Cellular Immunology 271 (2011) 474–479

Contents lists available at SciVerse ScienceDirect

Cellular Immunology
journal homepage: www.elsevier.com/locate/ycimm

Immunomodulatory activity of polyphenols derived from Cassia auriculata flowers

in 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, Malaysia
b
Stem Cell Research Laboratory, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, Malaysia
c
UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, Selangor, Malaysia
d
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: The immunomodulatory activity of Cassia auriculata (CA)-derived polyphenols was tested on aged rats.
Received 22 June 2011 Rats (24–26 months old) were given CA polyphenols supplementation at doses of 25, 50, and 100 mg/
Accepted 22 August 2011 kg for 28 days. Flow cytometry analysis of CA polyphenols-treated aged rats showed increased T and B
Available online 28 August 2011
cells percentage along with enhanced proliferation of splenocytes in both resting and LPS-stimulated
cells. Increased percentage of pan T cells is further supported by an elevation of CD4+, CD8+, and
Keywords: CD4+CD25+ regulatory cells. In terms of innate immune cell activity, CA polyphenol supplementation
Cassia auriculata
reduced the oxidative burst activity of neutrophils in response to PMA and Escherichia coli activation.
Immunosenescence
T cells
Our results collectively show that polyphenols derived from CA boost T cell immunity by increasing
Neutrophils the number of T cells and its sensitivity towards stimulants and decreasing ROS production by neutro-
Splenocytes phils that could potentially harm multiple biological systems in aged individuals.
Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction to rejuvenate age-affected immune functions. In line with this,

Immunosenescence is an age-related decline of immune func-
tion which affects both innate and adaptive immunity, whereby
the 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 and
inflammatory states that underlie the aging process are the basis
of 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 tissue
damage caused by infections in elderly individuals. The overall im-
pact of immunosenescence affects the primary lymphoid organs
which impede the production and maturation of T and B cells. This
includes 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 mucosal
immunity [6]. Synthetic and natural supplements have been
widely consumed as an alternative therapy to reduce the effect
of immunosenescence. It has been found that nutritional interven-
tions with polyphenolic antioxidants constitute a good alternative
⇑ Corresponding author at: Immunology Unit, Department of Pathology, Faculty
of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor, Malaysia. Fax: +60 3 8941 3802.
E-mail address: r.rajesh@medic.upm.edu.my (R. Ramasamy).
here we have reported the first time, the immunomodulatory
activity of polyphenols derived from Cassia auriculata flowers to
resuscitate the immune system of aged rats.
C. auriculata (Cesalpinaceae, common name: Tanner’s Cassia)
has been long used in traditional medicine by Asians. The ancient
Ayurvedic and Sidha medicines are often consume all components
(leaf, stem, root, and flower) of CA in their medicinal preparation. It
has been used as remedy for rheumatism, conjunctivitis, diabetes,
general well being and pre & postnatal treatments [7]. The current
literature 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 renal
injury was also reported, and is derived from the antioxidant and
free-radical-scavenging properties of CA [9]. Supplementation of
CA leaf extract has shown to protect the liver from free radical
mediated 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 immune
functions by utilising phytochemicals especially plant-derived
polyphenols. Ongoing research has highlighted the dynamic capac-
ity of polyphenols to protect against age-associated disorders
through a variety of important mechanisms [15]. Investigations
on exploring the immunomodulatory potential of plant-derived

0008-8749/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.cellimm.2011.08.017
 C.M. John et al. / Cellular Immunology 271 (2011) 474–479
polyphenol fraction revealed its effectiveness to inhibit the mito-
gen-stimulated proliferation of peripheral blood mononuclear cells
through LPS stimulation [16,17]. Moreover, Amrutesh et al. have
reported that CA contains a relatively higher level of polyphenols
as active ingredients and has been verified for its pharmacological
safety [18]. It also contains several active constituents such as
flavonoids, b-sitosterol- b-D-glucoside, polysaccharides, anthra-
cene, dimeric procyanidins and myristyl alcohol [19]. This study
was 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 Research
Institute Malaysia). Samples were stored in a herbarium as voucher
no. 18-1. The polyphenol compound of CA was extracted as previ-
ously described [20]. Briefly, flowers of CA were shade-dried for
few days; coarsely powdered; soaked in distiled water at the ratio
of 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 fraction
was collected from ethyl acetate and the total phenolic content
was 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 were
purchased from Sigma–Aldrich USA.
2.2. Animals observation
Female (350–380 g of 24–26 months old) SPF Sprague–Dawley
rats procured from Animal House of UiTM Puncak Alam, Malaysia
was used for the study. The animals were maintained as per OECD
guidelines [22].
2.3. Treatment
SD rats were randomly divided into five groups and each group
consists of at least six animals. The first two groups were assigned
as controls and respectively received a daily dose of vitamin C at
150 mg/kg and 0.5% CMC (solvent for polyphenols). Treatment
groups were divided into three dosages as 25, 50 and 100 mg/kg
and 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/day
dose. The dosage levels were selected based on the results of ear-
lier literatures [8,11]. All extract solutions were prepared freshly
and the volume of CA administered was adjusted to 1 ml/100 g
of body weight.
2.4. Measurement of ROS secretion
ROS secretion of neutrophils was measured by a commercially
available 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 and
incubated for 20 min at 37 °C. At end of incubation, 20 ll of the
fluorogenic substrate was added and cells were then lysed and
fixed by lysing solution for 10 min at room temperature. Cells were
washed and stained with DNA staining solution prior to flow
cytometer (FACS Canto) acquisition and thereafter analysed by
FACS Diva software for the DNA content and ROS secretion [23].
475
2.5. Splenocytes proliferation assay
Spleens were harvested from sacrificed rats and splenocytes
were isolated using a previously described standard procedure
[24]. Hundred thousand splenocytes were plated in each well of
96 well plates; stimulated separately with PHA and LPS with
respective concentrations for three days. Cultures were pulsed
with 10 ll (3H-TdR) 24 h prior to the measurement and the cells
were harvested onto glass filter mats by using a 96-well plate auto-
mated cell harvester (Harvester Mach III M, TOMTEC). The filter
mat was dried using oven (120 °C) for 10 min before adding 5 ml
scintillation 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 luminescent
Microbeta counter (Wallac). Results were expressed as counts
per minute (CPM).
2.6. Flow cytometer analysis
To analyse the degree of expression of cell surface proteins on
splenocytes, cells were incubated with anti rat antibodies against
CD3, CD4, CD8 and CD25. The total T, B and NK cells percentages
were measured by a commercially available TBNK cocktail. All
antibodies and cocktail kits were purchased from Becton Dickin-
son, USA. The stained samples were assessed by the FACS Canto
flow cytometer (Becton Dickinson, USA) and the data analysed
using FACS Diva software. The relevant isotype antibody controls
were 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 ANOVA
and Dunnet test. Significance levels were set at p value <0.05.
3. Results
3.1. CA supplementation increases the percentages of pan splenic T and
B cells
The flow cytometer analysis of total T, B and NK cell percentages
indicates that the CA polyphenols supplementation at all dosages
increases 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 and
100 mg dosages. Although vitamin C supplementation at 150 mg
significantly increased the T and B cell percentages, yet the magni-
tude of increase is much lower than the 50 and 100 mg dosage
groups. At all concentrations of CA polyphenols, the percentage
of NK cells remained similar to the untreated group.
3.2. CA supplementation increases the percentages of T helper, T
cytotoxic and regulatory T cells
The effect of CA polyphenol supplementation was further tested
on the percentage of helper and cytotoxic T cell populations. Help-
er and cytotoxic T cells were determined by expression of CD4 and
CD8 markers respectively. In line with a total increase in T cells by
measurement of CD3, both CD4 and CD8 expressions were found to
be 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+ and
CD8+ cells percentages with approximately two-fold increases.
Similarly, the percentage of CD4+CD25+ cells numbers was highest
476 C.M. John et al. / Cellular Immunology 271 (2011) 474–479

Fig. 1. Assessment of percentage of T, B and NK cells expression. Splenocytes from CA polyphenol supplemented and non-supplemented aged rats were assessed for
expression 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 cells
expression significantly. The results are expressed as mean ± SD from three independent experiments. ⁄ Significance was determined at p < 0.05.

at the 50 mg dosage of CA polyphenols (Fig. 2B). Although all dos-
ages of CA polyphenols increased the CD4+CD25+ T cells, yet the
significant differences could only be observed in 50 and 100 mg
dosages of CA polyphenols. Vitamin C supplementation did not al-
ter the percentage of CD4+CD25+ cells.
3.3. CA supplementation enhanced LPS stimulated splenocytes
proliferation
In order to determine whether the increased percentage of T
cells by CA polyphenol supplementation is accompanied by func-
tional enhancement; splenocyte proliferation assay was carried
out 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). The
highest proliferation was noted in the 50 mg dosage group and the
proliferation rate declined significantly at 100 mg dosage.
Although PHA did not induce substantial proliferation in all test
groups; 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 of
splenocytes 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 available
phagoburst assay. Among the three stimulators used, PMA exerted
a maximal secretion of ROS followed by E. coli. CA polyphenols at
50 and 100 mg dosages significantly reduced the ROS secretion in-
duced by PMA (Fig. 4). The inhibition of ROS secretion induced by
PMA reflected a dose dependent trend, with the highest inhibition
noted at 100 mg dosage. For ROS secretion induced by E. coli, the
highest inhibition occurred at 50 mg dosage. fMLP stimulation
did 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 to
vaccination [26]. It has been postulated that the distortion of
oxidative–anti oxidative balance, diminished function of lymphoid
organs such as bone marrow and thymus and declined stem cell
reservoirs play vital roles in affecting the immune integrity of aged
individuals [27]. Many studies have been conducted to restore nor-
mal immune function in the elderly by harmonising oxidative
stress, rejuvenating the thymus and consuming synthetic and nat-
ural supplements to ameliorate the putrefying biological system. In
our 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 chronic
illness, especially diabetes [11,14]. Most of the current literatures
on CA either in the forms of aqueous, ethanolic or polyphenolic
extractions are focused on its anti diabetic effect via correction of
hyperglycaemia and immune dysfunctions [8,11]. To date, there
are no reports indicating similar immunomodulatory activities of
CA in the immunosenescence paradigm, either using human or ani-
mal models. Therefore, our work will serve as the first report in
elaborating the immunomodulatory effect of CA polyphenols in
aged rats. We have utilised rats at age of 24–26 months old as this
range is comparably equivalent to the human age of 50–60 years
old [28].
Our results indicate that polyphenols derived from CA flowers
showed 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+ and
CD8+T cell populations (Fig. 2A). It has been documented that
aging causes reduced production of lymphocytes as the haemato-
poietic compartment of bone marrow shrinks with increasing age
and is replaced by adipose tissue [29]. The expression of CD4+ cells
were higher in aged rats compared to CD8 cells which reflect the
normal aging physiology of peripheral T cell compartments. It
has been also showed that reduced CD8+ T cells numbers and
the delayed cytolytic capability is observed in old mice compared
to young mice following in vivo inoculation with influenza virus
[30]. It would be extremely beneficial to boost the quantity and
composition of adaptive immune cells which may reduce the mor-
bidity, mortality and disability in elderly. Although, we have not
assessed the absolute cell counts of these adaptive cells, it is
deducible that CA supplementation augments the adaptive
immune cell compartment as the interplay between adaptive and
 C.M. John et al. / Cellular Immunology 271 (2011) 474–479 477

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 and
analysed by flow cytometer. CA polyphenol supplementation increases the composition of CD4+ and CD8+ cells (A) and CD4+CD25+ regulatory T cells. Supplementation of
vitamin C serves as positive control. The results are expressed as mean ± SD from three independent experiments. ⁄ Significance was determined at p < 0.05.

innate immune system is an important mechanism to provide
complete protection towards pathogens. To warrant an effective
immune response, a proper composition of immune cells at appro-
priate cell numbers is necessary. However, CA supplementation
does not alter the composition of NK cell as the percentage of
CD56+ cells were consistent with all dosages of CA polyphenols
(Fig. 2).
The increased percentage of CD4+ cells also markedly altered
the percentage of CD4+CD25+ regulatory T cells (Fig. 2B). Although
our findings report a significant small increase (1%) of
CD4+CD25+ regulatory T cells, however the current data on regula-
tory T cell number in the elderly remains unclear. It has been
shown that aged individuals have increased regulatory T cells in
peripheral blood [31]. However, our results should be interpreted
with caution as T regulatory cells were measured solely by
CD4+CD25+ expression and lacks Foxp3 analysis. Furthermore it
is not clear whether these regulatory T cells were derived from
the thymus or induced at the periphery.
We also demonstrated that the elevated T cell percentage
was strongly associated with its proliferative index, as CA
supplementation enhanced the proliferative rate of resting and
LPS stimulated T cells (Fig. 3). Reduced T cell proliferation is a
common feature of immunosenescence as the ability of T cells to
respond to external stimuli and their subsequent production of
cytokines were reported to be compromised [32]. CA supplementa-
tion profoundly increased the proliferation of T cells at a dosage of
50 mg/kg. Although vitamin C supplementation had incurred with
an elevated proliferation of T cells as well, yet the impact of CA
polyphenols on such proliferation is much greater. However, T cells
from aged rats did not respond towards PHA stimulation in both
control and treated groups. The limited ability of aged rats’ T cells
to respond to LPS but not to PHA may implicate the impaired
sensitivity of certain surface receptors or signalling pathways in
immunosenescenced T cells as both the ligands and signalling
pathways for LPS and PHA are different. Nonetheless, the effect
of CA polyphenols in both T cell percentage and proliferation in
aged rats promise a good herbal-based prophylactic therapy for
enhanced adaptive immunity.
Oxidative stress is considered as the most important overall
mechanism for cellular aging which leads to immunosenescence.
478 C.M. John et al. / Cellular Immunology 271 (2011) 474–479

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 cells
determined by 3H-thymidine assay. CA polyphenol supplementation profoundly increases the proliferation rate of splenocytes in response to LPS stimulation. The results are
expressed 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 polyphenol
supplementation inhibit the ROS production of neutrophils in response to PMA and E. coli stimulation. The results are expressed as mean ± SD from three independent
experiments. ⁄ Significance was determined at p < 0.05.

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; fragility
towards spontaneous release of ROS had made aged individuals
vulnerable towards tissue damages [33]. Our findings suggest that
generation of excessive and spontaneous ROS due to age-associ-
ated immune dysregulation can be modulated by supplementing
CA. The harnessing of oxidative stress exerted by CA is believed
to be derived from its high antioxidant activity through enzymes
like superoxide dismutase, catalase, glutathione peroxidise to as-
sist in detoxification of ROS [14,34].
In summary, our study provides scientific evidence to a series of
phenomena that are considered to contribute to the aging process
which can be altered by CA polyphenols. It clearly shows immuno-
modulatory effects of CA on various immune cell compositions and
their relevant functions. This CA polyphenol immunomodulatory
activity also could be exploited towards ameliorating pathological
conditions such as diabetic, cancer and AIDS. Furthermore, it
supports drug development from CA with the aim of creating
evidence-based plant medications in prevention and treatment of
immune diseases in the form of new single treatments or new
combinatory 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 Teknologi
MARA.
References
[1] I. Baeza, N.M. De Castro, L. Arranz, M. De la Fuente, Soybean and green tea
polyphenols improve immune function and redox status in very old
ovariectomized mice, Rejuvenation Res 13 (2010) 665–674.
[2] G.G. Steinmann, Changes in the human thymus during aging, Curr Top Pathol
75 (1986) 43–88.
 C.M. John et al. / Cellular Immunology 271 (2011) 474–479
[3] L. Haynes, S.M. Eaton, E.M. Burns, T.D. Randall, S.L. Swain, CD4 T cell memory
derived from young naive cells functions well into old age, but memory
generated from aged naive cells functions poorly, Proc Natl Acad Sci USA 100
(2003) 15053–15058.
[4] D. Frasca, A.M. Landin, S.C. Lechner, J.G. Ryan, R. Schwartz, R.L. Riley, B.B.
Blomberg, Aging down-regulates the transcription factor E2A Activation-
induced cytidine deaminase, and Ig class switch in human B cells, J Immunol
180 (2008) 5283–5290.
[5] A. Ademokun, Y.C. Wu, D. Dunn-Walters, The ageing B cell population:
composition and function, Biogerontology 11 (2010) 125–137.
[6] D.L. Schmucker, R.L. Owen, R. Outenreath, K. Thoreux, Basis for the age-related
decline in intestinal mucosal immunity, Clin Dev Immunol 10 (2003) 167–172.
[7] P.S. Vijayaraj, K. Muthukumar, J. Sabarirajan, V. Nachiappan, Evaluation of
antihyperlipidemic activity of ethanolic extract of Cassia auriculata flowers,
Indian J Biochem Biophys 48 (2011) 54–58.
[8] M. Latha, L. Pari, Preventive effects of Cassia auriculata L. Flowers on brain lipid
peroxidation in rats treated with streptozotocin, Mol Cell Biochem 243 (2003)
23–28.
[9] S. Annie, P.L. Rajagopal, S. Malini, Effect of Cassia auriculata Linn. root extract
on cisplatin and gentamicin-induced renal injury, Phytomedicine 12 (2005)
555–560.
[10] S. Kumar Rajagopal, P. Manickam, V. Periyasamy, N. Namasivayam, Activity of
Cassia auriculata leaf extract in rats with alcoholic liver injury, J Nutr Biochem
14 (2003) 452–458.
[11] M. Latha, L. Pari, Antihyperglycaemic effect of Cassia auriculata in
experimental diabetes and its effects on key metabolic enzymes involved in
carbohydrate metabolism, Clin Exp Pharmacol Physiol 30 (2003) 38–43.
[12] S.J. Surana, S.B. Gokhale, R.B. Jadhav, R.L. Sawant, J.B. Wadekar,
Antihyperglycemic Activity of Various Fractions of Cassia auriculata Linn. in
Alloxan Diabetic Rats, Indian J Pharm Sci 70 (2008) 227–229.
[13] S. Gupta, S.B. Sharma, K.M. Prabhu, Ameliorative effect of Cassia auriculata L.
leaf extract on glycemic control and atherogenic lipid status in alloxan-
induced diabetic rabbitss, Indian J Exp Biol 47 (2009) 974–980.
[14] S. Gupta, S.B. Sharma, K.M. Prabhu, S.K. Bansal, Protective role of Cassia
auriculata leaf extract on hyperglycemia-induced oxidative stress and its
safety evaluation, Indian J Biochem Biophys 46 (2009) 371–377.
[15] J. Gonzalez-Gallego, M.V. Garcia-Mediavilla, S. Sanchez-Campos, M.J. Tunon,
Fruit polyphenols, immunity and inflammation, Br J Nutr 104 (Suppl. 3) (2010)
S15–S27.
[16] C. Sanbongi, N. Suzuki, T. Sakane, Polyphenols in chocolate which have
antioxidant activity, modulate immune functions in humans in vitro, Cell
Immunol 177 (1997) 129–136.
[17] I. Rodriguez-Ramiro, S. Ramos, L. Bravo, L. Goya, M.A. Martin, Procyanidin B2
and a cocoa polyphenolic extract inhibit acrylamide-induced apoptosis in
human Caco-2 cells by preventing oxidative stress and activation of JNK
pathway, J Nutr Biochem 2011, accepted for publication (Epub ahead of print).
[18] G.H. Amrutesh S. Puranik, Sandeep Kumar, Ranjan Mogre, Kishori Apte, Ashok
D. B. Vaidya, Bhushan Patwardhan1, Cassia auriculata: aspects of safety
pharmacology and drug interaction, evidence-based complementary and
alternative medicine, 2011 (2011) 8.
479
[19] G. Nageswara Rao, P. Mahesh Kumar, V.S. Dhandapani, T. Rama Krishna, T.
Hayashi, Constituents of Cassia auriculata, Fitoterapia 71 (2000) 82–83.
[20] C.M. Ajila, S.K. Brar, M. Verma, R.D. Tyagi, S. Godbout, J.R. Valero, Extraction
and Analysis of Polyphenols: Recent trends, Crit Rev Biotechnol 31 (2011)
227–249.
[21] R. Ksouri, H. Falleh, W. Megdiche, N. Trabelsi, B. Mhamdi, K. Chaieb, A. Bakrouf,
C. Magne, C. Abdelly, Antioxidant and antimicrobial activities of the edible
medicinal halophyte Tamarix gallica L. and related polyphenolic constituents,
Food Chem Toxicol 47 (2009) 2083–2091.
[22] L. Institoris, O. Siroki, I. Desi, J. Lesznyak, P. Serenyi, E. Szekeres, I. Petri,
Extension of the protocol of OECD guideline 407 (28-day repeated dose oral
toxicity test in the rat) to detect potential immunotoxicity of chemicals, Hum
Exp Toxicol 17 (1998) 206–211.
[23] K. Parment, A. Zetterberg, J. Ernerudh, K. Bakteman, I. Steinwall, F. Sjoberg,
Long-term immunosuppression in burned patients assessed by in vitro
neutrophil oxidative burst (Phagoburst), Burns 33 (2007) 865–871.
[24] D.B. Millar, J.R. Thomas, N.D. Pacheco, F.M. Rollwagen, Natural killer cell
cytotoxicity and T-cell proliferation is enhanced by avoidance behavior, Brain
Behav Immun 7 (1993) 144–153.
[25] International Conference on Harmonisation; Guidance on S8 Immunotoxicity
Studies for Human Pharmaceuticals; availability. Notice, Fed Regist, 71 (2006)
19193–19194.
[26] D. Boraschi, G. Del Giudice, C. Dutel, B. Ivanoff, R. Rappuoli, B. Grubeck-
Loebenstein, Ageing and immunity: addressing immune senescence to ensure
healthy ageing, Vaccine 28 (2010) 3627–3631.
[27] I. Beerman, W.J. Maloney, I.L. Weissmann, D.J. Rossi, Stem cells and the aging
hematopoietic system, Curr Opin Immunol 22 (2010) 500–506.
[28] R. Quinn, Comparing rat’s to human’s age: how old is my rat in people years?,
Nutrition 21 (2005) 775–777.
[29] J.E. Compston, Bone marrow and bone: a functional unit, J Endocrinol 173
(2002) 387–394.
[30] R.B. Effros, R.L. Walford, The immune response of aged mice to influenza:
diminished T-cell proliferation, interleukin 2 production and cytotoxicity, Cell
Immunol 81 (1983) 298–305.
[31] P. Trzonkowski, E. Szmit, J. Mysliwska, A. Mysliwski, CD4+CD25+ T regulatory
cells inhibit cytotoxic activity of CTL and NK cells in humans-impact of
immunosenescence, Clin Immunol 119 (2006) 307–316.
[32] A.F. Majdalawieh, R. Hmaidan, R.I. Carr, Nigella sativa modulates splenocyte
proliferation, Th1/Th2 cytokine profile, macrophage function and NK anti-
tumor activity, J Ethnopharmacol 131 (2010) 268–275.
[33] C. Wenisch, S. Patruta, F. Daxböck, R. Krause, W. Hörl, Effect of age on human
neutrophil function, J Leukoc Biol 67 (2000) 40–45.
[34] A. Kumaran, R.J. Karunakaran, Antioxidant activity of Cassia auriculata flowers,
Fitoterapia 78 (2007) 46–47.
[35] G. Ulrich-Merzenich, D. Panek, H. Zeitler, H. Vetter, H. Wagner, Drug
development from natural products: exploiting synergistic effects, Indian J
Exp Biol 48 (2010) 208–219.
[36] B. Patwardhan, R.A. Mashelkar, Traditional medicine-inspired approaches to
drug discovery: can Ayurveda show the way forward?, Drug Discov Today 14
(2009) 804–811.