Adjuvant effects of Astragalus saponins Macrophyllosaponin B and Astragaloside VII

Journal of Ethnopharmacology 134 (2011) 897–903

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Adjuvant effects of Astragalus saponins Macrophyllosaponin B and Astragaloside VII Ays¸e Nalbantsoy a , Tuna Nesil b , Sec¸il Erden b , I˙ hsan C¸alıs¸ c , Erdal Bedir a,∗ a b c

Ege University, Faculty of Engineering, Bioengineering Department, 35100 Bornova, Izmir, Turkey Ege University, Graduate School of Natural and Applied Sciences, Department of Biotechnology, 35100 Izmir, Turkey Near East University, Faculty of Pharmacy, Department of Pharmacognosy & Pharmaceutical Botany, Nicosia, Cyprus

a r t i c l e

i n f o

Article history: Received 17 October 2010 Received in revised form 27 January 2011 Accepted 28 January 2011 Available online 4 February 2011 Keywords: Astragalus Saponin Cycloartane Adjuvant activity Astragaloside VII Macrophyllosaponin B

a b s t r a c t Aim of the study: The present study was undertaken to evaluate the hemolytic activities of two immunomodulator Astragalus saponins [Macrophyllosaponin B (MacB) from Astragalus oleifolius DC. and Astragaloside VII (Ast VII) from Astragalus trojanus Stev.], and their adjuvant potentials on the cellular and humoral immune responses of Swiss albino mice against BSA. Materials and methods: The hemolytic activity of Mac B and Ast VII was determined using 0.5% rabbit red blood cell. For adjuvant activity, Swiss albino mice were immunized subcutaneously with BSA 100 ␮g alone or with BSA 100 ␮g dissolved in saline containing Ast VII (30, 60, 120 and 240 ␮g), Mac B (30, 60, 90 and 120 ␮g) or Freund’s adjuvant on Days 1 and 15. Sera and splenocytes were collected 2 weeks after the last immunization for concanavalin A (Con A)-, lipopolysaccharide (LPS)- and BSA-stimulated splenocyte proliferation assay and measurement of BSA-specific antibodies in serum. Results: Mac B and Ast VII showed a slight hemolytic effect, with 0.42% and 0.54% values, respectively, at the highest concentration of 500 ␮g/ml. Mac B and Ast VII significantly enhanced the Con A-, LPS-, and BSA-induced splenocyte proliferation in the BSA-immunized mice especially at 120 and 240 ␮g (P < 0.001), and 60, 90 and 120 ␮g (P < 0.05, P < 0.01 or P < 0.001) doses, respectively. BSA-specific IgG, IgG1 and IgG2b antibody titers in serum were also significantly enhanced by Ast VII (120 ␮g), Mac B (90 ␮g) and Freund’s as compared to the control group (P < 0.01 or P < 0.001). Moreover, the IFN-␥ and IL-4 levels in the sera were detected using ELISA two weeks after the last immunization. Ast VII and Mac B were also found to stimulate IFN-␥ production such as Freund’s, two weeks after the last immunization at doses of 120 ␮g and 90 ␮g, respectively, as compared to the control. Conclusion: Results show that Ast VII and Mac B generate important specific antibody and cellular response against BSA in mice, proving their potentials as a new class saponin adjuvant. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The goal of vaccination is to stimulate a strong, protective and long-lasting immune response to the administered antigen. New generations of vaccines, particularly those based on purified recombinant proteins, synthetic peptides and plasmid DNA, are likely to be less reactogenic and immunogenic than traditional vaccines (Yang et al., 2005; Rajput et al., 2007; Ragupathi et al., 2008; Sun et al., 2008a; Xie et al., 2008b). The majority of these vaccines require association with adjuvants capable of increasing the potency or stimulating the appropriate immune response (Yang et al., 2005; Xie et al., 2008b; Sun et al., 2008b). Although a variety of adjuvants have been used in experimental vaccines, most of

∗ Corresponding author. Tel.: +90 232 388 4955; fax: +90 232 388 4955. E-mail addresses: [email protected], [email protected] (E. Bedir). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.01.054

these materials only elicit an antibody response and/or have undesirable side effects that have limited their potential application in vaccines (Edelman, 2000; Yang et al., 2005; Rajput et al., 2007; Sun et al., 2008a). Several important biological properties have been attributed to the saponins. Their use as humoral and cellular immunomodulators has been intensified in dogs, cattle, sheep, mice, pig and non-human primate experimental models (Yang et al., 2005; Rajput et al., 2007; Sun et al., 2008a,b; Xie et al., 2008a,b). The roots of Astragalus membranaceus are used in traditional Chinese medicine as an antiperspirant, diuretic and tonic drug. It has also been used in the treatment of diabetes mellitus, nephritis, leukemia and uterine cancer (Tang and Eisenbrand, 1992). Astragalus roots are known to be rich in two major classes of biologically active compounds, polysaccharides and saponins (Rios and Waterman, 1997; Bedir et al., 2000a). Astragalus saponins have shown interesting pharmacological properties, including

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immunostimulation (Calis et al., 1997; Bedir et al., 2000a; Yes¸ilada et al., 2005), anti-protozoal (Özipek et al., 2005), antiviral (Özipek et al., 2005), cytotoxic (Radwan et al., 2004) and cardiotonic activities (Khushbaktova et al., 1994; Luo et al., 2004; Chan et al., 2009). Moreover, Astragaloside IV, a widely encountered cycloartane-type saponin found in Astragalus species, has been proven to be a neuroprotective agent and proposed as a potential agent in the treatment of Parkinson’s disease (Luo et al., 2004; Chan et al., 2009). As part of our continuing studies to discover new pharmacologically active secondary metabolites, we undertook a detailed investigation on Astragalus genus and its saponins (Bedir et al., 2000a,b, 2001a,b; Tabanca et al., 2005; Polat et al., 2009, 2010). Overriding basis for our biological activity studies was traditional use of the Astragalus roots in South East Anatolia to cure leukemia. Therefore, our preliminary studies focused on the cytotoxicity of the Astragalus extracts and their saponins versus tumor cell lines, which resulted no significant activity (unpublished data). Based on that, it was hypothesized that the activity might be due to the stimulation of the immune system, and further studies were carried out (Calis et al., 1997; Bedir et al., 2000a; Yes¸ilada et al., 2005). In the most recent study (Yes¸ilada et al., 2005), our group screened fourteen Astragalus saponins and three different Astragalus crude saponin extracts for their activities on the immune system inducing cytokines at 3 ␮g/ml concentration. In this study, the cytokines responsible for the immune response against cancer (IL-2, IL-4, IL8, IFN-␥, IL-1␤, TNF-␣) were primarily targeted. Induction on IL-2 caused by the pure molecules was noteworthy ranging between 35.9% and 139.6%. Among the pure molecules, Astragaloside VII, tridesmosidic glycoside of cycloastragenol, was found to be the most remarkable compound with 139.6% induction compared to the control. Among the crude saponin extracts, Astragalus oleifolius DC. that is used for the treatment of leukemia in South East Anatolia showed the strongest activity on IL-2 induction with the value of 141.2%. Taking into account the results of our comprehensive studies and preliminary screenings in addition to recent progress in the literature, we have decided to carry out a series of in vivo studies to prove effectiveness of the Astragalus saponins. To investigate adjuvant properties of the Astragalus saponins, and their effects on the immune response cytokines (IL-1␤, TGF-␤, TNF-␣, IL-2, IL-4, and IFN-␥), we selected two cycloartane-type molecules based on the abovementioned in vitro studies: Macrophyllosaponin B (Mac B, the major saponin of the most active species Astragalus oleifolius), and Astragaloside VII. Here, we report the first part of our ongoing studies that is the hemolytic activity of Ast VII and Mac B and their adjuvant potentials on the cellular and humoral immune responses of Swiss albino mice against BSA.

2. Materials and methods 2.1. Materials Bovine serum albumin (BSA), 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), complete Freund’s (cFA) and incomplete Freund’s (iFA) adjuvant were purchased from Sigma Chemical Co., USA; concanavalin A (Con A), RPMI-1640 medium and fetal bovine serum (FBS) were from Biochrome, Berlin, Germany. The RMPI-1640 medium was supplemented with HEPES buffer 10 ␮mol/ml, gentamycine 10 ␮g/ml, l-glutamine 2 ␮mol/ml, 2mercaptoethanol 50 ␮mol/l and 10% fetal bovine serum, pH 7.2. Goat anti-mouse IgG, IgG1 and IgG2b peroxidase conjugate were from Southern Biotech. Assoc., Birmingham, AL, USA; lipopolysaccharide (LPS); prepared from Salmonella Enteritidis 64 K by hot water phenol extraction method (Nalbantsoy et al., 2010). All other chemicals were of AR grade.

OH

O

O HO

OH OH

O OH HO HO

O O

HO

O

OH

O HO

OH OH

Fig. 1. Astragaloside VII.

2.2. Experimental animals Male Swiss albino mice (4–5-weeks old) weighing 18–22 g were purchased from Bornova Veterinary Control and Research Institute (Bornova, Izmir, Turkey). Mice were maintained in groups of 5 under standard conditions of temperature 22 ± 1 ◦ C with regular 12 h light:12 h dark cycle and were allowed free access to standard laboratory food and water. The experimental protocol was approved by the Local Ethics Review Committee for Animal Experimentation of Ege University (number 2009-9). 2.3. Isolation of the saponins Triterpene glycosides, Ast VII (Bedir et al., 1999a) and Mac B (Calis et al., 1996) were previously isolated and chemically defined from Astragalus trojanus Stev. and Astragalus oleifolius DC., respectively (Figs. 1 and 2). 2.4. Hemolytic activity assay The hemolytic activity of Ast VII and Mac B was measured according to the modified method of Yang et al. (2005). Red blood cells were obtained from healthy New Zealand rabbit (Bornova Veterinary Control and Research Institute, Izmir, Turkey). Blood was collected with BD Vacutainer TM (NH 143 I. U., Belliver Industrial Estate, Plymouth, UK). Aliquots of 7 ml of blood were washed three times with sterile saline solution (0.89%, w/v NaCl, pyrogen free) by centrifugation at 2000 × g for 5 min. The cell suspension was prepared by finally diluting the pellet to 0.5% in saline solution. A volume of 0.01 ml of the cell suspension was mixed in U button 96-well microplate with 0.05 ml diluents containing 2.5, 12.5, 25, 50, 125, 250 and 500 ␮g/ml concentrations of Ast VII and Mac B in saline solutions The mixtures were incubated for 30 min at 37 ◦ C and centrifuged at 800 × g for 10 min. The free hemoglobin in the supernatants was measured spectrophotometrically at 412 nm. Saline and distilled water were included as minimal and maximal hemolytic controls. The hemolytic percent developed by the saline control was subtracted from all groups. Each experiment included triplicates at each concentration. O

OH

OH

O H3C HO

OH

OH

O HO

O HO

OH Fig. 2. Macrophyllosaponin B.

OH OH

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2.5. Immunization Four–six-weeks old male Swiss albino mice were divided into eleven groups, each consisting of five mice. Animals were immunized subcutaneously with BSA 100 ␮g alone or with BSA 100 ␮g dissolved in saline containing Ast VII (30, 60, 120 and 240 ␮g), Mac B (30, 60, 90 and 120 ␮g) or cFA–iFA on Day 1. Saline-treated animals were included as controls. A boosting injection was given 2 weeks later. Sera and splenocytes were collected 2 weeks after the second immunization for proliferation assay and measurement of BSA-specific antibody. 2.6. Measurement of BSA-specific antibody BSA-specific IgG, IgG1 and IgG2b antibodies in serum were detected by ELISA according to the method previously described by Yang et al. (2005) with some modifications. In brief, the wells of 96-well microtiter plates were coated with 100 ␮l of BSA solution (50 ␮g/ml in 50 mM carbonate buffer, pH 9.6) for overnight at 37 ◦ C. The wells were washed three times with 200 ␮l of PBS containing 0.05% (v/v) Tween 20. After washing three times with PBST, 100 ␮l of diluted serum samples (1:100 and 1:1000 for each antibody) or PBS as control were added to duplicate wells. The plates were then incubated for 1 h at 37 ◦ C, followed by washing three times. Aliquots of 100 ␮l of horseradish peroxidase-conjugated goat antimouse IgG, IgG1 or IgG2b (diluted 1:8000 in PBS) were added and incubated for 1 h at 37 ◦ C. After washing, the enzyme reaction was developed with the addition of H2 O2 (Merck, Darmstadt, Germany) and, O-phenylenediamine (Sigma, St. Louis, USA). The plate was incubated for 30 min at 37 ◦ C, and enzyme reaction was terminated by adding 50 ␮l/well of 4 M H2 SO4 (Riedel-de Haën, Seelze, Germany). The optical absorbance was measured at 492 nm. Data were expressed as the mean OD value of the samples minus the mean OD value of the control. Results were expressed as log 2 titers. Where sets of serum samples have been subjected to within and between group comparisons, ELISA assays of all the samples were performed on the same day. 2.7. Splenocyte proliferation assay in vivo The BSA-immunized mice were sacrificed by cervical dislocation under anesthesia with ether inhalation and spleen cells of mice were prepared by gently mincing and grinding the spleen fragment in RPMI-1640 medium on a fine steel mesh under aseptic conditions. After centrifugation (450 × g at 4 ◦ C for 7 min), the pelleted cells were washed three times. At last, the cells were resuspended in 2 ml RPMI-1640 complete medium, and cell numbers were done with a hemocytometer by trypan blue dye exclusion technique. Cell viability exceeded 95%. Splenocyte proliferation was assayed as previously described (Sun et al., 2007). Briefly, an aliquot of 100 ␮l of splenocytes at 5 × 106 cells/ml was seeded into each well of a 96-well flat bottom microtiter plate, thereafter Con A (final concentration 5.0 ␮g/ml), LPS (final concentration 10.0 ␮g/ml), BSA (final concentration 10.0 ␮g/ml), or medium was added giving a final volume of 200 ␮l. After preincubation for 68 h at 37 ◦ C in a humidified 5% CO2 incubator, 50.0 ␮l of MTT solution (2 mg/ml) was added into each well. The plate was incubated for another 4 h and centrifuged (1400 × g, 5 min) to remove the untransformed MTT carefully by pipetting. Then to each well a total of 200 ␮l DMSO was added to fully dissolve the colored material. The absorbance at 570 nm with a 630 nm reference was measured on an ELISA reader (Versa Max, Molecular Devices, USA). The stimulation index (SI) was calculated based on the following formula: SI = the absorbance value for mitogen − cultures divided by the absorbance value for non-stimulated cultures. Each experiment was performed in triplicate.

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Table 1 Hemolytic activities of Ast VII and Mac B saponins.a Group

Absorbance value

Hemolytic percent (%)

Distilled water Saline

1.557 ± 0.057 0.068 ± 0.001

100.00 ± 1.65*** 0.000 ± 0.32

Ast VII (␮g/ml) 500 250 125 50 25 12.5 2.5 Mac B (␮g/ml) 500 250 125 50 25 12.5 2.5

0.074 0.072 0.070 0.069 0.068 0.068 0.067

± ± ± ± ± ± ±

0.005 0.001 0.002 0.001 0.002 0.004 0.007

0.054 0.042 0.029 0.023 0.01 0.01 −0.01

± ± ± ± ± ± ±

0.21* 0.27 0.13 0.19 0.32 0.17 0.08

0.072 0.070 0.069 0.069 0.068 0.067 0.066

± ± ± ± ± ± ±

0.002 0.001 0.002 0.001 0.003 0.005 0.007

0.042 0.029 0.023 0.023 0.01 −0.01 −0.08

± ± ± ± ± ± ±

0.20 0.26 0.18 0.16 0.14 0.11 0.09

a Hemolytic percents of saline and distilled water were included as minimal and maximal hemolytic control. All values represent the mean ± standard deviation (n = 3 test). * Significant difference with saline group was designated as P < 0.05. *** Significant difference with saline group was designated as P < 0.001.

2.8. Measurement of IFN- and IL-4 The IFN-␥ and IL-4 production was determined by Enzyme Linked Immunosorbent Assay (ELISA) kits (Pierce Chemical Company, Rockford, IL, USA). Manufacturers’ guidelines were followed for each assay. Concentrations of IFN-␥ and IL-4 in the serum samples were then determined by comparing the average sample optical density readings with the concentrations from the assay standard curve. 2.9. Statistical analysis The data were expressed as mean ± standard errors (S.E.) and examined for their statistical significance of difference with Student t-test, ANOVA and the post hoc test. P-values of less than 0.05 were considered to be statistically significant. 3. Results 3.1. Hemolytic activities The hemolytic activities of Ast VII and Mac B on rabbit red blood cells were shown in Table 1. No hemolytic activity at concentrations of 2.5–500 ␮g/ml was observed. However, after centrifugation of the 96-well microplate, hemaglutination was detected by clumping of erythrocytes for both compounds only between 50 and 500 ␮g/ml concentrations with no hemolytic activity. 3.2. Effect of Ast VII and Mac B on mitogen- and BSA-stimulated splenocyte proliferation in BSA-immunized mice The effects of Ast VII and Mac B on mitogen- and BSA-stimulated splenocyte proliferation in BSA-immunized mice were shown in Figs. 3 and 5. Con A or LPS-stimulated splenocyte proliferation in the mice immunized with BSA/Ast VII and BSA/Mac B was significantly higher than that in the BSA control group (P < 0.001). Moreover, BSA-induced splenocyte proliferation in the BSA-immunized mice was also significantly enhanced by Ast VII and Mac B at 120 and 240 ␮g (P < 0.001), and 60, 90 and 120 ␮g (P < 0.05, P < 0.01 or P < 0.001) doses, respectively. However, no significant difference (P > 0.05) was observed between the BSA group and BSA/Freund’s

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Fig. 3. Effect of Ast VII on mitogen- and BSA-stimulated splenocyte proliferation in vivo. Groups of five male Swiss albino mice were immunized sc with BSA 100 ␮g alone or with BSA 100 ␮g dissolved in saline containing cFA, iFA, Ast VII (30, 60, 120 and 240 ␮g) on Days 1 and 14. Splenocytes were prepared 2 weeks after the last immunization and cultured with Con A, LPS, or BSA or RPMI 1640 for 72 h. Splenocyte proliferation was measured by the MTT method as described in the text, and shown as a stimulation index. The values are presented as mean ± S.E. (n = 5). Significant differences with BSA groups were designated as *P < 0.05, **P < 0.01 and ***P < 0.001.

group. Besides, there was no splenocyte proliferation difference (P > 0.05) between the BSA/Ast VII (30 and 60 ␮g) and BSA/Mac B (30 ␮g) groups.

with the BSA/Ast VII (120 ␮g) and BSA/Freund’s. Thus, based on the above findings, we can draw a conclusion that Ast VII and Mac B at proper doses can enhance serum antibody production in mice immunized with BSA.

3.3. Effects of Ast VII and Mac B on the BSA-specific serum antibody response The effects of Freund’s, Ast VII and Mac B on the induction of humoral immune response against BSA were studied on mice subjects by immunizing twice via sc routes. The BSA-specific IgG, IgG1 and IgG2b antibody levels in the sera were detected using ELISA two weeks after the last immunization. As shown in Figs. 4 and 6, the serum IgG, IgG1 and IgG2b antibody levels immunized with BSA were significantly increased by Ast VII (120 ␮g), Mac B (90 ␮g) and Freund’s as compared to control group (P < 0.01 or P < 0.001). Furthermore, the serum IgG2b level in Ast VII (120 ␮g), Mac B (90 and 120 ␮g) and Freund’s immunized mice were notably higher than those in the control (P < 0.01 or P < 0.001). Meanwhile, there was also no serum antibody response difference (P > 0.05) among the BSA-specific serum IgG and IgG1 levels in the groups immunized

3.4. Effect of Ast VII and Mac B on IFN- and IL-4 production To investigate the effects of Ast VII and Mac B on the production of IFN-␥ and IL-4 against BSA, groups of mice were immunized twice by sc route. The IFN-␥ and IL-4 levels in the sera were detected using ELISA two weeks after the last immunization as shown in Fig. 7. As shown in Fig. 7, the serum IFN-␥ level in mice immunized with BSA was considerably enhanced by Ast VII (120 ␮g), Mac B (90 ␮g) and Freund’s (P < 0.01 or P < 0.001). On the other hand, there was no serum IFN-␥ level difference (P > 0.05) among the BSA and physiologic saline whereas there was also no serum IL-4 difference in all the tested groups.

Fig. 4. Effect of Ast VII on BSA-specific IgG, IgG1 and IgG2b antibodies. Groups of five male Swiss albino mice were immunized sc with BSA 100 ␮g alone or with BSA 100 ␮g dissolved in saline containing Freund’s and Ast VII (30, 60, 120 and 240 ␮g) on Days 1 and 14. Sera were collected 2 weeks after the last immunization. BSA-specific IgG, IgG1 and IgG2b antibodies in the sera were measured by an indirect ELISA as described in the text. The values are presented as mean ± S.E. (n = 5). Significant differences with BSA groups were designated as ***P < 0.001.

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Fig. 5. Effect of Mac B on mitogen- and BSA-stimulated splenocyte proliferation in vivo. Groups of five male Swiss albino mice were immunized sc with BSA 100 ␮g alone or with BSA 100 ␮g dissolved in saline containing cFA, iFA, Mac B (30, 60, 90 and 120 ␮g) on Days 1 and 14. Splenocytes were prepared 2 weeks after the last immunization and cultured with Con A, LPS, or BSA or RPMI 1640 for 72 h. Splenocyte proliferation was measured by the MTT method as described in the text, and shown as a stimulation index. The values are presented as mean ± S.E. (n = 5). Significant differences with BSA groups were designated as *P < 0.05, **P < 0.01 and ***P < 0.001.

4. Discussion Adjuvants have been used to augment the immune response to antigens for more than 70 years. Mostly aluminum or oil adjuvants are used in vaccine, but these chemical adjuvants have many disadvantages, such as side effects, strong local stimulation and carcinogenesis, together with complicated preparations or failure to increase immunogenicity of weak antigen and so on (Edelman, 2000; Ragupathi et al., 2008). Some other group of adjuvants including oil emulsions, lipopolysaccharides, polymers, saponins, liposomes, cytokines, ISCOMs, Freund’s complete adjuvant, Freund’s incomplete adjuvant, alums, bacterial toxins, etc., have been evaluated and clinical trials are under investigation, although the mechanism of action of adjuvants often remain poorly understood (Edelman, 2000; Sun et al., 2008b; Ragupathi et al., 2008). However, there is still an urgent need for the development of new and improved vaccine adjuvants (Yang et al., 2005; Sun et al., 2008a; Xie et al., 2008b).

Many widely used botanicals are claimed to have immunostimulant effects, but clear evidence that they are able to augment immunological responses against defined antigens is lacking (Ragupathi et al., 2008). Saponins are natural products that are promising sources of adjuvants. Saponin based adjuvants have the ability to modulate the cell mediated immune system as well as to enhance antibody production and have the advantage that only a low dose is needed for adjuvant activity (Oda et al., 2000; Yang et al., 2005; Ragupathi et al., 2008; Sun et al., 2008a; Xie et al., 2008a,b). The most widely used saponin-based adjuvant is Quil A isolated from the bark of Quillaja saponaria Molina (Edelman, 2000; Xie et al., 2008a). Astragalus saponins are believed to induce the cellular and humoral immune responses with slight hemolytic activity. Yang et al. (2005) reported very low hemolytic effect (0.66%) with 500 ␮g/ml concentrations of Astragalus membranaceus saponins (AMS) induced in mice and significantly enhanced BSA-specific IgG, IgG1 and IgG2b antibody titers in mice serum.

Fig. 6. Effect of Mac B on BSA-specific IgG, IgG1 and IgG2b antibodies. Groups of five male Swiss albino mice were immunized sc with BSA 100 ␮g alone or with BSA 100 ␮g dissolved in saline containing Freund’s and Mac B (30, 60, 90 and 120 ␮g) on Days 1 and 14. Sera were collected 2 weeks after the last immunization. BSA-specific IgG, IgG1 and IgG2b antibodies in the sera were measured by an indirect ELISA as described in the text. The values are presented as mean ± S.E. (n = 5). Significant differences with BSA groups were designated as **P < 0.01 and ***P < 0.001.

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Fig. 7. Effect of Ast VII and Mac B on BSA-stimulated IFN-␥ and IL-4 production in the sera in vivo. Groups of five male Swiss albino mice were immunized sc with BSA 100 ␮g alone or with BSA 100 ␮g dissolved in saline containing Freund’s, Ast VII (120 ␮g) or Mac B (90 ␮g) on Days 1 and 14. Sera were collected two weeks after the last immunization. IFN-␥ and IL-4 in the sera were measured by an indirect ELISA as described in the text. The values are presented as mean ± S.E. (n = 5). Significant differences with BSA groups were designated as **P < 0.01 or ***P < 0.001.

In this study, Ast VII and Mac B that are the major constituents of Astragalus trojanus and Astragalus oleifolius, respectively, were investigated for their hemolytic effects and adjuvant activities in response to BSA antigen in vivo. The concentration of 500 ␮g/ml Ast VII and Mac B exhibited a slight hemolytic effect with 0.54% and 0.42% hemolysis on 0.5% rabbit red blood cell (Table 1). The chemical compositions of the saponins are directly related with their hemolytic properties. Previous studies proved steroidal saponins as superior hemolytic agents compared to triterpenes (Takechi and Tanaka, 1995). Among triterpenic saponins, the number and the complexity of the sugar moieties, presence of the acyl residues or the epoxy groups were found to be important for hemolytic index (Santos et al., 1997; Sun et al., 2008a). Ast VII, the first tridesmosidic molecule encountered in the plant kingdom, has three sugar units (␤-d-xylose at C-3, ␤-d-glucose units at C-6 and C-25) on the cycloartane framework (cycloastragenol) having 20,24-epoxy side chain. Mac B with macrophyllosapogenol aglycon possesses acyclic side chain at C-17 and two sugar moieties at C-3 and C-24 positions. Our findings, very low hemolytic activities observed for both compounds (Ast VII and MacB), were compatible with the previous studies, which revealed once more that fewer number of saccharide units and less complexity of the structures in regards to functional groups (missing aldehyde or acyl groups) results with negligible hemolytic action. Moreover, it should be emphasized that the tridesmosidic nature of the saponin (Ast VII), reported here for the first time, turned out to be an insignificant feature for the activity. Since Astragalus cycloartanes have been found mostly in di- or trisaccharidic forms with the same skeletons (20,24-epoxy side chain as in AST-VII or acyclic side chain as in MacB), one can assume that no or very low hemolytic activity could be derived from the Astragalus saponin preparations. In fact, Wang et al. (2002) investigated the protective effect of Astragalus crude saponin fraction (AMS) on human erythrocyte membrane damages caused by reactive oxygen species. The results showed that AMS could significantly inhibit the membrane lipid peroxidation generated by O2 − , H2 O2 and UV rays (Yang et al., 2005). Zheng et al. (1995) found that AMS could improve red blood cell deformability of human beings and mice (Yang et al., 2005). Baumann et al. (2000) reported that the interaction between AMS and the sterols of the erythrocyte membrane could not lead to damage to the lipid bilayer but decrease the chance of erythrocyte membrane bursts.

Based on our results and abovementioned studies, we deduce that the immune system stimulation caused by the Astragalus saponins is not due to their minor hemolytic activities. T-lymphocyte-mediated immunity plays an important role in combating intracellular microbial infections. Among the T lymphocytes, helper T-cells induces B-lymphocytes to secrete antibodies, and cytotoxic T-lymphocytes help phagocytes to destroy ingested microbes and to kill intracellular microbes. Humoral immunity, however, mediated by antibodies which are produced by B-lymphocytes, functions by neutralizing and eliminating extracellular microbes and microbial toxins. The capacity to elicit an effective T-cell immunity can be shown by the stimulation of lymphocyte proliferation response (Edelman, 2000; Marciani et al., 2000; Xie et al., 2008a). In the present study, we evaluated whether Ast VII or Mac B could enhance the cellular immune responses to BSA in mice when given together with BSA. As shown in Figs. 3 and 5, Ast VII and Mac B significantly augmented the mitogen- and BSA-stimulated splenocyte proliferations in BSA-immunized mice at a dose of 120 and 90 ␮g, respectively, compared to the BSA control group. However, no considerable difference was observed between the BSA and BSA/Freund’s groups. The results indicated that Ast VII and Mac B could notably increase the activation potential of T and B cells in BSA-immunized mice with no adverse effects, whereas Freund’s possesses poor adjuvant activity in cellular immune response, and causes adverse effects on the injection side. Thus, adjuvants based on Ast VII and Mac B might have a potential for use in vaccines against both pathogens and cancer. On the other hand, adjuvants should promote an appropriate immune response (Th1 or Th2). The Th1 response is required for protective immunity against intracellular infectious agents, such as viruses, certain bacteria and protozoa, and presumably against cancer cells, while Th2 immunity is effective for protection against most bacterial as well as certain viral infections (Edelman, 2000; Xie et al., 2008a). The immune responses in mice have been differentiated into either Th1- or Th2-type based on the cytokine profile induced. Different patterns of cytokine secretion can influence the type of immune response that develops. The Th1 profile is characterized by the initial production of IFN-␥ and IL-12 with an effector phase of T lymphocytes producing IL-2 and IFN-␥ and mediating cytotoxic activity (CTL). In contrast, Th2 responses are influenced by IL-4 with predominantly IL-5 and IL-10-producing T cells in

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the effector phase that drive antibody production. The isotype of antibody production depends on the ratio of Th1 and Th2 activation, with a Th1 bias giving preferentially IgG2a or IgG2b, and a Th2 preference resulting in more IgG1 (Edelman, 2000; Yang et al., 2005). Ast VII and Mac B significantly boosted BSA-specific IgG and IgG1 levels in mice immunized with BSA at doses of 120 ␮g, and 90, respectively, as compared to the controls. Ast VII and Mac B also notably enhanced total serum IgG2b level at doses of 120 ␮g, 90 ␮g and 120 ␮g, respectively, whereas no difference was observed between the BSA/Ast VII (120 ␮g) group and BSA/Freund’s group unlike Mac B (90 ␮g) group. In this study, Ast VII induced IFN-␥ production strongly two weeks after the last immunization at 120 ␮g, while Freund’s and Mac B were moderately active compared to the controls (Fig. 7). However, IL-4 was not detected two weeks after the last immunization in all the tested groups. Although IL-4 was absent on day 28, the presence of IgG1 implied that the Th2 mechanism was also active together with Th1 as described previously for Quillaja saponins (Sjölander et al., 1997). Mosmann and Coffman (1989) reported that since both Th types could activate B cells, which are then responsive to lymphokines produced by either Th type, antibody responses would be strongly supported in a mixed Th1 and Th2 response. Thus, it is likely that Ast VII and Mac B at suitable doses are primarily effective on Th1 and slightly on Th2 cells, as associated sensitively with an induction of IgG1 and IgG2b levels. Consequently, following sc immunization, BSA-Ast VII, -Mac B and -Freund’s induced high titers of BSA-reactive IgG1, and IgG2b. These observations suggest once more that adjuvants have varying effects on immunoglobulin isotype switching and can induce antibody responses with different isotype and IgG subclass profiles as described by Sjölander et al. (1997). Ast VII and Mac B have shown substantial specific antibody and cellular response against BSA in mice, proving their potentials as a new class saponin type adjuvant. Different from the other studies performed on the genus Astragalus, a well-known traditional medicine used to boost immune system, pure cycloartane-type saponins were shown for the first time as promising adjuvants. Further studies for probable use of Ast VII and Mac B in vaccination programs are in progress. Acknowledgments This work was financially supported in part by a grant from The Scientific and Technological Research Council of Turkey (TUBITAK, Project No: 109T637), and Ege University ScienceTechnology Application and Research Center (EBILTEM, Project No: 2010/BIL/016). References Baumann, E., Stoya, G., Volkner, A., Richter, W., Lemke, C., Linss, W., 2000. Hemolysis of human erythrocytes with saponin affects the membrane structure. Acta Histochemica 102, 21–35. Bedir, E., C¸alıs¸, I., Aquino, R., Piacente, S., Pizza, C., Trojanoside, H., 1999a. A novel cycloartane type glycoside from the aerial parts of Astragalus trojanus. Phytochemistry 51, 1017–1020. Bedir, E., Pugh, N., C¸alıs¸, I., Pasco, D.S., Khan, I.A., 2000a. Immunostimulatory effects of cycloartane-type triterpene glycosides from Astragalus species. Biological and Pharmaceutical Bulletin 23, 834–837. Bedir, E., C¸alıs¸, I., Khan, I.A., Macrophyllosaponin, E., 2000b. A novel compound from the roots of Astragalus oleifolius. Chemical and Pharmaceutical Bulletin 48, 1081–1083. Bedir, E., Tatlı, I.I., C¸alıs¸, I., Khan, I.A., 2001a. Trojanosides I–K: new cycloartane-type glycosides from the aerial parts of Astragalus trojanus. Chemical and Pharmaceutical Bulletin 49, 1482–1486. Bedir, E., C¸alıs¸, I., Dunbar, C., Sharan, R., Buolamwini, J.K., Khan, I.A., 2001b. Two novel cycloartane-type triterpene glycosides from the roots of Astragalus prusianus. Tetrahedron 57, 5961–5966. Calis, I., Zor, M., Sarac¸oglu, I., Isimer, A., Ruegger, H., 1996. Four novel cycloartane glycosides from Astragalus oleifolius. Journal of Natural Products 59, 1019–1023.

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