Evaluation of Antibacterial and Antitumor Activities of Some Turkish Endemic Plants

Purpose: To investigate the antibacterial and antitumor activities of the aerial parts of 8 different Turkish endemic plants (Phlomis russeliana, Phlomis armeniaca, Astragalus brachypterus, Astrantia maxima, Ptilostemon afer, Senecio castagneanus, Echium orientale and Arum euxinum). Methods: Two different bioassays were performed to evaluate the antibacterial and antitumor activities of the endemic plants. For each plant, 3 types of extracts (aqueous, methanol and ethanol) were prepared, giving a total of 24 extracts tested. The disc diffusion assay was used to screen for antibacterial activity against 10 bacteria including Streptococcus pyogenes, Staphylococcus aureus, Staphylococcus epidermidis, Escheria coli, Pseudomonas aeruginosa, Salmonella typhimurium, Serratia marcescens, Proteus vulgaris, Enterobacter cloacea, and Klebsiella pneumoniae. Five different antibiotics (ampicillin, carbenicillin, chloramphenicol, erythromycin and tetracycline) were used as positive controls. Antitumor activity was evaluated using potato disc diffusion bioassay with camptothecin as positive control. Results: The highest antibacterial activity was observed for all extracts of A. brachypterus against S. pyogenes (15.0 - 16.3 mm inhibition zone). The aqueous extract of S. castagneanus showed the strongest antibacterial activity against S. pyogenes (14.3 mm). Furthermore, alcohol extracts (ethanol and methanol) of P. russeliana exhibited moderate activity against S. epidermidis (9 and 8.5 mm, respectively) and S. pyogenes (9.5 mm). High antitumor activity was observed for all extracts of A. brachypterus (91.7 - 100 % tumor inhibition). In addition, ethanol extract of P. russeliana (75 % inhibition) exhibited strong antitumor activity. Conclusion: The present study reveals the strong antibacterial and antitumor activities of A. brachypterus. However, the active components of the plant extracts needs to be identified in future studies.


INTRODUCTION
Medicinal plants are the richest bioresource of drugs in traditional systems of medicine, modern medicines, nutraceuticals, food supplements, folk medicines, pharmaceutical intermediates and chemical entities for synthetic drugs [1].The use of most medicinal plants discovered by traditional societies has not been verified scientifically and bioassays can provide initial screening data about the biological activities of these plants.The scientific verification of the biological activity of endemic plants that are found exclusively in a particular area may be important in screening for the potential value of these peculiar plants [2].
The roots of Astragalus species are used to treat leukemia and for wound healing in Turkish folk medicine [12].Phlomis species have been used to treat various conditions such as diabetes, gastric ulcer, hemorrhoids, inflammation and wounds [13].Demirci et al [14] revealed that Phlomis essential oils might be an alternative to conventional antimicrobials in various foods.Ozcelik et al [15] determined the antiviral, antibacterial and antifungal effects as well as cytotoxicity of selected Turkish Phlomis species.Caffeic acid-containing phenylpropanoid glycolsides found in P. armeniaca showed activity against several kinds of cancer cells [16].
The aim of this study was to evaluate the antibacterial and antitumor activities of eight endemic plants found in Bolu, Turkey.

EXPERIMENTAL Plant material and extraction
The aerial parts of the eight endemic plants were collected from Bolu, Turkey [17].All plant samples and their treatments are presented in Table 1.
Three types of solvents -water, methanol (MeOH) and ethanol (EtOH)] were used for extraction.For aqueous extraction, each plant sample was extracted with water at 80 C for 12 h.Water was removed from the extract by freeze-drying (Christ Alpha 1-2 LD Freeze dryer).For alcohol extractions, the plant samples were Soxhlet extracted with MeOH or EtOH at 60 °C for 12 h.The extracts were then vacuum evaporated.For bioassays, each residue was dissolved in sterile distilled water to obtain a final concentration of 100 mg/ml.

Antibacterial bioassay
The disc diffusion assay was used to screen for antibacterial activity [18].All experiments were repeated three times.

Potato disc tumor induction assay
The antitumor activity of the extracts was assessed using potato disc method as modified by McLaughlin et al [19].A suspension of Agrobacterium tumefaciens (ATCC  23341) was standardized as determined by an absorbance value of 0.96  0.02 at 600 nm [20].All extracts and control solutions were filter sterilized.
Potatoes (Solanum tuberosum L.) were surfacesterilized by immersion in 10 % commercial bleach (Domestos ® ) for 20 min.Cylinders (10 mm diameter) were removed from the center of potato tissue using a cork borer and cut into 0.5 cm discs after excluding 1 cm end pieces.These discs were transferred to 24-well culture plates containing water-agar.Each disc was overlaid with 50 µl of appropriate inoculum.Plates were incubated at 28 °C in the dark.After 2 weeks, discs were stained with Lugol's reagent and tumors on each disc were counted.The experiments were repeated three times.

Bacterial viability testing
Bacterial suspension was serially diluted to 1 x 10 3 CFU.Bacterial viability was determined by incubating 1 ml of each plant extract with 1 ml of bacterial suspension.At 30 min after inoculation, 0.1 ml of inoculum (bacteria + extract) was removed and inoculated on YEM media with spread plate technique.After 24 h incubation of the inoculated plates at 28 °C, colony counts were made [20].

Data analysis
All data were analyzed by analysis of variance (ANOVA) and mean values were compared by Duncan's Multiple Range Tests using SPSS software, version 15 (SPSS Inc, Chicago, IL, USA).

RESULTS
Twenty-four different extracts prepared with three types of solvent (water, methanol and ethanol) of eight different endemic plant species were tested and the results are shown in Tables 1, 2 and 3. S. pyogenes was the most susceptible bacterium against tested extracts (Table 2).Good antibacterial activity was observed for all the tested extracts (water, EtOH and MeOH) of A. brachypterus against S. pyogenes (15, 17 and 16.3 mm, respectively).The aqueous extract of S. castagneanus also exhibited strong antibacterial activity against S. pyogenes (14.3 mm) while alcohol extracts of S. castagneanus and P.
russeliana showed moderate antibacterial activity against S. pyogenes (8.8 and 9 mm).Moderate inhibition was also observed for alcohol extracts of P. russeliana against S. epidermidis (8.5 and 9 mm).All the tested extracts of E. orientale showed little inhibition against K. pneumonia (7.5 -7.8 mm).Although E. cloacae was susceptible to only the ethanol extract of E. orientale (8.5 mm), this bacterium was not susceptible to other extracts (Table 2).All extracts of P. afer, P. armeniaca and A. maxima did not show any inhibitory activity against the pathogens (Table 2).S. aureus, S. marcescens, S. typhimurium, P. aeruginosa, P. vulgaris and E. coli were not sensitive to any of the extracts (data not shown).
Positive controls (reference antibiotics) generally showed antibacterial activity against the test microorganisms (Table 2).Since the final concentrations of all extracts were adjusted with distilled water, it was used as a negative control and the results show that there was no inhibition activity by the control solvent.
Good antitumor activity was observed with all extracts of A. brachypterus (91.7 -100 %) (Table 3).The alcohol extracts of P. russeliana and aqueous extract of P. armeniaca also exhibited strong antitumor activity (61.1 -75 %).Among the tested endemic plants, A. maxima showed least tumor inhibition (13.9 -30.6 %).No tumor formation was observed with positive control, camptothecin (100% inhibition) (Table 3).The final concentrations of all the extracts were adjusted with distilled water which also served as negative control.

DISCUSSION
Kirby-Bauer test (disc diffusion method) is the most widely used standard method for antibacterial bioassay.It is currently performed by National Committee for clinical laboratory standards on disc diffusion susceptibility testing [18].Gram-positive bacteria (S. epidermidis and S. pyogenes) were more susceptible to plant extracts than Gram-negative bacteria (Table 2).Susceptibility of Gram-positive bacteria may come from their cell wall structure consisting of a single layer, but the Gram-negative cell wall is a multi-layered structure and quite complex [2].
E. orientale extracts showed barely inhibitory activity against K. pneumonia and E. cloacae.Eight bacteria were not sensitive to E. orientale extracts in our study.Similarly, Mansouri [21] reported that ethanolic extract of Echium amoenum had no antibacterial activity against S. aureus isolates.Morteza-Semnani et al [22] demonstrated that Echium italicum essential oil exhibited concentration-dependent antimicrobial activity on all microorganisms tested.Kuruuzum-Uz et al [23] isolated kaempferol, uridine, lactic acid and rosmarinic acid from E. vulgare and compounds showed no detectable antimicrobial activity against E. coli, Bacillus subtilis, S. aureus and Candida albicans.E. vulgare contains pyrrolizidine alkaloids, allantoin, alkannins and mucilage.The alkannins are antimicrobial and allantoin help wounds to heal [4].
Strong antibacterial activity of A. brachypterus may explain why Astragalus species is used in folk medicine to treat skin conditions (caused by S. pyogenes) [12].All tested extracts of A. brachypterus showed strong inhibition against only S. pyogenes in our study (Table 2).Similarly, alcoholic extracts of Astragalus gymnolobus exhibited little inhibition against only S. pyogenes [2]. A. gymnolobus extracts were also effective only on Aeromonas hydrophila among tested five different fish pathogens [24].The moderate antibacterial activities of some members of Astragalus species (A.siculus, A. gummifer, A. membranaceus, A. malanophrurius and A. verrucosus) were recorded against Grampositive and Gram-negative bacteria [10,25]
All extracts of S. castagneanus showed strong antibacterial activity against only S. pyogenes in our study (Table 2).On the other hand, Albayrak et al [26] [13].
The inhibition of A. tumefaciens-induced tumors in potato disc tissue is an assay based on antimitotic activity and can detect a broad range of known and novel antitumor effects [19,20].The validity of this bioassay is predicated on the observation that certain tumorigenic mechanisms are similar in plants and animals.It was demonstrated that inhibition of crown gall tumor initiation on potato disc showed an apparent correlation with compounds and plant extracts known to be active in the 3PS (in vivo, murine leukemia) antitumor assay [20].
A prerequisite for potato disc tumor induction assay is that the extract or substance being tested should not have antibacterial activity toward A. tumefaciens [20].Inhibition of crown gall formation on potato discs is caused by two effects: by anti-tumorogenesis or decreasing the viability of the A. tumefaciens.Viability tests were carried out with all extracts to distinguish between these possibilities.There was no difference in bacterial growth across the plates between control (only A. tumefaciens) and tested extracts (A.tumefaciens + plant extracts) in terms of colony counts (ranged from 9.2 x 10 3 to 13 x 10 3 CFU).All the extracts did not affect the viability of the bacterium.Thus, the observed inhibition of tumor formation for these extracts was on the formation of tumors.Good tumor inhibition was observed with all the test extracts of A. brachypterus in our study.Triterpene saponins ingredient of Astragalus species may contribute to the strong immunomodulatory and anticancer activities

Mean diameter of inhibitory zone (mm ± SEM) S.epidermidis S.pyogenes K. pneumonia E. cloacae
.0 ± 0.9 de 9.5 ± 0.6 i ----M 8.5 ± 0.9 e 9.5 ± 0.  Note: W = aqueous extract; E = ethanol extract M = methanol [12].Similarly, strong antitumor activity of Astragalus membranaceous was recorded with some studies [27][28][29].Rittenhouse et al [27] reported that A. membranaceous may exert its antitumour activity by abolishing tumorassociated macrophage suppression.Tin et al [28] proposed that members of Astragalus may possess anti-tumorigenic potential in certain cancer cell types.The anti-carcinogenic effects of Astragalus saponin extracts were investigated in HT-29 human colon cancer cells and the results indicated that this extracts could be an effective chemotherapeutic agent in colon cancer treatment.Cho and Leung [29] isolated bioactive fractions from the roots of A. membranaceous.One of the fractions exhibited potent anti-tumor effects both in vitro and in vivo.Cho and Leung [30] also reported the immunomodulating and immunorestorative effects of same fraction.They concluded that A. membranaceous could exhibit both in vitro and in vivo anti-tumor effects, which might be achieved through activating the anti-tumor immune mechanism of the host [29,30].On the other hand, strong antitumor activity was not observed with Astragalus gymnolobus [2].P. armeniaca showed moderate antitumor activity in our study and Saracoglu et al [16] reported that phenyl propanoid caffeic acid, phenylethyl alcohol and phenylethylalcohol glycosides isolated from P. armeniaca were found to show cytotoxic activity against several kinds of cancer cells.Alcohol extracts of P. russeliana exhibited stronger antitumor activity than alcoholic extracts of P. armeniaca.Conversely, antitumor activity of aqueous extract of P. armeniaca was better than aqueous extract of P. russeliana in our study.

CONCLUSION
The antibacterial and antitumor activities of 24 different extracts obtained from eight different endemic plants grown in Turkey were evaluated.Results obtained herein revealed the strong antibacterial and antitumor activities of A. brachypterus.Future studies should focus on fractionation of the extracts of A. brachypterus in the hope of identifying active components.Furthermore, antiproliferative activity studies of A. brachypterus should be carried on using different types of cancer lines in the future.

Table 1 :
Family, botanical names, collection numbers, yields (%) and extraction solvents of tested endemic plants.Means with the same letter within columns are not significantly different at p>0.05.

Table 2 :
Antibacterial activities of used plant extracts.Means with the same letter within columns are not significantly different at p > 0.05.

Table 3 :
Mean number of tumors observed with used plant extracts.Means with the same letter within columns are not significantly different at p > 0.05.