FARMACIA, 2019, Vol. 67, 6 https://doi.org/10.31925/farmacia.2019.6.13 ORIGINAL ARTICLE STUDIES ON ACINOS ALPINUS L.: POLYPHENOLS AND TERPENOIDS COMPOUNDS PROFILE, ANTIMICROBIAL ACTIVITY, ANTIOXIDANT EFFECT AND RELEASE EXPERIMENTS ON THE ETHANOL AND PROPYLENE GLYCOL EXTRACTS LUCIA PÎRVU 1, FAWZIA SHA’AT 1, RAMONA PĂVĂLOIU 1, DENISA IOANA UDEANU 2, BUJOR ALBU 1* 1National Institute of Chemical Pharmaceutical R&D (INCDCF), 112 Vitan Road, 031299, Bucharest, Romania 2“Carol Davila” University of Medicine and Pharmacy, Faculty of Pharmacy, Department of Clinical Laboratory and Food Safety, 6 Traian Vuia Street, 020956, Bucharest, Romania *corresponding author: [email protected] Manuscript received: March 2019 Abstract The present study aims to evaluate the chemical profile, antioxidant and antimicrobial activities, as well as diffusion aspects of the active compounds (polyphenols and terpenoids) in a physiological saline solution, of two extracts from the aerial part of rock thyme, Acinos alpinus (L.) Moench. The extract in 70% ethanol (E18) was used to evaluate the chemical qualitative and quantitative composition of polyphenols and terpenoids, and to assess the scavenger activity and antioxidant properties (chemiluminescence method); the extract in 20% propylene glycol (P18) was used to test the antimicrobial effect on E. coli ATCC 8739, S. aureus ATCC 25923 and C. albicans ATCC 10231, and also in diffusion studies on Franz cell device through the use of a synthetic membrane of cellulose acetate type in physiological saline media, in order to evaluate the diffusion effectiveness of the active compounds supported on sugar and water, respectively. Rezumat Scopul acestui studiu a constat în evaluarea compoziției chimice calitative și cantitative, a potențialului antioxidant şi anti- microbian, precum și a unor caracteristici privind difuzia compușilor activi (polifenoli si terpenoide) în mediu de ser fiziologic, a două extracte obținute din partea aeriană de cimbrișor de stâncă, Acinos alpinus (L.) Moench, cunoscut îndeosebi pentru proprietățile aperitive și digestive. Astfel, extractul în etanol 70% (E18) a fost utilizat pentru evaluarea conținutului chimic în polifenoli și terpenoide, dar și pentru estimarea capacității de îndepărtare a radicalilor liberi din mediu, respectiv a efectului antioxidant (studii de chemiluminescență); extractul în propilen glicol 20% (P18) a fost utilizat pentru testarea efectului antimicrobian pe tulpini de E. coli ATCC 8739, S. aureus ATCC 25923 și C. albicans ATCC 10231, respectiv în studii de difuzie (pe celula Franz) utilizând o membrană sintetică de tip acetat de celuloză în mediu de ser fiziologic, cu scopul final al demonstrării eficacității cedării compușilor activi din suport apă, respectiv zahăr. Keywords: rock thyme, Acinos alpinus, chemical composition, antioxidant effect, diffusion studies Introduction campo, té de roca, poleo montesino, etc. Té de campo is very appreciated for the special aroma, due to the Acinos species (Lamiaceae family) are known to live specific content of pulegone, menthone, limonene and in cold places, in mountains or moorlands regions, in alpha-pinene(iv) volatile oil compounds [19]. Europe, Turkey and the north of Africa and America. Ethno-pharmaco-botanical records [19] in Spain Data [26] indicates that Acinos genus includes 11 describe Acinos alpinus plant species with antiseptic, species with numerous synonyms; Acinos alpinus (L.) antispasmodic, anti-inflammatory, cholesterol lowering, Moench, also known as rock thyme, can be found as and even anti-catarrhal properties, as well as having Satureja alpina (L.) Scheele, Acinos baumgartenii benefits on the gastrointestinal and urinary systems. It (Simonk.) Klokov, Acinos alpinus subsp. baumgartenii is recommended as digestive, aperitif, anti-diarrhoeic, (Simonk.) Pawl.l, Melissa baumgartenii (Simonk.), diuretic and slimming natural remedy. Clinopodium alpinum (L.) Merino, Calamintha alpina Romanian folk medicine does not have data or (L.) Lam., and Calamintha alpina (L.) Lam. subsp. recommendations on the use of the Acinos alpinus alpina. In the Romanian Carpathians, Acinos alpinus plant species. species can be found at altitudes above 1400 m. Scientific data on Acinos species is scarce and it mainly Regarding its medicinal use, in Spain, Acinos alpinus describes the volatile oils content and their antioxidant, plant species are included in the category of (alimentary) antimicrobial, and anti-inflammatory properties. For herbal teas; the popular name of the species is té de 1025 FARMACIA, 2019, Vol. 67, 6 instance, the volatile oil fraction from Acinos arvensis of ICCF. The plant material has been shading dried sp. was proved to be effective against E. coli, S. and minced into a fine plant powder, then used in aureus and K. pneumonia strains, while Acinos technological studies. suaveolens sp. volatile oil fraction proved efficacy Plant extracts’ preparation. Two charges of ten (10) against E. coli, S. aureus, S. epidermidis, S. hominis grams of plant powder were separately extracted with and K. pneumonia strains [26]. Ulukanli et al. [29] 200 mL of 70% (v/v) ethanol solution, one hour at also proved the high efficacy of acetone extracts 82°C. The two ethanolic extracts (150 mL each) were isolated from the roots and the aerial part of Acinos filtered and mixed. The resulted 70% ethanolic extract rotundifolius Pers. plant species on nine standard (codified E18) has been analysed as concerning bacterial strains; S. aureus Cowan 1, M. luteus LA chemical qualitative and chemical quantitative aspects. 2971, Mycobacterium smegmatis CCM 2067, Bacillus Further, fifty (50) mL of E18 has been hydrolysed subtilis IGM 22, Bacillus subtilis var. Niger ATCC 10, for 30 minutes in 4 N HCl environment. The resulted Aeromonas hydrophila ATCC 7966, K. pneumoniae filtrate sample has been evaporated to dryness and FMC 5, Pseudomonas aeruginosa ATCC 27853, and the solid matter obtained (spiss product) was re- C. albicans ATCC 10231. Similarly, the volatile oil dissolved into 98% ethanol solvent to a final volume fraction from Acinos arvensis Dandy plant species of 50 mL. The resulting (ethanolic) hydrolysate sample indicated antimicrobial activity against E. coli, S. (codified H18) was also analysed as concerning aureus and K. pneumoniae strains [11]. Jennan S. et al. qualitative (HP)TLC aspects. In parallel, one hundred [10] studied the antioxidant and anti-inflammatory (100) mL of E18 were concentrated at low pressure properties of the volatile oils fractions isolated from (Büchi Rotary Evaporator) and the resulted spiss Satureja alpina (syn. Acinos alpinus), Satureja briquetti product was solved into 20% (v/v) propylene glycol and Satureja atlantica Morocco plant species; the solution in order to assure the final content of 5 mg paw oedema carrageenan induced rat model in vivo total phenols content expressed as gallic acid using as standard a synthetic antioxidant compound equivalents (GAE) per 1 mL sample. The resulted namely butylated hydroxytoluene (BHT) indicated standardized 20% propylene glycol extract (codified that S. alpina volatile oils fraction provided the best P18) has been divided in 2 mL Eppendorf’s mono- anti-inflammatory effects; doses of 200 mg volatile doses and used for microbiological and diffusion oils per kg body weight were estimated with 91.4% experiments. inhibitory activity upon paw oedema induced Chemicals, reagents and references. Chemicals (sodium inflammation. Also, the leaves extract from Calamintha acetate, sodium carbonate and aluminium chloride), grandiflora Moench, C. nepeta Savi and C. sylvatica reagents (Folin-Ciocâlteu, Natural Product and Poly- Bromf. were reported to have antioxidant effects [2, 3]. ethylene Glycol 4000 - NP/PEG, and Vanillin-sulphuric This study aimed to evaluate the chemical qualitative acid - VS), solvents (ethanol, formic acid, glacial and quantitative aspects of the active compounds (poly- acetic acid, ethyl acetate, propylene glycol) and the phenols and terpenoids) found in ethanol and propylene reference products used in HPTLC studies (e.g., rutin glycol extracts from Acinos alpinus L., responsible for (min. 95%), chlorogenic acid (> 95%), caffeic acid the flavour and bitter properties of the species. Anti- (99%) and gallic acid (99%)) were purchased from oxidant activity and antimicrobial properties of the Merck (Fluka-Sigma-Aldrich) Co Bucharest, Romania. two extracts were also evaluated. Diffusion studies Qualitative analytic determination: Studies were done on Franz cell device through the use of a synthetic by (HP)TLC method according to Wagner et al. [25] membrane of cellulose acetate type, physiological and Reich et al. [31] using two solvent systems: saline media, and sugar and water supported Acinos system A (ethyl acetate:glacial acetic acid:formic acid: alpinus propylene glycol extracts have been done, water, 100:12:12:26) and system B (chloroform:glacial with the final purpose of designing new products acetic acid:methanol:water, 64:32:12:8) [21]; Silica gel beneficial for digestion processes. 60F254 (10 x 10) HPTLC plates (Merck, Darmstadt, Germany); reference compounds mixtures prepared as Materials and Methods 10-3 M solutions in 70% ethanol; E18 and H18 test vegetal extracts loaded as 8 mm band length; Hamilton Plant material. The fresh aerial part (herba et flores) syringe and Linomat 5 instrument (CAMAG, Muttentz, of Acinos alpinus L. (fam. Lamiaceae), commonly Switzerland). The loaded plates were kept in TLC named rock thyme, has been collected