Chemical and Antimicrobial Studies of Cupressus sempervirens L. and C. horizentalis Mill. Essential Oils

Document Type: Research Paper

Authors

1 Department of Medicinal Chemistry, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

2 Department of Pharmacognosy, Faculty of Pharmacy, and Biotechnology Research Center,Mashhad University of Medical Sciences, Mashhad, Iran

3 Department of Chemistry, Faculty of Sciences, Ferdowsi University, Mashhad, Iran

Abstract

      The essential oils of fresh fruits and terminal branches with adherent leaves of Cupressus sempervirens L. and C. horizentalis Mill., growing in Iran, were analyzed by gas chromatography-mass spectroscopy. The oils were also screened for bacteriostatic and fungistatic activities. Thirteen and ten components were identified in the essential oils of C. sempervirens and C. horizentalis, respectively. The main constituents of both fruits and leaves of C. sempervirens were a-pinene and -3-carene, while they were α-pinene and -2-carene for C. horizentalis. The essential oil of C. horizentalis leaves showed no antimicrobial activity against, Bacillus subtilis, Candida albicans, Escherichia coli and Staphylococcus aureus, while the essential oil of its fruits exhibited a weak effect on Bacillus subtilis. The essential oil of C. sempervirens leaves and fruits showed stronger antimicrobial activity against tested microorganisms compared to C. horizentalis essential oils. C. sempervirens fruits were relatively rich in tannins, and the leaves and fruits of C. horizentalis were quite rich in saponins and tannins. The amounts of flavonoids and alkaloids were not very high in both parts of these plants.  

Keywords


1. Introduction

 

     Cupressus sempervirens L. [C. fastigiata DC., C. sempervirens L. var. fastigiata Hansen], family Cupressaceae, is a monoecious and evergreen tree, up to 20-30 m high which grows in south and east of Europe, west part of Asia including various parts of Iran. Its Persian name is “Sarve Shirazi” or “Sarve Kashi” [1-8]. C. horizentalis Mill. [C.  sempervirens  L. var. horizentali (Mill.) Loud.] family Cupressaceae, is a monoecious and evergreen aromatic tree, up to 25 m high, broad and pyramidal growing; branches horizontal. This plant is widely grown from Crete to Iran. Its Persian name is “Zarbin” [1, 6-8]. C. sempervirens L. is a medicinal plant and act as expectorant and used externally for treating coughs and bronchitis [9]. There is no previous report on phytochemical characteristics and biological effects of these plants in Iran. However, a few phytochemical and biological studies on C. sempervirens L. in other parts of the world have been reported [10 -14].

 

     As a part of our systematic research on the chemical composition and antimicrobial properties of Iranian conifers, in this investigation, the essential oils content of fruits and terminal branches with adherent leaves (leaves) of both C. sempervirens and C. horizentalis was evaluated. In addition, the defatted ethanolic extracts of these organs were investigated qualitatively for the presence of alkaloids, flavonoids, saponins and tannins.

 

2. Materials and methods

 

2.1. Plant material

 

     The fruits and leaves of C. horizentalis Mill. were collected from Sorkesh forest at the height of 1000 m from the sea. This place is in province of Golestan which is located in the north of Iran. The fruits and leaves of C. sempervirens L. were collected from the National Botanical Garden of Iran (Tehran). The collected materials were stored at -20 °C in order to prevent changes in the chemical components [15].

 

      The plant material was identified by Dr. M. Assadi, Research Institute of Forest and Rangelands, Ministry of Jahad Keshavarzi, Iran. Voucher specimens have been deposited in the Herbarium of National Botanical Garden of Iran.

 

2.2. Isolation and analysis of non-volatile components

 

     The fruits and leaves of each plant (500 g) were dried at 50 °C and powdered separately. Each powder was defatted with petroleum ether (b.p. 40-60 °C) using Soxhlet apparatus (6 h). The chemical components of defatted powders were extracted by maceration with 70% ethanol (×4). The ethanolic extracts were concentrated at reduced pressure and analyzed for the presence of alkaloids [16], flavonoids [17], saponins [18] and tannins [19].

 

2.3. Isolation and analysis of volatile components

 

     Volatile oils were isolated from fresh plant material by wet steam distillation for 4 h [20]. The essential oils were separated from the aqueous layer and dried over anhydrous sodium sulfate and stored under nitrogen gas in a sealed vial and -20 °C until analyzed.

 

2.4. Gas chromatography-mass spectrometric analysis

 

     The  GC-MS  apparatus  consisted  of  a Varian 3400 GC equipped with a fused-silica column (DB-5, 30 m × 0.25 mm i.d., film thickness 0.25 mm; J&W Scientific Inc), and interfaced with a quadruple mass spectrometric detector (Incos50, Finnigan). The operating conditions were: Oven temperature 60-280 °C with the rate of 3 °C /min., injector mode: split injection; with the carrier gas, He; flow rate 2 ml/min.; ion source, 70 eV; ionization current, 750 mA; scan range, 40-300 u.

 

     The oil components were identified from their retention indices (RI) obtained with reference to n-alkane series (Sigma, UK) on DB-5 column, mass spectra with those of authentic samples, composition of their mass spectra and fragmentation patterns reported in literature [21], computer matching with MS-data bank (Saturn version 4). The peak area method was followed for quantitative determination of different constituents; the percentages were calculated relatively [21].

 

2.5. Antimicrobial activity determination

 

      The antimicrobial activity of each essential oil obtained from different parts of the plant was determined using five strains of microorganisms from the Persian Type Culture Collection (PTCC) [22]. The following microbial strains were used: Bacillus subtilis (PTCC 1023), Staphylococcus aureus (PTCC 1112), Escherichia coli (PTCC 1038), Pseudomonas aeroginosa (PTCC 1074) and Candida albicans (PTCC 5027). Minimum inhibitory concentrations (MICs) were determined using the agar dilution method [23]. Muller Hinton Agar medium (Oxoid, France) was prepared and sterilized by autoclaving for 20 min. at 121 °C. The medium was cooled to 50 °C and two-fold serial dilutions of the parent solution (essential oil plus 30% DMSO v/v) in adequate melted (50 °C) agar medium (1 ml) were prepared and added to 24-well plates (Greiner, France). The medium was allowed to solidify and then inoculated with the previously prepared microorganism suspension using the quadran streak method. For each strain tested, the adequacy of growth conditions, the effect of positive controls (gentamycin for bacteria and clotrimazole for fungus) and the sterility of the medium were tested in two wells. Plates were incubated for 24 h at 37 °C for the bacteria and 48 h at 25 °C for C. albicans.

 

3. Results

 

     The amount of non-volatile components in defatted ethanolic extracts of the fruits and leaves of C. sempervirens and C. horizentalis are shown in Table 1.

Table 1. Major non-volatile components of fruits and leaves of Cupressus sempervirens L. and C. horizentalis Mill.

 

Mill.

 

 

 

 

 

 

 

Chemical components

 

 

Average content*

 

 

 

 

 

 

C. sempervirens

C. horizentalis

 

 

 

Fruits

Leaves

Fruits

Leaves

 

Alkaloids

-

 

-

-

1+

 

Flavonoids

-

 

1+

1+

2+

 

Saponins

1+

1+

4+

4+

 

Tannins

2+

1+

4+

3+

 

 

 

 

 

* Average content was rated from - to 4+:1+, slightly positive; 2+, moderately positive; 3+, strongly positive; 4+, very strongly positive;

-,

not detected.

 

 

 

 

 

 

 


      The essential oils isolated separately from fruits and leaves of C. sempervirens and C. horizentalis were colorless with a strong odor. The fruits and leaves of C. sempervirens yielded 0.1% and 0.3% v/w of volatile oil while the fruits and leaves of C. horizentalis yielded 0.4% and 0.5% (v/w) of volatile oil, respectively.

 

     GC-MS analysis of leaves and fruits volatile oils of C. sempervirens led to the identification of 13 and 9 components, respectively (Table 2). However, for C. horizentalis leaves and fruits volatile oils 10 and 9 components were identified, respectively (Table 3).

Table 2. Chemical composition of the volatile oil of Cupressus sempervirens L. fruits and leaves.

 

Components

Retention

Leaves                        

Fruits    

 

Index*

oil (%)                         

oil (%)    


α-Pinene

940    

21.4                    

46.0     

Sabinene

974    

2.8                     

2.7       

β-Pinene

982    

   2.6                     

2.7       

Myrcene

997    

5.0                     

5.4       

-3-Carene

1018   

16.0                    

27.0      

Limonene

1035   

3.3                     

2.2        

Terpinolene

1092   

5.9                      

6.4        

α-Terpinyl acetate

1355   

5.9                      

2.7         

β-Caryophyllene

1416  

4.2                      

t           

α-Humulene

1454   

4.2                      

t           

Germacrene-D

1485  

13.0                     

2.1         

-Cadinene

1530  

1.6                      

t           

Cedrol

1601  

3.3                     

    t           

Grouped compounds:

 

 

 

Monoterpene hydrocarbons

 

57.0                    

92.4        

Oxygen-containing monoterpenes

 

5.9                     

2.7        

Sesquiterpene hydrocarbons

 

23.0                   

2.1       

Oxygen-containing sesquiterpenes

 

3.3                    

-


 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

t = trace (< 0.1 %)

* The retention indices were determined on DB-5 capillary column.


Table 3. Chemical composition of the volatile oil of Cupressus horizentalis. Mill. fruits and leaves.

Components

Retention

Leaves

Fruits

 

Index*

oil (%)

oil (%)

α-Pinene

941

46.2

59.2

Camphene

955

1.73

-

Sabinene

980

2.2

2.0

β-Pinene

985

3.3

2.8

Myrcene

990

4.6

3.4

-2-Carene

1005

22.7

14.9

Limonene

1034

2.8

2.4

Isoterpinolene

1080

3.7

3.2

α-Terpinyl acetate

1350

2.6

3.2

Germacrene-D

1482

6.3

2.1

Grouped compounds:

 

 

 

Monoterpene hydrocarbons

 

87.2

87.9

Oxygen-containing monoterpenes

 

2.6

3.2

Sesquiterpene hydrocarbons

 

6.3

2.1

 

 

 

 

 

 

 

 

 

 

 

 

 

* The retention indices were determined on DB-5 capillary column.


      The antimicrobial activity of essential oils of fruits and leaves of both plants were evaluated using the agar dilution method [23].Only, the essential oils of fruits of C. horizentalis had a weak effect on B. subtilis (Table 4). The essential oil of leaves and fruits of C. sempervirens exhibited mild antimicrobial activity against all of the tested microorganisms (Table 5).

 

Table 4. Antimicrobial activity of the essential oil of the fruits and leaves of Cupressus horizentalis. Mill.

 

Microorganism

 

Minimum inhibitory concentration

 

 

 

Fruits oil

Leaves oil

Gentamycin

Clotrimazole

 

 

(mg/ml)

(mg/ml)

(μg/ml)

(μg/ml)

Bacillus subtilis

50

100

0.5

-

 

Staphylococcus aureus

100

>100

0.8

-

 

Escherichia coli

100

>100

0.5

-

 

Pseudomonas aeroginosa

100

>100

0.7

-

 

Candida albicans

100

>100

-

8.0

 

 

Table 5. Antimicrobial activity of the essential oil of the fruits and leaves of Cupressus sempervirens L.

Microorganism

 

Minimum inhibitory concentration

 

 

Fruits oil

Leaves oil

Gentamycin

Clotrimazole

 

(mg/ml)

(mg/ml)

(μg/ml)

(μg/ml)

Bacillus subtilis

  25

50     

0.5

-

Candida albicans

  50

100    

-

8.0

Escherichia coli

  12.5

50     

0.5

-

Pseudomonas aeroginosa

  50

75    

0.7

-

Staphylococcus aureus

  6.25

25    

0.8

-








4. Discussion

 

     The presence of non-volatile components in defatted ethanolic extracts of leaves and fruits of both C. sempervirens and C. horizentalis were investigated. These extracts were examined for alkaloids, flavonoids, saponins and tannins. As it can be seen from Table 1, the amounts of non-volatile compounds in leaves of C. sempervirens were not significant but the fruits of this plant were relatively rich in tannins. Leaves and fruits of C. horizentalis were quite rich in saponins and tannins while the amounts of flavonoids and alkaloids were not very high in both parts of this species.

 

     In spite of the differences in the percentages of various components in the essential oils of fruits and leaves of these plants, the composition of the oils of leaves and fruits are fairly similar. Monoterpene hydrocarbons represented the most abundant constituents of the oil of the fruits and leaves of both plants (92.4% and 57.0% for fruits and leaves of C. sempervirens and 87.2% and 87.9% for leaves and fruits of C. horizentalis, respectively). The amounts of sesquiterpene hydrocarbons in leaves of these plants were higher than the amount in their fruits (23.0% and 2.1% for fruits and leaves of C. sempervirens and 6.3% and 2.1% for leaves and fruits of C. horizentalis, respectively). Oxygen-containing monoterpenes and sesquiterpenes were at relatively low levels in both fruits and leaves oils of C. sempervirens. No nonterpenic compounds were detected in these essential oils. Oxygen-containing monoterpenoids were low in leaves and fruits oils of C. horizentalis (2.6% for leaves and 3.2% for fruits), however, oxygenated sesquiterpenoids and nonterpenic constituents were not detected in the leaves and fruits of this species.

 

     The main components of C. horizentalis leaves and fruits essential oils were α-pinene (46.2%, 59.2%) and -2-carene (22.7%, 14.9%), respectively. On the other hand, the main components of both fruits and leaves essential oils of C. sempervirens were α-pinene, (46.0%, 21.4%) and -3-carene (27.0%, 16.0%), respectively.

 

     These findings are in general agreement with the previous reports [10, 11]. Although -3-carene was not a major compound in the reported investigation for C.  sempervirens [10], the amount of this compound in the essential  oil  of  this  plant  was  quite  high (27.0%  and  16.0%)  in  fruits  and  leaves, respectively.

      The antimicrobial activity of the examined essential oils varied at different concentrations against bacteria and yeast. The essential oil of leaves of C. horizentalis had no antimicrobial activity against B. subtilis, C. albicans, E. coli and S. aureus while the essential oil of fruits of this species showed a weak antimicrobial activity against B. subtilis. C. sempervirens leaves essential oil had a weak antimicrobial activity while the essential oil of fruits of this species exhibited stronger antimicrobial activity against all tested microorganisms. This is in general agreement with the Egyptian report indicating that the leave oil of C. sempervirens was active against P. aeroginosa and S. aureus, while it was inactive against B. subtilis and E. coil [12].

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