Food Chemistry 135 (2012) 2222–2228

Contents lists available at SciVerse ScienceDirect

Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Decreasing pro-inflammatory cytokine and reversing the immunosenescencewith extracts of Pu-erh tea in senescence accelerated mouse (SAM)

Liang Zhang a, Wan-fang Shao b, Li-feng Yuan c, Peng-fei Tu a, Zhi-zhong Ma d,⇑a Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University, Beijing 100191, Chinab College of Long Run Pu-erh Tea, Yunnan Agricultural University, Kunming, Yunnan 650201, Chinac Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, IN 47907-2063, USAd School of Basic Medical Science, Peking University, Beijing 100191, China

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

Article history:Received 4 April 2011Received in revised form 14 June 2012Accepted 4 July 2012Available online 15 July 2012

Keywords:ImmunosenescenceNaïve T lymphocytesNK cellsInterleukin-6Pu-erh teaImmune function

0308-8146/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2012.07.033

⇑ Corresponding author. Address: School of BasUniversity, Beijing 100083, China. Tel.: +86 10 82802

E-mail address: zhma@bjmu.edu.cn (Z.-z. Ma).

Immunosenescence, the progressive decline of adaptive immunity and chronic inflammation with ageinghas been demonstrated to be the main factor responsible for infections, cancer and autoimmuneconditions in the elderly. Senescence-accelerated mouse (SAM) was used to study the protective effectsof Pu-erh tea in the elderly. The senile-prone sub-strain, SAM-P8 mice were administered individuallywith ripened or crude Pu-erh tea at 125, 250 or 500 mg/kg. The results showed that Pu-erh tea signifi-cantly increased the fractions of naïve T lymphocytes, CD8+CD28+ T lymphocytes and NK cells in theperipheral blood, but decreased the levels of IL-6 in aged mice. These data suggested that the Pu-erhtea reversed the immunosenescence by restoring the immune deficiency and decreasing pro-inflamma-tory cytokine. Thus, long term drinking of Pu-erh tea may be beneficial for the aged population in terms ofincreasing the body’s resistance to infection and cancer.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Pu-erh tea was documented in ancient Chinese classics to helpthe human body in restraining from infections, anti-obesity anddelaying ageing when applied in the elderly. Pu-erh tea is producedusing leaves of Camellia sinensis (Linn.) var. assamica (Mast.) Kitam-ura, a tea variety from the Yunnan province of China. Pu-erh tea iscategorized into crude Pu-erh tea and ripened Pu-erh tea accordingto the fermentation process. Chemical studies revealed that itsmain molecular components were tea polyphenols, flavanols, andtea polysaccharides (Xie et al., 2009). In recent years, numerousbiomedical studies have shown that Pu-erh tea possesses multiplebioactivities such as hypolipoidemia, hypocholesterolaemia, anti-obesity, antioxidation and anti-inflammation (Hou et al., 2009;Jeng, Chen, Fang, Hou, & Chen, 2007; Kuo et al., 2004; Lin & Lin-Shi-au, 2006). However, the effects in reducing or limiting theinfection, especially for the aged population, have never beenaddressed and confirmed experimentally. The mechanisms associ-ated with these effects have not yet been clarified.

Ageing is associated with a decline in immune functions knownas immunosenescence (Aw, Silva, & Palmer, 2007), which is de-fined as the state of deregulated immune function that contributes

ll rights reserved.

ic Medical Science, Peking404; fax: +86 10 82802750.

to the increased susceptibility to infection, cancer and autoim-mune diseases observed in old people and various animals.

Although many kinds of cells and factors were involved in theprocess of immunosenescence, the key point was that the fractionof naïve T lymphocyte in adaptive immune system declinedprogressively with ageing, while the proportion of memory T lym-phocytes was expanded (Linton & Dorshkind, 2004). Additionally,the activated lymphocytes such as CD8+CD28+ T lymphocytesand natural killer cells (NKs), which are the front line of immunecells attacking and destroying the virus hosted cells, cancer cellsand various pathological microorganisms, also decline with ageing(Frahm et al., 2006).

The senescence-accelerated mouse (SAM) is inbred mouse mod-el of ageing derived from AKR/J strain. Among the strain, the senes-cence-accelerated mouse prone sub-strain (SAM-P8) has shorterlife span than senescence-accelerated mouse resistant sub-strain(SAM-R1) which is present with normal ageing process and lifespan (Frahm et al., 2006). In parallel with early onset of ageing,SAM-P8 mice illustrate the immunological deregulated alterationsand neurological senescence, which is associated with endothelialsenescence in the hippocampus and is ameliorated by testosteronereplacement. Then, it is widely used as naturally developed animalmodel to study ageing and anti-ageing therapeutics.

In this study, we examined the effects of aqueous extracts ofripened and crude Pu-erh tea on SAM. After oral administrationof Pu-erh tea extract for 4 weeks, the proportions of naive T

Table 1The contents of main polyphenol compounds of Pu-erh tea (mg/g).

Compoundsa GA GC EGC C EC EGCG GCG ECG

Crude Pu-erh tea 1.167 3.472 4.921 3.164 6.160 9.681 1.373 9.920Ripened Pu-erh tea 6.549 2.892 0.407 0.222 0.587 0.079 0.042 0.052

a GA: gallic acid; GC: gallocatechin; EGC: epigallocatechin; C: catechin; EC: epicatechin; EGCG: epigallocatechin gallate; GCG: gallocatechin gallate and ECG: epicatechingallate.

L. Zhang et al. / Food Chemistry 135 (2012) 2222–2228 2223

lymphocytes in the peripheral blood or spleen were analysed.Furthermore, activated CD8+CD28+ cells, NKs and cytokine inter-leukin-6 (IL-6) in SAM-P8 were measured.

2. Materials and methods

2.1. Materials

Crude and ripened Pu-erh tea was provided by China Academyof Pu-erh Tea Research. (Catalog No: 532721-616, Long Run Pu-erh, Yunnan, China). The tea leaves were collected from plantsgrown in the Yunnan highlands of China. Leaves were collectedand heated, dried at <60 �C, and moulded to make crude Pu-erhtea. To make ripened Pu-erh tea, the crude Pu-erh tea was damp-ened and ripened with a pure culture of Aspergillus niger for50 days at controlled temperature and humidity. Ripened Pu-erhtea was then dried at <60 �C and packed.

Fluorescein isothiocyanate (FITC), phycoerythrin (PE) andperidinin chlorophyll protein (Percp) conjugated monoclonal anti-bodies (FITC-CD4, Percp-CD3, Percp-CD8a, PC-CD28, PE-CD44,FITC-CD45RB, PE-CD49b/pank, and FITC-ly-6A/E (Sca-1)) were

Fig. 1. Flow cytometric analysis of Naïve T and memory T lymphocytes in the peripheral bT lymphocytes in the peripheral blood and splenic lymphocytes correlated with age and hT cells, and memory T cells, represented as CD3+CD44lowCD45RBhigh cells were measuredgenerated by gating on CD3+ T cells. The examples are representative for subjects belrepresent the thresholds used to distinguish CD3+, CD44high and CD45RBlow subsets. Thelymphocytes of peripheral blood lymphocytes (PBL). (B) FACS plots of naïve T lymphocymeasured in PBL. (D) The percentage of CD3+CD44highCD45RBlow T cells (naïve T cells) waCP-L: low dose of crude Pu-erh tea group, CP-M: medium dose of crude Pu-erh tea grougroup, RP-M: medium dose of ripened Pu-erh tea group and RP-H: high dose of ripened

purchased from BD Biosciences (San Diego, CA, USA). CBA Flexset including IFN-c, TNF-a, IL-12p70, IL-6, IL-10 and MCP-1 werealso purchased from BD Biosciences (San Diego, CA, USA).

2.2. Preparation of Pu-erh tea extracts

Pu-erh tea was minced and extracted with 15 times of boilingdistilled water for 20, 20 and 10 min consecutively. The extractedsolution was concentrated at 60 �C under reduced pressure to aconcentration of 0.1 mg raw material per ml solution. Concen-trated tea solution was subsequently kept in a home refrigerator(4 �C) for 1 month.

2.3. Determination of polyphenol contents in Pu-erh tea

One ml of extracted tea solution was filtered through a0.5 lm Millipore filter and then injected to HPLC for analysis.The contents of eight polyphenol compounds including gallicacid (GA), (+)-catechin (C), (�)-epicatechin (EC), (�)-gallocate-chin (GC), (�)-epigallocatechin (EGC), (�)-gallocatechin gallate(GCG), (�)-epigallocatechin gallate (EGCG) and (�)-epicatechin

lood and splenic lymphocytes of SAM-P8 and SAM-R1 mice. The percentage of naïveealth status. The percentage of naïve T cells, represented as CD3+CD44highCD45RBlow

in the samples of peripheral blood and splenic cells (SC). Presented FACS plots wereonging to the particular groups distinguished in the study. The lines in each plotnumbers are the mean number (% of the total cells) ± S.D. (A) FACS plots of naïve Ttes of SC. (C) The percentage of CD3+CD44highCD45RBlow T cells (naïve T cells) was

s measured in SC. R1: senescence-resistant group, P8: senescence-accelerated group,p, CP-H: high dose of crude Pu-erh tea group, RP-L: low dose of ripened Pu-erh teaPu-erh tea group. ⁄p < 0.05, ⁄⁄p < 0.01.

Fig. 2. Flow cytometric analysis of CD8+CD28+ T cells in the PBL of SAM-P8 and SAM-R1 mice. The data are the mean number (% of the total cells) ± S.D. Compared with SAM-R1, the CD8+CD28+ T cells of SAM-P8 were significantly decreased. After treatment with crude and ripened Pu-erh tea, the percentage of this subgroup of T lymphocytes wererecovered compared with SAM-P8. (A) FACS plots of CD8+CD28+ T cells in PBL. (B) The percentage of CD8+CD28+ T cells was measured in PBL. ⁄p < 0.05, ⁄⁄p < 0.01.

2224 L. Zhang et al. / Food Chemistry 135 (2012) 2222–2228

gallate (ECG) crude and ripened Pu-erh tea were determinedaccording to Zhang, Li, Ma, and Tu (2011).

2.4. Animals and treatment

Male SAM-P8 and their control SAM-R1 at the age of 8 monthswere purchased from the laboratory of Animal Breeding and Re-search Center of Peking University Health Science Centre (Beijing,China). This study was approved by the Peking University AnimalsResearch Committee and carried out according to the guidelines ofthe Care and Use of Laboratory Animals in Peking University. Thecertificate code of these animals was SYXK 2002-0002. These micewere kept in an environmentally controlled room (22 ± 2 �C, 45–60% relative humidity, and a 12 h light/dark cycle) and wereallowed free access to food and water. After 1 week of acclimation,the mice were divided into eight groups, which included six

treatment groups, SAM-R1 control group and SAM-P8 vehiclegroup. These six treatment groups were orally administrated witheither crude or ripened Pu-erh tea extract, which yielded doses of125, 250 or 500 mg/kg. The SAM-R1 control group and SAM-P8vehicle group were given oral administration of distilled water.After 28 days of treatment, the animals were fasted for 12 h andthen blood was drawn from orbit in the anaesthetized condition.The plasma was prepared by centrifugation of blood at 2500�gfor 30 min, and then aliquots of 100 ll of plasma were loaded intotwo eppendorf tubes separately and subsequently placed in ultralow temperature freezer (�60 �C) for 1 week.

2.5. Cytometric bead array immunoassay

Plasma cytokine levels were quantified using a cytometric beadarray (CBA) assay kit (BD Biosciences, San Diego, CA, USA), which is

Fig. 3. Flow cytometric analysis and the percentage of NK cells in the peripheral blood and splenic lymphocytes of SAM-P8 and SAM-R1 mice. The numbers are the mean (% ofthe total cells) ± S.D. Compared with senescence-resistant mice. The NK cells of senile SAM-P8 mice were significantly declined. After treatment with Pu-erh tea and ripenedPu-erh tea, NK cells of SAM-P8 in the peripheral blood and splenic lymphocytes were recovered. (A) FACS plots of NK cells in PBL. (B) FACS plots of NK cells in SC. (C) Thepercentage of NK cells (CD3+CD499+) was measured in PBL. (D) The percentage of NK cells was measured in SC. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.

L. Zhang et al. / Food Chemistry 135 (2012) 2222–2228 2225

capable of detecting IFN-c, TNF-a, IL-6, MCP-1, IL-12p70 or IL-10.Briefly, 50 ll of supernatants mixed with 50 ll of PE-conjugatedcytokine capture beads. After 1 h of incubation, samples werewashed, fixed with 1% paraformaldehyde, and analysed by FAC-SCalibur™ flow cytometer (BD Biosciences, San Diego, CA, USA).

2.6. Labelling flow cytometric analysis

Surface expression of various markers was determined usingFACSCalibur™ flow cytometer (BD Biosciences, San Diego, CA,USA) and BD CellQuest analysis software.

Peripheral blood mononuclear cells and splenic lymphocyteswere incubated with FITC-CD4, Percp-CD3, Percp-CD8a, PC-CD28,PE-CD44, FITC-CD45RB, PE-CD49b/pank, and FITC-ly-6A/E (Sca-1)monoclonal antibodies at room temperature for 30 min. Red cellswere then lysed by incubation with 1.0 ml FACS lysing solutionat room temperature for 5 min under dark. After centrifugationat 500�g for 5 min, the supernatant was decanted. Then washingwith PBS for three times, all of precipitant cells were re-suspended.The lymphocyte subpopulation was analysed by flow cytometryusing FACSCalibur™ flow cytometer (BD Biosciences, San Diego,CA, USA).

2.7. Statistical analysis

Data were given as mean ± S.D. Comparisons between differentgroups were done by one way ANOVA with Post-hoc test. Statisti-cal analysis was conducted using Statistical Package for SocialSciences for Windows (SPSS, Chicago, IL, USA). Difference was con-sidered significant if p value was less than 0.05.

3. Results

3.1. The contents of polyphenol compounds of Pu-erh tea

As shown in Table 1, crude and ripened Pu-erh tea showed thesignificant difference in their contents of polyphenol compounds.

Catechins were largely decreased after ripening by A. niger, butthe gallic acid was significantly increased. These results were sup-posed to be related to the metabolism of catechin gallate esterssuch as EGCG and GCG by fungi of fermentation (Qin, Li, Tu, Ma,& Zhang, 2012).

3.2. Determination of naïve T lymphocytes, NK cell, CD8+CD28+ Tlymphocytes and Sca-1 positive cells from SAM administered with Pu-erh tea (crude and ripened)

The decreasing of naïve T lymphocytes and the increasing ofmemory T lymphocytes in the peripheral lymphocyte pool arethe prominent features of immunosenescence, and also the mainunderlying reasons for ageing-related immunological abnormali-ties. The percentages of naïve T lymphocyte in peripheral bloodand splenic lymphocytes of senescence accelerated mouse (SAM-P8) were significantly lower than that of SAM-R1 (p < 0.001), whilethe memory T lymphocytes were accumulated in the aged mice.After being administered with the extract of Pu-erh tea (crude orripened), the proportion of naïve T lymphocytes in the peripherallymphocyte pool of SAM-P8 were significantly increased (Fig. 1),but the accumulated memory T cells in SAM-P8 mice were not af-fected by Pu-erh tea application (data not showed). Moreover, theCD8+CD28+ cells, which belong to the activated effect T lympho-cytes, were elevated by Pu-erh tea used in the aged mice (Fig. 2).Additionally, the NKs, the major cellular member of innate im-mune system, were heightened in SAM-P8 mice after usingextracts of Pu-erh tea statistically significantly (Fig. 3). Flow cyto-metric analysis of Sca-1 positive cells in the peripheral blood ofSAM-P8 and SAM-R1 mice is shown in Fig. 4.

3.3. Determination of inflammatory cytokines in plasma by CBA assay

CBA techniques were able to detect picogram levels of cytokinesin plasma. IFN-c, TNF-a, MCP-1, IL-12p70 and IL-10 did not showsignificant differences after being administered with Pu-erh tea in

Fig. 4. Flow cytometric analysis of Sca-1 positive cells in the peripheral blood of SAM-P8 and SAM-R1 mice. Intensity of fluorescence is plotted on the x axis and the numberof cells plotted on the y axis. Sca-1 expression of SAM-P8 was significantly lower compared with the SAM-R1. After treatment with crude and ripened Pu-erh tea, the Sca-1expression was enhanced markedly in peripheral blood of SAM-P8. (A) FACS plots of Sca-1 positive cells in PBL. (B) The percentage of Sca-1 positive cells in PBL. ⁄p < 0.05,⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.

2226 L. Zhang et al. / Food Chemistry 135 (2012) 2222–2228

SAM-P8, but IL-6 was significantly decreased (Fig. 5). The concen-tration of IL-6 was decreased from 49.32 ± 15.93 pg/ml to a valuein the range of 7.46 ± 8.29–28.37 ± 12.67 pg/ml in three dosegroups (p < 0.05–0.01).

4. Discussion

This study is the first to demonstrate beneficial effects of oraladministration of aqueous Pu-erh tea extract in an aged mousemodel. It indicates that Pu-erh tea is able to increase the propor-tions of naïve T lymphocytes significantly in the peripheral

lymphocyte pool. In addition, the depleted CD8+CD28+ effect Tlymphocytes in the aged mice is replenished remarkably. For theinnate immune system, NKs in the peripheral blood and spleenpool is also restored observably. On the contrary, the level of IL-6is reduced significantly.

Infectious diseases, malignancies, inflammatory and autoim-mune disorders are major causes of morbidity and mortality inthe elderly. Increased susceptibility to these ageing-related dis-eases may stem from underlying dysfunctions of the aged immunesystem. Ageing of the immune system involves the progressivealterations in both humoral and cellular-mediated immunity. The

Fig. 5. Concentrations of cytokine IL-6 in plasma from SAM-P8 and SAM-R1 mice byCBA testing. Statistical analysis showed that after treatment with crude and ripenedPu-erh tea, the pro-inflammatory cytokine IL-6 in plasma of SAM-P8 was decreasedcompared with SAM-P8 mice. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001.

L. Zhang et al. / Food Chemistry 135 (2012) 2222–2228 2227

principal changes in cellular immunity with ageing are thedepletion of naive T lymphocytes, which plays a critical and indis-pensable role in inducing adaptive immunity and antibody produc-tion. Therefore, lack of naïve T cells will pose the aged organismswith the threat of new pathogen’s invading and increased suscep-tibility to infection. It has been proven that the medicines andinterventions such as calorie restriction (Hursting, Lavigne, Berri-gan, Perkins, & Barrett, 2003; Nikolich-Zugich & Messaoudi,2005) and vitamin E supplementation (Meydani, Han, & Wu,2005), which increase the peripheral proportion of naïve T cellsalso demonstrated to decrease the frequency of infection, tumoursand inflammatory illnesses as well as to prolonging the life span.

In the present experiment, SAM-P8 shows significant deductionof naïve T lymphocytes compared with younger counterpart, SAM-R1 mice. After administration of Pu-erh tea (125, 250 and 500 mg/kg/daily), the naïve T cells both in peripheral blood and spleen arerestored to strengthen the aged immune function against infectionand cancer.

Besides the lymphocyte quantities, the bodies resistance againstinfection or tumours also rely on the activation status of lympho-cytes. Some studies have shown that obstruction of T lymphocyteactivation may contribute to the immunity defects (Meydaniet al., 2005) associated with ageing. Our data demonstrate thatthe activated effector CD8 T cells (CD8+CD28+ cells) were decreasedin SAM-P8. This sub-group of effector T lymphocytes plays animportant role in mediating immune destroying on virus infectedor cancer cells. The Pu-erh tea were able to augment the percentageof CD8+CD28+ T cells in the aged mice, which indicated this effectmay be another pathway for inhibiting infection and cancer of el-derly by Pu-erh tea.

In addition to adaptive immunity, NKs have been confirmed toexert the immune surveillance on invaded virus and emerged can-cer cells. Although the data about the alteration of NKs with ageingwere contradicted, however, the NKs in aged mice were declineddramatically in our studies. After administration of Pu-erh tea,the NKs in the SAM-P8 were regained in the peripheral poolremarkably. These data suggest that Pu-erh tea has the restorativeeffects on aged innate immune system by replenishing the NKs andresuming the naïve T and effect T cells in aged adaptive immunesystem.

Multiple studies illustrate that the haematopoietic stem cellsand progenitors in the bone marrow may suffer from the endoge-nous and microenvironment insults, undergone gradual decline offunction in self-renewal, differentiation and proliferation, thereby

leading to shrinking of output of cells including T lymphocytesand NKs. In our studies, we used the antibody of Sca-1, one of mainmarkers of haematopoietic stem cells and progenitors in mice, toobserve the new output cells in the blood from bone marrow.The results show that the Sca-1 positive cells into the blood ofSAM-P8 mice were reduced compared with SAM-R1 mice. Thisalteration is in parallel with alterations of naïve T lymphocytesand NKs in the peripheral. These results suggest that improvingthe bone marrow function may be one of underlining reasons torebuild the peripheral immune cellular components by Pu-erhtea. However, whether and how Pu-erh tea is able to affect the agedbone marrow function needs to be further addressed.

Recently, numerous studies have reported that teas includingPu-erh tea have been demonstrated to have beneficial effects inprophylaxis of cancer and limiting the progression of cancer. Fur-thermore, many reports have shown that tea and tea polyphenolswere able to inhibit oxidative stress-induced cancerous mutations(Wei, Zhou, Cai, Yang, & Liu, 2006; Yang, Wang, Lu, & Picinich,2009). Hernaez, Xu, and Dashwood (1998) reported that greentea showed better antimutagenic activity than black tea, becauseof the high contents of EGCG and EGC in green tea. However, ourstudies showed that the ripened Pu-erh tea also existed a similaractivity as crude Pu-erh tea in despite of the significant differenceof tea polyphenols contents. These results indicated that activecompounds of Pu-erh tea with respect to the developing immunityof the elderly needs further study.

Accumulating studies show that ageing is not only manifestedas immune deficiency, but chronic inflammation as well (Awet al., 2007; Giunta, 2009; Gupta, Agrawal, Agrawal, Su, & Gollapu-di, 2006). With ageing, the pro-inflammatory cytokines, such as IL-6, TNF-a, IL-1b and C-reactive protein increased significantly.These changes were further aggravated in the age related diseasessuch as Alzheimer’s disease, Parkinson’s diseases, amyotrophic lat-eral sclerosis and atherosclerosis. This phenomenon thereby istermed as ‘‘Inflamm-ageing’’ (Facchini et al., 1987). Thus, seekingthe agents that targeting at both reversing immunodeficiencyand decreasing chronic inflammation especially for the elderly isin high priority because the accumulation of aged populationworldwide.

These results supported that the Pu-erh tea has strong beneficialeffects for the health of the elderly. Pu-erh tea is always studiedand stressed for its hypolipidemic effect, and be marketed as aslimming product, while other pharmacological functions were ne-glected. Through the effects of Pu-erh tea on the immunologicalfunction, more healthcare functions of Pu-erh tea will be valued.

Acknowledgment

This work was supported by a Grant for ‘Study on the ActiveComponents and Healthcare Function of Pu-erh tea’ Topic ID:2007BAD58B04-2 and ‘National Tea Industrial Science and Tech-nology System’ from Modern Agriculture Industrial TechnologySystem.

References

Aw, D., Silva, A. B., & Palmer, D. B. (2007). Immunosenescence: Emerging challengesfor an ageing population. Immunology, 120(4), 435–446.

Facchini, A., Mariani, E., Mariani, A. R., Papa, S., Vitale, M., & Manzoli, F. A. (1987).Increased number of circulating Leu 11+(CD16) large granular lymphocytes anddecreased NK activity during human ageing. Clinical and ExperimentalImmunology, 68(2), 340–347.

Frahm, N., Kiepiela, P., Adams, S., Linde, C. H., Hewitt, H. S., Sango, K., et al. (2006).Control of human immunodeficiency virus replication by cytotoxic Tlymphocytes targeting subdominant epitopes. Nature Immunology, 7(2),173–178.

Giunta, S. (2009). Exploring the complex relations between inflammation and aging(inflamm-aging): Anti-inflamm-aging remodelling of inflamm-aging, fromrobustness to frailty. Inflammation Research, 58(2), 118.

2228 L. Zhang et al. / Food Chemistry 135 (2012) 2222–2228

Gupta, S., Agrawal, A., Agrawal, S., Su, H., & Gollapudi, S. (2006). A paradox ofimmunodeficiency and inflammation in human aging: Lessons learned fromapoptosis. Immunity & Ageing, 3(1), 5.

Hernaez, J. F., Xu, M., & Dashwood, R. H. (1998). Antimutagenic activity of teatowards 2-hydroxyamino-3-methylimidazo[4,5-f]quinoline: Effect of teaconcentration and brew time on electrophile scavenging. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 402(1–2),299–306.

Hou, Y., Shao, W. F., Xiao, R., Xu, K. L., Ma, Z. Z., Johnstone, B. H., et al. (2009). Pu-erhtea aqueous extracts lower atherosclerotic risk factors in a rat hyperlipidemiamodel. Experimental Gerontology, 44(6–7), 434–439.

Hursting, S. D., Lavigne, J. A., Berrigan, D., Perkins, S. N., & Barrett, J. C. (2003). Calorierestriction, aging, and cancer prevention: Mechanisms of action andapplicability to humans. Annual Review of Medicine, 54, 131–152.

Jeng, K. C., Chen, C. S., Fang, Y. P., Hou, R. C. W., & Chen, Y. S. (2007). Effect ofmicrobial fermentation on content of statin, GABA, and polyphenols in Pu-erhtea. Journal of Agricultural and Food Chemistry, 55(21), 8787–8792.

Kuo, K.-L., Weng, M.-S., Chiang, C.-T., Tsai, Y.-J., Lin-Shiau, S.-Y., & Lin, J.-K. (2004).Comparative studies on the hypolipidemic and growth suppressive effects ofoolong, black, Pu-erh, and green tea leaves in rats. Journal of Agricultural andFood Chemistry, 53(2), 480–489.

Lin, J. K., & Lin-Shiau, S. Y. (2006). Mechanisms of hypolipidemic and anti-obesityeffects of tea and tea polyphenols. Molecular Nutrition & Food Research, 50(2),211–217.

Linton, P. J., & Dorshkind, K. (2004). Age-related changes in lymphocytedevelopment and function. Nature Immunology, 5(2), 133–139.

Meydani, S. N., Han, S. N., & Wu, D. (2005). Vitamin E and immune response in theaged: Molecular mechanisms and clinical implications. Immunological Reviews,205, 269–284.

Nikolich-Zugich, J., & Messaoudi, I. (2005). Mice and flies and monkeys too: Caloricrestriction rejuvenates the aging immune system of non-human primates.Experimental Gerontology, 40(11), 884–893.

Qin, J. H., Li, N., Tu, P. F., Ma, Z. Z., & Zhang, L. (2012). Change in tea polyphenol andpurine alkaloid composition during solid-state fungal fermentation ofpostfermented tea. Journal of Agricultural and Food Chemistry, 60(5), 1213–1217.

Wei, Q. Y., Zhou, B., Cai, Y. J., Yang, L., & Liu, Z. L. (2006). Synergistic effect of greentea polyphenols with trolox on free radical-induced oxidative DNA damage.Food Chemistry, 96(1), 90–95.

Xie, G., Ye, M., Wang, Y., Ni, Y., Su, M., Huang, H., et al. (2009). Characterization ofPu-erh tea using chemical and metabolic profiling approaches. Journal ofAgricultural and Food Chemistry, 57(8), 3046–3054.

Yang, C. S., Wang, X., Lu, G., & Picinich, S. C. (2009). Cancer prevention by tea:Animal studies, molecular mechanisms and human relevance. Nature ReviewsCancer, 9(6), 429–439.

Zhang, L., Li, N., Ma, Z.-Z., & Tu, P.-F. (2011). Comparison of the chemicalconstituents of aged Pu-erh tea, ripened Pu-erh tea, and other teas usingHPLC-DAD–ESI-MSn. Journal of Agricultural and Food Chemistry, 59(16),8754–8760.