Anti-viral and Anti-inflammatory Isoflavonoids from Ukrainian Iris aphylla Rhizomes: Structure-Activity Relationship Coupled with ChemGPS-NP Analysis

• Abstract
• Introduction
• Results and Discussion
• Material and Methods
• Plant Material
• Extraction and isolation of compounds
• In vitro assessment of anti-viral activity
• Biology: cells, viruses, and chemicals
• Half-maximal effectiveness assay (EC50 assay)
• Cytotoxicity test (CC50 assay)
• Neuraminidase assay
• Coronavirus 229E assay
• In vitro assessment of the anti-inflammatory activity
• Preparation of human neutrophils
• Measurement of superoxide generation
• Measurement of elastase release
• ChemGPS-NP analysis
• Statistical analysis
• Contributorsʼ Statement
• References

Abstract

Dried Iris rhizomes have been used in Chinese and European traditional medicine for the treatment of various diseases such as bacterial infections, cancer, and inflammation, as well as for being astringent, laxative, and diuretic agents. Eighteen phenolic compounds including some rare secondary metabolites, such as irisolidone, kikkalidone, irigenin, irisolone, germanaism B, kaempferol, and xanthone mangiferin, were isolated for the first time from Iris aphylla rhizomes. The hydroethanolic Iris aphylla extract and some of its isolated constituents showed protective effects against influenza H1N1 and enterovirus D68 and anti-inflammatory activity in human neutrophils. The promising anti-influenza effect of apigenin (13, almost 100% inhibition at 50 µM), kaempferol (14, 92%), and quercetin (15, 48%) were further confirmed by neuraminidase inhibitory assay. Irisolidone (1, almost 100% inhibition at 50 µM), kikkalidone (5, 93%), and kaempferol (14, 83%) showed promising anti-enterovirus D68 activity in vitro. The identified compounds were plotted using ChemGPS-NP to correlate the observed activity of the isolated phenolic compounds with the in-house database of anti-influenza and anti-enterovirus agents. Our results indicated that the hydroethanolic Iris aphylla extract and Iris phenolics hold the potential to be developed for the management of seasonal pandemics of influenza and enterovirus infections.

Introduction

Viral infection represents a serious health issue for humanity with devastating complications and life-threatening conditions. Studies suggested that the prevention and treatment of early stages of viral infections are crucial to allow rapid recovery and to protect the general population [1], [2], [3], [4]. Influenza viruses such as H1N1, H3N2, H5N1, and H7N9 (Orthomyxoviridae) represent a serious threat to human health. These influenza viruses are highly infectious and result in a high rate of morbidity and mortality [5]. Enteroviruses, such as EV68, EV69, EV70, and EV71 (Picornaviridae), affect millions of people worldwide each year and are often found in respiratory secretions including saliva, sputum, or nasal mucus. However, enteroviruses are transmitted through ingestion (enteric) [6]. The emerging resistance of these viruses to drug substances urges the development of novel therapeutic strategies to combat their infections [7].

Influenza H1N1 (A/WSN/33) virus-infected Madin–Darby canine kidney epithelial (MDCK) cells or enterovirus D68-infected rhabdomyosarcoma (RD) cells are well-accepted cell-based models that are often used to assess the protective effects of newly tested samples [8]. Neuraminidase inhibition is one of the important mechanisms to block the release of the replicated virus particles from the infected cells [9]. This effect leads to a reduction in the spread of infection in the respiratory tract. Neuraminidase inhibitors including zanamivir and oseltamivir are non-toxic agents to cells and bind to a wide range of neuraminidase subtypes and, thus, are effective and crucial for the containment of influenza pandemics [10].

Inflammation, an innate immune response by the host defense mechanisms, is triggered by infection or tissue injury and leads to a series of complex biological processes. These include the accumulation of blood and immune cells together with the release of inflammatory mediators, which affect tissue structure and function. Untreated inflammation can lead to chronic inflammation, leading to various diseases, such as autoimmune diseases, neurodegenerative diseases, and cancers [11]. The inflammatory syndromes related to infections including influenza, enterovirus, or coronavirus involve uncontrolled function neutrophils with pathological and often lethal consequences [12]. Thus, neutrophils drive a plethora of diseases and contribute to the development of virus-associated complications including lung injury and acute respiratory distress syndrome (ARDS).

Despite the huge number of synthetic drugs, many viral infections lack preventive measures and effective anti-viral treatments. The search for new anti-viral drugs is still critical and natural products are excellent sources of novel anti-infective agents with potent activity and favorable safety profiles. The analysis of herbal medicinal products with anti-viral activity (influenza, colds, ARVI, and herpes virus) in leading databases showed that the number of such drugs is quite limited [13], [14], [15], [16]. The most common natural drugs with anti-viral activity belong to Echinacea sp.

Iris aphylla L. (synonyms Iris hungarica Waldst. et Kit., Iris aphylla L. subsp. hungarica (Waldst. et Kit.) Hegi) is a rhizomatous perennial plant from the Iridaceae Juss. family. It is a widely distributed plant in Central and Western Europe, especially in Ukraine, Germany, the Czech Republic, Slovakia, Hungary, and Poland [17], [18]. Previous phytochemical studies of I. aphylla revealed the presence of terpenoids, carboxylic acids, amino acids [19], and phenolic compounds [20]. The crude extracts of I. aphylla rhizomes showed potent anti-inflammatory, antioxidant, and cytotoxic activities [21]. However, compared with other Iris sp. extracts, individual I. aphylla components are still relatively understudied, and only a few compounds were identified including tectoridin, tectorigenin, iristectorigenin B, and iristectorin B [22]. Detailed investigation of the pharmacological mechanisms of action of I. aphylla rhizome extracts is still insufficient. Thus, this study focuses on the isolation of compounds from Iris rhizomes, as well as on the evaluation of the in vitro anti-inflammatory and anti-viral effects of the isolated compounds. We also evaluated those compounds with predicted bioactivities in accordance to their physico-chemical properties based on a chemical global positioning system for natural products (ChemGPS-NP) analysis [23], [24].

Results and Discussion

Compounds 1 – 11 and 13 – 17 ([Fig. 1]) were isolated from the EtOAc and CHCl₃ fractions of I. aphylla rhizomes by column chromatography; compound 12 was obtained from the butanol fraction (Scheme 1S). Compound 18 was obtained from the chloroform fraction. The rare natural compounds 1 – 5 were only found in Iris sp.; however, this is the first accurate description of the compoundsʼ structure elucidation (Fig. 1S-23S, Supporting Information).

The molecular formula of compound 1 was determined as C₁₇H₁₄O₆ from the quasi-molecular ion peak [M + H]⁺ at m/z 315 in the LC-MS spectrum, in conjunction with the ¹³C NMR data. In the ¹H NMR spectrum of 1 (Table 1S, Supporting Information), a pair of 2H signals at δ _H 7.49 (d, J = 8.5 Hz) and 7.00 (d, J = 8.5 Hz) indicated that the B-ring was substituted at C-4′. The characteristic isoflavone signal of H-2 was observed at δ _H 8.37 (s), together with only one aromatic proton at δ _H 6.50 (s) assigned to H-8 indicating that three substituents were linked to the A-ring. The substitution pattern was established from ¹³C NMR chemical shifts and 2D NMR spectra (NOESY, HSQC, and HMBC, [Fig. 2]; Table 1S, Supporting Information). The OH group at C-5 (δ _H 13.02, s) was deduced from the diagnostic resonance of C-4 (δ _C 180.9). The observed NOE effects between the OH group at C-5 and the methoxy group at C-6 (δ _H 3.74, s), as well as between the aromatic proton at H-8 and the OH group at C-7 (δ _H 10.79, brs), enabled the assignment of the full substitution pattern of ring A. Regarding ring B, the location of the methoxy group at C-4′ (δ _H 3.78, s) was inferred from the key HMBC correlations from the methoxy group to C-4′ and from the NOE effect between the methoxy group and H-3′,5′ ([Fig. 2 a]). Another NOE effect was detected between H-2 and H-2′,6′. Thus, compound 1 was identified as 5,7-dihydroxy-6,4′-dimethoxyisoflavone (irisolidone). The structure was corroborated by comparing its NMR data with the data reported for irisolidone [25]. It is a rare isoflavone and was isolated for the first time from the present species.

The LC-MS spectrum of compound 2 showed the quasi-molecular ion peak [M + H]⁺ at m/z 361. In conjunction with the ¹³C NMR data, the molecular formula of compound 2 was determined as C₁₈H₁₆O₈. The ¹H NMR spectrum of 2 (Table 2S, Supporting Information) showed the characteristic H-2 signal of an isoflavone at δ _H 8.36 (s), the signals of three methoxy groups (δ _H 3.79, s; 3.75, s; 3.70, s) and three OH groups (δ _H 13.03, s; 10.22, brs; 9.28, brs), the aromatic proton at δ _H 6.49 (s) assigned to H-8, and two isolated aromatic protons (δ _H 6.72, s and δ _H 6.66, s) of ring B. The substitution pattern of ring A was similar to that of compound 1 and was assigned by the interpretation of NOESY, HSQC, and HMBC spectra ([Fig. 2 b]; Table 2S, Supporting Information). The location of the aromatic protons of ring B was inferred from the key HMBC correlations from the H-2′ and H-6′ to C-3, and from the NOE effect between H-2′/H-6′ and H-2. The methoxy groups at δ _H 3.70 (s) and 3.79 (s) could be positioned at C-4′ and C-5′, respectively, by the fact that the NOE effect was detected between H-6′ and a methoxy group at C-5′. HMBC correlation was observed from the methoxy group to C-4′. Thus, compound 2 was identified as 5,7,3′-trihydroxy-6,4′,5′-trimethoxyisoflavone, or irigenin. The spectroscopic characteristics of compound 2 were identical to those of the literature data [26]. Irigenin was previously isolated from the dried rhizome of I. dichitoma, I. tectorum, and I. germanica.

During recrystallization from methanol, elongated-spherical-shaped crystals with pointed edges fell out (3, irisolone). The molecular formula of compound 3 was determined as C₁₇H₁₂O₆ from the quasi-molecular ion peak [M + H]⁺ at m/z 312 in the LC-MS spectrum, in conjunction with the ¹³C NMR data. In the UV spectrum, 3 showed the λ max absorptions at 265 and 323 nm (sh), suggesting an isoflavone skeleton. When adding a solution of sodium hydroxide, there was a bathochromic shift of the II band at 37 nm, indicating the presence of the -OH group at the C-4 position. The ¹H NMR spectrum of 3 exhibited the characteristic signal at δ 8.16 (s, 1Н, Н-2) that confirmed the isoflavone nature of the substance. The aromatic proton, which appeared at δ 6.93 as a singlet, was assigned to H-8. An additional three singlets were observed, and δ _H 9.52 (s) was assigned to the B ring hydroxy group (4′ОН). The singlet at δ _H 6.15 ppm was due to the methylenedioxy group in the C-6, C-7 positions, and the singlet at δ _H 3.88 ppm corresponded to the -OCH₃ group in the С-5 position. A group of aromatic proton signals was also detected at δ _H 6.93 (s, 1H, H-8), δ _H 7.32 (d, J = 8.5 Hz, 2H, H-2′, 6′,), and δ _H 6.79 (d, J = 8.5 Hz, 2H, H-3′, 5′). The ¹³C-NMR spectrum of 3 showed the presence of 17 carbon atoms in the molecule. Based on spectroscopic analysis, 3 was identified as irisolone or nigricin [27].

The LC-MS spectrum of compound 4 showed the quasi-molecular ion peak [M + H]⁺ at m/z 298. In conjunction with the ¹³C NMR data, the molecular formula of compound 4 was determined as C₁₆H₁₉O₆. The ¹H-NMR and ¹³C-NMR data of compound 4 showed characteristic signals for isoflavone. In the ¹H-NMR spectrum, it displayed a sharp singlet at δ _H 8.43 (s, 1H) assigned to H-2 of the isoflavone skeleton. A downfield single proton at δ _H 12.93 (s, 1H) indicated a 5-OH signal. A couple of ortho-coupled proton (AAʼBB' system) signals at δ _H 7.40 (d, J = 8.5 Hz, 2H) and 6.81 (d, J = 8.5 Hz, 2H) corresponded to H-2′,6′ and H-3′,5′ in the B-ring. The single sharp peak at δ _H 6.18 (s) was characteristic for a methylenedioxy group, which can be placed either at the 6,7 or 7,8 position. The singlet at δ _H 6.88 suggested a proton at C-8 because the NMR signal of the C-6 proton in isoflavones appeared slightly up-field compared with the C-8 proton. Therefore, the methylenedioxy group was established at the 6 and 7 positions. The ¹³C-NMR spectrum of 4 showed resonances for all carbon atoms. A signal at δ _C 89.9 suggested that there is no substituent at C-8, and a signal at δ _C 181.3 (C-4) indicated that C-5 was substituted by a hydroxy group. Thus, 4 was deduced as 5,4′-dihydroxy-6,7-methylenedioxyisoflavone or irilone [25].

The molecular formula of 5 was determined as C₂₃H₂₄O₁₁ from the quasi-molecular ion peak [M + H]⁺ at m/z 477 in the LC-MS spectrum along with the ¹³C NMR data. The proton resonance for isoflavone C-2 was located at δ _H 8.48 (s, 1Н) ppm, which also confirmed the isoflavone nature of the ring. Acid hydrolysis of 5 with 10% H₂SO₄ gave aglycone 1 (irisolidone) and D-glucose, which were identified by co-PC and co-TLC. In the ¹H NMR spectrum of 5 (Table 3S, Supporting Information), the characteristic H-2 signal of an isoflavone was found at δ _H 8.48 (s), and the 4-methoxyphenyl nature of the B-ring was deduced from the signals at δ _H 7.52 (d, J = 8.4 Hz, 2H, H-2′,6′) and 7.01 (d, J = 8.4 Hz, 2H, H-3′,5′). The J value (7.7 Hz) of the anomeric proton at δ _H 5.09 (d) (CD₃OD) indicated that the sugar was in the β-configuration. In the NOESY spectrum of 5, the position of the sugar was deduced to be at C-7 from the correlation between the anomeric proton of D-glc (δ _H 5.09) and a characteristic single proton H-8 (δ _H 6.90, s, 1H). The methoxy group at δ _H 3.77 was positioned at C-6 because the OH group at C-5 (δ _H 12.90) was correlated with the methoxy protons ([Fig. 2 c]). The spectrum also showed signals for two methoxy groups at δ _H 3.79 (s, 3Н, 4′-ОСН₃) and δ _H 3.77 (s, 3Н, 6-ОСН₃) ppm and a singlet for the hydroxy group at δ _H 12.90 (s, 1Н, 5-ОН) ppm. Based on these data, we concluded that 5 is irisolidone-7-O-β-D-glucopyranoside or kakkalidone, which was isolated from I. aphylla rhizomes for the first time. Kakkalidone and irisolidone were previously isolated only from the Pueraria lobata flower and I. germanica rhizomes [28].

The compounds were identified as ononin (7), genistein (8), daidzein (9), formononetin (10), nigricin 4′-О-β-D-glucopyranoside (germanaism B) (11), and xanthone mangiferin (12) on the basis of their physical and spectral data (Fig. 24S-34S, Supporting Information). Isoflavones 6, 16 – 18 and flavones 13 – 15 were identified as tectorigenin (6), tectoridin (16), iristectorigenin B (17) [29], 5,6-dihydroxy-7,8,3′,5′-tetramethoxyisoflavone (18) [30], apigenin (13), kaempferol (14), and quercetin (15) by comparing their spectral data with those available in the literature. Spectroscopic data of all compounds are presented in the Supporting Information. All phenolic compounds were isolated from I. aphylla for the first time.

In our previous investigation, we evaluated various bioactivities of Iris rhizomes extracts including anti-viral (coronavirus 229E), anti-inflammatory, antioxidant, anti-allergic, NRF2 expression, or lipid accumulation activities [21]. The potent antioxidant and immune-modulating effects of I. aphylla rhizomes extract motivated us to further explore the bioactivities of the major isolated phenolic components ([Fig. 1]). We believe that all Iris compounds deserve more attention, particularly the flavonoids that are also abundant in edible fruits and vegetables (13 – 15). All compounds were evaluated in vitro for their protective effects against enterovirus D68, influenza virus H1N1, and coronavirus 229E. The anti-inflammatory effect of the dedicated compounds was also tested in human neutrophils.

The anti-viral activity of I. aphylla extracts and isolated compounds was assessed by virus cytopathic assays against influenza H1N1 and enterovirus D68, as well as neuraminidase inhibitory activity assay ([Table 1]). Influenza H1N1 (A/WSN/33) virus-infected Madin–Darby canine kidney epithelial (MDCK) cells were used to evaluate the protective effects of Iris samples against influenza H1N1 infection. The initial screening revealed that none of the Iris samples showed a protective effect against the influenza virus at the concentration of 50 µg/mL. However, at a higher concentration (500 µg/mL), the water extract of I. aphylla rhizomes protected 80% of cells from the lethal effect of influenza H1N1 infection with 50% inhibitory effects at 125 µg/mL ([Table 1], Fig. 35S, Supporting Information). According to the preliminary analysis, I. aphylla rhizomes water extract contains a complex mixture of polyphenolics. We further evaluated the isolated compounds to provide insight into the bioactivity of the Iris rhizomes and their components. The isolated compounds were tested at 50 µM, and the results revealed that apigenin (13) inhibited cell death caused by H1N1 infection by almost 100%, kaempferol (14) by 92%, and quercetin (15) by 47.5% (Fig. 35S, Supporting Information). The inhibitory ratio of 13 and 14 was comparable to zanamivir (Relenza), which was used as a positive control resulting in 82.5% inhibition at 10 µM [31]. The results indicated that among all the tested flavonoids (1 – 3, 5, 8 – 11, 13 – 17), the presence of hydroxy groups in A- and B-rings (13 – 15) is essential for the anti-H1N1 activity, which is in accordance with the previous report [32]. However, we observed that the activity slightly decreased with the presence of higher numbers of the OH group in the B- and C-rings, with apigenin showing the highest activity followed by kaempferol (3-hydroxyapigenin; 14) and quercetin (3,3′-dihydroxyapigenin; 15). This bodes well, along with previous reports on the anti-influenza activities of apigenin [33] having relatively good bioavailability and ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties [34]. Mangiferin (glucoside of norathyriol) (12) showed poor bioavailability in previous studies [35]. The oral bioavailability of flavonoids is limited because of their low liposolubility and sometimes large molecular weights, which restrict their ability to pass across the lipid-rich biological membranes of the intestine. For glycosides, their hydrolysis would occur relatively fast after oral administration. The aglycones can be conjugated to glucuronides by several enzymes in the intestine or liver. Thus, it is important to study the bioavailability properties of glycosides or aglycones of active flavonoids such as irisolidone-7-glucoside (kikkalidone) (5) to be developed as potential drug leads.

Neuraminidase plays an important role in the release of the replicated virus particles from host cells [36]. The active anti-H1N1 compounds including kaempferol, quercetin, and apigenin, along with daidzein, formononetin, and irisolidone-7-glucoside (kikkalidone), were tested for their anti-neuraminidase effects (Fig. 36S, Supporting Information). Apigenin (13), kaempferol (14), and quercetin (15) showed a dose-dependent inhibitory effect at 25 – 200 µM with the peak inhibition of 29.7 – 38.1%. Moreover, apigenin (13) [34], kaempferol (14) [37], and quercetin (15) [37], [38] bound well to neuraminidase enzyme in silico as demonstrated by various docking models. This bodes well, along with our experimental data, and indicates the potent anti-virus activity of flavonoids (13 – 15) we commonly consume in fruits and vegetables.

Furthermore, the enterovirus D68-infected rhabdomyosarcoma (RD) cell model was used to assess the protective effect of Iris samples against enterovirus D68. The hydroethanolic extract of I. aphylla rhizomes protected 40% of cells from the lethal effect of enterovirus D68 at 500 µg/mL ([Table 1], Fig. 37S, Supporting Information); however, it showed approx. 50% toxicity to the RD cells. The isolated pure compounds were further evaluated, and the results revealed that irisolidone (1), a rare Iris isoflavone, exerted the most potent protective effect against the enterovirus D68 infection with almost complete inhibition at a concentration range from 12.5 µM to 50 µM ([Fig. 3]) with no toxic effects to the RD cells observed. Irisolidone-7-glucoside (kikkalidone) (5) (93%) and kaempferol (14) (83%) also showed inhibitory activity at 50 µM. Synthetic compound C-22 served as a positive control and showed 95% inhibition at 10 µM [39]. Irisolidone (1) and kikkalidone (5) contain the same aglycone, which may serve as an important pharmacophore for the enterovirus D68 activity. Interestingly, the activity of these compounds was observed specifically against the enterovirus infection but not against the influenza infection. In contrast, kaempferol (14) was active against both viruses. A previous study found apigenin, quercetin, and kaempferol active against the other enterovirus strain, EV71, in 293S cells [40]. These results revealed novel anti-viral bioactivity of I. aphylla and its characteristic components such as 1 and 5 that serve as marker compounds for the Iris species.

In another test, isolated compounds were evaluated for the protective effect against human coronavirus 229E (HCoV-229E) infection in vitro. Human coronavirus 229E (HCoV-229E) is a strain of coronavirus-family viruses that causes upper respiratory syndrome [41]. The results revealed no protective effect at 10 µM ([Table 1], Fig. 38S, Supporting Information) with low toxicity to Huh7 cells. Thus, a higher concentration of Iris flavonoids may be required.

Furthermore, we studied the anti-inflammatory activity of isolated Iris phenolic compounds. Respiratory burst and degranulation of neutrophils are important processes in the maintenance of human health but need to be well-regulated to prevent the development of chronic and auto-immune diseases, including post-infection complications. We evaluated the effect of Iris samples on superoxide anion generation and elastase release triggered by fMLF in CB-primed human neutrophils. The previous results revealed that the water extracts of I. aphylla rhizomes showed anti-inflammatory potential and inhibited superoxide anion generation at 10 µg/mL by 45.7% [21]. Among the tested compounds, apigenin (reaching 100% inhibition at 10 µM in both assays) and quercetin (61.9% at 10 µM in elastase release assay) revealed a potent inhibitory effect on superoxide anion generation or elastase release ([Table 2]) in a dose-dependent manner ([Fig. 4]). Interestingly, quercetin (10 µM) significantly interacted with ferricytochrome c (Fe³⁺), which indicated its antioxidant and possible heme-reducing effects. Mangiferin solely suppressed superoxide generation (65.9% at 10 µM), possibly because of its well-known antioxidant effects. Genistein (8), a known inhibitor of protein tyrosine kinase, inhibited superoxide (81.7% at 10 µM), while its effect on elastase release was weaker (29.4% at 10 µM). Both isoflavone genistein and flavone apigenin possess a hydroxyl substituent in the С-4′, C-5, and C-7 positions, which might contribute to the potent anti-neutrophilic effect. Interestingly, isoflavones lacking one of these hydroxyls (1 – 3, 5 – 7, 9 – 11, 16 – 18) were inactive. Meanwhile, isoflavone iristectorigenin B (17), which contains the С-4′, C-5, and C-7 hydroxyls with additional methoxy groups at C-3′ and C-6, was also inactive. Among the tested flavones with С-4′, C-5, and C-7 hydroxyl, similarly to the anti-influenza H1N1 results, the best anti-neutrophilic activity exerted apigenin (13), which lacks C-3 hydroxyl in comparison to quercetin (3,3′-dihydroxyapigenin; 15) and kaempferol (3-hydroxyapigenin; 14).

To further correlate the observed anti-viral effects of Iris flavonoids with clinical and experimental anti-viral drugs, in silico modeling with anti-influenza and anti-enterovirus drugs was performed using a chemical global positioning system relevant to natural products (ChemGPS-NP). ChemGPS is a tool to predict moleculesʼ bioactivity in the chemical space based on their physico-chemical properties. It is designed for natural products research and development [23]. It was utilized in the screening of specific bioactive natural compounds [42], as well as in comparing the properties of the tested compounds with clinical drugs [24].

The isolated flavonoids were positioned into the chemical space via the SMILES input into the web-based ChemGPS-NP system ([Fig. 5 a]). An in-house database of anti-influenza [43] and anti-enterovirus drugs [44] in different stages of clinical development (Supporting Excel Table) were plotted. According to the ChemGPS-NP analysis, the isolated compounds formed a cluster partly overlapping with the anti-influenza reference drugs (yellow dots) and neuraminidase inhibitors (orange dots) ([Fig. 5 b]). The flavonoids with higher aromaticity (toward the positive blue PC2, axis y) and hydrophilicity (toward the negative green PC3, axis z) revealed a protective effect against influenza H1N1 infection, while the other flavonoids were less active. There was no obvious correlation between the active flavonoids, apigenin (13), kaempferol (14), and quercetin (15) (red dots), and the neuraminidase inhibitors group (orange dots), indicating that the effect on neuraminidase contributed only in part to the anti-virus properties of the active Iris anti-influenza flavonoids (red dots; 13, 14, 15), which was also observed in vitro ([Table 1]).

The isolated Iris phenolics including the active anti-enterovirus irisolidone (1), irisolidone-7-glucoside (kikkalidone) (5), and kaempferol (14) (pink dots) and the inactive (blue dots) compounds were plotted together with the anti-enterovirus reference drugs (green dots) ([Fig. 5 c]). The overlapping cluster was observed with similarities in the aromaticity (blue axis, PC2, axis y) and lipophilicity levels (green PC3, axis z), while the size of the active molecules differed because of the presence of glucose in kikkalidone (5). Overall, the results demonstrated similarities in the physicochemical properties of the active Iris phenolics with the known anti-influenza and anti-enterovirus agents ([Fig. 5 d]).

In conclusion, the hydroethanolic extract of I. aphylla was subjected to chromatographic separation, which led to the isolation of compounds 1 – 18, not previously reported from I. aphylla rhizomes. Iris phenolics exerted anti-influenza, anti-enterovirus, and anti-inflammatory in vitro activities, and their physicochemical properties were correlated with the anti-viral drugs in a different stage of clinical development using a ChemGPS-NP global space map. The Iris phenolics showed interesting anti-viral activity suggesting their potential application in the management of influenza and enterovirus infections.

Material and Methods

Plant Material

I. aphylla rhizomes were collected from the Kharkiv Botanical Gardenʼs territory at the Kharkiv National University in September 2018. Plants were identified and authenticated by Dr. Buidin (Department of the Ornamental Plants, M. M. Gryshko National Botanical Garden of the National Academy of Sciences of Ukraine, Kyiv, Ukraine). A voucher specimen, number CWU0056534, verified by Dr. Gamulya was deposited at the Herbarium of V. M. Karazin Kharkiv National University (CWN), Kharkiv, Ukraine.

Extraction and isolation of compounds

I. aphylla air-dried rhizomes (1.0 kg) were crushed and macerated in 9.65 L of 70% (v/v) aqueous ethanol (EtOH) in a percolator for 24 h. Further details, general experimental procedures, and spectroscopic data of the isolated compounds from the I. aphylla hydroethanolic extract are presented in the “Supporting Information”. The purity of compounds was 95 – 98.0% based on HPLC, NMR, TLC, and melting point analyses.

In parallel, for activity-screening purposes, air-dried I. aphylla rhizomes powder was extracted with distilled water in a water bath at 100 °C (100 g, 1 L, 60 min × 3) and then evaporated to dryness.

In vitro assessment of anti-viral activity

Biology: cells, viruses, and chemicals

The information regarding the clinical enterovirus strain and influenza viruses is similar to a previous report [45]. MDCK cells were used for influenza A virus infection and RD cells were used to infect enterovirus D68. Both MDCK and RD cell lines and the influenza A (H1N1) virus were obtained from the American Type Culture Collection (ATCC). The clinical EV-D68 2795 used in this manuscript was from the Clinical Virology Laboratory of Chang Gung Memorial Hospital, Taiwan. For details, please see “Supporting Information”.

Half-maximal effectiveness assay (EC₅₀ assay)

The EC₅₀ value was calculated using the Reed–Muench method as the concentration of the compound that could inhibit the virus cytopathic effect by 50% [31]. For details, please see “Supporting Information””.

Cytotoxicity test (CC₅₀ assay)

The median cytotoxic concentration (CC₅₀) was calculated by the Reed–Muench method based on the concentration of the compound responsible for 50% of cell death utilizing MTT assay. For details, please see “Supporting Information””.

Neuraminidase assay

A baculovirus displayed neuraminidase NA9 on the surface (NA9-Bac) as a pseudotyped influenza virus was used. For details, please see “Supporting Information”.

Coronavirus 229E assay

The protective effect of samples on the Huh7 cells infected by human coronavirus 229E (HCoV-229) was determined as previously described [46]. For details, please see “Supporting Information”.

In vitro assessment of the anti-inflammatory activity

Preparation of human neutrophils

Neutrophils obtained from healthy human donors (approved by institutional review board at Chang Gung Memorial Hospital) were isolated using a standard method as previously described [47].

Measurement of superoxide generation

SOD inhibition was measured by the reduction in ferricytochrome c as described previously [48], [49]. For details, please see “Supporting Information”.

Measurement of elastase release

Elastase release was measured by degranulation of azurophilic granules [50]. For details, please see “Supporting Information”. Genistein served as positive control for both assays.

ChemGPS-NP analysis

ChemGPS-NP is a tool based on principal component analysis (PCA) that describes a comprehensive and biologically relevant chemical space [23]. The scores of these principal components (PC1, PC2, and PC3) were obtained by entering the SMILES (ChemBioDraw 17.0, or PubChem) of all isolated and reference compounds into the ChemGPS-NP system (http://chemgps.bmc.uu.se) [24], [42]. The in-house database of anti-influenza and anti-enterovirus drugs was created (Supporting Information). The drugs were classified into several groups including anti-influenza H1N1 drugs at different stages of clinical development (yellow dots, sources include review article [43] and ChemBL database ‘https://www.ebi.ac.uk/chembl/g/#search_results/all/query=influenza’), neuraminidase inhibitors (orange dots), or anti-enterovirus drugs in different stages of clinical development (green dots [44]). All drugs in the respective groups are listed in the Supporting Information file.

Statistical analysis

Results are expressed as value of mean ± SD of two independent measurements (influenza H1N1, enterovirus D68, and neuraminidase anti-viral assays) or three independent measurements (anti-inflammatory assay). Coronavirus assay screening (single measurement). Statistical analysis was performed using Studentʼs t-test (Sigma Plot, Systat software, Systat Software Inc.). Values with *P < 0.05, **P < 0.01, ***P < 0.001 were considered statistically significant.

Contributorsʼ Statement

The manuscript was written through contributions from all authors. All authors have given approval to the final version of the manuscript. Design of the study: O. Mykhailenko, M. Korinek, M. El-Shazly. Phytochemical study: O. Mykhailenko, V. Kovalyov, V. Georgiyants. Analysis and interpretation of the data: P. Shynkarenko, A. Nikishin, L. Ivanauskas. Statistical analysis: C.-F. Hsieh, T.-L. Hwang. Anti-inflammatory activity: T.-L. Hwang. Anti-viral activity assessment: C.-F. Hsieh, J.-T. Horng. Funding acquisition: B.-H. Chen, J.-T. Horng, T.-L. Hwang. Drafting the manuscript: O. Mykhailenko, M. Korinek. Critical revision of the manuscript: M. El-Shazly, V. Georgiyants, B.-H. Chen. ^◊ Olha Mykhailenko and Chung-Fan Hsieh contributed equally.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

We would like to express gratitude to distinguished Professor Fang-Rong Chang at the Graduate Institute of Natural Products, Kaohsiung Medical University for advice and technical support. The authors would like to thank the Center for Research Resources and Development, as well as the Drug Development and Value Creation Research Center at Kaohsiung Medical University, for providing instrumentation support. Dr. Mykhailenko is grateful for the CARA fellowship and the opportunity to continue scientific research at UCL School of Pharmacy, UK. The funders had no role in the study design, data collection, analyses, decision to publish, or preparation of the manuscript.

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Correspondence

Publication History

Received: 14 July 2022

Accepted after revision: 23 March 2023

Accepted Manuscript online:
28 March 2023

Article published online:
05 June 2023

© 2023. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

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  CrossrefPubMedSearch in Google Scholar
• 21 Mykhailenko O, Korinek M, Ivanauskas L, Bezruk I, Myhal A, Petrikaitė V, El-Shazly M, Yen CH, Chen BH, Georgiyants V, Hwang TL. Qualitative and quantitative analysis of Ukrainian Iris species: A fresh look on their antioxidant content and biological activities. Molecules 2020; 25: 4588
  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 25 Ibrahim SR, Mohamed GA, Al-Musayeib NM. New constituents from the rhizomes of Egyptian Iris germanica L. Molecules 2012; 17: 2587-2598
  CrossrefPubMedSearch in Google Scholar
• 26 Buch SM, Mir FA, Rehman S, Qurishi MA, Banday JA. Irigenin – an isoflavone: A brief study on structural and optical properties. Eur Phys J Appl Phys 2013; 62: 31201-31205
  CrossrefPubMedSearch in Google Scholar
• 27 Ayatollahi SAM, Moein MR, Kobarfard F, Nasim S, Choudhary MI. 1-D and 2D-NMR assignments of nigricin from Iris imbricate . Iran J Pharm Sci 2005; 4: 250-254
  PubMedSearch in Google Scholar
• 28 Zhou J, Xie G, Yan X. Encyclopedia of Traditional Chinese Medicines. Vol. 3. Berlin, Heidelberg, New York: Springer; 2011
  Search in Google Scholar
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  PubMedSearch in Google Scholar
• 30 Kovalev VN, Zatylʼnikova OA, Kovalev SV. A new isoflavone from Iris pseudacorus . Chem Nat Comp 2013; 49: 34-35
  CrossrefPubMedSearch in Google Scholar
• 31 Hsieh CF, Lo C, Liu CH, Lin S, Yen HR, Lin TY, Horng JT. Mechanism by which ma-xing-shi-gan-tang inhibits the entry of influenza virus. J Ethnopharmacol 2012; 143: 57-67
  CrossrefPubMedSearch in Google Scholar
• 32 Jeong HJ, Ryu YB, Park SJ, Kim JH, Kwon HJ, Kim JH, Park KH, Rho MC, Lee WS. Neuraminidase inhibitory activities of flavonols isolated from Rhodiola rosea roots and their in vitro anti-influenza viral activities. Bioorg Med Chem 2009; 17: 6816-6823
  CrossrefPubMedSearch in Google Scholar
• 33 Liu AL, Liu B, Qin HL, Lee SM, Wang YT, Du GH. Anti-influenza virus activities of flavonoids from the medicinal plant Elsholtzia rugulosa . Planta Med 2008; 74: 847-851
  Thieme ConnectPubMedSearch in Google Scholar
• 34 Alhazmi MI. Molecular docking of selected phytocompounds with H1N1 Proteins. Bioinformation 2015; 11: 196-202
  CrossrefPubMedSearch in Google Scholar
• 35 Mehta P, Shah R, Lohidasan S, Mahadik KR. Pharmacokinetic profile of phytoconstituent(s) isolated from medicinal plants-A comprehensive review. J Tradit Complement Med 2015; 5: 207-227
  CrossrefPubMedSearch in Google Scholar
• 36 Hsieh CF, Chen YL, Lin CF, Ho JY, Huang CH, Chiu CH, Horng JT. An extract from Taxodium distichum targets hemagglutinin- and neuraminidase-related activities of influenza virus in vitro . Sci Rep 2016; 6: 1-13
  CrossrefPubMedSearch in Google Scholar
• 37 Sadati S, Gheibi N, Ranjbar S, Hashemzadeh M. Docking study of flavonoid derivatives as potent inhibitors of influenza H1N1 virus neuraminidase. Biomed Rep 2018; 10: 33-38
  PubMedSearch in Google Scholar
• 38 Liu Z, Zhao J, Li W, Wang X, Xu J, Xie J, Tao K, Shen L, Zhang R. Molecular docking of potential inhibitors for influenza H7N9. Comput Math Methods Med 2015; 2015: 480764
  PubMedSearch in Google Scholar
• 39 Sethy B, Hsieh CF, Yeh C, Horng JT, Hsieh PW. Design, synthesis & structure-activity relationships of a new class of antihuman enterovirus D68 & A71 agents. Future Med Chem 2018; 10: 1333-1347
  CrossrefPubMedSearch in Google Scholar
• 40 Dai W, Bi J, Li F, Wang S, Huang X, Meng X, Sun B, Wang D, Kong W, Jiang C, Su W. Antiviral efficacy of flavonoids against enterovirus 71 infection in vitro and in newborn mice. Viruses 2019; 11: 625
  CrossrefPubMedSearch in Google Scholar
• 41 Zumla A, Chan JF, Azhar EI, Hui DS, Yuen KY. Coronaviruses – drug discovery and therapeutic options. Nat Rev Drug Discov 2016; 15: 327-347
  CrossrefPubMedSearch in Google Scholar
• 42 Rosen J, Lovgren A, Kogej T, Muresan S, Gottfries J, Backlund A. ChemGPS-NP(Web): chemical space navigation online. J Comput Aided Mol Des 2009; 23: 253-259
  CrossrefPubMedSearch in Google Scholar
• 43 Amarelle L, Lecuona E, Sznajder JI. Anti-influenza treatment: Drugs currently used and under development. Arch Bronconeumol 2017; 53: 19-26
  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 46 Hsieh CF, Jheng JR, Lin GH, Chen YL, Ho JY, Liu CJ, Hsu KY, Chen YS, Chan YF, Yu HM, Hsieh PW, Chern JH, Horng JT. Rosmarinic acid exhibits broad anti-enterovirus A71 activity by inhibiting the interaction between the five-fold axis of capsid VP1 and cognate sulfated receptors. Emerg Microbes Infect 2020; 9: 1194-1205
  CrossrefPubMedSearch in Google Scholar
• 47 Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl 1968; 97: 77-89
  PubMedSearch in Google Scholar
• 48 Korinek M, Hsieh PS, Chen YL, Hsieh PW, Chang SH, Wu YH, Hwang TL. Randialic acid B and tomentosolic acid block formyl peptide receptor 1 in human neutrophils and attenuate psoriasis-like inflammation in vivo . Biochem Pharmacol 2021; 190: 114596
  CrossrefPubMedSearch in Google Scholar
• 49 Yang SC, Chung PJ, Ho CM, Kuo CY, Hung MF, Huang YT, Chang WY, Chang YW, Chan KH, Hwang TL. Propofol inhibits superoxide production, elastase release, and chemotaxis in formyl peptide-activated human neutrophils by blocking formyl peptide receptor 1. J Immunol 2013; 190: 6511-6519
  CrossrefPubMedSearch in Google Scholar
• 50 Hwang TL, Su YC, Chang HL, Leu YL, Chung PJ, Kuo LM, Chang YJ. Suppression of superoxide anion and elastase release by C18 unsaturated fatty acids in human neutrophils. J Lipid Res 2009; 50: 1395-1408
  CrossrefPubMedSearch in Google Scholar

• Supplementary Material