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Advanced Science LettersVol. 5, 1–4, 2012

Evaluation of Antioxidant andAntimicrobial Activities of Torularhodin

Camelia Ungureanu∗ and Mariana Ferdes

Faculty of Applied Chemistry and Materials Science 1-7, University Politehnica of Bucharest,Polizu Street, 011061, Bucharest, Romania

The present study was designed to evaluate antioxidant activity and the antimicrobial efficacy of torularhodinagainst human pathogenic microbial strains assayed by using agar well diffusion assay. The extract in methanolof torularhodin was used during the study. The zone of inhibition in torularhodin extract was in the range of8.3 mm to 25 mm. The MIC values of ethanol extract of torularhodin for different pathogenic microbial strainsranged from 22.18 to 44.375 mg/mL. On the basis of this finding, the extracts demonstrating antimicrobialefficacy could result in the discovery of novel antimicrobial agent. The results obtained at the investigation ofantioxidant properties of the torularhodin extract revealed a strong antioxidant activity of the extract. Originallytorularhodin extract had an antioxidant activity of 96%. The novelty of this research consists in the orientationtowards a new carotenoid pigment preparation, the torularhodin, which will be used in therapeutic applicationsbecause of its important antioxidant and antimicrobial activities.

Keywords: Antioxidant, Torularhodin, Rhodotorula Rubra, Antimicrobial Activity.

1. INTRODUCTIONNatural colorants of microbial origin have attracted the world-wide commercial interest due to the potential toxicity causedby synthetic colors. With the help of biotechnology interven-tion, production of some food grade natural pigments suchas �-carotene from Rhodotorula glutinis, Rhodotorula rubra,riboflavin from Ashbya gossyppi; astaxanthin from Xathophyl-lomyces dendrorhous; red pigments from Monascus purpureus,Monascus ruber1�2 have gained considerable consumer accep-tance. Generally, these pigments are produced in cell bound stateand have low water solubility. Many natural pigments are alsosensitive to heat, pH change and light. Also low cost processesneed to be optimized for the production of microbial pigmentson an industrial scale.

Amongst pigments of natural origin, carotenoids seem to playa fundamental role, their presence in the human diet beingconsidered positively because of their action as pro-vitamin,3

antioxidant or possible tumors-inhibiting agents.4

It has been reported that yeast carotenoids may be impor-tant in the protection against oxidative stress,5 but the action ofindividual carotenoids has not yet been clarified, although mul-tiple carotenoids are produced. Rhodotorula rubra is yeast dis-tributed widely in nature and then can biosynthesize carotenoids;not only �-carotene, but also torularhodin having a non-cycled�-ionone ring is biosynthesized from �-carotene. The structure of

∗Author to whom correspondence should be addressed.

torularhodin (see Fig. 1) was elucidated by Ruegg et al. (1958)6

and it was shown to contain a single carboxyl group at position 1′.Torularhodin has a non-cycled �-ionone ring and a longer

polyene chain than that of �-carotene. It is likely that the morepotent quenching ability of torularhodin is due to its longerpolyene chain. Whereas oxidative stress is known to enhance thebiosynthesis of torularhodin or astaxanthin in other red yeastswhere they are associated with an antioxidant function, this isthe first report implicating plectaniaxanthin in a similar role.7

Oxidative stress resulted from an imbalance of oxidizing speciesand natural antioxidants in the body has been thought to havecontributed to aging, cell apoptosis, and severe diseases suchas cancer, Parkinson’s disease, Alzheimer’s disease, and evencardiovascular disorders.8 Epidemiological studies and interven-tion trials on prevention of cancer and cardiovascular diseasein people taking antioxidant supplements are suggestive thatdietary intake of antioxidants can help scavenge free radicals andoxidants and protect the body against diseases.9

Torularhodin is one of the carotenoid pigments produced bythe yeast Rhodotorula sp., with a terminal carboxylic group con-sidered now-a-days as a powerful antioxidant to be included infood and drugs formulations. As a red pigment of the microor-ganism Rhodotorula rubra, torularhodin has a more potenteffect on the scavenging of peroxyl radicals and inhibits sub-strate degradation by singlet oxygen more effectively than beta-carotene does. Torularhodin was not produced by either speciesunder any incubation conditions and does not directly affect theproductivity of superoxide anions.

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Fig. 1. Chemical structure of torularhodin.

Torularhodin exhibits antioxidation action by capturing activeoxygen, it is effective for preventing or suppressing the for-mation of lipid peroxide in the living body to prevent or sup-press the injury of the oxidation reduction system in the livingbody. It is useful for the prevention and treatment of the dam-age caused by ischemia and reperfusion, myocardial infarction,stenocardia, retinitis, angitis, diabetes, dermatitis, crystalline lensopacification including cataract, pigmentation, etc.

It is also useful as a stabilizing agent for foods, pharma-ceuticals, cosmetics, etc., susceptible to oxidation and withantimicrobial activity.10

2. EXPERIMENTAL METHODSSeveral experiments was realized for growth and carotenoids syn-thesis, including the extraction and quantification of torularhodinin the previously study.11�12

The novelty of this research consists in the orientation towardsa new carotenoid pigment preparation, the torularhodin, whichwill be used in therapeutic applications because of its importantantioxidant and antimicrobial properties.

2.1. Chemiluminescence MeasurementsThe in vitro antioxidant activity of samples has been determinedby chemiluminescence method (CL) using a ChemiluminometerTurner Design TD 20/20, USA, and luminol+H2O2 as generatorsystem, in tampon TRIS-HCl 0.2 M, pH = 8�6 at � = 420 nmand 10−3 M/L concentration compared with torularhodin at thesame concentration. All analyses were performed in triplicate.

The antioxidant activity of methanol solutions of samples wascalculated by using the Eq. (1):13

%AA= I0− IsI0

·100 (1)

where: I0 = the maximum CL for standard at t = 5 s; Is = themaximum CL for sample at t = 5 s.

The extracts were sonicated and vortexed to enhance theirsolubility in pure methanol.

2.2. Photochemiluminescence MeasurementsDetermination of torularhodin antioxidant capacity has beendetermined by photochemiluminescence method (PCL) usingPhotochem Analytic Jena.14 The antioxidant capacity (ACL) isexpressed in echivalent concentration of the Trolox.

In the Photochem, free radicals are generated photochemicallyby UV irradiation of a photosensitizer solution and registeredafter the transport of irradiated solution to the measuring cell ofthe measuring cell of the chemiluminometer.15 The calibrationcurve is constructed by measuring a series of standard solutions,1.0–3.0 nmol Trolox.

Photochemiluminescence measurements assay was employedto measure the antioxidant capacity of the torularhodin extracts.The assay was performed in triplicates. The results of the assaywere expressed relative to Trolox in terms of TEAC.16

Trolox is 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid, a water-soluble derivative of vitamin E. Trolox equivalentantioxidant capacity is a measurement of antioxidant strengthbased on Trolox, measured in units called Trolox Equivalents(TE), e.g., micromole TE/100 g.

2.3. Antimicrobial ActivityThe present study was designed to evaluate the antimicro-bial efficacy of torularhodin methanolic extract against humanpathogenic microbial strains such as bacteria included Staphylo-coccus aureus ATCC 25923, Enterococcus faecalis ATCC 29212,Escherichia coli K 12-MG1655 and the fungus Candida utilis,Aspergillus ochraceus and Fusarium oxysporum MUCL 791assayed by using agar well diffusion assay.17

The bacterial strains were grown in Luria Bertani Agar, LBAplates at 37 �C with medium composition (m1): peptone, 10 g/L;yeast extract 5 g/L, NaCl 5 g/L and agar 20 g/L.

Candida utilis was grown in Malt Extract Agar, MEA (Merck)with medium composition (m2): malt extract 17 g/L and agar20 g/L.

The fungus Aspergillus ochraceus and Fusarium oxysporumwere grown in Potato-Dextrose-Agar, PDA (Merck) with mediumcomposition (m3): potato infusion 4 g/L, dextrose 20 g/L andagar 15 g/L.

The stock culture was maintained at 4 �C.Evaluation of antimicrobial activity was performed by agar

well diffusion assay. Sterile LBA, MEA and PDA plates wereprepared by pouring the sterilized media in sterile Petri platesunder aseptic conditions. The test organism 1 mL was spread onagar plates. Wells were made at the size of 6 mm diameter, inthe agar plates using the sterile borer.

The wells with 50 �L of torularhodin methanolic extract wereplaced on the inoculated plates.

Similarly, each plate carried a blank well by adding solvent(alkaline methanol) alone to serve as a negative control.

All the plates containing bacteria were incubated at 37 �C for24 h and that of fungi at 28 �C for 48 h.

The sensitivity of the microorganism species to the toru-larhodin extracts was determined by measuring the sizes ofinhibitory zones (including the diameter of well) on the agar sur-face around the wells, and values <8 mm were considered asnot active against microorganisms.

The zone of inhibition was measured and expressed inmillimeters.

All of the experiments were performed in triplicate. The resultsare reported as the average of three experiments.

2.4. Determination of Minimum InhibitoryConcentration (MIC) of TorularhodinMethanolic Extract Against Microbial Strains

The minimum inhibitory concentration (MIC) is defined as thelowest concentration of the antimicrobial agent that will inhibitthe visible growth of a microorganism after overnight incuba-tion. MIC of methanol extract of torularhodin was determined bymacrodilution agar method. The MIC was determined followingthe methodology of Pundir and Jain.18

2.5. Macrodilution Agar MethodIn the macrodilution agar method, a two-fold serial dilution ofthe extract was prepared in methanol to achieve a decreasingconcentration ranging from 710 �g/L to 5.55 mg/mL in eight

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sterile tubes labeled 1 to 8. A sterile Durham tube 6 mm diameterwas used to bore well in the culture medium plates (m1, m2respectively m3) and 50 �L volume of each dilution was addedaseptically into the wells made in agar plates in triplicate thathad microbial strains seeded with the inoculum. Similarly, eachplate carried a blank well by adding solvent (methanol) alone toserve as a negative control.

All the test plates were incubated at 37 �C and were observedfor the growth after 24 hrs for bacteria and for fungi at 28 �C for48 hrs. The lowest concentration of an extract showing a clearzone of inhibition was considered as the MIC.19

3. RESULTS AND DISCUSSIONFor the tested Rhodotorula strain it has been determined the high-est pigments yield in the case of MS3 media; the total carotenoidpigments was 871 �g/L and the torularhodin concentration was710 �g/L.12

Antioxidant activity (AA) of torularhodin is determined bychemiluminescence (CL). The result given in Figure 2 presented

Fig. 2. Evolution of AA% in time.

Fig. 3. Variation of photochemical signal in time for torularhodin samples.

Fig. 4. Inhibition zone diameter for microbial strains tested.

(a)

(b)

(c)

Fig. 5. Antibacterial activity of methanol as negative control and toru-larhodin methanolic extract against: (a) Escherichia coli, (b) Staphylococcusaureus and (c) Enterococcus faecalis.

evolution of AA% for torularhodin in time (14 days) calculatedwith relation 1.

The results obtained at the investigation of antioxidant prop-erties of the torularhodin extract revealed a strong antioxidant

Fig. 6. Antifungal activity of methanol as negative control and torularhodinmethanolic extract against: (a) Candida utilis; (b) Aspergillus ochraceus and(c) Fusarium oxysporum.

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Table I. Minimum inhibitory concentration of tourlarhodin methanolic extract against test microbial strains.

Concentration of torularhodin methanolic extract (�g/L)

Microorganism 5.55 11.1 22.18 44.375 88.75 177.5 355 710 MIC

Escherichia coli + + NA NA NA NA NA NA 22�18Staphylococcus aureus + + NA NA NA NA NA NA 22�18Enterococcus faecalis + + + NA NA NA NA NA 44�375Candida utilis + + + NA NA NA NA NA 44�375Aspergillus ochraceus + + + NA NA NA NA NA 44�375Fusarium oxysporum + + + NA NA NA NA NA 44�375

activity of the extract. Originally torularhodin extract had anantioxidant activity of 96%. It decreased by 7% after 7 days andfurther on by 17% after 14 days.

The antioxidant capacity of the initial sample of toru-larhodin is 255.6 �g/mL Trolox equivalents, decreasing over time(it decreased by 4% after 7 days and further on by 26% after14 days). The decrease of antioxidant capacity (see also Fig. 3)indicates the reduction of the lipophilic compounds in the sample,these being subjected to the autooxidation process.

The results obtained for the ACL are according with theantioxidant activity, so there is a high probability that the sam-ple has a high content of lipophilic compounds (high antioxidantcapacity), thus it presents a high antioxidant activity.

It has been known for many years that carotenoids undergo“bleaching” i.e., lose their color, when exposed to radicals orto oxidizing species. This process involves interruption of theconjugated double bond system either by cleavage or by additionto one of the double bonds.

The main degradative reaction of carotenoids is oxidation.Oxygen may act either directly on the double bonds or throughthe hydroperoxides formed during lipid autoxidation.

Figure 3 shows the antioxidant activity decreases in time; toprevent this it is proposed that in future research to achieve stabi-lization of the torularhodin with solid lipid nanoparticles (SLN).

Since their first description by Müller et al.20 SLN haveattracted increasing attention as an efficient and non-toxic alter-native lipophilic colloidal drug carrier prepared either withphysiological lipids or lipid molecules used as common pharma-ceutical excipients.

The study of these materials represents an important step intheir utilization for various bio-medical applications.

Studies on the antimicrobial activities of torularhodin extractsare lacking in literature data.

Among treatments, according to Figure 4 maximum in vitroinhibition of tested microorganism E. coli, S. aureus and C. utilis(see also Figs. 5 and 6) was scored in methanol extract of toru-larhodin which offered inhibition zone of 25 mm, 18 mm and16 mm respectively.

The results for the minimum inhibitory concentration weretabulated in Table I.

From the present study it can be concluded that the methanolextract of torularhodin were highly effective against all thebacterial and fungal strains.

4. CONCLUSIONSIn conclusion, the methanol extract of torularhodin showed strongantioxidant activity, 96%. In addition, this extract possessed

noticeable antimicrobial activity against gram positive andgram-negative bacteria.

It is evident from the present study that the torularhodinextract could be utilized as a good natural source of antioxidantsand a possible food supplement or as an antimicrobial agent inpharmaceutical industry.

The present study of in vitro antimicrobial evaluation toru-larhodin forms a primary platform for further pharmacologicalstudies to discover new antibiotic drugs.

Acknowledgments: The work was financially supportedby the project POSDRU/89/1.5/S/52432 from 1.04.2010—Institutional organization of a postdoctoral school of nationalinterest “Applied biotechnology with impact in the Romanianeconomy;” the project was cofunded by the EU Social Fundin the framework of the Sectorial Operational Programme2007–2013 for Human Resources Development.

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Received: xx Xxxx xxxx. Accepted: xx Xxxx xxxx.

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