The Effect of Rheum ribes Extract Origin of Elazig Province on Ventilator-Associated Pneumonia and Antioxidant Capacity

Ventilator-associated pneumonia (VAP) is one of the most important infections in the intensive care unit (ICU) and contributes to increased mortality and morbidity in patients. In this study, we aimed to evaluate Rheum ribes (Elazig/Turkey) extract on bacterial isolates obtained from VAP patients the antimicrobial effects (agar well diffusion, MIC test) and antioxidant capacity (DPPH, FRAP and metal chelation). As a result of the analysis, the highest antimicrobial effect of R.ribes was observed in Klebsiella pneumonia-2 (K.pneumonia) and K.pneumonia-21 isolates, with zone diameters of 19.32 and 18.45 mm, respectively. Apart from these, Staph. aureus., K. pneumonia-20, K. pneumonia-16 and Pseudomonas aeruginosa (P. aeruginosa) were detected with zone diameters of 18.32, 15.14, 14.56 and 13.54 mm, respectively. R. ribes extract showed 98.3% and 94.88% inhibitory effect at 0.5 ppm in K.pneumonia isolates 16 and 20 while, it showed 100% inhibitory effect on the highest isolates 21 and 2. S. aureus showed a high inhibition effect of 50.36% at 0.25 ppm, and P. aeruginosa isolate at a rate of 82.82% at 0.5 ppm. Besides, DPPH, FRAP and metal chelation analyzes revealed a strong antioxidant effect. DPPH inhibition effect, FRAP analysis and chelating activity values of iron ions (Fe 2+ ) for the antioxidant effects of R.ribes extract were determined as 17.22%, 1.18 and 2.14%, respectively. As a result, strong effect and antioxidant capacity of R. ribes extract on pathogenic bacteria have been determined, and its beneficial properties can be deepened by in vivo and clinical studies


INTRODUCTION (GİRİŞ)
Infectious diseases, from past to present, are one of the most important factors that threaten human health.Lower respiratory tract infections are a common cause of death in developing countries and represent an important source of morbidity worldwide [1].A significant health issue, pneumonia is characterized by substantial morbidity and both short-and long-term mortality.Additionally, it is the major cause of infectious diseases that affect people of all ages globally.It is an acute respiratory infection that impairs breathing by causing edema in the lungs' alveoli, pus accumulation, and fluid accumulation [2][3].For empirical treatment, choosing antibiotics that cover the main bacterial causes of disease is the main focus.Antimicrobial resistance poses a challenge to both these temporary and permanent treatments, indicating that they will be more and more at risk over the next few decades [4].The most prevalent organisms in the globe that cause communityacquired pneumonia are S. pneumonia and S. aureus [2].
In the context of ICU-acquired pneumonia, VAP is characterized as lung parenchymal infection in patients subjected to invasive mechanical ventilation for at least 48 hours.Atelectasis, aspiration, venous thromboembolic event, pulmonary edema, acute respiratory distress syndrome and delirium are other adverse events that might potentially lengthen hospital stays and increase morbidity and mortality [5].VAP is just one of several adverse events.Although microbiological technologies have recently advanced, the epidemiology and diagnostic standards for VAP are still debatable.making treatment, prevention, and outcome studies difficult to interpret [6][7].
The main goal in the treatment of diseases aims to create a medication that has a lower effective dose and greater efficacy.The development of synthetic drugs often results in side effects such as angioedema, gastric irritation, ulceration, headache, hyperglycemia, hemolytic anemia, liver failure, and problems with immunodeficiency as well as others.Therefore, it is increasingly being thought about using natural medicines, which are typically regarded as safe, as an alternative therapeutic modality with little pharmacological response and side effects.There are numerous reports of the use of natural compounds extracted from herbs as successful antibiotics [8].These compounds, such as curcumin, resveratrol, epigallo-catechin-3gallate (EGCG), lycopene, capsaicin, genistein, sulforaphane are among the most researched [9][10].
Rheum ribes grows wild in China, India, Iran and Turkey.In Turkey, it grows especially around Southern and Eastern Anatolia.R. ribes it is the only rheum species growing wild in Turkey [11].R. ribes is called "uçkun, usgun or iskin" in Turkey.In the GC-MS component analyzes of R.ribes, important chemical contents such as palmitic acid (67.7%), 1octadecanol (4.3%), vitamin E (3.85%) were determined [12][13].In addition, approximately 30 major components were detected in the analysis of R. Ribes stem methanol extracts by HPLC-ESI-QTOF-MS.Among these components, the most researched health-related ones are gallic acid (m/z 169.0157), salicylic acid (m/z 137.026), epigallocatehin (m/z 305.0681), epigallocatechin gallate (m/z 457.083), cis-resveratrol (m/z 227.0707).), genistein (m/z 271.0608), quercetin (m/z 303.0499), rutin (m/z 609.1495), kaempferol-7-O-glucoside (m/z 449.1086) and emodol anthraquinones (m/z 271.0608) [14].Many plant species grown in Turkey are consumed by the people of the region to alleviate or eliminate the symptoms of various diseases [14].The studies investigating the therapeutic efficacy of R. ribes are mainly concerned with its antimicrobial and antiradical properties [15][16].Although the therapeutic properties of R. ribes grown in many regions have been investigated, there is no adequate study on ventilator-associated pneumonia of R. ribes originating from Aricak district of Elazig province.Previously, our team conducted research on gram-negative and positive bacteria related to R. ribes [17] and Lavandula angustifolia [18].The beneficial effects of herbal materials led to the investigation of pathogenic pneumonia bacteria and antioxidant structures of R. ribes plant.Therefore, in this study, it was aimed to determine the antimicrobial effects of methanol-chloroform extract of R. ribes obtained from VAP patients by agar well diffusion test and antioxidant capacity with different techniques on some pneumonia pathogens from Elazig province Aricak district.

R.ribes Extracts Obtaining (R.ribes
Ekstraktlarının Elde Edilmesi) 3. Rheum ribes plant, (38°32'52.7"N-40°05'29.9"E)coordinates of was collected in Elazig Province of Turkey (Saman Town, Aricak County).The collected R.ribes was immediately taken to -80 o C cold chain, untill analysis done.The area where the plant was collected and the images of the plant are given in Figure 1.The collected R. ribes samples were extracted by modifying the Tufekci [19] method.The residues were cleaned by washing with distilled water.Then, 150 grams of plant stem was weighed and kept in an oven at 60 o C for 24 hours.Then, IKA (A10) was ground in the mill with the help of mechanical grinder.100 grams of the ground sample was taken and 500 mL of Methanol-Chloroform (1:1 v/v) was added to it and kept in a desiccator for 3 days.At the end of the 3rd day, extracts Whatman No.It was filtered with 2 filter papers and evaporated at 35 °C.Thus, the entire solvent was removed and the extract was obtained.As a result, it was prepared from this extract with distilled water at a final concentration of 10 ppm [19].It was sent to the Microbiology Laboratory for 8 months (November 2016 to June 2017).All samples were first investigated for morphological and biochemical features, including gram staining, motility, and catalase.After adding 15% glycerol to a 1.5 mL microtube containing tryptic soy broth, the isolated strains were transferred there and kept at -80°C.This study was approved by the local ethics committee (protocol no: 54/2016).All critically ill patients diagnosed with VAP who needed invasive ventilation for at least 48 hours were included in the study.The diagnosis of pneumonia was made using new or progressive infiltration on a chest X-ray with at least two of the following criteria: fever leukocytosis (WBC>12000 cells/mL), (T>38°C) or hypothermia (T35.5°C) or leukopenia (WBC4000) cells/mL, or positive tracheal culture [20][21].

Pathogenic Microorganisms
Bacterial strain identification and antibiotic susceptibility tests were performed using conventional methods and the VITEK 2 system (bioMérieux SA, France).Acquired resistance profiles of pathogenic bacterial strains such as Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, and were determined as MDR according to the Centers for Disease Control and Prevention (CDC) and European Center for Disease Prevention Control (ECDC) definition.
Absence of (at least) sensitivity to an antibiotic in three or more antibacterial categories was defined as MDR.MDR pathogens were included in this study.

Antimicrobial Activity: strains and media
(Antimikrobiyal Aktivite: suşlar ve ortamlar) The antimicrobial effect of R.ribes on indicator microorganisms was investigated by well diffusion agar and MIC assay method were performed according to the methods designed by Bauer et al. [22], and Ericsson and Sherris [23].The effect of the R.ribes was evaluated for pneumoni bacteria K.pneumonia (20,21,16

Agar well diffusion test (Agar kuyu difüzyon testi)
Indicator microorganisms were stored at temperature below 5°C.They were removed andreactivated in tryptic soy broth at 37°C for 18 hour.12 ml of Mueller-Hinton agar (cooled 50 °C after otoclave) was poured into 90 mm Petri dishes and added 1ml fresh culture indicator bacteria with a density of 0.5 Mcfarland left to dry at 37 °C for 30 minutes.6 mm diameter wells were drilled in frozen agars.100 µL of R. ribes extract was added to the wells and incubated overnight at +4 °C for diffuse [24][25].
The petri dishes were then incubated under optimum conditions.The diameters of the inhibitory zones were measured in millimetres.Mueller Hinton Agar standard method procedure developed by Bauer et al. [22] and Stella and Marin [26] was used to eliminate or reduce variability in this test method.The procedure was adopted by the Clinical and Laboratory Standards Authority (CLSI, former NCCLS) as a consensus standard [27].In addition, bacterial isolates, Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa, were tested for antibiotic resistance/sensitivity (Table 1-3).

MIC test (Minimum inhibisyon konsantrasyonu testi)
MICs obtained with spectrophotometric microdilution method for R. ribes against indicator microorganisms were measured at 600 nm in a 96well plate reader (Multiskan™ FC Microplate Photometer, USA), 150 µL of dual-strength MHB and Dual-strength R. ribes were added to the first wells, dilutions (1:1 v/v) were then added to the other wells.Then, 30 µL of bacterial suspension of fresh culture indicator bacteria with a density of 0.5 Macfarland was mixed into prepared plates and incubated at optimum temperatures for 18-24 hours.Then, samples density were detected by plate reader MIC was expressed as the highest dilution inhibiting growth (turbidity max in tube is low).Among these MIC dilutions, those with positive detection of nonviable cells > 99% in the medium are considered as MBC [28].

DPPH Free Radical Scavenging Activity ( DPPH Serbest Radikal Temizleme Etkinliği)
Basic data on the extracts' antiradical activity is provided by the DPPH assay.Using a modified version of Blois [29] approach, the radical scavenging capacity of the R. ribes extract was assessed spectrophotometrically by observing the elimination of DPPH at 517 nm.In the presence of the sample, the bleaching rate of DPPH, a stable free radical, is observed at a distinctive wavelength.DPPH absorbs at 517 nm while it is a radical, but when it is reduced by an antioxidant or another radical species, its absorption drops.In a nutshell, 1.5 mL of R. ribes extract in ethanol at various concentrations (15-45 g/mL) were mixed to 0.5 mL of a 0.1 mM solution of DPPH (10 -3 M) in ethanol.These solutions underwent accurate vortexing and dark incubation.At 517 nm, the absorbance was measured against the blank samples after 30 minutes.The reaction mixture's lower absorbance denotes a higher level of DPPH free radical scavenging activity.
Standarts (BHT and Trolox) and R.ribes extract were compared against the absorbance of blank and then calculated of % inhibition values (Figure-2.A).

Chelating activity of ferrous ions (Fe 2+ )
(Demir iyonlarının (Fe2+) şelatlama aktivitesi) By using R. ribes extract and the standards, ferrous ions were chelated using the Dinis et al. [30] method.The reaction took place in an aquatic environment.Briefly, 0.4 mL R.ribes extract (10 μg/mL) was added to a solution of 0.2 mL FeCl2 (2 mM).The reaction was initiated by the addition of 0.4 mL ferrozine (5 mM) and the total volume was adjusted to 4 mL with ethanol.Then the mixture was shaken vigorously and left at room temperature for 10 min.Standarts (BHT and Trolox) (10 µg/mL) and R.ribes extract were compared against the absorbance of blank (contains FeCl2 and ferrozine) at 562 nm (Figure-2.B).

FRAP (FRAP testi)
FRAP method (suitable to determine hydrophilic and lipophilic antioxidants) was introduced to determine the total amount of antioxidants by the reduction capacity of iron (III).The oxidant in the FRAP assay was prepared by mixing 2.5 mL 10 mM TPTZ dissolved in 40 mM HCl, 25 mL acetate buffer, 20 mM 2.5 mL FeCl3 and water.This mixture is called as FRAP reagent.The final solution contained 1.67 mM Fe (III) and 0.83 mM TPTZ [31][32].R.ribes extract (10 µg/mL) and standarts (BHT and Trolox) (10 µg/mL) were added to final solution (1.67 mM Fe (III) and 0.83 mM TPTZ) and incubated in dark for 10 minutes at room temperature.Standarts and R.ribes extract were compared against the absorbance of blank at 595 nm (Figure-2.C).

Total phenolic content (Toplam Fenolik içerik)
The determination of the total amount of phenolic substance is generally carried out by measuring the absorbance of the blue color formed by the reduction of the Folin-Ciocalteu reagent [33].The color intensity formed is directly proportional to the phenolic substance concentration, and the total amount of phenolic substance can be calculated.Using this method, 0.5 N Folin-Ciocalteu reagent and 10% concentration Na2CO3 were prepared.Then, 30 min after pipetting, the absorbance was read at 760 nm.Gallic acid, a phenolic compound, was used in the preparation of the standard graph.Different concentrations of gallic acid, 1-0.031 mg mL -1 (50% percent to each dilution), were prepared with methanol, and their absorbances were read.A graph of absorbance versus concentration was constructed, and the total phenolic contents of the sample were determined as gallic acid equivalent.

Total flavonoid (Toplam Flavonoid)
The determination of the total flavonoids was achieved by observing the pink color formation, which is directly proportional to the flavonoid concentration.In this method, 10% AlCl3 was prepared in a fume hood.NaNO2 and 1 N NaOH were prepared at 5% concentrations.Absorbance was read at 510 nm, 15 min after pipetting.
Rutin was used in the preparation of the standard chart.Different concentrations of the rutin standard were prepared with methanol, and their absorbances were read.A graph of absorbance versus concentration was constructed, and the total flavonoid amounts of the sample were determined as rutin equivalent [34].The graph of the growth concentrations of R. ribes extract applied to 4 isolates of K. pneumonia bacteria at 600 nm compared to the control group is given in Figure 2.For all K.pneumonia isolates of R. ribes, at 0.5 ppm, 100% inhibitory effects of K.pneumonia 21 and 2 isolates were determined, while the highest 16 and 20 isolates were found to have 98.3% and 94.88%, respectively.It was observed that showed a strong inhibition effect at the rate of 50.36% at 0.25 ppm concentration in S. aureus (Figure 3A), and 82.82% at 0.5 ppm concentration in P.aeruginosa isolate (Figure 3B.).In addition, the total flavonoid and phenolic contents made are given in Table 5.According to the analyzes performed, the total flavonoid value was determined as 155.53 mg routine equivalent / mL extract, and the total phenolic value was determined as 69.57mg gallic acid equivalent / mL.Reactive oxidative species, which damages cells, are prevented by natural antioxidant molecules [46].By preventing the onset or spread of the oxidative chain reaction, which functions as free radical scavengers, singlet oxygen scavengers, and reducing agents, increasing the intake of exogenous antioxidants will lessen the harm caused by oxidative stress.Exogenous antioxidants come primarily from foods and medicinal plants, such fruits, flowers, spices and traditional herbs [47][48].
In an in vitro study where anthraquinones extracted from R.ribes were applied to bacteria, DPPH, FRAP and ferrous-ions chelating activities were found to be quite high as a result of the analyzes, and it is thought to be related to the secondary metabolites, flavonoids and phenolic substances in its structure [49].In a study on obese rats, it was reported that R.ribes root extract improved DNA damage and MDA levels in brain tissues and showed positive effects on antioxidant parameter activities in different tissues [50].Another study revealed that the whole plant butanol fraction of R. ribes collected from the Pakistan region exhibited significant anticancer (MCF-7) activity [51].

4.CONCLUSIONS (SONUÇLAR)
In conclusion, our research suggests that R. ribes extraction may reduce the severity of VAP in in vitro studies and that this effect may be mediated by altering bacterial infection mediators and oxidative stress parameters.In addition, it was confirmed that the R. ribes formulations applied in the study showed similar effects with antibiotic drugs with known benefits.We believe that herbal therapies, such as R. ribes, can provide a safe alternative to conventional treatments for the treatment of clinical VAP, which should be validated by carefully planned research.As a limitation of our study, experimental animal and human studies may compare with antibiotics often used to treat VAP symptoms.Long-term in vivo and clinical trials in humans with a focus on the VIP solution are required to elucidate the mechanism by which herbal supplements improve functional status for in vitro studies.

Figure 1 .
Figure 1.Aricak / Saman town localization of Rheum ribes (Rheum ribes'in Arıcak / Saman köyü lokalizasyonu) and Staphylococcus aureus isolates grown in tracheal aspiration cultures (TAK) of patients receiving mechanical ventilation therapy in Çukurova University Faculty of Medicine Reanimation unit (respectively 21 Pseudomonas, 21 Klebsiella, 6 Staphylococcus were identified in Çukurova University Hospital Central Laboratory)

Table 5 .
Total Values expressed are means ± SD of three parallel measurements.bRE,Rutinequivalent.cGA,Gallicacid equivalent.Although E. coli was the most effective bacteria in their study, it was found that K. pneumonia were the bacteria that had the greatest antimicrobial effect in our study when compared to the current study[43].Wijesinghe et al.(2021), in their in vitro study, found that cinnamon leaf essential oil showed antibacterial effects against P. aeruginosa, S. aureus and K. pneumoniae, in parallel with our current data[44].In addition, in a study on some resistant grampositive/negative bacteria; reported that treatment of drug-sensitive strains with a combination of ciprofloxacin and garlic extract was antimicrobial synergistic on S. aureus and P. aeruginosa bacteria[45].
[36]ruginosa and methicillin-resistant S.aureus are among the common etiological agents of VAP, A. baumannii and K. pneumoniae are known to be the most important causes of VAP in many intensive care units worldwide[36].In a clinical study, oral