Phytochemical characterization of phenolics by LC-MS/MS and biological evaluation of Ajuga orientalis from Turkey
Abstract
The aim of this study was to reveal the phytochemical constituents, antioxidant and antimicrobial activity of Ajuga orientalis. According to the antimicrobial results, the methanol extract of A. orientalis showed a MIC value of 312.5 µg/mL against the tested pathogenic bacterial strains. Anticandidal activity of extract was found as 156.3 µg/mL both against Candida albicans and C. parapsilosis strains. Whereas the extract was more effective against C. tropicalis with the MIC value of 78.1 µg/mL. The in vitro DPPH radical scavenging activity of the extract was determined as IC50=0.4 ± 0.02 mg/mL whereas the standard BHT IC50 was 0.01 ± 0.00 mg/mL. Trolox Equivalent Antioxidant Capacity (TEAC) of extract was determined 1.3 mM TEAC, while BHT, 1.9 mM TEAC.
Introduction
Ajuga L. is one of the largest genera of the Lamiaceae family with 301 species all over the world. In flora of Turkey, Ajuga is represented by 13 species and 23 taxa (Guner et al., 2012). Ajuga has some traditional therapeutic uses such as diuretic, antipyretic, tonic, diaphoretic and astringent in Turkey (Baytop, 1984). Ajuga species are used in traditional medicine all over the world against some illness such as gout, rheumatism, malaria, asthma and gastrointestinal diseases and have antibacterial, antitumor (Ben Jannet et al., 2006; Chen et al., 1996; Israili and Lyoussi, 2009, Setif, 2011), neuroprotective effects (Guo et al., 2011), anti-inflammatory and antioxidant (Makni et al., 2013; Turkoglu et al., 2010) activities among others biological effects.
Ajuga genus contains many important bioactive compounds like anthocyanins, diterpenoids, sterols, ionones, iridoids, phenylethanol and flavonoid glycosides (Maria et al, 1997, Chen et al., 1996; Shimomura et al., 1987; Terahara et al., 2001; Akbay et al., 2003).
Essential oil composition of A. orientalis was investigated and main compounds were germacren D (24.7%), beta-cubaben (18.3%) and beta-caryophyllene (16.9%) (Sajjadi and Ghanadi, 2004).
Even though several bioactive compounds have been well studied on some Ajuga species, there is not enough data on phytochemical and biological activities for A. orientalis. To the best of our knowledge, this is the first report on determination of the major phenolic compounds, antioxidant activity and antimicrobial properties of the methanol extract of A. orientalis.
Materials and Methods
Chemicals
All antimicrobial agents, Mueller Hinton II broth and RPMI medium were purchased from Sigma-Aldrich Chemical Co (St. Louis, MO). Solvents were used analytical grade.
Plant material and preparation of the extract A. orientalis was collected during flowering stage from Bursa, Turkey [A2 (A) Bursa: Bursa, Uludag,1773 m, 19.6.2008, K 400 06' 23.3" D 290 08' 11.1"].
Microbial strains
The antimicrobial activities of the extract were tested against a panel of microbial strains, including: Escherichia coli NRRL B3008, Staphylococcus aureus ATCC 6538, Salmonella typhimurium ATCC 13311, Bacillus cereus NRRL B-3711, Candida albicans ATCC 90028, C. tropicalis ATCC 1369 and C. parapsilosis ATCC 22019.
Determination of Minimum Inhibitory Concentration (MIC)
The MIC values of the strains were determined by broth microdilution methods (CSLI, 2006, 2008). The methanol extract of A. orientalis was examined against a panel of seven different human pathogenic bacteria and Candida strains using the microdilution method according to CSLI method compared with standard antimicrobial agents. Methanol extract of A. orientalis was studied between [1250-2.44 ug/mL] for MIC and the antimicrobial standard agents ampicillin, tetracycline, ketoconazole (64-0.125 µg/mL) and oxiconazole (16-0.03 µg/mL) were prepared in dimethyl sulfoxide (DMSO) and water.
The extract (100 µL) was added to wells of row A, while the remaining wells in rows B to H received 50 µL of cation adjusted Mueller-Hinton Broth-2 (MHB-2). Bacterial suspensions were grown overnight in double strength broth and were standardized to 105 cfu/mL for bacteria. Each bacterial suspension (50 µL) was added to the appropriate well. All Candida strains were inoculated on Patoto Dextrose Agar (PDA) prior the experiments at 35°C. After incubation grown microorganisms were inoculated sterile saline %0.85. And then standardized using a turbitometer (Biosan) (McFarland No: 0.5) to 5 x 103 cfu per well in RPMI medium under sterile conditions. Serial dilution series were prepared in 100 µL RPMI medium with an equal amount of the test samples. After serial dilution 100 µL each microorganism suspension was pipetted into each well and incubated at 35°C for 24 hours. Positive growth controls (to assess the presence of turbidity) were performed in wells not containing antimicrobial agents. In addition, negative growth control (medium) was applied in 96-well plate. After incubation at 35°C for 24 hours the first well without turbidity was determined as the MIC (µg/mL).
Antioxidant activity (total phenolics)
Total phenols were estimated as gallic acid equivalents (GAE), expressed as mg gallic acid/g extract (Singleton Vernon et al., 1999).
TEAC assay (Trolox Equivalent Antioxidant Capacity)
This test measures the ability of a compound to scavenge the ABTS (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) radical in comparison to antioxidant activity of trolox which is a water-soluble form of vitamin E, used as a standard. The blue-green ABTS radical was formed through the reaction of 7 mM ABTS with 2.5 mM sodium persulfate (Na2S2O8) in the dark at room temperature for 12-16 hours before use. ABTS solution was diluted with ethanol to a final absorbance of 0.7-0.8 at 734 nm. 10 µL portion of the sample was added to 990 µL of ABTS solution, and the reduction in absorbance was measured 1 min after addition of trolox up to 40 min after addition of the extract. The stock solution of trolox (2.5 mM) was prepared in ethanol. Absorbance was measured on a UV/spectrophotometer (Papandreou et al., 2006).
1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity
Serial dilutions were prepared with the stock solutions (4 mg/mL) of the extract to get the half concentration of previous one. Diluted solutions were added with DPPH (equal amounts). After 30 min UV absorbance was recorded at 517 nm. The experiment was performed in triplicate for extract and positive standard control, BHT (butylated hyroxytoluene). The average of the absorption was noted for each concentration. The percentage inhibition was calculated using Eq 1. The IC50 value, which is the concentration of the test material that inhibits 50% of the free radical concentration, was calculated as mg/mL using Sigma Plot statistical program (Kumarasamy et al., 2007).
Percentage Inhibition= [(Abscontrol-Abssample)/Abscontrol]x 100 ---------- Eq(1)
Phenolic compound determination
Experiments were performed with a Shimadzu 20A HPLC system coupled to an Applied Biosystems 3200 Q-Trap LC- MS/MS instrument equipped with an ESI ion source used in the negative ionization mode. Separations were performed on an ODS 150 x 4.6 mm, i.d., 3 µm particle sizes, octadecyl silica gel analytical column operating at 40ºC at a flow rate of 1 mL/min.
Powdered dried herbal parts of A. orientalis were macerated with methanol 70% at 25°C for 24 hours. After evaporation of the methanol part, the aqueous part freeze-dried and the dry extract was used in all experiments.
Result and Discussion
An antioxidant activity generally occurs a hydrogen donating procedure. Phenolic acids and flavonoids are well known hydrogen donating agents due to their high PKa values. Determination of the total phenol com pounds provides preliminary information about antioxidant activity results. The more total phenolic compounds means the more antioxidant activity.
According to results, extract of A. orientalis showed similar amount of gallic acid equivalent total phenol content in 1 g methanol extract. A. orientalis: 32 mg GAE/g ext.
According to the identification of phenolic profile by LC-MS/MS (Table I) 9 compounds were determined (Figure 1).
Figure 1: Ajuga orientalis LC chromatogram at 320 nm
Table I: Compounds determined with LC-MS/MS
| No. | Compound | Rt | (M-H) | Fragments | Reference |
|---|---|---|---|---|---|
| 1 | Galactonic acid | 2.8 | 195 | 177, 159, 129, 11 | Nist 14 |
| 2 | Ascorbic acid | 9.8 | 175 | 115 | Nist 14 |
| 3 | Luteolin glucuronide derivative | 14.6 | 513 | 487, 461, 285, 179, 135 | (Cvetkovikj et al., 2013) |
| 4 | Echinacoside | 15.4 | 785 | 623, 461, 161 | (Mitreski et al., 2014) |
| 5 | Verbascoside | 16.2 | 623 | 461, 315, 161 | (Mitreski et al., 2014) |
| 6 | Forsythoside A | 16.5 | 623 | 461, 315, 161 | (Guo et al., 2007; Mitreski et al., 2014) |
| 7 | Castanoside A | 18.5 | 799 | 623, 461 | (Mitreski et al., 2014) |
| 8 | 4"-O-methylisoscutellarein 7-O-allosyl(1-2)glucoside | 19.2 | 623 | 461, 315, 161, 135 | (Petreska et al., 2011) |
| 9 | Leucoseptoside A | 19.6 | 637 | 461, 175 | (Mitreski et al., 2014) |
Compound 1Rt =2.8 coeluted with [M-H]-molecular ions at m/z 195 (galactonic acid or gluconic acid) and m/z 341 (disaccharide like sucrose). High absorbance at 280 nm must be related to gradient changing of mobile phase. Product ions of m/z 341 similar with caffeoyl glucose fragments (m/z 179, m/z 161) but it is not possible to determine a caffeoyl glucose molecule at this retention time. Other fragments of galactonic acid can be seen in Table I.
Compound 2 was identified as ascorbic acid according to Nist 14 MS/MS library and comparison with authentic standard's fragmentation patterns.
Compound 3Rt = 14.6 displayed [M-H]- ion at m/z 513 and a base peak ion at m/z 461 further fragmentation showed several ions at m/z 487, 461, 285 (most probably a luteolin), 179 and 135. Following fragmentation of m/z 461, it was found as similar with luteolin glucuronide. However, compound 6 was exhibited 26 amu higher than luteolin glucuronide so this compound was labeled as luteolin glucuronide derivative.
Compound 4Rt = 15.4 displayed [M-’H]-’ ion at m/z 785 yielded a base peak ion at m/z 623 due to the loss of a hexose (or caffeoyl) moiety, the same loss of base peak ion was led to form ion at m/z 461. Other fragmentation corresponding with cinnamic acids, m/z 179 and 161, was also observed. The compound was identified as echinacoside by compression to compound previously reported (Petreska et al., 2011).
Two compound, compound 5 (Figure 2) and 6 were coeluted at Rt = 16.2 and Rt = 16.5 with a same deprotonated molecular ion at m/z 623 which showed same fragmentation pattern. Both of the spectrums yielded m/z 461 owing to loss of a caffeoyl moiety (-162) and m/z 315 due to the loss of a rhamnose unit (-146). Caffeoyl part of the compounds were also observed at m/z 179 and further fragmentation at m/z 161 and 135. Compound 5 at RT 16.2 was determined as verbascoside whereas compound 6 at Rt = 16.5 was identified as fosythoside A using similar mass spectrum and chromatographic separation of previously reported data (Guo et al., 2007; Mitreski et al., 2014).
Figure 2: Mass spectrum of compound 5: verbascoside
Compound 7Rt = 18.5 showed [M-H]- ion at m/z 799 its MS/MS spectrum yielded two main fragments at m/z 623 (loss of a feruloyl, -176) and 461 (loss of a caffeoyl moity). This compound was identified as castanoside A according to previous data (Mitreski et al., 2014).
Compound 8 showed MS fragmentation characteristic of verbascoside and forsythoside A. Pseudomolecular ion at m/z 623 and its MS2 experiment yielded in several ions at m/z 461, 315, 161 and 135. According to previously published data (Petreska et al., 2012) the compound, eluted after verbascoside and forsythoside A, with the same molecular weight is 4"-O-methylisoscutellarein 7-O-allosyl(1-2)glucoside. These data suggested that compound could be identified tentatively as 4"-O-methylisoscutellarein 7-O-allosyl(1-2)glucoside.
Compound 9 showed [M-H]- molecular ion at m/z 637 in MS2 spectrum a ferruloyl break at m/z 461 and the ferruloyl part at m/z 175 was also observed. This compound was labeled as leucoptoside A according to literature data (Mitreski et al., 2014; Petreska et al., 2011).
DPPH. is a radical that gives maximum absorption at 517 nm in alcoholic solutions because of its intense purple color. The color of radical converts pale yellow if neutralized via an antioxidant compound. DPPH radical was used for determination of the radical scavenging antioxidant potential of A. orientalis extract. The extract radical scavenging activity was not effective as a positive control standard BHT. IC50 value of the extract was 0.4 ± 0.02 mg/mL and the positive control of BHT 0.01 ± 0.00 mg/mL.
The calibration curve of trolox that is water soluble analog of vitamin E is used in the quantification. A radical form of ABTS is used as an indicator for evaluation of the antioxidant activities. This radical occurs after treatment of the non-radical ABTS salt with potassium persulfate after 16 hours. Alcoholic solution of radical gives maximum absorption at 734 nm owing to its green color. Green color of the radical converts to colorless and the absorption reduces if it encounters with antioxidant compound. In this test high TEAC values shows high antioxidant activity results. ABTS radical scavenging and DPPH radical scavenging effect showed a high correlation with each other. The extract at 1% concentration, showed ABTS radical scavenging effect. But the extract was not found as effective as standard BHT. TEAC values were found 1.3 mM for extract and 1.9 mM for BHT.
Oxidative attack of free radicals is the reason for many chronic diseases. Natural antioxidants such as flavonoids and phenolic acids help to protect human body from free radicals (Koolen et al., 2013). Antioxidant activity of this plant can be used as natural radical scavenger but not as good like as synthetic standard antioxidants. Determined phenolic compounds are the first reason of the antioxidant activities. Hydroxyl rich compounds is well known free radical scavengers. Obtained total phenolic compounds amount can be low for this plant material using this extraction procedure. However, high amount of hydroxyl groups of molecules that we determined showed moderate activity.
Discovering new antimicrobial agents are also important because of antimicrobial resistance of some microorganism. Synthetic antimicrobial agents are used in several years and today some of them are ineffective and researchers are focused on to investigate new natural antimicrobial agents.
Additionally phytochemical studies of the extract, in vitro antimicrobial activity was evaluated seven different strains in this study (Table II) using microdilution methods. A. orientalis extract exhibited antimicrobial action against all tested microorganisms, being more effective against yeasts than bacteria. The MIC was found at 312.5 µg/mL for Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and Bacillus cereus. MIC value of 156 µg/mL for C. albicans and C. parapsilosis for the extract. Our results showed that C. tropicalis was the most sensitive strain for the extract (MIC=78.12 µg/mL).
Table II: Minimum Inhibitory Concentrations (MIC, µg/mL)
| Microorganisms | Ajuga orientalis extract |
Ketoconazole | Oxiconazole | Ampiciline | Tetracycline |
|---|---|---|---|---|---|
| Escherichiacoli NRRL B-3008 | 312.5 | - | - | 4 | 4 |
| Staphylococcus aureus ATCC 6538 | 312.5 | - | - | 2 | 2 |
| Salmonella typhimurium ATCC 13311 | 312.5 | - | - | 2 | 2 |
| Bacillus cereus NRRL B-3711 | 312.5 | - | - | 0.5 | 0.5 |
| Candida albicans ATCC 90028 | 156.25 | 1 | 4 | - | - |
| Candida tropicalis ATCC 1369 | 78.12 | 0.5 | 2 | - | - |
| Candida parapsilosis ATCC 22019 | 156.25 | 2 | 2 | - | - |
There have been some reports for antimicrobial activities belong to A. orientalis. Ali-Shtayeh et al (2013) found that extract of A. orientalis inhibited the growth of some acne-inducing bacteria and aerobic bacteria (Ali-Shtayeh et al 2013). A. orientalis was also evaluated antifungal activity against Phytophthora capsici which is a pathogen fungus (Kiran et al, 2010).
Conclusion
As a result, the antimicrobial activity was determined the first time for A. orientalis extract, especially against Candida species. The extract can be used as an alternative natural prevention of Candida infections and candidal resistance.
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