BACKGROUND
among peripheral blood mononuclear cells , there are monocytes, natural killer cells, b cells and t cells.
naïve cd4+ t cells can differentiate into several distinct subpopulations including th <dig>  th <dig> and th <dig> cells.
identification of these t cells subsets is based mainly on their immunological functions that are supported by the specific cytokines these different cells produce.
th <dig> cells are characterized by high secretion of ifn-γ and il- <dig>  these cells are responsible for intracellular pathogen elimination  <cit> ; but are also involved in the development of organ-specific autoimmune diseases, as well as chronic inflammatory disorders  <cit> .
th <dig> cells are implicated in humoral immunity and provide protection against parasites.
they also play a major role in the initiation, maintenance, and amplification of human allergic inflammation  <cit> .
th <dig> cells produce il- <dig>  il- <dig> and il- <dig>  which are involved in allergic asthma  <cit> .
th <dig> cells produce mainly il-17a and il-17f and take a major part in the clearance of extracellular bacteria and fungi, due to their capacity to recruit and activate neutrophils  <cit> .
in contrast, they promote the pathogenesis of cancer  <cit> , several autoimmune and inflammatory diseases  <cit> , such as multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, psoriasis and contact dermatitis  <cit> .
new compounds could have a great interest, in several pathophysiological situations such as autoimmunity  <cit>  and cancer  <cit> , if they are able to suppress the expression of pro-inflammatory cytokines such as il- <dig>  these molecules could be synthetic or natural, extracted from plants.
capparis spinosa l.  is an aromatic plant growing wild in dry regions around the mediterranean basin  <cit> .
different parts of this plant, including the flower buds, fruits, seeds, and roots, were traditionally used in folk medicine to treat rheumatism, digestive problems, headaches, and toothaches.
c.s.
was also used as a diuretic, antihypertensive and tonic  <cit> .
it has also been shown that c.s.
extracts possess several bioactivities such as antioxidant, antifungal, anti-hepatotoxic and anti-inflammatory actions  <cit> .
reports showed that c.s.
contains alkaloids, lipids, polyphenols, flavonoids, indole and aliphatic glucosinolates  <cit> ; which are known to possess bioactive properties.
specific t cell subsets could be stimulated to produce specific cytokines after their interaction with different natural or synthetic molecules and cytokines .
their specific cytokines initiate and orientate the immune response.
on this basis, they are divided into pro-inflammatory  and anti-inflammatory  cytokines.
here, we aimed to evaluate potential anti-inflammatory properties of c.s.
leaves on human peripheral blood mononuclear cells.
our data indicate that, doses of c.s.
extracts, which are not toxic and do not affect cell proliferation, are able to suppress pro-inflammatory cytokines such as il- <dig> and promote expression of anti-inflammatory cytokines such as il- <dig>  c.s.
may therefore contain compounds that could be used as anti-inflammatory in certain pathophysiological situations.
studies are in progress in order to identify these compounds.
RESULTS
cell viability and metabolic activity upon treatment with capparis spinosa
in order to test whether c.s.
preparations could contain compounds that have the ability to regulate immune system cells and eventually suppress pro-inflammatory cytokines, we started by assessing cell toxicity.
human pbmcs were incubated with increasing doses of either c.s.
crud extract or the corresponding aqueous fraction, in the presence or absence of phytohaemagglutinin .
our results showed that both c.s.
preparations did not induce cell toxicity, even when used at relatively high doses  .
this absence of toxicity was observed also when the pha was added .
interestingly, we noticed unexpected and significant increase in succinat deshydrogenase  enzymatic activity upon pha stimulation in the presence of the aqueous  but not the crud  extract of c.s.
this enhancement of the enzymatic activity of mitochondrial sdh may reflect an increase in proliferation and/or other cell function as reported in previous papers  <cit> .
this effect was more pronounced in the presence of the aqueous fraction relative to the crud extract  and was statistically significant .fig.
<dig> increasing doses of capparis spinosa aqueous fraction enhanced mtt reduction in human pbmcs.
human pbmcs were cultured for 4 days with or without pha  in the presence of increasing doses of the aqueous fraction or the crud extract of capparis spinosa, panel a: pbmcs without pha in the presence of different doses of the aqueous phase of c.s.
panel b: pbmcs stimulated with pha in the presence of different doses of the aqueous fraction of c.s.
panel c: pbmcs without pha in the presence of different doses of the crude extract of c.s.
panel d: pbmcs stimulated with pha in the presence of different doses of the crud extract of c.s.
three to five independent experiences were performed.
data were analyzed using the one-way anova test.
indicate a p value of less than  <dig> ;  <dig>  and  <dig>  respectively
cell proliferation upon treatment with capparis spinosa
since c.s.
aqueous fraction showed enhanced sdh enzymatic activity on pbmcs, we tested whether this could be related to an enhancement of cell proliferation.
to assess this hypothesis, we used cfse and flow cytometry.
cfse-labeled pbmcs were stimulated with pha in the presence or absence of different doses of c.s.
preparations  for 96 h. while cfse-labeled, pha-treated pbmcs showed marked proliferation compared to control cells , neither of the two extracts of c.s.
induced proliferation either alone  or in the presence of pha .fig.
<dig>
capparis spinosa crud extract and aqueous fraction did not affect proliferation of human pbmcs.
a cfse-stained pbmcs stimulated with pha 5 μg/ml.
b cfse-stained pbmcs in the absence of stimulation.
c, d, and e an example of curves generated by cfse-stained pbmc, stimulated with pha 5 μg/ml and harvested with  <dig>   <dig> and 500 μg/ml respectively of aqueous fraction, f, g and h an example of curves generated by cfse-stained pbmc harvested with of  <dig>   <dig> and 500 μg/ml of aqueous fraction, for 4 days.
i, j and k an example of curves generated by proliferation of cfse-stained pbmc harvested with  <dig>   <dig> and 500 μg/ml of crud extract for 4 days of culture.
l and m an example of curves generated by cfse-stained and stimulated pbmc in the presence of  <dig> and 100 μg/ml respectively of the crud extract, for 4 days.
n and o cumulative data from five independent experiments analyzed statistically using one-way anova test.
indicate a p value of less  <dig>  respectively.
each peak represents a cycle cell division.
the curves generated by the cfse profile were analyzed using the proliferation platform of the flowjo software.
data shown represent results of five independent experiments
qualitative phytochemical analysis and antioxidant activity of capparis spinosa
in order to identify potential bio-active chemical classes present in c.s.
extracts, we proceeded to a qualitative chemical screening.
as depicted in table  <dig>  we found that our extract contained: tannins, sterols, alkaloids, polyphenols and flavonoids.
subsequently, we quantified total polyphenols and total flavonoids in c.s.
extract and found about  <dig>  mg galic acid equivalent /g of dried extract; and about  <dig>  mg quercitin equivalent /g of dried extract , total flavonoid represents  <dig>  % of total polyphenolic compounds.table  <dig> phytochemical analysis of capparis spinosa’s crud extract and aqueous fraction
- = negative result, + = positive result
on the other hand, antioxidant activity was assessed using dpph radical scavenging test.
the 50 % inhibition concentration  of scavenging free radical effect was evaluated and found to be  <dig>  mg of c.s.
crud extract per ml.
this value was far lower than that obtained with ascorbic acid, used as positive control, known by its high antioxidant activity  .fig.
<dig>
capparis spinosa’s low anti-oxidant activity by dpph radical scavenging test
the effect of c.s.
on pro and anti-inflammatory cytokines was then evaluated using doses of c.s., which were found to be not toxic.
figure  <dig> shows the effect of c.s.
aqueous fraction on the expression of  <dig> cytokines after 18 h treatment.
data in panel a shows increases in il- <dig> gene expression in cells treated with the aqueous fraction of c.s.
this increase was, however, statistically significant with 100 μg/ml but not with 500 μg/ml of the aqueous fraction of c.s.
this observation suggests that c.s.
promotes induction of the il- <dig> known as anti-inflammatory th <dig> cytokine.
in panel b, we found a clear decrease in il- <dig> gene expression when pbmcs were treated with 500 μg/ml of c.s.
aqueous fraction.
this decrease was statistically significant.
in contrast, cells treated with 100 μg/ml did not show any significant effect on il- <dig> gene expression.
these data suggested that c.s.
acts as an anti-inflammatory factor.
indeed, c.s.
consistently promoted il- <dig>  and inhibited il- <dig> .
regarding il- <dig>  we did not detect any significant effect on pbmcs treated with either  <dig> or 500 μg/ml of the aqueous fraction of c.s.
.
in fact, we observed a tendency of decrease using 100 μg/ml, and a tendency of increase at 500 μg/ml of the aqueous fraction of c.s.
however, none of these effects were statistically significant .
tendencies of increases in tgf-β with both  <dig> and 500 μg/ml and decreases in tnf-α were detected.
however, these effects were not statistically significant .
we also tried to quantify the expression of ifn-γ in our experiments, but we could detect its expression in only one out of five tested donors; thus no conclusions could be drawn.
when pbmcs were stimulated with pha and incubated with c.s.
aqueous fraction , we did not detect any significant effect in c.s.- treated relative to control cells for any of the cytokines tested, il- <dig>  il- <dig>  il- <dig>  tgf-β and tnf-α.
altogether our data suggest that 100 μg/ml of c.s.
aqueous fraction is efficient and sufficient to affect cytokine gene expression, by stimulating the expression of anti-inflammatory cytokines namely il- <dig> and tgf-β and inhibiting the expression of pro-inflammatory cytokine tnf-α.
on the other hand, c.s.
aqueous fraction at 500 μg/ml showed a clear inhibition of another pro-inflammatory cytokine, il- <dig>  these observations indicate that the c.s.
aqueous fraction may contain compounds endowed with anti-inflammatory properties.fig.
<dig> effects of capparis spinosa’s aqueous fraction on il- <dig>  il- <dig>  il- <dig>  tgf-β and tnf-α expression in pbmcs in culture.
a il- <dig>  b il- <dig>  c il- <dig>  d tgf-β, e tnf − α. incubated for 18 h, doses used  <dig> and 500 μg/ml.
data represent the mean ± s.d.
data from n =  <dig> separate experiments.
data were analyzed using the one-way anova , flowed by dunn’s post test  indicate a p value of less than  <dig>
analysis of capparis spinosa extract by hplc
in order to identify several known polyphenol and flavonoid compounds present in our c.s.
extract, we have analyzed it by hplc at 285 nm, using a mixture of  <dig> standards, which are catechin, vanillic acid, caffeic acid, syringic acid, rutin, ferulic acid, and vanillin .
five compounds were identified from the extract by matching their retention times to those of the standards .
these compounds are catechin, caffeic acid, syringic acid, rutin and ferulic acid.
peak assignment was confirmed by injection of standards.fig.
<dig> identification of several polyphenol and flavinoid compounds in capparis spinosa extract.
a chromatogram of  <dig> available polyphenol and flavonoid standards monitored at 280 nm and identified with retention times  1: catechin, 2: vanillic acid, 3: caffeic acid, 4: syringic acid, 5: rutin, 6: ferulic acid, 7: vanillin.
b hplc chromatogram of of capparis spinosa extract obtained under optimum conditions: 88 % water and 12 % acetonitril, 50 min.
peak no.
1: catechin, 3: caffeic acid, 4: syringic acid, 5: rutin and 6: ferulic acid were identified
discussion
immunomodulation using medicinal plants  can provide alternatives to the conventional therapy for a variety of diseases  <cit> .
it could be effective, for instance, in cases of immune deficiency, autoimmunity or cancer to appropriately regulate the immune response and maintain a disease free state.
in this study, we aimed to decipher immunomodulatory effects of c.s.
leaf extracts on human peripheral blood mononuclear cells using c.s.
plant from morocco.
in order to test whether c.s.
could exhibit potential anti-inflammatory or other beneficial immunomodulatory effects, non toxic doses of c.s.
extracts were determined using mtt assay.
subsequently, these non toxic doses were used to evaluate the c.s.
effect on cell proliferation using cfse.
our data revealed that doses up to 800 μg/ml of c.s.
preparations were not toxic to human pbmcs .
unexpectedly, we detected an increase in sdh enzymatic activity using doses of the c.s.
aqueous fraction ranging from  <dig> to 600 μg/ml .
this suggested either enhanced proliferation and/or enhanced metabolic activity of cells upon treatment with c.s.
consistent with our data, similar range of doses from  <dig> to 600 μg/ml were also shown to be non cytotoxic when lyophilized extracts of italian c.s.
buds were used  <cit> .
it has been demonstrated in previous studies, that c.s.
extracts elicit a protective effect in pathological conditions related to the oxidative stress status  <cit> .
this effect is likely due to effective antioxidant/free radical scavenging properties  <cit> .
indeed, c.s.
was shown to be rich in polyphenols, which are known to be effective antioxidant/free radical scavenging compounds  <cit> .
therefore, we tested whether our extracts, obtained from the moroccan c.s.
leaves, could show similar protective effects.
in this aspect, we began with a qualitative screening that confirmed the presence of flavonoids and polyphenols.
then we quantified their presence, and found that our c.s.
extract is not as rich in polyphenols as other c.s.
extracts used in other studies and obtained from different region of the world  <cit> .
in fact, in our extract, total polyphenol content was  <dig>  mg gae/g of dried extract which is far inferior to tunisian and indian c.s.
<cit> .
comparing the total polyphenol content of our leaf extract to that of extracts from other parts of the plant , ours is much lower than italian c.s.
flower buds  <cit> , tunisian flower buds  <cit>  and lower than bahrainian  <cit>  and turkish c.s.
fruit  <cit> .
on the other hand, in our c.s.
extract, total flavonoid content was  <dig>  mg quercitin equivalent /g of dried leaf extract.
in comparison to other c.s.
that are considered as a rich source of flavonoids, our values appear to be lower than those of bahrainian c.s.
fruit  <cit>  and those of indian c.s.
<cit> .
these variations and differences in total phenol and flavonoid contents might be explained by geographical factors and by distinct processing methods.
we also compared the ic <dig> of the scavenging free radical test obtained from our study to those obtained in previous studies.
the ic <dig> of our c.s.
leaf extract was  <dig>  mg/ml of crud extract, which is similar to the bahrainian c.s.
fruit  <cit> , and far weaker than the italian c.s.
bud extracts   <cit> ;  <dig>  mg/ml  <cit>  and the turkish caper fruit extract   <cit> .
our c.s.
leaf extract appears also to be by far a weaker scavenging free radical agent relative to the indian c.s.
leaf extract   <cit> .
worth specifying that direct comparison of data from the current study with those reported in the literature is difficult since different parts of the plant and different expression units were used.
however, in light of these data, the low anti-oxidant activity of the moroccan c.s.
leaf crud extract might be due to low amounts of total polyphenolic and flavonoid compounds known as effective anti-oxidant molecules.
cytokines are key elements of the innate and adaptive immune response and provide a window through which diseases can be monitored and eventually controlled  <cit> .
so we focused on the effects of c.s.
extract on cytokine expression.
in our present study, we showed that the aqueous fraction of c.s.
leaf extract seems to have a modulator effect on il- <dig>  il- <dig> in pbmcs from healthy donors.
this consists of a stimulation of il- <dig> expression  and an inhibition of il- <dig> expression .
additionally, we detected a tendency of stimulating tgf-β expression in parallel with a trend of inhibiting tnf − α expression in pbmcs treated with c.s.
extract though these effects were not statistically significant in our experiments.
finally, we could not detect consistent expression of ifn-γ gene in human pbmcs even in the absence of treatment with c.s.
extract.
the induction of il- <dig> and the inhibition of il- <dig> by c.s.
are interesting since these are anti and pro-inflammatory cytokines, respectively.
il- <dig> is produced mainly by th <dig> cells  and is a primary driver of the th <dig> phenotype; it is involved in down regulation of inflammatory and th1-mediated responses  <cit> .
on the other hand, il- <dig> is the hallmark of th <dig> cells.
it is considered a major pro-inflammatory cytokine, with pivotal roles in inflammatory and autoimmune diseases including rheumatoid arthritis and multiple sclerosis .
these data suggest an overall anti-inflammatory effect of c.s.
on the immune response.
compounds from this plant could eventually play, in the future, an interesting role in modulating several pathologies in which il- <dig> plays an important task.
for instance, an involvement of il- <dig> was also strongly suggested in tumor development and in both inflammation and sporadic cancers of the liver, stomach, and colon  <cit> .
in the same time, the interesting trends observed in our work; increased tgf-β expression and decreased tnf-α expression in cells treated with c.s., are consistent with the modulatory effect observed on il- <dig> and il- <dig>  indeed, tgf-β is mostly assigned as an anti-inflammatory cytokine , which achieves distinct immunosuppressive properties on various aspects of the immune response and also maintain self tolerance  <cit> .
on the other hand, tnf-α is a major pro-inflammatory cytokine, which plays relevant roles in pathogenesis of many autoimmune diseases and stimulates secretion of other inflammatory cytokines  <cit> .
high levels of tnf-α are indeed associated with various acute and chronic inflammatory conditions  <cit> .
our data seem to be different from those published in another report  <cit>  in which c.s.
extract failed to induce il- <dig> production and rather up-regulated, pro-inflammatory cytokines  in human pbmcs.
these effects were attributed to the richness of italian c.s.
in polyphenols and flavonoids.
in our experiments, the tendency of inhibiting tnf-α expression might be due either to a direct effect of extract compounds on the signaling cascade leading to tnf-α expression or to il- <dig> induction also known to inhibit tnf-α  <cit> .
tgf-β, which is an important anti-inflammatory cytokine, known to antagonize tnf-α and ifn-γ, was also stimulated by c.s.
the overall inhibition of il- <dig> and of tnf-α in addition to the stimulation of il- <dig> and tgf-β implies that there might be an enhancement of anti-inflammatory cytokine in the account of pro-inflammatory cytokines.
hplc analysis of c.s.
extract showed the presence of various bioactive polyphenolic and flavonoid compounds, known for their anticancer potential, anti-inflammatory and immunomodulatory effects.
for instance, catechin is a flavonoid and a major compound of the catechines family, which were famous for their ability to control lymphocyte proliferation and modulate il- <dig> and ifn-γ secretion in human pbmcs and mouse splenocytes  <cit> .
data from a previous work showed that catechin exerts an inhibition of immune activation and a regulation of unbalanced levels of il-17/il- <dig>  <cit> .
it reversed abnormal polarization of th <dig> as well as treg cells in rat peripheral blood and spleen, by oral administration.
it suppressed chronic heart failure induced by abdominal aorta ligation, which was closely associated with catechin modulation of th <dig> and treg in rats.
with regard to caffeic acid, a derivative of cinnamic acids which are part of phenolic acids, is structurally related to flavonoids and is a biologically active component.
it has been found to possess anti-mitogenic, anti-inflammatory and immunomodulatory effects  <cit> .
caffeic acid inhibited colonic il- <dig> expression and increased il- <dig> expression  <cit> .
it also decreased il-1β production by 40 % and did not affect tnf-α and il- <dig> in human blood  <cit> .
as for syringic acid, which is from the same family as the caffeic acid, it was found to suppress significantly pro-inflammatory cytokines  in serum of mice suffering immune-mediated liver inflammation  <cit> .
ferulic acid was found to have a chemopreventive activity against colon and rectal cancer  <cit> .
as for the compound rutin, also known as vitamin p, it is a very important plant phenolic compound because of its anti-inflammatory property  <cit> ;  <cit> .
since it was found to be capable of suppressing increased il- <dig>  and enhancing decreased il- <dig> expressions, a characteristic of dextran sulfate sodium - induced colitis, and presented a partial protection against dss-induced colitis in mice  <cit> .
rutin also, ameliorated tnbs-induced colitis in rats by inhibiting tnf-α-induced nf-kb activation  <cit> .
some reports also showed that rutin was effective on the chronic phase of rat adjuvant arthritis  <cit> .
rutin was found to possess, in addition to anti-inflammatory potential, an anti-carcinogenic effect  <cit> .
CONCLUSIONS
the anti-inflammatory response obtained on human pbmcs with moroccan c.s.
preparations suggests a potential beneficial effect of compounds present in this plant in diseases, where these pro- and anti-inflammatory cytokines are involved such as multiple sclerosis, rheumatoid arthritis and cancer.
this anti-inflammatory effect might result from the direct effect of one or more of the identified compounds such as catechin or caffeic acid or from a synergistic interaction between different compounds present in our preparations, such as tannins, sterols, and alkaloids; or from a single bio-active molecule.
at this point, our main focus is the identification of the compound underlying this effect.

 among peripheral blood mononuclear cells, there are monocytes, natural killer cells, b cells and t cells. naïve cd4+ t cells can differentiate into several distinct subpopulations including th th and th cells. identification of these t cells subsets is based mainly on their immunological functions that are supported by the specific cytokines these different cells produce. th cells are characterized by high secretion of ifn-γ and il- these cells are responsible for intracellular pathogen elimination; but are also involved in the development of organ-specific autoimmune diseases, as well as chronic inflammatory disorders. th cells are implicated in humoral immunity and provide protection against parasites. they also play a major role in the initiation, maintenance, and amplification of human allergic inflammation. th cells produce il- il- and il- which are involved in allergic asthma. th cells produce mainly il-17a and il-17f and take a major part in the clearance of extracellular bacteria and fungi, due to their capacity to recruit and activate neutrophils. in contrast, they promote the pathogenesis of cancer, several autoimmune and inflammatory diseases, such as multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, psoriasis and contact dermatitis. new compounds could have a great interest, in several pathophysiological situations such as autoimmunity and cancer, if they are able to suppress the expression of pro-inflammatory cytokines such as il- these molecules could be synthetic or natural, extracted from plants. capparis spinosa l. is an aromatic plant growing wild in dry regions around the mediterranean basin. different parts of this plant, including the flower buds, fruits, seeds, and roots, were traditionally used in folk medicine to treat rheumatism, digestive problems, headaches, and toothaches. c.s. was also used as a diuretic, antihypertensive and tonic. it has also been shown that c.s. extracts possess several bioactivities such as antioxidant, antifungal, anti-hepatotoxic and anti-inflammatory actions. reports showed that c.s. contains alkaloids, lipids, polyphenols, flavonoids, indole and aliphatic glucosinolates; which are known to possess bioactive properties. specific t cell subsets could be stimulated to produce specific cytokines after their interaction with different natural or synthetic molecules and cytokines. their specific cytokines initiate and orientate the immune response. on this basis, they are divided into pro-inflammatory and anti-inflammatory cytokines. here, we aimed to evaluate potential anti-inflammatory properties of c.s. leaves on human peripheral blood mononuclear cells. our data indicate that, doses of c.s. extracts, which are not toxic and do not affect cell proliferation, are able to suppress pro-inflammatory cytokines such as il- and promote expression of anti-inflammatory cytokines such as il- c.s. may therefore contain compounds that could be used as anti-inflammatory in certain pathophysiological situations. studies are in progress in order to identify these compounds.  cell viability and metabolic activity upon treatment with capparis spinosa in order to test whether c.s. preparations could contain compounds that have the ability to regulate immune system cells and eventually suppress pro-inflammatory cytokines, we started by assessing cell toxicity. human pbmcs were incubated with increasing doses of either c.s. crud extract or the corresponding aqueous fraction, in the presence or absence of phytohaemagglutinin. our results showed that both c.s. preparations did not induce cell toxicity, even when used at relatively high doses. this absence of toxicity was observed also when the pha was added. interestingly, we noticed unexpected and significant increase in succinat deshydrogenase enzymatic activity upon pha stimulation in the presence of the aqueous but not the crud extract of c.s. this enhancement of the enzymatic activity of mitochondrial sdh may reflect an increase in proliferation and/or other cell function as reported in previous papers. this effect was more pronounced in the presence of the aqueous fraction relative to the crud extract and was statistically significant.fig. increasing doses of capparis spinosa aqueous fraction enhanced mtt reduction in human pbmcs. human pbmcs were cultured for 4 days with or without pha in the presence of increasing doses of the aqueous fraction or the crud extract of capparis spinosa, panel a: pbmcs without pha in the presence of different doses of the aqueous phase of c.s. panel b: pbmcs stimulated with pha in the presence of different doses of the aqueous fraction of c.s. panel c: pbmcs without pha in the presence of different doses of the crude extract of c.s. panel d: pbmcs stimulated with pha in the presence of different doses of the crud extract of c.s. three to five independent experiences were performed. data were analyzed using the one-way anova test. indicate a p value of less than; and respectively cell proliferation upon treatment with capparis spinosa since c.s. aqueous fraction showed enhanced sdh enzymatic activity on pbmcs, we tested whether this could be related to an enhancement of cell proliferation. to assess this hypothesis, we used cfse and flow cytometry. cfse-labeled pbmcs were stimulated with pha in the presence or absence of different doses of c.s. preparations for 96 h. while cfse-labeled, pha-treated pbmcs showed marked proliferation compared to control cells, neither of the two extracts of c.s. induced proliferation either alone or in the presence of pha.fig. capparis spinosa crud extract and aqueous fraction did not affect proliferation of human pbmcs. a cfse-stained pbmcs stimulated with pha 5 μg/ml. b cfse-stained pbmcs in the absence of stimulation. c, d, and e an example of curves generated by cfse-stained pbmc, stimulated with pha 5 μg/ml and harvested with and 500 μg/ml respectively of aqueous fraction, f, g and h an example of curves generated by cfse-stained pbmc harvested with of and 500 μg/ml of aqueous fraction, for 4 days. i, j and k an example of curves generated by proliferation of cfse-stained pbmc harvested with and 500 μg/ml of crud extract for 4 days of culture. l and m an example of curves generated by cfse-stained and stimulated pbmc in the presence of and 100 μg/ml respectively of the crud extract, for 4 days. n and o cumulative data from five independent experiments analyzed statistically using one-way anova test. indicate a p value of less respectively. each peak represents a cycle cell division. the curves generated by the cfse profile were analyzed using the proliferation platform of the flowjo software. data shown represent results of five independent experiments qualitative phytochemical analysis and antioxidant activity of capparis spinosa in order to identify potential bio-active chemical classes present in c.s. extracts, we proceeded to a qualitative chemical screening. as depicted in table  we found that our extract contained: tannins, sterols, alkaloids, polyphenols and flavonoids. subsequently, we quantified total polyphenols and total flavonoids in c.s. extract and found about  mg galic acid equivalent /g of dried extract; and about  mg quercitin equivalent /g of dried extract, total flavonoid represents  % of total polyphenolic compounds.table phytochemical analysis of capparis spinosa’s crud extract and aqueous fraction - = negative result, + = positive result on the other hand, antioxidant activity was assessed using dpph radical scavenging test. the 50 % inhibition concentration of scavenging free radical effect was evaluated and found to be  mg of c.s. crud extract per ml. this value was far lower than that obtained with ascorbic acid, used as positive control, known by its high antioxidant activity.fig. capparis spinosa’s low anti-oxidant activity by dpph radical scavenging test the effect of c.s. on pro and anti-inflammatory cytokines was then evaluated using doses of c.s., which were found to be not toxic. figure  shows the effect of c.s. aqueous fraction on the expression of cytokines after 18 h treatment. data in panel a shows increases in il- gene expression in cells treated with the aqueous fraction of c.s. this increase was, however, statistically significant with 100 μg/ml but not with 500 μg/ml of the aqueous fraction of c.s. this observation suggests that c.s. promotes induction of the il- known as anti-inflammatory th cytokine. in panel b, we found a clear decrease in il- gene expression when pbmcs were treated with 500 μg/ml of c.s. aqueous fraction. this decrease was statistically significant. in contrast, cells treated with 100 μg/ml did not show any significant effect on il- gene expression. these data suggested that c.s. acts as an anti-inflammatory factor. indeed, c.s. consistently promoted il- and inhibited il-. regarding il- we did not detect any significant effect on pbmcs treated with either or 500 μg/ml of the aqueous fraction of c.s.. in fact, we observed a tendency of decrease using 100 μg/ml, and a tendency of increase at 500 μg/ml of the aqueous fraction of c.s. however, none of these effects were statistically significant. tendencies of increases in tgf-β with both and 500 μg/ml and decreases in tnf-α were detected. however, these effects were not statistically significant. we also tried to quantify the expression of ifn-γ in our experiments, but we could detect its expression in only one out of five tested donors; thus no conclusions could be drawn. when pbmcs were stimulated with pha and incubated with c.s. aqueous fraction, we did not detect any significant effect in c.s.- treated relative to control cells for any of the cytokines tested, il- il- il- tgf-β and tnf-α. altogether our data suggest that 100 μg/ml of c.s. aqueous fraction is efficient and sufficient to affect cytokine gene expression, by stimulating the expression of anti-inflammatory cytokines namely il- and tgf-β and inhibiting the expression of pro-inflammatory cytokine tnf-α. on the other hand, c.s. aqueous fraction at 500 μg/ml showed a clear inhibition of another pro-inflammatory cytokine, il- these observations indicate that the c.s. aqueous fraction may contain compounds endowed with anti-inflammatory properties.fig. effects of capparis spinosa’s aqueous fraction on il- il- il- tgf-β and tnf-α expression in pbmcs in culture. a il- b il- c il- d tgf-β, e tnf − α. incubated for 18 h, doses used and 500 μg/ml. data represent the mean ± s.d. data from n =  separate experiments. data were analyzed using the one-way anova, flowed by dunn’s post test indicate a p value of less than analysis of capparis spinosa extract by hplc in order to identify several known polyphenol and flavonoid compounds present in our c.s. extract, we have analyzed it by hplc at 285 nm, using a mixture of standards, which are catechin, vanillic acid, caffeic acid, syringic acid, rutin, ferulic acid, and vanillin. five compounds were identified from the extract by matching their retention times to those of the standards. these compounds are catechin, caffeic acid, syringic acid, rutin and ferulic acid. peak assignment was confirmed by injection of standards.fig. identification of several polyphenol and flavinoid compounds in capparis spinosa extract. a chromatogram of available polyphenol and flavonoid standards monitored at 280 nm and identified with retention times 1: catechin, 2: vanillic acid, 3: caffeic acid, 4: syringic acid, 5: rutin, 6: ferulic acid, 7: vanillin. b hplc chromatogram of of capparis spinosa extract obtained under optimum conditions: 88 % water and 12 % acetonitril, 50 min. peak no. 1: catechin, 3: caffeic acid, 4: syringic acid, 5: rutin and 6: ferulic acid were identified discussion immunomodulation using medicinal plants can provide alternatives to the conventional therapy for a variety of diseases. it could be effective, for instance, in cases of immune deficiency, autoimmunity or cancer to appropriately regulate the immune response and maintain a disease free state. in this study, we aimed to decipher immunomodulatory effects of c.s. leaf extracts on human peripheral blood mononuclear cells using c.s. plant from morocco. in order to test whether c.s. could exhibit potential anti-inflammatory or other beneficial immunomodulatory effects, non toxic doses of c.s. extracts were determined using mtt assay. subsequently, these non toxic doses were used to evaluate the c.s. effect on cell proliferation using cfse. our data revealed that doses up to 800 μg/ml of c.s. preparations were not toxic to human pbmcs. unexpectedly, we detected an increase in sdh enzymatic activity using doses of the c.s. aqueous fraction ranging from to 600 μg/ml. this suggested either enhanced proliferation and/or enhanced metabolic activity of cells upon treatment with c.s. consistent with our data, similar range of doses from to 600 μg/ml were also shown to be non cytotoxic when lyophilized extracts of italian c.s. buds were used. it has been demonstrated in previous studies, that c.s. extracts elicit a protective effect in pathological conditions related to the oxidative stress status. this effect is likely due to effective antioxidant/free radical scavenging properties. indeed, c.s. was shown to be rich in polyphenols, which are known to be effective antioxidant/free radical scavenging compounds. therefore, we tested whether our extracts, obtained from the moroccan c.s. leaves, could show similar protective effects. in this aspect, we began with a qualitative screening that confirmed the presence of flavonoids and polyphenols. then we quantified their presence, and found that our c.s. extract is not as rich in polyphenols as other c.s. extracts used in other studies and obtained from different region of the world. in fact, in our extract, total polyphenol content was  mg gae/g of dried extract which is far inferior to tunisian and indian c.s.. comparing the total polyphenol content of our leaf extract to that of extracts from other parts of the plant, ours is much lower than italian c.s. flower buds, tunisian flower buds and lower than bahrainian and turkish c.s. fruit. on the other hand, in our c.s. extract, total flavonoid content was  mg quercitin equivalent /g of dried leaf extract. in comparison to other c.s. that are considered as a rich source of flavonoids, our values appear to be lower than those of bahrainian c.s. fruit and those of indian c.s.. these variations and differences in total phenol and flavonoid contents might be explained by geographical factors and by distinct processing methods. we also compared the ic of the scavenging free radical test obtained from our study to those obtained in previous studies. the ic of our c.s. leaf extract was  mg/ml of crud extract, which is similar to the bahrainian c.s. fruit, and far weaker than the italian c.s. bud extracts;  mg/ml and the turkish caper fruit extract. our c.s. leaf extract appears also to be by far a weaker scavenging free radical agent relative to the indian c.s. leaf extract. worth specifying that direct comparison of data from the current study with those reported in the literature is difficult since different parts of the plant and different expression units were used. however, in light of these data, the low anti-oxidant activity of the moroccan c.s. leaf crud extract might be due to low amounts of total polyphenolic and flavonoid compounds known as effective anti-oxidant molecules. cytokines are key elements of the innate and adaptive immune response and provide a window through which diseases can be monitored and eventually controlled. so we focused on the effects of c.s. extract on cytokine expression. in our present study, we showed that the aqueous fraction of c.s. leaf extract seems to have a modulator effect on il- il- in pbmcs from healthy donors. this consists of a stimulation of il- expression and an inhibition of il- expression. additionally, we detected a tendency of stimulating tgf-β expression in parallel with a trend of inhibiting tnf − α expression in pbmcs treated with c.s. extract though these effects were not statistically significant in our experiments. finally, we could not detect consistent expression of ifn-γ gene in human pbmcs even in the absence of treatment with c.s. extract. the induction of il- and the inhibition of il- by c.s. are interesting since these are anti and pro-inflammatory cytokines, respectively. il- is produced mainly by th cells and is a primary driver of the th phenotype; it is involved in down regulation of inflammatory and th1-mediated responses. on the other hand, il- is the hallmark of th cells. it is considered a major pro-inflammatory cytokine, with pivotal roles in inflammatory and autoimmune diseases including rheumatoid arthritis and multiple sclerosis. these data suggest an overall anti-inflammatory effect of c.s. on the immune response. compounds from this plant could eventually play, in the future, an interesting role in modulating several pathologies in which il- plays an important task. for instance, an involvement of il- was also strongly suggested in tumor development and in both inflammation and sporadic cancers of the liver, stomach, and colon. in the same time, the interesting trends observed in our work; increased tgf-β expression and decreased tnf-α expression in cells treated with c.s., are consistent with the modulatory effect observed on il- and il- indeed, tgf-β is mostly assigned as an anti-inflammatory cytokine, which achieves distinct immunosuppressive properties on various aspects of the immune response and also maintain self tolerance. on the other hand, tnf-α is a major pro-inflammatory cytokine, which plays relevant roles in pathogenesis of many autoimmune diseases and stimulates secretion of other inflammatory cytokines. high levels of tnf-α are indeed associated with various acute and chronic inflammatory conditions. our data seem to be different from those published in another report in which c.s. extract failed to induce il- production and rather up-regulated, pro-inflammatory cytokines in human pbmcs. these effects were attributed to the richness of italian c.s. in polyphenols and flavonoids. in our experiments, the tendency of inhibiting tnf-α expression might be due either to a direct effect of extract compounds on the signaling cascade leading to tnf-α expression or to il- induction also known to inhibit tnf-α. tgf-β, which is an important anti-inflammatory cytokine, known to antagonize tnf-α and ifn-γ, was also stimulated by c.s. the overall inhibition of il- and of tnf-α in addition to the stimulation of il- and tgf-β implies that there might be an enhancement of anti-inflammatory cytokine in the account of pro-inflammatory cytokines. hplc analysis of c.s. extract showed the presence of various bioactive polyphenolic and flavonoid compounds, known for their anticancer potential, anti-inflammatory and immunomodulatory effects. for instance, catechin is a flavonoid and a major compound of the catechines family, which were famous for their ability to control lymphocyte proliferation and modulate il- and ifn-γ secretion in human pbmcs and mouse splenocytes. data from a previous work showed that catechin exerts an inhibition of immune activation and a regulation of unbalanced levels of il-17/il-. it reversed abnormal polarization of th as well as treg cells in rat peripheral blood and spleen, by oral administration. it suppressed chronic heart failure induced by abdominal aorta ligation, which was closely associated with catechin modulation of th and treg in rats. with regard to caffeic acid, a derivative of cinnamic acids which are part of phenolic acids, is structurally related to flavonoids and is a biologically active component. it has been found to possess anti-mitogenic, anti-inflammatory and immunomodulatory effects. caffeic acid inhibited colonic il- expression and increased il- expression. it also decreased il-1β production by 40 % and did not affect tnf-α and il- in human blood. as for syringic acid, which is from the same family as the caffeic acid, it was found to suppress significantly pro-inflammatory cytokines in serum of mice suffering immune-mediated liver inflammation. ferulic acid was found to have a chemopreventive activity against colon and rectal cancer. as for the compound rutin, also known as vitamin p, it is a very important plant phenolic compound because of its anti-inflammatory property;. since it was found to be capable of suppressing increased il- and enhancing decreased il- expressions, a characteristic of dextran sulfate sodium - induced colitis, and presented a partial protection against dss-induced colitis in mice. rutin also, ameliorated tnbs-induced colitis in rats by inhibiting tnf-α-induced nf-kb activation. some reports also showed that rutin was effective on the chronic phase of rat adjuvant arthritis. rutin was found to possess, in addition to anti-inflammatory potential, an anti-carcinogenic effect.  the anti-inflammatory response obtained on human pbmcs with moroccan c.s. preparations suggests a potential beneficial effect of compounds present in this plant in diseases, where these pro- and anti-inflammatory cytokines are involved such as multiple sclerosis, rheumatoid arthritis and cancer. this anti-inflammatory effect might result from the direct effect of one or more of the identified compounds such as catechin or caffeic acid or from a synergistic interaction between different compounds present in our preparations, such as tannins, sterols, and alkaloids; or from a single bio-active molecule. at this point, our main focus is the identification of the compound underlying this effect. 
using mtt assay, we identified a range of capparis spinosa doses, which were not toxic. this capparis spinosa also appeared to act as a weaker scavenging free radical agent as evidenced by dpph radical scavenging test. in this work, we aimed to evaluate immunomodulatory properties of capparis spinosa leaf extracts in vitro on human peripheral blood mononuclear cells from healthy individuals. finally, polyphenolic compounds including catechin, caffeic acid, syringic acid, rutin and ferulic acid were identified by hplc, in the capparis spinosa preparation. interestingly, capparis spinosa aqueous fraction appeared to induce an overall anti-inflammatory response through significant inhibition of il- and induction of il- gene expression when pbmcs were treated with the non toxic doses of and/or 500 μg/ml. unexpectedly, we found out that capparis spinosa aqueous fraction exhibited an increase in cell metabolic activity, even though similar doses did not affect cell proliferation as shown by cfse. capparis spinosaperipheral blood mononuclear cellsanti-inflammationcytokinesgene expressioncell proliferationcell viabilitymoroccan ministry of researchmoroccan ministry laboratory fundingbadou abdallah issue-copyright-statement© the author 2016 capparis spinosa l. is an aromatic plant growing wild in dry regions around the mediterranean basin. capparis spinosa was shown to possess several properties such as antioxidant, antifungal, and anti-hepatotoxic actions. phytoscreening analysis of the used capparis spinosa preparations showed that these contain tannins; sterols, alkaloids; polyphenols and flavonoids. surprisingly, quantification assays showed that our capparis spinosa preparation contains low amounts of polyphenols relative to capparis spinosa used in other studies. altogether, these findings suggest that our capparis spinosa preparation contains interesting compounds, which could be used to suppress il- and to enhance il- gene expression in certain inflammatory situations.
 capparis spinosa l. is an aromatic plant growing wild in dry regions around the mediterranean basin. capparis spinosa was shown to possess several properties such as antioxidant, antifungal, and anti-hepatotoxic actions. in this work, we aimed to evaluate immunomodulatory properties of capparis spinosa leaf extracts in vitro on human peripheral blood mononuclear cells from healthy individuals.  using mtt assay, we identified a range of capparis spinosa doses, which were not toxic. unexpectedly, we found out that capparis spinosa aqueous fraction exhibited an increase in cell metabolic activity, even though similar doses did not affect cell proliferation as shown by cfse. interestingly, capparis spinosa aqueous fraction appeared to induce an overall anti-inflammatory response through significant inhibition of il- and induction of il- gene expression when pbmcs were treated with the non toxic doses of and/or 500 μg/ml. phytoscreening analysis of the used capparis spinosa preparations showed that these contain tannins; sterols, alkaloids; polyphenols and flavonoids. surprisingly, quantification assays showed that our capparis spinosa preparation contains low amounts of polyphenols relative to capparis spinosa used in other studies. this capparis spinosa also appeared to act as a weaker scavenging free radical agent as evidenced by dpph radical scavenging test. finally, polyphenolic compounds including catechin, caffeic acid, syringic acid, rutin and ferulic acid were identified by hplc, in the capparis spinosa preparation.  altogether, these findings suggest that our capparis spinosa preparation contains interesting compounds, which could be used to suppress il- and to enhance il- gene expression in certain inflammatory situations. other studies are underway in order to identify the compound underlying this effect. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords capparis spinosaperipheral blood mononuclear cellsanti-inflammationcytokinesgene expressioncell proliferationcell viabilitymoroccan ministry of researchmoroccan ministry laboratory fundingbadou abdallah issue-copyright-statement© the author 2016 