Comparison of Biological and Antioxidant Activities of Above and Below-Ground Extracts of Endemic Heliotropium samolifolium subsp. erzurumicum

In this study, antioxidant, antimicrobial activities and effects on DNA damage of above and below-ground extracts of Heliotropium samolifolium subsp. erzurumicum were determined. This subspecies distributes only in the vicinity of Olur (Erzurum) in Turkey and is known as Erzurum Bambulu by the people. Heliotropium L. species include secondary metabolities such as; pyrrolizidine alkaloids, terpenoids, saponins, phenols, flavonoids, tannis, and steroids. The above and below-ground extracts of the subspecies were prepared using different organic solvents. For the antioxidant studies, DPPH and total phenolic content calculation methods were applied. The antimicrobial activity tests of the extracts were performed using four different standard strains, a yeast and MIC (Minimum Inhibition Concentration) method. The effects on DNA damage of plant extracts were explained using pBR322 plasmid DNA. The below-ground ethanol extract of the subspecies was seen to have stronger antimicrobial activity. According to antioxidant data, the highest activity was found in above-ground ethanol+aqueous, chloroform and below-ground ethanol extracts. Also, the below-ground aqueous and chloroform extracts had a greater effect on the open ring form of pBR322 plasmid DNA. It was determined that the below-ground extracts of the subspecies were more effective than the above ground extracts. It was suggested that the extracts obtained from this subspecies may be used in medicine industry and folk medicine. Research Article


INTRODUCTION
Turkey has a rich flora in terms of biodiversity in Europe. Medicinal and endemic plants in this rich flora constitute an important place. A large number of medicinal plants in Turkey have widely been used in many fields such as; tea, spices, dyes, ornaments, smell, taste industry, perfumes, cleaning, food and cosmetics by the people for many years (Başer, 2000;Toroğlu and Çenet, 2006). Nevertheless, medicinal plants have different effects on bacteria, fungi and viruses depending on the chemical structure and concentration of contain compounds. Thus, these plants have been a source of hope for many years in the treatment of many diseases caused by microorganisms.
In many studies, it has been reported that Heliotropium L. species contain various secondary metabolites such as saponins, tannins, steroids, terponoids, flavanoids, phenols and pyrrolizidine alkaloids (Singh et al., 2002;Goyal and Sharma, 2014;Santhosha et al., 2015;Roy, 2015). In particular, the pyrrolizidine alkoloids are one of the major secondary metabolites of this genus and have been identified more than 200 alkaloids. These alkaloids are extremely toxic and demonstrate anticancer activity and cytotoxic effects (Sharma et al., 2009;Singh and Sharma, 2019). Since secondary metabolites isolated from Heliotropium species have antimicrobial, antitumor, antiviral, anti-inflammatory, wound healing, cytotoxic and phytotoxic effects, these species have been used extensively in folk medicine, inflammation, gout, rheumatism, skin diseases (wart and rash), menstrual disorders, eye diseases, ulcer, febrile diseases, burns and poisonous animal bites for a long time (Singh et al., 2002;Reddy et al., 2002;Shoge et al., 2011;Ghaffari et al., 2013;Dash and Abdullah, 2013;Mourin et al., 2013;Yasmin, 2014;Ahmad et al., 2015;Roy, 2015). In particularly, the methanol and ethanol extracts of H. indicum have an important effect on the healing of wounds caused by S. aureus and P. aeruginosa (Yasmin, 2014).
Heliotropium is one of the important xerophytic and major genera of the Boraginaceae family. The vegetative diversity of the genus is seen in the widely different habitats and environments, and the species of genus spread out in tropical, subtropical, arid, semiarid regions, on dry soils, gypsum hills, eroded slopes and warm-temperate areas of world (Diane et al., 2002). The genus includes perennial and annual herbs, subshrubs or rarely shrubs (Riedl, 1978). Annual and perennial species are generally distributed in the mountains and deserts, very dry habitats, respectively. This genus is represented by 17 species in Turkey and more than 300 species in the world (Diane et al., 2002;Akhani, 2007;Luebert et al., 2011;Güner et al., 2012 (Güner et al., 2012). The Heliotropium genus is easily distinguished from allied genera of the family by its scorpioid cymes and highly modified stigma heads that are very different from the rest of the taxa of this family (Kandemir et al., 2020). H. samolifolium subsp. erzurumicum is herbaceous, annual, 10-50 cm high, dense villous hairy, inflorescence with 10-50 sessile flowered. The subspecies is known as "Erzurum Bambulu" by people and is generally distributed at altitudes of 900-930 m and in metamorphic rocky (Dönmez, 2008). Because of distribution only around Olur (Erzurum), it is among the endemic plants have a limited distribution in Turkey.
The aim of this study was to determine and compare the antioxidant, antimicrobial activities and effects on plasmid DNA of above and below-ground extracts of the subspecies. In addition, the antioxidant and antimicrobial aspects of this plant is to provide its usefulness in different fields consciously.

Collection and Identification of Plant Samples
Plant samples were collected from flowering periods (July and August) from metamorphic rocks around Buzluca Village between Olur and Yusufeli, which is the natural distribution area. The taxonomic description of the subspecies was made according to Dönmez (2008). The above and below-ground parts of the fresh plant samples were divided into small pieces and dried in the shade on the benches in the laboratory. Then, these dried plant samples were milled using a mill and used for biological activity studies.

Preparation of Plant Extracts
The above and below-ground parts of the plant were extracted with Soxhlet apparatus in the presence of different organic solvents ( Stock solutions of the used extracts were prepared at a concentration of 40 mg ml-1. Extracts were dissolved in DMSO. In broth dilution method, cultures were released to grow in 5 ml nutrient broth at 37°C for 18 h in 175 rpm shaker incubators.1 ml nutrient broth containing microorganisms was added to the test tubes. The compounds were added to the appropriate concentrations and half-serial dilution was performed. Tubes with serial dilution were allowed to incubate at 37°C in the incubator for 24 h. The last tube without bacterial growth was determined as MIC value. MIC values obtained in the study were shown as µg ml-1 (Table 1).

DPPH Free Radical Scavenging Activity
The antioxidant activities of the above and belowground extracts were tested by DPPH free radical scavenging activity. Butillated hydroxi anisole (BHA) was used as standard antioxidant. For this process, 50 µl of different concentrations (3-10 mg ml-1) of plant extracts were incubated with 2850 µl of DPPH solution (6x10 -5 M) in the dark and at room temperature for 30 minutes. At the end of this process, the absorbance was measured at 517 nm against the blank sample (Brand-Williams et al.,1995). DPPH % was calculated according to formula (I). The results were expressed as IC50 value (Table 2). IC50 value demonstrates to the concentration of plant at the moment when half of the DPPH amount is scavenging.
Inhibition %= (ADPPH-Asample)/ADPPH X 100 Respectively, ADPPH refers to· the DPPH radical in the absence of plant extract and Asample refers to the DPPH radical in the presence of plant extract absorbance (at 517 nm).

Total Phenolic Content Activity
The amount of phenolic contents of the above and below-ground extracts were determined according to the method reported by Singleton and Rossi (1965). Gallic acid was used as the standard phenolic compound. Stock solution was prepared by dissolving 1 mg gallic acid in 1 ml organic solvent (methanol). 10, 25, 50, 75 and 100 μl from stock solution were received and transferred to test tubes. The final volume was completed to 2400 μl with pure methanol. After, 50 µl of Folin-Ciocalteu reagent was added. Then, 150 µl from 2% (w/v) Na2CO3 solution was added to test tubes and incubated for two hours at room temperature. The absorbance of the samples was read at 760 nm against the blank, which did not contain a test sample. The results were determined as µg(GAE)ml-1(extract) ( Table 3).

DNA Interaction
The effects on plasmid DNA of the above and belowground extracts of plant were determined by agarose gel electrophoresis method (Babu et al., 2007). Initially, 1% agarose gel was prepared in TBE (1X) buffer. 120 µg ml-1 plant extracts were interacted with 0.5 µg ml-1 pBR322 plasmid DNA at 37 0 C for 2 h. After incubation, samples were mixed with 6X loading dye and loaded on 1% agarose gel. The electrophoresis was carried out at 100 v for 80 min. Then, gel was stained with EtBr (Ethidium Bromide) and the bands were imaged. Photographs were taken under UV light. The results were expressed and interpreted as the percentage of fragmentation of the DNA forms.

RESULTS and DISCUSSION
Plants have the ability to produce a large number of secondary metabolites. Most of these metabolites are necessary for defense systems in plants. Terpenes, quinone and tannins play an important role in odor and pigment formation and are used in antimicrobial research (Cowan, 1999;Silva and Fernandes, 2010). Cowan (1999) collected in 5 groups antimicrobial phytochemicals. Karou et al. (2007) reported that phenols constitute the largest group of herbal antimicrobial agents. In addition, antimicrobial activity of polyphenols and tannins in the plants were known for many years (Taguri et al., 2004).
The above-ground hexane extract of this subspecies did not demonstrate any antimicrobial activity on S. aureus, M. luteus, P. aeruginosa and C. albicans, while the above-ground hexane extract had moderate antimicrobial activity only on E. coli. The above- The chloroform extract was found to show greater activity against E. coli, whereas petroleum ether extract was found to have better effect against P. chrysogenum (Singh et al., 2002). Some sterol and triterpenoids isolated from H. ellipticum Ledeb. were applied to bacteria and fungi and some of them showed to have the highest level of antimicrobial activity (Jain et al., 2001).
The hexane extract of H. marifolium Koen. ex Retz. were tested on the pathogenic bacteria (E. coli, S. aureus) and fungi (A. niger ve P. chrysogenum). The antimicrobial results were showed to possess high activity against S. aureus P. chrysogenum E. coli and A. niger (Singh and Dubey, 2001 erzurumicum were seen to have moderate antimicrobial activity on selected yeast (C. albicans).
The below-ground ethyl acetate and ethanol extracts of subsp. erzurumicum exhibited antimicrobial activity on selected Gram-positive bacterium (S. aureus). Also, the below-ground ethanol extract of investigated subspecies possess strong antimicrobial activity on the selected Gram positive and negative bacteria, namely M. luteus and P. aeruginosa. In summary, in this study antimicrobial data are in harmony with Radha et al. (2003) and Sing and Dubey (2001) (Yasmin, 2014). In this study, the strongest and moderate antimicrobial activity on both Gram negative and positive bacteria were found in below-ground ethanol extracts. Yasmin (2014) and Rao et al. (2006) antimicrobial data almost support in this study antimicrobial data. This is thought to be due to the use of similar organic solvents. Jain and Sharma (1987) (Ahmad et al., 2009). In this study, the above-ground chloroform, ethyl acetate and ethanol extracts and all below-ground extracts of this subspecies possesses moderate antifungal avtivity on C. albicans. The reason why Ghaffari et al. (2015), Ahmad et al. (2009) and in this study results are different is due to the different Boraginaceae taxa, the use of different types of organic solvents and fungi in the studies.
In another research, the methanol, n-hexane and ethylacetate extracts of H. bacciferum Forssk. were found to have an excellent antimicrobial activity on E. coli, S. typhi, S. aureus, P. aeroginosa, E. carotovora, K. pneumoniae, B. atrophaeus and B. subtilis (Ahmad et al., 2015). However, n-butanol and aqueous extracts showed inactive activity on S. aureus and B. subtilis, respectively. The above-mentioned extracts of H. bacciferum were applied to C. albicans, Fusarium solani, A. niger, A. flavus, Trichoderma longibrachiantum and significant antifungal results were obtained. According to the above findings, Ahmad et al. (2015) reported that H. bacciferum would be important in the treatment of various diseases. In this study antimicrobial findings are close to those of Ahmad et al. (2015). This may result from the application of similar organic solvents because similar organic solvents reveal similar secondary metabolites in plants.
When DPPH results of above and below-ground extracts of subsp. erzurumicum examined, all above and below-ground extracts were seen to have high antioxidant activity (Figures 1 and 2, Table 2). Especially above ground ethanol+aqueous, chloroform and below-ground ethanol extracts demonstrated the highest antioxidant activity compared to other plant extracts. For this, above ground ethanol+aqueous, chloroform and below-ground ethanol extracts of subsp. erzurumicum may be preferred as natural antioxidant sources in the future.   (Modak et al., 2005(Modak et al., , 2009Goyal and Sharma, 2014). The dichloromethane and methanol, chloroform extracts of H. glutinosum Phil., H. taltalense Phil. and H. zeylanicum (Burm.) Lam. were seen to have antioxidant activity, respectively (Modak et al., 2007(Modak et al., , 2009Goyal and Sharma, 2014).  (Hussain et al., 2010). Also, the dichloromethane extracts of H. subulatum displayed significant antioxidant activity (Singh et al., 2017). Moreover, the flower, leaf, stem and root extracts of H. bacciferum exhibited noteworthy antioxidant activity (Al-Snafi, 2018). The above-mentioned antioxidant findings are consistent with in this study antioxidant findings. According to total phenolic content results, phenolic contents of both below and above-ground chloroform and below-ground ethanol+aqueous extracts of subsp. erzurumicum were the highest (Table 3). We think that the above and below-ground chloroform, below-ground ethanol+aqueous extracts of this plant may be used as antioxidant source.
The DNA interaction results are presented in Figures 3-5. 1 and 2 lanes in Figures 3-5 belong to pBR322 DNA+H2O and pBR322 DNA+DMSO control groups, respectively. According to data in Figure 3, the belowground aqueous, (Lane 3), ethyl acetate (Lane 4), ethanol (Lane 5) and above-ground aqueous (Lane 6) extracts have enhancing effect in the concentration of open ring form of pBR322 plasmid DNA. However, it was determined that below-ground aqueous extract was more effective than others in the formation of open ring form of pBR322 plasmid DNA. The above-ground ethanol (Lane 3), ethyl acetate (Lane 4), ethanol+aqueous (Lane 5) and below-ground ethanol+aqueous (Lane 6) and hexane (Lane7) extracts have no effect on pBR322 plasmid DNA (Figure 4). When the results of Figure 5 examined, the extracts of below and above-ground chloroform (Lanes 3 and 5) and above-ground hexane (Lane 6) have increasing effect in the concentration of open ring form of pBR322 plasmid DNA.  Kandemir, 2020). In a similar study with Leucojum aestivum L., below-ground ethanol extracts of L. aestivum displayed to have highly effect on pBR322 plasmid DNA (Hundur et al., 2018). However, the above and below-ground ethanol, ethyl acetate and dichloromethane extracts of Linaria corifolia Desf., were determined to have protective activity on pBR322 plasmid DNA (Gul et al., 2017).

CONCLUSION
Consequently, the below-ground extracts of this subspecies had more effective antioxidant and biological activity than the above-ground extracts. Therefore, it shows that extracts made with different solvents of this subspecies can be used as a source in the pharmaceutical industry and traditional medicine. Due to the above-mentioned features, we believe that the studied subspecies can take place in Turkey's medicinal plants. H. samolifolium subsp. erzurumicum is one of the rare endemic taxa for Flora of Turkey. Both the protection of this taxon and required sensitivity must be given the necessary importance to use it in the most efficient way.   Şekil 5. pBR322 plazmit DNA'nın etkileşimine dayalı agaroz jel elektroforez diyagramı Lane 1:pBR322 DNA+H2O control; Lane 2:pBR322 DNA+DMSO control; Lane 3:pBR322 DNA+BG chloroform extract; Lane 4: pBR322 DNA+BG hexane extract; Lane 5: pBR322 DNA+AG chloroform extract; Lane 6: pBR322 DNA+AG hexane extract.

ACKNOWLEDGMENTS
This study was produced within the scope of the project of FMB-BAP 17-0286 supported by Amasya University. This study is derived from master thesis Galip SAĞLAM's.

Statement of Conflict of Interest
Author has declared no conflict of interest.

Author's Contributions
The contribution of the authors is equal.