Parameters Influencing the Yield and Composition of the Essential Oil from Cretan Vitex agnus-castus Fruits

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1 Original Paper 245 Parameters Influencing the Yield and Composition of the Essential Oil from Cretan Vitex agnus-castus Fruits Janina M. Scarensen', Stavros Th. I<atsiotis2.* Mediterranean Agronomic Institute of Chania, Chania, Crete, Greece School of Pharmacy, Dept. Pharmaceutical Technology, Aristotle University, Thessalonilti, Greece Revision accepted: July 25, 1999; Received: June 22, 1999 Abstract: Mature and immature fruits of a Cretan Vitex agnuscastus L. population were chosen to investigate different parameters such as comminution, maturity, distillation period and extraction method influencing the essential oil yield and composition. The effect of the comminution and the maturity of the plant material showed highly significant differences in yield and composition of the essential oils obtained, as well as the distillation duration from one to five hours and the method applied (hydrodistillation and simultaneous distillation extraction). The variation of 36 essential oil components due to the parameters applied was studied. The results showed that many different essential oil qualities can be obtained from the same plant material according to the parameters employed in its extraction. Entire fruits hydrodistilled for one hour yielded an oil much richer in monoterpene hydrocarbons and oxygenated compounds whereas the best combination to obtain an oil rich in less volatile compounds is by SDE of comminuted fruits for five hours. For mature fruits the main components varied as follows due to the parameters studied: sabinene %, 1,8- cineole %, P-caryophyllene %. and trans-p-farnesene %. Key words: Vitex agnus-castus, Verbenaceae. fruit, essential oil, yield, chemical composition, maturation, comminution, distillation period, solvent extraction, sabinene, 1.8-cineole, trans-pfarnesene, fl-caryophyllene. Introduction Vitex agnus-castus (Verbenaceae, subfamily Viticoideae) is widely distributed in the Mediterranean area, up to Central Asia, the Tropics and the south of Europe (1). The fruit is a drupe, which upon maturity develops a hard endocarp (I), (2). The plant has a long tradition in folk medicine (3). The influence which Vitex agnus-castus fruits exert on the female hormonal household has recently received considerable attention, and clinical studies revealed an increase in nursing ability and positive results in the treatment of several female cycle anomalies (4). V. agnus-castus fruits increase the production Planta Medica 66 (2000) Ceorg Thierne Verlag Stuttgart. New York ISSN: of luteinizing hormone, and subsequently the net effect of the drug is a corpus luteum effect (4), (5), counter-acting estrogen domination. The compounds responsible for this action have not yet been identified (6). The fruits as well as leaves and flowers contain an essential oil, which is reported to vary in content and composition according to geographical origin (6-11). However, apart from geographical and environmental factors influencing yield and composition, most of the techniques employed in the extraction process as well have a significant quantitative and qualitative effect on the product obtained (12). The comminution of the plant material prior to distillation is generally recommended for tough materials, such as barks, seeds and roots (12), (13). The comminution was found the parameter of highest significance for the variation in essential oil yield and composition of Mentha x piperita and Juniperus commzuzis berries (14), (15). Regarding the yield, most plants liberate % of their oil content in the course of the first two hours of distillation. However, this practice has shown that certain constituents of any given oil are sometimes not distilled over, or only retrieved in small amounts and in many instances the quality of the oil depends on the presence of less volatile components. which are mainly obtained in the later stages of the distillation (16). On the other hand a prolonged distillation period can cause deterioration of the product (12). Oil batches obtained after various distillation periods showed considerable differences in composition in several studies on different species (15), (IG), (17). Even though hydrodistillation is the most common technique used to isolate essential oils from plant materials, distilled essential oils do not necessarily represent the true composition of the actual fragrance. In laboratory analyses of essential oils therefore, additional methods have been employed, such as solvent extraction or simultaneous distillation extraction (SDE), and considerable differences in chemical composition between hydrodistilled and solvent extracted oils have been noted (18). Previous investigations of Vitex essential oils have been carried out without any specific interest in the influence of the applied distillation conditions (G), (8), (9), (10). The objective of this study was to determine the parameters affecting the

2 yield and compositio~n of Vitex agnus-castus essential oils, such as the maturity and the comminution of the fruits, the distillation duration and the isolation method employed. Materials and Methods Sample collection Mature and immature fruits of Vitex agnus-castus were collected from a large natural population located around the lake of Cournas, district of Chania, Crete, Greece at 50 m altitude, 35" 20ilongitude, 24" 16' latitude. Voucher specimens are stored in the Herbarium of the Mediterranean Agronomic Institute of Chania (No. 2372), Crete, Greece. Plant material was collected randomly from a large number of plants. Immature fruits were immediately stored at -2O0C, dry mature fruits were cleaned of impurities and dust, and subsequently stored on filter paper in a dark room without air circulation at a temperature of approximately 20 "C. Comminution and isolation of the essential oil Mature and immature fruits were immersed in liquid nitrogen for 10 min prior to comminution in a closed type tecator mill (Cemoteca Sample Mill) for approximately 5 min, until all particles passed through a 0.8 mm laboratory test sieve. Distillation and/or extraction were commenced immediately after comminution of each batch. Hydrodistillations of entire and comminuted fruits were carried out with a Clevenger-type apparatus. Each distillation was carried out with 30g of plant material and 510 ml of distilled, deionised water in all cases (14). Series of distillations, each lasting exactly one hour longer, from 1 h to 5 h, were carried out with the mature entire and mature comminuted and the immature entire and immature comminuted fruits, each in threefold replication. Upon termination of the process, the essential oil yield was measured and the oil was collected, dried over anhydrous sodium sulphate (Na,SO,) and stored at -20 "C in 2 ml vials for further analysis. Simultaneous distillation--extraction was performed with a micro-steam version of a Likens-Nickerson Apparatus (Chrompaclz, The Netherlands) designed for solvents lighter than water. 5 g of plant material were introduced in the distillatioil site together with 80 in1 of distilled deionized water in all cases. The solvent used for the extraction site was n-pentane. Extraction period was 3 h for comminuted and 5 h for entire material. The extract obtained was dried over anhydrous sodium sulphate and stored as above. Quantitative and qualitative unalyses Quantitative analyses were performed by GC, Hewlett Paclzard 5890 Series 2 model controlled by the HP 3365 series 2 Chemstation software and equipped with an HP 7673 autosampler. Capillary column: FFAP (free fatty dcid phase) 50 m x 0.2 mm x 0.33pm, coated with a cross-linked polyethylene glycol-tpa phase. All samples were run continuously in order to minimize operation and column condition errors. Injection was performed by the autosampler. GC oven temperature program: 1 min at 60 C, then 3 "C/min to 210 "C and additional 20 min at 210 "C. All hydrodistilled samples were diluted with n-pentane to a I: 15 concentration, whereas SDE extracts were submitted as they were obtained. The amount injected was 0.5~1. Split ratio 1 : 25. Injector: 250 "C, detector (FID): 280 C. Carrier gas helium at a flow rate of 0.8 ml/min. The chromatograms and area percentage reports obtained by FFAP were evaluated after three chromatographic runs with a standard deviation of ir Qualitative analysis was carried out with a Hewlett Paclzard 5890 Series 2 model Gas Chromatograph, coupled to a VG Trio 2000 Mass spectrometer. Columns: 1) FFAP as above and programmed identically, and 2) HP5 30 m x 0.25 nlrn id., 0.25pm coating thickness. GC oven temperature program: 60 C to 240'C at 3 "C/miri. Injector: 250 C. Carrier gas helium at a flow rate of 1 ml/rnin. Mass spectra were taken at 70 ev at a scanning speed of 1 scanlsec from 40 to 240 m/z. Components were identified by comparisori j~ith the computerised MASS LYNX software from the Wiley Library, by their relative retention times (19) and by comparison with authen-. tic samples and literature. Results and Discussion All the parameters investigated, distillation period, maturity and comminution of the plant material, proved to be significant for the yield obtained. Entire and comminuted mature, as well as entire and comminuted immature fruits seemed to follow roughly the same pattern of essential oil release, in that yields within each group steadily increased. Already after 1 h a significant amount of oil was distilled over, comprising 72 %- 73 % of the oil obtained during the full 5 h distillation of mature entire and comminuted fruits respectively, 68% of the total yield from entire immature, and 81 % of the yield obtained from comminuted immature fruits (Table 1). Table 1 Essential oil yields of V. ngnus-casius fruits after variations periods of hydrodistillation. Maturity and Distillation Period, Oil Yield (% v/w) Comminution lh 2h 3h 4h Sh mature entire mature comminuted immature entire immature comminuted The comminution of the plant material did not have the same effect on mature and immature fruits. After 5 h in the case of the immature fruits, higher yields were obtained frorn the en-. tire (mean 0.47%), than from the comminuted fruits (mean 0.42%). Mature fruits on the other hand, gave a higher yield if comminuted (mean 0.71 %) than if distilled entire (mean 0.61 %). It is interesting to note that the comminution of this plant material did not influence the fruit essential oil yield to the same extent, as usually reported for plant materials which synthesise or store their essential oils in subcutaneous cavities, passages or ducts. An interesting feature is, neveitheless, that the immature fruits behaved quite differently, and gave a higher yield if left entire and in this respect seemed to behave more like leafy materials such as Mentha x piperita (I4), Salvia triloba and Salvia officinalis (20).

3 Parameters Influencing the Yield and Composition of the Essential Oil from Cretan Vitex agnus-costus Fruits Planta Med. 66 (2000) 247 Marked differences between mature and immature fruits were found in the percentages of the major components (Tables 2 and 3). Immature fruits contained a lesser concentration of the main component sabinene, whereas they showed elevated values of limonene and a-pinene. In the oxygenated monoterpene fractions, noticeable differences were detected between all of the ten investigated compounds. 1,s-Cineole reached a higher value in the mature fruits than in the immature fruits. Considering all the data obtained, regardless of the comminution and distillation period, both the hydrocarbon and oxygenated monoterpene fraction were elevated in the mature fruits, whereas sesquiterpenoids were elevated in immature fruits. Mature and immature fruits, entire or comminuted, followed generally the same trend of compositional changes during the distillation period (Tables 2 and 3). The present results confirm the observations by several researchers (14), (15). (16), (17) regarding the declining trend of monoterpene hydrocarbons in the time course of distillation. However, they do not confirm the observations that the hydrocarbons should elute later in the distillation process than the oxygenated compounds (12), neither do the compositional changes in V. agnus-castus essential oils follow the same pattern as observed in Melaleuca alternifolia, in which case the higher boiling alcohols were distilled before the hydrocarbons (13). Table 2 Rt. HP5 Compositional variation according to the distillation period. Essential oils obtained from mature fruits of Vitex agnus-costus. - Components Comminuted fruits. % in Oil Entire fruits. % in Oil 315 a-pinene 303 a-thujene 387 P-pinene 383 sabinene 443 6,-carene 407 P-myrcene 433 a-phellandrene 556 a-terpinene 480 limonene 541 yterpinene 471 p-cyrnene 606 terpinolene 486 1,&-cineole octanone 632 trans-sabinene hydrate 952 citronellol* 630 linalool 560 cis-sabinene hydrate 821 terpinen citronellyl acetate 854 a-terpineol 1269 a-terpinyl acetate 1489 a-bergamotene* 1447 P-caryophyllene 1541 oflo-aromadendrene 1536 trans-0-farnesene 1531 a-humulene 1640 gerrnacrene D 2055 P-bisabolol 1805 nerolidol* 1812 ledol 1836 spathulenol 1986 torreyol 1975 T-cadinol rnonoterpene hydrocarbons Oxygen containing rnonoterpenes 21.4 Sesquiterpenoids 18.0 Rt = Retention time, t = trace. * = correct isomer not determined.

4 Table 3 Compositional variation according to the distillation period. Essential oils obtained from immature fruits of Vitex agnus-castus. Components a-pinene /3-pinene sabinene 6,-carene P-myrcene a-phellandrene a-terpinene limonene yterpinene p-cymene terpinolene 1,%cineole 3-octanone trans-sa binene hydrate citronellol' linalool cis-sabinene hydrate terpinen-4-01 citronellyl acetate a-terpineol a-terpinyl acetate a-bergamotene* /3-caryophyllene 0110-aromadendrene trans-/3-farnesene a-humulene gerrnacrene D /3-bisabolol nerolidol" ledol spathulenol torreyol T-cadinol monoterpene hydrocarbons Oxygen containing monoterpenes Sesquiterpenoids Comminuted fruits % in Oil Entire fruits % in Oil t = trace. * = correct isomer not determined. Whereas the oxygenated compounds in Juniperus communis berries were reported to increase with the distillation period (15), the opposite trend was observed in I/. agnw-castus fruits. The main oxygenated compound 1,8-cineole in Eucalyptus globulus oil was found to decrease while terpinen-4-01 increased (17), as found for these compounds in the present study as well. The sesquiterpenoid compounds in the present study did not follow the pattern of general increase during the process as reported by several authors (15), (16), (17). Whereas Gauthier et al. (16) reported the sesquiterpenoids to distil over only from the second hour on, the sesquiterpenoids in the essential oils of V. agnus-castus were present with considerable percentages already in the oil obtained after one hour of distillation. and dramatic increases were not observed. Such individuality in the release behaviour of the chemical compounds present in the plant materials could suggest that probably both the species under investigation and the site of synthesis and storage of volatiles in plants influence the behaviour of these compounds during distillation. It could be suggested that the ratio of high, medium and low volatility components found in an oil presents a factor of interaction during the process of distillation. As such one could not expect components to behave similarly in an oil rich in monoterpenes compared to an oil rich in sesquiterpenes or an oil containing even higher boiling constituents. The comminution was found to influence the essential oil composition considerably (Tables 2 and 3). The monoterpene hydrocarbons and the oxygenated monoterpenes were present to a much higher degree, while the sesquiterpenoids com-

5 Table 4 Compositional variation according to extraction method. Mature fruits of Vitex ognus-castus. - Method Comminuted fruits Entire fruits SDE 3 h HD3h SDE 5 h % in Extract % in Oil % in Extract a-pinene a-thujene P-pinene sabinene &carene P-myrcene O a-terpinene limonene y-terpinene p-cymene terpinolene ,8-cineole octanone trans-sabinene hydrate citronellol* citronellyl acetate linalool cis-sabinene hydrate terpinen a-terpineol a-terpinyl acetate a-bergamotene* P-caryophyllene aromadendrene trans-p-farnesene a-humulene germacrene D nerolidol* ledol spathulenol torreyol T-cadinol rnonoterpene hydrocarbons Oxygen containing rnonoterpenes Sesquiterpenoids SDE = Simultaneous Distillation Extraction. HD = Hydrodistillation. * = correct isomer not determined. HD 5h % in Oil prise a smaller percentage in the oils obtained from the entire Vitex agnus-castus fruits compared to the oils obtained from comminuted fruits, as also found by Menary (13) and Chatzopoulou and Katsiotis (15), concerning Melaleuca alternifolia and Juniperus communis, respectively. The comminution however, influenced the quantitative composition of the mature fruits to a much greater extent. In the case of the immature fruits, less differences in compositional changes were detected between the entire and the comminuted fruits. The isolation method employed was shown to have a substantial influence on the essential oil composition of both mature entire and mature comminuted fruits. However, considering the data obtained (Table 4), the variation in comminuted fruits did not show as remarkable differences as found for entire fruits. It can be concluded that the monoterpene hydro- carbons are present to a n~uch higher degree in the hydrodistillate, compared with the SDE extract, while the oxygenated monoterpenes and the sesquiterpenoids are more abundant in the latter. In case of the entire fruits, many of the inonoterpene hydrocarbons, including the main component sabinene, were present with almost double values in the hydrodistilled oil, compared to the extract by SDE. Our results showed that an SDE extract using n-pentane as solvent differs from a hydrodistilled oil in the generally significantly elevated levels of higher boiling constituents, like sesquiterpenes and oxygenated compounds and lower levels of monoterpene hydrocarbons. However, the different behaviour of entire and comminuted fruits, respectively, due to the method employed has to be

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