FLAVOUR AND FRAGRANCE JOURNAL, VOL. 6, 129-134 (1991)

Estimation of the 1,8=Cineole Yield of Eucalyptus camaldulensis Dehnh. Leaves by Multiple Internal Reflectance Infrared

Spectroscopy

Ann Gibson Department of Forestry, The Australian National University, PO Box 4, Canberra, ACT 2601, Australia

J. C. Doran Division of Forestry and Forest Products, CSIRO, PO Box 4008 QVT, Canberra, ACT 2600, Australia

D. Bogsanyi Research School of Chemistry, The Australian National University, PO Box 4, Canberra, ACT 2601, Australia

Peaks characteristic of 1,8-cineole consistently ‘appeared in spectra obtained by multiple internal reflectance spectroscopy (MIR) from the surfaces of 1,8-cineole-rich Eucalyptus camaldulensis Dehnh. leaves. The absorbances derived from the peaks at wave numbers of 1050 and 1078 cm- ’ were well correlated (rZ = 0.87 and 0.91 respectively) with the 1,8-cineole yield of leaves from trees in the field known to have high, medium and low yields of this compound. Absorbances at these wave numbers were not recorded in spectra from a contrasting cineole-poor chemotype of high sesquiterpene content. The wave pattern of this chemotype was readily distinguishable from the pattern of the regular, 1,8-cineole-rich phenotype. Multiple internal reflectance spectroscopy may be useful for field- testing eucalypts for their potential 18-cineole production.

KEY WORDS Multiple internal reflectance spectroscopy 1,8-Cineole analysis Eucalyptus camaldulensis Dehnh.

INTRODUCTION

Eucalyptus trees with fresh foliage containing at least 1.5% by weight of essential oil are commer- cially interesting for pharmaceutical use, provided that at least 70% of the oil is 1,8-cineole (1,3,3- trimethyl-2-oxabicyclo[2.2.2.]octane; C1 ,H *O), the principal therapeutic agent. Within species, individual trees vary in the quantity and composi- tion (e.g. percentage of 1,8-cineole) of the oil pro- duced and the oil characteristics appear to be strongly inherited.”*

Eucalyptus camaldulensis Dehnh. is an extremely important forest crop plant. Seed from Petford, in northern Queensland, is used extensively through- out the wet/dry tropics for industrial plantations, shade and shelter, and agroforestry. Doran and Brophy3 found substantial intraspecific variation in the composition and yield of essential oils from the leaves of northern Australian populations of

this species, and highlighted the potential of Pet- ford plantations as a source of 1,8-cineole-rich Eucalyptus oil. In contrast, provenances from northwestern Queensland, Northern Territory and northwestern Western Australia were found to be low yielders of 1,8-cineole.

Typically, oil from trees at Petford (regular phenotypes) consists largely of 1,8-cineole (70 %), but the yield of this compound varies greatly be- tween trees. The range in yield of 1,8-cineole per 100 g of fresh leaf extends from trace amounts to greater than 2 g, with an overall average of 0.87 g (s.d. + 0.38 g). In addition there is a distinct che- motype present in the population, at a frequency of about 1 in 10 trees, which is characterized by low 1,8-cineole content (ca 10% of the oil) and a high proportion of sesquiterpenoids (ca 60%).

Chemical analysis of Eucalyptus oil is normally undertaken either directly on oil samples obtained by steam distillation of leaf material, the principal

0882-5734/91/020129-06%05.00 0 1991 by John Wiley & Sons, Ltd.

Received 12 July 1990 Accepted 22 November 1990

130 A. GIBSON, J. C. DORAN AND D. BOGSANYI

source of commercial oil, or on solvent extracts4 using analytical gas-liquid chromatography or other laboratory methods. These methods, while accurate, are time-consuming, specialized and largely confined to the laboratory. The screening of a large number of trees for 1,8-cineole yield, as would be necessary in selecting superior trees for this trait in a breeding program, is, therefore, costly. Clearly, a method of rapidly assessing the potential of individual trees as 1,8-cineole yielders, and especially one that could be employed in the field, would be highly advantageous in limiting the cost of extraction and laboratory analysis, and speeding up the selection process.

Multiple internal reflectance spectroscopy has been used for rapid non-destructive analysis of the components of cell walls for purposes as diverse as predicting forage crop quality' and assessing the weathering of wood.6 The material to be sampled is placed in optical contact with a prism made from a high refractive index material and an infrared beam is directed at the crystal at an angle larger than the critical angle, causing internal reflectance to occur. A static wave is established at the crystal-sample interface and there is some penetration of the infrared radiation into the sample to a depth up to 4 pm, depending on the system. The reflected radia- tion has decreased intensity at the frequencies at which the sample absorbs.

The suitability of multiple internal reflectance spectroscopy for determining the amount and type of oil present in the glands below the surface of eucalypt leaves was assessed in this study.

MATERIALS AND METHODS

About a dozen leaves from each of ten mature E. camaldulensis trees known to be low (four trees), medium (three trees) and high (three trees) in 1,8- cineole yield were collected at Petford on 17 June 1989. Three of the trees chosen for their low yield of 1,8-cineole were of the distinctive, low-cineole- high-sesquiterpene chemotype described earlier. Care was taken to sample only leaves that were estimated to be at a similar age and stage of maturity in each tree. The leaves were obtained from within the crown, below the immature leaves but above leaves showing signs of senescence. Other studies have shown this sampling technique to be appropriate for estimating l,8-cineole yield in various eucalypt specie^,^.' The leaves, initially held on ice, were received in Canberra on 20 June

(in winter) 1989 in plastic bags (one bag per tree) in a Styrofoam container with ice pack. The leaves had not sweated during transit and were assessed as being in excellent condition. They were then stored in the closed plastic bags in a cold room (3°C) awaiting analysis.

Spectroscopic Examination of Leaf Surfaces

Two leaves were chosen at random from each bag after 45 days in cold storage. An MIR spectrum was obtained from the adaxial side of each leaf. The long delay was regrettable but unavoidable at the time. All leaves had maintained their health and as changes in oil yield and composition were unlikely to be significant in these conditions it was decided to proceed with the test. Spectra from the abaxial and adaxial sides of recently matured, seedling, juvenile and adult leaves of E. camaldulensis do not differ significantly (Gibson and Bogsanyi, unpubl- ished data). Further spectra were obtained from a leaf chosen at random from each bag, after storage, with the bag open, in a refrigerator (3°C) for a further 6 days. Some deterioration in the leaves, mainly a general darkening in colour, was evident at this time.

A Perkin Elmer 1800 Spectrophotometer with a DTGS detector and a Specac # 1 loo0 MIR acces- sory with a 25 reflection KRS-5 MIR element was used for the measurements. The range of wave numbers used was 800-2000 cm-'. One hundred and twenty-eight scans were collected in single ratio mode at 4 cm-' resolution against the un- clamped element, held in a special holder.

A 50 x 20 mm rectangle along the lamina of the leaf was clamped to one side of the MIR element at a reproducible torque of 0.075 N m, while the other side was covered with aluminium foil to avoid interference from the material covering the clamp. The element was cleaned with dichloromethane- moistened tissue after each run to remove oil and wax, and a fresh background was collected after five runs.

Each spectrum was converted to absorbance, flattened using a 3-point algorithm (the points being computer selected), smoothed using a Gavitzky-Golay algorithm with a 13-point win- dow, and expanded so that the strongest band spanned the range of 0- 1.5 absorbance. Computer- selected bands and computer-calculated absor- bances were used throughout this study. The com- puter used was a Perkin Elmer 7500 running under

1,8-CINEOLE ANALYSIS BY MIR SPECTROSCOPY 131

idris o/s and using Perkin Elmer CDS-3 Applica- tions Software.

We were concerned that other terpenes com- monly found in Eucalyptus oil with infrared spectra similar to 1,8-cineole might be confounding the patterns ascribed to 1,8-cineole. Hence spectra were obtained for comparison from pure 1,8-cineole by MIR, and from 1,8-cineole and related terpenes (a- terpineol, terpinen-4-01, y-terpinene and terpino- lene) by transmission IR spectroscopy.

GLC Assay of l,&Cineole Yield

On arrival of the leaves in Canberra, leaf samples of 3 g and 50 ml of ethanol (ca 99.8%) were weighed accurately into sample bottles. At least 2 weeks were allowed for full extraction. The yields of 1,8-cineole in the ethanol extracts were determined using a Varian 3400 gas chromatograph fitted with a FID detector. The column was a bonded FSOT, 20 m x 0.32 mm id., coated with Superox-FA. The column temperature, initially 70"C, was held for 3 minutes and was programmed to rise from 70 to 90°C at 5°C min-' and 90 to 220°C at 20°C min-'. The maximum temperature was maintained for a further 5 min before cooling. The injection and detector temperatures were 220 and 280"C, respec- tively. The carrier gas was N, at 1.5 ml min-'. The sample size (solution in ethanol) was 0.5 p1 with a splitting ratio of 20:l. An internal standard, n-tetradecane (0.02 g), was added to each 50 ml sample and quantification of 1,8-cineole used a

response coefficient determined for this compound relative to the internal standard.

RESULTS

Peaks at wave numbers of 986, 1050, 1078, and 1165 cm-' characterized the spectra obtained from purified 1,8-cineole by transmission (IR) and MIR spectroscopy (Table 1). Each of the related terpenes had a distinctive pattern which combined 1,8- cineole peaks with other peaks. Moreover, the proportion of most of these compounds in Petford E. camaldulensis oil is low, so their effect on the key, 1,8-cineole peaks will be negligible.

The MIR spectra from leaves of the regular phenotype showed the peaks characteristic of 1,8- cineole in the wave numbers between 1000 and 1100 cm-' (Table 2). At the first sampling the peak at 1078 cm-' was present in all leaves and the peak at 1050 cm-' was present in the leaves with higher 1,8-cineole yields as determined by gas chromato- graph. The peaks at wave numbers 986 and 1165 cm- ' were absent from the spectra because of interference from other compounds. At the later sampling the peaks at 1050 and 1078 cm-' were present, but only in the samples of high 1,8-cineole yield and with reduced absorbances.

The range of 1,8-cineole yields was well corre- lated with the range of absorbances despite consid- erable variation between the duplicate samples from each tree. The regression coefficient, using the

Table 1 . Peaks found between wave numbers 986 and 1170 in transmission spectra from pure 1,8-cineole and some related terpenoid compounds commonly found in the leaf oils of various EucaZypfus species

~ ~~~~

Compound Wave number

(cm- ') 1,8-Cineole 1,8-Cineole0 a-Terpineol y-Terpinene Terpinolene Terpinen-4-01

986 1010 1020 1028 1050 1070 1078 1108 1123 1135 1158 1165 1170

0 0 0 0

0 0

0 0 0 0 0 0

0 0 0 0

0 0

0 0 0 0 0

0 0 0

~ ~~ ~ ~~

Comparative MIR x spectrum.

132 A. GIBSON, J. C. DORAN AND D. BOGSANYI

Table 2. Absorbances of peaks characteristic of 1,8-&eole in MIR spectra from leaves of Eucalyptus carnaldulensis Dehnh. The first spectra were obtained on 4 August from two leaves per tree, followed by another assay 6 days later (10 August) on a single leaf per tree. For comparison trees were ranked for 1,l-cineole yield by

GLC analysis of solvent extracts from matching samples of leaves

1,ICineole yield" Absorbance at characteristic wave numbers Tree (g per 100 g of 1078 cm-' 1050 cm-' no. fresh leaf) leaf no. 1 leaf no. 2 leaf no. 1 leaf no. 2

Date 4/8/89 lowest highest lowest highest reading reading reading reading

1 2 3 4 5 6 7 8 9

10

0.018b 0 . 2 w 0.299* 0.407 1.041 1.178 1.240 1.508 1.831 1.908

Date 10/8/89

1 2 3 4 5 6 7 8 9

10

O.ONb 0 . 2 w 0.299b 0.407 1.041 1.178 1.240 1.508 1.831 1.908

-

- - -

0.1782 0.2007 0.1779 0.3043 0.2776 0.3066

-

- - - -

0.043 1 0.1061 0.1458 0.2250 0.2133

-

- -

0.1147 0.2477 0.2121 0.2194 0.3263 0.3723 0.3430

- - - -

- 0.1873

0.2784 0.2596 0.2684

-

- -

- - -

0.2007

0.2919 0.3703 0.3407

-

As determined by GLC assay. High sesquiterpene chemotype.

mean of the duplicates, was 0.95 (r2 = 0.91) for wave number 1078 cm- and the regression equa- tion was y = 0 . 1 5 ~ + 0.05. The coefficient for 1050 cm-' was 0.93 (rZ 0.87) and the equation was y = 0.15~ + 0.03. The y intercept for both regres- sions is not significantly different from zero and the absorbances of the two key 1,8-cineole peaks in each spectrum were well correlated (r2 = 0.94).

The peaks in the region of wave numbers loo0 to 1100 an-' gave a characteristic appearance to the spectra from the higher and lower 1,8-cineole con- tent leaves of regular phenotype and distinguished them from the sesquiterpene chemotype (Figure 1).

DISCUSSION

Oil was released from the glands of E. camaldulensis leaves by pressure applied with the torque screw-

driver while attaching each leaf to the MIR ele- ment. The oil was squeezed on to the leaf surface/ prism interface in sufficient quantities to produce the absorbance peaks characteristic of l,&cineole (Table 1). The size of the peaks, using the algor- ithms described in 'Materials and Methods', was well correlated with the 1,8-cineole yield (Table 2) and, in addition, there appeared to be a strong relationship between the pattern of the spectra and the 1,8-cineole yield (Figure I), allowing one to broadly rank a regular phenotype as to its cineole- producing capacity.

Of further interest is the very obvious division of the spectra of the low 1,8-cineole yielding trees into two distinct types (Figure 1). These types were from the regular phenotype containing a high propor- tion of 1,8-cineole in the oil (about 70 %), as in the high- and medium-yielding trees, and from a chemotype containing a low proportion of 1,8-

1,8-CINEOLE ANALYSIS BY MIR SPECTROSCOPY 133

-7 A. HIGH CINEOLE B. MEDIUM/LOW CINEOLE r C CHEMOTYPE I

0.0 L 1 1 I 1 1 t I I I I I I 1200 1100 1000 goo i200 1100 1000 goo 1200 1100 1000 goo

WAVE NUMBER (ern-') Fig. 1. Examples of spectra obtained from the surfaces of Eucalyptus camaldulensis Dehnh. leaves from trees of the regular phenotype (A and B) and of the chemotype (C). 1,8-cineole yield (g per 100 g fresh leaf): A, 1.908 g (-) and 1.831 g (---); B, 1.240 g (. . ..),

1.178 g (-) and 0.407 g (---); C, 0.299 g (-) and 0.018 g (---)

cineole and high levels of sesquiterpenes. MIR spectra, therefore, would be useful in reducing the time spent on this adverse chemotype in a selection program aimed at improving the quality of E. camaldulensis oils for pharmaceutical purposes.

The absorbances of the spectra from deteriorat- ing leaves sampled 6 days after removal from the cold room were low, possibly due to evaporation of the oil, but the peaks characteristic of 1,8-cineole were still present in the high 1,8-cineole yielding samples. It appears from this test that meaningful results may be obtained from leaves stored in plastic bags in refrigerated conditions for lengthy periods (1 to 2 months) as long as the bags are not opened and the leaves retain their field colouration.

The spectra are obtained directly from the leaf surfaces, and 10 to 15 minutes are required for each spectrum, including time needed to clean the ele- ment between determinations. The time required compares favourably with the overall time needed for a GC determination on an ethanol extract of fresh leaves, which takes up to 2 weeks for full extraction, or steam distillation, which takes at least 4 h per sample and is limited by the number of stills operating. In addition to speed, simplicity and low labour costs, determinations in the field allow the user to map and label only those phenotypes of

future interest and to collect seed, scions or cuttings from the superior trees when they are identified, thus saving on time and on the number of visits to the field site.

CONCLUSION

These data suggest that MIR infrared spectroscopy could be a valid and practical technique for the initial screening of large numbers of trees of E. camaldulensis for oil type and 1,8-cineole yield, either in the field, using a robust, transportable instrument, or from leaves kept in cold storage, although the chemical techniques must still be used when an accurate oil analysis is required.

Further work needs to de done to field-test the method and to determine its suitability for the screening of other high- 1,8-cineole yielding Euca- lyptus species.

Acknowledgements-We thank W. F. Crow and J. C. G. Banks for helpful discussion and J. J. Brophy for review of the text. The leaf samples were collected in northern Queensland by J. G. Moriarty of CSIRO’s Australian Tree Seed Centre.

134 A. GIBSON, J. C. DORAN A N D D. BOGSANYI

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