MATERIALS AND METHODS

Iris sari extraction: The following materials were used for the extraction; dichloromethane (CH₂Cl₂) (Sigma Aldrich, Germany), methanol (CH₃OH) (Sigma Aldrich, Germany), hexane (C₆H₁₄) (Sigma Aldrich, Germany), distilled water (dH₂O), Soxhlet (Gerhardt EV 14) and evaporator (Heildolph Heizbad HB Digit). The dry root, flower and leaf parts of the plant were pulverized with a smasher. By Soxhlet method, dichloromethane, methanol, hexane and water were extracted from each part (root, flower, leaf) of I. sari. Dichloromethane, methanol and hexane were evaporated by rotary evaporator. The extractions were stored at +4°C until the experiment started.

Determination of antioxidant activity: The free radical removal capacity of the extracts was determined by using the free radicals of the 1,1-diphenyl-2-picrylhydrazyl (DPPH)¹⁵. The DPPH solution which was added to the extracts caused a decrease in the absorbance value of the optical density and the lightening of the colors of extracts showed that the extracts were radical scavengers.

Determination of total antioxidant activity with ABTS method: Commercially sold kits were used in order to determine the antioxidant capacity of the extracts obtained from the root, flower and leaf of the I. sari. The antioxidant agents of the sample turn the dark blue-green colored ABTS into a radical reduced ABTS and the change in the absorbance at the 600 nm determines the antioxidant level of the sample. The ABTS [2,2 azino bis (3-ethyl benzo thiazoline-6-sulfonic acid] was used as radical while synthetic antioxidant and Trolox which is the analogue of vitamin E were used as positive control¹⁶. Real Assay Diagnostics-TAA Assay Kit was used in the study.

Determination of total oxidant level and the antioxidant activity: This test is calibrated with hydrogen peroxide. In the presence of oxygen, the ferrous ion oxidizes the chelate complex into ferric ion. The density of the oxidation reaction increases depending on the presence of the oxygen within the environment of the reaction. The ferric ion forms a colored complex in acidic environment. The color intensity is measured spectrophotometrically. The absorbance value indicates the oxidant value in the example. The total oxidant level value was detected by comparing the obtained results with H₂O₂ Equiv (μmol L^–1)¹⁷. Vitamin C was used as a control.

Antibacterial activity of I. sari: For the antibacterial activity of root, flower and leaf extracts of I. sari, the following 9 different standard bacterial strains were used: Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 700603, Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25322, Escherichia coli ATCC 35218, Escherichia coli ATCC 10799, Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6538.

In order to determine the MIC values of the I. sari extracts, the CLS was performed for 3 times repeatedly with the microdilution method according to the guidelines¹⁸. The growth of the bacteria was observed in the presence of a white pellet or dimness.

DNA protective activity of I. sari: The DNA of the pBR322 plasmid was used to detect the effects of the extracts to protect the DNA from the UV and oxidative induced damages. The DNA of the plasmid was subjected to damage in the presence of the extracts by applying H₂O₂ and UV. The visualization was performed on 1.25% agarose gel. For the DNA protective activity test 0.1 mg was weighed from the extracts of the I. sari root, flower and leaf to prepare 0.1% stock concentration and it was dissolved in 100 μL DMSO (dimethyl sulfoxide, 10%). After ensuring the full dissolution of the extracts, the 0.1% I. sari root, flower and leaf extracts’ solutions were diluted in 1/5 ratio. Approximately 40 μL dH₂O was added on 10 μL extract and this ratio was used in the study¹⁹.

RESULTS AND DISCUSSION

Iris sari Schott ex Baker is an endemic plant and studies have not been revealed yet regarding its bioactive features. Therefore, in this study the biological activities of the I. sari root, flower and leaf parts were determined in dichloromethane, methanol, water and hexane solvents. The free radical remover effects of the plant extracts used in this study were tested on DPPH which is stable radical. In four extracts derived from the I. sari, the removing success of the DPPH from the environment has increased with the increase in the concentration in direct proportion. The Oxidative Stress Index (OSI) values of these four different extracts were determined by measuring the Total Antioxidant Levels (TAL) and Total Oxidant Levels (TOL). In Table 1, the low levels of the OSI values showed that the oxidant activity was rather low and it was detected that the dichloromethane, methanol and water extracts were good antioxidants but only the hexane extract did not demonstrate oxidant activity. Rigano et al.¹⁴determined that the isoflavones isolated from the rhizomes of I. pseudopumila showed high antioxidant activity. So, these results were correlated with the other Iris genus result. Table 2 and 3 show the reference values of total antioxidant and oxidant levels.

In this study, the inhibition effects of 4 different extracts obtained from the I. sari were investigated against 9 different bacteria by using the microdilution-broth method (Table 4).

Table 1:	Antioxidant, oxidant, OSI and IC50 values of I. sari
TAL: Total antioxidant level, TOL: Total oxidant level, OST: Oxidative stress index

The extracts obtained from the plant used in the study are thought to have wide-spectrum antibacterial effects against the used bacteria. Further pharmacologic researches are needed in order to use these extracts having antimicrobial activity as medicaments for the treatment of the diseases caused by pathogen microorganisms. The antibacterial results of this study have a preliminary nature for the identification of the extracts determinant compounds and for using these compounds as drug or medicine.

The DNA of the pBR322 plasmid demonstrated two bands in the agarose gel electrophoresis. These are the fast walking supercoiled DNA (scDNA; super stranded DNA; without fracture) which are the natural form of the plasmid and the slow walking open circular DNA (ocDNA; DNA containing a single fracture in the chain).

Table 2:	Total antioxidant level reference values
TAL: Total antioxidant levels

Table 3:	Total oxidant references values
TOL: Total oxidant levels

The exposure of the DNA to UV radiation in the presence of H₂O₂ caused fracture in the open-circular DNA and the emergence of the linear DNA (lin DNA; include one or more fractures in the two chains)²⁰. The results of the DNA protective activity showed linear DNA bands occurred in none of the extracts derived from the I. sari root, flower and leaf as shown in Fig. 2. In other words the water and methanol extracts were found to be protective against the damaging effects of UV and H₂O₂ to the DNA.

It is reported that phenyl propenyls, flavonoids, dihydroflavonols and glycozides are very common in Iris species. The roses of Iris species also abundant with anthocyanins. A glican flavonoids, xanthan iso-flavonoids and phenolics were also isolated from roots, flavor and rhizomes of Iris species²¹. Thus supported the present results that I. sari is one of the good anti microbial agent.

Moreover, anti bacterial, antifungal, insecticidal and phytotoxic activities of plant’s sap extracted from I. germanica were reported by Orhan et al.⁷.

The extracts of rhizomes of the Iris species have been reproted as antineoplastic, antioxidant, antitumoral, antiplasmodical, antituberculative, antispazmodic and diuretic effects of Iris sap by Kandemir¹².

On the other hand, a parralel work with present study was carried out by Rigano et al.¹⁴. In that study, iso-flavonoids isolated fromthe rhizomes of I. pseudopumila had a high antioxidant activity.

Table 4:	Antibacterial effects of I. sari
A: K. pneumoniae ATCC 700603, B: E. coli ATCC 35218, C: E. coli ATCC 10799, D: E. coli ATCC 8739, E: E. coli ATCC 25322, F: S. aureus ATCC 29213, G: S. aureus ATCC 6538, H: P. aeruginosa ATCC 27853 and I: S. aureus ATCC 25923

Fig. 2:	DNA protective activities of I. sari
	K1: Plasmid DNA+dH2O, K2: Plasmid DNA+dH2O+UV, K3: Plasmid DNA+dH2O+H2O2, K4: Plasmid DNA+dH2O+UV+H2O2, L1: Plasmid DNA+Flower water extract+UV+H2O2, L2: Plasmid DNA+Flower methanol extract+UV+H2O2, L3: Plasmid DNA+Flower dicloromethane extract+UV+H2O2, L4: Plasmid DNA+Flower hexane extract+UV+H2O2, M1: Plasmid DNA+Root water extract+UV+H2O2, M2: Plasmid DNA+Root methanol etract+UV+H2O2, M3: Plasmid DNA+Root dicloromethane extract+UV+H2O2, M4: Plasmid DNA+Root hexane extract+UV+H2O2, N1: Plasmid DNA+Leaf water extract+UV+H2O2, N2: Plasmid DNA+Leaf methanol extract+UV+H2O2, N3: Plasmid DNA+Leaf dicloromethane extract+UV+H2O2, N4: Plasmid DNA+Leaf hexane extract+UV+H2O2

It has been reported that there are alot of bioactive substans and the potentially important compounds in the sap of many species in Iris genus²¹.

Kassak²¹ worked on the phenolic and flavonoidal content of five Iris species (Iris pseudacorus, I. crocea, I. spuria, I. orientalis and I. ensata). Fresh dryed rrhizomes were exctracted and the maximum total flavonoids and maximal flavonoids were determined in Iris pseudacoru.

While there are many studies related especially to I. germenica from the Iridaceae family. Studies have been also conducted on the biological activities of the following species; I. nigricans, I. sibirica, I. carhaliniae, I. bungei, I. pseudopumila, I. spuria and I. junonia^20,22. In a study conducted by Uzun et al.²³ the effects of growth factor of the plant tissue cultures were investigated in vitro in the immature embrios of I. sari and I. schachti²⁴. However, any research was not revealed about I. sari in terms of its biological activity.

CONCLUSION

Iris sari was thought to have lot of bioactive compounds due to the fact that the other Iris species contain bioactive compounds and the rhizomes are used in the treatment of various diseases such as tuberculosis and plasmodia diseases among the people. The further investigations are needed for using this plant to some industrial areas such as drug or cosmetic.