FARMACIA, 2021, Vol. 69, 1 https://doi.org/10.31925/farmacia.2021.1.7 ORIGINAL ARTICLE PHYTOCHEMICAL EVALUATION AND CYTOTOXICITY ASSAY OF LYTHRI HERBA EXTRACTS IRINA MIHAELA IANCU 1, LAURA ADRIANA BUCUR 2*, VERGINICA SCHRODER 2, HORAȚIU MIREȘAN 2, MIHAI SEBASTIAN 2, VALERIU IANCU 2, VICTORIA BADEA 1 1“Ovidius” University of Constanța, Faculty of Dental Medicine, Department of Microbiology, 7 Ilarie Voronca Street, Constanța, Romania 2“Ovidius” University of Constanța, Faculty of Pharmacy, 6 Căpitan Al. Șerbănescu Street, Constanța, Romania *corresponding author: [email protected] Manuscript received: July 2020 Abstract Lythrum salicaria L. is a plant known in traditional European medicine for its healing effects for diseases such as dysentery and diarrhoea. The quantitative evaluation by spectrophotometric determinations of total polyphenols, tannins and anthocyanins content revealed values of 16.39% in polyphenols, 10.53% tannins and 0.3598% anthocyanosides, results comparable to the data in the literature. To determine the antioxidant activity of the aqueous extract the DPPH radical method was performed on the Lythri herba vegetal product. The aqueous extract shows an increased antioxidant activity (DPPH) of 94.39% for the concentration of 2.5 mg/mL, IC50 being registered at 0.2166 mg/mL. These results correlated with the effects of the biological activity of the extract on the Artemia salina L. biotester. Although the extract is non-toxic, cytological effects appear after 48 h (the accumulation of cytoplasmic inclusions, an increase of intercellular space and cell detachments at the level of the basement membrane). Rezumat Lythrum salicaria L. este una dintre plantele cunoscute în medicina tradițională europeană pentru efectele curative în afecțiuni precum dizenteria și diareea. Evaluarea calității prin determinări spectrofotometrice a conținutului de polifenoli totali, taninuri și antocianozide a scos în evidență valori de 16,39% polifenoli, 10,53% taninuri și 0,3598% antocianozide, rezultate comparabile cu datele din literatură. Determinarea activității antioxidante a extractului apos a fost realizată prin metoda radicalului DPPH din produsul vegetal Lythri herba. Extractul apos arată o activitate antioxidantă crescută (DPPH) de 94,39% pentru concentrația de 2,5 mg/mL, IC50 fiind înregistrat la 0,2166 mg/mL. Aceste rezultate au fost corelate cu efectele activității biologice ale extractului asupra biotesterului Artemia salina L. Deși extractul este non-toxic, după 48 h apar efectele la nivel citologic (acumulări de incluziuni citoplasmatice, spațiu mărit intercelular și detașări de celule la nivelul membranei bazale). Keywords: Lythri herba, tannins, Artemia salina, antioxidant activity Introduction determine its pharmacological effects such as anti- diarrheal, antimicrobial, anti-inflammatory and anti- Over the years, the popularity of medicinal plants oxidant properties [4, 28]. Important bioactive compounds has declined compared to the pharmaceutical chemical such as tannins, flavonoids, anthocyanins, phenolic industry and its multitude of synthetic compounds. The acids, alkaloids, steroids and triterpenes were revealed same can be said about Lythrum salicaria L. species by numerous studies [28]. Other evaluations have (Lythraceae), a plant well known in the literature for showed that Lythrum salicaria L. extracts possess its curative effects against dysentery and diarrhoea, hypoglycaemic effects and can also reduce the plasma even though Lythri herba is a standardized pharmacopoeial level of triglycerides [22, 28]. Ethanolic extracts of the plant material [1]. Lythrum salicaria L. species have shown antimicrobial Rehabilitating the importance of Lythrum salicaria L. activity against Gram-negative and Gram-positive species in the Romanian therapy is the main objective bacteria such as Staphylococcus aureus, Escherichia of this research. Lythrum salicaria L., known as purple coli, Pseudomonas aeruginosa and as well as on the loosestrife (răchitan in the Romanian language) in Candida albicans fungus [4]. The use of cytotoxicity Romanian folk medicine, is used internally for gastro- tests for plant extracts can be expensive, due to their intestinal disorders due to its rich tannin composition, complex composition, with effects that require repeated and externally for varicose veins, eczemas and bleeding tests while being correlated with various types of gums [27]. Previous phytochemical studies have shown solvents and testers. Among the easiest methods, that polyphenols are the main compound of the aerial frequently used in such tests, is brine shrimp lethality parts of the Lythrum salicaria L. species, which assay (BSLA). This is considered a preliminary test 51 FARMACIA, 2021, Vol. 69, 1 for cytotoxicity testing, an important evaluation stage edition [1] and in the Romanian Pharmacopoeia 10th before modelling [15] and optimizations of assays, in edition monography (FR X) [2]. pharmaceuticals studies. It is an in vivo test, performed The assay of water-soluble substances and 50% on Artemia salina larvae, a crustacean species used methanol study methods. This preliminary determination in toxicology [5, 12], nano-toxicology [17], plant is performed to assess the degree of extractability extracts testing and applications [8, 11, 14, 16, 24]. of the solvents to choose the optimal solvent with Meyer and Clarkson established the criteria for which the most active principles are extracted. The identifying plant extracts toxicity levels based on method used for these determinations is standardized relevant concentrations [13, 23]. Therefore, Artemia in FR X, and we used dried floral tips from Lythrum salina becomes a most useful test model for these salicaria L. species from Romania [2] as the plant types of tests [13, 23]. In addition, the possibility of material. 5.0167 g of pulverized plant material is highlighting cellular structures through transparency weighed on an analytical scale and transferred into a makes this test system an easy tool for evaluating vial with a ground-in stopper; we added 100 mL of the cytological changes [29, 31]. water, shook vigorously several times, left to macerate The aim of this study is to identify the phytochemical for 23 hours, stir again for 1 hour and filter, removing the content of the Lythrum salicaria L. (Lythri herba) first portions of the filtrate. 10 g of the filtrate are species, and to identify its cytotoxic potential. The evaporated to dryness on a water bath in a pre-weighed plant has been studied internationally [4, 6, 7, 18] and weighing ampoule. The weighing ampoule was dried is highly valued for its application of new extraction- in an oven at 105ºC for 3 hours, cooled in a desiccator induced epidermal regeneration data [18], but at the and weighed. The residue is made up of the soluble national level there is very little data or the data is substances and refers to 100 g of plant product [2]. incomplete in terms of active pharmacological Preparing the extracts activities. To obtain the dry extract, the air-dried plant material The novelty and originality of the study is given by the Lythri herba was milled to a powder (111.3081 g) fact that for the first time in Romania, an analysis of using an electric blender and extracted with water the correlation between the phytochemical composition (1000 mL) using a Soxhlet apparatus for 2 hours. and the biological activity of the extract is performed. After cooling, the extract was filtered and the filtrate In addition, the assessment of cytotoxicity involves was concentrated to evaporate the solvent with the the use of an in vivo test model, widely used for plant BUCHI R-215 rotary evaporator. The residue was extracts and standardized, which replaces other test dried (approximately 12 g), then transferred to a models just as effectively. This test is essential in sealed glass recipient and stored in an exicator until establishing preliminary cytotoxicity, as it increases we conducted the following experiments. the efficiency of determining which concentration The assay of total polyphenols, tannins and has toxic potential, used as an alternative for complex anthocyanins studies. The tannin content of the dry plant extract was determined using the Folin-Ciocâlteu method according Materials and Methods to the European Pharmacopoeia (EP) 10.0 edition [1]. To determine total polyphenols content, we performed Vegetal products the Folin-Ciocâlteu adapted method according to the Aerial parts of Lythrum salicaria L. (Lythri herba) EP 10.0 edition [1] while for determining the anthocyanins were collected in August 2019 from the Năvodari area, content we used the colorimetric method according Constanța city, Romania, while a voucher specimen to the EP 10.0 edition [1]. has been deposited in the exicata collection of the For total polyphenols and tannin determination, the Pharmacognosy discipline within the Faculty of amount of 0.5022 g dry extract was dissolved in Pharmacy, “Ovidius” University in Constanța, Romania. 250 mL of water. Then, the resulting solution (solution After harvesting, the plant material was sorted and A) was filtered through cotton wool in a 250 mL cleaned of any leaves or flowers that were altered. volumetric flask. 20 mL of water were added to Parts of the plant materials were kept in 70% alcohol solution A, thus creating solution B. Taking 2 mL of in a jar with a running stopper, to carry out the solution B, we added 1 mL of Folin-Ciocâlteu reagent, cross sections while other parts were dried at room 10 mL of water and 12 mL of Na CO 290 g/L solution. temperature, in the shade, for three weeks to use 2 3 The resulting solution is left to stand for 30 minutes. them for qualitative and quantitative examinations. The absorbance (A ) is read at a wavelength of 760 Loss on drying. We determined the loss of drying for 1 nm, using water as a compensation liquid. the dry extract, because all subsequent determinations In a 100 mL volumetric flask we brought 50 mg of will relate to the mass of dried plant material. The pyrogallol which is dissolved in 50 mL of water and method used for this preliminary determination is fill up to the mark with water (stock solution).