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

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.


Introduction
Salvia ceratophylla L. (S. ceratophylla) is a biennial lemonscented 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-tumour 6 and antidiabetic activities 7 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 mg mL À1 . 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 mg mL À1 . There are further data concerning antioxidant and chemical composition of the EO. [8][9][10][11] The review of Ulubelen 12 stated that the terpenoids present in S. ceratophylla exhibited interesting antibacterial activity. The work of Goren et al. 13 also showed that the diterpenoids identied 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 signicantly depressed antibutyrylcholinesterase activity with a percentage inhibition of 91.3%. 15 Based on ethnobotanical information, S. ceratophylla were used to treat cancers, infections, urinary complications, 8 inammation, 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 rst 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 avonoid content were quantied 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.

Plant material and preparation of extracts
Salvia ceratophylla samples were collected from natural population (Akyer village, Bozdag national park, 1020 m, and steppes areas) in the summer period of 2019. Botanical identi-cation 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 nhexane, dichloromethane (DCM), methanol, methanol-water (80%) (100 mL) under stirring for 24 h at 25 C. Aer 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 ltered and then lyophilized. All extracts were stored at 4 C in a refrigerator. The extraction yields (%) are given in Table 1.

Prole of bioactive compounds
The total phenolic and avonoid contents were determined using the Folin-Ciocalteu and aluminium chloride (AlCl 3 ) 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 Â 2.1 mm i. d., 2.6 mm) 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 prole and all exact analytical conditions have been published. 23

Determination of antioxidant and enzyme inhibitory effects
The metal chelating, phosphomolybdenum, ferric reducing antioxidant power (FRAP), cupric reducing antioxidant capacity (CUPRAC), 2,2 0 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) activities of the extracts (0.5-5 mg mL À1 ) 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 À1 ) against cholinesterases (by Ellman's method), tyrosinase, a-amylase and a-glucosidase were evaluated using standard in vitro bio-assays. 24 To provide comparison with standard antioxidants and inhibitors, IC 50 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).

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 Briey, 96-well plates were loaded with 100 mL Mueller Hinton Broth medium. Then 100 mL S. ceratophylla extracts were transferred to rst well of the plate and serial dilution was done by transferring of 100 mL volume mixture via multichannel pipette. When the extract-medium mixture was ready then fresh microorganism inoculum prepared from 0.5 Mc Farland turbidity and nal concentration 5 Â 10 5 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. Aer incubation period 20 mL 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

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 Modied Eagle medium (DMEM) supplemented with foetal bovine serum (10%), L-glutamine (2 mM, 1%), and penicillin (50 U mL À1 )/ streptomycin (50 mg mL À1 ) (1%), and kept under a humidied atmosphere at 37 C and 5% CO 2 .

Determination of cellular viability and selectivity
Cells were plated in 96-well plates at 5 Â 10 3 cells per well (HepG2 and S17) and 2 Â 10 3 cells per well (B16 4A5). Aer a 24 h incubation, cells were treated with the samples at the concentration of 100 mg mL À1 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)-2,5diphenyltetrazolium 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 ¼ CT/CNT, where CT and CNT stands for the cytotoxicity of the extract towards tumoral and non-tumoral cell lines, respectively. 28

Data analysis
Statistical calculations were done using Xlstat 2018 and R v 3.5.1 sowares. 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. Aerwards, 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.

Total bioactive compounds and phytochemical composition
Plants and herbs are known to be abounded with scads of phytochemicals possessing medicinal properties such as anti-inammatory, 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 avonoid 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 avonoids. The same outcomes were reported in previous studies whereby hexane solvent extracted the least amount of phenolic and avonoid content. [30][31][32] The methanolic aerial 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. Twentynine 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).

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 hydromethanolic, 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 AE 0.27 mg TE per g) and the highest reducing potential towards copper(II) (377.93 AE 2.38 mg TE per g). Among the different root extracts analysed, the hydromethanolic sample revealed to be the most potent ABTS radical scavenger (116.50 AE 1.65 mg TE per g) and displayed the highest reducing potential with both CUPRAC (250.03 AE 2.65 mg TE per g) and FRAP (142.00 AE 0.14 mg TE per g) assays. Similar ndings 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 AE 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 AE 0.34 mg EDTAE per g) followed by root (27.83 AE 0.49 mg EDTAE per g) extracts. Mounting evidence showed that natural products play a vital role in hindering b-amyloid bril aggregation due to their ability to bind metal ions with high affinities. 35

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 (a-amylase and a-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 diabetes 2 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 conrmed 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 AE 1.41 and 124.68 AE 4.47 mg KAE per g, respectively) and amylase (0.76 AE 0.02 and 0.84 AE 0.02 mmol ACAE per g, respectively) activities. To the best of our knowledge, it is the rst time S. ceratophylla was screened for tyrosinase, amylase and glucosidase activities. Therefore, comparison of our data with other work was not possible.

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 À1 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 À1 MIC and followed by the Bacillus cereus with 0.19 MIC value. For Citrobacter MIC was found as 1.56 mg mL À1 . Aerial part hexane extract had antifungal capacity at dose of 3.12 mg mL À1 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 À1 . Same as Pseudomonas, root extract was more effective than aerial part extract against Staphylococcus aureus with 0.39 mg mL À1 MIC value. The root hexane extract of S. ceratophylla manifested very signicant antibacterial activity against S. lutea and B. cereus at a dose of 0.048 mg mL À1 . It was effective against Proteus at 1.56 mg mL À1 MIC and Candida was more sensitive against root extract than aerial part hexane extract with 0.78 mg mL À1 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 À1 and affected from root extract at a concentration of 0.048 mg mL À1 . MIC values were determined as 0.097 mg mL À1 both for two extract against B. cereus. Root extracts was more effective against S. aureus than aerial part extract with 0.097 mg mL À1 . Two extracts of DCM affected P. aeruginosa at 1.56 mg mL À1 dose. Antifungal activity was observed at 3.12 mg mL À1 dose for two DCM extracts. The lowest MIC value was determined for  Methanol and water mixture aerial part extract of S. ceratophylla revealed MIC values between 3.12 to 1.56 mg mL À1 doses. Although MIC values for S. lutea and B. cereus were determined as 0.39 mg mL À1 , this extract was effective against S. typhimurium at a dose of 1.56 mg mL À1 when compared previous three extracts. Escherichia coli only affected from aerial part extract at 1.56 mg mL À1 dose. The lowest MIC reported for root extract was 0.097 mg mL À1 for B. cereus. Infusion aerial part extract manifested antibacterial activity against S. aureus at a dose of 1.56 mg mL À1 . Similarly, infusion root extract had antibacterial capacity against S. enteritidis (1.56 mg mL À1 ) only infusion extracts were effective against Y. enterocolitica with 6.25 mg mL À1 MIC value. The results showed that S. ceratophylla extracts had signicant antibacterial activities against Gram positive   Several Salvia species reported for their antimicrobial activity and pharmacological properties 37,38 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 signicant 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.Świsłocka 42 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 prooxidative properties and they can alter the hydrophobicity and aer 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 Signicant antimicrobial activities of extracts determined in this study can be attributed to presence of rosmarinic and caffeic acid in S. ceratophylla.

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.
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 signicant 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 ¼ 1.72).
The observed results could be attributed to the presence phytochemicals present in the latter extract. For instance, this nding may be linked to the presence of gallic acid, which has been claimed to inhibit carcinogenesis and induces apoptosis in previous studies. [45][46][47] Besides, the methanolic root extract contained luteolin, a avonoid, also known to possess anticancer effect. [48][49][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 specic bioactive compounds.

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 ve 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 nd 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 le side of the rst function while the roots extracts were aggregated on the positive and negative side of the rst two   50 (mg mL À1 ), 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.
function respectively. The model, had a great performance; in particular, incorporating the rst two function, it was able to discriminate the both parts with an accuracy of 96.89% (Fig. 3B). The loadings plot displayed the contribution of the biological activities on the rst 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 gure 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 antityrosinase as well as metal chelating ability.
Aerwards, 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 gure 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 Fe 2+ ion (Fig. 4).
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 specic 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.

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 signicant 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 Cu 2+ ion. Similarly, as regards the roots, the same extracts exhibited highest ABTS scavenging capacity and Fe 3+ and Cu 2+ reducing power. Both methanol and MeOH/water 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). 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

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 qualied 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.

Conflicts of interest
There are no conicts to declare.