RP-HPLC-UV Analysis of the Phenolic Compounds, Antimicrobial Activity Against Multi-Drug Resistant Bacteria and Antioxidant Activity of Fruit and Seed of Diospyros lotus L.

: The object of the present study was to investigate the antioxidant, antimicrobial activity and phenolic compounds of fruit and seeds of Diospyros lotus L. The fruits consumed by humans as nutrients were harvested from the plants that grow naturally in the province of Trabzon-Akçaabat, Eastern Black Sea Region of Turkey. The antioxidant activities of fruit and seeds of Diospyros lotus were determined by using four methods (% DPPH radical scavenging activity, FRAP antioxidant power determination, CUPRAC reducing antioxidant activity and total phenolic content (TPC) in five different proportion of methanol-water extracts. All extracts of fruit and seeds of D. lotus analyzed through whole antioxidant analysis methods showed significant antioxidant activity. In addition, antimicrobial activity of fruit and seeds extracted with DMSO was determined against seven standard bacteria and three multi-drug resistance clinical strains. Although fruit extracts did not have the antimicrobial activity against bacteria, seeds showed antimicrobial effect to both standard strains (Gram-negative and Gram-positive) and antibiotic resistance clinical isolates ( Klebsiella pneumoniae carrying bla KPC , Acinetobacter baumannii , harboring bla OXA-23 gene and resistance to colistin, and multi-drug resistant Pseudomonas aeruginosa ). MICs’ value of plant seed extracts for standard strains was 0.75-25 mg/mL and antibiotic resistant clinical bacteria were 12.5 and 25 mg/mL. Additionally, phenolic compounds in methanol extracts of fruit and seeds were also determined in by HPLC using 19 standards. Gallic acid and chrysin phenolic compounds were the major phenolic compounds in fruit and seeds, respectively.


INTRODUCTION
While organisms continue their cellular metabolic functions in the usual way, at the same activity against three Gram-positive, four Gram-negative bacterial strains and three multi-drug resistant bacteria. Also it was determined that the chemical composition of the fruit and seed.

Preparation of Plant Extraction
The fruits of the D. lotus were harvested in the Akçaabat district of Trabzon province in December 2017. The seeds (S) from fruit (F) of the plant were separated and both fruit and seeds are dried at room temperature. The dried fruits and seeds are milled in Waring Commercial Blender device.10 g of fruit powder and seed powder were weighed, separately and extracted in five different percentages of 100 mL methanol-pure water solutions (Table 1).
Thus, the extracts were mixed with a magnetic stirrer for 2 hours and were successively filtered through Whatman filter papers to obtain clear solutions. The extracts were stored in a cool, dark place at room temperature until analysis. In addition, in order to determine the antimicrobial activity, DMSO extracts were prepared for both fruits and seeds as in preparation of methanol-water extracts.  F1  100  -S1  100  -F2  75  25  S2  75  25  F3  50  50  S3  50  50  F4  25  75  S4  25  75  F5 -100 S5 -100

Plant Extraction for HPLC
Solvents of the methanol extracts of the fruit and seeds of the plant were evaporated with a rotary evaporator device at 40 °C. The residue dissolved in 10 mL pH 2 water was extracted, for the phenolic compounds, three times with 15 mL of diethyl ether then, three times with 15 mL of ethyl acetate consecutively. Organic phase was picked up in the same flask and evaporated till drying under reduced pressure in a rotary evaporator at 40 °C. Then the residues were weighed and dissolved with 2 mL of methanol for HPLC analysis. This solution was filtered with 0.45µm Whatman nylon filter and analyzed by HPLC-UV [30].

DPPH Radical Scavenging Activity
DPPH radical cleaning test is one of the most preferred methods in the determination of the antioxidant activity of the substances. The method developed by Cuendet et al. [31] was modified and applied to extracts. Firstly, a 100 µM methanolic DPPH• solution was mixed in the magnetic stirrer for 30 min and then a DPPH• reagent solution was prepared. Both the sample mixture and the reagent blank 3 were run in parallel. After 50 min, the absorbance values of the tubes mixed with DPPH reagent were determined at 517 nm and % inhibition (DPPH• cleaning) values were calculated using the following formula. % Inhibition (radical cleaning power) = [(ADPPH -ASample) / ADPPH] X 100 ADPPH : Absorbance value of the DPPH solution ASample : Absorbance value of the sample extract

Ferric Reducing Antioxidant Power (FRAP)
The antioxidant assay method was based on the principle of measuring the absorbance of the Fe 2+ -TPTZ (2,4,6-tris (2-pyridyl) -s-triazine complex in 595 nm [32]. The FRAP reagent consists of mixture of 10mM TPTZ in 40mM HCl and 20mM FeCl3.6H2O and 300mM acetate buffer (pH 3.6), in 1:1:10 ratio respectively. The calibration curve was created using the Trolox standard in different volumes (1000-500-250-125-62.5 µM). 50 µL of the plant extracts and standard Trolox solutions were vortexed with FRAP reagent (1.5 mL) and were kept at room temperature for 20 minutes. Then the values of absorbance were read at 595 nm against pure water. In addition, the absorbance of reagents and sample blanks was measured and these values were subtracted from the mean values of the three studied parallel samples. Antioxidant activity of FRAP was calculated based on Trolox calibration graph and expressed as TEAC, µM (Trolox equivalent antioxidant capacity). In the evaluation of the results, the high TEAC values of the samples also indicate high FRAP and therefore high antioxidant capacity.

Cupric Reducing Antioxidant Capacity (CUPRAC)
In CUPRAC method, 2,9-dimethyl-1,10-phenanthroline (Neocuproine-Nc) was used with Cu (II) to form Copper (II) -neocuproine complex [Cu (II) -Nc] [33]. The antioxidant capacity was determined with the reduction of the Cu(II)-Nc to Cu(I)-Nc chelate that has maximum absorbance at 450 nm [33]. Antioxidant standard Trolox® was studied at six different concentrations (0.03125-0.625-0.125-0.25-0.5-1-mg/mL) for plotting calibration curve. After pipetting, tubes were vortexed and kept in darkness for 30 minutes. Absorbance values of the solutions in each tubes transferred to plastic cuvettes were read at 450 nanometers. The results were compared with Trolox®, a standard antioxidant with a high reduction potential and were expressed as Trolox® equivalent antioxidant capacity (μM TEAC).

Total Phenolic Content (TPC)
The total phenolic content of the fruit and seed of the D. lotus was determined by modified Slinkard and Singleton [34]. method with use of Folin-Ciocalteu reagent. Firstly, 50 µL of the sample solution was diluted with 2.5 mL of distilled water, then 250 µL of 0.2 N Folin-Ciocalteu reagent and 750 µL of Na2CO3 (7.5%) was added and vortexed. The prepared tubes were incubated for 2 hours at room temperature and the absorbance values at 765 nm were determined. The amount of phenolic compounds in the samples was determined based on function of the line obtained from calibration graph of gallic acid standart (1000-500-150-125-62.5 µg/mL) in six different concentrations as gallic acid equivalent (GAE (µg/mL)).

Analysis of Phenolic Compounds by HPLC
The phenolic contents of the methanol extracts of the fruits and seed of D. lotus were determined by using the Elite LAChrom Hitachi, Japan HPLC with a UV-Vis detector. In total, nineteen of phenolic compounds standards (caffeic acid, catechin, chrysin, daidzein, epicatechin, ferulic acid, gallic acid, hesperetin, luteolin, myricetin, p-coumaric acid, 4hydroxybenzoic acid, pinocembrin, protocatechuic acid, resveratrol, rutin, syringic acid, tcinnamic acid and CAPE (caffeic acid phenethyl ester)) were used. The extracts were injected into an inverted phase C18 column (150 mm x 4.6 mm, 5 µm; Fortis). The mobile phase was formed within mixing of solvents A (2% AcOH in water) and B (acetonitrile/water in 70:30 ratio) which was sonicated before stirring and continuously degassed by the built-in HPLC system. The temperature of the column was fixed at 30 °C and 20 μL of extracts were injected. The mobile phase was composed of water (2% AcOH) and acetonitrile/water (70:30) filtered using a 0.45µm nylon filter (Whatman, Maidstone, The United Kingdom) degassed in a sonicator for 30 min. The mobile phase flow rate was 0.75 mL/min, and the elution in the gradient mode occurred as follow: 0 min 5% B in A; 8 min 15% B in A; 10 min 20% B in A; 12 min 25%B in A; 20 min 40%B in A; 30 min 80%B in A; 35 min 5% B in A; 50 min 5%B in A. Phenolic profile was determined according to Can et al. [30].

Determination of Minimum Inhibition Concentration
The minimum inhibitory concentration (MIC) of the plant extract against to 3 Grampositive (Bacillus subtilis ATCC 6633, Streptococcus pyogenes ATCC 19615 and Staphylococcus aureus ATCC 25923) and 4 Gram-negative bacterial strains (Escherichia coli ATCC 25922, Proteus vulgaris ATCC 13315, Pseudomonas aeruginosa ATCC 43288, Yersinia pseudotuberculosis ATCC 911) and antibiotic resistant strains (Acinetobacter baumannii, E. coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa) were determined with the use of the liquid microdilution method. DMSO was used as a solvent for extracts. The extracts concentration are 100 mg/mL (serial two-fold dilution was carried out) obtained from the fruit and seed of the plant. Ampicillin used as control. MIC values of the DMSO fruit and seed extracts were determined in the 96-well plates and triplicate.

Antioxidant Activity of D. lotus Extract
The antioxidant activities of fruits and seeds of D. lotus plant were investigated by using four different antioxidant determination methods in methanol-water extracts at different percentages. In the DPPH radical scavenging method, while F1 (100% methanol) had the highest antioxidant activity value with 79.04 and the lowest activity was measured as 59.94 for F4 sample (25% methanol-75% water ( Table 2). In the extracts of the seeds, the highest antioxidant activity was measured as 85.63 in S1 extract (100% methanol) as in fruits. However, the lowest activity was determined with 64.17 in S2 extract (75% methanol-25% water) ( Table  2). Similar results were obtained in FRAP, CUPRAC and TPC antioxidant detection methods. In all three methods, F2 (75 % methanol-25 %) and F5 (100 % water) showed the highest and lowest antioxidant activity respectively for both seed extracts and fruit extracts. It was determined that the antioxidant activity of the seed extracts was higher than the fruit extracts in all antioxidant determination methods.
In addition, the antioxidant activity of the extracts, which are generally 100 % water in the solvent, was found to be lower than the others. Except for the DPPH test, antioxidant activity was higher in fruits and seeds in 75 % methanol-25 % water and 50 % methanol-50 % water extracts for all three methods. In some studies conducted to determine DPPH radical scavenging activity values of D. lotus plant fruits (methanolic extract) grown in different regions of the world, determined values were significant [20,21,23]. Moreover, Lozzio et al. [20] also found high antioxidant activity using FRAP and ABTS methods.

Phenolic Compounds in D. lotus Extract
While five phenolic acids; gallic acid > ferulic acid >protocatechuic acid>syringic acid >t-cinnamic acid were determined in methanolic fruit extract (Table 3) eight phenolic compound; gallic acid> chrysin>CAPE> catechin>caffeic acid>p-coumaric acid >protocateuic acid>t-cinnamic acid were identified in the seed extract (Table 3). It was observed that the seed parts were richer in terms of phenolic content as well as antioxidant activity. The results support the antioxidant results. As it is seen in the Table 3, gallic acid, protocatechuic acid and tcinnamic acid were present in both fruit and seed. While gallic acid was measured in the highest quantity with 234.74 µg/g in fruit extract and it was second with 1065.54 µg/g in seed extract among the determined phenolic compounds. However, although the chrysin and CAPE was not present in fruit extracts, they reached quantitatively highest abundance with 1086.09 and 768.2 µg/g in seed extract respectively. Ayaz et al. [27] searched phenolic content of D. lotus fruits at different times during the development period of fruit in GC-MS and reported that eight phenolic acids (salicylic acid, 4hydroxybenzoic acid, vanillic acid, gentistic acid, 3,4-dihydroxybenzoic acid, syringic acid, pcoumaric acid, gallic acid). Gallic acid and syringic acid were also detected and gallic acid showed the highest amount the same as our study. Gao et al. [16] also used ten phenolic acid standards in HPLC analysis of phenolic compounds in different extracts of D. lotus. Seven of these standards (caffeic acid, ferulic acid, gallic acid, myricetin, p-coumaric acid, protocatechuic acid and rutin) were common with the present study. Similarly, rutin was not detected, while gallic acid was detected in the highest abundance [16]. Gallic acid, a phenolic compound of plant origin, is a powerful antioxidant [35]. In addition, it also shows the effects of antimutagenic, antitumor, antibacterial anti-inflammatory [35,36]. Rashed et al. [24] reported that among seven phenolic compounds obtained from D. lotus fruits, gallic acid was the most active compound against HIV-1 and inhibited HIV-1IIIB replication with EC50 value of 6.09 μg/mL. Again, the plant phenolic compounds were used against human cancer cells and gallic acid exhibit the highest anticarcinogenic effect against colorectal adenocarcinoma (IC50 2.6 μg/mL) and lung large cell carcinoma (IC50 4.66 μg/mL) [20]. Moghaddam et al. [23] conducted a study on the seed of this plant to determine its biological activity. However, the phenolic content of seed has not been studied. Although chrysin was present in some plants, honey, and propolis [37] it was first detected in the D. lotus plant in this study. It was also measured at the highest level among the identified phenolics in the seed. Chrysin is an important natural flavonoid having many biological activities such as aromatase inhibitor, antioxidant, anti-cancer, antiviral activities and anti-inflammatory effects [38,39].

Antimicrobial Activity
DMSO extract of plant seed has inhibited the growth of studied bacteria but DMSO extract of plant fruit did not have antimicrobial activity against bacteria. MICs of plant seed extracts were found to be between 0.75-25 mg/mL. Extract of plant seed showed very potent activity against E. coli and S. pyogenes with 0. 75 mg/mL MIC. MIC values of plant seed extract for Bacillus subtilis and P. aeruginosa were 12.5 mg/mL. Also, it was determined that the DMSO extract of plant seed had a lower MIC value (25 mg/mL) against S. aureus, Y. pseudotuberculosis and Proteus vulgaris. MIC values of plant extract against antibiotic resistant clinical bacteria were 12.5 and 25 mg/mL. DMSO extract of the plant seed was determined to be the highest MIC (12.5 mg/mL) against K. pneumonae carrying blaKPC, A. baumannii harboring blaOXA-23 gene and resistance to colistin, and multi-drug resistance P. aeruginosa. MIC value of the plant seed extract was found to be 25 mg/mL against NDM type metallo beta lactamase harboring K. pneumonia and multi-drug resistance E. coli.

CONCLUSION
Methanol and water extracts in different percentages of D. lotus plant fruits and seeds showed antioxidant activity in all antioxidant methods. Significantly, the content of phenolic compounds in plants contributes antioxidant activity. Among the phenolic compounds detected in this study, the highest measured gallic acid (in fruit and seed) and chrysin (only seed) may be determinant antioxidant activity. Chrysin flavonoid, which has high biological activity, can be studied in more detail in both naturally grown and cultivated D. lotus seeds. Antibioticresistant strains, especially multidrug-resistant isolates, are a serious threat to public health. The spread of beta-lactamases that hydrolyze antibiotics among bacteria has become a worldwide problem. To combat antibiotic resistance, many scientists are looking for both synthetic and natural products around the world. In this study, we show the antibacterial activity of plant seed extracts against multi-drug resistant strains that cause difficulties in the clinic.

Declaration of Conflicting Interests and Ethics
The authors declare no conflict of interest. This research study complies with research publishing ethics. The scientific and legal responsibility for manuscripts published in IJSM belongs to the author(s).