Available online at www.sciencedirect.com Chemico-Biological Interactions 173 (2008) 115–121 Immune up regulatory response of a non-caloric natural sweetener, stevioside Irum Sehar a,∗ , Anpurna Kaul a , Sarang Bani b , Harish Chandra Pal a , Ajit Kumar Saxena a a Pharmacology Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, 180001 Jammu, India b Cell Biology Laboratory, Indian Institute of Integrative Medicine (CSIR), Jammu, India Received 18 October 2007; received in revised form 11 January 2008; accepted 14 January 2008 Available online 31 January 2008 Abstract Immunomodulation is a process, which alters the immune system of an organism by interfering with its functions. This interference results in either immunostimulation or immunosuppression. An immunomodulator is any substance that helps to regulate the immune system. This “regulation” is a normalization process, so that an immunomodulator helps to optimise immune response. Immunomodulators are becoming very popular in the worldwide natural health industry as these do not tend to boost immunity, but to normalize it. Keeping this in view, major efforts have to be directed to modulate the immune responses, to permit effective treatment of various ailments associated with immune system and thus the development of a safe and effective immunomodulator for clinical us. Leaves of Stevia rebaudiana are a source of several sweet glycosides of steviol. The major glycoside, stevioside, diterpenoid glycoside—is used in oriental countries as a food sweetener. Its medical use is also reported as a heart tonic. Besides, it is used against obesity, hypertension, and stomach burn and to lower uric acid levels. Here in this study, stevioside was tested for its immunomodulatory activity on different parameters of the immune system at three different doses (6.25, 12.5 and 25 mg/kg p.o.) on normal as well as cyclophosphamide treated mice. Stevioside was found effective in increasing phagocytic activity, haemagglutination antibody titre and delayed type hypersensitivity. In parallel, stevioside substantially increase proliferation in the LPS and Con A stimulated B and T cells, respectively. Present study, therefore, reveals that the drug holds promise as immunomodulating agent, which acts by stimulating both humoral as well as cellular immunity and phagocytic function. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Stevioside; Immunomodulatory activity; Humoral response; Cellular immunity; Phagocytosis 1. Introduction Leaves of Stevia rebaudiana are a source of several sweet glycosides of steviol [1]. The major glycoside, stevioside, diterpenoid glycoside—is used in oriental countries as a food sweetener. Other major glycosides named rebaudioside, which is sweeter and more deli∗ Corresponding author. Tel.: +91 191 2569000 6x241; fax: +91 191 2569333. E-mail address: sehar irum@yahoo.co.in (I. Sehar). cious than stevioside, is utilised in beverages. To improve the sweetness and the taste, modifications of sugar moieties of both the glycosides were performed by enzymatic glycosylations and/or enzymatic trimming. Stevioside (Fig. 1) is a sweet-tasting glycoside occurring abundantly in the leaves of S. rebaudiana (Compositae). It has been used popularly in Japan and Brazil as a sugar substitute for decades. Previous study has shown that it lowered blood pressure in spontaneously hypertensive rats (SHRs) when administered intravenously. This study shows that intraperitoneal 0009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2008.01.008 116 I. Sehar et al. / Chemico-Biological Interactions 173 (2008) 115–121 experimental protocols (n = 6). Animals were housed and maintained following standard guidelines (CPCSEA, 2003). The study protocol was approved by Institutional Animal Ethics Committee. 2.2. Test sample Fig. 1. Structure of stevioside. injection of stevioside 25 mg/kg also has antihypertensive effect in SHRs [2]. For hundreds of years, indigenous people (Guaranis) native to Paraguay and Brazil have used its leaves as a sweetener, particularly to sweeten their tea (mate), which is very consumed among these tribes. Its medical use is also reported as a heart tonic. Besides, it is used against obesity, hypertension, and stomach burn and to lower uric acid levels [3]. The first written article about it dates back to 1900. In 1931, however, it has been found that the glycosides are the responsible constituents for the sweetening properties of its leaves [4]. Post phytochemical analyses of the results have recorded the presence of 5–10% of stevioside, 2–4% of rebaudioside and dulco-sides. Stevioside is the sweetest and its sweetening power was shown to have been 300 times greater than sacarosis and accounted for 18% of the leaf total composition. Today, Stevia sweetener has been traded almost all over the world and has been used to sweeten hundreds of diabetic products, particularly soft drinks. The Japanese are considered to be the greatest consumers. In addition to being a non-caloric sweetener, this plant has been reported to be hyploglycemiant, hypo-tensor, diuretic and cardiotonic. In Brazil, it has been successfully used as the most suitable sweetener for diabetic people. A great deal of clinical studies has validated such uses even in the USA, where its employment is forbidden due to the pressure and the lobbying led by the powerful industry of artificial sweeteners [5]. The present investigation was aimed to study the immunomodulatory activity of stevioside using reported methods in order to justify the drug as an immunomodulator. 2. Materials and methods 2.1. Animals Balb/c mice of either sex, 10–12 weeks old, weighing 20–25 gm were randomly distributed in groups as per Stevioside was prepared as freshly homogenized suspension in 1% (w/v) gum acacia and administered orally daily once a day for the duration of the experiment. The control animals were given an equivalent volume of gum acacia vehicle. Cyclophosphamide (200 mg/kg p.o.) was used to induce immunosuppression 2 days prior to immunization with SRBC. LPS and Con A were used as standard mitogens for in vitro lymphocyte proliferation. 2.3. Antigen Sheep red blood cells (SRBCs) were used as antigen. Fresh sheep blood was collected aseptically from the jugular vein of the sheep in sterile cold Alsever’s solution. Blood was washed three times with pyrogen free sterile normal saline (0.9% NaCl w/v) and 0.2 ml of 5 × 109 SRBCs/ml were used for immunization. Animals were challenged on 6th day from the day of immunization [6]. 2.4. Effect on general behaviour and acute toxicity The acute oral toxicity studies were carried out following OECD guidelines. Three different doses 6.25, 12.5 and 25 mg/kg body weight of test drug were administered orally to the groups of six mice. The animals were observed for 14 days for any change in gross behavior and mortality. 2.5. Humoral antibody (HA) titre The method described by Nelson and Midenhall was adopted [7]. Humoral immunity was determined in normal as well as immunosuppressed mice. For immunosuppression a single dose of cyclophosphamide (200 mg/kg, p.o.) was administered on 2-day prior to immunization (day 0). Animals were divided into groups of six mice each. The control group received 1% gum acacia solution only as vehicle; while animals in the treatment groups were given the test drugs (6.25, 12.5 and 25 mg/kg, p.o.) daily for 7 days. The animals were immunized by injecting 0.2 ml of 5 × 109 SRBC/ml suspension i.p. on 0 day. Blood samples were collected from individual animal by retro-orbital plexus on 7th day to obtain serum. Antibody levels were determined I. Sehar et al. / Chemico-Biological Interactions 173 (2008) 115–121 by haemagglutination technique. The reciprocal of the highest serum dilution of the test serum causing visible agglutination was taken as the antibody titre. 2.6. Delayed type hypersensitivity (DTH) response The method of Doherty was followed to determine SRBC induced DTH response in normal as well as immunosuppressed mice [8]. Mice were immunized by injecting 20 ␮l of 5 × 109 SRBC/ml suspension subcutaneously into the right hind footpad. Treatment groups received test drug (6.25, 12.5 and 25 mg/kg, p.o.) in 1% gum acacia daily for 7 days. On 7th day, the thickness of the right hind footpad was measured using digital vernier caliper (0.1 mm pitch) which served as control. The mice were then challenged by injection of same amount of SRBCs in left hind footpad. Foot thickness was again measured after 24 h of this challenge. The difference between the pre and post challenge foot thickness express in mm was taken as a measure of delayed type hypersensitivity (DTH). 2.7. Macrophage phagocytic response 2.7.1. In vitro The method of Lehrer was adopted [9]. Peritoneal macrophages (2 × 106 cells) allowed adhering to glass cover slip (22 mm × 22 mm) for 90 min in CO2 incubator (37 ◦ C, 5% CO2 and 95% humidity). The cover slip was washed gently with PBS to remove non-adherent cells. Simultaneously heat killed Candida albicans cells (100 ◦ C, 60 min) were opsonized for 90 min with 20% autologous serum to promote phagocytosis. An aliquot (100 ␮l) of opsonized candida cells were added to the cover slips and this mixture was allowed to incubate for 15 min in CO2 incubator (37 ◦ C, 5% CO2 and 95% humidity). The samples were washed with PBS and immediately evaluated microscopically. A total 100 macrophages were analyzed under microscope for C. albicans’ ingestion. Average value was compared with control and per cent phagocytosis was calculated. 2.7.2. In vivo The phagocytic function of the reticuloendothelial system was assayed in groups of six mice each by injecting i.v. 160 mg/kg of 1.6% suspension of gelatin stabilized carbon particles of 20–25 ␮m. Three different doses of stevioside (6.25, 12.5 and 25 mg/kg p.o. were administered daily for 7 days and 30 min prior to the carbon injection. Blood samples were collected before and at intervals varying between 2 and 90 min after carbon injection [10]. An aliquot (10 ␮l) of blood 117 sample was lysed with 2 ml of 0.1% acetic acid and the transparency was determined spectrophotometrically at 675 nm (Uvikon 810, spectrophotometer, Kontron Ltd., Switzerland). The phagocytic index K, was calculated by using the following equation: K= (ln OD1 − ln OD2 ) (t2 − t1 ) where OD1 and OD2 are the optical densities at times t2 and t1 , respectively. 2.8. Viability assay of murine spleenocytes The viability of cells was determined by the standard trypan blue exclusion assay. Approximately 2 × 106 cells/ml were treated with two-fold diluted concentration from 6.25 to 25 ␮g/ml of stevioside and controls with 0.1% of ethanol for 3 days at 37 ◦ C in a humidified atmosphere containing 5% CO2 . A visual count was then made of the number of live (white) versus dead (blue) cells using a hemocytometer following staining by trypan blue (0.4% in normal saline – 0.9% NaCl) and the percentage of live versus dead cells determined [11]. 2.9. Lymphocyte proliferation assay Spleen collected from the stevioside treated mice under aseptic conditions, in PBS (PBS, Sigma), was minced using a pair of scissors and passed through a fine steel mesh to obtain a homogeneous cell suspension, and the erythrocytes were lysed with ammonium chloride (0.8%, w/v). After centrifugation (380 × g at 4 ◦ C for 10 min), the pelleted cells were washed three times in PBS, and resuspended in complete medium [RPMI 1640 supplemented with 12 mM HEPES (pH 7.1), 0.05 mM 2-mercaptoethanol, 100 IU/ml penicillin, 100 ␮g/ml streptomycin, and 10% FCS]. Cell numbers were counted with a haemocytometer by trypan blue dye exclusion technique. Cell viability exceeded 95%. Briefly, splenocytes were seeded into three to four wells of a 96-well flat-bottom microtiter plate (Nunc) at 2 × 106 cells/ml in 100 ␮l complete medium. Suboptimal concentrations of LPS (10 ␮g/ml) and Con A (4 ␮g/ml) were added to each well separately for priming B and T cells, respectively. The plates were incubated at 37 ◦ C in a humid atmosphere with 5% CO2 . After 72 h, 50 ␮l of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide) solution (5 mg/ml) was added to each well and incubated for further 4 h [12]. The plates were centrifuged (1400 × g, 5 min) and the untransformed MTT was removed carefully by pipetting 200 ␮l of a DMSO (Sigma, USA) working solution 118 I. Sehar et al. / Chemico-Biological Interactions 173 (2008) 115–121 Table 1 Acute toxicity studies after stevioside treatment Treatment groups Control Stevioside Stevioside Stevioside Dose (mg/kg) p.o. – 6.25 12.5 25 Body weight (mean ± S.E.) Observations Initial Mortality Sign of toxicity 6/0 6/0 6/0 6/0 – – – – 21.66 22.00 21.41 21.91 Final ± ± ± ± 0.42 0.62 0.80 0.45 22.08 22.33 21.58 22.41 ± ± ± ± 0.61 0.70 0.80 0.50 Acute toxicity study of stevioside was done by administering different doses of stevioside orally once and animals were observed for mortality. Stevioside up to 25 mg/kg does not cause any mortality or any change in the behavior of mice. Data is represented as mean ± S.E.; (n = 6). (192 ␮l DMSO with 8 ␮l 1N HCl) was added to each well, and the absorbance was evaluated in an ELISA reader at 570 nm after 15 min. (P < 0.05) increase in antibody synthesis (15.38%) was obtained with stevioside at the dose of 12.5 mg/kg, p.o. in normal mice whereas in immunosuppressed mice antibody titre response was insignificant (Tables 2 and 3). 2.10. Statistical analysis The results were expressed as mean ± S.E. and presented as fold increase/decrease values compared with the untreated control. Experimental group were compared to the untreated and positive control group. 3.3. Delayed type hypersensitivity (DTH) response DTH response was checked by increased footpad thickness using digital vernier caliper. Administration of stevioside at the dose of 12.5 mg/kg, p.o. produced significant increase of 18% in DTH response in normal mice. In case of Cyclophosphamide treated mice, stevioside does not cause any significant change in DTH response (Tables 4 and 5). 3. Results 3.1. Effect on general behavior and acute toxicity A single dose of stevioside administered orally to each group of mice did not show any change in the general behaviour of the test animals. No mortality was observed over a period of 14 days up to a dose of 25 mg/kg in mice (Table 1). 3.4. Phagocytic response 3.4.1. In vitro Stevioside was tested at three concentrations of 6.25, 12.5 and 25 ␮g/ml against phagocytic function of peritoneal macrophages. A significant increase of 4.92% in the phagocytosis was observed at 12.5 ␮g/ml (Table 6). 3.2. Humoral antibody (HA) titre Humoral response to SRBCs was checked by haemagglutination antibody titre. A significant Table 2 Immunomodulatory effect of stevioside on humoral immune response in normal mice Treatment groups Control Stevioside Stevioside Stevioside Dose (mg/kg) p.o. – 6.25 12.5 25 Humoral immune response Antibody titre (mean ± S.E.) Immunomodulatory activity %change 6.50 6.83 7.50 6.66 – 5.07 ↑ 15.38↑ 2.46 ↑ ± ± ± ± 0.11 0.24 0.22* 0.19 Effect of stevioside on humoral response by antibody titre assay on normal mice. Groups of Balb/c mice (n = 6) were immunized intraperitoneally with 200 ml of 5 × 109 SRBCs/ml alone or associated with three different doses of stevioside. Serum antibody titre was determined after 7 days by heamagglutination technique. Reciprocal of highest dilution of the test serum causing visible agglutination was taken as antibody titre and percent change was calculated. A significant change (P < 0.05) in antibody titre was observed only at 12.5 mg/kg p.o. dose. Data is represented as mean ± S.E; (n = 6). * (P < 0.05) = significant. 119 I. Sehar et al. / Chemico-Biological Interactions 173 (2008) 115–121 Table 3 Immunomodulatory effect of stevioside on humoral immune response in immunesuppressed mice Treatment groups Dose (mg/kg) p.o. Control Cyclophosphamide Stevioside Stevioside Stevioside Humoral immune response Antibody titre (mean ± S.E.) Immunomodulatory activity %change 6.16 4.50 5.83 6.00 5.50 – 27 ↓ 81 ↑ 91 ↑ 61 ↑ – 200 6.25 12.5 25 ± ± ± ± ± 0.29 0.17* 0.35 0.32 0.47 Effect of stevioside on humoral response by antibody titre assay on immunosuppressed mice. Groups of Balb/c mice (n = 6) were treated with a single dose of cyclophosphamide (200 mg/kg p.o) to induce significant immunosuppression 2 days prior to sensitization with 200 ml of 5 × 109 SRBCs/ml. Antibody titre was determined after 7 days treatment with different doses of stevioside. Maximum stimulatory response was observed at 12.5 mg/kg p.o. dose of stevioside. Data is represented as mean ± S.E; (n = 6). * (P < 0.05) = significant. Table 4 Immunomodulatory effect of stevioside on cell mediated immune response (DTH response) in normal mice Treatment groups Control Stevioside Stevioside Stevioside Dose (mg/kg) p.o. – 6.25 12.5 25 Cell mediated immune (DTH) response Foot thickness (in mm after 24 h) (mean ± S.E.) Immunomodulatory activity %change 0.85 0.93 1.00 0.90 – 9.41 ↑ 18.00 ↑ 5.88 ↑ ± ± ± ± 0.02 0.03 0.05* 0.02 Cell mediated immune response (DTH) was determined in SRBC immunized normal mice treated with different doses of stevioside. On day 7th mice were challenged with antigen. The difference between the pre and post challenge foot thickness (in mm) after 24 h was taken as a measure of DTH and percent DTH response was calculated. Only 12.5 mg/kg p.o. dose of stevioside was able to exhibit significant (P < 0.05) cell mediated immune response. Data is represented as mean ± S.E.; (n = 6). * (P < 0.05) = significant. Table 5 Immunomodulatory effect of stevioside on cell mediated immune response (DTH response) in immune suppressed mice Treatment groups Control Cyclophosphamide Stevioside Stevioside Stevioside Dose (mg/kg) p.o. – 200 6.25 12.5 25 Cell mediated immune (DTH) response Foot thickness (in mm after 24 h) (mean ± S.E.) Immunomodulatory activity %change 1.03 0.60 0.96 1.01 0.92 – 42 ↓ 83 ↑ 95 ↑ 75 ↑ ± ± ± ± ± 0.05 0.05* 0.09 0.09 0.04 Delayed type hypersensitivity response on immunosuppressed mice was assessed in cyclophosphamide (200 mg/kg p.o two days prior to immunization) treated mice. DTH response was measured in terms of footpad thickness of mice 24 h after being challenged with SRBC. Data is represented as mean ± S.E.; (n = 6) * (P < 0.05) = significant. 3.4.2. In vivo Stevioside possess macrophage stimulatory activity as evidenced by increased phagocytic index in carbon clearance test. The phagocytic activity of reticuloendothelial is generally measured by the rate of removal of carbon particles from blood stream. The significant percent phagocytic index for stevioside at the dose of 12.5 mg/kg, p.o was found to be 6.5 (Table 6). 3.5. Effect of stevioside on the viability of splenic lymphocytes Unstimulated splenic lymphocytes were cultured with various concentrations of the stevioside (6.25–25 ␮g/ml). Up to 25 ␮g/ml, none of the stevioside group induced cell death (88–92% viability, results not included). However, at 25 ␮g/ml, stevioside does not rapidly affect splenic lymphocytes viability. Therefore, 120 I. Sehar et al. / Chemico-Biological Interactions 173 (2008) 115–121 Table 6 Immunomodulatory effect of stevioside on in vitro and in vivo phagocytic response of macrophages Treatment groups and dose (␮g/ml) Percent phagocytosis (in vitro study) (mean ± S.E.) Treatment groups and dose (mg/kg) p.o. Phagocytic index (in vivo study) (mean ± S.E.) Control Stevioside (6.25) Stevioside (12.5) Stevioside (25) 25.00 ± 1.26 26.07 ± 0.22 (4.10%) ↑ 26.23 ± 0.18* (4.92%) ↑ 26.18 ± 0.71 (4.72%) ↑ Control Stevioside (6.25) Stevioside (12.5) Stevioside (25) 1.22 ± 0.12 1.28 ± 0.32 (4.9%) ↑ 1.30 ± 0.22 * (6. 5%) ↑ 1.30 ± 0.12 (6. 5%) ↑ Influence of stevioside on phagocytic activity of peritoneal macrophages in vitro and in vivo was determined. For in vitro response peritoneal macrophages (2 × 106 cells) were incubated with C. albicans and percent phagocytic activity was determined microscopically as described in material and methods whereas in vivo effect of stevioside on phagocytosis was determined by carbon clearance method. Graded doses of stevioside were administered to mice for 7 consecutive days. 160 mg/kg of 1.6% suspension of gelatin stabilized carbon particles injected i.v. Blood samples collected and lysed with 2 ml of 0.1% acetic acid and transparency determined spectrophotometrically at 675 nm. Data is represented as mean ± S.E.; (n = 6), figures in paranthesis represents percentage change. * (P < 0.05) = significant. the concentrations of stevioside used in the different experiments were adjusted between 6.25 and 25 ␮g/ml. on B cells. Cells grown in the absence of mitogens did not show any proliferation. 3.6. Effect of stevioside on lymphocyte proliferation 4. Discussion Stevioside elicited substantial increase in proliferative response in the LPS stimulated B-lymphocytes. The increase in proliferation was observed in a dose dependent manner, the optimal significant effect being at the concentration of 12.5 mg/kg (Fig. 2). Similar effect was seen on Con A primed T cell proliferation but was insignificant while the relative effect was more apparent Immunomodulators are biological response modifiers; exert their effects by improving host defense mechanisms against diseases. Immune regulation is a complex balance between regulatory and effector cells and any imbalance in immunological mechanism can lead to pathogenesis. Stevia’s effects and uses as a heart tonic to normalize blood pressure levels, to regulate heartbeat, and for other cardiopulmonary indications first were reported in rat studies. In hypertensive rats the leaf extract increased renal plasma flow, urinary flow, and sodium excretion and filtration rate [2]. In other research, Stevia has demonstrated antimicrobial, antibacterial, antiviral and antiyeast activity [5]. Here in this study, immunomodulatory activity of stevioside was explored, by evaluating its effect on antibody titre, DTH response, phagocytic function and lymphocyte proliferation in mice. Administration of stevioside showed significant stimulation at the dose level of 12.5 mg/kg, p.o. with respect to these parameters. The increase in carbon clearance index reflects the enhancement of phagocytic function of mononuclear macrophage and non-specific immunity. Phagocytosis by macrophages is important against the smaller parasites and its effectiveness is markedly enhanced by opsonization of parasite with antibodies and complement C3b leading to more rapid clearance of parasite from blood. The antibody production of T-dependent antigen SRBCs requires the cooperation of T- and Blymphocytes and macrophages [13]. Results obtained during present investigation showed increased antibody production in response to SRBCs. Fig. 2. Influence of stevioside on proliferation of T- and Blymphocytes ex vivo. Mice were exposed to graded doses of stevioside p.o. daily for 15 days. Control mice received the vehicle only. Splenocytes were isolated and stimulated with sub-optimal amounts of mitogens: LPS (10 ␮g/ml), Con A (4 ␮g/ml) for B and T cell proliferation, respectively. Cells were incubated for 72 h and proliferation was measured by MTT reduction assay. Both B and T cells exhibited proliferative response with stevioside treatment but on response to B cells was significant (P < 0.05). I. Sehar et al. / Chemico-Biological Interactions 173 (2008) 115–121 DTH is antigen specific and the general characteristics are an influx of immune cells at the site of injection, macrophages and basophils in mice and induction becomes apparent within 24–72 h. T cells are required to initiate the reaction [14] and [15]. Increase in the DTH response indicates that drug has a stimulatory effect on lymphocytes and necessary cell types required for the expression reaction [16]. The present investigation therefore reveals that stevioside certainly possess immunomodulatory properties. It has been substantiated that functional foods containing physiologically active components, either from plant or animal sources, may improve health and longevity. The great interest in stevia as a non-caloric, natural sweetener has fueled many studies on it including immunomodulatory ones. The main sweet chemical, stevioside, has been found to be an immunostimulator as evidenced by the increase in B- and T-cell mediated humoral and DTH response, respectively. It also has been shown to enhance macrophage function and substantially modulate the T and B cell proliferation. Hence, it may be concluded that oral administration of stevioside is well-tolerated and effective modality that may be considered as an alternative or supplementary therapy. References [1] A.D. Kinghorn, D.D. Soejarto. Sweeteners: discovery, molecular design, and chemoreception, in: D.E. Walters, F. Orthoefer, G.E. DuBois (Eds.), American Chemical Society, Washington, DC, Symp. Ser. No. 450 (1991) 14-27. [2] P. Chan, D.Y. Xu, J.C. Liu, Y.J. Chen, B. Tomlinson, W.P. Huang, J.T. 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