Comparative Studies on Analytical, Antioxidant, and Antimicrobial Activities of a Series of Vegetal Extracts Prepared from Eight Plant Species Growing in Romania

Lucia Pirvu*, Cristina Hlevca, Ioana Nicu, and Corina Bubueanu

Key Words

(High-performance) thin-layer chromatographic screening East European plant species Polyphenols Antioxidant and antimicrobial activity

Summary 1 Introduction Given the large applicability in both pharmaceutical and cosmetic field, this work was aimed at analytical (high-performance thin-layer The use of the medicinal plants for prevention or cure of dis- chromatography [HPTLC] method), antioxidant (2,2-diphenyl- eases appears to be an ancient practice, dating from Paleolithic 1-picrylhydrazyl [DPPH] method), and antimicrobial (diffusion [1], and nonhuman primates [2] and sick animals also prove the method on Staphylococcus aureus ATCC 6538, Escherichia coli self-medication by using vegetal products [3]. Among potential ATCC 8739, and Candida albicans ATCC 10231) studies on a series threats, the infectious agents (including bacteria, viruses, fun- of whole vegetal extracts and corresponding aqueous, ethyl acetate gus, protozoa, parasites, etc.) come out as one of the most and chloroform fractions (selective extracts) prepared from eight important concerns in the living world. In support, biologists plant species growing in Romania. Briefly, it was revealed moderate reported lowland gorillas’ behavior that take 90% of their diet to certain activity against S. aureus ATCC 6538 and E. coli ATCC from Aframomum melegueta fruits, known for their potent 8739 in the case of greater burdock leaves (Arctium lappa), beech antimicrobial properties, also active against shigellosis, and leaves (Fagus sylvatica) and great willowherb aerial part (Epilobium hirsutum) whole extracts. Purple loosestrife aerial part (Lythrum sali- other similar infections [4]. On the other hand, 12 years of caria) and sea-buckthorn leaves (Hippophae rhamnoides) whole researches indicated that the chimpanzees, when sick, ingest extracts showed only weak activity against these bacteria, while tar- plant materials that are not generally eaten, thus, supporting the ragon aerial part (Artemisia dracunculoides), chokeberries leaves existence of self-medication among great apes [2]. Moreover, it (Aronia melanocarpa) and quince fruit (Cydonia oblonga) whole was noticed that even birds select nesting material rich in extracts have not shown activity. Subsequently studies on the selec- antimicrobial agents, thus, protecting their young from danger- tive extracts have revealed that combinations of glycosides of the ous bacteria [5], finally concluding that generally, sick animals same aglycones lead to very different antimicrobial properties indi- tend to forage with plants rich in secondary metabolites [6]. cating synergistic effects between polyphenols (demonstrated in the Regarding their chemical structure, most of them are aromatic case of Fagus sylvatica extracts) or the contribution of other phyto- substances belonging to polyphenols, coumarins, terpenoids, compounds to the final effect (demonstrated in the case of Arctium lappa extracts) and, secondly, that the glycosyl chain in which they essential oils, and alkaloids (sub)classes, but also to lectins or occur may also contribute to the final antimicrobial effect (demon- poly-peptides, each with a specific mechanism of inhibition [7]. strated by the fact that the most active vegetal extracts emphasized Given their high interest in promoting human health, the struc- the dominance of flavonoid monoglycosides). Moreover, inhibitory tural features related to flavonoids antimicrobial effectiveness antimicrobial effects were revealed (Arctium lappa extracts). Finally, were intensely studied, and the results were reviewed as follows: none of the studied extracts acted against C. albicans ATCC 10231. 2,4- or 2,6-dihydroxylation of the B (phenyl) ring and 5,7-dihy- Concerning antioxidant activity, DPPH tests indicated high potency droxylation of the A (flavan) ring in the flavanone structure are of all tested extracts (IC50 measuring from 2.66 to 4.80). Further important for their anti-methicillin-resistant Staphylococcus studies on the most active vegetal extracts which aim to reveal MICs aureus (MRSA) activity; substitution at the 6th or 8th position values and potential inhibitory or activatory effects in combination with a long chain aliphatic group such as lavandulyl (5-methyl- with chemical antibiotics are ongoing. 2-isopropenyl-hex-4-enyl) or geranyl (trans-3,7-dimethyl-2,6- octadienyl) enhances antimicrobial activity; substitution with C8 and C10 chains enhances the activity of flavan-3-ol class; 5- hydroxy-flavanones and 5-hydroxyisoflavanones with hydroxyl group at position 2 are very active; chalcones are more effective L. Pirvu, C. Hlevca, I. Nicu, and C. Bubueanu, National Institute of Chemical- against MRSA than flavanones or flavones, hydroxyl groups at Pharmaceutical R&D, Department of Pharmaceutical Biotechnologies, Plant Extract Laboratory, Vitan Avenue No. 112, Bucharest, Romania. the 2nd position being very important for their anti-staphylococ- E-mail: [email protected] cal activity; methoxy groups lead to drastically decreasing in the

Journal of Planar Chromatography 27 (2014) 5, 346–356 DOI: 10.1556/JPC.27.2014.5.4 346 0933-4173/$ 20.00 © Akadémiai Kiadó, Budapest

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Table 1

Plant material description.

No. Common name Latin name Family Plant part Harvest method Location, source

P1 Chokeberries Aronia melanocarpa Rosaceae Folium Cultivated Prahova and Ilfov*, Romania P2 Tarragon* Artemisia dracunculoides Asteraceae Herba Cultivated P3 Sea-buckthorn Hippophae rhamnoides Elaeanaceae Folium Wild-crafted P4 Quince* Cydonia oblonga Rosaceae Fructus Cultivated P5 Purple losestrife Lythrum salicaria Lythraceae Herba Wild-crafted P6 European beech Fagus sylvatica Fagaceae Folium Wild-crafted P7 Greater burdock Arctium lappa Asteraceae Folium Wild-crafted P8 Great willowherb Epilobium hirsutum Onagraceae Herba Wild-crafted

*Purchased from the vegetable market antibacterial activity of flavonoids [8]. In view of all these, this into 20% propylene glycol reagent (prepared with deionized work was aimed at analytical (HPTLC method), antioxidant water) in a manner to obtain identical total phenol content (2,2-diphenyl-1-picrylhydrazyl [DPPH] method), and antimi- (5 mg/1 mL extract, expressed as gallic acid equivalents crobial (diffusion method on three microbial strains – S. aureus [GAE]). The resulting eight 20% propylene glycol vegetal ATCC 6538P, Escherichia coli ATCC8739, and Candida albi- extracts (measuring from 20 to 30 mL), also called whole cans ATCC 10231) screening on a series of vegetal extracts pre- extracts (codified P1–P8), were stored in amber glasses bottles pared from eight plant species growing in Romania. Plant at 4°C until microbiological studies. species were selected on basis of the final practical goal, the obtaining of some hygiene and cosmetic plant-derived products, 2.3 Preparation of Selective Vegetal Extracts respectively, for which some less used, valuable, and also abun- dant plant materials (ex., leaves of Arctium lappa and Fagus syl- Other 250 mL of each (E1–E8) ethanolic extracts were concen- vatica; herba of Epilobium hirsutum, Lythrum salicaria, and trated at residue, and the residues were separately solved into Artemisia dracunculoides; and fruits of Cydonia oblonga) or 100 mL distilled water. The resulting aqueous solutions were plant parts (ex., leaves of Hippophae rhamnoides and Aronia manually extracted, first with (3 × 100 mL) chloroform and then melanocarpa) were selected. with (3 × 100 mL) ethyl acetate solvents, 24 h separation time per respective extraction. The resulting aqueous (codified AQ), ethyl acetate (codified EA), and chloroform1 (codified CHL) 2 Experimental fractions were (separately) concentrated at residue, and the residues were solved into 20% propylene glycol reagent in a manner to also assure 5 mg total phenols (GAE) per 1 mL 2.1 Plant Materials sample. The eight medicinal plants (S1–S8) used in this study were cul- tivated or harvested from the wild (Table 1); taxonomic identi- 2.4 Preparation of Extracts Hydrolysates2 fication was done by the botanist’s team of National Institute of Other 50 mL of each (E1–E8) ethanolic extracts were (separate- Chemical-Pharmaceutical R&D (ICCF), Bucharest, Romania, ly) hydrolyzed for 30 min in 4 N HCl environment. The result- and voucher specimens are deposited in ICCF Plant Material ing filtrates were next partitioned with ethyl acetate (50 mL × 3) Storing Room. Except quince fruit prepared as a fresh smashed and, after that, evaporated to dryness and redissolved in 98% paste, all other (seven) plant materials were shade dried and ethanol to a final volume of 50 mL. The resulting ethanolic ground (using a sieve mill) to finally prepare medium-sized hydrolysates (codified H1–H8) as well as the original ethanolic (3–5 mm) vegetal powders. (E1–E8) extracts were used for comparative analytical (HP)TLC studies. 2.2 Preparation of Whole Vegetal Extracts Eight charges of 50 g vegetal raw material were twice (heat- 2.5 Chemicals assisted) extracted with 500 mL of 70% ethanol (prepared with Chemicals (sodium carbonate and 2,2-diphenyl-1-picrylhy- deionized water). The (eight) pairs of extracts were filtered drazyl/DPPH), Folin and Natural Product reagents, and solvents through paper filter resulting in 850–900 mL ethanolic extract (methanol, ethanol, ethyl acetate, formic acid, acetic acid, and (codified E1–E8). chloroform) were purchased from Sigma-Aldrich Co. and Fluka Two hundred fifty milliliters of each (E1–E8) ethanolic extract (Bucharest, Romania) as well as (95%), (min. were concentrated at residue. The (eight) residues were passed 95%), isoquercitrin (>90%), hyperoside (>97%),

1Due to lack of polyphenols compounds, chloroform residue was prepared using the corresponding whole extract algorithm. The obtained selective extracts (in fact, aque- ous, ethyl acetate, and chloroform fractions) were also stored into amber glasses bottles at 4°C until analytical and microbiological studies. 2Basically, this study intended vegetal extracts polyphenols aglycones releasing, much more accessible on the reference compounds market compared to their glycosides.

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Table 2

Polyphenols references comparative RF values in studied systems.

Reference compounds (polyphenols) Spot color RF System A System B

Chlorogenic acid Light-blue fl. 0.48–0.55 0.28 Quercetin-3-O-rhamnoglucoside/rutin Yellow-orange fl. 0.40–0.45 0.29–0.30 -7-O-apioglucoside/apiin Green fl. 0.50–0.55 0.31–0.32 Quercetin-3-O-glucoside/isoquercitrin Orange fl. 0.65–0.67 0.34–0.36 Quercetin-3-O-galactoside/hyperoside Orange fl. 0.68–0.69 0.38–0.40 Luteolin-7-O-glucoside/cynaroside Yellow fl. 0.68–0.69 0.42–0.44 Apigenin-8-C-glucoside/vitexin Light-green fl. 0.70–0.72 0.52–0.54 Apigenin-7-O-glucoside/cosmosiin Green fl. 0.72–0.74 0.61–0.63 Gallic acid Blue-indigo fl. 0.94 0.42–0.48 Rosmarinic acid Blue fl. 0.96 0.47–0.50 Protocatechuic acid Blue-indigo fl. 0.97 0.70 Gentisic acid Blue-marine fl. 0.98 0.74 Caffeic acid Light-blue fl. 0.96 0.78 Ferulic acid Blue-Indigo fl. 0.99 0.98 Quercetin Yellow-orange fl. 0.98 0.68 Luteolin Yellow fl. 0.98 0.78 Kaempferol Blue-green fl. 0.98 0.86 Apigenin Green fl. 0.98 0.92 Umbelliferone Intense-blue fl. 0.97 0.96 Scopoletin Intense-blue fl. 0.98 0.98

(95%), apigenin (>97%), apigenin-7-O-glucoside/cosmosiin loaded in 2-3 μL per 8 mm band. The loaded plate was then kept (97%), apigenin-8-C-glucoside/vitexin (>96%), vitexin- in TLC twin developing chamber at 18–19°C with respective 2˝O-rhamnoside (>98%), apigenin-7-O-apiosylglucoside/apiin mobile phase (System A or System B) up to 90 mm. The devel- (>99%), luteolin (>98%), luteolin-7-glucoside (analytical stan- oped plate was dried using a hair dryer and then immersed into dard), chlorogenic acid (>95%), caffeic acid (99%), rosmarinic identification reagents (Natural Product followed by PEG4000). acid (97%), gallic acid (95%), protocatechuic acid (>97%), and The dried plate was next disposed in photo-documentation gentisic acid (>99%) as well as umbelliferone (>99%) and chamber, and the images were captured at UV 366 nm. scopoletin (>99%) reference substances3. Polyphenol spots were assigned by comparison with literature and own reference product data as well as individual plant prod- uct literature data. 2.6 Qualitative Analysis of Polyphenols Qualitative analysis of polyphenols was performed using TLC 2.7 Estimation of Total Phenolics Content technique according H. Wagner et al. [9] and E. Reich et al. [10] indication. The HP(TLC) screening has been done using two Total phenolics content was measured by standard Folin– study models; system ethyl acetate–acetic acid–formic acid– Ciocalteau method (Romanian Pharmacopoeias, Xth edn. [11]). water/100:12:12:26 (System A) recommended for flavonoids Briefly, (three) aliquots of 50–500 μL tested vegetal sample glycosides (polyphenols) screening and system chloroform– were mixed with 200 μL of Folin–Ciocalteau reagent and accu- acetic acid–methanol–water/64:32:12:8 (System B) recom- rately finished at 5000 μL volumetric flasks with (5% w/v) sodi- mended for saponins, but also very useful for polyphenols agly- um carbonate. Flasks were mixed and left in the dark place cones study (Table 2). Briefly, variable volumes (0.5–5 μL) of at room temperature for 5 min, and then the absorbance was read the vegetal samples/extracts were loaded as 8 mm band length in at λ = 750 nm with a UV/vis (Hélios γ, Thermo Electron the 10 × 10 cm silica gel 60F HPTLC plate (Merck, Darmstadt, Corporation) spectrophotometer. Total phenol content was esti- Germany) using Hamilton syringe and Linomat 5 instrument mated using gallic acid standard calibration curve (r2 = 0.9992). (CAMAG, Muttenz, Switzerland). Similarly, the reference Results were expressed as mg gallic acid equivalents (GAE) per compounds (10−3 M in 70% ethanol solutions) mixtures were 1 mL extract.

3All reference substances were prepared as 10−3 m solution in (70%) ethanol.

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2.8 Free Radical Scavenging Assay 2.9.4 Antimicrobial Assay Free radical scavenging potency was measured by using The tests were performed in sterile Petri dishes, each containing Sanchez-Moreno et al.’s method [12]. Precisely, the capacity of 15–20 mL of appropriate culture medium (CaSoA for bacteria the eight (20%) propylene glycol extracts (the whole extracts) and SDA for fungus) previously inoculated with 104–105 CFU with identical total phenols content (5 mg GAE/1 mL) to scav- mL−1. On each dish, 4 stainless steel cylinders of 8 mm diameter enge 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals has were placed on the solidified surface of the medium. been measured. Briefly, the eight vegetal extracts as well as Afterwards, in each cylinder, 0.2 mL sample was added in the gallic acid and rutin references were prepared as four dilution following manner: one cylinder contained 20% propylene gly- series (methanol solvent), 5, 0.5, 0.05, and 0.005 mg (GAE)/ col reagent (the solvent in which the plant extracts were passed), 1 mL sample. Aliquots of 50 μL of each diluted sample were and the other 3 contained triplicates of each vegetal sample mixed with 2950 μL of DPPH solution (0.025 g L−1, also pre- (P1–P8 extracts and their aqueous, ethyl acetate, or chloroform pared in methanol solvent). A blank solution containing the fractions). The Petri dishes were then incubated 18–24 h at same amount of methanol and DPPH was also prepared. After 30–35°C for bacterium and 2 days at 20–25°C for the fungus. 30 min standing at room temperature, the radical scavenging After the incubation period, the growth inhibition zones were activity of the test samples and the extract series against 2,2- measured, and the results were expressed as the arithmetic mean diphenyl-1-picryl hydrazyl radical, respectively, were deter- of three measurements for the same sample (triplicates). mined by measuring the absorbance at λ = 517 nm. Gallic acid Antimicrobial activity was calculated on basis of the diameter of and rutin references were measured as positive controls. Each the growth inhibition as follows: (<10 mm) – no antimicrobial sample was measured in triplicate and averaged. The percent- activity; (10–15 mm) – weak antimicrobial activity; (16–20 age radical scavenging activity (RSA), also known as antioxi- mm) – moderate antimicrobial activity; (>20 mm) – certain dant activity (AA) was calculated using the following formula: antimicrobial activity. % RSA = {(A – B)/A} × 100 (where A is the absorbance of the control, and B is the absorbance of samples). The free radical scavenging activities of the eight whole extracts, rutin, and 3 Results and Discussion gallic acid references were expressed as IC50 (defined as con- centration [in μg mL−1] of sample that inhibits 50% of the for- mation of DPPH radical). 3.1 Qualitative Analytical Screening A. melanocarpa L. (Fam. Rosaceae), common chokeberry, is 2.9 Microbiological Test described as one of the richest plant sources of polyphenols compounds, mainly cyanidin-3-galactosides. Chlorogenic acid 2.9.1 Test Organisms isomers and quercetin glycosides were also reported in aronia berries [13, 14]. As for the aronia leaves raw material, literature The tests were carried out using cylinder method in plates on reports about the occurrence of quercetin and caffeic acid three microorganisms: a Gram-negative bacterium (E. coli derivates including hyperoside, rutin, quercetin 3-O-α-arabino- ATCC 8739), a Gram-positive bacterium (S. aureus ATCC pyranosil-(1→6)-β-D-glucopyranoside, and chlorogenic acid 6538), and a fungus (C. albicans ATCC 10231); test organisms isomers [15]. In the present study, the TLC screening in System were purchased from Mecconti s.à r.l. through their distributor A (allowing flavonoids glycosides separation) indicated aronia in Romania (Merck Romania S.R.L.). leaves (70%) ethanolic extract (Figure 1A, T1 tracks) as con- taining at least eleven polyphenols compounds as follows: six 2.9.2 Test Extracts yellow-orange fluorescent (fl.) spots attributed to quercetin The tests were fulfilled on the eight 20% propylene glycol derivates (s3, s4, s5, s6, s9, and s10) including rutin (s6), hyper- (P1–P8) extracts (the whole extracts), and the corresponding oside (s9), isoquercitrin (10), and, the most likely, 3-O-arabino- aqueous (AQ), ethyl acetate (EA), and chloroform (CHL) frac- glucopyranoside (s5); two blue-fl. spots attributed to caf- tions (the selective extracts) were prepared from E6, E7, and E8 feoylquinic acid derivates including chlorogenic (s7) and ethanolic extracts. neochlorogenic (s8) acids and three blue-green fl. spots (s1, s2, and s11), the most probable to belong to kaempferol glycosides. 2.9.3 Inoculum Preparation In support, the next studies in System B setting (Figure 1B) con- The bacterial strains were activated by passaging the cells on firmed hyperoside and isoquercitrin (T1 track, orange, fl. spots at R = 0.40 and 0.35) attendance, aronia leaves hydrolysate Casein soya bean digest agar medium (CaSoA) and incubated F for 18–24 h at 30–35°C. In the case of microorganisms used in a sample (T1h track) also proving chlorogenic acid and caffeic acid (blue fl. spots at R = 0.28 and 0.78) abundancy as well as sporulate form (C. albicans ATCC 10231), the strain was acti- F umbelliferone (intense blue fl. zone at the R = 0.96), and vated by passaging it on Sabouraud dextrose with chloramfeni- F kaempferol (green fl. zone at the R = 0.86) attendancy, the last col medium (SDA) and left for 5 days at 20–25°C. After the F incubation period, 1-2 colonies were collected with a sterile loop one supporting kaempferol derivates apartness of the s1, s2, and and added in sterile distilled water in order to obtain microbial s11 spots. Additionally, aronia leaves hydrolysate sample have suspensions. The turbidity of these suspensions was adjusted to revealed quercetin derivates sensibility to acidic (4 N HCl) match the turbidity standard of 0.5 McFarland units correspond- hydrolysis. ing to 1-2 × 10(8) colony-forming units per milliliter A. dracunculoides L. (Fam. Asteraceae), common (Russian) tar- (CFU mL−1) [11]. ragon, is mainly known for its volatile oils richness measuring

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Figure 1 Figure 2

TLC aspects of aronia leaves 70% (v/v) ethanolic extract and corre- TLC aspects of tarragon herba 70% (v) ethanolic extract and corre- sponding hydrolysate sample. System A: Tracks T1, aronia leaves sponding hydrolysate sample. System A: Track T1, chlorogenic acid ethanolic extract – duplicate sample; Track T2, rutin, chlorogenic (ref.); Track T2, rutin, chlorogenic acid, apigenin-7-O-glucoside/cos- acid, apigenin-7-O-glucoside/cosmosiin, quercetin (ref.); Track T3, mosiin, quercetin (ref.); Tracks T3, tarragon herba ethanolic extract – luteolin-7-O-glucoside, luteolin (ref.); System B: Track T4, chloro- duplicate sample; System B: Track T4, luteolin-7-O-glucoside, lute- genic acid, quercetin-3-O-galactoside/hyperoside, rosmarinic acid olin, kaempferol (ref.); Track T5, apigenin-7-O-apiosyl-glucoside/ (ref.); Track T1, aronia leaves ethanolic extract sample; Track T1h, apiin, quercetin-3-O-galactoside/hyperoside, rosmarinic acid, kaemp - aronia leaves ethanolic extract hydrolysate sample; Track T5, rutin, ferol (ref.); Track 3, tarragon herba ethanolic extract sample; Track apigenin-8-C-glucoside/vitexin, and umbelliferone (ref.); Track T6, T3h, tarragon herba ethanolic extract hydrolysate sample; Track T6, protocatechuic acid and apigenin (ref.). rutin, apigenin-7-O-glucoside/cosmosiin, protocatechuic acid, and apigenin (ref.).

from 0.15 to 3.1%, function of plant origin. As for phenolic con- the present work, sea buckthorn leaves (70%) ethanolic extract tent, tarragon herba was assigned with numerous polyphenols has been confirmed as a mixture of numerous (at last 15 compounds such as coumarins (e.g., herniarin, esculetin, polyphenols) quercetin, apigenin, kaempferol, and gallic and esculin, umbelliferone, scopoletin, scopanone, scoparin, etc.), ferulic acid derivates. Thus, the TLC study in System A (Figure phenyl carboxylic acids (e.g., chlorogenic acid and its isomers, 3A, T2 tracks) emphasized at least six yellow-orange fl. zones, caffeic, chicoric, gentisic, anisic, hydroxybenzoic, and ferulic mostly quercetin polyglycosides (s1, s2, s3, and s5) added to acids), and flavonoids (e.g., rutin, kaempferol, quercetin, lute- rutin (s7) and hyperoside (s11) compounds aside at least five olin, , and ) [16]. In the present work, green and blue-green fl. zones, mainly in the upper part of the TLC qualitative study in System A setting (Figure 2A, T3 chromatogram attributed to apigenin and kaempferol glycosides tracks) indicated tarragon herba (70%) ethanolic extract as being (s4, s8, s12, s13, and s14). The two intense blue fl. spots (s9 and characterized by four main polyphenol compounds as follows: s10) in the middle of the chromatogram as well as the deep blue rutin (s1), chlorogenic acid (s2), and two other caffeoylquinic and purple (s6 and s15) fl. spots situated in lower front part were acid derivates, like 4,5-di-O-caffeoylquinic acid (s3) and cichor- assigned to gallic and ferulic acid derivates, respectively. ic acid (s4). Next, analysis in System B (Figure 2B) confirmed Subsequently, screening in System B setting (Figure 3B) con- System A spot ascription and also quercetin derivates destruc- firmed rutin and hyperoside attending (T2 track, yellow-orange tion in 4 N HCl, the hydrolysate sample (T3h track) clearly fl. zone at RF = 0.32 and 0.41) as well as vitexin and cosmosiin revealing caffeic acid (blue fl. spot at R = 0.78) basis of the F (T2 track, green fl. zone at RF = 0.54 and 0.63) presence. In addi- three (s2, s3, and s4) phenolic acids. tion, the hydrolysate sample (T2h track) clearly revealed gallic acid (indigo fl. zone at R = 0.44), apigenin (green fl. zone at R H. rhamnoides L. (Fam. Elaeagnaceae), commonly sea buck- F F = 0.92), and kaempferol (green fl. zone at R = 0.86) aglycones thorn, is an important medicinal species mainly owing to its fruit F valuable chemical content. As for the sea buckthorn leaves raw attending, thus, sustaining System A qualitative appraisals. material, studies also revealed valuable phytocompounds Cydonia oblonga Mill. (Fam. Rosaceae), quince fruit in particu- such as (e.g., quercetin-3-O-galactoside/hyperoside, lar, was reported with high contents of glucides, organic acids, quercetin-3-O-glucoside/, kaempferol, and isor - pectins, and proteins added to polyphenols and also some small hamnetin), leucoanthocianidols, and (e.g., (−)-epicat- quantities of fats, vitamins (C, A, B, and PP), and minerals (Ca, echin, (+)-gallocatechin, (−)-epigallocatechin) as well as pheno- Fe, P, K, Cu, Mg, and S) [18]. As for polyphenols profile, analy- lic acids, gallic and ferulic acids derivates. Besides, carotenes, ses on quince pulp part indicated several caffeoylquinic acid vitamin E, free and esterified sterols (triterpenic acids), iso- derivates such as 3-, 4-, and 5-O-caffeoyl-quinic acids and 3,5- prenols, and minerals (Ca, Mg, and K) attendance were report- dicaffeoyl- aside small quantities of rutin. Quince ed, and the hydrolysated samples also reveal , isostric- peel part screening indicated, besides upper phenolics, several tinin, , and presence [17]. In quercetin 3-galactoside, kaempferol 3-glucoside, kaempferol 3-

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Figure 3 Figure 4

TLC aspects of the sea-buckthorn leaves 70% (v/v) ethanol extract TLC aspects of the quince fruit 70% (v) ethanolic extract and corre- and corresponding hydrolysate sample. System A: Track T1, rutin, sponding hydrolysate sample. System A: Track T1, rutin, chlorogenic chlorogenic acid, apigenin-7-O-apiosylglucoside/apiin, quercetin-3- acid, apigenin-7-O-glucoside/cosmosiin, and quercetin (ref.); Tracks O-galactoside/hyperoside, rosmarinic acid, and apigenin (ref.); T2, quince fruit ethanolic extract – duplicate sample; Track T3, rutin, Tracks T2, sea-buckthorn leaves ethanolic extract – duplicate sam- chlorogenic acid, apigenin-7-O-apiosylglucoside/apiin, quercetin-3- ple; Track T3, rutin, vitexin-2-O-rhamnoside, quercetin-3-O-rhamno- O-galactoside/hyperoside, rosmarinic acid, and apigenin (ref.); side/, and quercetin (ref.); System B: Track T4, rutin, api- System B: Track T4, chlorogenic acid, rutin, gallic acid, and caffeic genin-8-C-glucoside/vitexin, and umbelliferone (ref.); Track T5, proto- acid (ref.); Track T2, quince fruit ethanolic extract sample; Track T2h, catechuic acid and apigenin (ref.); T2, sea-buckthorn leaves ethano- quince fruit ethanolic extract hydrolysate sample; Track T5, luteolin- lic extract sample; Track T2h, sea-buckthorn leaves extract hydroly- 7-O-glucoside, luteolin, and kaempferol (ref.); Track T6, apigenin-7-O- sate sample; Track T6, luteolin-7-O-glucoside, luteolin, and kaempfer- apiosylglucoside/apiin, quercetin-3-O-galactoside/hyperoside, ros- ol (ref.). marinic acid, and kaempferol (ref.). rutinoside, and other unidentified glycosides likely acylated derivates resistance at acidic attack (T3h track, green fl. zone at with p-coumaric acid incidence [19]. In the present work, TLC RF = 0.34). screening using System A setting (Figure 4A, T2 tracks) indi- F. sylvatica L. (Fam. Fagaceae), exactly European beech leaves, cated quince fruit (70%) ethanolic extract as characterized by were ascribed with polysaccharides (xylanes and pectins struc- three main caffeoylquinic acid derivates (blue fluorescent spots ture), polyphenols, triterpenic acids of ginsenosid Ro-type, vita- situated at R = 0.51, 0.58 and 0.88) attributed to chlorogenic F mins (C, E, K1, and a-tocopherols), carotenes, and minerals acid (s1), neochlorogenic acid (s2), and, most probably, 3,5- (Mn, Mo, Cu, Zn, Fe, Co, and sulphur). Of polyphenols, pheno- dicaffeoylquinic acid (s3). System B setting (Figure 4B) study lic acids such as p-hydroxibenzoic, syringic, gallic, abscisic, and on the quince fruit hydrolysate sample (T2h track) confirmed cinnamic acid and flavonoids such as quercetin, kaempferol, chlorogenic acid and caffeic acid aglicone (blue fl. zone at R = F chrysin, , and (epi) derivates were previously 0.28 and 0.78) attendancy, thus, confirming caffeoylquinic acid revealed [22, 23]. More recent (HP)TLC studies on a series of polyphenols dominant in quince fruit. ethanol extracts have revealed a dynamic of the beech leaves L. salicaria L. (Fam. Lythraceae), or purple loosestrife, is polyphenols content along the vegetation time; it was revealed described with high amounts of polyphenols compounds (up to that if spring leaves are dominated of one major kaempferol 18%) including gallotannins (e.g., 1,6-di-O-galloyl-, diglycoside aside only traces of chlorogenic acid and apigenin 1- and 6-O-galloylglucose), ellagitannins (e.g., vescalagin and derivates, summer leaves abound in chlorogenic acid and api- ), anthocyanins (e.g., malvidin and cyaniding genin derivates next to small quantities of quercetin and cate- types), and flavon C-glycosides (e.g., vitexin, isovitexin, ori- chin derivates; in contrast, autumn leaves indicated that chloro- entin, and isoorientin) added to triterpenic acids (e.g., betulinic genic acid isomers decline versus apigenin, quercetin, and cate- acid, betulinic acid methyl ester, and vanoleic acid dilactone), chin derivates augmentation [24]. In the present work, System A polysaccharides (mucilages type), fatty acids, volatile oils, setting study (Figure 6A1, T2 tracks) on (70%) ethanol extract carotenes, vitamin C, glucose, and fructose [20, 21]. The present of beech leaves harvested in September time indicated the fol- TLC study in System A setting (Figure 5A, T3 tracks) indicated lowing polyphenols profile: at least eight blue fl. spots disposed purple loosestrife herba (70%) ethanolic extract as being charac- along the entire chromatogram owing to chlorogenic acid iso- terized by two luteolin derivates, intense yellow fl. spots situat- mers (s1, s3, s4, s6, s8, s9, s11, and s12) including chlorogenic ed in the center of the chromatogram attributed to orientin (s4) (s1) and neochlorogenic (s3) acids, numerous isochlorogenic and isoorientin (s2) compounds. Also, two blue fl. zones (s3 and acids (s5, s6, s8, s9, and s11) and caffeic acid (s12) as well; at s6) belonging to phenolic acids and rutin (s1), and vitexin (s5) least three green fl. spots owing to apigenin derivates (s5/over- flavonoids were detected. The next study in System B setting lapped by hyperoside, s7 and s10) including vitexin (s7); three (Figure 5B) disclosed chlorogenic acid isomers (T3 track, blue yellow-orange fl. spots (s2, s5, and s13) owing to quercetin fl. zone at RF = 0.15–0.28) and gallic acid (T3 and T3h tracks, derivates including hyperoside (s5) and quercetin (s13); and one ′ indigo fl. zone at RF = 0.42) occurrence as well as luteolin big (blue)green fl. spot likely owing to kaempferol-4 -p-

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Figure 6

TLC aspects of beech leaves 70% (v/v) ethanolic extract and corre- sponding hydrolysate sample. System A: (A1) Track T1, rutin, chloro- genic acid, apigenin-7-O-apiosylglucoside/apiin, quercetin-3-O- galactoside/hyperoside, rosmarinic acid, and apigenin (ref.); Tracks Figure 5 T2, beech leaves ethanolic extract – duplicate sample; Track T3, rutin, TLC aspects of purple loosestrife herba 70% (v/v) ethanolic extract vitexin-2-O-rhamnoside, luteolin-7-O-glucoside, quercetin-3-O-rham- and corresponding hydrolysate sample. System A: Track T1, rutin, noside/quercitrin, and quercetin (ref.); (A2) Tracks T2aq, aqueous vitexin-2-O-rhamnoside, luteolin-7-O-glucoside, quercetin-3-O-rham- fraction/P6AQ – duplicate sample; Track T2ea, ethyl acetate fraction/ noside/quercitrin, and quercetin (ref.); Track T2, rutin, chlorogenic P6EA – duplicate samples; Track T2chl, chloroformic fraction/ acid, apigenin-7-O-apiosylglucoside/apiin, quercetin-3-O-galacto- P6CHL – single sample. System B: Track T4, rutin, apigenin-7-O-glu- side/hyperoside, rosmarinic acid, and apigenin (ref.); Tracks T3, pur- coside/cosmosiin, protocatechuic acid, and apigenin (ref.); Track T5, ple loosestrife herba ethanolic extract – duplicate sample; System B: chlorogenic acid, rutin, gallic acid, and caffeic acid (ref.); Track T2, Track T4, chlorogenic acid, rutin, gallic acid, and caffeic acid (ref.); beech leaves ethanolic extract sample; Track T2h, beech leaves etha- Track T3, purple loosestrife 70% ethanolic extract sample; Track T3h, nolic extract hydrolysate sample; Track T6, apigenin-7-O-apiosylglu- purple loosestrife ethanolic extract hydrolysate sample; Track T5, coside/apiin, quercetin-3-O-galactoside/hyperoside, rosmarinic acid, luteolin-7-O-glucoside and luteolin (ref.); Track T6, apigenin-7-O-apio- and kaempferol (ref.). sylglucoside/apiin, quercetin-3-O-galactoside/hyperoside, ros- marinic acid, and kaempferol (ref.).

coumaryl-3-monoglycoside (s4), the major compound. Further compounds of germacrane-type furan sesquiterpenes such as proved with real antimicrobial potency, auxiliary qualitative arctiopicrin and polyphenols such as chlorogenic acid and rutin (HP)TLC studies on the separate (aqueous, ethyl acetate, and (the major compounds) are reported [21]. Our System A TLC chloroform) fractions (Figure 6A2) clearly revealed that caf- study (Figure 7A1, T2 tracks) on burdock leaves (70%) ethanol feoylquinic acid isomers focus in aqueous fraction (T2aq tracks) extract indicated chlorogenic acid (s2) and isoquercitrin (s4) as while flavonoids derivates focus in ethyl acetate fraction (T2ea the major phenolics. Further shown with certain antimicrobial track), and chloroformic fraction (T2chl track) did not show any properties, supplementary qualitative studies on the three sepa- phenolic compound. Finally, System B setting study (Figure rate (aqueous, ethyl acetate, and chloroform) fractions (Figure 6B) clearly revealed caffeic acid and kaempferol (T2h track, 7A2) clearly revealed three new quercetin derivates (s1, s3, and blue and green fl. zone at RF = 0.78/0.86) aglycones presence, s5) previously overlapped by blue fl. phenolic acids; spots were the nonhydrolized sample (T2 track) also confirming hyperoside attributed to rutin (s1) and two other quercetin monoglycosides

(orange fl. zone at RF = 0.39), and apigenin-7-O-glucoside/cos- (s3 and s5). Studies on the separate fractions also revealed mosiin (green fl. zone at RF = 0.63) attendance in beech leaves quercetin glycosides partition between aqueous and ethyl (70%) ethanolic extract. acetate (polar) fractions as well as the lack of the phenolics com- A. lappa L. (Fam. Asteraceae), common greater burdock, is pounds at the level of chloroform fraction. System B setting mainly known for its roots’ benefits; in Asian regions, these (Figure 7B) study confirmed isoquercitrin (T2 track, orange fl. zone at R = 0.35) presence as well as caffeic acid basis (T2h roots are used as vegetable. Bardanae radix contains high levels F track, R = 0.78) of the greater burdock phenolic acids. of carbohydrates (69%) including inulin (27–50%) and F mucilages, dietary fibers, fats, proteins, vitamins (B1, B2, B3, E. hirsutum L. (Fam. Onagraceae), commonly great wil- B5, B6, B9, C, E, and k), and minerals (Ca, Fe, Mg, Mn, P, K, lowherb, another valuable and abundant phytomedicine in Na, and Zn). Besides, there were reported some tannin–iron Romania, is known with high content in flavonoids (up to 5.8%) complexes, numerous caffeoyl quinic acids (such as caffeic, [26]; thus, there were reported some unique ellagitannins of chlorogenic, isochlorogenic, and other isomers), various poly- 2-O-galloyl 3-O-valoneoyl dilactone-(α/β)-4C1-gluco-pyra- acetylenes (about 15) and sulphuric acetylene compounds nose, 1′-mono-decarboxy- dilactone, and valoneic (about 10), essential oil, guainolides, costic acid, dehydrocostus dilactone dioxine type aside gallic, ellagic, protocatechuic, val- lactone and 11,13-dehycrodihydrocostus-lactone (the root's bit- oneic dilactone and p-coumaric acids, methyl gallate, p- ter principles), lignans (e.g., neoarctin, arctigenin, arctiin, dau- methoxy gallic acid methyl ester as well as kaempferol, costerol, matairesinol, and lappaol) as well as sitosterol, stig- quercetin and 6-O-galloylglucose, 1,6-di-O-galloyl- masterol, and gamma-guanidino-n-butyric acid [25]. As for the glucose, 2,3-di-O-galloyl-glucose, 1,2,6-tri-O-galloylglucose greater burdock leaves raw material, literature is poorer; bitter plus their 3-O-glucuronides, 3-O-arabinosides, and 3-O-rham-

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Figure 7 Figure 8

TLC aspects of burdock leaves 70% (v/v) ethanolic extract and corre- TLC aspects of great willowherb (70%) ethanolic extract and corre- sponding hydrolysate sample. System A: (A1) Track T1, rutin, chloro- sponding hydrolysate sample. System A: (A1) Track T1, rutin, chloro- genic acid, apigenin-7-O-apiosylglucoside/apiin, quercetin-3-O- genic acid, and caffeic acid (ref.); Track T2, great willowherb ethano- galactoside/hyperoside, rosmarinic acid, and apigenin (ref.); Tracks lic extract – duplicate sample; (A2) Track T2aq, aqueous fraction/ T2, burdock leaves ethanolic extract – duplicate sample; Track T3, P8AQ – duplicate sample; Track T2ea, ethyl acetate fraction/P8EA – rutin, vitexin-2-O-rhamnoside, luteolin-7-O-glucoside, quercetin-3-O- duplicate sample; Track T2chl, chloroform fraction/P8CHL sample; rhamnoside/quercitrin, and quercetin (ref.); (A2) Tracks T2aq, System B: Track T3, chlorogenic acid, rutin, gallic acid, and caffeic aqueous fraction/P7AQ – duplicate samples; Track T2ea, ethyl aceta- acid (ref.); Track T2, great willowherb ethanolic extract sample; Track te fraction/P7EA – duplicate samples; Track T2chl, chloroformic frac- T2h, great willowherb ethanolic extract hydrolysate sample; Track T4, tion/P7CHL – single sample. System B: Track T4, rutin, apigenin-7-O- rutin, apigenin-7-O-glucoside/cosmosiin, protocatechuic acid, and glucoside/cosmosiin, protocatechuic acid, and apigenin (ref.); Track apigenin (ref.); Track T5, apigenin-7-O-apiosylglucoside/apiin, quer - T5, apigenin-7-O-apiosylglucoside/apiin, querectin-3-Ogalactoside/ cetin-3-O-galactoside/hyperoside, rosmarinic acid, and kaempferol hyperoside, rosmarinic acid, and kaempferol (ref.); Track T2, burdock (ref.). leaves ethanolic extract sample; Track T2h, burdock leaves ethanolic extract hydrolysate sample; Track T6, chlorogenic acid, rutin, gallic acid, and caffeic acid (ref.). nosides [27]. Moreover, some neutral and acidic saponins, indicated that all tested extracts are potent antioxidant agents; anthocianidins, volatile oils, vitamin C, and essential amino aronia leaves (IC50 = 2.66 μg mL−1) and purple loosestrife herba acids (e.g., cysteine, valine, serine, threonine, leucine, pheny- (IC50 = 2.83 μg mL−1) whole extracts show the best DPPH scav- lalanine, proline, isoleucine, tyrosine, etc.) as well as minerals enging activity, even better than that of references (IC50 rutin = and microelements (e.g., Co, Cu, Zn, K, S, P, Mg) have been 5.08 μg mL−1, IC50 gallic acid = 4.00 μg mL−1). Scavenger identified [18]. The present TLC study on great willowherb potency of the other vegetal extracts decreased as follows: sea (70%) ethanolic extract (Figure 8A1, T3 tracks) and System A buckthorn leaves (IC50 = 3.0 μg mL−1), quince (IC50 = 3.01 setting analysis, respectively, revealed five intense red-orange μg mL−1), tarragon herba (IC50 = 3.08 μg mL−1), beech leaves fl. spots attributed to myricetin derivates (s2˝, s3, s5, s6, and s7), (IC50 = 3.25 μg mL−1), great willowherb (IC50 = 4.66 μg mL−1), among which two could be of myricetin-3-O-rhamnoside/ and burdock leaves (IC50 = 4.80 μg mL−1). and myricetin-3-O-glucuronide [28] compounds aside one blue-green fl. spot (s2′′′) attributed to a kaempferol 3.3 Antimicrobial Activity Screening glycoside and one blue fl. (s2′) plus three indigo fl. (s1, s4, and s8) spots likely owing to caffeic acid and, respectively, gallic The studies were aimed at the antimicrobial activity assessment acid derivates. TLC study in System B setting (Figure 8B) con- of the eight whole (P1–P8) vegetal extracts prepared into (20%) firmed four of the five miricetin derivates (T3 track, red fl. spots Table 3 at RF = 0.28, 0.33 and 0.39 and 0.48), the hydrolysate sample (T3h track) also confirming kaempferol (blue-green fl. zone at IC50 values of the eight whole extracts comparatively to gallic acid

RF = 0.87), and gallic acid (indigo fl. zone at RF = 0.46/0.52) and rutin references. aglycones attending. Additionally, this study clearly revealed −1 2 myricetin derivates damage in acidic (4 N HCl) medium. Also Plant material/whole extracts IC50 (yg mL ) R proved with antimicrobial activity, the auxiliary qualitative stud- Aronia leaves (P1) 2.66 0.9966 ies on the separate (aqueous, ethyl acetate, and chloroform) frac- Tarragon herba (P2) 3.08 0.9908 tions (Figure 8A2) had to reveal that myricetin glycosides focus in ethyl acetate fraction (T3ea tracks) while kaempferol glyco- Sea-buckthorn leaves (P3) 3.00 0.9923 side attends in aqueous fraction (T3aq tracks), and chloroform Quince fruit (P4) 3.01 0.9875 fraction has not shown any phenolic compound. Purple loosestrife (P5) 2.83 0.9805 Beech leaves (P6) 3.25 0.9894 3.2 Antioxidant Activity Screening Greater burdock leaves (P7) 4.80 0.9916

Table 3 presents comparative IC50 values of the eight whole Great willowherb herba (P8) 4.66 0.9929 vegetal extracts comparatively to rutin and gallic acid refer- Gallic acid 4.00 0.9994 ences; tests were fulfilled on five dilution series (5–5000 μg mL−1), and results are presented as mean ± SD (n = 3). Data Rutin 5.08 0.9991

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Table 4

Antimicrobial assay results on the eight whole extracts.

Plant material/whole extracts Microbial strain Inhibition zone diameter (mm)

Aronia leaves (P1) Staphylococcus aureus ATCC 6538 <8 Escherichia coli ATCC 8739 <8 Tarragon herba (P2) Staphylococcus aureus ATCC 6538 8 ± 0.06 Escherichia coli ATCC 8739 8 ± 0.13 Sea-buckthorn leaves (P3) Staphylococcus aureus ATCC 6538 12 ± 0.15 Escherichia coli ATCC 8739 <8 Quince fruit (P4) Staphylococcus aureus ATCC 6538 <8 Escherichia coli ATCC 8739 <8 Purple loosestrife (P5) Staphylococcus aureus ATCC 6538 12 ± 0.05 Escherichia coli ATCC 8739 8 ± 0.16 Beech leaves (P6) Staphylococcus aureus ATCC 6538 17 ± 0.10 Escherichia coli ATCC 8739 12 ± 0.10 Greater burdock leaves (P17) Staphylococcus aureus ATCC 6538 21 ± 0.29 Escherichia coli ATCC 8739 <8 Great willowherb herba (P8) Staphylococcus aureus ATCC 6538 17 ± 0.06 Escherichia coli ATCC 8739 17 ± 0.15

Values are mean inhibition zone (mm) ± S.D. of three replicates, where diam. <10 mm means no activity, diam. 10–15 mm means weak activity, diam. 16–20 mm means moderate activity, and diam. >20 mm means certain activity

propylene glycol solution. Also, in the case of the most active (P5), similar to sea buckthorn leaves whole extract (P3), exhib- whole extracts, additional studies on the corresponding selective ited only weak activity on S. aureus ATCC 25923 and no activi- extracts (represented of their separate aqueous/AQ, ethyl aceta- ty on E. coli ATCC 8739. In contrast, beech leaves whole extract te/EA, and chloroform/CHL fractions) have been done; all, (P6) indicated moderate activity on S. aureus ATCC 25923 and whole and selective extracts, were at identical concentration weak activity on E. coli ATCC 8739, greater burdock leaves level, 5 mg total phenols/1 mL sample (GAE). Antimicrobial whole extract (P7) indicated certain activity on S. aureus ATCC activity screening was purchased on three organisms: a Gram- 25923 and no activity on E. coli ATCC 8739, while great wil- negative bacterium (E. coli ATCC8739), a Gram-positive bac- lowherb whole extract (P8) exhibited moderate activity on both, terium (S. aureus ATCC25923), and a fungus (C. albicans S. aureus ATCC 25923 and E. coli ATCC 8739. Results are ATCC10231). Briefly, the sterile Petri dishes, each containing detailed in Table 4. Subsequently, studies on the most effective 15–20 mL of appropriate culture medium (CaSoA for bacteria plant species selective extracts (respectively on the and SDA for fungus), were first inoculated with 104–105 CFU aqueous/AQ, ethyl acetate/EA, and chloroform/CHL fractions mL−1. Further, on each dish, 4 stainless steel cylinders of 8 mm prepared through processing beech leaves/P6, greater burdock diameter were placed on the solidified surface of the medium leaves/P7, and willowherb aerial part/P8 ethanolic extracts) and, afterwards, in each cylinder, 0.2 mL of test sample was have revealed interesting relationships between separate frac- added in the following manner: one cylinder contained (20%) tions in making final antimicrobial activity (Table 5 and 6). propylene glycol solvent, and the other three, the vegetal extract Thus, compared to beech leaves whole extract (P6) shown with (triplicate sample). The Petri dishes were then incubated 18–24 moderate activity on S. aureus ATCC 25923 and weak activity h at 30–35°C for bacterium and 2 days at 20–25°C for the fun- on E. coli ATCC 8739, chloroformic fraction (P6CHL) as well gus. After the incubation period, the growth inhibition zones as aqueous fraction (P6AQ) did not reveal activity against these were measured, and the results were expressed as the arithmetic bacterial strains; in contrast, ethyl acetate fraction (P6EA) mean of three measurements for the same sample (triplicate revealed weak activity on S. aureus ATCC 25923 and no activi- sample). Antimicrobial activity was calculated on the basis of ty on E. coli ATCC 8739, likely suggesting that aqueous or chlo- the diameter of the growth inhibition (<10 mm – no activity; roformic fraction induces a potency activity (synergistic effects) 10–15 mm – weak activity; 16–20 mm – moderate activity; upon ethyl acetate fraction in the making antimicrobial activity. >20 mm – certain antimicrobial activity). It must be noted that In the case of greater burdock leaves extracts, if whole extract (20%) propylene glycol solvent showed no antimicrobial (P7) presented certain activity against S. aureus ATCC 25923 activity. and no activity against E. coli ATCC 8739, chloroform fraction The obtained results indicated that the whole extracts of aronia (P7CHL) indicated moderate activity against S. aureus ATCC leaves (P1), tarragon herba (P2), and quince fruit (P4) have not 25923 and no activity against E. coli ATCC 8739. Anticipated shown antimicrobial activity on E. coli ATCC 8739 and S. by the HPTLC data (quercetin glycosides partition between the aureus ATCC 25923. Purple loosestrife herba whole extract two fractions, respectively), ethyl acetate fraction (P7EA), sim-

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Table 5

Antimicrobial assay results on Staphylococcus aureus ATCC 6538 strain.

Plant material Inhibition zone diameter (mm) per tested extract Whole extract Selective extracts Chloroform fraction (CHL) Ethyl acetate fraction (EA) Aqueous fraction (AQ)

Beech leaves (P6) 17 ± 0.10 <8 13 ± 0.10 <8 Greater burdock leaves (P7) 21 ± 0.29 17 ± 0.06 17 ± 0.03 12 ± 0.13 Great willowherb (P8) 17 ± 0.06 <8 15 ± 0.03 14 ± 0.15

Values are mean inhibition zone (mm) ± S.D. of three replicates, where diam. <10 mm means no activity, diam. 10–15 mm means weak activity, diam. 16–20 mm means moderate activity, and diam. >20 mm means certain activity

Table 6

Antimicrobial assay results on Escherichia coli ATCC 8739 strain.

Plant material Inhibition zone diameter (mm) per tested extract Whole extract Selective extracts Chloroform fraction (CHL) Ethyl acetate fraction (EA) Aqueous fraction (AQ)

Beech leaves (P6) 12 ± 0.10 <8 <8 <8 Greater burdock leaves (P7) <8 <8 16 ± 0.10 12 ± 0.10 Great willowherb (P8) 17 ± 0.15 <8 15 ± 0.08 16 ± 0.09

Values are mean inhibition zone (mm) ± S.D. of three replicates, where diam. <10 mm means no activity, diam. 10–15 mm means weak activity, diam. 16–20 mm means moderate activity, and diam. >20 mm means certain activity

ilar to aqueous (P7AQ) fraction, presented moderate activity on respectively, thus, allowing the appraisal of the relationship S. aureus ATCC 25923 but also low activity on E. coli ATCC between qualitative chemical composition and biological effect. 8739, thus, suggesting the ability of the greater burdock chloro- HPTLC and microbiological studies on S. aureus ATCC 6538, form fraction to inhibit the antimicrobial activity of the aqueous E. coli ATCC 8739, and C. albicans ATCC 10231 carried on and ethyl acetate fractions against E. coli ATCC 8739 strain these whole and selective vegetal extracts revealed two major (inhibitory effects). Furthermore, if the whole willowherb aerial aspects: first of all, that combinations of glycosides of the same part extract (P8) exhibited moderate activity against both bacte- aglycones lead to very different antimicrobial properties indica- rial strains, the corresponding ethyl acetate fraction (P8EA) as ting synergistic effects between polyphenols species (demons- well as aqueous (P8AQ) fraction induced weak to moderate trated in the case of F. sylvatica extracts on both S. aureus ATCC activity against S. aureus ATCC 25923 and E. coli ATCC8739; 6538 and E. coli ATCC 8739) or the contribution of other phy- chloroform fraction (P8CHL) has not shown antimicrobial tocompounds to the final effect (demonstrated in the specific activity. Taking also into account the HPTLC data (polyphenols case of A. lappa extracts tested on S. aureus ATCC 6538) and, partition between the three fractions, respectively), these results second, that the glycosyl chain in which they occur may also clearly demonstrated kaempferol and myricetin derivates’ inde- contribute [8] to the antimicrobial effect (demonstrated by the pendent antimicrobial activity. Finally, none of these fractions fact that the most active vegetal extracts revealed the dominance (selective extracts) exceeded the antimicrobial potency of the of flavonoids, apigenin, quercetin, myricetin and kaempferol, original, whole extract, and none of the whole or selective monoglycosides, aside phenolic acids, gallic and caffeoyl quinic extracts acted against C. albicans ATCC10231 strain. Current acid derivates). Moreover, there were inhibitory antimicrobial data were used also as the scientific basis of a new patent [29]. effects revealed (demonstrated in the specific case of A. lappa extracts tested on E. coli ATCC 8739 strain). In support of these, the whole extracts from greater burdock leaves (combining 4 Conclusion quercetin monoglycosides with caffeoyl quinic acid derivates), beech leaves (combining apigenin and quercetin monoglyco- Given the large applicability of vegetal polyphenols, this work sides also with caffeoyl quinic acid derivates), and great wil- was aimed at analytical, antioxidant, and antimicrobial activity lowherb aerial part (combining myricetin and kaempferol mono studies on a series of whole and selective vegetal extracts isolat- and diglycosides respectively, with caffeoyl quinic and gallic ed from eight East European plant species. The whole extracts, acid derivates) indicated moderate to good efficacy against S. as well as their corresponding selective extracts (in fact the aureus ATCC 6538 and E. coli ATCC8739. In contrast, the aqueous/AQ, ethyl acetate/EA, and chloroform/CHL fractions whole extracts prepared from purple loosestrife aerial part prepared through processing the origin, whole ethanolic (dominated by luteolin derivates aside caffeoyl quinic and gallic extracts) were finally passed into (20%) propylene glycol sol- acid derivates) and sea buckthorn leaves (dominated by vent so as to achieve identical total phenols content, 5 mg/1 mL quercetin polyglycosides aside caffeoyl quinic, gallic and also

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Unauthenticated | Downloaded 09/29/21 02:44 AM UTC Comparative Studies on Analytical, Antioxidant, and Antimicrobial Activities of Vegetal Extracts ferulic acid derivates) showed only weak activity against these [10] E. Reich, A. Schibli, HPTLC for the Analysis of Medicinal Plants, bacteria, while the whole extracts from chokeberries leaves Thieme, New York–Stuttgart, 2008. (also dominated by quercetin polyglycosides aside caffeoyl [11] Romanian Pharmacopoeia, Xth edn., Ed. Medicala, Bucharest, quinic acid derivates), tarragon herba (containing rutin and caf- 1993. feoyl quinic acid derivates), and quince fruit (containing caf- [12] C. Sanchez-Moreno, J.A. Larrauri, F. Saura-Calixto, J. Agric. feoyl quinic acid derivates only) showed no antimicrobial activ- Food Chem. 76 (1998) 270–276. ity at all. It must be noted that none of these selective extracts [13] R. Taheri, B.A. Connolly, M.H. Brand, B.W. Bolling, J. Agric. Food were superior to the corresponding whole extract, also support- Chem. (2013) [Epub ahead of print]. ing plant compounds cooperation in making final antimicrobial activity and that none of the whole or selective extracts acted [14] B. Olas, B Wachowicz, P. Nowak, M. Kedzierska, A. Tomczak, against C. albicans ATCC10231. Finally, DPPH tests indicated A. Stochmal, W. Oleszek, A. Jeziorski, J. Piekarski, J. Physiol. Pharmacol. 59 (2008) 823–835. that all tested extracts are potent antioxidant agents (IC50 mea- suring from 2.66 to 4.80), aronia leaves (IC50 = 2.66 μg mL−1), [15] I. Hideyuki, Y. Yoshihiro, K. Tae-Hoon, Y. Takashi, Nat. Med. 59 and purple loosestrife herba (IC50 = 2.83 μg mL−1) whole (2005) 52. extracts showing the best DPPH scavenging activity, even better [16] D. Obolskiy, I Pischel, B. Feistel, N Glotov, M Heinrich, J. Agric. than that of references (IC50 rutin = 5.08 μg mL−1; IC50 gallic Food Chem. 59 (2011) 11367–11384. acid = 4.00 μg mL−1). Summing up, the results have revealed [17] G. Suryakumar, A. Gupta, J. Ethnopharmacol. 138 (2011) activatory but also inhibitory effects, thus, proving phytocom- 268–278. pounds cooperation in making final effect and plant compounds [18] C. Parvu, Universul Plantelor. Mica Enciclopedie, Enciclopedica antimicrobial activity complexity. Ed., Bucharest, 1997. Further studies on the most active vegetal extracts which aim to [19] S.M. Branca, A.B. Paula, F.F. Domingues, A.L. Seabra, reveal minimum inhibitory concentrations (MICs) values and R.M. Ferreira, A. Margarida, J. Agric. Food Chem. 50 (2002) potential inhibitory or activatory effects in combination with 4615–4618. chemical antibiotics are ongoing. [20] J.P. Rauha, J.L. Wolfender, J.P. Salminen, K. Pihlaja, K. Hos- tettmann, H. Vuorela, Z. Naturforsch. 56 (2001) 13–20. [21] V. Istudor, Farmacognozie Fitochimie Fitoterapie, Medicala Ed., Bucharest, 2001. References [22] J.M. Duran, C.L. Augustin, Phytochemistry 44 (1984) 25–35. [1] J. Sumner, The Natural History of Medicinal Plants, foreword by [23] G. Romussi, G. Bignardi, Arch. Pharm. 320 (1987) 153–158. Mark J. Plotkin, Timber Press, Portland, OR, 2000. [24] L. Pirvu, A. Grigore, C. Bubueanu, E. Draghici, J. Planar [2] S. Krief, J.M. Krief, J. Kasenene, T. Sévenet, C.M. Hladik, Chromatogr. 26 (2013) 237–242. G. Snounou, J. Guillot, Bull. Acad. Natl. Med. 195 (2011) 1927–1935. [25] Y.S. Chan, L.N. Cheng, J.H. Wu, E. Chan, Y.W. Kwan, S.M.Y. Lee, G.P.H. Leung, P.H.F. Yu, S.W. Chan, Inflammopharmacology 19 [3] M.A. Huffman, Proc. Nutr. Soc. 62 (2003) 371–81. (2011) 245–254. [4] C. Engel, Wild Health: How Animals Keep Themselves Well and [26] H.H. Barakat, A.M. Sahar Hussein, S.M. Marzouk, I. Merfort, What We Can Learn from Them, Houghton Mifflin Harcourt, M. Linscheid, A.M. Mahmoud Nawwar, Phytochemistry 46 (1997) Boston, 2003. 935–941. [5] J. Ichida, BJS, Science Blog, Wed, 2004-05-26. [27] https://sites.google.com/site/icbcluj/Home/programul-cercetare-de- [6] M.R. Hutchings, S. Athanasiadou, I. Kyriazakis, I.J. Gordon, Proc. excelenta---ceex/epitrans-ro Nutr. Soc. 62 (2003) 361–70. [28] L. Battinelli, B. Tita, M.G. Evandri, G. Mazzanti, Farmaco [7] M.M. Cowan, Clin Microbiol. Rev. 12 (1999) 564–582. (Lausanne) 56 (2001) 345–348. [8] T.P.T. Cushnie, A.J. Lamb, Int. J. Antimicrob. Agents 26 (2005) [29] L. Pirvu, C. Hlevca, C. Bubueanu, I. Nicu, M. Panteli, I. Rasit, 343–356. Patent application No. A/00458/18.06.2014. [9] H. Wagner, S. Bladt, Plant Drug Analysis, 2nd edn., Springer, Ms received: February 21, 2014 Berlin, 1996. Accepted: July 31, 2014

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