Composition and biological activities of Libyan Salvia

South African Journal of Botany 117 (2018) 101–109

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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb

Composition and biological activities of Libyan Salvia fruticosa Mill. and S. lanigera Poir. extracts S. Duletić-Laušević a, A. Alimpić Aradski a,⁎, K. Šavikin b, A. Knežević a, M. Milutinović c, T. Stević b, J. Vukojević a, S. Marković c, P.D. Marin a a b c

University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden “Jevremovac”, Takovska 43, 11000 Belgrade, Serbia Institute for Medicinal Plant Research “Dr Josif Pančić”, Tadeuša Košćuška 1, 11000 Belgrade, Serbia University of Kragujevac, Faculty of Science, Department of Biology and Ecology, Radoja Domanovića 12, 34000 Kragujevac, Serbia

a r t i c l e

i n f o

Article history: Received 31 March 2017 Received in revised form 27 April 2018 Accepted 8 May 2018 Available online xxxx Edited by GI Stafford Keywords: Salvia fruticosa Salvia lanigera Extracts Phenolics composition Biological activities

a b s t r a c t This study was aimed to analyze the chemical composition and biological activities of extracts of Salvia fruticosa and S. lanigera originated from Libya. Dichloromethane, ethyl acetate, methanol, ethanol and water extracts obtained from wild growing plants were analyzed for the composition using HPLC-DAD, which revealed presence of phenolic acids and flavonoids, mainly in alcoholic and water extracts. Total phenolic and flavonoid contents, as well as anti-oxidant activity using 2.2-diphenyl-1-picrylhydrazyl (DPPH), 2.2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid diammonium salt (ABTS), ferric reducing anti-oxidant potential (FRAP) and β-carotene bleaching (β-CB) assays were determined spectrophotometrically. Extracts of S. fruticosa exhibited stronger activity in all applied assays, especially ethanol extract in DPPH assay (IC50, 29.55 μg/mL) and in β-CB assay (85.11%). The ethanol and water extracts were selected for further investigation, because of their wide usage in phytotherapy. The extracts were screened for the antimicrobial activity against 11 bacteria (six Gramnegative and five Gram-positive) and seven fungi using microdillution method. Ethanol extracts showed stronger activity than water extracts, particularly against Gram-positive bacteria. Trichophyton mentagrophytes was the most sensitive to the water extract of S. lanigera (MIC, minimal inhibitory concentration and MFC, minimal fungicidal concentration, 8 mg/mL). Cytotoxic activity on human carcinoma cell line HCT-116 was determined by 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide (MTT) cell viability assay, where only ethanol extract of S. fruticosa demonstrated certain activity (IC50, 375.96 μg/mL). The testing of anti-neurodegenerative activities of extracts showed better tyrosinase inhibiting effect (55.26–74.66%) than standard kojic acid (33.93–51.81%), while the extracts were less effective against acetylcholinesterase compared with standard galanthamine. According to the obtained results, S. fruticosa and S. lanigera originated from Libya have proved to be the promising source of natural compounds possessing a range of biological activities. © 2018 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Numerous species of Salvia, the largest genus of the Lamiaceae family, are used in traditional medicine all around the world since the ancient times. In modern times those species are subjected to detailed chemical and pharmacological analyses aimed to identify biologically active compounds. Diverse studies have focused on the biological properties of the essential oils and extracts of various Salvia species, which have shown antimicrobial, anti-oxidant, antidiabetic, antitumor, antiinflammatory, and anticholinesterase activities (Tepe et al., 2006; Orhan et al., 2007; Şenol et al., 2010; Shaheen et al., 2011; Hamidpour et al., 2014; Salari et al., 2016). ⁎ Corresponding author at: University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden “Jevremovac”, Takovska 43, 11000 Belgrade, Serbia. E-mail address: [email protected] (A. Alimpić Aradski).

https://doi.org/10.1016/j.sajb.2018.05.013 0254-6299/© 2018 SAAB. Published by Elsevier B.V. All rights reserved.

Growing concern is noted in the recent years regarding undesirable side effects and potential health risks of synthetic medicaments which enforced the search for efficient, non-toxic natural compounds enabling their potential usage in food, pharmaceutical and cosmetic products. Medicinal plants, including many Salvia species, having a long history of use in the treatment of various health disorders, represent important source for development of new natural drugs possessing beneficial health effects. Salvia fruticosa Mill. (Syn. S. triloba L., Greek sage) is endemic species, native to the eastern Mediterranean basin. It is distributed from Italy and Cyrenaica through the South Balkan Peninsula to West Syria (Hedge, 1982). It is one of the most economically important Salvia species valued for its beauty, medicinal and culinary properties, along with its sweet nectar and pollen, having especially long tradition in application in Greece. Salvia fruticosa was studied from different aspects, such as chemical composition, antimicrobial, antiviral, anti-inflammatory, cytotoxic,

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anti-oxidant, as well as neuroprotective effects of essential oils and extracts (Savelev et al., 2004; Tawaha et al., 2007; Papageorgiou et al., 2008; Şenol et al., 2011; Giweli et al., 2013; Topcu et al., 2013). Salvia lanigera Poir. (wooly sage) is herbaceous perennial shrub distributed in North Africa, from northern Egypt and Arabia, to the south of Turkey and Iran (Jafri and El-Gadi, 1985). The plant is covered with short erect hairs, and this feature gave the name to the species (“lanigera” means “wool-bearing” or “fleecy”). Salvia lanigera is used by Bedouins as a condiment for tea (Bailey and Danin, 1981). The composition of essential oil, antimicrobial, anti-oxidative and antiproliferative properties of oils and different extracts were reported for plants collected in Jordan (Fiore et al., 2006; Flamini et al., 2007), Egypt (Hawas and El-Ansari, 2006; Shaheen, 2011; Shaheen et al., 2011; Ibrahim et al., 2013), Saudi Arabia (Al-Howiriny, 2003) and Cyprus (Tenore et al., 2011). The genus Salvia is represented by ten species in the Flora of Libya (Jafri and El-Gadi, 1985), which are very scarcely investigated. Libyan S. fruticosa, appreciated for medicinal and culinary features due to essential oils produced in glandular trichomes, was studied from micromorphological and ultrastructural points of view (Al Sheef et al., 2013) and also for the chemical composition, antimicrobial and anti-oxidant activities of the essential oil (Giweli et al., 2013). Libyan S. lanigera was not analyzed for phytochemical constituents and biological effects till now. The results on anti-oxidant and cytotoxic effects of Libyan S. fruticosa and S. lanigera water extracts were previously published (Alimpić et al., 2015), and the results are included in Tables 1 and 3, containing data about yield, total phenolic and flavonoid content, and anti-oxidant activity. The aim of the present study was to carry out the comprehensive research and provide data on the chemical composition and anti-oxidant, antimicrobial, cytotoxic and anti-neurodegenerative activities of Libyan S. fruticosa and S. lanigera extracts (dichloromethane, ethyl acetate, methanol, ethanol and water).

(L-DOPA) were purchased from Sigma Chemicals Co. (USA), while Tween 40 and linoleic acid were purchased from Acros Organics (Belgium). The phenolic compounds standards (caffeic acid, rosmarinic acid, apigenin, luteolin, genkwanin, hyperoside, rutin, coumarin), were purchased from Merck (Germany). 2.2. Plant material Aerial parts were collected in the flowering phase from the natural localities (S. fruticosa in Biadda, on the Green Mountain in eastern Libya; S. lanigera in Zintan on the Western Mt., Aljabel Algarbi) in March 2010. Total sample of plant material consisted of about 50 individuals was air-dried (approximately 500 g of dry material). Voucher samples are deposited in the Herbarium of the Institute of Botany and Botanical Garden “Jevremovac”, Faculty of Biology, University of Belgrade (BEOU: 16702 and BEOU: 16880, respectively). 2.3. Preparation of plant extracts Air-dried plant material, randomly taken from whole collected sample, were milled. Extracts were prepared using two extraction procedures. One portion of 10 g of plant material was successively extracted by 100 mL of dichloromethane, ethyl acetate and methanol (Şenol et al., 2010; Alimpić et al., 2015, 2017). Other two portions per 10 g of plant material were individually extracted by 100 mL of solvent (ethanol and hot distilled water) as it was described before (Alimpić et al., 2015, 2017). In both cases, extraction was performed by maceration during 24 h at room temperature. Ultrasonic bath was used during the first and the last hour of extraction to improve the extraction process. Thereafter, extracts were filtered and evaporated under reduced pressure (Buchi rotavapor R-114) and obtained crude extracts were stored at +4 °C for further experiments.

2. Materials and methods

2.4. Determination of total phenolic and flavonoid contents

2.1. Chemicals

The total phenolic and flavonoid contents were quantified using spectrophotometer (JENWAY 6305UV/Vis) according to the previously described procedure (Alimpić et al., 2015, 2017). Phenolic content of extracts was calculated from gallic acid curve equation and expressed as gallic acid equivalents (mg GAE/g dry extract). Flavonoid content of extracts was calculated from quercetin curve equation and expressed as quercetin equivalents (mg QE/g dry extract). Values were presented as mean ± standard deviation averaged from three measurements.

Methanol, ethanol, distilled water, glacial acetic acid, hydrochloric acid, dichloromethane, chloroform and ethyl acetate were purchased from Zorka Pharma, Šabac (Serbia). Gallic acid, quercetin, ascorbic acid, 2(3)-t-butyl-4-hydroxyanisole (BHA), 3.5-di-tert-butyl-4 hydroxytoluene (BHT) 2.2-dyphenyl-1-picrylhydrazyl (DPPH), 2.2′-azino-bis(3ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS), 2.4.6tripyridyl-s-triazine (TPTZ), potassium acetate (C2H3KO2), potassiumpersulfate (K2S2O8), dimethylsulfoxide (DMSO), sodium carbonate anhydrous (Na2CO3), aluminum nitrate nonahydrate (Al(NO3)3 × 9H2O), sodium acetate (C2H3NaO2), iron (III) chloride (FeCl3), iron (II)sulfate heptahydrate (FeSO4 × 7H2O), beta-carrotene, Folin-Ciocalteu reagent, sodium phosphate monobasic (NaH2PO4), sodium phosphate dibasic (Na2HPO4), 5.5′-dithiobis (2-nitrobenzoic acid) (DTNB), acetylcholinesterase from Electrophorus electricus (electric eel) (AChE), acetylcholine iodide, galanthamine hydrobromide from Lycoris sp., kojic acid, tyrosinase from mushroom and 3.4-dihydroxy-L-phenylalanine

2.5. HPLC analysis of extracts The HPLC analyses of phenolic components were performed using the Agilent 1100 Series and UV-DAD (UV-Diode Array Detector) as described before (Alimpić et al., 2017). Peak detection in UV region at 350 nm was used. Quantification of phenolic components of extracts was performed by normalization method based upon the area percent reports obtained by HPLC-DAD.

Table 1 The yield, total phenolic content (TPC) and total flavonoid content (TFC) of S. fruticosa (SF) and S. lanigera (SL) extracts. Extracts

Yield (%) SF

Ethanol Water Dichloromethane Ethyl acetate Methanol (pt)

8.92 7.62(pt) 3.60 3.43 6.90

TPC (mg GAE/g) SL 7.40 7.32(pt) 2.29 0.98 7.60

SF

TFC (mg QE/g) SL

x

154.18 ± 2.79 67.68 ± 0.62(pt)x 164.71 ± 0.73x 105.10 ± 0.91x 132.00 ± 3.51x

SF x

43.04 ± 0.85 58.47 ± 0.20(pt)x 67.39 ± 1.60x 64.99 ± 1.84x 51.11 ± 2.20x

SL x

29.08 ± 0.54 21.73 ± 0.16(pt)x 38.38 ± 0.54x 17.00 ± 0.77x 22.69 ± 0.71x

previously tested (Alimpić et al., 2015); number of replicates: yield (n = 1), TPC and TFC (n = 3); x Values within column are significantly different (p b 0.05).

26.42 ± 0.93x 17.18 ± 0.54(pt)x 35.87 ± 0.63x 51.59 ± 1.08x 28.42 ± 1.47x


2.6. Evaluation of anti-oxidant activity 2.6.1. DPPH assay DPPH free radical scavenging assay was performed using previously described experimental protocol (Alimpić et al., 2015, 2017). BHA, BHT and ascorbic acid were used as anti-oxidant standards. Results were expressed as IC50 values (μg/mL) averaged from three measurements. 2.6.2. ABTS assay ABTS free radical scavenging assay was performed according to procedure described by Alimpić et al. (2015, 2017). The extracts and antioxidant standards (BHA and BHT) were tested in concentration of 1 mg/mL and 0.1 mg/mL, respectively. ABTS activity was calculated from ascorbic acid calibration curve (0–2 mg/L) and expressed as ascorbic acid equivalents per gram of dry extract (mg AAE/g), averaged from three measurements.

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after re-inoculation on SDA was defined as minimal fungicidal concentration (MFC). 2.8. Cytotoxic activity Cytotoxic activity of extracts was measured by microculture tetrazolium test (MTT) for cell viability, using human colon tumor HCT-116 cells according to previously described experimental design (Alimpić et al., 2017). The extracts were tested at the concentrations from 1 to 500 μg/mL. The absorbances were measured on Microplate Reader (ELISA 2100 C). Cell proliferation was calculated as the ratio of absorbance of treated group divided by the absorbance of control group, multiplied by 100 to give a percentage proliferation. As positive control for cytotoxicity 5-Fluorouracil was used under the same experimental conditions on HCT-116 cell line according to Žižić et al. (2013). 2.9. Anti-neurodegenerative activity

2.6.3. FRAP assay FRAP assay was performed according to previously used experimental protocol (Alimpić et al., 2015, 2017). The extracts and standards BHA, BHT and ascorbic acid were tested in concentration of 0.5 mg/mL and 0.1 mg/mL, respectively. FRAP values of samples were calculated from standard curve equation and expressed as μmol FeSO4 × 7H2O/g dry extract, and presented as mean ± standard deviation averaged from three measurements. 2.6.4. β-Carotene bleaching (β-CB) assay β-CB assay, designed to evaluate the capacity of the antioxidants to reduce the oxidative loss of beta-carotene in a beta-carotene linoleic acid emulsion, was applied as described before (Alimpić et al., 2017). Crude extracts and standards BHA, BHT and ascorbic acid were tested in concentration of 0.5 mg/mL. The absorbances were measured using Tecan Sunrise SN microplate reader at 490 nm immediately and after 120 min incubation. The anti-oxidant activity of the samples was evaluated in terms of inhibition of beta-carotene bleaching using the following equation: [(A120 − C120) / (C0 − C120)] × 100%, where A120 and C120 are the absorbances measured at 120 min for sample and control, respectively, while C0 is absorbance of control at 0 min.

2.9.1. Acethylcholinesterase (AChE) inhibitory activity assay AChE inhibitory activity assay was performed spectrophotometrically using 96-well plates (Orhan et al., 2007; Şenol et al., 2010) according to previously described procedure (Alimpić et al., 2017). The extracts and standard galanthamine were tested in concentration of 25, 50 and 100 μg/mL. Absorbances were measured at 412 nm using Tecan Sunrise SN microplate reader. Percentage of inhibition of AChE was determined using the formula [(C × S) / C] × 100%, where C - the activity of enzyme in control (without test sample) and S - the activity of enzyme with test sample. 2.9.2. Tyrosinase inhibitory activity assay Tyrosinase inhibitory activity assay was performed spectrophotometrically as described before (Alimpić et al., 2017). Samples (the extracts and standard kojic acid) were tested in concentration of 25, 50 and 100 μg/mL. The absorbances were measured at 475 nm using Tecan Sunrise SN microplate reader. Percentage of inhibition of tyrosinase was determined using the formula: [(A − B) − (C − D) / (A − B)] × 100%, where A - contained buffer and tyrosinase, B - contained only buffer, C - contained buffer, tyrosinase and sample and D contained buffer and sample.

2.7. Antimicrobial activity 2.10. Statistical analysis 2.7.1. Antibacterial assay The antibacterial activity of ethanol and water extracts, as well as standard antibiotic streptomycin was tested against six Gramnegative: Escherichia coli (ATCC 25922), Salmonella typhimurium (ATCC 14028), Salmonella enteritidis (ATCC 13076), Pseudomonas tolaasii (NCTC 387), Pseudomonas aeruginosa (ATCC 27853), Proteus mirabilis (ATCC 14273) and five Gram-positive bacteria: Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 10876), Micrococcus flavus (ATCC 14452), Sarcina lutea (ATCC 10054) and Listeria monocytogenes (ATCC 15313) as it was described in the previous study (Alimpić et al., 2017). The lowest concentrations without visible growth were defined as concentrations that completely inhibited bacterial growth - minimal inhibitory concentrations (MICs). 2.7.2. Antifungal assay Antifungal activity of ethanol and water extracts, as well as standard antimycotic ketoconazole was tested against pathogenic micromycetes (human isolates): Candida krusei (Castell.) Berkhout, Candida albicans (C.P. Robin) Berkhout, Candida parapsilosis (Ashford) Langeron & Talice, Aspergillus glaucus (L.) Link, Aspergillus fumigatus Fresen., Aspergillus flavus Link and Trichophyton mentagrophytes (C.P. Robin) Sabour. The experimental procedure was performed as described before (Alimpić et al., 2017). The lowest concentration of extract without visible fungal growth was defined as minimal inhibitory concentration (MIC). The lowest concentration of extract which inhibited fungal growth

All measurements were carried out in triplicate and expressed as mean ± standard deviation. Since five solvents were used for extraction, one-way analysis of variance (ANOVA) and Tukey's HSD post-hoc test were performed to test the significance of differences among their mean values. Pearson's correlation coefficients were calculated between content of phenolic components and values obtained applying different anti-oxidant assays and interpreted according to Taylor (1990). All statistical analyses were performed using the MS Office Excel, 2007. 3. Results and discussion 3.1. The yield of extracts, total phenolic and flavonoid content The results for yield, total phenolic and flavonoid content are presented in Table 1 where the results for water extracts, obtained earlier (Alimpić et al., 2015), are also included in order to compare previous and results obtained in the current study. Ethanol extract of S. fruticosa and methanol extract of S. lanigera provided the highest yields (8.92% and 7.60%, respectively), while the lowest yields were obtained for ethyl acetate extracts of both species. It can be observed that more polar solvent extracts yielded more than extracts obtained by less polar solvents and this finding is consistent with Şenol et al. (2010), who applied the same extraction procedure. Different yields of extracts were obtained in the other studies of S. fruticosa (Bozan et al., 2002;

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differences among populations of S. fruticosa originated from Albania, with higher content of rosmarinic acid, and from Greece, with higher content of caffeic acid. According to Dincer et al. (2012), differences could be attributed to developmental stage of plants. Shaheen (2011) and Shaheen et al. (2011) found phenolic acids derivatives including one caffeic acid dimer and p-coumaric acid ester in Egyptian S. lanigera. Apigenin and luteolin were abundant in ethyl acetate and ethanol extracts of S. fruticosa, while apigenin was present in the highest amount in ethyl acetate extract of S. lanigera. Apigenin, luteolin and genkwanin glycosides were mostly present in S. fruticosa methanol, water and ethanol extracts, respectively, while genkwanin glycosides were absent in the same extracts of S. lanigera. Kaempferol glycosides were predominant among flavonols (9.69–58.39%), in water, methanol and ethanol extracts of both species. Dincer et al. (2012) also identified kaempferol in the 80% methanolic extract of S. fruticosa from Turkey. Among other polyphenols, coumarin was present at the highest amount in the ethyl acetate extract of both Salvia species. There are different results in previous studies of S. fruticosa samples originated from other Mediterranean countries. Myricetin and morin were pointed as the most abundant in the study of Dincer et al. (2012), quercetin in Papageorgiou et al. (2008), apigenin and luteolin in Askun et al. (2009), while apigenin and luteolin glycosides were reported by Cvetkovikj et al. (2013) as the most abundant flavonoids. Those differences in the results could be affected by geographic conditions and seasonal variations. This study revealed that Libyan S. fruticosa have significant amount of phenolic compounds, particularly kaempferol glycosides, which are reported before as powerful anti-oxidant and antimicrobial agents (Tatsimo et al., 2012). Hawas and El-Ansari (2006) found 12 flavonoid compounds (5 flavone glycosides, 2 aglycones and 5 methylated flavones) in several S. lanigera fractions from plant material collected in Egypt. Acetone and n-butanol extracts of leaves and stems of Egyptian S. lanigera plants contained flavones, apigenin and salvigenin (Shaheen et al., 2011), and in aceton extract Shaheen (2011) also found luteoline-7-O-glucoside and 7-methoxyapigenin.

Pizzale et al., 2002; Papageorgiou et al., 2008; Ibrahim and Aqel, 2010; Şenol et al., 2010), which can be explained by the influence of plant development stage, extraction conditions, geographical region, collecting season, or ecological conditions. Results revealed that solvents significantly affected the amount of total phenolic and flavonoid contents in tested extracts (p b 0.05). In the present study, both dichloromethane extracts had the highest contents of phenolics (164.71 mg GAE/g and 67.39 mg GAE/g, respectively) (Table 1). Dichloromethane extract of S. fruticosa and ethyl acetate extract of S. lanigera showed the highest amount of flavonoids (38.38 mg QE/g and 51.59 mg QE/g, respectively). Salvia fruticosa extracts were previously studied for total phenolic and/or flavonoid content using different methods and the literature survey (Tawaha et al., 2007; Papageorgiou et al., 2008; Şenol et al., 2010; Dincer et al., 2012; Gonaid et al., 2012; Alimpić et al., 2015) revealed that these contents varied in a broad range. These differences could be attributed to plant origin, ecological conditions of growing, plant parts used for analysis, harvesting period, storage conditions, extraction methods employed (Papageorgiou et al., 2008; Dincer et al., 2012). Total phenolic and flavonoid contents of S. lanigera extracts have not been yet studied. 3.2. HPLC analysis of extracts Qualitative and quantitative composition of five S. fruticosa and S. lanigera extracts was studied using HPLC analysis and results are presented in Table 2. The water and methanol extracts showed the highest percentage of identified phenolic components, which differs from TPC results (Table 1), probably due to low percentage of identified components in dichloromethane extract by HPLC method. Polyphenolic components were classified according to Bolton et al. (2008) and Neveu et al. (2010) in the phenolic acids, flavonoids, including flavones and flavonols and other polyphenols. Among phenolic acids, caffeic acid, which is precursor of rosmarinic acid (Petersen et al., 2009), was present in methanol, ethanol and water extracts of both species (0.24–16.07%), while rosmarinic acid was detected only in methanol extract of S. fruticosa (1.41%), although many researchers reported rosmarinic acid as the most abundant phenolic acid in Salvia species (Lu and Foo, 2002; Pizzale et al., 2002; Dincer et al., 2012; Orhan et al., 2012). Askun et al. (2009) also found higher level of caffeic acid in methanol extract of S. fruticosa, while Papageorgiou et al. (2008) reported hydroxibenzoic acid as the major phenolic acid. The study of Cvetkovikj et al. (2013) demonstrated

3.3. Anti-oxidant activity Anti-oxidant activity of S. fruticosa and S. lanigera extracts was evaluated using four test-systems: DPPH, ABTS, FRAP and β-carotene bleaching assays. The values are presented in Table 3 as IC50 (μg/mL), mg AAE/g, μmol Fe(II) and % of inhibition, respectively. The results for

Table 2 Composition of S. fruticosa (SF) and S. lanigera (SL) extracts. Extracts

Dichloromethane

Ethyl acetate

Constituents

SF

SL

SF

SL

SF

SL

SF

SL

SF

SL

– –

– –

– –

– –

0.24 1.41

0.88 –

1.39 –

0.30 –

4.02 –

16.07 –

0.96 – – – –

– – – – 1.21

8.34 – 6.79 – –

4.36 – 1.35 – –

5.57 5.98 6.22 6.10 3.60

0.46 4.17 1.80 33.25 –

5.68 – 7.93 – 9.51

– 1.07 0.71 8.29 –

– 5.37 1.51 9.70 7.99

– 3.76 3.89 6.81 –

– – 18.06

– – 7.71

– – 11.24

– – 6.63

43.93 4.21 1.40

38.91 – 1.19

42.56 – –

9.69 – 5.93

58.39 1.67 –

23.07 – –

– 19.02

0.82 9.74

4.21 30.58

1.27 13.61

1.96 80.62

0.86 81.52

– 67.07

– 25.99

– 88.65

– 53.60

Phenolic acids Caffeic acid Rosmarinic acid Flavonoids Flavones Apigenin Apigenin glycosides Luteolin Luteolin glycosides Genkwanin glycosides Flavonols Kaempferol glycosides Rutin Hyperoside Other polyphenols Coumarin Total of identified components (%) Number of replicates: (n = 1).

Methanol

Ethanol

Water


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Table 3 Anti-oxidant activity of S. fruticosa (SF) and S. lanigera (SL) extracts. Extracts/standards DPPH activity (IC50, μg/mL)

ABTS activity (mg AAE/g)

FRAP assay (μmol Fe(II)/g)

Extracts

SF

SL

SF

SL

SF

SL

SF

SL

Ethanol Water Dichloromethane Ethyl acetate Methanol Standards BHT BHA Ascorbic acid

29.55 ± 0.36x 48.11 ± 1.50(pt),x 33.23 ± 0.17x 62.68 ± 2.59x 36.37 ± 1.18x

448.49 ± 1.56x 205.45 ± 5.03(pt),x 614.23 ± 7.03x 406.86 ± 4.11x 222.16 ± 5.50x

2.31 ± 0.03a,x 1.98 ± 0.01a,(pt),x 2.41 ± 0.02a,x 1.59 ± 0.02a,x 1.83 ± 0.09a,x

0.68 ± 0.05a,x 1.77 ± 0.09a,(pt),x 0.64 ± 0.02a,x 0.55 ± 0.02a,x 1.06 ± 0.10a,x

834.10 ± 7.64b,x 1191.51 ± 8.11b,(pt),x 653.67 ± 4.87b,x 342.32 ± 10,54b,x 332.19 ± 5.77b,x

137.61 ± 2.29,b,x 79.13 ± 5.25b,(pt),x 154.43 ± 4.03b,x 80.28 ± 3.24b,x 200.11 ± 2.43b,x

85.11 ± 1.84b,x 69.68 ± 2.76b,x 50.53 ± 4.22b,x 32.98 ± 1.60b,x 12.23 ± 4.79b,x

76.06 ± 2.76,b,x 77.66 ± 2.60b,x 10.64 ± 2.26b,x 18.62 ± 2.20b,x 12.77 ± 1.84x

17.94 ± 0.17 13.37 ± 0.43 5.11 ± 0.14

2.75 ± 0.02c 2.82 ± 0.01c nt

445.34 ± 5.77c 583.72 ± 5.26c 180.81 ± 8.61c

β-CB assay (% of inhibition)

57.71 ± 3.39b 53.72 ± 2.26b 17.82 ± 1.13b

Number of replicates: (n = 3); nt –not tested. a At concentration of 1 mg/mL. b 0.5 mg/mL. c 0.1 mg/mL. pt previously tested (Alimpić et al., 2015). x Values within column are significantly different (p b 0.05).

anti-oxidant activity of water extracts, obtained earlier by Alimpić et al. (2015), are included in the Table 3 in order to compare activity of water and other extracts obtained in the current study. Salvia fruticosa extracts performed significantly higher anti-oxidant effect compared with S. lanigera. From obtained results it can be seen that solvents significantly affected anti-oxidant capacity of tested extracts (p b 0.05), except for methanolic extract of tested species in β-carotene bleaching assay. Ethanol extract of S. fruticosa and water extract of S. lanigera showed the strongest activity in all applied assays, except FRAP assay. All of the S. fruticosa extracts performed DPPH activity (ranged from 29.55–62.68 μg/mL), but weaker than positive controls. DPPH activity of S. lanigera extracts was many-fold weaker (205.45–614.23 μg/mL). In applied sample concentration, S. fruticosa ethanol extract showed ABTS activity (2.31 mg AAE/g) close to synthetic antioxidants BHA and BHT. Salvia lanigera water and methanol extracts display ABTS activity above 1 mg AAE/g. Water and ethanol extracts of S. fruticosa were the most powerful in FRAP assay (1191.51 μmol Fe (II)/g and 834.10 μmol Fe (II)/g, respectively). Salvia lanigera methanol extract manifested the most expressive ability to reduce Fe (III) to Fe (II) ion (200.11 mg Fe (II)/g). In β-carotene-linoleic acid system, the ethanol (85.11%) and water (69.68%) extracts of S. fruticosa exhibited even stronger inhibition than synthetic antioxidants (17.82– 57.71%). S. lanigera ethanol and water extracts also showed stronger inhibition compared with positive controls (76.06% and 77.66%, respectively). Literature survey demonstrated that anti-oxidant activity of S. fruticosa extracts was studied using different assays, mostly by Turkish (Bozan et al., 2002; Şenol et al., 2010, 2011; Dincer et al., 2012; Topcu et al., 2013; Ozcan and Ozkan, 2015), Greek (Exarchou et al., 2002; Papageorgiou et al., 2008; Pasias et al., 2010; Stagos et al., 2012) and Jordanian (Tawaha et al., 2007; Al-Mustafa and Al-Thunibat, 2008) authors. Anti-oxidant activity of Libyan S. fruticosa essential oil was investigated by Giweli et al. (2013), who reported low DPPH activity (IC50 = 15.53 mg/mL). Shaheen et al. (2011) tested three extracts of Egyptian S. lanigera and obtained the highest activity of acetone extract, close to α- tocoferol and stronger than BHT. Pizzale et al. (2002) pointed out the difficulty of comparing the results of many different methods used for testing anti-oxidant activities, as well as the difficulty of assessing the anti-oxidant activity basis on a single method. Different tests provide more comprehensive understanding of anti-oxidant potential of tested species, and it is expected that anti-oxidant capacity of samples depends on used plant parts, locality, harvesting period, extraction solvent and procedure (Pizzale et al., 2002; Orhan et al., 2007, 2012; Al-Mustafa and Al-Thunibat, 2008; Papageorgiou et al., 2008; Pasias et al., 2010; Şenol et al., 2010; Dincer et al., 2012; Stagos et al., 2012).

3.4. Correlation among content of phenolic components and anti-oxidant capacity of extracts Pearson's correlation coefficients (r) were calculated between content of polyphenols and anti-oxidant activity of extracts measured by four different assays, and the results are presented in Table 4. All assays were more strongly correlated to total flavonoid contents determined by HPLC than to those spectrophotometrically measured. DPPH assay was weakly to moderate correlated to content of identified phenolic components. ABTS assay was the most strongly correlated to content of luteolin, genkwanin and genkwanin glycosides. Content of the genkwanin, and especially, kaempferol glycosides were the most strongly correlated to ABTS activity (r N 0.68). Presence of genkwanin glycosides (r = 0.81) could contribute to FRAP activity of extracts. Also, genkwanin glycosides and caffeic acid are moderately correlated to inhibition expressed in the β-CB assay. Anti-oxidant tests were weakly to strongly correlated among each other (correlation coefficients from 0.29 to 0.70). The other researchers have also found positive correlation between anti-oxidant activity and the amount of total phenolic content in S. fruticosa extracts (Pizzale et al., 2002; Tawaha et al., 2007; Al-Mustafa and Al-Thunibat, 2008; Papageorgiou et al., 2008; Stagos et al., 2012).

Table 4 Pearson's correlation coefficients between anti-oxidant activity and content of phenolic components of S. fruticosa and S. lanigera extracts. DPPH assay ABTS assay FRAP assay β-CB assay Caffeic acid Apigenin Apigenin and its glycosides Luteolin Luteolin and its glycosides Genkwanin glycosides Kaempferol glycosides Hyperoside Coumarin TPC (HPLC) TFC (HPLC) TPC (spectrophotometrically) TFC (spectrophotometrically) DPPH assay ABTS assay FRAP assay β-CB assay

−0.10a −0.43 −0.35a −0.59b −0.02a −0.46b −0,52b −0.63 −0.15a −0.60b −0.60b −0.66b 0.44b 1.00 −0.70c −0.64b −0.29a

According to Taylor (1990): a r ≤ 0.35 weak correlation. b 0.36 b r b 0.67 moderate correlation. c 0.68 b r b 1 strong correlation.

0.38b 0.37b 0.46b 0.80c −0.01a 0.74c 0,75c 0.04a 0.06a 0.75 0.71c 0.32a −0.68c

−0.10a 0.03a 0.18a 0.16a −0.11a 0.81 0.58b 0.29a −0.26a 0.48b 0.51b 0.46b −0.18a

0.48 −0.20a −0.05a 0.17a −0.19a 0.43b 0.26a −0.17a −0.54b 0.16a 0.09a 0.09a −0.36b

1.00 0.55b 0.43b

1.00 0.45b

1.00

106


Table 5 Antibacterial activity of S. fruticosa and S. lanigera extracts and standard antibiotic streptomycin. Bacteria

Code

MIC (mg/mL) S. fruticosa

Gram-negative bacteria Escherichia coli Salmonella typhimurium Salmonella enteritidis Pseudomonas tolasii Pseudomonas aeruginosa Proteus mirabilis Gram-positive bacteria Staphylococcus aureus Bacillus cereus Micrococcus flavus Sarcina lutea Listeria monocytogenes

S. lanigera

Streptomycin

Ethanol

Water

Ethanol

Water

ATCC 25922 ATCC 14028 ATCC 13076 NCTC 387 ATCC 27853 ATCC 14273

15 15 15 20 25 15

25 30 25 35 35 30

30 25 20 30 35 35

50 40 40 50 N50 N50

0.012 0.010 0.010 0.016 0.016 0.005

ATCC 25932 ATCC 10876 ATCC 14452 ATCC 10054 ATCC 15313

10 10 12 15 10

25 25 20 30 25

20 20 15 25 15

30 40 30 40 30

0.016 0.005 0.010 0.012 0.010

Number of replicates: (n = 1).

The ethanol and water extracts were chosen for further tests of antimicrobial activity, cytotoxicity, and enzyme inhibition, since those solvents are usually recommended in phytotherapy (Stagos et al., 2012). 3.5. Antimicrobial activity Antibacterial activity of S. fruticosa and S. lanigera ethanol and water extracts was tested against six Gram-negative and five Gram-positive pathogenic bacteria and results are presented in Table 5. All of the examined extracts inhibited bacterial growth with MIC values ranged from 10 to 50 mg/mL. Ethanol extract generally showed stronger activity (MICs 10–35 mg/mL) than water extract (MICs from 20 to 50 mg/mL), although inhibitory effect of extracts was weaker comparing to streptomycin (MICs ranged as 0.005–0.016 mg/mL). Giweli et al. (2013) analyzed essential oil of Libyan S. fruticosa from the same locality and their results revealed considerable bacteriostatic (MIC 0.125–1.5 mg/mL) and bactericidal effects (MBC 0.5–2.0 mg/mL) against eight bacteria. In the present study Gram-positive bacteria were more sensitive, which is consistent with previous studies on a range of herbs and spices, among them sage extracts and oils (Shelef et al., 2006; Shan et al., 2007), which could be explained by differences in membranes between bacteria. The cell wall of Gram-negative bacteria possess differences in the outer membrane arrangement which serves as penetration barrier towards macromolecules (Nikaido, 2003). The most sensitive bacteria were S. aureus, B. cereus, L. monocytogenes and M. flavus, which is in accordance with study of Gonaid et al. (2012), but not with Askun et al. (2009). The most resistant bacterium to examined extracts was P. aeruginosa. Using disc diffusion method, Gonaid et al. (2012) demonstrated that the ethanol extract of Libyan S. fruticosa had moderate inhibitory effects against E. coli and P. aeruginosa, but higher than those

obtained for B. subtilis and S. aureus. On the other hand, Askun et al. (2009) found that methanol extract of S. fruticosa displayed high activity on Gram-negative bacteria growth (S. typhimurium and E. aerogenes), which could be probably attributed to the significant amount of carvacrol in the extract. Hawas and El-Ansari (2006) tested ether, chloroform, ethyl acetate, n-butanol, ethanol, and water extracts of Egyptian S. lanigera, among which the ether soluble fraction was found to be highly active against Gram-positive bacteria (Streptomyces viridochromogenes), while the other fractions and pure compounds showed low antimicrobial activities. The essential oils of S. lanigera originated from different countries were studied for the antibacterial activity. The growth of Bacillus species was the most affected by essential oils from Cyprian (Tenore et al., 2011) and Egyptian (Ibrahim et al., 2013) samples. In the study of Al-Howiriny (2003) Staphylococcus strains were the most sensitive to the essential oil obtained from S. lanigera collected in Saudi Arabia. Antifungal activity of S. fruticosa and S. lanigera extracts was investigated using microdilution assay and results are presented in Table 6. The activity of tested extracts was evaluated as low comparing to values obtained for ketoconazole (MIC/MFC = 0.0078–0.0156 mg/mL). Fungistatic and fungicidal effects of S. fruticosa extracts at applied concentrations were not observed for tested Candida species. Salvia lanigera ethanol extract inhibited the growth of all tested Candida species (MIC were ranged from 16 to 64 mg/mL) while water extract affected only C. parapsilosis. Fungicidal effect was not detected for Candida species. Similar to the previous studies (Dulger and Hacioglu, 2008; Salari et al., 2016), Candida species were generally sensitive to Salvia extracts. Aspergillus glaucus was the only sensitive Aspergillus species to tested extracts (MIC/MFC ranged from 8 to 64 mg/mL), particularly to the water extract of S. fruticosa and ethanol extract of S. lanigera. Water extract of both species showed the most expressive effect on T. mentagrophytes growth (MIC/MFC ranged from 8 mg/mL to

Table 6 Antifungal activity of S. fruticosa and S. lanigera extracts and commercial antimycotic ketoconazole. Micromycetes

Strain code

S. fruticosa

S. lanigera

Ethanol

Candida krusei Candida albicans Candida parapsilosis Aspergillus glaucus Aspergillus fumigatus Aspergillus flavus Trichophyton mentagrophytes

BEOFB 821m BEOFB 811m BEOFB 831m BEOFB 301m BEOFB 232m BEOFB 221m BEOFB 1320m

Water

Ketoconazole

Ethanol

Water

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

na – – 64 – – –

na – – 64 – – –

na – – 16 – – 32

na – – 32 – – 32

64 32 16 8 – – 32

na – – – – – –

na – 32 64 – – 8

na – – – – – 8

0.0078 0.0078 0.0078 0.0078 0.0078 0.0078 0.0019

0.0156 0.0156 0.0156 0.0078 0.0156 0.0078 0.0039

MIC — minimum inhibitory concentration (mg/mL), MFC — minimum fungicidal concentration (mg/mL); na — not assessed at tested concentrations; number of replicates: (n = 1).

S. Duletić-Laušević et al. / South African Journal of Botany 117 (2018) 101–109 Table 7 Cytotoxic effects of S. fruticosa and S. lanigera extracts and commercial cytostatic against HCT-116 cell line. Extracts S. fruticosa Ethanol Water S. lanigera Ethanol Water 5-Fluorouracila

24 h

72 h

N500 N500

375.96 ± 2.550 N500

N500 N500 0.018 ± 0.004

N500 N500 0.81 ± 0.49

107

S. lanigera (IC50 values from 9.83 to over 100 μg/mL), especially when acetone extract was applied. The best results were obtained for the liver hepatocellular carcinoma cell lines, for all tested extracts. In the previous study (Alimpić et al., 2015) of water extracts of five Salvia species, S. lanigera extract exhibited low cytotoxic effect on K562 cells (IC50 = 200–400 μg/mL).

3.7. Enzyme inhibition activity

Number of replicates: (n = 3). a According to Žižić et al. (2013).

32 mg/mL). In the research of Gonaid et al. (2012) the ethanol extract of Libyan S. fruticosa demonstrated moderate inhibitory activity against C. albicans, with no effect on A. flavus. In the study of Libyan S. fruticosa, essential oil showed MIC at 0.125–1.0 mg/mL and MFC at 0.125– 1.5 mg/mL against eight fungal species (Giweli et al., 2013), with higher sensitivity of C. albicans and A. fumigatus than those obtained in this study. The essential oils of S. lanigera from Cyprus (Tenore et al., 2011), and from Saudi Arabia (Al-Howiriny, 2003) were effective against Candida species. 3.6. Cytotoxic activity Cytotoxic activity of S. fruticosa and S. lanigera ethanolic and water extracts on human colon carcinoma cell line (HCT-116) was determined by MTT cell viability assay after 24 and 72 h of treatments and results are presented in Table 7. Obtained results showed that only S. fruticosa ethanol extract exhibited cytotoxic activity on HCT-116 cells after 72 h. Although in this study analyzed extracts did not show significant cytotoxic activity on investigated cell line at tested concentrations, several studies pointed out cytotoxic potential of S. fruticosa in other cell lines. In the previous study, the water extract of S. fruticosa was active against human leukemia K562 cell line (IC50 = 386 μg/mL), (Alimpić et al., 2015). Other researchers reported remarkable antiproliferative and cytotoxic effects of S. fruticosa extracts against different cell lines such as human colorectal carcinoma cell lines, HCT15 and CO115 (Xavier et al., 2009), human carcinoma malignant cell lines, HEp-2, RD and AMN3 (Ibrahim and Aqel, 2010), five breast cancer cell lines, MCF-7, T47D, ZR-71-1, BT474 and Vero (Abu-Dahab et al., 2012), colon adenocarcinoma (HCA, HepG2, MCF-7) and human pancreatic carcinoma (HPC) (Badisa et al., 2005). In the study of Fiore et al. (2006), methanol extracts of six Jordanian Salvia species were screened in human cancer cell lines from different histological types. The extract of S. lanigera did not show a dose response effect, but displayed toxic activity at all concentrations tested in each cell line. Shaheen et al. (2011) tested methanol, acetone and n-butanol extracts of S. lanigera and S. splendens from Egypt against selected human tumor cell lines. The obtained results varied depending on the species, extract type and cell line used in the study. S. lanigera extracts in the present study were less active than extracts of Egyptian

Enzyme inhibition activity of S. fruticosa and S. lanigera extracts was tested against two enzymes, acetylcholinesterase (AChE) and tyrosinase (TYR), and results are presented in Table 8. Significant difference in AChE inhibition assay between ethanol and water extracts was observed only for S. fruticosa at concentration of 100 μg/mL. In AChE inhibition assay, S. lanigera extracts, especially ethanol one, performed slightly higher inhibition than both S. fruticosa extracts. Extracts exhibited weaker activity (16.28–32.42%) than standard galanthamine (42.38–57.11%). Although galanthamine inhibited AChE in dosedependent manner, the extracts acted the best at concentration of 50 μg/mL, as it was reported before for some Salvia species (Şenol et al., 2010; Orhan et al., 2013). With concentration increasing, significant difference in AChE inhibition was detected only for S. fruticosa water extract. Ethanol extracts of fourteen Salvia species were tested by Orhan et al. (2013) at the concentrations of 25, 50 and 100 μg/mL and AChE inhibition ranged from 1.00 to 27.95%. Using the same concentrations, Şenol et al. (2010) found that successively obtained extracts of Turkish S. fruticosa performed AChE inhibition from 26.73 to 51.07% for dichloromethane and 34.27–35.78% for ethyl acetate extract, while methanol extract showed no activity. In the study of Orhan and Aslan (2009), ethanol extract of S. fruticosa exhibited a mild inhibiton of AChE in in vitro experiment (IC50 = 0.71 mg/mL) compared with galanthamine, but high in vivo anti-amnesic activity. The essential oil of Cyprian S. fruticosa inhibited AChE similar to S. lavandulifolia (IC50 = 0.04–0.05 mg/mL) after 5 min of incubation (Savelev et al., 2004). As shown in Table 8, both water extracts at the concentration of 50 μg/mL exhibited the highest inhibition of TYR (N70%). The significant difference in TYR inhibition between ethanol and water extracts of S. lanigera at all applied concentrations and for S. fruticosa at concentration of 50 μg/mL was noticed. The increase of concentration significantly affected the inhibition rate of both S. lanigera extracts and S. fruticosa water extract. Salvia fruticosa and S. lanigera extracts performed potent activity (55.26–74.66%) in TYR inhibition assay in comparison with standard kojic acid (33.93–51.81%) at tested concentrations. On the other hand, Orhan et al. (2012) found no or very low inhibition of TYR by 16 Salvia species from Turkey at concentration of 100 μg/mL. In research of Süntar et al. (2011), ethanol extracts of S. cryptantha and S. cyanescens performed TYR inhibition weaker than those obtained in our study. Caffeic acid (Roseiro et al., 2012), kaempferol, quercetin and its glycosides (Chang, 2009), identified in our samples, were previously recognized as strong AChE and TYR inhibitors, while literature data on genkwanin are not available till now.

Table 8 Anti-neurodegenerative activities of S. fruticosa and S. lanigera extracts and commercial inhibitors. Conc. (μg/mL)

AChE inhibition (%) S. fruticosa

25 50 100

TYR inhibition (%) S. lanigera

Galanthamine

Ethanol

Water

Ethanol

Water

24.44 ± 3.72 25.95 ± 5.46 25.73 ± 3.70x

23.17 ± 1.00 26.82 ± 2.04 16.28 ± 0.59x

31.54 ± 3.51 32.42 ± 2.64 31.03 ± 0.43

31.21 ± 0.04 31.79 ± 0.29 30.97 ± 1.15

Number of replicates: (n = 3). x Values within column are significantly different (p b 0.05);

42.38 ± 0.74 50.56 ± 0.51 57.11 ± 1.67

S. fruticosa

S. lanigera

Kojic acid

Ethanol

Water

Ethanol

Water

62.25 ± 0.65 62.35 ± 1.08x 62.99 ± 0.68

59.38 ± 5.75 74.66 ± 1.58x 57.89 ± 7.38

60.61 ± 1.81x 56.35 ± 2.08x 55.26 ± 1.66x

69.97 ± 2.14x 70.20 ± 2.14x 63.98 ± 0.41x

35.73 ± 5.46 33.93 ± 3.78 51.81 ± 2.55

108


4. Conclusion Based on the results obtained by HPLC analysis, it can be concluded that polar extracts (water, methanol and ethanol) of Salvia fruticosa and S. lanigera from Libya were more abundant in polyphenolic components than non-polar ones. Among phenolics, kaempferol glycosides dominated. Alcoholic and water extracts of examined species showed the strongest activity in the most of the applied anti-oxidant assays. Only ethanol extract of S. fruticosa showed cytotoxic activity. Salvia fruticosa extracts displayed stronger antibacterial and tyrosinase inhibition activities, while S. lanigera exhibited better antifungal properties and AChE inhibition. Especially significant result was obtained for tyrosinase inhibition activity, which was even higher than standard kojic acid. 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