Part 1:Extracts Of Cistanche Deserticola Can Antagonize Immunosenescence And Extend Life Span in Senescence-Accelerated Mouse Prone 8 (SAM-P8) Mice
Mar 12, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
Ke Zhang,1 Xu Ma,1 Wenjun He,1 Haixia Li,1 Shuyan Han,1 Yong Jiang,1
Hounan Wu,2 Li Han,3 Tomohiro Ohno,3 Nobuo Uotsu,3 Kohji Yamaguchi,3 Zhizhong Ma,1,4 and Pengfei Tu1
1 Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Beijing 100191, China
2 Medical and Healthy Analytical Center, Peking University, No. 38 Xueyuan Road, Beijing 100191, China
3 Fundamental Research Faculty, Fancl Research Institute, FANCL Corporation, 12-13 Kamishinano, Totsuka-ku, Yokohama, Kanagawa 244-0806, Japan
4 Department of Integration of Traditional Chinese and Western Medicine, School of Basic Medical Sciences, Peking University, No. 38 Xueyuan Road, Beijing 100191, China
Correspondence should be addressed to Zhizhong Ma; zhma@bjmu.edu.cn and Pengfei Tu; pengfeitu@bjmu.edu.cn Received 19 July 2013; Revised 23 October 2013; Accepted 11 November 2013; Published 9 January 2014; Academic Editor: Adair Santos
Introduction
Cistanche deserticola, one of the most popular traditional Chinese herbal medicines/health products, has been described in a number of historical Chinese herbal pharmacopeias as having antiaging properties. Consequently, it has been widely used in China for treating various age-related disorders including senile dementia, impotence, infertility, chronic infection, and hematopoietic disorders in the elderly [1]. Modern chemical approaches have allowed two principal types of compounds, phenylethanoid glycosides, and oligosaccharides, to be isolated as the main active ingredients of Cistanche deserticola [1]. In the last decade, Cistanche deserticola and its extracts have been studied intensively and shown to be capable of protecting neurons from injury induced by neurotoxins[2], inhibiting carbon tetrachloride-induced hepatotoxicity [2], and promoting the recovery of bone marrow cells from Co induced radiation damage [3]. It has also been shown to have anti-inflammatory, antioxidant, and anti-aging effects [4]. However, whether Cistanche deserticola can increase life span and what are the underlying molecular mechanisms [3] associated with its anti-aging properties have not been rigorously tested.
Anti-aging Cistanche deserticola extracts
Immunosenescence, that is, alteration of the immune system with age forms the background against which increased susceptibility to infections, cancer, neurodegenerative diseases, and autoimmune diseases in the elderly has been noted [5]. Therapeutic interventions, such as caloric restriction [6] and vitamin E supplementation, have been reported to be effective at delaying the progression of immunosenescence and hence reducing the morbidity of some age-related diseases as well as prolonging the life span of both humans and rodents [7, 8]. However, studies in this area are complicated by the fact that aging is associated with the paradox of simultaneous immune deficiency and chronic inflammation [9]. This means that simple stimulation of lymphocyte pro- liberation or anti-inflammation does not represent ideal therapeutic interventions in dealing with aging and age-related conditions [10]. Consequently in searching for medical interventions capable of preventing or alleviating age-related conditions including infections, cancer, autoimmune diseases, atherosclerosis, and neurodegenerative diseases which are leading causes of death and disability, and repair of immune system defects must be accompanied by an inhibition of inflammatory responses.
The senescence-accelerated mouse [11] is an inbred mouse model, derived from the AKR/J strain, that is widely used in studies of aging. The P8 substrain (SAM-P8) of these mice has a markedly shortened life span when compared to the R1 substrain (SAM-R1), which also shows a slower aging process [12]. In parallel with their premature aging, SAM-P8 mice also exhibit increased neurological senescence, immunosenescence, and age-related hematopoietic deficits which closely mimic typical human aging characteristics [13, 14]. Analysis of the underlying mechanisms responsible for the accelerated aging process and age-related disorders indicates that mitochondrial dysfunction [15], oxidative stress, and increased somatic DNA mutation rate all appear to be involved [16, 17]. This mouse system, with its homogeneous genetic background, therefore, provides an excellent experimental model for studying aging and antiaging therapeutics [12] been deposited in the herbarium of the School of Pharmaceutical Sciences, Peking University, China.
cistanche can anti-aging
2. Materials and Methods
2.1. Materials.
Fresh Cistanche deserticola Y. C. MA was collected from various areas in northwest China including Xinjiang, Neimenggu, and Ningxia autonomous regions. The samples used to prepare the extracts were authenticated by Professor Pengfei Tu, a specialist of pharmacognosy at the Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University. The voucher specimen of Cistanche deserticola (number CD-2007-03-08) used had Science Centre (Beijing, China). This study was approved by the Peking University Animals Research Committee and carried out according to the guidelines for the Care and Use of Laboratory Animals at Peking University. The certification number of these mice was SCXK2001-2008. The mice were kept in standard metabolic cages in environmentally
C, 45–60% humidity, and 12 h light/dark cycle) and allowed free access to food and water. After 2 weeks of acclimation, animals were divided into five groups: 3 ECD treatment groups, a SAM-R1 control group, and a no-treatment SAM-P8 control group. The diet of the 3 treatment groups was supplemented with a diet mixed with different concentrations of ECD: yield of low (50 mg/kg), medium (150 mg/kg), and high (450 mg/kg) average doses of ECD daily. The SAM-R1 and SAM-P8 no treatment control groups were fed with the same diet without ECD. Food intake measurement of all animals was carried out every three days throughout the experiment. Blood pressure and heart months for the duration of the experiment to determine the health status of the mice. The life span of each mouse was Viable apoptotic and necrotic lymphocytes were quantified using the Annexin V-FITC kit (Beijing Biosea Biotechnology Co., Ltd., Beijing, China). The lymphocytes were subjected to double staining with fluorescein isothiocyanate conjugated labeled annexin V (Annexin V-FITC) and propid-suspended in 200 of binding buffer (10 mM HEPES/NaOH,
2.2 Preparation of Extracts of Cistanche deserticola (ECD) and HPLC Analysis.
Air-dried and sliced Cistanche deserticola (2.0 kg) was powdered and extracted twice with 70% ethanol for 1 hour. The ratios of plant/ethanol used were 1/6 (w/w) in the first extraction and 1/4 (w/w) in the second. The two extracts were combined and filtered before being concentrated to a relative density of 1.10–1.15 under the reduced pressure at 60 C. This concentrate was then vacuumed- dried and the resulting powder was the extract of Cistanche deserticola (ECD) used throughout this study.
The components of ECD were analyzed using HPLC as previously described [1]. Briefly, 100 mg of ECD powder was dissolved in 10.0 mL H O and after filtration injected into the HPLC. HPLC analysis was performed on an Agilent 1100 liquid chromatography system (Agilent Co., USA) The mobile phase consisted of a mixture of methanol (A) and 0.10% methanoic acid (B).A gradient chromatography program was employed; this was 26.5% (A) and 73.5% (B) in 0–7 min, 26.5–29.5% (A) and 73.5–70.5 (B) in 8–10 min, and 29.5% (A) and 70.5% (B) in 20–27 min. The flow rate was held constant at 1.0 mL/min, the injection volume was 10 mL, and the column temperature was maintained at 25 C. A UV detector set at 330 nm was used to monitor the column outflow and generate chromatograms.
Dried and sliced Cistanche deserticola
Phenylethanoid glycosides and oligosaccharides of cistanche were identified from their retention times and absorption spectra. Quantification was carried out by external standard calibration curves. The yield of extracts of Cistanche deserticola (ECD) was about 3.33% and the content of the phenylethanoids was 17.94%. Acteoside and echinacoside were two major constituents in this fraction, with their contents being 3.80% and 8.25%, respectively. The oligosaccharides account for 82% of total ECD. The concentrations of the main active components of ECD are shown in Table 1.
2.3 Analysis of Animal Life Spans.
This study has focused on investigating whether extracts of Cistanche deserticola are able to extend the life span of SAM-R1 mice were purchased from the Laboratory Animal Breeding and Research Center of Peking University Health SAM-P8 mice and reverse their immunosenescence status.
2.4. Statistical Analysis.
Data are shown as mean values S.D. Comparisons between different groups were done by one-way ANOVA with a post hoc test. In the life span study, the data underwent Kaplan-Meier survival analysis, which included the use of both the Log-rank (Mantel-Cox) and Gehan-Breslow- Wilcoxon tests. Statistical analysis was conducted using the Statistical Package for Social Sciences for Windows (SPSS, phosphate-buffered saline (PBS, pH 7.4) containing 0.01% sodium azide, and subjected to FCM analysis. Lymphocyte subpopulations were analyzed by flow cytometry using a FACS Calibur flow cytometer (BD Biosciences, San Diego, CA, USA) and BD CellQuest analysis software. Chicago, IL) and a to be significant.
Phenylethanoid glycosides and oligosaccharides of cistanche
3. Results
A value of less than 0.05 was considered. Assessment of Cellular Viability of Peripheral T Lymphocytes by Annexin V-FITC and PI Double Staining and FACS Analysis. Following dietary supplementation of SAM- P8 mice with three doses of ECD for 4 weeks, the animals were fastened for 12 hours and then anesthetized, and blood was collected and analyzed.
3.1. Analysis of Potential Active Components in Extracts of Cistanche deserticola (ECD). As shown in Table 1, ECD is composed mainly of two types of compound, phenylethanoid glycosides, and oligosaccharides. In the phenylethanoid glycosides, the echinacoside, acteoside, and 8-epiloganic acid have been identified, whilst in the oligosaccharides, only galactitol was identified.
3.2.TheImpactofCistanchedeserticolakbdO4ctson the Average Life Span of SAM-P8 Mice. For this and subsequent studies eight-month-old male SAM-P8 mice were divided into 4 groups. Among them, one groupofmice werefed witha normal diet without ECD, the other 3 groups have separately ingested the diets which contain different proportions of ECD. Mice from the SAM-R1 substrain, which have anormal aging process and lifespan, wereused as acontrolgroup in all experiments.
Compared to the control SAM-R1 group, the average life span of SAM-P8 mice was significantly shortened (Figure1(a);<0.001). Although supplementation of diet with the low dose(50mg/kg)of ECD failed to produce a significant increase in life spanofSAM-P8mice, at the medium (150 mg/kg)and high(450 mg/mL) supplementary doses there was a dose-dependent increase in life span (Figures 1(a)and 1(b);<0.05 0.0thatwasconfirmedby Kaplan-Meier survival analysis, which included use of both the log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests.
3.3. Reversal of Immunosenescence in SAM-P8 Mice by Extracts of Cistanche deserticola (ECD). A decrease of peripheral naive T lymphocytes and concomitant increase of peripheral memory T lymphocytes are prominent features of immunosenescence which are widely regarded as the main underlying reasons for age-related immunological abnormalities. This immunosenescence in SAM-P8relative to SAM-R1
aniftalsv0agtlearly evident when FACS analysis was to enumerate(CD3+CD44wCD4Bigh)lymphocytes as an indicator of naive T cells and(CD3+ CD44gh CD45 BR lymphocytes a san indicator of memory T-cells. Thus,inbpth peripheral blood(Figure2)and spleen cell(Figure 3)popu-lationsreducedlevelsofnaive Tcells andincreasedlevelsof memory T cells were seen in SAM-P8 mice. Supplementation of the diet of SAM-P8mice with ECD was found to be able to reverse these indicators of immunosenescence in a dose-dependent fashion in both peripheral blood (Figure2) and spleen cell (Figure 3)populations. As an additional indicator of the reversal of immunosenescence by ECD supplemen-tation of diet, thelevelof naturalkiller [18]cells, amajor cellular marker of the innate immune system, wasanalyzed. This showed that ECD diet supplementation resulted in a dose-dependent increase in NK(CD3+ CD49+)cells in both peripheral blood (Figure4) and spleen cell(Figure 5)lymphocyte populations.
3.4. Extracts of Cistanche deserticola Strengthen the Relatice Fluorescence Intensituy of Sca-1 Positive Cells in SAM-P8 Mice. Stem cell antigen-1(Sca-1)is one of the most prominent biomarkers of hematopoietic stem cells (HSC)in bone marrow cell populations.Sca-1 positive cells also represent lymphocyte progenitors that have been newly exported from bone marrow into peripheral blood where they undergo.
Figure2:FACSanalysis of the the affect of dietary supplementation with ECD onnaiveand memory Tlymphocytesin peripheralblood.Eight-month-old male SAM-P8 and control SAM-RI mice were divided into 5 groups. The 3 treatment groups ofSAM-P8 animals were fed for four weeks with diets supplemented with low(100mg/kg), medium(500mg/kg), and high (2500 mg/kg) doses of ECD, and the two control animals
by gatingon CD3+Tcels.The linesshownoneachplotindicate the thresholdsused to distinguishCD44x*and CD45 be used total low identification of naive(CD3+Cp b"C4RB→)and memory(CD3+CD4hCD 4Bl)T-cells. b)Histogram showing groups were fed on the same dietwithoutsupplementation. Following fasting for 12 hours, peripheral blood was collected from anesthetized RB Tcellsubsets animals and subjected to FACS analysis all as described in Section 5. （a） Representative FACSplots of the different groups of miæ generated the percentage of naive T cells in peripheral blood of the different animal groups. (c)Histogram showingthe percentage ofmemory Tcells in peripheral blood of the differentanimalgroups. Bars represent the mean±SD (in each group, = 10).<0.05SAM-P8versus SAM-R1.
Figure 3: FACSanalysis of theeffectof dietary supplementation with ECD onnaive andmemory Tlymphocytes inspleen.Eight-month-old maleSAM-P8 and controlSAM-RI mice were divided, into5 groups.The3 treatment groups of SAM-P8 animals were fed for four weeks with diets supplemented with low (100mg/kg), medium (500mg/kg), and high (2500mg/kg)doses of ECD and thetwocontrolanimal groups by gatingon CD3+Tcells. The lines shown on each plot indicate that there should be used to distinguishCD44zhand CD45 bw used to allow identification of naive（CD3+CD "CsRB动）and memory（CD3+CD42CD I'm）Tcels.（b）Histogram showing were fed on the same diet without supplementation. Following fasting for 12 hours, splenic lymphocytes were collected from anesthetized RB T cell subsets animals and subjected to FACS analysis all s described in Section 5. (a) Representative FACSplots of the different groups of mic generated.
Figure4:The effectsof ECDdietary supplementationonnaturalkiller cells(NKcells, CD3+ CD49+ Tcells)in the peripheral blood lymphocytes populations of SAM-P8 mice. Eight-month-old male SAM-P8 and control SAM-RI mice were divided into 5 groups. The 3 treatment groups of SAM-P8 animals were fed for four weeks with diets supplemented with low (100mg/kg), medium (500mg/kg), and high(2500mg/kg)doses of ECDand the two control animal groups were for the same diet without supplementation. Following fasting for 12 hours, peripheral blood was collected from anesthetized animals and subjected to FACSanalysis all as described in Section 5.(a)RepresentativeFACS plots of the differentgroups of mice generated by gating on CD3+CD49+Tcells. (b) Histogram showing the percentage ofnaturalkiller Tcellsin peripheralbloodof the differentanimalgroups.Barsrepresentthe mean±SD (ineach group,=10).= <0.01 SAM-P8versusSAM-Rl;**<0.01 each treatedgroup versusSAM-P8group (byone way ANOVAwith post hoc test).
Figure 5: The effects of ECD dietary supplementation onnatural killer cells (NKcells, CD3+ CD49+ T-cells)in the splenic lymphocytes of SAM-P8 mice. Eight-month-old male SAM-P8 and control SAM-RI mice were divided into5 groups. The 3treatmentgroups of SAM-P8 animals were fed for four weekswith diets supplemented withlow (100mg/kg), medium(500mg/kg), and high(2500mg/kg) doses ofECD and the two control animal groups were fed on the same diet without supplementation. Following fasting for 12 hours.splenic lymphocytes were collected from anesthetized animals and subjected toFACSanalysisallasdescribed in Section 5.(a)Representative FACSplotsofthe different groups of mice generated by gating on CD3+ CD494 T cells.(b)Histogram showing the percentage of natural killer T cells in splenic lymphocytes of the different animal groups.Bars represent the mean SD (in each group,= 10), # < 0.01 SAM-P8 yersus SAM-R1;**<0.01 eachtreatedgroup versusSAM-P8group(by oneway ANOVAwithposthoctest).
Further differentiation into various types of mature lymphocytes. Consequently, these Sca-1 positive cells represent the main source of naive T lymphocytes in peripheral blood. Figure6 shows that compared with SAM-R1 control mice, the relative fluorescence intensity of Sca-1 positive cells in SAM-P8 mice was substantially lower. Diet supplementation of SAM-P8mice with three different doses of ECD was shown to significantly enhance the relative fluorescence intensity of Sca-1 positive cells (Figure 6).
Benefit of cistanche herb: anti-aging
