Introduction

Helicobacter pylori (H. pylori) is a Gram negative bacterium that colonizes 50% of the world population stomach1. It is considered as a main factor of chronic gastritis and gastric/duodenal ulcers2. A marked reduction in gastric or duodenal ulcers recurrence rates associated with H. pylori infection was achieved by curing the infection3. H. pylori infection is the major risk factor associated with gastric cancer development4. H. pylori infection triggers the transition from normal mucosa to non-atrophic gastritis and initiates precancerous lesions which after that progress to intestinal metaplasia and atrophic gastritis5. H. pylori is classified by the International Agency for Research on Cancer6 and World Health Organization (WHO) as group one carcinogen.

Noticeable infiltration of inflammatory cells along with release of the inflammatory mediators occur due to H. pylori colonization in the gastric mucosa7. Also, H. pylori infection activates the mucosal defense mechanisms2. Prostaglandins (PGs) play an important role as mucosal defense factors protecting the gastric mucosa against injury by inducing gastric mucus and bicarbonate secretion, inhibiting acid secretion and decreasing gastric motility8. PGs are synthesized through cyclooxygenase (COX) enzymes.

COX-1 is constitutively expressed in the stomach, while COX-2 expression is induced at inflammation sites9. COX-1 participates in PGs production under normal physiological conditions. Thus, it is regarded as the isoform generating PGs associated with housekeeping functions, as protective action on the gastric mucosa against ulceration. On the other hand, COX-2 is involved in the PGs production associated with the development of several diseases10 such as autoimmune diseases including rheumatoid arthritis11,12, allergic sign and symptoms13, Alzheimer’s disease14, and the development and prognoses of numerous cancers15. Moreover, COX-2 was proved to be involved in gastrointestinal cancer16. H. pylori increases COX-2 expression consequently stimulating the release of PGs in H. Pylori associated premalignant and malignant gastric lesions17.

Thus, selective COX-2 inhibitors decrease PGs levels in inflammatory sites only and they have no effect on gastric mucosal PGs levels or integrity . On the other hand, specific COX-2 inhibitors, which cause no significant inhibition of COX-1, have been reported to delay healing of ulcers18,19.

Inflammation predisposes to the development of cancer and promotes all stages of tumorigenesis. inflammation drives tumor initiation, growth, progression, and metastasis20. Therefore, NSAIDs can aid in cancer prevention21 and a dual anti-H. pylori/anti-inflammatory compound could both help in eradicating the microorganism and avoid the development of H. pylori-related cancers.

Safe NSAID COX-2-inhibitors must have selectivity indices (IC50 COX-2/IC50 COX-1) ranging from 5 to 5022. Since super selective COX-2-inhibitors like Rofecoxib were withdrawn from the market because of the increased risk of heart attack and stroke. Thus, it becomes essential to find alternative safe NSAID COX-2-inhibitors. Most of the natural products tend to be more selective towards COX-1 than COX-2 but they can be further modified to increase COX-2 selectivity23.

Antibiotics such as amoxicillin, tetracycline, metronidazole or clarithromycin in addition to proton pump inhibitors or bismuth salts are considered as the only known current therapy for H. pylori infection24. This triple therapy is not always successful in eradication of the infection. Reduced treatment efficacy can occur due to emergence of antibiotic resistant H. pylori. Moreover, non-steroidal anti-inflammatory drugs (NSAIDs), which have been commonly used for COX-2 inhibition, resulted in serious adverse effects as gastrointestinal bleeding and gastric mucosa damage after prolonged intake25. H. pylori infection and NSAIDs are the main causative risk factors for gastroduodenal ulcers26. Therefore, discovering alternative therapy for management of H. pylori infection and the associated inflammation is crucial to overcome the undesirable effects of the currently used therapy.

Since ancient times, the use of medicinal plants has been adopted and considered the origin of modern medicine27. Many plants are not deeply investigated, although they have been reported to contain various secondary metabolite classes of well known anti-microbial and anti-inflammatory potentials.

Iris is the largest genus in the family Iridaceae with the greatest taxonomic diversity28. Numerous interesting secondary metabolites such as triterpenoids and phenolics; flavonoids and xanthones were detected in genus Iris. Extracts and pure compounds of the irises were reported to have several biological activities such as antimicrobial, anti-oxidant, anti-inflammatory, phytoestrogenic, anti-diabetic, anti-tumor, and anti-cholinesterase activities29,30.

Long time ago irises were used for the treatment of bacterial infections in the Mongolian folk medicine31. Irises have been found in Karnak temple engraved on a marble panel of the ancient Egyptians32. In traditional Chinese medicine, the rhizomes of Iris confusa, one of the irises native to western China, were used extensively to treat tonsillitis, acute bronchitis, colitis, and chronic atrophic gastritis33.

Reviewing the current literature, very few reports were traced concerning I. confusa Sealy (bamboo iris) phytochemical constituents29,34. In addition, nothing was traced about the biological activity of its isolated compounds except the study conducted by Chen et al. 2018 who studied their anti- hepatitis B potential34. In a continuation of our interest in exploring irises metabolites and their possible activities35, I. confusa was selected for this study. Its polar and non-polar fractions were subjected to chromatographic fractionation and purification with the aim of isolation of its major constituents and testing their anti H. pylori and selective anti-inflammatory potencies.

Material and methods

Plant material, extraction and fractionation

Iris confusa Sealy was collected from Al-Mansouria, Giza, Egypt in the flowering stage after permission from Agricultural Research Center, Giza, Egypt in compliance with the national guidelines. The plant material identity was kindly authenticated and verified by Dr Nina Davies, the curator of the African Iridaceae, Royal Botanic Gardens, Kew, London, UK. Specimen was deposited in Pharmacognosy Department Herbarium, Cairo University (registration no. 15.1. 2019I). The air-dried powdered underground parts of I. confusa were extracted and fractionated according to Salem et al. yielding polar fraction (PF) and non-polar fraction (NPF)36. Collection of plant material, complied with the institutional, national, and international guidelines and legislation.

Major constituents isolation

The PF and NPF were screened on pre-coated silica gel 60 F254 using solvent system S1 (methylene chloride:methanol:formic acid 85:15:0.2 v/v/v) and S2 (methylene chloride:methanol 97:3 v/v), respectively. The spots were examined in UV light before and after ammonia vapour exposure and AlCl3 spraying and after being sprayed with p-anisaldehyde/H2SO4 followed by heating at 110 °C. PF and NPF purification using a vacuum liquid chromatography column (VLC) packed with silica gel H 60, ion exchange resin (diaion HP-20), sephadex LH 20 and silica gel 60 were illustrated in detail in Fig. S1.

Similar fractions were pooled together and evaporated to dryness under reduced pressure yielding compounds P1 (yellow powder, Rf = 0.28, S1), P2 (yellow powder, Rf = 0.18, S1), P3 (yellow crystals, Rf = 0.69, S1) and P4 (yellow crystals, Rf = 0.55, S1) from the PF and N1 (white crystals, Rf = 0.62, S2) and N2 (white crystals, Rf = 0.43, S2) from the NPF.

Determination of anti-Helicobacter pylori activity

The anti-Helicobacter pylori activity of the PF and NPF as well as the isolated compounds was evaluated against H. pylori ATCC 700392 (type strain, obtained from the American Type Culture Collection unit (ATCC), using microwell dilution method and clarithromycin as standard drug37. The detailed procedures were described in the supplementary file.

Determination of COX-1 and COX-2 inhibitory activity

The tested samples and the standard drugs (ibuprofen and celecoxib) were prepared in dimethyl sulfoxide and subsequent eight twofold dilutions (125–0.98 µg) were carried out in a 96-well plate. The inhibitory COX activity was assayed colourimetrically by examining the inhibition of the ovine COX-1 and human recombinant COX-2 enzymes as described by George et al.38, using a COX inhibitor screening assay kit. The detailed procedures were described in the supplementary file.

Determination of nitric oxide (NO) inhibitory activity

The macrophage cell line, RAW 264.7 was obtained from Vaccines, Sera and Drugs Egyptian Company (VACSERA). It was cultured in (RPMI, 1640) medium supplemented with 10% fetal bovine serum and 1% gentamicin39. The detailed procedures were described in the supplementary file.

In vitro HpIMPDH inhibition assay

The most potent isolated compound, as revealed from the previous assays, was screened at different concentrations (10–0.078 µM) in triplicates. The assay was carried out according to Galal40 and was described in detail in the supplementary file.

In vitro hIMPDH2 inhibition assay

Human inosine-5′-monophosphate dehydrogenase (hIMPDH2) was purchased from NovoCIB SAS (Lyon, France). It was used for the in vitro screening of the most potent isolated compound, as revealed from the previous assays, against H. pylori and the standard drug at the concentrations (10–0.078 µM) in triplicates40. The detailed procedures were described in the supplementary file.

Quantitative determination of the main phytochemical classes and antioxidant assay

Total phenolic content (TPC)

Determination of TPC was carried out according to the European Pharmacopeia procedure41, using the Folin–Ciocalteu colourimetric method. It depends on measuring the intensity of the blue colour produced in correlation to the reducing power of existing phenolics. Determinations were carried out in triplicates; results are the mean values ± standard deviations and expressed as μg gallic acid equivalent (GAE) per mg dry fraction (μg GAE/mg).

Total flavonoid content (TFC)

TFC was determined based on measuring the intensity of the yellow colour developed upon reaction of flavonoids with aluminium chloride reagent42. Quercetin was used to compute the standard calibration curve. Determinations were carried out in triplicates; results were the mean values ± standard deviation and expressed as μg quercetin equivalent (QE) per mg dry fraction (μg QE/mg).

Total triterpene content (TTC)

TTC was determined based on measuring the intensity of the red–purple colour developed upon reaction of perchloric acid-oxidized triterpenes in glacial acetic acid with vanillin43. Ursolic acid was used to compute the standard calibration curve. Determinations were carried out in triplicates; results were the mean values ± standard deviations and expressed as μg ursolic acid equivalent (UAE) per mg dry fraction (μg UAE/mg).

DPPH assay

The DPPH anti-oxidant assay was carried out as described by Romano et al.44 with some modifications. The PF and NPF of I. confusa underground parts were separately dissolved in methanol by the aid of sonication to give a set of serial dilutions for each sample. Experiments were performed in triplicates using a 96-wells plate. The reaction mixture in each case consisted of 22 μL of the tested sample and 200 μL of 0.004% DPPH in methanol. The non-quenched DPPH radicals were assessed spectrophotometrically at λmax = 492 nm using a micro-plate reader.

Ethical statement

Collection of the plant material, complied with relevant institutional, national, and international guidelines and legislation.

Results

Purification of PF and NPF

Chromatographic purification of I. confusa underground parts PF allowed the isolation of three isoflavonoids; tectoridin (P1), irigenin (P3), and tectorigenin (P4), and one flavone, orientin (P2). On the other hand, purification of the NPF led to the isolation of one triterpene, isoarborinol (N1), and one sterol, stigmasterol (N2).

The isolated compounds (P1–P4 and N1–N2) were identified through their physicochemical characters, spectroscopic analysis, UV spectral data and via comparing their 1H and 13C NMR data with the published data (Tables S1–S4 in the supplementary file). Their structures are shown in Fig. 1.

Figure 1
figure 1

Structure of the isolated compounds. P1 tectoridin, P2 orientin, P3 irigenin, P4 tectorigenin, N1 isoarborinol, N2 stigmasterol.

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Helicobacter pylori inhibitory activity

I. confusa underground parts PF inhibited the growth of H. pylori with MIC value of 62.50 μg/mL (Table 1 and Fig. 2). Compounds isolated from PF showed more potent activity than their crude fraction (irigenin: MIC 3.90 μg/mL; orientin: MIC 15.53 μg/mL and tectorigenin: MIC 15.63 μg/mL) except tectoridin which showed MIC > 125 μg/mL. The most potent isolated compound was irigenin (MIC 10.82 μM) followed by orientin (MIC 34.64 μM) and tectorigenin (MIC 52.05 μM). I. confusa underground parts NPF as well as its isolated compounds (isoarborinol and stigmasterol) exhibited no H. pylori inhibitory potential.

Table 1 Minimum inhibitory concentration of I. confusa underground parts PF, NPF and isolated compounds against Helicobacter pylori and their IC50 on COX-1, COX-2, and nitric oxide (NO).
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Figure 2
figure 2

(A) Percentage inhibition and (B) MIC values of I. confusa underground PF and isolated compounds against H. pylori. All determinations were carried out in a triplicate manner and values are expressed as the mean ± SD; PF polar fraction.

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COX-1 and COX-2 inhibitory activity

I. confusa underground parts PF inhibited COX-1 and COX-2 by an IC50 of 112.08 ± 0.60 and 47.9 ± 1.50 µg/mL, respectively (Table 1 and Fig. 3). On the other hand, the NPF did not show any inhibition to COX-1 and IC50 > 125 µg/mL against COX-2.

Figure 3
figure 3

Effect of I. confusa underground parts PF, NPF and isolated compounds on (A) COX-1 and (B) COX-2 inhibition, (C) IC50 compared to reference drugs. All determinations were carried out in triplicate and values are expressed as the mean ± SD; NPF non-polar fraction, PF polar fraction, IC50 concentration at which the tested sample produces 50% inhibition, Standard ibuprofen for COX-1, celecoxib for COX-2.

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The isolated compounds were more potent than the corresponding crude fractions except for tectorigenin (COX-1 IC50 of 122.30 ± 0.90 and COX-2 IC50 of 56.70 ± 1.50 µg/mL) and its glycoside tectoridin which showed no inhibition against both COX-1 and COX-2. Among the isolated compounds, irigenin was the most potent COX-1 (IC50 of 35.25 ± 1.40 μM) and COX-2 (IC50 of 10.83 ± 0.86 μM) inhibitor followed by isoarborinol (COX-1 IC50 of 73.35 ± 1.00 μM and COX-2 IC50 of 51.79 ± 2.10 μM).

The isolated compounds showed higher inhibitory potential against COX-2 than COX-1 enzyme. Among all tested samples, irigenin showed the highest selectivity for COX-2 (SI = 3.26).

Nitric oxide assay

In the same manner, I. confusa underground parts PF exhibited nitric oxide inhibition by an IC50 of 47.80 ± 0.89 µg/mL while the NPF was inactive (Table 1 and Fig. 4). Irigenin was the most active tested compound. It showed nitric oxide inhibition by an IC50 of 42.46 ± 1.80 μM followed by isoarborinol (IC50 of 44.29 ± 1.70 μM).

Figure 4
figure 4

Effect of I. confusa fractions and isolated compounds on NO inhibition. All determinations were carried out in triplicate and values are expressed as the mean ± SD; L-NIL L-N6-(1-iminoethyl) lysine hydrochloride, PF polar fraction.

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IMPDH inhibition assays

On the basis of the previous in vitro assays on I. confusa underground parts PF and NPF as well as their isolated compounds (Table 1), the most active compound was irigenin. Thus, irigenin was selected to test its HpIMPDH inhibitory activity by monitoring NADH production at concentration range of 10–0.078 µM compared to clarithromycin as standard drug. Also, to examine irigenin selectivity toward the bacterial IMPDH, human hIMPDH2 inhibition potential was assayed.

Results showed that irigenin (P3) exhibited potent inhibitory potential against HpIMPDH enzyme with IC50 of 2.07 ± 1.90 μM (Table 2). Whereas the IC50 value of clarithromycin (standard drug) was 0.51 ± 0.58 μM. In addition, irigenin was less active and safer against hIMPDH2 with IC50 > 10 μM, compared with clarithromycin (IC50 3.10 ± 2.10 μM), which make sure that its selectivity is toward HpIMPDH in contrast of clarithromycin. I. confusa underground parts PF exhibited high DPPH scavenging activity with EC50 of 41.68 ± 6.67 μg/mL. The polar fraction TPC amounted 99.05 ± 0.02 μg GAE/mg dried fraction and TFC amounted 98.31 ± 0.04 μg QE/mg dried fraction while, the non-polar fraction TTC amounted 126.30 ± 0.04 μg UAE/mg dried (Table 3).

Table 2 HpIMPDH and hIMPDH2 inhibitory activity of irigenin (P3) and the standard drug clarithromycin.
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Table 3 Total phenolic, flavonoid, and triterpene content of the polar and non-polar fractions of I. confusa underground parts and their DPPH scavenging activity.
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Discussion

The eradication of H. pylori infection has been proven to prevent gastric or duodenal relapses. Direct correlation was observed between the development of gastric adenocarcinoma and the long-term infection with H. Pylori. Therefore, antibiotics such as clarithromycin have been used for treatment. The declining eradication rates provoked the search for alternative therapeutic options which should be more effective and safer.

Exploring medicinal plants along with their phytoconstituents for H. pylori infection control is not just a way to discover safer pharmaceutical alternatives, but also is a trial to discover a natural affordable effective drug especially in developing countries.

The selection of Iris in this study was based on our previous experience with this genus diverse metabolites content and antibacterial potential29,35. As well as, I. confusa has been used a long time ago in the treatment of bacterial infections and gastritis in traditional Chinese medicine33. Hence, the in vitro H. pylori, COX-1, COX-2, and NO inhibition potency of I. confusa underground parts PF, NPF, and their isolated compounds were assessed. Additionally, the selective IMPDH inhibition activity of the most potent isolated compound, irigenin, was examined.

Purification of the PF led to the isolation of four metabolites (P1–P4) while two compounds (N1–N2) were purified from the NPF. Compounds P1–P4 spectral data were in agreement with the reported data of tectoridin45, orientin46, irigenin47, and tectorigenin45. Compounds N1–N2 spectral data were in agreement with the reported data of isoarborinol48 and stigmasterol49. This is the first report for the presence of isoarborinol (N1) in genus Iris. P1–P4 and N2 are herein isolated from I. confusa for the first time.

I. confusa underground parts PF and its isolated compounds showed significant inhibitory effects on H. pylori. Irigenin was superior over tectorigenin as anti-H. pylori. This was attributed to the presence of methoxy group at C-4′ in irigenin, which increases the H. pylori inhibitory effect than the hydroxyl group in C-4′ of tectorigenin as previously reported by Park et al.50.

Although tectorigenin (P4) exhibited certain inhibitory activity against H. pylori, its corresponding O-glycoside; tectoridine (P1) exhibited no activity. This was attributed to the effect of glycosylation at 7-OH of tectoridin which caused dramatic decrease in the H. pylori inhibitory activity compared to its aglycone tectorigenin. This dramatic decrease in the activity of the isoflavone glycosides was well documented before50. The observed anti-H. pylori activity of orientin matched that previously reported by Król-Kogus et al.51.

The inflammation usually associated with H. pylori infection drained the authors interest to explore the COX-1 and COX-2 inhibitory potential. The need to explore drugs with selective COX-2 inhibitory potential that are able to decrease PGs dependent inflammation while maintaining protective gastric mucosal PGs synthesis intact has increased in recent decades. Finding COX-2 inhibitors, but not specific and still having a degree for COX-1 inhibition18,52 was the main target.

Irigenin was found to exhibit COX-2 selective inhibitory activity three times more than COX-1 (SI = 3.26) followed by tectorigenin (SI of 2.16) and orientin (SI of 1.58).

Nitric oxide is free oxygen radical and has cytotoxic effect in pathological processes, particularly in inflammatory disorders. Nitric oxide is a potent proinflammatory molecule secreted during inflammation and causes vasodilation and cellular migration. At higher concentrations, it downregulates adhesion molecules and induces apoptosis of inflammatory cells. Inhibition of NOS (inducible nitric oxide synthase) is beneficial for the treatment of inflammatory disease53,54,55,56. Irigenin exhibited significant potential in inhibiting nitric oxide followed by isoarborinol.

Many microbial infections are characterized by rapid proliferation that is supported by guanine nucleotide pool expansion in the rapidly dividing cells. Inosine 5ʹ‐monophosphate dehydrogenase (IMPDH), an important enzyme required for the new synthesis of guanine nucleotides, is an interesting target for antimicrobial drug development57. This enzyme, IMPDH, catalyzes the oxidation of inosine 5ʹ‐monophosphate (IMP) to xanthosine 5ʹ‐monophosphate (XMP) leading to reduction of nicotinamide adenine dinucleotide (NAD+), which is an important step in guanine nucleotides de novo synthesis. IMPDH inhibition surely leads to major fall in guanine nucleotide pool which consequently blocks proliferation57.

The most active anti H. pylori and anti-inflammatory tested compound; irigenin (P3) was chosen to test its inhibitory potential against the bacterial HpIMPDH enzyme and to evaluate its selectivity relative to the host enzyme (hIMPDH2). It was found to have promising activity against the HpIMPDH enzyme, and to be safer and less active against the hIMPDH2, which reflected its selectivity.

Therefore, I. confusa underground parts and its isolated compounds can be used as excellent therapy to control gastric ulcers via eradication of H. pylori and exerting its anti-inflammatory potentials.

The PF of I. confusa underground parts exhibited high DPPH scavenging activity which was in accordance with its high TPC and TFC. The DPPH scavenging activity could be attributed to its total phenolic and flavonoid contents58. The observed anti-oxidant potential of the NPF could be attributed to the recently detected I. confusa xanthones and triterpenoids29. Xanthones and triterpenoids metabolites are well documented anti-oxidants59,60.

In recent years, irigenin, isolated from Belamcanda chinensis (Iridaceae) has been a hot research topic due to its important bioactivities. Irigenin controlled the metastatic progression in lung carcinoma cells61. It was recently proved to exhibit beneficial potentials in management of cardiac injuries. It alleviated doxorubicin (DOX)-induced cardiotoxicity62 and protected HUVECs (human umbilical vein endothelial cell lines) from angiotensin II-induced oxidative stress and apoptosis injury63. Irigenin exhibited inhibitory effects on prostaglandin E2 and nitric oxide production in murine macrophage RAW 264.7 cells64.

Our results presented irigenin (P3) as a new anti‐H. pylori, COX, NO and HpIMPDH inhibitor beside being previously reported to significantly enhance TRAIL-regulated apoptosis in Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistant gastric cancer cells65. This highlights the promising expected results upon future testing of its in vivo potential in gastric ailments.

It is noteworthy to mention that, this is the first time to isolate irigenin from I. confusa by such classical chromatographic techniques. Thus, we herein present I. confusa as another natural source of irigenin other than Belamcanda chinensis (Iridaceae).

Besides irigenin, isolated in this study, the anti-H. pylori activity of I. confusa underground parts is attributed to its content of flavonoids, isoflavonoids, and xanthones which were recently reported in the PF29. Flavonoids and isoflavonoids react with superoxide anion, lipid peroxy, and hydroxyl radicals thus can protect the gastric mucosa against reactive O2 species formed during the infection50. In addition, several natural flavonoids were reported to exhibit potent bactericidal potential against antibiotics resistant H. pylori strains66. Xanthones also are well documented to exhibit anti H. pylori activity67,68. Mangiferin xanthone reported in I. confusa PF29 is a very potent gastroprotective69,70 and anti inflammatory71 compound.

It is worthy to note that, the H. pylori, COX-1, COX-2, and NO inhibitory potentials of irigenin (P3), were much higher than that of PF from which it was isolated. Thus, fractionation and purification of I. confusa crude fractions caused significant increase in the aforementioned activities which may indicate a chance for isolation of more active constituents.

To the best of our knowledge, this is the first study that demonstrates the anti-H. pylori activity as well as COX-2 and IMPDH selective inhibitory activity of I. confusa underground parts and its isolated compounds.

Novel strategies are urgently required to control H. pylori infection. Our findings recommend further studies on I. confusa bioactive metabolites and also suggest irigenin as an important lead for management of H. pylori infection after detailed in vivo and clinical studies.

Conclusion

Irigenin exhibited promising activity against HpIMPDH enzyme with low activity against human hIMPDH2 than clarithromycin, assuring its selectivity in addition to it’s selective COX-2 inhibitory potential. This study scientifically validated the claimed anti-bacterial activity of I. confusa and has put strong focus on exploring traditional Chinese medicine for novel anti- H. pylori infections controlling drugs.