Introduction
Genus Stachys. L, or woundworts, comprises more than 300 herbs and shrubs and is considered one of the largest genera from Lamiaceae family (Tomou et al. 2020). Most of the species are distributed in temperate and tropical regions of the world especially in the Mediterranean. There are 22 species from Stachys genus in Bulgaria, 5 of which are under the protection of the Bulgarian Biodiversity Law. The natural habitats of some of these species are located in some of the Bulgarian national parks and others are within localities included in NATURA 2000. The plants from genus Stachys have a long history of use in ethnomedicine for various diseases, coughs, ulcers, genital tumors, inflammatory conditions, and infected wounds (Tundis et al. 2007; Conforti et al. 2009; Goren 2014).
It is reported that woundworts exhibit various biological effects such as antioxidant, antibacterial, anti-inflammatory, wound healing, cytotoxic, hepatoprotective properties (Khanavi et al. 2005; Vundać et al. 2007; Háznagy-Radnai et al. 2012; Tundis et al. 2014; Tomou et al. 2020). According to different phytochemical studies, the plants from the Stachys genus are sources mainly of phenylethanoid glycosides (Karioti et al. 2010; Delazar et al. 2011), iridoids (Murata et al. 2008; Tundis et al. 2014) and phenolic acids (Venditti et al. 2014).
Taking into account the research done so far, Stachys species may be considered a favourable subject for exploration and discovery of secondary metabolites with antimicrobial potential.
The inconsistent application of antibiotics poses a great risk of antibiotic resistance in most of the microbial species that cause human infections (Ventola 2015). This creates an urgency for the research and discovery of alternative sources of antimicrobial agents.
Plants have been used by humanity since ancient times for the treatment of various bacterial infections even without scientific proof of their effectiveness. As a potential source of numerous biologically active substances, plant species have always been potential candidates for alternative agents with antimicrobial activity.
In recent years the antimicrobial potential of some Stachys species was a great point of interest among different research groups. Published data indicate that different polar extracts, as well as essential oils, show antimicrobial activity against human pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa (Dulger et al. 2005; Aleebrahim-Dehkordy et al. 2016; Cüce et al. 2016).
Stachys thracica Davidov (The Plant List) or Thracian woundwort is a Balkan endemic plant distributed in Bulgaria, Greece and Turkey. In Bulgaria, it is classified as “rare” and some of its localities are within Natura 2000 ecological network. The populations of the Thracian woundwort are comprised of a small number of individuals and are located in the Strandja Mountain, Black Sea coast and Sofia region. There is no available data on ex-situ conservation of the species and its chemical composition and biological activity are not well studied.
The aim of the present research is a comparative determination of the antimicrobial activity of methanolic extracts obtained from in situ wild, in vitro cultivated and ex vitro adapted Stachys thracica plants.
Materials and methods
Plant material
S. thracica Davidov plants grew in situ in their natural habitat near the village of Sinemorets, Tsarevo municipality, Bulgaria. A small set of samples from aerial parts of the plants in the period of active blooming (in June) and seeds (in September) were collected with the permission of the Ministry of Environment and Water of Bulgaria. A voucher specimen SO107847 was deposited in the Herbarium of Sofia University “St. Kliment Ohridski”.
In vitro shoot culture from S. thracica was induced by sterilisation of seeds with 70% ethanol for 5 min. The sterilised seeds were placed on a germination medium containing water and agar (WA) and further, the sprouting seedlings were transferred on MS medium (Murashige and Skoog 1962) supplemented with 3% sucrose and 0.7% agar,without growth regulators. The in vitro collection was maintained under controlled environmental conditions (16 h light/8 h dark, 60 mmol/(m2s) photosynthetic photon flux density, Philips TLD-33, temperature 25 °C and 60–70% relative air humidity).
Ex vitro adaptation was performed in three stages with plants having well-developed root systems. At the first step, the regenerated plants were planted in pots and subjected to acclimation in a phytotron chamber for a period of one month. After that, they were transferred to a greenhouse for another month and at a final stage were planted on the experimental field of Sofia University “St. Kliment Ohridski”.
Methanolic extracts preparation
Three grams (3 g) of finely powdered dry plant material from aboveground parts of in situ grown, in vitro cultivated and ex vitro adapted S. thracica were subjected to triple sonication extraction with 30 ml chloroform (Sigma-Aldrich, Spain) in ultrasonic bath for 10 minutes. In the next step, the dried biomass was extracted three times with methanol for 30 minutes. The final plant extract from each variant was concentrated through a vacuum evaporator (IKA, Germany) and dried to constant dry weight. The yields of extracts from in situ, in vitro cultivated and ex vitro adapted plants were 13.8%, 28.46% and 13.6% respectively. For the current study, each methanolic extract was dissolved in 5% DMSO.
Antimicrobial activity
Microbial strains
The methanolic extracts from in situ, in vitro cultivated and ex vitro adapted S. thracica plants were individually tested against seven Gram-negative microbial strains – Pseudomonas aeruginosa NBIMCC 3700, Proteus mirabilis NBIMCC 8690, Proteus hauseri NBIMCC 1393, Enterobacter cloacea NBIMCC 8570, Acinetobacter calcoaceticus NBIMCC 3730, Escherichia coli NBIMCC 8954, Klebsiella pneumoniae NBIMCC 3670 and three Gram-positive bacteria – Staphylococcus aureus ATCC 25923, Staphylococcus epidermitis NBIMCC 3360, Enterococcus faecalis NBIMCC 1093 microbial species and the yeast Candida albicans NBIMCC 74. The microbial specimens were purchased from The Bulgarian Collection for Industrial Microorganisms and Cell cultures (NBCIMCC). The bacterial strains were cultured overnight at 37 °C on Muller-Hinton agar (MHA) and the yeast was cultured on Sabouraud Dextrose Agar (SDA).
Disk diffusion assay
An initial screening of the antimicrobial activity of the dried methanolic extracts from S. thracica was performed by agar disk diffusion method according to the guidelines of CLSI (Clinical and Laboratory Standards Institute). The dried extracts from in situ, in vitro and ex vitro adapted plants were dissolved in 5% DMSO to a final concentration of 200 mg/ml and filtered by 0.45 µm Millipore filters for sterilization. Briefly, 100 µl of each suspension containing 107 cell/ml was inoculated in 25 ml MHA for bacterial strains and SDA for the yest respectively. Sterile paper disks (6 mm diameter) were impregnated with the extracts (8 mg/disk) and allowed to dry under aseptic conditions before placing them on the inoculated agar. DMSO at concentration 5% was used as a negative control. The antibiotics tetracycline and amikacin were used as a positive control for the bacterial strains and nystatin for C. albicans. The samples were incubated at 37 °C for 24 hours and 48 hours for bacterial strains and C. albicans respectively. The antimicrobial activity of the extracts was related to the inhibition zones.
Micro-well dilution assay
Bacterial strains which were sensitive to the methanolic extracts in the disk diffusion assay were studied for their minimal inhibitory concentration (MIC) using the micro-well dilution assay (Wiegand et al. 2008, EUCAST). For the experiment, the bacterial suspension was prepared in a liquid MH-Muller Hinton medium with a density of 0.5 on the McFarland scale, corresponding to 107 cells/ml. The 96-well plates were prepared by dispersing 50 µl MH broth in each well. The serial dilutions of each extract were prepared directly in the wells as the starting concentration was 64 mg/ml. Then, 50 µl of each well was transferred to the next and the final dilution of each extract was 2 mg/ml. Finally, 50 µl of the bacterial suspension was added to each well and the final volume of each well was 150 µl.
Prior to incubation, the absorbance of each microplate was measured using ELISA reader (Uscn Kit Inc., China) at λ=630 nm and this was considered the absorption at 0 h.
Discussion
In vitro multiplication and ex vitro adaptation of <italic><tp:taxon-name><tp:taxon-name-part taxon-name-part-type="genus" reg="Stachys">S.</tp:taxon-name-part> <tp:taxon-name-part taxon-name-part-type="species" reg="thracica">thracica</tp:taxon-name-part></tp:taxon-name></italic>
The conservational status of the Thracian woundwort and its limited distribution enforced us to apply an alternative method which would allow simultaneously the conservation of the species and the determination of its biological activity. The in vitro micropropagation is a reliable method for ex-situ conservation of endemic and threatened plant species and it is successfully applied for the investigation of such, without disturbing their natural population and habitats.
For the current study we successfully initiated in vitro shoot cultures of S. thracica from sterilised ripe dried seeds. The in vitro culture is successfully grown on MS medium without the addition of plant growth regulators and the micropropagated plants are characterised with vigorous growth, plentiful leaf biomass and very well-developed root system. This in turn led to the successful ex vitro acclimation of the Thracian woundwort with 83% survival rate.
Similar to our results, the successfully initiated in vitro culture from the Balkan endemic S. maritima was maintained on a hormone-free MS medium and the plants showed an excellent regeneration rate (Panayotova et al. 2008).
Antimicrobial activity
There is a high possibility that different growth conditions would affect the biological activity of plants extracts. S. thracica is a source of pharmacologically active secondary metabolites such as phenylethanoid glycosides (Bankova et al. 1999). To evaluate the changes in the antimicrobial activity of methanolic extracts from S. thracica, a comparison between the in situ grown, in vitro cultivated and ex vitro adapted plants was made.
The antimicrobial activity was evaluated against 10 bacterial strains and 1 yeast strain – C. albicans. The disk diffusion method was used for preliminary study of the antibacterial activity and the microdilution assay was applied afterwards for determination of MIC and verification of the results obtained by the initial screening. All the extracts were more active against Gram-negative bacteria which may be due to the different structure of the cell wall of gram-negative and gram-positive bacteria. We observed no visible trend in the antimicrobial activity of the methanolic extracts obtained from in situ, in vitro cultivated and ex vitro adapted plants with the exception that all the extracts were equally active against A. calcoaceticus showing inhibitory zones of 8 mm and MIC values – 8 mg/ml.
Typically, A. calcoaceticus is a soil bacterium but it is very frequently associated with infections within hospitals due to its ability to form a complex with another species – Acinetobacter baumannii and it is usually used in laboratory testing instead of Acinetobacter baumannii (Mancilla-Rojano et al. 2020).
Ebrahimabadi et al. (2010) reported that the polar fraction of Stachys inflata Benth. was active against only two microbial species which in parts overlaps with our results. In another study, Dulger et al. (2004) demonstrated that methanolic extracts from Stachys species were active against E. coli, P. aeruginosa, S. aureus and B. cereus but no activity was established against the tested yeast cultures – C. albicans, K. fragilis and R. rubra. Contrary to our results, Cüce et. al. (2017) reported that methanolic and hexane extracts from in situ and in vitro cultivated S. annua plants were active against S. aureus and the methanolic extract showed activity against P. aeruginosa.