A review on phytochemical and therapeutic potential of Iris germanica

Abstract

Objectives

Iris germanica L. is a medicinal plant, which has a long history of uses, mainly in medieval Persia and many places worldwide for the management of a wide variety of diseases. In this study, we aimed to review ethnopharmacological applications in addition to phytochemical and pharmacological properties of I. germanica.

Key findings

Ethnomedical uses of I. germanica have been reported from many countries such as China, Pakistan, India, Iran and Turkey. The medicinal part of I. germanica is the rhizome and the roots. Based on phytochemical investigations, different bioactive compounds, including flavonoids, triterpenes, sterols, phenolics, ceramides and benzoquinones, have been identified in its medicinal parts. Current pharmacological studies represent that the plant possesses several biological and therapeutic effects, including neuroprotective, hypoglycaemic, hypolipidaemic, antimicrobial, antioxidant, antiproliferative, anti-inflammatory, antiplasmodial, antifungal, immunomodulatory, cytotoxic and antimutagenic effects.

Summary

Although the majority of preclinical studies reported various pharmacological activities of this plant, however, sufficient clinical trials are not currently available. Therefore, to draw a definitive conclusion about the efficacy and therapeutic activities of I. germanica and its bioactive compounds, further clinical and experimental studies are required. Moreover, it is necessary to focus on the pharmacokinetic and safety studies on the extracts of I. germanica.

Graphical Abstract

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Introduction

Bioactive molecules in plant species are considered rich sources to provide new therapeutic approaches for different diseases. Approximately, 80% of the world population is thought to rely on traditional plant-based medicines principally in developing countries.^{[1, 2]}Iris contains a large genus with 300 species of flowering perennial plants, which belongs to the Iridaceae family^{[3, 4]} and is considered to be the largest and most complicated genus in this family.^{[3, 5]} Different species of this genus are widely distributed throughout the world.^[3] The origin of Iris species has been reported in the temperate regions of the Northern Hemisphere mostly in Eurasia, North Africa and North America. These species can also be found in wetland, desert, semi-desert or dry and rocky environments.^{[3, 6, 7]} Some species of Iris are found in China,^[3] Turkey,^[1] Pakistan,^[1] India,^[8] Egypt,^[9] Morocco^[10] and Iran.^[11] They have elegant flowers with violet-like fragrance.^[6] The showy and fragrant blossoms of irises have a variety of colours, which have been used as ornamental plants since ancient times.^[5]Iris is commonly known as ‘rainbow’. This word has a Greek root, which refers to a wide spectrum of flower’s colours that exist among its species in nature.^{[1, 12]} Over the past years, different species of this plant have increased popularity in the perfume and cosmetic industries due to their sweet aroma.^{[1, 4, 13]} Pharmacological studies have provided scientific evidence for the presence of secondary metabolites on the species of Iris genus.^[4]

Flowering plants in the Iridaceae family have excellent medicinal importance in folk medicine. They have been used in the treatments of cold, flu, malaria, toothache, bruise, cancer, inflammation bacterial and viral infections.^{[1, 14]} A variety of chemical compounds such as flavonoids and their glycosides, triterpenoids, benzoquinones and stilbene glycosides have been recognised in Iris species, which have antineoplastic, antioxidant, anti-inflammation, antiulcer, antiplasmodial and antituberculosis activities.^{[1, 3, 4, 14]} Among Iris species, Iris germanica L. is well-known and horticulturally the most tall-bearded one, which has been thoroughly investigated.^{[6, 14]}

Different parts of I. germanica are used for several purposes. The rhizomes exhibit potent pesticidal and anticancer activities, they have blood-purifying ingredients and are used to treat venereal diseases. The leaves are a rich source of vitamins, especially vitamin C (ascorbic acid). The underground parts and rhizomes of this plant have been identified as its medicinal parts. Moreover, a number of secondary metabolites have been introduced for I. germanica.^{[6, 13–15]} It has been shown that the aqueous extract of I. germanica has several biological properties, including a reduction in smooth muscle activity, central antiserotonin activity, induction of transient hypotension and stimulation of breathing.^{[6, 14, 16]} Numerous beneficial effects such as diuretic, emmenagogue, anti-inflammatory, antispasmodic and intensive cathartic have been attributed to its root decoction.^{[1, 6, 9, 12, 13]} Phytochemical investigations have revealed the presence of several constituents including flavonoids, triterpenes, sterols, phenolics, ceramides and benzoquinones in I. germanica.^{[6, 9, 10, 14]}

Recent studies demonstrated the antimicrobial,^[14] antioxidant,^[16] antiproliferative,^[7] anti-inflammatory,^[14] antiplasmodial,^[2] immunomodulatory,^[8] cytotoxic^[15] and antimutagenic^[7] activities of I. germanica. Furthermore, the therapeutic effects of this plant such as neuroprotective,^{[17, 18]} hypoglycaemic^[19] and hypolipidaemic^[20] activities have been studied. Reviewing the available literature shows that there is no comprehensive review on phytochemical, pharmacological and therapeutic aspects of this plant. In this review, we intend to provide an overview of botanical, phytochemical and pharmacological activities of the rhizomes of I. germanica as well as its traditional uses in different ethnomedical systems. This information may be beneficial by providing a background for future research, with a view to developing new treatments approaches for a wide range of ailments.

Materials and Methods

In this study, we searched the available data in electronic databases such as PubMed, ScienceDirect, Scopus, Google Scholar and other relevant ones to find articles describing ethnomedicinal uses and pharmacological and phytochemical aspects of I. germanica. Data were collected from June 1990 up to June 2020. The search was performed with the keywords ‘Iris germanica’ and ‘Iris’. We did not consider any restrictions on the language of the articles and evaluated all published articles. Moreover, we surveyed the most famous ancient traditional Persian medicine (TPM) textbooks such as Al-Qānūn fī al-Tibb )The Canon of Medicine) by Avicenna (11th century) and Zakhireye Kharazmshahi by Jorjani (12th century) and the most comprehensive TPM pharmacopeia, namely, Makhzan al-Adviah by Aghili Shirazi (18th century) to find traditional uses and medicinal properties of Iris. Finally, we collected and classified the items related to the subject. All members of the research team participated in the discussion and analysis of data. Data on isolated compounds from I. germanica and their drug-like properties and also the pharmacological activities of the plant were extracted in individual tables.

Plant description

According to ‘The Plant List’, I. germanica is the only accepted name for this plant with 18 synonyms.^[21]I. germanica is used as an ornamental plant and has extensive geographical distribution worldwide except the coldest regions.^{[1, 13, 18, 20]} Although the origin of this plant has not been well defined, it seems that I. germanica is native to southern Europe and Eurasia. It is mainly cultivated in China,^[15] Pakistan,^[13] Egypt,^[14] Turkey,^[16] Morocco^[10] and Iran.^[11]I. germanica is a flowering perennial plant with very thick underground stems.^[11] This plant grows to the height of 30–100 cm with long-pedicled and perfumed flowers consisting of four to five blossoms, the outer parts of the flower are white or slightly blue, which seem to be darker with a yellow beard (Figure 1). Its fruit is a large capsule about 5 cm long with brown seeds, which are lined up in a number of sections like rolls of coins. The leaves of the plant are broad, sword-like and usually curved and are gray-green in colour with 2.5–4.5 cm width. The medicinal part of I. germanica is the rhizome and the roots; it has a thick and short rhizome.^{[11, 22]}

Traditional applications

Reviewing traditional medicine manuscripts shows that medicinal uses of Iris species (Iridaceae) have a long history in many places in the world.^[3] For the first time, they were used in traditional and folk medicine; later, they were commonly cultivated in gardens or parks as ornamental plants.^[12] According to literature, different parts of Iris species have been used in different ethnomedical systems, especially their underground parts. In traditional European folk medicine, they have been used for their diuretic, expectorant, cathartic and emetic properties.^[23] Moreover, topical and oral applications of the rhizomes of I. germanica have been prescribed for the treatment of sores and freckles, respiratory diseases, liver and spleen ailments and as a pain relief for teething-associated pain in children.^{[1, 7, 20, 24, 25]} The diuretic, antispasmodic, stimulants and laxative activities of its rhizomes are considered as folk remedies in Pakistan. Also, it was used traditionally for dropsies and gall bladder diseases.^{[1, 15]} In Anatolian folk medicine, the diuretics, carminative and laxative activities of the rhizomes of some Iris species like I. germanica have been well documented. Moreover, they have a long history of medicinal uses in China and Mongolian traditional medicine.^{[1, 3, 12]}I. germanica which is known as ‘Irsa’ in TPM has been considered an important medicinal plant in medieval Persia. Medicinal properties of this plant have been mentioned in medical textbooks of some scientists in TPM, including the Canon of Medicine by Avicenna (980–1037 AD) and Zakhireye Kharazmshahi by Jorjani (1042–1136 AD). This plant is recommended as a medication for some types of headaches; it has a hypnotic effect and relieves rhinorrhea and sinus discharges.^{[26, 27]} The useful properties of Iris for the treatment of all diseases caused by phlegm in the respiratory tract, especially asthma, productive cough and pneumonia, have been described. According to TPM, Iris is an aromatic plant, and the aroma of the plant is responsible for its potent exhilarant and cardiac tonic effects.^[26–29] Administration for gastrointestinal problems is the other important therapeutic effect of I. germanica; it is suggested that Iris can relieve gastric, liver and spleen pain as well as treat obstructions of the liver and spleen. Interestingly, oral administration of a mixture of Iris with pure vinegar is prescribed as a cleansing agent for the spleen.^{[26, 28]} This plant is useful for some kidney and bladder diseases due to its diuretic activity. It is an emmenagogue and has also been described useful for the treatment of male and female genitourinary infections; for this purpose, I. germanica is first dissolved in water and then applied as an immersion bath.^[26–29]

Phytochemical constituents

Phytochemical investigations on the roots and rhizomes of I. germanica have revealed the diverse distribution of phytoconstituents including flavonoids, isoflavones, iridal-type triterpenoids and steroids.^{[3, 12]} Isoflavonoids are the main active constituents of this plant.^[1]Table 1 shows the major phytochemicals that have been isolated and characterised from I. germanica. Chemical structures of secondary metabolites of this plant are shown in Figures 2–7. In addition, these secondary metabolites were further screened for drug-like properties (absorption, distribution, metabolism and excretion). The drug-like properties consisted of small molecules that were orally active in humans and the success of small molecules to convert a drug. This analysis is an essential assessment for the selection of drug candidates in the early stages of drug discovery. Based on Lipinski’s rule of 5 (Ro5), an orally active drug molecule should have a molecular mass less than 500 Da, maximum 5 H-donor bonds or less, maximum 10 H-acceptor bonds or less and log P (octanol/water partition coefficient) not greater than 5. If a compound has more than two outrange of these criteria, it would not probably be orally active.^{[30, 31]} The drug-like properties of secondary metabolites of I. germanica are presented in Table 1. Further studies are needed to meet the rule of secondary metabolites in this plant.

Isoflavones and glycosylated isoflavones

Isoflavonoids contain a 3-phenylchroman skeleton that is biogenetically derived from 2-phenylchroman skeleton of flavonoids. The most important effect of isoflavonoids is dietary antioxidants and protection against oxidative stress linked to inflammation and free radicals of nitrogen and oxygen.^[32] Many therapeutic effects of the rhizomes of I. germanica have been mainly related to the presence of isoflavones. A great diversity of isoflavones is present in different species of Iris such as aglycone and glycoside forms. The isoflavones are the first category of compounds extracted from I. germanica. Structures of major isoflavones and glycosylated isoflavones (compounds 1–40) are shown in Figures 2 and 3.

Flavonoids and glycosylated flavonoids

Flavonoids are a wide group of secondary plant metabolites that contain a benzopyran heterocycle linked to a benzene ring accompanied by various significant biological effects.^[33] Some types of flavonoids and c-glycosylated flavonoids are reported from I. germanica. Structures of flavonoids and glycosylated forms (compounds 41–48) are presented in Figure 4.

Iridal-type terpenoids

Terpenoids are the largest and most widespread class of secondary metabolites of plants with a wide variety of biological activities. Iris extracts are rich sources of terpenoids.^[12]Iris extract contains iridal-type triterpenoids, a group of C₃₁-triterpenoids derived from squalene by a unique biosynthetic pathway. These compounds have usually a seco-ring-A residue and are divided into monocyclic, bicyclic and spirocyclic derivatives.^[34] In addition, I. germanica is a source of iridal-type terpenoids (compounds 49–65) that are shown in Figure 5.

Steroids

Steroids are also found in the extracts of Iris plants. The structure of steroids found in I. germanica (compounds 66–69) is shown in Figure 6.

Miscellaneous compounds

Some simple phenols, benzoquinone structures and miscellaneous compounds were obtained from the rhizomes of I. germanica. The structures of this group (compounds 70–76) are summarised in Figure 7. One of these compounds is ceramides. Ceramides are a group of waxy lipid molecules composed of sphingosine and a fatty acid. Mohamed et al.^[9] have isolated two new ceramides, irisamides A (75) and B (76), from the methanol extract of the rhizomes of I. germanica.

Pharmacological activities

Various in-vitro and in-vivo studies evaluated biological activities of I. germanica. The pharmacological and biological activities of this plant are summarised in Table 2. Further discussion about the main pharmacological and medicinal effects of I. germanica will be made below.

Antioxidant and antinociceptive effects

Many medicinal plants demonstrated antioxidant properties according to their polyphenolic constituents, such as crocin in saffron,^{[35, 36]} rutin in apples, onions and berries,^[37] rosmarinic acid in rosemary,^[38] galbanic acid in Ferula,^[39] quercetin in vegetables^[40] and so on. Iris germanica is recognised to be a rich source of isoflavonoids (natural estrogen mimics, phytoestrogens), which are accumulated mainly in the rhizomes.^[41] Owing to their phenolic nature, isoflavonoids can give hydrogen atoms from their phenolic hydroxyl group(s) to the harmful oxyradicals, thereby forming the less-reactive phenoxyl radicals. Also, they can chelate metal ions and exhibit potent antioxidant activity.^[6]

The methanolic extracts of the rhizomes of I. germanica showed antioxidant activity under different storage conditions using sunflower and soybean oils as oxidation substrates. It may be attributed to a high concentration of phenolics, avenanthramides and antioxidants which are well known to act as excellent natural substances.^[42]

Anti-inflammatory and immunomodulatory effects

Current evidence confirmed that herbal-derived natural products had anti-inflammatory and immunomodulatory effects and indicated special activity on inflammatory mediators.^[43] The anti-inflammatory activity of nine isoflavonoids isolated from methanolic extracts of the rhizomes of I. germanica was evaluated by a spectrophotometric assay using the activated human neutrophils. These compounds showed different anti-inflammatory activities compared with anti-inflammatory drugs used in clinical practice (e.g. aspirin and indomethacin). Structures of these compounds were identified by spectral comparison with the reported data and the active ones categorise as non-steroidal anti-inflammatory agents.^[1]

I. germanica is a rich source of isoflavones. Irisolidone and irilone are two isoflavones isolated from this plant which act as immunomodulators. Their impact on production of T-lymphocytes cells (CD4+ and CD8+) and cytokines (IFN-γ, IL-2, IL-4 and IL-5) were reported in the Balb/c mice model. Oral administration of irilone revealed immunosuppressant activity through down-regulation of these cytokines; therefore, it can be useful in settings such as organ transplantation. Use of irisolidone can be beneficial in pathological conditions in which the balance of T lymphocyte cells changes.^[8]

The antiulcer effects of I. germanica have also been evaluated. The methanol extract obtained from the root of I. germanica on the ethanol-HCl-induced ulceration in rats showed potent antiulcer activity. Further assessment of this extract leads to obtaining γ-irigermanal (55) as a potent antiulcer agent.^[44]

Another study showed that among the six flavones isolated from I. germanica, compounds with hydroxyl groups in rings A and B had intense stimulatory activity on the proliferation of the osteoblast-like UMR106 and SaSO-2 cell lines compared with standard drug diazedine, whereas the other members did not exhibit significant effect because of the conversion of the hydroxyl group to either acetoxy or methoxyl groups. In addition, these compounds and their analogues have shown inhibitory activity nearly identical to the Elcitonin on the formation of osteoclast-like cells (RAW 263.7). Furthermore, all isolated isoflavones represented notable interactions with NF-κB and established a significant relationship between in-silico score and in-vitro anti-osteoporotic study.^[45]

Neuroprotective effects

The prevalence of neurodegenerative disorders such as Alzheimer’s disease (AD) is growing worldwide and it is expected to double till 2050.^[46] There are many medicinal plants having neuroprotective effects.^{[47, 48]} The cholinergic activity is altered in neurodegenerative diseases like AD.^[49] Acetylcholine is hydrolysed by acetylcholinesterase (AChE) and butyrylcholinesterase enzymes, and the cholinergic activity is elevated by inhibiting these enzymes.^[50] Furthermore, oxidative stress has an important key role in the pathogenesis of these disorders.^[51] In the study by Ullah et al.,^[17] the metabolites isolated from I. germanica showed AChE effects. Furthermore, I. germanica isolated fractions are rich in flavonoids, phenolics and saponins, which exhibited high antioxidant properties. Iris germanica extracts with high amounts of phenolics, flavonoids and saponins are responsible for the antioxidant and AChE inhibitory activities and potential remedies for the treatment of AD. Therefore, it was concluded that I. germanica has a beneficial effect on AD. This hypothesis was tested in an in-vivo experiment of a rat model of AD.^[18] The cognitive function of Aβ-induced AD rats became better with I. germanica. Besides, histopathological evaluations of the hippocampus confirmed a significant decrease in the number of Aβ plaques in the I. germanica-treated groups.^[18] Therefore, I. germanica has been considered a promising candidate for the treatment of neurodegenerative diseases such as AD.

Hypoglycaemic effects

Globally, diabetes mellitus (DM) is one of the fastest-growing diseases and its incidence is progressively increasing.^[52] So, it is very important to find new prevention and treatment methods. In this regard, α-amylase is one of the enzymes in human saliva and in the small intestinal brush border. The hydrolysis of starch into oligosaccharides, disaccharides and monosaccharides is done by this enzyme; thus α-amylase inhibitors are considered ancillary medications in DM. At the early stage of digestion of carbohydrates, α-amylase inhibitors can effectively postpone the digestion and assimilation. Therefore, glucose absorption is decreased, leading to the suppression of postprandial hyperglycaemia.^[53] Some studies demonstrated that polyphenols reduce the postprandial plasma glucose by inhibiting α-amylase.^[54]

The antioxidant and α-amylase inhibitory effects of flavonoids from the methanolic extracts of the rhizomes of I. germanica spectrophotometrically were investigated using 2,2′-diphenylpicrylhydrazyl (DPPH) and α-amylase inhibitory tests. Compounds such as 8-hydroxyirilone 5-methyl ether (11), 8-hydroxyirilone (12) and irilone (16) exhibited prominent antioxidant activities compared with propyl gallate (IC₅₀ 7.11 μm). Moreover, 8-hydroxyirilone 5-methyl ether (11), 8-hydroxyirilone (12), irilone (17) and irisolidone (6) possessed the highest α-amylase inhibitory activity in comparison to acarbose.^[19] In another study, iridin A (1), irilone 4′-methyl ether (18), irilone (17), irisolidone (6), irigenin S (13), irigenin (7), irilone 4′-O-β-d-glucopyranoside (36), iridin S (35) and iridin (33) were estimated for their α-amylase and 1,1-diphenyl-2-picrylhydrazyl inhibitory capacities. Irisolidone (6), irigenin (7) and iridin A (1) showed α-amylase inhibitory activities compared with acarbose, and only iridin A (1) exhibited moderate antioxidant activity with IC₅₀ of 8.91 μm.^[19] It was proved that antioxidant and α-amylase inhibitory effects were closely related to the structural properties of the compounds. Iris germanica is a rich source of antioxidants.^{[16, 17]} Previous studies demonstrated that the antioxidant intake could reduce DM risk by reducing oxidative stress.^{[55, 56]}

Hypolipidaemic effects

Coronary heart diseases (CHDs) are a leading cause of morbidity and mortality worldwide. Hyperlipidaemia has the main role in the development of atherosclerosis and CHDs.^[57] Several studies have revealed that phenolic compounds of medicinal plants could potentially protect the cardiovascular system and reduce hyperlipidaemia.^[58–61] In the study of Iqbal et al., the effects of ethanolic extract of I. germanica on lipid profiles were evaluated on rats fed on a high-fat diet.^[20] The serum levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were examined after 10 weeks. The results revealed that ethanolic extract of I. germanica significantly decreased the serum levels of TC, TG and LDL-C. Furthermore, it also reduced the ratio of TC to HDL-C concomitantly increased HDL-C/LDL-C ratio, which is a reliable risk assessment factor for CHDs.^[20] Therefore, I. germanica may be considered a good cardioprotective agent.

Antimicrobial effects

Medicinal plants have been documented as beneficial sources of antimicrobial agents.^{[62, 63]} Different studies evaluated the antimicrobial and antifungal effects of various components of I. germanica. The antiplasmodial and antifungal activities of iridal (60), a triterpenoidic compound extracted from I. germanica, was studied by Benoit-Vical et al. They evaluated the antimicrobial effect of iridal (60) in vitro on Plasmodium falciparum chloroquine-resistant and chloroquine-sensitive strains, in vivo on Plasmodium vinckei and on some Candida albicans and Candida arapsilosis strains too. The IC₅₀in vitro (human malaria strain) was 1.8–26.0 mg/ml and the ED₅₀in vivo (mice i.p. route) was 85 mg/kg/day. The minimal inhibitory concentrations in the strain of yeast were higher than 50 mg/ml. The results demonstrated that iridal (60) has an antiplasmodial activity similar to that of Azadirachta indica extracts (a reference in malaria phytomedicine). However, iridal (60) showed no important antifungal activity.^[2]

The Biomphalaria alexandrina snails were used for evaluating the molluscicidal activity of I. germanica (leaf and rhizome). The rhizome chloroform extract showed the highest molluscicidal activity (LC₉₀ = 1.26 mg/l). Also, it has been shown to have a significant cercaricidal potential in the concentration- and time-dependent manner. Importantly, 5,2′-dihydroxy-3-methoxy-6,7-methylenedioxyflavone (43) and 5,7,2′-trihydroxy-6-methoxyflavanone (44) were two compounds, which were isolated from chloroform extraction of Iris rhizome that displayed molluscicidal activity.^[64] Triterpenens such as irisgermanical A (49), B (50), C (51) and iridal-type triterpenes such as iripallidal (58) and iriflorental (57) that were extracted from the rhizome of I. germanica showed piscicidal activities.^[65] Many studies demonstrated that most of the piscicidal substances in natural products have many other biological activities such as insecticidal, aphrodisiac as well as antitumour properties,^[66] and I. germanica rhizome extract has many of these compounds.^[67]

One study evaluated the antimicrobial, antioxidant and antimutagenic properties and also phenolic compounds of ethanolic extracts of the aerial parts and rhizomes of the I. germanica. According to the findings, I. germanica showed only a limited antimicrobial activity against the Bacillus subtilis ATCC 6633. The total antioxidant activity of both extracts was found to be higher than that of ascorbic acid and butylated hydroxytoluene at similar concentrations. The radical scavenging activities of the ethanolic extracts of the aerial parts and rhizomes of I. germanica were found to have IC₅₀ values of 5.38 and 12.3 mg/ml, respectively, whereas the total antioxidant activity of the extracts (at 3.15 mg/ml) was 98.7% and 97.4%, respectively; the total phenolic content was 267.36 ± 2.05 and 331.96 ± 1.78 mg gallic acid equivalent/g extract, respectively. The ability of I. germanica extracts to scavenge free radicals using DPPH can be mainly attributed to the phenolic content of the plant. Moreover, high-performance liquid chromatography analysis of phenolic compounds identified protocatechuic acid (70), chlorogenic acid (71) and ferulic acid (72) as the primary phenolic acids contained in the extract of the aerial parts of I. germanica, whereas chlorogenic acid (71), (+)-catechin (45) and ferulic acid (72) were identified as the primary phenolic acids in the extract of the rhizome. The antimutagenic activity using the Ames Salmonella/microsome mutagenicity test was found to be dose-dependent. The most active antimutagenic activity was observed at 1.5 mg/plate concentration of the rhizome extract against Salmonella typhimurium TA 100 strain.^[7]

Antiproliferative and cytotoxic effects

Polyphenolic compounds of flowers and rhizomes of some types of Iris plant had significant antiproliferative activity against human skin and lung cancer cell lines.^[68] Flavonoids and isoflavanoids are the most essential class among secondary metabolites of Iris, which are potentially effective in limiting the cancer growth. Methanolic extracts of the rhizome of different species of Iris plant including I. germanica demonstrated inhibitory effect on proliferation of both A549 and Caco-2 cell lines in a dose-dependent manner and could be promising anticancer reagents.^[69]

The cytotoxicity of 10 isoflavones isolated from the rhizomes of I. germanica was tested using Ehrlich’s ascites carcinoma (EAC) cancer cell line by MTT and ATP assays. The results showed that these compounds possessed tremendous potential antiproliferative effect on EAC cell line.^[15]

Six different triterpenoids (known iridals) extracted from I. germanica were purified and bioassayed on two cultured human tumour cell lines: A2780 and K562 (and for each one a drug-sensitive and a drug-resistant cell line). All of the tested iridals showed cytotoxicity comparable to that of doxorubicin on the multidrug resistance (MDR) phenotype. Some of them were shown to be more effective than doxorubicin. Toxicity of iridals on MDR cell lines seems to indicate that the effects of these triterpenoids are less affected by MDR than those of doxorubicin or taxol. Structural and functional roles of iridals in cell membranes could vindicate its cytotoxicity against tumour cells. Finally, the authors suggested a mechanism at the sphingomyelin cycle level inducing accumulation of ceramides that are involved in cell cycle inhibition.^[70]

In another study, six known isoflavones were isolated from the rhizomes of I. germanica (irisolidon (6), irisolidone 7-O-alpha-D-glucoside (37), irigenin (7), irilone (17), iriflogenin (10) and iriskashmirianin (14)) and were shown to be potent inhibitors of cytochrome P450 1A activity that is involved in the metabolic conversion of procarcinogens into carcinogens. The isoflavones displayed moderate or weak activity as inducers of NAD(P)H: quinone reductase in cultured mouse Hepa 1c1c7 cells and the radical (DPPH) scavenging bioassay, respectively. Concerning antitumour promoting potential based on anti-inflammatory mechanisms, none of the compounds demonstrated significant activity in the concentration range tested.^[23]

Halpert et al.^[71] found that a lipidic extract from I. germanica was able to increase HeLa cell spreading by increasing cell adhesion and augment the formation of actin stress fibres by activation of Rac1 (a small GTPase whose activation is involved in cell adhesion, migration and division). Biochemical verification of the extract revealed the presence of iripallidal (58), iriflorental (57), 17-hydroxyiridal (64) and 16-hydroxyiridal (65) in the active fractions, which can integrate into liposomal membranes containing phosphatidylcholine and affect their fluidity, similar to cholesterol. Iripallidal (58) was found to bind to protein kinase Cα and the guanylnucleotide exchange factors such as phorbol esters, but it is not yet clear how this binding contributes to their antiproliferation activity. Iridals were shown to activate protein kinase C and induce differentiation of HL-60 promyelocytic leukaemia cells, thereby releasing tumour necrosis factor-α in mouse skin and promoting mouse skin tumours. It can be concluded that different iridals’ signals lead to either growth inhibition or promotion through different pathways.

Structure-related activity of I. germanica isoflavonoids

According to different studies, isoflavonoids’ structure and position of functional groups are important for biological activities (Figure 8). The position and number of OH groups were crucial to antioxidant activity. The existence of C-4′ and C-5 hydroxyl leads to high antioxidant potentials and its replacement by methoxy or loss of them reduces the abilities of these compounds; however, hydroxylation of C-7 has no significant effect on the activity of these compounds. In contrast, hydroxylation at C-5 enhances the amylase inhibitory activities, whereas glycosylation diminished their activities. However, methylation of the hydroxyl groups in rings A and B improved the activities. Furthermore, the presence of OH group at C-8 significantly increases the antioxidant and α-amylase inhibitory activity.^{[6, 19]}

It was proved that the presence of the hydroxyl groups in ring A of flavonoids coupled with the absence of hydroxyls in ring B stimulates the proliferation of human peripheral blood mononuclear cells and the IFN-γ production, whereas the presence of the hydroxyl groups on ring B represses them. The immunomodulatory activity of methylated products is drastically decreased after its O-methylation, showing that free phenolic groups at the 5′ and/or 4′ position have an essential role in such activities possibly due to the change in the partitioning of the compounds between water and lipid phases in biological systems.^[8]

Generally, compounds with hydroxyl groups in rings A and B showed intense stimulation activity, whereas the other members did not indicate significant effect because of the conversion of the hydroxyl group to either acetoxy or methoxyl group. Cytotoxicity studies suggested that the cytotoxic potential of isoflavonoids was dependent on the substitution patterns of pharmacophores (hydroxyl, methoxy, acetoxy) of rings A and B in the nucleus of flavones and the interaction of pharmacophore with the active site of the receptors.

Side effects and toxicity

Although the long history of medicinal uses of Iris species in many places in the world can suggest their safety, it should be considered that their long-term use may cause some problems. Currently, there is little evidence about toxicity of I. germanica. The aqueous extract of I. germanica showed toxicity and psychotropic activity in mice.^[72] Methanolic extract of I. germanica was found to exhibit potent toxicity to Oryzias latipes. Therefore, it could be considered a pesticide.^[67] Furthermore, bactericidal, fungicidal and insecticidal activities as well as phytotoxicity of I. germanica were also confirmed in in-vitro studies.^[1] Reviewing the current literature demonstrates that there is not enough evidence related to serious adverse effects and toxic reactions for the consumption of I. germanica in human subjects. However, further animal and human studies are needed to verify its potential adverse effects.

Future Perspectives and Conclusions

In summary, I. germanica is a medicinal plant, which has a long history of applications in several ethnomedical systems. The present study reviewed the botany, traditional uses, phytochemistry and pharmacology of I. germanica. The underground parts and rhizomes of this plant have been identified as its medicinal parts. Based on phytochemical investigations, the roots and rhizomes of I. germanica contain different bioactive compounds, including flavonoids, isoflavones, iridal-type triterpenoids and steroids. Its main active constituents are isoflavonoids. Several in-vivo and in-vitro investigations have revealed that I. germanica has many biological activities such as neuroprotective, hypoglycaemic, hypolipidaemic, antimicrobial, antioxidant, antiproliferative, anti-inflammatory, antiplasmodial, immunomodulatory, cytotoxic and antimutagenic properties. In a rat model of AD, administration of I. germanica showed an ameliorative effect on cognitive function. Iris germanica has antioxidant and AChE inhibitory activities due to the presence of high amounts of phenolics, flavonoids and saponins. Therefore, it could be a promising candidate for AD.

The flavonoids that are isolated from I. germanica showed antioxidant and α-amylase inhibitory activities. They have inhibitory effect on carbohydrate-hydrolyzing enzymes that reduce the postprandial plasma glucose. These findings represent the antidiabetic potential of this plant. Ethanolic extract of the rhizomes of I. germanica showed therapeutic effects against the experimentally induced hyperlipidaemia. Different studies proposed that I. germanica and its components such as iridal, triterpens and iridal-type triterpenes are potent antimicrobial, antifungal and antiplasmodial agents. The presence of high levels of isoflavonoids in the rhizomes may be attributed to the antioxidant and antinociceptive properties of I. germanica. Moreover, irisolidone and irilone are two isoflavones isolated from this plant, which have anti-inflammatory and immunomodulatory properties. Their impact on production of T lymphocytes cells and cytokines was reported. Although recent studies mentioned the antiproliferative and cytotoxic effects of I. germanica and its metabolites in cancer cell lines, there is a controversy among the findings of these studies. Further studies are needed to confirm or rule out the antitumour effects of this plant.

There are insufficient pharmacokinetic and safety studies on the extracts of I. germanica; so, it is necessary to focus on them. Even though the majority of preclinical studies reported various pharmacological activities of I. germanica, clinical trial reports are not currently available. Therefore, further studies are needed to validate its clinical application in the herbal formulations for human uses.

Conflict of Interest

The authors declare that they have no conflict of interest to disclose.

Acknowledgement

This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.

References