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Chemical characterization, cytotoxic, antioxidant, antimicrobial, and enzyme inhibitory effects of different extracts from one sage (Salvia ceratophylla L.) from Turkey: open a new window on industrial purposes

Sengul Uysal*ab, Gokhan Zenginc, Kouadio Ibrahime Sinanc, Gunes Akc, Ramazan Ceylanc, Mohamad Fawzi Mahomoodallyd, Ahmet Uysale, Nabeelah Bibi Sadeerd, József Jekőf, Zoltán Cziákyf, Maria João Rodriguesg, Evren Yıldıztugayh, Fevzi Elbasanh and Luisa Custodiog
aErciyes University Halil Bayraktar Health Services Vocational College, Kayseri, Turkey. E-mail: senguluysal@erciyes.edu.tr
bDrug Application and Research Center, Erciyes University, Kayseri, Turkey
cPhysiology and Biochemistry Research Laboratory, Department of Biology, Science Faculty, Selcuk University, Campus, Konya, Turkey
dDepartment of Health Sciences, Faculty of Medicine and Health Sciences, University of Mauritius, Réduit, Mauritius
eDepartment of Medicinal Laboratory, Vocational School of Health Services, Selcuk University, Konya, Turkey
fAgricultural and Molecular Research and Service Institute, University of Nyíregyháza, Nyíregyháza, Hungary
gCentre of Marine Sciences, University of Algarve, Faculty of Sciences and Technology, Ed. 7, Campus of Gambelas, 8005-139 Faro, Portugal
hDepartment of Biotechnology, Science Faculty, Selcuk University, Campus, Konya, Turkey

Received 27th November 2020 , Accepted 19th January 2021

First published on 28th January 2021


Abstract

In the present study, the methanolic, hydro-methanolic, dichloromethane, hexane and aqueous extracts of Salvia ceratophylla L. (Family: Lamiaceae), a lemon-scented herb, were tested for total phenolic (TPC) and flavonoid content (TFC) and antioxidant activities were evaluated using a battery of assays (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), cupric reducing antioxidant capacity, total antioxidant capacity (TAC) (phosphomolybdenum) and metal chelating). Enzyme inhibitory effects were investigated using acetyl- (AChE), butyryl-cholinesterase (BChE), tyrosinase, α-amylase and α-glucosidase as target enzymes. Regarding the cytotoxic abilities, HepG2, B164A5 and S17 cell lines were used. The phytochemical profile was conducted using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Our data showed that the methanolic aerial extracts possessed the highest phenolic (72.50 ± 0.63 mg gallic acid equivalent per g) and flavonoid (43.77 ± 1.09 mg rutin equivalent per g) contents. The hydro-methanolic aerial extract showed significant DPPH radical scavenging activity (193.40 ± 0.27 mg TE per g) and the highest reducing potential against CUPRAC (377.93 ± 2.38 mg TE per g). The best tyrosinase activity was observed with dichloromethane root extract (125.45 ± 1.41 mg kojic acid equivalent per g). Among the tested extracts, hexane root extract exerted the highest antimicrobial potential with a minimum inhibitory concentration value of 0.048 mg mL−1. Methanolic root extract showed the lowest cytotoxicity (28%) against HepG2 cells. Phytochemical analysis revealed the presence of important polyphenolic compounds including luteolin, gallic acid, rosmarinic acid, to name a few. This research can be used as one methodological starting point for further investigations on this lemon-scented herb.


1. Introduction

Salvia ceratophylla L. (S. ceratophylla) is a biennial lemon-scented herb belonging to one of the largest genera of the Lamiaceae comprising of about 900 species distributed worldwide.1 The herb is native to numerous places such as Afghanistan, Iran, Iraq, Lebanon-Syria, Palestine, the Transcaucasus, Turkey, and Turkmenistan.2 Published literature reported that a number of different Salvia species and their respective essential oils have showed promising pharmacological propensities namely antioxidant, cytotoxicity,3 antibacterial, anti-neurodegenerative,4 anti-enzymatic (anticholinesterase, anti-urease, anti-tyrosinase, anti-elastase),5 anti-tumour6 and antidiabetic activities7 to name a few. The herb, S. ceratophylla, in particular, is aromatic and in a recent analysis its essential oil (EO) was reported to possess anti-trypanosomal effects resulting with an inhibitory concentration (IC) 50 of 2.65 μg mL−1. The hexane extract demonstrated cytotoxicity activity against mouse erythroleukemia (MEL), KB (containing human papillomavirus 18 (HPV-18)), BT-549 (human breast cancer cell line), SK-OV-3 (human ovarian cancer cell line), LLC-PK1 (renal epithelial cell line) and VERO (kidney epithelial cell line) cell lines with IC50 values ranging from 60 to 100 μg mL−1. There are further data concerning antioxidant and chemical composition of the EO.8–11 The review of Ulubelen12 stated that the terpenoids present in S. ceratophylla exhibited interesting antibacterial activity. The work of Goren et al.13 also showed that the diterpenoids identified from the root of the herb exhibited strong antibacterial activity against Staphylococcus epidermidis and Proteus mirabilis. Furthermore, two seco-4,5-abietane diterpenoids showed cytotoxic effects against MOLT-4 (human acute T lymphoblastic leukaemia cells) and MCF-7 (human breast cancer cell line) cell lines.14 In another study, the chloroform extract of S. ceratophylla significantly depressed anti-butyrylcholinesterase activity with a percentage inhibition of 91.3%.15 Based on ethnobotanical information, S. ceratophylla were used to treat cancers, infections, urinary complications,8 inflammation, and even nociceptive disorders.8,16,17 The World Health Organisation outlines that cancer is the second leading cause of death across the globe with 9.6 million deaths recorded in the year 2018.2 Despite cancer is one of the most studied disease and the clinical care and technology have advanced greatly, yet cancer remains still incurable.18 Natural products have been the only storehouse of pharmaceuticals for decades and have contributed enormously in human health through effective and unique bioactive compounds. Oxidative stress involving free radicals is the onset of several chronic diseases including cancers, neurological disorders, and cardiovascular diseases.19 Medicinal plants act as a major reserve of pharmaceuticals since the early days of mankind. Today, more than 80% of medicines are directly or indirectly linked to medicinal plants due to their strong pharmacological properties, low toxicity and low cost.20 On many occasions, natural enzyme inhibitors isolated from medicinal plants have been acknowledged as useful therapeutic tools for the management of numerous human pathologies.

Therefore, the quest for novel and efficient drugs from medicinal plants should be an ongoing process and a continuing need. For this reason, we evaluated the aerial part and root extracts of S. ceratophylla prepared from polar and non-polar solvents for their antioxidant, anti-enzymatic [acetylcholinesterase (AChE), butyrylcholinesterase (BChE), amylase, glucosidase, tyrosinase], anti-microbial and cytotoxicity activities. To the best of our knowledge, this is the first time the polar and non-polar extracts of this plant will be evaluated for the aforementioned studies and compiled in one single research work. The total phenolic and flavonoid content were quantified and the prepared extracts were screened for phytochemicals using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) in order to correlate the observed biological activities with the biomolecules present. We believe that this study will add information on S. ceratophylla that can be used for further investigations.

2. Materials and methods

2.1. Plant material and preparation of extracts

Salvia ceratophylla samples were collected from natural population (Akyer village, Bozdağ national park, 1020 m, and steppes areas) in the summer period of 2019. Botanical identification was performed by one of the co-authors (Dr Evren Yıldıztugay) and a voucher specimen was kept at the herbarium of Selcuk University (EY-3005). Aerial parts and roots were carefully separated, dried in a shade for ten days, and then grinded by using a laboratory mill.

Different extracts were used in this study. To this end, powdered aerials parts and roots (5 g) were extracted in n-hexane, dichloromethane (DCM), methanol, methanol–water (80%) (100 mL) under stirring for 24 h at 25 °C. After that, the solvents were removed by a rotary evaporator and the extracts stored at 4 °C until analysis. Regarding aqueous extracts, we used traditional infusion technique and the plant material (5 g) were kept with 100 mL of boiled water. The extracts were filtered and then lyophilized. All extracts were stored at 4 °C in a refrigerator. The extraction yields (%) are given in Table 1.

Table 1 Extraction yields (%), total phenolic and flavonoid content of Salvia ceratophylla extractsa
Parts Solvents Yield (%) TPC (mg GAE per g) TFC (mg RE per g)
a Values are reported as mean ± SD. DCM: dichloromethane; MeOH: methanol; TPC: total phenolic content; TFC: total flavonoid content; GAE: gallic acid equivalent; RE: rutin equivalent. Different letters indicate significant differences in the extracts (p < 0.05).
Aerial parts Hexane 4.0 17.33 ± 0.10h 5.25 ± 0.12f
DCM 4.96 21.72 ± 0.20f 28.79 ± 1.34b
MeOH 12.11 72.50 ± 0.63a 43.77 ± 1.09a
MeOH/water (80%) 14.26 72.26 ± 0.39a 23.69 ± 0.19c
Aqueous 16.70 69.16 ± 0.56b 18.04 ± 0.25d
Roots Hexane 3.81 19.58 ± 0.04g 2.13 ± 0.10g
DCM 1.75 39.17 ± 0.58f 8.70 ± 0.60e
MeOH 10.26 44.27 ± 0.11e 8.75 ± 0.48e
MeOH/water (80%) 12.04 50.61 ± 0.40c 3.33 ± 0.06g
Aqueous 11.45 45.50 ± 0.24d 2.52 ± 0.02g


2.2. Profile of bioactive compounds

The total phenolic and flavonoid contents were determined using the Folin–Ciocalteu and aluminium chloride (AlCl3) assays, respectively.21,22 Results were expressed as gallic acid (mg GAEs per g extract) and rutin equivalents (mg REs per g extract) for respective assays.

Chromatographic separation was accomplished with a Dionex Ultimate 3000RS UHPLC instrument, equipped with Thermo Accucore C18 (100 mm × 2.1 mm i. d., 2.6 μm) analytical column for separation of compounds. Water (A) and methanol (B) containing 0.1% formic acid were employed as mobile phases, respectively. The total run time was 70 minutes, the elution profile and all exact analytical conditions have been published.23

2.3. Determination of antioxidant and enzyme inhibitory effects

The metal chelating, phosphomolybdenum, ferric reducing antioxidant power (FRAP), cupric reducing antioxidant capacity (CUPRAC), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) activities of the extracts (0.5–5 mg mL−1) were assessed following the methods described by Grochowski et al.24 The antioxidant activities were reported as trolox equivalents, whereas ethylenediaminetetraacetic acid (EDTA) was used for metal chelating assay. The possible inhibitory effects of the extracts (0.5–5 mg mL−1) against cholinesterases (by Ellman's method), tyrosinase, α-amylase and α-glucosidase were evaluated using standard in vitro bio-assays.24 To provide comparison with standard antioxidants and inhibitors, IC50 values were also given (this is extract concentration required for scavenging 50% of radicals, ferrous ion-ferrozine and enzyme inhibitory assays; this is effective concentration at which the absorbance was 0.5 for CUPRAC, FRAP and PBD assays).

2.4. Antimicrobial evaluation

In this study totally twelve microorganisms (eleven bacteria and one yeast) were used to elucidate of antimicrobial potential of S. ceratophylla extracts. Standard microorganisms were obtained from Microbiology Research Laboratory of Vocational School of Health Services, Selcuk University. Broth micro dilution method was conducted for antimicrobial activity of extracts according to Balouiri et al.25

Briefly, 96-well plates were loaded with 100 μL Mueller Hinton Broth medium. Then 100 μL S. ceratophylla extracts were transferred to first well of the plate and serial dilution was done by transferring of 100 μL volume mixture via multichannel pipette. When the extract-medium mixture was ready then fresh microorganism inoculum prepared from 0.5 Mc Farland turbidity and final concentration 5 × 105 were added to each well. Plates were sealed and incubated in an incubator at 37 °C for 18–24 hours. Gentamicin was used as positive control. After incubation period 20 μL of 2,3,5 tri phenyl tetrazolium chloride solution (0.5%) loaded to each well for detecting of minimum inhibitory concentration (MIC) of S. ceratophylla extracts. The MIC is the lowest concentration of antimicrobial agent that completely inhibits growth of the organism in tubes or microdilution wells as detected by the unaided eye.26

2.5. Cell culture

The human hepatocarcinoma HepG2 cells and murine bone marrow stromal S17 cells were kindly provided by the Centre for Molecular and Structural Biomedicine of Biomedical and Molecular BME, University of Algarve, Portugal), while mouse melanoma B16 4A5 cells was purchased from Sigma-Aldrich (Germany). All cell lines were cultured in Dulbecco's Modified Eagle medium (DMEM) supplemented with foetal bovine serum (10%), L-glutamine (2 mM, 1%), and penicillin (50 U mL−1)/streptomycin (50 μg mL−1) (1%), and kept under a humidified atmosphere at 37 °C and 5% CO2.

2.6. Determination of cellular viability and selectivity

Cells were plated in 96-well plates at 5 × 103 cells per well (HepG2 and S17) and 2 × 103 cells per well (B16 4A5). After a 24 h incubation, cells were treated with the samples at the concentration of 100 μg mL−1 for 72 h. Cells incubated with DMSO at 0.5% (the highest DMSO concentration used in the test wells) were used as control. The cellular viability was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, as described formerly.27 The percentage of viable cells was calculated relative to the control (DMSO, 0.5%). Selectivity index (SI) was calculated by the formula SI = CT/CNT, where CT and CNT stands for the cytotoxicity of the extract towards tumoral and non-tumoral cell lines, respectively.28

2.7. Data analysis

Statistical calculations were done using Xlstat 2018 and R v 3.5.1 softwares. Firstly, the one-way ANOVA with Tukey post-hoc test was performed for comparisons among samples. Pearson correlation coefficients were calculated among total bioactive compounds and biological activities. Afterwards, the biological activities dataset was analysed by supervised Partial Least Square Discriminant Analysis PLS-DA. The accuracy of model was recorded by calculating the AUC average. Finally, line plot was used following one-way ANOVA to investigate the effect of extraction solvents on the biological activities of each studied parts respectively.

3. Results and discussion

3.1. Total bioactive compounds and phytochemical composition

Plants and herbs are known to be abounded with scads of phytochemicals possessing medicinal properties such as anti-inflammatory, anticancer, and antioxidant, to name a few.29 The prepared aqueous, hexane, DCM, hydro-methanolic (80%) and methanolic root and aerial part extracts were evaluated for their total phenolic and flavonoid content using colorimetric methods. Results obtained are summarized in Table 1. Upon comparison between the different extracts, hexane root and aerial extracts were found to yield the least amount of phenolic and flavonoids. The same outcomes were reported in previous studies whereby hexane solvent extracted the least amount of phenolic and flavonoid content.30–32 The methanolic aerial extract possessed the highest phenolic (72.50 ± 0.63 mg GAE per g) and flavonoid content (43.77 ± 1.09 mg RE per g). In terms of roots, phenolic content was higher in the hydro-methanolic extract (50.61 ± 0.40 mg GAE per g) in contrast to the methanolic extract (44.27 ± 0.11 mg GAE per g). It can be said that phenolic and flavonoid compounds were better extracted in hydro-methanol and methanol solvents compared to the other extraction solvents.

The LC-MS/MS analysis allowed the characterization of the chemical composition of all the studied extracts obtained from S. ceratophylla. In total, 54 major compounds occurring in the aerial methanolic extract were detected, 47 in methanolic root, 48 in aqueous aerial and 37 in aqueous root extracts. The detailed chromatographic results are given Tables 2–5. Twenty-nine compounds were found in common between the aqueous root and aerial extracts (Fig. 1a) while 38 were common between methanolic root and aerial extracts (Fig. 1b). Fig. 2 shows that a total of 29 phytochemicals were found in common in all four analysed extracts (methanolic root and aerial, aqueous root and aerial).

Table 2 Chemical composition of aerial parts-MeOH
No. Name Formula Rt [M + H]+ [M − H] Fragment 1 Fragment 2 Fragment 3 Fragment 4 Fragment 5
a Confirmed by standard.
1a Gallic acid (3,4,5-trihydroxybenzoic acid) C7H6O5 2.64   169.01370 125.0230 97.0281 69.0331    
2 Dihydroxybenzoic acid C7H6O4 5.50   153.01879 123.0437 109.0281 108.0202 81.0331  
3 Pantothenic acid C9H17NO5 6.06 220.11850   202.1079 184.0973 174.1133 116.0346 90.0556
4 Caftaric acid (2-O-Caffeoyltartaric acid) C13H12O9 8.50   311.04031 179.0340 149.0080 135.0440 87.0072  
5 Dihydroxycoumarin-O-hexoside C15H16O9 12.85 331.15455   179.0342 151.0390 133.0284 123.0444 85.0291
6 Kynurenic acid C10H7NO3 13.80 190.05042   162.0552 144.0444 116.0500 89.0392  
7 Caffeic acid C9H8O4 15.12   179.03444 135.0439 107.0489      
8 Unidentified alkaloid C10H11NO3 16.17 194.08172   166.0865 136.0760 108.0449 87.0447 80.0502
9 Naringenin-6,8-di-C-glucoside C27H32O15 17.31   595.16630 505.1357 475.1238 415.1028 385.0929 355.0821
10 Phaselic acid (2-O-Caffeoylmalic acid) C13H12O8 18.62   295.04540 179.0340 135.0439 133.0130 115.0022 71.0122
11 4-O-Feruloylquinic acid C17H20O9 18.93   367.10291 193.0499 173.0444 134.0360 93.0330  
12 Loliolide C11H16O3 19.99 197.11777   179.1070 161.0963 135.1171 133.1015 107.0860
13 Rosmarinic acid-di-O-hexoside C30H36O18 22.30   683.18234 521.1315 359.0995 323.0777 197.0449 179.0340
14 Luteolin-O-glucuronide isomer 1 C21H18O12 22.49   461.07201 285.0407 217.0501 199.0396 151.0024 133.0280
15 Luteolin-O-hexoside isomer 1 C21H20O11 22.61   447.09274 327.0501 285.0407 284.0329 256.0376 151.0025
16 Luteolin-O-glucuronide isomer 2 C21H18O12 22.71   461.07201 285.0406 217.0500 199.0393 151.0024 133.0279
17 Luteolin-7-O-glucoside (cynaroside) C21H20O11 22.86   447.09274 327.0507 285.0407 284.0330 256.0381 151.0026
18 Rosmarinic acid-O-hexoside C24H26O13 23.38   521.12952 359.0730 323.0772 197.0448 179.0340 161.0232
19 Methoxy-tetrahydroxy(iso)flavone-O-glucuronide C22H20O13 23.40   491.08257 315.0513 300.0277 272.0327 151.0024 113.0230
20 Apigenin-O-glucuronide C21H18O11 24.36   445.07709 269.0456 225.0554 175.0237 117.0332 113.0230
21a Cosmosiin (Apigenin-7-O-glucoside) C21H20O10 24.44 433.11347   271.0603 153.0183 119.0501    
22 Rosmarinic acid (labiatenic acid) C18H16O8 24.65   359.07670 197.0449 179.0340 161.0232 135.0439 133.0283
23 Methyl caffeate C10H10O4 24.67 195.06574   163.0392 145.0287 135.0444 117.0339 89.0392
24 Chrysoeriol-7-O-glucuronide C22H20O12 24.82   475.08766 299.0562 284.0329 256.0376    
25 Apigenin-O-hexoside C21H20O10 24.89   431.09782 311.0562 269.0456 268.0377 151.0021 117.0336
26 Luteolin-O-hexoside isomer 2 C21H20O11 25.10   447.0974 285.0407 284.0330 255.0297 151.0024 133.0279
27 N-trans-feruloyltyramine C18H19NO4 25.12 314.13924   194.0816 177.0548 149.0600 145.0286 121.0651
28 Abscisic acid C15H20O4 25.75   263.12834 219.1385 204.1151 201.1281 152.0831 151.0752
29 Martynoside or isomer C31H40O15 26.20   651.22890 475.1822 193.0500 175.0390 160.0154 134.0361
30 Pentahydroxy(iso)flavone C15H10O7 26.26   301.03483 273.0401 257.0444 151.0023 107.0121  
31 3-O-Methylrosmarinic acid C19H18O8 26.57   373.09235 197.0449 179.0340 175.0390 160.0154 135.0439
32 Dihydroactinidiolide C11H16O2 27.07 181.12286   163.1119 145.1015 135.1171 121.1016 107.0860
33 Methoxy-trihydroxy(iso)flavone isomer 1 C16H12O6 28.06   299.05556 284.0328 283.0252 256.0378 228.0422 227,0345
34a Luteolin (3′,4′,5,7-Tetrahydroxyflavone) C15H10O6 28.37   285.03991 217.0495 199.0393 175.0387 151.0024 133.0282
35 N1,N5,N10-Tricoumaroylspermidine C34H37N3O6 29.46   582.26042 462.2038 436.2245 342.1458 145.0283 119.0488
36 Apigenin (4′,5,7-Trihydroxyflavone) C15H10O5 30.22   269.04500 225.0547 201.0557 151.0024 149.0232 117.0330
37 Chrysoeriol (3′-methoxy-4′,5,7-trihydroxyflavone) C16H12O6 30.44   299.05556 284.0329 283.0251 256.0376 227.0344 151.0018
38 Dihydrololiolide C11H18O3 30.50 199.13342   181.1226 163.1119 135.1172 111.0445 107.0860
39 Methoxy-tetrahydroxy(iso)flavone C16H12O7 30.54   315.05048 300.0277 272.0326 227.0335 151.0026 149.0233
40 Undecanedioic acid C11H20O4 31.32   215.12834 197.1176 153.1272 125.0959 57.0332  
41 Dihydroxy-trimethoxy(iso)flavone C18H16O7 31.83 345.09743   330.0735 329.0663 315.0495 312.0631 284.0682
42 Dihydroxy-dimethoxy(iso)flavone C17H14O6 32.42 315.08686   300.0632 272.0678 257.0447 229.0487  
43 Methoxy-trihydroxy(iso)flavone isomer 2 C16H12O6 33.02   299.05556 284.0328 283.0237 256.0375 227.0346 151.0030
44 Hydroxy-tetramethoxy(iso)flavone C19H18O7 33.31 359.11308   344.0891 343.0810 326.0790 315.0862 298.0838
45 Dodecanedioic acid C12H22O4 33.75   229.14399 211.1334 185.1539 167.1431    
46a Genkwanin (4′,5-dihydroxy-7-methoxyflavone) C16H12O5 35.05 285.07630   270.0525 242.0574 213.0543 167.0341 119.0493
47 Hydroxy-trimethoxy(iso)flavone C18H16O6 35.34 329.10252   314.0788 313.0701 299.0547 296.0683 268.0731
48 Apigenin-4′,7-dimethyl ether (4′,7-dimethoxy-5-hydroxyflavone) C17H14O5 38.71 299.09195   284.0682 256.0731 167.0338 133.0649  
49 Stearidonic acid C18H28O2 40.13   275.20111 231.2107 177.1633 59.0124    
50 Hydroxyoctadecatrienoic acid C18H30O3 40.21   293.21167 275.2020 235.1700 231.2117 171.1018 121.1008
51 Unidentified terpene 1 C20H30O2 41.92 303.23241   285.2215 267.2123 257.2264 247.1695 201.1644
52 Unidentified terpene 2 C30H48O4 43.42 473.36309   455.3521 437.3416 419.3310 401.3207 359.2582
53 Unidentified terpene 3 C30H48O4 43.59 473.36309   455.3523 437.3418 419.3314 401.3216 359.2582
54 Unidentified terpene 4 C30H48O4 44.26 473.36309   455.3520 437.3418 419.3313 401.3202 109.1016


Table 3 Chemical composition of aerial parts-aqueous
No. Name Formula Rt [M + H]+ [M − H] Fragment 1 Fragment 2 Fragment 3 Fragment 4 Fragment 5
a Confirmed by standard.
1 Dihydroxybenzoic acid C7H6O4 5.47   153.01879 123.0439 109.0281 108.0203 81.0331  
2 Pantothenic acid C9H17NO5 6.03 220.11850   202.1088 184.0973 174.1128 116.0347 90.0555
3 Caftaric acid (2-O-Caffeoyltartaric acid) C13H12O9 8.48   311.04031 179.0340 149.0079 135.0439 87.0072  
4 Kynurenic acid C10H7NO3 13.77 190.05042   162.0552 144.0448 116.0497 89.0394  
5 Caffeic acid C9H8O4 15.10   179.03444 135.0439 107.0489      
6 Unidentified alkaloid C10H11NO3 16.15 194.08172   166.0865 136.0760 108.0449 87.0447 80.0502
7 Naringenin-6,8-di-C-glucoside C27H32O15 17.28   595.16630 505.1334 475.1242 415.1036 385.0932 355.0826
8 Phaselic acid (2-O-Caffeoylmalic acid) C13H12O8 18.60   295.04540 179.0340 135.0440 133.0130 115.0022 71.0122
9 Loliolide C11H16O3 19.97 197.11777   179.1070 161.0963 135.1172 133.1016 107.0861
10 Rosmarinic acid-di-O-hexoside C30H36O18 22.28   683.18234 521.1299 359.0994 323.0775 197.0449 179.0340
11 Rosmarinic acid-O-hexoside isomer 1 C24H26O13 22.37   521.12952 359.0753 323.0766 197.0449 179.0340 161.0232
12 Luteolin-O-glucuronide isomer 2 C21H18O12 22.65   461.07201 285.0407 217.0501 199.0389 151.0024 133.0280
13 Luteolin-7-O-glucoside (cynaroside) C21H20O11 22.84   447.09274 327.0524 285.0407 284.0329 256.0371 151.0023
14 Rosmarinic acid-O-hexoside isomer 2 C24H26O13 23.36   521.12952 359.0772 323.0775 197.0448 179.0340 161.0232
15 Methoxy-tetrahydroxy(iso)flavone-O-glucuronide C22H20O13 23.39   491.08257 315.0514 300.0278 272.0326 151.0024 113.0230
16a Cosmosiin (apigenin-7-O-glucoside) C21H20O10 24.45 433.11347   271.0604 153.0186 119.0491    
17 Apigenin-O-glucuronide C21H18O11 24.49   445.07709 269.0457 225.0549 175.0235 117.0332 113.0230
18 Methyl caffeate C10H10O4 24.63 195.06574   163.0392 145.0287 135.0444 117.0339 89.0392
19 Rosmarinic acid (labiatenic acid) C18H16O8 24.66   359.07670 197.0449 179.0340 161.0232 135.0439 133.0282
20 Chrysoeriol-7-O-glucuronide C22H20O12 24.82   475.08766 299.0562 284.0328 256.0385    
21 N-trans-Feruloyltyramine C18H19NO4 25.12 314.13924   194.0816 177.0548 149.0600 145.0286 121.0651
22 Luteolin-O-hexoside isomer 2 C21H20O11 25.13   447.09274 285.0407 284.0328 255.0298 151.0025 133.0280
23 Abscisic acid C15H20O4 25.77   263.12834 219.1385 204.1150 201.1279 152.0830 151.0752
24 Martynoside or isomer C31H40O15 26.22   651.22890 475.1835 193.0499 175.0390 160.0154 134.0362
25 Pentahydroxy(iso)flavone C15H10O7 26.28   301.03483 273.0401 257.0452 151.0025 107.0126  
26 3-O-Methylrosmarinic acid C19H18O8 26.57   373.09235 197.0449 179.0340 175.0389 160.0153 135.0439
27 Dihydroactinidiolide C11H16O2 27.08 181.12286   163.1120 145.1014 135.1172 121.1016 107.0860
28 Martynoside or isomer C31H40O15 27.56   651.22890 475.1806 193.0501 175.0389 160.0152 134.0358
29 Methoxy-trihydroxy(iso)flavone isomer 1 C16H12O6 28.09   299.05556 284.0329 283.0256 256.0375 228.0427 227.0342
30a Luteolin (3′,4′,5,7-Tetrahydroxyflavone) C15H10O6 28.38   285.03991 217.0494 199.0392 175.0392 151.0024 133.0282
31 N1,N5,N10-Tricoumaroylspermidine C34H37N3O6 29.48   582.26042 462.2035 436.2205 342.1466 145.0282 119.0488
32 Apigenin (4′,5,7-Trihydroxyflavone) C15H10O5 30.24   269.04500 225.0550 201.0555 151.0024 149.0233 117.0331
33 Chrysoeriol (3′-methoxy-4′,5,7-trihydroxyflavone) C16H12O6 30.44   299.05556 284.0329 283.0245 256.0378 227.0351 151.0027
34 Dihydrololiolide C11H18O3 30.49 199.13342   181.1226 163.1119 135.1171 111.0445 107.0861
35 Undecanedioic acid C11H20O4 31.32   215.12834 197.1177 153.1273 125.0961 57.0333  
36 Dihydroxy-trimethoxy(iso)flavone C18H16O7 31.82 345.09743   330.0737 329.0654 315.0501 312.0631 284.0682
37 Dihydroxy-dimethoxy(iso)flavone C17H14O6 32.41 315.08686   300.0631 272.0682 257.0448 229.0487  
38 Methoxy-trihydroxy(iso)flavone isomer 2 C16H12O6 33.03   299.05556 284.0329 283.0239 256.0371 227.0346 151.0031
39 Hydroxy-tetramethoxy(iso)flavone C19H18O7 33.31 359.11308   344.0887 343.0818 326.0790 315.0881 298.0839
40 Dodecanedioic acid C12H22O4 33.76   229.14399 211.1334 185.1530 167.1430    
41a Genkwanin (4′,5-dihydroxy-7-methoxyflavone) C16H12O5 35.04 285.07630   270.0526 242.0577 213.0543 167.0342 119.0494
42 Hydroxy-trimethoxy(iso)flavone C18H16O6 35.33 329.10252   314.0786 313.0719 299.0546 296.0682 268.0732
43 Apigenin-4′,7-dimethyl ether (4′,7-dimethoxy-5-hydroxyflavone) C17H14O5 38.71 299.09195   284.0683 256.0732 167.0344 133.0654  
44 Stearidonic acid C18H28O2 40.15   275.20111 231.2120 177.1633 59.0126    
45 Hydroxyoctadecatrienoic acid C18H30O3 40.22   293.21167 275.2019 235.1700 231.2110 171.1016 121.1008
46 Unidentified terpene 1 C20H30O2 41.94 303.23241   285.2216 267.2104 257.2267 247.1689 201.1644
47 Unidentified terpene 2 C30H48O4 43.42 473.36309   455.3527 437.3422 419.3322 401.3216 359.2585
48 Unidentified terpene 4 C30H48O4 44.30 473.36309   455.3526 437.3422 419.3319 401.3214 109.1017


Table 4 Chemical composition of root-MeOH
No. Name Formula Rt [M + H]+ [M − H] Fragment 1 Fragment 2 Fragment 3 Fragment 4 Fragment 5
a Confirmed by standard.
1a Gallic acid (3,4,5-trihydroxybenzoic acid) C7H6O5 2.69   169.01370 125.0230 97.0279 69.0331    
2 Dihydroxybenzoic acid C7H6O4 5.55   153.01879 123.0438 109.0281 108.0203 81.0331  
3 Pantothenic acid C9H17NO5 6.17 220.11850   202.1077 184.0973 174.1124 116.0346 90.0555
4 Caftaric acid (2-O-Caffeoyltartaric acid) C13H12O9 8.56   311.04031 179.0341 149.0080 135.0439 87.0070  
5 Salicylic acid-2-O-glucoside C13H16O8 13.50   299.07670 137.0232 113.0230 93.0330 85.0280 71.0123
6 Kynurenic acid C10H7NO3 13.82 190.05042   162.0552 144.0447 116.0498 89.0392  
7 Caffeoylhexose C15H18O9 14.88   341.08726 179.0340 135.0440 107.0486 89.0229 71.0124
8 Caffeic acid C9H8O4 15.13   179.03444 135.0439 107.0489      
9 Phaselic acid (2-O-Caffeoylmalic acid) C13H12O8 18.61   295.04540 179.0341 135.0440 133.0130 115.0022 71.0122
10 4-O-Feruloylquinic acid C17H20O9 18.92   367.10291 193.0498 173.0445 134.0361 93.0330  
11 Loliolide C11H16O3 19.98 197.11777   179.1070 161.0963 135.1172 133.1015 107.0861
12 Rosmarinic acid-di-O-hexoside C30H36O18 22.28   683.18234 521.1306 359.1000 323.0775 197.0449 179.0341
13 Luteolin-O-glucuronide isomer 2 C21H18O12 22.74   461.07201 285.0407 217.0495 199.0393 151.0025 133.0281
14 Luteolin-7-O-glucoside (cynaroside) C21H20O11 22.83   447.09274 327.0510 285.0408 284.0330 256.0377 151.0023
15 Rosmarinic acid-O-hexoside C24H26O13 23.38   521.12952 359.0770 323.0774 197.0450 179.0341 161.0233
16a Cosmosiin (apigenin-7-O-glucoside) C21H20O10 24.46 433.11347   271.0603 153.0184 119.0495    
17 Apigenin-O-glucuronide C21H18O11 24.49   445.07709 269.0457 225.0544 175.0235 117.0330 113.0230
18 Rosmarinic acid (labiatenic acid) C18H16O8 24.64   359.07670 197.0450 179.0341 161.0233 135.0440 133.0283
19 Methyl caffeate C10H10O4 24.65 195.06574   163.0392 145.0287 135.0444 117.0339 89.0391
20 Luteolin-O-hexoside isomer 2 C21H20O11 25.10   447.09274 285.0408 284.0336 255.0304 151.0025 133.0283
21 N-trans-Feruloyltyramine C18H19NO4 25.12 314.13924   194.0820 177.0549 149.0602 145.0287 121.0652
22 Martynoside or isomer C31H40O15 26.21   651.22890 475.1812 193.0500 175.0390 160.0154 134.0361
23 3-O-Methylrosmarinic acid C19H18O8 26.57   373.09235 197.0449 179.0340 175.0390 160.0154 135.0439
24 Dihydroactinidiolide C11H16O2 27.08 181.12286   163.1120 145.1016 135.1172 121.1016 107.0861
25 Methoxy-trihydroxy(iso)flavone isomer 1 C16H12O6 28.07   299.05556 284.0330 283.0243 256.0378 228.0424 227.0351
26a Luteolin (3′,4′,5,7-Tetrahydroxyflavone) C15H10O6 28.36   285.03991 217.0499 199.0395 175.0390 151.0025 133.0282
27 Apigenin (4′,5,7-Trihydroxyflavone) C15H10O5 30.22   269.04500 225.0553 201.0557 151.0026 149.0233 117.0331
28 Chrysoeriol (3′-methoxy-4′,5,7-trihydroxyflavone) C16H12O6 30.43   299.05556 284.0329 283.0255 256.0377 227.0352 151.0023
29 Undecanedioic acid C11H20O4 31.30   215.12834 197.1178 153.1273 125.0959 57.0332  
30 Dihydroxy-trimethoxy(iso)flavone C18H16O7 31.81 345.09743   330.0736 329.0659 315.0503 312.0631 284.0682
31 Dihydroxy-dimethoxy(iso)flavone C17H14O6 32.42 315.08686   300.0633 272.0681 257.0439 229.0487  
32 Methoxy-trihydroxy(iso)flavone isomer 2 C16H12O6 32.99   299.05556 284.0329 283.0252 256.0375 227.0344 151.0029
33 Dodecanedioic acid C12H22O4 33.75   229.14399 211.1334 185.1556 167.1431    
34a Genkwanin (4′,5-dihydroxy-7-methoxyflavone) C16H12O5 35.04 285.07630   270.0526 242.0576 213.0552 167.0342 119.0495
35 Hydroxy-trimethoxy(iso)flavone C18H16O6 35.32 329.10252   314.0788 313.0718 299.0540 296.0683 268.0732
36 Unidentified terpene 5 C20H30O3 36.08 319.22732   301.2169 291.2325 289.2166 277.1802 165.0914
37 Unidentified terpene 6 C20H26O4 38.49 331.19094   313.1800 295.1698 267.1746 229.1226 211.1121
38 Apigenin-4′,7-dimethyl ether (4′,7-dimethoxy-5-hydroxyflavone) C17H14O5 38.69 299.09195   284.0682 256.0732 167.0340 133.0650  
39 Unidentified terpene 7 C21H28O4 39.90 345.20658   327.1961 313.1799 295.1696 267.1746 229.1226
40 Unidentified terpene 8 C20H26O4 40.00 331.19094   313.1802 295.1700 267.1744 229.1226 211.1121
41 Hydroxyoctadecatrienoic acid C18H30O3 40.21   293.21167 275.2020 235.1692 231.2117 171.1012 121.1012
42 Unidentified terpene 9 C21H28O4 41.96 345.20658   327.1966 313.1802 295.1700 267.1746 229.1226
43 Viridoquinone C20H24O2 42.23 297.18546   279.1748 269.1896 239.1433 237.1277 197.0966
44 Unidentified terpene 2 C30H48O4 43.39 473.36309   455.3525 437.3420 419.3312 401.3196 359.2586
45 Unidentified terpene 3 C30H48O4 43.56 473.36309   455.3528 437.3425 419.3318 401.3213 359.2586
46 Unidentified terpene 4 C30H48O4 44.25 473.36309   455.3527 437.3424 419.3318 401.3228 109.1017
47 Unidentified terpene 10 C30H50O2 46.23 443.38891   425.3799 407.3697 217.1951 203.1799 191.1799


Table 5 Chemical composition of roots-aqueous
No. Name Formula Rt [M + H]+ [M − H] Fragment 1 Fragment 2 Fragment 3 Fragment 4 Fragment 5
a Confirmed by standard.
1 Dihydroxybenzoic acid C7H6O4 5.51   153.01879 123.0438 109.0280 108.0203 81.0332  
2 Pantothenic acid C9H17NO5 6.15 220.11850   202.1077 184.0973 174.1128 116.0347 90.0556
3 Caftaric acid (2-O-Caffeoyltartaric acid) C13H12O9 8.53   311.04031 179.0340 149.0079 135.0439 87.0071  
4 Salicylic acid-2-O-glucoside C13H16O8 13.49   299.07670 137.0232 113.0230 93.0330 85.0279 71.0123
5 Kynurenic acid C10H7NO3 13.80 190.05042   162.0552 144.0446 116.0499 89.0393  
6 Caffeoylhexose C15H18O9 14.88   341.08726 179.0340 135.0439 107.0486 89.0228 71.0123
7 Caffeic acid C9H8O4 15.13   179.03444 135.0439 107.0489      
8 Phaselic acid (2-O-Caffeoylmalic acid) C13H12O8 18.61   295.04540 179.0340 135.0439 133.0130 115.0022 71.0122
9 Loliolide C11H16O3 19.99 197.11777   179.1070 161.0963 135.1172 133.1016 107.0861
10 Rosmarinic acid-di-O-hexoside C30H36O18 22.30   683.18234 521.1307 359.1003 323.0774 197.0449 179.0340
11 Luteolin-O-glucuronide isomer 2 C21H18O12 22.73   461.07201 285.0406 217.0501 199.0387 151.0025 133.0280
12 Luteolin-7-O-glucoside (cynaroside) C21H20O11 22.82   447.09274 327.0513 285.0407 284.0329 256.0371 151.0023
13 Rosmarinic acid-O-hexoside C24H26O13 23.38   521.12952 359.0762 323.0773 197.0449 179.0340 161.0232
14a Cosmosiin (Apigenin-7-O-glucoside) C21H20O10 24.45 433.11347   271.0603 153.0183 119.0496    
15 Apigenin-O-glucuronide C21H18O11 24.48   445.07709 269.0457 225.0553 175.0238 117.0327 113.0230
16 Rosmarinic acid (labiatenic acid) C18H16O8 24.67   359.07670 197.0449 179.0340 161.0232 135.0439 133.0283
17 Methyl caffeate C10H10O4 24.68 195.06574   163.0392 145.0287 135.0444 117.0339 89.0391
18 N-trans-Feruloyltyramine C18H19NO4 25.11 314.13924   194.0822 177.0547 149.0598 145.0286 121.0653
19 Martynoside or isomer C31H40O15 26.21   651.22890 475.1839 193.0501 175.0390 160.0154 134.0361
20 3-O-Methylrosmarinic acid C19H18O8 26.57   373.09235 197.0449 179.0340 175.0390 160.0154 135.0439
21 Dihydroactinidiolide C11H16O2 27.07 181.12286   163.1119 145.1014 135.1172 121.1015 107.0860
22 Martynoside or isomer C31H40O15 27.56   651.22890 475.1825 193.0500 175.0390 160.0154 134.0361
23a Luteolin (3′,4′,5,7-Tetrahydroxyflavone) C15H10O6 28.38   285.03991 217.0509 199.0388 175.0390 151.0023 133.0282
24 Apigenin (4′,5,7-Trihydroxyflavone) C15H10O5 30.23   269.04500 225.0549 201.0553 151.0024 149.0229 117.0332
25 Undecanedioic acid C11H20O4 31.31   215.12834 197.1177 153.1273 125.0959 57.0332  
26 Dihydroxy-dimethoxy(iso)flavone C17H14O6 32.43 315.08686   300.0630 272.0682 257.0434 229.0487  
27 Dodecanedioic acid C12H22O4 33.76   229.14399 211.1334 185.1533 167.1430    
28a Genkwanin (4′,5-dihydroxy-7-methoxyflavone) C16H12O5 35.05 285.07630   270.0528 242.0575 213.0552 167.0342 119.0497
29 Hydroxy-trimethoxy(iso)flavone C18H16O6 35.34 329.10252   314.0787 313.0709 299.0543 296.0683 268.0732
30 Unidentified terpene 5 C20H30O3 36.07 319.22732   301.2164 291.2324 289.2161 277.1803 165.0913
31 Unidentified terpene 6 C20H26O4 38.51 331.19094   313.1800 295.1696 267.1747 229.1226 211.1121
32 Apigenin-4′,7-dimethyl ether (4′,7-dimethoxy-5-hydroxyflavone) C17H14O5 38.72 299.09195   284.0682 256.0732 167.0346 133.0650  
33 Unidentified terpene 8 C20H26O4 40.00 331.19094   313.1799 295.1694 267.1748 229.1227 211.1121
34 Hydroxyoctadecatrienoic acid C18H30O3 40.22   293.21167 275.2019 235.1702 231.2116 171.1014 121.1009
35 Viridoquinone C20H24O2 42.24 297.18546   279.1748 269.1897 239.1433 237.1276 197.0965
36 Unidentified terpene 2 C30H48O4 43.42 473.36309   455.3525 437.3422 419.3309 401.3203 359.2583
37 Unidentified terpene 4 C30H48O4 44.28 473.36309   455.3522 437.3423 419.3311 401.3228 109.1017



image file: d0ra10044g-f1.tif
Fig. 1 Venn diagrams displaying common compounds between different (a) aqueous (b) methanolic extracts.

image file: d0ra10044g-f2.tif
Fig. 2 Venn diagram showing number of common compounds found in all four analysed extracts (methanolic root and aerial, aqueous root and aerial).

3.2. Antioxidant activities

Six methods namely DPPH, ABTS, FRAP, CUPRAC, phosphomolybdenum and metal chelating were used to assess the antioxidant activities of the prepared extracts. Table 6 details the data gathered in this work. The remarkably high antioxidant activity was found to be distributed among the hydro-methanolic, methanolic and aqueous extracts while the hexane extracts exhibited the lowest antioxidant activity with all methods irrespective of the plant part used. For instance, the hydro-methanolic aerial extract showed the maximum DPPH radical scavenging activity (193.40 ± 0.27 mg TE per g) and the highest reducing potential towards copper(II) (377.93 ± 2.38 mg TE per g). Among the different root extracts analysed, the hydro-methanolic sample revealed to be the most potent ABTS radical scavenger (116.50 ± 1.65 mg TE per g) and displayed the highest reducing potential with both CUPRAC (250.03 ± 2.65 mg TE per g) and FRAP (142.00 ± 0.14 mg TE per g) assays. Similar findings were recorded in previous work showing that hydro-alcoholic extracts possessed substantially higher antioxidant activity compared to other extracts derived from low polarity solvents.33,34 In a study conducted by Orhan et al.,15 the methanolic extract displayed a high percentage inhibition of 84.8 ± 1.11 against DPPH radicals, corroborating our results. The total antioxidant capacity of the aerial and root extracts ranged from 1.81–2.48 and 0.97–2.41 mmol TE per g, respectively. The metal chelating ability was higher with aqueous aerial (28.25 ± 0.34 mg EDTAE per g) followed by root (27.83 ± 0.49 mg EDTAE per g) extracts. Mounting evidence showed that natural products play a vital role in hindering β-amyloid fibril aggregation due to their ability to bind metal ions with high affinities.35
Table 6 Antioxidant properties of Salvia ceratophylla extractsa
Parts Solvents DPPH ABTS CUPRAC FRAP MCA PBD
(mg TE per g) IC50 (mg mL−1) (mg TE per g) IC50 (mg mL−1) (mg TE per g) IC50 (mg mL−1) (mg TE per g) IC50 (mg mL−1) (mg TE per g) IC50 (mg mL−1) (mmol TE per g) IC50 (mg mL−1)
a Values are reported as mean ± SD. DCM: dichloromethane; MeOH: methanol; TE: trolox equivalent; EDTAE: EDTA equivalent; MCA: metal chelating ability; PBD: phosphomolybdenum.; nt: no tested. Different letters indicate significant differences in the extracts (p < 0.05, the letter “a” indicates strong ability). IC50 (mg mL−1), effective concentration at which the absorbance was 0.5 for CUPRAC, FRAP and PBD assays and at which 50% of the DPPH and ABTS radicals were scavenged and the ferrous ion-ferrozine complex were inhibited.
Aerial parts Hexane 6.47 ± 0.80h >5 3.88 ± 0.34i >5 48.30 ± 0.57i 2.68 ± 0.03k 23.91 ± 1.41h 1.97 ± 0.12i na na 1.81 ± 0.08bc 1.44 ± 0.06cd
DCM 11.83 ± 1.37g 4.66 ± 0.54h 10.40 ± 1.38h >5 79.73 ± 1.13h 1.62 ± 0.02i 31.27 ± 0.51h 1.50 ± 0.02i na na 2.20 ± 0.19ab 1.19 ± 0.11bc
MeOH 188.81 ± 0.68b 0.29 ± 0.01c 125.36 ± 0.43c 0.60 ± 0.01d 324.13 ± 11.42c 0.40 ± 0.01d 172.49 ± 6.32b 0.27 ± 0.01c 17.89 ± 0.59e 1.15 ± 0.04f 2.48 ± 0.22a 1.06 ± 0.10b
MeOH/water (80%) 193.40 ± 0.27a 0.28 ± 0.01b 155.43 ± 1.38b 0.48 ± 0.01c 377.93 ± 2.38a 0.34 ± 0.01b 217.46 ± 3.46a 0.22 ± 0.01b 19.38 ± 0.29c 1.06 ± 0.02d 2.40 ± 0.21a 1.09 ± 0.10b
Aqueous 187.33 ± 0.86b 0.29 ± 0.01c 191.93 ± 2.42a 0.39 ± 0.01b 342.83 ± 2.43b 0.38 ± 0.01c 219.20 ± 1.72a 0.21 ± 0.01b 28.25 ± 0.34a 0.73 ± 0.02b 1.93 ± 0.09bc 1.36 ± 0.06cd
Roots Hexane 46.13 ± 0.73f 1.18 ± 0.02g 37.03 ± 0.51g 2.03 ± 0.03h 84.29 ± 2.93h 1.54 ± 0.05i 47.72 ± 0.10g 0.99 ± 0.01h 1.63 ± 0.07f >5 0.97 ± 0.05d 2.68 ± 0.15e
DCM 80.61 ± 0.46e 0.68 ± 0.01f 92.76 ± 1.00f 0.81 ± 0.01g 183.12 ± 0.85g 0.71 ± 0.01h 98.33 ± 2.67f 0.48 ± 0.01g 23.43 ± 0.31b 0.87 ± 0.01c 2.41 ± 0.08a 1.08 ± 0.04b
MeOH 97.60 ± 0.32c 0.56 ± 0.01d 105.25 ± 1.97e 0.71 ± 0.01f 229.95 ± 0.63e 0.56 ± 0.01f 128.91 ± 0.83d 0.36 ± 0.01e 17.91 ± 0.24d 1.14 ± 0.02e 1.81 ± 0.12bcd 1.45 ± 0.10cde
MeOH/water (80%) 96.95 ± 0.04c 0.56 ± 0.01d 116.50 ± 1.65d 0.65 ± 0.01e 250.03 ± 2.65d 0.52 ± 0.01e 142.00 ± 0.14c 0.33 ± 0.01d 18.96 ± 0.31c 1.08 ± 0.02d 1.73 ± 0.05cd 1.51 ± 0.05de
Aqueous 89.70 ± 1.51d 0.61 ± 0.01e 105.46 ± 0.64e 0.71 ± 0.01 200.52 ± 1.28f 0.64 ± 0.01 115.01 ± 1.97e 0.41 ± 0.01f 27.83 ± 0.49a 0.74 ± 0.01b 1.51 ± 0.14d 1.73 ± 0.15e
Standards Trolox 0.05 ± 0.01a   0.07 ± 0.01a 0.13 ± 0.01a 0.05 ± 0.01a nt 0.65 ± 0.01a
EDTA nt   nt nt nt 0.02 ± 0.01a nt


3.3. Enzyme inhibitory effects

In this research work, the extracts of S. ceratophylla were screened for possible enzyme inhibitory effects against several non-communicable diseases including diabetes mellitus type II (α-amylase and α-glucosidase), Alzheimer's disease (AChE and BChE) and skin hyperpigmentation (tyrosinase). These aforementioned diseases were targeted since no cure has been found yet to combat such pathological disorders and the statistics presented by the World Health Organisation (WHO) is alarming. For instance, more than 420 million people have been diagnosed with diabetes2 and about 50 million people have dementia.2 Hence, searching for treatment and novel drugs should be an ongoing process. The WHO has approved drugs derived from plants to combat diabetes for various reasons, such as: (i) non-toxicity, (ii) negligible adverse effects compared to synthetic antidiabetic drugs, (iii) economically viable and, (iv) their safety has been confirmed through traditional medicine.36

Results obtained from the enzyme inhibitory effects of S. ceratophylla are shown in Table 7. All samples exhibited inhibitory activities against tyrosinase, amylase and glucosidase. Both aqueous root and aerial extracts were ineffective against cholinesterase enzymes. The petroleum ether and ethyl acetate extracts were also found inactive against BChE according to the study of Orhan et al.15 The DCM root and aerial extracts showed the highest tyrosinase (125.45 ± 1.41 and 124.68 ± 4.47 mg KAE per g, respectively) and amylase (0.76 ± 0.02 and 0.84 ± 0.02 mmol ACAE per g, respectively) activities. To the best of our knowledge, it is the first time S. ceratophylla was screened for tyrosinase, amylase and glucosidase activities. Therefore, comparison of our data with other work was not possible.

Table 7 Enzyme inhibitory properties of Salvia ceratophylla extractsa
Parts Solvents AChE inhibition BChE inhibition Tyrosinase inhibition Amylase inhibition Glucosidase inhibition
(mg GALAE per g) IC50 (mg mL−1) (mg GALAE per g) IC50 (mg mL−1) (mg KAE per g) IC50 (mg mL−1) (mmol ACAE per g) IC50 (mg mL−1) (mmol ACAE per g) IC50 (mg mL−1)
a Values are reported as mean ± SD. DCM: dichloromethane; MeOH: methanol; GALAE: galantamine equivalent; KAE: kojic acid equivalent; ACAE: acarbose equivalent; na: not active.; nt: not tested. Different letters indicate significant differences in the extracts (p < 0.05, the letter “a” indicates strong ability). IC50 (mg mL−1), inhibition concentration at which 50% of the enzyme activities were inhibited.
Aerial parts Hexane 3.78 ± 0.36c 0.71 ± 0.07d 5.65 ± 0.45a 1.06 ± 0.09b 96.32 ± 4.09cd 0.90 ± 0.04de 0.75 ± 0.05b 1.78 ± 0.12c 2.13 ± 0.01cd 0.55 ± 0.01de
DCM 3.22 ± 0.04d 0.84 ± 0.01e 6.55 ± 1.33a 0.94 ± 0.19b 124.68 ± 4.47a 0.69 ± 0.02b 0.84 ± 0.02a 1.59 ± 0.03b 2.17 ± 0.01c 0.54 ± 0.01d
MeOH 4.37 ± 0.27ab 0.62 ± 0.04bc 2.81 ± 0.36b 2.14 ± 0.25c 107.99 ± 8.04bc 0.80 ± 0.06cd 0.72 ± 0.03bc 1.85 ± 0.07cd 2.16 ± 0.02c 0.55 ± 0.01d
MeOH/water (80%) 2.58 ± 0.03e 1.04 ± 0.01f na na 111.50 ± 4.42abc 0.78 ± 0.03bcd 0.73 ± 0.01bc 1.83 ± 0.03cd 0.24 ± 0.01g 4.99 ± 0.30h
Aqueous na na na na 82.68 ± 8.12de 1.05 ± 0.11ef 0.14 ± 0.01d >5 0.05 ± 0.01h >5
Roots Hexane 3.93 ± 0.15bc 0.68 ± 0.03cd 6.99 ± 0.42a 0.85 ± 0.05b 112.10 ± 1.73ab 0.77 ± 0.01bc 0.68 ± 0.01c 1.95 ± 0.01d 2.21 ± 0.01bc 0.53 ± 0.01cd
DCM 4.62 ± 0.13a 0.58 ± 0.02b na na 125.45 ± 1.41a 0.69 ± 0.01b 0.76 ± 0.02b 1.76 ± 0.04c 2.07 ± 0.01c 0.57 ± 0.01d
MeOH 4.17 ± 0.03abc 0.64 ± 0.01bcd 6.19 ± 0.29a 0.96 ± 0.04b 116.23 ± 7.23ab 0.74 ± 0.05bc 0.70 ± 0.01bc 1.90 ± 0.03cd 2.31 ± 0.01a 0.51 ± 0.01b
MeOH/water (80%) 2.73 ± 0.22de 0.99 ± 0.08ef 3.67 ± 0.25b 1.63 ± 0.11c 106.56 ± 4.50bc 0.81 ± 0.04cd 0.75 ± 0.01b 1.77 ± 0.02c 1.02 ± 0.07d 1.16 ± 0.08e
Aqueous na na na na 73.36 ± 1.85e 1.18 ± 0.03f 0.13 ± 0.01d >5 0.79 ± 0.03e 1.49 ± 0.07f
Standards Galantamine 0.0027 ± 0.001a 0.006 ± 0.001a nt nt nt
Kojic acid nt nt 0.09 ± 0.01a nt nt
Acarbose nt nt nt 0.86 ± 0.01a 0.76 ± 0.01a


3.4. Antimicrobial evaluation

The broth microdilution assay results were given in Table 8. According to the results obtained from test, hexane extracts of aerial parts of S. ceratophylla revealed MIC values ranging between 3.12–0.019 mg mL−1 doses. It was seen that Sarcina lutea was the most sensitive bacteria against to aerial hexane extract with a dose of 0.097 mg mL−1 MIC and followed by the Bacillus cereus with 0.19 MIC value. For Citrobacter MIC was found as 1.56 mg mL−1. Aerial part hexane extract had antifungal capacity at dose of 3.12 mg mL−1 against Candida albicans. While Pseudomonas aeruginosa was resistant to aerial part hexane extract it affected from root extracts at a concentration of 1.56 mg mL−1. Same as Pseudomonas, root extract was more effective than aerial part extract against Staphylococcus aureus with 0.39 mg mL−1 MIC value. The root hexane extract of S. ceratophylla manifested very significant antibacterial activity against S. lutea and B. cereus at a dose of 0.048 mg mL−1. It was effective against Proteus at 1.56 mg mL−1 MIC and Candida was more sensitive against root extract than aerial part hexane extract with 0.78 mg mL−1 MIC. When the dichloromethane extracts were evaluated it was determined that S. lutea affected from DCM aerial part extract at a dose of 0.097 mg mL−1 and affected from root extract at a concentration of 0.048 mg mL−1. MIC values were determined as 0.097 mg mL−1 both for two extract against B. cereus. Root extracts was more effective against S. aureus than aerial part extract with 0.097 mg mL−1. Two extracts of DCM affected P. aeruginosa at 1.56 mg mL−1 dose. Antifungal activity was observed at 3.12 mg mL−1 dose for two DCM extracts. The lowest MIC value was determined for methanol aerial part extract against B. cereus and S. lutea at a dose of 0.78 mg m−1. For root methanol extract B. cereus was the sensitive bacterium with 0.19 mg mL−1 MIC value. Salmonella enteritidis, which was resistant to hexane and DCM extracts, affected from aerial part methanol extract at 1.56 mg mL−1 concentration. Similarly, Klebsiella pneumoniae was sensitive to root methanol extract at a dose of 1.56 mg mL−1 while this bacterium resistant to hexane and DCM extracts. Except for Yersinia enterocolitica and Salmonella typhimurium, most of the bacteria showed MIC value ranging between 3.12-1.56 mg mL−1 concentrations against methanol extracts.
Table 8 Minimum inhibitory concentrations of Salvia ceratophylla extracts against pathogenic microorganisms
Strains MIC values of Salvia ceratophylla extracts (mg mL−1) Gentamicin (μg mL−1)
Hexane DCM Methanol Methanol/water Aqueous
Aerial Root Aerial Root Aerial Root Aerial Root Aerial Root
Escherichia coli ATCC 25922 1.56 6.25 1.95
Pseudomonas aeruginosa ATCC 27853 1.56 1.56 1.56 1.56 1.56 1.56 1.56 <0.97
Klebsiella pneumoniae ATCC 70603 1.56 1.56 1.56 7.81
Staphylococcus aureus ATCC 43300 3.12 0.39 3.12 0.097 1.56 1.56 1.56 0.78 1.56 3.12 1.95
Salmonella enteritidis ATTC 13076 1.56 1.56 1.56 1.56 1.95
Sarcina lutea ATCC 9341 0.097 0.048 0.097 0.048 0.78 1.56 0.39 0.19   1.95
Salmonella typhimurium NRRLE 4463 1.56 1.56 1.95
Yersinia enterocolitica ATCC 1501 6.25 6.25 1.95
Proteus mirabilis ATCC 25933 3.12 1.56 3.12 3.12 3.12 1.56 1.56 1.56 3.12 3.12 1.95
Bacillus cereus ATTC 11778 0.19 0.048 0.097 0.097 0.78 0.19 0.39 0.097 1.95
Citrobacter freundii ATCC 8090 1.56 1.56 1.56 1.56 1.56 1.56 1.56 1.56 6.25 6.25 1.95
Candida albicans ATCC 26555 3.12 0.78 3.12 3.12 3.12 3.12 1.56 3.12 3.12 7.81


Methanol and water mixture aerial part extract of S. ceratophylla revealed MIC values between 3.12 to 1.56 mg mL−1 doses. Although MIC values for S. lutea and B. cereus were determined as 0.39 mg mL−1, this extract was effective against S. typhimurium at a dose of 1.56 mg mL−1 when compared previous three extracts. Escherichia coli only affected from aerial part extract at 1.56 mg mL−1 dose. The lowest MIC reported for root extract was 0.097 mg mL−1 for B. cereus. Infusion aerial part extract manifested antibacterial activity against S. aureus at a dose of 1.56 mg mL−1. Similarly, infusion root extract had antibacterial capacity against S. enteritidis (1.56 mg mL−1) only infusion extracts were effective against Y. enterocolitica with 6.25 mg mL−1 MIC value. The results showed that S. ceratophylla extracts had significant antibacterial activities against Gram positive bacteria (B. cereus, S. lutea and S. aureus) than Gram negative bacteria. Especially hexane and DCM root extracts revealed very good antibacterial activity against Gram positive bacteria at 0.048 mg mL−1 dose. The lowest MIC values were determined against S. lutea and B. cereus. The study showed that Y. enterocolitica and E. coli were the most resistant bacteria. K. pneumoniae affected from methanol-based extracts. Also extracts had antifungal capacity against Candida albicans. Hexane root extract showed the lowest antifungal activity at a dose of 0.78 mg mL−1.

Several Salvia species reported for their antimicrobial activity and pharmacological properties37,38 revealed that Salvia species contain caffeic acids, major group of phenolic acids, and derivatives. Caffeic acid plays a central role in the biochemistry of Lamiaceae and occurs predominantly in the dimer form as rosmarinic acid.39 The trimers and tetramers are also interesting from a therapeutic point of view as they have demonstrated various biological activities such as anti-oxidant, antimicrobial and anticancer.40 Chemical composition analyses showed that S. ceratophylla extracts tested in this assay included phenolic compounds such as rosmarinic acid and caffeic acid. In a study conducted by Matejczyk et al.,41 it was determined that caffeic acid revealed significant antimicrobial action against tested pathogens. Also, Li and Na salts of caffeic acid had an important activity, too. In that study also rosmarinic acid and its Li, Na and K salts were tested and better results were observed. Świsłocka42 reported that rosmarinic acid had bactericidal activity against Staphylococcus epidermidis, Stenotrophomonas maltophilia, and Enterococcus faecalis. Antimicrobial mechanisms of rosmarinic acid has not been explained clearly yet. But there were several studies about antibacterial mechanism of phenolic acids. The possible explanation for this situation could be as follows: the phenolic acids have pro-oxidative properties and they can alter the hydrophobicity and after the charging of the cell surface cellular cracking and formation can occur. The main mechanism of action of rosmarinic acid is its ability to damage the cell membrane.43 Significant antimicrobial activities of extracts determined in this study can be attributed to presence of rosmarinic and caffeic acid in S. ceratophylla.

3.5. Cytotoxicity effects

Plant-derived natural products have been considered as promising and potent chemotherapeutic agents for more than 40 years.44 In this study, the extracts of S. ceratophylla were evaluated against HepG2 (a human liver cancer cell line) and B164A5 (a skin melanoma cell line). The effects of the extracts on the viability of S17 cells, from non-tumoral origin, were also determined. Results are shown in Table 9.
Table 9 Cellular viability (%) of HepG2, B16 4A5 and S17 cell lines after application of the extracts of Salvia ceratophylla at the concentration of 100 μg mL−1a
Cell line DMSO 0.5% Aerial parts-MeOH Aerial parts-aqueous Roots-MeOH Roots- aqueous
a Values represent the mean ± standard error of the mean (SEM) of six replicates (n = 6). HepG2 – human hepatocellular carcinoma cells; B16 4A5 – murine melanoma cells; S17 – murine bone marrow cells (normal cells); SI – selectivity index. In the same line, values marked by different letters are significantly different according to the Tukey HSD test (P < 0.05).
HepG2 101 ± 7a 75.3 ± 2.6b 89.4 ± 6.3ab 30.9 ± 2.5c 34.5 ± 1.7c
B16 4A5 88.2 ± 2.1a 90.4 ± 2.8a 57.3 ± 1.5b 95.1 ± 2.8a 91.1 ± 3.7a
S17 79.3 ± 4.9b 33.8 ± 2.7c 98.4 ± 1.0a 42.0 ± 1.2c 39.3 ± 3.4c
SI – HepG2 0.79 0.45 1.10 1.36 1.14
SI – B16 4A5 0.90 0.37 1.72 0.44 0.43


Root-MeOH and root-aqueous were the most toxic towards HepG2 cells (30.9 and 34.5% of cell viability), while extract aerial part-water was more active against B16 4A5 cells (57.3% of cell viability). Regarding the non-tumoral S17 cells, all samples showed significant toxicity, except extract aerial part-water that showed higher cell viability than the control (P < 0.05). Therefore, aerial part-aqueous although displaying moderate cytotoxic activity on B16 4A5 melanoma cells, exhibited the highest selectivity index for (SI = 1.72).

The observed results could be attributed to the presence phytochemicals present in the latter extract. For instance, this finding may be linked to the presence of gallic acid, which has been claimed to inhibit carcinogenesis and induces apoptosis in previous studies.45–47 Besides, the methanolic root extract contained luteolin, a flavonoid, also known to possess anti-cancer effect.48–50 However, as a future work, further assays should be conducted with the aim to isolate and identify the phytochemicals responsible for the observed cytotoxic properties and ensure if the toxicity towards cancerous cell lines is related to specific bioactive compounds.

3.6. PLS-DA based methods to discriminate between studied parts

The present study was focused upon two parts from S. ceratophylla including aerial part and roots and it was undertaken to assess the total antioxidant and selected five enzyme inhibitory activities of diverse extracts derived from said parts. For the purpose of evaluating the variation of antioxidant and enzyme inhibitory activities between the different studied parts, the supervised partial least squares discriminant analysis (PLS-DA) was applied to the data. PLS-DA is a multivariate regression analysis aiming at find the optimal linear combinations of variables being able accurately to discriminate the sample groups. In particular, latent function emanating from the linear combinations of variables summarize as much as possible the information and reduce the dimension of the original data. Thus, to perform the model, the factor “Parts” as used as class membership criteria and the results were reported in Fig. 3. By viewing Fig. 3A, we noted a clear discrimination between the two parts. The majority of aerial parts extracts were grouped on the left side of the first function while the roots extracts were aggregated on the positive and negative side of the first two function respectively. The model, had a great performance; in particular, incorporating the first two function, it was able to discriminate the both parts with an accuracy of 96.89% (Fig. 3B).
image file: d0ra10044g-f3.tif
Fig. 3 Partial least square discriminant analysis on biological activities of Salvia ceratophylla. (A) projection of samples into the subspace spanned by the first two function of PLS-DA. (B) The ROC (Receiver Operating Characteristic) curves assessing the prediction accuracy of a classification model. (C) Loadings plot showing the contribution of biological activities on the two function and the biological activities abundance among each parts. (D) discriminant biological activities identified by Variable Important in Projection (VIP).

The loadings plot displayed the contribution of the biological activities on the first two function. Function 1 was positively related to MCA, glucosidase, AChE, BChE and tyrosinase and negatively bound to the other activities (PPBD, DPPH, FRAP, CUPRAC, Amylase and ABTS). While function 2 was positively determined by BChE, PPBD, amylase, glucosidase and AChE and negatively associated to MCA, ABTS, CUPRAC, FRAP, DPPH, and tyrosinase. On the other hand, this figure allowed to determine the biological activities characterizing each part. In general, antioxidant activities and anti-amylase recorded the highest value in aerial parts in contrast to roots that exhibited the best anti-cholinesterase, anti-glucosidase and anti-tyrosinase as well as metal chelating ability.

Afterwards, the biological activities which mostly varied from one part to another were observed. In this regard, the VIP score of each bioactivity was calculated and reported in figure AC. On the basis of the value above 1, it emerged that four activities including PPBD, MCA, DPPH and ABTS, differed considerably across parts. Thus, aerial parts were characterized by an excellent total antioxidant capacity and ability to scavenging ABTS and DPPH radicals while roots were distinguished by a high ability to chelate Fe2+ ion (Fig. 4).


image file: d0ra10044g-f4.tif
Fig. 4 Effect of extraction solvents on the antioxidant activities of the tested extracts of each parts. TE: trolox equivalent; EDTAE: EDTA equivalent. (a–d) Column wise values with same superscripts of this type indicate no significant difference among extracts (P > 0.05).

The results of the present study indicated high levels of bioactivities variability between the areal parts and roots of S. ceratophylla. The reason is that the concentration and type of secondary metabolites involve in the evaluated bioactivities, vary according to the plants parts. This outcome are in agreement with our previous work on the topic, which has reported that different parts of the same plant are characterized by different content of secondary metabolites.51,52 Further, this variability may be due to ordered expression of the genome such that specific enzymes or group of enzymes are activated for the biosynthesis of certain molecules at particular tissue or organ of plant, and not in another. For instance, Yosr et al.53 reported that the amount in leaves of phenolic compounds compared to the other plant organs may be due to the interaction between organs and multiple processes of synthesis or degradation and transport implied in the distribution of these phenolic compounds at the plant level.

3.7. Effect of extraction solvents on the antioxidant and enzyme inhibition activities of each parts

Multiple solvents extraction condition was used with the purpose of achieving the best method to obtain a higher antioxidant and enzyme inhibitory activities of aerial parts and roots of S. ceratophylla (Fig. 3A and B). In general, a significant difference was observed between the extracts of each parts, for all biological activities. In aerial part, the extraction procedure using MeOH/water (80%) was highly efficient to scavenge DPPH radical and reduce Cu2+ ion. Similarly, as regards the roots, the same extracts exhibited highest ABTS scavenging capacity and Fe3+ and Cu2+ reducing power. Both methanol and MeOH/water (80%) extracts of roots and aerial parts scavenged DPPH radicals more effectively and presented highest total antioxidant capacity respectively. The extracts of aerial parts obtained using water possessed excellent ABTS and MCA activities while the water and MeOH/water (80%) showed a better reducing Fe3+ activity. Total antioxidant capacity of roots was ranged in order of DCM > MeOH > MeOH/water (80%) > water MeOH/water (80%) > hexane, whereas metal chelating activity increased as follows: water > DCM, MeOH/water (80%) > methanol > hexane. When it comes to enzyme inhibitory activities, hexane extract of aerial parts and roots had the highest anti-tyrosinase activity. In addition, the same extract exhibited strongest anti-BChE activity. However, in aerial parts, the activity of hexane extract was similar to that of DCM. For the second enzyme involved in the management of neurodegenerative disease, methanol (aerial parts) and DCM (roots) extractions showed the best anti-AChE activity. Regarding the anti-amylase assay, the strongest activity was shown by DCM for aerial parts and DCM and MeOH/water (80%) for roots. Furthermore, three extracts derived from aerial parts i.e., DCM, hexane and MeOH showed the highest anti-glucosidase activity while regarding the roots the best activity was presented by MeOH (Fig. 5).
image file: d0ra10044g-f5.tif
Fig. 5 Effect of extraction solvents on the enzyme inhibitory activities of the tested extracts of each parts. GALAE: galatamine equivalent; KAE: kojic acid equivalent; ACAE: acarbose equivalent. (a–d) Column wise values with same superscripts of this type indicate no significant difference among extracts (P > 0.05).

Historically, it is well known that extraction of secondary metabolites from plant matrix is impacted by multiple factors such as their chemical nature, the presence of interfering substances without forgetting the extraction solvent and technique used. In fact, the polarities of secondary metabolites in plants greatly vary and therefore, it is necessary to select an adequate solvent for efficient extraction in quantity and quality of the molecules of interest. As it is well known that secondary metabolites have diverse nature, concentration ranges and physicochemical properties. Accordingly, no single solvent able to recovery efficiently all of the classes of secondary metabolites from a plant matrix, simultaneously. This lends support our observations that the different solvent used, had showed each at least good result on all the evaluated biological activities. Moreover, outside the conventional extraction solvents, several researchers have employed combination of organic solvent-water for the extraction of secondary metabolites from plant. According to Cheng et al.,54 solvent mixtures allow to extract different molecules values, thanks to their differing efficacies in the penetration of plant matrixes and solubilization of the secondary metabolites. Much more, the presence of water enhance the permeability of cell membrane and therefore enables efficiently mass transfer by molecular diffusion as well as the extraction of the water soluble compounds.54

4. Conclusion

In the current work, all extracts of S. ceratophylla exhibited activity against amylase and glucosidase which are the key clinical enzymes related to diabetes, a disease affecting millions of people across the globe. A particular interest is the tyrosinase inhibitory activity displayed by the DCM root extract which can be qualified as a potent and promising activity. Thus, extract can further be examined for potential epidermal hyperpigmentation processes. Additionally, data amassed herein demonstrated that the hydro-methanolic aerial extract may act as a good antioxidant. From the antimicrobial analysis, it can be concluded that S. ceratophylla can be a potential source of bioactive compounds to combat Bacillus cereus infections. Methanolic root extract demonstrated a relatively low cytotoxicity. However, further toxicological studies should be conducted to ascertain its safety. The present study provides rationale for further in vivo/ex vivo pharmacological investigations.

Author contributions

Sengul Uysal: conceptualization, data curation, formal analysis, writing – original draft. Gokhan Zengin: formal analysis, writing – original draft, supervision. Kouadio Ibrahime Sinan: methodology. Gunes Ak: methodology. Ramazan Ceylan: methodology. Mohamad Fawzi Mahomoodally: data curation, investigation, writing – original draft. Ahmet Uysal: methodology. Nabeelah Bibi Sadeer: data curation, investigation, writing – original draft. József Jekő: data curation, investigation. Zoltán Cziáky: data curation, investigation. Maria João Rodrigues: data curation, investigation. Evren Yıldıztugay: methodology. Fevzi Elbasan: methodology. Luisa Custodio: data curation, investigation.

Conflicts of interest

There are no conflicts to declare.

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