INTRODUCTION

The Lamiaceae family includes more than 245 genera and 7886 species distributed worldwide.¹ Ajuga L. is a genus of annual and perennial herbaceous flowering plants in the Lamiaceae family, with most species native to Asia, Africa, and Europe. Ajuga is represented by 14 species and 27 taxa in Turkey.²

Ajuga has a long history of use for wound healing preparation, and although little used today, it is well known in Anatolia as “Mayasil otu”. Some Ajuga species are widely consumed as diuretic, diaphoretic, astringent, antipyretic, and tonic in Turkish traditional medicine.³

Ajuga sp. plants are reported for their in vitro antimalarial,⁴ antimicrobial,^5,6 antioxidant,⁵ anti-inflammatory,⁷ lipoxygenase, acetylcholinesterase, and butyrylcholinesterase inhibition,⁸ antipyretic,⁹ and antiproliferative¹⁰ activities.

Phytochemical constituents diterpenoids, such as phenylethanoid glycosides, sterols, phytoecdysteroids, flavonoids, and iridoids were reported as the main active compounds in Ajuga L. species.¹¹

Ajuga salicifolia sterol glycosides were isolated and tested for antimicrobial and cytotoxic activity.¹² Iridoid, ionone, and phenylethanoid glycosides from the same group were also reported for this species.¹³

Phytochemical profile of Romanian Ajuga genevensis L. and A. reptans reptans were recently reported.⁶ A summary of phytochemical investigations on A. salicifolia, A. reptans, and A. genevensis species are listed in Table 1.^{6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36} In vitro antioxidant and antimicrobial activity of different extracts of Romanian A. genevensis L. and A. reptans were recently reported.⁶ Previous antimicrobial activity results of A. reptans, A. genevensis and A. salicifolia are listed in Table 2.^37,38,39,40

A. genevensis was used in traditional Austrian medicine and consumed as medicinal tea in treating respiratory tract disorders,¹⁴ and in vitro anticancer activity studies were reported from Europe, Asia, and America.¹⁵

A. reptans grows natively in Europe and have bluish-purple flowers colored with anthocyanin pigments.¹¹ It was used in Mediterranean traditional medicine for cardiovascular complications and skin disorders¹⁶ and in traditional Austrian medicine as a medicinal tea for the treatment of respiratory tract disorders.¹⁴ A previous study showed that A. reptans L. is used due to the anti-inflammatory effects of its polyphenols, its wound healing properties, and antidiarrhea, antiulcerogenic, and hepatoprotective effects due to the presence of iridoids.¹⁷

In the present study, 70% methanol extract of aerial parts of A. reptans L., A. salicifolia (L.) Schreber, and A. genevensis L. from Turkey were evaluated for their phytochemical profiles, total phenol, and total flavonoid contents, as well as their in vitro antioxidant and antimicrobial activities. LC-MS/MS techniques were used for phytochemical analyses. In vitro 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and trolox equivalent antioxidant capacity (TEAC) assays were performed. Additionally, antimicrobial properties of Ajuga extracts were assessed against microbial strains of Escherichia coli NRRL B3008, Staphylococcus areus ATCC 6538, Salmonella thyphimurium ATCC 13311, Bacillus cereus NRRL B-3711, Candida albicans ATCC 90028, Candida tropicalis ATCC 1369, and Candida parapsilosis ATCC 22019.

To the best of our knowledge, this is the first study to perform the TEAC antioxidant activity for all extracts and the in vitro antimicrobial activity of A. salicifolia.

MATERIALS AND METHODS

Chemicals

Antimicrobial standards, Mueller Hinton Broth, and RPMI-1640 medium were purchased from Sigma-Aldrich Chemical Co (Sigma-Aldrich Corp., St. Louis, MO). All chemicals and solvents used were of analytical grade.

Plant materials

A. reptans L.: [A1(E) Kırklareli: İğneada, Fidanlık kavşağı, 350 m, N 41 52’ 25.3’’ E 27 56’ 11’’, 21 ıv 2009], A. salicifolia (L.) Schreber: (B3, Eskişehir: Çağlan köyü, 1000 m, K 390 39’ 971’’ D 300 31’ 185’’, 31 vi 2010), and A. genevensis L. [A1(E) Kırklareli: Dereköy yolu, 449 m, K 410 50’ 6.13 D 270 18’ 3.18’’, 22 ıv 2009] were collected and identified by Dr. Y.B Köse, and herbarium materials were deposited in the Herbarium of Anadolu University, Faculty of Pharmacy under herbarium code YBK1560, YBK1575, and YBK 1561, respectively.

Preparation of extracts

The aerial parts of the plants were dried in the shade at room temperature and ground to powder in a mechanical grinder. Each species (1 g) was extracted with methanol (70%, 100 mL) for 24 h, three times a day. After filtration, the solvents were evaporated under vacuo.

Phytochemical analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS)

The phytochemical analyses were performed using LC-MS/MS techniques.⁵

Determination of phenolic compounds

The total phenols contained in the extracts were calculated using the Folin-Ciocalteu method equivalent to gallic acid (GA).³⁰ A sample solution (100 µL) and Folin-Ciocalteu reagent (500 µL) were added to a 10 mL scale vessel containing 6 mL of distilled water. After 1 min, 1.5 mL of 20% aqueous Na₂CO₃ was added and completed with water to reach 10 mL. The reagent-free of extracts was used as the control. After incubation at 25ºC for 2 h, the absorbance was read at 760 nm and compared with the GA calibration curve. The total amount of phenolic was calculated as equivalent to GA. Three parallel experiments were performed, and the results were reported as mean values.

Biological activity

DPPH radical scavenging assay

The DPPH radical scavenging activity was performed according to Kumarasamy et al.³¹ For this purpose, 100 µL of methanol and samples were transferred to the first column of 96-well microtiter plates. A 10-well dilution was made in an equal amount of MeOH via a multi-channel pipette and stirred in the vortex for 5 min. The DPPH stock solution was prepared by dissolving 2 mg of DPPH^● in 25 mL of MeOH, and solution was added to each well and left in a dark place for 30 min. Butylated hydroxy toluene (BHT) and GA at the same concentration were used as positive controls, and ultraviolet (UV) absorbance was measured at room temperature using a Biotek microplate spectrophotometer at 517 nm.

The following equations using 50% inhibition concentration (IC₅₀) (equation 1) and percentage (%) inhibition values (equation 2) were calculated as follows:

IC₅₀ = [(A₀-A₁)/A₀] × 100                                                  equation (1)

$\mathrm{Percentage} \mathrm{Inhibition}=\left[\frac{(\mathrm{Abs} \mathrm{control} - \mathrm{Abs} \mathrm{sample})}{\mathrm{Abs} \mathrm{control}}\right] \mathrm{x}100 \mathrm{equation} \left(2\right)$

TEAC assay

Experiments were performed as declared by Papandreou et al.³² sweeping ABTS^●+ (2,2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) radical and vitamin E. It is based on the comparison of water-soluble analog with trolox. The mixture of 7 mM ABTS^●+ and 2.5 mM sodium persulfate was kept in the dark for 12-16 h, resulting in the formation of blue-colored radicals. A sample (10 µL) and ABTS^●+ solution (990 µL) were mixed, and absorbance was measured at 734 nm per minute intervals for 30 min. To find out the TEAC activity results, the ABTS^●+ radical was plotted using Trolox’s 2.5-2-1.5-1-0.5-0.1 (mM) concentrations, according to the % inhibition values. For quantification, a Trolox calibration curve was used where all experiments were repeated in triplicates.

Antimicrobial activity

Antimicrobial activity testing was performed according to the guidelines of broth microdilution methods.^33,34,35 Standard strains, E. coli NRRL B3008, S. areus ATCC 6538, S. thyphimurium ATCC 13311, B. cereus NRRL B-3711, C. tropicalis ATCC 1369, C. parapsilosis ATCC 22019, and C. albicans ATCC 90028, as well as antimicrobial standards, such as ampicillin, tetracycline, ketoconazole, and oxiconazole, were used in this study. Methanol extracts were prepared at 1250-2.44 µg/mL concentrations and dissolved in dimethylsulfoxide and initial test solutions. Serial dilutions were prepared at 64-0.125 µg/mL for ampicillin, tetracycline, and ketoconazole. All experiments were evaluated in triplicates, and mean values were reported.

Statistical analysis

Data obtained from antioxidant and total phenolic content experiments were expressed as mean standard error. IC₅₀ values were estimated using a non-linear regression algorithm.

RESULTS AND DISCUSSION

LC-MS/MS analysis of the extracts

Screening of the extracts by LC-MS/MS enabled the identification of phenolic acids, such as coumaroyl glucoside, flavonoids, and phenylethanoid glycosides. Figures 1, 2, 3 show the 280 nm UV chromatograms of A. reptans, A. genevensis and A. salicifolia, respectively. The compounds detected from Ajuga sp. methanol extracts are listed in Table 3.

As a result, the only substance commonly identified in all Ajuga species was forsythoside A and luteolin glucuronide.

The coumaroyl glucose and its glucoside isomer (Figure 4, 5) were determined for A. salicifolia extract. The echinacoside (Figure 6) was detected in A. reptans extract. The LC-MS/MS sprectrum of luteoline derivative (Figure 7), forsytoside A (Figure 8), and luteoline glucuronide (Figure 9) were observed in both A. reptans and A. genevensis extracts.

The phenolic acids as caffeic acid and flavonoids; apigenin-C-hexoside-C-pentoside, quercetin glucuronide, luteolin, and apigenin were identified only for A. genevensis. Furthermore, the phenylethanoid glycosides forsythoside B and leonosides A were identified only for A. salicifolia. The phytochemical research on Ajuga species focus on the isolation of flavonoids, caffeic and chlorogenic acid type derivates, phenylethanoid glycosides, phytoecdysteroids, iridoids, and diterpenes.^{12,13,17,18,19,20,21,22,23,24} Some anthocyanins, delphinidin, and cyanidin 3-O-sophoroside-5-O-glucosides, were acylated with p-coumaric acid, while ferulic acid and malonic acid were isolated from the flowers of cell cultures of A. reptans.^25,26,27

Total phenolic amounts of the extracts

The amount of total phenolics ranged from 30.0 to 42.2 mg GA equivalent (GAE)/g of the extracts. The phenolic amounts equivalent to GA in all three methanol extracts are shown in Table 4. The highest total phenolic level was found in the methanol extract of A. reptans. In previous studies, the total phenolic content of methanol extracts of A. reptans and A. genevensis has been evaluated to be 20.86±0.53 mg RE/g dw²⁸ and 22.63±0.61 mg GAE/g.²⁹

DPPH radical scavenging activity

DPPH radical scavenging activity results are presented in Table 4. The positive control, BHT with IC₅₀ value of 0.06 mg/mL, was found as the most potent antioxidant. The highest radical scavenging activity were obtained for A. salicifolia (IC₅₀: 0.28±0.01 mg/mL) and A. reptans (IC₅₀: 0.30±0.01 mg/mL) extracts. A correlation was also found between radical scavenging capacity and total phenol content. Previous studies showed the antioxidant activity of the methanol extract of A. genevensis flowers as IC₅₀: 72.08±6.02 µg/mL²⁹ and A. reptans as IC₅₀: 83.16±5.21.²⁸

However, there have been no reports on the antioxidant activity of A. salicifolia. This study is the first to determine the antioxidant activity of A. salicifolia.

TEAC assay

The results obtained for the evaluation of the antioxidant activity using TEAC assay are presented in Table 4. ABTS^●+ radical sweeping impact results are in parallel with the results of the DPPH radical scavenging effect. Extracts from all three plants show ABTS^●+ radical scavenging activity at 1% concentrations, but these effects are not as high as the BHT used as the standard.

There have been no reports on the antioxidant activity performed by a TEAC assay. Antioxidant activity was performed for the first time in this study for A. reptans L., A. salicifolia (L.) Schreber, and A. genevensis L. species found in Turkey.

Antimicrobial activity

The seven different strains tested in this study are presented in Table 5. Ajuga L. extracts showed antimicrobial effect against all microorganisms tested and were more effective against yeasts than bacteria [minimum inhibitory concentration (MIC): 156.25 µg/mL]. The MIC was 312.5 µg/mL for E. coli NRRL B-3008, S. areus ATCC 6538, S. thyphimurium ATCC 13311, and B. cereus NRRL B-3711 for methanol Ajuga extracts. As a result, the antimicrobial activity was observed in Ajuga extracts, especially against Candida strains.

Data from previous studies on the antimicrobial activity of A. reptans, A. genevensis, and A. salicifolia are listed in Table 2. To the best of our knowledge, this is the first report on the antimicrobial evaluation for methanol extract of A. salicifolia, which was more effective against yeast than bacteria.

CONCLUSION

We disclose the phytochemical profiles of A. reptans, A. salicifolia, and A. genevensis collected from Turkey.

The extracts were found to contain valuable metabolites; phenolics acids, coumaroyl glucoside, flavonoids, and phenylethanoid glycosides. The phytochemicals could be employed as potential chemotaxonomic markers because different phytochemicals were observed between the three Ajuga species.

The scope of this study included the biological potential of methanol extracts of A. reptans, A. salicifolia, and A. genevensis evaluated for the first time against some pathogenic strains.

Ajuga species may be considered a valuable natural source against Candida infections and candidal resistance. However, further in vitro and in vivo experiments using different alternative Candida and fungal species are required to validate these screening results.

ACKNOWLEDGMENTS

This work was supported by the Anadolu University Research Funding (project number: BAP: 080307).

*This work is dedicated to late Hulusi Malyer Prof. MD. for his outstanding contributions to pharmaceutical botany.

Conflict of interest: No conflict of interest was declared by the authors. The authors are solely responsible for the content and writing of this paper.