Concerning four Stachys spp. (Lamiaceae) from the Oltenia flora, the paper highlights the polyphenolic content of aerial parts through high-performance thin-layer chromatography coupled with photodensitometry. Chlorogenic acid (CA) was identified and quantified in all 20% methanolic extracts of Stachydisherba. The highest CA amount was determined in S. recta (59.88 mg%), followed by S. sylvatica (32.90 mg%), S. officinalis (31.34 mg%), and S. germanica (25.16 mg%), respectively

APOSTOLESCU, Florian George

BEJENARU, Cornelia

BEJENARU, Ludovic Everard

BIŢĂ, Andrei

MOGOŞANU, George Dan

NEAMŢU, Johny

RADU, Antonia

English

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

Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru (Annals of the University of Craiova - Agriculture, Montanology, Cadastre Series)Vol. 52/1/2022   PRELIMINARY CHROMATOGRAPHIC INVESTIGATION OF FOUR STACHYS SPP. (LAMIACEAE) FROM OLTENIA FLORA  Florian George APOSTOLESCU1, Andrei BIŢĂ2, Ludovic Everard BEJENARU2*,  Cornelia BEJENARU3, Antonia RADU3, George Dan MOGOŞANU2, Johny NEAMŢU1,4  (1) Doctoral School, University of Medicine and Pharmacy of Craiova, 2 Petru Rareş Street, 200349 Craiova, Romania; E-mail: apostolescugeorge@yahoo.com (2) Department of Pharmacognosy & Phytotherapy, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, 2 Petru Rareş Street, 200349 Craiova, Romania;  E-mail: ludovic.bejenaru@umfcv.ro, andrei.bita@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 Physics, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova,  2 Petru Rareş Street, 200349 Craiova, Romania; E-mail: johny.neamtu@umfcv.ro  Corresponding author email: ludovic.bejenaru@umfcv.ro  Abstract Concerning four Stachys spp. (Lamiaceae) from the Oltenia flora, the paper highlights the polyphenolic content of aerial parts through high-performance thin-layer chromatography coupled with photodensitometry. Chlorogenic acid (CA) was identified and quantified in all 20% methanolic extracts of Stachydis herba. The highest CA amount was determined in S. recta (59.88 mg%), followed by S. sylvatica (32.90 mg%), S. officinalis (31.34 mg%), and S. germanica (25.16 mg%), respectively. Key words: high-performance thin-layer chromatography, Lamiaceae, photodensitometry, polyphenols, Stachys spp.  INTRODUCTION Commonly known as hedge nettle, Stachys genus, Lamiaceae family, contains more than 300 different annual or perennial species (herbs or small shrubs) originating from Mediterranean region, Asia, Americas, and southern Africa. In the Romanian flora, 12 Stachys spp. are also found (Ciocârlan, 2009; Imbrea et al., 2011). The flowering aerial part of Stachys spp. contains important active principles, such as: essential oil (germacrene D, limonene, β-caryophyllene, β-pinene, nerolidol, linalyl acetate, linalool, caryophyllene oxide, spathulenol, α-cadinene, α-pinene, β-myrcene, α-terpineol, β-bourbonene, β-ylangene); iridoids (ajugoside, aucubin, harpagide, acetyl-harpagide, harpagoside); flavonoids (apigenin, luteolin, chrysoeriol, isoscutellarein, kaempferol, quercetin, isorhamnetin, naringenin and their glycosides); diterpenes with labdane, neo-clerodane, rosane and ent-kaurene type (deoxyandalusol, stachaegyptins, roseo-stachone, stachyrosanes); triterpenes (ursolic acid and oleanolic acid derivatives); phytosterols; phenylethanoid glycosides (acteoside, verbascoside, martynoside, leucosceptoside A, lavandulifoliosides); phenylpropanoid glycosides (coniferin, syringin); lignans (sesamin, paulownin, urolignoside); phenolic acids (chlorogenic acid, caffeic acid); fatty acids (linoleic, oleic and palmitic acids): oligosaccharides (Giuliani et al., 2009; Goren et al., 2011; Hajdari et al., 2012; Háznagy-Radnai et 13Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru (Annals of the University of Craiova - Agriculture, Montanology, Cadastre Series)Vol. 52/1/2022   al., 2006 & 2012; Khanavi et al., 2009; Kiliç et al., 2017; Lazarević et al., 2013; Sarikurkcu et al., 2021; Stegăruş et al., 2021; Tomou et al., 2020; Tundis et al., 2014; Venditti et al., 2013). In Turkey, Stachys spp. are used as wild (mountain) tea mainly against stomatitis, diabetes, rheumatism, cough and cold, or as functional foods for a healthy diet (Carović-Stanko et al., 2016; Gören, 2014; Satıl & Açar, 2020; Tomou et al., 2020). Stachys spp. exhibited significant pharmacological actions, such as: antimicrobial (against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Candida albicans), antioxidant, anti-inflammatory (useful for polycystic ovary syndrome), hepatoprotective, anti-diabetes, wound healing, antiviral, cytotoxic and antiproliferative, enzyme inhibitory (Alizadeh et al., 2020; Goren et al., 2011; Hajdari et al., 2012; Háznagy-Radnai et al., 2012; Khanavi et al., 2009 & 2012; Lazarević et al., 2010 & 2013; Paun et al., 2018; Sarikurkcu et al., 2021; Tomou et al., 2020; Tundis et al., 2014). The purpose of our research was the preliminary analysis of polyphenols in the aerial parts of four Stachys spp. from Oltenia flora, by high-performance thin-layer chromatography (HPTLC)–densitometry.  MATERIALS AND METHODS Plant material The plant material was harvested during the flowering period, in May–June 2022, from the Oltenia Region, South-West of Romania. It was represented by the aerial parts of four Stachys spp. (S. germanica L., S. officinalis (L.) Trevis. sin. Betonica officinalis L., S. recta L., and S. sylvatica L.) Our study did not involve endangered or protected herbal species. The voucher specimens (StG-20220602/1, StO-20220623/1, StR-20220524/1, and StS-20220607/1, respectively) were deposited in the Herbarium of the Department of Pharmaceutical Botany, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova.  HPTLC analysis Starting from the aerial parts of four Stachys spp. (Stachydis herba), preliminary HPTLC–densitometric analysis of polyphenols was made on CAMAG (Muttenz, Switzerland) system, according with some specific experimental conditions (Altemimi et al., 2015; Bojić et al., 2013; Gîrd et 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 Stachydis herba; reference compounds (Merck): 0.1% methanolic solutions of caffeic acid, chlorogenic acid and rutin; migration distance: 62 mm (sample application line 8 mm, solvent front 70 mm); application of sample (2 μL) 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 situ UV light (280 nm) spectra, without derivatization, deuterium–tungsten lamp, scanning speed 20 mm/s, data resolution 100 μm/step, measurement 14Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru (Annals of the University of Craiova - Agriculture, Montanology, Cadastre Series)Vol. 52/1/2022   mode absorbance; visionCATS ver. 3.1 software package (CAMAG).  DPPH in situ qualitative assay In the CAMAG TLC Spray Cabinet 2, the HPTLC plates were sprayed with 0.5 mM methanolic solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH); then, the plates were dried at room temperature, in the dark, for 90 seconds, heated at 60°C, in an oven, for 30 seconds, and analyzed in white light illumination (Pozharitskaya et al., 2008).  RESULTS AND DISCUSSIONS The experimental results on the preliminary HPTLC analysis of polyphenols from the aerial parts of four Stachys spp. (Stachydis herba) were highlighted in Figures 1–9. Through linear regression mode, with 5% range deviation, 1.68% coefficient of variation (CV), and 0.9961 correlation coefficient (R), the calibration curve for chlorogenic acid (Figure 9) was used for quantitative analysis: y = 3.571×10-9x + 4.636×10-3. In the 20% methanolic extracts of Stachydis herba, the highest amount of chlorogenic acid (Rf 0.22) was quantified in S. recta (59.88 mg%), followed by S. sylvatica (32.90 mg%), S. officinalis (31.34 mg%), and S. germanica (25.16 mg%) aerial parts, respectively. After the chromatographic separation, using HPTLC–DPPH assay, for Stachydis herba methanolic extracts, the screening of the antioxidant activity was performed in situ. For the HPTLC bands, the intensity of the yellow color is directly proportional with the concentration of polyphenols (chlorogenic acid) in the examined samples (Figure 3). Starting from the polyphenolic content of Stachys spp., our results agree with the specialized research in the field of natural products chemistry (Khanavi et al., 2009; Lazarević et al., 2010; Stegăruş et al., 2021; Tomou et al., 2020).   Figure 1. HPTLC chromatogram of polyphenols from Stachydis herba 20% methanolic extracts:  UV 254 nm, without derivatization. B: Blank;  Lanes 1–3: Caffeic acid, Rf 0.76; Lanes 4–6: Chlorogenic acid, Rf 0.22; Lanes 7–9: Rutin,  Rf 0.08; Lanes 10–13: Samples (S. germanica,  S. officinalis, S. recta, and S. sylvatica, respectively).   Figure 2. HPTLC chromatogram of polyphenols from Stachydis herba 20% methanolic extracts:  UV 365 nm, without derivatization. B: Blank;  Lanes 1–3: Caffeic acid, Rf 0.76; Lanes 4–6: Chlorogenic acid, Rf 0.22; Lanes 7–9: Rutin, not visualized; Lanes 10–13: Samples (S. germanica, S. officinalis, S. recta, and S. sylvatica, respectively).  Figure 3. HPTLC chromatogram of polyphenols from Stachydis herba 20% methanolic extracts:  white light illumination, derivatization with DPPH. B: Blank; Lanes 1–3: Caffeic acid, Rf 0.76; Lanes 4–6: Chlorogenic acid, Rf 0.22; Lanes 7–9: Rutin, Rf 0.08; Lanes 10–13: Samples (S. germanica,  S. officinalis, S. recta, and S. sylvatica, respectively). 15Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru (Annals of the University of Craiova - Agriculture, Montanology, Cadastre Series)Vol. 52/1/2022     Figure 4. Densitogram of chlorogenic acid  (UV 280 nm, without derivatization) separated  from the Stachys germanica aerial parts  20% methanolic extract.   Figure 5. Densitogram of chlorogenic acid  (UV 280 nm, without derivatization) separated  from the Stachys officinalis aerial parts  20% methanolic extract.    Figure 6. Densitogram of chlorogenic acid  (UV 280 nm, without derivatization) separated  from the Stachys recta aerial parts  20% methanolic extract.   Figure 7. Densitogram of chlorogenic acid  (UV 280 nm, without derivatization) separated  from the Stachys sylvatica aerial parts  20% methanolic extract.   Figure 8. In situ UV spectra (280 nm) of chlorogenic acid reference (a) and compound separated from the analyzed samples (b).   Figure 9. Chlorogenic acid reference calibration curve.  CONCLUSIONS Preliminary chromatographic analysis of the polyphenols in the aerial parts of four Stachys spp. from the Oltenia flora was achieved by HPTLC coupled with photo-densitometry. Identified and quantified in 16Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru (Annals of the University of Craiova - Agriculture, Montanology, Cadastre Series)Vol. 52/1/2022   the 20% methanolic extracts of Stachydis herba, the amount of chlorogenic acid was fluctuating (mg%): S. recta (59.88) > S. sylvatica (32.90) > S. officinalis (31.34) > S. germanica (25.16).  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PRELIMINARY CHROMATOGRAPHIC INVESTIGATION OF FOUR STACHYS SPP. (LAMIACEAE) FROM OLTENIA FLORA

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PRELIMINARY CHROMATOGRAPHIC INVESTIGATION OF FOUR STACHYS SPP. (LAMIACEAE) FROM OLTENIA FLORA

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