Evaluation of anti-inflammatory and anti-viral activities of herbal drugs Anosohip and Votalek

F I N A L  R E P O R T

In connection with the implementation of paragraph 2 of contract with “UNAVITA”, Republic of Macedonia and the National Centre of Infectious and Parasitic Diseases (NCIPD), Bulgaria

For the Study of “Evaluation of cytotoxic, anti-inflammatory and anti-anti- tumor activities of two nutritional supplements Anosohip and Votalek”

 Performers: Assoc. Prof. Andrei Tchorbanov; Assos. Prof. Petia Genova-Kalou Ph.D. Introduction


Cancer is one of the major reasons for human death in the last decade following cardiovascular diseases, infectious diseases and ischemic heart diseases. Current therapies of cancer include chemotherapy and radiotherapy although they both have severe side effects. Each type of cancer requiresspecific treatment, whichexplains the need for developmentof highly specific targeted anti-cancer agents [1].

Various immunotherapeutic approaches have been used for the treatment of cancer. The BCG (Bacillus Calmette-Guérin) vaccine for tuberculosis that contains attenuated Mycobacterium bovis is one of the most extensively used immunotherapeutics based on its strong non-specific immunostimulatory properties [2, 3]. BCG is widely administrated in cases of colorectal, lung cancers and melanoma.

In the last years, a number of therapeutic antibodies have been approved for clinical treatment in cases of breast cancer (Trastuzumab), non-Hodgkin (Rituximab) and Hodgkin lymphoma (Brentuximabvedotin), colorectal cancer (Panitumumab, Cetuximab), chronic lymphocyte leukemia (Alemtuzumab), and acute myelogenousleukemia (Gemtuzumabozogamicin) [4]. Immunotherapy with cytokines is another option for cancer treatment. IL-2 in metastatic melanoma and renal cell carcinoma and IFN-alpha in Stage III melanoma have been permitted for cancer therapy [5].

Different cancer vaccines have been developed for prevention and treatment of malignant diseases but only Sipuleucel-T is approved for therapy of advanced prostate cancer in case hormonal treatment is ineffective [6]. Many of the developed vaccines include cancer carbohydrate antigens chemically conjugated to carrier protein with or without other adjuvant [7-9].

Today natural products and their compounds derived from fungi, plants or microbes are of great interest for an anti-cancer research. This huge variety of chemical structures provides different mechanisms of action and specific effects used for anti-tumor therapy. A number of natural compounds are designed to repair, stimulate, or enhance the immune  system response. Most of them are small molecules, inhibitors of key enzymes for carcinogenesis like matrix metalloproteinazes (MMPs), HIFs, topoisomerase, protein kinase C (PKC) or transcripion factors like NFkB [10]. Special attention is played on marine-derived anti-angiogenesis products, which suppress and prevent the succesful formation of vascular system, supporting tumor growth and invasion. The majority of these substances act via inhibition of enzymes or factors, crucial for the process of angiogenesis [11].

C-26 murine model of colorectal carcinoma is classical tumor model, developed in 1975 and determined as an undifferentiated Grade IV carcinoma [40]. C-26 cells display high tumorigenicity and low tendency to metastasize mainly in the lungs. Inoculated in syngeneic Balb/c mice C-26 cells cause high mortality. This model is used more than 35 years for studying the process of carcinogenesis and anti-cancer therapy.

The aim of the present work was to develop an experimentalmurine model of colon carcinoma and to investigate the anti-tumor activity of new generated therapy.



Female 10 weeks old Balb/c mice were obtained from Harlan Farm, Blackthorn, UK. The animals were kept under specific pathogen free (SPF) conditions and the manipulations were approved by the Animal Care Commission at the Institute of Microbiology in accordance with the international regulations.


FITC (Fluorescein isothiocyanate)-conjugated anti-mouse CD8 and CD335, PE (Phycoerythrin)-conjugated anti-mouse -APC, and CD19 and APC (Allophycocyanin)– conjugated anti-mouse CD4 and CD11bmAbs (eBioscience, Frankfurt, Germany) were used for FACS (Fluorescence-activated cell sorting) experiments.

Cell line

Murine colon carcinoma cell line C-26 (CT26.WT (ATCC® CRL-2638™)) was cultured in complete RPMI (Roswell Park Memorial Institute medium) 1640 medium (Gibco, Gaithersburg, MD) containing 10% FCS (fetal calf serum), 4 mM L-glutamine, 50 μM 2- mercaptoethanol and antibiotics at 37 °С / 5% CO2. The confluent monolayer cells (90%) were trypsinized and used for animal administration and solid tumor establishment.

Tumor model establishment

For selection of proper C-26 murine colon carcinoma model 2 groups of mice were  challenged with 5×105 cells per mouse. The animals were observed for 14 weeks. Palpable solid tumors were observed 2 weeks after C-26 challenge followed by 0% survival 3 month safter cell challenge, which is acceptable for the model requested. A group of mice injected with PBS only was used as a control group.

Treatment schedule

A group of female Balb/c mice (5 animals per group) was inoculated with C-26 cells (5x105cells/mouse) and the mice were fed three times per day with test substances (TS group).The TS contains 0.075 mg Anosohip and 0.2µl Votalek per dose. Control groups of mice were challenged with C-26 cells without treatment or PBS only (Fig. 1). Tumor   growth

was monitored by measuring palpable solid tumors once a week with a microcaliper and the tumor volume was determined. Every two weeks the animals from all groups were bled and the sera were kept frozen at –70 °C. All experiments were duplicated.

Figure 1.  Treatment schedule

Phenotyping of spleen and tumor cell suspensions

We have performed an immune system modulation analysis during tumor development when the tumor model was established to demonstrate the immune reaction to the tumor growth. Organs from mice challenged with 5×105 C-26 carcinoma cells or PBS treated only were isolated andanalyzed by flow cytometry. Spleens and solid tumors from representative  number of animals (n = 4 to 6) were taken and monocellular suspension was isolated by cell strainers  (BD  Biosciences,  Erenbodegem,  Belguim).  The  cells  were  washed  with PBS (containing 2.5% FCS and 0.05% sodium azide) and incubated with one of the following anti- mouse antibodies – anti-CD4-APC, anti-CD8-FITC, anti-CD19-PE, or anti- CD11bAPC/CD335-FITC, or CD14-APC. Each incubation step was performed for 30 min at 4°C. Finally, the cells were washed twice and kept at 4°C. Ten thousand cells were analyzed from each sample with a BD LSR II flow cytometer using the Diva 6.1.1. software (BD Biosciences, San Jose, CA).

Figure 2A.FACS analysis of spleen and tumor suspensions.Spleens (upper) and solid tumors (lower) from representative number of animals (n = 4 to 6) were excised and the cell suspensions were incubated with one of the following anti-mouse antibodies – anti-CD4-APC, anti-CD8-FITC, anti-CD19-PE, anti-CD335-FITC/CD11b-APC or anti-CD14-APC. Ten thousands cells were analyzed from each sample by flow cytometry. Data are representative of at least 5 experiments.

We observed an increase of CD4+, CD8+, CD19+ as well as CD14+ cells in the spleens of  the treated animals compared to PBS injected Balb/c mice (Figure 2A – upper panels).A cell suspension prepared from solid tumors of the studied mice showed presence of CD4+, CD8+, CD19+, CD335+/CD11b+ as well as CD14+ cell populations in the tumor microenvironment (Figure 2A – lower panel).

Figure 2B. Percent of cell subtypes from FACS analysis of spleen and tumor cell  suspensions.

An increase of CD8+, CD19+, CD335+/CD11b+as well as CD14+ cells, and decrease of CD4+ cells was observed into the treated group compared to the control animals. These results suggest a movement of immune cells populations to the developed tumors and treatment (Figure 2B).

Cytokine quantification

IFNγ, IL10 and IL4 levels were measured in mouse sera using ELISA sets (BD OptEIATM, BD Biosciences Pharmingen, Erenbodegem, Belguim) according tothe manufacturer’s instructions.

Challenge with 5×105  C-26 carcinoma cells and treatment with PBS only induced high  serum levels of IFNγ and IL4 in experimental animals while the treatment with TS reduced the levels of these cytokines (Fig. 3). However, administration of the TS did not affect the levels of IL10 compared to the control group.

Figure 3.Administration of TS to C-26 carcinoma cells challenged mice modulates cytokines production. Serum levels of IL4, IL10 and IFNγ in all groups were measured by sandwich ELISA.All samples were triplicated and average values were used for analysis. Mean ± SD values were calculated for each group; p values were calculated using the two-way ANOVA test.(*p<0.05) in comparison to PBS-treated controls.

Survival and tumor manifestation

The survival of all tested groups was compared with the survival of animals inoculated with C-26 cells only. The results showed 80% survival of the treated group on day 49 compared to the C-26 group without treatment (Figure 4A).

Figure 4A.Survival curves of studied animal groups.TS treatment significantly prolonged the survival of C-26 inoculated mice (10 animals per group). The data are presented as mean ± SD. The p-values are calculated using the Log-rank test (*p<0.05), in comparison to PBS treated mice.

Spleens were taken from the treated animals and were compared with organs from untreated sick mice for size differences. The dynamics of tumor growth was monitored in the treated group compared to the control group (C-26 only).

Splenomegaly is a characteristic feature of C-26 murine tumor model. The mice from each group were sacrificed for organ comparison and examination. We observed a more than 2- fold significant increase of the spleens in none-treated C-26-bearing mice compared to TS animals. Animals treated with TS developed small size tumors within the whole period of observation (Figure 4B).

Figure 4B. Organ comparison. Spleen and tumor size comparison ofC-26 bearing  mice with C-26 bearing TS treated animals (upper figure); The dynamics of tumor growth  was monitored in all TS treated animals and compared to control group (C-26 only) – lower figure.The data are presented as mean ± SD. The p-values are calculated using the Two-way ANOVA test (***p<0.001), in comparison to C-26 bearing mice. Data are representative of at least 3 independent experiments.

Histology and organ comparison

Figure 5. Histological tumor analyses.Kidney sections stained with haematoxylin/eosin from C-26 bearing mice (A), and C-26 bearing TS treated animals (B). Data are representative of 5 experiments. Original magnification x 250.

We have examined morphological and histological features of C-26 tumor model. Solid tumors were resected from terminal animals. After fixation in phosphate-buffered formalin (10%) the tissues were embedded in paraffin and 7µm sections were analyzed using a standard haematoxylin/eosin staining technique.

The paraffin sections of solid tumors from all studied groups were stained with heamatoxilin- eosin histological staining technique and were analyzed by light microscope. The tumor necrosis indicates a poor prognosis for different cancers and is associated with the survival.

Tumor growth and metastasis depend on angiogenesis and lymphangiogenesis triggered by chemical signals from tumor cells in a phase of rapid growth. In the absence of vascular support, tumors may become necrotic or even apoptotic. Therefore, angiogenesis is an important factor in the progression of cancer. We observed differences within the tumor mass of the studied animals at 6th  week of the treatment schedule. The micrographs of tumors without TS treatment showed visible process of angiogenesis and erythrocytes presence into the tumor structure. It was not observed among the TS treated animals (Figure 5). These results could be associated with prolonged survival of these animals.

Statistical analysis

The values in figures correspond to mean ± SD. The Two-way ANOVA test was used to determine differences between each two groups. A value of P < 0.05 was considered as statistically significant. Survival significance was determined via analysis of survival curves with Prism software from GraphPad (San Diego, CA).


  1. Vanneman, M., Dranoff, G. Combining immunotherapy and targeted therapies in cancer treatment. Rev. Cancer. 2012, 12: 237–251
  2. Morales, A.,Eidinger, D., Bruce, A.Intracavitary Bacillus Calmette-Guerin in the treatment of superficial bladder tumors. Urol. 1976, 116: 180–183.
  3. Alexandroff, A., Jackson, A., O’Donnell, M., James, K.BCG immunotherapy is used to prevent and treat superficial bladder cancer. BCG immunotherapy of bladder cancer: 20 years on. 1999, 353: 1689–1694.
  4. Scott, A., Wolcho, J., Old, L.Antibody therapy of cancer. Rev. 2012, 12: 278–287.3893.
  1. Lee S,Margolin K: Cytokines in Cancer Cancers 2011, 3: 3856–
  1. Small, ,   Schellhammer,   P.,   et   al.   Placebo-Controlled   Phase   III   Trial  of Immunologic    Therapy    with    Sipuleucel-T     (APC8015)    in    Patients    with    Metastatic, Asymptomatic Hormone Refractory Prostate Cancer. J. Clin. Oncol.2006, 24: 3089–3094
  1. Sabbatini, P., Kudryashov, V., Ragupathi, G., et al. Immunization of ovarian cancer patients with a synthetic Lewisy-protein conjugate vaccine: Clinical and serological Int. J. Cancer 2000, 87: 79–85.
  2. Ragupathi, G., Slovin, S., Adluri, S., Sames, D., Kim, I.J., Kim, H.M., Spassova, M., Bornmann, W.G., Lloyd, K.O., Scher, H.I., Livingston, P.O., Danishefsky,S.J. A fully synthetic globo H carbohydrate vaccine induces a focused humoral response in  prostate cancer patients: A proof of principle. Chem. Int. Ed. Engl. 1999, 38: 563–566.
  3. Adluri, S., Helling, F., Ogata, S.,Zhang, S., Itzkowitz, S.H., Lloyd, K.O., Livingston, P.O. Immunogenicity of synthetic TF-KLH (keyhole limpet hemocyanin) and sTn-KLH conjugates in colorectal carcinoma patients. Cancer Immunol. Immunother. 1995, 41: 185–192.
  4. Bhatnagar, I., Kim, S.K. Marine Antitumor Drugs: Status, Shortfalls and Mar. Drugs. 2010, 8: 2702–2720.
  1. Wang YQ, Miao ZH: Marine-Derived Angiogenesis Inhibitors for Cancer Therapy. Mar.Drugs 2013, 11: 903–933.