Evidence-Based Complementary and Alternative Medicine

Evidence-Based Complementary and Alternative Medicine / 2013 / Article
Special Issue

Natural Products for the Treatment of Obesity, Metabolic Syndrome, and Type 2 Diabetes

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Review Article | Open Access

Volume 2013 |Article ID 782461 | 10 pages | https://doi.org/10.1155/2013/782461

Metabolic Syndrome and Inflammation: A Critical Review of In Vitro and Clinical Approaches for Benefit Assessment of Plant Food Supplements

Academic Editor: Ravirajsinh N. Jadeja
Received13 Dec 2012
Revised21 Jan 2013
Accepted21 Jan 2013
Published27 Feb 2013


Metabolic syndrome is defined as the clustering in an individual of several metabolic abnormalities associated with insulin resistance, type 2 diabetes, and obesity, in which low-grade chronic inflammatory activity is commonly observed. Part of the European Project PlantLIBRA is concerned with methods to assess the benefits of plant food supplements (PFSs) in countering inflammatory activity and metabolic syndrome. This paper summarizes the current methods used for benefit assessment of PFS, taking into consideration only in vitro, in silico, and clinical methodologies used to investigate the anti-inflammatory properties of plants. No in silico studies (using computer simulation) related to metabolic syndrome were found; these methods appear to be used exclusively for identifying or testing potentially effective compounds in drug development. Most in vitro methods for the assessment of beneficial effects of botanicals or plant food supplements in diabetes were based on a quantitative polymerase chain reaction (PCR), whereas the preferred kind of clinical study was the double-blind randomized controlled clinical trial. Only two parameters were observed to change after treatment with botanicals in both in vitro and in vivo studies: interleukin-6 and tumour necrosis factor-α, and these biomarkers should be carefully considered in future studies for PFS benefit assessment.

1. Introduction

Metabolic syndrome (MS) defines the clustering in an individual of multiple metabolic abnormalities [1]. World Health Organization and programs including the National Cholesterol Education Program (NCEP) and Adult Treatment Program III (ATP III) have now agreed [2, 3] to consider MS as a disease characterized by five traits: (1) increased abdominal girth, (2) low levels of high-density lipoprotein cholesterol (HDL-C), (3) hypertriglyceridemia, (4) hypertension, and (5) fasting hyperglycemia. A low-grade chronic inflammatory activity is commonly observed in metabolic diseases such as obesity and type 2 diabetes (T2D).

A major shortcoming of current definitions of MS is the lack of inclusion of measures of a proinflammatory state and oxidative stress [4, 5]. The multicenter Insulin Resistance Atherosclerosis Study had shown a linear relation between the inflammatory marker C-reactive protein (CRP) and a number of metabolic disorders [6]. Other proinflammatory markers known to increase in patients with MS include fibrinogen [2], cytokines such as interleukin-6 (IL-6), and tumour necrosis factor- (TNF- ).

Type 2 diabetes (T2D) is considered an MS-related disease and an inflammatory disease. As with MS, patients with T2D show higher levels of circulating CRP, fibrinogen, plasminogen activator inhibitor (PAI), and proinflammatory cytokines such as interleukin-1 (IL-1 ) and IL-6. Circulating levels of IL-18 have been reported to be elevated in subjects with the metabolic syndrome and closely associated with the biomarkers of the syndrome to predict cardiovascular events and mortality in populations affected by MS-related diseases [7]. In patients with T2D, metabolic stress promotes insulin resistance and activation of IkB kinase- (IKK ) and JUN N-terminal kinase (JNK), which suggests that these kinases have key roles in the pathogenesis of this disease [8]. IKK activates nuclear factor- B (NF- B) which induces expression of NF- B-dependent genes such as proinflammatory cytokines (e.g., TNF and IL-1 ), so that the suppression of this transcription factor could reduce metabolic disorders and the complications occurring in diabetes (retinopathy, nephropathy, and neuropathy) [8].

In the last 10 years, the link between inflammation and nutrition has become increasingly apparent [810]. It has been shown that excessive macronutrient intake can contribute to the inflammatory response occurring in MS [11], whereas some dietary polyphenols are able to reduce the incidence of MS, including diabetes [12]. Many of the metabolites occurring in plants are now recognized as useful for the maintenance of human health, hence the recommended use of plant food supplements (PFSs). A methodology for the safety assessment of botanicals has been promoted by EFSA (European Food Safety Authority), but major bottlenecks remain in its implementation.

The European Project PlantLIBRA (acronym for plant food supplements: levels of intake, benefit, and risk assessments) aims to promote the safe use of PFS or botanicals and the measurement of the risk/benefit ratio related to their consumption. Part of the project is devoted to the discovery of methods for the evaluation of the benefits of PFS and their application and validation. The first step was to review the evidence for PFS benefit in epidemiological, clinical, and intervention studies, in particular the value of PFS as anti-inflammatory agents [13, 14].

The aim of the present paper was to identify the in vitro and in vivo methods that can detect a decrease of the inflammatory biomarkers that play a key role in metabolic syndrome and diabetes.

2. Methods

2.1. Source and Search Strategy

The following databases were searched electronically to identify relevant articles published up to September 2011: PubMed/Medline, SciFinder Scholar, and Cochrane Library. Search limits were in vitro, in silico, clinical methodologies, and the European languages, without limits of year of publication.

A search strategy was developed for each electronic database using specific medical subject heading (MeSH) terms (e.g., inflammation mediators, C reactive protein CRP, and metabolic X syndrome) in addition to relevant text keywords (plant extract, plant preparation, methods, and analytical approaches).

The same MeSH terms were used in the T2D area and in the MS area to search for inflammatory biomarkers and plant extracts, and the specific terms diabetes mellitus, noninsulin-dependent OR diabetes mellitus, and type II were combined with relevant keywords (plant extract, plant preparation, methods, and analytical approaches).

Titles and abstracts of retrieved citations were first screened to identify publications reporting in vivo methods developed in humans, in vitro and in silico methods used in inflammation conditions related to MS or diabetes. Animal studies were not considered since PlantLIBRA neither uses nor promotes in vivo experiments on animals. Other exclusion criteria were the use of plant ingredients for homeopathy, topical use, aerosol/inhalation, and hygiene products. Reviews, commentaries, and patents were also discarded.

3. Results and Discussion

The search by title and abstract retrieved 46 papers for MS and 68 for diabetes. After removal of duplicates and application of the inclusion/exclusion criteria, the total number of papers was 43. Papers were also rejected if they were not in a European language. In silico methods for assessing PFS benefit in MS-related diseases were not found. All the studies selected for diabetes were related to T2D.

3.1. Metabolic Syndrome Studies

Neither in silico nor in vitro studies for assessing inflammation in metabolic syndrome were found. Although diabetes is one of the features of MS, the methods related to diabetes have been considered separately from those relating to MS, because of the complexity of the metabolic syndrome, which includes several alterations of metabolic conditions not specifically associated with diabetes.

3.1.1. In Vivo Methods

Table 1 reports the in vivo methods used in clinical trials. The preferred type of clinical study to evaluate the anti-inflammatory effect of PFS in humans was the double-blind randomized controlled clinical trial.

Method1Botanical or botanical derivatives usedNo. of participants and length of treatmentParameters measuredResultsReference

Double-blind randomized controlled cross-over trialOlive oil (Olea europaea L.)20 (6-week washout period plus 4-h study session)Expression of inflammatory genes (CCL3, CXCL1, CXCL3, CXCR4, IL-1β, IL-6, and OSMExpression of inflammatory genes was reducedCamargo et al., 2010 [15]
Randomized double-blind controlled trialsProAlgaZyme (freshwater algae infusion)
60 (10 weeks)hs-CRP, IL-6, and TNF-αSignificant reduction of all parametersOben et al., 2007 [16]
Single-blind randomized controlled trialPlant sterol margarines (30 g/day) 53 (5 weeks)CRP, IL-6, CD 40 ligand (CD40L), and E-selectinNo changesGagliardi et al., 2010 [17]
Randomized open controlled cross-over trialsSoy nut/soy proteins482 (8 weeks)Serum endothelin-1, sICAM-1, sVCAM-1, E-selectin, IL-2, IL-6, IL-18, TNF-α, SAA, and CRPReduction of CRP for soy protein, E-selectin, TNF-α, IL-18, and CRP for soy nutsAzadbakht et al., 2007 [18]
Randomized open controlled clinical trialsBerries and derivatives*61 (20 weeks)TNF-α, ICAM, VCAM, E-selectin, and hs-CRPNo significant differences Lehtonen et al., 2010 [19]
Open nonrandomized noncontrolled clinical trialsFreeze-dried strawberry extract 35 (4 weeks)Malondialdehyde (MDA), hs-CRP, and adiponectinNo significant differencesBasu et al., 2009 [20]
Cross-sectional studiesFruit food group** 
Vegetable fruit group***
486 (1 year)CRPReduction of CRP for both groupsEsmaillzadeh et al., 2006 [21]

*Participants consumed lingonberry, bilberry, black currant, sea buckthorn as such or as derivatives (juice, oil, and powder).
**Pears, apricots, cherries, apples, raisins or grapes, bananas, cantaloupe, watermelon, oranges, grapefruit, kiwi, strawberries, peaches, nectarines, tangerines, mulberry, plums, persimmons, pomegranates, lemons, pineapples, fresh figs, and date.
***Vegetable fruit group: cabbage, cauliflower, Brussels sprouts, kale, carrots, tomatoes, spinach, lettuce, cucumber, mixed vegetables, eggplant, celery, green peas, green beans, green pepper, turnip, corn, squash, mushrooms, and onions.
1Clinical studies used to assess inflammatory parameters are listed from the best to the worst method applied.

Several publications reported in the present review used randomization in clinical trials, but in a few cases, randomization was not described in detail or incompletely applied (e.g., no randomization for age or gender). Positive aspects of randomization include the elimination of biases, balanced arms, and the capacity to form the basis for statistical tests.

A method of randomization should be considered appropriate if it allows each study participant to have the same chance of receiving the intervention [22]. Methods of allocation using the date of birth or of admission, hospital numbers, or alternation are not appropriate. Suitable methods for randomization include using a table of random numbers or computer generation. In most studies, the subjects enrolled included people of both sexes. Studies of only male or female subjects do not reflect the whole population and the results are not reliable. Almost all studies reported the number of dropouts, but it was not always clear if this was due to lack of efficacy or due to adverse effects. The analytical method used to quantify inflammatory markers such as CRP, cytokines, malondialdehyde (MDA), and adhesion molecules was reported in most studies. In several cases, methods were well described, and manufacturers’ protocols were appropriate. The methods most frequently applied were the ELISA test, immunoturbidimetry, and real-time PCR.

3.1.2. Inflammatory Biomarkers Affected by PFS in Metabolic Syndrome

The inflammatory parameters decreasing after PFS treatment were also reviewed. Although changes after PFS treatment depend on the plant used and on the bioavailability of the active compounds and the method conditions, parameters that change in a short period of time can be selected as useful biomarkers of the anti-inflammatory properties of PFS. In patients affected by MS (Table 1), CRP levels decreased in three of the seven studies measuring it: a randomized open controlled cross-over study involving 482 patients for eight weeks [18], a 10-week randomized double-blind controlled clinical trial involving 60 patients [16], and a 1-year cross-sectional study involving 486 patients [21].

On the contrary, the other four of these studies [17, 19, 20, 23] reported no changes in CRP values. This discrepancy could be due to the nature of the botanicals used, the bioavailability of active compounds, the number of patients enrolled in the study, the type of study, and/or the laboratory methods for biomarker measurement. A larger number of studies is necessary to assess if CRP is a suitable parameter to evaluate the decrease of inflammatory status in MS after treatment with PFS.

E-selectin levels were evaluated in four studies [1719, 23], but a decrease was recorded in only one [18], which rules out this biomarker as suitable for the purpose. TNF- levels were evaluated in three studies [16, 18, 19], and two of them [16, 18] reported a significant reduction after treatment with soy proteins (8 weeks) and fresh algae infusion (10 weeks). No significant differences before and after treatment with berry derivatives were found [19]. IL-18 was evaluated in only one study [18], and no conclusions can be drawn. IL-6 levels: two studies [17, 23] reported no significant differences after 5 and 9 weeks; another three studies [16, 18, 20] reported a significant reduction in both gene expression and serum after 8, 10, and 20 weeks.

We may conclude that TNF- and IL-6 may be suitable biomarkers to evaluate in a rather short time (6–10 weeks) the improvement of the inflammatory status in humans after PFS treatment, but careful consideration of the botanical formulation used in the study (i.e., kind of extract, occurrence of active principles, and their bioavailability) and the amount of PFS taken before should be given before ruling a proinflammatory biomarker in or out. Additional studies are needed before considering CRP and IL-18 as biomarkers modulated by PFS treatment, since the data occurring in the literature do not allow us to draw clear conclusions. The importance of IL-6, TNF- , and CRP as proinflammatory biomarkers is well documented. TNF- is released by adipose tissue and is overexpressed in obesity, and TNF- can modulate insulin resistance in a variety of clinical trials related to obesity [6, 15, 24]. CRP and IL-6 are peripheral inflammatory markers, and their measurement improves the prediction of the risk of cardiovascular events [24].

3.2. Diabetes Studies
3.2.1. In Vitro Methods

Table 2 reports the in vitro methods applied to investigate inflammatory markers in diabetes. The method mostly used for the assessment of beneficial effects of botanicals/plant food supplements on diabetes were based on quantitative PCR (qPCR), a real-time PCR coupled with reverse transcriptase. This is a very sensitive and specific method for analyzing mRNA levels as a marker of gene expression. Uemura et al. [25] reported the use of SYBR Green assay, while Chuang et al. [26, 27] and Cao et al. [28] reported the use of the highly specific and sensitiveTaQman assay. Other frequently used methods are ELISA assay, immunoblotting, and transfections, in a variety of cell cultures, with a plasmid containing the luciferase reporter gene under the control of the NF- B responsive element(pNF- B-luc);the latter method is widely used for in vitro assays because it assesses events upstream of the inflammatory cascade which activates the NF- B pathway.

MethodBotanical usedParameters measuredResultsReference

Flow cytometryPeanut oil (Arachis hypogaea)Apoptosis induced by TNF-α in rat β-pancreatic cell line (INS-I)Reduction of apoptosisVassiliou et al., 2009 [29]
Luciferase assaySinocrassula indica Berger (Shilianhua) extractTranscriptional activity of NF-kB induced by LPS in RAW264.7 macrophages Reduction of transcriptional activity of NF-kBYin et al., 2009 [30]
Quantitative polymerase chain reaction (qRT-PCR)Zizyphus lotus L. Desf. extractmRNA levels of IL-2 in human (Jurkat) T cells stimulated with anti-CD3 antibodiesReduction of mRNA and IL-2 levels Uemura et al., 2010 [25]
Lyophilized grape powder (V. vinifera) Gene expression of IL-6, IL-8, IL-1β, MCP-1, COX-2, and TLR-2 in primary human adipocytes stimulated with TNF-αAttenuation of all parameters measuredChuang et al., 2011 [26]
White grape seed extract (V. vinifera)Gene expression of CYP, PPARγ, LEP, APM1, IL-6, and MCP-1 in THP-1 cells and human adipocytes stimulated with LPS and TNF-α, respectivelyReduction of IL-6 and MCP-1 expressions; modulation of APM1 and LEP (adipokine) gene expressionsKar et al., 2009 [31]
Lyophilized grape powder (V. vinifera) Gene expression of IL-6, IL-8, IL-1β, and TNF-α in human macrophages and adipocytes stimulated with LPSReduction of IL-6, IL-8, IL-1β, and TNF-α gene expressions Chuang et al., 2010 [27]
Cinnamomum burmannii extractExpression of tristetraprolin (TTP) in mouse RAW264.7 macrophages treated with LPSInduction of TTPCao et al., 2008 [28]
Crataegus pinnatifida Bunge var. typica Schneider and C. pinnatifida Bunge Gene expression of iNOS and COX-2 in murine RAW264.7 macrophages treated with LPSmRNA levels of iNOS and COX2 were inhibited by C. pinnatifida BungeLi et al., 2010 [32]
Fructus XanthiiProtection from IL-1β and IFN-γand NF-kB induced in pancreatic cell line RINM5FComplete protectionSong et al., 2009 [33]
Lyophilized grape powder (V. vinifera) obtained from red, green, and blue-purple seeded and seedless California table grapesConcentration of IFN-γ inducible protein-10 (IP-10) in human adipocytes and macrophages after treatment with LPSReduction of IP-10Chuang et al., 2010 [27]
Palmitic acid, oleic acid, or DHAProduction of TNF-α and IL-10 in 3T3-L1 murine adipocytesIncrease of
IL-10, no effect on TNF-α
Bradley et al., 2008 [34]
Lyophilized grape powder, (V. vinifera)Activation of NF-kB factor and Ikα degradation in primary cultures of human adipocytes treated with TNF-αReduction of NF-kB activity and mediation of Ikα degradationChuang et al., 2011 [26]
Lyophilized grape powder, (V. vinifera)Activation of NF-κB factor in primary cultures of human adipocytes and macrophages treated with LPSInhibition of NF-kB activationChuang et al., 2010 [27]
Cinnamomum burmanii Cytokine (TNF-α, IL-6, and COX-2) production by mouse RAW 264.7 macrophages treated with LPSReduction of cytokine (TNF-α, IL-6, and COX-2) productionCao et al., 2008 [28]
Chromogenic assay (TMPD) Extract of rhizome of Zingiber officinale Roscoe Enzymatic activity of COX
in C2C12 cells
Reduction of enzymatic activity Priya Rani et al., 2011 [35]
ELISA Diosgenin from seeds of Trigonella foenum-graecum Levels of adiponectin and MCP-1 in 3T3-L1 preadipocytesIncrease of adiponectin levels and decrease of MCP-1Uemura et al., 2010 [25]
Caspase-3 activity luminometric assay Hypericum perforatum L.Determination of apoptosis in rat insulinoma cell line (INS-1E) stimulated with cytokinesInhibition of apoptosisMenegazzi et al., 2008 [36]
White grape seed extract (V. vinifera)P65 translocation and PIKBα protein in human monocyte cell line THP-1 and human adipocyte treated with LPS and TNF-αPartial inhibition Chac n et al., 2009 [37]
No production colorimetric assay Crataegus pinnatifida Bunge var. typica Schneider and C. pinnatifida BungeCytotoxicity and no inhibitory activity in murine RAW 264.7 macrophages stimulated with LPSInhibition of cytotoxicity and no activity only by C. pinnatifida BungeLi et al., 2010 [32]
Nitrite measurement colorimetric assayFructus XanthiiNo production in RINm5F cells treated with IL-1β and IFN-γInhibition of no productionSong et al., 2009 [33]
NF-kB binding assayDietary fatty acidsBinding activity of the P65 subunit of NF-kB in nuclear extracts from 3T3-L1 murine adipocytesDecrease of binding activityBradley et al., 2008 [34]

In the luciferase transfection assay only, Chuang et al. [26, 27] used primary cultures (from humans or animals) in their experiments; they are considered the most predictive, as primary cells retain the characteristics of the starting tissue. However, the isolation of appropriate cells from primary cultures can be difficult as the cell population is heterogeneous. Moreover, primary cultures have a limited life. Considering the challenges associated with modelling a chronic disease such as diabetes, it is encouraging to see that several in vitro methods for investigating inflammation have been developed, but few of them have been applied to the evaluation of PFS benefit assessment. In this sense, it would be interesting to develop methods to evaluate the following inflammatory events:(1)nuclear translocation of NF- B factor in the nucleus (usually evaluated by ELISA test) and the consequent transcription of inflammatory genes and the expression of adhesion molecules on endothelium, leading to the vascular complications of diabetes;(2)expression of adhesion molecules (i.e., sICAM-1, sICAM-2 and sVCAM-1, and E-selectin), which are typically overexpressed in diabetes and in cardiovascular disease [38]. Adhesion molecule expression is usually measured by RT-PCR (mRNA levels) and ELISA (protein expression on the cell surface);(3)evaluation of metalloproteinase-9 (MMP-9) secretion and gene expression. MMP-9 is induced by hyperglycemia and accelerates some diabetic complications such as retinopathy [39]. MMP-9 gene expression is usually measured by RT-PCR, secretion and enzymatic activities by zymography or western blotting;(4)evaluation of monocyte-macrophage chemotaxis in the endothelium. Indeed, monocytes and macrophages play a role in accelerating diabetes and in the development of atherosclerosis. They express specific receptors for advanced glycation end products (AGEs), which are proteins or lipids that become nonenzymatically glycated and oxidized after contact with aldose sugars.

After the binding of AGEs and intracellular processing, monocytes/macrophages synthesize and secrete growth-promoting cytokines such as TNF- , interleukin 1, and insulin-like growth factor, responsible for vascular complications in diabetes [40]. Furthermore, circulating AGEs may interact with endothelial receptors, which leads to perturbation of cellular properties, such as upregulation of the transcription and the translocation of nuclear factor NF- B [41]. Chemotaxis is generally measured under agarose [42, 43] or by chemotaxis assay, based on evaluation of cell migration through apposite filters after inflammatory stimuli have been placed in chambers (Boyden Chamber Assay) [44].

3.2.2. In Vivo Methods

Table 3 reports the in vivo methods developed for human trials.

Method1Botanical or botanical derivatives usedNo. of participants
and length of treatment
Parameters measuredResultsReference

Double-blind randomized controlled cross-over trialGrape seed extract (V.vinifera)32 (4 weeks) hs-CRPDecreased hs-CRPKar et al., 2009 [31]
Double-blind randomized controlled multicenter trialPycnogenol (extract of bark from the French Maritime Pine, Pinus pinaster)77 (12 weeks)Endothelin-1 and ketoprostaglandin F1-αDecreased endothelin-1 and increased ketoprostaglandin F1-αLiu et al., 2004 [45]
Randomized double-blind controlled trialPomegranate (Punica granatum L.), green tea (C. sinensis L.) extract114 (12 weeks)Plasma MDADecrease of MDA Kutan Fenercioglu et al., 2010 [46]
Blueberry (Vaccinium arctostaphylos L.) leaves water extract42 (4 weeks)Serum CRPDecreased hs- CRPAbidov et al., 2006 [47]
Single-blind randomized controlled trialCoffee 47 (1-month washout, 1 month 4 cups/day, and 1 month 8 cups/day)CRP, IL-6, IL-1, IL-18, 8-isoprostane, MIF, adiponectin, leptin, and SSADecreased IL-18, 8-isoprostane and increased adiponectin and other markers unchangedKempf et al., 2010 [48]
Randomized controlled cross-over trialGreen tea (C. sinensis L.) aqueous extract55 (4 weeks) hs-CRP and major cytokine mediator (IL-6)Both mediators unchangedRyu et al., 2006 [49]
Randomized open controlled clinical trialBlack tea (C. sinensis)46 (4 weeks: 150 mL week 1, 300 mL week 2, 450 mL week 3, and 600 mL week 4)Serum CRP, MDA, and fibrinogenDecreased serum CRP after consumption of 600 mL and MDA after consumption of 300 mL;
fibrinogen unchanged
Neyestani et al., 2010 [50]
Open noncontrolled nonrandomized clinical trialGinkgo biloba extract (24% ginkgo flavone glycosides and 6% terpenes47 (12 weeks)Urinary metabolites of thromboxane B2 (TXB2)and prostacyclin (PGI2)Decreased urinary TXB2and PGI2Kudolo et al., 2003 [51]
Prospective cohort studiesCoffee2040 (6 years)
AdiponectinHigher in drinkers of >4 cups/dayWilliams et al., 2008 [52]

Macrophage migration inhibitory factor (MIF) and malondialdehyde (MDA).
1Clinical studies used to assess inflammatory parameters are listed from the best to the worst method applied.

The majority of clinical trials evaluating the effect of botanicals on inflammatory biomarkers in diabetic patients were randomized, double-blind randomized controlled trials and randomized, open and controlled studies. The lack of blinding is particularly critical when the treatment is applied versus placebo. In fact, the placebo effect is a major cause of bias due to patient or doctor awareness [22]. Several studies have indicated that nonrandomized trials are more likely to yield a positive result for a new treatment than for an established conventional one. In some of the clinical trials considered, randomization was not described in detail or was unsuitable. In two papers [53, 54], the number of subjects was limited. In some studies [49, 51, 55], the dropout rate and the reasons for it were not critically discussed. The most often used methods for biomarker quantification were ELISA assay and HPLC analysis.

3.2.3. Inflammatory Biomarkers Affected by PFS in Diabetes

The parameters most frequently measured in clinical trials were as follows: CRP MDA endothelin-1 urinary thromboxane metabolites IL-6, IL-18, TNF- , and , 8-isoprostane. Six studies [31, 4750, 55] examined CRP levels; among them, three [31, 47, 50] reported a significant reduction in serum CRP levels after 4 weeks of treatment, two studies reported no significant changes [48, 49], and one reported increased levels of CRP [55]. As for MS, further studies are mandatory before conclusions on the efficacy of in vivo CRP measurement in diabetes after PFS treatment can be drawn.

Serum levels of malondialdehyde (MDA) were measured in three studies [46, 50, 56], and all reported a significant reduction after 4, 8, and 12 weeks of PFS consumption. MDA is an important biomarker of oxidative stress in diabetes as a consequence of persistent hyperglycemia and lipid peroxidation. These trials establish serum MDA measurement as appropriate for the purpose.

IL-6 levels were measured in three studies [48, 49, 56] and one reported a significant reduction after PFS treatment [56]. IL-18 [48] and TNF- [56] each were measured in only one study, and in both cases, a significant decrease was found. Nevertheless, further studies are needed to evaluate IL-6 and IL-18 as suitable inflammatory parameters. Endothelin-1, generally overexpressed in diabetes models [57], was found to decrease in two studies [45, 56], after 12 and 8 weeks of treatment, respectively.

Two studies showed a significant reduction of thromboxane ( ) urinary metabolites after 4 and 12 weeks of treatment, respectively [51, 54]. [51] and 8-isoprostane [48] were each evaluated only in one study; both decreased after PFS treatment. , , and 8-isoprostane are key proinflammatory biomarkers in diabetes. They are massively released in the wake of hypercoagulation and the vascular modifications that are typical for this disease. The paucity of studies in which these mediators were assayed in response to PFS treatment does not allow us to establish their usefulness in investigating the benefit of PFS treatment.

4. Conclusions

The aim of the present paper was to collect and critically discuss the existing experimental approaches used in vitro, in silico, and in vivo for benefit assessment of botanicals or plant food supplements (PFSs) in decreasing inflammation in MS-related diseases. PFS often consist of a complex mixture of compounds, which makes benefit assessment difficult and subject to interferences and false positives. The development of reliable methods for evaluating benefit assessment and their application and validation is therefore crucial.

No in silico methods were found in MS-related diseases. qRT-PCR was the in vitro method most widely applied for measuring the expression of inflammatory cytokines in diabetes, but no in vitro methods were found for testing PFS benefits in MS. Data from the in vivo studies (clinical trials) show that the inflammatory markers CRP, MDA, and are likely to be useful in judging the efficacy of PFS treatment, although more studies are needed to validate this conclusion. In addition, two proinflammatory biomarkers, IL-6 and TNF- , were the only two parameters to change in both in vitro and in vivo systems, and these biomarkers should be carefully considered in future studies.


The writing of this paper was funded by the European Community’s Seventh Framework Programme under Grant agreement no. 245199. It has been carried out within the PlantLIBRA project (website: http://www.plantlibra.eu). This paper does not necessarily reflect the Commission views of its future policy on this area. The fellowship of Elisa Colombo is partially funded by FSE, Regione Lombardia.


  1. A. Onat, “Metabolic syndrome: nature, therapeutic solutions and options,” Expert Opinion on Pharmacotherapy, vol. 12, no. 12, pp. 1887–1900, 2011. View at: Publisher Site | Google Scholar
  2. J. C. Pickup and M. A. Crook, “Is type II diabetes mellitus a disease of the innate immune system?” Diabetologia, vol. 41, no. 10, pp. 1241–1248, 1998. View at: Publisher Site | Google Scholar
  3. J. M. Fernández-Real and J. C. Pickup, “Innate immunity, insulin resistance and type 2 diabetes,” Trends in Endocrinology and Metabolism, vol. 19, no. 1, pp. 10–16, 2008. View at: Publisher Site | Google Scholar
  4. J. I. Cleeman, “Executive summary of the third report of the National Cholesterol Education Program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel III),” JAMA, vol. 285, no. 19, pp. 2486–2497, 2001. View at: Google Scholar
  5. S. M. Grundy, J. I. Cleeman, C. N. Merz et al., “Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III Guidelines,” Journal of the American College of Cardiology, vol. 44, no. 3, pp. 720–732, 2004. View at: Google Scholar
  6. A. Festa, R. D'Agostino, G. Howard, L. Mykkänen, R. P. Tracy, and S. M. Haffner, “Chronic subclinical inflammation as part of the insulin resistance syndrome: the insulin resistance atherosclerosis study (IRAS),” Circulation, vol. 102, no. 1, pp. 42–47, 2000. View at: Google Scholar
  7. M. Trøseid, I. Seljeflot, and H. Arnesen, “The role of interleukin-18 in the metabolic syndrome,” Cardiovascular Diabetology, vol. 9, article 11, 2010. View at: Publisher Site | Google Scholar
  8. G. Solinas and M. Karin, “JNK1 and IKKβ: molecular links between obesity and metabolic dysfunction,” The FASEB Journal, vol. 24, no. 8, pp. 2596–2611, 2010. View at: Publisher Site | Google Scholar
  9. T. Pischon, S. E. Hankinson, G. S. Hotamisligil, N. Rifai, W. C. Willett, and E. B. Rimm, “Habitual dietary intake of n-3 and n-6 fatty acids in relation to inflammatory markers among US men and women,” Circulation, vol. 108, no. 2, pp. 155–160, 2003. View at: Publisher Site | Google Scholar
  10. G. Zhao, T. D. Etherton, K. R. Martin, S. G. West, P. J. Gillies, and P. M. Kris-Etherton, “Dietary α-linolenic acid reduces inflammatory and lipid cardiovascular risk factors in hypercholesterolemic men and women,” Journal of Nutrition, vol. 134, no. 11, pp. 2991–2997, 2004. View at: Google Scholar
  11. I. Abete, A. Astrup, J. A. Martínez, I. Thorsdottir, and M. A. Zulet, “Obesity and the metabolic syndrome: role of different dietary macronutrient distribution patterns and specific nutritional components on weight loss and maintenance,” Nutrition Reviews, vol. 68, no. 4, pp. 214–231, 2010. View at: Publisher Site | Google Scholar
  12. F. Visioli, “Nutritional support in the pharmacological treatment of metabolic syndrome,” European Journal of Pharmacology, vol. 668, supplement 1, pp. S43–S49, 2011. View at: Publisher Site | Google Scholar
  13. M. Dell'agli, C. Di Lorenzo, M. Badea, E. Sangiovanni, L. Dima, and P. Restani, “Plant food supplements with anti-inflammatory properties: a systematic review (I),” Critical Reviews in Food Science and Nutrition, vol. 53, no. 4, pp. 403–413, 2013. View at: Publisher Site | Google Scholar
  14. M. Dell'agli, C. Di Lorenzo, M. Badea et al., “Plant food supplements with anti-inflammatory properties: a systematic review (II),” Critical Reviews in Food Science and Nutrition, vol. 53, no. 4, pp. 403–413, 2013. View at: Publisher Site | Google Scholar
  15. A. Camargo, J. Ruano, J. M. Fernandez et al., “Gene expression changes in mononuclear cells in patients with metabolic syndrome after acute intake of phenol-rich virgin olive oil,” BMC Genomics, vol. 11, no. 1, article 253, 2010. View at: Publisher Site | Google Scholar
  16. J. Oben, E. Enonchong, D. Kuate et al., “The effects of ProAlgaZyme novel algae infusion on metabolic syndrome and markers of cardiovascular health,” Lipids in Health and Disease, vol. 6, article 20, 2007. View at: Publisher Site | Google Scholar
  17. A. C. M. Gagliardi, R. C. Maranho, H. P. D. Sousa, E. J. Schaefer, and R. D. Santos, “Effects of margarines and butter consumption on lipid profiles, inflammation markers and lipid transfer to HDL particles in free-living subjects with the metabolic syndrome,” European Journal of Clinical Nutrition, vol. 64, no. 10, pp. 1141–1149, 2010. View at: Publisher Site | Google Scholar
  18. L. Azadbakht, M. Kimiagar, Y. Mehrabi, A. Esmaillzadeh, F. B. Hu, and W. C. Willett, “Soy consumption, markers of inflammation, and endothelial function: a cross-over study in postmenopausal women with the metabolic syndrome,” Diabetes Care, vol. 30, no. 4, pp. 967–973, 2007. View at: Publisher Site | Google Scholar
  19. H. M. Lehtonen, J. P. Suomela, R. Tahvonen et al., “Berry meals and risk factors associated with metabolic syndrome,” European Journal of Clinical Nutrition, vol. 64, no. 6, pp. 614–621, 2010. View at: Publisher Site | Google Scholar
  20. A. Basu, M. Wilkinson, K. Penugonda, B. Simmons, N. M. Betts, and T. J. Lyons, “Freeze-dried strawberry powder improves lipid profile and lipid peroxidation in women with metabolic syndrome: baseline and post intervention effects,” Nutrition Journal, vol. 8, no. 1, article 43, 2009. View at: Publisher Site | Google Scholar
  21. A. Esmaillzadeh, M. Kimiagar, Y. Mehrabi, L. Azadbakht, F. B. Hu, and W. C. Willett, “Fruit and vegetable intakes, C-reactive protein, and the metabolic syndrome,” American Journal of Clinical Nutrition, vol. 84, no. 6, pp. 1489–1497, 2006. View at: Google Scholar
  22. A. R. Jadad, R. A. Moore, D. Carroll et al., “Assessing the quality of reports of randomized clinical trials: is blinding necessary?” Controlled Clinical Trials, vol. 17, no. 1, pp. 1–12, 1996. View at: Publisher Site | Google Scholar
  23. J. Plat, G. Brufau, G. M. Dallinga-Thie, M. Dasselaar, and R. P. Mensink, “A plant stanol yogurt drink alone or combined with a low-dose statin lowers serum triacylglycerol and non-HDL cholesterol in metabolic syndrome patients,” Journal of Nutrition, vol. 139, no. 6, pp. 1143–1149, 2009. View at: Publisher Site | Google Scholar
  24. P. Dandona, A. Aljada, A. Chaudhuri, P. Mohanty, and R. Garg, “Metabolic syndrome: a comprehensive perspective based on interactions between obesity, diabetes, and inflammation,” Circulation, vol. 111, no. 11, pp. 1448–1454, 2005. View at: Publisher Site | Google Scholar
  25. T. Uemura, S. Hirai, N. Mizoguchi et al., “Diosgenin present in fenugreek improves glucose metabolism by promoting adipocyte differentiation and inhibiting inflammation in adipose tissues,” Molecular Nutrition and Food Research, vol. 54, no. 11, pp. 1596–1608, 2010. View at: Publisher Site | Google Scholar
  26. C. C. Chuang, A. Bumrungpert, A. Kennedy et al., “Grape powder extract attenuates tumor necrosis factor α-mediated inflammation and insulin resistance in primary cultures of human adipocytes,” Journal of Nutritional Biochemistry, vol. 22, no. 1, pp. 89–94, 2011. View at: Publisher Site | Google Scholar
  27. C. C. Chuang, K. Martinez, G. Xie et al., “Quercetin is equally or more effective than resveratrol in attenuating tumor necrosis factor-α-mediated inflammation and insulin resistance in primary human adipocytes,” American Journal of Clinical Nutrition, vol. 92, no. 6, pp. 1511–1521, 2010. View at: Publisher Site | Google Scholar
  28. H. Cao, J. F. Urban, and R. A. Anderson, “Cinnamon polyphenol extract affects immune responses by regulating anti- and proinflammatory and glucose transporter gene expression in mouse macrophages,” Journal of Nutrition, vol. 138, no. 5, pp. 833–840, 2008. View at: Google Scholar
  29. E. K. Vassiliou, A. Gonzalez, C. Garcia, J. H. Tadros, G. Chakraborty, and J. H. Toney, “Oleic acid and peanut oil high in oleic acid reverse the inhibitory effect of insulin production of the inflammatory cytokine TNF- both in vitro and in vivo systems,” Lipids in Health and Disease, vol. 8, article 25, 2009. View at: Publisher Site | Google Scholar
  30. J. Yin, A. Zuberi, Z. Gao, D. Liu, Z. Liu, and J. Ye, “Shilianhua extract inhibits GSK-3β and promotes glucose metabolism,” American Journal of Physiology, vol. 296, no. 6, pp. E1275–E1280, 2009. View at: Publisher Site | Google Scholar
  31. P. Kar, D. Laight, H. K. Rooprai, K. M. Shaw, and M. Cummings, “Effects of grape seed extract in Type 2 diabetic subjects at high cardiovascular risk: a double blind randomized placebo controlled trial examining metabolic markers, vascular tone, inflammation, oxidative stress and insulin sensitivity,” Diabetic Medicine, vol. 26, no. 5, pp. 526–531, 2009. View at: Publisher Site | Google Scholar
  32. C. Li, H. J. Son, C. Huang, S. K. Lee, J. Lohakare, and M. H. Wang, “Comparison of Crataegus pinnatifida Bunge var. typica Schneider and C. pinnatifida Bunge fruits for antioxidant, anti-α-glucosidase, and anti-inflammatory activities,” Food Science and Biotechnology, vol. 19, no. 3, pp. 769–775, 2010. View at: Publisher Site | Google Scholar
  33. M. Y. Song, E. K. Kim, H. J. Lee et al., “Fructus Xanthii extract protects against cytokine-induced damage in pancreatic β-cells through suppression of NF-κB activation,” International Journal of Molecular Medicine, vol. 23, no. 4, pp. 547–553, 2009. View at: Publisher Site | Google Scholar
  34. R. L. Bradley, F. M. Fisher, and E. Maratos-Flier, “Dietary fatty acids differentially regulate production of TNF-α and IL-10 by murine 3T3-L1 adipocytes,” Obesity, vol. 16, no. 5, pp. 938–944, 2008. View at: Publisher Site | Google Scholar
  35. M. Priya Rani, K. P. Padmakumari, B. Sankarikutty, O. Lijo Cherian, V. M. Nisha, and K. G. Raghu, “Inhibitory potential of ginger extracts against enzymes linked to type 2 diabetes, inflammation and induced oxidative stress,” International Journal of Food Sciences and Nutrition, vol. 62, no. 2, pp. 106–110, 2011. View at: Publisher Site | Google Scholar
  36. M. Menegazzi, M. Novelli, P. Beffy et al., “Protective effects of St. John's wort extract and its component hyperforin against cytokine-induced cytotoxicity in a pancreatic β-cell line,” International Journal of Biochemistry and Cell Biology, vol. 40, no. 8, pp. 1509–1521, 2008. View at: Publisher Site | Google Scholar
  37. M. R. Chacón, V. Ceperuelo-Mallafré, E. Maymó-Masip et al., “Grape-seed procyanidins modulate inflammation on human differentiated adipocytes in vitro,” Cytokine, vol. 47, no. 2, pp. 137–142, 2009. View at: Publisher Site | Google Scholar
  38. C. Urso, E. Hopps, and G. Caimi, “Adhesion molecules and diabetes mellitus,” La Clinica Terapeutica, vol. 161, no. 1, pp. e17–e24, 2010. View at: Google Scholar
  39. R. A. Kowluru, “Role of matrix metalloproteinase-9 in the development of diabetic retinopathy and its regulation by H-Ras,” Investigative Ophthalmology and Visual Science, vol. 51, no. 8, pp. 4320–4326, 2010. View at: Publisher Site | Google Scholar
  40. M. Kirstein, J. Brett, S. Radoff, S. Ogawa, D. Stern, and H. Vlassara, “Advanced protein glycosylation induces transendothelial human monocyte chemotaxis and secretion of platelet-derived growth factor: role in vascular disease of diabetes and aging,” Proceedings of the National Academy of Sciences of the United States of America, vol. 87, no. 22, pp. 9010–9014, 1990. View at: Publisher Site | Google Scholar
  41. A. Goldin, J. A. Beckman, A. M. Schmidt, and M. A. Creager, “Advanced glycation end products: sparking the development of diabetic vascular injury,” Circulation, vol. 114, no. 6, pp. 597–605, 2006. View at: Publisher Site | Google Scholar
  42. L. S. Martin, T. J. Spira, S. L. Orloff, and R. C. Holman, “Comparison of methods for assessing chemotaxis of monocytes and polymorphonuclear leukocytes isolated from patients with AIDS or AIDS-related conditions,” Journal of Leukocyte Biology, vol. 44, no. 5, pp. 361–366, 1988. View at: Google Scholar
  43. R. D. Nelson, P. G. Quie, and R. L. Simmons, “Chemotaxis under agarose: a new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes,” The Journal of Immunology, vol. 115, no. 6, pp. 1650–1656, 1975. View at: Google Scholar
  44. R. Snyderman, L. C. Altman, M. S. Hausman, and S. E. Mergenhagen, “Human mononuclear leukocyte chemotaxis: a quantitative assay for humoral and cellular chemotactic factors,” The Journal of Immunology, vol. 108, no. 3, pp. 857–860, 1972. View at: Google Scholar
  45. X. Liu, J. Wei, F. Tan, S. Zhou, G. Würthwein, and P. Rohdewald, “Antidiabetic effect of Pycnogenol French maritime pine bark extract in patients with diabetes type II,” Life Sciences, vol. 75, no. 21, pp. 2505–2513, 2004. View at: Publisher Site | Google Scholar
  46. A. Kutan Fenercioglu, T. Saler, E. Genc, H. Sabuncu, and Y. Altuntas, “The effects of polyphenol-containing antioxidants on oxidative stress and lipid peroxidation in Type 2 diabetes mellitus without complications,” Journal of Endocrinological Investigation, vol. 33, no. 2, pp. 118–124, 2010. View at: Publisher Site | Google Scholar
  47. M. Abidov, A. Ramazanov, M. Jimenez Del Rio, and I. Chkhikvishvili, “Effect of Blueberin on fasting glucose, C-reactive protein and plasma aminotransferases, in female volunteers with diabetes type 2: double-blind, placebo controlled clinical study,” Georgian Medical News, no. 141, pp. 66–72, 2006. View at: Google Scholar
  48. K. Kempf, C. Herder, I. Erlund et al., “Effects of coffee consumption on subclinical inflammation and other risk factors for type 2 diabetes: a clinical trial,” American Journal of Clinical Nutrition, vol. 91, no. 4, pp. 950–957, 2010. View at: Publisher Site | Google Scholar
  49. O. H. Ryu, J. Lee, K. W. Lee et al., “Effects of green tea consumption on inflammation, insulin resistance and pulse wave velocity in type 2 diabetes patients,” Diabetes Research and Clinical Practice, vol. 71, no. 3, pp. 356–358, 2006. View at: Publisher Site | Google Scholar
  50. T. R. Neyestani, N. Shariatzade, A. Kalayi et al., “Regular daily intake of black tea improves oxidative stress biomarkers and decreases serum C-reactive protein levels in type 2 diabetic patients,” Annals of Nutrition and Metabolism, vol. 57, no. 1, pp. 40–49, 2010. View at: Publisher Site | Google Scholar
  51. G. B. Kudolo, S. Dorsey, and J. Blodgett, “Effect of the ingestion of Ginkgo biloba extract on platelet aggregation and urinary prostanoid excretion in healthy and Type 2 diabetic subjects,” Thrombosis Research, vol. 108, no. 2-3, pp. 151–160, 2002. View at: Publisher Site | Google Scholar
  52. C. J. Williams, J. L. Fargnoli, J. J. Hwang et al., “Coffee consumption is associated with higher plasma adiponectin concentrations in women with or without type 2 diabetes: a prospective cohort study,” Diabetes Care, vol. 31, no. 3, pp. 504–507, 2008. View at: Publisher Site | Google Scholar
  53. L. Axelrod, J. Camuso, E. Williams, K. Kleinman, E. Briones, and D. Schoenfeld, “Effects of a small quantity of ω-3 fatty acids on cardiovascular risk factors in NIDDM: a randomized, prospective, double-blind, controlled study,” Diabetes Care, vol. 17, no. 1, pp. 37–44, 1994. View at: Google Scholar
  54. R. Takahashi, J. Inoue, H. Ito, and H. Hibino, “Evening primrose oil and fish oil in non-insulin-dependent-diabetes,” Prostaglandins Leukotrienes and Essential Fatty Acids, vol. 49, no. 2, pp. 569–571, 1993. View at: Publisher Site | Google Scholar
  55. Y. Fukino, M. Shimbo, N. Aoki, T. Okubo, and H. Iso, “Randomized controlled trial for an effect of green tea consumption on insulin resistance and inflammation markers,” Journal of Nutritional Science and Vitaminology, vol. 51, no. 5, pp. 335–342, 2005. View at: Google Scholar
  56. P. Usharani, A. A. Mateen, M. U. R. Naidu, Y. S. N. Raju, and N. Chandra, “Effect of NCB-02, atorvastatin and placebo on endothelial function, oxidative stress and inflammatory markers in patients with type 2 diabetes mellitus: a randomized, parallel-group, placebo-controlled, 8-week study,” Drugs in R and D, vol. 9, no. 4, pp. 243–250, 2008. View at: Publisher Site | Google Scholar
  57. A. G. Minchenko, M. J. Stevens, L. White et al., “Diabetes-induced overexpression of endothelin-1 and endothelin receptors in the rat renal cortex is mediated via poly(ADP-ribose) polymerase activation,” The FASEB Journal, vol. 17, no. 11, pp. 1514–1516, 2003. View at: Google Scholar

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