Microbiology Research 2018; volume 9:7583

In vitro bactericidal activities of recently, some authors have identified Crete various extracts of saffron as the primary centre of domestication dur- Correspondence: Beniamino T. Cenci-Goga, ing the Late Bronze Age.2-5 In fact, even Department of Veterinary Medicine, (Crocus sativus L.) stigmas though the botanical origins of C. sativus University of Perugia, via San Costanzo, from Torbat-e Heydarieh, have not yet been completely clarified, 06126 Perugia, Italy. Gonabad and Khorasan, Iran some authors have identified C. cartwright- E-mail: [email protected] ianus (2n = 16), which grows in the south- Key words: Saffron; Antimicrobial activity; ern Greece and in the neighbouring islands, 1 Enterococcus spp.; Staphylococcus spp.; Beniamino T. Cenci-Goga, and C. oreocreticus (ibid.), which grows in 2 Escherichia coli. Renzo Torricelli, Crete, as putative fertile diploid progeni- 2 Yousef Hosseinzadeh Gonabad, tors.6 Saffron was introduced in to the 2 Contributions: the authors contributed equally. Nicoletta Ferradini, Greek and Roman world by Phoenicians 3 1 Roberto Venanzoni, Paola Sechi, and later Arabs and Moors brought it to Conflict of interest: the authors declare no Maria Francesca Iulietto,1 Spain.2,5,7 Many Greek, Roman and potential conflict of interest. Emidio Albertini2 Egyptian historians, described the use of Funding: none. 1Department of Veterinary Medicine; saffron as a precious component to stain 2 cloth, to enrich the taste of food and drinks Department of Agricultural, Food and 6,8,9 Received for publication: 17 January 2018. 3 and for use in medical therapy. The Environmental Sciences; Department of Revision received: 26 January 2018. pharmacological effects of saffron compo- Chemistry, Biology and Biotechnology, Accepted for publication: 26 January 2018. nents such as safranal and crocin are well University of Perugia, Perugia, Italy known and widely used in medicine. This work is licensed under a Creative Several studies on the biological activities Commons Attribution NonCommercial 4.0 of these constituents have highlighted the License (CC BY-NC 4.0). Abstract beneficial therapeutic effects and are often used to support traditional medical thera- only©Copyright B.T. Cenci-Gogaet al., 2018 Saffron is one of the most expensive pies in various diseases.10 Crocin, and Licensee PAGEPress, Italy Microbiology Research 2018; 9:7583 spices in the world (20,000 €/kg) and this is safranal have displayed important antioxi- doi:10.4081/mr.2018.7583 due not only to the high demand for its var- dant properties that prevent gastric disor- ious uses such as cooking, production of ders like ulcer formation and stomach staining medicines, cosmetics etc., but also 11,12 lesion. These compounds are alsouse used most expensive spices in the world (20,000 for the high costs of cultivation and produc- to prevent cardiovascular disease, insulin €/kg),31 and this is due not only to the of the tion. Several studies have demonstrated that resistance, depression, premenstrual syn- high demand for its various uses such as differences in saffron quality are mainly due drome, insomnia and anxiety.13-21 Studies cooking, or for the production of staining to the methodology followed in the process- on the antimicrobial properties of saffron medicines, cosmetics, etc.7 but also for the ing of stigmas, and environmental condi- active compounds are few, several authors high costs of cultivation and production. In tions independent of the origin. Some have been reported the anti-bacterial activi- order to gather the saffron, stigmas of the C. authors found phenotypic variations within ty of aqueous and methanolic extracts of sativus flowers must be handpicked. This is cultivated saffron, but very limited genetic crocin and safranal against Helicobacter time consuming; usually to obtain 1 gram of diversity. The reason for the very limited pylori.22,23 An interesting anti-bacterial saffron, an average of 150 flowers must be genetic diversity in cultivated saffron is effects of the same active compounds on collected.32 explained by its asexual mode of reproduc- stigma contamination by Salmonella during Many researches have demonstrated tion (propagation). The aim of this study storage at room temperature was that differences in saffron quality are main- was to assess the antimicrobial activity of observed.24 The absence of Salmonella on ly due to the methodology used in the pro- stigma saffron, which were tested against saffron spice coming from the main produc- cessing stigmas and, independent from the different bacteria strains. The results er countries (Greece, Iran, Italy, Morocco, origin of the species quality is also influ- obtained from the antimicrobial activity and Spain) has also been observed.25 enced by sowing time and environmental study indicate that stigmas ofNon-commercial C. sativus Despite the high production costs, saf- conditions.33-35 Several studies have found have some antimicrobial effect. fron cultivation is widespread throughout phenotypic variations within cultivated saf- the world and it is currently cultivated not fron, such as flower size, shape of the only in Anatolia but also in China, India, tepals, differences of colour and intensity in Turkey, Europe, Africa and more recently in the tepals of samples collected from differ- Introduction Mexico and Australia.26 ent areas, but very limited genetic diversity Crocus sativus L. (saffron) belongs to Total world saffron production is esti- has been observed.34,36-38 the Iridaceae (Liliales, Monocots) family, it mated at about 220,000 tons, of which The very reduced genetic diversity exis- is a sterile triploid (2n=3x=24) species that about 90% is produced in Iran (Khorasan tent in cultivated saffron is attributed to its is propagated vegetatively via its corms Province) and the rest in Greece, Spain, asexual propagation, and by successive which undergo a period of dormancy.1 At Italy and India (Kashmir).27-29 The compo- selection during breeding.36 However, other least 85 species are known to belong to the nents of the spice saffron are localized in factors that may have contributed to the low Crocus genus and C. sativus is the most the red stigmatic lobes of C. sativus flower level of molecular polymorphisms found interesting one, not only as source of spice and these are responsible for its distinct are related to the marker techniques that for cooking but also as basic compound in color, flavor and smell.30 The commercial have been applied.39,40 cosmetics and medicine. Several archeolog- spice is obtained from dried stigmas and In an attempt to determine possible ical studies locate the probable saffron cen- marketed as saffron filaments or as powder antimicrobial properties of saffron, the aim tre of origin in Iran and Kashmir even if, from milled stigmas. Saffron is one of the of this study was to assess the antimicrobial

[Microbiology Research 2018; 9:7583] [page 43] Article activity of saffron stigma, which was tested UK) was used for the growth of bacterial sample extracts “A”, “B”, and “C” respec- against different bacterial strains. strains. Each organism was separately sus- tively, were added and left in contact again pended in a normal saline solution and for the selected times at the test tempera- transmittance (T) of 75-77% at 530 nm was ture. At the end of the contact time (5 min- made, which is equal to 106 cfu ml-1. Each utes), 1 ml of mixture was transferred into a Materials and Methods plate was inoculated with 0.2 ml of micro- test tube containing 8 ml of distilled water. bial suspension. 100μl of “A”, “B”, and “C” After 5 minutes of neutralization procedure, Plant material sample extracts, respectively, were added to the mixture was vortex-stirred and a double Iranian (6 accessions) and Italian corms each well and incubated at 37 ºC for 24h. count was performed by inclusion in agar. (2 accessions) were planted in September The positive antimicrobial activity was The number of cfu per plate was deter- 2012 on a sandy-clay soil in Umbria, evaluated based on growth inhibition zone mined following incubation for 48 hours at Central Italy (220 m a.s.l.). Sowing was and compared with the control. The extracts 37°C, and Na value was then calculated. executed in raised beds at 20 cm depth 50 were then diluted in a two-fold manner to cm between rows and 20 cm between corms make different concentrations. 100μl of the Statistical data analysis in rows. A complete randomized block active extracts were then added to each For antimicrobial activity, the count design with two replicates was adopted. well. All the tests were repeated in dupli- was performed using the number of Fifteen corms per accession per replication cates. colonies counted on both plates. Only the were used; no fertilizer and no irrigation plates showing a number of colonies includ- were applied. Weeds were managed by Vitality reduction ed in a 15-300 range were used to perform hand. Vitality reduction activity was per- the result calculation. A deviation of 10% is For antimicrobial activity, stigmas of C. formed for E. coli, strain CSH26 K-12; E. sativus collected from Torbat-e Heydarieh accepted, so the limits are 14 and 330. In the faecalis, strain NCTC 12201; and S. aureus, (Iran) (sample “A”), Gonabad (sample “B”) assay, where the number of cfu on every strain 27R, using the BS EN 1040:2005.42 and Khorasan (sample “C”), dried with the plate counted is <14, the number of cfu/ml Experimental conditions were validated as was recorded as < 1.4 102. Where the num- traditional technique of flow of hot air emit- follows: 1 ml of sterile water and 1 ml of ted from a ventilated oven were used. ber ofonly cfu on every plate counted was >330, bacterial validation suspension containing the number of cfu/ml was recorded as >3.3 Water-washed stigmas were shade dried at 2 3 -1 3.0 10 to 1.6 10 cfu ml were placed in a 103. room temperature and powdered by manual test tube. The components were left in con- blender. 1g of each of the dried and pow- Vitality reduction is expressed in loga- tact for 2 minutes; then 8 ml of water were rithm and was calculated for each organism dered materials were macerated separately added and left in contact at the temperatureuse with sterile water at a concentration of 100 and test concentration using the following adopted during the assay for the longest formulas: mg/100 ml. period to be tested (see below). At the end The bacterial strains used for the test of the contact time, the mixture was vortex- N(cfu/ml) = c/(n1 + 0.1n2)d (1) were taken from the collection of the stirred and a double count was performed Laboratorio di Ispezione degli Alimenti di by inclusion in agar. The number of colony- where N = bacterial counting; c = sum of Origine Animale (University of Perugia: forming units per ml of the mixture was Escherichia coli, strain CSH26 K-12; colonies counted on both plates; n1 = num- determined following incubation for 48 ber of counted plates in the lower dilution; Enterococcus faecalis, strain NCTC 12201; hours at 37 ± 1°C. The bacterial suspension n2 = number of counted plates in the higher and Staphylococcus aureus, strain 27R. The 5 5 showing concentrations in a 1.5 10 to 5 10 dilution; d = dilution factor corresponding microorganisms were grown aerobically in -1 6 cfu ml range were diluted up to 10 and to the lower dilution. Nutrient Broth (NB; CM0001, Oxoid, 107. A double counting through inclusion in Basingstoke, UK) at 37 °C for 24 h. The agar was performed. The number of colony- 1gR = 1gN0 - 1gNa (2) total viable cell count on Nutrient Agar forming units per ml of the suspension was (NA; CM0003, incubated at 37 °C on air for determined following incubation for 48 9 where: R = Reduction of vitality; N0 = 24 h; Oxoid) at 24 h was approximately 10 hours at 37 ± 1°C. For the assay, the bacte- cfu ml-1. Decimal dilutions were performed N/10; Na = bacterial counting for the test Non-commercialrial suspension showing concentrations in a mixture al the end of the contact time. The to obtain the following concentrations in 1.5x108 to 5x108 cfu ml-1 range were dilut- 6 -1 5 -1 4 -1 higher the value, the higher the vitality NB: 10 cfu ml , 10 cfu ml , 10 cfu ml , ed up to 106 and 107. 3 -1 reduction. and 10 cfu ml . The total viable cell counts A double counting through inclusion in for all dilutions were recorded as controls agar was performed. The number of colony- on NA and on the following media. Violet forming units per ml of the suspension was red bile glucose agar (CM0485, Oxoid), determined following incubation for 48 was used for counts of E. coli, Enterococcus hours at 37°C ± 1°C and N value was calcu- Results agar (CM0984, Oxoid) was used for E. fae- lated. For the initial antimicrobial screening calis, Oxacillin Resistance Screening Agar The assay sample and the bacterial sus- with the well plate test (Table 1), S. aureus base (ORSAB; CM1008, Oxoid) with pensions had previously been stabilised at growth was inhibited by plant extract A and ORSAB selective supplement (SR019, the test temperature of 20°C ±1°C. For each B at the concentration of 1:1 and 1:2; E. fae- Oxoid) was used for S. aureus. bacterial strain and for each concentration calis growth was inhibited by extract A and of the test substance, one test tube contain- B at the highest concentration. No activity Antimicrobial screening with a mod- ing 1 ml of sterile water and 1 ml of bacte- was detected against E. coli and plant ified well plate test rial test suspension showing concentrations extract C had no detectable antimicrobial Antimicrobial activity was determined in a 1.5x108 to 5x108 cfu range, was pre- activity. using a modified well plate method.41 pared at the temperature adopted during the Based on the results of antimicrobial Muller Hinton agar (Oxoid, Basingstoke, assay. Alter 2 minutes of contact, 8 ml of the screening, the three tester strains were chal-

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Table 1. Minimum inhibitory concentration of aqueous extract of saffron stigmas (C. sativus from Torbat-e Heydarieh (sample “A”), Gonabad (sample “B”) and Khorasan (sample “C”), Iran. Sample A Sample B Sample C 1 2 4 8 16 C 1 2 4 8 16 C 1 2 4 8 16 C E. coli + + + + + + + + + + + + + + + + + + S. aureus - - + + + + - - + + + + + + + + + + E. faecalis - + + + + + - + + + + + + + + + + + MIC: minimum inhibitory concentration. 1 = 100 mg/100 mL, 2, 4, 8, 16 are the serial dilutions, C: negative control.

Table 2. Vitality reduction expressed in logarithm (LogR), for the three tester strains chal- lenged with several dilution of aqueous extract of saffron stigmas (C. sativus from Torbat- e Heydarieh, Iran). References 1. Fernández J. Biology, biotechnology Whole 1:2 1:4 1:8 1:16 and biomedicine of saffron. Recent Res E. coli 6.56 3.76 0.46 0.79 0.83 Develop Plant Sci 2004;2:127-65. E. faecalis 6.85 5.92 1.40 0.71 0.71 2. Alberini M. Saffron: sapore e colore. Lo zafferano. Proceedings of the S. aureus 4.44 2.47 3.83 0.77 0.77 International Conference on Saffron (Crocus sativus L). L’Aquila, Italy; 1990;39-46. 3. Frello S, Heslop-Harrison J. Repetitive DNA sequences in Crocus vernus Hill (Iridaceae): the genomic organization onlyand distribution of dispersed elements in the genus Crocus and its allies. Genome 2000;43:902-9. 4.Grilli Caiola M, Canini A. Ultrastructure of chromoplasts and use other plastids in Crocus sativus L. (Iridaceae). Plant Biosyst 2004;138:43- 52. 5. Winterhalter P, Straubinger M. Saffron- renewed interest in an ancient spice. Food Rev Int 2000;6:39-59. 6. Basker D, Negbi M. Uses of saffron. Econ Bot 1983;37:228-36. 7. Grilli Caiola M, Canini A. Looking for Figure 1. Vitality reduction activity of aqueous extract of saffron stigmas (C. sativus from saffron’s (Crocus sativus L.) parents In: Torbat-e Heydarieh, Iran). Saffron (A.M. Husaini, ed.). Funct Plant Sci Biotechnol 2010;4:1-14. 8. Porter R. The flora of the Theran wall lenged with aqueous solutions of saffron antimicrobial activity. In conclusion, the painting: living plants and motifs- Sea stigmas, which showed modest, yet evident, degree of antibacterial property for aqueous lily, Crocus, Iris e Ivy. In: The wall antibacterial activities as demonstrated by extracts of saffron, yet mild, has been paintings of thera. Proceedings of the the test tube analysis. R values forNon-commercial the high- demonstrated. Saffron may be selected for first- International Symposium, Thera est concentration are in the range 4.4-6.84 use as potential anti-microbial agent in var- Foundation. Vol II. 2000;603-630. (Table 2 and Figure 1). ious food products depending on the desired 9. Tammaro F. Notizie storico-colturali flavour of the products. However, there are sullo zafferano (Crocus sativus L. some limitations in using saffron in certain Iridaceae) nell’area mediterranea. food matrix, such as the decreasing of Micol Veget Medit 1987;2:44-59. Discussion and Conclusions antimicrobial activity when diluted and the 10. Melnyk J, Wang S, Marcone M. strong flavour. The overall flavour of the The results obtained from the antimi- Chemical and biological properties of products may not be acceptable if a large crobial activity study indicate that stigmas the world’s most expensive spice: amount of saffron is needed to achieve a of C. sativus are a good source of antimicro- Saffron. Food Res Int 2010;43:1981-9. significant inhibition of pathogens and bial agents. Based on these results, it is pos- 11. Inoue E, Shimizu Y, Shoji M, et al. hygiene indicators, especially when the sible to conclude that aqueous extracts of Pharmacological properties of N-095, a effect of other preservatives is weak. A pos- stigmas of saffron had a moderate antimi- drug containing red ginseng, polygala sible way is to use saffron in combination crobial activity. These results might be con- root, saffron, antelope horn and aloe with other preservatives such as acid, salt, sidered sufficient for further studies on the wood. Am J Chin Med 2005;33:49-60. sugar, and other chemical preservatives, or isolation and identification of the active 12. Xu G, Li G, Ma H, et al. Preventive other food preservation systems such as principles and to evaluate of possible syner- effect of crocin in inflamed animals and thermal processing, freezing or cold stor- in LPS-challenged RAW 264.7 cells. J gism among extract components for their 43 age. Agric Food Chem 2009;57:8325-30.

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