GC Volatile Components Analysis of Different Parts of Litchi chinesis.

UNIVERSITEIT GENT FACULTEIT FARMACEUTISCHE WETENSCHAPPEN Erasmus onderzoeksstage Geneesmiddelenleer Faculty of Pharmacy, University of Camerino Laboratory of Food Chemistry Academiejaar 2008-2009 GC Volatile Components Analysis of Different Parts of Litchi chinesis. Ysaure Custers Eerste Master in de Farmaceutische Zorg Ugent Promotor Prof. dr. apr. S. Van Calenbergh Camerino Promotor Prof. S. Vittori Commissarissen Prof. dr. apr.b. De Spiegeleer Prof. dr.apr. S. De Saeger

UNIVERSITEIT GENT FACULTEIT FARMACEUTISCHE WETENSCHAPPEN Erasmus onderzoeksstage Geneesmiddelenleer Faculty of Pharmacy, University of Camerino Laboratory of Food Chemistry Academiejaar 2008-2009 GC Volatile Components Analysis of Different Parts of Litchi chinesis. Ysaure Custers Eerste Master in de Farmaceutische Zorg Ugent Promotor Prof. dr. apr. S. Van Calenbergh Camerino Promotor Prof. S. Vittori Commissarissen Prof. dr. apr.b. De Spiegeleer Prof. dr.apr. S. De Saeger

AUTEURSRECHT De auteur en de promotor geven de toelating deze masterproef voor consultatie beschikbaar te stellen en delen ervan te kopiëren voor persoonlijk gebruik. Elk ander gebruik valt onder de beperkingen van het auteursrecht, in het bijzonder met betrekking tot de verplichting uitdrukkelijk de bron te vermelden bij het aanhalen van de resultaten uit deze masterproef. 25 mei 2009 Promotor Prof. dr.apr. Serge Van Calenbergh Auteur Ysaure Custers

Acknowledgements The material in this paper could not have been developed without my internship in the laboratory of the department of Food Chemistry in Camerino, Italy. It has been immensely enjoyable to have been learning about other cultures at the same time as I was completing my master test at the university of Camerino. Thanks to Fabrizio Papa for giving me professional direction during my research and for providing me with the opportunity to learn new scientific techniques. Thanks to Prof. Sauro Vittori for being my tutor and for reviewing the editing of my results. Thanks to Yangting Zuo for giving me moral support and company during my long days in the laboratory. Thanks also to all of them for all the Chinese food and delicious Italian desserts which they shared with me to keep me going. I would also like to thank Prof. Serge Van Calenbergh for reviewing the final result of my research abroad. A last thank you goes out to all the friends I made during my Erasmus term in Camerino who helped me keep the balance between pleasure and work.

TABLE OF CONTENTS 1. INTRODUCTION... 1 2. OBJECTIVES... 6 3.1 FRUIT MATERIAL... 7 3.3 HYDRODISTILLATION... 8 3.4 SPME FIBER SCREENING... 10 3.5 HS-SPME... 10 3.6 HEADSPACE GAS-CHROMATOGRAPHY... 13 3.7 GC-FID CONDITIONS... 14 3.8 GC-MS CONDITIONS... 15 3.9 IDENTIFICATION AND QUANTIFICATION OF VOLATILE COMPOUNDS.... 16 4. RESULTS AND DISCUSSION... 21 4.1 HEADSPACE-GAS CHROMATOGRAPHY... 21 4.2 HS-SPME ANALYSIS... 22 4.3 ESSENTIAL OIL ANALYSIS... 33 4.3 ESSENTIAL OIL ANALYSIS OF COMMERCIAL SAMPLE... 45 5.CONCLUSIONS... 48 References... 49

1. INTRODUCTION The lychee (Litchi Chinesis) is the sole member of the genus Litchi in the soapberry family Sapindaceae. It is native from China, but it is also grown in few other tropical areas such as India, Madagascar, Nepal, Bangladesh, Pakistan, southern Taiwan, northern Vietnam, Indonesia, Thailand, the Philippines, and Southern Africa. It is a tropical medium-sized evergreen fruit tree, 9 to 20 m tall and equally broad, with alternate pinnate leaves. Each leaf is 15 25 cm long, with 2-8 lateral leaflets 5 10 cm long and the terminal leaflet is absent. They are somewhat leathery, smooth and glossy. The flowers are small, greenish-white or yellowish-white, produced in panicles up to 30 cm long. http://www.lycheesonline.com/lycheedescription.cfm http://www.hort.purdue.edu/newcrop/morton/lychee.html The fruit of the lychee has a white, juicy aril which is surrounded by a reddish, prickly leather-like skin as illustrated in figure 1.1 and contains a shiny brown, usually large seed (Johnston et al.,1980). Figure 1.1 Lychee fruit 1

Because there is only a short production period and the fruit has a short shelf life, the fresh fruit is only available during a few months. However, there are canned lychees available all year but they have a different taste. There have been studies about the antioxidant effects of compounds in the fruit and the flowers of the lychee, the improvement of the storage and cultivation and possible involvement of lychees in food allergies. However, the amount of studies that identifies and quantifies the volatile compounds in the different parts of the lychee is limited. To the best of our knowledge only four other studies have been reported in which the volatile fraction in the lychee has been examined. Johnston et al. in 1980; Ong, Acree in 1998; Chyau et al. in 2003 and Mahattanatawee et al. in 2007. The study by Johnston et al. only identified the volatile compounds in the fresh fruit and demonstrated that the citrus note of the lychee is due to the presence of limonene, geranial and neral and that it owes its floral character to β-phenethyl alcohol. The second study performed a Gas Chromatography/Olfactory analysis of the volatile compounds in the fresh fruit and the juice of lychee. They confirmed the origin of the citric and floral character and determined that the aroma of the lychee is due to the interaction between compounds with floral, nutty, citrus and fruity aromas (Ong, Acree, 1998). Chyau et al. made a comparison between free and glycosidically bound aroma compounds and obtained similar volatile profiles. An important difference between the two fractions was that the free volatile fraction had a fresh-fruity, lychee-like aroma whereas the glycosylated fraction was odorless. The forth study compared three major lychee cultivars and found twenty-four common volatile compounds (Mahattanatawee et al., 2007). However, it is important to clarify that the previous studies used different extraction methods and solvents than those employed in the current study. The purpose of this study is not only to examine the volatile compounds in the edible parts (pulp and juice) of the lychee but, in particular, also the other parts that are considered waste, like the peel and seed. No significant medicinal effects are demonstrated yet for any part of the lychee. The fruit and juice contain a high level of vitamin C, plus calcium, iron, and vitamin B. http://www.groentenfruit.nl/index_product.asp?pg=2&cpg=15&p=102&r=9 2

Obviously, it would be interesting to identify some volatile compounds in the peel or the seed that could be of any use. Two different samples will be analyzed, namely fresh and dried sample, using headspace, headspace-solid-phase microextraction and hydrodistillation for the identification of essential oils, followed by gas chromatography-flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Essential oils, also know as ethereal or volatile oils, are very complex, usually goodsmelling, mixtures with low solubility in water and soluble in organic solvents. They are predominated by fenylpropanoids and terpenoids, but also other classes of compounds, like sulfur containing compounds, are frequently found. Plants rich of essential oils (0.01-10% dry weight) are found in about 30% of plant families, namely Apiaceae, Laminaceae, Lauraceae, Myrtacea and Rutaceae. The essential oils can be located in many different parts of the plants, like in special oil cells, elimination channels, glandular hairs or flowers. Insulation is usually performed by steam distillation or hydrodistillation, extraction with organic solvents or supercritical carbon dioxide. In this project we used hydrodistillation. Plants that contain essential oils are of great economic importance, for example for applications in the perfume industry, cosmetics, food industry and pharmaceutical industry. In alternative medicine aroma therapy, based on essential oils, is important, while in the pharmaceutical industry the oils are used to improve the smell and taste of the medicine. As mentioned above, essential oils are composed of a very complex mixture of hundreds (sometimes thousands) different substances. Many of the components of an essential oil are multiple derived from the hydrocarbon isoprene, the structure of which is shown in figure 1.2. CH 3 CH H 2 C 2 FIGURE 1.2 structure of isoprene 3

The components of an essential oil can be roughly classified into the following different types: terpens (monoterpens and sesquiterpens), oxygenated compounds, alcohols, phenols, aldehydes, ketons, esters, coumarines, lactons, ethers and oxides. Monoterpens contain ten carbon atoms (formed by two isoprene units) and at least one double bound. Some terpens are thought to exhibit different medicinal properties, like anti-inflammatory, antiseptic, antiviral, antibacterial and analgesic. Sesquiterpens normally contain fifteen carbon atoms and are considered to have very complex pharmacological activity. An example is shown in figure 1.3. CH 3 CH 3 CH 3 CH 2 H 3 C FIGURE 1.3 Farnesene The monoterpene alcohols, for example alpha Terpineol (fig. 1.4), have antiseptic, antiviral and antifungal properties and are claimed to have an energizing impact on the user. CH 3 C H 3 CH 3 OH FIGURE 1.4 α-terpineol Sesuiterpene alcohols are rarely found in essential oils but, like bisabolol (fig. 1.5), they are antiallergen, anti-inflammatory, and can also act as liver and glandular stimulants. 4

C H 3 OH H CH 3 C H 3 CH3 FIGURE 1.5. α-bisabolol Aldehydes have antifungal, anti-inflammatory, and disinfectant properties, together with some sedative effects. Ketons can be very toxic in general, e.g. thujone, the structure of which is shown in figure 1.6. However, at appropriate concentrations they have useful therapeutic properties. They promote the secretion of mucous and also induce cell and tissue regeneration. O H 3 C H 3 C CH 3 FIGURE 1.6 β-thujone The fruity and fragrant character of the oil is mostly due to the presence of esters. Some of those compounds are reported to have antifungal and antimicrobial activity. Others have been shown to act as sedatives and antispasmodics. Very few ethers are found in essential oils and those that do occur are phenolic in nature. The oxides in essential oils are largely responsible for the expectorant characteristics of such oils.(scott, 1995) 5

2. OBJECTIVES To the best of our knowledge, there have been no previous studies that investigated the volatile compounds in the peel and the seed of litchi chinesis and only a few that investigated the volatile compounds in the edible part. Hence, the goal of this work was to investigate the presence of useful volatiles in the parts that are considered waste, to fully exploit this fruit. In this study, sample preparation on all the different parts of the lychee will be executed using headspace, headspace-solid phase micro-extraction (HS-SPME) and hydrodistillation (HD). We used two kinds of sample, namely fresh and dried samples, as shown in the following pictures, Figure 2.2 and 2.3. The characterization of the volatile compounds in all these parts was performed using gas chromatography - flame ionization detector (GC-FID) and GC-mass spectrometry (GC-MS). FIGURE 2.1 fresh lychee sample FIGURE 2.2 dried lychee sample 6

3. METHODS AND MATERIALS 3.1 FRUIT MATERIAL Two different samples of lychee fruit were examined. At first fresh lychee, bought from a local supermarket in Matelica, Marche, Italy, during the month of December, were stripped into their different parts: peel, whole seed and edible part. The edible part and the peel were dried in the oven at a temperature of 35 C. The peel was kept in the oven (Bicasa, beta-oven, Italy) for one day and the edible part for three days. The seeds were dried at room temperature (+/- 25 C). After this process the different parts were kept in closed glass jars. Before submitting the dry parts to hydrodistillation and SPME, they were shredded (Janke & Kunkel IKA-WERK MFO nr: 162559, Germany) to a powder with a particle size of 1mm, using the shredder shown in figure 3.1. The second sample was purchased from a Chinese supermarket located in Civitanova, Marche, Italy, and was already dried as a whole. From now on, we will refer to this second sample using the term commercial sample. Also this sample was homogenized to a particle size of 1 mm. FIGURE 3.1 Shredder 7

3.2 WATER DETERMINATION For both, the fresh and the commercial sample, we determined the amount of water in all different parts as follows. We weighted a small amount of the different parts on glass containers and put them in the oven at 110 C for 24 hours. After 24 hours we weighed the samples and from this obtained weight we subtracted the weight of the containers to obtain the weight of the dried sample, witch in turn was subtracted from the weighed sample. To obtain the percentage of water in the sample we divided this result by the weight of the sample and multiplied this by 100. This was performed in duplicate and the average was taken as final result. 3.3 HYDRODISTILLATION Hydrodistillations (HD) were performed with two Clevenger-type apparatus of different sizes, a small and big one, pictured in fig 3.2, to extract the essential oils from the peel and the seeds of the Litchi chinesis using n-hexane (Carlo Erba reagents, Italy) as extraction solvent (1-3ml). The shredded fruit material (20-100g) was completely immersed in water (1-3 liter) and the temperature was raised to the boiling point. Since this technique is based on azeotropy, this point will be lower than the boiling point of pure water and pure compounds. From the moment the mixture goes into vapor phase they will act like pure compounds again. Once they arrive in the cooler the oil and water will condense and the oil will be separated from the water. After three hours of boiling the instrument was cooled off, and the water was eluted and the essential oil recovered as solution in the n-hexane. 8

FIGURE 3.2 small and big size Clevenger-type apparatus. A small amount of anhydrous sodium sulphate (J.T. Baker, Holland) was added to adsorb the remaining water in the organic phase. The next step was to filtrate the sample in order to separate the essential oil from the water adsorbed to the anhydrous sodium sulphate. After we obtained the filtrate, we washed the Erlenmeyer flask, used to collect the essential oil from the distillatory, with some n-hexane, to make sure no essential oil was left behind. Finally, the solvent was evaporated under a N 2 flow. For each part of the lychee we performed three hydrodistillations. Before using the obtained essential oil for GC-FID and GC-MS analyses, it was kept in sealed vials protected from the light at - 20 C. The oil yield was estimated on a dry weight basis. 9

3.4 SPME FIBER SCREENING Before starting the analysis with the extraction technique SPME, it is necessary to do a fiber screening in order to choose the fiber with the most appropriate affinity for the volatile compounds in Litchi chinesis. Three fibers with different coatings of different polarities were screened: polydimethylsiloxane (PDMS, 100 µm), polydimethylsiloxane-divinylbenzene (PDMS- DVB, 65 µm) and Stableflex divinylbenzene-carboxen-polydimethylsiloxane (DVB- CAR-PDMS, 50/30 µm), with PDMS-DVB being the most apolar, DVB-CAR-PDMS the most polar and PDMS being of intermediate polarity. The silica fibers and the manual SPME holder were purchased from Supelco (Bellefonte, PA, USA). The coating of all fibers was 1 cm long. 3.5 HS-SPME Solid phase microextraction (SPME) is a fast and solvent free sample preparation method in which coated fibers are used to isolate and concentrate analytes. After extraction, the fibers are transferred to an analytical instrument for separation and quantification of the target analytes (Pawliszyn, 1997). Headspace Solid Phase Microextraction (SPME) utilizes a short, thin, solid rod of fused silica, coated with an adsorbent polymer. The extraction technique consists of two processes: (1) the partition between fiber and sample, and (2) the desorption of the concentrated analytes from the coated fiber to the analytical instrument. (Pawliszyn, 1997) The sample, dry and fresh fruit materials (5 mg of ground peel, whole seed, internal part of the seed, external part of the seed and 100-150 mg of pulp and juice), was put in a 4 ml vial closed by a polypropylene hole cap and PTFE/silicone septum (Supelco, Bellefonte, PA, USA). To perform the extraction, we manually pierced the septum with the protective shear of the SPME fiber and subsequently lower the plunger in order to expose the fiber to the sample headspace, as shown in figure 3.4. 10

FIGURE 3.3 vial placed in water-oil bath at 60 C FIGURE 3.4 protective shear pierced true the septum of the vial and fiber exposed to the sample Besides headspace sampling, you can also use direct liquid sampling. In this case the fiber is immersed directly into the aqueous sample. The vial, containing the sample, was placed in an oil bath with an extraction temperature of 60 C and an extraction time of 30 minutes. This process is shown in figure 3.3. During this time there will arise an equilibrium between three phases: (1) the solid phase to the headspace, (2) the headspace to the fiber coating, and (3) the solid phase to the fiber coating. Extraction is complete when the analyte has reached equilibrium distribution between the matrix and the fiber coating. Equilibrium condition can be described as: n = K V (3.1) fs f C 0 11

where: n : amount of compounds extracted by the coating K fs : distribution coefficient between the fiber coating and the sample matrix V f : volume of the fiber coating C 0 : initial concentration of analyte in the sample After the absorption period, the fiber was withdrawn back into the protective sheath, then the sheath was pulled out of the sampling vial. Immediately after, the sheath was inserted in the GC injector and the plunger again lowered to expose the fiber, as shown in figure 3.5. Now the desorption can take place. For each part of the fruit, the process was performed in triplicate. No reconditioning was needed for each fiber before sampling. FIGURE 3.5 fiber inserted in the GC. 12

3.6 HEADSPACE GAS-CHROMATOGRAPHY A third method we used in terms of extraction was static headspace gas-chromatography (HD-GC). Analyses were performed using an Agilent 7694E head sampler (California, USA). This method consists of two steps. First, the sample, in our case solid, with a gas phase above it is put in a closed vial (60ml). Secondly, the vial is warmed up to a constant temperature until equilibrium is reached between the two phases. When equilibrium is reached an aliquot of the gas phase is carried away by a gas stream towards the column of the gas chromatographer, where the volatile compounds are analyzed (Kolb, Ettre, 1997). This method is based on the same principles as HS-SPME. The most important differences are: 1) In HS-GC there is only one equilibrium that takes place instead of three, namely between the solid sample and the gas phase above the sample. 2) The analytes are not captured by a fiber in HS-GC but they are directly transferred to the gas chromatographer by the transfer line between both instruments. In other words, no selection process related to the polarity takes place in HS-GC. This ensures that we will obtain a high number of compounds using the HS-GC, but they will be less concentrated than when obtained by HS-SPME. The temperature program to follow: 60 C for the vial, 70 C for the loop and 90 C for the transfer line. The vial equilibration time was set at 30 min, pressurization time was 0.20 min, loop fill time at 0.20 min, loop equilibration time at 0.05 min and injection time of 1 min. In combination with the headspace sampler we used GC equipped with an Innowax column (0.32 mm i.d., 0.25 µm film thickness). The inlet and detector temperature were set at 250 C. The temperature program was the following: 60 C for 3 min, then ramp at 10 C/min to 220 C, then 20 C/min up to 240 C, held for 20 min. 13

3.7 GC-FID CONDITIONS The volatile compounds were analyzed using an Agilent 4890D instrument, shown in fig 3.6, with a flame ionization detector (FID). The FID measures the difference in electrical conductivity between an H 2 /O 2 flame and carrier gas loaded with a component loaded carrier gas. The response is proportional to the amount of carbon compounds. Two types of injections were performed: direct injection of the essential oil obtained from the hydro distillations, using a splitless injector, and head space injection using a split injector. FIGURE 3.6 Agilent 4890D instrument with FID detector. The separation of the compounds was performed on a HP-5 capillary column (5% phenylmethylpolysiloxane, 25 m, 0.32 mm i.d.; 0.17 µm film thickness) (J & W Scientific, Folsom, CA), working with the following temperature programs: a) 5 min at 60 C, and subsequently at 4 C/min up to 220 C, then 11 C/min up to 280 C, held for 15 min for the direct injection of the essential oil and b) 3 min at 60 C, subsequently 10 C/min up to 220 C and finally 20 C/min up to 280 C held for 20 min; injection volume of 1 µl, split ratio, 1:34 ; injector and detector temperatures, 280 C for the direct injection and 250 C and 280 C, respectively, for SPME ; carrier gas, helium (1.4 ml/min). 14

In order to calculate the retention indices (as Kovats indices) of each compound extracted with HS-SPME or hydrodistillation, a mixture of aliphatic hydrocarbons (C 8 -C 30 ) (Sigma, Milan, Italy) in hexane was directly injected into the GC injector or loaded onto the SPME fiber and injected under the above temperature program. 3.8 GC-MS CONDITIONS GC-MS analysis was performed on a Agilent 6890N gas chromatograph, equipped with a HP-5MS capillary column (5% phenylmethylpolysiloxane, 30 m, 0.25 mm i.d., 0.1 µm film thickness), coupled to a 5973N mass spectrometer (MS) (J & W Scientific, Folsom). The temperature program for the direct injection was the following: 60 C for 5 min, then ramp at 4 C/min to 220 C, then 11 C/min up to 280 C held for 15 min, finally 11 C/min up to 300 C, held for 5 min; the program for head space injection: 60 C for 3 min, then ramp at 10 C/min up to 220 C, directly after another ramp at 20 C/min up to 280 C, held for 20 min; carrier gas: helium; flow rate: 1.0 ml/min; injector and transfer line temperatures for direct injection and headspace injection: 280 C-250 C; injection volume: 2 µl; split ratio: 1:50; scan time: 75 min; acquisition mass range: 29-400 m/z. The direct injection was performed in split, while the headspace injection in splitless mode. The first step in an MS analysis is the formation of ions in an ionization process. In our study the ionization and fragmentation was executed in electron impact (EI) mode with an ionization voltage of 70 ev. After the formation of the molecular ion and the fragmentation of the molecule to fragment ions, the ions are separated according to mass/charge (also referred to as m/z). The final steps are the detection of the ions using an electron multiplier and the registration of the relative quantity of each type of ion present. Since the nature and number of formed ions is a characteristic of the composition and structure of the organic molecule, one can argue that the mass spectrum is its fingerprint. This is the reason why mass spectrometry is a very useful technique for structural clarification and confirmation. 15

3.9 IDENTIFICATION AND QUANTIFICATION OF VOLATILE COMPOUNDS. The identification of the volatile compounds was performed through retention indices calculated with the Kovats method using equation (3.2) for compounds extracted with HS-SPME as for the compounds present in the essential oil obtained from the hydro distillation. The Kovats retention index is a logarithmic scale on which the adjusted retention time of a peak is compared with those of linear n-alkanes as reference compounds. http://www.chromtech.net.au/kovats_ri.cfm True the Kovats indices the retention values become independent from the GC conditions (Adams, 1989). I log t' r ( x) log t' r ( n) = 100 n + ( N n) (3.2) log t' r ( N ) log t' r ( n) where : I : Kovats index n : number of carbon atoms in the smaller alkane N : number of carbon atoms in the larger alkane t r (n) : the adjusted retention time of the smaller alkane t r (N) : the adjusted retention time of the larger alkane t r (x) : the adjusted retention time of the unknown compound After the calculation of the indices the chromatogram obtained with GC-FID was compared with the chromatogram obtained with GC-MS since it is possible that some compounds only produce a signal in either one detector. Thereafter, for each peak present in the mass chromatogram, we examined the mass spectra using specific computer software. These mass spectra and calculated retention indices (RI) were compared with other spectra found in commercially available libraries, such as NIST05, WILEY275 and ADAMS. These are non-specialized collections of spectra, mainly taken from the literature. Adams library is dedicated to the essential oil field using an ion trap mass analyzer (Scott, 1995). 16

For the quantification, we calculated the relative amount, expressed as percentages, of each volatile compound, using FID peak area normalization. Usually the response factor is calculated using only one concentration of each compound using following equation: RF C analyte = CIS (3.3) Aanalyte A IS where C analyte is the concentration of the different analytes, A analyte its absolute peak area, A is is the absolute peak area of one alkane and C is its concentration. We on the other hand calculated the response factor (RF) from the FID analyses of eight different classes of volatiles, as shown in table 3.2. These eight different classes correspond to the different classes of volatiles identified. Owing to the large number of compounds present and the non-availability of commercial standards of many of them, compounds of each of the eight classes were assumed to have the same quantitative GC correction factor. For each class, more than one standard was considered in order to improve the reliability. Five replicates of mixtures, each prepared with four different concentrations (0.04-0.08-0.16-0.4 mg/ml) with equal amounts of two internal standards (octane and octadecane) and representative compounds were prepared; taking into account the purity determined for each of them at GC-FID. Compounds used as references were purchased from Sigma-Aldrich (Milan, Italy). We calculated the slopes for each compound using regression lines, as shown below in figure 3.7 for E-β- Caryphyllene and α-humulene, belonging to the class of sesquiterpens hydrocarbons. In order to obtain the slopes we calculated the ratio s shown in table 3.1. For the compounds that could not be divided into any of these classes, we considered the correction factor of octane until the compounds that contain thirteen carbons, and in case they contain more carbons we used the correction factor of octadecane. 17

TABLE 3.1 Report from excel, showing the data needed for the calculation of the slopes; I S: internal standard, C:compound, Conc: concentration. E β Caryophyllene 1e conc. 2e conc. 3e conc. 4e conc. Slope I S Area/ C Area 3.993 2.129 1.108 0.495 I S Conc/ C Conc 4.161 2.080 1.040 0.416 1.0722 α Humulene Slope I S Area/ C Area 11.095 5.218 2.592 1.116 I S Conc/ C Conc 10.084 5.042 2.521 1.008 1.105 Media St Dev CV% 1.088 0.0231 2.13 FIGURE 3.7 Regression lines of E-β-Caryophyllene and α-humulene. 18

TABLE 3.2. Determinations of response factor (RF) for different classes of volatiles occurring in Lychee Grouped compounds Mean ± SD RF Terpenoids Monoterpenes hydrocarbons β-pinene 1.04±0.01 limonene 1.15±0.01 γ-terpinene 1.04±0.01 p-cymene 1.14±0.01 1.09±0.06 Oxygenated monoterpenes 1,8-cineole 1.29±0.01 linalool 1.33±0.01 camphor 1.32±0.01 terpinen-4-ol 1.29±0.01 verbenone 1.42±0.01 nerol 1.53±0.01 carvone 1.35±0.01 1.36±0.09 Sesquiterpenes hydrocarbons (E)-caryophyllene 1.07±0.01 α-humulene 1.08±0.01 1.10±0.02 Oxygenated sesquiterpenes caryophyllene oxide 1.20±0.01 1.20±0.00 Aliphatics Alkanes octane octadecane 0.91±0.01 1.35±0.01 1.13±0.01 1-octen-3-ol 1.30±0.01 dodecanol 1.45±0.01 1.38±0.11 Esters isobornyl acetate 1.39±0.01 isobornyl isovalerate 1.38±0.01 1.39±0.01 19

Aldehydes octanal 1.50±0.01 dodecanal 1.47±0.01 1.49±0.03 Aromatics benzaldehyde 1.28±0.01 p-vinylanisole 1.36±0.01 1.32±0.05 20

4. RESULTS AND DISCUSSION In this study, three methods were considered for the extraction of the volatiles compounds from lychee chinesis: headspace, HS-SPME and HD. In particular, SPME was performed on all different parts of the lychee fruit, while HD only on the whole seed and the peel. 4.1 HEADSPACE-GAS CHROMATOGRAPHY We started the analysis using this technique for the extraction of the volatile compounds, but, as shown in figure 4.1, this technique gives us good results for the peel but not for the seed. As for the peel, we started with hundred milligrams for the seed, but we only observed relatively small peaks for selected compounds. Increasing the amount to five hundred milligrams, did not really improve the result. Based on those facts, we moved forward using HS-SPME. FIGURE 4.1 HS-GC chromatograms; from top to bottom: peel and seed. 21

4.2 HS-SPME ANALYSIS After a comparison of the three previous mentioned fibers, namely PDMS (100 µm), PDMS-DVB (65 µm) and DVB-CAR-PDMS ( 50/30 µm), we found that the fiber coated with PDMS had the greatest affinity for the five most abundant volatile compounds present in the different parts of the lychee. This is illustrated in the histogram shown in figure 4.2, were we can see that, with the fiber of intermediate polarity (PDMS coating), we obtained the largest peak areas. The extraction with SPME was continued using only the fiber with PDMS coating. 2500000 Peak Area (pa 2000000 1500000 1000000 500000 E-Caryophyllene ar-curcumene β-selinene α-zingiberene β-sesquiphellandrene Total Area 0 PDMS DVB-PDMS DVB-CAR- PDMS FIGURE 4.2. Comparison of three fibers of different polarity. In table 4.1. all compounds identified using HS-SPME-GC-FID are shown, together with their retention indices, percentage composition and the method by which they were identified. In total, one hundred and twelve different volatile components were identified. Sesquiterpen hydrocarbons were the main class of volatile compounds in all different parts of the lychee, as reported in figure 4.3, showing a percentage ranging from 74.77% in the pulp to 99.01% in the internal part of the seed. 22

In all different parts E-β-Caryophyllene (3.40-13.73%), ar-curcumene (6.18-36.68%), β-selinene (7.83-31.73%), α-zingiberene (13.08-45.87%) and β-sesquiphellandrene (2.92-5.63%) are the five major volatiles. The abundance of these five volatile compounds in the different parts of the lychee, as illustrated in figures 4.3-4.5 is very different, except for the juice and the pulp, where β-selinene is the most abundant. In the whole seed and the internal part of the seed α-zingiberene is the major compound, whereas in the cuticula and peel ar-curcumene is predominant. % 50,00 45,00 40,00 35,00 30,00 25,00 20,00 15,00 10,00 5,00 0,00 E-Caryophyllene ar-curcumene β-selinene α-zingiberene β-sesquiphellandrene pulp juice peel cuticula internal part whole seed FIGURE 4.3 The five major volatiles in the different parts of the lychee in terms of percentage. 23

14000000 12000000 Peak area (pa* 10000000 8000000 6000000 4000000 2000000 0 whole Seed internal part cuticula Peel juice pulp E-Caryophyllene ar-curcumene β-selinene α-zingiberene β-sesquiphellandrene Total area FIGURE 4.4 The five major volatiles in the different parts of the lychee in terms of absolute peak area. (SPME) Peak area (pa* 16000 14000 12000 10000 8000 6000 4000 2000 0 juice pulp E-Caryophyllene ar-curcumene β-selinene α-zingiberene β-sesquiphellandrene Total area FIGURE 4.5 The five major volatiles in the juice and the pulp of the lychee in terms of absolute peak area. (SPME) 24

25 Table 4.1 Chemical composition as percentage of volatile compounds obtained with HS-SPME of the different parts of the lychee fruit. Alcohols Compound name RI RI Adams RI Nist Whole Seed Internal seed Cuticula Peel Juice Pulp % CV% % CV% % CV% % CV% % CV% % CV% Identification Methods 1-octenol 1065 1066 - - - - tr tr MS,RI,Std p- Cymen-8-ol 1182 1182 - - - - tr tr MS,RI,Std α -Terpineol 1187 1188 - - - - tr tr MS,RI,Std Myrtenol 1199 1195 - - - - tr - MS,RI Dihydro carveol <iso> 1212 1214 tr tr - - - - MS,RI,Std Ethanol - - - - 6.35 7.26 4.06 18.26 MS,RI,Std Total % of alcohols tr tr tr tr 6.35 4.06 Aldehydes/ Ketons 5-Hepten-2-one-6-methyl 983 985 - - - tr - - MS,RI 2-Nonanone 1091 1090 - - - - tr tr MS,RI n-nonanal 1100 1100 tr tr tr tr tr tr MS,RI cis-pinocamphone 1177 1175 - - - - tr tr MS,RI n-decanal 1210 1201 0.02 19.20 tr tr tr - - MS,RI n-undecanal 1300 1306 0.02 tr - - - - MS,RI Di-tert-Butylquinone 1466 1462 - - - - 0.33 18.08 tr MS,RI Total % of aldehydes/ketons 0.03 tr tr tr 0.33 tr Alkanes and others Nonanoic acid 1267 1270 tr - - - - - MS,RI Heptadecane 1701 1700 - - 0.06 6.04 - - - MS,RI,St Total % of Alkanes and others tr tr 0.06 tr tr tr

26 Aromatics Compound name RI RI Adams RI Nist Whole Seed Internal Seed Cuticula Peel Juice Pulp % CV% % CV% % CV% % CV% % CV% % CV% Identification Methods Benzen acetaldehyde 1039 1042 - - - tr - tr MS,RI Phenyl ethyl alcohol 1108 1107 tr tr - - tr tr MS,RI p-methyl-acetophenone 1183 1182 - - - tr - - MS,RI Benzothiazole 1223 1225 - - - - tr tr MS,RI Total % of Aromatics tr tr tr tr tr tr Esters cis-chrysanthenyl acetate 1277 1265 - - - - tr - MS,RI Linoleic acid ethylester 2169 2162 - - - - - 0.13 18.00 MS,RI Ethyl Oleate 2173 2171 - - - - - 0.18 16.24 MS,RI Total % of Esters tr tr tr tr tr 0.32 Monoterpen Hydrocarbons Limonene 1033 1029 tr tr - - tr tr MS,RI,Std Total % monoterpens h. tr tr tr tr tr tr Oxygenated Monoterpens Linalol 1096 1096 tr tr - - tr tr MS,RI,Std cis-linalol oxide (furanoid) 1071 1072 - - - - tr tr MS,RI cis-rose oxide 1107 1108 - - - - tr tr MS,RI trans-pinocarveol 1139 1139 - - - - tr - MS,RI Terpinen-4-ol 1174 1177-0.02 3.95 - - tr 0.07 22.12 MS,RI,Std trans-dihydro carvone 1201 1200 - - - - tr - MS,RI,Std n-decanal 1202 1200 - - - - - 0.10 5.18 MS,RI

27 Compound name RI RI Adams RI Nist Whole Seed Internal seed Cuticula Peel Juice Pulp % CV% % CV% % CV% % CV% % CV% % CV% Identification Methods Verbenone 1203 1205 - - - - tr tr MS,RI trans-carveol 1218 1216 - - - - tr - MS,RI Citronellol 1230 1225 - - - tr 5.03 1.34 11.20 1.13 5.67 MS,RI Geraniol 1258 1252 - - - - - 1.31 7.31 MS,RI,Std Geranial 1265 1264 - - - - 1.59 13.55 0.15 19.29 MS,RI Z Citral 1277 1271 - - - - - tr MS,RI Total % o. monoterpens tr 0.02 tr tr 2.94 2.77 Sesquiterpens Hydrocarbons Bicycloelemene 1325 1333 0.10 17.59 0.10 11.13 - - - - MS,RI δ-elemene 1336 1338 0.88 8.04 0.58 4.38 0.11 13.85 0.02 7.70 - - MS,RI α Cubebene 1348 1348 0.31 12.12 0.14 8.66 - - - - MS,RI,Std α Longipinene 1349 1352 - - 0.18 13.27 0.16 11.07 - - MS,RI,Std (-)Isoledene 1364 1376 1373 0.35 15.37 0.18 9.26 - - - - MS,RI Cyclosativene 1364 1371 - - 0.15 9.70 0.22 12.26 - - MS,RI α-ylangene 1370 1375 0.18 18.07 0.15 9.93 0.13 10.17 0.14 11.60 - - MS,RI α Copaene 1374 1376 1.39 15.47 1.62 9.26 0.57 10.18 0.34 11.91 - - MS,RI,Std β Cubebene 1383 1388 0.04 2.48 - - - - - MS,RI 7-epi-Sesquithujene 1386 1391 1.28 6.95 2.33 9.49 1.67 11.14 1.72 15.07 0.33 7.71 0.43 14.12 MS,RI Sibirene 1399 1400 0.43 12.12 0.13 8.91 0.46 8.66 0.46 13.31 0.16 14.81 0.23 19.67 MS,RI,Std Longifolene 1405 1407 - - - 0.06 2.52 - - MS,RI Italicene 1407 1405 - - 0.15 20.74 - - - MS,RI α Gurjunene 1408 1409 0.34 3.85 0.19 12.58 - - - - MS,RI,Std Acora-3,7(14)-diene 1412 1408 - - 0.28 9.17 - - - MS,RI α Cedrene 1413 1411 0.19 12.04 0.10 10.70-0.29 8.21 - - MS,RI,Std E-Caryophyllene 1418 1419 13.02 11.57 13.73 6.94 5.04 10.71 4.30 10.95 3.40 5.62 3.90 13.37 MS,RI,Std β Copaene 1428 1432 0.88 18.34 1.00 7.47 0.59 14.24 0.45 8.95 0.31 11.71 0.40 15.11 MS,RI

Compound name RI RI Adams RI Nist Whole Seed Internal seed Cuticula Peel Juice Pulp % CV% % CV% % CV% % CV% % CV% % CV% Identification Methods γ-elemene 1432 1436 - - - - 0.26 0.19 16.10 MS,RI trans-α-bergamotene 1432 1434 0.90 9.06 1.59 6.39 0.97 10.68 0.89 12.68 0.29 4.54 0.20 19.30 MS,RI Aromadendrene 1441 1441 0.28 6.76 0.46 9.00 0.23 8.71 - - - MS,RI,Std α-himachalene 1449 1451 0.66 6.76 0.40 9.28 0.51 10.89 0.46 4.10 0.90 6.14 0.92 16.89 MS,RI α-humulene 1454 1454 3.31 9.28 3.11 7.47 2.34 10.45 1.80 19.17 2.15 5.71 2.45 14.81 MS,RI,Std β-farnesene 1454 1456 - - - - tr tr MS,RI Sesquisabinene 1460 1459 - - 0.87 10.02 - - - MS,RI allo Aromadendrene 1461 1460 1.51 10.32 1.16 8.78 0.35 9.96 1.12 15.55 - - MS,RI,Std α-acoradiene 1467 1466 0.20 16.37 0.12 8.56 - - - - MS,RI β Acoradiene 10-epi 1475 1475 - - 1.19 6.52-0.17 1.19 13.14 MS,RI γ Gurjunene 1475 1477 - - - tr 1.06 14.41 - MS,RI β-acoradiene 1476 1470 1.05 11.67 1.36 7.94 - - - - MS,RI γ Muurolene 1477 1479 - - 0.47 18.46 - - - MS,RI Ar Curcumene 1481 1480 17.49 11.16 11.89 7.87 29.16 10.66 36.68 10.56 6.18 5.19 8.06 12.71 MS,RI β-selinene 1488 1490 22.63 5.80 4.73 4.60 26.71 8.82 7.83 14.32 27.53 5.97 31.73 15.36 MS,RI α-zingiberene 1492 1493 23.83 7.08 45.87 6.38 17.08 12.07 33.41 11.71 13.08 6.54 17.34 23.55 MS,RI β-himachalene 1501 1500 - - - - - 2.11 13.38 MS,RI α-muurolene 1502 1500 1.41 19.16 0.60 8.65 1.18 9.89-2.30 2.93 - MS,RI β-bisabolene 1506 1505 0.89 8.06 0.60 5.57 1.60 10.77 1.09 3.38 0.92 3.20 1.10 15.70 MS,RI β-curcumene 1510 1515 0.39 13.43 0.44 11.54 - - - MS,RI α-muurolene 1511 1500 - - - 0.96 3.08 - - MS,RI γ Cadinene 1514 1513 - - 0.40 10.25 - - - MS,RI Sesquicineole 1515 1516 0.45 4.96 tr - - - - MS,RI δ Cadinene 1516 1523-0.41 5.95 - - - - MS,RI (E,Z)-α-Farnesene 1518 1498 - - 0.63 8.49 - - MS,RI β-sesquiphellandrene 1522 1522 3.88 9.34 5.63 6.43 5.07 10.58 5.02 11.93 2.92 3.05 3.39 17.73 MS,RI γ (E)-Bisabolene 1530 1531 0.18 8.33 0.11 9.64 0.13 14.90 0.15 8.37 0.26 19.49 0.24 18.28 MS,RI γ-cuprenene 1537 1533 - - - tr - - MS,RI 28

Compound name RI RI Adams RI Nist Whole Seed Internal seed Cuticula Peel Juice Pulp % CV% % CV% % CV% % CV% % CV% % CV% Identification Methods γ-vetivenene 1540 1533-0.14 7.13 - - - MS,RI Z-α-Bisabolene 1540 1507 1555 0.14 10.08 0.08 13.34 0.05 8.23 0.14 11.63 - - MS,RI α-calacorene 1545 1545 0.05 8.04 0.04 5.71 tr 0.05 8.09 0.36 3.70 0.30 21.76 MS,RI Germacrene B 1559 1561 0.22 3.86 0.15 10.00 0.16 10.69 0.16 7.48 tr 0.36 15.63 MS,RI β-calacorene 1564 1565 0.04 16.32 0.03 2.76-0.03 9.09 0.16 7.47 0.23 17.92 MS,RI Total % sesquiterpens h, 98.89 99.01 97.95 98.54 62.75 74.77 Oxygenated Sesquiterpens MS,RI Sesquisabinene 1454 1459 - tr - - - - - MS,RI (+)Spathulenol 1579 1578 0.04 10.18 0.17 7.20 0.27 5.04 - - - MS,RI ar Turmerol 1579 1583 - - - 0.02 19.15 0.41 5.89 0.20 14.30 MS,RI Caryophyllene oxide 1583 1583 0.02 7.42 0.07 12.61 0.06 3.26 0.02 6.18 0.30 0.30 18.96 MS,RI,St Guaiol 1602 1600 - - - - 0.56 7.41 0.35 20.66 MS,RI Humulene epoxide II 1609 1608 0.01 11.81 0.05 5.01 - - - - MS,RI 1,10-di-epi-Cubenol 1611 1619 - - - 0.03 18.62 - - MS,RI β Acorenol 1630 1637 0.01 0.95 0.03 7.92-0.04 18.07 - - MS,RI 1-epi-Cubenol 1630 1628 - - - - 1.15 7.60 0.78 23.05 MS,RI Isospatulenol 1640 1638 0.01 17.07 0.07 12.49 - - - - MS,RI Himachalol 1652 1653 0.01 22.49-0.07 4.89 0.17 12.07 6.76 3.40 4.17 14.02 MS,RI (neo)intermedeol 1662 1660 0.02 15.23 0.14 13.19 - - - - MS,RI α-bisabolol 1685 1685 0.02 15.07 - - 0.06 18.40 3.31 19.71 2.08 18.63 MS,RI 6S,7R-Bisabolone 1753 1749 - - - 0.02 19.84 0.98 4.82 0.68 19.16 MS,RI Xhanthorrizol 1758 1753 tr - - 0.04 19.33 0.54 9.30 0.29 11.74 MS,RI Total % o. sesquiterpens 0.15 0.53 0.40 0.40 14.01 8.84 Total identified (%) 99.07 99.56 98.41 98.95 86.36 90.76 29

30 a Values are means of three determinations. They were obtained at FID by peak area normalization calculating the relative response factor (see Table 3.1). b Compounds belonging to each class are listed in order of their elution from a HP-5 column. c RI, retention indices as determined on HP-5 column using homologous series of C8-C30 alkanes. d Identification methods: MS, by comparison of the mass spectrum with those of the computer mass libraries Wiley, NIST 05 and ADAMS; RI, by comparison of RI with those reported from Adams (2007) and NIST 05 (2005); std, by comparison of the retention time, mass spectrum and retention index of authentic standard. e tr, traces (mean value below 0.01%).

The highest contribution of identified volatiles, in terms of the sum of SPME-GC-FID peak areas, was given by the peel, as reported in figure 4.6, that proved the richest in sesquiterpenes hydrocarbons (98.89%) (figure 4.7). Parts Lychee Peak area (pa* 10000000 9000000 8000000 7000000 6000000 5000000 4000000 3000000 2000000 1000000 0 whole seed internal part cuticula peel juice pulp FIGURE 4.6 Comparison of identified volatiles in parts of lychee in terms of peak areas. Among all parts of the investigated fruit, the juice and the pulp (edible part) were the richest in oxygenated monoterpens, oxygenated sesquiterpens and alcohols, with respectively Citronellol ( 1.34-1.13%), Himachalol ( 6.76-4.17%) and Ethanol (6.35-4.06%) being the major components; on the other hand, they do not contain any alkane (figure 4.7). Esters were only found in the pulp in very small amounts; Linoleic acid ethyl ester (0.13%) and Ethyl oleate (0.18%) were the most abundant. 31

% 100,00 80,00 60,00 40,00 20,00 0,00 whole seed internal part cuticula peel juice pulp Alcohols Aldehydes Alkanes Aromatics Esters M.H. M.O. S.H. S.O. FIGURE 4.7 Classes of compounds found in all different parts of the lychee; M.H.:monoterpen hydrocarbons; M.O.: oxygenated monoterpens; S.H.: sesquiterpens hydrocarbons; S.O.: oxygenated sesquiterpens Noteworthy is the fact that in some cases we identified compounds in the internal part of the seed or in the cuticula that were not found in the whole seed. This was the case for Heptadecane (0.06%), α-longipinene (0.18%), Cyclosativene (0.15%), Italicene (0.15%), Acora-3,7(14)-diene (0.28%), Sesquisabinene (0.87%), β-acoradiene 10- epi (1.19%), γ-muurolene (0.47%) and γ-cadinene (0.40%) that were found in the cuticula; Terpinen-4-ol (0.02%) and δ-cadinene (0.41%) were instead found in the internal part of the seed. The reverse also occurred, namely that we identified compounds in the whole seed, i.e. Nonanoic acid (trace), β-cubebene (0.04 %) and α-bisabolol (0.02%), that did not appear, neither in the internal part of the seed nor in the cuticula. The cause of this phenomenon can be, in some cases, small contaminations caused by the contact between the seed and the peel or fruit. 32

4.3 ESSENTIAL OIL ANALYSIS. The mean percentages of essential oil obtained with hydrodistillation and calculated on a dry weight basis of the peel and the whole seed of the lychee, together with the percentage of water calculated in all different parts, are shown in table 4.2. TABLE 4.2. percentage of essential oil (HD) and percentage of water. Part % essential oil % water fresh peel 0.34 26.03 cuticula 22.32 internal part 18.30 whole seed 0.04 19.50 pulp 79.07 juice 80.95 commercial peel 0.54 13.07 seed 0.04 9.23 The percentage composition of the components identified in the essential oil, obtained from the whole seed and the peel using GC-MS, is shown together with their retention indices, in table 4.3. The percentage composition of each compound is calculated from nine peak areas, which are the result of three hydrodistillations of both the whole seed and the peel, whereof each obtained essential oil was analyzed in triplicate. 33

TABLE 4.3 Chemical composition as percentage a of the essential oil obtained by HD from whole seed and peel of the fruit of Lychee Chinesis. Seed Peel Compound name b RI c FID RI Adams RI Nist % CV% % CV% Identification Methods d Alkanes Octan 800 MS,RI,Std Tetradecane 1401 1400 0.22 19.13 0.11 11.07 MS,RI Pentadecane 1496 1500 0.91 13.02 - MS,RI Hexadecane 1601 1600 0.10 17.24 0.08 17.62 MS,RI Heptadecane 1699 1700 tr e 0.05 19.28 MS,RI Octadecan 1800 MS,RI,Std Total % Alkanes 1.23 0.25 Esters Ethyl hexadecanoate 1992 1993 2.75 19.55 - MS,RI Total % Esters 2.75 tr MS,RI Sesquiterpens Hydrocarbons δ Elemene 1333 1338 0.27 22.34 - MS,RI α Longipinene 1343 1352 0.11 18.57 0.10 17.45 MS,RI,Std α Cubebene 1346 1348 0.09 21.20 - MS,RI,Std Cyclosativene 1358 1371 0.25 19.61 0.13 14.16 MS,RI Ylangene 1366 1375 0.11 21.61 0.09 17.31 MS,RI α Copaene 1370 1376 0.90 19.32 0.22 17.23 MS,RI,Std β-cubebene 1384 1388 tr - MS,RI 7-epi-Sesquithujene 1387 1391 1.51 13.93 1.30 14.68 MS,RI Sibirene 1394 1400 1.46 19.83 0.33 12.91 MS,RI Acora-3,7(14)-diene 1404 1408 tr tr MS,RI E-Caryophyllene 1411 1419 10.35 18.21 3.58 10.94 MS,RI,Std β-duprezianene 1416 1422 0.19 16.35 - MS,RI β-cedrene 1422 1420 0.56 16.09 - MS,RI β-copaene 1422 1432-0.30 10.39 MS,RI γ Elemene 1430 1436 0.15 8.18 tr MS,RI trans-α-bergamotene 1432 1434 0.53 19.31 - MS,RI β Gurjunene 1432 1433-0.59 10.85 MS,RI 6,9-Guaiadiene 1441 1444 tr - MS,RI α-himachalene 1441 1451 0.75 19.91 1.04 10.60 MS,RI α-humulene 1447 1454 2.26 18.62 0.94 9.64 MS,RI,Std (-)-allo-aromadendrene 1453 1460 0.99 18.29 0.39 9.20 MS,RI,Std Amorpha-4,11-diene 1457 1451-1.29 9.04 MS,RI Cadina-1(6),-diene<cis> 1457 1463 1.20 15.54 - MS,RI trans-β-farnesene 1461 1456 0.12 15.72 0.11 8.30 MS,RI 34

Seed Peel Compound name b RI c RI RI Identification % CV% % CV% FID Adams Nist Methods d β-acoradiene 1468 1470 0.69 22.85 0.63 8.45 MS,RI γ-himachalene 1471 1482 0.89 17.40 1.20 9.75 MS,RI β Selinene 1478 1490 12.21 7.74 16.23 8.59 MS,RI Ar-Curcumene 1481 1480 13.32 17.30 16.93 9.41 MS,RI α-zingiberene 1494 1493 34.15 11.13 38.67 10.36 MS,RI β-bisabolene 1505 1505 1.04 18.43 1.11 13.01 MS,RI (Z)-γ-Bisabolene 1512 1515 0.36 14.16 - MS,RI δ-cadinene 1520 1523 tr - MS,RI β-sesquiphellandrene 1520 1522 5.05 11.38 5.94 8.09 MS,RI (E)-γ-Bisabolene 1529 1531 0.18 17.40 0.15 13.93 MS,RI α-calacorene 1539 1545 0.07 10.01 0.07 16.77 MS,RI Germacrene B 1551 1561 0.25 18.90 0.05 12.67 MS,RI β-calacorene 1561 1565 0.12 21.36 - MS,RI Total % Sesquiterpens H. 90.13 91.39 Oxygenated Sesquiterpens Spathulenol 1572 1578 0.68 20.21 - MS,RI Caryophyllene Oxide 1576 1583-0.05 22.55 MS,RI,Std Globulol 1578 1590 0.84 19.54 - MS,RI Ar-Turmerol 1579 1583-0.14 18.04 MS,RI Viridiflorol 1585 1592 0.28 19.99 - MS,RI Cubeban-11-ol 1588 1595 0.15 8.30 - MS,RI Rosifoliol 1595 1600 0.16 17.61 0.19 21.72 MS,RI Agarospirol 1620 1648 1631-1.43 20.65 MS,RI 1,10-di-epi-Cubenol 1621 1619 0.41 14.70 - MS,RI α-acorenol 1632 1633-0.08 21.34 MS,RI β-acorenol 1638 1637 0.23 19.56 1.95 8.47 MS,RI α-cadinol 1650 1654 0.55 16.70 - MS,RI Allohimachalol 1656 1662 0.09 17.78 0.55 17.88 MS,RI epi-β-bisabolol 1669 1671 0.32 17.06 - MS,RI β-bisabolol 1669 1675-0.47 17.54 MS,RI epi-α-bisabolol 1679 1683 0.61 10.57 0.09 5.92 MS,RI α-bisabolol 1681 1685-0.88 18.95 MS,RI Curcuphenol 1719 1718-0.06 13.19 MS,RI 2E,6Z-Farnesal 1734 1741-0.45 13.17 MS,RI (6S, 7R) Bisabolone 1745 1749 0.12 16.07 0.23 17.41 MS,RI Xanthorrhizol 1754 1753 0.88 18.76 0.96 16.93 MS,RI Total % O. Sesquiterpens 5.33 7.54 Total identified (%) 99.44 99.17 Total components 63 52 43 35

a Values are means of three determinations. b Compounds belonging to each class are listed in order of their elution from a HP-5 column. c RI, retention indices as determined on HP-5 column using homologous series of C8-C30 alkanes. d Identification methods: MS, by comparison of the mass spectrum with those of the computer mass libraries Wiley, NIST 05 and ADAMS; RI, by comparison of RI with those reported from Adams (2007) and NIST 05 (2005); std, by comparison of the retention time, mass spectrum and retention index of authentic standard e tr, traces (mean value below 0.01%). We identified a total of sixty-three compounds, whereof fifty in the whole seed and fortytwo in the peel, representing 99.17-99.44% of the oils under study (table 4.3). The amount of compounds identified in the whole seed and peel are comparable with the amounts found with SPME, where we found fifty-one different compounds in the whole seed and forty-five in the peel. The sesquiterpene hydrocarbons are in the essential oil the major fraction of identified compounds, with 90.13-91.39% of the total percentage identified. The major components were E-β-Caryophyllene (3.58-10.35%), β-selinene (12.21-16.23%), ar-curcumene (13.32-16.93%), α-zingiberene (34.15-38.67%) and β-sesquiphellandrene (5.05-5.94%). Figure 4.8 is an illustration of the sequence of the major volatiles in the chromatograms obtained with GC-FID for HS-SPME and essential oil with HP-5column and essential oil with innowax column. 36

A B C FIGURE 4.8 A.Essential oil (HP-5), B.HS-SPME (HP-5), C.Essential oil (innowax); 1.E-β- Caryophyllene,2. β-selinene, 3.ar-Curcumene, 4.α-Zingiberene, 5. β-sesquiphellandrene. 37

The structure of the five most abundant volatiles, together with the mass spectra obtained by the identification and the mass spectra found in Wiley or at http://www.pherobase.com. for the corresponding compounds are reported in figure 4.10. The peel is the richest in every major component, except for E-β-Caryophyllene, as illustrated in figure 4.9. We can also see that, in both the peel and the seed, α- Zingiberene, that is the major component of commercially available oil derived from rhizomes of the ginger plant Zingiber officinale Roscoe, widely used in cosmetics and fragrances (Millar, 1998), is by far the most abundant component. On the other hand, α- Humulene, identified with both SPME and HD and not belonging to the category of the most abundant components, has proven cytotoxic effect. (Legault et al., 2003) % 45,00 40,00 35,00 30,00 25,00 20,00 15,00 10,00 5,00 0,00 E-Caryophyllene ar-curcumene seed peel FIGURE 4.9 The five most abundant components found in whole seed and peel. 38

ar-curcumene CH 3 CH 3 H 3 C CH 3 (A1) Abundance m/z--> Abundance Average of 12.181 to 12.189 min.: 170309-MF-3.D (-) 119 9000 132 8000 7000 6000 105 5000 4000 91 202 3000 145 41 2000 55 77 161 1000 189 29 66 175 281 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 (A2) (A3) m/z--> #87321: AR-CURCUMENE 119132 9000 8000 7000 105 6000 5000 4000 91 3000 145 202 69 2000 1000 159 56 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 α-zingiberene C H 3 H H CH 3 H 3 C CH 3 (B1) Abundance 9000 8000 7000 6000 Average of 12.337 to 12.363 min.: 170309-MF-3.D (-) 119 93 (B2) 5000 4000 3000 2000 m/z--> Abundance 41 77 204 56 1000 161 133 147 175 189 355 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 #89151: Zingiberene $$ 1,3-Cyclohexadiene, 5-(1,5-dimethyl... 93 9000 8000 7000 41 119 (B3) 6000 5000 4000 69 3000 2000 55 m/z--> 1000 204 133147161 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 39

E-β-Caryophyllene (C1) CH CH 3 H 3 H 3 C H CH 2 (C2) Abundance Average of 11.382 to 11.386 min.: 170309-MF-3.D (-) 93 133 9000 8000 79 7000 41 6000 105 5000 120 161 4000 67 147 3000 55 189 2000 175 1000 29 204 221 267 0 20 40 60 80 100 120 140 160 180 200 220 240 260 m/z--> Abundance m/z--> #89246: trans-caryophyllene $$ Bicyclo[7.2.0]undec-4-ene,... 93 69 9000 133 8000 41 7000 6000 5000 105 81 120 4000 55 148161 3000 2000 189 27 204 175 1000 0 20 40 60 80 100 120 140 160 180 200 220 240 260 (C3) β-sesquiphellandrene CH 3 CH3 (D1) CH 2 H 3 C CH 3 Abundance 9000 Average of 12.680 to 12.697 min.: 170309-MF-3.D (-) 69 (D2) 8000 7000 93 161 6000 5000 41 4000 3000 2000 55 77 133 204 120 105 1000 147 189 29 85 175 0 20 30 40 50 60 70 80 90 100110120130140150160170180190200210 m/z--> Abundance 9000 #89700:.BETA.-SESQUIPHELLANDRENE 69 8000 7000 6000 m/z--> 5000 93 4000 77 3000 57 161 2000 120 133 109 1000 204 147 0 20 30 40 50 60 70 80 90 100110120130140150160170180190200210 (D3) 40

γ-gurjunene (E1) β-selinene (F1) CH 2 CH 2 CH 2 H CH H 3 C 3 CH 3 H CH 3 CH 3 (E/F2) Abundance m/z--> Abundance Average of 12.215 to 12.228 min.: 170309-MF-3.D (-) 93 9000 133 8000 105 161 204 7000 79 119 6000 5000 41 189 4000 67 55 147 3000 2000 1000 29 175 0 281 20 40 60 80 100 120 140 160 180 200 220 240 260 280 (F3) m/z--> 9000 #89380:.beta.-Selinene $$ Naphthalene, decahydro-4a-methy... 79 93 105 204 8000 189 7000 133 161 6000 121 147 5000 4000 3000 2000 1000 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 41

γ-gurjunene (E4) β-selinene (F4) FIGURE 4.10 structure (A1- E1); mass spectra obtained by our identification (A2-F2), mass spectra found in wiley (A3-D3,F3) or pherobase data bank (E4-F4). 42

Remarkable in the analysis of essential oil is that we identified a compound that eluted before ar-curcumene and had a mass spectrum very similar to the mass spectrum of γ- Gurjunene and β-selinene found in Adams, as shown in figure 4.10. This particular difference in retention time is illustrated in figure 4.11. The retention index we obtained with FID was more similar to the index of γ-gurjunene found in Adams (table 4.3). With SPME we identified β Selinene as one of the major compounds eluting right after ar- Curcumene. Abundance 1.3e+07 1.2e+07 1.1e+07 1e+07 9000000 8000000 TIC: 070509-VS-4.D 14.43 2 14.26 3 14.35 4 7000000 6000000 5000000 4000000 3000000 14.76 2000000 14.56 1000000 13.8413.91 13.95 13.72 13.78 14.02 14.07 14.10 14.52 14.66 13.70 13.80 13.90 14.00 14.10 14.20 14.30 14.40 14.50 14.60 14.70 14.80 Time--> Abundance 5500000 5000000 4500000 4000000 3500000 3000000 21.85 TIC: 190209-SG-13.D 1 2 21.99 4 22.42 2500000 2000000 1500000 1000000 22.22 23.15 Time--> 500000 23.09 21.06 21.26 22.73 21.52 21.60 21.17 22.58 21.40 22.82 22.84 22.88 23.37 23.46 21.20 21.40 21.60 21.80 22.00 22.20 22.40 22.60 22.80 23.00 23.20 23.40 1. γ-gurjunene, 2. ar- Curcumene, 3. β-selinene and 4. α-zingiberene FIGURE 4.11 Chromatograms obtained with GC-MS of the peel 43

In order to find an explanation for this we performed three tests. First, we used mass spectrometry to check the purity of the peaks. Therefore we inserted certain ion fragments, using the computer software, and, as illustrated in figure 4.12 the investigated peak contains only one compound, hence there was no interference between different compounds. Abundance 200000 180000 160000 140000 120000 100000 80000 60000 40000 20000 0 Time--> Ion 82.00 (81.70 to 82.70): 190209-SG-13.D Ion 93.00 (92.70 to 93.70): 190209-SG-13.D Ion 91.00 (90.70 to 91.70): 190209-SG-13.D 21.60 21.65 21.70 21.75 21.80 21.85 21.90 21.95 TIC: 190209-SG-13.D Peak 71 Ret time 21.85 Components 2 scan ion ion 1 4669.3 82 2 4670.5 93 91 Abundance 1000000 Ion 91.00 (90.70 to 91.70): 070509-VS-4.D Ion 133.00 (132.70 to 133.70): 070509-VS-4.D 900000 800000 700000 600000 500000 400000 300000 200000 100000 0 Time--> 14.2214.2414.2614.2814.3014.3214.3414.3614.3814.4014.4214.4414.46 TIC: 070509-VS-4.D Peak 26 Ret time 14.35 Components 1 scan ion ion 1 3288.6 91 133 FIGURE 4.12 GC-MS chromatograms checked for peak purity; from bottom to top: essential oil and SPME. 44

Secondly, we performed SPME using essential oil as a sample, in order to check the possibility that SPME inverted the order of the elution of compounds. Based on the chromatograms obtained from this analysis, we can say that the SPME could invert the eluting order of compounds. For our last investigation, we changed the column of the GC to a more polar one, namely innowax. This analysis, as illustrated in figure 4.8C, showed us that peak 2 can be identified as β-selinene instead of γ-gurjunene. This considering the very similar retention index obtained, namely 1710 obtained with FID against 1711 found in the pherobase data bank. 4.3 ESSENTIAL OIL ANALYSIS OF COMMERCIAL SAMPLE. As a last research activity, we decided to do an ultimate analysis on the peel of the commercial sample that has already been mentioned in chapter 3.1. To our surprise, we identified different volatiles compared to those identified in the fresh sample. All identified compounds are reported in table 4.4. The four major volatiles in this sample are α-copaene (10.77%), trans-β Caryophyllene (33.70%), α-humulene (9.96%) and δ- Cadinene (6.57%). α-zingiberene, β-sesquiphellandrene and ar-curcumene, three of the major compounds found in the fresh sample cannot be found in this sample. β-selinene is identified, but is only present in very small amount. To explain this discrepancy further research is needed. 45

TABLE 4.4 Chemical composition as percentage of the volatiles in the essential oil obtained from the peel of the commercial sample. Compound name RI Fid RI Adams % Identification Methods Aromatics 10-nor-Calamenen-10-one 1698 1702 0,15 MS,RI 0,15 Sesquiterpens Hydrocarbons δ-elemene 1334 1338 0,11 MS,RI α-cubebene 1345 1348 0,48 MS,RI,Std Cyclosativene b 1357 1371 0,98 MS,RI Cyclosativene 1360 1371 0,05 MS,RI α-ylangene 1366 1375 0,17 MS,RI α-copaene 1370 1376 10,77 MS,RI β-cubebene 1385 1388 0,09 MS,RI β-elemene 1387 1390 0,36 MS,RI α-gurjunene 1401 1409 0,35 MS,RI trans-β-caryophyllene 1412 1416 33,70 MS,RI,Std β-copaene 1422 1432 2,15 MS,RI γ-elemene 1431 1436 0,16 MS,RI α-humulene 1447 1454 9,96 MS,RI,Std allo-aromadendrene 1454 1460 0,86 MS,RI cis-cadina-1(6),4-diene 1457 1463 0,26 MS,RI Dauca-5,8-diene21 1469 1472 0,30 MS,RI trans-cadina-1(6),4-diene 1472 1476 2,35 MS,RI α-amorphene 1475 1484 0,41 MS,RI β-selinene 1479 1490 0,49 MS,RI δ-selinene 1482 1492 0,14 MS,RI trans-muurola-4(14),5-diene 1484 1493 0,20 MS,RI α-selinene 1488 1498 1,59 MS,RI α-muurolene 1495 1500 4,13 MS,RI Epizonarene 1501 1501 0,14 MS,RI α-amorphene 1507 1511 1,30 MS,RI γ-cadinene 1513 1513 0,10 MS,RI δ-cadinene 1519 1523 6,57 MS,RI trans-cadina1,4-diene 1527 1534 0,18 MS,RI α-cadinene 1533 1538 0,21 MS,RI α-calacorene 1538 1544 1,94 MS,RI Germacrene B 1550 1561 0,32 MS,RI β-calacorene 1559 1564 0,93 MS,RI α-corocalene 1619 1623 1,02 MS,RI 46

Compound name RI Fid RI Adams % Identification Methods Cadalene 1671 1676 1,19 MS,RI 83,93 Sesquiterpens Oxygenated Caryophyllenyl alcohol 1563 1572 0,21 MS,RI Spathulenol 1572 1578 2,16 MS,RI Caryophyllene oxide 1576 1583 4,76 MS,RI,Std Gleenol 1581 1587 0,19 MS,RI Viridiflorol 1585 1592 0,17 MS,RI Rosifoliol 1595 1600 0,44 MS,RI Humulene epoxide II 1601 1608 1,53 MS,RI 1-epi-Cubenol 1623 1627 1,07 MS,RI γ-eudesmol 1627 1632 0,68 MS,RI Caryophylla-4(12),8(13)-dien-5β-ol Caryophylla-4(12),8(13)-dien-5α-ol 1631 1640 0,52 MS,RI α-epi-cadinol 1638 1640 1,39 MS,RI α-muurolol 1643 1646 0,59 MS,RI α-cadinol 1650 1654 1,26 MS,RI 14-hydroxy-9-epi-(E)-Caryophyllene oxide 1667 1669 0,95 MS,RI 15,92 Total compounds 49 Total identification (%) 96,92 b tentative of identification 47

5.CONCLUSIONS The volatile compounds in the different parts of the litchi chinesis fruit were identified and a comparison between identification with HS-SPME and HD was made. For both extraction methods a high percentage of identification was obtained, i.e. 86.39-99.56% with HS-SPME and 99.17-99.44% with HD. The five major volatiles, E-β- Caryophyllene, ar-curcumene, β-selinene, α-zingiberene and β-sesquiphellandrene, were the same for both extraction methods and also the amount of different compounds identified in the whole seed and the peel were comparable, 51-49 with SPME and 52-43 with HD, respectively. Another similarity between both extraction methods was that the highest percentage of identified volatiles belonging to the class of sesquiterpene hydrocarbons. In the analysis with SPME we saw that the whole seed gave the highest contribution of identified volatiles and the lowest contribution was given by the edible part. The highest contribution of oxygenated sesquiterpens was identified in the juice and the pulp. In general, the percentage of oxygenated sesquiterpens was higher upon hydrodistillation. Since the peel and the seed are richer in volatile compounds than the edible part and since they contain several interesting volatile compounds, like α-zingiberene and α-humulene they could be of great potential for the perfumery and pharmaceutical industry. The commercial sample contains a high concentration of volatiles different from those identified in the fresh sample. There, the four major volatiles are α-copaene, trans-β- Caryophyllene, α-humulene and δ-cadinene. Further investigation is needed to explain the difference between the two samples and to unravel the biochemical processes that might be responsible changes in the composition of volatile compounds. 48

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