Hematopoietic effect of water-soluble polysaccharides from Angelica sinensis on mice with acute blood loss

Experimental Hematology 2010;38:437–445

Hematopoietic effect of water-soluble polysaccharides from Angelica sinensis on mice with acute blood loss Pei-Jou Liua, Wen-Ting Hsieha, Shih-Hao Huangb, Hui-Fen Liaoc, and Been-Huang Chianga a

Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan; bDepartment of Food and Beverage Management, Taipei College of Marine Technology, Taipei, Taiwan; cDepartment of Biochemical Science and Technology, National Chiayi University, Chiayi, Taiwan (Received 14 October 2009; revised 1 March 2010; accepted 16 March 2010)

Objective. To assess the hematopoietic effects of Angelica sinensis and to investigate the possible mechanism related to its hematopoietic activity. Materials and Methods. The crude extract of Angelica sinensis (AS) was separated into two fractions, polysaccharides (ASPS) and small molecular weight compounds. The AS, ASPS, and small molecular weight compounds were incubated with mice spleen cells to obtain conditioned mediums, and then their hematopoietic activities were evaluated by granulocyte macrophage (GM) colony-forming assay in vitro. During in vivo test, we used mice that were bled approximately 0.5 mL by retro-orbital bleeding at day 0 as our anemia model. Results. We found that polysaccharide (ASPS) was the major component responsible for the hematopoietic effect of Angelica sinensis. The hematopoietic activity was through the stimulation of secretion of interleukin-6 and GM colony-stimulating factor, and the amounts of these hematopoietic growth factors secreted, in general, agreed with the number of GM colony formations. Administration of low-dose ASPS (2.3 mg ASPS/kg body weight per day) could significantly accelerate the recovery of hemoglobin level of the blood-loss mice to its original value, as compared to the control (p ! 0.05). Moreover, the colony-forming ability of bone marrow cells that were removed from mice that received ASPS was also markedly increased (p ! 0.05) during ex vivo test. Conclusions. Results of this study demonstrated the potential of ASPS for treatment of anemia. Ó 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Anemia is a widespread public health problem, particularly among females. Angelica sinensis is an herb commonly used in Chinese medicine for ‘‘blood supplementation.’’ According to the World Health Organization, anemia is a condition in which hemoglobin concentrations are !13 g/dL and !11 12 g/dL in adult men and women, respectively. Blood loss, decrease of red cell production, and increase of red cell destruction are three main causes of anemia. Based on the surveys conducted between 1993 and 2005, approximately 468.4 million (30.2%) nonpregnant women and 56.4 million (41.8%) pregnant women suffered from anemia [1]. Therefore, methods for the improvement of hematopoiesis among women have attracted tremendous attention.

Offprint requests to: Been-Huang Chiang, Ph.D., Institute of Food Science and Technology, National Taiwan University, 1, Section 4, Roosevelt Road, Taipei, Taiwan, ROC; E-mail: [email protected]

Hematopoiesis is a process that is described as hematopoietic stem cells or progenitors in the bone marrow differentiating into mature blood cells by regulation through secretion of various hematopoietic growth factors (HGFs), such as interleukin (IL)-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colonystimulating factor (M-CSF), and other cytokines secreted from the surrounding cells existing in the microenvironment of bone marrow [2 7]. The root of Angelica sinensis (Oliv.) Diels, belonging to Umbelliferae family, is one of the most often used traditional Chinese medicine [8,9]. The earliest use of Angelica sinensi appeared in Shen Nong Bencao Jing O2,000 years ago, and its traditional functions include replenishing blood, treating abnormal or painful menstruation, uterine bleeding, and other diseases affecting women. Because of its medicinal qualities, Angelica sinensis is often referred to as ‘‘female ginseng’’ in China [10,11].

0301-472X/$–see front matter. Copyright Ó 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. doi: 10.1016/j.exphem.2010.03.012

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The bioactive constituents in the root of Angelica sinensis are complicated, including nonvolatile and volatile compounds. The polysaccharides and phenolics, such as nicotinic acid, phthalic acid, p-coumaric acid, and ferulic acid, are the major nonvolatile compounds. Ligustilide, butylidene phthalide, and butyl phthalide are the major volatile compounds found in Angelica sinensis [12]. Ferulic acid has been reported to provide anti-inflammatory [13] and antioxidant [14,15] effects. The Angelica sinensis extract and its major components, including ligustilide, butylidene phthalide, and senkyunolide A, are found to have vasorelaxing [16 18], neuroprotective [12,19 21], and anti-tumor [22 24] effects. The polysaccharides found in the root of Angelica sinensis (ASPS) also received great interest because of their gastrointestinal protective [25 28], anti-tumor [29], and immunomodulatory [30 33] effects. Moreover, the chemoprotective effects of ASPS [25] on aplastic anemic mice have been considered to be related to its blood tonic effect. However, the various components in Angelica sinensis that concern its hematopoietic activity and the mechanism of hematopoietic enhancement by Angelica sinensis still need to be investigated. In the present study, using in vitro assay, we tried to clarify whether the water-soluble polysaccharides derived from Angelica sinensis are the major hematopoietic compounds. The effect of ASPS on mice after acute bleeding was then determined. In addition, we investigated the secretion of pertinent HGFs to understand the possible mechanism of the hematopoietic activity of ASPS by using in vitro and ex vivo assays.

Materials and methods Sample preparation The Angelica sinensis (Oliv.) Diels used in this study was from Sichuan province in China and identified by the Center of Herbal Authentication of Taipei Medical University. Sliced roots of Angelica sinensis (AS; 40 g) were extracted by boiling distilled water (2 L) in a pressure cooker for 3 hours to obtain crude extract (AS). Most of the small molecules in the AS extract had molecular weights between 100 and 300 Dalton, except folinic acid (473 Da). To separate these small compounds from macromolecules, the crude extract was dialyzed by hollow fiber ultrafiltration membrane, which had a molecular weight cutoff of 300 Da to obtain permeate, which was rich in small molecules (molecular weight !300 Da [ASsp]), and retentate containing macromolecules (molecular weight O300 Da). The retentate was further precipitated twice by 95% ethanol (ethanol: retentate 5 4: 1) to obtain crude AP polysaccharides (ASPS). All samples were lyophilized and then stored at 20 C for later uses. The endotoxin contents of AS (1 mg/mL) and ASPS (1 mg/mL) were determined using LAL chromogenic assay kit (Associates of Cape Cod, Inc, East Falmouth, MA, USA) according to manufacturer’s instruction. We found that endotoxin contamination in AS and ASPS was 0.0012% (0.1193 Eu/mL) and 0.0108% (1.0828 Eu/ mL) indicating that LPS contamination in samples were negligible.

Experiments in vitro Preparation of murine spleen cell conditioned medium. Preparation of murine spleen cell conditioned medium (SCM) was as described by Liao et al. [34]. In brief, spleen cells (1 107 cell/ mL) removed from mice were cultured in 6-cm dishes containing RPMI-1640 medium (supplemented with 2 mM L-glutamine, streptomycin/penicillin, and 10% fetal bovine serum), and treated with phosphate-buffered saline (PBS), AS, ASsp, or ASPS at different concentrations. The spleen cells were then incubated in a 37 C 5% CO2-humidified chamber. After 3 days of incubation, the supernatants were individually collected by the 0.22-mm filters (Millipore, Bedford, MA, USA) to obtain PBS-SCM (control), AS-SCM, ASsp-SCM, and ASPS-SCM, respectively, and stored in 1.5-mL centrifugation tubes at 20 C until use. Granulocyte-macrophage/monocyte colony-forming assay. After obtaining various SCMs, we proceeded with the colony-forming unit granulocyte-macrophage/monocyte (CFU-GM) assay. The CFU-GM assay using methyl cellulose or agarose culture was performed to evaluate the hematopoietic ability of bone marrow stem cells through formation of granulocyte-macrophage/monocyte colonies [35]. Bone marrow cells removed from normal BALB/c mice were cultured in 10-cm dishes containing RPMI-1640 medium (supplemented with 2 mM L-glutamine, streptomycin/penicillin, and 10% fetal bovine serum) at 37 C. After 1.5 to 2 hours of incubation, adherent cells were removed. The nonadherent cells (1  106 cell/mL, containing hematopoietic stem cells and hematopoietic progenitor cells) were then plated in 1-mL layer of 1.18% agar in McCoy’s 5A medium (supplemented with 2 mM L-glutamine, nonessential amino acids, vitamin C, sodium pyruvate, streptomycin/penicillin, and 10% fetal bovine serum) with PBS-SCM (control), AS-SCM, ASPS-SCM, as well as ASsp-SCM, and incubated at 37 C. After 5 to 7 days, we counted the number of granulocyte-macrophage/monocyte colonies under inverted microscope. Measurement of cytokine. The concentrations of IL-3, IL-6, M-CSF, and GM-CSF in various SCMs were measured by enzyme-linked immunoassay (R&D Systems, Minneapolis, MN, USA). PBS-SCM group was used as control. Experiments in vivo Animals. Male BALB/c mice (4 weeks of age) were purchased from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan, ROC) and were maintained in a pathogen-free environment. All animal procedures were performed in accordance with the guidelines described in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (DHHS publication no. NIH 85-23, revised 1996). Grouping and administration of ASPS. Mice were allowed free access to food (Rodent Chow Diet; Nutrition International, Brentwood, MO, USA) and distilled water until 7 weeks of age. They were then randomly divided into three groups: one group was treated with distilled water (normal group), another one with 2.3 mg ASPS/kg body weight per day (low-dose group), and the last with 6.9 mg ASPS/kg body weight per day (high-dose group). The entire animal experiment was completed within 10 days.

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On day 0, all groups were acutely bled about 0.5 mL by retroorbital bleeding. After 24 hours, in addition to normal diet, each group received distilled water or ASPS by tube-feeding daily. On day 11, the mice were sacrificed and their bone marrows, hearts, livers, lungs, kidneys, and spleens were collected and weighed prior to further ex vivo experiments. In addition, the total number of spleen cells removed from sacrificed mice was determined. During the period of animal experiment, we recorded the body weight of mice and measured the levels of hemoglobin every 2 days. Measurement of hemoglobin content. The hemoglobin level was estimated by the Drabkin’s method. In brief, the blood (0.01 mL) was mixed with Drabkin’s reagent (2.5 mL) to form cyano-methemoglobin complex and then detected at 540 nm. Experiments ex vivo CFU-GM assay. The bone marrow cells were individually removed from BALB/c mice that received distilled water, low and high dosages of ASPS, and then cultured in 10-cm dishes containing RPMI-1640 medium supplemented with 2 mM L-glutamine, streptomycin/penicillin, and 10% fetal bovine serum at 37 C. After 1.5 to 2 hours of incubation, the adherent cells were removed. The nonadherent cells (1  106 cell/mL) were then plated in 1-mL layer of 1.18% agar in McCoy’s 5A medium (supplemented with 2 mM L-glutamine, nonessential amino acids, vitamin C, sodium pyruvate, streptomycin/penicillin, and 10% fetal bovine serum) and incubated at 37 C. After 5 to 7 days, we counted the number of granulocyte-macrophage/monocyte colonies under inverted microscope. Measurement of cytokine and hemoglobin level. Spleen cells removed from BALB/c mice received distilled water, low dosage, and high dosage ASPS were cultured in RPMI-1640 medium (supplemented with 2 mM L-glutamine, streptomycin/penicillin, and 10% fetal bovine serum) at 37 C for 3 days and filtrates were then collected by 0.22-mm filters. The concentrations of IL-3, IL-6, M-CSF, and GM-CSF in the filtrates were determined as described here. Statistical analysis The statistical significance was determined by one-way analysis of variance, and the comparisons between means were performed by Duncan’s multiple range test. Differences were considered significant at a value of p ! 0.05.

Results Effect of ASPS-SCM, AS-SCM, and ASps-SCM on CFU-GM colony formation To determine whether Angelica sinensis possesses hematopoietic activity and the active components responsible for its hematopoietic activity, mice bone marrow cells were cultured with PBS-SCM (the control group), AS-SCM, ASPS-SCM, and ASsp-SCM, respectively, and the hematopoietic activity was estimated by CFU-GM colony-forming assay. As shown in Table 1, the GM-colony formation ability of mice bone marrow cells was significantly increased with

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the treatments of AS-SCM or ASPS-SCM at concentrations 50, 100, and 200 mg/mL, as compared with the control group (p ! 0.05). In addition, the ASPS-SCM treated groups had significantly higher GM-colony formation ability than the AS-SCM treated groups at all concentrations (p ! 0.05). Contrarily, the ASsp-SCM treated group did not show any hematopoietic activity (p O 0.05). Direct and indirect effects of ASPS on CFU-GM colony formation Additional experiments verified the direct and indirect effects of ASPS on the hematopoietic function of mice bone marrow cells in vitro. In this experiment, bone marrow cells were treated with either ASPS or ASPS-SCM. As shown in Figure 1, the ASPS-SCM treated group raised the GM-colony formation of mice bone marrow cells markedly, as compared with the control and ASPS-treated groups (p ! 0.05). Furthermore, the maximal number of CFU-GM colony was obtained when mice bone marrow cells were treated with ASPS-SCM at the concentration of 2 mg/mL. It is interesting to note that among the ASPS-treated groups, the 2-mg/mL treated group was the only group that had significantly higher hematopoietic activity than the control group (PBS-treated group) (p ! 0.05). Levels of hematopoietic growth factor in ASPS-SCM The major cytokines that support growth and differentiation of GM colony include IL-3, IL-6, GM-CSF, and M-CSF. In the preliminary study, we cultured the mice spleen cells with ASPS for 3 or 5 days to obtain ASPS-SCM, and then used the conditioned medium to treat mice bone marrow cells. We found that there was no significant difference between 3 and 5 days of incubation on the formation of CFU-GM colonies (data not shown). In the SCMs incubated for 3 days, we found that the amounts of GM-CSF and IL-6 in ASPS-SCM were significantly higher than those in the control group at every tested concentration (Fig. 2), and their maximal amounts were 94.24 6 2.00 pg/mL at 1.5 mg/mL and 299.11 6 2.06 pg/mL at 6.25 mg/mL, respectively. However, M-CSF and IL-3 could not be detected at any dosage. Effect of ASPS on hemoglobin level in BALB/c mice after acute bleeding The total circulating blood volume of adult mice is 1.6 to 3.2 mL, and the total removable blood volume is about 1 to 1.5 mL. Therefore, each mouse was bled for about 0.5 mL, approximately 1/5 of total removable blood, to decrease its hemoglobin level in our study. During experiments, the body weight changes of three groups (the control, low-dose, and high-dose groups) were similar, as shown in Figure 3, demonstrating that administration of ASPS had no adverse influence on the normal growth of the tested BALB/c mice. As shown in Figure 4, due to acute bleeding, the amount of hemoglobin was

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Table 1. Effect of ASPS-SCM, AS-SCM, and ASsp-SCM on CFU-GM colony formation of mice bone marrow cells No. colonies/1  105 bone marrow cells Concentration (mg/mL) 0 50 100 200

ASPS-SCM 1 7 6 7

6 6 6 6

1Z,x 1Y,x 1Y,x 2Y,x

AS-SCM 1 3 3 3

6 6 6 6

1Z,x 1Y,y 1Y,y 2Y,y

ASsp-SCM 1 1 1 1

6 6 6 6

1Y,x 1Y,z 1Y,z 1Y,y

Values are mean 6 standard deviation (n 5 3). AS 5 Angelica sinensis extracts; ASPS 5 Angelica sinensis polysaccharides (molecular weight O300 Da Angelica sinensis extracts and proceeded ethanol precipitation); ASsp 5 molecular weight !300 Da Angelica sinensis extracts; CFU-GM 5 colony-forming unit granulocytemacrophage; SCM 5 spleen cell conditioned medium. Y,Z Data with different uppercase letters are significantly different among columns (p ! 0.05). x,y,z Data with different lowercase letters are significantly different among rows (p ! 0.05).

markedly decreased on day 1 for all three groups, as compared with that on day 0 (p ! 0.05). The hemoglobin levels of the two ASPS-treated groups significantly increased (p ! 0.05) at day 3, while no significant change in hemoglobin level could be observed for the control group until day 5. On day 3 and 5, both of the low-dose and high-dose groups had significantly higher hemoglobin levels than the control group (p ! 0.05). However, the hemoglobin level of the low-dose group was markedly

higher than the high-dose group on day 5 (p ! 0.05). Moreover, the low-dose group maintained a significantly higher hemoglobin level than the other two groups throughout the experimental period (p ! 0.05). And the low-dose group was the only group fully recovered from blood loss on day 9. Effect of ASPS on CFU-GM colony formation of bone marrow cells of BALB/c mice Besides observing the level of hemoglobin, we also removed bone marrow cells from tested BALB/c mice and proceeded with CFU-GM colony-forming assay ex vivo. As shown in Table 2, the total number of CFU-GM colony in the high-dose group was significantly higher than the control and low-dose groups (p ! 0.05), while the low-dose group was significantly higher than the control group (p ! 0.05). Effect of ASPS on HGFs in the spleen cells’ culture from BALB/c mice To better understand the possible mechanism for the hematopoietic activity of ASPS in vivo, we analyzed the pertinent HGFs in spleen cells’ cultures from BALB/c mice (after acute bleeding and received distilled water or ASPS). Moreover, we weighed the major organs (i.e., hearts, livers, lungs, kidneys, and spleens) removed from these mice and counted the number of spleen cells. As shown in Table 3, the weights of major organs were not significantly different among three groups, indicating that

Figure 1. Effect of Angelica sinensis polysaccharides (ASPS) (direct stimulation) and ASPS spleen cell conditioned medium (SCM) (indirect stimulation) on colony-forming unit granulocyte-macrophage (CFU-GM) colony formation of mice bone marrow cells. Mice spleen cells (1  107 cells/mL) were treated without (control) or with ASPS (0.5, 1, 1.5, 2, 2.5, 3, 3.125, 6.25 mg/mL) for 3 days and then collected as SCM. Mice bone marrow cells (1  106 cells/mL) were then cultured with ASPS or ASPS-SCM. After 5 to 7 days of incubation, the hematopoietic activity was determined by formation of CFU-GM. Values are mean 6 standard deviation (n 5 3). A FData with different uppercase letters are significantly different (p ! 0.05).

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Figure 2. Level of hematopoietic growth factors in Angelica sinensis polysaccharides (ASPS) spleen cell conditioned medium (SCM) cultured for 3 days. Mice spleen cells (1  107 cells/mL) were treated without (control) or with various concentrations of ASPS (0.5, 1, 1.5, 2, 2.5, 3, 3.125, and 6.25 mg/mL) for 3 days and then collected as various SCM. Levels of hematopoietic growth factors in SCM were estimated by enzyme-linked immunoassay. Values are mean 6 standard deviation (n 5 3). A FData with different uppercase letters are significantly different in levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin (IL)-6 treated with various concentrations of ASPS (p ! 0.05).

ASPS did not affect the normal growth of mice as described previously. However, the high-dose group significantly increased the number of spleen cells compared with the control and low-dose groups (p ! 0.05), while the number of spleen cells in the low-dose group was higher than that in

the control group (p ! 0.05). Furthermore, as shown in Table 4, the levels of GM-CSF and IL-6 of high-dose group were significantly higher than those of the low-dose and control groups (p ! 0.05), but M-CSF and IL-3 were not detectable in all three groups.

Figure 3. Body weight changes of BALB/c mice (after acute bleeding) administrated without (normal control) and with low (2.3 mg Angelica sinensis polysaccharides [ASPS]/kg body weight per day) or high (6.9 mg ASPS/kg body weight per day) dosages of ASPS for 10 days. Values are mean 6 standard deviation (n 5 8 10).

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Figure 4. Hemoglobin changes of BALB/c mice (after acute bleeding) administrated without (normal control) and with low (2.3 mg Angelica sinensis polysaccharides [ASPS]/kg body weight per day) or high (6.9 mg ASPS/kg body weight per day) dosages of ASPS for 10 days. Values are mean 6 standard deviation (n 5 8 10). A EData with different uppercase letters are significantly different among days in each group (p ! 0.05). a cData with different lowercase letters are significantly different among three groups of each day (p ! 0.05).

Discussion The first strategy of our study was to identify the major hematopoietic components of Angelica sinensis. We found that crude extracts of Angelica sinensis could significantly increase colony formation as compared to the control group (Table 1), demonstrating that Angelica sinensis possesses hematopoietic activity. This result is consistent with its traditional use. Moreover, polysaccharides extracted from Angelica sinensis were found to have at least twice the hematopoietic activity of the crude extract (Table 1), suggesting that polysaccharides were the major hematopoietic components of the root of Angelica sinensis. Besides determining the major hematopoietic component, we tried to understand the pathway of ASPS to Table 2. Effect of lowa and highb dosages of Angelica sinensis polysaccharides (ASPS) on colony-forming unit granulocyte macrophage colony formation of bone marrow cells from BALB/c mice (after acute bleeding) treated without (normal group) or with ASPS for 10 days Treatment

Total no. of colonies

Control Low dose High dose

2 6 1z 11 6 6y 21 6 7x

Values are mean 6 standard deviation (n 5 8 10). x,y,z Data with different lowercase letters are significantly different (p ! 0.05). a 2.3 mg ASPS/kg body weight per day. b 6.9 mg ASPS/kg body weight per day.

stimulate the differentiation of mice bone marrow cells. Direct stimulation [36] and stimulation by secreted HGFs from bone marrow stromal cells, such as fibroblasts, blood cells, and other cells [37], are two pathways related to promotion of hematopoiesis by natural products [34]. Results of this study showed that ASPS-SCM had a significantly higher stimulating effect on the formation of GM colonies as compared to ASPS alone (p ! 0.05) (Fig. 2), suggesting that ASPS might enhance production of GM colonies through indirect stimulation. For the sake of further understanding the possible mechanism of the hematopoietic activity of ASPS, we analyzed the pertinent HGFs in ASPS-SCM. HGFs are groups of cytokines that can support growth and differentiation of different blood cell lineages. IL-3, IL-6, GM-CSF, and M-CSF are principle HGFs involved in various stages of development of granulocyte-monocyte/macrophage lineage [3,38]. Even though both IL-3 and GM-CSF have pleiotropic effects on hematopoietic cells and exhibit overlapping activities with each other, IL-3 targets the earliest progenitors to induce their proliferation, differentiation, survival, and progeny [39 41]. M-CSF primarily regulates the survival, growth, and differentiation of macrophages and their progenitors [42,43], while IL-6 synergizes with IL-3 to induce the multilineage progenitors from murine spleens [44], and with M-CSF to stimulate the number and size of CFU-M [45]. In our results, although the maximal secretions of GM-CSF (94.24 6 2.00 pg/mL) and IL-6 (299.11 6 2.06 pg/mL) in ASPS-SCM were detected at dosages

P.-J. Liu et al./ Experimental Hematology 2010;38:437–445 Table 3. Effect of lowa and highb dosages of Angelica sinensis polysaccharides (ASPS) on the weight of major organs and total number of spleen cells of BALB/c mice (after acute bleeding) treated without (normal group) or with ASPS for 10 days Organ weight (g)

Treatment

(Heart, liver, lung, kidney)

Spleen

No. of spleen cells (108/spleen)

Control Low dose High dose

2.4 6 0.2 2.5 6 0.3 2.3 6 0.2

0.1 6 0.0 0.1 6 0.0 0.1 6 0.0

1.4 6 0.1z 1.8 6 0.2y 2.8 6 0.2x

Values are mean 6 standard deviation (n 5 8 10). x,y,z Data with different lowercase letters are significantly different (p ! 0.05). a 2.3 mg ASPS/kg body weight per day. b 6.9 mg ASPS/kg body weight per day.

of 1.5 and 6.25 mg/mL of ASPS, the highest total number of GM-CFU colony formation was obtained by stimulating with 2 mg/mL ASPS-SCM (Figs. 1 and 2). This inconsistent result suggested that other HGFs might also contribute to the hematopoietic activity of ASPS. The hematopoiesis is an intricate process regulated by various HGFs. Therefore, besides the four major HGFs affecting the formation of GM colonies, there are still other HGFs, such as IL-1 and stem cell factor, that may act on early hematopoietic lineages [46]. Moreover, we could not detect M-CSF and IL-3 in the ASPS-SCM, which might be due to the short half-life of M-CSF and IL-3 [47 49], which makes it difficult to detect them. Because polysaccharides were identified as the major hematopoietic components in Angelica sinensis through in vitro assay, we would like to further confirm their effect in vivo. Our findings showed that hemoglobin levels of all experimental groups increased gradually after acute bleeding, indicating that healthy animals could cope with acute blood loss by themselves. However, we found that the mice given a low dose (2.3 mg/kg body weight per day) of ASPS had significantly higher hemoglobin levels Table 4. Measurement of GM-CSF, M-CSF, IL-6, and IL-3 of spleen cells cultured for 3 days from BALB/c mice (after acute bleeding) treated without (normal group) or with Angelica sinensis polysaccharides for 10 days

Treatment

GM-CSF value (pg/mL)

M-CSF value (pg/mL)

IL-6 value (pg/mL)

IL-3 value (pg/mL)

Control Low dose High dose

16.7 6 5.4 22.9 6 7.3 58.1 6 29.0*

ND ND ND

122 6 20 128 6 11 164 6 12*

ND ND ND

Values are mean 6 SD (n 5 8-10). GM-CSF 5 granulocyte-macrophage colony-stimulating factor; IL 5 interleukin; M-CSF 5 macrophage colony-stimulating factor; ND 5 not detectable. *p ! 0.05, significant difference compared with control group.

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than the control group (p ! 0.05) (Fig. 4). This observation suggested that although mice might naturally recover from the blood loss, the administration of ASPS could accelerate the rate of recovery. In 2006, Hui et al. [25] injected cyclophosphamide subcutaneously to induce cytotoxicity of male ICR mice and then administrated different dosages of ASPS. They also found that 5 mg/kg ASPS could accelerate recovery of white blood cell levels, and this result is similar to what we have found. In addition, we found that administration of ASPS did not affect the normal growth of mice (Fig. 3 and Table 3). However, the high-dose group was found to be less effective than the low-dose group (Fig. 4). We suspect that ASPS might have some adverse effect on the bone marrow of mice at high dosage, suppressing the production of hemoglobin. Further research is needed to clarify this point. Nevertheless, results of this study have demonstrated that the proper dosage of ASPS could accelerate hemoglobin recovery rate of mice with acute blood loss. Furthermore, in this study, we also removed the spleen and bone marrow cells from treated mice to perform the colony-forming assay and analyzed the pertinent HGFs. We found that the numbers of colonies and spleen cells were significantly higher in the high-dose group than that in the low-dose and control groups (p ! 0.05). Moreover, the levels of IL-6 and GM-CSF of the high-dose group were also the highest among all groups (Table 4). These results indicated that the spleen cells might be stimulated by ASPS in a dose-dependent manner to secrete relevant hematopoietic growth factors to activate the hematopoietic stem cells existing in the bone marrow. Several other studies have found that Angelica sinensis could enhance proliferation of lymphocytes and macrophages. For example, Wilasrusmee et al. [50] revealed that AS stimulated proliferation of lymphocytes from spleen cells of C57 strain mice. Yang et al. [31] found that ASPS could specifically promote the number of helper T cells (Th1 cells) from spleen cells of BALB/c mice and enhance the secretion of IL-2 and interferon-g. The findings mentioned previously and the result of this study demonstrated similar ASPS functions. It is interesting to note that on days 7 and 9, only the low-dose group mice had significantly higher hemoglobin level than the control group (p ! 0.05), but the hemoglobin level of the high-dose group was essentially the same as the control group (Fig. 4). The determination of hemoglobin level and the colony-forming assay are two different evaluation methods for hematopoietic activity, and they are based on different mechanisms. The hemoglobin, which is produced by immature red blood cells, consists of heme and globin parts [51]. The heme part is synthesized by a complicated series of steps in the mitochondrion of red blood cells, and it contains iron ion to bind oxygen. The globin part is synthesized by ribosomes, and it has four polypeptide chains, two a with 141 amino acid residues

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and two b with 146 amino acid residues [52,53]. During in vivo test, we examined the hemoglobin content that is associated with red blood cells. However, for ex vivo test, we observed the formation of granulocyte-macrophage colonies by colony-forming assay, which is associated with white blood cells. This might be the reason that optimal doses of ASPS were different in these two experiments. Nevertheless, extension of animal experiment period and adjustment of ASPS dosages should be considered in future experiments. In addition, several extended themes also require further investigations. For example, the characteristics of ASPS, such as its molecular weight and monosaccharide composition, should be determined. Furthermore, the bioavailability of ASPS is another subject to be discussed. In general, polysaccharides are nondigestible carbohydrates, resisting the hydrolysis of gastrointestinal tract [54]. Therefore, the hematopoietic mechanism of ASPS in vivo requires better understanding.

Acknowledgments This study was supported by grants from the National Science Council of Republic of China (Taipei, Taiwan, ROC) (NSC962221-E-002-059).

Conflict of Interest Disclosure No financial interest/relationships with financial interest relating to the topic of this article have been declared.

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