Chemical composition of the essential oils of Stevia rebaudiana ...

Chemical composition of the essential oils of Stevia rebaudiana Bertoni leaves 

/ Asian Journal of Traditional Medicines, 2010, 5 (2) 

Regular Articles 

Chemical composition of the essential oils of Stevia 

rebaudiana Bertoni leaves 

M. Amzad Hossain a * , A. B. Siddique b , S.M. Mizanur Rahman b , M. Amzad Hossain c 

a. Chemistry Division, Atomic Energy Centre, GPO Box. 164, Ramna, Dhaka-1000, Bangladesh; 

b. Department of Chemistry, University of Dhaka, Dhaka-100, Bangladesh; 

c. Bangladesh Sugarcane Research Institution, Ishurdi, Pabna, Bangladesh. 

Abstract 

The chemical composition of the hydrodistilled essential oils of Stevia rebaudiana Bertoni leaves, grown for the first time in an 

experimental field in Bangladesh, was analysed by GC–MS. Sixty two compounds representing 92.27 % of the total leaf oils were 

identified, of which α-cadinol (2.98 %), caryophyllene oxide (1.23 %), (-)-spathulenol (2.21 %) and β-Guaiene (0.32 %), were the 

major components. Thus, the monoterpenes and sesquiterpenes were the predominant constituents of the oils. The leaves contained 

some new essential oil compounds, such as ledene oxide-(ΙΙ), beta.-guaiene, geranyl vinyl ether, tricyclo [5.2.2.0 (1, 6)] undecan-3-ol, 

indole, aristolene epoxide, 1, 2, 3, 5, 6, 7, 8, 8a-octahydro-1, 4-dione and 2, 6, 6-trimethyl-2-cyclohexene-1, 4-dione identified for the 

first time. 

Key words: Stevia rebaudiana Bertoni; hydrodistillation; essential oil composition; GC-MS analysis 

Introduction 

There is an ever increasing shortage of food 

supplies because of population growth, particularly 

in the developing countries. The search for novel 

high quality, but inexpensive, sources of food has 

remained a major concern of all agencies involved 

in providing adequate food supplies and ensuring 

the proper nutritional status [1] . Many indigenous 

medicinal plants are used as foods [2] . India has a 

rich biodiversity of such foods although few studies 

have been conducted to document the nutritive 

* Author to whom correspondence should be addressed. Address: 

Dr. Md. Amzad Hossain, Principal Scientific Officer, Chemistry 

Division, Atomic Energy Centre, Ramna, Dhaka-1000,; Tel: 

+88-29663742; Fax: +88-29664856; E-mail: dramzadh@gmail.com 

Received: 2009-10-18 Accepted: 2010-03-12 

value of less familiar foods [3-4] and the available 

information is quite poor. Hence, in an attempt to 

explore such untapped sources of good nutrition, the 

present investigation has been undertaken to identify 

the chemical composition of the essential oils in the 

Bangladeshi species Stevia rebaudiana Bertoni. 

Stevia rebaudiana Bertoni belongs to the family 

Compositae (Asteraceae) [5-6] that is indigenous to 

Paraguay and Brazil and is also cultivated in some 

regions of Asia, Europe and Canada [7] . Recently, 

cultivation of Stevia rebaudiana started in India and 

Bangladesh. The main sweet component of the leaves 

of Stevia rebaudiana Bertoni is stevioside [8] . It is 

300 times sweeter than sucrose [9] and has recently 

become important as natural non-calorific sweetener. 

Stevia leaf extracts are used in Japan, Korea and 

some countries in South America to sweeten soft 

drinks, soju, soy sauce, yogurt and other foods, 

56



while in the United States it is used as a dietary 

supplement and in Bangladesh as an antidiabetic 

tea. Stevia rebaudiana Bertoni sweetener extracts 

have been suggested to have a beneficial effect on 

human health because of their antihypertensive [10-11] , 

antihyperglycemic [12] , anticariogenic [13] and antihuman 

rotavirus activities [14] . These sweeteners are 

also thought to influence glucose metabolism [15-16] 

and renal function [17] . The increasing importance 

of essential oils in various areas of human activity 

(pharmacy, cosmetics as well as the food and drink 

industries) has made this field very interesting 

for chemical investigators. Current research into 

Stevia rebaudiana Bertoni essential oil, is very 

active. However, it should be remembered that the 

sweetening effect is simply a pleasant by-product. The 

primary reason that stevia is combined with other 

herbs is to enhance the nutritive value of these other 

herbs! Stevia is, after all, nutrient-rich, containing 

substantial amounts of protein, calcium, phosphorous 

and other important nutrients [18-19] . 

Carrying the above considerations a step further, 

there are many very legitimate reasons for using 

stevia as a medicinal food. In spite of the prominence 

stevia has attracted as a flavor enhancer, it contains 

a variety of constituents besides the steviosides 

and rebaudiosides, including the nutrients specified 

above and lots of sterols, triterpenes, flavonoids, and 

tannins, as well as being an extremely rich volatile 

oil containing lots of aromatic agents, aldehydes, 

monoterpenes and sesquiterpenes [20] . These and other, 

as yet unidentified constituents, probably have a variety 

of effects on human physiology and may help explain 

some of the reported therapeutic uses of stevia. 

Therefore, the aim of the present study is to 

examine the chemical composition of the essential oils 

isolated from the leaves of Stevia rebaudiana Bertoni 

by GC–MS. 

Material and methods 

Plant material 

The green leaves of Stevia rebaudiana Bertoni 

were collected from the Bangladesh Sugarcane 

Research Institute, Ishurdi, Pabna, Bangladesh. The 

plants were harvested during the month of September, 

2009. The leaves were collected at 2:00 pm-3:00 pm 

on September 2, 2009 and packed in polyethylene 

bags and stored at 4 °C until required. All chemicals 

used were of research grade purity. 

GC-MS analysis 

The GC–MS analysis of the essential oil samples 

of Stevia rebaudiana Bertoni was performed using a 

Varian GC–MS instrument (Model Varian CP 3800, 

USA) equipped with a VF-5 fused silica capillary 

column (30 m×0.25 i. d. mm film thickness 0.25 µm, 

Varian, USA). For GC–MS detection, an electron 

ionization system with ionization energy of 70 eV 

was used. Helium gas was used as a carrier gas at 

a constant flow rate of 1 ml/min. The injector and 

mass transfer line temperatures were set at 250 °C 

and 300 °C, respectively. The oven temperature was 

programmed from 50 °C to 200 °C at 8 °C/min, and 

then kept isothermal for 20 min and finally raised to 

300 °C at 10 °C/min. Diluted samples (1/100 v/v, in 

methanol) of 0.2 µl were manually injected in splitless 

mode. Identification of compounds of the essential oil 

was based on GC retention times on the VF-5 capillary 

column, with computer matching of mass spectra with 

those of standards (Mainlab, Replib and Tutorial data 

of GC–MS systems). 

Extraction and isolation of essential oil 

The air-dried leaves and stems (250 g each) 

of Stevia rebaudiana Bertoni were subjected to 

hydrodistillation for 3h using a Clevenger type 

apparatus. The distillate was mixed with 1g NaCl and 

20 ml dichloromethane (DCM) in a separatory funnel, 

and shaking was continued by hand for 40 min and the 

mixture was then allowed to stand for 15 min. Finally, 

the DCM layer was collected in a round bottom flask 

for rotary evaporation and evaporated at 30 °C until 

57



5 ml remained. The oil was dried over anhydrous 

sodium sulphate and stored in a sealed vial at 4 o C 

until further analysis. 

Results and discussion 

Chemical composition of essential oils 

The GC-MS total ion chromatogram (TIC) 

of the essential oils from the leaves of Stevia 

rebaudiana Bertoni is shown in Fig. 1. GC–MS 

analyses of the oils led to the identification of 62 

different compounds, representing 92.27 of the total 

oils from the leaves. The identified compounds are 

listed in Table 1 according to their elution order 

on a VF-5 capillary column. The oils contained a 

complex mixture consisting of mainly oxygenated 

monoterpene and sesquiterpene hydrocarbons. The 

major compounds detected in the leaves and stems 

oils, respectively, were (-)-spathulenol, caryophyllene 

oxide, caryophyllene, ledene oxide-(ΙΙ), β.-guaiene, 

geranyl vinyl ether, tricyclo [5.2.2.0 (1, 6)] undecan- 

3-ol, indole, aristolene epoxide, 1, 2, 3, 5, 6, 7, 8, 

8a-octahydro-1, 4-dione and 2, 6, 6-trimethyl-2- 

cyclohexene-1,4-dione (Table 1). In the terpene 

group, seven compounds, ledene oxide-(ΙΙ), β-guaiene, 

geranyl vinyl ether, tricyclo [5.2.2.0 (1,6)] undecan- 

3-ol, indole, aristolene epoxide, 1, 2, 3, 5, 6, 7, 8, 

8a-octahydro-1, 4-dione and 2, 6, 6-trimethyl-2- 

cyclohexene-1, 4-dione, were present that had not 

previously been reported as constituents of Stevia 

rebaudiana Bertoni. All the essential oils exhibited 

very potent biological activity. 

Hydrodistillation of the leaves of Stevia 

rebaudiana Bertoni gave dark yellowish oils with the 

major components being oxygenated monoterpenes 

and sesquiterpenes, and their respective hydrocarbons. 

In recent years, several researchers have reported 

that monoterpene and sesquiterpene hydrocarbons 

and their oxygenated derivatives are the major 

components of essential oils of plant origin, and these 

have enormous potential to strongly inhibit microbial 

pathogens [21-22] . In general, the active antimicrobial 

compounds of essential oils are terpenes, which are 

phenolic in nature, it would seem reasonable that their 

antimicrobial or anti-fungal mode of action might be 

related to that of other compounds. 

e constituents of essential oil from the leaves of Stevia 

Counts 

igure 1. A typical chromatogram of the constituents of essential oil from the leaves of Stevia 

Rebaudiana Bertoni 

t (min) 

Figure 1. A typical chromatogram of the constituents of essential oil from the leaves of Stevia 

Fig. 1. A typical Figure chromatogram 1. A typical chromatogram of the constituents of the Rebaudiana Figure constituents of 1. essential A typical Bertoni of oil essential chromatogram from the oil from leaves of the of leaves constituents Stevia of rebaudiana Stevia of essential Bertoni oil from the leaves of 



 

 

Figure 1. A Figure typical 1. chromatogram A typical chromatogram of the constituents of the constituents of essential of oil essential from theoil leaves from of theStevia 

leaves o 

Rebaudiana Rebaudiana 

Figure Bertoni Bertoni 

1. A typical chromatogram of the constituents of essential oil from the le 

58 Rebaudiana Bertoni



Table 1. Percentage composition of the volatile leaf oils of Stevia 

Compound RT Leaves (%) 

5, 4-Benzyloxy-2-fluoro-5-hydroxy benzaldehyde 3.412 4.68 

3-Bromobutyric acid 4.024 4.15 

Undecanol-5 6.377 0.18 

1-Hepten-3-ol 6.782 0.03 

Cyclohexene, 4-bromo- 7.810 0.05 

Benzyl-diseryl phosphate 7.954 4.82 

1, 3, 5-Cycloheptatriene 8.120 3.65 

α-Methyl-α-[4-methyl]-3-pentene 8.523 0.60 

1, 6-Octadien-3-ol, 3, 7-dimethyl- 9.023 2.22 

Cis-3-hexenyl-iso-butyrate 9.109 0.38 

Cyclopentasiloxane, decamethyl- 9.372 0.88 

Phenylethyl alcohol 9.421 1.28 

2, 6, 6-Trimethyl-2-cyclohexene-1, 4-dione 9.975 0.13 

3-Cyclohexene-1-methanol 10.884 0.53 

2-Isopropenyl-5-methylhex-4-enal 11.343 0.29 

Bicyclo [2.2.1] hept-2-ene, 1, 7, 7-trimethyl- 11.478 0.13 

5-Amino-2-methoxyphenol 11.598 0.09 

Geranyl vinyl ether 11.778 0.14 

Cyclohexasiloxane, dodecamethyl- 12.318 4.40 

Indole 12.778 0.17 

2-Methoxy-4-vinylphenol 12.960 0.18 

Phenol, 2-methoxy-3-(2-propenyl)- 13.624 0.19 

1-(2, 6, 6-Trimethyl-1, 3-cyclo)-2-buten-1-one 14.028 0.06 

Cyclohexane, 1-ethyl-1-methyl-2, 4-bis- 14.164 0.09 

Caryophyllene 14.715 0.08 

Tricyclo [3.3.0.0 (2, 8)] octan-3-one, 4-methyl 14.822 0.07 

1, 6, 10-Dodecatriene, 7, 11-dimethyl-3-methyl 15.048 0.02 

3-Buten-2-one, 4-(2, 6, 6-trimethyl-1-cyclo)- 15.615 0.03 

3-Buten-2-one, 4-(2, 6, 6-trimethyl-7-oxab)- 15.677 0.06 

Naphlalene, 1, 2, 3, 5, 6, 7, 8, 8a-octahydro- 15.822 0.05 

Heptasiloxane, hexadecamethyl- 16.092 0.04 

Berjomotol, Z-α-trans- 16.294 0.08 

Lanceol, cis- 16.352 0.09 

2 (4H)-Benzofuraone, 5, 6, 7, 7a-tetrahydro 16.584 0.15 

1, 6, 10-Dodecatrien-3-ol, 3, 7, 11-trimethyl 16.747 2.26 

Epoxy-α-terpenyl acetate 16.916 0.76 

Benzene, 1, 1´-(1, 1, 2, 2-tetramethyl-1, 2-ethanediyl) bis- 17.078 0.08 

(-)-Spathulenol 17.195 2.21 

Caryophyllene oxide 17.282 1.23 

Cyclooctasiloxane, hexadecamethyl- 17.374 2.23 

Santalol, cis-α- 17.527 0.07 

5α-Hydroxy-4α-8, 10. 11-tetram 17.605 0.09 

Isoaromadendrene epoxide 17.678 0.35 

β-Guaiene 17.975 0.30 

Ledene oxide-(ΙΙ) 17.735 0.21 

59



Continued Table 1 

Naphthalene, 1, 2, 3, 4, 4a, 5, 6, 8a-octa-hydro- 18.085 3.15 

α-Cadinol 18.292 2.98 

Tricyclo [5.2.2.0 (1, 6)] undecan-3-ol, 2-methyl 18.513 1.24 

6-Isopropenyl-4, 8a-dimethyl-1, 2, 3, 5, 6, 7-hexahydroxyl 18.828 0.11 

Aristolene epoxide 19.657 0.18 

Z, Z, Z-4, 6, 9-Nodadecatriene 20.035 0.06 

2-Pentadecanone, 6, 10, 14-trimethyl- 20.611 0.04 

Phallic acid, cyclohexyl isohexyl ester 21.078 0.06 

Pentadecacanoic, 14-methyl-, methyl- 22.048 0.17 

1, 2-Benzenedicarboxylic acid, butyl 2-ethyl 22.888 0.34 

3-Methyl-2-pent-2-enyl-cyclopent-2-enon 22.484 0.33 

1H-Naphtho [2, 1-b] pyrane, 3-ethenydodecane 24.040 9.83 

9H-Naphtho [2, 1-b] pyran-9-one, 3-ethenyl 31.735 0.71 

Pregnanetriol 36.284 2.23 

Tritetracontane 41.810 0.08 

1, 2-Benzenedicarboxylic acid, diisooctyl 45.078 0.18 

Benzaldehyde 6.615 0.35 

Total (Sixty two) 92.27 

The essential oils of Stevia rebaudiana Bertoni 

showed marked antifungal effects against all the 

fungi tested [23] . This activity could be attributed to 

the presence of α-cadinol (2.98 %), caryophyllene 

oxide (1.23 %), (-)-spathulenol (2.21 %), β-guaiene 

(0.32 %), ledene oxide-(ΙΙ), geranyl vinyl ether, 

tricyclo [5.2.2.0 (1, 6)] undecan-3-ol, indole, 

aristolene epoxide, 1, 2, 3, 5, 6, 7, 8, 8a-octahydro-1, 

4-dione and 2, 6, 6-trimethyl-2-cyclohexene-1, 

4-dione which significantly inhibited the growth of 

all the phytopathogens tested, and/or other major and 

minor oxygenated monoterpenes and sesquiterpenes 

present in the oils [23] . 

In spite of this, most of these oils are available for 

purchase on their own or as a part of pharmaceutical 

or cosmetic products, indicating that toxic effects 

do not prevent their use. However, the ongoing 

investigation of their toxic or irritant properties is 

imperative, especially when considering any new 

products for human use, be it medicinal or otherwise. 

In our opinion, the major components of 

Stevia rebaudiana Bertoni essential oils are 

α-cadinol (2.98 %), caryophyllene oxide (1.23 %), 

(-)-spathulenol (2.21 %) and β-guaiene (0.32 %) 

and they exhibit important anti-fungal activities. 

These anti-fungal activities have also been 

reported by others [24] . However, the more minor 

components,such as ledene oxide-(ΙΙ), geranyl vinyl 

ether, tricyclo [5.2.2.0 (1, 6)] undecan-3-ol, indole, 

aristolene epoxide, 1, 2, 3, 5, 6, 7, 8, 8a-octahydro-1, 

4-dione and 2, 6, 6-trimethyl-2-cyclohexene-1, 

4-dione, also contribute to the antifungal activity of 

these oils [23] . 

Conclusion 

A quantitative analysis of the essential oil from 

the leaves of Bangladeshi Stevia rebaudiana Bertoni 

showed that mono and sesquiterpenes are major known 

constituents (about 50 compounds) [21-23] out of the 

62 compounds identified. Some of these compounds 

were identified for the first time in this plant (ledene 

oxide-(ΙΙ), geranyl vinyl ether, tricyclo [5.2.2.0 (1, 6)] 

undecan-3-ol, indole, aristolene epoxide, 1, 2, 3, 5, 6, 

7, 8, 8a-octahydro-1, 4-dione and 2, 6, 6-trimethyl- 

2-cyclohexene-1, 4-dione). It is obvious that Stevia 

rebaudiana Bertoni grown in Bangladesh possesses 

60



certain specific characteristics due to the cultivation 

conditions of the local plantations i.e., location, soil, 

climate and plant growing conditions. 

Acknowledgement 

We are grateful to Dr. M. Ali, Chief Scientific 

Officer and Head, Chemistry Division, Atomic 

Energy Centre, Ramna, Dhaka, for his continuous 

encouragement during the work and all laboratory 

facilities. We also thank Mr. Zahidul Islam and Mr. 

Ayub Ali for their help with sample preparation. 

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