-

0.05 - -

0.8 1

0.12 0.20

0.18 0.17

-

-

-

- 2.70 -

-

0.62

0.19 -

-

-

-

0.56 0.83

1.29 1.36

10.73

-

- -

- -

- -

1.07 tr - - - -

0.15 tr - - - -

1.08 0.15

0.17 tr 0.48 tr

0.25 0.20

- -

- -

0.33 - __ - - -

4.36 0.10 - -

- -

0.8 tr

0.44 -

- -

- -

- -

- -

0.89 0.55 1.26 0.12

1.88 tr 1.43 1.25

14.61 2.37

- -

by means of GC-MS. The composition of the glycosidic bound volatiles of flowers, leaves, stems, and roots differed in some respects. Table 2 shows that the bicyclic terpenoid skeleton, which was expected to be present in the glycosidic fraction, only occurred in the leaves (verbenol and myr- tenol). The myrtenol content was the same (1 %) in all the three stages of development, whereas the amount of verbenol increased from 1 % to 15 %.

The other glycosidic bound volatiles did not show any special structural relationship to the components of the essential oils, neither of the oils of the aerial parts (flowers, leaves, stems) nor of that of the roots. One of the dominating com- pounds was eugenol, which is considered to be connected with the lignin biosynthesis.* Eugenol is

neither present in the essential oils nor as glycoside in the roots, but occurs in relatively large amounts as glycoside in the aerial parts.

In contrast to other Lamiaceae species,' hyssop shows little correlation between the structures of the free and glycosidic bound volatiles. Such a result has also been found for other species.lo3" Thus, in the case of hyssop, only a restricted correlation between the biogenesis of the essential oil components and the glycosides can be assumed, e.g. with regard to geraniol, nerol, linalol, a-terpin- eol and terpinen-4-01, which are key intermediates in the terpene biosynthesis. Their occurrence as glycosides has led to the hypothesis of a 'mobile terpene p o ~ l ' . ' ~ The results described in the present paper neither support nor disprove this hypothesis.

VOLATILES

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FROM H Y S S O P U S OFFICINALIS 73

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