International Immunopharmacology 18 (2014) 333–339

Contents lists available at ScienceDirect

International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp

Haemolytic activity and adjuvant effect of soyasaponins and some of their derivatives on the immune responses to ovalbumin in mice Na Qiao a, Qi Liu a, He Meng b,c,⁎, Dayun Zhao a,d,⁎⁎ a

Department of Food Science and Technology, Shanghai Jiao Tong University, Shanghai 200240, China Department of Animal Science, Shanghai Jiao Tong University, Shanghai 200240, China Shanghai Key Laboratory of Veterinary Biotechnology, Shanghai 200240, China d Bor S. Luh Food Safety Research Center, Shanghai Jiao Tong University, Shanghai 200240, China b c

a r t i c l e

i n f o

Article history: Received 8 October 2013 Received in revised form 13 December 2013 Accepted 15 December 2013 Available online 27 December 2013 Keywords: Soyasaponin Soyasaponin derivative Immunologic adjuvant Haemolysis Ovalbumin

a b s t r a c t Immunological adjuvants are agents that enhance specific immune responses to vaccines. At present more studies are needed to identify a suitable adjuvant for a particular vaccine with maximum safety and efficacy. In this study, six soyasaponins (Aa, Ab, Af, Ba, Bb, and Bb′) and three soyasaponin Ab-derivatives (AbDs) were selected to evaluate their toxicities and adjuvant activities. The haemolytic activity assay was performed to evaluate the toxicity of the tested soyasaponins and AbDs. Immunoadjuvant activity was investigated in vivo and in vitro using a splenocyte proliferation assay and sera indirect ELISA. Our results demonstrated that soyasaponins and AbDs showed a slight haemolytic effect to 0.5% red blood cell. Except for the Af and Ba groups, other soyasaponins and AbDs groups stimulated by concanavalin A (ConA) and lipopolysaccharide (LPS) showed a greater proliferative response at appropriate doses (0.01–10 μg/ml) compared with the control and Alum groups. Anti-OVA IgG, IgG1, IgG2a, and IgG2b were significantly enhanced by the soyasaponins (Ab, Ba, Bb, and Bb′), QS and AbDs groups (p b 0.05 or p b 0.01). In addition, three AbDs indicated a tendency of immunoadjuvant potential improvement after structural modification. Moreover, Ab-D2 showed adjuvant activity at the lowest injection dose among the three AbDs. In conclusion, these results suggested that soyasaponins together with their derivatives may represent viable candidates for effective vaccine adjuvants due to their higher immune response and lower or non-haemolytic effects. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Adjuvants, derived from the Latin word “adjuvare,” means “to help,” and have been used to improve vaccine efficacy from the early 1920s [1,2]. Many antigen formulations require co-administration with an adjuvant, a substance that is in itself not necessarily immunogenic but functions in concert with the antigen to enhance/prolong the immune response [3]. Until recently, there has been a number of different adjuvants that can be categorised into two major groups: particulate and non-particulate. These adjuvants include aluminium salts, waterin-oil (W/O) emulsions, oil-in-water (O/W) emulsions, immunestimulating complexes (ISCOM), liposomes, cytokines, lipid A, bacterial toxins, carbohydrate polymers and saponins. Despite a plethora of options, only aluminium salts have gained acceptance as human vaccine adjuvants, and veterinary vaccines are largely dependent upon the use

⁎ Correspondence to: H. Meng, Department of Animal Science, Shanghai Jiao Tong University, Shanghai 200240, China. Tel./fax: +86 21 3420 6146. ⁎⁎ Correspondence to: D. Zhao, Department of Food Science and Technology, Shanghai Jiao Tong University, Shanghai 200240, China. Tel./fax: +86 21 3420 5711. E-mail addresses: [email protected] (H. Meng), [email protected] (D. Zhao). 1567-5769/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.intimp.2013.12.017

of aluminium salts because of their safe and strong Th2 response [2]. Thus, great efforts have been made to identify ideal adjuvants for vaccine application. It has been demonstrated that saponins induced strong Th1 and Th2 responses and moderate CTL responses to some proteins most likely as a result of forming mixed protein–saponin micelles [2]. Many types of plant saponins, including Achyranthes bidentata saponins, Panax notoginseng saponins, Acacia concinna saponins, Bupleurum Chinese saponins and ginsenoside have shown adjuvant effects on purified protein antigens [4–10]. The most widely used plant-saponin adjuvants are quillaja saponin (QS) and its derivative QS-21, isolated from the bark of the South American tree Quillaja saponaria Molina [11]. These adjuvants are still currently under clinical evaluation in various vaccines and have been tested in many clinical trials [12]. Quillaja saponin not only promotes humoral immunity but also induces the Th1 as well as Th2 immune responses against exogenous antigens [13], which make it more effective than aluminium in stimulating an antibody response [14]. However, their high toxicity, instability in the aqueous phase and undesirable haemolytic effect have limited their use as adjuvant in human vaccination [15]. Thus, there is increased interest to identify ideal saponin-based adjuvants with high immune activity and low haemolytic effects.

334

N. Qiao et al. / International Immunopharmacology 18 (2014) 333–339

Soyasaponins are bioactive components in soybean and other legumes. They belong to the family of pentacyclic triterpenoid oleanane saponins [16]. They contain a sapogenol, which is linked to one or more sugar chains or oligosaccharide moieties [17]. Chemical saponin studies in soy can be traced back to the 1930s [18,19]. Within the past few decades, large research efforts, particularly those conducted by Japanese scholars, have been made to investigate the chemical structure and biological activities of soyasaponins [20,21]. It has been reported that approximately 36 soyasaponins can be identified in soybean, which on the basis of their sapogenol structures, can be mainly subdivided into four groups: A, B, E and DDMP, respectively [22–24]. These soyasaponins have been shown to exhibit a wide range of bioactivities, such as antioxidant activities [25], anti-cancer effects [26], cardiovascular protective effects [27], antiviral activities and hepatoprotective activities [28,29], and among these, the adjuvant effect [30] has aroused a strong interest in researchers. The present studies on the immunological properties of soyasaponins have focused on the field of crude extracts [20,21]. A comprehensive investigation of the adjuvant effect and haemolytic activity of soyasaponin individuals has not yet been clearly reported. In addition, there has been no clinical trial of the adjuvant potential of soyasaponin individual derivatives. In this study, we selected six soyasaponins (Aa, Ab, Af, Ba, Bb, and Bb′) and three AbDs (Ab-phenylethylamine (Ab-D1), Ab-dodecylamine (Ab-D2) and Ab-ethylenediamin e-phenylpropionic acid (Ab-D3)) (see their structures in Fig. 1) to analyse their haemolytic activity on red blood cells and to compare their activities to those commonly used in animal and human vaccination: aluminium hydroxide gel (Alum) and QS. The adjuvant potential of soyasaponins and AbDs on the cellular and humoral immune responses of ICR mice against OVA was measured. Data of select non-haemolytic saponins may provide a foundation for future research studies and further applications of soyasaponins and their derivatives, which could potentially be used as new adjuvants in future large-scale clinical vaccination trials. 2. Materials and methods 2.1. Soyasaponins and other reagents Soyasaponins Aa (HPLC purity N99%), Ab (N98%), Af (N85%), Ba (N 91%), Bb (N 98%), and Bb′ (degraded from γg) were obtained as previously described by our laboratory [32]. The chemical structures of these saponins were determined on the basis of chemical and physicochemical evidence, including UPLC-MS and NMR. Soyasaponin Ab was catalysed using carbodiimide, linked with different functional groups and derivatives in the form of Ab-phenylethylamine (Ab-D1, 99%), Ab-dodecylamine (Ab-D2, 99%) and Ab-ethylenediaminephenylpropionic acid (Ab-D3, 99%), were obtained (see their structures in Fig. 1). Detailed synthesising methods are as follows [31]. Briefly, hydrophobic dicyclohexylcarbodiimide (DCC) or hydrophilic N-(3Dimethylamino propy1)-N-ethylcarbodiimide hydrochloride (EDC) was dissolved in pyridine or dimethylformamide (DMF), respectively. After the addition of N-hydroxysuccinimide (NHS) and soyasaponin Ab, various compounds containing functional groups were induced. The derivatives were then tested using HPLC, and their structures were confirmed using IR, EMI/MS and NMR [33]. Stock solutions of soyasaponins at a concentration 1 mg/ml were prepared by dissolving in phosphate-buffered saline (PBS) and sterilised by passing it through a 0.22 μm Millipore filter. For the soyasaponin derivatives, after induction of the hydrophobic groups, the solubility of the derivatives was decreased in PBS. Prior to the preparation of the 5% (v/v) DMSO/PBS stock solution, solubilisation tests were performed to detect the maximal concentration of AbDs. Finally, all derivatives were prepared at their maximum concentrations (80 μg/ml) in 5% (v/v) DMSO/PBS stock solutions. Ovalbumin (OVA, grade V), concanavalin A (ConA), lipopolysaccharide (LPS) and saponins from quillaja bark (QS, CFAD-S4521-10G) as

partially purified extract with 20–35% of sapogenin content were purchased from Sigma Chemical Co. RPMI-1640 medium was obtained from HyClone Inc. Foetal calf serum (FCS) was purchased from Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. Goat anti-mouse IgG, IgG1, IgG2a or IgG2b horseradish peroxidase (HRP)-conjugates were obtained from Proteintech Group, Inc. Aluminium hydroxide gel (Alum) was purchased from Zhejiang Wanma Pharm Co. Ltd., Hangzhou, Zhejiang, China.

2.2. Haemolytic activity assay The haemolytic activity assay was determined as previously described [7] with minor modifications. Red blood cells were obtained from healthy female ICR mice (6 to 8 weeks, Shanghai Lab. Animal Research Center, PR China). The blood cell suspension was prepared by diluting the pellet to 0.5% with PBS. The free haemoglobin in the supernatants was tested spectrophotometrically at 405 nm. Each experiment included triplicates at each concentration. The 50% haemolysis dose (HD50) was defined as the concentration of saponins inducing 50% of maximum haemolysis.

2.3. Splenocyte proliferation assay Splenocyte proliferation was assayed as previously described [6]. Female ICR mice were sacrificed by cervical dislocation, and the splenocytes at 1 × 107 cell/ml were seeded in sextuplicates in a 96well flat-bottom microtiter plate (Shanghai Winhong Biology Technology Co., Ltd). Concanavalin A (ConA, Sigma, final concentration 5 μg/ml), lipopolysaccharide (LPS, Sigma, final concentration 10 μg/ml), soyasaponin samples (final concentration 0.01–10 μg/ml) or complete medium was added to a volume of 200 μl. After the addition of the Cell Counting Kit-8 (CCK-8 solution, Shanghai qcbio Science & Technologies Co., Ltd, 20 μl/well), the optical densities (OD) of each well were evaluated using an ELISA reader at 450 nm (TECAN, Switzerland). The stimulation index (SI) was calculated based on the  following formula: SI ¼ ðODtc −ODbl Þ ðODn −OD Þ . (ODtc is the mean OD of tc

bl

the treated cultures, ODntc is the mean OD of the non-treated cultures, and ODbl is the mean OD of the blank wells) [6].

2.4. Measurement of sera OVA-specific antibody Groups of six female ICR mice were immunised subcutaneously with either ovalbumin alone in PBS or with OVA mixed with six soyasaponins, three AbDs, QS or Alum. Immunisations were performed twice on days 1 and 15. After 2 weeks of the second immunisation, sera samples were collected to measure the level of anti-OVA IgG, IgG1, IgG2a and IgG2b. All procedures were performed according to the China legislation on the use and care of laboratory animals and according to the guidelines established by the Shanghai Animal Care and Use Committee and university committee for animal experiments. Indirect ELISA was used to detect OVA-specific antibodies in serum as previously described with slight modifications [8]. The absorbance was tested at a wavelength of 450 nm using an ELISA reader. Data were reported as the mean optical density (OD) of the serum samples minus the mean OD of the control. The experiment was performed in triplicate.

2.5. Statistical analysis The data were expressed as the mean ± standard error (S.E.). SPSS statistics (version 17.0) were performed. Student's t-test and one-way ANOVA were used to examine the statistical significance of the difference. P-values of less than 0.05 were considered to be statistically significant.

N. Qiao et al. / International Immunopharmacology 18 (2014) 333–339

335

Fig. 1. Chemical structure of six soyasaponins and three AbDs.

3. Results 3.1. Haemolytic activity The haemolytic activity of soyasaponins, AbDs and QS is listed in Table 1. The haemolytic activities of mouse red blood cell treated with

soyasaponins were Bb 6.57% at a concentration of 200 μg/ml, whereas those of AbDs were Ab-D1 5.66% and 5.47% at a concentration of 64 μg/ml and 48 μg/ml, respectively. Ab-D2 was 4.09% at a concentration of 44 μg/ml. No haemolytic activity at other concentrations for other soyasaponins was observed, which demonstrated the safety of soyasaponins. Thus, the HD50 values for soyasaponins and their

336

N. Qiao et al. / International Immunopharmacology 18 (2014) 333–339

difference between the Af groups (0.01, 0.1, 1, and 10 μg/ml) and control group.

Table 1 Haemolytic activities of six soyasaponins, three AbDs and QS. Group

Concentration (μg/ml) (nmol/ml)

Absorbance value

Haemolytic percentage (%)

Distilled water PBS Aa

nd nd 200 (147) 150 (110) 100 (73) 50 (37) 200 (139) 150 (104) 100 (70) 50 (35) 200 (157) 150 (118) 100 (79) 50 (39) 200 (209) 150 (157) 100 (104) 50 (52) 200 (212) 150 (160) 100 (106) 50 (53) 200 (251) 150 (188) 100 (126) 50 (63) 64 (42) 48 (31) 32 (21) 16 (10) 44 (28) 33 (21) 22 (14) 11 (7) 76 (47) 57 (35) 38 (23) 19 (12) 40 (20) 20 (10) 10 (5) 1 (0.5)

0.981 0.183 0.213 0.207 0.211 0.199 0.213 0.209 0.188 0.184 0.209 0.209 0.191 0.178 0.184 0.186 0.190 0.158 0.235 0.203 0.177 0.154 0.209 0.205 0.195 0.202 0.228 0.227 0.217 0.146 0.227 0.216 0.217 0.180 0.205 0.197 0.197 0.211 0.980 0.731 0.351 0.193

100.00 ± 1.56⁎⁎⁎ 0.000 ± 0.66 3.71 ± 1.04 2.97 ± 1.05 3.50 ± 0.71 1.99 ± 1.09 3.75 ± 1.02 3.32 ± 0.88 0.61 ± 0.66 0.08 ± 0.51 3.24 ± 0.71 3.22 ± 1.12 1.03 ± 0.58 0.63 ± 0.57 0.14 ± 0.39 0.37 ± 0.89 0.86 ± 0.70 0.17 ± 0.46 6.57 ± 0.38⁎⁎⁎ 2.52 ± 0.45 0.79 ± 0.70 0.61 ± 1.95 3.29 ± 0.85 2.78 ± 1.15 1.47 ± 0.57 2.35 ± 1.57 5.66 ± 1.54⁎⁎⁎ 5.47 ± 1.57⁎⁎⁎

Ab

Af

Ba

Bb

Bb′

Ab-D1a

Ab-D2a

Ab-D3a

QS

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.012 0.005 0.008 0.008 0.006 0.009 0.008 0.007 0.005 0.004 0.006 0.009 0.005 0.004 0.003 0.007 0.006 0.004 0.003 0.004 0.006 0.015 0.007 0.009 0.005 0.013 0.012 0.013 0.008 0.006 0.008 0.007 0.008 0.005 0.007 0.007 0.009 0.010 0.004 0.008 0.002 0.001

4.21 ± 1.02 0.69 ± 0.80 5.51 ± 0.90 4.09 ± 0.89⁎⁎⁎ 4.21 ± 0.96 0.41 ± 0.59 2.79 ± 0.88 1.75 ± 0.90 1.75 ± 1.15 3.49 ± 1.20 100.00 ± 0.49⁎⁎⁎ 68.81 ± 1.33⁎⁎⁎ 21.07 ± 0.28⁎⁎⁎ 1.21 ± 0.01

Haemolytic percentages of PBS and distilled water were included as the minimal and maximal haemolytic controls. All values represent the mean ± SE (n = 6). Significant differences with PBS group were designated as ***p b 0.001. a AbDs are at the maximum concentrations. Data in parentheses are the concentrations of compounds in molarity.

derivatives may be very high and need not be detected. However, the haemolytic activity of QS was 100% at a concentration of 40 μg/ml, and the HD50 value for QS was 17.35 μg/ml, as determined under the same conditions (Curve Expert 1.3 was used to calculate the HD50).

3.2. Effect of soyasaponins and AbDs on splenocyte proliferation The effect of soyasaponins and AbDs on splenocyte proliferation in pathogen-free ICR mice is shown in Fig. 2. After exposure to ConA, the splenocyte stimulation index (SI) was significantly enhanced by the Aa (0.1 μg/ml), Ab (0.01, 0.1, 1, and 10 μg/ml), Bb (0.01, 0.1, 1, and 10 μg/ml), Bb′ (0.1, 1, and 10 μg/ml), Ab-D1 (0.01, 0.1, 1, and 10 μg/ml), Ab-D2 (0.01, 0.1, 1, and 10 μg/ml), Ab-D3 (0.1, 1, and 10 μg/ml) and QS (0.01, 0.1, 1, and 10 μg/ml) groups (p b 0.05 or p b 0.01). Except for the Af groups, each saponin group stimulated by LPS showed a greater proliferative response compared to the control group (p b 0.05 or p b 0.01). However, no significant difference (p N 0.05) was observed between the Alum groups (0.01, 0.1, 1, and 10 μg/ml) and control group. In addition, there was also no significant

3.3. Effect of soyasaponins and AbDs on the OVA-specific serum antibody response The effect of six soyasaponins and three AbDs on the induction of humoral immune responses in OVA-immunised mice was investigated on day 14 after the secondary immunisation. The OVA-specific serum IgG, IgG1, IgG2a and IgG2b levels were detected using indirect ELISA and the results are shown in Fig. 3. Except for Af, all of the other groups significantly enhanced the OVA-specific IgG and IgG1 levels in OVAimmunised mice (p b 0.05 or p b 0.01). Moreover, the enhancing effects of Ab, Ab-D2, and QA on serum IgG levels and Ab, Bb, Ab-D2, and QA on IgG1 levels were significant compared to the OVA/Alum group (p b 0.05 or p b 0.01). Significant enhancements in OVA-specific IgG2a were observed in groups of Ab, Ba, Bb, Bb′, Ab-D2, Ab-D3 and QS-immunised mice compared with the OVA group and OVA/Alum group (p b 0.05 or p b 0.01). The serum IgG2b level in mice was also significantly enhanced by Ab, Bb, Ab-D1, Ab-D2, Ab-D3 and QS compared with the OVA-immunised mice group (p b 0.05 or p b 0.01). However, no significant differences (p N 0.05) were observed between the OVA group and OVA/Alum group on serum IgG2a and IgG2b levels. Thus, some soyasaponins and AbDs at experimental doses significantly enhanced the serum antibody production in mice immunised with OVA and were more effective in enhancing the IgG2a and IgG2b induction responses of mice compared to Alum. 4. Discussion Effective inactivating vaccines require both an appropriately chosen antigen and an adjuvant capable of stimulating the significant and appropriate immune response [34]. In addition, low toxicity is regarded as an important property of the adjuvant. 4.1. Lower haemolytic activity of soyasaponins and AbDs compared with QS In this investigation, we found that soyasaponins and AbDs showed a slight haemolytic effect, with 6.57% haemolysis at a concentration of 200 μg/ml for Bb, 5.66% and 5.47% at a concentration of 64 μg/ml and 48 μg/ml for Ab-D1, respectively, and 4.09% at a concentration of 44 μg/ml for Ab-D2 (Table 1). In contrast, QS expressed 100% haemolysis at a concentration of 40 μg/ml, and the HD50 value of QS was 17.35 μg/ml as determined under the same conditions. This finding indicated a higher haemolytic activity for QS compared to soyasaponins and AbDs. Sherif et al. found that commercial saponin-rich quillaja extract demonstrated a significantly higher haemolytic activity compared to soybean extracts at the same saponin concentration using an ELISA [35]. It was also reported that quillaja saponin was lethal to mice in the dose range of 100 to 125 μg [36]. Our experimental results were consistent with these findings. Moreover, the haemolytic activities of saponins were related to their chemical structures [5]. It was also demonstrated that the haemolytic activity of steroidal saponins was greater than those of the triterpenoids after analysing the haemolytic activity of 27 saponins out of 75 synthetic steroid and triterpenoid glycosides [37]. Although QS is triterpenoidal, it exhibits serious disadvantages due to their high toxicity and undesirable haemolytic effects, which limit their application as an adjuvant in human vaccinations [38]. The haemolytic activity of QS was shown to be correlated to the long carbohydrate chains [4] and the presence of side chains bearing aglycone (sugar chains), acyl residues or the epoxyframework system [11]. Moreover, QS was not stable in the aqueous phase due to hydrolysis of their ester moieties on the C-28 fucose, which is regarded as a restriction in their use as a vaccine adjuvant [36,39]. In contrast with all of the QS-fractions from QS, the soyasaponins and AbDs contain between two to five sugars (xylose, glucose, and

N. Qiao et al. / International Immunopharmacology 18 (2014) 333–339

337

Fig. 2. Effect of soyasaponins and AbDs on splenocyte proliferation in pathogen-free ICR mice. Splenocytes of ICR mice were prepared and cultured with ConA or LPS together with saponins or Alum samples (final concentration 0.01–10 μg/ml) for 48 h. Splenocyte proliferation was measured using the CCK-8 method as described in the text and shown as the stimulation index (SI). The values are presented as the mean ± SE (n = 6). Significant differences with control groups (samples blank) were designated as ⁎p b 0.05 and ⁎⁎p b 0.01.

rhamnose residues) attached to the carbon C-3 and/or C-22 in the oleanane-type triterpene skeleton (soyasapogenol A or soyasapogenol B) to form monodesmoside or bisdesmosides. This is in the absence of

distinct acyl residues attached to its aglycone [17,40], which make them less hydrophobic, and may account for the less haemolytic activity of soyasaponins and AbDs.

Fig. 3. Effect of six soyasaponins and three AbDs on serum OVA-specific IgG, IgG1, IgG2a and IgG2b antibody responses. Groups of six female ICR mice were immunised subcutaneously on days 1 and 14 with OVA (100 μg) alone in PBS or with OVA 100 μg mixed with QS (50 μg), Alum (50 μg), Aa (50 μg), Ab (50 μg), Af (50 μg), Ba (50 μg), Bb (50 μg), Bb′ (50 μg), Ab-D1 (16 μg), Ab-D2 (11 μg) or Ab-D3 (19 μg). PBS-treated mice were used as negative controls. OVA-specific IgG, IgG2, IgG2a and IgG2b antibodies in sera were measured using indirect ELISA as described in the text 14-days after the second immunisation. The values are presented as the mean ± SE (n = 6). Significant differences with OVA groups were designated as ⁎p b 0.05 and ⁎⁎p b 0.01; those with OVA/Alum groups as ap b 0.05 and bp b 0.01. *AbDs are at the maximum solubilities.

338

N. Qiao et al. / International Immunopharmacology 18 (2014) 333–339

4.2. Stronger cellular immune responses of soyasaponins and AbDs compared to Alum T cell-mediated immunity results from the complex interactions between the lymphoid and phagocytic cells, which plays an important role to combat intracellular microbe infections, and can stimulate the lymphocyte proliferative response [9,41]. In addition, it is generally known that ConA stimulates T cells while LPS is a polyclonal B-cell mitogen [42]. As shown in Fig. 2, Aa, Ab, Bb, Bb′, Ab-D1, Ab-D2, Ab-D3 and QS could significantly enhance splenocyte proliferation at an appropriate dose compared with the control and Alum groups (p b 0.05 or p b 0.01). Interestingly, there was an optimal concentration of each type of saponins. Either low or high concentrations are disadvantageous in stimulating splenocyte proliferation. These results indicated that Aa, Ab, Bb, Bb′, Ab-D1, Ab-D2, Ab-D3 and QS could significantly increase the activation potential of T and B cells in ICR mice, while Alum demonstrates poor adjuvant activity at inducing cellular immune responses. 4.3. Significant enhancement of specific antibody of soyasaponins and AbDs compared to Alum OVA-specific serum IgG, IgG1, IgG2a and IgG2b antibodies in OVA-immunised ICR mice were expressed in Fig. 3. Ab, Ba, Bb, Bb′, Ab-D1, Ab-D2, Ab-D3 and QS significantly enhanced OVA-specific IgG, IgG1, IgG2a, and IgG2b in OVA-immunised mice compared with the control group (p b 0.05 or p b 0.01). The OVA/Alum group could induce specific serum IgG and IgG1. However, there was no detectable effect in activating the production of IgG2a and IgG2b. T lymphocytes mediate the defence against some extracellular microbes and help B lymphocytes to produce antibodies. Helper T (Th) cells may differentiate into subsets of effector cells, which produce restricted sets of cytokines and perform different functions [43]. Th1 cells secrete cytokines, such as tumour necrosis factor-β (TNF-β), interleukin-2 (IL-2) and interferon-γ (IFN-γ), and stimulate the production of IgG2a, IgG2b and IgG3 in mice. They are also responsible for cytotoxic T lymphocyte (CTL) production, the function of which is to kill cells producing cytoplasmic microbial antigens. Th2 cells, which produce IL-4, IL-5 and IL-10, stimulate IgG1 and IgA production and activate eosinophils, which function mainly in the defence against helminthes [2,44–46]. The Th1 response against malignant cells and intracellular pathogens is superior to the Th2 response [9]. Previous studies have shown that the most commonly used adjuvants (water/oil emulsions and Alum) elicit only a Th2 immune response [2]. In this study, it is likely that several soyasaponins (Ab, Ba, Bb, and Bb′) and three AbDs are effective on both Th1 and Th2 cells, as shown with an enhancement of IgG1, IgG2a and IgG2b levels. It is also implicated that the adjuvant potential of soyasaponins and AbDs was superior to that of Alum. However, compared with QS, both soyasaponins and their derivatives showed weak immunological activity (see Fig. 3). QS is a representative adjuvant with stronger adjuvant activity compared to many other plant saponins. It was reported that quillaja saponin extract expressed higher antibody activity at a dose of 20 μg compared to ARS saponins with doses of 100 μg [9]. Oda et al. demonstrated a positive correlation of QS between the effect to activate IgG subclass and injection doses at a range of 0 to 100 μg [30]. Moreover, QS (50 μg) exhibited a much higher activation of the production of IgG and IgG subclass compared to soyasaponins (50 μg) in this study. The high HLB value of QS may account for its strong adjuvanticity on the basis that several saponins reported to be strong adjuvants demonstrated higher HLB values [30,47,48]. Furthermore, additional consideration of the other chemical structure factors might secondarily explain its adjuvant activity [49]. The in vivo and in vitro experimental results indicated the high adjuvant activity of soyasaponins and AbDs with a significant enhancement in the specific antibody and cellular response.

4.4. Adjuvant activity potent of three AbDs′ In the development of formulations, solubility of the active component in the drug product has great importance. To determine a safe concentration that is effective for a new drug at room temperature, the saturated concentration (Cs) or saturated solubility and the median effective dose (ED50) are usually measured [50]. As previously described, after the derivation and induction of hydrophobic groups, the solubility of AbDs decreases. In this study, we determined the saturated solubilities of three AbDs using the HPLC external standard method and used saturated solubilities as injection doses of the following derivatives: Ab-D1 (16 μg), Ab-D2 (11 μg) and Ab-D3 (19 μg). Three AbDs exhibited the same effect with their non-modified form Ab on enhancing a specific antibody compared to the OVA and OVA/Alum groups, whereas their injection doses were much lower than that of Ab (50 μg). These results indicated a tendency of a potential immunoadjuvant improvement after the modification of the natural form of soyasaponin. Moreover, Ab-D2 induced total IgG, IgG1, IgG2a and IgG2b responses with a lower dose than Ab-D1 or Ab-D3, which indicated that Ab-D2 might possess a higher adjuvant activity at the same dose of other AbDs. One study on soyasaponins [30] revealed that the adjuvant activity increased with the HLB value and the amphipathic structure may define the fundamental adjuvanticity of saponins. Ab-D1, Ab-D2, and Ab-D3 were synthesised by its linkage with different functional groups: phenylethylamine, dodecylamine and ethylenediamine-phenylpropionic acid to soyasaponin Abs C-3-O glucose carboxyl acid group. Due to the higher hydrophobic property of dodecylamine, Ab-D2 showed stronger hydrophobicity compared with Ab-D1 and Ab-D3. This may be the reason for its greater adjuvanticity compared to the other two AbDs. However, it was reported that cationic (positively charged) particles are more likely to induce immunostimulatory response than anionic (negatively charged) particles and neutral species [51,52]. Our experimental results demonstrated that that the positively charged molecular structure of Ab-D2 makes it easy for the cell membrane to deliver and/or stimulate the immunogenic activity due to the negatively charged membrane. 4.5. Conclusion In conclusion, several soyasaponins (Ab, Bb, and Bb′) and three AbDs are capable of enhancing both cellular and humoral immune responses against OVA in mice. In addition, they might be effective on Th1 and Th2 cells. Furthermore, they expressed a lower haemolytic side-effect compared to QS. These results suggest that soyasaponins could be safely used as an adjuvant superior to Alum and QS. AbDs exhibit a tendency of immunoadjuvant potential improvement after modification. Moreover, Ab-D2 showed adjuvanticity at the lowest dose among the three AbDs, which might result from its highest hydrophobicity and the positively charged molecular structure. However, further studies are required to elucidate the relationship between the chemical structure and adjuvant activity of soyasaponins. Modification of the sugar chains of natural soyasaponins also warrants further studies to develop the derivatives as a new type of immunological adjuvant. Furthermore, it is also very important to enhance the delivery of antigens to antigen-presenting cells (APCs), particularly dendritic cells (DCs), in the development of effective vaccines using soyasaponin derivatives as novel adjuvants. Ongoing investigations are being undertaken to examine the regulation of the immune response by ISCOMs or antigen-loaded nanoparticles with these derivatives. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 31071630/C200303). The authors are grateful for the financial funding. All authors declare no conflicts of interest in this study.

N. Qiao et al. / International Immunopharmacology 18 (2014) 333–339

References [1] Sanders MT, Brown LE, Deliyannis G, Pearse MJ. ISCOM-based vaccines: the second decade. Immunol Cell Biol 2005;83(2):119–28. [2] Cox JC, Coulter AR. Adjuvants—a classification and review of their modes of action. Vaccine 1997;15:248–56. [3] Kim YJ, Wang PF, Navarro-Villalobos M, Rohde BD, Derryberry JM, Gin DY. Synthetic studies of complex immunostimulants from Quillaja saponaria: synthesis of the potent clinical immunoadjuvant QS-21Aapi. J Am Chem Soc 2006;128:11906–15. [4] Song XM, Hu SH. Adjuvant activities of saponins from traditional Chinese medical herbs. Vaccine 2009;27:4883–90. [5] Sun HX. Haemolytic activities and adjuvant effect of Bupleurum chinense saponins on the immune responses to ovalbumin in mice. Vaccine 2006;24:1324–31. [6] Kukhetpitakwong R, Hahnvajanawong C, Homchampa P, Leelavatcharamas V, Satra J, Khunkitti W. Immunological adjuvant activities of saponin extracts from the pods of Acacia concinna. Int Immunopharmacol 2006;6:1729–35. [7] Sun HX, Pan HJ, Pan YJ. Haemolytic activities and immunologic adjuvant effect of Panax notoginseng saponins. Acta Pharmacol Sin 2003;24(11):1150–4. [8] Sun HX. Adjuvant effect of Achyranthes bidentata saponins on specific antibody and cellular response to ovalbumin in mice. Vaccine 2006;24:3432–9. [9] Sun YX, Li M, Liu JC. Haemolytic activities and adjuvant effect of Anemone raddeana saponins (ARS) on the immune responses to ovalbumin in mice. Int Immunopharmacol 2008;8:1095–102. [10] Xie Y, Pan HJ, Sun HX, Li D. A promising balanced Th1 and Th2 directing immunological adjuvant, saponins from the root of Platycodon grandiflorum. Vaccine 2008;26:3937–45. [11] Sun HX, Xie Y, Ye YP. Advances in saponin-based adjuvants. Vaccine 2009;27:1787–96. [12] Liu G, Anderson C, Scaltreto H, Barbon J, Kensil CR. QS-21 structure/function studies: effect of acylation on adjuvant activity. Vaccine 2002;20:2808–15. [13] Boyaka PN, Marinaro M, Jackson RJ, Van Ginkel FW, Cormet-Boyaka E, Kirk KL, et al. Oral QS-21 requires early IL-4 help for induction of mucosal and systemic immunity. J Immunol 2001;166:2283–90. [14] Nardin EH, Oliveira GA, Calvo-Calle JM, Castro ZR, Nussenzweig RS, Schmeckpeper B, et al. Synthetic malaria peptide vaccine elicits high levels of antibodies in vaccines of defined HLA gegotypes. J Infect Dis 2000;182:1486–96. [15] Ragupathi G, Gardner JR, Livingston PO, Gin DY. Natural and synthetic saponin adjuvant QS-21 for vaccines against cancer. Expert Rev Vaccines 2011;10:463–70. [16] Vincken JP, Heng L, Groot AD, Gruppen H. Saponins, classification and occurrence in the plant kingdom. Phytochemistry 2007;68:275–97. [17] Yoshiki Y, Kudou S, Okubo K. Relationship between chemical structures and biological activities of triterpenoid saponins from soybean. Biosci Biotechnol Biochem 1998;62(12):2291–9. [18] Sumiki Y. Studies on the saponin of soy bean part I. Bull Agric Chem Soc 1929;5:27–32. [19] Sumiki Y. The saponin of soy bean part II. Bull Agric Chem Soc 1930;6:49–51. [20] Kudou S, Tsuizaki I, Uchida T, Okubo K. Purification and some properties of soybean saponin hydrolase from Aspergillus oryzae KO-2. Agric Biol Chem 1991;55:31–6. [21] Ireland PA, Dziedzic SZ. Effect of hydrolysis on sapogenin release in soy. J Agric Food Chem 1986;34(6):1037–41. [22] Kang J, Badger TM, Ronis MJJ, Wu XL. Non-isoflavone phytochemicals in soy and their health effects. J Agric Food Chem 2010;58:8119–33. [23] Haralampidis K, Trojanowska M, Osbourn AE. Biosynthesis of triterpenoid saponins in plants. In: Dutta NN, Hammar K, editors. History and Trends in Bioprocessing and Biotransformation. New York: Springer; 2002. p. 31–49. [24] Kitagawa I, Yoshikawa M, Wang HK, Saito M, Tosirisuk V, Fujiwara T, et al. Revised structures of soyasapogenols A, B, and E, oleanene-sapogenols from soybean. Chem Pharm Bull 1982;30(6):2294–7. [25] Yoshiki Y, Okubo K. Active oxygen scavenging activity of DDMP (2,3-dihydro-2,5dihydroxy-6-methyl-4H-pyran-4-one) saponin in soybean seed. Biosci Biotechnol Biochem 1995;59:1556–7. [26] Kim HY, Yu R, Kim JS, Kim YK, Sung MK. Antiproliferative crude soy saponin extract modulates the expression of IκBα, protein kinase C, and cyclooxygenase-2 in human colon cancer cells. Cancer Lett 2004;210:1–6. [27] Xiao JX, Peng GH, Zhang SH. Prevention effects of soyasaponins on hyperlipidemia mice and its molecular mechanism. Acta Nutr Sin 2005;27:147–50.

339

[28] Kingjo J, Okawa M, Udayama M, Sohno Y, Hirakawa T, Shii Y, et al. Hepatoprotective and hepatotoxic actions of oleanolic acid-type triterpenoidal glucuronides on rat primary hepatocyte cultures. Chem Pharm Bull 1999;47(2):290–2. [29] Yang XS, Dong C, Ren G. Effect of soyasaponins-rich extract from soybean on acutealcohol-induced hepatotoxicity in mice. J Agric Food Chem 2011;59:1138–44. [30] Oda K, Matsuda H, Murakami T, Katayama S, Ohgitani T, Yoshikawa M. Relationship between adjuvant activity and amphipathic structure of soyasaponins. Vaccine 2003;21:2145–51. [31] Marciani Dante J, Brimingham M, Ala. Immunostimulating and vaccine compositions employing saponin analog adjuvants and uses thereof; 2000-07-27 (America. US, 6080725A). [32] Zhao DY, Yan MX, Huang YA, Sun XJ. Efficient protocol for isolation and purification of different soyasaponins from soy hypocotyls. J Sep Sci 2012;35:3281–92. [33] Guo WX, Yan XB, Zhao DY. Development of semisynthetic soyasaponin derivatives. Food Sci 2013;34(22):123–7 (in Chinese). [34] Marciani DJ, Press JB, Reynolds RC, Pathak AK, Pathak A, Gundy LE, et al. Development of semisynthetic triterpenoid saponin derivatives with immune stimulating activity. Vaccine 2000;18:3141–51. [35] Hassan Sherif M, Byrd James A, Cartwright Aubry L, Bailey Chris A, et al. Hemolytic and antimicrobial activities differ among saponin-rich extracts from guar, quillaja, yucca, and soybean. Appl Biochem Biotechnol 2010;162:1008–17. [36] Kensil Charlotte R, Patel Usha, Lennick Michael, Marciani Dante. Separation and characterization of saponins with adjuvant activity from Quillaja saponaria Molina cortex. J Immunol 1991;146(2):431–7. [37] Takechi M, Tanaka Y. Haemolytic time course differences between steroid and triterpenoid saponins. Planta Med 1995;61:76–7. [38] Waite DC, Jacobson EW, Ennis FA, Edelman R, White B, Kammer R, et al. Three double-blind, randomized trials evaluating the safety and tolerance of different formulations of the saponin adjuvant QS-21. Vaccine 2001;19(28–29):3957–67. [39] Oliveira-Freitas E, Casas CP, Borja-Cabrera GP, Santos FN, Nico D, Souza LO, et al. Acylated and decylated saponins of Quillaja saponaria mixture as adjuvants for the FML-vaccine against visceral leishmaniasis. Vaccine 2006;24(18):3909–20. [40] Gurfinkel DM, Rao AV. Soyasaponins: the relationship between chemical structure and colon anticarcinogenic activity. Nutr Cancer 2003;47(1):24–33. [41] Lopez C, O’Reiliy RJ. Cell-mediated immune responses in recurrent herpesvirus infections. J Immunol 1977;118(3):895–902. [42] Okamura M, Lillehoj HS, Raybourne RB, Babu US, Heckert RA. Cell-mediated immune responses to a killed Salmonella enteritidis vaccine: lymphocyte proliferation, T-cell changes and interleukin-6 (IL-6), IL-1, IL-2, and IFN-γ production. Comp Immunol Microbiol Infect Dis 2004;27:255–72. [43] Abbas AK, Lichtman AH, Pillai S. Basic Immunology: Functions and Disorders of the Immune System. 2nd ed. Philadelphia: Elsevier Health Sciences; 2012. [44] Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL. Two Types of Murine Helper T Celll Clone. J Immunol 1986;136(7):2348–56. [45] Mosmann TR, Sad S. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol Today 1996;17(3):138–45. [46] Constant SL, Bottomly K. Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches. Annu Rev Immunol 1997;15:297–322. [47] Bomford R, Stapleton M, Winsor S, Beesley JE, Jessup EA, Price KR, et al. Adjuvanticity and ISCOM formation by structurally diverse saponins. Vaccine 1992;10:572–7. [48] Nagai T, Suzuki Y, Kiyohara H, Susa E, Kato T, Nagamine T, et al. Onjisaponins, from the root of Polygala tenuifolia Willdenow, as effective adjuvants for nasal influenza and diphtheria-pertussis tetanus vaccines. Vaccine 2001;19:4824–34. [49] Oda K, Matsuda H, Murakami T, Katayama S, Ohgitani T, Yoshikawa M. Adjuvant and haemolytic activities of 47 saponins derived from medicinal and food plants. Biol Chem 2000;38:67–74. [50] Zhou JX, Luo NF, Liang XM, Liu J. The efficacy and safety of intravenous emulsified isoflurane in rats. Anesth Analg 2006;102:129–34. [51] Dobrovolskaia MA, Mcneil SE. Immunological properties of engineered nanomaterials. Nat Nanotechnol 2007;2:469–77. [52] Shakweh M, Ponchel G, Fattal E. Particle uptake by Peyer's patches: a pathway for drug and vaccine delivery. Expert Opin Drug Deliv 2004;1:141–63.

Haemolytic activity and adjuvant effect of soyasaponins and some of their derivatives on the immune responses to ovalbumin in mice.

Immunological adjuvants are agents that enhance specific immune responses to vaccines. At present more studies are needed to identify a suitable adjuv...
895KB Sizes 0 Downloads 0 Views