Infusion of aerial parts of Salvia chudaei Batt. & Trab. from Algeria: Chemical, toxicological and bioactivities characterization

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Introduction
The use of traditional medicines, particularly herbal medicines, has been widely increasing during the recent decades (Popović et al., 2016).Since the old times, people used medicinal plants for healing and other health care, probably due to their wide availability, as well as the high cost and difficult access to drugs in some regions of the world.Their use represents also the preservation of the cultural heritage, considering the traditional medicine practice as a part of peoples culture (Van Wyk and Wink, 2018).
Phenolic compounds include a multitude of molecules (e.g.flavonoids, phenolic acids, stilbenes, tannins, and lignans), responsible for the main organoleptic properties of plant derived-foods and beverages, and contributing also to the vegetables and fruits nutritional properties (Tapas et al., 2008).Simultaneously, phenolic compounds are getting a large interest thanks to their association with many bioactivities, probably related to their antioxidant power (Shahidi and Yeo, 2018).
S. chudaei is an endemic plant of the central Sahara massif (Ozenda, 1991), known as Aouit in Tamahaq (indigenous language of the Touareg population in the South of Algeria) and as Tagrouft in Arabic.This plant is used to relieve numerous diseases and disorders such as ulcer, diarrhea, rheumatism, kidney diseases, dysmenorrhea, abdominal pain, spasms, urinary retention, urinary tract infection, and prostate pain, consumed as infusion, decoction, or powder (Hammiche and Maiza, 2006;Ramdane et al., 2015;Sekkoum et al., 2011).However, besides the use of this plant to relieve dysfunctions, no studies were carried out about its acute toxicity, anti-inflammatory activity, analgesic effect as well as its phenolic compound profile.Yet, some studies reported the composition of essential oil from aerial parts along with the antimicrobial and antioxidant activities of some organic extracts of S. chudaei (Hammoudi et al., 2017a;Krimat et al., 2015aKrimat et al., , 2015b)).
In order to confirm some of the reported healing virtues of S. chudaei infusion obtained from aerial parts, this research aimed to identify and quantify the molecules responsible for those medicinal benefits.To do that, different doses of infusion were tested in what concerns its toxicity, in vivo analgesic and anti-inflammatory effects, as well as its antioxidant activity, total phenolic and flavonoid contents, and phenolic profile by Liquid Chromatography coupled to Diode Array Detector and Electrospray Ionization tandem Mass Spectrometry (LC-DAD-ESI/MSn).

Plant material
S. chudaei was harvested in January 2017 in the Ilamen station (longitude: 005 • 29 30.6 E, latitude: 23 • 14 26.9 N, altitude: 2055 m), located in the region of Tamanrasset in Southern Algeria.The authentication of the plant species was certified by the Higher National Agronomic School, El Harrach, Algiers, Algeria, according to Quezel and Santa (1963).A Standard specimen is conserved in the herbarium of Montpellier (voucher N • : MPU 010317).The plant aerial parts were air shade dried, at room temperature (22 ± 2 • C), then ground in a mill to small particles for further use.

Preparation of the infusion
The infusion (5% w/v) was prepared by adding boiling deionized water to the sample powder and left to stand for 5 min.Three independent extractions were prepared, the content filtered via Whatman paper N • 3. The prepared infusion was thereafter used in the assays.For the LC-DAD-ESI/MSn analysis, the infusion extract was firstly lyophilized then dissolved in methanol/water (80:20, v/v).

Experimental animals
20-30g Swiss albino mice (NMRI) purchased from the Pasteur Institute of Algiers (Algeria) were acclimatized for 2 weeks before experiments, housed at 24 ± 1 • C, 50 ± 5% humidity, and 12/12 h lightdark cycle.They were fed with rodent chow from the Animals Food National Office (ONAB) Bejaia, Algeria, and had free access to water.The animals were handled following the care and use of the laboratory animals guide (Council, 2010).All procedures involving animals were approved by the University Animal Experiment Ethics Committee (approval ref no.162/2011/8).

Acute oral toxicity
The toxicity was evaluated in animals of both sexes, according to the Organization for Economic Co-operation and Development (OECD) guideline 423 with slight modifications (OECD, 2001).3 groups of NMRI mice, consisting of 3 males and 3 females each, were used for this evaluation.Each group received 0.5 mL of S. chudaei infusion, in different doses as follows: group 1-2000 mg/kg b.w.; group 2-3000 mg/kg b.w.; and group 3-5000 mg/kg body weight (b.w.).After administration, the animals were carefully observed in the first 4 h for any abnormal behavior or toxicity sign and after, daily during 14 days to observe mortality.

Analgesic activity
To determine the analgesic activity two tests were evaluated: the Writhing and Formalin tests.

Writhing test
The writhing test using acetic acid (Liu et al., 2015) proceeded as follows.5 groups, of 6 mice each, were used in this evaluation, the different groups receive orally as follows: group 1-0.5 mL saline solution; group 2-0.5 mL of paracetamol® at 100 mg/kg b.w.; group 3, 4, and 5-0.5 mL of S. chudaei infusion at 250, 500 and 1000 mg/kg b.w., respectively.After 30 min they were intraperitoneally injected with μl of 0.6% acetic acid.5 min after, the abdominal constrictions number was counted for 15 min.The results were expressed as protection percentage using the following formula: where NCc represents constrictions number in control (saline solution) and NCt the constrictions number in tested drug (paracetamol® and the infusion at different doses).

Formalin test
The antinociceptive activity of the sample extracts was assessed by the formalin test (Hunskaar et al., 1985).For that, 5 groups containing mice each were formed.The different groups received per os: 1-0.5 mL of saline; 2-50 mg/kg b.w.paracetamol®; 3-250 mg/kg b.w. of the infusion; 4-500 mg/kg b.w. of the infusion; 5-1000 mg/kg b.w. of the infusion.After 1h, 20 μl of formalin (2.5% formaldehyde in 0.9% NaCl saline) were subcutaneously injected into the right paw dorsal surface.Immediately, the licking and biting time of the injected paw was recorded for 5 min (early phase), and after 15-30 min (late phase).The protection percentage was calculated as follows: in which, Tc represents the time of licking and biting in the control (saline solution), Tt the time of licking and biting in the tested drug (paracetamol® and the infusion at different doses).

Anti-inflammatory activity
The evaluation of the anti-inflammatory activity was carried out by paw edema provocation with carrageenan (Levy, 1969).1% carrageenan in saline solution (0.9% NaCl), was injected into the mice right hind paw plantar surface.In this experiment 5 groups of 6 mice each were used; the control group received 0.5 mL of saline solution; the reference group received 50 mg/kg b.w. of diclofenac; and the other three groups received 250, 500, and 1000 mg/kg b.w. of S. chudaei infusion.The edema was induced after 30 min of the drug administration.4 h later, mice were sacrificed by cervical dislocation and immediately both right and left hind paws were cut at the talocrural joint and weighted.
The results are expressed as inhibition edema percentage (IEP) using the formula below: in which, EP represents edema percentage in control (c, saline solution) and in tested drug (t, diclofenac and infusion at different doses).
The edema percentage (EP) is calculated as follows: where RPW representing the right paw weight and LPW the left paw weight.

Total phenolic content (TPC)
Total phenolics were determined by spectrophotometry (BioTek Instruments, Inc.) (Alves et al., 2010) with some modifications.Briefly, 30 μl of the sample were mixed with 150 μl of Folin-Ciocalteu reagent (1:10, v/v) and 120 μl of sodium carbonate (7.5%, m/v).The mixture was then incubated during 15 min at 45 • C, followed by 30 min at room temperature, protected from light.The absorbance was measured at 765 nm.The calibration curve was prepared with gallic acid (5-80 mg/L; r = 0.9998) and the results expressed as mg of gallic acid equivalents/100g of sample dry weight (mg GAE/100g DW).

Total flavonoid content (TFC)
TFC were determined according to Costa et al. (2014).In brief, 100 μL of sample was mixed with 400 μL distilled water and 30 μL NaNO 2 5%; after 5 min at room temperature 30 μL AlCl 3 were added to the mixture; 1 min after, 200 μL NaOH 1M and 250 μL distilled water were added.Absorbance measurements were performed at 510 nm.A calibration curve was prepared with epicatechin (2.5-400 mg/L; r = 0.9946), and results expressed as mg of epicatechin equivalents/100g of sample dry weight (mg ECE/100g DW).

Antioxidant activity 2.9.1. DPPH • radical scavenging activity
This method is based on the reduction of the free radical DPPH • (2,2diphenyl-1-picrylhydrazyl) by the antioxidants present in the infusion.The evaluation of the scavenging power was performed according to Costa et al. (2014), with some modifications.Briefly, 30 μl of infusion was mixed with 270 μl of DPPH • (6.0 × 10 − 5 mol/L in ethanol).The absorption decrease of DPPH • was measured at 525 nm in intervals of 2 min, in order to observe the kinetics reaction.The reaction endpoint was reached in 20 min.The results were expressed as mg trolox equivalent/g of sample dry weight (mg TE/g DW) using a standard curve (5.62-75.87mg/L, r = 0.9978).

Ferric reducing antioxidant power (FRAP)
The FRAP assay was carried out according to Costa et al. (2014).In brief, 35 μl of sample was mixed with 265 μl of FRAP reagent (0.3 M acetate buffer, 10 mM TPTZ solution, and 20 mM of ferric chloride).The mixture was incubated for 30 min at 37 • C in dark.Absorbance was measured at 595 nm.A calibration curve was prepared with ferrous sulfate (25-500 μmol/L; r = 0.9997) and the results expressed as mg of ferrous sulfate/g of sample DW (mg FSE/g DW).

LC-DAD-ESI/MSn profiling of phenolic compounds
LC-DAD-ESI/MSn (Dionex Ultimate 3000 UPLC, Thermo Scientific, San Jose, CA, USA) was used for the phenolic profile determination, according to Bessada et al. (2016) protocol.The obtained extract was lyophilized then dissolved in methanol:water (80:20, v/v) at 10 mg/mL concentration.Compounds were detected using a DAD with (370, 330, and 280 nm) as favored wavelengths and MS in negative mode, via a Linear Ion Trap LTQ XL mass spectrometer (Thermo Finnigan, San Jose, CA, USA) coupled to ESI source.The compounds identification was based on their chromatographic behavior and UV-vis, and mass spectra, comparing with standards and reported data in literature via Xcalibur® data system (Thermo Finnigan, San Jose, CA, USA).The quantification was based on the UV-vis signal using the calibration curve for the available standards or the most similar for the non-available ones.
Values were expressed as mg/g of extract.

Statistical analysis
Results were presented in mean ± standard deviation.Comparisons between groups were performed by one-way ANOVA (analysis of variance) followed by post-hoc Tukey's HSD (Honestly Significant Difference) test.p<0.05 was considered statistically significant.All statistics were carried out using R statistical software (version 3.6.0).

Phytochemical analysis
In this work, we sought to investigate the antioxidant, antiinflammatory, and analgesic activities of S. chudaei infusion from the region of Tamanrasset (south of Algeria).It is well-known that establishing a correlation between phytochemical composition and potential biological activities has an essential role to figure out the potential compounds responsible for those activities.Hence, we have performed profiling and quantification of S. chudaei infusion phenolic compounds.
The comparison of TPC and TFC values of S. chudaei infusion with other reported results allows having an idea about the richness in phenolic compounds of S. chudaei infusion.Nevertheless, it is important to identify the type of phenolic compounds present in the sample, here identified by LC-DAD-ESI/MSn.The compounds identification was achieved through the UV-vis behavior and mass spectra fragmentation in negative ion mode, comparing with standards, when available, and Xcalibur® data system.According to literature review, no work on the profiling of phenolic compounds concerning S. chudaei from the Tamanrasset region (southern Algeria) or elsewhere, has been reported previously.Ten compounds from two classes, phenolic acids and flavonoids, were identified.Among the phenolic acids, one hydroxybenzoic acid (syringic acid hexoside derivative (2)) and four hydroxicinnamic acids (4-O-caffeoylquinic acid (1), caffeic acid (3), rosmarinic acid hexoside (7), and rosmarinic acid (10)) were detected.For the flavonoids class, five compounds were putatively identified, being three flavonols (kaempferol-O-diglucuronide (4), kaempferol-O-deoxyhexoside-hexoside (8), and kaempferol-O-glucuronide (9)), one flavanone (eriodictyol-O-glucuronide (5)), and one flavone (apigenin-O-diglucuronide (6)).
Quantitatively, the rosmarinic acid was the major compound with an amount of 18.3 ± 0.5 mg/g of extract, followed by another rosmarinic acid derivative, the rosmarinic acid hexoside with 6.58 ± 0.05 mg/g of extract, representing together about 73% of identified phenolics.4-Ocaffeoylquinic acid, kaempferol-O-diglucuronide, and apigenin-Odiglucuronide are present in considerable amounts of 1.783 ± 0.005, 1.42 ± 0.03, and 1.40 ± 0.02 mg/g of extract, respectively.The less abundant were eriodictyol-O-glucuronide, caffeic acid, and syringic acid hexoside derivative with 0.81 ± 0.02, 0.74 ± 0.03, and 0.51 ± 0.02 mg/ g of extract, respectively.The phenolic compounds extracted are almost all in the glycosylated form; glycosylation increases the solubility in water, the stability, and the promotion of the biological activity of various natural compounds including phenolics (Moon et al., 2017;Torres et al., 2011).
Several studies about phenolic compounds in Salvia species, using different extraction solvents (water, methanol, and ethanol), are in agreement with the present results.They reported rosmarinic acid as the dominant phenolic compound.This is the case of S. cadmica (Kocak et al., 2016), S. officinalis (Pedro et al., 2016), S. brachyantha and S. aethiopis (Tohma et al., 2016), and S. farinacea Var.Victoria Blue (Afonso et al., 2019).Nevertheless, some researchs have highlighted other abundant compounds than rosmarinic acid: luteolin-glucuronide in the infusion of S. officinalis (Martins et al., 2015;Schnitzler et al., 2008), and decoction and methanol/water extract of S. officinalis (Martins et al., 2015), kaempferol in an ethanolic extract of S. microstegia (Tohma et al., 2016).These molecules may function as genus markers of this species.Several factors can affect the chemical composition of plants, for example the harvesting season, geographical origin, and the extraction method (Moon et al., 2017).The reported findings indicate that S. chudaei can be a good source of rosmarinic and caffeic acid, as well as apigenin and kaempferol.
The FRAP assay consists in TPTZ-Fe (III) complex reduction to TPTZ-Fe (II) by the compounds present in tested samples.The reducing power is a useful determinant of potential antioxidant activity.S. chudaei infusion showed a value of 437.53 ± 17.32 μmol FSE/g DW, this value seems better than the ones previously reported by other authors.According to Grzegorczyk-Karolak and Kiss (2018) a FRAP activity between 890 and 1575 μmol FSE/g extract were obtained for decoction, infusion, and ethanolic extracts of S. viridis.Alimpić et al. (2017) evaluated the decoction and ethanolic extract of S. jurisicii (277.14 ± 8.6,  (Loizzo et al., 2014).
In both tests, DPPH • and FRAP, S. chudaei infusion exhibit excellent values for antioxidant activity, taking into account the reported results in the literature.It is also well-known that this feature has a strong correlation with TPC (Fernandes et al., 2016;Grzegorczyk-Karolak and Kiss, 2018;Kamatou et al., 2010), demonstrating the strong involvement of phenolics in antioxidant activity.Indeed, rosmarinic acid, non-exhaustively, could be responsible for the high antioxidant power of the sample.Rosmarinic acid extracted from Perilla frutescens leaves has displayed a higher DPPH • radical scavenging activity (SC50 = 5.5 ± 0.2 μg/mL) than P. frutescens leaves extract (10.8 ± 0.1 μg/mL).Additionally, rosmarinic acid SC50 compared to standards was slightly close to that obtained by Trolox (5.1 ± 0.1 μg/mL) and higher than that of butylated hydroxytoluene (BHT) (6.6 ± 0.3 μg/mL) (Zhu et al., 2014).
Moreover, rosmarinic acid showed the greatest activity potential among various phenolic acids tested in many antioxidant methods; caffeic acid has also shown an excellent antioxidant activity (Sevgi et al., 2015).Lu and Foo (2001) working on S. officinalis polyphenols have found that caffeic acid and rosmarinic acid derivatives exhibited potent antioxidant activity than flavone glycosides, and suggested that this strong activity could be due to their catechol moieties and conjugated unsaturation presence.
Reactive oxygen species (ROS) play a crucial role in the inflammatory response.They proceed as inflammatory mediators and signaling molecules produced by polymorphonuclear neutrophils (PMNs).Besides, reactive nitrogen species (RNS) can be rapidly formed by combination of nitrogen oxide (NO) and superoxide (O 2

Acute toxicity
Table 3 displays the results (mortality and toxicity sign) of the determination of acute toxicity of the infusion of S. chudaei aerial parts.The different infusion tested doses (2000, 3000, and 5000 mg/kg b.w.) did not show any toxicity sign (salivation, sleep, diarrhea, coma, etc.) in the first 4 h after oral administration.In the 14 days after, no mortality was observed for either male or female mice.This feature allows supposing the safety of S. chudaei infusion at the tested doses.Likewise, it is expected that LD50 (lethal dose causing 50% of tested animals death) will be superior to 5000 mg/kg b.w., taking into account the absence of any toxicity sign/mortality.Nevertheless, it will be very interesting to complete this test with a sub-acute oral toxicity test including biochemical and histological studies.

Anti-inflammatory activity
The anti-inflammatory effect of S. chudaei infusion was evaluated via carrageenan test, one of the most popular tests to screen the antiinflammatory properties of drugs and plant extracts.Table 4 shows the effect of three doses of S. chudaei infusion orally administered on mice paw induced inflammation.The doses (250, 500, and 1000 mg/kg b.w.) of S. chudaei infusion and diclofenac® (50 mg/kg b.w.) reduced significantly (p<0.001) the paw edema after 4 h of administration, comparing to the control.The lower edema percentage was obtained with the more concentrated infusion (1000 mg/kg b.w.) with an inhibition percentage of 58%, showing a strong anti-inflammatory effect.This effect was better than the effect obtained with diclofenac® (50 mg/ kg b.w.) that allowed an inhibition percentage of 53%.The other assayed infusion doses (250 and 500 mg/kg b.w.) allowed obtaining lower percentages but not so far from the mentioned values (45 and 48%, respectively).Many Salvia species have been screened for their anti-inflammatory activity (Akram et al., 2015;Kamatou et al., 2010).Choi et al. (2018) reported that some caffeic acid derivatives, especially rosmarinic acid methyl ester, extracted from S. miltiorrhiza exhibit potent anti-inflammatory effects through the inhibition of inflammatory mediators (COX-2: cyclooxygenase-2, NO: nitric oxide, and iNOS: inducible nitric oxide synthase) and the induction of HO-1 (heme oxygenase-1) which have for mission to inhibit the oxidative stress.On the other hand, Kamatou et al. (2010) tested the anti-inflammatory potential of sixteen Salvia species, reporting a poor inhibition of the 5-lipoxygenase enzyme (IC50 > 100 μg/mL), with exception of one species that displayed a moderate inhibition.The administration of Rosmarinus officinalis extract and its rosmarinic acid in similar dose (25 mg/kg b.w.) resulted the same paw edema inhibition of about 60% volume reduction compared with control (Rocha et al., 2015).
Taking into account the results of the phytochemical analysis performed in this study revealing the presence of rosmarinic acid as the main compound, and its described anti-inflammatory and antioxidant potential, it is expectable the ability of S. chudaei infusion to inhibit paw edema.This activity has rosmarinic acid as the responsible, probably due to its action on the COX-2 inhibition pathway.Many studies involve the flavonoids in the in vitro and in vivo anti-inflammatory properties.This is the case of apigenin, a compound with a significant presence in the studied sample, that is linked to its ability to inhibit cyclooxygenase and lipoxygenase enzymes (Kim et al., 2004;Narayana et al., 2001).

Acetic acid-induced writhing test
The analgesic effect of S. chudaei infusion was evaluated by the acetic acid test and Table 5 summarizes the obtained results.All tested doses (250, 500, and 1000 mg/kg b.w.) showed highly significant (p<0.001)In the acetic acid test, after intraperitoneal injection of the acid, an increase in the number of polymorphonuclear neutrophils into the peritoneal fluid was detected; this may raise the release of several inflammatory mediators, leading to the inflammatory pain in the inflammation site through nociceptive neurons stimulation.The acetic acid activates directly visceral and somatic nociceptors, as a general (nonselective) nociceptive model.It is a sensitive test to evaluate the peripheral analgesic action of drugs (Abbate et al., 1990;Le Bars et al., 2001;Satyanarayana et al., 2004).

Formalin test
The antinociceptive action of S. chudaei infusion was determined by measuring the spent time in licking/biting mice paw, after formalin injection, and the results are displayed in Table 6.Three doses (250, 500, and 1000 mg/kg b.w.) of infusion were used to evaluate the neurogenic (early) and inflammatory (late) phases.In both phases, the licking/ biting time was dose-dependent, decreasing with the dose increasing.In the early phase, the licking/biting time of the tested doses decreased significantly, comparing to the control (Table 6).The highest protection percentage was exhibited by the 1000 mg/kg b.w.dose (61%).This value is near to the one presented by paracetamol® 50 mg/kg b.w.(63%).In the late phase, a significant decrease in licking/biting time was also observed in all tested doses, comparatively to control, and in a similar way as described for the early phase.The 1000 mg/kg b.w.infusion dose showed a protection percentage of 58%, lower than the paracetamol 50 mg/kg b.w.protection percentage (65%) but an interesting antinociceptive action.
In the formalin test, there is a biphasic nociceptive response: an immediate and intense response characterized by direct activation of sensory fibers (early phase: 0-5 min); a prolonged tonic response phase related to the functional changes in the spinal cords dorsal horn and the production and release of numerous inflammatory mediators in the peripheral tissue (late phase: 15-30 min) (Dubuisson and Dennis, 1977;Tjølsen et al., 1992).In general, drugs acting on the central system inhibit these phases, whereas the peripherally acting ones inhibit only the early phase.
The results obtained from these analgesic tests suggest that S. chudaei infusion has peripheral and central analgesic activity.Rosmarinic acid may be the responsible, once it was proven to attenuate both central and peripheral pain involving opioid receptors.It may reduce the liberation of inflammatory mediators (see anti-inflammatory part (3.4.)) or directly block receptors (Boonyarikpunchai et al., 2014).Moreover, rosmarinic acid orally administered produces a significant and dose-dependent nociceptive response inhibition in mice paw glutamate-induced nociception (Guginski et al., 2009).In addition, rosmarinic acid showed a decrease of inflammatory and oxidative markers (COX-2, PGE-2, NO, IL-1β) in the spinal cord levels (Rahbardar et al., 2017).
Another study reported rosmarinic acid pain reduction only in the formalin test late phase (Falcão et al., 2016).Furthermore, kaempferol glycosides may display also an analgesic effect.After oral administration, they showed a potent analgesic and anti-inflammatory effect in mice (De Melo et al., 2009).

Conclusions
This is the first work about phenolic compounds composition, antiinflammatory, and analgesic effect of the infusion of Salvia chudaei Batt.& Trab.The infusion has a remarkable antioxidant and antiinflammatory activities, with analgesic effects in both central and peripheral models.The rosmarinic acid seems to be the main compound justifying such effects.Thus, the biological activities of S. chudaei aerial parts infusion may be due to the ability of phenolic compounds to inhibit inflammatory mediator generation, to act as radical scavenging, and to block directly the nociceptive receptors.The results of this study support the folk use of Salvia chudaei to treat inflammation and pain.However, more tests are needed to completely understand the mechanisms of action.Mean ± SD, different letters indicate statistically significant differences between groups (p<0.05).

Table 1
Table 2 shows the results obtained with TPC, TFC, DPPH • , and FRAP values of S. chudaei infusion.

Table 3
Effect of S. chudaei infusion on acute oral toxicity in mice.

Table 4
Effect of S. chudaei infusion on carrageenan-induced paw edema in mice.

Table 5
Effect of S. chudaei infusion on constrictions number of acetic acid-induced writhing test in mice.

Table 6
Effect of S. chudaei infusion on early and late phase of the formalin test in mice.