Hematopoietic and myeloprotective activities of an acidic Angelica sinensis polysaccharide on human CD34+ stem cells

Journal of Ethnopharmacology 139 (2012) 739–745

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Hematopoietic and myeloprotective activities of an acidic Angelica sinensis polysaccharide on human CD34+ stem cells Ji-Gua Lee a , Wen-Ting Hsieh a , Shee-Uan Chen b , Been-Huang Chiang a,∗ a b

Institute of Food Science and Technology, National Taiwan University, Taipei 106, Taiwan Department of Obstetrics and Gynecology, National Taiwan University Hospital, National Taiwan University, Taipei 106, Taiwan

a r t i c l e

i n f o

Article history: Received 26 June 2011 Received in revised form 20 November 2011 Accepted 27 November 2011 Available online 6 December 2011 Keywords: Angelica sinensis polysaccharides Hematopoiesis Myeloprotection Human umbilical cord blood CD34+ stem cells

a b s t r a c t Ethnopharmacological relevance: Angelica sinensis (AS) is a Chinese herbal medicine traditionally used in prescriptions for replenishing blood and treating abnormal menstruation and other women’s diseases. Aim of the study: This study aimed to separate and identify the major hematopoietic fraction from Angelica sinensis polysaccharides (ASPS), and to investigate the myeloprotective activity of the major bioactive fraction of ASPS as a possible supporting agent for cancer treatments. Materials and methods: The ASPS was fractionated with DEAE-Sepharose CL-6B column to obtain four fractions (F1, F2, F3 and F4). Each fraction was cultured with human peripheral blood mononuclear cells (MNCs) to collect conditioned medium (CM). The hematopoietic ability of various MNC-CM was then evaluated by the colony-forming assay on CD34+ cells collected by the MACS method from human umbilical cord blood (UCB). In myeloprotective experiment, Adriblastina was used to act as the myelosuppressive agent. The monosaccharide composition of ASPS was analyzed by high-performance anion-exchange chromatography-pulse amperometric detector. Results: The F2 fraction, which was found to have the highest hematopoietic activity, stimulated the human peripheral blood MNCs to secret GM-CSF and IL-3. F2 could also protect the hematopoietic function of CD34+ cells from Adriblastina. F2 occupies 19% of ASPS and contains 0.53% protein. The monosaccharide composition of F2 was arabinose (51.82%), fructose (1.65%), galactose (29.96%), glucose (4.78%) and galacturonic acid (14.80%), with molecular weight 2.5–295 kDa. Conclusions: The bioactive fraction identified and fractionated from ASPS may be used as a healthpromoting agent for anemia patients and cancer patients under chemoradiation treatment. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction As a standard treatment nowadays for neoplastic diseases, chemoradiation is however not without side effects. Myelosuppression, for one, is serious in that it may cause serious morbidity and mortality problems (Henning et al., 2000), making stimulation of hematopoiesis an important issue in clinical cancer treatments. Out of the various supporting therapies for cancer treatments, food supplement remains a promising one for it is relatively harmless to human health. Among the several herbs used for hematologyrelated diseases, we will focus in the present study on the root of Angelica sinensis (Oliv.) Diels, a Chinese herbal medicine for over 2000 years. Angelica sinensis (AS) is traditionally used in relation to gynecology, hinting at its hematopoietic functions in replenishing

∗ Corresponding author at: 1 Section 4, Roosevelt Road, Taipei, Taiwan. Tel.: +886 2 33664119; fax: +886 2 23620849. E-mail address: [email protected] (B.-H. Chiang). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.11.049

blood, treating abnormal or painful menstruation, and other women’s diseases (Saker and Nahar, 2004). Specific biological functions have been reported to be associated with the different components of AS. For example, the volatile compounds such as ligustilide, butylidene phthalide and butyl phthalide are said to have antitumor (Kan et al., 2008; Harn et al., 2011; Liu et al., 2011), neuroprotective (Huang et al., 2008; Chen et al., 2011), immunomodulatory (Su et al., 2011) and cardiovascular protective (Yeh et al., 2011) effects. The ferulic acid, the most abundant phenolic non-volatile compound of AS, has been reported for its anti-inflammatory and antioxidant potency (Ozaki, 1992; Ronchetti et al., 2006; Su et al., 2011). Other nonvolatile compounds such as polysaccharides have been claimed for their gastrointestinal protective, antitumor, and immunomodulatory functions (Shang et al., 2003; Hui et al., 2006; Yang et al., 2007, 2008a,b; Cao et al., 2010a,b; Chen et al., 2010). Plant polysaccharides have also been used as biological response modifiers (Leung et al., 2006). For example, those extracted form Aloe vera (Talmadge et al., 2004), Angelica acutiloba Kitagawa (Hatano et al., 2004), Glycine max L. Merr. (Liao et al., 2005), Grifola

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frondosa (Lin et al., 2004), and Panax ginseng (Song et al., 2003) have been confirmed to display hematopoiesis-enhancing or/and myeloprotective activities. Polysaccharides have been identified as the major component responsible for the hematopoietic effect of AS in our previous research (Liu et al., 2010a). Although studies on cyclophosphamide-induced myelosuppression mice (Hui et al., 2006) and radiation-treated mice (Sun et al., 2005; Liu et al., 2010b) confirmed that polysaccharides extracted from the AS roots could protect the experimented from chemoradiation injury, it nevertheless remains unknown as to the actual types of AS-derived polysaccharides attributing to the hematopoietic function. The present study therefore aims to separate and identify the major hematopoietic fraction from Angelica sinensis polysaccharides (ASPS), and to further investigate the myeloprotective activity of its major bioactive fraction as a possible supporting agent for cancer treatments. In conjunction, we would modify the in vitro hematopoietic evaluating method used in the study. Considering that human umbilical cord blood (UCB) cells are, due to its ease of collection with less ethical complication, better candidate for hematopoietic stem cells (HSCs), we will perform colony-forming assay on HSCs sourced from human UCB to evaluate the hematopoietic and myeloprotective effects of ASPS and those in the various fractions we isolated from within. 2. Materials and methods 2.1. Sample preparation The Angelica sinensis (Oliv.) Diels used in our research was provided by Sichuan Province in China and its DNA was verified by the Industrial Technology Research Institute (Hsinchu, Taiwan). The sliced roots of Angelica sinensis (80 g) were extracted by being placed in boiling water (4 L) for 3 h. The extract was then precipitated by 95% ethanol (extract:ethanol = 1:4) overnight at 4 ◦ C. Above precipitating procedure was repeated twice to obtain crude Angelica sinensis polysaccharide (ASPS). The yield of ASPS was 3.31%, and its endotoxin level of 0.0108% (1.0828 Eu/mL), as detected by LAL chromogenic assay kit (Associates of Cape Cod, Inc., MA, USA), a level which is considered negligible. For fractionation of ASPS, 10 mL of ASPS (6000 ␮g/mL) was applied to DEAE-Sepharose CL-6B column (Amersham Bioscience, Uppsala, Sweden). Each fraction was eluted with 300 mL of eluent at the rate of 1 mL/min, and collected by monitoring the carbohydrate content in the eluate via phenol–sulfuric acid method. The four major factions, F1, F2, F3 and F4, were respectively eluted with 0, 0.15, 0.2 and 0.25 M of NaCl solutions. All samples were lyophilized and then stored at −20 ◦ C for later uses. The specific rotation of F2 dissolving in dH2 O under 27◦ C is +32.35 ([˛]27 = +32.35), as determined by JASCO P-1000 digital polarimeter (Tokyo, Japan). 2.2. Monosaccharide composition analysis of ASPS fractions F1, F2, F3 and F4 were first hydrolyzed by methanolysis combined with trifluro acetic acid (TFA) hydrolysis (de Ruiter et al., 1992). After that, we analyzed monosaccharide compositions of the hydrolyzed samples by high-performance anion-exchange chromatography-pulse amperometric detector (HPAEC-PAD). The HPAEC-PAD system was equipped with a CarboPac PA1 column (4 mm × 250 mm, Dionex, California, USA) in combination with a CarboPac guard column (4 mm × 50 mm, Dionex, California, USA) together in a 20 ␮L Bioscan sample loop (Bioscan 812 unit, Metrohm, Herisau, Swizerland). We prepared three kinds of eluents, including eluent A (10 mM NaOH containing 1 mM Ba(OAc)2 ), eluent B (75 mM NaOH containing 150 mM NaOAc as well as 1 mM Ba(OAc)2 ), and eluent C (0.5 N NaOH), with the gradient of above

eluents set as follows: (1) 100% eluant A for 15 min; (2) 100% eluant B for 20 min; (3) 100% eluant C for 15 min; and (4) 100% eluant A for 70 min. The separation of hydrolyzed samples in eluents was done at the rate of 1 mL/min. And the monosaccharides of the samples were detected by pulse amperometric detector (PAD, Metrohm Bioscan 817, Metrohm, Herisau, Switzerland), with electrode pulse potentials and durations set as following: E1 = 0.05 V, t1 = 0.4 s; E2 = 0.75 V, t2 = 0.2 s; and E3 = 0.15 V, t3 = 0.4 s. Data were collected and analyzed by the computer equipped with Metrohm Metrodata IC Net 2.1 (Metrohm, Herisau, Switerland). 2.3. Preparation of human mononuclear cell-conditioned medium (MNC-CM) Human white blood cell concentrates of healthy donors were obtained from Taiwan Blood Services Foundation-Taipei Blood Center (Taipei, Taiwan) during 2007–2008. The blood cell concentrates were diluted with phosphate buffer (PBS) and separated by the density gradient (Ficoll-Paque Plus, Pharmacia, Uppsala, Sweden) under the centrifugation at 1500 rpm for 30 min, and then washed twice by Hanks balanced salt solution (HBSS) under the centrifugation at 1500 rpm for 5 min to obtain mononuclear cells (MNCs). After that, the MNCs (1.6 × 106 cells/mL) were cultured with PBS (the control group) or different samples in 12-well plates containing RPMI 1640 medium (GIBCO BRL, Gaitherburg, Mo, USA) supplemented with 10% fetal bovine serum (FBS) at 37 ◦ C. After 5 days, the supernatants were collected and filtered through 0.22␮m filters to acquire MNC-conditioned mediums (MNC-CMs). The MNC-CMs were stored in 1.5-mL centrifugation tubes at −20 ◦ C until use. 2.4. Isolation of CD34+ cells (hematopoietic progenitor cells) from human umbilical cord blood After having obtained the informed consents, we collected human umbilical cord blood (UCB) samples of healthy infants at the time of delivery in the National Taiwan University Hospital (Taipei, Taiwan). The collected UCB was firstly diluted with PBS at the ratio of 1:4, with the UCB-MNCs separated by density gradient under the centrifugation at 400 × g for 35 min. After that, CD34+ stem cells were first targeted by the major surface marker of hematopoietic progenitor cells-CD34+ magnetic microbeads and then isolated using magnetic force as described in the manual (MACS, MiltenyiBiotec, Bergisch Glabach, Germany). 2.5. Colony-forming assay The isolated CD34+ cells were, before being plated into three 3cm dishes, first mixed with Iscove’s MDM medium (IMDM, Sigma, St. Louis, MO, USA) plus 2% FBS to adjust the cell density to 5 × 103 to 2 × 104 cells/mL, before the cells (0.3 mL) were added to the tubes containing 3 mL Methocult 4434 medium (StemCell Technologies Inc., Vancouver, British Columbia, Canada) with 0.3 mL PBS (the control group), or with various MNC-CMs we obtained previously. After 14–16 days of incubation at 37◦ C, the number of colonies was counted under inverted microscope. 2.6. Cytokine analysis The concentrations of interleukin-3 (IL-3) and granulocyte macrophage-colony stimulating factor (GM-CSF) in various MNCCMs were measured by enzyme-linked immunoassay (R&D Systems, Minneapolis, MN). The MNC-CM derived from MNC treated with PBS was chosen as the control group.

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Table 1 Effects of MNC-CM treated with ASPS and its fractions on colony formation of CD34+ cells of human umbilical cord blood. Concentration (␮g/mL)

No. of CFU/103 CD34+ cell ASPS

Control 1 2 3 20 40 60 80 100 200

34 42 43 43 56 73 76 105 90 83

± ± ± ± ± ± ± ± ± ±

F1 1G 2F,b 2F,b 1F,b 3E,b 3D,a 1D,a 4A,a 2B,b 4C,b

34 36 36 37 47 52 57 56 65 42

F2 ± ± ± ± ± ± ± ± ± ±

1F 1F,c 1F,c 1EF,c 2D,c 3C,b 4B,b 2BC,c 5A,c 3E,d

34 51 53 53 70 74 76 93 123 89

F3 ± ± ± ± ± ± ± ± ± ±

1F 2E,a 2E,a 1E,a 3D,a 3CD,a 2C,a 7B,b 4A,a 3B,a

34 31 31 30 30 32 33 50 60 56

F4 ± ± ± ± ± ± ± ± ± ±

1D 2D,d 2D,d 3D,d 3D,d 4D,c 1D,c 1C,cd 3A,c 4B,c

34 41 41 42 58 67 60 41 47 44

± ± ± ± ± ± ± ± ± ±

1E 1D,b 3D,b 2D,b 3B,b 2A,a 3B,b 2C,d 1C,c 1CD,d

Each value presents mean ± SD (n = 3). Means with different uppercase letters are significantly different within each column (p < 0.05). a–c Means with different lowercase letters are significantly different within each row (p < 0.05). Abbreviations: MNC-CM: mononuclear cells-conditioned medium; ASPS: polysaccharide of Angelica sinensis; F1: the fraction of ASPS eluted with 0 M NaCl solution; F2: the fraction of ASPS eluted with 0.15 M NaCl solution; F3: the fraction of ASPS eluted with 0.2 M NaCl solution; F4: the fraction of ASPS eluted with 0.25 M NaCl solution. A–C

2.7. Protective effect of F2 fraction on myelosupression Dissolved in NaCl solution, Adriblastina (Adriblastina Rapid Dissolution Vial, Pfizer, Italy), the myelosuppression agent, was adjusted to different concentrations (10, 20, 40, 60, 80, and 100 ng/mL) and added to the mixtures containing CD34+ cells (0.3 mL), 100 ␮L of PBS (the control group) or 100 ␮L of F2-treated MNC-CM (at 100 ␮g/mL), and 3 mL of Methocult 4434 medium. The colony formation after 14–16 days of incubation at 37 ◦ C was observed under inverted microscope. 2.8. Estimation of molecular weight of F2 fraction The F2 of ASPS (1.5–2.0 mg/mL), collected at the flow rate of 3 mL/min and monitored for carbohydrate content through phenol–sulfuric acid method, was applied onto the column (2.6 cm × 120 cm) packed with Tosoh TSK gel HW-65F gel (Tosoh, Tokyo, Japan) and eluted with NaCl solution of 0.1 M. The results were then adjusted to eliminate experimental errors with distribution coefficient, estimated by the equation (Vx − V0 )/(Vt − V0 ), where Vx is the elution volume, and V0 and Vt are, respectively, values of void and total permeation volumes, designated as elution volumes of large molecular weight blue dextran (MW: 2000 kDa) (4 mg/mL) and small molecular weight glucose (0.1–0.2 mg/mL). The molecular weight of F2 was estimated by the calibration curve from glucose, a series of pullulans (P5, P10, P50, P100, P200, P400 and P800) and blue dextran with molecular weights as well as distribution coefficients (in parentheses) as follows: 0.18 kDa (1.00), 5.9 kDa (0.87), 11.8 kDa (0.83), 47.3 kDa (0.70), 112 kDa (0.57), 212 kDa (0.46), 404 kDa (0.34), 788 kDa (0.19) and 2000 kDa (0.0). 2.9. General analytical methods Total carbohydrate content was measured by phenol–sulfuric acid method at 490 nm (Dubois et al., 1956), taking d-glucose (0–100 ␮g/mL) as standard. Total protein content was determined by the Lowry method at 750 nm (Lowry et al., 1951), with bovine serum albumin (BSA) (0–1.0 mg/mL) as standard. To detect uronic acid contents, samples were reacted with m-hydroxybiphenyl at 520 nm by using d-galacturonic acid as the standard (Blumenkrantz and Asboe-Hansen, 1973). 2.10. Statistical analysis Each experiment was repeated three times. The statistical significance was determined by one-way ANOVA, and comparison done

by Duncan’s multiple range test. The result is considered statistically significant when P < 0.05. 3. Results 3.1. Effects of ASPS on the colony formation of CD34+ stem cells from umbilical cord blood cells In our previous study (Liu et al., 2010a), hematopoietic stem cells (HSCs) from mouse bone marrow were used to establish polysaccharides as the major hematopoietic compounds of Angelica sinensis. Different from our 2010 study, HSCs collected from human umbilical cord blood (UCB) were used here to re-investigate the hematopoietic activities of Angelica sinenesis polysaccharides (ASPS). Having verified the hematopoietic activities of Angelica sinensis extracts via indirect stimulation (Liu et al., 2010), we treated in this study human MNCs with PBS (the control group) or with various ASPS concentrations to obtain conditioned mediums (MNC-CMs). CD34+ cells from human UCB with MNC-CMs were cultured and hematopoietic activities were evaluated by the colony forming assay. ASPS demonstrated, as expected, significantly higher hematopoietic activity than the control group (p < 0.05) (Table 1). The maximal number of colony formation occurred at the dosage level of 80 ␮g/mL. It was also found that the number of colony formed was dosage-dependent. It increased when the dosage level was lower than 80 ␮g/mL, but decreased when the level went higher than 80 ␮g/mL. 3.2. Fractionation of ASPS In order to identify the kind of polysaccharides in Angelica sinensis responsible primarily for hematopoietic activity, we separated ASPS into four fractions (F1, F2, F3, and F4) by eluting NaCl solutions of various concentrations on the DEAE-Sepharose CL-6B column. The elution profile is shown in Fig. 1. The fractions F1, F2, F3 and F4 were respectively obtained at 0, 0.15, 0.2, and 0.25 M of NaCl solutions, with the peak ratio at 19:19:36:18. 3.3. Effects of ASPS fractions on colony formation For the four fractions isolated from ASPS, we used colony forming assay on human UCB-derived CD34+ cells to examine the hematopoietic activities of these fractions. As shown in Table 1, F2 and F4 significantly enhanced the colony forming abilities at all the dosage levels tested, whereas F1 and F3 could only increase the number of colony formation with high concentrations (>20 ␮g/mL). Among the four fractions, F2 demonstrated

n.d. n.d. n.d. n.d. n.d. 16.69 ± 1.29c ± ± ± ± ± ± Each value presents mean ± SD (n = 3). The level of GM-CSF of control (PBS group) was 35.42 ± 1.64 pg/mL, and IL-3 was not detectable for the control group. A–C Means with different uppercase letters are significantly different within each column (p < 0.05). a–c Means with different lowercase letters are significantly different within each row (p < 0.05). n.d.: not detectable. The detection limit is 15 pg/mL.

37.21 39.30 41.54 41.88 43.94 44.90 45.39 42.05 44.83 43.67 48.75 48.09 20 40 60 80 100 200

46.19 48.88 49.00 55.67 57.39 59.46

± ± ± ± ± ±

B,b

1.78 0.96B,b 6.56B,b 4.99A,b 3.65A,b 2.91A,b

F1

± ± ± ± ± ±

D,b

1.05 0.75C,bc 0.77B,bc 0.98BC,c 1.53A,c 1.10A,c

52.16 62.23 81.32 89.17 89.82 86.79

± ± ± ± ± ±

1.31 1.93C,a 3.73B,a 1.32A,a 2.19A,a 4.65A,a

D,a

36.01 35.63 37.36 40.77 42.05 43.66

± ± ± ± ± ±

A,c

3.09 7.39A,c 2.80A,d 4.62A,c 2.06A,d 5.18A,c

F4 F3 F2 Concentration (␮g/mL)

The myeloprotective activity of F2 was also evaluated by colony-forming assay on human UCB-derived CD34+ cells, with Adriblastina, also known as doxorubicin, used as the myelosuppresive agent. As shown in Fig. 2, the numbers of colony formation for the PBS-treated groups (the control groups) were significantly decreased with the increase of Adriblastina concentrations (p < 0.05). And the hematopoietic function of CD34+ cells was significantly enhanced by F2, suggesting that F2 may have protected CD34+ cells from being damaged by Adriblastina. At 100 ␮g/mL, F2 doubled the hematopoietic activity of the CD34+ cells, when it was able to fully recover the hematopoietic function of CD34+ cells when the Adriblastina concentrations were lower than 20 ng/mL.

Table 2 Levels of GM-CSF and IL-3 in MNC-CMs after stimulated by the ASPS or the fractions of ASPS for 5 days.

3.5. Myeloprotective activity of F2

46.12 52.38 69.57 73.22 70.03 53.93 3.31 4.72AB,c 2.20AB,cd 3.77AB,c 0.74A,d 3.52A,c

B,b

3.4. Levels of hematopoietic growth factors in MNC-CM Granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) are two important hematopoietic growth factors involved in the process of hematopoiesis (Kindt et al., 2007). To understand the possible hematopoietic mechanism, we need to measure the levels of GM-CSF and IL-3 in various MNC-CMs we obtained by treating MNCs with PBS or with different samples. As shown in Table 2, all MNC-CMs contained GM-CSF. Significant increase in GM-CSF levels was observed in F1, F2 and F4 with the increase of the dosage levels, but not in F3 (p > 0.05). Moreover, higher levels of GM-CSF were seen in the F2-treated MNC-CMs at all concentrations tested (p < 0.05), with the highest level detected when the dosage was increased to 80 ␮g/mL. No such difference in GM-CSF levels of the F2-treated MNC-CMs was however displayed when the dosage was increased from 80 to 200 ␮g/mL. IL-3 was detected in the F2-treated MNC-CMs at all dosage levels, but not in the ASPS-treated MNC-CMs. For the F2-treated MNC-CMs, the level of IL-3 was generally increased with the increase of the dosage, although at the highest dosage level in this study (200 ␮g/mL), IL-3 was significantly reduced (p < 0.05). For F1 and F3-treated MNC-CMs, IL-3 could only be detected at the highest dosage level (200 ␮g/mL), as clearly shown in Table 2.

n.d. n.d. n.d. n.d. n.d. 31.27 ± 3.56b

ASPS

F1

Level of IL-3 (pg/mL)

ASPS

the highest hematopoietic-enhancing ability. When comparing the hematopoietic activities of F2 and ASPS, we found that the colony counts of the F2-treated groups were, in general, significantly higher than those of the ASPS-treated groups. Besides, the maximal number of colony formation (123 ± 4 colonies) of F2-treated groups was at 100 ␮g/mL, although lower number of colonies (93 ± 7 colonies) was found for the F2-treated groups than for the ASPS-treated groups (105 ± 4 colonies) at 80 ␮g/mL.

Level of GM-CSF (pg/mL)

Fig. 1. The elution profile of Angelica sinensis polysaccharides on anion-exchange DEAE-Sepharose CL-6B column eluted by stepwise gradient of sodium chloride solution.

n.d. n.d. n.d. n.d. n.d. n.d.

F2

± ± ± ± ± ±

B

5.37 3.37B 2.23A 4.37A 2.22A 2.583B,a

F3

n.d. n.d. n.d. n.d. n.d. n.d.

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F4

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Fig. 2. Protective effect of F2 fraction on colony formation of CD34+ stem cells suppressed by Adriblastina. Data are presented as the mean colony counts ± SD (n = 3). (A–E) Columns with different uppercase letters are significantly different within the PBS and F2-treated groups (p < 0.05). # Significant difference between the F2-treated group and the PBS-treated group (p < 0.05).

3.6. Chemical characteristics of F2 In view of the major hematopoietic fraction of F2, we tried to measure its molecular weight by Tosoh TSK gel HW-65F column so as to analyze its monosaccharide composition and protein content. As shown in Fig. 3, the molecular weight of F2 was between 2.5 and 295 kDa. As 19% of ASPS, F2 consists of 51.82% arabinose, 1.65% fructose, 29.96% galactose, 4.78% glucose, 14.80% galacturonic acid, and 0.53% protein (Table 3). Besides F2, the composition of remaining three fractions was also studied. Arabinose was discovered to be the predominant monosaccharide in all the ASPS fractions except F4, whose principle monosaccharide was galacturonic acid, an oxidized form of galactose not found in F1. F3, as the major fraction of ASPS (36%), displayed the simplest monosaccharide composition, consisting of arabinose, galactose and galacturonic acid. All the four ASPS fractions contained protein ranging from 0.16 to 0.53%. 4. Discussion Even though polysaccharides in Angelica sinensis were identified via the mice model in our earlier study (Liu et al., 2010a) as the major component with blood-enriching ability, we are well aware that various biological functions such as anticancer (Shang et al., 2003), immuno-modulation (Yang et al., 2007, 2008a,b) and radioprotection (Hui et al., 2006; Liu et al., 2010b)

Fig. 3. The elution profile of the F2 fraction on gel filtration Tosoh TSK gel HW-65F column. The standards are glucose, pullulan P5, P10, P50, P100, P200, P400, P800 and blue dextran.

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are associated with different polysaccharides from the root of AS. Therefore, in this study, crude Angelica sinensis polysacchrides (ASPS) were further fractionized on the DEAE-Sepharose CL6B column, and HSCs from human umbilical cord blood (UCB) were sampled to estimate the hematopoietic function of ASPS. Hematopoietic growth factors (HGFs) were investigated in order to determine the possible hematopoietic mechanism of ASPS. Considering plant polysaccharides as potential hematopoietic modifiers against myelosuppression after cancer treatments, we also investigated the myeloprotective ability of the ASPS fractions. It is hoped that an ASPS-derived agent may be discovered as an aid in cancer treatments. We first used the hematopoietic colony-forming assay to confirm that the hematopoietic activity of ASPS could be examined on HSCs from human UCB. The hematopoietic colony-forming assay is a hematopoietic activity evaluation method based on colony formation of HSCs on semi-solid medium, with HSCs being the stem cells capable of self-renewal, proliferation and differentiation. Major sources of HSCs in humans include bone marrow and UCB, but the latter is a better candidate because it is immature and can be collected via non-invasive methods to avoid possible ethical concerns (Parent-Massin et al., 2010). CD34+ being one of the most important antigen expressed in the earliest progenitors of human HSCs (Watt and Vissert, 1992), it was used as the marker to separate HSCs from UCB. Table 1 clearly indicates that the hematopoietic activities, for ASPS as well as for the four ASPS fractions (F1–F4), reached the peak at a certain dosage level, but decreased with an increased dosage level, suggesting that the conditioned ASPS mediums may have toxic effects on CD34+ cells at high dosage levels. As a result, dosage levels should be carefully evaluated during the process. Note that the number of colonies formed in the ASPS-treated group was lower only than that of the F2 treated group, but it was higher than the other groups treated with F1, F3 and F4. We suspect that there is a synergistic effect for the four ASPS fractions. Second, we determined the possible hematopoietic mechanism of ASPS and its fractions, with hematopoiesis defined as a process by which hematopoietic stem cells or progenitors in the bone marrow differentiate into mature blood cells, a process regulated by various HGFs secreted by the surrounding cells such as macrophages, fat cells, fibroblasts and other cells in the bone marrow (Kindt et al., 2007). For example, IL-3 and GM-CSF are two principle HGFs that exhibit pleiotropic as well as overlapping effects in the various stages of hematopoeisis. Unlike GM-CSF, IL-3 targets only the earliest progenitors (Koike et al., 1987; Leary et al., 1987). The levels of GM-CSF and IL-3 in the F2-treated MNC-CMs of this study were both higher than those in other groups (p < 0.05) (Table 2), consistent with the results of the colony forming assay (Table 1). Note that IL-3 could not be detected in the F2-treated MNC-CMs, except at the highest dosage level. In view of the secreting profiles of both growth factors in the MNC-CMs (Table 2) matched in general the number of colonies formed during treatment of the CD34+ cells with F2 (Table 1), we claim F2 as the major hematopoietic fraction in ASPS. However, the fact that F2 occupies only 19% of ASPS probably explains why we could not detect IL-3 in the ASPS-treated MNCCMs. In addition, since botanical polysaccharides are non-digestible carbohydrates, resisting the hydrolysis of he gastrointestinal tract (Delzenne and Roberfroid, 1994), plant polysaccharides may act on immune cells, such as macrophages, to stimulate hematopoietic effects in the intestine. In fact, such immuno-activation and modulation by botanical polysaccharides occurs through binding specific receptors of immune cells, such as toll-like receptors (Yoon et al., 2003; Balachandran et al., 2006; Kim et al., 2007). What should be further investigated is the actual in vivo function of purified ASPS. After identifying F2 as the most active ASPS fraction, we also examined the monosaccharide compositions of F1–F4. Indeed, similar attempts to identify the biological functions of Angelica sinensis

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Table 3 The yield, protein content and monosaccharide composition of ASPS and each fraction of ASPS separated by DEAE-Sepharose CL-6B column. Sample

Yield (%)

F1 F2 F3 F4

19 19 36 18

Molar percentage (%)

Protein content (%)

Ara

Fru

Gal

Glc

Man

GalA

53.46 51.82 42.55 13.52

2.56 1.65 – 0.06

10.31 29.96 18.70 3.55

27.29 4.78 – 1.03

6.38 – – –

– 14.80 38.75 81.84

0.16 0.53 0.16 0.50

Abbreviations: Ara: arabinose; Fru: fructose; Gal: galactose; Glc: glucose; Man: mannose; GalA: galacturonic acid.

polysaccharides have been made in recent years. For instance, Sun et al. (2005) separated from Angelica sinensis polysaccharide (ASP) three fractions (ASP1, ASP2 and ASP3) by the DEAE-Sepharose CL6B column and discovered that ASP3, which had radioprotective activity, comprised galacuronic acid (58.27%), galactose (24.93%) and arabinose (10.5%), with a molecular weight of 3.4 × 104 Da. Yang et al. (2007) reported a series of studies regarding the composition and the related bioactivities of three fractions separated from Angelica sinensis polysaccharides (APF1–APF3). APF2, composed of mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose at a ratio of 0.44:1.00:10.52:7.52:8.19:14.43, could activate macrophages through toll-like receptor 4. Yang et al. (2008a,b) further pointed out the immuno-stimulating properties of these three fractions, and singled out the highest thermal stability of APF2 with an uronic acid content of 39.2%. We in the present study confirmed hematopoietic activities in all the four ASPS fractions obtained through anion-exchange DEAE-Sepharose CL-6B column and identified F2, composed of arabinose (51.82%), fructose (1.65%), galactose (29.96%), glucose (4.78%), and galacturonic acid (14.80%), exhibited the highest activity when molecular weight was around 2.5–295 kDa. Similar to APF2 in Yang et al. (2007), F2 was also found to contain high amount of arabinose, galactose and galacturonic acid. What remains to be further investigated is whether its bioactivity is related to the polysaccharide as seen in these monosaccharides. Finally, the myeloprotective potential of F2 was clarified. Many previous attempts have been made to make use of myeloprotective functions of plant polysaccharides, used as biological response modifiers to enhance hematopoiesis (Lin et al., 2004; Liao et al., 2005; Leung et al., 2006) to minimize the side effect of bone marrow toxicity after chemo-radiotherapy for cancer treatments (Tubiana et al., 1993). We in this study examined further the myeloprotective effect of F2 in terms of its major hematopoietic fraction on adriblastina-induced myelosuppressive human CD34+ cells from UCB, since the myeloprotective (Hui et al., 2006) and radioprotective (Sun et al., 2005; Liu et al., 2010b) activities were reported with reference to polysaccharides of Angelica sinensis. Adriblastina, commonly known as doxorubicin, a highly effective anti-cancer agent and a kind of anthracycline antibiotic, was used here to establish our myelosuppressive model. The cytotoxic effects of anthracycline antibiotics possibly occur due to the disturbance of nucleic acid synthesis through interacting with DNA double-helix, or the inhibition of topoisomerase II and the prevention of transcription, leading to the breakdown of double-strand DNA and cell death (Tan et al., 2009). Moreover, because of accumulation in bone marrow cells, doxorubicin may affect hematopoietic precursors (Cocke et al., 1993). As shown in Fig. 2, Adriblastina was found to cause myelosuppresion on the CD34+ cells in a dosage-dependent manner and it reduced, at a concentration of 40 ng/mL, approximately 59% of the hematopoietic activity. Nevertheless, the hematopoietic activity of CD34+ cells could be fully restored at low doses of Adriblastina, hinting at the myeloprotective potential of F2 (Fig. 2). Although the mechanism of the myeloprotective effect of F2 was not investigated in this study, Lin et al. (2004) suggested such effects might be due to growth factors, such as IL-3 and GM-CSF.

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