The Effect of Crocus sativus Extract on Human Lymphocytes’ Cytokinesand T Helper 2/T Helper 1 Balance
Mohammad Hossein Boskabady,1 Seyedh Masoumeh Seyedhosseini Tamijani,1
Houshang Rafatpanah,2 Abadolrahim Rezaei,2 and Azam Alavinejad1
1Department of Physiology and Pharmaceutical Research Center; 2Immunology Research Center;Buali Institute, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
ABSTRACT The effects of macerated extracts of Crocus sativus (Family Iridaceae) (saffron) on cell viability and cytokine
release of stimulated peripheral blood mononuclear cells by phytohemagglutinin (PHA) and nonstimulated cells were ex-
amined. The effects of three concentrations of macerated extract, dexamethasone, and saline on cell viability and production
of cytokines, including interleukin (IL)-4, IL-10, and interferon-c (IFN-c) were evaluated. In cells stimulated with PHA,
different concentrations of the extract significantly inhibited cell viability of lymphocytes (P < .001 for all concentrations).
High concentrations of the extract (500 lg/mL) also inhibited secretion of IFN-c in stimulated cells and IL-10 secretion in both
stimulated and nonstimulated cells (P < .05 for all cases). The effects of high and low concentrations of the extract (500 and
50 lg/mL, respectively) on IL-4 secretion were lower than that of dexamethasone (P < .05 to P < .001). The extract showed a
stimulatory effect on IFN-c and IL-4 secretion in nonstimulated cells. The ratios of IFN-c to IL-4 in the presence of all
concentrations of saffron on stimulated cells were significantly higher than for the control group (P < .05 to P < .01). These
results indicated that the extract of saffron leads to increased ratio of IFN-c to IL-4.
KEY WORDS: � cell viability � Crocus sativus � cytokine � human lymphocyte � Iridaceae � peripheral mononuclear cell
INTRODUCTION
Crocus sativus L., a small perennial plant from the irisfamily (Family Iridaceae), is cultivated in many regions,
particularly in Iran. It has green, hairy leaves about 30 cmlong and a funnel-shaped, reddish-purple flower. The part ofthe plant used medicinally is its stigma, also called the style(the central part of the flower, the female sexual organ).1,2
The main constituents of the stigma are crocins, safranal,picrocrocin, ketoisophorone, isophorone, and glycosidicterpenoids.3
Anti-inflammatory,4,5 radical scavenger, and antioxi-dant6–10 properties and antitumor effects1,2,11–13 for C. sa-tivus have been shown in previous studies. The effect of theplant and crocin on apoptosis and mutagenicity was alsodemonstrated.14,15 Saffron extract also has chemopreventiveand genoprotective effects and protects against genotoxin-induced oxidative stress in mice.16,17 The antitumor activityof saffron and its constituents was also documented in dif-ferent studies.5,15,18–20
The pathological characteristic of asthma is airway in-flammation.21 Many inflammatory cells are involved in the
pathogenesis of airway inflammation in asthma.22 One of theimmunologic features of asthma is a balance shift from Thelper 1 (Th1) to T helper 2 (Th2). It is evident that Th2 isactivated in asthma disease, and its mediators cause manyfeatures of asthma such as airway inflammation, mucussecretion, and airway hyperresponsiveness.23 Th1 proved toinhibit Th2 responses, and thus one goal of asthma therapyshould be focused on increasing the activity of Th1.24
Therefore, in the present study, the effect of the extract ofC. sativus on cell viability and Th1/Th2 balance was studiedby measuring levels of the secreted cytokines interleukin(IL)-4, interferon-c (IFN-c), and IL-10.
MATERIALS AND METHODS
Plant and extracts
C. sativus was collected from Torbat Heydarieh (EasternIran) and identified by Mrs. M. Molaei (University of Fer-dowsi, Mashhad, Iran). A voucher specimen was preservedin the Herbarium of the School of Agriculture, University ofFerdowsi (Herbarium Number 143-0319-1). The wholeplant was collected and identified, but the stigma of theidentified plant was isolated and used in the study. Themacerated hydro-ethanolic extract of the isolated stigmawas prepared as follows: 3 g of chopped, dried, and isolatedstigma of the plant was mixed with 50 mL of 70% ethanol
Manuscript received 8 January 2011. Revision accepted 18 July 2011.
Address correspondence to: Mohammad Hossein Boskabady, M.D., Ph.D., Departmentof Physiology, School of Medicine, Mashhad, Post Code 9177948564, Iran, E-mail:[email protected] or [email protected]
JOURNAL OF MEDICINAL FOODJ Med Food 14 (12) 2011, 1538–1545# Mary Ann Liebert, Inc. and Korean Society of Food Science and NutritionDOI: 10.1089/jmf.2011.1697
1538
(15 mL of distilled water and 35 mL of ethanol) for 72 hoursat 25�C and shaken intermittently. The solvent was thenremoved under reduced pressure. An extract of 2.5 g wasobtained from 10 g of C. sativus stigma. The plant ingredientconcentration in the final extract was adjusted to 0.1 g/mLby adding distilled water to the dried extract.
Characterization of C. sativus extract
The quality of the extract of the same stigma from C.sativus was characterized by high-performance liquidchromatography (HPLC) (Waters model 474 chromato-graph, Waters Corp., Milford, MA, USA) fingerprinting.The extract was dissolved in a distilled water:methanolmixture (50:50 vol/vol) and then filtered through a mem-brane filter (pore size, 0.22 lm). Twenty microliters ofsample (5 mg/mL) was injected into the reverse-phaseHPLC column (C18). The mobile phase consisted of meth-anol:water:glacial acetic acid (50:44.5:0.5 by volume) withan isocratic elution at the flow rate of 1 mL/minute. Thepeaks were monitored at 440 nm (Fig. 1). The crocineconstituent of the extract was also evaluated using a spec-trophotometric method as previously described.25
Peripheral blood mononuclear cell isolation
Ten healthy male subjects (20–60 years old) participatedin the study. Ten milliliters of peripheral whole blood wascollected into an EDTA-anticoagulated tube. For peripheralblood mononuclear cell (PBMC) separation, whole bloodcells were layered onto a Ficoll density gradient (Sigma, St.Louis, MO, USA) and centrifuged for 15 minutes at 360 g atroom temperature. The layer of PBMCs was removed, andcells were washed three times with phosphate-buffered sa-line at 130 g for 10 minutes. PBMCs were cultured as above(control) or incubated with phytohemagglutinin (PHA) at aconcentration at 5 lg/mL for 72 hours (stimulated cells withPHA).
Cell viability
Cell viability was assessed using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) as-say.26 In brief, 2 · 105 cells were seeded at 5,000 cells perwell in flat-bottom 96-well culture plates and allowed togrow for 72 hours followed by treatment with C. sativuswith or without PHA as mentioned above. The medium wasremoved, cells were incubated with MTT solution (5 mg/mLin phosphate-buffered saline) for 4 hours, and the resultingformazan was solubilized with dimethyl sulfoxide (100 lL).The optical density was measured at 570 nm with a refer-ence of 620 nm.
Measurement of IL-4, IL-10, and IFN-c in supernatantof cell culture
The samples were centrifuged at 560 g at 4�C for 5 min-utes. The supernatant was collected and immediately storedat - 20�C until the time of analysis. The amounts of IL-4, IL-10, and IFN-c were measured using enzyme-linked immu-nosorbent assay kits (Bender MedSystems Inc., Vienna,Austria) according to the manufacturer’s instructions.
Experimental groups
All the above measurements (MTT, IL-4, IL-10, and IFN-c) were examined in the following groups: (1) nonstimulatedlymphocytes (control group); (2) PHA-stimulated lympho-cytes; (3) control group treated with dexamethasone(0.1 mM) (Sigma); (4) control group treated with C. sativusextract at a concentration of 50, 250, or 500 lg/mL; and (5)PHA-stimulated lymphocytes treated with C. sativus extractat a concentration of 50, 250 and 500 lg/mL. In experi-mentally treated Groups 3 and 4, separated lymphocyteswere treated with dexamethasone or the extract and cul-tured. In experimentally treated Group 5, separated lym-phocytes were treated with the extract, then incubated withPHA, and cultured.
FIG. 1. High-performance liquidchromatography fingerprint of theaqueous-ethanolic extract of stigmafrom C. sativus illustrating crocin (re-tention time, 2.6 minutes) and safranal(retention time, 7.56 minutes).
EFFECT OF C. SATIVUS ON TH2/TH1 BALANCE 1539
Table 1. Determination of Crocin and Safranal in Various Saffron Samples
Using High-Performance Liquid Chromatography and Ultraviolet-Visible Spectrophotometry
Saffron (mg/g) Crocin (mg/g) Safranal (mg/g)
Direct readinga UV-visible UV-visible HPLC HPLC
Geographic location ISO extractb Modified extractc ISO extractb Modified extractc Modified extractc Modified extractc
Torbat 300 333 34.8 38.8 41.3 0.22Torbatjam 291 323 33.9 33.9 40.2 0.26Sabzevar 298 330 36.3 40.2 42.9 0.07Kashrnar 277 308 32.6 36.2 38.6 0.06Mashhad 279 311 32.3 36.1 38.3 0.17Neishaboor 266 495 31.2 32.6 36.9 0.29
aData are extinction cofficients at 1% concentration (pathlength = 1 cm, k = 440 nm).bExtraction according to ISO 3632 method with water.cExtraction with ethanol and water (50:50 vol/vol) and sonication.25
HPLC, high-performance liquid chromatography; UV, ultraviolet.
FIG. 2. Photographs of cultured lymphocytes in (a) the control group, (b) cultured lymphocytes stimulated with phytohemagglutinin, (c) treatedwith dexamethasone, (d–f) treated with three concentrations of safranal (50, 250, and 500 lg/mL saffron, respectively), and (g–i) stimulated withphytohemagglutin and treated with three concentrations of safranal (50, 250, and 500 lg/mL saffron, respectively). Magnification, · 10. Colorimages available online at www.liebertonline.com/jmf
1540 BOSKABADY ET AL.
Statistical analysis
All data were expressed as mean – SEM values. Percen-tage changes in the values of MTT, IFN-c, IL-10, and IL-4in proportion to control values in cultured lymphocytestreated with different concentrations of saffron were calcu-lated. The ratio of IFN-c to IL-4 was also calculated inall experimental groups. Statistical comparisons betweentreated groups with extract, dexamethasone, and controlwere made using analysis of variance with Tukey–Kramermultiple post hoc test. Comparison of the effect of eachconcentration of saffron between stimulated and non-stimulated lymphocytes treated with PHA was done usingunpaired t test. Statistical comparisons of the proportion ofIFN-c to IL-4 between treated and control groups were doneusing the nonparametric analysis of variance (Kruskal–Wallis) test. Significance was accepted at P < .05.
RESULTS
Characterization of the extract
Safranal (0.26%) and crocin (41.3%, crocin 2) levels ofthe same plant (C. sativus) were measured at the School ofPharmacy, Mashhad University of Medical Sciences, byHPLC (Table 1).25
Effect of stimulation of PBMCs by PHA
PHA stimulation of lymphocytes significantly increasedthe percentage viability of lymphocytes (256.12 – 25.65%vs. 100 – 0.0%) and secretion of IFN-c (87.02 – 27.39% vs.0.911 – 0.34%), IL-10 (13.033 – 2.10% vs. 2.65 – 0.61%),and IL-4 (15.59 – 3.17% vs. 7.39 – 1.78%) compared withthe control group (P < .05 to P < .001) (Figs. 2–6).
Effect of dexamethasone on percentage viabilityand cytokine secretion by lymphocytes
Dexamethasone did not cause a significant change inpercentage viability of lymphocytes (93.12 – 6.22% vs.100 – 0%) and secretion of IFN-c (7.58 – 4.456% vs. 0.91 –0.34%), IL-10 (3.66 – 0.96% vs. 2.65 – 0.61%), and IL-4(1.6 – 1.31% vs. 7.39 – 1.78%) compared with the controlgroup (Figs. 2–6).
Effect of the extract of C. sativus on percentage viabilityand cytokine secretion by lymphocytes
Different concentrations of saffron did not cause a sig-nificant change in the percentage viability of nonstimulatedlymphocytes and secretion of IFN-c, IL-10, and IL-4 exceptat a low concentration, which caused a significant increasein IL-10 compared with the control group (8.03 – 2.279% vs.2.65 – 0.61%) (P < .05) (Figs. 2–6).
In stimulated cells with PHA, different concentrations ofsaffron extract caused significant inhibition in percentageviability of lymphocytes (P < .001 for all concentrations).The high concentration of the extract (500 lg/mL) alsosignificantly inhibited secretion of IFN-c (21.73 – 8.7% vs.
87.02 – 27.39%) and IL-10 (0.51 – 0.29% vs. 13.03 –2.95%). However, the extract did not affect the IL-4 secre-tion from the stimulated lymphocytes (Figs. 2–6).
Comparison of the effect of C. sativus extractwith dexamethasone
In nonstimulated cells, only the inhibitory effects of highand low concentrations of the extract (500 and 50 lg/mL) on
FIG. 3. Percentage viability of cultured lymphocytes in the controlgroup (C) (open columns), stimulated with phytohemagglutinin (P)(columns with vertical lines), treated with dexamethasone (D) (col-umns with cross-hatched lines), and stimulated with phytohemag-glutinin and treated with three concentrations of saffron (P + Saffron)(fine-, medium-, and coarse-lined columns are 50, 250, and 500 lg/mL concentrations, respectively). Data are mean – SEM values(n = 50). Significant difference between group C and the other groups:***P < .001. Significant difference between group P and the othergroups: + + + P < .001. Statistical comparisons were made usinganalysis of variance with the Tukey–Kramer multiple post hoc test.
FIG. 4. Values of interleukin (IL)-4 in cultured lymphocytes ingroup C (open columns), group P (columns with vertical lines), groupD (columns with cross-hatched lines), and group P + saffron (fine-,medium-, and coarse-lined columns are 50, 250, and 500 lg/mLconcentrations, respectively). Data are mean – SEM values (n = 6).Significant difference between group C and the other groups:*P < .05. Significant difference between group P and the other groups:+ + P < .01. Statistical comparisons were made using analysis of var-iance with the Tukey–Kramer multiple post hoc test.
EFFECT OF C. SATIVUS ON TH2/TH1 BALANCE 1541
IL-4 secretion were lower than the effect of dexamethasone(P < .001 and P < .01 respectively, Table 2).
Comparison of the effect of three concentrationsof C. sativus extract
In nonstimulated cells, the effects of a low concentrationof the extract (50 lg/mL) on lymphocyte viability weresignificantly lower than the effect of the other two concen-trations (P < .05 vs. 250 lg/mL saffron extract and P < .001vs. 500 lg/mL saffron extract, Table 2). In addition, theinhibitory effect of the low concentration extract (50 lg/mL)on IL-10 secretion was lower than that of its high concen-tration (500 lg/mL) (P < .05, Table 2).
Comparison of the effect of C. sativus extract betweenstimulated and nonstimulated lymphocytes
The inhibitory effect of all concentrations of the extracton percentage viability of nonstimulated lymphocytes and
its high concentrations (500 lg/mL) on IL-4 secretion weresignificantly lower than in stimulated lymphocytes (P < .001for cell viability and P < .05 for IL-4, Table 3). In fact, theextract showed a stimulatory effect on IFN-c and IL-4 se-cretion in nonstimulated cells (Table 3).
Comparison of the ratio of IFN-c to IL-4 between treatedand control groups
The ratios of IFN-c to IL-4 obtained in the presence of allconcentrations of saffron only on stimulated cells with PHAwere significantly higher than in the control group (P < .01for all cases) (Table 4).
DISCUSSION
The present study showed significant inhibition byall three concentrations of saffron on viability of PHA-stimulated lymphocytes. The results showed that the ex-tract did not inhibit IL-4 secretion in either stimulated or
FIG. 5. Values of IL-10 in cultured lymphocytes in group C (opencolumns), group P (columns with vertical lines), group D (columnswith cross-hatched lines), and group P + saffron (fine-, medium-, andcoarse-lined columns are 50, 250, and 500 lg/mL concentrations,respectively). Data are mean – SEM values (n = 9). Significant dif-ferences between group C and the other groups: *P < .05, **P < .01.Significant difference between group P and the other groups:+ P < .05. Statistical comparisons were made using analysis of vari-ance with Tukey–Kramer multiple post hoc test.
FIG. 6. Values of interferon-c (IFN-c) in cultured lymphocytes ingroup C (open columns), group P (columns with vertical lines), groupD (columns with cross-hatched lines), and group P + saffron (fine-,medium-, and coarse-lined columns are 50, 250, and 500 lg/mLconcentrations, respectively). Data are mean – SEM values (n = 9).Significant difference between group C and the other groups:**P < .01. Significant difference between group P and the othergroups: + P < .05. Statistical comparisons were made using analysis ofvariance with the Tukey–Kramer multiple post hoc test.
Table 2. 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyl Tetrazolium, Interferon-c, Interleukin-10, and Interleukin-4 Levels
in Nonstimulated Cultured Lymphocytes, Treated with Three Concentrations of Saffron
Variable PZ3 PZ2 PZ1 Z3 Z2 Z1 D
MTT 115.92 – 12.71 120.16 – 11 145.2 – 13.03 69.90 – 4.34 85.15 – 4.71 107.26 – 6.6{{{{ 93.12 – 6.22IFN-c 21.73 – 8.7 25.1 – 10.11 46.1 – 14.66 1.11 – 0.34 2.96 – 0.88 4.26 – 1.09 7.583 – 4.456IL-10 0.516 – 0.30 6.12 – 2.73 11.19 – 3.57{ 0.66 – 0.38 2.68 – 1.059 8.03 – 2.28{{ 3.655 – 0.960IL-4 7.13 – 2.32 8.36 – 1.73 7.15 – 2.54 13.56 – 1.09*** 8.1 – 1.37 10.37 – 2.51** 1.6 – 1.306
Data are mean – SEM values. For 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) (n = 50), for IFN-c and IL-10 (n = 9), and for IL-4 (n = 6)
measurements, Z1, Z2, and Z3 represent 50, 250, and 500 lg/mL concentrations of saffron, respectively; PZ1, PZ2, and PZ3 represent the same respective
concentrations in phytohemagglutinin-stimulated lymphocytes.
Statistical comparisons were made using analysis of variance with the Tukey–Kramer multiple post hoc test. Significant difference between group D and saffron-
treated groups: **P < .01, ***P < .001. Significant difference between low versus medium and high saffron concentrations: {{P < .01, {{{P < .001. Significant
difference between medium versus high saffron concentration: {P < .05.
1542 BOSKABADY ET AL.
nonstimulated lymphocytes. The inhibitory effect of allconcentrations of the extract was significantly less than theeffect of dexamethasone. The results also showed a stim-ulatory effect on IFN-c and IL-4 secretion in non-stimulated cells. The two higher concentrations of saffronincreased the ratio of IFN-c to IL-4 in nonstimulatedlymphocytes. Therefore, the results of the present studyshowed a stimulatory effect of the extract of C. sativus(saffron) on IFN-c secretion in nonstimulated cells. Inaddition, the extract caused an increase in the ratio of IFN-c to IL-4. The extract also showed a potent inhibitoryeffect on cell viability on both nonstimulated and PHA-stimulated human lymphocytes.
The increased ratio of IFN-c to IL-4 seen with C. sativus(saffron) suggests a suppressive effect of the plant on theTh2 lymphocyte subtype. In fact, anti-inflammatory1,2 andantioxidant6–10 effects for C. sativus have been shown inprevious studies. Garcıa-Olmo et al.27 also showed the ef-fect of saffron on T cells, which also support the findings ofthe present study.
It has been shown that inflammatory conditions such asairway inflammation in asthma are regulated by the balance
of two helper cell types, Th1 and Th2.23,24 Th2 cells pro-mote the activity of macrophages and regulate the pro-inflammatory response, whereas Th1 cells inhibit theactivity of Th2 and regulate the anti-inflammatory re-sponse.28 Th1 cells produce IL-2 and IFN-c, whereas Th2cells produce IL-4 and IL-10.27 Therefore, the results of thepresent study suggest that the extract from C. sativus causedan inhibitory effect on IL-4 but enhanced the production ofIFN-c, indicating an inhibitory effect on Th2 cells andstimulatory effect on Th1 cells.
The effects of the extract on suppressing lymphocyte vi-ability and IL-10 secretion but increased secretion of IFN-cand ratio of IFN-c to IL-4 were comparable with the effectof dexamethasone. Although the effect of dexamethasoneon IFN-c secretion was higher than those of the extract,its effect on lymphocyte viability was less than withhigher concentrations of the plant, indicating that C. sativusinhibited Th2 and promoted Th1 cells, similar to dexa-methasone.
The inhibitory effect of the extract on cell viability andthe enhancing effect of the extract on IFN-c secretion wereconcentration dependent. However, the effects of the extracton IL-4 and IL-10 secretion were not concentration depen-dent. These results could suggest that the maximum effect ofthe extract on IL-4 and IL-10 secretion may occur at lowerconcentration.
The results of the present study also showed that the ef-fects of the extract on PHA-stimulated lymphocytes aregreater than on nonstimulated cells. These findings indicatethat the effect of the plant on increasing Th1/Th2 balance isgreater in stimulated lymphocytes such as those that exist inasthmatic inflamed airways.
The HPLC results also indicate that the extract of saffroncontains only 0.26% safranal, whereas the amount of crocinin the extract was 41.3%.25 Therefore, the inhibitory effectof crocin and safranal on Th1/Th2 should be evaluated infurther studies.
Our previous study showed a potent relaxant effect of theextract of C. sativus (saffron) on tracheal chains,29 a stim-ulatory effect of the plant on b2-adrenoceptors,30 and aninhibitory effect on histamine (H1) receptors.31 In addition,an antitussive effect of C. sativus (saffron) and safranal onguinea pigs was also demonstrated.32 The results of thepresent study also showed an increased Th1/Th2 balance for
Table 3. Percentage Change in Levels of 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyl Tetrazolium, Interferon-c,
Interleukin-10, and Interleukin-4 in Proportion to Control Values in Nonstimulated and Stimulated Lymphocytes
Variable Z1 Z2 Z3 PZ1 PZ2 PZ3
MTT - 10.32 – 6.31 - 31.13 – 6.22 - 70.18 – 10.55 - 93.75 – 11.63*** - 35.25 – 14.05*** - 165.52 – 18.21***IFN- c 145.38 – 21.83 166.19 – 118.41 85.97 – 116.22 - 693.16 – 448.7 - 1,441.98 – 854.7 - 2,537.61 – 1,568.2Il-4 50.23 – 135.37 55.15 – 90.30 150.38 – 82.01 - 217.28 – 94.55 - 109.26 – 80.5 - 201.89 – 95.83*Il-10 204.09 – 106.57 - 176.27 – 157.12 - 367.46 – 72.22 - 98.33 – 291.15 - 783.35 – 534.74 - 869.98 – 231.56
Data are mean – SEM values. For MTT (n = 50), IFN-c and IL-10 (n = 9), and IL-4 (n = 6) measurements, Z1, Z2, and Z3 are 50, 250, and 500 lg/mL
concentrations of saffron, respectively; PZ1, PZ2, and PZ3 represent the same respective concentrations in phytohemagglutinin-stimulated lymphocytes.
Statistical comparisons were made using unpaired t test. Significant difference between different concentrations of saffron in cultured lymphocytes versus those
cultured and stimulated with phytohemagglutinin: *P < .05, **P < .01, ***P < .001.
Table 4. Proportion of Interferon-c to Interleukin-4
in Nonstimulated Cultured Lymphocytes
Treated with Either Dexamethasone
or Three Concentrations of Saffron
Group IFN-g/IL-4
C 10.7 – 4.6Z1 100 – 80Z2 19.2 – 6.75Z3 5.75 – 2.2PZ1 371.4 – 119.7**PZ2 226 – 49.2**PZ3 283.7 – 36**D 411.97 – 66.24***
Data are mean – SEM values. The dose of dexamethasone used was 0.1 mM.
Z1, Z2, and Z3 are 50, 250, and 500 lg/mL concentrations of saffron,
respectively; PZ1, PZ2, and PZ3 represent the same respective concentrations
in phytohemagglutinin-stimulated lymphocytes.
Statistical comparisons were made using the nonparametric analysis of
variance (Kruskal–Wallis) test. Significant difference in different concentra-
tions of saffron between stimulated and nonstimulated cultured lymphocytes
versus those of group C: **P < .01, ***P < .001.
EFFECT OF C. SATIVUS ON TH2/TH1 BALANCE 1543
the extract. Therefore the stigma of C. sativus (saffron),which is used in traditional medicine for treatment of vari-ous disorders,1,2 could have a therapeutic effect on ob-structive pulmonary diseases such as asthma by causingboth bronchodilation and changing the balance of Th1/Th2toward increasing activity of Th1 and suppressing Th2.However, more experiments are needed to evaluate andconfirm the effect of C. sativus and its major constituents onTh1/Th2 balance. In addition, in further studies, the effect ofthis plant and its constituents should be examined on airwayinflammation in an animal model of asthma and on asth-matic patients.
In conclusion, the results of this study showed that saffronextract increased the ratio of IFN-c to IL-4, which indicatesan increased Th1/Th2 balance. Therefore the results suggestthat saffron may have therapeutic effects on inflammatorydisorders that are associated with increasing Th2 cytokineproduction, such as asthma.
ACKNOWLEDGMENTS
This study was financially supported by the ResearchDepartment of Mashhad University of Medical Sciences.The authors would also like to thank Dr. F. Hadizadeh, Dr.A. Emami, and Dr. Hassanzadeh Khiat for their help inHPLC measurement of safranal and crocin and Dr. HR.Sadeghnia for characterization of the extract by HPLC.
AUTHOR DISCLOSURE STATEMENT
No competing financial interests exist.
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