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Immunomodulation mediated by a herbal syrup containing a standardized Echinacea root extract: a pilot study in healthy human subjects on cytokine gene expression.

ABSTRACT

In this study, the immunomodulatory effect of a triply standardized Echinacea angustifolia root extract (Polinacea[R]) was evaluated in 10 healthy subjects. Ten ml of syrup containing one hundred mg of extract (corresponding to 4.7 mg of Echinacoside and 8.0 mg of a high molecular weight-20,000 Da- polysaccharide) were administered as a herbal syrup once a day for one month. The immunomodulatory effect was evaluated before and after herbal syrup administration evaluating the expression levels of the cytokines IL-2, IL-8, IL-6 and TNF-[alpha]. Cytokine expression was studied in lympho-monocytes and in plasma samples measuring the mRNA and protein levels, respectively. The results were analysed by ANOVA and non-parametric Friedman rank sum tests; when possible it was adopted a pair-wise comparisons at different post-treatment times, using the paired t-tests with Holm correction. The correlation between the variations of cytokine plasma levels and the respective mRNA was carried out using a linear regression model.

In lympho-monocytes our data indicate the up-regulation of the mRNA levels of IL-2 and IL-8 and the down regulation of the mRNA levels of the pro-inflammatory cytokines TNF-[alpha] and IL6. The differential regulation was maximal after 14 days of treatment. IL-2 up-regulation and IL-6 down-regulation were also confirmed at the protein level in plasma. Finally, the up-regulation of the mRNA of IL-2/IL-8 and the down-regulation of IL-6 positively correlated with the protein levels detected in the plasma.

In conclusion, this pilot study suggests a relevant role for the standardized Echinacea angustifolia root extract in the control of cytokine expression. This first demonstration of the immuno-modulating activity of Echinacea angustifolia root extract in the healthy subject, supports at least in part the common use of such products as health promoting supplement.

Keywords:

Echinacea angustifolia root standardized

extract (Polinacea[R])

Herbal syrup

Immune stimulating activity

Human gene expression

Cytokines

Introduction

Echinacea is one of the most used medicinal plants in herbal medicines and food supplements. The Native American Populations such as Cheyenne, Choctaw, Comanche, Crow, Dakota, Delaware, Pawnee, Hidatsa, Sioux and others used Echinacea, for the treatment of several diseases such as cold, flu, burns, wounds, snakes bites, general inflammatory condition, etc. (Morazzoni et al., 2005; Barnes et al., 2005; Barrett, 2003; Goel et al., 2004; Shah et al., 2007). Nowadays, Echinacea spp. are largely used mainly in supplements and herbal products for the prevention and treatment of common cold, flu and upper respiratory infections, as well as for the treatment of urinary infections (Wagner et al., 1999; Morazzoni et al. 2005; Barnes et al., 2005). Many published works have described both the chemistry and bioactivity in vitro and in vivo of these plants and some of their phytoconstituents (Bauer, 1999; Binns et al., 2002; Barnes et al., 2005; Woelkart et al., 2008). However, contrasting results about Echinacea efficacy (Schoop et al., 2006; Woelkart et al., 2008) have been reported. This is mainly attributable to the fact that the products under study were not comparable because they belonged to different Echinacea species or different commercial/botanical preparations or even different Echinacea components. An additional source of uncertainty derives from the fact that some authors have proven the biological Echinacea activity in in vitro studies (Randolph et al., 2003; Burger et al., 1997; Steinmuller et al., 1993), while others have conducted animal (Jia et al., 2009) or clinical studies (Guiotto et al., 2008; Woelkart et al., 2008; Di Pierro et al., 2012).

To provide a rational comparison among Echinacea products, in this work a standardized extract obtained from the roots of Echinacea angustifolia was administered to 10 healthy volunteers for a period of 1 month evaluating its immunomodulatory activity. This extract, whose immunomodulatory activity was proven both in vitro and in vivo in mice by Morazzoni et al. (2005) and in humans by Di Pierro et al. (2012), is a triply standardized extract from the roots of Echinacea angustifolia and is marketed with the name of Polinacea[R]. Polinacea[R], as it appears by HPLC analysis, is reported to be composed of [greater than or equal to] 4.0% of echinacoside, of a high molecular weight polysaccharide with ca. 20,000 Da, ([greater than or equal to] 5.0%) and of alkamides (isobutylamides) ([less than or equal to] 0.1%) (Di Pierro et al., 2012). In particular, this polysaccharide has a highly branched galacturonic structure and was highlighted for the first time in the root of Echinacea Angustifolia.

The use of such extract also meets the increasing demand of the phytotherapeutical industry to develop standardized extracts, to offer to the market a product with a constant and certified quality.

The present investigation is a pilot scale human clinical trial involving 10 healthy subjects orally dosed once a day for one month with a herbal syrup containing the above mentioned standardized extract. As immunomodulation markers a panel of 4 cytokines IL-2,1L-8, IL-6 and TNF-[alpha], demonstrating good responsiveness in initial screen, was considered. Their expression was studied in lympho-monocytes and plasma samples measuring the mRNA and protein levels, respectively, before and after the administration of the herbal syrup.

[FIGURE 1 OMITTED]

Materials and methods

Human pilot trial methodology

The in vivo study was conducted at the University of Trieste, Italy. The protocol, approved by the Human Research Ethics Committee of the University of Trieste, was conducted in compliance with the Declaration of Helsinki and the International Conference on Harmonization Guidelines. A number N = 10 of healthy volunteers, non-smoking, normally active of both genders (5 male and 5 female) between 26 and 53 years of age participated in the trial. All the volunteers were with normal liver function and without diagnosed allergy or sensitivity to Compositae or Crossolariaceae plants. None of the volunteers was on a special diet. No medicines were taken during the study (wash out period: 1 week before the beginning of the present study). All subjects gave their written consent to participate in this study.

On the first 5 weeks (from Day -35 to Day -1 Fig. 1) a fasting baseline blood draw was taken. On the following 4 weeks (from day 0 to day 28), 10 ml syrup was taken between meals. Ten ml syrup (corresponding to the maximal daily dose) containing 100 mg of Polinacea[R] root dry extract (Indena, Milano, Italy). The dry extract consists of; 4% of echinacoside, 12.7% of high molecular weight polysaccharide. HPLC analysis of polysaccharide and echinacoside content were performed and chromatograms are reported in supplementary materials with other analytical details. Results showed that syrup contains 4.7 mg/10 ml of echinacoside and 8.0 mg/10 ml of high molecular weight polysaccharides (expressed as dextran MW 12,000).

Blood samples (2x5 ml) collected in EDTA-tubes were taken once a week on days -35, -28, -21, -14, -7, 0, 7, 14, 21, 28 (for a total of 10 blood sampling). After sample withdrawal, one ml of plasma was immediately separated by centrifugation and frozen at -80[degrees]C for subsequent cytokine analysis assay. Within 4 h after blood withdrawal, lympho-monocytes were isolated by Ficoll-Paque[TM] method (Boyum, 1968) from 5 ml of complete blood.

Quantitative PCR

Total RNA was extracted, quantified and the quality evaluated as described (Baiz et al., 2014; Zanetti et al., 2008). Reverse transcription was performed using 500 ng of total RNA in the presence of random examers and MuLv reverse transcriptase (Applera Corporation, USA). To detect the mRNA levels of the cytokines IL-2, IL-8, IL-6 and TNF-[alpha], Real-Time polymerase chain reaction was performed as described (Baiz et al., 2014; Zanetti et al., 2008). The primers (MWG Biotech, GA, USA, 300 nM) and the annealing temperatures used are reported in Table 1. The relative amounts of each target mRNA were normalized by 28S rRNA content according to Pfaffl (2004).

Cytokine analysis

The protein levels of cytokines IL-2, IL-8, IL-6 and TNF-[alpha] were measured by flow cytometry, using commercially available CBA Inflammation kit from BD Biosciences (San Jose, CA, USA) according to the manufacturer's instructions. Flow cytometry analysis was performed using a FACScan flow cytometer from Becton Dickinson (Franklin Lakes, NJ, USA); data analysis was performed with the CBA software. A total of 3000 events were analysed for each sample.

Statistical analysis

The results were statistically analysed by ANOVA and nonparametric Friedman rank sum test, also conducting, when possible, pair-wise comparisons at different post-treatment times, using paired t-tests with Holm correction. The correlation between the variation of the plasmatic cytokine values and the pertinent mRNA was carried out using a linear regression model. Data are reported as mean [+ or -] SD.

Results

Cytokine mRNA level evaluation in lympho-monocytes

The effect of formulated product on the cytokine expression in circulating lymphocytes was firstly evaluated at the mRNA level. For each mRNA (out of 4) the control value was defined as the mean of all the pertinent 6 x 10 "blank" data, and, for each mRNA (out of 4) and each time t (out of 10, from t= -35 to t= 28, step size = 7) the mean m(t) and the s.d. (over the 10 volunteers) of the differences to the assumed control value have been computed. Fig. 1 shows the control value as dotted horizontal line, drawn at T=0; the mean differences m(t) at times t are joined with line segments, while the vertical bars represent [+ or -] S.D.

A first visual inspection clearly shows that the treatment induces an increase of IL-2 and IL-8, while it induces a decrease of IL 6 and TNF-[alpha] mRNAs. The standard statistical tool to test this hypothesis is the one-way ANOVA with repeated measures (within subjects), taking time as a 5-level factor, more precisely, the pertinent control level and the levels measured 7, 14, 21 and 28 days after starting the drug treatment. The ansatz that the theoretical assumptions of ANOVA (normality, etc.) are satisfied was assumed. For the sake of safeness and for comparison, also the non-parametric version of ANOVA with repeated measures, i.e., the Friedman rank sum test, was run; this additional analysis gave results coherent with those of ANOVA at the classical significance level of [alpha] = 0.05.

Table 2 reports both the p-values of the F-distribution for ANOVA and for the Friedman rank sum test for cytokine mRNA levels, obtained using the standard statistical package R (R Development Core Team, 2010). These results show that the treatment has a significant effect on the mRNA levels of the cytokines considered. The only partial exception is represented by IL-6, being the variation in the mRNA level significant for ANOVA analysis, but just borderline for the more conservative Friedman test.

A post-hoc test is needed after ANOVA test in order to determine which groups differ from each other. In particular it could be of interest to find at what time (selected from 7,14,21 and 28 days of treatment) the difference with respect to the control are more significant, or when the time effect is more important. Unfortunately, a post-hoc test as the Tuckey HSD (honestly significant difference) is not available in the R package for repeated measures; thus a pairwise comparison using paired f-tests with the Bonferroni-Holm correction, or, shortly, Holm (1979) p-value adjustment for multiple comparisons was chosen. Table 3 reports the pertinent results, showing that the highest modification in the expression levels of the considered cytokines occurred at t= 14, that means two weeks after the beginning of syrup dosing.

Worth of note, among the data reported in Table 3, is the marked up regulation of IL-2.1L-2, primarily produced by activated CD4+ T cells and by naive CD8+ T cells and DCs (Nelson, 2004), is a growth factor for antigen-stimulated T lymphocytes and is responsible for T-cell clonal expansion after antigen recognition in adaptive immunity. This suggests that, by up-regulating IL-2, the active constituents of the syrup, namely Polinacea[R] may promote lymphocyte proliferation, consistently with the data obtained by Morazzoni et al. (2005). Additionally, IL-2 up-regulation may contribute to explain the findings obtained by Di Pierro et al. (2012), who observed a significant increase of the lymphocyte percentage following Polinacea[R] administration in patients previously treated with influenza vaccine. In our case serum chemistry and hematological values for each subject did not significantly differ from baseline during the observation period (data not shown).

Also the mRNA of CXCL8 (formerly known as interleukin-8) was significantly up-regulated by Polinacea[R]. IL-8 is a strong chemoactractant and an angiogenic factor produced/secreted by a variety of cell types including monocytes, lymphocytes, granulocytes, fibroblasts, endothelial, and epithelial cells. Additionally, T cells may directly orchestrate neutrophilic activation by releasing CXCL8 which is a neutrophil-attracting chemokine CXCL8 (Britschgi and Pichler, 2002). The IL-8 up-regulation observed here did not confirm what observed by Randolph et al. (2003), who demonstrated a modest decrease of gene expression of IL-8 in human lymphocytes. However, these latter data were referred to an oral administration in tablets of a commercial blended Echinacea product whose qualitative and quantitative composition was not reported. Despite this discrepancy, our data suggests for Echinacea syrup a role in the activation of the immune-system favoring lymphocyte proliferation and neutrophil activation, possibly via the up-regulation of the mRNA levels IL-2 and IL-8, respectively.

The data presented in Table 3 also show that the syrup induces the down-regulation of the mRNA levels of the pro-inflammatory cytokines IL-6 and TNF-[alpha]. These are two of the most important mediators of fever and of the acute phase response, responsible for the elevation of the classical inflammation marker C-reactive protein. Elevated IL-6 concentrations are associated with several inflammatory diseases and malignancies (Rincon, 2012) and TNF-[alpha] has been recognized to have a central pathogenic role in many immune-inflammatory diseases. The fact that these two cytokines are down regulated at the mRNA level by Polinacea[R] may contribute to explain a mechanism by which Polinacea[R] exerts its anti-inflammatory effects. Finally, the fact that TNF-[alpha] regulates the expression of IL-6 (Bhatnagar et al., 2010) opens the possibility that Polinacea[R] induces the decrease of IL-6 mRNA via the down regulation of TNF-[alpha].

Cytokine protein level evaluation in plasma

The second part of the research encompassed the monitoring of cytokine protein levels in plasma. For each examined cytokine the control value has been defined as the mean value (over the volunteers) at time t=0. The mean (over the volunteers) differences of the measured data to the above defined baseline have been investigated--in the same way as the mRNA case--employing the one-way ANOVA with repeated measures (within subjects), taking the time as a 5-level factor: t = 0, 7, 14, 21, 28 (taking t = 0 as the control level), and with the non-parametric Friedman rank sum test. The ANOVA and non-parametric Friedman rank sum for cytokine analysis test showed that the drug treatment had a very significant effect on the plasma levels of IL-2 and IL-6, while the tests gave borderline p-values for IL-8, and n.s. for TNF-[alpha]. These results only partially confirmed the mRNA data (Table 4). This outcome is however not completely surprising for different reasons. First, the quantification of the cytokine mRNA levels was evaluated in the lympho-monocyte population only. In contrast, the quantification of the protein levels was performed in the plasma, thus reflecting cytokine production by many more different cell types. Second, the protein plasma levels of the cytokines also depends on their clearance from the blood, a fact depending by many variables. Third, the detection systems used, quantitative real time PCR and flow cytometry for the quantification of mRNAs and proteins, respectively, may not necessarily have the same sensibility. Despite the above considerations, the decrease of IL-6 both at the protein and mRNA levels, strengthens the concept that the anti-inflammatory effect of Polinacea[R] may occur via IL-6 down regulation.

Finally, since the post-hoc pair-wise comparisons using paired t-tests with Holm correction for the plasma data at times t=0,7,14, 21,28 (where t = 0 is the control level) did not give a clean output, the results have been omitted in the present paper.

Correlation between gene expression and plasma cytokines results

In spite of the complexity of the dynamics regulating the protein levels of the considered cytokines in plasma, a very basic correlation study was carried out between protein and mRNA levels. For this purpose, we used a linear regression model in which the variation of the plasmatic values of the considered cytokines is assumed linearly proportional to the variation of the respective mRNA. The observed regression lines of plasma cytokines were computed to the mRNAs, taking into account the mean values at time t=0, ..., 28. Fig. 2 reports on the X-axis the variation of the mRNA levels (with respect to its baseline), and on the Y-axis the variation of the plasma levels measured at same time points. The variable appeared positively correlated for the IL 2,6 and 8, but not for TNF-[alpha] for which the correlation resulted to be non-significant. Thus this analysis indicates that the up-regulation of the mRNA of IL-2/IL-8 and the down-regulation of IL-6, is positively correlated with the protein levels detected in the plasma.

[FIGURE 2 OMITTED]

Conclusions

In this pilot study, 10 human volunteers have been treated for four weeks with a herbal syrup product containing 100 mg of Polinacea[R] (corresponding to 4.7 mg of Echinacoside and 8.0 mg of high molecular weight polysaccharide). Limitations of this study include the small size of the sample population and the lack of comparison with other Echinacea extracts with different composition. Despite this, the presented data suggests possible mechanisms by which the formulated syrup influences the immune system functions, in particular via the down regulation of IL-6 expression. Additionally this study demonstrates the immuno-modulating activity of Polinacea[R] in the healthy subject, supporting at least in part its use as health promoting supplement. Future perspectives include a systematic study in a larger trial, the inclusion of cold affected individuals and the effects of seasonality.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at http://dx.doi.Org/10.1016/j.phymed. 2014.04.034.

Conflicts of Interest

The authors declare absence of conflict of interest.

ARTICLE INFO

Article history:

Received 25 February 2014

Received in revised form 3 March 2014

Accepted 20 April 2014

Acknowledgments

The authors wish to thank Alessandro Portolan, Federica Pasqualetto and Marco Riva--Unifarco S.P.A. (Santa Giustina, Italy) for the active cooperation in the project "Valutazione farmacocinetica di integratori alimentari--Immunofluid". B. Dapas is supported by the Italian Minister of Instruction, University and Research (MIUR), PRIN 2010, number 20109PLMH2.

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B. Dapas (a), S. Dall'Acqua (b), R. Bulla (c), C. Agostinis (c, d), B. Perissutti (e), S. Invernizzi (c), G. Grassi (a), D. Voinovich (e),*

(a) Department of Life Sciences, Cattinara Hospital, University of Trieste. Strada di Fiume 447, Trieste, Italy

(b) Department of Pharmaceutical Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova, Italy

(c) Department of Life Sciences, University of Trieste, Via Valerio 28, 34127 Trieste, Italy

(d) Institute for Maternal and Child Health lRCCSBurlo Carofolo, Via dellTstria 65/1, 34100 Trieste, Italy

(e) Department of Chemical and Pharmaceutical Sciences, University of Trieste, P.le Europa 1, 34127 Trieste, Italy

* Corresponding author. Tel.: +39 0405583106; fax: +39 04052572.

E-mail address: vojnovic@units.it (D. Voinovich).

http://dx.doi.org/10.1016/j.phymed.2014.04.034
Table 1
Primer sequences (probes) used in the realtime PCR experiments.

GeneBank
number      Protein   Primer pair

NM 000586   IL2       (F) 5'-nTGAATGGAA1TAATAATTACAAGAATCC-3'
                      (R) 5'-TTCTAGACACTGAAGATGTTTCAGTTC-3'
NM.000594   TNF-      (F) 5'-CrrCTTCCTCAGCaCrTCT-3'
            [alpha]   (R) 5'-AAAGTGCAGCAGGCAGAAGA-3'
NM_000584   IL8       (F) 5'-TTCCTGAnTCTGCAGCTCT-3'
                      (R) 5'-TGTCTTTATGCACTGACATC-3'
NM.000572   IL10      (F) 5'-GATTTCTrCCCTGTGAAAAC-3'
                      (R) 5'-TTGTAGATGCCiTTCTCTTG-3'
M54894      IL6       (F) 5'-GCTGAAAAAGATGGATGCTTC-3'
                      (R) 5'-ACTCCAAAAGACCAGTGATG-3'
M11167      28S       (F) 5'-TGGGAATGCAGCCCAAAG-3'
                      (R) 5'-CCTTACGGTACTTCTTGACTATGC-3'

GeneBank    Tm             Amplification    Length
number      ([degrees]C)   region           (bp)

NM 000586   60             186-295          109

NM.000594   60             265-334          69

NM_000584   60             138-218          80

NM.000572   60             442-526          84

M54894      60             336-414          78

M11167

Table 2
Results of ANOVA and non-parametric Friedman rank
sum test for gene expression.

mRNA           ANOVA        Friedman

IL-2           <0.001 ***   <0.001 ***
IL-6            0.014 *      0.09
IL-8           <0.00 ***    <0.001 ***
TNF-[alpha]    <0.001 ***   <0.001 ***

Significance codes ([alpha]):

* p = 0.05.

*** p = 0.001.

Table 3
Pairwise comparisons at different post-treatment
times, using paired t-tests with Holm correction.

Cytokine   day   mRNA

                 Control      7          14           21

           7     0.002 **
IL-2       14    <0.001 ***   0.74
           21    0.04 *       0.45       0.03 *
           28    0.37         0.33       0.02 *       0.37

           7     1
IL-6       14    0.19         0.43
           21    0.75         1          0.02 *
           28    1            1          0.19         0.83
           7     <0.001 ***
IL-8       14    <0.001 ***   0.12
           21    0.02 *       0.002 **   <0.001 ***
           28    0.12         0.006 **   0.001 **     0.34
           7     0.02 *
TNF-       14    0.002 **     0.79
[alpha]    21    0.02 *       0.79       0.05'
           28    0.58         0.21       0.02'        0.79

Significance codes (a):

* p = 0.05.

** p = 0.01.

*** p = 0.001.

Table 4
Results of ANOVA and non-parametric Friedman rank
sum for cytokine production.

Cytokine   ANOVA       Friedman

IL-2       0.005 **    0.005 **
IL-6       0.007 **    <0.001 ***
IL-8       0.08        0.08
TNF-       0.57 n.s.   0.58 n.s.
[alpha]

Significance codes ([alpha]):

* p = 0.01.

*** = 0.001.
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Author:Dapas, B.; Dall'Acqua, S.; Bulla, R.; Agostinis, C.; Perissutti, B.; Invernizzi, S.; Grassi, G.; Voi
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Sep 25, 2014
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