Preliminary Evaluation of the Antimicrobial Activity of Total Extract and Fractions of Chloroform, Methanol, and Aqueous from the Aerial Parts of <i>Salvia aegyptiaca</i>

Mojdeh Hakemi-Vala; Ayda Eslamzadeh; Fatemeh Bagheri Bejestani; Jinous Asgarpanah; Mohsen Heidary; Saeed Khoshnood

doi:10.5812/ajcmi.43457

Preliminary Evaluation of the Antimicrobial Activity of Total Extract and Fractions of Chloroform, Methanol, and Aqueous from the Aerial Parts of Salvia aegyptiaca

Mojdeh Hakemi-Vala ^{1 , *} , Ayda Eslamzadeh ² , Fatemeh Bagheri Bejestani ² , Jinous Asgarpanah ² , Mohsen Heidary ³ and Saeed Khoshnood ⁴

Authors Information

¹ Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

² Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran

³ Department of Microbiology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran

⁴ Department of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

Article Notes and Dates

Avicenna Journal of Clinical Microbiology and Infection: August 2017, 4 (3); e43457.Published Online: May 7, 2017

Article Type: Research Article; Received : October 29, 2016; Revised : February 22, 2017; Accepted : March 29, 2017
DOI: http://dx.doi.org/10.5812/ajcmi.43457

To Cite : Hakemi-Vala M, Eslamzadeh A, Bagheri Bejestani F, Asgarpanah J, Heidary M, et al. Preliminary Evaluation of the Antimicrobial Activity of Total Extract and Fractions of Chloroform, Methanol, and Aqueous from the Aerial Parts of Salvia aegyptiaca, Avicenna J Clin Microb Infec. 2017 ;4(3):e43457. doi: 10.5812/ajcmi.43457.

Abstract

Copyright: Copyright © 2017, Avicenna Journal of Clinical Microbiology and Infection. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.

1. Background

Due to extending microbial resistance to chemical medicines and also increase of the side effects of the existing antibiotics, studies on herbs to recognize their antimicrobial effects are increasing (1). Iran is one of the few areas with special geographical situation and climate suitable for the growth of medicinal plants used in traditional medicine (2, 3). Many different documents show the antimicrobial activity of medicinal plants or herbs such as garlic as a natural source of healing (4). Due to the adverse side effects of synthetic drugs and emergence of resistant microbes armed with different intrinsic or acquired antibiotic resistance mechanisms, there is a need to find new natural compounds with antimicrobial activity (5). Salvia is one of the medicinal plants with about 900 species in various parts of the world such as Europe, Asia, and America except the cold areas; 56 species of them are reported in Iran (6, 7). The most known Salvia species are S. officinalis, S. macro sipho Boiss, S. mirzayuni and S. aegyptiaca. Most of the differences between the species are in their growth conditions, morphology, and physical appearance (8). Salvia aegyptiaca, is a floral, angiosperm, and dicotyledon plant that pertains to Tubiflorales class, Verbenales (Lamiaceae) phylum, Labiatae family and Salvia genus (9). Salvia aegyptiaca, is native to Iran and has dispersed in the South of Iran, especially Hormozgan province (9). Based on the documents, this plant is used in traditional medicine to improve nervous disorders, dizziness, and diarrhea (6, 7).

Since other species of Salvia previously showed significant antibacterial effects, the current study aimed at evaluating the antimicrobial activity of total extract and the chloroform, methanol, and aqueous fractions of aerial parts of Salvia aegyptiaca against some Gram-positive and -negative bacteria and Candida albicans by cup plate, and determination of the minimum inhibitory concentration (MIC) with micro-dilution methods for the first time.

2. Methods

2.1. Plant Collection

Salvia aegyptiaca collected from Bandar Abbas (Hormozgan province) in April 2015 and identified by Dr. J. Asgarpanah. Aerial parts of the plant were washed and dried for 14 days at room temperature under the shade and kept away from direct sunlight and moisture. Then, dried plants were powdered by electric mill and stored in a dark, caped, and waterproof glass jar (2).

2.2. Extraction by Maceration

Extraction was done by maceration method and using a percolator apparatus. Following that, 500 g of plant’s powder weighed by a digital scale to the nearest 0.01 g and extraction was done by methanol solvent. After 4 or 5 days, the extract was collected in a glass container. This procedure was repeated 3 times. Finally, the extracts were pooled and concentrated using a rotary evaporator. The concentrated extract was poured into a container and kept away from light and heat to dry completely. Finally 60 g extract was obtained from 500 g of powdered plant and more than half of it was kept for the preparation of fractions. Part of the extract was poured into a large beaker and 25 to 30 mL of chloroform solvent was added. Then, all the fractions were concentrated using a rotary evaporator (10).

2.3. Preparation of Extracts

Two grams of the total methanol extract and each of its different fractions were weighed by an analytical scale to the nearest 0.0001 g, and then, solved in 2% dimethyl sulfoxide (DMSO) as solvent and a concentration of 1000 mg/mL of the total extract and fractions were prepared. All of the extracts were filtered by a 0.45 micron syringe filter and further concentrations of 125, 250, and 500 mg/mL were prepared after double dilution.

2.4. Microbial Strains

The microbial strains were tested in the study were Staphylococcus aureus (PTCC 1431), Staphylococcus epidermidis (PTCC 1435), Bacillus cereus (PTCC 1015), Escherichia coli (PTCC 1399), Pseudomonas aeruginosa (PTCC 1430), and Candida albicans (PTCC 5027). All were purchased from Iranian research organization for science and technology (IROST).

2.5. Cup Plate Method

Cup plate method was used to study the antibacterial activity of S. aegyptiaca extract and the fractions. For this purpose, based on the clinical and laboratory standards institute (CLSI) 2013 (11) protocol, the MHA (Mueller-Hinton agar) plates containing microorganisms were used. The wells with 6 mm diameter were created by a sterile Pasteur pipet. A microbial suspension was prepared from each microorganism with a turbidity of 0.5 McFarland (1.5 × 10⁸ CFU/mL). Each well was filled by 80 µL of prepared total extract and fractions of chloroform, methanol, and aqueous of concentrations of 125 to 1000 mg/mL. Erythromycin and gentamycin as positive controls and 2% DMSO as the negative control were tested, simultaneously. This antimicrobial procedure was repeated 3 times (2, 11).

2.6. Minimum Inhibitory Concentration by Microdilution Method

To determine the MIC of each extract and fractions, micro-dilution method was used based on CLSI 2013 guidelines12. The 96-well micro-plates (Extra Gene-Company-product No. EL- 1190-FCS) were used and microbial suspensions with the turbidity of 0.5 McFarland (1.5 × 10⁸ CFU/mL) were prepared. Then, the bacterial suspensions were diluted 1:20 to yield 5 × 10⁶ CFU/mL. When 0.01 mL of this suspension was inoculated into the broth, the final concentration of the microorganism was approximately 5 × 10⁵ CFU/mL. All the microplates were incubated at 37°C for 24 hours. The eerythromycin and gentamycin (Chemie Darou and Alborz Daru, Iran) and 10% DMSO as controls were tested, simultaneously. This antimicrobial procedure was repeated 3 times.

2.7. Statistical Analysis

Statistical analysis was performed by SPSS software version 16.1 and the paired t test was conducted for analogy extracts and antibiotic analysis.

3. Results

In the current study, the antimicrobial effects of different concentrations (125 to 1000 mg/mL) of total extract and chloroform, methanol, and aqueous fractions of S. aegyptiaca in 2% DMSO solvent were determined against some Gram-positive and -negative bacteria and Candida albicans by micro-dilution (MIC) and cup plate methods (Tables 1 - 3). To validate the evaluation of inhibition zone, cup plate method was conducted 3 times.

Table 1. Minimum Inhibitory Concentration of Total Extract and Different Fractions of Salvia aegyptiaca

Bacteria	MIC, mg/mL
	Total extract	Chloroform fraction	Methanol fraction	Aqueous fraction	Gentamicin	Erythromycin	Nystatin	2% DMSO
*Escherichia coli*	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 1000.0	500.0 ± 0.0	0.0 ± 1000.0	-	-	0.0 ± 0.0
*Pseudomonas aeruginosa*	0.0 ± 1000.0	0.0 ± 1000.0	500 ± 0.0	500 ± 0.0	0.0 ± 1000.0	-	-	0.0 ± 0.0
*Staphylococcus aureus*	250.0 ± 0.0	0.0 ± 1000.0	500 ± 0.0	0.0 ± 1000.0	-	0.0 ± 1000.0	_	0.0 ± 0.0
*Staphylococcus epidermidis*	0.0 ± 0.0	0.0 ± 0.0	500 ± 0.0	0.0 ± 1000.0	-	0.0 ± 1000.0	-	0.0 ± 0.0
*Bacillus cereus*	0.0 ± 0.0	0.0 ± 1000.0	125.0 ± 0.0	0.0 ± 1000.0	-	0.0 ± 1000.0	-	0.0 ± 0.0
*Candida albicans*	0.0 ± 0.0	0.0 ± 1000.0	0.0 ± 0.0	0.0 ± 0.0	-	0.0 ± 250.0	0.0 ± 1000.0	0.0 ± 0.0

Table 2. Mean ± Standard Deviation of Effect of Salvia aegyptiaca Total Extract by the Cup Plate

Bacteria	Total Extract (Concentrations), mg/mL				Gentamicin	Erythromycin	Nystatin	2% DMSO
	1000	500	250	125
*Escherichia coli*	5.3 ± 5.3	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	1.66 ± 38.33	-	-	0.0 ± 0.0
*Pseudomonas aeruginosa*	0.88 ± 11.33	1.45 ± 11.33	4.67 ± 4.67	3.33 ± 3.33	0.0 ± 40.0	-	-	0.0 ± 0.0
*Staphylococcus aureus*	11.67 ± 0.88	6.67 ± 3.33	0.0 ± 0.0	0.0 ± 0.0	-	35.0 ± 0.0	-	0.0 ± 0.0
*Staphylococcus epidermidis*	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	-	35.0 ± 0.0	-	0.0 ± 0.0
*Bacillus cereus*	10.0 ± 0.0	10.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	-	43.0 ± 0.0	-	0.0 ± 0.0
*Candida albicans*	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	-	-	35.0 ± 0.0	0.0 ± 0.0

Table 3. Mean ± Standard Deviation of Effect of Salvia aegyptiaca Different Fractions in the Cup Plate Method

Bacteria	Tested Extracts and Antibiotics
	Chloroform fraction, mg/mL				Methanol extract, mg/mL				Aqueous extract, mg/mL				Gentamycin	Erythromycin	Nystatin	2% DMSO
	1000	500	250	125	1000	500	250	125	1000	500	250	125
*Escherichia coli*	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	38.33 ± 1.66			0.0 ± 0.0
*Pseudomonas aeruginosa*	17.33 ± 1.45	17.33 ± 1.86	16.33 ± 2.96	15.0 ± 3.61	10.67 ± 0.67	11.3 ± 0.67	10.33 ± 0.33	10.67 ± 0.67	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	40.0 ± 0.0	-	-	0.0 ± 0.0
*Staphylococcus aureus*	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	4.0 ± 0.0	3.3 ± 3.3	0.0 ± 0.0	0.0 ± 0.0	-	35.0 ± 0.0	-	0.0 ± 0.0
*Staphylococcus epidermidis*	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0					-	30.0 ± 2.0	-	0.0 ± 0.0
*Bacillus cereus*	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	2.67 ± 2.6	6.67 ± o.33	8.0 ± 0.0	10.33 ± 1.2	11.33 ± 0.67	11.00 ± 0.0	12.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	-	40.0 ± 1.50	-	0.0 ± 0.0
*Candida albicans*	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	-	-	35.00 ± 0.0	0.0 ± 0.0

4. Discussion

Salvia aegyptiaca is one of the native plants in Iran that its antibacterial effects are not studied yet (12). The current study determined the antimicrobial effect of different concentrations (125 to 1000 mg/mL) of total extract and chloroform, methanol, and aqueous fractions of S. aegyptiaca fractions in DMSO10% solvent against some Gram-positive and -negative bacteria and Candida albicans by cup plate and micro-dilution methods.

In the study by Farjam et al., the antimicrobial effect of S. aegyptiaca oil was evaluated by determining MIC using the agar dilution method (9). They showed that the MIC ranged from 64 to 256 μg/mL and the best results were against Klebsiella oxytoca (64 μg/mL), followed by S. aureus and Fusarium solani (128 μg/mL) and Aspergillus niger and A. alternaria (256 μg/mL). However, there were differences among the studies by Farjam et al. and the current study regarding the method of MIC determination (agar vs. broth dilution) and also evaluation of oil in the Farjam vs. methanol extract and the fractions of S. aegyptiaca in the current study, both had similar antimicrobial effects against some Gram-negative and -positive bacteria and also fungi.

As mentioned in the results, in the cup plate method, there was no inhibition zone for the total extract against P. aeruginosa, S. aureus, and B. cereus, for chloroform fraction against P. aeruginosa, for methanol fraction against B. cereus and P. aeruginosa, and for aqueous fractions against S. aureus, and B. cereus. In contrast, in micro-dilution method, significant results were obtained against all tested bacteria and also Candida albicans. With regard to no inhibition zone in the cup plate method, it can be concluded that the total extract and its fractions had no good diffusion on Mueller-Hinton agar. Similarly, according to the study by Chang in 2011, to investigate the antimicrobial activity of chitosan, failure diffusion of chitosan extracts on solid media seemed logical. They concluded that in such conditions, a direct collision method should be used (12). Also, other studies evaluated (13-15) the antioxidant property and the components of Salvia spp. and S. aegyptiaca. Fifteen phenolic compounds were identified by high-performance liquid chromatography (HPLC). Existence of different phenolic compounds in the methanol extract of this plant may be related to its antimicrobial effect detected in the current study.

Finally, it is hoped that by proven non-toxicity of different extracts of S. aegyptiaca, the product could be useful as a medicinal plant in one of the pharmaceutical forms in future. Therefore, the following topics are recommend for further studies: a) Evaluation of the antibacterial effect of S. aegyptiaca fraction against other microbial strains, b) Identification and purification of the active substances in each extract of the mentioned plant, c) Survey of S. aegyptiaca mutagenicity, d) Isolation of other plant extracts, e) Performing in vivo tests in animal models and also cell cultures.

Footnotes

References

1. Eum JS. Activity of nature plants extracts against pseudomonas aeruginosa ATCC 27853. J Korean Inst Inform Commun Eng. 2012; 16(8): 1799-804[DOI]
2. Hakemi Vala M. Preliminary phytochemical screening and evaluation of the antibacterial and mutagenic activity of Rhynchocorys elephas (L.) Griseb. J Med Plants Res. 2012; 6(2)[DOI]
3. Zeinalian M, Eshaghi M, Naji H, Marandi SM, Sharbafchi MR, Asgary S. Iranian-Islamic traditional medicine: An ancient comprehensive personalized medicine. Adv Biomed Res. 2015; 4: 191[DOI][PubMed]
4. Mozaffari Nejad AS, Shabani S, Bayat M, Hosseini SE. Antibacterial Effect of Garlic Aqueous Extract on Staphylococcus aureus in Hamburger. Jundishapur J Microbiol. 2014; 7(11)[DOI][PubMed]
5. Singh K, Panghal M, Kadyan S, Chaudhary U, Yadav JP. Green silver nanoparticles of Phyllanthus amarus: as an antibacterial agent against multi drug resistant clinical isolates of Pseudomonas aeruginosa. J Nanobiotechnology. 2014; 12: 40[DOI][PubMed]
6. Firuzi O, Miri R, Asadollahi M, Eslami S, Jassbi AR. Cytotoxic, antioxidant and antimicrobial activities and phenolic contents of eleven salvia species from iran. Iran J Pharm Res. 2013; 12(4): 801-10[PubMed]
7. Gorai M, Gasmi H, Neffati M. Factors influencing seed germination of medicinal plant Salvia aegyptiaca L. (Lamiaceae). Saudi J Biol Sci. 2011; 18(3): 255-60[DOI][PubMed]
8. Ibrahim TA. Chemical composition and biological activity of extracts from Salvia bicolor Desf. growing in Egypt. Molecules. 2012; 17(10): 11315-34[DOI][PubMed]
9. Farjam MH, Kavoosifar A, Joukar M. Salvia aegyptiaca oil extracted by microwave-assisted and normal hydrodistillation: composition and antibacterial activity. Adv Environ Biol. 2014; 8(21): 277-81
10. Mehrara M, Halakoo M, Hakemi-Vala M, Hashemi SJ, Asgarpanah J. Antibacterial and antifungal activities of the endemic species Glaucium vitellinum Boiss. and Buhse. Avicenna J Phytomed. 2015; 5(1): 56-61[PubMed]
11. Clinical and Laboratory Standards Institute (CLSI) . Performance standard for antimicrobial susceptibility testing; seventeenth informational supplement. CLSI document M100-S17. 2013;
12. Chang HL, Chen YC, Tan FJ. Antioxidative properties of a chitosan–glucose Maillard reaction product and its effect on pork qualities during refrigerated storage. Food Chemistry. 2011; 124(2): 589-95[DOI]
13. Pourhosseini M, Asgarpanah J. Essential and fixed oil chemical profiles of salvia aegyptiaca L. Flowers and seeds. J Chilean Chem Soc. 2015; 60(4): 2747-8[DOI]
14. Forouzin F, Jamei R, Heidari R. Comparison of essential oil components and antioxidant activity between salvia syriaca and salvia aristata in their natural habitats in west azerbaijan province, Iran. J Pharm Pharmacol. 2015; 3: 404[DOI]
15. Ben Farhat M, Jordán MJ, Chaouch-Hamada R, Landoulsi A, Sotomayor JA. Changes in phenolic profiling and antioxidant capacity of Salvia aegyptiaca L. by-products during three phenological stages. LWT - Food Sci Technol. 2015; 63(1): 791-7[DOI]

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