Raeseri Boiss Research Articles

Establishment of an in vitro method for micropropagation of ironwort, (Sideritis raeseri Boiss. & Heldr.)

Ironwort / Mountain Tea (Sideritis raeseri Boiss & Heldr.) is an endangered (EN) plant species in Albania. This study aimed to develop a rapid clonal propagation protocol using in vitro methodologies. The ironwort seeds were pre-treated with three concentrations of GA3 (250, 500, and 1000 mg l-1). During the inoculation stage, two types of culture media, Murashige & Skoog (MS) and Woody Plant Medium (WPM), were tested, and the effects of both GA3 concentration and culture media used were evaluated. For the subculture stage, three cytokinins (6-benzylaminopurine / BAP, kinetin, zeatin) at four concentrations (0.5; 1.0; 1.5; 2.0 mg l-1), were compared for the RGR index, while for the rooting stage, two different auxins (1-naphthaleneacetic acid / NAA and indole-3-butyric acid / IBA) at four concentrations (0.5; 1.0; 1.5; 2.0 mg l-1) were tested. GA3 at 500 mg l-1 and MS medium resulted as more effective. The highest value of the RGR index during the subculture stage was obtained in the MS nutrient medium supplemented with BAP at 1.5 mg l-1. For rhizogenesis response, IBA was more effective for roots and length number. Based on these results, in vitro methodologies can be a promising tool for the mass production of this endangered plant species and with possible applications for enhancing the production of valuable nutraceuticals.

Bioassay-Guided Skin-Beneficial Effects of Fractionated Sideritis raeseri subsp. raeseri Extract.

This study aimed to evaluate the potentials for skin-beneficial effects of the hydroethanolic extract of the aerial parts of ironwort (Sideritis raeseri Boiss. & Heldr. subsp. raeseri) and its fractions (petroleum ether, ethyl acetate, n-butanol and water). For these purposes, chemical analysis and in vitro antioxidant, anti-tyrosinase and antimicrobial assays, as well as determination of sun protection factor (SPF), were performed. The highest total phenolic content and quantity of individual flavonoids, chlorogenic acid and verbascoside were determined in the n-butanol fraction, which is in line with results obtained for antioxidant activity. The greatest antimicrobial activity against Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa, the most common causative agents of the skin infections in humans, was exhibited by the ethyl acetate fraction. The strongest anti-tyrosinase activity was shown by the hydroethanolic extract (52.64% of inhibition at 100 μg/mL). Almost all tested samples showed photoprotective activity with SPF higher than 6 obtained at a low concentration of 0.1 mg/mL, which was more than 15 for the butanol fraction. These findings revealed that the hydroethanolic extract of S. raeseri aerial parts could be a valuable ingredient in the formulation of cosmetic and medicinal products intended to overcome various skin disorders.

Microencapsulation of Sideritis raeseri Boiss. & Heldr. subsp. raeseri Extract Using Spray Drying with Maltodextrin and Whey Protein

Multiple medical properties and beneficial influence on health attributed to the aerial parts of Sideritis raeseri Boiss. & Heldr. subsp. raeseri indicated the need for further investigation. S. raeseri extracts were subjected to microencapsulation by the spray drying process in order to disperse and preserve unstable active compounds within a protective matrix. Two inlet air temperatures (120 and 140 ºC) were applied for the encapsulation of S. raeseri extract in a matrix composed of maltodextrin (10, 20, and 40%) or whey protein (40%). The effects of spray drying on physico-chemical properties, contents of total phenols and flavonoids, as well as of individual flavonoid glycosides of the obtained powders were determined. The 40% whey protein treatment increased bulk density (238.46 mg/mL) while it decreased hygroscopicity (14.27%). In addition, the high maltodextrin concentration of the S. raeseri powder resulted in the highest process efficiency (63.46%), the highest water solubility index (86.40%), and the lowest water absorption index (5.71%). Moreover, powders produced without maltodextrin were characterized by greater content of flavonoid glycosides. Overall, the results suggested that S. raeseri powders produced using spray-dried technique under adequate conditions could be considered as a novel functional and pharmaceutical ingredient.

Genetic diversity and structure of Sideritis raeseri Boiss. & Heldr. (Lamiaceae) wild populations from Balkan Peninsula

Sideritis raeseri Boiss. & Heldr is a subalpine/alpine plant species endemic to the southern part of the Balkan Peninsula. Mountain tea (S. raeseri) is a traditional medical remedy as a decoction of high value and a source of essential oils beneficial to human health. Recent findings of Sideritis active constituents in preventive therapy of osteoporosis, cancer and neurodegenerative diseases have increased public interest with pronounced impact on natural populations and species diversity. Morphological descriptors and chemotypes are commonly used for species description and diversity evaluation; however, these are affected by environmental perturbations. The present study assessed the genetic diversity and relationships of nine wild S. raeseri populations, using 12 URP molecular markers. In total, 283 distinct and reliable bands in all populations were amplified, including 86–122 polymorphic bands within each population. AMOVA analysis revealed a percentage of 66.30 % inter-population genetic diversity. Genetic structure analysis of the individual samples indicated that nine relatively independent gene pools were represented in the data and each individual sample was correctly assigned to its population of origin. Geographic distance was not correlated to diversity among populations. Conservation and breeding strategies are suggested as appropriate management measures for protection of germplasm resources against genetic erosion.

Insight into the Mechanisms Underlying the Tracheorelaxant Properties of the Sideritis raeseri Extract.

Sideritis raeseri Boiss. and Heldr. (Lamiaceae), known as “mountain tea,” is a native plant from the Mediterranean region, which is widely used in traditional medicine. This study evaluates the effects of the ethanol extract of Sideritis raeseri (SR) on airway smooth muscle activity and identifies the underlying mechanism. The S. raeseri extract (SRE) was extracted from air-dried parts of the shoot system of SR. The SRE (0.3–2 mg/mL) was tested in isolated rabbit tracheal rings, suspended in the organ bath, filled with Krebs solution, and bubbled with the carbogen mixture (95% O2/5% CO2) under a resting tension of 1 g in 37°C. In in vitro experiments, the SRE relaxed against acetylcholine-induced constriction in tracheal rings. Furthermore, SRE inhibited Ca2+-induced contractions in carbachol (CCh, 1 μM) as well as in the K+-depolarized trachea (80 mM). Our findings showed the NO/cGMP involvement in tracheorelaxant effects of SR. To this end, the effect of the SRE was potentiated by bradykinin (nitric oxide (NO) synthase activator, 100 nM), whereas it was inhibited by ODQ (inhibitor of NO-sensitive guanylyl cyclase, 10 μM) and L-NAME (NO synthase inhibitor, 100 μM), as well as indomethacin (cyclooxygenase inhibitor, 10 μM). These data suggest that the tracheorelaxant effect of the SRE is mediated at least partly by NO/cyclic guanosine monophosphate and cyclooxygenase-1-prostaglandin E2-dependent signaling. These findings indicate that the SRE may be used in various respiratory disorders.

The Stimulating Effects of Imazalil and Carbendazim Fungicides on in Vitro Propagation and ex Situ conservation of the Medicinal Balkan Range-Restricted Sideritis Raeseri Boiss & Heldr. Subsp. Raeseri

Sideritis raeseri Boiss & Heldr. subsp. raeseri, known in Greece as Mountain tea of Parnassus or Velouchi is a range restricted medicinal plant of the Balkan peninsula. Conventional propagation methods did not allow the mass production of plant material in a short period of time due to both low seed germination rate and rooting of cuttings. Therefore, the aim of this study was to establish a reliable, reproducible and efficient regeneration protocol for mass and large-scale micropropagation, germplasm and ex situ conservation of S. raeseri Boiss & Heldr. using Imazalil and Carbendazim fungicides. After 9 weeks, 2.5-10 mg/l Imazalil stimulated root length by 1 cm but diminished root number and rooting percentage. Optimum shoot number (5.58), shoot length (24.91 mm), shoot multiplication (100%), root number (20.63) and rooting (66.67%) were recorded with 0.5 mg/l kinetin (KN) + 0.05 mg/l ?-napthaleneacetic acid (NAA) (Imazalil-free). After 4 ½ weeks, 1 mg/l Carbendazim + 0.5 mg/l 6-benzyladenine (BA) yielded 5.77 shoots/explant, 16.1 mm shoot length and 100% shoot multiplication. However, Carbendazim did not result in rooting. The ex vitro survival percentage of rooted shoot-tip explants derived from Imazalil experiment was 95%. Carbendazim proved useful in shoot proliferarion and Imazalil in root elongation of S. raeseri Boiss & Heldr. subsp. raeseri micropropagation system.

Effect of glycine betaine, polyvinylpyrrolidone and D-mannitol on micropropagation of Sideritis raeseri Boiss and Heldr. subsp. Raeseri.

Despite being protected, the number of plants that belong to Sideritis genus has decreased in the last years due to the urbanization of its natural habitat. Sideritis raeseri Boiss and Heldr. subsp. raeseri known as mountain tea of Parnassus or Velouchi is a valuable genetic resource of the Greek flora because of its special characteristics, so, the development of a micropropagation protocol is crucial to its conservation and use in breeding programs. The objectives of the study were to evaluate the effects of glycine betaine (GB), polyvinylpyrrolidone (PVP-10) and D-mannitol applied in different concentrations in combination with auxins [α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA)] on shoot proliferation and rooting of shoot-tip explants of the studied species. Better shoot proliferation results were obtained in the following two combination treatments: (1) 100 mg/l GB + 0.5 mg/l NAA (5.5 shoots/explant 2.34 cm long, 75% shoot multiplication), and (2) 2.5 g/l D-mannitol + 3 mg/l IBA (2.92 shoot/explant 83.33% shoot multiplication percentage). In relation to rooting, better results were performed only with PVP at 0.5 g/l applied simultaneously with 0.5 mg/l NAA (12 roots/rooted microcutting, 1.47 cm root length, 90% rooting). The ex vitro survival percentage of rooted microcuttings derived from all 5 experiments was ranged between 68% and 93%.

Local Knowledge of Past and Present Uses of Medicinal Plants in Prespa National Park, Albania

Ethnobotanical studies have highlighted the need to address temporal dynamics of local knowledge in response to socio-economic changes. The southwestern Balkans are a hotspot of folk botanical knowledge and represent a unique region to study such dynamics. The present study focused on changes in ethnomedicinal knowledge in the rural mountains of Prespa National Park, Albania. The resident ethnic Macedonian minority was fairly isolated under communism (1946–1991), with a long tradition of wild medicinal plant collection. We identified 80 adults and 20 young community members through snowball sampling based on knowledgeability of medicinal plants. In-depth open and semi-structured interviews, free-listing, and participant observation elicited ethnomedicinal knowledge on past and present uses. We recorded 82 botanical taxa belonging to 39 families. Cultural importance analyses showed that Sideritis raeseri Boiss. & Heldr. was by far the culturally most salient species. Informants perceived a steep increase in home consumption of medicinal plants compared to the communist period, despite increased globalization and market liberalization. Trade had significantly decreased but remained an important fallback option in times of economic uncertainty. We observed the phenomenon of knowledge hybridization through access to “modern” knowledge and homogenization through political influences, pointing towards a both resilient and dynamic body of knowledge.

Effect of acidic and enzymatic pretreatment on the analysis of mountain tea (Sideritis spp.) volatiles via distillation and ultrasound-assisted extraction

A number of beneficial medicinal properties are attributed to the extract and essential oil of the aerial parts of Sideritis species (Lamiaceae). Hydrodistillation of the aerial parts of wild Sideritis clandestina ssp. peloponnesiaca (an endemic taxon in northern Peloponnesus, Greece) gave a low essential oil yield (<0.12%); about 65 components, mainly α-pinene, β-caryophyllene, β-pinene, globulol, caryophyllene oxide, were identified via GC–MS. Internal and external standards were used for quantification. For miniaturization of the procedure, we studied side-by-side maceration (MAC) and ultrasound-assisted extraction (UAE) methods, as well as the effect of preincubation in acidic medium (pH 4.8) for 75min at 37°C with or without a mixture of cellulase, hemicellulase and pectinase. Maceration and UAE provide consistent chemoprofiling of the main volatile compounds (about 20); UAE has lower demands on time, solvent, plant material (3g) and results in higher yields. Pretreatment with enzymes can increase the respective yields of hydrodistillation and UAE, but this effect is definitely attributed to the concurrent acidic pretreatment. In conclusion, incubation of plant material prior to hydrodistillation or UAE in citrate buffer, pH 4.8, significantly enhances the overall yield and number of components obtained and is recommended for the analysis of Sideritis volatiles. The acidic pre-treatment method was also successfully applied to analysis of cultivated Sideritis raeseri Boiss. & Heldr. in Boiss. ssp. raeseri; α-pinene, α- and γ-terpinene and β-thujene were predominant albeit in different percentages in flowers and leaves.

Effect of the NO donor “sodium nitroprusside” (SNP), the ethylene inhibitor “cobalt chloride” (CoCl2) and the antioxidant vitamin E “α-tocopherol” on in vitro shoot proliferation of Sideritis raeseri Boiss. &amp; Heldr. subsp. raeseri

In the present study, the effects of the NO donor “sodium nitroprusside” (SNP) and the ethylene inhibitor “CoCl2” combined with 2.2 μM 6-benzyladenine (BA), and the antioxidant vitamin E “α-tocopherol” in combination with 2.2 μM zeatin (ZT) and 0.2 μM α-naphthaleneacetic acid (NAA) on in vitro shoot proliferation of Sideritis raeseri Boiss. & Heldr. subsp. raeseri, one of the seven endemic mountain tea species of the Greek flora were investigated. The basal nutrient culture medium used in all experiments was the MS (Murashige, Skoog, Physiol Plant 15:473–497, 1962). Taking simultaneously into consideration all shoot proliferation parameters evaluated, the explants performance was optimum and significantly better among others when treated with 2.2 μM BA + 10 μM SNP or 2.2 μM BA + 2.5 μM CoCl2, or 2.2 μM ZT + 0.2 μM NAA + 11.6 μM α-tocopherol. In particular, 10 μM SNP gave 4.6 ± 0.6 shoots/explant and 100% shoot multiplication percentage while 20 μM SNP exhibited the greatest shoot length (17.5 ± 1.8 mm). However, SNP (10–40 μM) caused hyperhydricity symptoms to the 30–100% of explants. CoCl2 applied at 2.5 μM led to the production of 5.5 ± 0.7 shoots/explant, 11.8 ± 1.1 mm shoot length and 100% shoot multiplication percentage and concurrently decreased the percentage of vitrified explants from 33.3 to 20%. 11.6 μM α-tocopherol gave 6.7 ± 0.8 shoots/explant, 14.5 ± 0.7 mm shoot length, 100% shoot multiplication percentage, diminishing vitrification percentage from 41.6 to 8.3% (33.3% decrease) and completely inhibiting the appearance of necrotic explants, indicating its antioxidant properties as a vitamin. Shoot-tip explants responded better and exhibited better shoot proliferation results when cultured in MS medium with 2.5 μM CoCl2 for 5 weeks or 11.6 μM α-tocopherol for 9 weeks rather than with SNP for 4 weeks. In this study, three new different protocols were established for the in vitro shoot proliferation stage of S. raeseri.

Traditional uses, chemical composition and biological activities of Sideritis raeseri Boiss. & Heldr.

Sideritis species have been used in folk medicine for their antimicrobial, antiulcerogenic, digestive and anti-inflammatory properties. Over the years, the phytochemistry of the genus Sideritis has been studied, and various terpenoids, sterols, coumarins and especially flavonoid aglycones and glycosides have been identified. In particular, species from the Balkan Peninsula have been studied and were found to be rich in flavonoids, with valuable antioxidant activity. In the folk medicine of the Balkan countries, Sideritis raeseri is used as a herbal tea in the treatment of inflammation, gastrointestinal disorders and coughs, and also as a tonic, whereas extracts are used as a component of dietary supplements for anaemia. Its dried inflorescences are used to prepare a beverage called 'mountain tea'. In light of the considerable interest generated in the chemistry, pharmacological properties and commercial value of S. raeseri Boiss. & Heldr., we review and summarise the available literature on these plants. The review details the chemical composition of the essential oil, its mineral and polyphenol contents, the naming of these plants and their physicochemical characterisation, and the nuclear magnetic resonance spectral data and biological properties associated with the plant extracts, with a focus on their potential chemotherapeutic applications. © 2016 Society of Chemical Industry.

Aroma Compounds of Mountain Tea (Sideritis scardica and S. raeseri) from Western Balkan

The composition of the volatile aroma components was defined in the dried and fresh aerial parts of Sideritis scardica Griseb. from R. Macedonia and S. raeseri Boiss. & Heldr. from R. Macedonia, Albania and Greece. Analysis was made by gas chromatography (GC/FID/MS) equipped with a headspace (HS) sampler. Thirty-two components (15 monoterpenes representing 33.2- 62.8% and 17 sesquiterpenes representing 25.2- 51.2% of the entire volatiles) were identified as aroma components of dried plant material of S. scardica. Thirty components {14 monoterpenes (19.3-74.2%), 2 alcohols (6.2- 38.4%) and 14 sesquiterpenes (18.2- 33.5%)} were identified as aroma components in the fresh aerial parts of S. scardica. The predominant components were trans-caryophyllene, β-pinene, α-pinene and 1-octen-3-ol, which were found only in the fresh samples. In the aerial parts of S. rteseri, 43 components were identified in the dried samples {22 monoterpenes (65.7-94.3%) and 21 sesquiterpenes (5.4- 27.8%)} and 29 components {15 monoterpenes (77.3-90.7%) and 14 sesquiterpenes (6.3- 18.2%)} in the respective fresh samples. Prevailing components in all tested samples of S. raeseri were β-pinene, α-pinene, α-copaene, sabinene and limonene. Only minor differences were revealed in the qualitative composition of the aroma volatiles between the dried and fresh plant material of both species. Furthermore there was almost no difference in the chemical profiles of the aroma compounds between S. scardica and S. raeseri, except for 1- octen-3-ol, which was present only in fresh S. scardica.

Volatile aroma compounds in infusions of stems and rosette leaves of Sideritis raeseri Boiss. &amp; Heldr. from R. Macedonia, Albania and Greece

The composition of the volatile aroma components was defined in the infusion prepared by stems of Sideritis raeseri (SR-S) from R. Macedonia, Albania and Greeсe and in the infusion of rosette leaves of S. raeseri (SR-R) from R. Macedonia. Analysis were made by gas chromatography (GC/FID/MS) equipped with a headspace (HS) sampler. Twenty components (12 monoterpenes representing 76.70- 94.84% and 8 sesquiterpenes representing 5.16-18.55% of the entire volatiles) were identified as aroma components in SR-S samples. The predominant components in all samples were β-pinene, α-pinene and trans-сaryophyllene. The high abundance of limonene and δ-3-сarene were characteristic for the samples from Greeсe and 1,8-сineole was for the samples from R. Maсedonia and Albania. In the infusions of SR-R, ten components were identified, 5 monoterpenes (73.20-83.89%) and 5 sesquiterpenes (16.10-26.80%). Prevailing components in all tested samples of SR-R were β-pinene, α-pinene, 1,8-cineole and α-epi-murolol. There was almost no difference in the chemical profiles of the aroma compounds between SR-S and SR-R. Infusion of rosette leaves exhibit very similar aroma compounds profile with the infusion of stems of S. raeseri.

Spasmolytic Activity of the Ethanol Extract of Sideritis raeseri spp. raeseri Boiss. &amp; Heldr. on the Isolated Rat Ileum Contractions

Sideritis raeseri spp. raeseri Boiss. & Heldr., known as "mountain tea," has been widely used in the Mediterranean region as a spice and in folk medicine as a very popular decoction because of its anti-inflammatory, carminative, analgesic, antitussive, stomachic, and antimicrobial properties. The study was aimed to investigate the effects of an ethanol extract of S. raeseri on intestinal activity. Air-dried and powdered aerial parts were extracted with 96% ethanol. The rat ileum preparations were incubated in Tyrode's solution gassed (95% O(2)/5% CO(2)) at 37°C. The ethanol extract of S. raeseri (0.03-0.3 mg/mL) relaxed spontaneous contractions in isolated rat ileum, similar to that produced by papaverine. The plant extract in a concentration-dependent manner (0.015-0.15 mg/mL) significantly inhibited the contractile response to acetylcholine (P<.01). Atropine inhibited the response to acetylcholine. A similar relaxation-inducing effect of the S. raeseri extract was observed on the precontracted ileum by histamine and barium chloride. Plant extract (0.03-0.3 mg/mL) significantly shifted the histamine concentration-response curve to the right and down (P<.01). The S. raeseri extract (0.03-0.3 mg/mL) significantly inhibited the contractions induced by barium chloride (P<.01). The results show that the ethanol extract of S. raeseri can produce inhibition of the the spontaneous rat ileum contractions and contractions induced by different spasmogens. These data indicate that S. raeseri acts as a spasmolytic on intestinal smooth muscle, which justifies its use in gastrointestinal disorders.

Chemical composition of ultrasonic-assisted n-hexane extracts of Sideritis scardica Grieseb. and Sideritis raeseri Boiss. &amp; Heldr. (Lamiaceae) from Macedonia and Albania

Chemical composition of n-hexane extracts obtained from dried over-ground parts of two species of Sideritis, S. scardica Grieseb. and S. raeseri Boiss. &amp; Heldr. (Lamiaceae) was analyzed using GC/FID/MS. The collection of plants was made on different locations in the western part of Macedonia and the southern part of Albania, comprising twelve different samples of plant material. The ultrasonic-assisted extraction process was used for preparation of the n-hexane extracts yielded 0.73-3.33 % and 9.11-10.44 % of extracts for S. scardica and S. raeseri, respectively. Over one hundred constituents of the extracts were identified, belonging to several classes of components: diterpenes, hydrocarbons, dominantly present in each of the extracts, followed by fatty acids, aliphatic and aromatic alcohols, sterols, triterpene alcohols, and monoterpenes and sesquiterpenes, which were found in much smaller amounts or only in traces. The most abundant constituents of the extracts of both species of Sideritis were two diterpene components, both with M=286, which were not fully identified. Large percentages of nonacosane (1.71-12.22% and 7.46-19.68% for S. scardica and S. raeseri, respectively) and hentriacontane (4.48-20.79% and 8.09-30.31 % for S. scardica and S. raeseri, respectively) were also found in the extracts of both species.

Chemical constituents of the essential oils of Sideritis scardica Griseb. and Sideritis raeseri Boiss and Heldr. from Bulgaria and Macedonia

The essential oils of two species of Sideritis growing spontaneously in Bulgaria and Macedonia are reported, Sideritis scardica and Sideritis raeseri. The oils of S. scardica from different locations differed significantly: in the Macedonian sample α-cadinol (20%) predominated, while in the oil of Bulgarian samples the main components were diterpenic compounds and octadecenol (over 20%). This is the first report of ditrpenoids in essential oil of S. scardica. The oil of S. raeseri demonstrated a distinct chemical profile with its high concentration of sesquiterpenes, main components being germacrone (25%) and elemol acetate (15.9%). The observed qualitative variability of the oil composition of S. scardica of different geographic origin could be a result of different ecologic conditions but might also reflect the well-known tendency of some Sideritis species to hybridize.

Antioxidant activity of flavonoids from Sideritis raeseri

Nine 7- o-allosyl glucosides of 5,8-dihydroxy substituted flavones were isolated from a fraction of the methanol extract of the aerial parts of Sideritis raeseri Boiss et Heldr. subsp. raeseri. The structures were identified by spectroscopic methods. The antioxidant activity of this fraction, evaluated by the Co(II) EDTA-induced luminol chemiluminescence and by the DPPH scavenging activity, was found to be moderate. The activity can be related with the presence of 5- and 8- o-disubstituted flavones.