Al- Mustansiriya J.

Sci

Vol. 21, No 4, 2010

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

of Active Compounds Produced from Rosemary
(Rosmarinus Officinalis L.) Callus and Leaf Extracts
Salah K. Mahmoud
Foundation of Technical Education
Received 28/9/2009 Accepted 5/10/2010

( Rosmarinus officinalis L.)

.
. / BA 2,4-D MS
. 1780 BA / 0.5 2,4-D / 2
GC-MS
(%2.66) cineole (%4.53) ferruginol (%3.88) camphor(%5.85) isocarnosol:
.(%1.50) borneol( %2.50) verbenone

quinoline .
.

ABSTRACT
The yield of rosemary (Rosmarinus officinalis L.) from active compounds was
investigated. Yield of callus tissue was compared with the intact plant production.
Callus was induced in leaf explants and maintained on Murashige and Skoog
medium (MS) supplemented with 2,4-D and BA. Maximum callus fresh weight was
obtained in the combination of 2 mg/l 2,4-D and 0.5 mg/l BA under 16/8 hrs
photoperiod, which reached 1780 mg. The GC-MS analysis of leaf ethanolic
extracts revealed the most abundant components; isocarnosol (5.85%), camphor
(3.88%), ferruginol (4.53%), cineole (2.66%), verbenone (2.50%) and borneol
(1.50%). The chemical analysis of callus ethanolic extracts showed the most
compounds but at higher percentages than in leaf extracts. Quinoline alkaloids, were
found for the first time in callus have been of great therapeutic value in
pharmaceutical industries.

INTRODUCTION
Rosemary (Rosmarinus officinalis) is a very important medicinal
plant which belongs to the family Lamiaceae, has been cultivated for a
long time [1]. Rosemary is widely used as a culinary spice, and is also
used for its fragrance in soaps and cosmetics. Traditionally, rosemary
has been used medicinally to improve memory, relieve muscle pain and
spasm, stimulate hair growth, and support the circulatory and nervous
systems [2]. The family Lamiaceae contains an extremely wide variety
of aromatic plants, among this rich array of plants yielding essential oils
[3].

111

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Active Compounds Produced From

Rosemary (Rosmarinus Officinalis L.) Callus and Leaf Extracts
Salah

Labiates are known for their essential oils common to many

members of the family. Many of the active essential oils have been
isolated from various members of this family. Plant cells and callus
cultures have been extensively used to explore the possibility of
producing useful secondary metabolites through biotechnology
methods. The induction of callus growth and subsequent differentiation
is accomplished by the differential application of growth regulators and
the control of conditions in the culture medium. With the stimulus of
endogenous growth substances or by addition of exogenous growth
regulators to the nutrient medium, cell division, cell growth and tissue
differentiation are induced [4].
Plants are the traditional source for many chemicals used as
pharmaceuticals. Medicinal plants are used in crude or purified form in
the preparation of drugs in different systems [5].
The leaves of rosemary contain between (1.0-2.5)% essential oil,
such composition may markedly vary according to the chemo type and
the development stage at which the plant has been harvested. It is an
almost colorless to pale yellow liquid with a characteristics, refreshing
and pleasant odor [6].
Some of the more commonly lavender, sage, rosemary, eucalyptus,
jasmine, lemon, orange, rose and tea tree oil [7]. Rosemary oils are
largely used in traditional medicine, in perfumery, phytocosmetic and in
liquor manufacturing. EOs composition of rosemary comprise more
than sixty individual components of which major substances can
constitute up to 89% of the EOs [8].
Essential oils are produced using several techniques. Distillation uses
water and steam to remove the oils from dried or fresh plants, and the
expression method uses machines to squeeze the oil out of the plants.
Other techniques may use alcohol or solvents to remove essential oils
from plant materials [9].

MATERIALS AND METHODS

This study was carried out in the plant tissue culture lab., Biotech.
Dept., Al-Nahrain University during the period 1/3/2009-1/9/2009.
Rosemary plant was collected from the gardens belongs to College of
Science-Baghdad University.
Callus induction and maintenance
After surface sterilization, leaf explants were cut at the ends into
sections of approximately 1cm in length. Explants were placed on the
medium in Petri-dishes (100 x 15 mm). For callus induction the basal
medium was supplemented with various concentrations 0.0, 0.5, 1.0 or
2.0 mg/l of 2,4-D, BA at the concentrations 0.0, 0.2, 0.5 or 1.0 mg/l.
For maintenance of callus cultures, the friable callus obtained from leaf
112

Al- Mustansiriya J. Sci

Vol. 21, No 4, 2010

explants of rosemary were subcultured in 16 hrs photoperiod at 252C

on agar solidified MS medium containing 2.0 mg/l 2,4-D and 0.5 mg/l
BA and 3% sucrose was added.
Preparation of leaf ethanol extracts
Powdered rosemary leaves (50 g) were soaked into 250 ml of 70%
ethanol. The mixture was kept for 24 hrs in tightly sealed vessels at
room temperature, protected from sunlight and mixed several times with
a sterile glass rod, the mixture was then filtered through Wattman no.1
filter paper. The extracted liquid was subjected to rotary evaporation in
order to remove the ethanol [10].
Preparation of callus ethanol extracts
Callus was dried at 40C for 24 hrs, ground into powder using
grinding machine or a mortar, then subjected to extraction. A quantity
of 10 g of callus powder was extracted with 50 ml of 70% ethanol by
soxhlet apparatus for 6 hrs at 60C. The solution then evaporated to
dryness as mentioned above, then the extract was stored at 4C in
refrigerator for future use [11].
Gas Chromatography/ Mass Spectrometry Analysis (GC-MS)
Analysis of the essential oils was performed using GC-MS (Agilent
6780-USA), equipped with mass selective detector and HP-5 MS
capillary column (30m x 0.25mm, film thickness 0.25 m). For GC-MS
detection electron ionization system with an ionization energy of 70 eV
was used. Samples were injected automatically in the split mode at split
ratio of 1:100. All the tests were performed in triplicate.

RESULTS AND DISCUSSION

Maintenance of callus cultures
Table (1) revealed that the addition of 2,4-D exhibited a positive
effect on rosemary callus growth at the concentration of 2.0 mg/l in
combination with 0.5 mg/l of BA. Inclusion of 2,4-D at the
concentration of 2.0 mg/l gave significantly higher callus fresh weight
(1099.2) mg than other concentrations, while the lowest fresh weight
(75.5) mg obtained in 2,4-D free medium. Addition of 0.5 or 1.0 mg/l of
2,4-D gave 450.7, 814.2 mg of callus fresh weight.

113

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Active Compounds Produced From

Rosemary (Rosmarinus Officinalis L.) Callus and Leaf Extracts
Salah

Table-1: Effect of different concentrations of 2,4-D and BA and their interactions on

rosemary callus fresh weight (mg) grown on MS maintenance medium at 16 hrs
photoperiod (initial callus weight was 50 mg)
2,4-D
(mg/l)
0.0
0.5
1.0
2.0
Mean
LSD 0.05

BA (mg/l)
0.0
0.2
0.5
52.8
56.4
112.0
406.4
492.1
776.4
559.2
878.5
1228.9
667.1
1103.5
1780.0
421.4
632.6
974.3
BA: 21.205*
2,4-D: 21.205*

1.0
Mean
81.0
75.5
127.
450.7
590.2
814.2
846.4
1099.2
411.3
BA x 2,4-D: 42.41*

The highest callus fresh weight obtained from BA treatment (974.3)

mg was at the concentration 0.5 mg/l. Treatment with 1.0 or 2.0 mg/l of
BA gave 411.3 or 632.6 mg of callus fresh weight as compared to the
control which recorded 421.4 mg. The interaction between 2,4-D and
BA at the treatments 2.0 mg/l or 1.0 mg/l of 2,4-D with 0.5 mg/l of BA,
resulted in maximum callus fresh weight (1780.0) mg or (1228.9) mg
respectively.
The interaction between 2,4-D at the concentration of
2.0 or 1.0 mg/l and BA at the concentration of 1.0 mg/l gave 846.4 or
590.2 mg of callus fresh weight. The interaction between 0.5 mg/l of
2,4-D and 1.0 mg/l of BA gave 127.0 mg of callus fresh weight, which
was less than the control treatment, whereas the interaction between 0.5
mg/l of 2,4-D and 0.2 mg/l of BA gave higher callus fresh weight which
reached 492.1 mg. All the treatments were significantly higher than the
control (52.9) mg. The results are in agreement with [12] who proved
that rosemary callus grown on solid MS medium containing 2.0 mg/l
2,4-D increases in size. [13] found that induction and maintenance of
callus was obtained in MS basal medium supplemented with 2.0 mg/l
2,4-D and 0.5 mg/l BAP, and the leaf segment showed the best response
for callus Induction of Flacourtia jangomas Lour. [14] also studied the
effect of different concentrations of 2,4-D in the presence and absence
of calcium silicate on callus formation of three rice genotypes. Among
the combinations (1-2) mg/l of 2,4-D were found the best for callus
induction (100%) and maintenance. Cultures grown on MS medium
under a photoperiod of 16 hrs were mostly, friable, grew fast and large
in size. Light has been shown to be an important regulator in various
plant species, light has significantly enhanced callus initiation, callus
fresh weight was light dependent, this may be due to the influence of
both irradiation and light quality on callus cultures which is in
agreement with [15]. The daylight fluorescent tubes give important
emission bands at red and blue spectral wavelength. These conditions
have been found to give the best culture growth rates [16].

114

Al- Mustansiriya J. Sci

Vol. 21, No 4, 2010

GC-MS analysis of rosemary leaf extracts

Rosemary leaf ethanol extracts were tested by GC-MS, their chemical
composition showed about 12 components. Table (2) and figure (1)
showed that the most abundant component was isocarnosol (5.85%).
The extract also contain camphor (3.88%), ferruginol (4.53%), cineole
(2.66%), verbenone (2.50%) and borneol (1.56%) as a major
components. The composition of rosemary leaf extracts was
qualitatively similar to those obtained by other authors [17, 18] but with
different quantitative composition.
Table -2: Retention time and peak area (%) of the different compounds found in
rosemary leaf extracts analyzed by GC-MS
Compound
Cineole
-caryophyllene
Camphor
Borneol
Terpinen-4-ol
Non-identified
Verbenone
2-methyl-4-vinylphenol
Eugenol
Pyrazolobis (bbn) thiolium
Ferruginol
Isocarnosol
Non identified components

RT (min)
8.80
11.71
13.36
14.44
14.88
15.59
16.26
21.09
22.85
57.58
58.70
61.69

Peak area%
2.66
0.40
3.88
1.56
0.25
0.59
2.50
0.15
0.09
9.65
4.53
5.85
67.89

These differences in the chemical composition could be attributed to

climatic effects on the plants, besides the following factors should be
considered when observing differences between studies; genotypic and
environmental differences within species, sample extraction time and
extraction technique used to obtain the rosemary extract [19].

115

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Active Compounds Produced From

Rosemary (Rosmarinus Officinalis L.) Callus and Leaf Extracts
Salah

Figure -1: Chromatogram showing the chemical composition of rosemary leaf

ethanolic extract analyzed by GC

GC-MS analysis of rosemary callus extracts

The GC-MS analysis of untreated callus of rosemary plant was
revealed the main constituents, their relative percentage of the total peak
area and retention times as shown in Table (3) and figure (2). The data
indicate that 18-21 components were identified in callus ethanol extract.
The major constituents being, cineole (4.44%), camphor (2.51%),
verbenone (2.30%), borneol (1.91%) and caryophyllene (0.64%). Callus
extract also contained Ferruginol (1.59%), isocarnosol (1.27%), Stearic
acid (0.71%), phytol (9.12%),qiunoline (16.78%) and many other nonidentified components.
Table -3: Retention time and peak area (%) of the different compounds found in
rosemary callus extracts analyzed by GC-MS
Compound
Cineole
Camphor
Borneol
Verbenone
5-oxymethylfurfurol
Eugenol
Caryophyllene
Ferruginol
Isocarnosol
Stearic acid
Bornyl acetate
Phytol
Quinoline
Dihydronomorphinone
Sclareol
Non identified components

RT (min)
9.36
15.10
16.58
19.04
22.29
23.66
31.59
58.07
61.89
62.35
64.41
64.82
67.11
67.47
67.78

116

Peak area%
4.44
2.51
1.91
2.30
0.72
1.66
0.64
1.59
1.27
0.71
1.66
9.12
16.78
3.73
0.71
53.98

Al- Mustansiriya J. Sci

Vol. 21, No 4, 2010

The data showed that callus extract produced high percent for some
constituents as compared with the same constituents in leaf extract.
Cineole percentage was reached 4.44% in callus extract of rosemary,
while it was found 2.66% in leaf extract. Borneol and caryophyllene
percentage was 1.91%, 0.64% in callus extract, while it was recorded
1.56%, 0.40% in leaf extract respectively. Eugenol percentage was
1.66% in callus, while it was trace in leaf (0.09%). Other constituents
such as, isocarnosol, bornyl acetate, phytol, sclareol and quinoline were
found only in callus extract at the percentages of 1.27, 1.66, 9.12, 0.71
and 16.68% respectively, while they were absence in rosemary leaf
extract. It was clear that callus extract of rosemary plant contain more
constituents than that found in leaf extract and at higher percentages.
Quinoline alkaloids (which was found in callus) have been of great
therapeutic value, the antimalarial activity of these alkaloids has been
documented, besides their pharmaceutical uses, they are used frequently
in the food and soft drink industry [20].

min
Figure -2: Chromatogram showing the chemical composition of rosemary callus
ethanol extract analyzed by GC

REFERENCES
1- Eva, S. B.; Maria, H. T.; Attila, H.; Csilla, R.; and Szollosi, V. Antioxidant
effect of various rosemary (Rosmarinus officinalis L.) clones. Acta
Biologica szegediensis., 47(1-4): 111-113(2003).
2- Foster, S. and Tyler, V. The honest herbal; A sensible guide the use of
herbs and related remedies. 4th ed. Haworth Herbal Press., 321-322(1999).
3- Chalchate, J. C. O.; Garry, R. P.; Michet, A.; Benjilali, B. and Chabart, J. I.
Essential oils of rosemary (Rosmarinus officinalis L.). The chemical

117

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Active Compounds Produced From

Rosemary (Rosmarinus Officinalis L.) Callus and Leaf Extracts
Salah

composition of oils of various origins (Morocco, Spain, France). J.

Essential Oil Rosmarinus., 5: 613-618(1993).
4- Leena, T. and Jaindra, N. T. Role of biotechnology in medicinal plants.
Tropical Journal of Pharma. Res., 2(2): 243-253(2003).
5- Robins, R. L. Secondary products from cultured cells and organs:
Molecular and cellular approaches. In: Dixon, R. A., Gonzates, R. A. (eds).
Plant Cell Culture. Oxford; IRL, Press(1999).
6- Bauer, K.; Garbe, D. and Surburg, H. Common Fragrance and Flavor
Materials. 3rd ed. Germany: Wiley-VCH(1997).
7- Hayashi, K.; Kamiya, M. and Hayashi, T. Virucidal effects of the steam
distillate from Houttuynia cardata and its components on HSV-1,
influenza virus and HIV. Planta. Med., 61: 237-241(1995).
8- Burt, S. Essential oils; their antibacterial properties and potential
application in foods. A review. International J. of Food Microbiol., 94:
223-253(2004).
9- Mounchid, K.; Bourjilat, F.; Dersi, N.; Aboussaauira, T.; Rachidai, A.;
Tantaoul,-Elaraki, A. and Alaoui-Ismaili, M. Toxicity of south Morocco
Rosmarinus officinalis essential oil: antibacterial and histopathological
effects. Les Actes del'institut Agronomique et Veterinaire., (2-3): 139144(2004).
10- Akueshi, C. O.; Kadiri, C. O.; Akueshi, E. U.;Agina, S. E. and
gurukwem, B. Antimicrobial potential of Hyptis sauvedens Poit
(Lamiaceae), Nigeria. J. Bot., 15: 37-41(2002).
11- Harborne, J. B. Phytochemical Methods. A guide to modern technique of
plant analysis, Chapman Hall, London(1984).
12- Caruso, J. L.; Callahan, J.; DeChant, C.; Jayasimhulu, K. and Winget, G.
D. (2000). Carnosic acid in green callus and regenerated shoots of
Rosmarinus officinalis. Plant Cell Rep. Vol., 19: 500-503.
13- Chandra, I. and Bhanja, P. Study of organogenesis in vitro from callus
tissue of Flacourtia jangomas (Lour.). Current Science, 83. Burdwan
University, India(2002).
14- Islam, M. M.; Ahmed, M. and Mahaldar, D. In vitro callus induction and
plant regeneration in seed explants of rice (oryza sativa L.). Research J. of
Agric. and Biol. Sci., 1: 72-75(2005).
15- Senger, H. The effect of blue light on plants and microorganisms.
Phytochem., 35: 911-920(1982).
16- Roland, R.; Davin, C. and Zryd, J. Betalain production cell cultures of
Beta vulgaris L. Var. Bikores monogerm (Red beet). In vitro Cell. Dev.
Biol., 28: 39-45(1992).
17- Panizzi, L. Composition and antimicrobial properties of essential oils of
four Mediterranean Lamiaceae. J. of Ethnopharm. Livorno / Pisa., 39:
167-170(1993).
18- Aziza, K. G.; Haiko, H.; Artur, S. J. and Simone, M. S. Rosemary
(Rosmarinus officinalis) a study of composition, antioxidant and
antimicrobial activities of extracts obtained with supercritical carbondioxide. Cienc. Technol. Ailment., Campinas., 28(2): 463-469(2008).
19- Gachkar, L. Chemical and biological characteristics of Cuminum
cyminum and Rosmarinus officinalis essential oils. Food Chem., 102: 898902(2007).
20- Ramawat, K. G. Plant Biotechnology. S. Chand and company LTD, Ram
Nagar, New Delhi(2008).

118