CHEMICAL COMPOSITION CHANGES DURING STORAGE OF LEMON BASIL ESSENTIAL OIL (Ocimum citriodorum sp.

) Dwiarso Rubiyanto*#, Chairil Anwar** dan Hardjono Sastrohamidjojo** Department of Chemistry, Faculty of Math and Natural Sciences, Islamic University of Indonesia, Jogjakarta, Indonesia ** Department of Chemistry, Faculty of Math and Natural Sciences, Gadjah Mada University, Jogjakarta, Indonesia # e-mail : iwdarso@yahoo.com
*

ABSTRACT Difference in post harvest treatment due to the pre distill process of some essential oil resources can lead to differences in chemical compositions included Daun Kemangi (Lemon basil (Ocimum citriodorum sp.)) essential oil. Specific compounds that taking the role of major constituents in lemon basil oil and control the special aromatic scents could be identified during storage time. Minor components in lemon basil essential oil took place as contributors that couldnt be forgotten too. The unique characteristic and manner of each major and minor substituent in the presence of specific pre distillation process can lead to providing useful information to next step due to this essential oil utility. In this research, essential oil sample from lemon basil grown in 4 x 6 m2 garden was taken from Jogjakarta, Indonesia has been produced by steam distillation (SD) method. Five different storage time were conducted before distillation process running. Variation of storage time for pre distillation process including 0 day (fresh leaf), 1 day (stale condition), 7 days (very stale), 14 days (dry condition) and 30 days (very dry). The analysis of its main components has been performed using gas chromatography- mass spectrometry (GC-MS). Chemical composition vary due to increasing time although it could be seen that some compounds were remain chemically stable during storage. Some identical compounds could be figured out from each storage time period applied and some compounds could be potentially used as storage-finger print-compound of each time. There were approximately 71 accumulative compounds with vary concentration could be observed in lemon basil essential oil during storage. Citral which consist of citral A and citral B were the one of the primary compound with concentration of 63.17% - 79.08%.

Keywords: lemon basil essential oil, Kemangi, composition change, storage time, Ocimum citriodorum sp.

Presented in the International Seminar on Essential Oil 2009, (ISEO 2009) 26th-28th Oct 2009, IPB International Convention Center, Bogor

Introduction Extensive analytical and sensory evaluation is necessary to select the plant cultivar/chemotype, the part of the plant growth and the stage of development for harvest that gives the best yield of the particular oil character desired. The quality and price of some oils are based on the percentage content of a single chemical component, so separation and measurement of individual components is very important. Indonesia was among of few countries that had very large variety of aromatic plants and cultivars. Some of them had been known as an established essential oil resources but many hadnt been developed yet. There many other plants could be potentially germinated. Its mega-biodiversity on types and varieties of them could bring to prosperities and benefits to the nation and citizens. The Ocimum genus belonging to the Lamiaceae family is characterized by a great variability of both morphology and chemo-types, the ease of crosspollination leads to a large number of subspecies, varieties and forms, which differ in essential oil composition and morphological characters. (Omer, et.al., 2008) Basils (Ocimum spp., Lamiaceae) contain a wide range of essential oils rich in mono terpenoids and phenolic compounds (Simon et. al., 1990; Phippen and Simon, 2000). The basil family includes about 150 species from Asia, Africa, and South America. Here are a few examples : Ocimum basilicum (Sweet basil); Ocimum basilicum var. citriodorum (Lemon basil); Ocimum gratissimum (East Indian basil); Ocimum basilicum var. crispum (Curly basil); Ocimum basilicum var. minimum (Greek basil); Ocimum sanctum (Holy or sacred basil). Basil has culinary and ornamental value as a herbs. Fresh or dried leaves can be used in soups, stews, and sauces; meat, fish, and egg dishes; salads; and vegetables. The leaves are used in flavored vinegars and teas. There was such opinion that these are herbs that are best used in their fresh state since their flavors are easily lost soon after picking from plants. It is best to pick these herbs when they are needed for use. They also can be processed to produce flavored oils using blended with other vegetables oil. Cultural methods which cause changes in basil essential oil content and composition thus have the potential to also affect the aroma and taste of the fresh product. (Johnson, et. al., 1998)

Presented in the International Seminar on Essential Oil 2009, (ISEO 2009) 26th-28th Oct 2009, IPB International Convention Center, Bogor

Basil essential oils production still limited because most people used in fresh or blended conditions. As a part of Ocimum species, compared to other like basils leaf, lemon basil essential oil was rarely found in market because most people used this fresh herb as part of culinary to complete of food processing and as an ornamental herb. But, this pungent-strong lemon odor and taste-leaves can produced to such an essential oil having great chemical component and physicochemical properties that converted to another chemical compounds. One of Ocimum citriodorum (Lemon basil) variety was known as Kemangi in Indonesia had been long time used in culinary. Its cheap and easily founded makes people plant them in their yard or garden because its colors and smells that natural and fresh. It is believed that Kemangi has physiology activity in traditional healing methods like reducing muscle tension, stomachache, headache, and giving pleasant-relaxing scents. Using Kemangi as source of essential oil production was very potential way to get new ingredient for some purpose. The essential oil of Ocimum basilicum was screened for its composition and some central nervous system activities (viz., sedative, hypnotic, anticonvulsant, local anesthetic). When tested in mice, OB essential oil had no effect on motor activity up to a dose of 1.2 mLkg-1 at 90 min post administration. (Ismail, 2006) People knew that some essential oils could act as a specific chemical that responded by other species like insects or mammals as pheromone, deterrent, attractant, and having some biologically action or effects as antifungal, antimicrobial, and etc. Many researches aimed to find the effective way on pest control especially using deterrent or antifeedant effect against insects (Aerts and Mordue, 1997; Ahmad, 1993; Carrol, 1994; Cowles, et. al., 1990; Govindacharl, 1996; Rubiyanto, 2008). So, composition of the essential oil of Daun Kemangi should be evaluated and controlled to find out whether differences in pre distill treatment could impact those chemical composition. It has been considered that usually people using Daun Kemangi in freshly state. In a point of view that concerning lemon basil oil as source of chemical substances, it is important to examine if there were any changes of chemical composition of different time of storage in a post harvest treatment comparing them in the fresh state. can be

Presented in the International Seminar on Essential Oil 2009, (ISEO 2009) 26th-28th Oct 2009, IPB International Convention Center, Bogor

Materials and methods Fresh samples of lemon basil (O. citriodorum) or daun Kemangi were collected from 4 x 6 m2 garden taken from specific location in Jogjakarta Indonesia which was planted in rainy season in October 2008. This plant was harvested each time which particular storage time would be done. The samples (leaf and stem) were cut and left to air dry in 0 day (fresh leaf), 1 day (stale condition), 7 days (very stale), 14 days (dry condition) and 30 days (very dry). Plant materials were then subjected to distillation using semi macro steam distillation (SD) apparatus that consist of stainless steel boiler tank (20 L of capacity) as steam production, connected to sample Kemangi leaves container (4 to 5 kg dry sample maximum capacity). Vapor produced was passed out through water condenser and outlet to oil collector. The oily layers obtained were separated and purified using sodium sulphate anhydrous. Analysis of components has been carried out using GCMS-QP2010S SHIMADZU (EI, Rtx-5MS, 30 m x 0.25 mm ID, He). Results and discussion Steam distillation methods were very common in means of obtaining esential oils. Essential oils producer must take a best effort to collect essential oils as much as they can. The key to get optimum yield of essential oil collection is to maximize water steam penetrated to samples and discovered suitable state or condition of the samples like fresh, stale, dry or frozen . Even there are hundreds of Ocimum species, but mostly only 2 3 types that can be found in daily life in Jogjakarta province as well as used in cullinary and dishes or just an ornamental herbs. (Rubiyanto, 2006)

Figure 1. Left to right : Ocimum citriodorum (lemon basil), Ocimum basilicum Canum sims (purple basil) and Ocimum basilicum Lime (sweet basil)

Presented in the International Seminar on Essential Oil 2009, (ISEO 2009) 26th-28th Oct 2009, IPB International Convention Center, Bogor

In this research, all harvesting was done after lemon basil plants reached enough age for cropping, means between 4 6 months. Each plant approximately needs 2 3 months after seeding to reach strong rod and leaf form completely blossomed. Leaf harvesting should be set continuously to prevent flowering because it could lead to retard leaf developing that affected oil content. Post harvest treatment including mechanically (stripping, cutting, abrasive), thermally (drying, sunning, freezing), or aging for periods would affect the oil contents. (Gang, et.al., 2001) After storage for certain period then the lemon basil samples were subjected to steam distillation for 3 - 4 hours. The essential oil yield and its density then measured in each process. Data showed that lemon basil essential oil could get the optimum yield and density (compared to basil essential oil in common with density : 0.89 0.90 g/mL) in 3 7 days of storage. Detail results as follows :
1.1000 1.0000 0.9000 0.8000 0.7000 0.6000 0.5000 0.4000 0.3000 0.2000 0.1000 0.0000
0 1.00 0.9136 0.6818 0.5125 0.57 0.52 0.35 0.16 1 0.12 3 7
Storage days Density, g/mL Yield, %

0.8989 0.8380

0.8688

14

30

Figure 2. Correlation between essential oil yield and density in each storage time Two classes of compounds, terpenoids and phenylpropenes, make up the bulk of essential oils and contribute to the particular properties of many spices and herbs. The analysis of chemical composition of lemon basil essential oil was run on GC-MS using Rtx-5-MS as the stationary phase and electron impact (EI) ionization. Some compounds showed small traces (below 1%) as a characteristic in storage periods (Table 1) which meant that there was substituent showed up in first periode of storage ( 0 7 days) but the other hand, there was showed up in middle (7-14 days) or end period (14 30 days). These changes could be caused by inter conversion among substituent that be induced by oxidation or microorganism activities. Aging by leaving lemon basil samples to dry would bring in decreasing physical form of leaf surface that could be indicated with pale color, growing darkness and lack of humidity. Flexibility
Presented in the International Seminar on Essential Oil 2009, (ISEO 2009) 26th-28th Oct 2009, IPB International Convention Center, Bogor

and freshness would be replaced by rough and crisp texture. This process could be continued by changing in chemical composition of essential oil materials. Day to day changing also showed us that some compounds remained stable and some higher quantities of constituent could be potential for scientific reason due to explore new sources from plants material as well as an individual compound or as an aggregate. Table 1. Chemical composition of between periods of storage
Period, days 0-7 Cis-geraniol Trans-geraniol 2,6-dimethyl1,6-octadiene Neric acid 2,3-dimethyl3-buten-2-ol Geranyl acetate Alpha-cadinol 0-14 Betapinenoxide 2,6-dimethyl heptanal 1-14 Phelandral Alphagurjunene 1-30 Beta- Ocimene Trans -3,5dimethyl-1,6octadiene Beta-sesqui phellandrene Alphahumulene Germacrene D Beta-bisabolene Alpha.copaene 14 - 30

Compounds

Chromatograms showed some characteristic profiles and amount of substituent influenced by time of storage that could be associated to lemon basil essential oil composition. Identification of compounds were obtained from mass spectra (MS) analysis. Totals compounds in accumulation of difference storage time should be 71 compounds. Thus, those chemicals would contribute the overall characteristic of lemon basil essential oil storage. From each chromatogram of storage condition would be summarized that there were identical compounds found in all days of storage (Figure 4) which most of them present in higher concentration (above 1%) and some compounds could be called as storage-finger print-compound meant only specific compounds should be discovered in particular storage time (Figure 5) although they revealed in small quantities (below 1%). It showed 12 compounds that identically in figure 4 which are : (a) 6-methyl-5-hepten-2-one, (b) Linalool, (c) 2,2-Dimethyl-3,4-octadienal, (d) Trans-4,5-epoxi- caran, (e) Alpha terpineol, (f) Cis-citral, (g) Trans-citral, (h) Transcaryophyllene, (i) Alpha.-bergamotene, (j) (E)-beta-Farnesene, (k) Cis-alphabisabolene, (l) (-)-Caryophyllene oxide

Presented in the International Seminar on Essential Oil 2009, (ISEO 2009) 26th-28th Oct 2009, IPB International Convention Center, Bogor

9 33

7 6

2 12 2

0 - 7 days 0 - 14 days 0 - 30 hari days 1 - 14 days 1 - 30 days 14 - 30 days accumulation of specific day spread

Figure 3. Amounts of chemical compounds in each period of storage

Figure 4. Chromatographic profiles of identical components in all storage time; A:0 day, B: 1 day, C: 7 days, D: 14 days and E: 30 days.

Presented in the International Seminar on Essential Oil 2009, (ISEO 2009) 26th-28th Oct 2009, IPB International Convention Center, Bogor

Figure 5. Specific compounds that found in individual storage time

References

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Presented in the International Seminar on Essential Oil 2009, (ISEO 2009) 26th-28th Oct 2009, IPB International Convention Center, Bogor

Gang, D.R., Wang, J., Dudareva, N., Nam, K.H., Simon, J.E., Lewinsohn, E., and PicherskyAn, E., 2001, Investigation of the Storage and Biosynthesis of Phenylpropenes in Sweet Basil, Plant Physiol., Vol. 125 GovindacharI, T.R.,1996, Insect antifeedant and growth-regulating activities of salanin and other c-seco limonoids from neem oil in relation to azadirachtin. J.Chem. Ecol., 1996, v. 22, n. 1, p. 1453-61, New York Ismail, M., 2006, Central Properties and Chemical Composition of Ocimum basilicum, Pharmaceutical Biology, Vol. 44, No. 8, pp. 619626 Egypt Omer, E.A., Said- Al Ahl, H.A.H. and Hendawy, S.F., 2008, Production, Chemical Composition and Volatile Oil of Different Basil Species/ Varieties Cultivated under Egyptian Soil Salinity Conditions Research Journal of Agriculture and Biological Sciences, 4(4): 293-300, INSInet Publication Phippen, W.B. and Simon, J.E., 2000, Anthocyanin inheritance and instability in purple basil (Ocimum basilicum L.), J. Hered, 91:289296 Rubiyanto, D. and Istiqomah, N., 2006, Chromatographic and infra red spectra profiles sweet basil leaf oils of Ocimum basilicum Lime and Ocimum basilicum Canum sims , Jurnal EKSAKTA, Jogjakarta. Rubiyanto, D., 2008, Chemical composition of Daun Kemangi (Ocimum citriodorum sp.) essential oil and its potential anti feedant on the grasshopper, Malaysian International Conference on Essential Oils, Flavor and Fragrance Materials (MICEOFF5), Kuala Lumpur, Malaysia. Simon, J.E., J. Quinn, and R.G. Murray, 1990, Basil: A source of essential oils. p: 484 489, Timber Press, Portland

Presented in the International Seminar on Essential Oil 2009, (ISEO 2009) 26th-28th Oct 2009, IPB International Convention Center, Bogor