Herbal Medicine Department College of Food and Drug, Shahrekord Branch, Islamic Azad University, Shareakord Iran

Article Publishing History

Introduction

In despite of the high development of medical sciences, treatment of infectious diseases caused by pathogenic agents like bacteria, fungi and viruses is in trouble. These problems are mainly occurring due to the occurrence of antibiotic resistances (Dehkordi et al., 2012, 2014). Antimicrobial resistance threatens the effective prevention and treatment of an ever-increasing range of infections caused by bacteria, parasites, viruses and fungi. It is an increasingly serious threat to global public health that requires action across all government sectors and society (Momtaz et al., 2013; Dormanesh et al., 2014).

Resistant microorganisms are able to withstand attack by antimicrobial drugs, such as antibacterial drugs (e.g. antibiotics), antifungals, antivirals, and antimalarials, so that standard treatments become ineffective and infections persist, increasing the risk of spread to others (Davies and Davies 2010). Occurrence of these antimicrobial resistances caused chemical and pharmacological factories to use from novel sources for antibiotic producing. Application of medicinal plants for producing of antimicrobial agents had an ancient history.

Medicinal plants are a suitable sources of antimicrobial agents. Calendula officinalis (C. officinalis) is one of the most commonly used medicinal plants among Iranian people which is native to the Mediterranean regions (Pan et al., 2013). C. officinalis, commonly known as pot marigold, is an annual herb and belongs to Asteraceae family. Flowers are monoecious (individual flowers are either male or female, but both sexes can be found on the same plant) and are pollinated by Bees. It is noted for attracting wildlife. C. officinalis can be broadly applied as an antiseptic, anti-inflammatory and cicatrizing as well as a light antibacterial and antiviral agent (Pan et al., 2013; Arora et al., 2013; Efstratiou
et al., 2012; Butnariu et al., 2012; Martins et al., 2014). The plant contains esquiterpenes glycosides, saponins, xanthophylls, triol triterpenes, flavonoids, volatiles, ä- cadinene, á-cadinol, 1,3,5-cadinatriene and á-muurolol which show anti-oxidative and antimicrobial effects (Pan et al., 2013; Arora et al., 2013; Efstratiou et al., 2012; Butnariu et al., 2012; Martins et al., 2014).

officinalisis used as anti-bacterial, analgesic, anthelmintic, anti-fungal, cholagogue, anti-spasmodic, anti-pyretic, hemostatic, antiseptic, anti-emetic, candidicide, anti-viral, astringent, bitter, anti-inflammatory, lymphatic, cardiotonic, carminative, diaphoretic, dermagenic, diuretic, immunostimulant, and uterotonic agent (Pan et al.,2013; Arora et al., 2013; Efstratiou et al., 2012; Butnariu et al., 2012; Martins et al., 2014). According to the high prevalence of pathogenic bacteria in Iranian cases of hospital infections, economic, cosmetic, and pharmaceutical values of C. officinalis and lack of published data on the identification of chemical components and antimicrobial activities of C. officinalis, the present study was carried out to evaluate the chemical components and antimicrobial effects of C. officinalis on standard strains of Pseudomonas aeruginosa, Escherichia coli, Salmonella typhi, Bacillus cereus and Staphylococcus aureus.

Materials And Methods

The present study was accepted by the ethical committees of the Islamic Azad University of Shahrekord, Iran. Written consent was signed by the Research Adjutancy of the Islamic Azad University of Shahrekord (IAUSHK 202542). Permission of this work was also taken from the Head of the Islamic Azad University of Shahrekord.

officinalisflowers were collected from the plains and mountains of the Zagros zone, Chaharmahl Va Bakhtiary province, Iran. Genus of collected flowers were identified and confirmed by the professor of the Medicinal Plants Research Center of the Islamic Azad University of Shahrekord, Iran. All flowers were collected on the February of 2015. Five-hundred grams of fresh flowers were hydro distilled separately for 3 h in an all-glass Clevenger apparatus in accordance with the British pharmacopoeia method (British Pharmacopoeia 1980).

In order to study the chemical compositions of C. officinalis flowers, the GC-mass analysis method was used using an Agilent 6890 Series II gas chromatograph (Palo Alto, USA) coupled to an Agilent 5973 quadrupole mass spectrometer with electron ionization mode (EI) generated at 70 eV (ion source at 230 °C and transfer line at 280 °C). The GC was performed using a J&W DB-5 (5% diphenyl- 95% dimethyl silicone) capillary column (30 m x 0.25 mm i.d. x 0.25 µm film), and helium was used as a carrier gas (1 mL min-1). The initial temperature was programmed from 35 °C to 60 °C (at 1 °C min-1), to 170 °C (3 °C min-1), to 200 °C (8 °C min-1), and to 280 °C (15 °C min-1), and maintained at 280 °C for 5 min. The injector port (splitless mode, 0.5 min) was at 250 °C. Retention indexes were calculated with reference to nalkanes. All compounds were identified by comparison of both the mass spectra (Wiley 275 library) and the retention index data found in the literature (Adams 1995).

The bacterial cultures were purchased from the Pasteur Institute of Iran. They were subculture onto Petri plate containing nutrient agar media (Merck, Germany). The strain of bacteria selected to assess susceptibility pattern against C. officinalis extract were Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 8739), Salmonella typhi (ATCC 14028), Bacillus cereus (ATCC 10987) and Staphylococcus aureus (ATCC 6538). Each of the microorganisms was reactivated prior to susceptibility testing by transferring them into a separate test tubes containing broth and incubated overnight at 37°C at shaker.

Agar disc diffusion method was used for screening of antibacterial activity of C. officinalis extract (Efstratiou et al., 2012). Bacterial strains were spread on to Nutrient Agar (NA, Merck, Germany) medium. Paper discs were separately impregnated with 25µl of the 0.5 mg/mL plant essential oil and placed on the inoculated agar plates. All the plates were allowed to stay at room temperature for 30 min to allow diffusion of the essential oil then incubated at 37 ºC for 24 hrs. Interpreting of the diameter of the zone of inhibition was done according to the protocol of the Clinical Laboratory Standard Institute (CLSI 2012).

Antimicrobial effects of the C. officinalis essential oil were tested 3 times. Results were transferred to a Microsoft Excel spreadsheet (Microsoft Corp., Redmond, WA) for analysis. Statistical analysis was performed using SPSS/20.0 software (SPSS Inc., Chicago, IL) for significant relationship between antimicrobial effects of C. officinalis essential oil on tested bacteria. The chi-square test and Fisher’s exact 2-tailed test analysis were performed in this study. Statistical significance was regarded at a P value < 0.05.

Results And Discussion

Frequency of chemical components in C. officinalis essential oil is shown in table 1. Totally, 40 different chemical components were detected in the essential oil of the C. officinalis essential oil. Totally, 1,8-cineole (30.456%), ®-Terpinene (25.547%), Terpinolene (4.584%), ©-Terpineol (4.490%) and Trans-â-ocinene (4.153%) were the most commonly detected components in the essential oil of the C. officinalis. Significant statistical differences were seen between the frequency of chemical components (P < 0.05).

Table 1: Frequency of chemical composition of Calendula officinalis essential oil.
Number	Chemical components	Frequency of components (%)
1	á-Thujene	0.459
2	á-Pinene	3.032
3	Camphene	0.256
4	Sabinene	0.293
5	P-Pinene	0.490
6	1-Octen-3-ol	0.291
7	1,8-cineole	30.456
8	p-cymene	2.495
9	á-Terpinene	0.283
10	â-caryophyllene	1.289
11	Trans-â-ocinene	4.153
12	Benzene acetaldehyde	1.354
13	ã-Terpinene	25.547
14	cis-Sabinene hydrate	2.928
15	Terpinolene	4.584
16	á-phellandrene	0.390
17	á-terpineol	0.676
18	Carvacrol	3.146
19	Terpinene-4-ol	0.369
20	á-Terpineol	4.490
21	n-Dodecane	0.279
22	Carvacrol methy ether	0.301
23	á-copaene	0.318
24	á-bourbonene	0.510
25	á-Terpinyl acetate	0.520
26	Eugenol	0.676
27	n-Tetradecane	3.146
28	(E)-Caryophyllene	0.369
29	á-muurolene	2.805
30	â-Bisabolene	0.622
31	(E)-ã-Bisabolene	0.301
32	Spathulenol	0.318
33	â-Eudesmol	0.510
34	á-Cadinol	0.520
35	ã-Cadinene	3.319
36	Cadina 1,4-diene	0.950
37	á-cadinene	1.434
38	á-Bisabolol	0.024
39	á-cadinol	0.616
40	T-muurolol	0.324

Table 2 represents the antibiotic susceptibility pattern of bacterial strains against essential oil of the C. officinalis. We found that the C. officinalis essential oil harbored the highest levels of antibiotic effects on the Gram-negative than Gram-positive bacteria (P < 0.05). The highest amount of diameter of the inhibition zone was seen for the E. coli (13.31±1.24 mm), followed by P. aeruginosa (10.22±0.83 mm). The lowest amount of diameter of the inhibition zone was seen for the S. aureus (3.14±0.27 mm). Statistically significant differences were seen between the types of bacteria and antibiotic effects of C. officinalis essential oil (P < 0.05).

Table 2: Antibiotic susceptibility pattern of bacterial strains against Calendula officinalis essential oil.
Bacteria	Mean zone of inhibition (mm)
Pseudomonas aeruginosa	10.22±0.83a*
Escherichia coli	13.31±1.24a
Salmonella typhi	7.34 ±0.62b
Bacillus cereus	4.10±0.30c
Staphylococcus aureus	3.14±0.27c
*Dissimilar leathers in this column shows significant differences about P < 0.05.

As far as we know, the present investigation is the first prevalence report of chemical composition and antimicrobial effects of the C. officinalis on the pathogenic bacterial strains in Iran. We found that the essential oil of the C. officinalis had low antibacterial effects on tested bacteria. Unauthorized and indiscriminate prescription of antibiotics are the main reasons for the high prevalence of resistance (low zone of inhibition) in the bacterial strains of our study.

aureusstrains had the highest levels of resistance against C. officinalisessential oil. S. aureus strains of various previously published works harbored the highest levels of resistance against strong antibiotic agents such as penicillin, tetracycline, gentamycin, ampicillin, cefexime and ciprofloxacin which was similar to our results on the C. officinalis (Tokajian et al., 2011; Virdis et al., 2010; Udo et al., 2008; Rijal et al., 2008; Deng et al., 2013). All of these researches have recommended synthesis, formulation and application of novel antimicrobial agents to overcome occurrence of high antibiotic resistance in the S. aureus strain of human and even animal clinical samples, but we found that the C. officinalis essential oil is not appropriate approach for synthesis of anti-S. aureus antibiotic.

In despite of the S. aureus and B. cereus which had the low diameter inhibition zone, C. officinalis essential oil had a high antibacterial effects on E. coli and P. aeruginosa. Probably, chemical components of this plant make it effective on the Gram-negative bacteria. Similar results have been reported previously.

In a study which was conducted by Efstratiou et al. (2012) (Efstratiou et al., 2012), results showed that the C. officinalis extracts represented exceptional antibacterial activity against P. aeruginosa, E. coli, K. aerogenes, E. faecalis and K. pneumonia which was similar to our findings. Chakraborthy (2008) (Chakraborthy 2008) reported that the lowest Minimum Inhibitory Concentration (MIC) values of C. officinalis were observed for ethanol extract, chloroform extract, water extract and petroleumether extract against the bacteria. They showed that the extracts of C. officinalis leaves were significantly effective against both Gram-positive and especially Gram-negative organisms. High antimicrobial effects of the C. officinalis is due to its antimicrobial chemical components. Recent study revealed that triterpenoid like calendulaglycoside, triterpenoid saponin like faradiol, asorhamnetin3-O-neohesperidoside, quercetin and isorhamnetin are the main chemical components of the C. officinalis which are responsible for antioxidative, anti-cancer, antimicrobial, anti-inflammatory and wound healing effects (Muley et al., 2009).

We found that, 8-cineole (30.456%), ã-Terpinene (25.547%), Terpinolene (4.584%), á-Terpineol (4.490%) and Trans-â-ocinene (4.153%) chemical components had a high quantity in the C. officinalis essential oil. These components are anti-oxidant and antimicrobial materials of the C. officinalis. Results of the documented reports revealed that the main compounds within Calendula are the triterpenoids (Arora et al., 2013; Butnariu et al., 2012) which are claimed to be the most important anti-inflammatory and antimicrobial components within the plant. Other constituents identified in Calendula such as the saponins, micronutrients, flavonoids, and polysaccharides, may also be responsible for the antimicrobial, anti-inflammatory, antioxidant, and wound healing effect of the plant (Arora et al., 2013; Butnariu et al., 2012; Faria et al., 2011).

The antimicrobial activity of essential oil of C. officinalis is attributed to its main chemical components including citral (aldehyde), geraniol (primary alcohol), eugenol (phenol), menthol (secondary alcohol) and cinnamic aldehyde (aldehyde) (Hartman and Coetzee 2002). Compounds such as linalool, citral, geraniol, or thymol are more antiseptic agents in the essential oil of the C. officinalis (Bruneton 2001). Butnariu and Coradini (2012) (Butnariu and Coradini 2012) reported that marigold is renowned for its antibacterial, anti-oxidant, cholagogic, diaphoretic and vulnerary properties. They showed that marigold extract is full of phenolic and saponin components which warranty its antibacterial and anti-oxidative activities. Rigane et al. (2013) (Rigane et al., 2013) showed that Rutin, quercetin-3-O-glucoside, scopoletin-7-O-glucoside, isorhamnetin-3-O-glucoside and gallic acid were the most commonly flavonoid-based chemical components. They reported that C. officinalis (leaf extract) exhibited a better MIQ against E. coli and S. aureus than aqueous-methanolic flower extract having strong activity against S. typhimurium at lower quantities. The phenolic compounds and flavonoids found in C. officinalis could be responsible for its antimicrobial activity against E. coli, S. typhimurium, S. aureus, C. albicans and A. niger. Their results are in agreement with our findings.

Conclusion

In conclusion, we identified a large numbers of chemical components in the essential oil extracted from C. officinalis. Totally, flavonoids and phenols are the main chemical components of C. officinalis. Good antibacterial effects of the C. officinalis especially on E. coli and P. aeruginosa but it was not effective on the S. aureus and B. cereus. Therefore, we recommended production of anti-E. coli and P. aeruginosa agent for treatment of the diseases caused by these two bacterium such as urinary tract infections, food-poisoning and burn and wound infections. Judicious prescription of antibiotics can control and eliminate the occurrence of antibiotic resistance in pathogenic bacteria.

Acknowledgements

The authors would like to thank Dr. F. Safarpoor Dehkordi of the Department of Food Hygiene and Quality Control, University of Tehran, Iran and all the staff members of the Medicinal and Aromatic Plants Research Center of the Islamic Azad University of Shahrekord, Iran for their important technical and clinical support. The present study was supported by the Islamic Azad University of Shahrekord, Iran (IAUSHK 1991394).

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