Metabolic profiling of rhizomes of native populations of the strictly endemic Croatian species Iris adriatica

Abstract

Iris adriatica Trinajstić ex Mitić (Iridaceae L.) is a strictly endemic taxon from Croatia. It is a rhizomatous dwarf plant from the I. pumila complex with a distribution area limited to the Croatian part of the Mediterranean area, mainly central Dalmatia. The genus Iris is known for its richness in isoflavonoids which also play a significant role in chemotaxonomy and biological activity. Hence, in the current study, different plant batches of I. adriatica collected in early spring of 2016 were analysed for their phytochemical profiles and qualitatively compared. UHPLC-PDA-ESI-MS analyses of methanolic rhizome extracts were performed. Altogether, 36 compounds, representing isoflavonoids (including 6,7-methylendioxy derivatives), benzophenones and xanthones were found as aglycones or in glycosidically bound form to be the main constituent groups of I. adriatica rhizomes. Qualitative results were identical between different batches of plant material from collection sites in central Dalmatia, they differed only in quantity. For some phenolic compounds of I. adriatica, chemotaxonomic relevance was detected.

Keywords:

• Subgenus Iris
• series Iris
• Iris pumila complex
• isoflavonoids
• benzophenones
• xanthones
• UHPLC-MS

Introduction

Iris adriatica Trinajstić ex Mitić is an accepted name describing the strictly endemic taxon from Croatia (Mitić 2002; The international plant names index 2012; The Plant List 2013). The taxonomic position of I. adriatica has been clarified relatively lately (Mitić 2002). This diploid (2n = 16), rhizomatous dwarf plant, together with I. pseudopumila Tineo, I attica Boiss. et Heldr. and I. pumila L., belong to the so-called I. pumila complex (Mitra 1956; Randolph and Mitra 1959; Trinajstić et al. 1980; Mitić 2002) of the series Pumilae Lawr., section Iris, subgenus Iris (so called “bearded” or Pogon irises) (Lawrence 1953; Mathew 1981). The short stems (1–5 cm) are overtopped by narrow, gentle and often falcate leaves (3–10 cm), which are overtopped by relatively large and decorative solitary flowers. Greeny, slightly keeled, spathes, surrounded by scarious margins are slightly longer than or as long as the hypanthial tube which is 4 to 7 cm long. Tepals are usually of different shades of yellow and less frequent of violet or purple colour. The outer tepals have multicellular coloured hairs (so called “beard”). Plants bloom very early in the spring, in March and April. Its distribution area is limited to the Croatian part of the Mediterranean area – central Dalmatia, the surroundings of the cities of Drniš, Unešić, Zadar, Šibenik and Split, as well as the nearby islands (e.g. Vir, Čiovo, Brač, Kornati etc.). Populations of I. adriatica are generally small, and in the past few years, some of them have disappeared or decreased. For this reason, I. adriatica belongs to the NT (near threatened) IUCN category in the Croatian Red Book of Vascular Plants (Nikolić 2017). However, the plant is suitable for cultivation in greenhouses and in vitro (Vršek et al. 2004; Kereša et al. 2009), and therefore, besides an ornamental use, it has a good perspective to become a useful medical plant, as it was shown for some other Iris taxa (Kukula-Koch et al. 2015; Venditti et al. 2017).

Furthermore, phytochemical analysis of native populations of I. adriatica could also have possible taxonomic implications on different classification levels. Namely, chemical constituents are genetically controlled traits that are sometimes considered to have advantages over morphological ones in systematic research (Harborne and Turner 1984), and taxonomically useful phenolic variations have been established in the family Iridaceae (Harborne and Williams 2000), genus Iris (Wang et al. 2010), and especially in some other taxa of the subgenus Iris (Williams et al. 1997, 2000; Rusak et al. 2005; Venditti et al. 2017; Abdel-Mageed et al. 2018).

Isoflavones, flavones, xanthones and benzophenones have been discovered as major phenolic constituents in Iris spp. (Williams et al. 1997; Mizuno et al. 2012; Wei et al. 2012; Kaššák 2014; Xie et al. 2014; Kukula-Koch et al. 2015). Rhizomes are known from traditional usage and medicine, obviously because of their phenolic compounds (Kaššák 2012). Moreover, some recent surveys have revealed medically useful phenolic compounds in rhizomes of some other Iris taxa (Conforti et al. 2009; Rigano et al. 2009; Wei et al. 2012; Xie et al. 2014; Venditti et al. 2017). Recently, a number of isoflavones together with two benzophenones, a xanthone and the ubiquitous β-sitosterol have been isolated and identified from a sample of I. adriatica of undefined origin bought at an open market (Bukvički et al. 2017).

Hence, the detailed investigation of phenolic compounds from samples of I. adriatica collected at the wild habitats were chosen for three purposes: to provide the first results of phytochemical analysis of I. adriatica from different collection sites of its original habitats, to validate whether the established phenolic compounds have already been found earlier to have a useful phytotherapeutic potential and to provide new evidence for possible taxonomic implications of established phenolic compounds.

Material and methods

Plant material

The Iris plants were collected at the beginning of April of 2016 on several different native localities of the species I. adriatica (Table S1, Supplementary data). Collections were made with the Permission of the Croatian Ministry of Environment and Nature Protection (Decision No. UP/1-612-07/15-48/23). Collecting of particular plant parts and their quantity depends on the fitness condition of the specific population. Different plant parts were separated and dried at room temperature for one month. Live voucher specimens are deposited within the Iris-collection of the Botanical Garden Zagreb (the codes presented in the Table S1), and voucher specimen no. 14341 in the herbarium collection ZA, as well.

Sample preparation

Air-dried samples (rhizomes) were extracted with methanol in a shaking water bath at 50 °C (0.5 g, 10 ml, 60 min), the solvent was evaporated to dryness. Samples for LC-MS analysis were prepared by dissolving the extracts in MeOH at a concentration of 5 mg/ml and centrifugation (5 min., 3000 U/min.).

In order to isolate selected compounds for structural confirmation by NMR, remaining amounts of rhizomes from all collection sites were combined (261 g) and extracted in a Soxhlet apparatus with methanol in two batches (4.5 h extraction time) with an extraction yield of 16.3%.

UHPLC-PDA-ESI-MS analysis

Analysis of extracts was carried out on a Dionex Ultimate 3000 RS LC system coupled to a LTQ XL linear ion-trap mass spectrometer equipped with an ESI ion source (all components Thermo Scientific). Separation was done on a Zorbax SB-C18 Rapid Resolution HD column, 100 × 2.1 mm, 1.8 μm particle size (Agilent). A gradient elution using 0.1% formic acid in water (A) and gradient grade acetonitrile (B) was performed, starting with 8% B, increasing to 45% B 0–9 min.; 9–13 min. 45% B to 100% B; 15–15.5 min. 100% B to 8% B, equilibrating for 8 min; flow rate 0.39 ml/min. Column temperature was 35 °C; injection volume 2 μl. PDA detection was performed in the 190 nm to 500 nm wavelength range. An estimate of quantitative relationships of major compounds was made on basis on PDA total scan chromatograms. The mass spectra were recorded in negative and positive ion mode in the m/z range of 50 to 2000 amu. Mass spectral conditions were set as follows: Source voltage 3.5 kV (ESI pos), 5.0 kV (ESI neg); capillary temperature 300 °C; source temperature 350 °C; sheath gas flow 65 arb (arbitrary units), auxiliary gas flow 15 arb.

Isolation of compounds

20.2 g of extract was separated by VLC on 200 g silica gel (Silicagel 60 (Merck), 0.04–0.063 mm) with gradient elution of different hexane – ethyl acetate – methanol ratios into 31 fractions. Compounds 11, 15, 19 and 24 were obtained from fractions F21–24 (eluted with MeOH – EtOAc 50:50, v/v), followed by semipreparative HPLC (Shimadzu CBM-20A controller, LC-20AT solvent delivery module, SIL-10AF autosampler, CTO-20AC column oven, SPD-M20A diode array detector, FRC-10A fraction collector) using a Luna C18(2) column, 250 × 10 mm, 10 μm (Phenomenex), water – acetonitrile 75:25 (v/v) isocratic; 4 ml/min; 25 °C column temperature; 200 μl injection volume. Compounds 31, 32, 34 and 36 were isolated from F9–10 (eluted with hexane – EtOAc 50:50, v/v) by CC on Sephadex LH-20 eluting with EtOAc – MeOH 9:1 (v/v), followed by semipreparative HPLC (Shimadzu system, see above) eluting with water – MeOH (35:65, v/v) (36) and water – MeOH (45:55, v/v) (31, 32, 34), respectively.

Results

Combining information of UV spectra and mass spectrometric detection, altogether 36 compounds, representing 22 isoflavones, 7 xanthones, 6 benzophenones and 1 acetophenone, could be identified in the methanolic extracts of I. adriatica rhizomes (see Table 1). Representative chromatograms are presented in Figure 1 and Figure S1 (Supplementary data), structures of compounds can be depicted from Figure 2. Comparison of samples of all accessions showed only differences in relative proportions of the identified compounds, which could be detected in all samples (see Figure S2, Supplementary data). Seven isoflavones (15, 19, 24, 31, 32, 34, 36) and irisxanthone (11) could be isolated and fully structure elucidated by additional NMR analysis, thereby confirming structural proposals deduced from UV spectral and mass spectrometric data and defining type and linkage of monosaccharides in isoflavone glycosides 15, 19 and 24, as well as irisxanthone (11).

Table 1. Compounds in rhizomes of Iris adriatica identified by UHPLC-PDA-ESI-MS analysis.

Compound	Mol. mass	[M − H]^−	[M + H]^+	Aglycone	Group	Proposed structure
1	584	583	585		Xanthone	Neomangiferin
2	408	407	n.d.	14	Benzophenone	Iriflophenone-2-O-hexoside
3	408	407	409	14	Benzophenone	Iriflophenone-C-hexoside
4	598	597	599		Xanthone	7-O-Methyl(iso)mangiferin-O-hexoside
5	328	327	n.d.		Acetophenone	3-Hydroxy-5-methoxyacetophenone-3-O-hexoside or 4-Hydroxy-3-methoxyacetophenone-4-O-hexoside
6	584	583	n.d.	25	Benzophenone	2,4′,6-Trihydroxy-4-methoxybenzophenone-O-dihexoside
7	598	597	599		Xanthone	7-O-Methyl(iso)mangiferin-O-hexoside
8	422	421	423		Xanthone	Mangiferin
9	598	597	599		Xanthone	Irisxanthone-8-O-hexoside
10	422	421	423	25	Benzophenone	2,4′,6-Trihydroxy-4-methoxybenzophenone-O-hexoside
11	436	435	437		Xanthone	Irisxanthone
12	816	815	817	31	Isoflavone	Iristectorigenin A-O-trihexoside
13	436	435	437		Xanthone	7-O-Methyl(iso)mangiferin
14	246	245	247	14	Benzophenone	Iriflophenone
15	654	653	655	31	Isoflavone	Iristectorigenin A-7-O-β-D-glucopyranosyl-(1→6)- β-D-glucopyranoside
16	798	797	799	30	Isoflavone	Irisolone-O-trihexoside
17	684	683	685	32	Isoflavone	Irigenin-O-dihexoside
18	462	461	463	28	Isoflavone	Tectorigenin-O-hexoside
19	636	635	637	30	Isoflavone	Irisolone-4′-O-[β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (Germanaism E)
20	492	491	n.d.	31	Isoflavone	Iristectorigenin A-O-hexoside
21	492	491	493	31	Isoflavone	Iristectorigenin A-O-hexoside
22	522	521	523	32	Isoflavone	Irigenin-O-hexoside
23	636	635	637	30	Isoflavone	Irisolone-O-dihexoside
24	474	473	475	30	Isoflavone	Irisolone-4′-O-β-D-glucopyranoside (Germanaism B)
25	260	259	261	25	Benzophenone	2,4′,6-Trihydroxy-4-methoxybenzophenone
26	460	459	461	34	Isoflavone	Irilone-O-hexoside
27	680	679	681	30	Isoflavone	Irisolone-O-sinapoylhexoside
28	300	299	301	28	Isoflavone	Tectorigenin
29	650	649	651	30	Isoflavone	Irisolone-O-feruloylhexoside
30	312	311	313	30	Isoflavone	Irisolone
31	330	329	331	31	Isoflavone	Iristectorigenin A
32	360	359	361	32	Isoflavone	Irigenin
33	356	n.d.	357	33	Isoflavone	Iriskumaonin methyl ether
34	298	297	299	34	Isoflavone	Irilone
35	374	373	n.d.	35	Isoflavone	Irigenin S
36	386	n.d.	387	36	Isoflavone	Irisflorentin

Figure 1. Representative UV chromatogram (320 nm) of the methanolic extract of Iris adriatica rhizome (Sample 1). For compound identities see Table 1, for experimental conditions refer to Materials and methods.

Figure 2. Structures of xanthones, benzophenone and isoflavone aglycones identified in Iris adriatica rhizomes.

Structures of isoflavones

Description of mass spectral fragments of isoflavonoids due to retro-Diels-Alder reaction follows nomenclature of Ma et al. (Ma et al. 1997). Characteristic ring fissions are presented in Figure S3 (Supplementary data).

Compound 36 could be identified as irisflorentin (Arisawa et al. 1973a) which was deduced from the ^1,3A⁺ fragment of m/z 195 and loss of 4 methyl groups and NMR data. Compound 35 was designated as irigenin S (Ibrahim et al. 2012) due to loss of 3 methyl groups and fragments ^0,4B^- at m/z 221 and ^0,3B^-+H at m/z 194. Irilone (34) (Rigano et al. 2007) was identified by its fragment ^1,3A⁺ at m/z 181 and NMR spectral data. Loss of a hexose unit (−162 u) and identical aglycone fragments as in 34 led to the assignment of an irilone-O-hexoside for compound 26. The assignment of iriskumaonin methyl ether (33) (Pailer and Franke 1973) was based on loss of 3 methyl groups, loss of an oxymethylene fragment (−30 u) and loss of a ^1,3B⁺-H (−161 u) unit. Irigenin (32) (Pailer and Franke 1973) showed consecutive loss of 3 methyl groups and representative ^1,3A⁺ (m/z 183) and ^1,3B⁺ (m/z 178) fragments. NMR data confirmed the structural proposal. Irigenin glycosides 17 and 22 were characterised as O-dihexoside (17) and O-hexoside (22) due to losses of −162 u and aglycone fragments matching with those of irigenin (32). According to mass spectral fragmentation compound 31 was identified as one of the isomers iristectorigenin A or B. Detailed analysis of HMBC correlations of H-6′ (7.28 ppm) to C-4′ (148.9 ppm) and methoxy protons at 3.78 ppm led to the unequivocal assignment of 31 to iristectorigenin A (Hanawa et al. 1991). Compounds 12, 15, 20 and 21 could be characterised as iristectorigenin A glycosides. In compound 15 the glycosidic moiety was shown to be a β-d-glucopyranosyl-(1→6)- β-d-glucopyranoside attached to the hydroxyl group at position 7 of the aglycone (Schütz et al. 2011). The consecutive losses of 3 hexosyl units (−162 u) indicated that compound 12 was a O-trihexoside of iristectorigenin A, which has not been described before in Iris spp. Similarly, an O-trihexoside of irisolone (16) could be found, which is a new report for Iris spp. In fact, the highest number of isoflavone glycosides in I. adriatica rhizomes was depicted as irisolone glycosides, their aglycone (30) (Dhar and Kalla 1972) could also be detected in free form. Glycosides 19 and 24 could be describe as irisolone-4′-O-[β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside (19, Germanaism E) (Rigano et al. 2007) and irisolone-4′-O-β-d-glucopyranoside (24, Germanaism B) (Rigano et al. 2007) based on UV, ESI-MS and NMR data. Compounds 27 and 29 proved to be acylated hexosides of irisolone which was deduced from the loss of −338u (29) and −368u (27) in ESI^- spectra and a consecutive loss of −176u (29) and –206u (27) as well as −162u (27, 29) in ESI⁺ mode (Cuyckens and Claeys 2004), assigning compound 27 tentatively to irisolone O-sinapoylhexoside and compound 29 to irisolone O-feruloylhexoside, respectively. Acylation with phenylpropanoic acids is also in accordance with the high retention times on reversed phase columns compared to corresponding dihexosides as well as an additional UV absorbance band at ca. 326 nm compared to irisolone 30. Aglycone 28 was identified as tectorigenin (Pailer and Franke 1973) due to loss of one methyl group in ESI^- mode and a characteristic loss of a ^1,3B⁺ fragment (−118 u) in MS³ spectrum of ESI⁺ mode. Its corresponding O-hexoside 18 showed a loss of a hexose unit (−162 u) and matching aglycone fragments.

Structures of xanthones

For xanthones, fragmentation pathways proposed by Yu et al. (2013) and Schieber et al. (2003) were used. In case of xanthones, UV spectra were useful for identifying this class of compounds due to 4 characteristic absorption bands at 230–245 nm, 250–265 nm, 305–330 nm and 340–400 nm (Harborne 1998).

Compounds 11 and 13 proved to be xanthone-C-glycosides based on UV and mass spectral data. According to congruence with mass spectra of mangiferin 8 (Schieber et al. 2003; Yu et al. 2013) but mass fragments increased by 14u, 13 was assigned to 7-O-methylmangiferin or 7-O-methylisomangiferin (Fujita and Inoue 1981). Xanthones 4 and 7 revealed to be O-hexosides of either isomer due to loss of a hexose unit (−162 u). Based on mass spectral similarity but a higher UV absorption band II close to 280 nm, compound 11 was designated as irisxanthone (Arisawa et al. 1973b), in addition its O-hexoside compound 9 could be identified. The structural proposal of 11 was confirmed by NMR data of the isolated substance. MS² spectrum of glycoside 1 (ESI^- mode) was in congruence with that of neomangiferin (Yu et al. 2013), the 7-O-β-d-glucoside of mangiferin 8. So far, this xanthone was only found outside subgenus Iris, i.e. in I. dichotoma and I. domestica (Zhang et al. 2011; Wei et al. 2012).

Structures of benzophenones and acetophenones

Benzophenones showed fission of the C–C bond between central carbonyl group and one of the adjacent aromatic rings leading by proton migration to either substituted benzyl rings or bicyclic β-lactones, the latter were characterised by a further loss of CO₂. According to that, compound 25 could be identified as 2,4′,6-trihydroxy-4-methoxybenzophenone (Roger et al. 2012). O-Glycosidically bound hexoses attached to aglycone 25 were found in compounds 6 and 10, the di-O-hexoside (6) eluting before the mono-O-hexoside (10). Compound 14 proved to be another benzophenone with an identical loss of the hydroxylated ring B, but showing ring A of 14 mass units less than compound 25. Hence, for compound 14 the structure of iriflophenone was proposed (Arisawa et al. 1973b). Glycosides 2 and 3 could be designated as hexosides of iriflophenone, with the hexose moiety attached to a hydroxyl (most likely in position 2) of the aglycone in case of 2, and a C-glycosidyl hexoside in case of compound 3. The latter showed a typical fragmentation of C-glycosidically bound hexoses in ESI^- mode (Cuyckens and Claeys 2004; de Rijke et al. 2006). Tentative identification of compound 5 as 3-hydroxy-5-methoxyacetophenone or 4-hydroxy-3-methoxyacetophenone hexoside was deduced from fragmentation in ESI^- mode due to losses of a hexose unit (−162 u), a methyl group (−15 u) and a further loss of CO (−28 u). 3-Hydroxy-5-methoxyacetophenone was identified before in I. germanica (Ibrahim et al. 2012).

Quantitative estimate of major compounds in I. adriatica rhizomes

Germanaism B (24) represented the major compound with 18.5–25.6% of total peak area (based on PDA total scan chromatograms) in extracts of samples 1–5 and 7–9. On the contrary, in sample 6 compounds 30, 31 and 32 summed up to 50.4% of total peak area, and the relative proportion of compound 24 was only 12.1%. Also germanaism E (19, 0.4%) and irigenin-O-hexoside (22, 0.9%) showed exceptionally low levels in sample 6, compared to all other samples in which these compounds were present in proportions of 1.4–5.0% (19) and 2.0–3.0% (22), respectively. The major xanthones were mangiferin (8, 1.7–5.3%) and irisxanthone (11, 3.6–5.6%, in a mixture with 12). Among benzophenones, compound 25 (2,4′,6-trihydroxy-4-methoxybenzophenone) was dominating (2.7–6.3%). Table S2 summarises a quantitative estimate of major phenolic compounds in the analysed samples.

Discussion

This is the first LC-PDA-MS analysis performed on taxa of the I. pumila complex in order to get a more detailed insight into the pattern of phenolic compounds (xanthones, benzophenones, isoflavones) in the native rhizomes of Croatian stenoendemic taxon I. adriatica. Complete or partial phenolic metabolite profiles of Iris spp. have been published before, and some researched species belong to the subgenus Iris, e.g. I. albicans Lange (Abdel-Mageed et al.2018), I. germanica L. (Schütz et al. 2011), I. hookeriana (Dar et al. 2016), I. marsica (Venditti et al. 2017), I. nigricans (Huwaitat et al. 2013) and I. pseudopumila (Rigano et al. 2007; Conforti et al. 2009).

Isoflavones were the main group of phenolic compounds in I. adriatica rhizomes of both native and planted collections. Similar to the report of Bukvički et al. (2017), likewise in our research isoflavonoids showing a 6,7-methylendioxy group were dominating in terms of number (compounds 16, 19, 23, 24, 26, 27, 29, 30, 33, 34). Among them six glycosides of irisolone (30), including the major compound in all extracts except that of sample 6 (cf. Table S1 and Table S2), i.e. compound 24, could be identified. In sample 6, collected at Brnjica-Pokrovnik, iristectorigenin A (31) and irigenin (32) dominated. This population was the only one growing within the Festuco-Koelerietum splendentis Horvatić 1963 association (an extensive open calcareous meadow), whilst the rest of the researched populations belong to the Stipo-Salvietum officinalis Horvatić 1985 association (hills, limited rocky pastures, endangered by the succession of garrigue). If further research proves that our finding for this population of I. adriatica is of chemotaxonomic value, this could point to a more distinct position of this particular population, which might be a specific ecotype of the typical species. The occurrence of methoxylated isoflavones with and without 6,7-methylendioxy structural element is in accordance with the findings for rhizomes of I. pseudopumila Tineo (Rigano et al. 2007), belonging to the I. pumila complex. However, in contrast to the report of Rigano et al. (2007), in none of our investigated I. adriatica rhizomes flavones and flavonol derivatives like isoscutellarein and kaempferol glycosides could be detected. This finding would be of chemotaxonomic value as well, especially in case of such closely related taxa, often mistakenly mixed in the past (Randolph and Mitra 1959; Webb and Chater 1980; Mitić 2002). Though, this difference in flavonoid composition between the closely related taxa should be considered with care, as the flavonoid composition of the aerial parts of plants of the subgenus Iris with typical occurrence of C-glycosyl flavones as reported by others (Williams et al. 1997; Conforti et al. 2009) and according to our preliminary investigations of a few leave samples (data not shown here), can be quite different from rhizomes. Also, in reports of Bukvički et al. (2017) no flavones/flavonols as constituents of I. adriatica rhizomes were mentioned. Hence, careful separation of leaves and rhizomes before analysis seems to be essential. On the other hand, the absence of 7-O-methyltectorigenin derivatives in I. adriatica compared to I. pseudopumila rhizomes (Rigano et al. 2007, 2009) should also be considered as taxonomical marker and investigated further.

During our study of I. adriatica for the first time for representatives of the subgenus Iris, two isoflavone triglycosides were described. In addition, acylated irisolone glycosides (27, 29) have never been reported before in the genus Iris, and are potential candidates of chemotaxonomic importance on taxonomic levels higher than species level. Yet, they were present only in low amounts which makes them difficult to isolate and get their detailed structures. Actually, isoflavone glycosides with cinnamic acids attached to the glycosidic moiety are rare and exemplified only by a few reports from Juglans sigillata (Hu et al. 2017), Pterocarpus santalinus and Pterocarpus marsupium (Veitch 2007). Since mentioned plants are phylogenetically very distant from the genus Iris, these results require further investigation of isoflavone glycosides within the genus Iris and family Iridaceae, as well.

Mangiferin (8) and irisxanthone (11) are the dominating xanthones in I. adriatica rhizomes. The occurance of 8, isomangiferin and methylated derivatives thereof has also been described for Italian taxa of the subgenus Iris including I. pseudopumila (Williams et al. 1997).

According to our studies, benzophenones are represented in I. adriatica by iriflophenone (14) and 2,4′,6-trihydroxy-4-methoxybenzophenone (25) not only as aglycons, but also in their glycosidic form, including a possible C-glycosyl derivative of 14. This is a new finding for taxa of the I. pumila complex, however, the 2-O-β-d-glucopyranoside of 25 has been reported for I. germanica (subgenus Iris, series Iris) (Xie et al. 2014).

The inter- and intra-population morphological, karyological and molecular variations within the subgenus Iris are sometimes difficult to detect (Williams et al. 2000; Mitić et al. 2001; Wilson 2011), and in such cases phytochemical features might help in providing additional taxonomical information. However, for more precise conclusion about the role of chemical markers in the taxonomy of the I. adriatica and its closely related taxa, further phytochemical analysis should be carried out.

Biological activity of some of the established phenolic compounds from the rhizomes of I. adriatica was already tested and proved in some other Iris taxa (Kaššák 2012). Compared to activities reported for I. germanica rhizomes, it seems that a number of isoflavones which are also present in I. adriatica could be active as cancer chemopreventors (Wollenweber et al. 2003). The methanolic extract of rhizomes of I. pseudopumila showed significant anti-inflammatory activity based on inhibition of LPS induced NO production in RAW 264.7 macrophages (Conforti et al. 2009), IC₅₀ values of 67.5 and 55.1 μM were determined for compounds 19 and 22. Similarly, inhibition of NO release was also reported for 2,4′,6-trihydroxy-4-methoxybenzophenone (25) (Pan et al. 2016) which additionally showed DPPH radical scavenging effects (Pan et al. 2016) as well as induction of apoptosis in HT-29 colon carcinoma cells (Lay et al. 2014). Mangiferin (8), the main xanthone in I. adriatica rhizomes, revealed several activities related to antidiabetic effects, like inhibiting endothelial insulin resistance (Xu et al. 2017) or α-glucosidase inhibition (Vo et al. 2017) as well as anti-inflammatory and antidepressant action (Cao et al. 2017). Irilone (34) was shown to possess α-amylase inhibitory effects which would support antidiabetic action (Ibrahim et al. 2017). The same compound (34) could also play a role as phytoestrogen as it was contributing to the oestrogenic activity of red clover extracts ((Lutter et al. 2014). Hence, there is remarkable potential of I. adriatica constitutents to be explored further for antiinflammatory, chemopreventive, antidiabetic and phytoestrogenic effects.

Conclusions

To conclude, I. adriatica proved to be a rich source of phenolic compounds of great pharmacological and taxonomical value. Although it is an endemic species, it is also easy to grow both in greenhouses and in vitro (Vršek et al. 2004; Kereša et al. 2009), and therefore is suitable for further exploration and utilisation for both scopes – pharmaceutical and chemotaxonomical.

Supplemental data

Supplemental data for this article can be accessed https://doi.org/10.1080/11263504.2018.1478906

Disclosure statement

The authors declare that they are not in a potential conflict of interest.

Acknowledgements

The authors are grateful to several colleagues who have been assisting us in the organisation of the field work (Milenko Milović, Miroslav Mitić, Radnić family, Dalibor Vladović and Nediljko Ževrnja) and to Elvira Knauder for assistance in phytochemical work. The Croatian Ministry of Environment and Nature Protection is acknowledged for giving us the permission to collect I. adriatica plant material (Decision No. UP/1-612-07/15-48/23).