Allium ursinum has a distinct garlic-like scent associated with the presence of sulfur-containing compounds which are the most characteristic constituents in Allium plants.
These are undoubtedly the most important ramson’s constituents, both in terms of chemotaxonomic value and pharmacological activity. Their qualitative and quantitative profile is subject to great variation, the predominant reason for which is their lability. Of the various sulfur compounds present in this, as well as other Allium species, glutamyl peptides and sulfoxides are considered as primary. Usually, these plants contain a high concentration of S-alk(en)yl-l-cysteine-sulfoxides—odorless, non-volatile sulfur secondary metabolites, which, following subsequent hydrolysis, give rise to many volatile compounds, including thiosulphinates and (poly)sulfides responsible for specific Allium flavor and odor (Boscher et al. 1995; Yoo and Pike 1998).
Ramson belongs to a methiin/alliin-type Allium species, which means it contains mainly a mixture of (+)-S-methyl-l-cysteine-sulfoxide (methiin) and (+)-S-allyl-l-cysteine-sulfoxide = (+)-S-2-propenyl-l-cysteine-sulfoxide (alliin) (Schmitt et al. 2002; Kubec et al. 2000). However, isoalliin—(+)-S-(1-propenyl)-l-cysteine-sulfoxide and propiin—(+)-S-propyl-l-cysteine-sulfoxide are present as well (Fig. 1) (Schmitt et al. 2002). Also, ethiin (S-ethyl-cysteine-sulfoxide) was reported in the sample of fresh leaves collected in April in Czech Republic (Kubec et al. 2000).
The quantitative profile of cysteine-sulfoxides depends on the plant organ and time of harvest. Their total content in the leaves collected in April, expressed as mg/100 g of fresh weight, was 101.9 (in this: methiin—60.0, ethiin—0.4, propiin—1.2, alliin—40.3, isoalliin—traces) (Kubec et al. 2000). The content of total cysteine sulfoxides in the bulbs harvested in late summer, calculated as alliin, was 0.26 % (amount was related to the fresh weight) (Keusgen et al. 2003). In water extracts from cloves and leaves containing hydrolytic enzyme inhibitor, the alliin content ranges were: for cloves—0.65–1.10 and for leaves 0.20–0.72; while methiin: 0.60–1.40 and 0.30–0.95, respectively (Sendl 1995). The relative proportions of cysteine sulfoxides in ramson are presented in Table 1 (Fritsch and Keusgen 2006).
The analysis of the changes in the total cysteine sulfoxides content in different parts of A. ursinum collected in Germany throughout the vegetation period (focused on the months from March to June) showed that the highest amounts in leaves, storage leaves and bulbs (0.42, 0.26, 0.38 % respectively) were reached in March and April, that is before flowering time (Schmitt et al. 2002, 2005). Samples of fruits and leaf stalks collected in June contained 0.25 and 0.15 % of cysteine sulfoxides (fw) respectively. Furthermore, the relative quantitative profile of the investigated sulfoxides (alliin, methiin, isoalliin and propiin) differed during the vegetation period. In March the bulb contained almost the same amounts of alliin and methiin. In the following weeks alliin became the main component (73 ± 18 %), while methiin content decreased to 15 ± 9 % in mid-May (Schmitt et al. 2005). Then, the rise of methiin levels was observed. The relative content of propiin was always below 5 %, while of isoalliin ~10 %.
As was mentioned above, cysteine sulfoxides are subject to hydrolytic cleavage leading to a formation of a number of characteristic volatile secondary products. This is performed by specific enzymes, named C,S-lyases, which catalyse the cleavage of the Cβ–Sγ bond of the sulfoxides. In the intact cell, cysteine sulfoxides are localized in the cytoplasm, while the hydrolytic enzyme is found in vacuoles. Cellular compartmentalization damage results in its release, and subsequent hydrolysis of sulfoxides (Boscher et al. 1995; Sendl 1995). This reaction occurs upon tissue damage, e.g. when the organ is crushed, minced, or otherwise processed, or in case of the pathogens attack (Ankri and Mirelman 1999).
C,S-lyase isolated from ramson has a molecular mass of 150,000 Da and consists of three subunits (Landshuter et al. 1994). The optimal pH and temperature for its activity were established at 6.0 and 35 °C, respectively. After 30 min. of incubation at pH 3.0 ramson’s C,S-lyase lost its activity by 90 %. Nevertheless, its enzymatic properties are maintained at low temperatures, what was observed even after ramson cloves were freezed at −20 °C. It seems that such feature of the C,S-lyase system provides all year long protection for the underground parts of the plant after disruption of the parenchyma. In contrast to similar enzymes isolated from A. sativum, A. cepa or A. porrum, ramson’s C,S-lyase is not a glycoprotein. This was revealed as no interaction with Concanavalin-A-Sepharose, and no staining by periodic acid Schiff-reagent, were observed. The enzyme was however sensitive to low hydroxylamine concentrations. The ramson’s C,S-lyase catalyses S-alk(en)yl-cysteine-sulfoxides hydrolysis, with alliin as the most preferred substrate (Landshuter et al. 1994). Its comparison with common garlic’s alliinase showed that even though it is less specific to alliin, it still shows higher relative activity towards other cysteine sulfoxides, especially methiin and isoalliin (Schmitt et al. 2005). The primary products formed as a result of C,S-lyases action are thiosulfinates, pyruvic acid, and ammonia.
Thiosulfinates are regarded as primarily responsible for odor and flavor of freshly prepared ramson macerates, and of the products obtained by both the extraction and an unorthodox kind of distillation conducted at room temperature (Block et al. 1992). The results showed that room temperature steam distillation provides half the amount of thiosulfinates obtained by direct extraction (Block et al. 1992).
The major thiosulfinates found in ramson extracts are allicin (diallyl thiosulfinate = di-2-propenyl thiosulfinate), and methyl-allyl- or dimethyl thiosulfinates (Fig. 2) (Sendl and Wagner 1991). According to Sendl they constitute 75–90 % of all the compounds formed immediately after hydrolysis of cysteine sulfoxides (Sendl 1995). Data from quantitative HPLC determination of thiosulfinates in chloroform extracts from leaves and bulbs, refered to dry weight, is presented in Table 2 (Sendl and Wagner 1991). In a ramson freeze-dried powder total thiosulfinates concentrations expressed as molar percentage of total was 21 (based on the weight of powder) (Block et al. 1992).
Thiosulfinates are unstable, reactive compounds, that easily decompose to (poly)sulfides, dithiins, ajoenes, and other volatile and non volatile compounds. This takes place on storage, during processing, e.g. in the presence of organic solvents, and also when heat-treated. Allicin is very unstable even at room temperature. Studies by Bagiu et al. (2010) showed that after 20 h at 20 °C it decomposed completely resulting in di-2-propenyl disulfide, di-2-propenyl trisulfide, di-2-propenyl sulfide, and sulfur dioxide.
Vinyldithiins are cyclic compounds which are another group of degradation products of allicin. They are formed as reaction products in solvents less polar than 2-propanol, e.g. hexane (Sendl 1995). It was also observed that pure allicin due to its thermolabile nature degraded to vinyldithiins during GLC analysis. In the hexane extract from the bulbs of A. ursinum Wagner and Sendl (1990) identified 2-vinyl-4H-1,3-dithiin and 3-vinyl-4H-1,2-dithiin (Fig. 3). Vinyldithiins (3,4-dihydro-3-vinyl-1,2-dithiin and 2-vinyl-4H-1,3-dithiin) were identified also in the essential oil isolated from the leaves and flowers of ramson samples collected in Bulgaria (Ivanova et al. 2009).
Another group of thiosulfinates degradation products, namely ajoene, methyl- and dimethyl ajoenes, were identified in acetone–chloroform extracts from ramson bulbs (Fig. 3). Comparative analysis of A. sativum and A. ursinum extracts showed that ajoene dominated in garlic, while its methyl- and dimethyl homologues were the main components in ramson extract (Wagner and Sendl 1990). It should be mentioned that ajoenes and vinyldithiins were found as well in oil-macerated garlic (Benkeblia and Lanzotti 2007).
Apart from the above-mentioned sulfur-containing sulfoxides degradation products, various sulfur compounds have also been detected as constituents of ramson’s essential oil. This was obtained (0.007 %) for the first time already in 1887 by Semmler, who identified alkyl sulfides, alkyl polysulfides, and trace amounts of mercaptan (Sendl 1995).
The amount of oil varies depending on soil condition, geographical location, and part used. For example, isolation of essential oil from fresh and air-dried leaves and flowers of ramson collected in Bosnia yielded 0.011 % (w/w) and 0.024 % (w/w) respectively (Copra-Janicijevic et al. 2008). The results of qualitative analyses of essential oils from ramson collected from different sites in Europe showed significant differences in their composition (Table 3). Furthermore, different time of harvest and analytical method applied influenced the profile of investigated oils.
From among over 20 components identified in the volatile oil of A. ursinum collected in Serbia, the most abundant fraction was disulfides (54.7 %), followed by trisulfides (37.0 %), tetrasulfides (4.7 %), and the non-sulfur components (1.0 %) (Godevac et al. 2008). The composition of essential oils of three ecotypes of A. ursinum collected in Poland also differed significantly in the dominant components (Błażewicz-Woźniak and Michowska 2011). The Roztocze ecotype contained methyl-2-propenyl disulfide (16.05 % on average), 6,10,14-trimethyl-2-pentadecanone (13.55 %), nonanal (11.93 %), and dimethyl trisulfide (12.07 %) as main components. The Dukla ecotype oil was composed mainly of phytol (17.03 %) and n-hexadecanoic acid (16.57 %), while in the oil of the Bieszczady ecotype phytol acetate (16.40 %) and (E)-β-ionone (13.33 %) dominated (Błażewicz-Woźniak and Michowska 2011). The results of SPME-GC analysis of ramson oil, from the leaves collected in the area of Quedlinburg (Germany) showed that diallyl disulfide was the major component, amounting to ~50 % (Schmitt et al. 2005). Allyl-methyl disulfide, allyl-methyl sulfide, diallyl sulfide, and (E)-allyl-1-propenyl disulfide were abundant, as well. However, according to the authors, the SPME method did not supply information on some other volatile substances, such as 2-hexenal or hex-3-en-1-ol, which were detected by an SDE-GC method. GC/MS analysis of samples from fresh flowers collected in the vicinity of Ihtiman (Bulgaria), showed that the main components of the volatile fraction were (E)-methyl-2-propenyl disulfide, methyl-2-propenyl trisulfide, dimethyl trisulfide, 3,4-dihydro-3-vinyl-1,2-dithiin and 2-vinyl-4H-1,3-dithiin (Ivanova et al. 2009). Schmitt et al. (2005) detected the decrease of the relative yield of oil (sum of volatile substances) during the vegetation period, and observed that this was in agreement with the decreasing amounts of cysteine sulfoxides. An especially significant decrease in allyl methyl disulfide and dimethyl disulfide levels was seen, while the relative content of (E)-allyl-1-propenyl disulfide increased.
Apart from studies on the composition of steam-distilled essential oil from ramson, a very interesting aspect is investigation of the atmospheric emission rate of organic sulfur compounds. Such studies were conducted in a Viennese suburban forested park in which A. ursinum was grown as ground cover (Puxbaum and König 1997). Sulfur emission rates (μg S) per gram of dry weight and per unit of ground area were 1 μg/g × h and 60 μg/m2 × h, respectively. The authors claimed this was the highest rate ever reported for such substances emitted from a terrestrial plant.
Apart from sulfur-containing substances A. ursinum has been also reported to be a good source of phenolic compounds. It should be mentioned, however that the extraction method may substantially alter the level of active compounds isolated. Total polyphenol content, expressed as gallic acid equivalents (GAE), in the leaf extract obtained by a 12-day maceration with 70 % ethanol at room temperature (20 °C) was higher in comparison with the one prepared by the ultrasound-assisted extraction: 27.9 g GAE/100 g dry basis versus ~10 g GAE/100 g (Gîtin et al. 2012). Total free phenolics content in the leaves was determined as 3.24 mg/g, while in the bulbs 2.30 mg/g. The amount of bound forms was about the same in the leaves and in the bulbs (1.10 and 1.00 respectively) (Djurdjevic et al. 2004). Gross differences were also noted in studies on gallic acid levels. Its qualitative and quantitative analysis in hydroalcoholic extracts from A. ursinum leaves showed that: 96 % methanol extract had a gallic acid content 0.0576 mg/ml, 80 % methanol extract—0.0165 mg/ml; while 96 % ethanol extract—0.0076 mg/ml (Condrat et al. 2010).
The studies on the content of phenolic acids in fresh leaves and bulbs of ramson collected in an experimental forest situated in West Serbia exhibited some differences between free and bound compounds in these plant parts (Djurdjevic et al. 2004). The amounts of free phenolic acids in leaves and bulbs were 119.75 and 180.91 μg/g, respectively, while of bound forms—135.30 and 248.97 μg/g, respectively. The leaves contained free forms of ferulic and vanillic acids, and bound forms of p-coumaric, ferulic and vanillic acids. In the bulbs free ferulic, p-hydroxybenzoic and vanillic acids, and bound forms of p-coumaric and ferulic acids were detected.
The flavonoid content (expressed as mg quercetin equivalent—QE) determined in fresh leaves collected in March, from the Bacau city forests (Romania), using ultrasound-assisted extraction was ~7.3 mg QE/kg fresh plant; while using conventional maceration—2.7 mg QE/kg (Gîtin et al. 2012). Also, the total content of flavonoids in the different parts of A. ursinum collected in June in the forest area near Wrocław (Poland) differed significantly: seeds—73.14 mg/100 g of dry mass, stalks—206.07 mg/100 g, green leaves—1,856.31 mg/100 g, yellow leaves 2,362.96 mg/100 g (Oszmiański et al. 2013).
As far as qualitative profile is concerned, ramson is abundant predominantly in kaempferol derivatives. The ethanol extract from the leaves collected near Laceno Lake (Italy) yielded: 3-O-β-neohesperidoside-7-O-[2-O-(trans-p-coumaroyl)]-β-d-glucopyranoside (1), 3-O-β-neohesperidoside-7-O-[2-O-(trans-p-feruloyl)]-β-d-glucopyranoside (2), 3-O-β-neohesperidoside-7-O-[2-O-(trans-p-coumaroyl)-β-d-glucopyranosyl]-β-d-glucopyranoside (3), 3-O-β-neohesperidoside-7-O-β-d-glucopyranoside (4), 3-O-β-neohesperidoside (5) (Carotenuto et al. 1996). From the n-butanol fraction of the dry leaves of ramson collected in Denmark seven flavonoid glycosides were isolated. Three of them have been previously reported by Carotenuto et al. (compounds 1, 4, 5). The remaining were also identified as kaempferol derivatives: 3-O-β-d-glucopyranoside, 3-O-β-d-glucopyranosyl-7-O-β-d-glucopyranoside, 3-O-α-l-rhamnopyranosyl-(1 → 2)-[3-acetyl]-β-d-glucopyranoside and 3-O-α-l-rhamnopyranosyl-(1 → 2)-[6-acetyl]-β-d-glucopyranoside (Wu et al. 2009). Compounds 4, 5, kaempferol 3-O-β-d-glucopyranoside and kaempferol 3-O-β-d-glucopyranosyl-7-O-β-d-glucopyranoside were isolated from the n-butanol extract from fresh flowers (Ivanova et al. 2009). The analysis of flavonoid content in acidified methanol extracts from green and yellow leaves, stalks and seeds collected in June in the forest area near Wrocław (Poland) led to the isolation of 21 compounds, all kaempferol derivatives (Oszmiański et al. 2013).
Similarly to organosulfur compounds, steroidal saponins are also commonly found in the Allium genus. The following were reported in the bulbs of A. ursinum: diosgenin 3-O-α-l-rhamnopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 4)-[α-l-rhamnopyranosyl-(1 → 2)]-β-d-glucopyranoside and (25R)-spirost-5,25(27)-dien-3β-ol 3-O-α-l-rhamnopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 4)-[α-l-rhamnopyranosyl-(1 → 2)]-β-d-glucopyranoside (Fig. 4) (Sobolewska et al. 2006). A pregnane glycoside: 3-hydroxy-pregna-5,16-dien-20-on 3-O-α-l-rhamnopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 4)-[α-l-rhamnopyranosyl-(1 → 2)]-β-d-glucopyranoside has been identified as well (Fig. 4) (Sobolewska et al. 2006).
Diosgenin content in A. ursinum depended on the part of the plant and the time of harvest (Sobolewska et al. 2009). Methanol extract prepared from fresh bulbs collected in April, prior to flowering, yielded the highest content of diosgenin (0,137 %). In the extract made from leaves collected at the same time, the amount of diosgenin was 10 times lower, while in the one from leaves collected in March and June it was not detectable. Low diosgenin content in ramson does not make this plant species a valuable source for the isolation of this sapogenin.
In an ethanol extract from fresh leaves β-sitosterol 3-O-β-d-glucopyranoside was found (Sabha et al. 2012).
Other interesting constituents identified in A. ursinum include lectins, which were isolated from bulbs, roots and leaves collected in April (Smeets et al. 1997). Root compounds were identical to those found in bulbs: AUAI, which is a heterodimeric lectin composed of polypeptides of 12.5 and 11.5 kDa, and AUAII a homodimeric lectin composed of polypeptides of 12 kDa. Both lectins are mannose-specific, and show a good reaction with synthetic (1 → 3) and (1 → 6) mannans. The ramson leaf lectin (AUAL) differs from the bulb lectins, and also from the leaf-specific lectins identified in other Allium species. It is a dimer composed of 12 kDa subunits.
The bulbs are also rich in polysaccharides. According to Hegnauer, they may contain as much as 30–90 % of mostly fructans (Hegnauer 1963). In the bulbs harvested between May and August the content of fructan U, consisting of fructose residues only, was estimated as 75–90 % (Meier and Reid 1982). Unfortunately, no modern structure elucidation studies on this compound have been performed to date. The studies on reserve carbohydrates in A. ursinum from the Sheffield flora (United Kingdom) resulted in determination of the maximum fructan concentration in the bulbs harvested in summer, as 139 mg/g fresh weight (Hendry 1987).
A number of fatty acids were reported in the hexane extract from the bulbs. These were palmitic, linoleic, oleic, palmitoleic, stearic, α-linolenic, and myristic acid (Wiater et al. 1998). Moreover, water extracts yielded fairly rare, but pharmacologically valuable γ-glutamylpeptides, and many amino acids, such as: asparagine, glutamine, aspartic acid, glutamic acid, arginine, alanine, glycine, threonine (Wagner and Sendl 1990). In an ethanol extract from fresh leaves 2-di-O-α-linolenoyl-3-O-β-d-galactopyranosyl-sn-glycerol (DLGG) (Fig. 5), was identified (Sabha et al. 2012).
Ramson leaves seem to be relatively abundant in pigments, as compared to other Allium plants, the content of which amounts to: 2.87 ± 0.03 mg/g of chlorophyll a, 1.35 ± 0.01 mg/g of chlorophyll b, and as much as 9.99 ± 0.01 mg/g of carotenoids (Štajner and Szöllosi Varga 2003). Comparative analysis of some macro- and microelements in A. ursinum and A. sativum showed that ramson contained higher levels of magnesium (7,000 mg/kg), manganese (1,600 mg/kg) and iron (230/mg/kg) than garlic (6,114, 952, 14 mg/kg, respectively). A. ursinum is a rich source of adenosine (120 mg/kg) (Nagori et al. 2010).
Allium ursinum L. ssp. ucrainicum floral nectar volume and concentration were investigated in three different habitats in the Mecsek hills (South Transdanubia, Hungary) (Farkas et al. 2012). The study revealed that ramson produces low to medium volumes (ranged 0.1–3.8 μl) of highly concentrated nectar (25–50 % sugar concentration). Freely sun-exposed flowers produced lower quantity of nectar than covered flowers at a given time. The higher volume of nectar with higher sugar content was observed in populations living in optimal life conditions for A. ursinum (the sessile oak-hornbeam association). The plants living in the silver lime-flowering ash rock forest, where the lack of sufficient nutrients was observed, produced lower quantities of nectar.