FIZIOLOGIA
physiology
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Fiziologia - Physiology • 2015.25.2(86)FIZIOLOGIA

physiology
CONTENTS
1. Effect of Magnegita on Selected Blood Parameters ...................................................................................................................... 4
Minca DM, Brad S, Tatu C, Cobzariu FI
2. Analysis of Sterolic Compounds from Vegetal Extracts of Ribes Nigrum, Rosa Canina, Betula Pubescens,
Carpinus Betulus, Viburnum Lantana and Propolis Tincture .......................................................................................................... 10
Orodan M, Vlase L, Istudor V
3. Antibacterial Effects of Some Plant Extracts on Staphylococccus Aureus Strains .................................................................... 16
Simon LM, Pepelea L, Junie LM
4. Acute Oral and Dermal Effects of Spirulina in Mice ..................................................................................................................... 22
Andrica FM, Dehelean C, Serban MC, Pânzaru I, Coricovac D, Drăgan S
5. The Role of Spirulina Platensis in the Control of Type 2 Diabetes Mellitus ................................................................................ 27
Serban MC, Stoichescu-Hogea G, Gurban C, Petcu F, Jeyakumar D, Andrica F, Munteanu C, Dragan S
6. Effects of Resveratrol and Coenzyme Q10 Supplementation in Metabolic Syndrome ............................................................... 32
Ardelean F, Stoichescu-Hogea G, Gurban C, Şerban MC, Petcu F, Antal DS, Drăgan S
7. Novel Insights into the Role of Methylene Blue in Mitochondrial Protection................................................................................ 38
Duicu OM, Sturza A, Scurtu-Mytiko I, Privistirescu A, Dănilă M, Noveanu L, Muntean DM

CUPRINS
1. Efectul Magnegita asupra unor parametri sanguine ...................................................................................................................... 4
Minca DM, Brad S, Tatu C, Cobzariu FI
2. Analiza compusilor sterolici din extractele vegetale de Ribes Nigrum, Rosa Canina, Betula Pubescens,
Carpinus Betulus, Viburnum Lantana și tinctura de propolis ....................................................................................................... 10
Orodan M, Vlase L, Istudor V
3. Efectele antibacteriene ale unor extracte din plante asupra tulpinilor de Staphylococccus Aureus .......................................... 16
Simon LM, Pepelea L, Junie LM
4. Efectele acute orale și cutanate ale spirulinei pe model animal de șoarece ............................................................................... 22
Andrica FM, Dehelean C, Serban MC, Pânzaru I, Coricovac D, Drăgan S
5. Rolul Spirulina Platensis în controlul diabetului zaharat tip 2 .................................................................................................... 27
Serban MC, Stoichescu-Hogea G, Gurban C, Petcu F, Jeyakumar D, Andrica F, Munteanu C, Dragan S
6. Efectele suplimentelor alimentare conținând resveratrol și coenzima Q10 la pacienții cu sindrom metabolic ......................... 32
Ardelean F, Stoichescu-Hogea G, Gurban C, Şerban MC, Petcu F, Antal DS, Drăgan S
7. Noi perspective asupra rolului albastrului de metilen în protecția mitocondrială ...................................................................... 38
Duicu OM, Sturza A, Scurtu-Mytiko I, Privistirescu A, Dănilă M, Noveanu L, Muntean DM

Fiziologia - Physiology • 2015.25.2(86)3EFFECT OF MAGNEGITA ON SELECTED BLOOD PARAMETERS
DELIA MARIA MINCA1, SILVIU BRAD2, CARMEN TATU2, FLORIN IOSIF COBZARIU2
1Pius Branzeu County Clinical Emergency Hospital Timisoara
2Victor Babes University of Medicine and Pharmacy Timisoara

ABSTRACT
Magnetic resonance imaging (MRI) is a noninvasive diagnostic test widely used in clinical practice. Signal resolution
and sensitivity can be further enhanced by adding contrast agents in order to make abnormalities or disease processes
more visible. Gadopentetate dimeglumine was the first approved gadolinium-based contrast agent (GBCA) and showed
good diagnostic value and high levels of safety and tolerance. However, if the free gadolinium (Gd) ion is released invivo, cardiovascular and neurologic toxicity may occur. The plasma half-life of the Gd chelates is dependent upon thevolume of distribution and the glomerular filtration rate of the agent, and it is higher in patients with impaired renal
function. This is why both FDA and EMEA recommended caution when using GBCA-enhanced MRI in patients withmoderate renal impairment and banned their use in those with severe renal impairment. The aim of this study was toassess the effect of Magnegita on certain laboratory parameters in patients with normal renal function undergoing
cerebral and abdominal MRI scans. Twenty-one patients were included from whom we collected three blood samples(prior to MRI scan, at 30 minutes and 24 hours after). We have assessed 27 parameters by the means of hematology,
biochemistry and ionogram tests. Our results showed minor and transient changes in some of the parameters, but mostof them remained unchanged throughout the study. Further studies are needed to determine the effect ofgadopentetate dimeglumine on the same laboratory parameters in patients with impaired renal function.
Key words: Magnegita, gadopentetate dimeglumine, laboratory parameters

INTRODUCTION
Magnetic resonance imaging (MRI) is a noninvasive
diagnostic test widely used in clinical practice as it allows
for the simultaneous examination of the anatomy andphysiological parameters of tissues and organs bymeasuring the radio-frequency signals emitted by water
protons in living tissues, without using harmful radiation[1,2]. In order to make abnormalities or disease
processes more visible, the signal resolution andsensitivity need to be enhanced and this is achieved byadding contrast agents (CAs); therefore approximately
35% of MRI scans are currently performed with the useof CAs [3, 4]. Gadolinium (Gd), a rare-earth metal of thelanthanide series, is the most widely used metal for the
production of CAs, being administered in the form ofchelate complexes in order to prevent the release of toxic
metal ion [5] The free gadolinium ion has cardiovascularand neurologic toxicity, being deposited in liver, bone andlymph nodes [6].
Gadopentetate dimeglumine (Gd-DTPA) was the first
approved gadolinium-based contrast agent (GBCA) [7,8].
Early studies [9-11] evaluating the safety and efficacy of
Gd-DTPA have concluded it has higher diagnostic valuethcompared to the non contrast-enhanced MRI and
exhibits high levels of safety and tolerance. In a
multicenter double-blind randomized clinical trial [9],Gd-DTPA improved diagnostic ability in 65% of patientsand led to changes in presumptive diagnosis, along withvisualization of more lesions in some of those patients.
Adverse reactions in some patients were reported, suchas asymptomatic rise in serum iron and bilirubin levels,
hyper- or hypotension, weakness, conjunctivitis, local
burning at injection site, headache, nausea and vomiting,all of which were minor and transient. These adverse
reactions were comparable to those related toadministration of iodinated non-ionic roentgen contrast
media [12].
However, the plasma half-life of the Gd chelates isdependent upon the volume of distribution and theglomerular filtration rate of the agent, and it is higher
in patients with impaired renal function [13].
Contrast-induced nephropathy, defined as increase increatinine concentration [14] or decrease inglomerular filtration rate [15] has been associated with
GBCAs, particularly at high doses and in patients withrenal impairment. Moreover, nephrogenic systemic
fibrosis (NSF), a life-threatening fibroproliferativethReceived 10 of May 2015. Accepted 5 of June 2015. Address for correspondence: Delia Minca, MD, PhD student, Pius
Branzeu County Clinical Emergency Hospital Timisoara, Iosif Bulbuca No. 10 Street, RO-300736, Timisoara, Romania; phone:+40356433121; fax: +40256486956; e-mail: delia.minca@yahoo.com

4

Fiziologia - Physiology • 2015.25.2(86)disease seen in people with severe renal impairment,
was first observed in 1997 [16] and it has been
associated with the use of GBCAs. NSF is thought to becaused by the deposition of free gadolinium in tissues ofpatients with severe renal dysfunction and thesubsequent inability of kidneys to eliminate it because ofpoor water solubility of the gadolinium [17]. Consequently,
in 2007 the USA Food and Drug Administration
requested manufacturers to add a warning box to thelabels of all GBCAs, stating the increased risk of NSF inpatients with acute or chronic severe renal insufficiency[18], while the European Medicines Agency (EMEA) has
released in 2009 a set of recommendations [19],classifying the active substances in the GBCAs into three
categories of risk: high-, medium-, and low-risk. EMEA’s
Committee for Medicinal Products for Human Use hasrecommended contraindications in patients with severe
kidney problems, in those scheduled for or who had
recently received a liver transplant and in newborn
babies up to four weeks of age for high-risk
gadolinium-containing contrast agents, including
Magnegita. Additionally, patients with unknown kidney
problems should always be screened for kidney
problems using laboratory tests before the use of a
GBCA [19]. Finally, a wide variety of changes inphysiology may occur following the administration ofgadolinium salts and chelates [20, 21]. However, most of
the studies have focused on the effects of GBCAs on
kidneys, while just a few have specifically addressed thephysiological changes triggered by these agents.
The aim of this study was to assess the effect ofMagnegita on certain laboratory parameters in patientswith normal renal function undergoing cerebral andabdominal MRI scans.

40 years of age, while the other three (2 women, 1 man)were aged over 40 (Figure 1). The number of men
included was slightly higher (Figure 2).

Fig. 1. Distribution of patients by age group

Fig. 2. Distribution of patients by gender

MATERIALS AND METHODS
The study group was composed of 21 patients who
underwent cerebral and abdominal MRI scans in theRadiologica Plus Medical Imagistics Centre Timisoara in
the period January - March 2015. No patient has had
kidney or liver impairment.
Inclusion criteria: patients aged 20-60, both genders; notpregnant or breastfeeding, in case of women; had not
had an organ transplant; had not received any
medications within 48 hours of participation; had notreceived any contrast agent within 7 days; willing to sign
Informed Consent Form; willing to provide three blood
samples.
Exclusion criteria: patients with impaired renal function;
patients who have had a contrast-enhanced MRI or CT
seven days prior to inclusion in the study; under chemoor radiation therapy; highly elevated or decreasedlaboratory parameters.
Most of the patients (18; 7 women, 11 men) were under
Fiziologia - Physiology • 2015.25.2(86)All the patients in this study received 0.1 ml/kg bodyweight gadopentetate dimeglumine (Magnegita 500
micromol/ml, Biokanol Pharma GmbH) by intravenous
injection once and the exposure to the contrast agent
was not repeated in any of the patients. As the use of
GBCAs has been linked to the risk of developing
nephrogenic systemic fibrosis, all the patients arescreened for renal dysfunction prior to be administered
Magnegita. None of the patients included in our study
suffered from moderate (GFR 30–59 ml/min/1.73 m2) orsevere (GFR < 30 ml/min/1.73m2) renal impairment.
After the patients have signed the Informed Consent
Form to be included in this study, two blood samples of 3
ml (for complete blood count) and 6 ml (for biochemistry
tests), respectively, were collected from each patient forthree times, namely prior to MRI, then 30 minutes and 24
hours after the MRI scan. We have analysed the same
27 parameters (see Table I) by the mean of complete
blood count, biochemistry and ionogram tests. We haveused reagents from Diagon for hematology tests, DiaSys
5

Diagnostic Systems for biochemistry tests and Medica for
ionogram tests. All reagents were used according to the
manufacturer’s instructions.
Table I. Methods and parameters studied

one of the patients who had subnormal values prior to
injection of Magnegita, but dropped again at 24 hours,
when we also found one more patient with increased
values. Distribution of patients with lower, normal or
higher neutrophil and lymphocyte percentages across thethree measurements is shown in Figure 3.

Fig. 3. Distribution of patients according to neutrophil andlymphocyte percentages

RESULTSAt baseline, the WBC level was normal in 20 patients andlower-than-normal in one; no changes were seen in the
WBC level of the latter patient following theadministration of the contrast agent at the subsequent
blood tests. Thirty minutes after the injection ofMagnegita, the WBC level slightly increased in one
patient and then returned to normal after 24 hours.
Additionally, a slightly decreased level was recorded in
one patient at 24 hours, although this patient has had
normal values both at baseline and 30 minutes
post-injection. As mentioned above, the changes wereminor, with no clinical significance.
The white blood cell differential has yielded various
results. The absolute count of neutrophils, lymphocytes
and monocytes has remained unchanged throughout thethree measurements. However, the neutrophil
percentage was above the normal range limit in threepatients at baseline and it remained high after thecontrast agent was injected; the only difference noted
was in one patient with lower values at 30 minutes and24 hours. The lymphocyte percentage was in the normal
range in 16 patients at baseline (lower values - 3 patients;
higher values -2 patients); 30 minutes post-injection, thelymphocyte percentage has reached the normal range in

6

The absolute count of eosinophils revealed 11 patients had
normal values at baseline, but only five after 30 minutes
and 24 hours, respectively, while the other patients (10 atbaseline, 16 at the other two measurements) had slightly
decreased levels. Nevertheless, the eosinophil
percentages exhibited values in the normal range for 17
patients (higher - 4 patients) at the baseline, for 19 patients(higher - 2 patients) 30 minutes after the administration of
the GBCA, and for 18 patients (higher - 3 patients) 24
hours post-injection (Figure 4).
Both the absolute count and the percentage of basophils
were in the normal range for 20 patients (higher - 1
patient) at the first and second measurements, while atthe third measurement, values of 19 patients (higher - 2
patients) remained normal. The monocyte percentage
has shown no variations, with one of the patients having
decreased values throughout the study.

Fig. 4. Distribution of patients according to absolute count andpercentage of eosinophils
Fiziologia - Physiology • 2015.25.2(86)Red blood cells count showed three patients had lower
levels at baseline and remained unchanged at the othertwo measurements, while the other 18 patients had normal
levels. After the injection of Magnegita, RBC dropped inanother three patients at the 30 minutes post-injection
measurement, but returned to normal after 24 hours. We
have also found one patient with higher levels at the 24 hblood test (see Fig. 5). Lower levels of hemoglobin andhematocrit were also seen in four and five patients,
respectively, at all three measurements. In three of thepatients who had normal levels at baseline, HGB has
decreased 30 minutes post-injection, but returned tonormal after 24 hours. Similarly, the HCT level has
decreased at the second measurement in two patients, but
it was normal after 24 hours. The mean platelet volume
was higher in three patients at baseline, but it decreased in
one of them to a value within the normal range at thesubsequent measurements. The platelet distribution width
has slightly decreased in one patient 24 h after the
injection of the contrast agent. No changes were recorded
in mean corpuscular volume, mean corpuscularhemoglobin, mean corpuscular hemoglobin concentration,
red cell distribution width, and platelets.

Fig. 6. Distribution of patients according to serum calcium levels

Fig. 7. Distribution of patients according to serum chloride levels

DISCUSSION

Fig. 5. Distribution of patients according to RBC levels

The biochemistry tests showed no changes in serum urea,
serum
creatinine,glycemia,
andaspartateaminotransferases. Two patients had higher-than-normal
levels of alanine aminotransferases at baseline, but only
one at the following measurements. The total serum
proteins were also higher at baseline in three patients, andthen decreased in two of them at the subsequent tests.
The serum calcium level was higher in one patient
throughout the study. At the second measurement, it
decreased in another patient, while 24 hours later twopatients had lower levels (Figure 6). The serum chloride
was found to be higher than normal in 12 patients atbaseline, and then in 13 and 14 patients at 30 minutes
and 24 hours, respectively, after the injection ofMagnegita (Figure 7). Serum magnesium, sodium andpotassium remained unchanged during the study.
Fiziologia - Physiology • 2015.25.2(86)Gadolinium chelates are routinely used nowadays inmagnetic resonance imaging, mostly for CNS pathology,
followed by contrast-enhanced MR angiography, liver
imaging, and breast evaluation for malignancy [22]. After
intravenous injection, they rapidly distribute in the bloodand the extracellular space [13, 23] and are normallyeliminated unmetabolized via kidneys through glomerular
filtration [13, 24], with half-lives of approximately 1.5
hours in people with normal renal function [5, 13, 23].
Unfortunately, toxic Gd(III) ions may be released in vivo
by transmetallation with other metal ions, thus the kinetic
stability of the gadolinium chelates is very important [3].
The ionic linear Gd chelates (such as gadopentetate
dimeglumine) were shown to be more stable than the
nonionic linear ones under normal and particularly underaltered physiological conditions [25]. Although earlystudies have found gadopentetate dimeglumine is safe
and well tolerated [9-11] in people with normal renal
function, it has been later proved that GBCAs may trigger
nephrogenic systemic fibrosis in patients with kidney
disorders [6, 26, 27]. Therefore, for safety reasons, the7benefits and risks of GBCA-enhanced MRI should be
carefully balanced in patients with impaired renal function
and also in patients with liver impairment.
A
double-blind,randomized,
crossover,placebo-controlled study designed to evaluate the safety
and tolerance of gadopentetate dimeglumine 0.1
mmol/kg when administered to normal subjects at a fourtimes higher bolus injection rate than that recommended
by the producer found no clinically significant changes inlaboratory parameters, even a few values outside thespecified normal ranges were recorded [28].
Nevertheless, other data in the literature show
gadolinium may interfere with various laboratory
parameters, although changes are minor and transient.
In a retrospective study, data regarding serum calcium,magnesium and creatinine levels were reviewed before
and within 1 day after gadodiamide or gadopentetate
dimeglumine administration [29]. A transient decrease inserum creatinine was seen in patients with renal
impairment who received gadopentetate dimeglumine,
but no changes were recorded in serum calcium andmagnesium. Conversely, 36.7% of the patients withhigh-dose gadodiamide injection and renal insufficiency
had spurious critical hypocalcemia and minor increases
in serum magnesium. Moreover, other in vivo and in vitro
studies have documented the interference withcolorimetricassaysofgadodiamide
andgadoversetamide, resulting in spurious hypocalcemia,
but gadopentetate dimeglumine has not been shown tocause this interference [20, 30].
The study by Proctor et al. [20] on the effects ofgadodiamide,
gadoversetamide,
gadopentetate
dimeglumine, and gadoteridol has shown that GBCAs
can produce positive or negative analytic interferencewithmultipleserum
assays,includingangiotensin-converting
enzyme,calcium,iron,magnesium, total iron binding capacity, and zinc, with thefirst two producing the most interference. Gadopentetate
dimeglumine showed interference with iron, while amanual colorimetric zinc assay showed interference withall the GBCAs studied. However, the other parameters
measured (41 in total, including some of the parameters
we have studied) showed no significant interference withany of the four GBCAs.
Our study results are mostly consistent with these reports.
Furthermore, we have found minor and transient or nochanges in the other parameters evaluated in this study.

CONCLUSION
Although the risk of developing nephrogenic systemic
fibrosis or contrast-induced nephropathy is currently aconcern in people with impaired renal function, the use of
GBCAs in patients with normal renal function remains
safe. Moreover, gadopentetate dimeglumine is among8

the safest GBCAs and it triggered no clinically significant
changes in the laboratory parameters of the patients
included in this study. Further studies are needed todetermine the effect of gadopentetate dimeglumine on
the same laboratory parameters in patients with impaired
renal or hepatic function.

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12. Niendorf HP. Tolerance and safety of Gd-DTPA in 7000
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13. Ersoy H, Rybicki FJ: Biochemical safety profiles ofgadolinium-based extracellular contrast agents andnephrogenic systemic fibrosis. J Magn Reson Imaging 2007;
26(5): 1190–1197.
14. Briguori C, Colombo A, Airoldi F, et al. Gadolinium‐based
contrast agents and nephrotoxicity in patients undergoingcoronary artery procedures. Catheterization and CardiovascularInterventions 2006; 67(2): 175-180.
15. Erley CM, Bader BD, Berger ED, et al. Gadolinium-based
contrast media compared with iodinated media for digital
subtraction angiography in azotaemic patients. Nephrology
Dialysis Transplantation 2004; 19(10): 2526-2531.

Fiziologia - Physiology • 2015.25.2(86)16. Tavernaraki A, Skoula A, Benakis S, and Exarhos D. Side
Effects and Complications of Magnetic Resonance Contrast
Media. Hospital Chronicles 2012; 7(4): 208-214.
17. Penfield JG and Reilly RF. What nephrologists need toknow about gadolinium. Nature Clinical Practice Nephrology
2007; 3(12): 654-668.
18. http://www.fda.gov/downloads/safety/medwatch/safetyinf
ormation/safetyalertsforhumanmedicalproducts/ucm154532.pdf
19. http://www.ema.europa.eu/docs/en_GB/document_library/Press_release/2009/11/WC500015569.pdf
20. Proctor KAS, Rao LV and Roberts WL. Gadolinium
magnetic resonance contrast agents produce analyticinterference in multiple serum assays. American Journal of
Clinical Pathology 2004; 121(2): 282-292.
21. Adding LC, Bannenberg GL, Gustafsson LE. Basicexperimental studies and clinical aspects of gadolinium salts
and chelates. Cardiovascular drug reviews 2001; 19(1):41-56.
22. Runge VM. Current Technological Advances in Magnetic
Resonance with Critical Impact for Clinical Diagnosis andTherapy. Investigative Radiology 2013; 48(12): 869-877.
23. Aime S, Caravan P. Biodistribution of gadolinium-based
contrast agents, including gadolinium deposition. J Magn
Reson Imaging. 2009; 30: 1259–67.
24. Canga A, Kislikova M, Martinez-Galvez M et al. Renalfunction, nephrogenic systemic fibrosis and other adverse

reactions associated with gadolinium-based contrast media.
Nefrologia 2014; 34(4): 428-38.
25. Frenzel T, Lengsfeld Ph, Schirmer H, Hütter J, andWeinmann HJ. Stability of Gadolinium-Based Magnetic
Resonance Imaging Contrast Agents in Human Serum at 37[degrees] C. Investigative Radiology 2008; 43(12): 817-828.
26. Thomsen HS, Marckmann P, Logager VB. Nephrogenic
systemic fibrosis (NSF): a late adverse reaction to some of thegadolinium based contrast agents. Cancer Imaging 2007; 7:
130-137.
27. Zou Z, Zhang H, Roditi GH, Leiner T, Kucharczyk W, andPrince MR. Nephrogenic systemic fibrosis: review of 370
biopsy-confirmed cases. JACC: Cardiovascular Imaging 2011;
4(11): 1206-1216.
28. Kashanian FH, Goldstein HA, Blumetti RF, Holyoak WF,
Hugo FP, Dolker M. Rapid bolus injection of gadopentetate
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29. Zhang HL, Ersoy H, Prince MR. Effects of gadopentetate
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2006;23(3):383-387.
30. Moore CD, Newman RC, Caridi JG. Spurious
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resonance imaging: a case report and review of theliterature. Reviews in Urology 2006; 8(3):165.

EFECTUL MAGNEGITA ASUPRA UNOR PARAMETRI SANGUINIREZUMAT
Imagistica prin rezonanță magnetică (IRM) este o metodă non-invazivă de diagnostic larg utilizată în pracica
clinică. Rezoluția și sensibilitatea semnalelor poate fi sporită prin adăugarea de agenți de contrast, cu scopul dea crește vizibilitatea anomaliilor sau a proceselor patologice. Gadopentetatul de dimeglumină a fost primul
agent de contrast pe bază de gadoliniu (GBCA) aprobat și a dovedit o valoare diagnostic bună, precum și unnivel ridicat de siguranță și tolerabilitate. Totuși, dacă ionul liber de gadoliniu (Gd) este eliberat in vivo, poateapărea toxicitatea cardio-vasculară și neurologică. Timpul de înjumătățire în plasmă a chelaților de Gd depindede volumul de distribuție și de rata de filtrare glomerulară a fiecărui agent de contrast și este mai mare la pacienții
cu suferință renală. De aceea, atât FDA, cât și EMEA au recomandat prudență în utilizarea IRM cu GBCA lapacienții cu insuficiență renală moderată și au interzis utilizarea lor la cei cu insuficiență renală severă. Scopul
acestui studiu a fost de a evalua efectul Magnegita asupra anumitor parametri de laborator la pacienți cu funcție
renală normală care au fost evaluați prin IRM cerebral sau abdominal. În studiu au fost incluși 21 de pacienți, dela care s-au recoltat trei probe de sânge (înainte de IRM, la 30 de minute și la 24 de ore după). Am evaluat 27 deparametri prin intermediul testelor de hematologie, biochimie și ionogramă. Rezultatea noastre au arătat că uniiparametri au suferite modificări minore și tranzitorii, majoritatea rămânând neschimbați. Sunt necesare alte studii
pentru a determina efectul pe care gadopentetatul de dimeglumină îl are asupra parametrilor de laborator lapacienții cu disfuncție renală.
Cuvinte cheie: Magnegita, gadopentetat de dimeglumină, parametri de laborator

Fiziologia - Physiology • 2015.25.2(86)9ANALYSIS OF STEROLIC COMPOUNDS FROM VEGETALEXTRACTS OF RIBES NIGRUM, ROSA CANINA, BETULAPUBESCENS, CARPINUS BETULUS, VIBURNUM LANTANAAND PROPOLIS TINCTURE
MIHAELA ORODAN1, LAURIAN VLASE2, VIORICA ISTUDOR3
1Clinical Emergency Hospital Arad and “Carol Davila” University of Medicine and Pharmacy Bucharest
2Deptartment of Pharmaceutical Technology and Biopharmaceutics, Faculty of Pharmacy, University of Medicine andPharmacy "Iuliu Hatieganu" Cluj-Napoca
3“Carol Davila” University of Medicine and Pharmacy Bucharest

ABSTRACT
Gemmotherapic vegetal extracts in DH1 dilution of Ribes nigrum, Rosa canina, Betula pubescens, Carpinus betulus,
Viburnum lantana, 30% propolis tincture were analyzed through high performance chromatography coupled with mass
spectrometry (LC/MS) to analyze the sterols, specifically: betasitosterol, stigmasterol, campesterol, ergosterol,brassicasterol.
Gemmotherapic vegetal extracts in the DH1 dilution of Rosa canina (X1), Betula pubescens (X2), Ribes nigrum (X3),
Viburnum lantana (X5), Carpinus betulus (X6) and propolis tincture 30% (X4) are frequently used in integrative
medicine for their anti-inflammatory properties. Due to the fact that the anti-inflammatory effect of the vegetal extracts
may also be induced by the sterols, we proposed to analyze these extracts (obtained from Plantextract, Cluj), throughhigh performance chromatography coupled with mass spectrometry (LC/MS)[1,2,3]. The extracts analyzed werebetasitosterol, stigmasterol, campesterol, ergosterol, brassicasterol [1,3]. The concentrations of sterols found throughthe method above mentioned (expressed in µg/mL) were significant only for betasitosterol in: X1 = 15.859, X2 = 17.251,
X3 = 21.598, X4 = 1.516; X5 = 2.033, X6 - unidentifiable.
Keywords: Gemmotherapic vegetal extracts, sterols and propolis, LC/MS/MS analysis

INTRODUCTION
Gemmotherapic vegetal extracts in the DH1 dilution of
Rosa canina (X1), Betula pubescens (X2), Ribes nigrum(X3), Viburnum lantana (X5), Carpinus betulus (X6) andpropolis tincture 30% (X4) are frequently used in
integrative medicine for their anti-inflammatory andantiallergic properties. Due to the fact that we didn’t findresearch regarding their chemical composition in
specialty literature, in this study we analyzed the sterolcompounds using high performance chromatography
coupled with mass spectrometry (LC/MS), as they are
known for anti-inflammatory effect.

MATERIALS AND METHODS
Devices: HPLC coupled with HP1100 Series mass
spectrometer with binary pump; HP1100 Series
Autosampler, HP1100 Series Thermostat, Agilent Ion
thTrap 1100 SL Mass spectrometer; Working conditions
HPLC: Zorbax SB-C18 analytic column 100 mm x 3.0mmi, d = 0.5 microns.
Mobile Phase: methanol-acetonitrile mixture, 10:90 (v/v),
isocratic elution mode; debit: 1mL/min, temperature: 45C,
detection MS/MS, mode MRM; injection volume =5 L.
Working conditions of MS: ion source: APCI (atmosphericpressure chemical ionization); ionization mode: positive;
nebulizer: nitrogen; pressure 60 psi; vaporizer 400C,
drying gas: nitrogen; debit 7L/min; temperature = 325C;
capillary potential: -4000V.
Analysis mode: monitoring of the sterol specific ions.
Standards: beta-sitosterol, stigmasterol, campesterol andergosterol
Monitored ions detected through MS methods arepresented in Table I. Due to the fact that throughionization all the sterols lose on molecule of water, ionsdetected by the MS are always in the following form
[M-H2O+H]+.

th

Received 12 of March 2015. Accepted 20 of April 2015. Address for correspondence: Mihaela Orodan, MD, PhD student,„Carol Davila” University of Medicine and Pharmacy Bucharest, Clinical Emergency Hospital Arad, Andrenyi Caroly No.2-4
Street, Arad, Romania; phone/fax: 0040257211233; e-mail: mihaela_noni@yahoo.com

10

Fiziologia - Physiology • 2015.25.2(86)Table I. Monitored sterol specific ions in ascending orderaccording to retention time (r.t.)
Sterolr.t. (min)

M

M-H2O

M-H2O+H+

Ergosterol

2.6

396

378

379

Brassicasterol

3.3

398

380

381

Stigmasterol

4.0

412

394

395

Campesterol

4.0

400

382

383

Sitosterol

4.6

414

396

397

MS detection revealed parent-ions of the analyzed
sterols (parent-ions = ions which are formed after
ionization before their eventual fragmentation) in eachsterol expected ions were revealed (Table I).
Mass spectrum (full-scan type) of the four sterols arepresented in Figure 1 (a-e).

This method of analysis (also named MS/MS) is
extremely specific as it only registers the intensity of themain ion. An isomer or isobar compound – with the same
molecular mass – may give a false positive signal. The
analysis on the basis of the fragments from the MS
spectrum, which are specific to each structure in turn andwhich are not the same from one isomer to another, willdetect only the compound of interest without interference
from others. As well as this, the ion intensity from the MS
is proportional with the concentration of the substance inthe compound analyzed, so the method may be appliedfor quantification in place of qualitative analysis.
MS/MS spectrums of the analyzed compounds arepresented in Figure 2 (a-e).

Fig. 1. Full -scan spectrums of the analyzed sterols: a. ergosterol, b.brassicasterol, c.campesterol, d. stigmasterol, e. β-sitosterol

Fiziologia - Physiology • 2015.25.2(86)11

MS/MS spectrums of the analyzed compounds arepresented in Figure 2 (a-e).

In order to quantify the analyzed sterols, we built achromatogram for each of the analyzed sterols,
according to the intensity of the principle ions from theMS. The ions used for quantification are numbered inTable II.
Table II. Ions from the MS spectrum of analysed sterols
No.

Compound nameIons specific for identification

Ion [M-H2O+H+] > Ions from spectrum
1

Ergosterol379> 253.3,295.3,309.3

2

Brassicasterol381>201.3,203.3,215.2,217.3,241.
2,255.3,257.4,271.1,297.3,299.3

3

Campesterol383>147.3,149.3,161.3,175.3,189.
3,203.3,215.3,229.3,243.3,257.3

2

Stigmasterol395>163.3,173.2,187.3,199.3,227.
2,241.3,255.3,269.2,283.4,297.3

4

Sitosterol397>160.9,174.9,188.9,202.9,214.
9,243.0,257.0,287.1,315.2

12For the construction of calibration curves, standard
sterols were dissolved in acetone (concentration 1
mg/mL), from which successive dilution in acetonitrile
were made in order to obtain the calibration solution. The
concentration of calibration solution of each analyzed
sterol, concentrations found and the accuracy of thedetermination are presented in Tables III-VII.
Table III. Concentration of calibration solutions of ergosterol
and the accuracy of the determination
Concentration
level
1
2
3
4
5
6
7

Ergosterol
Standard
concentration(ng/ml)69.00
138.00276.00552.001104.00
2208.00
3312.00

Concentration
found (ng/ml)

Accuracy(%)

70.29
132.21
293.67514.051084.812229.773534.03101.8795.80
106.4093.12
98.26
100.99106.70Fiziologia - Physiology • 2015.25.2(86)Table IV. Concentration of calibration solutions of brassicasterol
and the accuracy of their determination

Concentrationlevel

Brassicasterol
Standard
concentration(ng/ml)Table VII. Concentration of calibration solutions of sitosterol
and the accuracy of their determination
Sitosterol

Concentration
Concentration
found (ng/ml)

Accuracy %standardlevel

concentration(ng/mL)

Concentration

Accuracyfound (ng/mL)

(%)

1

62.00

60.71

97.92

1

132.00139.48105.672

123.00132.74107.922

264.00244.5892.65

3246.00258.38105.03

3528.00528.91

100.174492.00489.8699.56

41056.00

1094.38103.635984.00913.6692.85

52112.00

1943.1392.00

61968.00

1971.57

100.1864224.00

4250.72

100.6372952.00

2931.1699.29

76336.00

6996.62110.43

Table V. Concentration of calibration solution of campesterol
and the accuracy of the determination

A chromatogram of the 5 analyzed sterols in the above
conditions, is shown in Figure 3.
Intens.
x10 5Concentration
level

Campesterol
Standard
concentration(ng/ml)ST_ID___000005.D: TIC +All MSn, Smoothed (0.4,1, GA)

4
2

Concentration
found (ng/ml)

0x10 5 ST_ID___000005.D: EIC 145.2; 159.2; 171.2; 185.2; 199.3; 201.3; 211.3; 213.3; 225.3; 239.2; 253.3; 267.3; 271.4; 283.3; 295.3; 309.3 +MS2(379.4), Smoothed (0.0,1, G

Accuracy (%)

1.5
1.0
0.5

1

59.00

60.58

102.682

117.00111.1194.97

3234.00235.06100.454468.00491.72105.075936.00912.9897.54

61872.00

1761.1494.08

72808.00

3046.94108.51

0.0
x10 4 ST_ID___000005.D: EIC 159.2; 176.4; 187.2; 201.1; 215.3; 217.3; 233.3; 243.3; 255.2; 297.3; 299.3; 301.3; 311.3 +MS2(381.4), Smoothed (0.0,1, GA)
8
6
4
2

0
x10 5 ST_ID___000005.D: EIC 147.2; 149.2; 161.3; 173.2; 175.2; 187.3; 189.3; 203.3; 215.2; 217.2; 233.3; 243.3; 247.3; 257.3; 259.3; 261.3; 273.4; 285.4; 287.4; 301.4 +MS
2.0

1.5
1.0
0.5
0.0
x10 5 ST_ID___000005.D: EIC 161.2; 173.2; 179.2; 187.2; 189.2; 213.3; 215.3; 227.2; 241.2; 269.6; 281.3; 283.3; 297.3; 311.3 +MS2(395.4), Smoothed (0.0,1, GA)
1.00
0.75
0.50
0.25

0.00
x10 4 ST_ID___000005.D: EIC 147.2; 161.2; 163.2; 173.2; 175.3; 177.2; 187.2; 189.2; 203.2; 215.3; 243.3; 257.3; 261.4; 285.4; 289.3; 299.3; 301.3; 315.4 +MS2(397.4), Smo

6
4

Table VI. Concentration of calibration solutions of stigmasterol
and their accuracy of determination

Concentration
level

Stigmasterol
Standard
concentration(ng/ml)Concentration
found (ng/ml)

Accuracy (%)

1

136.00144.6409

106.352272.00241.255288.70

3544.00535.529798.44

41088.00

1152.767

105.9552176.00

2294.101105.4364352.00

4311.8599.08

76528.00

6656.755

101.97

Fiziologia - Physiology • 2015.25.2(86)

2
0
1

2

3

4

5 Time [min]Fig. 3. Chromatograms of the analysed sterols (top to bottom,
superimposed signals of standards. a. ergosterol, b.
brassicasterol, c. campesterol, d. stigmasterol, e. sitosterol.

Calibration curves of each sterol, on the basis of which
the quantification was made are presented in Figure 4(a-e).

13

RESULTS AND DISCUSSION
The concentrations of the sterols found are presented inTable VIII. Concentrations according to the dilutions
car
Table VIII. Sterol concentrations from analyzed sterols
SampleErgosterol

Stigmasterol

Beta-Sitosterol

Campesterol

Brassicasterol

X1

0.000

0.000

15.859

0.000

0.000

X2

0.000

0.000

17.251

0.000

0.000

X3

0.000

0.000

21.598

0.000

0.000

X4

0.000

0.000

1.516

0.000

0.000

X5

0.000

0.000

2.033

0.000

0.000

X6

0.000

0.000

0.000

0.000

0.000

Key: X1= RC DH1, X2=BP DH1 , X3=RN DH1, X4= P 30%, X5=VL DH1,X6=CB DH1

Of all the analyzed sterols, the only sterol detected was-sitosterol. With the exception of the gemmotherapeutic
product of Carpinus betulus (X6), all samples analyzed
contain betasitosterol.

14-sitosterol are found in thegemmotherapeutic products of the following Ribes
nigrum, Betula pubescens and Rosa canina. Smallquantities are found in Viburnum lantana and in propolis
tincture.
Fiziologia - Physiology • 2015.25.2(86)CONCLUSIONSREFERENCES

Due to the fact that in our bibliography we didn`t find the-sitosterol, we consider that our researchbrings a modest contribution to the knowledge of thechemical composition of the gemmotherapeutic products
obtained from Ribes nigrum, Betula pubescens, Rosa
canina.and Carpinus betulus.

1. Pop G, Galuscan A, Peev C, Militaru A, Vlase L, Ardelean
L, Rusu LC. HPLC-MS Identification of Sterol Fractios from
Vegetable Oil. Revista de chimie, 2012; 63(10): 1046-1050.
2. Khalaf I, Vlase L, Ivanescu B, Lazar D, Corciova A. HPLC
Analysis of polyphenolic compounds, phytoestrogens andsterols from Glycyrrhiza Glabra L. Tincture. Studia Ubb Chemia,
2012; 57(2): 113-118.
3. Khalaf I, Corciovă A, Vlase L, Ivănescu B, Lazăr D.
LC/MS Analysis of sterolic compounds from Glycyrrhiza Glabra.
Studia Ubb Chemia, 2011; 56 (3): 97-102.

ANALIZA COMPUSILOR STEROLICI DIN EXTRACTELE VEGETALE DE RIBESNIGRUM, ROSA CANINA, BETULA PUBESCENS, CARPINUS BETULUS,
VIBURNUM LANTANA SI TINCTURA DE PROPOLISREZUMAT
Extractele vegetale de tip gemoterapic in dilutie DH1 de Rosa canina (X1), Betula pubescens (X2), Ribes nigrum(X3), Viburnum lantana (X5), Carpinus betulus (X6) și tinctura 30% de propolis (X4) sunt frecvent folosite în
medicina integrativă pentru acțiune antiinflamatoare. Intrucat efectul antiinflamator al unui extract vegetal poate fi
imprimat și de steroli, ne-am propus analiza acestora produse (de proveniență Plantextract, Cluj), princromatografie de lichide de înalta performanță cuplată cu spectrometrie de masa (LC/MS). S-au analizat:
betasitosterolul, stigmasterolul, campesterolul, ergosterolul, brassicasterolul. Concentratiile de steroli gasite, prin
metoda mai sus mentionata (exprimate in µg/mL) au fost semnificative doar pentru betasitosterol, în: X1 =15.859, X2 = 17.251, X3 = 21.598, X4 = 1.516; X5 = 2.033, X6 neidentificabil.
Cuvinte cheie: extracte vegetale de tip gemoterapic, steroli si propolis, analiza LC/MS/MS

Fiziologia - Physiology • 2015.25.2(86)15

ANTIBACTERIAL EFFECTS OF SOME PLANT EXTRACTSON STAPHYLOCOCCCUS AUREUS STRAINSLAURA M SIMON, LIA PEPELEA, LIA M JUNIE
Microbiology Department, UMF ’’Iuliu Haţieganu”, Cluj-Napoca, Romania

ABSTRACT
Background & Aims: The purpose of this study was to investigate the antibacterial properties of alcoholic extracts andessential oils derived from plants against S. aureus strains isolated from patients.
Material and Methods: We studied 50 strains of S. aureus isolated from various clinics in Cluj-Napoca, as well as fromoutpatients and six natural products: Calendula tincture (Calllendulae flos), tincture of hawthorn (Folium Crataegi as
flowers), Melissa officinalis essential oil, tincture of sage (Salviae folium), propolis tincture and Biosept forte containing
80% thyme essential oil (Thyme aetheroleum) and 20% cloves essential oil (Caryophyllorum aetheroleum). For eachstrain susceptibility testing was performed using disk diffusion method on subculture standardized Kirby-Bauer withmanual reading.
Results: MSSA strains showed inhibition zone diameters of ≥ 10 mm for Melissa officinalis oil at 100% of strains, for
propolis tincture in proportion of 93.33% of strains, for Salviae folium at a rate of 86.66% of strains and in 46.66% ofstrains for both calendula tincture and for hawthorn. MRSA strains showed inhibition zone diameters of ≥ 10 mm, incase of Melissa officinalis oil and propolis tincture to 100% of strains, for Salviae folium in the proportion of 97.14% ofstrains, a rate of 28.57% of strains for tincture of hawthorn and the rate of 14.28% of strains for Calendula tincture.
Conclusion: All six plant extracts tested strains of S. aureus had antibacterial effects. The best antibacterial effects
was observed in case of Biosept forte product. Data showed significant antibacterial effect of Melissa officinalis
essential oil and propolis. Moderate antibacterial effect was observed for sage tincture (Salviae folium), hawthorn(Folium crataegi) and marigold (Calllendulae flos).
Key words: plant extracts, MRSA, antibacterial effect.

INTRODUCTION

MATERIALS AND METHODS

Despite the fact that the pharmaceutical industry produce
new antibiotics, in recent years the resistance tochemotherapy is growing. Therefore, there are serious
questions over the future use of chemotherapy, which
requires taking measures to identify new antibacterial
substances.
Currently are evaluated various plant extracts forpossible antibacterial effects which covers many bacterial
species, including those responsible for causing
nosocomial infections, as Staphylococccus aureus (S.
aureus).
The evaluation of antimicrobial activity is based on
quantification of bacterial development after the contact
with natural extracts.
The purpose of this study was to investigate theantibacterial properties of alcoholic extracts and essential
oils derived from plants against S. aureus strains isolated
from patients.

We studied 50 strains of S. aureus isolated from various
clinics in Cluj-Napoca, as well as from outpatients.
The identification of isolates was based on morphologicalcharacters, cultural, biochemical and antigenic tests. For
each strain susceptibility testing was performed using
disk diffusion method on subculture standardized
Kirby-Bauer with manual reading (according to CLSI).
The natural products tested were: Calendula tincture(Calllendulae flos) from Hofigal, tincture of hawthorn(Folium Crataegi as flowers) from Fares, Melissa
officinalis essential oil from Fares, tincture of sage
(Salviae folium) from Fares, propolis tincture and Biosept
forte (natural antibiotic) from the company Fares
containing 80% thyme essential oil (thyme aetheroleum)and 20% cloves essential oil (Caryophyllorum
aetheroleum). In vitro antibacterial activity of the natural
products was tested on solid culture media (Mueller
Hinton) distributed in Petri dishes (90 mm).

thth

Received 15 of March 2015. Accepted 20 of April 2015. Address for correspondence: Laura M Simon, Microbiology
Department, UMF ’’Iuliu Haţieganu” Cluj-Napoca, 6th Pasteur Street, RO-400349, Cluj-Napoca, Romania; phone/fax:+40264597257, e-mail: lauramihaelasimon@yahoo.com

16

Fiziologia - Physiology • 2015.25.2(86)The preparation of inoculum was made by suspending
the colonies in 3 ml saline to the density of a McFarland
0.5 turbidity standard. We used several morphologicallysimilar colonies. The inoculation of the agar plates wasmade with a sterile cotton swab by removing the excess
fluid and by spreading the inoculum over the entire
surface of the plate.
After 15 minutes we applied the sterile disks of 5 mmdiameter at a distance of 20-25 mm between two
neighboring disks. The disks were impregnated with 20μlof each plant extract. We worked with pairs samples, foreach strain 2 tests were performed with the same
substance. Reading was done after incubation (for 18-20
hours at 37 ˚C) with a ruler measuring the diameter of theinhibition zones (in mm). We noted the mean (arithmetic
average) of the two determinations. We started from theprinciple that bacteria are more sensitive to thesubstance studied as the inhibition zone diameter islarger.

Fig. 3. Effects of natural extracts D (tincture of sage), E(propolis tincture) and F (Biosept forte) on S. aureus strain
ATCC 25923

Statistical methods. To describe qualitative variables weused absolute and relative frequencies plotted by sector
and column charts. In case of quantitative variables weused the average as centrality parameter and standard
deviation as the dispersion parameter.

RESULTS

Fig. 1. Effects of natural extracts: A (Calllendulae flos), B(tincture of hawthorn) (Folium crataegi as flowers) and C(Melissa officinalis essential oil) on a MSSA strain

In case of MSSA strains, using the tincture of calendula(Calllendulae flos), we obtained diameters of theinhibition zones <10 mm for 18% of strains (9 strains)and in case of MRSA strains we recorded values <10 mm
were in 62% of strains (31 strains). The inhibition zone
diameters between 11 and 20 mm were recorded in 12%
of MSSA (6 strains) and 8% MRSA (4 strains) (Figure 4).

Fig. 4. Diameters of the inhibition zones for Calendula tincture

Fig. 2. Effects of natural extracts: D (tincture of sage), E(propolis tincture) and F (Biosept forte) on a MSSA strain

Fiziologia - Physiology • 2015.25.2(86)From 50 strains studied (15 strains MSSA and 35 MRSA
strains), 12 strains (24%) showed inhibition zone
diameter ≥ 10 mm (7 MSSA strains (14%) and 5 MRSA
strains (10%)), of which 4 MSSA strains (8%) showed
inhibition zone diameter ≥ 15 mm (Table I).

17

Table I. Diameters of the zones of inhibition ≥ 10 mm for
Calendula tincture
Inhibition zone diameter≥ 10 mmCalendula tincture
18
17
16
15
14
13
11
10
Total

MSSA

1 (2%)
1 (2%)
1 (2%)
1 (2%)
1 (2%)
1 (2%)
1 (2%)
7 (14%)

MRSA

2 (4%)
1 (2%)
1 (2%)
1 (2%)
5 (10%)

TOTAL
%
1(2%)
1(2%)
1(2%)
1(2%)
3 (6%)1(2%)
2 (4%)
2 (4%)
12 (24%)

We recorded 38 strains (76%) who had the inhibitionzone diameters <10 mm, of which 8 MSSA strains (16%)
and 30 MRSA strains (60%) (Table II).
Table II. Diameters of the inhibition zones <10 mm for
Calendula tincture
Inhibition zone
diameter <10 mm
Calendula tincture
Total

MSSA

MRSA

TOTAL

8 (16%)

30 (60%)38 (76%)

Calendula tincture (Calllendulae flos) had no effect on 13
strains (26%): 9 MRSA strains (18%) and 4 MSSA strains(8%). Thus, Calendula tincture showed antibacterial
effect of the 12 strains (24%) of the 50 strains tested.
Calendula tincture (Calllendulae flos) had no effect on the
38 strains (76%), 30 MRSA strains (60%) and 8 MSSA
strains (16%). For hawthorn tincture (Folium crataegi) thevalues of the diameters of the inhibition zones for MSSA
were less than 10 mm in 18% of strains (9 strains) and inin 60% of MRSA strains (30 strains). The diameters of
the of inhibition zones were between 11 and 20 mm in
12% of MSSA strains (6 strains) and in 10% of MRSA
strains (5 strains) (Figure 5).

From all studied strains, 17 strains (34%) showed an
inhibition zone diameter ≥ 10 mm: 7 MSSA strains (14%)
and 10 MRSA strains (20%). 3 MSSA strains (6%)
showed inhibition zone diameters ≥ 15 mm (Table III).
Table III. Diameters of the inhibition zones ≥ 10 mm for
Hawthorn tincture
Inhibition zone
diameter ≥ 10 mm
Hawthorn tincture(Folium crataegi)
18
17
16
15
14
13
12
11
10
Total

MSSA

1 (2%)
1 (2%)
1 (2%)
2 (4%)
1 (2%)
1 (2%)
7 (14%)

MRSA

4 (6%)
1 (2%)
5 (10%)
10 (20%)TOTAL

1 (2%)1(2%)
6 (12%)2(4%)
6 (12%)
17 (34%)Thus, the hawthorn tincture showed antibacterial effecton 17 strains (34%) of the 50 tested strains and had noeffect on 33 strains(66%): 25 MRSA strains (50%) and 8
MSSA strains (16%).
For Melissa officinalis essential oil, the diameters of theinhibition zones for MSSA ranged from 21-40 mm in 12%
of strains (6 strains) and they were less than 20 mm in
18% of strains (9 strains). In case of MRSA inhibitionzone diameter values were less than 20 mm in 64% ofstrains (32 strains) and were between 21 and 40 mm in 6
strains (Figure 6).

Fig. 6. Diameters of the inhibition zones for Melissa officinalis
essential oil

Fig. 5. Diameters of the inhibition zones for hawthorn tincture

18

In case of sage tincture (Salviae folium), the diameters of
the inhibition zones were for MSSA strains between 9
and 27 mm, and for MRSA strains they were between
8-18 mm. 9 strains were resistant. Diameters ranging
from 15 to 18 mm have been reported to 6 MSSA strains(12%) and to 14 MRSA strains (28%) (Figure 7).

Fiziologia - Physiology • 2015.25.2(86)propolis tincture in proportion of 93,33% of strains, for
Salviae folium at a rate of 86,66% of strains and in
46,66% of strains for both Calendula tincture and forhawthorn.
MRSA strains showed inhibition zone diameters of ≥ 10mm, in case of Melissa officinalis oil and propolis tincture
to 100% of strains, for Salviae folium in the proportion of
97,14% of strains, a rate of 28,57% of strains for tinctureof hawthorn and the rate of 14,28% of strains for
Calendula tincture (Figure 10).
Fig. 7. Diameters of the inhibition zones for sage tincture

For propolis tincture, the diameters of the inhibition zones
for MSSA strains were between 9-26 mm. For MRSA
strains, the diameters were between 16 and 24 mm.
Diameters between 21 and 26 mm have been reported
for 7 strains of MRSA (14%) and 6 MSSA strains (12%)(Figure 8).

Fig. 10. Diameters of the inhibition zones ≥ 10 mm for MSSA
and MRSA strains

DISCUSSION

Fig. 8. Diameters of the inhibition zones for propolis tincture

In case of Biosept forte, the diameters of the zones ofinhibition for MSSA were between 51-61 mm. For MRSA
the diameters of the zones of inhibition ranged between
40-65 mm. We reported diameters larger than 51 mm at
12 MSSA strains (24%) and at 28 MRSA strains (56%)(Figure 9).

Fig. 9. Diameters of the zones of inhibition for Biosept forte

MSSA strains showed inhibition zone diameters of ≥10 mm for Melissa officinalis oil at 100% of strains, for
Fiziologia - Physiology • 2015.25.2(86)Given the alarming rise of antibiotic resistance, there is a
growing concern among researchers for the discovery of
new antibacterial substances. Thus, the study ofantibacterial effects of plant extracts is very important.
The use of plants in therapy has many advantages,
including: low cost and low toxicity when administered athigh doses.
Plants contain components with antibacterial activity
whose use is increasing worldwide. Antioxidants are
considered protective agent, serving to enhance theoxidative processes. The components with antioxidant
effects are phenolic compounds, especially flavonoids,
which interact with free radicals. Antibacterial effect could
be the result of induction of free radicals [6,10].
In our study, the most intense antimicrobial product canbe considered Biosept forte, which presented inhibitionzone diameters of 65 mm maximum, followed byessential oil of Melissa officinalis with maximum 40 mm
inhibition zone diameters.
Propolis tincture had also an important antimicrobial
effect, for which the maximum diameter of the inhibition
zones was 26 mm and 28% from all strains presented adiameter between 21 and 26 mm.
In case of these 50 strains tested strains, the inhibitionzone diameters for sage tincture ranged between 15 and
18 mm for 40% of strains, showing an antimicrobial
effect.
19

Hawthorn tincture (Folium crataegi) had no effect on 78%
of S. aureus strains tested, and in case of marigold
tincture (Calllendulae flos), 80% of strains were found to
be resistant.
Based on these diameters we can conclude that mostplant extracts showed antibacterial effect.
A recent study in Brazil reveals anti-inflammatory,
antibacterial and angiogenic properties of C. officinalis[14].
A study based on several aerobic and facultative
anaerobic bacterial species including S. aureus
indicates a moderate inhibitory activity with MIC> 2048
mg / L in all bacteria tested, except Prevotella spp [9].
In Portugal, a study on several herbs highlights in caseof Crataegus monogyna a highest concentration ofphenolic acids (5.5 mg/gdw), which proves an importantantioxidant activity [2].
Melissa officinalis has sedative [20], antiviral andantibacterial effects [5]. Good antimicrobial effects wererecorded on a multidrug-resistant strains of Shigellasonei.
Antimicrobial effects of sage is due to its components:
oleanolic acid, triterpenoid and were identified MRSA andStreptococcus pneumoniae [8,22].
A study on Salvia corrugator Vahl identified antibacterial
activity on Gram-positive bacteria of two components:
fruticuline A and demetilfruticuline. The first presented
bacteriostatic activity on all tested strains and the two
showed significant bactericidal activity against S. aureus
and S. epidermidis and bacteriostatic activity on
Enterococcus faecalis and Enterococcus faecium [4].
A study made in Tunisia shows antimicrobial andantioxidant effect of methanolic extracts of sage [16],while another study in Jordan on several plant extracts,
including sage showed no antibacterial effect on S.
aureus and P. aeruginosa [1].
Takaisi & Schilcher have suggested that the antimicrobial
action of propolis is due to the combined action of someconstituents that inhibit the bacterial RNA polymerase(pinocembrine,
galangineandcaffeicacid–phenethyl-ester) [21].
There are studies that show the antibacterial effect ofpropolis on MRSA [3,15], it is higher than the
antibacterial effect of honey [13] and more pronouncedon Gram-positive [18] bacteria compared togram-negative bacteria and fungi [19]. Propolis increases
the antibacterial effect of antibiotics such as Ampicillin,
Gentamicin, Netilmicin, Chloramphenicol, Vancomycin [7]and Streptomycin [17]. The antibacterial effect against S.
aureus was evidentiated in Japan, but data obtainedemphasises its variation depending on the area and themonth in which this was collected [12].
The essential oil of Thymus vulgaris showed an
antibacterial effect on E. coli [11].

20

CONCLUSIONSAll six plant extracts tested strains of S. aureus had
antibacterial effects;
The best antibacterial effects was observed in caseof Biosept forte product;
Data showed significant antibacterial effect ofMelissa officinalis essential oil and propolis;
Moderate antibacterial effect was observed for sage
tincture (Salviae folium), hawthorn (Folium crataegi)
and marigold (Calllendulae flos).

REFERENCES
1. Abu-Darwish MS, Al-Ramamneh EA, Kyslychenko VS, et al.
The antimicrobial activity of essential oils and extracts of somemedicinal plants grown in Ash-shoubak region - South of
Jordan. Pak J Pharm Sci 2012; 25(1): 239-46.
2. Barros L, Dueñas M, Carvalho AM, et al. Characterization ofphenolic compounds in flowers of wild medicinal plants from
Northeastern Portugal. Food Chem Toxicol 2012;50(5):1576-82.
3. Berretta AA, Nascimento AP, Bueno PC, et al. Standardized
Extract (EPP-AF®), an Innovative Chemically and Biologically
Reproducible Pharmaceutical Compound for Treating Wounds.
Int J Biol Sci 2012; 8(4): 512-21.
4. Bisio A, Romussi G, Russo E, et al. Antimicrobial activity ofthe ornamental species Salvia corrugata, a potential new cropfor extractive purposes. J Agric Food Chem 2008; 56(22):10468-72.
5. Canadanović-Brunet J, Cetković G, Djilas S, et al. Radical
scavenging, antibacterial, and antiproliferative activities ofMelissa officinalis L. extracts. J Med Food 2008; 11(1): 133-43.
6. Dawidowicz, A.L., Wianowska D, Baraniak B. The
antioxidant properties of alcoholic extracts from Sambucus
nigra L (antioxidant properties of extracts). LWT-Food Sci.
Technol 2006; 39: 308-15.
7. Fernandes Júnior A, Balestrin EC, et al. Propolis:
anti-Staphylococcus aureus activity and synergism with
antimicrobial drugs. Mem Inst Oswaldo Cruz 2005; 100(5): 563-6.
8. Horiuchi K, Shiota S, Hatano T, et al. Antimicrobial activity ofoleanolic acid from Salvia officinalis and related compounds on
vancomycin-resistant enterococci (VRE). Biol Pharm Bull 2007;30(6): 1147-9.
9. Iauk L, Lo Bue AM, Milazzo I, et al. Antibacterial activity of
medicinal plant extracts against periodontopathic bacteria.
Phytother Res 2003; 17(6): 599-604.
10. Inci E, Civelek S, Seven A, et al. Laryngeal cancer: In
relation with oxidative stress. Tohoku J Exp Med 2003; 200:
17-23.
11. Jugl-Chizzola M, Spergser J, Schilcher F, et al. Effects ofThymus vulgaris L. as feed additive in piglets and against
haemolytic E. coli in vitro. Berl Munch Tierarztl Wochenschr
2005; 118: 495-501.
12. Lu LC, Chen YW, Chou CC. Antibacterial activity of propolis
against Staphylococcus aureus. Int J Food Microbiol 2005;
102(2): 213-20.
13. Miorin PL, Levy Junior NC, Custodio AR, et al. Antibacterial
activity of honey and propolis from Apis mellifera andFiziologia - Physiology • 2015.25.2(86)Tetragonisca angustula against Staphylococcus aureus. J Appl
Microbiol 2003; 95(5): 913-20.
14. Parente LM, Lino Júnior RS, Tresvenzol LM, et al. Wound
Healing and Anti-Inflammatory Effect in Animal Models of
Calendula officinalis L. Growing in Brazil. Evid BasedComplement Alternat Med 2012: 375671.
15. Raghukumar R, Vali L, Watson D, Fearnley J, Seidel V.
Antimethicillin-resistant Staphylococcus aureus (MRSA) activityof 'pacific propolis' and isolated prenylflavanones. PhytotherRes 2010; 24(8): 1181-7.
16. Salah KB, Mahjoub MA, Ammar S, et al. Antimicrobial andantioxidant activities of the methanolic extracts of three Salvia
species from Tunisia. Nat Prod Res 2006; 20(12): 1110-20.
17. Scazzocchio F, D'Auria FD, Alessandrini D, et al.
Multifactorial aspects of antimicrobial activity of propolis.
Microbiol Res 2006; 161(4): 327-33.
18. Seidel V, Peyfoon E, Watson DG, et al. Comparative studyof the antibacterial activity of propolis from different

geographical and climatic zones. Phytother Res 2008; 22(9):1256-63.
19. Silici S, Kutluca S. Chemical composition and antibacterial
activity of propolis collected by three different races ofhoneybees in the same region. J Ethnopharmacol 2005; 99(1):69-73.
20. de Sousa AC, Alviano DS, Blank AF, et al. Melissa officinalis
L. essential oil: antitumoral and antioxidant activities. J Pharm
Pharmacol 2004; 56(5): 677-81.
21. Takaisi NB, Schilcher H. Electron microscopy andmicrocalorimetric investigations of the possible mechanism of
the antibacterial action of a defined propole provenance. Planta
Med 1994; 60: 222-7.
22. Weckesser S, Engel K, Simon-Haarhaus B, et al. Screeningof plant extracts for antimicrobial activity against bacteria andyeasts with dermatological relevance. Phytomedicine 2007;
14(7-8): 508-16.

EFECTELE ANTIBACTERIENE ALE UNOR EXTRACTE DIN PLANTEASUPRA TULPINILOR DE STAPHYLOCOCCCUS AUREUS
REZUMAT
Scop: Scopul acestui studiu a fost de a investiga proprietatile antibacteriene ale extractelor alcolice si uleiurilor
esentiale derivate din plante asupra tulpinilor de S. aureus isolate de la pacienti.
Materiale si metode: Am studiat 50 de tulpini de S. aureus isolate din diferite institutii spitalicesti din Cluj-Napoca,
precum si de la pacienti tratati ambulator, si 6 produse naturale: tinctura de galbenele (Calllendulae flos), tincturade paducel (Folium Crataegi as flowers), ulei esential de Melissa officinalis, tinctura de salvie (Salviae folium),
tinctura de propolis si Biosept forte, care contine 80% ulei esential de cimbru (Thyme aetheroleum) si 20% ulei
esential de cuisoare (Caryophyllorum aetheroleum). Pentru fiecare tulpina, testarea suscetibilitatii a fost efectuatafolosind metoda difuzimetrica standardizata a subculturilor Kirby-Bauer cu citire manuala.
Rezultate: Tulpinile de MSSA au prezentat zone de inhibitie cu diametrul ≥ 10 mm la uleiul de Melissa officinalis
pentru 100% dintre tulpini, la tinctura de propolis in proportie de 93,33% dintre tulpini, la Salviae folium 86,66%
dintre tulpini si 46,66% dintre tulpini la tinctura de galbenele si paducel. Tulpinile de MRSA au prezentat zone deinhibitie cu diametrul ≥ 10 mm in cazul uleiului de Melissa officinalis si a tincturii de propolis pentru 100% dintre
tulpini, 97,14% tulpini pentru Salviae folium, 28,57% tulpini pentru tinctura paducel si 14,28% tulpini pentru
tinctura de galbenele.
Concluzii: Toate cele sase extracte din plante testate pe tulpinile de S. aureus au prezentat efecte antibacteriene.
Cele mai bune efecte antibacteriene au fost observate in cazul produsului Biosept forte. Datele arata un effectantibacterian semnificativ al uleiului esential de Melissa officinalis si propolis. Efect antibacterian moderat a fostobservat in cazul tincturii de salvie (Salviae folium), paducel (Folium crataegi) si galbenele (Calllendulae flos).
Cuvinte cheie: extracte din plante, plant extracts, MRSA, efect antibacterian.

Fiziologia - Physiology • 2015.25.2(86)21ACUTE ORAL AND DERMAL EFFECTS OF SPIRULINA IN MICEFLORINA-MARIA ANDRICA1, CRISTINA DEHELEAN2, MARIA-CORINA SERBAN3*,
IULIA PÂNZARU2, DORINA CORICOVAC2, SIMONA DRĂGAN4
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy
2Department of Toxicology, Faculty of Pharmacy
3Department of Functional Sciences, Faculty of Medicine
4Department of Cardiology, Faculty of Medicine“Victor Babes” University of Medicine and Pharmacy, Timisoara

ABSTRACT
Spirulina (SP) is a spiral-shaped, blue-green microalga included in bacteria kingdom. SP has a rich content in essential
amino acids, phycobiliproteins (phycoerythrin, phycocyanin), minerals, vitamins, carotenoids, polyphenols andessential fatty acids. The aim of this study consists in a safety evaluation of SP treatment using oral and dermal toxicity
studies. SP has revealed no significant changes in skin parameters, behavior or other locomotion problems when was
administered at limited doses of 2000 mg/bw/24 h and 5000 mg/bw/24h on SKH1 mice. Consequently, SP is a natural
product that can be used both by adults and children, being characterized by multi-target beneficial effects for all wholehuman body and, nevertheless, by lack of toxicity.
Keywords: spirulina, acute toxicity, skin toxicity, subchronic toxicity, oral toxicity

INTRODUCTION
Spirulina (SP) is a spiral-shaped, blue-green alga [1]which belongs to the Oscillateriaceae family [2]. Firstly,
SP was included in the plant kingdom, although, in thepresent it belongs to the bacteria kingdom because of its
biochemical, physiological and genetics properties [3].
The most commonly used species of SP are: Arthrospiraplatensis, Arthrospira maxima and Arthrospira fusiformis[3,4]. SP has a rich content in essential amino acids,
phycobiliproteins (red phycoerythrin, blue phycocyanin)[5], minerals, vitamins (B1, B2, B3, B5, B6, B7, B9, B12, D, E),
carotenoids (alpha-carotene, beta-carotene, zeaxanthin)[6,7] and essential fatty acids (3,6 gamma-linolenic acid,linoleic acid, stearidonic acid and arachidonic acid) [8-12].
The methanolic extract of SP has also revealed highamounts of phenolic compounds including salicylic,trans-cinnamic, chlorogenic and caffeic acids that havebeen responsible for its antioxidant effects [13].
Furthermore, the microelements of SP depend both on
the type and the mineral pollution [14] of producing areas[15]. SP and its derivatives are widely used for theirantioxidant, anticancer, anti-inflammatory, anti-diabetic,
antimicrobial, as well as for their immunostimulant effects[16-18]. In this regard, SP is generally recommended invarious chronic pathologies including arterialthhypertension, hypercholesterolemia, malnutrition, obesity,
sugar diabetes and cancer [19,20]. SP is considered to
be one of the most complex natural products that is
widely used not only by adults but also by children [21].
The aim of this study consists in a safety evaluation of
SP treatment using oral and dermal toxicity studies.

MATERIALS AND METHODSThis experimental study was done on healthy young
adult SKH1 mice. The females were non-pregnant. All theanimals involved in the study were between 8 and 12
weeks old. The mice were housed in standard conditions:room temperature 22ºC (+ 3ºC), relative humidity
between 30% and 70 % and artificial lighting
characterized by 12 hours of light and 12 hours dark.
During the experiment the mice have received foodconsisting in special pellets and water ad libitum. The
animals were randomly selected, marked for individual
identification and acclimated to the laboratory conditionsfor 5 days before to begin the experiment. The animals
that were included in the study have appropriate size andage [22].
Spirulina, a dark blue-green powder was purchased fromFAVISAN Laboratories, Lugoj, Romania.

thReceived 16 of May 2015. Accepted 10 of June 2015. Address for correspondence: Lecturer Maria-Corina Serban, MD,
PhD, MSc, Department of Functional Sciences, University of Medicine and Pharmacy “Victor Babes” Timisoara, Eftimie Murgu
Square No. 2A, RO-300041, Timisoara, Romania; phone: +40752444900, fax : +40256220479; e-mail:dr.corinaserban@yahoo.com

22

Fiziologia - Physiology • 2015.25.2(86)Acute toxicity studies
Hairless SKH1 mice weighing 25 – 30 g were includedin the study. Acute oral and dermal toxicities studied
were performed according to OECD- 425 andOECD-434 guidelines. Because SP extract is widelyused for centuries all around the world with no major
side effects we concluded to apply the limit test forevaluating the acute oral and dermal toxicity of thisnatural product. According to OECD-425, only one
mouse was firstly selected for the acute oral study. It was
fasted overnight and in the next morning it was dosedwith a test dose of 2000 mg/bw/24h. The animal has
survived, and we continued to dose other four female
mice with the same dose of SP. The animals have also
survived. After that, we continued to give a dose of 5000
mg/bw/24 h to one mouse firstly and after that to theother three mice. The animals were observed for side
effects for 14 days and after that were sacrificed.
According to OECD-434, for acute dermal toxicity test
were selected 18 mice having approximately the sameage and weight. These mice were divided in 4 groups: 5
female mice (group 1), 5 male mice (group 2), 4 healthy
mice treated with diluted solution of alcohol (2 males, 2
females) (group 3), 4 healthy untreated mice (2 males,
2 females). SP was dissolved in a diluted alcoholic
solution (67%) and, then, was applied uniformly over
10 % of the total body surface area. The skinparameters (melanin levels and erythema) weredecelated using a mexameter MX18 (Courage&
Khazaka Electronics, Germany) and the skinhydratation was evaluated using a corneometer CM825
MX18 (Courage&Khazaka Electronics, Germany). All
animals were observed for 14 days after application of
SP extract and after that were sacrificed.
The experimental study protocol was designed
according to the Universal Declaration of Animal Rights
proclaimed in Paris in 1978 and to the Declaration of
Helsinki, the European Convention (ETS No. 123)
amended in 1998 (ETS No. 170) and Council Directive
86/609/EEC on the protection of vertebrate animals
used for experimental and other scientific purposes.
The animal study protocol was approved by the Ethics
Committee of the Faculty of Pharmacy, “Victor Babes”
University of Medicine and Pharmacy, Timisoara,
Romania.

Fig. 1. Mice – 24 hours after the skin application of SP extract:A) male mice treated with alcoholic solution of SP; B) female
mice treated with alcoholic solution of SP; C) skin of female
mice treated with alcoholic solution of SP; D) female micetreated with alcoholic solution; E) female mice untreatedFig. 2. Evolution of melanin levels in mice

There were no significant differences between the valuesof melanin before and after SP application in female mice(p = 0.2097) p>0.05 and in male mice (p = 0.7517)
p>0.05.

RESULTS
It was observed that all the mice that received SP in bothdoses of 2000 mg/bw/24 h and 5000 mg/bw/24 h havesurvived and they did not manifest any behavioral
changes, convulsions, locomotion problems or mortality(Figure 1). Consequently, the LD50 for SP was greaterthan 5000mg/bw/24h.
Fiziologia - Physiology • 2015.25.2(86)Fig. 3. Evolution of erythema in mice

23

The erythema values increased in the first 48 h after local
administration of SP both in female (p = 0.035) p<0.05
and male mice (p = 0.05222) p>0.05 but significant
changes were seen only in female mice treated with SP.
The alcoholic solution that was used as solvent did notcaused significant differences in female (p =0.1000)
p>0.05 and in male groups (p = 0.071) p>0.05.
Consequently, the significant changes in females grouptreated with SP were caused only by SP and not by thesolvent.

methylsulfonylmethane, organic cayenne pepper, thealgae Aphanizomenon flos-aquae and SP was described[27]. Moreover, SP caused anaphylaxis to a 17-year-old
male after one ingestion of a SP tablet [28] and acuterhabdomyolysis to another patient which consumed a
dietary supplement containing SP [29].
According to Health Food Organization, 4g of SP is daily
recommended for an adult of 50 kg body weight [30].The
beneficial effects of SP are recognized by World HealthOrganization (WHO), Food and Agriculture Organizationof the United Nations, United Nations Industrial
Development Organization and United Nations Children's
Fund [2]. The WHO projects considered SP to be one of
the most curative and prophylactic components ofnutrition in the 21st century [31] due to its remarkable
nutrient profile, lack of toxicity and therapeutic effects [32].
The Dietary Supplements Information Expert Committeeof the United States Pharmacopeia Convention assigned
a class A safety rating for SP maxima and SP platensis,
including these dietary supplement ingredients as
monographs in United States Pharmacopeia andNational Formulary [33].

Fig. 4. Evolution of skin hydratation levels in mice

There were no significant differences regarding the skinhydratation measurements before and after the local
application of SP in female (p = 0.060) and in male group(p = 0.3830), both p > 0.05. Consequently, according to
our toxicity studies on mice, we concluded that SP has
lack oral and dermal toxicity, because the majority of theskin parameters that were measured after the local
administration of SP extract did not suffer significant
changes. The erythema levels were significantly higher
only in the female group treated with SP. However, theinformation regarding the side effects of SP treatments isstill limited. According to another experimental study,
higher dose of SP (1000 mg/kg) did not cause importantchanges in cardiac parameters in mice [23].
Some clinical trials tested the toxicity of SP in humans.
There have been reported only minor hypersensitivity
reactions including urticarial, rash, diaphoresis andbronchospasm [24]. Other side effects observed inpatients treated daily with 1g of SP were: flushing of theface, headache, muscle pain, sweating and difficulty
concentrating [25]. Moreover, it was reported a higher
rate of protidemia and creatinemia in HIV patients treatedwith SP for 6 month [26]. It has been reported a clinicalcase of an 82-year-old healthy woman who manifested
bullae, partly hemorrhagic, on the trunk and extremities,
secreting erosions and submammary macerations after a
2-year period of SP intake. Another 57-year-old man
diagnosed with pemphigus vulgaris has developed asevere flare-up of the disease after consumption of a
supplements containing Ginseng, Ginkgo biloba and SP.
Another case of a 45-year-old woman who presented
dermatomyositis after intake of a supplement based on
24

CONCLUSION
The acute oral and dermal toxicity studies that weremade on hairless SKH1 mice using limit doses of 2000
mg/bw/24 h and 5000 mg/bw/24 h of SP revealed nosignificant changes in skin parameters, behavior or otherlocomotion problems. Moreover, all the animals included
in study have survived; in female group it has been
observed a higher level of erythema parameters. We
consider that SP is a complex natural product withmulti-target beneficial effects in rats characterized by lackof toxicity that can be used both by adults and children.
COMPETING INTERESTS
The authors declare that they have no competing interests.
ACKNOWLEDGEMENT
This work was supported by the POSDRU grant no.
159/1.5/S/136893: “Strategic partnership for the increaseof the scientific research quality in medical universities
through the award of doctoral and postdoctoral
fellowships – DocMed.Net_2.0”

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25

EFECTELE ACUTE ORALE ȘI CUTANATE ALE SPIRULINEIPE MODEL ANIMAL DE ȘOARECEREZUMAT
Spirulina (SP) este o microalgă albastră-verde, cu formă spiralată, fiind inclusă în regnul bacteriilor. SP are unconţinut bogat în aminoacizi esenţiali, ficobiliproteine (ficoeritrină şi ficocianină), minerale, vitamine, carotenoizi,
polifenoli şi acizi graşi esenţiali. Scopul acestui studiu constă în evaluarea siguranţei tratamentului cu SP prinaplicarea unor texte de toxicitate acută orală şi cutanată. În urma tratamentului oral şi local cu SP utilizându-sedoze limită de 2000 mg/kg corp/24h şi 5000 mg/kg corp/24 h nu s-au observat modificări semnificative în ceea cepriveşte parametrii studiaţi, şi comportamentul animalelor şi nu au fost semnalate alte probleme de mobilitate. În
concluzie, SP este un produs natural care poate fi utilizat în siguranţă atât de adulţi cât şi de copii, avândnumeroase efecte benefice asupra organismului şi nu in ultimul rând datorită toxicităţii sale foarte reduse.
Cuvinte cheie: spirulina, toxicitate acută, toxicitate cutanată, toxicitate subcronică, toxicitate orală

26

Fiziologia - Physiology • 2015.25.2(86)THE ROLE OF SPIRULINA PLATENSIS IN THE CONTROLOF TYPE 2 DIABETES MELLITUS
MARIA-CORINA SERBAN, GHEORGHE STOICHESCU-HOGEA, CAMELIA GURBAN,FLORINA PETCU, DINESHA JEYAKUMAR, FLORINA ANDRICA, CALIN MUNTEANU,SIMONA DRAGAN
University of Medicine and Pharmacy “Victor Babes”, Timisoara, Romania

ABSTRACTIn recent years, many experimental and clinical studies evaluated blue-alga Spirulina platensis for the treatment ofdiabetes mellitus (DM). The objective of this study was to evaluate the effects of Spirulina supplements in the control oftype 2 diabetes mellitus. The study included patients aged 30-70 years with type 2 DM for at least two years and thathave been treated with a stable dose of Metformin for at least 3 months. This randomized, single blinded on parallelgroups study included 30 patients with DM type 2, divided in two groups: 15 patients treated with Metformin andplacebo (group 1) and 15 patients treated with Metformin and Spirulina (group 2). Spirulina was administered twice per
day during 2 months, in a total dose of 800 mg daily. The following parameters were evaluated: blood pressure, totalcholesterol (TC), triglycerides (TG), LDL-cholesterol, HDL-cholesterol, fasting serum glucose, and glycated hemoglobin(HbA1c). After 2 months, the mean glycemic value significantly decreased from 124.5±29.28 mg/dl to 108.3±12.53
mg/dl (p<0.003) in group 1 and from 149.5 ±19.89 mg/dl to 121.1± 10.66 mg/dl in group 2 (p<0.001).The mean valueof HbA1c significantly decreased from 7.2±0.63% to 6.3 ± 0.69% in group 1 (p=0.008) and from 7.1±0.92% to 6.6±0.33 % in group 2 (p=0.009).
Keywords: Spirulina platensis, lipids, type 2 diabetes mellitus

INTRODUCTION
Diabetes Mellitus (DM) is a chronic disease that requires
long term medical attention to limit and manage thedevelopment of devastating complications [1]. An early
initiation of pharmacological therapy was associated withimproved glycemic control and reduced long termcomplications in type 2 DM. Many experimental andclinical studies have shown the beneficial effects of foodsupplements in DM and, particularly, the beneficial effectsof Spirulina platensis in the control of type 2 DM. Spirulina
is a filamentous cyanobacterium worldwide used as afood supplement [2]. Spirulina platensis (Arthrospiraplatensis) is one of the three species investigated mostoften [3]. Spirulina has many nutritional components andit is a rich source of phycocyanin, vitamins, especiallyvitamin B12 and provitamin A (ß-carotene), minerals, andcarotenoids [4]. The lack in cellulose of cell walls in favorof easy digestion is another great advantage of Spirulina
consumption [5].
Many experimental and clinical studies have shown thebeneficial
metabolic,antiviral,liver-protecting,blood-vessel relaxing, anti-cancer, anti-inflammatory andthantioxidant properties of Spirulina supplements [6]. In
Korea, a double blind, randomized clinical trial evaluatedthe effects of Spirulina consumption on lipid parameters,
immune markers and antioxidant capacity in elderly men.
As compared to placebo, Spirulina users had lower lipid
parameters, increased serum interleukin-2, but
decreased serum interleukin-6, researchers concludingthat Spirulina is suitable as a functional food [7]. Anotherstudy conducted in Greece showed the beneficial effectsof spirulina supplements on lipid profile in 52 dyslipidemic
patients. The lipid fractions total cholesterol (TC),triglycerides (TG), LDL-cholesterol (LDL-C) andnon-HDL-cholesterol (non-HDL-C) were significantly
reduced after 3 months of Spirulina supplementation, themajor reduction being observed in TG levels (-16.3%) in
women. However this reduction reached 21.3% in
women aged > 47 years was 21.3% and 18.6% in
women with TG levels >150 mg/dl [8]. These findings are
extremely valuable considering the importance ofelevated TG levels as a risk factor for cardiovasculardisease in women [9]. This study concluded that Spirulina
supplementation at a dose of 1g daily has strong
hypolipidemic effects especially on TG concentration,

thReceived 10 of May 2015. Accepted 6 of June 2015. Address for correspondence: Lecturer Maria-Corina Serban, MD, PhD,
MSc, Department of Functional Sciences, University of Medicine and Pharmacy “Victor Babes” Timisoara, Eftimie Murgu SquareNo. 2A, RO-300041, Timisoara, Romania; phone: +40752444900, fax : +40256220479; e-mail: dr.corinaserban@yahoo.com

Fiziologia - Physiology • 2015.25.2(86)27being proposed as a dietary supplement in dyslipidemic
patients [8]. Another study done by Parikh et al on type 2 DM
patients evaluated the hypolipidemic and hypoglycemic
effect of Spirulina [10]. The efficacy of Spirulina
supplementation (2g/day for 2 months) in both placebo andSpirulina groups was evaluated by determining serum lipid
concentrations, blood glucose levels and HbA1c [10].
Spirulina significantly decreased blood glucose, HbA1c TC,
TG and apolipoprotein B levels, while HDL-C andapolipoprotein A1 levels marginally increased. This study
concluded that Spirulina supplementation has beneficial
effects in controlling glycemia and improving the lipid profile
of type 2 DM patients [10].
Despite de evidence, the effects of Spirulina
supplementation as add-on therapy in diabetic patients
have not been conclusive. The objective of this study wasto evaluate the effects of Spirulina supplements on lipid
profile and glycemic parameters in patients with type 2
DM.

MATERIAL AND METHODSThis interventional study (800 mg Spirulina/daily) was arandomised, single blinded study on parallel groups with
2 months duration. The patients included in the study
were aged 30-70 years, diagnosed with type 2 DM for atleast two years and under treatment with a stable dose of
Metformin for at least 3 months. The exclusion criteria
were BMI > 40 kg/m², any acute or chronic inflammatory
disease, cancer, kidney disease, moderate or severe
hepatic disease and frequent use of antioxidants andanti-inflammatory. The study included 30 patients with
type 2 diabetes mellitus (DM), divided as follows: Group
1: 15 patients with DM type 2 + Metformin+ Placebo andGroup 2: 15 patients with DM type 2 + Metformin+
Spirulina supplements.
All subjects were instructed to maintain an isocaloric dietand continue with their eating habits throughout the study.
The physical examination, chest radiograph, 12-leadelectrocardiogram, anthropometric measurements, bloodpressure and laboratory tests was carried out at the
beginning and after two months of treatment. A venous
blood sample was drawn from an antecubital vein in all
subjects after an overnight fast to determine totalcholesterol (TC), triglycerides (TG) and HDL-cholesteroland uric acid using standard enzymatic method on a
COBAS Integra 400 plus analyzer. Bayer diagnostic kit
was used to estimate the blood glucose levels. The
glycated haemoglobin was determined by the variant
haemoglobin A1c using the high performance liquid
chromatography technique, using Randox diagnostic kits.
Hypertension was diagnosed according to 2013
European Guidelines for the Management of Arterial
Hypertension [11]. Diabetes was defined by the current
intake of oral hypoglycemic treatment or use of insulin.
28

For this interventional clinical trial, FAVISAN Company(www.Favisan.ro) provided spirulina and placebo
capsules. During this study all subjects were asked torefrain from any source related supplements other thanthose provided by the study. The compliance of all
patients was evaluated weekly by telephone.
Statistical analysis was carried out with the programme
SPSS-13, which is a software package used forstatistical analysis by comparison of data from two
samples, paired t test was used and unpaired t-test wasused respectively when appropriate (1 and 2 tail tests).
The study was conducted according to the guidelines forclinical investigation for the treatment and prevention ofdiabetes CPMP / EWP / 1080 / 00Rev1 delivered on 14
May 2012 by the European Medicines Agency, respecting
the Charter of Fundamental Rights of the EU and WMA
Declaration of Helsinki (1964) last amended in 2008. This
study was conducted in accordance with the Good
Clinical Practice of the European Community,
CPMP/ICH/135/95 and the ICH Guidelines for Good
Clinical Practice: Consolidated Guidance (GCP E-6, April
1996) / and the Declaration of Helsinki and the applicable
local laws and regulations.

RESULTS
The study included 30 patients, divided in two groups,
similar in terms of age and sex: 15 patients with DM type 2
treated with Metformin and placebo (group 1) and 15
patients with DM type 2 treated with Metformin and the
supplement Spirulina twice daily, in a total dose of 800 mg(group 2). No significant differences between
anthropometric measurements, mean blood pressure (BP),lipids and glycemic parameters were observed between
the two groups at inclusion in the study (Table I).
Table I. Characteristics of the two groups at inclusion in the
study (n=30)
Studied variables

Age
Weigh (kg)Height (cm)Body mass index
Waist
circumference(cm)
Visceral fat area
(cm2)

Group

DM
CONTROLDMCONTROLDMCONTROLDMCONTROLDMCONTROLDMCONTROL

Mean±Standard
deviation(SD)
61.7 ±6.85
61.6±8.90
103.1±16.09
100.7±22.04
168.0±7.23167.0±5.82
36.6±6.05
36.2±6.93
115.3±8.55
113.1±8.29
195.3±42.97
194.0±37.45

p value andsignificance0.956ns
0.738ns
0.686ns
0.861ns
0.503ns
0.931ns

Fiziologia - Physiology • 2015.25.2(86)Systolic bloodpressure (mmHg)
Diastolic bloodpressure (mmHg)
Glycemia (mg/dl)
TC (mg/dl)
TG (mg/dl)
HDL (mg/dl)
LDL (mg/dl)
Uric acid (mg/dl)

DM
CONTROLDMCONTROLDMCONTROLDMCONTROLDMCONTROL
DZ
CONTROLDMCONTROLDMCONTROL141.9±15.57
142.4±15.80
82.0±8.82
83.2±8.68
149.5±19.89
124.5±29.28
203.3±64.02
211.25±66.43
191.2±72.28
198.7±85.09
43.9±12.68
45.8±16.37
121.9±51.31
130.6±46.76
6.2±1.73
6.8±1.84

0.933ns
0.712ns
0.705ns0.266ns
0.580ns
0.702ns0.259ns
0.373ns
0.08nsFig. 1. Evolution of glycemia before and after 2 months
compared between groups

Key: ns – non-significant differences (p>0.05)
s – Significant differences (p<0.05)

Table II. The comparison between the parameters studied atinclusion and after two months of Spirulina supplementation in
the placebo group (n=15) and Spirulina group (n=15)
The variables
studiedWeight (kg)Weight after 2
monthsGlycemia (mg/dl)
Glycemia after 2
monthsHbA1c %
HbA1c after 2
monthsTC (mg/dl)

PLACEBO
GROUP
Mean±
Standard
deviation

p value

100.7 ±22.04
96.5±14.2

0.008s<0.003s211.25±66.43

TC after 2 months

144.58±38.58

TG (mg/dl)

198.7±85.09

TG after 2 months

139.9±45.09

HDL (mg/dl)
HDL after 2
monthsLDL (mg/dl)

45.8±16.37
48.5±15.36

0.008s0.04s

6.6±0.33

0.009s

168.5±37.58

Fig. 2. The evolution of glycated hemoglobin before and after
two months compared between groups

0.004s154.5±33.71
43.9±12.68
45.5±12.45

0.019ns

0.113ns121.9±51.31

130.6±46.76

99.3±18.98

0.059ns6.2±1.73

6.8±1.84
5.58±0.84s

191.2±72.28
0.02s

119.4±40.45

Uric acid after 2
months<0.001203.3±64.02<0.001s

0.07nsLDL after 2
monthsUric acid121.1±10.66

0.001s

7.1±0.92

7.2±0.63
6.3±0.69

100.8±14.57
149.5±19.89

124.5±29.28
108.3±12.53

SPIRULINA
GROUP
Mean±
p
Standard
value
deviation
103.1 ±16.09

0.008s

5.04±0.86

Key: ns – non-significant differences (p>0.05)
s – Significant differences (p<0.05)

Fiziologia - Physiology • 2015.25.2(86)0.471nsFig. 3. Evolution of total cholesterol before and after two
compared between groups29

Fig. 4. The evolution of TG before and after two months
compared between groups

Fig. 5. The evolution of HDL before and after two months
compared between groups

DISCUSSION
Our study showed that the supplementation with Spirulina
platensis tablets has beneficial effects in controlling blood
glucose levels and improving the lipid profile of patientswith type 2 DM. The lowering effects of Spirulina on blood
lipids are not completely known. Spirulina could act onserum lipids by a similar mechanism as statins, inhibiting
the hydroxmethylglutaryl coenzyme A reductase andconsecutive reduction of cholesterol synthesis [10].
Although the exact biochemical mechanism by which
Spirulina reduces lipid levels is not well understood, somestudies have presumed that a high C-phycocyanin content
might inhibit the activity of pancreatic lipase activity [7].
The content rich in C-phycocyanin is presumed to act
together with glycolipid hemoglobin (Hb)-2 to decrease injejunal cholesterol absorption and ileal bile acidreabsorption [12]. The results showed that spirulina isprobably functioning like an insulin-like protein or may
stimulate type beta cells of Langerhans to increase theoutput of insulin, stimulating the decrease of serum30

glucose levels. Spirulina has an effect which leads to thedecrease in level of lipoproteins rich in triacylglycerols. In
addition, spirulina could act by a similar mechanism as
resins, where reabsorption of bile acids in intestine, andcholesterol conversion to bile acids is inhibited [13]. By an
unspecified mechanism, spirulina could affect liver cells
through which it intervenes directly in cholesterol
formation and thus influences its level in serum.
Furthermore, spirulina could affect the metabolism of fatty
acids, reduce synthesis of VLDL in the liver and as a result
minimize the formation of LDL-C in the circulation.
Through antioxidative effect, spirulina has shown to
reduce lipoperoxidation in liver [14].
Many studies have shown that during diabetes, excess of
glucose present in blood reacts with haemoglobin to form
HbA1c [15]. The level of HbA1c is always marked as areliable index of glycaemic control in diabetes. The reason
there is an increase in the level of HbA1c is due todecreased level haemoglobin and increased level of blood
glucose. The patients who were given spirulina showed adecrease in level of HbA1c, the results in the spirulina
group showed an average value of 7.1± 0.92% before useof spirulina and 6.6 ±0.33% after, (p=0.009), the value of p
showed a significant difference in HbA1c. The reason forthis could be explained by the fact that spirulina is rich iniron and this contributes to the elevated levels of
haemoglobin.
Spirulina platensis is considered a safe product, only few
patients reported diarrhea after consumption, being
Generally Recognized as Safe (GRAS) compound. Our
study, in accordance with previous experimental andclinical trials have shown promising data in the control oftype 2 DM and therefore it is expected that spirulina,
which acts like an insulin like protein can also be used in
the future as an anti-diabetic natural substance,
eventually replacing oral medication or even insulin.

CONCLUSION
The results obtained in this study showed a significant
reduction of weight, glycemia, total cholesterol andtriglycerides values after administration of Spirulina
platensis supplements at dose of 800 mg daily for twomonths in patients with type 2 DM. These results revealthat Spirulina platensis might be a promising dietary
supplement for type 2 DM management. Future
well-designed larger trials are required to identify theactive ingredients beside Phycocyanin and establish thesafety profile of Spirulina platensis in humans.
COMPETING INTERESTS
The authors declare that they have no competinginterests.

Fiziologia - Physiology • 2015.25.2(86)ACKNOWLEDGEMENT
This work was supported by the POSDRU grant no.
159/1.5/S/136893: “Strategic partnership for the increaseof the scientific research quality in medical universities
through the award of doctoral and postdoctoral
fellowships – DocMed.Net_2.0”.

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ROLUL SPIRULINA PLATENSIS ÎN CONTROLUL DIABETULUI ZAHARAT TIP 2
REZUMATÎn ultimii ani, numeroase studii experimentale și clinice au evaluat alga albastră Spirulina platensis pentru
tratamentul diabetului zaharat (DZ). Scopul acestui studiu a fost evaluarea efectelor suplimentelor de Spirulină în
controlul DZ de tip 2. Studiul a inclus pacienți cu vârste de 30-70 ani, cu DZ tip 2 diagnosticat de cel puțin doi ani,care au fost trataţi cu o doză stabilă de Metformin pentru cel puțin 3 luni. Acest studiu randomizat, singur orb pe
grupuri paralele a inclus 30 de pacienți cu DZ tip 2, 15 de pacienți tratați cu Metformin şi placebo (grupul 1) și 15
pacienți tratați cu Metformin și Spirulină (grupul 2). Spirulina a fost administrată de două ori pe zi timp de 2 luni,
într-o doză totală de 800 mg pe zi. Următorii parametri au fost evaluați: tensiunea arterială, colesterolul total (TC),trigliceridele (TG), LDL-colesterolul, HDL-colesterolul, valorile serice ale glicemiei și hemoglobinei glicozilate(HbA1c). După 2 luni, s-a observat că valoarea medie a glicemiei a scăzut semnificativ de la 124,5 ± 29,28
mg/dl la 108,3 ± 12,53 mg/dl (p <0,003) în grupul 1 și de la 149,5 ± 19,89 mg/dl la 121,1 ± 10,66 mg/dl în grupul
2 (p <0,001). Valoarea medie a HbA1c scăzut semnificativ de la 7,2 ± 0,63% la 6,3 ± 0,69% în grupul 1 (p =0,008) și de la 7,1 ± 0,92% până la 6,6 ± 0,33 % în grupul 2 (p = 0,009).
Cuvinte cheie: Spirulina platensis, lipide, diabet zaharat tip 2

Fiziologia - Physiology • 2015.25.2(86)31EFFECTS OF RESVERATROL AND COENZYME Q10
SUPPLEMENTATION IN METABOLIC SYNDROMEFLORINA ARDELEAN*, GHEORGHE STOICHESCU-HOGEA, CAMELIA GURBAN,
MARIA-CORINA ŞERBAN, FLORINA PETCU, DIANA S. ANTAL, SIMONA DRĂGAN
University of Medicine and Pharmacy “Victor Babeş” Timişoara

ABSTRACT
Resveratrol and coenzyme Q10 are two nutraceuticals intensively studied for their benefits in several diseases. Theirlipid-lowering, anti-inflammatory and antioxidant activities could improve the condition of patients with metabolic
syndrome. Our study aimed to evaluate the effects of the association of a dietary supplement containing resveratroland coenzyme Q10 to the prescribed medication in patients with metabolic syndrome and statin therapy. The study
included 29 patients randomized in two groups, one receiving the dietary supplement (n=15) and one receiving placebo(n=14). The serum levels of total cholesterol (TC), triglycerides (TG), LDL-cholesterol (LDL-C), HDL-cholesterol(HDL-C), transaminases and high sensitivity C-reactive protein (hsCRP) have been evaluated at the beginning of thestudy and after three months. A decrease of serum levels of TC, TG, LDL-C and hsCRP and an increase of HDL-C
were noticed. The greater increase of HDL-C in the group receiving the dietary supplement (from 43.80±13.18 to
46.67±10.40 mg/dl) almost reached a statistical significance (p=0.057). The results indicate that the association of
resveratrol and coenzyme Q10 in patients with metabolic syndrome could moderately decrease TC, LDL-C, TG andsignificantly increase HDL-C. Further studies are needed to elucidate the effects of resveratrol combined withcoenzyme Q10 on lipid profile parameters.
Key words: metabolic syndrome, resveratrol, coenzyme Q10

INTRODUCTION
Nowadays, metabolic syndrome (MS) is a condition withan increased prevalence, determining a high risk for
cardiovascular disease and type 2 diabetes mellitus (DM).
Many factors such as the excess calorie diet, thesedentary life habits, and genetic factors could contributeto the development of this disease [1,2]. Several
definitions have been proposed for this complex disease
by different organizations. The criteria proposed in thesedefinitions for the diagnosis of MS present similarities,
but insulin resistance, lipid and blood pressure (BP)levels are present in most of them [3,4]. The central
obesity, dyslipidemia, high BP, proinflammatory andprothrombotic state and insulin resistance are the main
factors responsible for the evolution to cardiovasculardisease (CVD) and type 2 DM [5]. The chronic low-grade
inflammation state in metabolic syndrome might becaused by the generation of leptin and adiponectin anddifferent others adipokines by the adipose tissue [6,7].
The main approach in controlling the risk factors of MS isrepresented by lifestyle changes (increased physicalactivity, anti-atherogenic diet) [1]. When lifestyle changesth

are insufficient, the drug intervention combines
lipid-lowering, hypotensive and hypoglycemic drugs in
MS patients [8]. The polymedication in the same patient
increases costs, decreases the compliance and is
responsible for many side effects [5]. One of thecommonly prescribed classes of drugs to control
dyslipidemia in MS patients is represented by statins,
leading to a reduction of cardiovascular events [9]. Manyside effects like muscle and liver toxicity [10], increase in
the incidence of type 2 DM, cognitive impairment havebeen observed in statin users [11]. It has been shownthat statins inhibit cholesterol biosynthesis throughdecreasing the synthesis of mevalonate and consecutive
reduction of coenzyme Q10 levels in the organism [12].
Therefore, an association of statins with coenzyme Q10
supplements might be a good strategy in statin users to
improve metabolic parameters [13].
Resveratrol (3, 4’, 5 trihydroxystilbene) is a polyphenolic
compound that has attracted great interest because of its
presence in red wine and its multiple benefits [14-16]. The
antioxidant [17], cardioprotective [18], anticancer [19],anti-inflammatory [20], lipid-lowering, anti-hyperglycemic[21] or neuroprotective [22] effects of resveratrol havethReceived 16 of April 2015. Accepted 12 of May 2015. Address for correspondence: Florina Ardelean, Department ofPharmaceutical Botany, Faculty of Pharmacy, “Victor Babes” University of Medicine and Pharmacy Timisoara, Eftimie Murgu
Square No. 2A, RO-300041, Timisoara, Romania; phone/fax: +40256 220479; e-mail: florina_ardelean87@yahoo.com

32

Fiziologia - Physiology • 2015.25.2(86)been explored in many experimental and clinical trials.
However, the benefits of resveratrol supplementation are
still controversial, some recent studies indicating thatresveratrol is overestimated [23].
The aim of our study was to evaluate the effect on lipid
and inflammation parameters of a dietary supplement
containing both resveratrol and coenzyme Q 10 in MS
patients on statin therapy.

MATERIAL AND METHODThis study was randomized, single-blinded with parallelgroups and included 29 patients with MS on statin therapy
for at least 3 months. The exclusion criteria from this study
were represented by any type of cancer or degenerative
disease, moderate to severe renal or hepatic disease,acute or chronic inflammatory disease, pregnancy orbreastfeeding, abnormal pulmonary X-ray, abnormal
ecography, abnormal ECG, deterioration of hematological,
hepatic or renal function, hypersensitivity or allergy to anyof the compounds of the product administrated in this study,
the use of anti-oxidative and anti-inflammatory substancesless than two weeks before the start of the study.
The patients enrolled have been distributed in two groups.
During three months, the first group (15 patients)
received Procor Forte, 1 capsule/day and the second
group (14 patients) received placebo. Procor Forte is adietary supplement containing coenzyme Q10 (60 mg),resveratrol (15 mg), vitamin B6 (3 mg) and magnesiumoxide (250 mg) providing 150 g magnesium. For thisinterventional clinical trial, Sun Wave Pharma Company
provided the dietary supplement and the Favisan
Company provided the placebo capsules.
The patients have been diagnosed with MS according to
the criteria of NCEP-ATP III, presenting 3 or more of thefollowing: waist circumference > 102 cm (men) or > 88cm (women), triglycerides ≥150mg/dl, HDL cholesterol <40mg/dl (men) or < 50mg/dl (women), blood pressure ≥130/85 mmHg, fasting glucose ≥ 110 mg/dl [8]. Patients
had increased cardiovascular risk factors (risk SCORE≥5). The prescribed medication including statin therapy
was respected during the intervention period. Before thebeginning of the study patients respected a wash-out
period of two weeks in order to eliminate the effects ofother dietary supplements or plant extracts consumed
before.
At the beginning of the study and after three months thefollowingparameters
have
been
evaluated:
anthropometric parameters: weight, height, waist
circumference, body mass index (BMI). The serum levelsof total cholesterol, triglycerides, LDL, HDL,
transaminases, blood glucose, hsCRP and bloodpressure were determined in all patients. Bayerdiagnostic kit was used to determine the levels of
biochemical parameters.
Fiziologia - Physiology • 2015.25.2(86)This study was conducted in accordance with the Good
Clinical Practice of the European Community,
CPMP/ICH/135/95 and the ICH Guidelines for Good
Clinical Practice: Consolidated Guidance (GCP E-6, April
1996) / and the Declaration of Helsinki and the applicable
local laws and regulations. Written consent was obtained
from the patients included in the study. The study wasapproved by the Ethics Committee of the Institute ofCardiovascular Diseases.
Statistical analysis was done using with the programme
SPSS-13, a software package used for statisticalanalysis by comparison of data from two samples, pairedt test was used and unpaired t-test was used respectively
when appropriate (1 and 2 tail tests).

RESULTS
The MS patients on statin therapy included in the study
presented similar baseline characteristics regarding age,sex, weight, height, BMI. Of the 29 patients 14 were men
and 15 women, 6 male and 9 female in the Procor groupand 8 male and 6 female in the placebo group. No
significant differences have been noted for age (p =0.289), weight (p = 0.268), height (p = 0.982), BMI (p =0.222), waist circumference (p = 0.902) as shown in
Table I.
Table I. The anthropometric parameters at inclusion

The majority of the patients included in the study
received atorvastatin (10 patients in the Procor groupand four patients in the placebo group), followed byrosuvastatin (7 patients in the placebo group and 4 in the
Procor group). Five patients included in the studied
presented side effects to statins (hepatic cytolysis,
artralgia and myalgia).
The values of the evaluated parameters in both groups
are presented in Table II. For the Procor group (n = 15)
significant improvements have been observed for TC witha decrease from 230.40 ± 56.11 mg/dl to 186.13 ± 31.86
mg/dl, LDL-C from 137 ± 38.68 to 114.13 ± 23.39 mg/dl,
TG from 167.53 ± 75.99 to 127.07 ± 38.29 mg/dl, ALTfrom 51.47 ± 50.07 to 18.55±10.33 and hsCRP from 3.73
33

± 2.19 to 2.67 ± 1.68 mg/dl. No significant improvement
was observed for AST levels. The serum values of
HDL-C increased from 43.80 ± 13.18 mg/dl to 46.67 ±10.40 mg/dl (p = 0.057) in Procor group (Table II).
Table II. The parameters in the two groups at inclusion andafter 3 months

Fig.2. Evolution of TG levels in Procor and placebo group

HDL: high density lipoprotein; LDL: low density lipoprotein;
TG: triglyceride; ALT: alanine aminotransferase, AST:
aspartate aminotransferase, hs CRP: high sensitivity
C-reactive protein

In the placebo group (n=14), the serum levels of lipid
parameters, transaminases and hsCRP decreased after
3 months of supplementation with placebo: TC
decreased from 233.93 ± 80.89 to 182.14 ± 47.25 mg/dl,
LDL-C from 144.40 ± 54.40 to 119.86 ±38.36 mg/dl and
TG from 207.21 ± 81.73 to 149 ± 48,39 mg/dl, ALT from36.07 ± 19.84 to 22.79 ± 8.47 and hsCRP from 3.43 ±1.7 to 2.57 ± 1.16 mg/L. A decrease of serum levels of
HDL-C, from 45.93±15.97 mg/dl to 45.57±13.96 has
been observed.

Fig.3. Evolution of LDL-C levels in Procor and placebo group

Fig.4. Evolution of HDL-C levels in Procor and placebo group

Fig.1. Evolution of total cholesterol levels in Procor and placebo
group

34

Fiziologia - Physiology • 2015.25.2(86)Fig.6. Evolution of hs CRP levels in Procor and placebo groupand oxidized LDL after six months of administration witha grape supplement enriched in resveratrol [27]. A
decrease of serum levels of LDL-C was also noted after
the administration of 10 mg of resveratrol daily in patientswith coronary artery disease [18].
Our results are in accordance with previous studies on
diabetic patients, which point out an increase of serum
levels of HDL-C after supplementation with resveratrol(1g/day) [21]. Moreover, the association of coenzyme
Q10 with statin therapy might also increase the serum
levels of HDL-C. A study evaluated the benefits of theassociation of coenzyme Q10 and statins, and revealed
increased serum levels of HDL-C in patients on
rosuvastatin, compared to patients on atorvastatin [28].
Atorvastatin seems to determine a more evident
decrease of ubiquinol levels than rosuvastatin does [28].
Our results indicated an increase of serum levels of
HDL-C in the Procor group, supporting the evidence thatthe association of coenzyme Q10 to statin can improve
serum lipid parameters.
The dose of resveratrol present in the dietary supplement
was 10 mg. Although other clinical trials have usedhigher doses of resveratrol, benefits have been observed
even al lower doses. Brasnyó et al used in their study
capsules containing 5 mg of resveratrol (twice daily) andobserved an improvement in insulin sensitivity [29].
The small number of patients included in the study andthe short period of intervention could explain themoderate effects of this dietary supplement containing
mainly coenzyme Q10 (60 mg) and resveratrol (15 mg)
on lipid profile parameters.

DISCUSSIONCONCLUSION

Our study showed positive effects following theadministration of a dietary supplement (Procor Forte)containing resveratrol and coenzyme Q10 in patients with
MS on statin therapy. The dietary supplement determined
an improvement of lipid profile, decreasing TC, LDL-C,
and TG and increasing HDL-C (Figures 1-4). The
modifications on transaminases and inflammatory
parameters like hsCRP were similar in both groups, after
Procor or placebo supplementation during three months(Figures 5, 6). The lipid-lowering effects of resveratrol
have been noticed in many experimental studies [24,25].
Numerous clinical studies have been performed forresveratrol and coenzyme Q10 to establish the potential
benefit of these compounds for health. Even though
results indicated by these studies are sometimes
controversial, positive effects have been noticed inpatients with hypercholesterolemia and type 2 DM
[18,26]. A recent study has focused on resveratrol
benefits as a dietary supplement in patients with type 2
DM and hypercholesterolemia on statin treatment. The
results revealed a decrease of LDL-C, apolipoprotein B

The association of a dietary supplement containing
resveratrol and coenzyme Q10 in patients with metabolic
syndrome on statin therapy can improve the lipid profile,mainly through increasing the levels of HDL-C. Further
studies conducted on a larger period of time andincluding an increased number of patients are needed toestablish the benefit of the association of resveratrol andcoenzyme Q10 in patients wih MS.

Fig.5. Evolution of ALT levels in Procor and placebo group

Fiziologia - Physiology • 2015.25.2(86)

COMPETING INTERESTS
The authors declare that they have no competinginterests.
ACKNOWLEDGEMENT
This work was supported by the POSDRU grant no.
159/1.5/S/136893: “Strategic partnership for the increaseof the scientific research quality in medical universities
through the award of doctoral and postdoctoral
fellowships – DocMed.Net_2.0” (“Parteneriat strategic
pentru creșterea calității cercetării științifice din35universitățile medicale prin acordarea de burse doctoraleși postdoctorale – DocMed.Net_2.0”).

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Fiziologia - Physiology • 2015.25.2(86)EFECTELE SUPLIMENTELOR ALIMENTARE CONȚINÂND RESVERATROLȘI COENZIMA Q10 LA PACIENȚII CU SINDROM METABOLICREZUMAT
Resveratrolul şi coenzima Q10 sunt două nutraceutice intens studiate pentru beneficiile lor în mai multe boli.
Activitățile lor hipolipemiante anti-inflamatorii şi antioxidante ar putea îmbunătăți starea pacienților cu sindrom
metabolic. Studiul nostru a urmărit evaluarea efectelor asocierii un supliment alimentar care conține resveratrol șicoenzima Q10 la medicamentele prescrise la pacienţii cu sindrom metabolic şi tratament cu statine. Studiul a
inclus 29 de pacienţi randomizaţi în două grupuri, unul care a primit suplimentul alimentar (n = 15) și un grup carea primit placebo (n = 14). Nivelurile serice ale colesterolului total (TC), trigliceridelelor (TG), LDL-colesterolului(LDL-C), HDL-colesterolului (HDL-C), transaminazelor și proteinei C reactive cu sensibilitate crescută (hsCRP)
au fost evaluate la începutul studiului și după trei luni. S-a observat o scădere a nivelurilor serice ale TC, TG,
LDL-C și hsCRP și o creștere a HDL-C. Creşterea HDL-C observată în grupul care a primit suplimentul alimentar(de la 43.80 ± 13.18 la 46.67 ± 10.40 mg / dl), s-a apropiat de valoarea semnificativă din punct de vedere
statistic (p = 0,057). Rezultatele prezentului studiu indică faptul că asocierea resveratrolului și coenzimei Q10 lapacienţii cu sindrom metabolic ar putea scădea moderat TC, LDL-C, TG și crește semnificativ valoarea HDL-C.
Sunt necesare studii suplimentare pentru a elucida efectele combinaţiei resveratrol şi coenzima Q10 asupraparametrilor profilului lipidic.
Cuvinte cheie: sindrom metabolic, resveratrol, coenzima Q10

Fiziologia - Physiology • 2015.25.2(86)37NOVEL INSIGHTS INTO THE ROLE OF METHYLENE BLUEIN MITOCHONDRIAL PROTECTIONOANA M. DUICU*, ADRIAN STURZA*, ILEANA SCURTU-MYTIKO, ANDREEAPRIVISTIRESCU, MARIA DĂNILĂ*, LAVINIA NOVEANU*, DANINA M. MUNTEAN*
Department of Pathophysiology, *Center for Translational Research and Systems Medicine, “Victor Babeș” University of
Medicine and Pharmacy, Timișoara, Romania

ABSTRACT
Mitochondrial dysfunction and reactive oxygen species (ROS) generation are critical events in the pathophysiology ofageing and chronic disorders, in the past years modulation of mitochondrial function becoming thus an importanttherapeutic target. Pharmacological agents able to improve mitochondrial function and/or to decrease ROS production
are nowadays highly investigated. Among them an emerging compound is methylene blue (MB), a tricyclic
phenothiazine, used for more than a century to treat a variety of disorders. Due to its redox activity, MB has a highaffinity for numerous tissue oxidases, including those within mitochondria. Here we briefly review the use of MB in
medicine and biology and discuss its beneficial effects as a mild-redox agent able to prevent mitochondria-driven
disorders. Methylene blue has a significant potential to improve mitochondrial metabolism and represents a promisingtherapeutic approach in several conditions associated with mitochondrial dysfunction.
Key words: mitochondria, methylene blue, redox potential

INTRODUCTION
Historically, methylene Blue (MB), a tetramethylthionehydrochloride (C16H18ClN3S) is a heterocyclic aromaticcompound firstly synthesized by Heinrich Caro and used
as a cotton staining dye [1]. In the 19th century, Peter
Ehrlich demonstrated its beneficial effects in thetreatment of malaria [2]; this report represented thestarted point for investigating the role of MB in the field ofbacterial and viral infections [3] and cancer [3,4]. MB was
further used both as therapeutic (e.g., antiseptic, cancer
chemotherapy drug) and diagnostic compound (e.g.,
staining in neuroanatomy and bacteriology, redox agent
in biochemical studies) [4,6].
US Food and Drug Administration (FDA) approved MB intreating methemoglobinemia, either inborn [7] or after anoverexposure to drugs, nitrophenols, and environmental
poisons (e.g., nitrates or cyanides) [8]. The beneficial
effects of MB in methemglobinemia therapy are due to its
unique autooxidyzing property: indeed, as stipulated by
Rojas et al. (2012), "its reduction-oxidation capacity allows
electron cycling, without MB gaining any permanent
stoichiometric or net reduction" [9](Figure 1). This property
explains how MB is acting as an electron donor in the
nonenzymatic reduction of methemoglobin: the NADPH
methemoglobin reductase reduces MB to LeucoMB
th(Figure 1), with a nonenzymatic transfer of electrons tomethemoglobin, and by thus restoring the functionalhemoglobin and MB [10]. Therefore, same Paul Ehrlich
named it the "magic bullet" due to its autooxidyzing
property for electron cycling, which was firstly identified in
the nervous tissue [11].

Fig. 1. Methylene blue: a redox active agent. NADPH
reduces MB (blue color) to MBH2 (colorless) which is further
oxidized by O2 (modified after 12).

MB has a high-affinity for the nervous tissue [13], being
used firstly in 1938 in the experimental treatment ofschizophrenia [14]. Since then, MB has proved to be aneuroprotective agent in numerous neurodegenerative
disorders such as Alzheimer’s disease (AD), Parkinson’s
disease (PD), Leber’s optic neuropathy [7, 15-22]. The
sulfur atom within the structure of MB compound has
been suggested to be responsible for this high-affinity toneural structures, yet the underlying mechanism is notcompletely understood [23].

thReceived 12 of March 2015. Accepted 17 of April 2015. Address for correspondence: Danina Muntean, MD, PhD,
Department of Pathophysiology, “Victor Babeș” University of Medicine and Pharmacy of Timişoara, 14 Tudor Vladimirescu st.,300173 Timisoara, RO, Tel/Fax: +40-256-493085; e-mail: daninamuntean@umft.ro38

Fiziologia - Physiology • 2015.25.2(86)In the past decade, an increasing number of studies
investigated the neuroprotective role of MB in various
pathological conditions: accordingly, MB reduced the
cerebral damage induced by ischemia/reperfusion (I/R) ina model of transient focal cerebral ischemia andattenuated the behavioral and neurochemical impairment
in a PD model [24]. Furthermore, MB prevented thedecline of the cognitive function in transgenic models of
AD and slowed the progression of the disease in aclinical trial [25–27].
MB’s protective effects have been also studied in relation
with ageing [28] and in pathological states associatedwith liver failure due to intestinal I/R injury [29]. Thus, MB(at nM levels) was proven to extend the life span of human
IMR90 fibroblasts in tissue culture by enhancing
mitochondrial function: it increased mitochondrial complex
IV, enhanced cellular oxygen consumption, increased
heme synthesis, and reversed premature senescencecaused by H2O2 or cadmium [28]. Collange et al. (2013)
demonstrated that the intra-peritoneally injection of MB
before reperfusion restored the oxidative capacity of liver
mitochondria after I/R injury of the gut, a result which
could have beneficial clinical implications during vascularsurgery by decreasing the incidence of postoperative
multiple organ dysfunction [29].
Nowadays, MB is clinically used in multiple disorders anddiagnostic procedures (see Table I).
Table I. Clinical indications of MB and corresponding
dosage (after 25 and 30)
Clinical indications of

MB dosage/Ref.

MBInherited1 x 50–250 mg/day (for lifetime) (7)methemoglobinemia
Acute

1–2 x 1.3 mg/kg (i.v. over 20

methemoglobinemia

minutes) (30)

Ifosfamid-induced

4 x 50 mg/day p.o. or i.v. (18)neurotoxicity
Prevention of urinary tract

3 x 65 mg/day p.o. (30)infections in elderly
patients
Vasoplegic

200 mg i.v. over 1 hour followed byadrenaline-resistant shock

infusion (0.25–2 mg/kg/hour) (31)Alzheimer’s disease3 x 60 mg/day (Rember™ according
to 32)

Pediatric malaria

2 x 12 mg/kg p.o. for 3 days (30)

Parathyroid imaging

3–7.5 mg/kg i.v. (25)Sentinel lymph node

Local application of 1–5mL of 1% MBbiopsy(25)

Methylene Blue and Mitochondrial Function
An important approach to improve mitochondrial
function is represented by agents with mild redox
activity. It is well known that mitochondria are rich in theredox centers (heme, flavin mononucleotide, flavinadenine dinucleotide, iron-sulfur, copper, or naturalcoenzyme Q) which represent the major source of free
radicals production [33]. Thus, since mitochondria
physiology depends on major redox activities, redox
active agents have been recently proposed as
mitochondrial modulators able to enhance energy
production and/or to decrease oxidative stress [33].
The mitochondrial electron transport chain (ETC) at the
inner mitochondrial membrane is composed of fivemultimeric complexes (complexes I to V) and twointermediary substrates (coenzyme Q and cytochromec). The reduced NADH+H+ and FADH 2 produced by theintermediate metabolism are further oxidized by the
mitochondrial ETC to induce an electrochemical
gradient of protons from the mitochondrial matrix to theinter-membrane space (Figure 2), which is finally usedby the F1F0-ATP synthase (complex V) to produce ATP[34].
Atamna et al. (2012) suggested that redox agents witha mild redox potential (-0.1 to 0.1 V) are able to improvethe mitochondrial function via "bypassing the productionof superoxide radicals and the cycling between the
reduced and oxidized form": mild redox agents enter
the cell in the oxidized form, are reduced by thephysiologic redox centers (e.g., NADH-dehydronase ofcomplex I), and are further re-oxidized by a second
physiologic center (e.g., cytochrome c) [33]. The redox
potential of MB is ~ 10 mV, which is considered mild vs.
the redox potential of the ETC components [28],thereby MB serves as an electron carrier increasing themitochondrial energy production and inhibiting thesuperoxide production [35].
As several other drugs, MB presents hormetic
pharmacological effects: at low doses elicits beneficial
effects, whereas in high concentrations might becomedeleterious [9]. To enter cytosol, MB is reduced at the cellsurface to its lipophilic form, MBH2. Inside the cell, MBH2
is re‐oxidized to MB by the heme proteins. At low doses,
there is an MB‐MBH2 equilibrium (electron cycling) andMBH2 can donate electrons to ETC complexes andoxygen, leading to enhanced energy metabolism anddecreased superoxide formation. At high doses, this
MB‐MBH2 equilibrium is impaired and MB can takeelectrons away from ETC complexes, leading todecreased activity of these complexes and promoting
oxidative stress [9].

i.v.: intravenous; p.o., oral.
Fiziologia - Physiology • 2015.25.2(86)39CONCLUSIONS

Fig. 2. Schematic representation of methylene blue action
within mitochondria (modified after 9). Methylene blue acts as
an artificial electron donor to: (i) mitochondria electron transport
chain complexes, increasing thus the energy production, and (ii)oxygen, preventing superoxide formation. (IMM - innermitochondrial membrane; OMM – outer mitochondrial
membrane). Illustration realized thanks to Servier Medical Art.

MB hormesis is strongly linked to the mitochondrial
membrane potential: a high membrane potential will be
responsible for an important accumulation of MB and thedimerization of MB molecules [9]. Gabrielli et al. (2004)
demonstrated that oxidative radicals are highly produced in
the presence of MB monomers and in a lesser extent in
the presence of MB dimers [36]. Thus, low concentrations
rather than high concentrations of MB, are associated withenhanced mitochondrial metabolism [37]. In line with theseobservations, we recently investigated the effects of 5
progressive concentrations of MB (0.05, 0.1, 1, 5, and 10µM, respectively) on oxygen consumption rate (OCR) andthe extracellular acidification rate (ECAR), respectively inH9c2 cardiomyoblasts [38]. We clearly demonstrated thehormetic effect of the acute administration of MB: 0.1 µM
induced an increase in both OCR and ECAR, indicating an
overall stimulatory impact on cellular bioenergetics.
However, all OCR linked parameters and ECAR weredecreased in a concentration-dependent manner when MB
was administered in the higher concentrations (1, 5, and10 µM), and were not influenced by 0.05 µM MB,
respectively [38]. Our results are in agreement with thosereported by Atamna et al. (2008) who showed that human
IMR90 fibroblasts in tissue culture exposed to high(micromolar range) but not to low (nanomolar range)
concentrations of MB showed a compensatory
upregulation of antioxidant enzymes with decreased heme
expression and iron uptake [28]. Moreover, highconcentrations of MB induced the alteration of neural
structure or function in vivo, including humans [37, 39-43].
Another important finding is that not only acuteadministration of MB is beneficial; chronic administration of
low doses of MB increased complex IV activity in brain andheart of old mice, explaining thus the preservation of
mitochondrial function and energy metabolism [9, 28, 44,
45].

40

It is widely accepted that preventing mitochondrial
dysfunction in both acute and chronic pathologies has
significant clinical benefits, an observation that rendered
mitochondria in the past decades an attractive
therapeutic target. In this regard, when administered in
an appropriate doses, methylene blue has a significant
potential to improve mitochondrial respiration anddecrease superoxide radicals formation, via its mild
redox potential. Further studies aimed at dissecting themolecular and cellular mechanisms underlying thebeneficial effects of methylene blue are definitely
warranted.
ACKNOWLEDGMENTS
This work was supported by the POSDRU grant no.
159/1.5/S/136893 titled “Strategic partnership for theincrease of the scientific research quality in medical
universities through the award of doctoral andpostdoctoral fellowships – DocMed.Net_2.0”. (O.M.D.).
We would like to thank Adrian Wolf (undergraduate
student and tutor at the Department of Pathophysiology,“Victor Babes” University of Medicine and Pharmacy,Timișoara) for his valuable contribution to the study.

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NOI PERSPECTIVE ASUPRA ROLULUI ALBASTRULUI DE METILEN
ÎN PROTECȚIA MITOCONDRIALĂREZUMAT
Disfuncția mitocondrială și producerea de specii reactive de oxigen (SRO) constituie evenimente critice în
fiziopatologia procesului de îmbătrânire precum și a numeroase afecțiuni cronice, astfel că modularea funcției
mitocondriale a devenit o importană țintă terapeutică în ultimii ani. În consecință, agenții farmacologici capabili săîmbunătățească funcția mitocondrială și/sau să inducă reducerea producției de SRO sunt acutalmente intens
investigați. Printre aceștia, un compus extrem de important este albastrul de metilen (AM), un compus
fenotiazinic triciclic, folosit de mai bine de un secol în tratarea unor afecțiuni diverse. Datorită potențialului său detip redox, AM prezintă o afinitate crescută pentru numeroase oxidaze tisulare, incuzându-le și pe cele de la nivel
mitocondrial. Vom prezenta pe scurt utilizarea AM în medicină și biologie, precum și rolul benefic al AM ca șiagent de tip redox capabil de a preveni afecțiunile induse de disfuncția mitocondrială. Albastrul de metilen
prezintă un potențial semnificativ de îmbunătățire a metabolismului mitocondrial, reprezentând astfel o abordare
terapeutică promițătoare în afecțiunile induse de disfuncția mitocondrială.
Cuvinte cheie: mitocondrii, albastru de metilen, potențial redox

42

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