- Email: [email protected]
Analysis of Mount Atlas mastic smoke: A potential food preservative
Fitoterapia 81 (2010) 577–580
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Analysis of Mount Atlas mastic smoke: A potential food preservative Abdolali Mohagheghzadeh ⁎, Pouya Faridi, Younes Ghasemi Pharmaceutical Sciences Research Center and Department of Pharmacognosy and Traditional Pharmacy, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
a r t i c l e
i n f o
Article history: Received 1 August 2009 Accepted in revised form 23 January 2010 Available online 4 February 2010 Keywords: Antimicrobial activity Flavor Medicinal smokes Mount Atlas mastic Pistacia terebinthus Preservative Volatile oil
a b s t r a c t Pistacia terebinthus L. smoke has been used traditionally in Iran as disinfectant and air purifier. Smoke was collected by a simple method, and the chemical constituents and antimicrobial activity of the smoke were analyzed. The chemical constituents of the smoke were α-pinene (65.1%), limonene (11.5%) and allo-ocimene (2.8%). The non polar phase of smoke noticeably inhibited the growth of different microorganisms. MIC test shows that non polar fraction of smoke can inhibit the growth of some bacteria. The results indicating that the properties of the smoke as a flavoring and preservative agent could be a potential subject for future studies. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Historically, the application of smoke has been successfully used for food preservation, often in conjunction with other processes, such as cooking and drying [1]. In our recent review, we reported 10 plant species whose smoke is used in folk medicine as a preservative [2]. However, there is very little information available on the chemical composition and pharmacological activities of medicinal smokes [2]. One study identified the chemical composition of Peganum harmala (Esfand in Persian) seeds, used as an antiseptic in Persian traditional medicine. Furthermore its smoke showed good antimicrobial activity against twelve microorganisms [3]. In other study, Havan samagri, an important disinfectant medicinal smoke in Indian traditional medicine, showed very good antifungal activity [4]. Pistacia terebinthus (Anacardiaceae) is a perennial plant that grows widely in different parts of Iran and is called “chatlanghoush” in Persian [5]. Mount Atlas mastic (terebinth or Saghghez in Persian) is P. terebinthus oleo gum resin which is ⁎ Corresponding author. P.O. Box: 71345-1583. Tel.: + 98 711 2425374; fax: + 98 711 2426070. E-mail address: [email protected] (A. Mohagheghzadeh). 0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2010.01.022
a well known natural product used in medicine and food preparation. It has been used as a breath-sweetener and is used today to flavor a sweet confection used by smokers as a breath freshener [6]. It has been used for a long time in Achaemenid Persia and has been an important element in the traditional diet of young Persians [7]. This gum is also mentioned in the Bible [8]. Avicenna suggested that this oleo gum resin is an appetizer that dissolves phlegm and is astringent, rarefying, laxative, demulcent, diuretic, emmenagogue, and carminative. It is useful for beautifying skin and teeth, for treating visceral inflammation and scabies, and for fortifying the function of the stomach, liver and kidneys [9]. Today it is used by natural healers as a remedy for cancer, gastrointestinal disorders, motion sickness, and as a chewing gum in Iran [10,11]. Smoke from the Mount Atlas mastic or mastic is used, with or without Peganum harmala fruit, as an air purifier and antiseptic in Iran [1], while in Arabia and Morocco mastic smoke is used for flavoring water and water jars [12,13]. Because of the widespread use of this oleo gum resin and its smoke in traditional medicine as an antiseptic and flavoring, we decided to develop a simple method for producing and collecting its' medicinal smoke. Furthermore, we have analyzed the chemical composition of Mount Atlas mastic smoke and volatile oil markers for their products
578
A. Mohagheghzadeh et al. / Fitoterapia 81 (2010) 577–580
standardization, and the antimicrobial activity of the smoke and volatile oil were tested.
according to the method recommended in the British Pharmacopoeia [14].
2. Materials and methods
2.4. Gas chromatography/mass spectrometry
2.1. Plant material
The gas chromatography/mass spectrometry (GC/MS) analyses were carried out using a Hewlett Packard 6890 GC equipped with a HP-5 M capillary column (phenyl methyl siloxan, 25 m × 0.25 mm id, Hewlett–Packard Part no. 190915.433, USA). For the smoke, the oven temperature was programmed to rise from 30 °C (4 min.) to 300 °C at the rate of 3 °C/min. and finally held for 10 min. at 300 °C. For the volatile oil, the oven temperature was programmed to rise from 50 °C (3 min.) to 250 °C at the rate of 3 °C/min. and finally held for 10 min. at 250 °C. The carrier gas was helium with a flow rate of 1.2 ml/min. The MS (Hewlett–Packard 5973, USA) was operating in EI mode at 70 eV. The interface temperature was 250 °C; the mass range was 30–600 m/z. Identification of components was based on a comparison of their retention indices (RI) and mass spectra with Wiley (275) and Adams libraries spectra [15].
The oleo gum resin of Pistacia terebinthus L. was collected from plants growing wild in Kavar, some 70 km from Shiraz, Iran, in April, 2005. The plant material was identified by S. Khademian, and a voucher specimen was deposited in the Shiraz Faculty of Pharmacy herbarium (no. Pm7). 2.2. Smoke collection For the manufacture of smoke extracts, smoke from smoldering plant material (100 g) was conducted to an apparatus (Fig. 1). In this method, the temperature is ca. 300 °C. There is non-continuous air flow (1 puff per 10 min.) produced by Speedivac pump (England). The smoke produced (with an acidic pH) is trapped in a mixture of distilled water and n-hexane, and the mixture is continuously shaken. The solvent mixture was surrounded by a water and ice mixture that kept the temperature between 0–4 °C. The resulting extract in the hydrophobic phase was evaporated to yield 1% dark-brown viscous residue and reserved for chemical composition analysis. 2.3. Volatile oil distillation P. terebinthus oleo gum resin of (25 g) was hydrodistilled (250 ml water) for 4 h using a Clevenger-type apparatus
2.5. Antimicrobial activity 2.5.1. Disk diffusion method Bacillus subtilis, Salmonella typhi, Escherichia coli, Staphylococcus epidermidis, Pseudomonas aeruginosa had been produced in a suspension of physiological saline solution (0.9% w/v). Inoculums' concentration was 0.5 MacFarland (1.5 × 108 CFU/ml). The bacterial suspension was prepared to match the turbidity of the 0.5 McFarland turbidity standards. Nutrient agar was used as a growth medium, inoculated with
Fig. 1. Apparatus for smoke production and collection.
A. Mohagheghzadeh et al. / Fitoterapia 81 (2010) 577–580 Table 1 Chemical composition of Pistacia terebinthus smoke and volatile oil.
579
Table 3 Results of minimum inhibitory concentration (MIC) for Pistacia terebinthus oleo gum resin smoke (non polar phase).
Component
Smoke %
Volatile oil %
RI
α-Thujene α-Pinene Camphene Verbenene Sabinene β-Pinene β-Phellandrene γ-Terpinene p-Cymene Limonene 1,3,8-p-menthatriene β-Campholene-aldehyde Allo-ocimene trans-Pinocarveol cis-Verbenole trans-Verbenole Karahanaenone p-Mentha-1,5-dien 8-ol p-Cymene-8-ol Myrtenal Verbenone Monoterpene hydrocarbons Oxygen-containing monoterpenes Oxygen-containing sesquiterpenes Total
0.5 65.1 0.7 2.3 – 1.6 0.7 0.5 2.3 11.5 2.1 0.9 2.8 – – – – – – – 0.3 90.1 0.3 0.9 91.3
– 78.7 1.0 0.7 1.0 2.4 – – 0.7 1.6 – 1.4 – 1.9 1.2 5.3 0.6 1.3 0.4 0.4 1.2 86.1 11.7 2.0 99.8
935 942 956 972 979 985 1009 1020 1029 1036 1115 1129 1133 1142 1147 1149 1158 1169 1185 1197 1208
Microorganism
MIC, μL/mL
Pseudomonas aeruginosa Salmonella typhi Bacillus subtilis Shigella lexneri Escherchia coli Staphylococus epidermidis Proteus vulgaris Shigella dysenteriae
1.5625 3.125 1.5625 6.25 1.5625 3.125 6.25 1.5625
tion for 12 h at 37 °C, the first tube without turbidity was determined as the MIC. 3. Results and discussion
a lawn of test microorganisms, and 10 µl of smoke or volatile oil were placed in each disk. Gentamicin (10 µl) or ampicillin (10 µl), with corresponding solvents, were used as positive and negative controls. Plates were incubated at an appropriate temperature for bacteria (37 °C) for a period of 18–24 h. Studies were performed in triplicate. Samples with antimicrobial activity produced a distinct, clear, and circular zone of inhibition around the disc [16].
2.5.2. Minimum inhibitory concentration (MIC) Briefly, for minimum inhibitory concentration (MIC) according to reference [16], a microdilution broth susceptibility assay was used to evaluate antimicrobial activity of the non polar phase of smoke. The microorganisms were B. subtilis, S. typhi, E. coli, S. epidermidis, P. aeruginosa, Shigella lexneri, Proteus vulgaris, S. dysenteriae. To do this, 2 ml of a microbial suspension containing 5 × 105 CFU/ml of nutrient broth was prepared. Then according to the serial dilution different amounts of the non polar phase of smoke was added to each tube. One of the tubes contained no smoke and it was kept as positive control and the other one as a negative one which contained no microorganism. After bacterial incuba-
Here a simple method was developed (Fig. 1) for producing and collecting Mount Atlas mastic smoke simulated according to the folk culture. Corresponding volatile oil obtained with a yield of 1.7% (v/w). The constituents of the smoke and volatile oil are shown in Table 1. The components identified account for 91.3% of the smoke and 99.8% of the oil. The major components of Mount Atlas mastic smoke were α-pinene (65.1%), limonene (11.5%) and allo-ocimene (2.8%). Monoterpene hydrocarbons were also the major group in the smoke (90.1%). Our results showed that the major components of Mount Atlas mastic volatile oil were α-pinene (78.7%), trans-verbenole (5.3%) and β-pinene (2.4%) (Table 1). In a study [17], α-pinene (42.9%) and β-pinene (13.2%) were the major components, and in another study on the oil of air-dried resinous gum of P. terebinthus of Greek origin, major compounds were α-pinene (39.6%), β-pinene (19.5%), and sabinene (6.5%) [18]. In all three studies cited above, α-pinene is the major compound and Monoterpene hydrocarbons is the major group (86.1% in our study), but there are some differences in the concentration of α-pinene. A lower concentration of α-pinene and an increased proportion of limonene were found in the smoke as compared to the volatile oil. Comparison of the constituents of the smoke and volatile oil showed that 71.6% of the constituents and 8 compounds are similar. The possible of conversion of α-pinene to limonene (in an acidic pH) is similar to what occurs when the oil is processed using cold sulphuric acid in turpentine [19]. Limonene is used as a flavoring agent in food manufacturing and medicine, so the pleasant odor of the smoke may be due to limonene production in the smoke process.
Table 2 Antimicrobial activity of Pistacia terebinthus smoke and volatile oil. Sample
Smoke fraction Nonpolar Aqueous Volatile oil Gentamicin Ampicillin
Zone of inhibition Bacillus subtilis
Salmonella typhi
Escherichia coli
Staphylococcus epidermidis
Pseudomonas aeruginosa
14 mm 0 mm 8 mm 8 mm –
12 mm 0 mm 6 mm – 11 mm
15 mm 0 mm 9 mm – 9 mm
12 mm 0 mm 7 mm 8 mm –
14 mm 7 mm 11 mm – 7 mm
580
A. Mohagheghzadeh et al. / Fitoterapia 81 (2010) 577–580
In a study, the volatile oil of this oleo gum resin has shown good antimicrobial activity against resistant gram positive and negative clinical bacteria [20]. Table 2 shows the antimicrobial activity of the smoke and volatile oil of Mount Atlas mastic smoke. The non-polar smoke fraction had good antimicrobial activity in comparison with standards and volatile oil. So, minimum inhibitory concentration tests for non-polar phase were done. Results have shown that this material can inhibit gram positive and negative bacterial growth in a lower concentration than standard and it means that it has a good antimicrobial activity. As it is shown in Table 3 the smoke have good antimicrobial activity on all of microorganism especially on S. dysenteriae, E. coli, B. subtilis and P. aeruginosa. Today, liquid smoke, used as a preservative agent in food processing, is the aqueous phase of smoke [1], but in this study, the non-polar phase possessing considerable content of α-pinene and limonene had a better antimicrobial activity. The long use of P. terebinthus oleo gum resin smoke in traditional medicine and our findings indicate its potential as a future object of studies focusing on its use as a naturally origin material as a air purifier and for flavoring and preservation of foods. For further works, the carcinogenic activity of this smoke should be investigated. Acknowledgments This work was made possible by financial support from Shiraz University of Medical Sciences and Health Services. The authors wish to acknowledge the helpful assistance of Dick Edelstein and AuthorAid of the Eastern Mediterranean. This work was a part of a Pharm.D thesis. References [1] Holley RA, Patel D. Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials. Food Microbiol 2005;22:273–92.
[2] Mohagheghzadeh A, Faridi P, Shams-Ardakani M, Ghasemi Y. Medicinal smokes. J Ethnopharmacol 2006;108:161–84. [3] Shahverdi AR, Monsef-Esfahani HR, Nickavar B, Bitarafan L, Khodaee S, Khoshakhlagh N. Antimicrobial activity and main chemical composition of two smoke condensates from Peganum harmala seeds. Z Naturforsch C 2005;60:707–10. [4] Nautiyal CS, Chauhan PS, Nene YL. Medicinal smoke reduces airborne bacteria. J Ethnopharmacol 2007;114:446–51. [5] Davis PH. Flora of Turkey and The East Aegean Islands, vol. 2. Edinburgh: Edinburgh University Press; 1967. [6] Shipley G. Human landscapes in classical antiquity: environment and culture. Routledg; 1996. http://books.google.com/books?id=3kYhEehfAAC, accessed 17 June 2009. [7] Dalby A. Food in the ancient world from A to Z. Routledge; 2003. p. 323–4. http://books.google.com/books?id=FtIXAe2qYDgC&printsec= frontcover#PPR6,M1, accessed 17 June 2009. [8] Duke JA, Duke PK, duCellie JL. Duke's Handbook of Medicinal Plants of The Bible. New York: CRC Press; 2008. [9] Avicenna, Al Qanun Fil Tibb, vol. 2. English translation by H.A. Hameed (1998). S.Waris Nawab, Senior Press Superintendent, New Delhi: Jamia Hamdard Printing Press; 1024. [10] Hooper D, Field H. Useful plants and drugs of Iran and Iraq. Botanical Series, vol. 9. Chicago: Field Museum of Natural History; 1937. [11] Amin G. Popular medicinal plants of Iran. Tehran: Iranian Research Institute of Medicinal Plants; 2005. [12] Mastic. Wikipedia. http://en.wikipedia.org/wiki/Mastic, 2009 accessed 17 June 2009. [13] Encyclopedia of Spices. Mastic. http://www.theepicentre.com/Spices/ mastic.html2008 accessed 17 June 2009. [14] British Pharmacopoeia, vol. 2. London: HMSO; 1988. p. 137–8. [15] Adams RP. Identification of Essential Oil Components by Gas Chromatography Quadrupole Mass Spectroscopy. Carol Stream: Allured Publishing Co; 2004. [16] Mahon CR. Textbook of Diagnostic Microbiology. London: W. B. Saunders; 1995. [17] Barrero AF, Herrador MM, Arteaga JR, Akssira M, Mellouki F, Belgarrabe A, Blizquez MA. Chemical composition of the essential oils of Pistacia atlantica Desf. J Essent Oil Re 2005;17:52–4. [18] Papageorgiou V, Assimopoulou AN, Yannovits-Argiriadis N. Chemical composition of the essential oil of Chios turpentine. J Essent Oil Res 1999;11:367–8. [19] Evans WC. Treas and Evans’ Pharmacognosy. 15th ed. W.B. Edinburg: Saunders; 2002. [20] Ghalem BR, Mohamed B. Essential oil from gum of Pistacia atlantica Desf.: Screening of antimicrobial activity. Afr J Pharm Pharmacol 2009;3:87–91.
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Analysis of Mount Atlas mastic smoke: A potential food preservative Abdolali Mohagheghzadeh ⁎, Pouya Faridi, Younes Ghasemi Pharmaceutical Sciences Research Center and Department of Pharmacognosy and Traditional Pharmacy, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
a r t i c l e
i n f o
Article history: Received 1 August 2009 Accepted in revised form 23 January 2010 Available online 4 February 2010 Keywords: Antimicrobial activity Flavor Medicinal smokes Mount Atlas mastic Pistacia terebinthus Preservative Volatile oil
a b s t r a c t Pistacia terebinthus L. smoke has been used traditionally in Iran as disinfectant and air purifier. Smoke was collected by a simple method, and the chemical constituents and antimicrobial activity of the smoke were analyzed. The chemical constituents of the smoke were α-pinene (65.1%), limonene (11.5%) and allo-ocimene (2.8%). The non polar phase of smoke noticeably inhibited the growth of different microorganisms. MIC test shows that non polar fraction of smoke can inhibit the growth of some bacteria. The results indicating that the properties of the smoke as a flavoring and preservative agent could be a potential subject for future studies. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Historically, the application of smoke has been successfully used for food preservation, often in conjunction with other processes, such as cooking and drying [1]. In our recent review, we reported 10 plant species whose smoke is used in folk medicine as a preservative [2]. However, there is very little information available on the chemical composition and pharmacological activities of medicinal smokes [2]. One study identified the chemical composition of Peganum harmala (Esfand in Persian) seeds, used as an antiseptic in Persian traditional medicine. Furthermore its smoke showed good antimicrobial activity against twelve microorganisms [3]. In other study, Havan samagri, an important disinfectant medicinal smoke in Indian traditional medicine, showed very good antifungal activity [4]. Pistacia terebinthus (Anacardiaceae) is a perennial plant that grows widely in different parts of Iran and is called “chatlanghoush” in Persian [5]. Mount Atlas mastic (terebinth or Saghghez in Persian) is P. terebinthus oleo gum resin which is ⁎ Corresponding author. P.O. Box: 71345-1583. Tel.: + 98 711 2425374; fax: + 98 711 2426070. E-mail address: [email protected] (A. Mohagheghzadeh). 0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2010.01.022
a well known natural product used in medicine and food preparation. It has been used as a breath-sweetener and is used today to flavor a sweet confection used by smokers as a breath freshener [6]. It has been used for a long time in Achaemenid Persia and has been an important element in the traditional diet of young Persians [7]. This gum is also mentioned in the Bible [8]. Avicenna suggested that this oleo gum resin is an appetizer that dissolves phlegm and is astringent, rarefying, laxative, demulcent, diuretic, emmenagogue, and carminative. It is useful for beautifying skin and teeth, for treating visceral inflammation and scabies, and for fortifying the function of the stomach, liver and kidneys [9]. Today it is used by natural healers as a remedy for cancer, gastrointestinal disorders, motion sickness, and as a chewing gum in Iran [10,11]. Smoke from the Mount Atlas mastic or mastic is used, with or without Peganum harmala fruit, as an air purifier and antiseptic in Iran [1], while in Arabia and Morocco mastic smoke is used for flavoring water and water jars [12,13]. Because of the widespread use of this oleo gum resin and its smoke in traditional medicine as an antiseptic and flavoring, we decided to develop a simple method for producing and collecting its' medicinal smoke. Furthermore, we have analyzed the chemical composition of Mount Atlas mastic smoke and volatile oil markers for their products
578
A. Mohagheghzadeh et al. / Fitoterapia 81 (2010) 577–580
standardization, and the antimicrobial activity of the smoke and volatile oil were tested.
according to the method recommended in the British Pharmacopoeia [14].
2. Materials and methods
2.4. Gas chromatography/mass spectrometry
2.1. Plant material
The gas chromatography/mass spectrometry (GC/MS) analyses were carried out using a Hewlett Packard 6890 GC equipped with a HP-5 M capillary column (phenyl methyl siloxan, 25 m × 0.25 mm id, Hewlett–Packard Part no. 190915.433, USA). For the smoke, the oven temperature was programmed to rise from 30 °C (4 min.) to 300 °C at the rate of 3 °C/min. and finally held for 10 min. at 300 °C. For the volatile oil, the oven temperature was programmed to rise from 50 °C (3 min.) to 250 °C at the rate of 3 °C/min. and finally held for 10 min. at 250 °C. The carrier gas was helium with a flow rate of 1.2 ml/min. The MS (Hewlett–Packard 5973, USA) was operating in EI mode at 70 eV. The interface temperature was 250 °C; the mass range was 30–600 m/z. Identification of components was based on a comparison of their retention indices (RI) and mass spectra with Wiley (275) and Adams libraries spectra [15].
The oleo gum resin of Pistacia terebinthus L. was collected from plants growing wild in Kavar, some 70 km from Shiraz, Iran, in April, 2005. The plant material was identified by S. Khademian, and a voucher specimen was deposited in the Shiraz Faculty of Pharmacy herbarium (no. Pm7). 2.2. Smoke collection For the manufacture of smoke extracts, smoke from smoldering plant material (100 g) was conducted to an apparatus (Fig. 1). In this method, the temperature is ca. 300 °C. There is non-continuous air flow (1 puff per 10 min.) produced by Speedivac pump (England). The smoke produced (with an acidic pH) is trapped in a mixture of distilled water and n-hexane, and the mixture is continuously shaken. The solvent mixture was surrounded by a water and ice mixture that kept the temperature between 0–4 °C. The resulting extract in the hydrophobic phase was evaporated to yield 1% dark-brown viscous residue and reserved for chemical composition analysis. 2.3. Volatile oil distillation P. terebinthus oleo gum resin of (25 g) was hydrodistilled (250 ml water) for 4 h using a Clevenger-type apparatus
2.5. Antimicrobial activity 2.5.1. Disk diffusion method Bacillus subtilis, Salmonella typhi, Escherichia coli, Staphylococcus epidermidis, Pseudomonas aeruginosa had been produced in a suspension of physiological saline solution (0.9% w/v). Inoculums' concentration was 0.5 MacFarland (1.5 × 108 CFU/ml). The bacterial suspension was prepared to match the turbidity of the 0.5 McFarland turbidity standards. Nutrient agar was used as a growth medium, inoculated with
Fig. 1. Apparatus for smoke production and collection.
A. Mohagheghzadeh et al. / Fitoterapia 81 (2010) 577–580 Table 1 Chemical composition of Pistacia terebinthus smoke and volatile oil.
579
Table 3 Results of minimum inhibitory concentration (MIC) for Pistacia terebinthus oleo gum resin smoke (non polar phase).
Component
Smoke %
Volatile oil %
RI
α-Thujene α-Pinene Camphene Verbenene Sabinene β-Pinene β-Phellandrene γ-Terpinene p-Cymene Limonene 1,3,8-p-menthatriene β-Campholene-aldehyde Allo-ocimene trans-Pinocarveol cis-Verbenole trans-Verbenole Karahanaenone p-Mentha-1,5-dien 8-ol p-Cymene-8-ol Myrtenal Verbenone Monoterpene hydrocarbons Oxygen-containing monoterpenes Oxygen-containing sesquiterpenes Total
0.5 65.1 0.7 2.3 – 1.6 0.7 0.5 2.3 11.5 2.1 0.9 2.8 – – – – – – – 0.3 90.1 0.3 0.9 91.3
– 78.7 1.0 0.7 1.0 2.4 – – 0.7 1.6 – 1.4 – 1.9 1.2 5.3 0.6 1.3 0.4 0.4 1.2 86.1 11.7 2.0 99.8
935 942 956 972 979 985 1009 1020 1029 1036 1115 1129 1133 1142 1147 1149 1158 1169 1185 1197 1208
Microorganism
MIC, μL/mL
Pseudomonas aeruginosa Salmonella typhi Bacillus subtilis Shigella lexneri Escherchia coli Staphylococus epidermidis Proteus vulgaris Shigella dysenteriae
1.5625 3.125 1.5625 6.25 1.5625 3.125 6.25 1.5625
tion for 12 h at 37 °C, the first tube without turbidity was determined as the MIC. 3. Results and discussion
a lawn of test microorganisms, and 10 µl of smoke or volatile oil were placed in each disk. Gentamicin (10 µl) or ampicillin (10 µl), with corresponding solvents, were used as positive and negative controls. Plates were incubated at an appropriate temperature for bacteria (37 °C) for a period of 18–24 h. Studies were performed in triplicate. Samples with antimicrobial activity produced a distinct, clear, and circular zone of inhibition around the disc [16].
2.5.2. Minimum inhibitory concentration (MIC) Briefly, for minimum inhibitory concentration (MIC) according to reference [16], a microdilution broth susceptibility assay was used to evaluate antimicrobial activity of the non polar phase of smoke. The microorganisms were B. subtilis, S. typhi, E. coli, S. epidermidis, P. aeruginosa, Shigella lexneri, Proteus vulgaris, S. dysenteriae. To do this, 2 ml of a microbial suspension containing 5 × 105 CFU/ml of nutrient broth was prepared. Then according to the serial dilution different amounts of the non polar phase of smoke was added to each tube. One of the tubes contained no smoke and it was kept as positive control and the other one as a negative one which contained no microorganism. After bacterial incuba-
Here a simple method was developed (Fig. 1) for producing and collecting Mount Atlas mastic smoke simulated according to the folk culture. Corresponding volatile oil obtained with a yield of 1.7% (v/w). The constituents of the smoke and volatile oil are shown in Table 1. The components identified account for 91.3% of the smoke and 99.8% of the oil. The major components of Mount Atlas mastic smoke were α-pinene (65.1%), limonene (11.5%) and allo-ocimene (2.8%). Monoterpene hydrocarbons were also the major group in the smoke (90.1%). Our results showed that the major components of Mount Atlas mastic volatile oil were α-pinene (78.7%), trans-verbenole (5.3%) and β-pinene (2.4%) (Table 1). In a study [17], α-pinene (42.9%) and β-pinene (13.2%) were the major components, and in another study on the oil of air-dried resinous gum of P. terebinthus of Greek origin, major compounds were α-pinene (39.6%), β-pinene (19.5%), and sabinene (6.5%) [18]. In all three studies cited above, α-pinene is the major compound and Monoterpene hydrocarbons is the major group (86.1% in our study), but there are some differences in the concentration of α-pinene. A lower concentration of α-pinene and an increased proportion of limonene were found in the smoke as compared to the volatile oil. Comparison of the constituents of the smoke and volatile oil showed that 71.6% of the constituents and 8 compounds are similar. The possible of conversion of α-pinene to limonene (in an acidic pH) is similar to what occurs when the oil is processed using cold sulphuric acid in turpentine [19]. Limonene is used as a flavoring agent in food manufacturing and medicine, so the pleasant odor of the smoke may be due to limonene production in the smoke process.
Table 2 Antimicrobial activity of Pistacia terebinthus smoke and volatile oil. Sample
Smoke fraction Nonpolar Aqueous Volatile oil Gentamicin Ampicillin
Zone of inhibition Bacillus subtilis
Salmonella typhi
Escherichia coli
Staphylococcus epidermidis
Pseudomonas aeruginosa
14 mm 0 mm 8 mm 8 mm –
12 mm 0 mm 6 mm – 11 mm
15 mm 0 mm 9 mm – 9 mm
12 mm 0 mm 7 mm 8 mm –
14 mm 7 mm 11 mm – 7 mm
580
A. Mohagheghzadeh et al. / Fitoterapia 81 (2010) 577–580
In a study, the volatile oil of this oleo gum resin has shown good antimicrobial activity against resistant gram positive and negative clinical bacteria [20]. Table 2 shows the antimicrobial activity of the smoke and volatile oil of Mount Atlas mastic smoke. The non-polar smoke fraction had good antimicrobial activity in comparison with standards and volatile oil. So, minimum inhibitory concentration tests for non-polar phase were done. Results have shown that this material can inhibit gram positive and negative bacterial growth in a lower concentration than standard and it means that it has a good antimicrobial activity. As it is shown in Table 3 the smoke have good antimicrobial activity on all of microorganism especially on S. dysenteriae, E. coli, B. subtilis and P. aeruginosa. Today, liquid smoke, used as a preservative agent in food processing, is the aqueous phase of smoke [1], but in this study, the non-polar phase possessing considerable content of α-pinene and limonene had a better antimicrobial activity. The long use of P. terebinthus oleo gum resin smoke in traditional medicine and our findings indicate its potential as a future object of studies focusing on its use as a naturally origin material as a air purifier and for flavoring and preservation of foods. For further works, the carcinogenic activity of this smoke should be investigated. Acknowledgments This work was made possible by financial support from Shiraz University of Medical Sciences and Health Services. The authors wish to acknowledge the helpful assistance of Dick Edelstein and AuthorAid of the Eastern Mediterranean. This work was a part of a Pharm.D thesis. References [1] Holley RA, Patel D. Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials. Food Microbiol 2005;22:273–92.
[2] Mohagheghzadeh A, Faridi P, Shams-Ardakani M, Ghasemi Y. Medicinal smokes. J Ethnopharmacol 2006;108:161–84. [3] Shahverdi AR, Monsef-Esfahani HR, Nickavar B, Bitarafan L, Khodaee S, Khoshakhlagh N. Antimicrobial activity and main chemical composition of two smoke condensates from Peganum harmala seeds. Z Naturforsch C 2005;60:707–10. [4] Nautiyal CS, Chauhan PS, Nene YL. Medicinal smoke reduces airborne bacteria. J Ethnopharmacol 2007;114:446–51. [5] Davis PH. Flora of Turkey and The East Aegean Islands, vol. 2. Edinburgh: Edinburgh University Press; 1967. [6] Shipley G. Human landscapes in classical antiquity: environment and culture. Routledg; 1996. http://books.google.com/books?id=3kYhEehfAAC, accessed 17 June 2009. [7] Dalby A. Food in the ancient world from A to Z. Routledge; 2003. p. 323–4. http://books.google.com/books?id=FtIXAe2qYDgC&printsec= frontcover#PPR6,M1, accessed 17 June 2009. [8] Duke JA, Duke PK, duCellie JL. Duke's Handbook of Medicinal Plants of The Bible. New York: CRC Press; 2008. [9] Avicenna, Al Qanun Fil Tibb, vol. 2. English translation by H.A. Hameed (1998). S.Waris Nawab, Senior Press Superintendent, New Delhi: Jamia Hamdard Printing Press; 1024. [10] Hooper D, Field H. Useful plants and drugs of Iran and Iraq. Botanical Series, vol. 9. Chicago: Field Museum of Natural History; 1937. [11] Amin G. Popular medicinal plants of Iran. Tehran: Iranian Research Institute of Medicinal Plants; 2005. [12] Mastic. Wikipedia. http://en.wikipedia.org/wiki/Mastic, 2009 accessed 17 June 2009. [13] Encyclopedia of Spices. Mastic. http://www.theepicentre.com/Spices/ mastic.html2008 accessed 17 June 2009. [14] British Pharmacopoeia, vol. 2. London: HMSO; 1988. p. 137–8. [15] Adams RP. Identification of Essential Oil Components by Gas Chromatography Quadrupole Mass Spectroscopy. Carol Stream: Allured Publishing Co; 2004. [16] Mahon CR. Textbook of Diagnostic Microbiology. London: W. B. Saunders; 1995. [17] Barrero AF, Herrador MM, Arteaga JR, Akssira M, Mellouki F, Belgarrabe A, Blizquez MA. Chemical composition of the essential oils of Pistacia atlantica Desf. J Essent Oil Re 2005;17:52–4. [18] Papageorgiou V, Assimopoulou AN, Yannovits-Argiriadis N. Chemical composition of the essential oil of Chios turpentine. J Essent Oil Res 1999;11:367–8. [19] Evans WC. Treas and Evans’ Pharmacognosy. 15th ed. W.B. Edinburg: Saunders; 2002. [20] Ghalem BR, Mohamed B. Essential oil from gum of Pistacia atlantica Desf.: Screening of antimicrobial activity. Afr J Pharm Pharmacol 2009;3:87–91.