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filing and in vitro biological properties of two Lamiaceae species
(Salvia modesta and Thymus argaeus): A comprehensive evaluation
Gokhan Zengin
a,⁎, Bayram Atasagun
b, Muhammad Zakariyyah Aumeeruddy
c,
Hammad Saleem
d,e, Adriano Mollica
f, Mir Babak Bahadori
g, Mohamad Fawzi Mahomoodally
caDeparment of Biology, Faculty of Science, Selcuk University, Campus, Konya, Turkey bDeparment of Biology, Faculty of Science, Erciyes University, Kayseri, Turkey
cDepartment of Health Sciences, Faculty of Science, University of Mauritius, 230, Réduit, Mauritius
dSchool of Pharmacy, Monash University, Jalan Lagoon Selatan, 47500, Bandar Sunway, Selangor Darul Ehsan, Malaysia eInstitute of Pharmaceutical Sciences (IPS), University of Veterinary & Animal Sciences (UVAS), Lahore, 54000, Pakistan fDepartment of Pharmacy, University“G. d’Annunzio” Chieti-Pescara, 66100, Chieti, Italy
gResearch Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran
A R T I C L E I N F O Keywords: Salvia modesta Thymus argaeus Antioxidant Enzyme inhibition Phytochemical A B S T R A C T
The genus Salvia and Thymus have gained much popularity as an alternative therapy in Turkish folk medicine for abdominal pain, cold, nausea, among others. Nonetheless, some species are yet to be further explored for their bioactivities. We investigated the biological activities of 3 extracts (dichloromethane, methanol, and water (decoction)) of Salvia modesta Boiss. and Thymus argaeus (Fisch. & C.A.Mey.) Boiss. & Balansa. based on anti-oxidant and enzyme inhibition along with the determination of polyphenolic content. Antianti-oxidant potential was assessed using six assays namely: 2,2-diphenyl-1-picrylhydrazyl, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sul-phonic acid), cupric ion reducing antioxidant capacity, ferric reducing antioxidant power, phosphomolybdenum, and metal chelating. Moreover, enzyme inhibition activities of the extracts were studied against acet-ylcholinesterase, butyracet-ylcholinesterase, tyrosinase,α-amylase, and α-glucosidase. Results revealed that the de-coction of both plants was the strongest antioxidants. The methanol extracts displayed the highest tyrosinase inhibition while the dichloromethane extracts of both plants were the most effective butyrylcholinesterase and α-glucosidase inhibitors. In addition, the total phenolic and total flavonoid content was highest in the decoction and methanolic extract of Thymus argaeus, respectively. The most abundant phenolic compound was rosmarinic acid (6574μg/g and 5390 μg/g in T. argaeus and S. modesta methanolic extracts, respectively). PASS prediction analysis revealed that chlorogenic acid showed the highest Pa value for antioxidant activity (0.809) including the mechanism of free radical scavenging (0.856), while rosmarinic acid showed the highest Pa value (0.798) for antidiabetic activity. To conclude, both Salvia modesta and Thymus argaeus can be regarded as new sources of antioxidants and enzyme inhibitors to manage oxidative stress and their complications.
1. Introduction
Plants play an essential part of human's life in terms of survival, food, shelter, and medicines. Today, there is a great attention towards the investigation of medicinal plants as novel sources of antioxidants and enzyme inhibitors to manage oxidative stress-associated diseases. However, the scientific exploration of the vast amount of plant species is a complex process, requiring time, specialists, and funding (Phumthum et al., 2018). Consequently, researchers have been using an easier and cost effective approach by observing medicinal plants which are traditionally used among different ethnic groups followed by their
pharmacological validation. This strategy has ultimately lead to the discovery of new lead compounds; e.g. the isolation of salicin from Salix alba L. bark, a plant traditionally used for pain and fever. Salicin was converted into salicylic acid, and was then modified into aspirin to reduce side effects. Other popular plant-derived pharmaceuticals in-clude the antimalarial quinine from Cinchona officinalis L. and the painkiller, morphine, from Papaver somniferum L. (McRae et al., 2007). The Lamiaceae is a large family of plants, with more than 200 genera and 7000 species (approximately 245 genera and 7886 accepted species recorded in The Plant List). They are suitable for pharmaceutical, food, and cosmetic industries due to their goodflavor, easy cultivation,
https://doi.org/10.1016/j.indcrop.2018.11.027
Received 11 October 2018; Received in revised form 6 November 2018; Accepted 12 November 2018
⁎Corresponding Author.
E-mail address:gokhanzengin@selcuk.edu.tr(G. Zengin).
Available online 20 November 2018
0926-6690/ © 2018 Elsevier B.V. All rights reserved.
and wide medicinal benefits (Bekut et al., 2017). In this work, we report the biological potential and chemical profile of two Turkish Lamiaceae species, Salvia modesta and Thymus argaeus. The genus Salvia has a great diversity in Turkey. Ninetyfive species are now recognized in Turkey of which 45 are endemic (Kotan et al., 2008;Özler et al., 2011). Salvia species are used in Turkish traditional medicine for the treatment of abdominal pain, menstrual pain, common cold, nausea, and also used as an expectorant in the region of Acipayam (Bulut et al., 2017). In Mar-maris, they are consumed to manage stomach ache, flatulence, cold, tonsillitis, and used as a laxative and antipyretic (Gürdal and Kültür, 2013). The genus is rich in terpenoids, essential oils, and phenolic compounds (Bahadori et al., 2018).
The genus Thymus (Turkish name: Kekik) is characterized by 59 taxa in Turkey with 53% endemism (Karaman et al., 2001). They are widely utilized as herbal tea andflavoring agents (Sagdic et al., 2009). For instance, they are also consumed to manage gastrointestinal diseases, cold,flu, high cholesterol, stomach ache, and respiratory tract diseases (Güneş et al., 2017;Gürdal and Kültür, 2013;Polat and Çakılcıoğlu, 2018;Sargin et al., 2015).
Recent pharmacological investigations have evidenced wide biolo-gical activities of many Salvia (Alimpić et al., 2017; Bahadori et al., 2017;Orhan et al., 2013;Tepe, 2008;Zengin et al., 2018b) and Thymus species (Habashy et al., 2018; Hamdani et al., 2014; Tohidi et al., 2017). In an attempt to further investigate these two genera, this work aimed to evaluate the antioxidant activity, key enzymes inhibitory properties, and phenolic profile of two less exploited species, Salvia modesta Boiss. and Thymus argaeus (Fisch. & C.A.Mey.) Boiss. & Balansa. Few studies have explored the above mentioned bioactivities of these species. For example, a previous study by (Şenol et al., 2010) in-vestigated the acetylcholinesterase inhibitory capacity of 55 Turkish Salvia members including Salvia modesta and also screened their DPPH radical scavenging and iron-chelating potential. With regards to T. ar-gaeus,Sagdic et al. (2009)determined the antiradical effect of its es-sential oil and methanolic extract together with the chemical compo-sition of the essential oil. Nonetheless, the detailed antioxidant activity of these important species should be explored based on different me-chanisms of action (scavenging, reducing, chelating). So, to the best of our knowledge, this study could be considered as the first compre-hensive report on their in-depth enzyme inhibitory activities and anti-oxidant potentials.
2. Materials and methods 2.1. Plant material
The studied plant materials were collected from Kayseri (central Anatolia region, Turkey) in July 2016 (Thymus argaeus: Erciyes Mount., above Tekir plateau, 2316 m, 38° 32′ 533″ N, 035° 31′ 105″ E; Salvia modesta: Erciyes Mount., above Tekir plateau, 2250 m, 38° 32′ 564″ N, 035° 31′ 306″ E). The scientific identification was performed by Dr. Bayram Atasagun (botanist at Erciyes University, Department of Biology, Kayseri, Turkey). Voucher specimens were deposited in the herbarium of Erciyes University (voucher numbers: 1098 (S. modesta) and 1087 (T. argaeus)). The aerial parts were separated and kept for 10 days at room temperature for drying. Then, samples were powdered with a laboratory mill and preserved at + 4 °C until processing. 2.2. Extraction
For organic extracts (dichloromethane and methanol), samples were stirred overnight (24 h) at room temperature (5 g in 100 mL solvent). After filtration (by Whatman no. 1 filter paper), the extracts were concentrated using a rotary evaporator under vacuum at 40 °C. A de-coction was also prepared (5 g of plants were added to 100 mL of water and boiled for 20 min) and then were lyophilized. All extracts were stored at + 4 °C until analyses.
2.3. Spectrophotometric and chromatographic analysis for metabolites The total phenolic content (TPC) was determined using the Folin-Ciocalteu method andflavonoids content (TFC) by the AlCl3method.
Thefindings obtained were expressed as equivalents of standard com-pounds (gallic acid (mg GAE/g sample) for TPC and rutin (mg RE/g sample) for TFC), respectively).
All extracts were analyzed using reverse phase high-performance liquid chromatography-photodiode array detector (Shimadzu Scientific Instruments, Kyoto, Japan) for quantitative analysis of phenolic com-pounds. The system was equipped with a C-18 column (Eclipse XDB, 250 mm × 4.6 mm, 5μm, Agilent, Santa Clara, CA, USA). The column temperature was 30 °C. All chromatographic details are given in our previously published work (Movahhedin et al., 2016). Standard phe-nolic compounds were used for identifying and quantifying individual phenolic components in the samples asμg/g dry extract.
2.4. Antioxidant assays
Antioxidant propensities of the extracts were screened by distinct chemical assays including antiradical methods (ABTS and DPPH), re-ductive power (CUPRAC and FRAP), phosphomolybdenum, and che-lating effect. The results were expressed as standard compounds equivalents (mg TE/g sample and mg EDTAE/g sample). All methods are given in our previous paper (Grochowski et al., 2017).
2.5. Inhibition effects against key enzymes
The inhibition effects of the extracts were investigated on key en-zymes, namely cholinesterases, α-amylase, α-glucosidase, and tyr-osinase. The experimental protocols were reported in our previous paper (Grochowski et al., 2017). Standard inhibitors were galantamine (for AChE and BChE), kojic acid (for tyrosinase), and acarbose (for α-amylase andα-glucosidase).
2.6. In silico prediction using PASS
Prediction of Activity Spectra for Substances (PASS) was employed for prediction of biological effects of the major compounds identified in the tested extracts. This software predicts the desirable pharmacolo-gical effect, mechanisms of action, and adverse effects of chemical structures, on the basis of structure-activity relationship with a known chemical entity. The activity is estimated in terms of probable activity (Pa) and probable inactivity (Pi) (Jamkhande et al., 2016). The PASS results could be used in aflexible manner: (a) only activities having Pa value > Pi value are considered as possible for a particular compound; (b) when Pa > 0.7, the probability of experimental pharmacological effect is high; and (c) when Pa < 0.5, in experiment, the probability of pharmacological effect is low, but the chance for finding novel chemical structures is high (Pramely and Raj, 2012).
2.7. Statistical analysis
All tests were carried out in triplicates andfindings are expressed as mean value ± SD. SPSS v. 17.0 was employed for statistical analysis. One-way ANOVA was done to determine any differences between the tested samples following by Tukey’s test. p < 0.05 were assigned to be statistically significant. The principal component (PCA) and Pearson linear correlation were employed to recognize any relationship between phytochemical contents and the observed biological activities. 3. Results and discussion
3.1. Phytochemical contents
isolation and characterization of compounds. These plant-derived sub-stances have gained considerable importance owing to their versatile applications in the development of nutraceuticals, food supplements, functional foods, pharmaceutical intermediates, and chemical entities for drugs (Ahmed et al., 2017). Herein, the extracts of S. modesta and T. argaeus were evaluated for their phenolic andflavonoid contents. The results are summarized inTable 1.
Among the extracts of S. modesta, the decoction had the highest TPC (78.63 mg GAE/g sample), followed by the methanol extract (60.50 mg GAE/g sample). In contrast, the methanolic extract possessed the highest TFC (27.64 mg RE/g sample). A similar pattern was observed for T. argaeus extracts whereby TPC order was as follows decoction (107.48) > methanol (85.67) > dichloromethane (11.52). On the other hand, theflavonoid content order was methanol (52.11) > decoction (42.76) > dichloromethane (17.44). Overall, the polar extracts of T. argaeus have the highest TPC and TFC.
Additionally, HPLC-DAD analysis of the extracts of S. modesta and T. argaeus indicated that a number of important phenolic components are present in these herbs (Table 2). (+)-catechin, p-hydoxybenzoic acid, caffeic acid, and p-coumaric acid were identified in all extracts. The most abundant compound in the methanol extracts of T. argaeus and S.
modesta was rosmarinic acid (6574μg/g and 5390 μg/g sample, re-spectively). Chlorogenic acid was another compound abundant in T. argaeus (methanolic: 572μg/g; decoction: 346 μg/g). Luteolin was also present in notable amount in the methanolic extract of S. modesta (312μg/g sample). In contrast, gallic acid and sinapic acid were de-tected only in the decoction of T. argaeus (2μg/g and 68 μg/g sample, respectively). On the other hand, epicatechin, benzoic acid, hesperidin, and quercetin were not identified in neither of the tested extracts.
It is to be noted that a previous phytochemical study byHatipoglu et al. (2016)found 76 volatile organic components in the aerial parts of S. modesta, including oxygenated monoterpenes, sesquiterpene hydro-carbons, oxygenated sesquiterpenes, and monoterpene hydrocarbons. The major components were α-pinene (6.46%), caryophyllene oxide (5.31%), borneol (4.39%), sclareol (4.16%), β-(E)-caryophyllene (3.78%), and Z-caryophyllene (3.55%). With respect to T. argaeus, Sagdic et al. (2009)previously analyzed its chemical composition of essential oil and found linalool,α-terpineol, linalyl acetate, and thymol as the main components. They also reported the total phenolics, fla-vanol, andflavonol components of the plant. The TPC of the essential oil (83.31 mg GAE/g) was lower compared to that observed in the de-coction and methanol extracts in our study, which indicates variation in the chemical composition of the volatile and non volatile fraction of these plants.
3.2. Antioxidant activity
It is worth to mention that any antioxidant assay cannot completely reflect the “total antioxidant capacity” of a plant extract. A compre-hensive antioxidant assay must reflect both the capacity of both lipo-philic and hydrolipo-philic compounds based on their mechanism (hydrogen transfer, electron transfer, and metal chelating). In this context, dif-ferent antioxidant methods are required for profiling the total anti-oxidant capacity of a natural extract. In our work, the antianti-oxidant po-tential of S. modesta and T. argaeus were tested on the basis of radical quenching, reductive power, and metal chelating (Table 3).
With regards to the scavenging properties of S. modesta, the me-thanolic extract showed the greatest DPPH scavenging activity (97.76 mg TE/g sample) and the decoction exhibited the strongest ABTS scavenging effect (258.45 mg TE/g sample). It is to be noted that Table 1
Total bioactive components of the tested extracts*.
Plant species Solvents Total phenolic content (mg GAE/g sample) Totalflavonoid content (mg RE/g sample) Salvia modesta DCM 24.18 ± 1.23b 14.73 ± 0.97a MeOH 60.50 ± 0.86c 27.64 ± 0.50c Water decoction 78.63 ± 1.51d 26.91 ± 0.33c Thymus argaeus DCM 11.52 ± 0.24a 17.44 ± 0.08b MeOH 85.67 ± 1.16e 52.11 ± 0.35e Water decoction 107.48 ± 1.35f 42.76 ± 0.91d
* Values expressed are means ± S.D. of 3 parallel measurements. DCM:
Dichloromethane; MeOH: Methanol; GAE: Gallic acid equivalent; RE: Rutin equivalent. Different letters in the same columns indicate a significant differ-ence (p < 0.05).
Table 2
Phenolic components of the tested extracts (μg/g sample) (mean ± S.D).
Phenolic compounds S. modesta- DCM S. modesta- MeOH S. modesta- Water decoction T. argaeus -DCM T. argaeus - MeOH T. argaeus–
Water decoction Gallic acid – – – – – 2 ± 0.9 Protocatecheuic acid – 43 ± 0.3b 48 ± 0.6c – 64 ± 0.3e 55 ± 0.6d (+)-Catechin 7 ± 0.4a 157 ± 2e 82 ± 2b 6 ± 0.4a 147 ± 10d 113 ± 2c p- hydoxybenzoic acid 2 ± 0.1a 41 ± 0.3d 20 ± 0.6b 3 ± 0.1a 40 ± 0.3d 28 ± 0.6c Chlorogenic acid 5 ± 0.1a 63 ± 1c 57 ± 2b – 572 ± 40e 346 ± 20d Caffeic acid 5 ± 0.2a 44 ± 2c 45 ± 4c 8 ± 0.2b 171 ± 3e 109 ± 10d Epicatechin – – – – – – Syringic acid – – – 2 ± 0.1a – 24 ± 0.1b Vanilin – 13 ± 1b 17 ± 0.3a 2 ± 0.1c – – p- coumaric acid 1.2 ± 0.1b 22 ± 0.9e 17 ± 0.1c 0.5 ± 0.01a 18 ± 0.8d 15 ± 0.1b Ferulic acid – 29 ± 0.2c 37 ± 0.4d 2 ± 0.01a 29 ± 0.2c 27 ± 0.4b Sinapic acid – – – – – 68 ± 1 Benzoic acid – – – – – – o-coumaric acid – 11 ± 0.4b 3 ± 0.01a – – – Rutin 59 ± 0.7b 44 ± 0.6a – – – – Hesperidin – – – – – – Rosmarinic acid 13 ± 1a 5390 ± 162d 2758 ± 66b – 6574 ± 163e 3017 ± 48c Eriodictyol – 42 ± 0.3c 17 ± 0.6a – 45 ± 0.3b – Cinnamic acid 2 ± 0.1a 13 ± 0.5b – 2 ± 0.01a 16 ± 0.5c 31 ± 1d Quercetin – – – – – – Luteolin 37 ± 3a 312 ± 14d 100 ± 6b – 137 ± 4c 37 ± 3a Kaempferol – 50 ± 2a – – 50 ± 2a – Apigenin 75 ± 4b 266 ± 5e 103 ± 2c 21 ± 1a 113 ± 4d –
a previous study byŞenol et al. (2010)also found that the methanol extract of S. modesta exhibited the strongest DPPH scavenging activity, with a percentage inhibition of 89.43 at 100μg/mL, compared to the ethyl acetate extract (64.33% inhibition) and dichloromethane extract (22.90% inhibition). As for T. argaeus, the presentfindings revealed that the decoction was the most efficient ABTS (302.78 mg TE/g sample) and DPPH (221.50 mg TE/g sample) scavenger. The weakest scaven-ging activity was displayed by the dichloromethane extracts of both plants.
The reducing potential of the tested extracts on Cu2+and Fe3+are
shown inTable 3. Among the S. modesta extracts, the decoction showed the most effective reducing power (CUPRAC: 381.35 mg TE/g sample; FRAP: 238.95 mg TE/g sample). As regards to T. argaeus, the metha-nolic extract showed the highest cupric reducing capacity (504.84 mg TE/g sample) while the decoction was most efficient in reducing Fe3+
(312.75 mg TE/g sample). Overall, the dichloromethane extract of both tested plants had the lowest reducing effect in both assays.
Besides the above mentioned assays, we conducted two additional antioxidant assays, metal chelating and phosphomolybdenum tests. Table 3shows that the decoction of both species exhibited the strongest antioxidant activity in the phosphomolybdenum test (2.26 and 2.52 mmol TE/g, respectively), while the dichloromethane extracts showed the least activity. Although the decoction of S. modesta exerted the strongest metal chelating effect (29.94 mg EDTAE/g) compared to its counterpart solvent extracts, the dichloromethane extract of T. ar-gaeus showed the highest effect (36.41 mg EDTAE/g) followed by its decoction and methanol extracts.
From the abovefindings, it is clear that the decoction and methanol extracts of both plants have high antioxidant activities in most assays which is in proportion to their phenolics and flavonoids content, re-spectively. PCA and correlation analysis confirmed these results (Fig. 1). We noted two principal components as 82.82% of total variability in PCA. The variables regarding antioxidant parameters contribute strongly to the formation of the axis 1 (63.29%), while the variables including enzyme inhibition assays are strongly correlated to the axis 2 (19.47%). As Pearson correlation shows, a strong positive relationship was observed between TPC (R = 0.888-0.987), TFC (R = 0.664-0.943), andfive out of the six antioxidant (DPPH, ABTS, FRAP, CUPRAC, and phosphomolybdenum) assays. The strongest correlation of TPC was observed with the FRAP assay (R = 0.987) while TFC showed the strongest correlation with DPPH scavenging (R = 0.943).
The positive correlation between polyphenolic content and anti-oxidant effects have been reported previously (Acosta-Quezada et al., 2015; Khan et al., 2016; Llorent-Martínez et al., 2017). Moreover, rosmarinic acid, luteolin, and protocatechuic acid were among the most abundant phenolic compounds in the methanolic and decoction sam-ples compared to the dichloromethane extract. Indeed, the antioxidant potential of these compounds has been evidenced by previous studies. For instance, the DPPH scavenging effect of rosmarinic acid (EC50= 0.23 mM) was proven byAdomako-Bonsu et al. (2017), which
was found to be comparable to quercetin (EC50= 0.21 mM).Kasala
et al. (2016)also observed that a luteolin treatment with 15 mg/kg body weight could counteract variations in enzymatic and non-enzy-matic antioxidants on induced carcinogenesis in mice. Moreover,Li et al. (2011)found that in eight different antioxidant assays, proto-catechuic acid exhibited higher antioxidant ability in comparison with standard antioxidant Trolox, in a dose-dependent manner.
However, it should be noted that the metal chelating ability of di-chloromethane extract of T. argaeus is not in conformity with its total phenolic andflavonoid amounts. In fact, the present work revealed that both TPC (R = 0.189) and TFC (R = 0.121) were weakly correlated with metal chelating activity (Fig. 1). This view was not supported by (Arya and Yadav, 2010;Sowndhararajan and Kang, 2013) since they observed a positive correlation between TPC, TFC, and metal chelating activity. A possible explanation for the present observation might be that there are other classes of compounds present in the di-chloromethane extract which are responsible for its metal chelation activity. Also, it is possible that a combination of specific components, though might be present in small amounts, may exert a synergistic ef-fect. The antioxidant effect of individual phenolic has been widely re-ported, but there is not enough knowledge about the mechanisms of action and possible antagonistic, synergistic, and additive effects.
Taking into consideration the PASS predictions of the major com-pounds identified in the two tested plants (Table 5), chlorogenic acid showed the highest Pa value for antioxidant activity (0.809) including the mechanism of free radical scavenging (0.856). Luteolin and api-genin also displayed Pa value > 0.7. Rosmarinic acid showed high Pa for free radical scavenging effect (0.745). This predicts the high con-tribution of these compounds to the antioxidant properties of S. modesta and T. argaeus.
3.3. Enzyme inhibitory activity
Enzyme inhibition has become an important approach in drug dis-covery for the treatment of various conditions. AChE is a major target since it is the main enzyme which hydrolyses acetylcholine and ends neurotransmission. Acetylcholine alters neuronal excitability, affects synaptic transmission, stimulates synaptic plasticity, and also co-ordinatesfiring of groups of neurons (Picciotto et al., 2012). The in-hibition of AChE can thus prevent the hydrolysis of acetylcholine thereby enhancing the cholinergic signaling and improve the cognitive symptoms which occur, for example, in Alzheimer’s disease. In addi-tion, although the function of BChE, the second enzyme of the choli-nesterase family, is not fully clear, it is believed to be linked to many disorders amongst Alzheimer’s disease (Karlsson, 2013). In fact, selec-tive BChE inhibition was found to elevate brain acetylcholine, improve learning and lower Alzheimerβ-amyloid peptide in rodent (Greig et al., 2005). AsTable 4shows, Among the S. modesta extracts, the methanolic one showed the strongest AChE inhibition (2.93 mg GALAE/g sample) and the dichloromethane extract exerted the most efficient BChE Table 3
Antioxidant potentials of the tested extracts*.
Plant species Solvents DPPH
(mg TE/g sample) ABTS (mg TE/g sample) CUPRAC (mg TE/g sample) FRAP (mg TE/g sample) Phosphomolybdenum (mmol TE/g)
Metal chelating ability (mg EDTAE/g) Salvia modesta DCM 14.43 ± 0.13b 11.61 ± 1.01a 56.03 ± 2.37a 24.03 ± 0.90b 1.79 ± 0.16b 13.90 ± 1.67a MeOH 97.76 ± 1.75c 153.66 ± 4.79b 330.58 ± 8.60b 193.05 ± 4.56c 2.21 ± 0.04c 25.74 ± 0.52b Water decoction 89.52 ± 6.39c 258.45 ± 3.11d 381.35 ± 9.49c 238.95 ± 4.21d 2.26 ± 0.05c 29.94 ± 0.06c Thymus argaeus DCM 5.30 ± 0.12a 15.16 ± 2.72a 55.93 ± 0.62a 16.41 ± 0.37a 0.78 ± 0.05a 36.41 ± 2.68 MeOH 189.97 ± 2.08d 216.28 ± 4.60c 504.84 ± 6.05d 283.38 ± 9.43e 2.30 ± 0.10c 22.89 ± 2.77b Water decoction 221.50 ± 11.31e 302.78 ± 7.29e 501.14 ± 8.75d 312.75 ± 2.85f 2.52 ± 0.02d 33.86 ± 1.01d
*Values expressed are means ± S.D. of 3 parallel measurements. DCM: Dichloromethane; MeOH: Methanol; TE: Trolox equivalent; EDTAE: EDTA equivalent.
inhibitory activity (5.66 mg GALAE/g sample). No AChE and BChE inhibition were displayed by the decoction. In contrast to our study, Şenol et al. (2010) observed no AChE inhibition by the di-chloromethane extract of S. modesta at 100μg/mL. With regards to T. argaeus, the present study revealed that the dichloromethane extract was the most effective AChE (3.58 mg GALAE/g sample) and BChE (4.96 mg GALAE/g sample) inhibitor.
Additionally, pancreaticα-amylase is an important enzyme in the initial step of the starch hydrolysis to smaller carbohydrates including maltose and maltotriose. α-Glucosidase then further degrades these products to glucose which enters the blood stream. Consequently, in-hibition ofα-amylase and α-glucosidase activity is the main goal for slowing glucose liberation and glucose absorption, which in turn, re-duce blood glucose level and thus suppress hyperglycemia and diabetes (Isaac et al., 2014;Kajaria et al., 2013). Among the S. modesta extracts tested, the dichloromethane extract was the most effective anti-diabetic
agent (Table 4), displaying an inhibition of 0.64 and 9.48 mmol ACAE/ g sample againstα-amylase and α-glucosidase, respectively. As for T. argaeus, the methanol extract showed the strongestα-amylase inhibi-tion (0.77 mmol ACAE/g sample) and the dichloromethane extract ex-erted the strongest α-glucosidase inhibition (19.27 mmol ACAE/g sample). In contrast, the decoction of both plants showed the weakest amylase inhibition while the methanol extracts were the weakest α-glucosidase inhibitor. Although the dichloromethane extracts showed high inhibitory activities against cholinesterases, amylase, and α-glucosidase, they possessed the lowest total levels of phenolics and flavonoids, which indicates the presence of other compounds mod-ulating the observed activities as previously mentioned. A negative correlation was observed between TPC of the tested plants and their inhibitory effect against these enzymes (R values in the range -0.572 to -0.768). Similarly, TFC was negatively correlated with enzyme inhibi-tion (R values in the range -0.085 to -0.860). This suggests the presence Fig. 1. Statistical evaluations (A&B: Distribution of the tested extracts on the factorial plan and representation of biological activities on the correlation circle based on PCA C: Correlation coefficients between total bioactive compounds and biological activities (Pearson Correlation Coefficient (R), p < 0.05); DCM: Dichloromethane; MeOH: Methanol; TPC: Total phenolic content; TFC: Totalflavonoid content).
Table 4
Enzyme inhibitory properties of the tested extracts*.
Plant species Solvents AChE (mg GALAE/g
sample)
BChE (mg GALAE/g sample)
Tyrosinase (mg KAE/g sample)
α-amylase (mmol ACAE/g sample)
α-glucosidase (mmol ACAE/g sample) Salvia modesta DCM 2.84 ± 0.14b 5.66 ± 0.50d 59.91 ± 0.18b 0.64 ± 0.03b 9.48 ± 0.01e MeOH 2.93 ± 0.09b 3.99 ± 0.50b 125.50 ± 0.54d 0.57 ± 0.01b 1.75 ± 0.12a Water decoction na na 51.70 ± 2.69a 0.10 ± 0.01a 2.64 ± 0.05b Thymus argaeus DCM 3.58 ± 0.26c 4.96 ± 0.12c 118.16 ± 2.42c 0.66 ± 0.07c 19.27 ± 0.18f MeOH 3.16 ± 0.10c 3.03 ± 0.32a 121.52 ± 1.46d 0.77 ± 0.07d 3.51 ± 0.42c Water decoction 0.31 ± 0.03a na 55.29 ± 0.29a 0.13 ± 0.01a 5.19 ± 0.04d
*Values expressed are means ± S.D. of 3 parallel measurements. DCM: Dichloromethane; MeOH: Methanol; GALAE: Galatamine equivalent; KAE: Kojic acid
of other compounds modulating the observed activities as previously mentioned. Data obtained from PASS prediction analysis revealed that rosmarinic acid showed the highest Pa value (0.798) for antidiabetic activity while caffeic acid and chlorogenic acid exhibited low Pa values (0.384 and 0.340, respectively), thus indicating the potential of ros-marinic acid as an effective α-amylase and α-glucosidase inhibitor.
The present study also investigated into the tyrosinase inhibitory activity of the tested plant extracts (Table 4). Tyrosinase is a key en-zyme in melanin synthesis. Today, several tyrosinase inhibitors are utilized in the treatment of hyperpigmentation disorders in humans (Kao et al., 2013). In this work, among the solvent extracts of S. mod-esta, the methanolic one showed the strongest tyrosinase inhibition (125.50 mg KAE/g sample) followed by the dichloromethane (59.91 mg KAE/g sample) and decoction (51.70 mg KAE/g sample). Similarly, the methanolic extract of T. argaeus exerted the highest tyrosinase inhibi-tion (121.52 mg KAE/g sample) while the decocinhibi-tion exhibited the least inhibitory activity (55.29 mg KAE/g sample). AsFig. 1shows, a weak positive correlation was observed between TFC and tyrosinase inhibi-tion (R = 0.150) while TPC was negatively correlated (R= -0.268) (Fig. 1). Similarly, some researchers reported no correlation among these parameters (Chiocchio et al., 2018;Zengin et al., 2018a). In fact, PASS prediction analysis (Table 5) revealed that all studied compounds showed Pa value lower than 0.7 for melanin inhibition, which is directly linked to tyrosinase inhibition. At this point, non-phenolic in-hibitors could be attributed to the tyrosinase inhibitory effects. 4. Conclusion
In this study, we evaluated the antioxidant ability, enzyme in-hibitory activity, and phytochemical composition of Salvia modesta and Thymus argaeus. Rosmarinic acid was identified as the most abundant phenolic compound present in both plants. PASS prediction analysis revealed that chlorogenic acid is most likely to contribute to the anti-oxidant activity of the extracts, while rosmarinic acid contributed mostly to antidiabetic activity, which is related toamylase and α-glucosidase inhibition. This study presented new findings on the bio-logical properties of these plants, which tend to justify their efficacy in the management of public health problems including oxidative stress and its complications. Nevertheless, in vivo studies are required to de-termine if the observed in vitro activities of these extracts produce si-milar effects in model organisms, which in turn, will provide a com-prehensive conception on the pharmacological properties of these species.
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