Effect of Danggui Buxue Tang on immune-mediated aplastic anemia bone marrow proliferation mice.
To investigate the pharmacological effects of Danggui Buxue Tang (DBT) on immune-mediated aplasia anemia mice. The model of immune-mediated aplasia anemia mice was induced by means of [sup.60]Co [gamma]-ray irradiation and mixed cells of thymus and lymphnode of DBA/2 mice infusion through tail vein, the parameters tested indices were as following: blood picture, bone marrow nucleated cell count (BMNC), murine colony-forming unit-megakaryocytes (CFU-GM) of bone marrow cells, murine colony-forming unit-erthroid (CFU-E) and burst forming unit-erythroid (BFU-E). The results showed that DBT could not only withstand significantly decreation of blood cells by immune-mediated, but also stimulate on the growth of bone marrow colony cell and increase the weight of hemopoietic progenitor of bone marrow. Therefore, DBT had an obvious treat effect on immune-mediated aplasia anemia models mice.
Danggui Buxue Tang
Aplastic anemia mice
Bone marrow colony cell
Aplastic anemia is a very common disease of the blood system, but it is also one of the severe case in hematopoietic system, its incidence showed an increasing trend. Aplastic anemia is still the medical profession as a "difficult challenge" (Rodrigo et al., 2005; Young et al., 2010). Its morbidity is characterized by bone marrow hematopoietic function failure although the exact pathophysiological mechanism is not yet fully clear and definite. In recent years, with the development of immunology, molecular biology and cell biology techniques, it is widely believed that aplastic anemia is connected with hematopoietic pluripotent stem cell damage, abnormal bone marrow hematopoietic microenvironment and immune dysfunction by domestic and foreign scholars (Rosenthal et al., 2011; Rauff et al., 2011; Gouthamchandra et al., 2010; Scheinberg and Young, 2012).
The immunosuppressive agents (such as cyclosporine A, etc.) are using to treat aplastic anemia in the clinical therapy which had achieved good clinical efficacy, however, after long-term use of the immunosuppressive agents there had certain side effects. In recent years, it was reported that traditional Chinese medicines (TCMs) could treat aplastic anemia because of their unique efficacy and less toxicity (Scheinberg et al., 2009; Marsh et al., 2013).
Danggui Buxue Tang (DBT) is a Chinese medicinal decoction used commonly for treating women's ailments. According to historical usages in traditional Chinese medicine (TCM), at least three DBT formulae, namely DBT1247, DBT1155 and DBT1687, have been recorded. In a book entitled Neiwaishang Bianhuo Lun in 1247 AD, DBT1247, contains Radix Angelicae Sinensis (Danggui, RAS) and Radix Astragali (Huangqi, RA) in a weight ratio of 5:1, was first described by Li Dongyuan in China (Yi et al., 2009; Wei et al., 2008; Li et al., 2009; Yan et al., 2010). However, at least two other Angelica herbal decoctions recorded as DBT were prescribed in Song (1155 AD) and Qing dynasties (1687 AD). The first record of DBT1155 was revealed, which composed of four herbs: ASR, AR, Jujuba Fructus (JF) and Zingiberis Rhizoma Recens (ZRR) in a weight ratio of 12:10:5:4. DBT1687 was recorded in "BianzhengLu" by Chen Shiduo in Qing dynasty (1687 AD), which composed of ASR, AR and Rehmanniae Radix Praeparata (RRP) in a 2:1:1 ratio (Zhang et al., 2012).
According to Chinese medicinal theory, DBT1247 (Hereinafter short for DBT) is frequently prescribed in herbal clinics today; we selected DBT for further study for several reasons. First, DBT is a simple formula that consists of two medicinal plants: RAS and RA. Thus, the identification of the active compounds and their interactions is less complex. Second, the extraction process of DBT has been standardized, and several chemical compounds such as ferulic acid and ligustilide from RAS, and total saponins of Astragalus (TSA) (including astragaloside I-IV et al.) and formononetin from RA have been used as marker compounds for quality control. Their structures are shown in Fig. 1. These ensured the chemical consistencies among different preparations (Yang et al., 2009; Gao et al., 2008,2011).
Commonly used in clinical Astragalus, Angelica compatibility for the treatment of aplastic anemia, confirmed that after many years of clinical efficacy, and to further explore its mechanism, provide the basis for the clinical application of this study will be the use of immune-mediated aplastic anemia mouse model experiments.
Several previous studies showed DBT can promote hematopoiesis, stimulate cardiovascular circulation, prevent osteoporosis and posses anti-oxidation activity (Gao et al., 2011; Mak et al., 2006; Gao et al., 2007). However, there is a lack of scientific studies on its potential pharmacological properties. Therefore, it is necessary to study the Effect of DBT on immune-mediated aplastic anemia bone marrow proliferation. Fufang Zaofan Pill (FZP, Chinese Pharmacopoeia recommendations 2010 edition) is one of the most well-known formulations. It consists of six herbs: Ferrous sulphate, Panax quinquefolium L, sea horse, Cinnamomum cassia Presl, Ziziphus jujuba Mill (enucleation), and Juglans regia L. The pillt promote powerful hematopoiesis effects, and it was used as the positive control drug in the following studies.
Materials and methods
Chemicals and materials
The herbal materials, RAS and RA were provided by Chongqing Academy of Chinese Materia Medica, Professor Chang-hua Wang (Chongqing Academy of Chinese Materia Medica, China) identified all of the raw medicinal herbs. The raw medicinal herbs and the specimens were deposited at the Herbarium of Chongqing Normal University (voucher no. 2013010). Ferulic acid, formononetin, ligustilide, astragaloside I, astragaloside II, astragaloside 111 and astragaloside IV were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Their structures are shown in Fig. 1. Chromatography-grade acetonitrile used for HPLC-MS analysis were purchased from Alltech Scientific (Beijing, China). HPLC-grade water was obtained from a Milli-Q system (Millipore, Billerica, MA, USA). Formic acid was A.R.-grade and purchased from the First Chemical Company of Nanjing (Jiangsu, China). RPMI1640 nutrient solution, 20% newborn calf, serum horse serum and recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF) were purchased from Sigma (St. Louis, MO, USA). Fufang Zaofan Pill (FZP) was used as a positive control in the tests and was provided by Shanxi Haoqijun Pharmaceutical Co., Ltd. (Shanxi Province, China). All other chemicals used in these studies were analytical grade reagents and purchased from Promega Corporation (Madison, WI, USA).
Preparation of DBT extract
DBT was prepared using traditional methods. Briefly, dried and mixed Astragalus membranaceus var. mongholicus and Astragalus sinensis powder was extracted by refluxing for 2.0 h with 10 volumes of 80% ethanol (1:10, w/v). After 2.0 h, the solution was filtered using filter paper (Whatman no. 4), and the residue was re-extracted two additional times with same volume of 80% ethanol. Finally, the entire filtered solution of mixture was concentrated under reduced pressure and then dried with a spray dryer. The final DBT product was stored at -20[degrees]C. A yield of 37.14% was obtained.
For the chemical analysis, an appropriate amount of the drying powder DBT were accurately weighed and transferred to calibrated, amber flasks and extracted with 50 ml of methanol in an ultrasonic bath for 30 min. Additional methanol was added after sonication to compensate for any lost volume, and the resulting solution was filtered through a 0.45-[micro]m membrane filter prior to HPLC injection.
Chemical analysis of active compositions by LC-ESI-MS/MS
The HPLC analyses were performed using a Waters HPLC system (Waters, USA) equipped with a dual pump, an autosampler, and a Waters Acquity[TM] BEH [C.sub.18] analytical column (150 mm x 2.1 mm x 1.7 [micro]m) in this study. The mobile phase was initially composed of acetonitrile (component A) and 0.1% formic acid aqueous solution (component B), where it was held for 2 min. From 3 to 8 min, the concentration of component A was increased linearly to 66%, where it was held for 4 min. From 13 to 17 min, the concentration of A was linearly decreased from 66% down to 32%, where it was held for 3 min. The mobile phase flow rate was 0.2 ml/min, and the injection volume was 10 [micro]l. The MS/MS system consisted of a Quattro LC triple quadrupole tandem mass spectrometer (Micromass, Manchester, UK) equipped with a the electrospray ionization (ESI) source. The ESI source was operated at a source voltage of 3.6 kV and with a tube compensation lens voltage of 32 V. Nitrogen was used for both the sheath gas (precolumn pressure: 7Mpa) and the auxiliary gas (precolumn pressure: 0.56 Mpa, flow rate: 10 L/min). The capillary was heated to 330[degrees]C and maintained within a voltage range of 16-52 V. Using the full-scan, positive-ion mode, we monitored ions in the 100-1000 m/z range.
A standard stock solutions of each of the 7 compounds were directly prepared in methanol. The standard solutions containing the 7 compounds were prepared and diluted with methanol to appropriate concentrations for establishment of calibration curves. The standard stock solutions were all prepared in dark brown calibrated flasks and stored at 4[degrees]C. The linearity of the responses was determined for six concentrations.
Adult Blab/C mice (18-22 g) were obtained from the Experimental Animal Center of Third Military Medical University. The animals were housed in temperature-controlled rooms with access to water and food ad libitum until they were used. They were fed in a 12-h light/dark cycle. The study was approved by the Animal Research Welfare Committee (certificate no. SCXK 20130005), Third Military Medical University, Chongqing, China.
Dosage regimen design
These studies followed Chinese Pharmacopoeia recommendations (2010 edition) for the effective and safe dosage of DBT and FZP (Zheng 2005). Adult Blab/C mice received DBT as follows: 1.25g/kg in the low-dose, 2.50 g/kg in the mid-range dose and 5.00g/kg in the high-dose. The dose received FZP as follows: adult Blab/C mice received 1.00g/kg. All doses were administered intragastrically (i.g.) 3 times per day.
Model test research on immune-mediated aplastic anemia bone marrow proliferation
Sixty Blab/C mice (half male and half female) were randomly and equally divided into a control group, a model group, a FZP group or one of three doses of DBT treatment groups (n = 10 for each group). All groups except the control group were radiated one time by [sup.60]Co (5.5 Gy) [gamma] ray. Subsequently, Balb/c mice were given the mixed cells (thymus of micedymph gland 1:2, Concentration: 1.0 x [10.sup.6]/mice) through tail vein to establish the immune-mediated aplastic mice model after 4 h. The control group and model group received an equivalent volume of saline (i.g), and the other groups (three doses of DBT treatment groups) received treatment as described above.
Peripheral hemogram assay
The animals were housed in a temperature-controlled room with access to water and food until 1 h after the final administration. The blood were sampling from tail vein to assay the blood routine by Sysmex F-820 semi-automatic blood analysor (Japan's east Asia Company) which included white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT) and platelets (PLT).
Prepare of the single cell suspension and investigation of nucleated cells in bone marrow
After the last administration, Balb/c mice were sacrificed by cervical, their femur were taken out in the case of sterile, cutting off the both ends of femur, exposing the bone marrow cavity, the marrow cavity was repeatedly washed 2 ml RPMI1640 nutrient solution by using a sterile syringe with no. 4 needle and the bone marrow cell were collected. The cells were repetitive beat by pipette and mechanical separation. The optimal centrifuge condition is temperature 4[degrees]C, rotation speed 1000 r/min and time 10 min. The cell suspension was made into and more than 95% survival rate of the cells were detected by 4% trypanblue.
Murine colony-forming unit-megakaryocytes (CFU-GM) assay
The CFU-GM training system was made into 0.2 mi cell suspension (2 x [10.sup.5]), 0.6 ml horse serum, 0.2 ml GM-CSF and 0.7 ml methylcellulose (27 g/1) by well mixing, after that the mixed solution was adjusted to 2.0 ml by using RPMI 1640 nutrient solution. The CFU-GM training solution was transferred 1 ml to each culture dish and incubated 7 d at 5% carbon dioxide incubator and 37[degrees]C. The more than 50 number of the cell colonies were count by using inverted biological microscope (Japan's optical industry Jurassic type society).
The culture of murine colony-forming unit-erthroid (CFU-E) and burst forming unit-erythroid (BFU-E) in vitro
The CFU-E and BFU-E training system was made into 0.2 ml cell suspension (2 x [10.sup.5]), 0.3 ml newborn calf (concentration: 20%), 0.2 ml mercaptoethanol (10mol/l), 0.05 ml EPO (CFU-E: 0.2 [micro]/ml, BFU-E:1 [micro]/ml) and 0.1ml methylcellulose (27g/1) by well mixing, after that the mixed solution was adjusted to 1.0 ml by using RPMI 1640 nutrient solution. The training solution was incubated 3d (CFU-E) and 7d (BFU-E) at 5% carbon dioxide incubator and 37[degrees]C, respectively. The cell mass with orange cells ([greater than or equal to] 8) was CFU-E, and The more than 50 number of the cell colonies was BFU-E.
The pathological observation of sternum marrow
The sternum marrow was fixed after organization decalcifiedin and conventional dehydration. Resulting pathomorphological changes in sternum marrow were studied with conventional hematoxylin-eosin (HE) staining of samples.
The data obtained were analyzed using the GraphPad software program Version 4.0 and expressed as the mean [+ or -] standard error of the mean (S.E.M.). Statistically significant differences between the treatment groups were calculated by analysis of variance (ANOVA) followed by the Newman-Keuls test. A value of probability (p < 0.05) was considered to be statistically significant.
Preparation of DBT extract and quantitative analysis of the main compounds
Calibration curve linearity was examined using standard solutions. The linear regression equations determined by plotting peak area (Y) versus concentration (X) for each analyte are given in Table 1. All calibration curves showed good linearity ([r.sup.2] [greater than or equal to] 0.99) within the concentration ranges tested.
The limits of detection (LOD) and quantification (LOQ) were defined as the concentrations of a compound with signal-to-noise ratios (S/N) of 3:1 and 10:1, respectively. 7 Analytes were determined by serial dilution of a standard solution using the described HPLC-MS/MS conditions. The LODs and the LOQs for the analytes were found to be less than 0.054 [micro]g/l (LOD) and 0.177 [micro]g/l (LOQ) for all 7 analytes under the described HPLC-MS/MS conditions (Table 1).
To test the recoveries of the methods, six portions of DBT were spiked with the mixed standard solution. The samples were processed as described above, and the results are summarized in Table 1. All recoveries were between 99.03% and 100.01%, with RSDs of 1.55% or less, which is well within acceptable limits (see Table 1).
The developed assay was subsequently applied to the simultaneous determination of the 7 compounds in the DBT. The contents of ferulic acid, formononetin, ligustilide, astragaloside I, astragaloside II, astragaloside III and astragaloside IV were respectively 1312.54 [+ or -] 6.24 [micro]g/g, 152.64 [+ or -] 0.58 [micro]g/g, 1208.27 [+ or -] 0.29 [micro]g/g. 168.34 [+ or -] 9.76 [micro]g/g, 315.16 [+ or -] 13.45 [micro]g/g, 76.82 [+ or - ] 2.73 [micro]g/g and 242.38 [+ or -] 0.13 [micro]g/g. HPLC-MS chromatograms of the standards and DBT sample are shown in Fig. 2A and B, respectively.
Effects of peripheral hemogram
Blab/C mice in the model group that received [sup.60]Co exhibited changes in WBC, RBC, HGB, HCT and PLT as compared to animals in the control group (Fig. 3). The WBC of the model group significantly decreased to 0.74 [+ or -] 0.56 from 6.47 [+ or -] 1.61 (p < 0.05) as compared to the control group (Fig. 3). However, The RBC, HGB, HCT and PLT of the model group significantly decreased to 7.73 [+ or -] 1.56, 116.13 [+ or -] 24.09, 0.384 [+ or -] 0.085 and 28.88 [+ or -] 18.13 from 11.08 [+ or -] 0.66, 169.90 [+ or -] 8.29, 0.559 [+ or -] 0.040 and 769.50 [+ or -] 110.65 (p < 0.01), respectively, as compared to the control group (Fig. 3). As demonstrated in Fig. 3, Levels of PLT exhibited an decreasing trend while WBC, RBC, HGB and HCT had a increasing trend in the low-dose group as compared to the model group; however, these changes were not statistically significant. Levels of WBC, RBC, HGB, HCT and PLT exhibited an increasing trend in the mid-range dose group as compared to the model group; these changes were not statistically significant, too. The WBC and HGB of the high-dose group significantly increased to 9.03 [+ or -] 1.12 and 131.43 [+ or -] 15.44 from 7.73 [+ or -] 1.56 and 116.13 [+ or -] 24.09 (p < 0.05) as compared to the model group (Fig. 3); however, the changes of WBC, HCT and PLT were not statistically significant.
Effects of BMNC, CFU-GM, CFU-E and BFU-E
Blab/C mice in the model group that received [sup.60]Co exhibited changes in bone marrow nucleated cell count (BMNC), CFU-GM, CFU-E, BFU-E as compared to animals in the control group (Fig. 4). The BMNC, CFU-GM, CFU-E and BFU-E of the model group very significantly decreased to 9.86 [+ or -] 10.06, 15.75 [+ or -] 14.54, 8.25 [+ or -] 4.53 and 2.63 [+ or -] 3.02 (p < 0.01) from 530.80 [+ or -] 106.75, 142.30 [+ or -] 38.92, 32.00 [+ or -] 10.69 and 10.60 [+ or -] 4.72, respectively, as compared to the control group (Fig. 4). As demonstrated in Fig. 4, The BMNC, CFUGM, CFU-E and BFU-E of the low-dose group and the mid-dose group exhibited an increasing trend while the Level of CFU-GM significantly increased to 39.33 [+ or -] 29.81 from 15.75 [+ or -] 14.54 as compared to the model group (p < 0.05, Fig. 4); the of BMNC, CFU-E and BFU-E increased to 14.33 [+ or -] 15.28,14.33 [+ or -] 4.92 and 4.56 [+ or - ] 3.05 while the changes of them were not statistically significant as compared to the model group (p>0.05, Fig. 4). The BMNC of the high-dose group significantly increased to 24.86 [+ or -] 16.95 (p < 0.05) from 9.86 [+ or -] 10.06 as compared to the model group (Fig. 4) while the CFU-GM, CFU-E and BFU-E of the high-dose group very significantly increased to 126.25 [+ or -] 79.68,26.13 [+ or -] 7.34 and 7.63 [+ or -] 3.54 from 15.75 [+ or -] 14.54, 8.25 [+ or -] 4.53 and 2.63 [+ or -] 3.02, respectively, as compared to the model group (p < 0.01, Fig. 2).
Pathological study on effects of sternum marrow
As demonstrated in Fig. 5A, pathological changes showed that the blood sinus and hemopoietic tissue of the sternum marrow in control group were rich, however, adipose tissues were rare. As demonstrated in Fig. 5B, the hemopoietic tissue in the model group were obviously atrophia, the number of blood sinus was a significant reduction, too. They were replaced by hyperplastic adipose tissues. In the part of the adipose tissue stroma, the reticulum cell was proliferative accompany with a large number of lymphocytes and the proliferative phlogocyte infiltrates. The proportion of adipose tissue in adipose tissue were 70%, 70%, 50%, 90%, 30% and 50%, respectively. The average amount was 60% which was very significantly increased as compared to the control group. As demonstrated in Fig. 5C, the hematopoietic tissue in the low-dose group decreased compared with the normal group, however, the blood sinus were rich as compared to the model group. The proportion of adipose tissue in adipose tissue were 25%,5%, 5%, 40%, 15% and 30%, respectively. The average amount was 20% which was very significantly decreased as compared to the model group. Similarly, As demonstrated in Fig. 5D and E, the average amount of the proportion of adipose tissue in adipose tissue in the mid-range dose group and high dose group were 25% and 38%,respectively.They were very significantly decreased as compared to the model group, too. As demonstrated in Fig. 5F, the hematopoietic tissue in the FZP group (positive control group) decreased as compared to animals in the control group, however, it increased as compared to animals in the model group, the blood sinus were rich. The proportion of adipose tissue in adipose tissue were 30%, 50%, 60%, 40% and 45%, respectively. The average amount was 45% which was very properly decreased as compared to the model group.
As demonstrated in Fig. 6, the proportion of bone marrow hematopoietic tissue was 48.75 [+ or -] 20.49 (V/V) in the model group. The low-dose treatment group had an increasing trend (62.00 [+ or -] 17.67) as compared to the model group; however, these changes were not statistically significant (p>0.05). However, the mid-dose treatment group and the positive control group respectively and significantly increased to 75.00 [+ or -] 22.36 and 72.50 [+ or -] 16.69 (p < 0.05); furthermore, the high-dose treatment group very significantly increased to 80.00 [+ or -] 14.14 as compared to the model group (p < 0.01).
The present results provided evidence for the potential use of DBT on immune-mediated aplasia anemia mice (Yetgin and Elmas, 2010; Heckl et al., 2011). The findings indicated that three doses of DBT could not only withstand decreation of blood cells by immune-mediated, but also DBT administered at high dose group significantly increased the level of the WBC and HGB (p < 0.05).
Similarly, DBT administered at middle dose group and high dose group could significantly increased the CFU-GM and BMNC of aplastic anemia mice bone marrow cell colonies, respectively (p < 0.05); furthermore, DBT administered at high dose group could very significantly increased the CFU-GM, CFU-E and BFU-E of aplastic anemia mice bone marrow cell colonies, respectively (p < 0.01) as compared to the model group. The mid-dose treatment group significantly increased the proportion of bone marrow hematopoietic tissue (p < 0.05); furthermore, the high-dose treatment group very significantly increased as compared to the model group (p < 0.01).
Received 23 June 2013
Received in revised form 5 August 2013
Accepted 17 October 2013
This study was supported by the Funds for the Chongqing Normal University (Project No. 12XLB028). At the same time, it was supported by the Fundamental Research Funds for the Science and Technology research projects of Chongqing Shi Jiaoyu Weiyuanhui, Project No KJ130618.
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Xian Yang (a,b), Chong-Gang Huang (c), Shou-Ying Du (a), *, Shui-Ping Yang (d), Xue Zhang (c), Jian-Yi Liu (c), XianQinLuo (c), Jia-Hong Xu (c)
(a) School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, PR China
(b) College of Life Sciences, Chongqing Normal University, Chongqing 401331, PR China
(c) Chongqing Academy of Chinese Materia Medica, Chongqing 400065, PR China
(d) College of Resources and Environment, Southwest University, Chongqing 400716, PR China
* Corresponding author at: School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, PR China. Tel.: +13911053905.
E-mail address: firstname.lastname@example.org (S.-Y. Du).
TABLE 1 Linearity, LOD, LOQ, recovery and recovery of 7 control compositions. Compound Linear range Linear equation ([micro]g/ml) Ferulic acid 0.021-10.8 Y=9613.3X + 3997.3 Ligustilide 0.081-24.30 y=7865.8X + 2965 Formononetin 0.017-2.040 V=4140.5X - 162.23 Astragaloside IV 0.031-12.40 y=3960.3X - 2136.4 Astragaloside III 0.089-10.68 y=1792.1X - 686.67 Astragaloside II 0.094-45.12 y=854.68X + 466.47 Astragaloside I 0.034-12.24 y=2211.3X - 1016 Compound Regression LOD [r.sup.2] (n = 5) ([micro]g/ml) Ferulic acid r=0.9948 0.002 Ligustilide r=0.9942 0.054 Formononetin r=0.9934 0.031 Astragaloside IV r=0.9923 0.009 Astragaloside III r=0.9949 0.013 Astragaloside II r=0.9982 0.012 Astragaloside I r=0.9933 0.011 Recovery Compound LOQ ([micro]g/ml) Average(%) RSD(%) Ferulic acid 0.006 99.76 0.62 Ligustilide 0.177 100.01 1.04 Formononetin 0.095 99.81 0.86 Astragaloside IV 0.026 99.13 1.13 Astragaloside III 0.037 99.12 0.97 Astragaloside II 0.035 99.03 1.21 Astragaloside I 0.034 99.31 1.55
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|Author:||Yang, Xian; Huang, Chong-Gang; Du, Shou-Ying; Yang, Shui-Ping; Zhang, Xue; Liu, Jian-Yi; Xian-QinLuo|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Apr 15, 2014|
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