İpolamiidin Lactobacillus acidophilus and Lactobacillus rhamnosus Bakterilerinin Probiyotik Özellikleri Üzerine Etkileri

Iridoid glikozitler, dogal olarak yapraklarda, meyvelerde, tohumlarda, agac kabugunda, bitki koklerinde bulunan terpenin 2-siklopentanoid turevleridir. Tibbi olarak onemlidir, cunku normalde bitkileri biyotik ve abiyotik ataklardan korurken bircok hastaligin tedavisinde kullanilir. Anti-mikrobiyal, anti-tumor, anti-kardiyak ve anti-enflamatuar etkilere sahiptirler. Ipolamiid, iridoid glikozitlerden biridir ve bircok bitkide dogal olarak bulunur. Ipolamiid ​​biyolojik aktiviteleri acisindan cok az bilinen bir bilesiktir. Vucuttaki faydali mikroorganizmalar ve bitki kokenli bilesikler, insan gastrointestinal kanalinda etkilesime girebilir. Probiyotikler, bagirsaklarin mikrobiyal dengesini gelistirerek bircok saglik yararina sahip olan canli mikroorganizmalardir. En bilinen ve en cok calisilan probiyotikler arasinda Lactobacillus acidophilus ve Lactobacillus rhamnosus bulunur. Bu calismanin amaci, ipolamiidin probiyotik bakteriler Lactobacillus rhamnosus GG (GG) ve Lactobacillus acidophilus LA-5 (LA-5) uzerindeki etkilerini arastirmaktir. Bu amacla probiyotiklerin buyumesine farkli konsantrasyonlarda ipolamiid eklenmis ve bakteriyel buyume kinetigi, bakteriyel yuzey hidrofobisitesi (Solventlere Mikrobiyel Yapisma - MATS Testi) ve bakteriyel agregasyona (Oto-Agregasyon Testi) etkileri incelenmistir. Sonuclar, ipolamidin probiyotik bakterilerin yuzey hidrofobisitesinde onemli bir degisiklige sebep olmadigini gostermistir. LA-5 ve GG'nin oto-agregasyon ozelliklerinde doza bagli artislar gozlenmistir. Bununla birlikte, ipolamiidin diger olasi biyolojik aktiviteleri hakkinda bilgi edinmek icin daha ayrintili calismalara ihtiyac vardir.


Introduction
Plantago species are represented by about 275 species that grown all over the world. The most common use of Plantago species as herbal remedy is in the treatment of skin disorders and wound healing. In addition, several studies were reported previously analgesic (Das et al., 1984), anti-inflammatory (Barua et al., 2011), antiviral (Chiang et al., 2002), antioxidant  and anticancer (Moon & Zee, 1999) activity of various member of Plantago species. According to the literature; Plantago species include many secondary metabolites such as phenyl ethanoids (Nishibe et al., 1993;Murai et al., 1995), flavonoids (Jurišić Grubešić et al., 2013), iridoids (Handjieva et al., 1991;Jensen et al., 1996;Darrow & Bowers, 1997;Fuchs & Bowers, 2004), terpenoids (Venditti et al., 2015) and steroids (Zacchigna et al., 2009;Najib et al., 2012). Plantago euphratica is one of the endemic species of Turkey. To the best of our knowledge, no previous studies have been reported the biological activity and phytochemical content of Plantago euphratica. Probiotics are defined as living microorganisms that have a beneficial effects on the host by improving the microbial balance of the intestinal system (Hill et al., 2014). Among these, Bifidobacterium and Lactobacillus species are widely used. Lactic acid bacteria are among the group of microorganisms that possess effects on human health and constitute the most important group of probiotic microorganisms (Uymaz, 2010). Foods containing probiotic microorganisms constitute an important part of the food market. Increasing consumer awareness and better understanding of the importance of diet for a healthy life result in increasing demand for probiotic foods (Kleerebezem & Vaughan, 2009). Lactobacillus plantarum, L. rhamnosus, L. paracasei, L. acidophilus and L. salivarius are commonly found in the mucosa from the mouth to the rectum (Alp & Ertürkmen, 2017). In order to use a microorganism in food as probiotic; it should be preferably of human origin, able to survive in the gastrointestinal tract, not be pathogen, not have antibiotic resistance, produce antimicrobial compounds, stimulate the immune system, be resistant to stomach acid and bile salt (Collins & Gibson, 1999;Dunne et al., 2001;Erem, Küçükçetin, & Certel, 2013;Kechagia et al., 2013;Uymaz, 2010). Probiotic microorganisms compete with pathogenic microorganisms for adherence to the intestinal wall and the consumption of nutrients contained in the lumen, produce substances with antimicrobial properties and regulate the immune functions associated with the intestinal mucosa to demonstrate these beneficial effects (Alp & Ertürkmen, 2017).
The beneficial microorganisms in the body and the compounds of plant origin interact in the human digestive tract. The aim of this study was to investigate the in vitro interaction of ipolamiide and probiotic. For this purpose, L. acidophilus LA-5 and L. rhamnosus GG bacteria were grown in the presence of different concentrations of ipolamiide and the effects of ipolamiide on bacterial growth kinetics, surface hydrophobicity and autoaggregation were investigated.

Plant Collection and Ipolamiide Isolation
Plantago euphratica was collected from Erzincan (Kemah-İliç Road 32 th km, jipsy rock) at June 2018. The plant sample was dried under room conditions without direct exposure sunlight. 100 g of well-grounded aerial parts of Plantago euphratica were extracted with 2X600 mL of methanol for 12 h. After removing solvent, a dark green slurry extract (9.8 g) was obtained. In order to remove chlorophylls, extract was dissolved in hot water then cooled to rt., non-soluble parts were filtrated, and the filtrate lyophilized overnight to give 4 g of pale-yellow solid. 1 g of extract dissolved in 20 mL of water and repeatedly injected to semi prep-HPLC ten times with 2 mL sample loop using a gradient elution from 90:10 to 50:50 (water: ACN) with 8 mL/min flow rate. The main peaks were collected according to the absorbance at 235 nm. The collected fractions between 16-18 minutes were purified using recycling mode with isocratic elution with 85:15 (Water: ACN), with 8 mL/min flow rate. After 10 th cycle, a clear separation was observed then peaks were collected. The solvents were evaporated to give ipolamiide (128 mg) with high purity.

Growth of probiotic bacteria in the presence of ipolamiide and bacterial growth kinetics
Lactobacillus acidophilus LA-5 and Lactobacillus rhamnosus GG, which are kind gifts of Chr. Hansen, Turkey, were grown in Man, Rogosa and Sharpe (MRS) medium without shaking, at 37ºC (Celebioglu, Delsoglio, Brix, Pessione, & Svensson, 2018). The bacteria were divided into groups and treated with ipolamiide. Ipolamiide was not added to the control group (MRS only), and 5 µg/mL, 10 µg/mL, and 20 µg/mL ipolamiide was added to the treated groups in MRS medium. Bacterial optical density was determined by densitometry. Reading the densitometry every four hour, the effects of ipolamiide on bacterial growth were investigated.

Microbial Adhesion to Solvents (MATS)
Bacterial surface hydrophobicity was measured by the method of microbial adhesion to solvents (MATS) (Kos et al., 2003). The bacteria (control and treated groups) were harvested in the stationary phase (3200xg, 15 min), washed with PBS (Phosphate-saline buffer), and suspended in 0.1 M KNO3 (pH 6.2) to have OD600 of 0.5. One mL of xylene (nonpolar solvent) was added to 3 mL of bacterial suspension and incubated at RT for 10 min. The two-phase system was vortexed for 2 min, the aqueous phase was separated and incubated for another 20 min at RT. Absorbance was measured at 600 nm and the bacterial adhesion to the solvent was calculated using the formula where, A1 is the absorbance measured after the incubation and A0 is the absorbance measured before the incubation (Kos et al., 2003).

Probiotic Auto-Aggregation
Bacterial cells were collected in the stationary phase (3200xg, 15 min), washed with PBS and re-suspended in PBS to OD600 0.5 (Kos et al., 2003). Auto-aggregation was determined by adding 4 mL of bacterial suspensions to the test tubes after vortex for 10 sec. for one hour-incubation at room temperature. After incubation, 100 µL of suspension was taken, added to the tube containing 900 µL of PBS, and the absorbance was measured at 600 nm. The percentage of auto-aggregation was calculated with the formula where At is the absorbance measured after incubation and A0 is the absorbance measured at 0 th hour (Kos et al., 2003).

Statistical Analysis
Every experiments were performed with at least three replicates and the results were expressed as mean ± Standard deviation and compared using Students's t-test. p<0.05 was considered as statistically significant. The NMR spectra correspond to the ipolamiide. Thus, this confirms that the isolation of ipolamiide was successful.

Bacterial Growth Kinetics
In this study, 0, 5, 10, and 20 µg/mL concentrations of ipolamiide were used. Ipolamiide showed a statistically significant decrease in Lactobacillus acidophilus LA-5 at 5 and 10 µg/ml, whereas 20 µg/ml was not observed. When Lactobacillus rhamnosus GG was examined, no significant change was observed (Fig. 2). This means ipolamiide did not Show very effective against the probiotic bacteria, and even higher concentrations could have positive effects on the growth. It is important that such compounds have no effect on the growth of beneficial microorganisms, while their anti-bacterial activities against pathogenic bacteria are present. Thus, this compound can selectively affect on beneficial bacteria.

Bacterial Surface Hydrophobicity
Bacterial surface hydrophobicity plays an important role in the adhesion of bacteria to the mucosa in the intestines (Liu et al., 2004). Therefore, the more surface hydrophobicity increases, the more likely the beneficial bacteria will bind to the mucosa in the gastraointestinal tract. Only at 10 µg/mL concentration of ipolamiide applied on probiotic bacteria, the surface hydrophobicity of Lactobacillus rhamnosus GG bacteria was significantly (p <0.05) decreased, while no other concentrations showed any alteration on either Lactobacillus acidophilus LA-5 or Lactobacillus rhamnosus GG surface hydrophobicity (Fig. 3). This means that no changes observed on the hydrophobicity could not alter the adhesion of the probiotic bacteria to the host components, such as mucus layer and epithelial cells.

Bacterial Auto-Aggregation
Bacterial aggregation is also of great importance for attachment of probiotics to the intestinal mucosa (Etzold et al., 2014). High aggregation is an indication of better adhesion. When the auto-aggregation of the bacteria was examined (Fig. 4), significant increases (10 and 20 µg/mL) were observed in Lactobacillus acidophilus LA-5, as well as in the auto-aggregation of Lactobacillus rhamnosus GG bacteria (p <0.05). however, a significant decrease was observed in the auto-aggregation of Lactobacillus rhamnosus GG when the concentration of 5 µg/mL was used.

Conclusions and Recommendations
Probiotics and plant-derived compounds are great canditates for the functional foods, defined as food or food ingredients that have posivitive health benefits on human. Thus, interplay between probiotics and plant compounds should be studied in terms of more bioactive compounds could be produced by the metabolism of the beneficial microorganisms. Furthermore, such compounds could affect the probiotic activities of the microorganisms. Thus, the present study was conducted as preliminary screening of this interaction between ipolamiide and very well known probiotic bacteria Lactobacillus acidophilus and Lactobacillus rhamnosus.
Further studies are required in order to investigate how these bacteria can metabolize the ipolamiide and when it is metabolized, whether its biological activity is increased.

Acknowledge
Chr. Hansen, Turkey is acknowledged for their supplying the probiotic strains.