© Borgis - Postępy Fitoterapii 3/2014, s. 127-135
*Karina Schönknecht1, Frank Conrad2, Hartwig Sievers2, Jerzy Jambor1, Andrzej M. Fal3,4
Przeciwwirusowa Aktywność Biostyminy® (wodnego wyciągu ze świeżych liści aloesu drzewiastego) przeciwko wirusom powodującym infekcje górnych dróg oddechowych w badaniach in vitro
Anti-viral activity of Biostymina® (Aloe arborescens folii recentis extractum fluidum) against viruses causing upper respiratory tract infections tested in vitro
1Phytopharm Klęka SA, Nowe Miasto on Warta, Poland
President of the Board: Wojciech Skrobański
2PhytoLab GmbH & Co. KG, Vestenbergsgreuth, Germany
Management board, Medical Affairs: dr Hartwig Sievers
3Department of Public Health, Wrocław Medical University, Poland
Professor & Chair: Andrzej M. Fal, MD, PhD
4Department of Internal Medicine and Allergology, Central Clinical Hospital MSW, Poland
Professor & Head: Andrzej M. Fal, MD, PhD
Wstęp. Infekcje górnych dróg oddechowych, przeważnie o etiologii wirusowej, stanowią częsty problem zdrowotny we wszystkich grupach wiekowych. Stosowanie leków przeciwwirusowych w tych schorzeniach jest ograniczone zarówno ze względu na niewielką ilość substancji aktywnych dostępnych na rynku, jak i przez ich działania niepożądane i wysoki koszt terapii. Istotnym pozostaje więc badanie kolejnych substancji, szczególnie o większej dostępności cenowej i bardziej korzystnym profilu bezpieczeństwa.
Cel pracy. Celem badań było określenie aktywności przeciwwirusowej preparatu Biostymina® (będącego wodnym wyciągiem z A. arborescens), przeciwko wirusowi grypy typu A (hFluA), wirusowi RSV (Respiratory syncytial virus), wirusowi Coxsackie (CA9) i adenowirusowi (Adeno 5), w warunkach in vitro.
Materiał i metody. Dla oceny żywotności komórek przeprowadzono na wstępie badanie aktywności cytotoksycznej preparatu Biostymina® in vitro na linii komórkowej HEp-
Upper respiratory tract infections (URTIs) are among the most common reasons to visit a primary care provider. URTIs (known also as a “cold”) are estimated to be the direct cause of seeking medical consultation in 60-90% of children (1). The easy transmission of viral respiratory pathogens, which can be carried in secretions as aerosols and droplets or via mucosal contact, allows for the rapid spread of the disease, especially among the family members of the infected person (1-3). Uncomplicated URTIs last usually 7 to 10 days, and include a variety of symptoms, such as cough, sneezing, sore throat, rhinitis, and sometimes fever. In over 90% of cases URTIs are caused by viral infections and the systemic inflammatory response to them (2, 4). Rhinoviruses are considered to be responsible for up to 80% of URTIs. Other infectious agents include respiratory syncytial virus (RSV), influenza virus (hFluA), adenovirus (Adeno), and Coxsackie virus (CA) (1, 2, 5-10).
Unfortunately, despite the large scale of URTIs, which also has a serious negative economic impact (11), there are only a few antiviral drugs used for its treatment (including ribavirin – a nucleoside analog, adamantanes, and neuraminidase inhibitors (for hFluA), palivizumab – a monoclonal antibody (for RSV), pleconaril – an inhibitor of enterovirus replication (for CA), and cidofovir, a nucleoside phosphonate analog (for Adeno) (3, 7, 9, 10, 12)). However, the use of these therapeutics is limited by the high cost and limited effectiveness of therapy, as well as the potential health risk due to side-effects (11, 12). On the other hand, widely overused antibiotics have not been shown to treat URTI or even to prevent secondary bacterial infections. Moreover, the excessive use of antibiotics results in an increased risk of antibiotic resistance (2, 4, 11). Taken together, this illustrates that new drugs are needed in this area. One of their potential sources is herbal medicines with significant antiviral, immunomodulatory, or anti-inflammatory activity. They could be a good alternative in primary or adjunctive therapy and prophylaxis of URTI (13).
Aloe arborescens Mill. (Asphodelaceae: Alooideae) is a large, evergreen succulent, endemic to the mountainous regions of Southern Africa (14). It is known also as candelabra aloe or krantz aloe. This much-branched shrub grows up to 5 m tall. Its usually greyish green leaves with yellowish teeth form apical dense rosettes, whereas inflorescences consist of a characteristic elongated inverted-conical dense raceme with cylindrical flowers. However, A. arborescens hybridizes easily with other aloe species, which leads to many morphological variations (15). The aqueous extract from the leaves of this plant has been used to treat and prevent URTI in Central and Eastern Europe since the 1950s, when the first product Biostymina® was officially registered in Poland as an immunomodulatory agent, also for children. A. arborescens used for the production of Biostymina® is not collected from its natural habitat, but from the drug manufacturer’s greenhouses where the cultivation is under controlled conditions. This procedure prevents from the formation of hybrids and provides a genetically uniform species through the years. A. arborescens, unlike other Aloe species, is characterized by a very low anthranoid content, which prevents the unwanted laxative effect (13). On the other hand, it contains many other substances responsible for its therapeutic value, such as glycoproteins, lectins P-2 and S-1, heteropolysaccharides, and phenolic compounds:, aloenin, and phenolic acids (15-18). The results of several studies have indicated immunomodulatory activity of A. arborescens, associated with stimulating B and T lymphocytes (13). The recent results confirm that Biostymina®, obtained in the current manufacturing process, influences the cellular response of the immune system, and accelerates the maturation of thymocytes and the acquisition by it of immune competence. These studies also demonstrated that stimulation of a cellular response by Biostymina® is comparable to that of synthetic immunostimulants: Levamisole and Isoprinosine (19). Candelabra aloe was also shown to inhibit cancer-cell-induced-angiogenesis, which suggests its possible role in immunocompromised neoplastic patients (20-22). A. arborescens is regarded also as an excellent appetite stimulant, demulcent, and allergy reducer (13, 23). Furthermore, the anti-inflammatory, antibacterial, antifungal, antioxidant, antidiabetic, radioprotective, and wound-healing properties of A. arborescens have been demonstrated (23-33). Recently, the antiviral activity of Biostymina® was shown in vitro against human rhinovirus (HRV14) (5). Its influence on other respiratory viruses has not been established yet, however. The aim of this study was an in vitro evaluation of Biostymina® with regard to its possible dose-dependent anti-viral activity against four human pathogenic RNA and DNA viruses: Influenza A (hFluA), Respiratory Syncytial Virus (RSV), Coxsackie (CA9), and Adenovirus (Adeno 5).
Materials and methods
Biostymina®, Aloe arborescens folii recentis extractum fluidum (0.25:1) from fresh leaves, in vials for oral intake, was provided by the manufacturer Phytopharm Klęka S.A. For the in vitro assays this test substance was diluted as described with the respective cell culture media.
Non-treated virus-infected cells cultivated in cell culture medium alone (MEM – Minimum Essential Medium Eagle), without active components, served as controls (virus-controls). Negative controls were non-infected cells, and Ribavirin Viracole® (ICN Pharmaceuticals) (4-6 μg/ml) in RSV-infected HEp-2 or in CA9-infected BGM cell cultures and Amandatin hydrochloride (Glaxo Welcome) (4 μg/ml) used in hFluA-infected MDCK cells, and in Adeno 5 an internal standard-infected HEp-2 cells (7.5μg/ml) served as positive controls. The efficacy of all reference substances was confirmed in the anti-viral tests.
The cytotoxicity and cell viability of the test substance diluted in cell culture medium was investigated in untreated HEp-2 cells via MTT tests and morphological examinations.
Ribavirin (Virazole®), (3-6μg/ml), ICN Pharmaceuticals, effective against the RNA-viruses e.g. RSV and CA9, Amandatin hydrochloride (4 μg/ml), Glaxo Welcome, active in influenza virus (hFluA), and a laboratory standard (7.5μg/ml) against Adeno5 were used as positive controls. The efficacy of all reference substances was confirmed in the test systems.
Virus Strains and Cells
Four viruses responsible for URTI were used in the study:
– Human Influenza A/Chile/01/83 (H1N1, hFluA),
– Respiratory Syncytial Virus (RSV) – strain long,
– Coxsackie virus (CA9),
– Adenovirus C subtype 5 (Adeno 5).
The following cell lines were used in the anti-viral study:
– Madin Darbey Canine Kidney (MDCK) cells – selective for hFluA,
– Human-Epidermoid-Carcinoma (HEp-2) cells – selective for RSV and Adeno 5,
– Borgio Green Monkey (BGM) cells selective for CA9.
Cells used in the cytotoxicity study were Human-Epidermoid-Carcinoma (HEp-2) cell line.
All virus strains and cells were obtained from the Department of Medical Virology and Epidemiology of Viral Diseases of the Hygiene Institute of the University of Tübingen, Germany and Friedrich-Schiller-University, Jena, Germany.
Cytotoxicity tests in vitro
In order to determine the optimal concentration of Biostymina® for the anti-viral activity assay, a cytotoxicity study was performed to exclude its cytotoxic effects. Therefore, Biostymina® was tested for its in vitro cytotoxicity and metabolic effects on HEp-2 cell cultures (MTT test and morphological examination).
For determination of the highest non-toxic concentrations, the test substance (100%) was diluted 10:1 with ten-fold concentrated cell culture medium, and further diluted in log 2 and log 10 dilution steps. For analysis of the cytotoxicity of test substance, all examined dilutions were added in four replicates to growing HEp-2 cells. Thereafter, cells were incubated for 6 days at 37°C and 5% CO2.
Analysis of the in vitro cytotoxicity was performed at days 1, 3 or 4, and 6 with the MTT test (34). For this purpose, cells were incubated for 1-2 hours with an MTT solution and after dissolution of the formazan crystals in DMSO, the optical density (OD) of the cell culture supernatants was analyzed with a photometer at 570 nm. OD values of the non-treated medium controls defined 100% viability (0% cytotoxicity). Cytotoxicity of the test substance determined in the MTT assay (% cytotoxicity) was the basis of the dose-response (toxicity) curves for the determination of the inhibitory concentrations showing 50% cytotoxicity (IC50).
Additionally, via phase contrast microscopy (magnification of 10x for general and 40x for intracellular view), the analysis of the cell cultures (HEp-2) was performed at day 6 after addition of the test substance, in order to verify the cytotoxic effects. The criteria for the microscopic analysis were as follows: altered cell morphology, generation of intracellular vacuoles, and destruction of cell monolayers.
Determination of anti-viral activity in vitro
The anti-viral activity of Biostymina® was assessed using plaque-reduction assays (FluA, RSV, CA9) and analyses of cytopathogenic effects (CPE) (Adeno 5). In addition, the amount of newly synthesized Adenovirus viral proteins was measured in a virus-specific enzyme-linked immunosorbent assay (ELISA), in a so-called “therapeutic approach”, i.e. after adding the test substance to the cell cultures in semi-solid medium containing agarose 1 hour after virus infection. This part was run in four replicates and two (RSV, CA9, Adeno 5) or three (FluA) independent experiments. Experiments differed in the amount of virus used for the infection (multiplicity of infection, M.O.I.).
Plaque-reduction and CPE-based assays were performed on MDCK, HEp-2, BGM, and HeLa-cell cultures using standard procedures for the detection of infectious particles. The antiviral activity of the test substance was quantified in plaque-reduction assays by counting virus-derived plaques (plaque-forming units/ml, PFU/ml) for hFluA, RSV, and CA9. Furthermore, the cytopathogenic effect (CPE) for Adeno 5 was analyzed with a computer-based image processing system (AIDsystems). Virus plaques and CPE were quantified by employing an optical evaluation system (ELISpot reader and AID Diagnostika, respectively). The number of plaques or percent CPE of the test substance-treated cell cultures were compared to the number of plaques or percent CPE lesions of the virus controls, which determined 100% infection. For quantification of Adeno 5 antigens in the infected cell-culture supernatants, enzyme immunoassays (Merlin Diagnostika) were used.
Cytotoxic properties in vitro
In order to exclude the cytotoxic effects of Biostymina®, the analysis of its in vitro cytotoxicity and metabolic effects on the virus-permissive cells HEp-2 was conducted. As previous research showed, Biostymina® did not cause cytotoxic effects on HeLa cells even in the 90% solution (4). Therefore, in this study a similarly high concentration of the extract was used.
Initially, the cytotoxicity study on HEp-2 cells did not show any cytolytic effect of Biostymina® in 90% to 5.6% solutions at day 1 or day 3. Therefore, the anti-viral studies with the test substance against the examined viruses, performed on MDCK, BGM, and HEp-2 cells, were started with solutions of 50% (1:2, FluA and CA9) and 90% (9+1, RSV and Adeno 5), followed by further log2 and log10 dilutions (tab. 1).
Table 1. Summary of the concentrations of the test substance (Biostymina® – 100% solution in water) used in the experiments for the quantification of the cytotoxicity on HEp-2 cells in vitro.
| ||Dilution (1/x) / concentrations (% solution, v/v) in cell culture media (MEM)|
|Percent of solution||90||45||22.5||11.3||5.6||2.8||1.4||0.8||0.4||0.04|
However, a highly sensitive reaction to Biostymina®, observed after using solutions of 50% (1:2) to 6.25% (1:16) on FluA-infected MDCK cells, as well as on CA9-infected BGM cells, was associated with the cytotoxic effects. The cytotoxic side effects of the test substance in solutions of 90% (9+1) to 5.6% (1:16) were revealed also in RSV- and Adeno 5-infected HEp-2 cells, which needed longer incubation times (6-7 days) until virus-caused lesions in the cell monolayer were visible. Therefore, an additional cytotoxicity study with Biostymina® on non-infected HEp-2 cells and longer incubation times (4-6 days) was performed to separate clearly cytolytic and anti-viral effects in the assays for the anti-viral activity.
The remarkable reduction of the HEp-2 cells metabolic activity from 33.9% to 12.1% was noticed at day 4 and day 6 while using dilutions of 90% (9+1) to 5.6% (1:16). Although no typical cytotoxic side effects were observed, the microscopic examination showed a slight but clear cellular modification of the cell monolayer. Only the solution of 3.3% (dilution of 1:30) when used for virus-infected cells did not cause visible cytotoxic side effects. Therefore, the final anti-viral studies with Biostymina® against hFluA, CA9, RSV, and Adeno 5, conducted on the MDCK, BGM, and HEp-2 cells, respectively, were performed with the 3.3% starting solution followed by further log 2 and log10 dilutions.
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