Salvia genus, representative for the Lamiaceae family as it comprises approximately one thousand different species, is considered for a wide plethora of therapeutic actions, such as antioxidant, anti-inflammatory, hepatoprotective, antitumoral and antidiabetic activity. The paper presents the preliminary chromatographic investigations of the polyphenols in the aerial parts of some Salvia spp. Using high-performance thin-layer chromatography (HPTLC) coupled with photodensitometry, caffeic acid was identified and quantified in the 20% methanolic extracts of Salviaeherba, in descending order, as follows: S. nemorosa (3.096%) &gt; S. verticillata (3.041%) &gt; S. sclarea (2.663%) &gt; S. glutinosa (1.962%) &gt; S. aethiopis (0.926%)

BEJENARU, Cornelia

BEJENARU, Ludovic Everard

BIŢĂ, Andrei

MARINESCU, Roxana Maria

MOGOŞANU, George Dan

PISOSCHI, Cătălina Gabriela

RADU, Antonia

RĂU, Gabriela

English

Annals of the University of Craiova - Agriculture, Montanology, Cadastre Series

AnaleleUniversităţii din Craiova, seriaAgricultură – Montanologie – Cadastru (Annals of the University of Craiova - Agriculture, Montanology, Cadastre Series)Vol. 52/1/2022   PRELIMINARY CHROMATOGRAPHICRESEARCH ON SOME  SALVIA SPP. (LAMIACEAE)  Roxana Maria MARINESCU1, Ludovic Everard BEJENARU2, Andrei BIŢĂ2*,  Cornelia BEJENARU3, Antonia RADU3, Gabriela RĂU4,  George Dan MOGOŞANU2, Cătălina Gabriela PISOSCHI1,5  (1)Doctoral School, University of Medicine and Pharmacy of Craiova, 2 Petru Rareş Street, 200349 Craiova, Romania; E-mail: roxana.balasoiu@umfcv.ro (2)Department of Pharmacognosy& Phytotherapy, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, 2 Petru Rareş Street, 200349 Craiova, Romania;  E-mail: andrei.bita@umfcv.ro, ludovic.bejenaru@umfcv.ro, george.mogosanu@umfcv.ro (3)Department of Pharmaceutical Botany, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, 2 Petru Rareş Street, 200349 Craiova, Romania;  E-mail: cornelia.bejenaru@umfcv.ro, antonia.radu@umfcv.ro (4)Department of Organic Chemistry, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, 2 PetruRareş Street, 200349 Craiova, Romania; E-mail: gabriela.rau@umfcv.ro (5)Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, 2 Petru Rareş Street, 200349 Craiova, Romania;  E-mail: catalina.pisoschi@umfcv.ro  Corresponding author email:andrei.bita@umfcv.ro  Abstract Salvia genus, representative for the Lamiaceae family as it comprises approximately one thousand different species, is considered for a wide plethora of therapeutic actions, such as antioxidant, anti-inflammatory, hepatoprotective, antitumoral and antidiabetic activity. The paper presents the preliminary chromatographic investigations of the polyphenols in the aerial parts of some Salvia spp. Using high-performance thin-layer chromatography (HPTLC) coupled with photodensitometry, caffeic acid was identified and quantified in the 20% methanolic extracts of Salviaeherba, in descending order, as follows: S. nemorosa (3.096%) > S. verticillata (3.041%) > S. sclarea (2.663%) > S. glutinosa (1.962%) > S. aethiopis (0.926%). Key words:photodensitometry, polyphenols, Salvia spp., thin-layer chromatography  INTRODUCTION Salvia genus, commonly known as sage, Lamiaceae family, includes almost one thousand different annual, biennial, and perennial herbaceous species originating mainly from the Central America, South America, Asia, and Mediterranean region. Fifteen Salvia spp. are also found in the Romanian flora (Ciocârlan, 2009).   From the phytochemical point of view, the aerial parts and leaves of Salvia spp. contain various active principles, such as:   essential oil (β-caryophyllene, germacrene-B, spathulenol, and cis-β-farnesene, β-ocimene, α-gurjunene, germacrene-D, hexyl acetate, aromadendrene); flavonoids (acacetin, kaempferol, pinocembrin, catechin, quercetin, glycosidesof apigenin, luteolin and scutellarein); diterpenoids of abietane, clerodane, pimarane and labdanetypes (carnosol, rosmanol, salvinolone, horminone); steroids (brassicasterone); triterpenoids of ursane, AnaleleUniversităţii din Craiova, seriaAgricultură – Montanologie – Cadastru (Annals of the University of Craiova - Agriculture, Montanology, Cadastre Series)Vol. 52/1/2022  oleanane, and lupane types; polyphenolcarboxylic acids (caffeic acid, rosmarinic acid, chlorogenic acid, salvianolic acid, ferulic acid); tannin; resins and oleoresins (Aćimović et al., 2018; Afonso et al., 2019; Bidabadi et al., 2020; Boufadiet al., 2021; Gîrdet al., 2014; Hanganu et al., 2019; Ivanov et al., 2022). In ethnopharmacology, Salvia spp. are used for rheumatism, diabetes, inflammation, ulcers, dizziness, seizures, and in various brain tonic recipes (Boufadi et al., 2021; Lopresti, 2017). For Salvia spp., up to date research highlighted some important pharmacological properties: anti-inflammatory, antioxidant, hypoglycemic, antimicrobial, wound healing, enhancer of the cognitive activity, protective towards neurodegenerative diseases, immuno-modulatory, hepatoprotective, cytotoxic in vitro against HepG2 (hepatocellular), K562 (chronic myelogenous leukemia), HL60 (human acute promyelocytic leukemia cells), MCF-7 (human breast), HeLa (cervical) carcinoma cells, enzyme inhibitory, anti-leishmanial (Aćimović et al., 2018; Afonso et al., 2019; Bahadori et al., 2018; Boufadiet al., 2021; Firuziet al., 2013; Gülçinet al., 2004; Hanganu et al., 2019; Ivanov et al., 2022; Lopresti, 2017; Tosun et al., 2009; Vasilache et al., 2021; Wu et al., 2012). The aim of our paper was the preliminary chromatographic analysis of polyphenols in the aerial parts of five Salvia spp. from Oltenia flora, using high-performance thin-layer chromatography (HPTLC) coupled with photodensitometry.  MATERIALS AND METHODS Plant material The plant material (aerial parts of five Salvia spp. – S. aethiopis L., S. glutinosa L., S. nemorosa L., S. sclarea L., and S. verticillata L.) was collected during the flowering period, in May–June 2022, from the Oltenia Region, South-West of Romania. The research did not involve endangered or protected herbal species. Voucher specimens (SaA-20220624/1, SaG-20220607/1,SaN-20220524/1, SaS-20220602/1, and SaV-20220607/1) were stored in the Herbarium of the Department of Pharmaceutical Botany, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova.  HPTLC analysis Preliminary HPTLC analysis of polyphenols from the aerial parts of five Salvia spp.(Salviae herba) was performed on CAMAG (Muttenz, Switzerland) system, in thefollowing experimental conditions (Altemimiet al., 2015; Bojić et al., 2013; Gîrdet al., 2014; Jug et al., 2018): stationary phase: HPTLC silica gel 60 F254 (Merck, Darmstadt, Germany) 20×10 cm precoated glass plates; mobile phase: ethyl acetate–formic acid–methanol–water (15:1:0.1:1, in volumes);10 mL of mobile phase were added in the developing twin-chamber (CAMAG) and then oversaturated for 20 minutes; sample: five 20% methanolic extracts of Salviae herba;reference compounds (Merck): 0.1% methanolic solutions of caffeic acid, chlorogenic acid and rutin;migration distance: 62 mm (sample application line 8mm, solvent front 70 mm);application of sample (2μL) and reference solutions(2 μL, 3 μl, 4 μL): CAMAG Linomat 5 semi-automatic system – spraying gas nitrogen, syringe volume 100 μL, dosage speed 150 nL/s, pre-dosage volume 0.2 μL, bands length of 8 mm;plate drying: 5 minutes, at 25ºC (cold air dryer);plate shooting: ultraviolet (UV) light (254 nm and 365 nm);detection:CAMAG TLC Scanner 3 photodensitometer, for densitogram and in AnaleleUniversităţii din Craiova, seriaAgricultură Montanology, Cadastre Series)Vol. 52/1/2022  situ UV light (280 nm) spectra, without derivatization, deuterium–tungsten lamp, scanning speed 20 mm/s, data reso100 μm/step, measurement mode absorbance;visionCATS ver. 3.1package (CAMAG).  DPPH in situ qualitative assay HPTLC plates were sprayed with0.5 mM methanolic solution of 2,2-diphenylpicrylhydrazyl (DPPH), in the CAMAG TLC Spray Cabinet 2, and dried at room temperature, in the dark, for 90 seconds, then heated at 60°C, in an oven, for 30 seconds. Chromatograms were documentedat white light illumination (Pozharitskayaal., 2008).  RESULTS AND DISCUSSIONS Figures 1–10exhibited the experimental data on the preliminary HPTLC analysis of polyphenols from the aerial parts of five Salvia spp. (Salviaeherba). Caffeic acid calibration curve (Figuwas achieved by linear regression with 5% range deviation, 1.5% coefficient ofvariation and 0.9927 correlation coefficient (R): y = 3.752×10-9x + 1.351×10-Caffeic acid (Rf 0.76) was quantified in the 20% methanolic extracts of Salviaeherbaas follows: S. nemorosa (3.096%) >verticillata (3.041%) >S. sclarea>S. glutinosa (1.962%) >S. aethiopis(0.926%). The screening of the antioxidant activity for Salviaeherbamethanolic extracts was determined in situ, after chromatographic separation (HPTLC–DPPH assay). The intensity of the yellow color for the HPTLC bands is directly correlated with the amount of polyphenols (e.g., caffeic acid) identified and quantified in the analyzed samples (Figure 3). Our results are comparable to those obtained in specialty papers regarding the – Montanologie – Cadastru (Annals of the University of Craiova lution  software -1- et re 10) mode,  2. , S.  (2.663%)  polyphenolic content of (Aćimović et al., 2018; Hanganu 2019; Lu & Foo, 2002; Mocan Wu et al., 2012).  Figure 1. HPTLC chromatogram of polyphenols from Salviaeherba 20% methanolic extractUV 254 nm, without derivatization. B: Blank; Lanes 1–3: Caffeic acid, Rf 0.76; Lanes 4Chlorogenic acid, Rf 0.22; Lanes 7Rf 0.08; Lanes 10–14: Samples (S. glutinosa, S. nemorosa, S. sclareaS. verticillata, respectively) Figure 2. HPTLC chromatogram of polyphenols from Salviaeherba 20% methanolic extractsUV 365 nm, without derivatization. B: Blank; Lanes 1–3: Caffeic acid, Rf 0.76; Lanes 4Chlorogenic acid, Rf 0.22; Lanes 7visualized; Lanes 10–14: Samples (S. glutinosa, S. nemorosa, S. sclareaS. verticillata, respectively). Figure 3. HPTLC chromatogram of polyphenols from Salviaeherba 20% methanolic extractswhite light illumination, derivatization with DPPH. B: Blank; Lanes 1–3: Caffeic acid, R- Agriculture, Salvia spp. et al., et al., 2020;  s:  –6: –9: Rutin,  S. aethiopis,   and  .  :  –6: –9: Rutin, not S. aethiopis,   and    : f 0.76; Lanes AnaleleUniversităţii din Craiova, seriaAgricultură Montanology, Cadastre Series)Vol. 52/1/2022  4–6: Chlorogenic acid, Rf 0.22; Lanes 7Rf 0.08; Lanes 10–14: Samples (S. aethiopisS. glutinosa, S. nemorosa, S. sclareaS. verticillata, respectively).Figure 4. Densitogram of caffeic acid (UV 280 nm, without derivatization) separated from the aethiopis aerial parts 20% methanolic extract.  Figure 5. Densitogram of caffeic acid (UV 280 nm, without derivatization) separated from the glutinosa aerial parts 20% methanolic   Figure 6. Densitogram of caffeic acid (UV 280 nm, without derivatization) separated from the – Montanologie – Cadastru (Annals of the University of Craiova –9: Rutin, ,   and    Salvia   Salvia  extract.  Salvia nemorosa aerial parts 20% methanolic extract. Figure 7. Densitogram of caffeic without derivatization) separated from the sclarea aerial parts 20% methanolic extract.   Figure 8. Densitogram of caffeic acid (UV 280 nm, without derivatization) separated from the verticillata aerial parts 20% methanol   Figure 9. In situ UV spectra (280 nm) of caffeic acid reference (a) and compound separated from - Agriculture,   acid (UV 280 nm, Salvia   Salvia ic extract.  AnaleleUniversităţii din Craiova, seriaAgricultură Montanology, Cadastre Series)Vol. 52/1/2022  the analyzed samples (b).  Figure 10. Caffeic acid reference calibration curve. CONCLUSIONS Salvia genusincludesabout 1000 different species, being considered for therapeutic properties (anti-inflammatory, antioxidant, antitumoral, hepatoprotective, antidiabetic). Preliminary chromatographic analysis of the polyphenols in the aerial parts of fiveSalvia spp. was madeHPTLC–photodensitometry.In the 20% methanolic extracts, caffeic acid was identified and quantified: S. nemorosa(3.096%) >S. verticillata (3.041%) >sclarea (2.663%) >S. glutinosa >S. aethiopis (0.926%).  ACKNOWLEDGEMENTS This work was supported by the POCU/993/6/13/153178, “Performanţăîncercetare” – “Research performance” co-financed by the European Social Fund within the Sectorial Operational Program Human Capital 2014–2020.  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PRELIMINARY CHROMATOGRAPHICRESEARCH ON SOME  SALVIA SPP. (LAMIACEAE)

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PRELIMINARY CHROMATOGRAPHICRESEARCH ON SOME SALVIA SPP. (LAMIACEAE)

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Annals of the University of Craiova - Agriculture, Montanology, Cadastre Series