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Immunorestoration and augmentation of mitogen lymphocyte response in Graffi tumor bearing hamsters by purified saponin mixture from Astragalus corniculatus.


Investigations on the effect of purified saponin mixture (PSM) obtained from the aerial parts of Astragalus corniculatus Bieb. (Fabaceae) on mitogen response of the spleen cells in Graffi tumor bearing (GTBH) and healthy hamsters were reported. The saponin mixture in a doses of 50 mg/kg b.w. was injected i.p. 4 times starting simultaneously with implantation of tumor cells. Stimulation indices to phytohemagglutinine (PHA) and lipopolysaccharide (LPS) of lymphocytes in spleens of tumor bearing hamsters (TBH) were significantly decreased during the whole period of the observation. It was established that PSM stimulated the functions of spleen cells in Graffi-TBH, resulting in increased mitogen response to PHA and LPS. The stimulation was better expressed in healthy PSM-treated hamsters. The proliferation response of spleen lymphocytes to PSM was also found. PSM did not change the in vitro proliferation ability of Graffi tumor cells.

The results obtained proved the immunostimulating and immunorestorating activity of PSM on the T- and B-spleen cells in healthy and GTBH hamsters, as well as the proliferative response of it to PSM.

[c] 2007 Elsevier GmbH. All rights reserved.

Keywords: Astragalus corniculatus; Saponins; Graffi myeloid tumor; Mitogen response; Spleen lymphocytes; PHA; LPS


One of the major needs in cancer prevention is the development of effective and safe new agents for chemoprevention targeted at mechanisms known to be involved in the process of carcinogenesis (Hong and Sporn, 1997).

Species of genus Astragalus are known to have numerous pharmacological activities and are used for medicinal purposes in many countries. The properties of these plants have been associated with its triterpene saponins and polysaccharides (Pistelli, 2002; Sinclair, 1998). The reason for the rising interest towards the triterpene saponins is due to their immunomodulatory, anti-cancer and antiviral activities (Rios and Waterman, 1997; Lacaille-Dubois, 1999, 2005).

A number of reports have demonstrated that extracts from Astragalus species stimulated immune functions both in vivo and in vitro (Kemper and Small, 1999; Tan and Vanitha, 2004; Yesilada et al., 2005).

Astragalus extracts restored local graft versus host immune response to normal levels and enhanced PHA-induced lymphocyte proliferation in a dose-dependent fashion in immunocompromissed cancer patients (Chu et al., 1994). In mice spleen cells, astragalus markedly stimulated normal cells to proliferate and also enhanced activation of macrophages (expressed by production of cytokines, THF and IL-6) and B cells (production of Ig G antibodies), but had no effect on NK-cell activity (Yoshida et al., 1997). Astragalus extracts modulate the function of the immune cells - Mos, NK cells, T and B lymphocytes (Sun, 1986; Marinova et al., 1994; Kajimura et al., 1997; Rios and Waterman, 1997; Kemper and Small, 1999; Sinclair, 1998; Verotta et al., 2001, 2002; Block et al., 2003). The antitumor effect of Astragalus was supposed to be the results of its immune-related mechanism or possible direct cytotoxic activity mechanisms (Cheng et al., 2004). Lau et al. (1994) have shown the direct inhibition of the tumor growth of murine and renal carcinoma after treatment with Astragalus membranaceus extract.

The experimental data demonstrate the protective effect of purified saponin mixture (PSM) from Astragalus corniculatus on the survivability of GTBH as expressed by decrease of the tumor transplantation, inhibition of tumor growth and reduced mortality percentage (Krasteva et al., 2004) as well as the immunostimulating activity on the blood PMNs and pMos in GTBH (Toshkova et al., 2007). Phytochemical analysis of the PSM led to the isolation of three new oleanane-type triterpene saponins (Krasteva, 2006; Krasteva et al., 2006).

The aim of the present work was to investigate the effect of the PSM from A. corniculatus on the proliferative activity of spleen cells from PSM-treated healthy and GTBH and in this way to provide evidence for the implication of its immunomodulatory potential for the overall protective effect in the lethal Graffi experimental tumor in hamsters.

Materials and methods

Experimental animals

Forty males and females "Golden Syrian" hamsters (8 weeks old), weighing 80-100g, were used for the experiments. The animals were bred and grown under conditions accepted by the Bulgarian Veterinary Control Service in the animal house of Institute of Experimental pathology and parasitology, Bulgarian Academy of Sciences. Hamsters were separated in four experimental groups, each containing 10 animals as follows: group 1-hamsters treated i.p. 4 (consecutive days) times with PSM 50 mg/kg b.w. per animal starting simultaneously with s.c. tumor transplantation of 2 x [10.sup.4] on day 1 (PSM + T); group 2-hamsters treated only with tumor cells on 1 day (T); group 3-healthy hamsters treated 4 times with PMS as pointed for the 1st group (PSM); group 4 - control healthy hamsters without any treatment (C).

Plant material and preparation of PSM

The aerial parts of the plant were collected in Northern Bulgaria and identified by Dr. D. Pavlova from the Department of Botany, Faculty of Biology, Sofia University, where voucher specimen has been deposited (SO95265). Details of obtaining of PSM have been published previously (Krasteva et al., 2004). PSM was analysed by thin-layer chromatography on Kieselgel 60 [F.sub 254] (0.24 mm thick, Merck) plates, using the solvent systems n-BuOH-AcOH-[H.sub.2]O (4:1:1), [CHCl.sub.3]-EtOH-EtOAc-[H.sub.2]O (30:40:15:10), n-BuOH-MeOH-[H.sub.2]O (5:3:1) and EtOAc-EtOH-[H.sub.2]O (70:21:9). The spots were visualized by spraying with anisaldehyde/conc. [H.sub.2][SO.sub.4], followed by heating. The results show that PSM contains three triterpene saponins. Phytochemical investigation of PSM led to the isolation of three new oleanane-type triterpene saponins (Fig. 1), which were identified by chemical and spectral analysis as 3[beta]-0-[0-4-oxo-pentopyranosyl-(1 [right arrow] 2)-[beta]-D-glucopyranosyl]-21[alpha]-hydroxyolean-12-ene-28-oic acid (1); 21 [alpha]-hydroxyolean-12-ene-28-oic acid 3 [beta]-4-oxo-pentopyranoside (2) and 19[alpha]-hydroxyolean-12-ene-28, 21[beta]-olide 3-[beta]-D-xylopyranoside (3) (Krasteva, 2006; Krasteva et al., 2006).

For the animal study, PSM was dissolved extempore in redistilled water. Endotoxin was determined by limulus amoebocyte lysate assay (QCL-1000, Cambrex). The endotoxin content was < 0.06 EU/ml.

PSM in a dose of 50 mg/kg b.w. was injected four times (1, 2, 3 and 4 days) starting simultaneously with implantation of tumor cells. The dose was optimal with respect to the immunostimulating activity and toxicity in mice (Marinova et al., 1994).

Experimental tumor

Graffi mouse leukemia virus was adapted and maintained in vivo in hamsters as solid tumor by s.c. inoculation of 1-2 x [10.sup.6] viable tumor cells. Transplantation of the tumor in all experimental animals was achieved by single s.c. inoculation of 2 x [10.sup.4] viable trypan blue excluded tumor cells in interscapular area. The cells were obtained by mechanical desegregation of tumor mass, extirpated under sterile conditions. Our previous studies showed that such quantity of tumor cells induced 100% transplantibility and mortality in hamsters according to Toshkova (1995).

In vitro proliferation of spleen lymphocytes

The proliferation responses of lymphocytes in the presence of mitogens as well as PSM were determined on days 7, 14 and 21 after tumor inoculation by the method described by Masson and Gwanzura (1990).

Briefly, hamsters were euthanized by carbon dioxide according to institutional guidelines. Spleens were harvested immediately in an aseptic way, cooled in ice-cold phosphate buffered saline (PBS) supplemented with 5.0% FCS and suspended by passing through a stainless steel sieve. A single cell lymphocyte suspension was prepared. The spleen cells were diluted in PBS, layered on 3 ml Ficoll-Paque (Pharmacia, Uppsala, Sweden) in ratio 2:1. The gradient was centrifuged at 1850 rpm for 40 min at 20 [degrees]C. The lymphocytes were collected from the inter-phase and washed three times with RPMI-1640 medium. The viability of lymphocytes tested by trypan blue exclusion test was about 95%. Cell suspensions with concentrations of 5 x [10.sup.6] cells/ml in complete RPMI-1640 medium were adjusted and subsequently used for proliferation test.

Suspensions of 5 x [10.sup.5] cells in 0.1 ml of complete RPMI-1640 medium were distributed in triplicate in 96 wells of flat bottom tissue culture plates. The cells were stimulated by 20 [micro]g/ml phytohemagglutinin (Sigma) or 20 [micro]g/ml Escherichia coli lipopolysaccharide (LPS) (Sigma) or 20 [micro]g/ml PSM. The cells were cultivated in a [CO.sub.2] incubator at 37 [degrees]C for 72 h. Following incubation, the plates are pulse labeled with 1 [micro]Ci/well of[.sup.3] H thymidine and incubated for additional 18 h. The cells were collected on nitrocellulose filters using a semi-automatic manual cell harvester and were distributed in scintillation flasks. After a supplementation with 5 ml scintillation mixture, the isotope uptake of each sample was determined in a Beckman scintillation counter. Stimulation index (SI) of lymphocytes was calculated according to the formula: SI = number of cpm of lymphocytes incubated with mitogen: number of cpm of lymphocytes incubated without mitogen.


In vitro tumor cell proliferation

In vitro tumor cell proliferation in the presence of PSM is evaluated for quantitative measurement of direct cytotoxicity of PSM. Briefly, tumor cells are obtained from the Graffi tumor, extirpated under sterile conditions. Non-necrotic tumor pieces were passed through a stainless steel seeve. The viability of the tumor cells examined by the Trypan blue exclusion test was 95-98%. The tumor cells are washed (2 times), resuspended in RPMI-medium (complete), plated in Falkon-96-well microtiter plates (2 x [10.sup.5] cells/well) and incubated for 72 h at 37 [degrees]C and 5% [CO.sub.2] in the presence or absence of PSM (20 [micro]g/ml). Cells were pulsed with 1 [micro]Ci/well of [.sup.3]H thymidine for the final 18 h of incubation. Following incubation the cells were harvested on NC membranes transferred to scintillation vials and supplemented with 5 ml scintillation mixture. The samples were counted in Beckman scintillation counter. Thymidine incorporation (cpm/min) is employed as index of tumor cell proliferation.


Each set of experiments was carried out three times and Student's t-test was performed to analyze the significance of the differences between the control and experimental groups. Differences were considered significant at p < 0.05 *; p < 0.01 **; p < 0.001 ***.


Effect of application of PSM isolated from Astragalus corniculatus on the mitogen responsibility of the spleen lymphocytes in hamsters with progressing Graffi myeloid tumors were followed during 21 days after tumor transplantation.

The results from estimations of PHA-mitogen reactivity of spleen lymphocytes are presented in Fig. 2.

It was established that Graffi tumor suppressed significantly the mitogen activity of spleen lymphocytes to PHA during the whole period of observation (SI = 0.18 [+ or -] 0.06, 0.61 [+ or -] 0.08 and 0.39 [+ or -] 0.07, established on days 7, 14 and 21, respectively, control healthy being 1.24 [+ or -] 0.016) (Fig. 2, group T). In the group of PSM-treated TBH SIs were 3-5-fold increased with a maximum on day 14 (0.88 [+ or -] 0.1, 1.99 [+ or -] 0.26 and 0.96 [+ or -] 0.15, determined on days 7, 14 and 21, respectively). PSM treatment augmented most noticeably the SI to PHA of spleen lymphocytes in the healthy hamsters (SI-1.96 [+ or -] 0.1; 2.56 [+ or -] 0.22 and 1.84 [+ or -] 0.13, found on days 7, 14 and 21, respectively) (Fig. 2).


The Graffi tumor suppressed also the mitogen response of spleen lymphocytes to LPS. SI were 1.26 [+ or -] 0.15; 0.78 [+ or -] 0.15 and 0.59 [+ or -] 0.1 on days 7, 14 and 21, control healthy being 1.68 [+ or -] 0.14) (Fig. 3). The application of PSM stimulated the mitogen response of spleen lymphocytes both in TBH and healthy hamsters. PSM treatment of GTBH restored the mitogen response on days 7, 14 and 21, and SI reached those of controls (SI were 1.58 [+ or -] 0.18, 1.98 [+ or -] 0.26 and 1.39 [+ or -] 0.12 on days 7, 14 and 21, respectively). The mitogen response of spleen lymphocytes of healthy hamsters to LPS was enhanced markedly after the treatment with PSM on day 14 (SI-2.9 [+ or -] 0.5 were established). SI on days 7 and 14 were near those of control (SI-1.8 [+ or -] 0.23 and 1.62 [+ or -] 0.17, respectively, control healthy being 1.68 [+ or -] 0.14) (Fig. 3).


The proliferation ability of spleen lymphocytes cultivated in vitro in the presence of PSM was also studied. As shown in Fig. 4, the lymphocyte response to the PSM is most pronounced in healthy hamsters, treated with PSM (group H + PSM) (SI of 1.58 [+ or -] 0.23 was determined). The lymphocytes from PSM-treated GTB animals showed lower SIs (1.12 [+ or -] 0.29). The stimulation indices of lymphocytes from GTBH are lower compared with the controls (SI = 0.85 [+ or -] 0.22; control healthy being 1.02 [+ or -] 0.2) (Fig. 4).


It was established that during the in vitro cultivation of tumor cells in the presence of PSM (20 [micro]g/ml), the proliferation activity (cpm/min) of tumor cells remained similar to that of tumor cells cultivated without PSM (cpm 8110 [+ or -] 599 and cpm 8096 [+ or -] 530 were established, respectively).


Graffi tumor is an example of a disease associated with immunosuppression. However, immune response to the tumor is complex and more accurately reflect an immunofocusing of host defense capabilities.

The results from the present experiments showed that the Graffi tumor in hamsters induced a suppression of mitogen response of spleen lymphocytes during tumor development. The application of the PSM from A. corniculatus four times (4 consecutive days) produced a restoration effect on the suppressed functions of both kinds of spleen lymphocytes (T- and B-). Meanwhile, the application of PSM induced a significant and continuous (until day 21) increase of the proliferation activity of lymphocytes in healthy hamsters. Particularly affected was the PHA ability of lymphocytes; a 1.5-2.0-fold rise was observed in the group of healthy hamsters under the action of the preparation (Fig. 2). PSM restored also the suppressed mitogen response to LPS of spleen lymphocytes in GTB hamsters and improved SIs of healthy PSM-treated animals with maximum on day 14 (Fig. 3). The experimental data also provide evidence that the PSM ameliorate the proliferation of spleen lymphocytes, without any cytotoxicity on Graffi tumor cells in vitro. PSM did not change the proliferation ability of tumor cells. The proliferation of tumor cells cultivated in vitro in the presence or absence of PSM are approximately the same.

The enhancing effect of PSM on pMO and PMN functions in GTBH hamsters and its beneficial action on these functions in healthy and GTBH hamsters has been reported previously (Toshkova et al., 2007).

Saponins of A. membranaceus significantly enhanced the Con A-, LPS- and OVA-induced splenocyte proliferation as well as IgG, IgG1 and IgG2 antibody titers in serum of OVA-immunized ICR mice (Yang et al., 2005).

All triterpene saponins isolated from Turkish species Astragalus showed a prominent IL-2-inducing activity (Yesilada et al., 2005).

The saponins isolated from Astragalus spp. endemic to Egypt were not cytotoxic against a variety of human cancer cells. However, dose-related modulation of lymphocyte proliferation was observed, and structure-activity relationships are described (Verotta et al., 2001, 2002). Cheng et al. (2004) show that incubation of K562 or HEL cells with A. membranaceus extract for 3-5 days results in the expression of beta-globin gene in both cell lines and leads to terminal differentiations. Moreover, the apoptosis in HEL cells can be induced by increasing concentrations of Astragalus extract.

Treatment with tumor antigen peptide sensitized DCs plus Astragalus decreased the tubercle of lung cancer, increased proportion of subsets CD4 + T and CD8 + T in mice's splenic cells and serum IL-2/IL-4 ratio and tumor developing rate was lower than that in mice treated with DCs alone (Dong and Dong, 2005).

Nonetheless, the presented data are not sufficient to draw any conclusion about the mechanism of action of PSM on the immune response. We suppose that the immunorestorating effect of the PSM on immune cells contributes to its overall antitumor effect in GTBH. This is consistent with the findings that phagocytes (mainly M[empty set]s and PMNs) and lymphocytes are the crucial elements of immune response to tumors in hosts.

In conclusion, the presented results demonstrate convincingly that the PSM from A. corniculatus interfered with Graffi experimental tumor through potentiation and restoration of the host immune response as well as the immunostimulating activity in healthy animals. Based on our results we could suggest that the PSM is a promising candidate as an immune modifier, useful for the treatment of immunosuppression in experimental tumors.


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* Corresponding author. Tel.: + 35929236552; fax: +359 2 9879874. E-mail address: (I.N. Krasteva).

0944-7113/$-see front matter [c] 2007 Elsevier GmbH. All rights reserved. doi: 10.1016/j.phymed. 2007.11.026

R.A. Toshkova (a), I.N. Krasteva (b), *, S.D. Nikolov (b)

(a) Department of Immunology, Institute of Experimental Pathology and Parasitology, BAS, G. Bonchev St., Bl. 25, 1113 Sofia, Bulgaria

(b) Department of Pharmacognosy, Faculty of Pharmacy, Medical University, 2 Dunav Street, 1000 Sofia, Bulgaria
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Author:Toshkova, R.A.; Krasteva, I.N.; Nikolov, S.D.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EXBU
Date:Oct 1, 2008
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