Cellular Immunology 271 (2011) 474–479

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Cellular Immunology

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Immunomodulatory activity of polyphenols derived from Cassia auriculata flowersin aged rats

Cini M. John a,d, Pratheep Sandrasaigaran a, Chih Kong Tong a, Aishah Adam d, Rajesh Ramasamy a,b,c,⇑a Immunology Laboratory, Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, Malaysiab Stem Cell Research Laboratory, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, Malaysiac UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, Selangor, Malaysiad Pharmacology and Toxicology Laboratory, Faculty of Pharmacy, Universiti Teknologi MARA, Selangor, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history:Received 22 June 2011Accepted 22 August 2011Available online 28 August 2011

Keywords:Cassia auriculataImmunosenescenceT cellsNeutrophilsSplenocytes

0008-8749/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.cellimm.2011.08.017

⇑ Corresponding author at: Immunology Unit, Depaof Medicine and Health Sciences, Universiti Putra MSelangor, Malaysia. Fax: +60 3 8941 3802.

E-mail address: r.rajesh@medic.upm.edu.my (R. Ra

The immunomodulatory activity of Cassia auriculata (CA)-derived polyphenols was tested on aged rats.Rats (24–26 months old) were given CA polyphenols supplementation at doses of 25, 50, and 100 mg/kg for 28 days. Flow cytometry analysis of CA polyphenols-treated aged rats showed increased T and Bcells percentage along with enhanced proliferation of splenocytes in both resting and LPS-stimulatedcells. Increased percentage of pan T cells is further supported by an elevation of CD4+, CD8+, andCD4+CD25+ regulatory cells. In terms of innate immune cell activity, CA polyphenol supplementationreduced the oxidative burst activity of neutrophils in response to PMA and Escherichia coli activation.Our results collectively show that polyphenols derived from CA boost T cell immunity by increasingthe number of T cells and its sensitivity towards stimulants and decreasing ROS production by neutro-phils that could potentially harm multiple biological systems in aged individuals.

� 2011 Elsevier Inc. All rights reserved.

1. Introduction

Immunosenescence is an age-related decline of immune func-tion which affects both innate and adaptive immunity, wherebythe decrease in cell-mediated and humoral immune responses pre-disposes to an increased prevalence and severity of infectious dis-eases in elderly patients. It has been reported that oxidative andinflammatory states that underlie the aging process are the basisof immunosenescence [1]. Aging is also characterised by a consti-tutive pro-inflammatory environment (inflamm-aging) with per-sistent low-grade of immune activation that may trigger tissuedamage caused by infections in elderly individuals. The overall im-pact of immunosenescence affects the primary lymphoid organswhich impede the production and maturation of T and B cells. Thisincludes T and B cell-related changes such as thymic involution[2]; skewed T cell composition from naive to memory T cells [3];altered T-cell activation; changes in B cell percentage [4]; ineffi-cient immunoglobulin class switching [5] and reduced mucosalimmunity [6]. Synthetic and natural supplements have beenwidely consumed as an alternative therapy to reduce the effectof immunosenescence. It has been found that nutritional interven-tions with polyphenolic antioxidants constitute a good alternative

ll rights reserved.

rtment of Pathology, Facultyalaysia, 43400 UPM Serdang,

masamy).

to rejuvenate age-affected immune functions. In line with this,here we have reported the first time, the immunomodulatoryactivity of polyphenols derived from Cassia auriculata flowers toresuscitate the immune system of aged rats.

C. auriculata (Cesalpinaceae, common name: Tanner’s Cassia)has been long used in traditional medicine by Asians. The ancientAyurvedic and Sidha medicines are often consume all components(leaf, stem, root, and flower) of CA in their medicinal preparation. Ithas been used as remedy for rheumatism, conjunctivitis, diabetes,general well being and pre & postnatal treatments [7]. The currentliterature reveals that the aqueous extract of CA prevents lipid per-oxidation in brains of diabetic rats [8]. Moreover, the nephro-pro-tection role by CA against cisplatin- and gentamicin-induced renalinjury was also reported, and is derived from the antioxidant andfree-radical-scavenging properties of CA [9]. Supplementation ofCA leaf extract has shown to protect the liver from free radicalmediated oxidative stress in experimental hepatotoxicity [10].The flower and leaf extracts of CA exert an anti hyperglycaemic ef-fect in streptozotocin-induced experimental diabetes [8,10–14].Furthermore, histopathological analysis of the liver and brain con-firms the non toxicity of CA extracts [10].

In recent years, there is growing interest in modulating immunefunctions by utilising phytochemicals especially plant-derivedpolyphenols. Ongoing research has highlighted the dynamic capac-ity of polyphenols to protect against age-associated disordersthrough a variety of important mechanisms [15]. Investigationson exploring the immunomodulatory potential of plant-derived

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polyphenol fraction revealed its effectiveness to inhibit the mito-gen-stimulated proliferation of peripheral blood mononuclear cellsthrough LPS stimulation [16,17]. Moreover, Amrutesh et al. havereported that CA contains a relatively higher level of polyphenolsas active ingredients and has been verified for its pharmacologicalsafety [18]. It also contains several active constituents such asflavonoids, b-sitosterol- b-D-glucoside, polysaccharides, anthra-cene, dimeric procyanidins and myristyl alcohol [19]. This studywas thus initiated with the aim of evaluating the immuno protec-tive role of CA against immunosenescence in aged female rodents.

2. Materials and methods

2.1. Preparation and standardization of polyphenols

C. auriculata flowers were collected freshly from Kuala Lumpur,Malaysia; identified and authenticated at FRIM (Forest ResearchInstitute Malaysia). Samples were stored in a herbarium as voucherno. 18-1. The polyphenol compound of CA was extracted as previ-ously described [20]. Briefly, flowers of CA were shade-dried forfew days; coarsely powdered; soaked in distiled water at the ratioof 30 g in 100 ml for 72 h at 4 �C. Extract was separated by centri-fugation at 2000 rpm for 20 min and washed with hexane to re-move the lipo-soluble compounds. The polyphenol-rich fractionwas collected from ethyl acetate and the total phenolic contentwas determined by using the Folin–Ciocalteu reagent as a stan-dard. HPLC was used for standardization [21]. All chemicals uti-lised for preparation, extraction and HPLC standardisation werepurchased from Sigma–Aldrich USA.

2.2. Animals observation

Female (350–380 g of 24–26 months old) SPF Sprague–Dawleyrats procured from Animal House of UiTM Puncak Alam, Malaysiawas used for the study. The animals were maintained as per OECDguidelines [22].

2.3. Treatment

SD rats were randomly divided into five groups and each groupconsists of at least six animals. The first two groups were assignedas controls and respectively received a daily dose of vitamin C at150 mg/kg and 0.5% CMC (solvent for polyphenols). Treatmentgroups were divided into three dosages as 25, 50 and 100 mg/kgand animals were fed by oral gavages daily. All animals were trea-ted for 28 days. Individual dosages were based on the most re-cently recorded body weights to provide the correct mg/kg/daydose. The dosage levels were selected based on the results of ear-lier literatures [8,11]. All extract solutions were prepared freshlyand the volume of CA administered was adjusted to 1 ml/100 gof body weight.

2.4. Measurement of ROS secretion

ROS secretion of neutrophils was measured by a commerciallyavailable Phagoburst assay kit (ORPEGEN Pharma, Germany). Hun-dred microlitres of fresh heparinised blood was treated with Esch-erichia coli bacteria, PMA and fMLP in separate assays andincubated for 20 min at 37 �C. At end of incubation, 20 ll of thefluorogenic substrate was added and cells were then lysed andfixed by lysing solution for 10 min at room temperature. Cells werewashed and stained with DNA staining solution prior to flowcytometer (FACS Canto) acquisition and thereafter analysed byFACS Diva software for the DNA content and ROS secretion [23].

2.5. Splenocytes proliferation assay

Spleens were harvested from sacrificed rats and splenocyteswere isolated using a previously described standard procedure[24]. Hundred thousand splenocytes were plated in each well of96 well plates; stimulated separately with PHA and LPS withrespective concentrations for three days. Cultures were pulsedwith 10 ll (3H-TdR) 24 h prior to the measurement and the cellswere harvested onto glass filter mats by using a 96-well plate auto-mated cell harvester (Harvester Mach III M, TOMTEC). The filtermat was dried using oven (120 �C) for 10 min before adding 5 mlscintillation fluid (OptiPhase SuperMix Cocktail; Perkin Elmer, Bos-ton USA). The filter mat was then sealed and fitted into a scintilla-tion cassette for radioactive measurement using the luminescentMicrobeta counter (Wallac). Results were expressed as countsper minute (CPM).

2.6. Flow cytometer analysis

To analyse the degree of expression of cell surface proteins onsplenocytes, cells were incubated with anti rat antibodies againstCD3, CD4, CD8 and CD25. The total T, B and NK cells percentageswere measured by a commercially available TBNK cocktail. Allantibodies and cocktail kits were purchased from Becton Dickin-son, USA. The stained samples were assessed by the FACS Cantoflow cytometer (Becton Dickinson, USA) and the data analysedusing FACS Diva software. The relevant isotype antibody controlswere used in parallel to all measurements to set negative gating[25].

2.7. Statistical analysis

Values for all measurements are presented as mean ± SD.Comparisons for all pairs were performed by one way ANOVAand Dunnet test. Significance levels were set at p value <0.05.

3. Results

3.1. CA supplementation increases the percentages of pan splenic T andB cells

The flow cytometer analysis of total T, B and NK cell percentagesindicates that the CA polyphenols supplementation at all dosagesincreases the relative percentages of T and B cells (Fig. 1). A signif-icant increase in B cell percentage was noted in all dosages; how-ever T cell percentage increased significantly only at 50 and100 mg dosages. Although vitamin C supplementation at 150 mgsignificantly increased the T and B cell percentages, yet the magni-tude of increase is much lower than the 50 and 100 mg dosagegroups. At all concentrations of CA polyphenols, the percentageof NK cells remained similar to the untreated group.

3.2. CA supplementation increases the percentages of T helper, Tcytotoxic and regulatory T cells

The effect of CA polyphenol supplementation was further testedon the percentage of helper and cytotoxic T cell populations. Help-er and cytotoxic T cells were determined by expression of CD4 andCD8 markers respectively. In line with a total increase in T cells bymeasurement of CD3, both CD4 and CD8 expressions were found tobe increased in treatment groups including vitamin C supplemen-tation (Fig. 2A). In comparison to all CA polyphenols supplementa-tions, dosage at 50 mg/kg governed the highest yield of CD4+ andCD8+ cells percentages with approximately two-fold increases.Similarly, the percentage of CD4+CD25+ cells numbers was highest

Fig. 1. Assessment of percentage of T, B and NK cells expression. Splenocytes from CA polyphenol supplemented and non-supplemented aged rats were assessed forexpression of T, B and NK cells via flow cytometer analysis. One million of cells were labelled with a commercially available TBNK staining kit. At dosages 50 and 100 mg/kg,the expression of pan T cells was elevated significantly and B cells expression in increased significantly for all dosages. CA polyphenol supplementation does not alter NK cellsexpression significantly. The results are expressed as mean ± SD from three independent experiments. ⁄ Significance was determined at p < 0.05.

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at the 50 mg dosage of CA polyphenols (Fig. 2B). Although all dos-ages of CA polyphenols increased the CD4+CD25+ T cells, yet thesignificant differences could only be observed in 50 and 100 mgdosages of CA polyphenols. Vitamin C supplementation did not al-ter the percentage of CD4+CD25+ cells.

3.3. CA supplementation enhanced LPS stimulated splenocytesproliferation

In order to determine whether the increased percentage of Tcells by CA polyphenol supplementation is accompanied by func-tional enhancement; splenocyte proliferation assay was carriedout in response to PHA and LPS stimulation. Among all stimuli,LPS stimulation induced a significant proliferation with all CA pol-yphenol dosages including vitamin C supplementation (Fig. 3). Thehighest proliferation was noted in the 50 mg dosage group and theproliferation rate declined significantly at 100 mg dosage.Although PHA did not induce substantial proliferation in all testgroups; yet a noticeable increased was noted at CA polyphenols-treated groups. Furthermore, supplementation of 50 mg CA poly-phenols significantly increased the base line proliferation ofsplenocytes in the resting state.

3.4. CA supplementation reduced ROS production of neutrophils

Reactive oxygen species (ROS) production by resting and acti-vated neutrophils was measured by a commercially availablephagoburst assay. Among the three stimulators used, PMA exerteda maximal secretion of ROS followed by E. coli. CA polyphenols at50 and 100 mg dosages significantly reduced the ROS secretion in-duced by PMA (Fig. 4). The inhibition of ROS secretion induced byPMA reflected a dose dependent trend, with the highest inhibitionnoted at 100 mg dosage. For ROS secretion induced by E. coli, thehighest inhibition occurred at 50 mg dosage. fMLP stimulationdid not induced ROS production in all control and test groups.

4. Discussion

Immunosenescence is an inevitable phenomenon and it con-tributes to an increased risk for health ailments due to infections,malignancies, auto immune diseases and declined responses tovaccination [26]. It has been postulated that the distortion of

oxidative–anti oxidative balance, diminished function of lymphoidorgans such as bone marrow and thymus and declined stem cellreservoirs play vital roles in affecting the immune integrity of agedindividuals [27]. Many studies have been conducted to restore nor-mal immune function in the elderly by harmonising oxidativestress, rejuvenating the thymus and consuming synthetic and nat-ural supplements to ameliorate the putrefying biological system. Inour present study, we have tested the ability of a local herb, C.auriculata and its flower-derived polyphenols in improving age-re-lated immune dysfunctions.

C. auriculata (CA) is a local herb commonly found in Asian coun-tries that has been consumed for many centuries to treat chronicillness, especially diabetes [11,14]. Most of the current literatureson CA either in the forms of aqueous, ethanolic or polyphenolicextractions are focused on its anti diabetic effect via correction ofhyperglycaemia and immune dysfunctions [8,11]. To date, thereare no reports indicating similar immunomodulatory activities ofCA in the immunosenescence paradigm, either using human or ani-mal models. Therefore, our work will serve as the first report inelaborating the immunomodulatory effect of CA polyphenols inaged rats. We have utilised rats at age of 24–26 months old as thisrange is comparably equivalent to the human age of 50–60 yearsold [28].

Our results indicate that polyphenols derived from CA flowersshowed a significant expansion of pan T and B cell percentages(Fig. 1). Moreover, a further dissection of pan T cell population re-vealed that CA supplementation raised the percentage of CD4+ andCD8+T cell populations (Fig. 2A). It has been documented thataging causes reduced production of lymphocytes as the haemato-poietic compartment of bone marrow shrinks with increasing ageand is replaced by adipose tissue [29]. The expression of CD4+ cellswere higher in aged rats compared to CD8 cells which reflect thenormal aging physiology of peripheral T cell compartments. Ithas been also showed that reduced CD8+ T cells numbers andthe delayed cytolytic capability is observed in old mice comparedto young mice following in vivo inoculation with influenza virus[30]. It would be extremely beneficial to boost the quantity andcomposition of adaptive immune cells which may reduce the mor-bidity, mortality and disability in elderly. Although, we have notassessed the absolute cell counts of these adaptive cells, it isdeducible that CA supplementation augments the adaptiveimmune cell compartment as the interplay between adaptive and

Fig. 2. Assessment of T cell subpopulation and regulatory T cells. One million of splenocytes were stained with CD4, CD8 and CD25 fluorochrome conjugated antibodies andanalysed by flow cytometer. CA polyphenol supplementation increases the composition of CD4+ and CD8+ cells (A) and CD4+CD25+ regulatory T cells. Supplementation ofvitamin C serves as positive control. The results are expressed as mean ± SD from three independent experiments. ⁄ Significance was determined at p < 0.05.

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innate immune system is an important mechanism to providecomplete protection towards pathogens. To warrant an effectiveimmune response, a proper composition of immune cells at appro-priate cell numbers is necessary. However, CA supplementationdoes not alter the composition of NK cell as the percentage ofCD56+ cells were consistent with all dosages of CA polyphenols(Fig. 2).

The increased percentage of CD4+ cells also markedly alteredthe percentage of CD4+CD25+ regulatory T cells (Fig. 2B). Althoughour findings report a significant small increase (�1%) ofCD4+CD25+ regulatory T cells, however the current data on regula-tory T cell number in the elderly remains unclear. It has beenshown that aged individuals have increased regulatory T cells inperipheral blood [31]. However, our results should be interpretedwith caution as T regulatory cells were measured solely byCD4+CD25+ expression and lacks Foxp3 analysis. Furthermore itis not clear whether these regulatory T cells were derived fromthe thymus or induced at the periphery.

We also demonstrated that the elevated T cell percentagewas strongly associated with its proliferative index, as CA

supplementation enhanced the proliferative rate of resting andLPS stimulated T cells (Fig. 3). Reduced T cell proliferation is acommon feature of immunosenescence as the ability of T cells torespond to external stimuli and their subsequent production ofcytokines were reported to be compromised [32]. CA supplementa-tion profoundly increased the proliferation of T cells at a dosage of50 mg/kg. Although vitamin C supplementation had incurred withan elevated proliferation of T cells as well, yet the impact of CApolyphenols on such proliferation is much greater. However, T cellsfrom aged rats did not respond towards PHA stimulation in bothcontrol and treated groups. The limited ability of aged rats’ T cellsto respond to LPS but not to PHA may implicate the impairedsensitivity of certain surface receptors or signalling pathways inimmunosenescenced T cells as both the ligands and signallingpathways for LPS and PHA are different. Nonetheless, the effectof CA polyphenols in both T cell percentage and proliferation inaged rats promise a good herbal-based prophylactic therapy forenhanced adaptive immunity.

Oxidative stress is considered as the most important overallmechanism for cellular aging which leads to immunosenescence.

Fig. 3. Assessment of splenocytes proliferation. Splenocytes from CA treated rats were cultured for 72hr with PHA and LPS. At end of incubation, the proliferation of cellsdetermined by 3H-thymidine assay. CA polyphenol supplementation profoundly increases the proliferation rate of splenocytes in response to LPS stimulation. The results areexpressed as mean ± SD from three independent experiments. ⁄ Significance was determined at p < 0.05.

Fig. 4. Assessment of neutrophil mediated ROS production. Neutrophil mediated ROS production was measured by a commercially available phagoburst assay. CA polyphenolsupplementation inhibit the ROS production of neutrophils in response to PMA and E. coli stimulation. The results are expressed as mean ± SD from three independentexperiments. ⁄ Significance was determined at p < 0.05.

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Our results show that, CA significantly down regulates the produc-tion of ROS by neutrophils at 50 mg/kg dosage in aged rats (Fig. 4).Although the numbers of neutrophils were claimed to be not al-tered, however other functional aspects of neutrophils such as re-duced microbicidal activity and chemotactic ability; fragilitytowards spontaneous release of ROS had made aged individualsvulnerable towards tissue damages [33]. Our findings suggest thatgeneration of excessive and spontaneous ROS due to age-associ-ated immune dysregulation can be modulated by supplementingCA. The harnessing of oxidative stress exerted by CA is believedto be derived from its high antioxidant activity through enzymeslike superoxide dismutase, catalase, glutathione peroxidise to as-sist in detoxification of ROS [14,34].

In summary, our study provides scientific evidence to a series ofphenomena that are considered to contribute to the aging processwhich can be altered by CA polyphenols. It clearly shows immuno-modulatory effects of CA on various immune cell compositions andtheir relevant functions. This CA polyphenol immunomodulatoryactivity also could be exploited towards ameliorating pathologicalconditions such as diabetic, cancer and AIDS. Furthermore, it

supports drug development from CA with the aim of creatingevidence-based plant medications in prevention and treatment ofimmune diseases in the form of new single treatments or newcombinatory drug regimes exploiting its synergy-effects as a poly-phenol [35,36].

Acknowledgments

The study was partially supported by a research grant from Re-search University Grant Scheme (04-01-09-0781RU) from Univer-siti Putra Malaysia and research grant from MOSTI [100-IRDC/BIOTEK 16/6/2/(6/2006)] which was held by Universiti TeknologiMARA.

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