Phytochemical characterization and biomedical potential of Iris kashmiriana flower extracts: a promising source of natural antioxidants and cytotoxic agents

Abstract

Iris kashmiriana belongs to the family Iridaceae and is an important endemic medicinal plant of Kashmir. The current study was designed to determine the phytoconstituents, antioxidant, and cytotoxic potential of ethyl acetate (IRK-ETH) and methanol (IRK-MTH) extracts of Iris kashmiriana flowers. IRK-MTH extract demonstrated maximum radical scavenging activity in DPPH, ABTS, and Superoxide anion radical antioxidant assays with IC₅₀ values of 73.15 μg/ml, 79.05 μg/ml, and 86.52 μg/ml respectively. IRK-ETH and IRK-MTH extracts possessed phenolic (70.9 and 208.5 mgGAE/gdw) and flavonoid (487.7 and 40.55 mgRE/gdw) contents respectively. In MTT assay IRK-ETH demonstrated the highest cytotoxicity towards the MCF-7 cell line with a GI₅₀ value of 49.13 μg/ml. Phase contrast and fluorescence microscopic studies in MCF-7 cells revealed that IRK-ETH extract caused condensation of chromatin, rounding of cells, and nuclear condensation in cells which shows the apoptotic potential of the extract. GCMS analysis for phytochemical characterization revealed the presence of 9 compounds in both extracts which have been reported to possess antibacterial, cytotoxic, and anti-oxidant activities. HPLC analysis confirmed the presence of different polyphenols in both extracts with IRK-MTH extract having maximum polyphenols like epicatechin, rutin, quercetin, vanillic acid, sinapic acid, caffeic acid, chlorogenic acid and ellagic acid. These findings suggest that the flowers of Iris kashmiriana possess very good antioxidant and cytotoxic potential owing to its rich phytoconstituents.

Introduction

Cancer, a condition caused by pathophysiological changes in the natural process of cell division, has become a serious disorder responsible for the deaths of people every year around the globe¹. Recently, more than 19.3 million (19,300,000) cases of cancer were identified and reported; and are expected to rise at a fast pace in the coming years with almost 10 million fatalities in 2020². The majority of cancer treatments come with side effects including hair loss, numbness or chronic pain in certain body areas, harm to vital organs, chemoresistance, and tumor recurrence³. Cancer research, therefore, continues to be focused on the development of new therapies with little to no side effects and improvement of existing therapies⁴. Plant products and their derivatives have been rich sources of therapeutically effective medications for thousands of years⁵ There are four classes of plant-derived anticancer agents being used in the market, the vinca alkaloids (vinblastine, vincristine, and vindesine), the epipodophyllotoxins (etoposide and teniposide), the taxanes (paclitaxel and docetaxel) and the camptothecin derivatives (camptotecin and irinotecan). Plants still hold vast potential to facilitate effective drugs as they are a rich reservoir of chemical compounds that possess chemoprotective potential against cancer⁶. There is ample evidence linking oxidative stress caused by free radicals to inflammation and cancer development. The primary cause of oxidative stress is the excessive generation of reactive oxygen species or free radicals, which is linked to a reduced ability of the body’s own defensive system. Drug candidates with anti-inflammatory and free radical scavenging properties are highly valued as potential anticancer treatments^7,8. They neutralize radicals by donation of their own electrons and termination of the electron “stealing” reaction. Thus, antioxidants are pivotal in the prevention of cancer, cardiac, and other diseases⁹. Numerous studies have reported that consuming medicinal plants, whether as chemical ingredients or raw extracts, is generally linked to a decreased risk of degenerative illnesses brought on by oxidative stress since these plants include antioxidants such as phenolics, flavonoids, vitamins, and carotenoids. Therefore, research and development are needed to create natural antioxidants with higher effectiveness and fewer negative effects¹⁰.

Iris kashmiriana belongs to the family Iridaceae, and is commonly known as ‘Mazarmund’ and ‘Kabriposh’ in the Kashmir region of India¹¹. It is used traditionally by the local Bakarwal community of the Jammu Kashmir region of India for several treatments. The dried rhizome is used in powdered form to treat joint pains and mixture of Iris kashmiriana and Jaggery (Gur) is given to animals to increase their milk production and to overcome general body weakness^11–13. Biological activities of Iris kashmiriana plant extracts include anticancer, antimicrobial, antifungal, and antioxidant activity^14–17. Several compounds have been isolated from Iris kashmiriana like Iriflogenin, Kashmigenin, Iriskashmirianin, Isoirisolidone, and Irisolidone¹⁸. Flavonoids like Iriflogenin, Iriskashmirianin, and Irilone have been demonstrated to possess chemopreventive effects. The genus Iris has historically been used to treat respiratory illnesses and eczema, as well as to lessen discomfort and inflammation^19,20. The current study was planned to evaluate the antioxidant and cytotoxic potential of the Iris kashmiriana flowers.

Materials and method

Plant collection and identification

Iris kashmiriana flowers (Fig. 1) were collected from the graveyard of village Lajoora, district Pulwama Kashmir, northern state of India (Latitude 33°52′21.1296″, longitude 74°53′ 34.2708″). The plant species was identified by Akhter Malik, the plant taxonomist and Botanist affiliated with the University of Kashmir. The plant voucher specimen No.: 8757-KASH was deposited at the Centre of taxonomy and biodiversity University of Kashmir. All methods were carried out in accordance with relevant guidelines and legislation for plant based studies.

Figure 1.

Iris kashmiriana.

Preparation of plant extracts

Flowers were collected, cleaned, air-dried at room temperature and pulverized into powder for extraction. The powder (250 g) was macerated²¹ in different organic solvents in order of increasing polarity viz. hexane, chloroform, ethyl acetate, and methanol, and allowed to stand for 48 h at room temperature with shaking at intervals to yield n-hexane fraction IRK-HEX (1.6%), chloroform fraction IRK-CHL (1.4%), ethyl acetate fraction IRK-ETH (0.56%) and methanol fraction IRK-MTH (5.2%), respectively. The polarities of the solvents were capable of partitioning and separating the secondary metabolites of the fractions as per their solubility. The mixture was filtered with Whatman No. 1 filter paper and the filtrate was concentrated using Buchi Rotavapor R-210, Flawil, Switzerland at a temperature of 40 °C to avoid thermal decomposition of volatile compounds. Concentrated extracts were obtained and stored at 4 °C for subsequent assays and analysis. The yield percent was calculated using the formula given below:

$$\text{Yield}\text{(\% )}=W_1/W_2\times 100,$$

where W₁ is the dry weight of the extract (g) and W₂ is the weight of dried plant material (g)²².

Qualitative phytochemical screening

The phytochemical substances that are extracted using polar solvents have more medicinal significance because they exhibit relatively higher levels of antioxidant activity, reducing characteristics, and free radical scavenging activity²³. Thus, the polar extracts IRK-ETH and IRK-MTH were chosen for the current study. Iris kashmiriana IRK-ETH and IRK-MTH fractions were subjected to qualitative analysis for detection of plant metabolites including phenolic compounds-ferric chloride test, potassium dichromate test; tannin-10% NaOH test; alkaloids-picric acid test; cardiac glycosides-keller-killani test; phytosterols-salkowski’s test; carbohydrates-molisch’s reagent test; proteins-ninhydrin test, xanthoproteic test; flavonoid-concentrated H₂SO₄ test, alkaline reagent test; coumarins-NaOH test; saponins-foam test using standard chemical procedures^24,25 with little modifications.

Determination of total phenolic content (TPC)

The total phenolic content of Iris kashmiriana IRK-ETH and IRK-MTH flower extracts was determined according to the method of Noshad et al.²⁶ with little modifications. To the 100 µl of test solution (200 μg/ml), 900 µl of double distilled water, 0.5 ml Folin Ciocalteu reagent, and 1.5 ml of 20% sodium carbonate were added. The final volume was made up to 10 ml by the addition of double distilled water. Absorbance was read at 765 nm using a multi-well plate reader (BioTek Synergy, HT) after 2 h of incubation. Gallic acid was used as standard compound and TPC was presented as mg of gallic acid equivalent per gram of dry weight of plant extract (mgGAE /gdw).

Determination of total flavonoid content (TFC)

The total flavonoid content of the extracts of Iris kashmiriana flowers was assessed by the method of Kim et al.²⁷. Briefly to the 1 ml of extract solution 4 ml of double distilled water was added followed by the addition of 300 µl of 5% NaNO₂ and 300 μl of 10% AlCl₃. After 5 min of incubation 2 ml of 1 M NaOH was added to the reaction mixture and the final volume was made up to 10 ml. The absorbance was read at 510 nm. Rutin was used as a standard to make standard calibration curve. The TFC was expressed as mg of Rutin equivalents (RE) per gram of dry weight of plant extract (mgRE /gdw).

Antioxidant activity

DPPH assay

DPPH free radical scavenging assay was carried out according to the method of Blois et al.²⁸ with minor modifications. To the 0.3 ml of test solutions (50–800 μg/ml) of IRK-ETH and IRK-MTH extract, 2 ml of 0.1 mM methanolic solution of DPPH was added and shaken well. The reaction mixture was incubated in the dark at 37 °C for half an hour. Finally, the absorbance was recorded at 517 nm. Decreasing absorbance of the reaction mixture indicated higher free radical scavenging capacity. Rutin was used as a standard compound and % radical scavenging activity was calculated using the equation given below:

$$Scavenging(\%)=\left[\left(A_{\mathit{control}}-A_{\mathit{sample}}\right)/A_{\mathit{control}}\right]\times 100$$

where A is the absorbance.

ABTS assay

Free radical scavenging activity of the extracts was also determined using ABTS assay²⁹. ABTS radical cations were produced by reacting 7 mM ABTS solution in deionised water with 2.45 mM potassium persulfate solution and the mixture was kept in the dark for 12–16 h at room temperature. For the experiment, ABTS radical cation solution (ABTS^•+) was diluted with methanol to an absorbance of 0.7 at 734 nm. To the 0.1 ml solution of different extract concentrations (50–800 μg/ml), 3.9 ml of the ABTS^•+ dilution was added. ABTS^•+ was used to prepare the blank. The decrease in absorbance was recorded at 734 nm using a multi-well plate reader (BioTek Synergy, HT) after 6 min of incubation. The percentage inhibition was calculated using the above formula.

Superoxide anion radical scavenging assay (SARS)

The superoxide anion radical scavenging ability of extracts of Iris kashmiriana was carried out by the reduction of nitro-blue tetrazolium³⁰. To the 500 μl extract solution (50–800 μg/ml), 1 ml of 156 μM nitro-blue tetrazolium and 1 ml of 468 μM NADH solution both prepared in 100 mM of phosphate buffer of pH 7.4 were added and mixed gently. The reaction was initiated by adding 100 µl of 60 μM phenazine methosulphate. Then the incubation was given for 5 min at 25˚C. Rutin was used as standard antioxidant. Absorbance decrease was recorded at 560 nm and percentage inhibition was calculated using following formula:

$$\text{\% Scavenging}=\left(\left(A_0-A_1\right)/A_0\right)\times 100$$

where A₀ is the absorbance of control and A₁ is the absorbance of sample.

Cytotoxic activity

The cytotoxic activity of Iris kashmiriana flower extracts was evaluated against brain glioblastoma (LN-18), Human cervix carcinoma (HeLa), and Human adenocarcinoma (MCF-7) cancer cell lines through colorimetric MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay³¹. Different cell lines were purchased from the National Centre for Cell Science (NCCS), Pune, India, cultured in DMEM medium, maintained at 37˚C and 5% CO₂ using a CO₂ incubator. For the MTT assay cells were seeded in 96 well plate at a density of 8 × 10³ cells/well and allowed to attach for 24 h. After 24 h of cell seeding, cells were treated with different concentrations of extract (31.25–500 μg/ml). 20 µl of 5 mg/mL MTT was added to each well after 24 h of treatment and incubated at 37 °C for 4 h. 100 µL of DMSO was added to each well to solubilize formazan crystals. The absorbance was measured at 570 nm and percentage growth inhibition was calculated using the following formula:

$$\text{\% Growth Inhibition}=\left(\left(A_0-A_1\right)/A_0\right)\times 100$$

where A₀ is the absorbance of control (untreated cells) and A₁ is the absorbance of treated cells.

Morphological assessment of cancerous cells

IRK-ETH was found to be more potent than IRK-MTH as revealed by MTT assay so IRK-ETH extract was used for further studies. After the treatment of MCF-7 cells with the IRK-ETH extract, the morphological changes were observed using a phase-contrast microscope and fluorescence microscope.

Phase-contrast microscopy

For analyzing the morphological changes in the MCF-7 cell line, the cells were seeded at a density of 2 × 10⁵ cells/well in 24 well plate and kept in a CO₂ incubator. The following day, cells were treated with GI₅₀ (49.13 μg/ml) of IRK-ETH extract. Morphological changes in cells were observed 24 h after treatment under an inverted phase contrast microscope.

Nuclear staining with DAPI

The nuclear morphology and apoptosis-inducing potential of IRK-ETH extract was assessed in the MCF-7 cell line by DAPI staining³². Briefly, MCF-7 cells were seeded in 6 well plate (4 × 10⁵ cells/well) and after 24 h treatment was given with a GI₅₀ concentration of IRK-ETH extract. Cells were washed with 1X PBS after a treatment period of 24 h followed by fixation using paraformaldehyde. After fixation cells were incubated with 4 μg/ml of DAPI for 30 min in the dark. After staining, the excess dye was removed by washing the cells twice with 1X PBS and cells were examined after mounting a coverslip on a glass slide and viewed under the Nikon A1R Laser Scanning Confocal Microscope system (Nikon Corporation, Tokyo, Japan).

Determination of Mitochondrial Membrane Potential (MMP) using fluorescence microscopy

The effect of IRK-ETH fraction on the changes in mitochondrial transmembrane potential in MCF-7 cells was observed by Rhodamine-123 (Rh-123) staining using a fluorescence microscope³³. MCF-7 (4 × 10⁵ cells/well) were treated with 49.13 μg/ml of IRK-ETH extract for 24 h. The cells were then washed twice with 1 × PBS, followed by fixation with 70% ethanol, and incubated for 20–30 min with Rh-123 solution (2 μg/ml) in the dark. After staining, traces of dye were removed by washing with 1 × PBS, and imaging of the cells was done under a fluorescent microscope (Nikon Eclipse E200, Japan).

Detection of apoptosis by AO/EtBr staining

MCF-7 cells were seeded in 6 well plate at a density of 4 × 10⁵ cells/well. After 24 h cells were treated with GI₅₀ concentration of IRK-ETH extract. Then, attached as well as suspended cells were collected and centrifuged for 5 min at 1500 rpm to form a pellet. The pellet obtained was resuspended in 100 μl of 1 × PBS after decanting off the supernatant followed by incubation in the dark with a mixture of AO/EtBr (5 μl) for 5 min. 20 μl of the stained cell suspension was added to a glass slide, covered with a coverslip, and viewed under a fluorescence microscope immediately³⁴ (Nikon Eclipse Ci, Japan).

GC–MS analysis

Agilent 7890A gas chromatogram coupled to an Agilent 5975C inert XL MSD mass spectrometer with triple axis detector was used to perform GC–MS analysis of the samples. The GC column DB-5(30 m × 0.25 mm × 0.25 µm) temperature was initially kept at 50 °C for 1 min, then 50–250 °C at the rate of 50 °C/min and held for 5 min. Carrier gas helium was used at a flow rate of 0.5 ml/min. Mass spectra were recorded in electron impact mode (EI) with a scan range of 50–600 amu and ionization energy of 70 eV. The temperature of the inlet and transfer line was set at 250 °C. Compounds on GC chromatogram were identified by comparison with Wiley and NIST libraries.

FTIR analysis

The IR spectra of the IRK-ETH and IRK-MTH extract of Iris kashmiriana was acquired with the help of Shimadzu Fourier Transform (FTIR) spectrometer (Model IR Tracer-100). IR spectra was recorded in absorbance mode in the frequency range of 400–4000 cm⁻¹ at a spectral resolution of 4 cm⁻¹.

HPLC analysis

Chromatography study was performed on an HPLC–DAD (Agilent Technologies 1100 series, CA, USA). Briefly, 3 µl of the sample was injected into the C18 column (1.8 µm particle size, 2.1 × 50 mm, CA, USA) maintained at 38 °C. For chromatographic analysis, the continuous gradient of solvent A (0.1% formic acid in water), and solvent B (acetonitrile) was used as elution of the mobile phase. The setting of gradient program was as follows: (0–2 min) 0.5% B, (2–12 min) 10% B, (12–25 min) 21% B, (25–30 min) 32% B, (30–38) 60% B, and (38–40 min) 0.5% B. The flow rate was constant at 0.5 ml/min and the total run time was 40 min. Absorbance was automatically detected by a DAD (UV–Vis) detector at 254 nm.

Statistical analysis

All the experiments were carried out in triplicate and the results were expressed as Mean ± Standard error. The significant differences among all the groups were calculated using One-way Analysis of Variance at p ≤ 0.05 level of significance and the Means were compared using Tukey’s HSD (Honestly significant difference) test.

Results

Qualitative phytochemical screening

The presence of different phytochemicals viz. phenolic acids, alkaloids, cardiac glycosides, phytosterols, tannins, flavonoids, tannins, and coumarins in both the IRK-ETH and IRK-MTH flower extract in Iris kashmiriana was revealed by preliminary phytochemical screening (Table 1).

Table 1.

Qualitative analysis of phytochemicals presents in Iris kashmiriana IRK-ETH and IRK-MTH extracts.

Phytochemical	Phytochemical test	IRK-ETH	IRK-MTH
Phenolic acids	Potassium dichromate test Ferric chloride test	+ +	+ +
Tannins	10% NaoH test	+	+
Alkaloids	Picric acid test	−	+
Cardiac glycosides	Keller–Killani test	+	+
Proteins	Ninhydrin test Xanthoproteic test	− −	+ +
Flavonoids	Conc.H2SO4 test Alkaline reagent test	+ +	+ +
Phytosterols	Salkowski test	+	+
Coumarins	NaOH test	+	+
Carbohydrates	Molisch’s Reagent test	+	+
Saponins	Foam test	−	−

Negative sign (−) shows the absence and positive sign (+) shows the presence of phytochemical.

Total phenolic and flavonoid content

The total phenolic and flavonoid content of both the extracts of Iris kashmiriana flowers are presented in Table 2. The result demonstrated that IRK-ETH extract exhibited higher flavonoid content (487.7 mgRE /gdw) in comparison to the IRK-MTH extract (40.55 mgRE /gdw) while the phenolic content was found to be higher in methanol extract (208.5 mgGAE /gdw).

Table 2.

Total phenolic and flavonoid content of the extracts of Iris kashmiriana flower.

Plant extract	Phenolic content (mgGAE/gdw)	Flavonoid content (mgRE/gdw)
IRK-ETH	70.9 ± 1.362b	487.7 ± 5.68b
IRK-MTH	208.5 ± 0.71a	40.55 ± 4.53a

GAE Gallic acid equivalent, RE Rutin equivalent, gdw gram dry weight.

Values are expressed as Mean ± S.E. (p ≤ 0.005).

Data labels with different letters represent significant difference among the values.

Antioxidant activity

The antioxidant activity of Iris kashmiriana flower extracts was assessed using different antioxidant assays viz. DPPH assay, ABTS assay, and Superoxide anion radical scavenging assay.

DPPH assay

Both the extracts of Iris kashmiriana scavenged the DPPH free radicals in a dose-dependent way (Fig. 2). The IRK-MTH extract was more potent in scavenging the radicals with 50% inhibition at a concentration of 73.15 μg/ml and the extract demonstrated maximum inhibition (87.59%) at a concentration of 800 μg/ml. The IRK-ETH extract exhibited less antioxidant activity with an IC₅₀ value of 125.90 μg/ml (Table 3).

Figure 2.

Antioxidant activities of Iris kashmiriana flower extracts at various concentrations in different assays.

Table 3.

IC₅₀ values of extracts of Iris kashmiriana flower in different antioxidant assays.

Antioxidant assay	IC50 μg/ml
	Rutin	IRK-ETH	IRK-MTH
DPPH	21.34	125.90	73.15
ABTS	38.09	133.06	79.05
(SARS)	31.97	127.19	86.52

IC₅₀ 50% inhibitory concentration.

ABTS assay

In the ABTS assay it was found that IRK-ETH extract exhibited 86.56% inhibition of free radicals at the maximal tested concentration of 800 μg/ml while Rutin and IRK-MTH extract showed 88.8% inhibition at the same concentration (Fig. 2). The 50% inhibition was demonstrated by IRK-ETH and IRK-MTH extract at a concentration of 133.06 μg/ml and 79.05 μg/ml respectively (Table 3).

Superoxide anion radical scavenging assay (SARS)

The scavenging capacity of IRK-ETH and IRK-MTH extract is shown in Fig. 2. The results indicated a concentration-dependent activity against superoxide anion radicals with IC₅₀ values of 31.97, 127.09, and 86.52 μg /ml for rutin (standard compound), IRK-ETH and IRK-MTH extract respectively (Table 3). It is evident from this observation that the Iris kashmiriana flower possesses active components that seem to contribute to radical scavenging activity.

Cytotoxic activity

In this study, the cytotoxic activities of IRK-ETH and IRK-MTH extracts of Iris kashmiriana were tested against HeLa, LN-18, and MCF-7 cancer cell lines (Fig. 3) at an incubation period of 24 h. A wide range of concentrations of both extracts were used ranging from 31.25 to 500 µg/ml. IRK-ETH extract showed better cytotoxic activity with reduction of over 50% cell growth of LN-18 and HeLa cells at a concentration of 75.63 and 91.72 µg/ml respectively. The IRK-ETH extract showed excellent cytotoxicity against MCF-7 cells with a GI₅₀ value of 49.13 µg/ml (Table 4). The IRK-MTH extract also markedly inhibited the cell growth in MCF-7 and HeLa cells with GI₅₀ value of 123.68 and 135.30 µg/ml respectively while the IRK-MTH extract inhibited the 50% cell growth of LN-18 at a concentration of 176.17 µg/ml.

Figure 3.

Cytotoxic potential of Iris kashmiriana extracts IRK-ETH and IRK-MTH against HeLa, LN-18 and MCF-7 cell lines at different concentrations (31.25–500 μg/ml). Values are represented in mean ± S.E. Data labels with different letters represent significant difference among the values.

Table 4.

GI₅₀ values of Iris kashmiriana flower extracts against HeLa, MCF-7 and LN-18 cell lines.

Plant extract	GI50 μg/ml
	HeLa	MCF-7	LN-18
IRK-ETH	91.72	49.13	75.63
IRK-MTH	135.30	123.68	176.17

GI₅₀ growth inhibitory concentration.

Morphological changes visualized in MCF-7 cell line

Following treatment with the IRK-ETH extract, the morphological alterations in MCF-7 cells were examined under the phase-contrast microscope and the fluorescence microscope.

Phase-contrast microscopy

Under a phase-contrast microscope, it was discovered that after treatment with ethyl acetate extract, the MCF-7 cells had detached from the substrate, their cytoplasmic edges had rounded off, and their cell membranes had blebbed which is one of the most significant morphological changes in apoptotic cells while control MCF-7 cells did not show any such characteristics (Fig. 4a).

Figure 4 .

(a) Phase contrast images of MCF-7 cells. (b) DAPI staining of MCF-7 cells visualized under fluorescence microscope. (c) Rhodamine-123 staining of MCF-7 cells visualized under fluorescence microscope. (d) Acridine orange and ethidium bromide (AO/EtBr) staining of MCF-7 cells visualized under fluorescence microscope cells. Cells in green, orange and red colour denoted by the arrows represent live cells, proapoptotic cells and dead cells respectively. (A) Untreated MCF-7 cells, (B) MCF-7 cells treated with IRK-ETH extract (49.13 μg/ml) for 24 h.

Fluorescence microscopy

DAPI, Rh-123, and AO/EtBr dyes were used to stain cells and to visualize them under a fluorescence microscope. DAPI is a fluorescent dye that binds to the AT region of a minor groove of DNA and enhances the nuclear alterations that take place during apoptosis. The MCF-7 cells exposed to GI₅₀ concentration of IRK-ETH extract displayed dramatic morphological alterations with evident apoptotic features, including nuclear fragmentation and condensation of chromatin as indicated by arrows in (Fig. 4b) while untreated MCF-7 cells were normal in appearance. Changes in the mitochondrial membrane potential were analysed using Rh-123 staining. Regulation of Mitochondrial membrane potential (ΔΨm) is important for the survival of cells and any abnormality in ΔΨm indicates cell death by apoptosis³⁵. It was found that control untreated MCF-7 cells had intact ΔΨm as indicated by efficient cellular uptake of Rh-123 and high fluorescent intensity while cells treated with IRK-ETH extract (49.13 μg/ml) exhibited drop in cellular uptake of dye with less fluorescent intensity resulting from the dissipation of ΔΨm (Fig. 4c). Capacity of IRK-ETH extract to induce apoptosis was validated by AO/EtBr dual staining. An increase in apoptosis was seen in comparison to control after treatment with GI₅₀ (49.13 μg/ml) of the IRK-ETH extract as demonstrated in the Fig. 4d. Control cells i.e., viable cells were observed green in color as AO can penetrate the normal cell membrane while cells treated with IRK-ETH extract showed orange and red color indicating early and late apoptosis respectively as indicated by the arrows.

GCMS

The phytochemicals present in the IRK-ETH and IRK-MTH extract of Iris kashmiriana were identified by GC–MS analysis. The compounds with their retention time, percentage area, molecular formula, and molecular structure are presented in Table 5. The prevalent phytochemicals of IRK-ETH flower extract include Benzene, 1,3-bis(1,1-dimethyl ethyl) (4.16%), 2,4-Di-tert-butylphenol (16.60), 9-Eicosene (14.61), Ethyl 14-methyl-hexadecanoate (40.54), n-Propyl 9,12-octadecadienoate (12.24), 1-[3,3-Dimethyl-2-(3-methyl-buta-1,3-dienyl)-cyclopentyl]-2-hydroxy-ethanone (11.84).

Table 5.

GCMS analysis of IRK-ETH extract of Iris kashmiriana flower.

Peak	Retention time	Area%	Compound name	Molecular formula	Structure
1	12.521	4.16	Benzene, 1,3-bis(1,1-dimethylethyl)	C14H22
2	15.995	16.60	2,4-Di-tert-butylphenol	C14H22O
3	20.575	14.61	9-Eicosene, (E)-	C20H40
4	23.505	40.54	Ethyl 14-methyl-hexadecanoate	C19H38O2
5	25.474	12.24	n-Propyl 9,12-octadecadienoate	C21H38O2
6	25.580	11.84	1-[3,3-Dimethyl-2-(3-methyl-buta-1,3-dienyl)-cyclopentyl]-2-hydroxy-ethanone	C14H22O2

The GC–MS investigation of the IRK-MTHextract of flower revealed 4 compounds namely 2-Hexanol, 5-methyl (Area % 36.59), Benzene,1,3-bis(1,1-dimethyl ethyl) (5.51), Di-n-decyl sulfone (25.86%) and N-Methyl nicotinimidate, O-trimethylsilyl (32.04) as demonstrated in Table 6.

Table 6.

GCMS analysis of IRK-MTH extract of Iris kashmiriana flower.

Peak	Retention time	Area%	Compound name	Molecular formula	Structure
1	4.048	36.59	2-Hexanol, 5-methyl	C7H16O
2	12.523	5.51	Benzene, 1,3-bis(1,1-dimethylethyl)	C14H22
3	27.398	25.86	Di-n-decyl sulfone	C20H42O2S
4	29.350	32.04	N-Methyl nicotinimidate, O-trimethylsilyl	C10H16N2OSi

FTIR

Functional groups present in compound of Iris kashmiriana extracts were evaluated through Fourier Transfer Infrared (FTIR) spectroscopic study by their peak values. The absorption spectra of IRK-ETH and IRK-MTH extracts are provided in Fig. 5. The first two peaks in the region of 2850–3000 cm⁻¹ are due to C–H stretching of alkanes³⁶ while peaks in the area 1600–1760 cm⁻¹ correspond to C=O stretching of acetones, aldehydes, esters, and free fatty acids also they represent N–H bending vibrations of amino acids³⁷. The next peaks in the region of 1500–1600 cm⁻¹ correspond to bending vibrations of N–H. The bands in the range of 1500–1000 cm⁻¹ are caused by C–O, O–H, C–H, and C=C stretching, and they may be related to the family of chlorogenic acids, which includes acids like ferulic, caffeic, and coumaric³⁸. The peaks around 910–665 cm⁻¹ are attributed to the stretching vibration of N–H indicating the presence of primary and secondary amines³⁶. Peaks around 518 cm⁻¹ and 601.2 cm⁻¹ correspond to stretching vibrations of organhalogens³⁹.

Figure 5.

FTIR spectra of IRK-ETH and IRK-MTH extracts of Iris kashmiriana flower.

HPLC

Iris kashmiriana extracts were analyzed for the presence of polyphenolic compounds using HPLC. The HPLC chromatograms for the various identified phenolic compounds in both extracts are shown in Figs. 6 and 7 respectively. There are numerous peaks in both the chromatograms, but only those that were found by comparing their retention times to those of the 12 standards available were studied. Table 7 lists the identified compounds. It was found that IRK-MTH extract was rich in polyphenolic compounds with the highest content of epicatechin (923 mg/L) followed by rutin, quercetin, vanillic acid, sinapic acid, caffeic acid, and chlorogenic acid while the ellagic acid content was found to be least among all the polyphenolic standards studied. The IRK-ETH extract showed the presence of fewer phytoconstituents with lesser content which were as follows rutin, vitexin, caffeic acid, vanillic acid, and ellagic acid.

Figure 6.

HPLC chromatogram of IRK-ETH extract of Iris kashmiriana flower.

Figure 7.

HPLC chromatogram of IRK-MTH extract of Iris kashmiriana flower.

Table 7.

Amount (mg/L) of different phytochemicals quantified by HPLC in IRK-ETH and IRK-MTH extracts of Iris kashmiriana.

Standard compound	Concentration (mg/L)
	IRK-ETH	IRK-MTH
Ascorbic acid	–	–
Gallic acid	–	–
Vanillic acid	7.87 ± 0.40	71.53 ± 2.26
Chlorogenic acid	–	42.07 ± 3.29
Caffeic acid	20.42 ± 1.69	44.06 ± 3.63
Coumaric acid	–	–
Epicatechin	–	923 ± 24.81
Sinapic acid	–	44.14 ± 3.87
Ellagic acid	5.0 ± 0.03	11.28 ± 2.64
Vitexin	21.92 ± 0.16	–
Rutin	54.79 ± 4.57	238.56 ± 6.43
Quercetin	–	138.98 ± 7.27

Values are expressed as mean ± SE.

Discussion

The phytoconstituents found in plants like saponins, tannins, phenolic compounds, glycosides, carbohydrates, flavonoids, terpenoids, triterpenes, amino acids, proteins, glucose, and isoflavones also referred to as secondary metabolites give them their unique physical, chemical, and also therapeutic properties like antibacterial, antifungal, anti-inflammatory and anticancer, etc⁴⁰. The results of qualitative phytochemical screening of IRK-ETH and IRK-MTH flower extracts confirmed the presence of various classes of bioactive chemical constituents including alkaloids, cardiac glycosides, proteins, phytosterols, coumarins, saponins, carbohydrates, tannins, phenols, and flavonoids.

While evaluating the quality and biological potential of natural plant extract the levels of total phenolic and flavonoid compounds are thought to be important factors⁴¹. In the present study remarkable amount of phenolics up to 208.5 mg/g GAE and TFC values up to 487.7 mg/g QE were observed for IRK-MTH and IRK-ETH extract, respectively. Previous studies on Iris kashmiriana also emphasize that it is a rich source of phenols and flavonoids as methanolic extract of rhizomes of this plant has been reported to have TPC content of 114.1 mg/g GAE and TFC content up to 131 mg/g QE⁴².

Further identification of various phytochemicals in extracts of Iris kashmiriana was performed using GCMS, FTIR, and HPLC analysis. Nine compounds, belonging to hydrocarbons, alcohols, fatty acids and esters, etc. were identified and characterized in IRK-ETH and IRK-MTH extracts using GCMS analysis that are known to possess important bioactivities like 2,4-Di-tert-butylphenol has antioxidant and anticancer properties⁴³. It also shows antifungal and anti-biofilm activity against Candida albicans⁴⁴. A study by Choi et al.⁴⁵ reported the protective effect of this compound against myloid-beta peptide (Aβ_1–42)-induced neurotoxicity by reducing oxidative stress. 9-Eicosene is known to possess antimicrobial and cytotoxic properties. Benzene, 1,3-bis(1,1-dimethyl ethyl) identified in IRK-ETH and IRK-MTH extract has antibacterial properties⁴⁶. FTIR is an analytical technique that offers rapid and non-destructive fingerprinting of powdered samples or plant extracts allowing to identify the chemical constituents and determining the structure of compounds⁴⁷. So, FTIR analysis was employed to determine the functional groups present in IRK-ETH and IRK-MTH extracts which revealed the presence of alkyl halides, esters, primary and secondary amines, amino acids, chlorogenic acids, alkanes, and free fatty acids, corresponding to the compounds detected in GCMS and HPLC analysis.

Numerous studies have shown that plant compounds like polyphenols are advantageous because of their comparatively low toxicity, effectiveness in preventing cancer, and regulating DNA damage induced by oxidative stress⁴⁸. HPLC analysis was carried out to identify the polyphenols in extracts using 12 standard polyphenolic compounds. Eight polyphenolic compounds were found in IRK-MTH extract in appreciable amounts viz. epicatechin, rutin, quercetin, vanillic acid, sinapic acid, caffeic acid, chlorogenic acid and ellagic acid while 5 polyphenolic compounds viz. rutin, vitexin, caffeic acid, vanillic acid and ellagic acid were identified in IRK-ETH extract. Polyphenols like Epicatechin, Vitexin, and Rutin have also been reported in other species of the genus Iris like I. pseudacorus L., I. schachtii Markgr. and I. germanica L., etc. thus validating our results¹⁹.

Many diseases affecting humans are thought to be influenced by reactive oxygen species which easily combine with various biomolecules like carbohydrates and lipids etc. and oxidise them making them docile and causing damage to cells or tissues leading to the development of disease. This harmful impact of reactive oxygen species in biological processes makes radical scavenging activities extremely important⁴⁹. Since several processes contribute to antioxidant capacity, using a technique based on one mechanism could not accurately reflect antioxidant activity⁵⁰. Therefore, several antioxidant assays were utilized to assess the antioxidant activity. It was found that IRK-MTH extract showed higher antioxidant capacity in all scavenging assays with IC₅₀ values of 73.15, 79.05, and 86.52 μg/ml in DPPH, ABTS, and Superoxide anion radical scavenging assay respectively. The scavenging power increased with the increase in the extract concentrations. These results were in agreement with previous studies that found higher antioxidant properties in the ethanolic leaf extract of Iris kashmiriana¹⁷. Also, the ethyl acetate fraction of rhizomes of Iris kashmiriana has been reported to show 95.12% DPPH radical scavenging activity at a concentration of 250 µg/mL⁵¹.

The above activity can be attributed to the redox characteristics of phenolic or flavonoid molecules found in extracts which are crucial in absorbing and neutralizing free radicals, thereby stopping the chain reaction of free radicals. According to recent research, diets high in polyphenols contribute significantly to illnesses linked to oxidative stress because of their capacity to fight free radicals⁵². Iris kashmiriana is also a rich source of polyphenolic compounds as confirmed by the HPLC analysis of extracts which may contribute towards its radical scavenging activity.

Additionally, Iris kashmiriana flower extracts demonstrated potent cytotoxicity against brain glioblastoma (LN-18), Human cervix carcinoma (HeLa), and Human adenocarcinoma (MCF-7) cancer cell lines. IRK-ETH extract showed the highest antiproliferative activity against MCF-7 cells, also pronounced effect was seen on other cell lines. Potent anticancer activity has been reported by Abdullah in the methanolic extract of flowers of Iris barnumiae on HT-29. PC3, MCF-7 cancer cell lines⁵³. Similar results have been obtained in methanolic rhizome extract against adenocarcinoma cell line (A549) and colon adenocarcinoma (Caco-2)⁵⁴ .

Iris species have huge reserves of secondary metabolites, especially flavonoids and isoflavonoids which are known to have different therapeutic properties like anti-bacterial, anti-inflammatory, and anti-tumour activity so this cytotoxic activity may be attributed to these classes of phytochemicals⁵⁵. To further study anti-cancer effects, IRK-ETH extract was used because it demonstrated greater cytotoxic efficacy against MCF-7 cells among all tested cell lines. Comparing treated and untreated cells, the phase contrast images demonstrated that IRK-ETH extract reduced the number of MCF-7 cells, and caused cell shrinkage and cell separation from the monolayer surface. The IRK-ETH extract was also investigated for its potential to induce apoptosis, one of the main causes of cell death. As seen using DAPI, AO/EtBr, and Rh-123 dye staining, the MCF-7 cells treated with IRK-ETH extract displayed typical apoptotic hallmarks including condensation of chromatin, DNA fragmentation, cell shrinkage, formation of apoptotic bodies, and loss of cell membrane integrity which were absent among the untreated cells. Our research indicated that IRK-ETH extract-treated cells had an increase in the number of cells going through apoptosis, as evidenced by the presence of orange/red chromatin in the MCF-7 cells (Fig. 4d). After treating A549 cells with the ethanolic extract of I. taochia, Kandemir et al. observed a rise in apoptotic cells by staining with annexin V⁵⁶. Numerous studies have confirmed that phenolic and flavonoid compounds induce apoptosis in cancer cells⁵⁷. The presence of coumarins, glycosides, phenolic acids, flavonoids, and other phytoconstituents may be responsible for the  Iris kashmiriana flower’s potent antioxidant and cytotoxic effects.

Conclusion

In the current study, Iris kashmiriana extracts demonstrated promising antioxidant and anti-cancer properties. The polyphenolics and other phytoconstituents of these extracts as identified by GC–MS analysis were believed to have contributed to these biological activities. Ultimately, the findings offer a biochemical justification for further chemical analysis in addition to animal and human research centered on chemotherapeutic and antioxidant approaches.

Author contributions

Chandni—investigation, data curation, methodology, analysis, interpretation, validation, writing—original draft. Sheikh Showkat—investigation, analysis, interpretation, software, editing the manuscript. Ambika Saloni, Gulshan Bhagat, Sajad Ahmad—analysis, software, interpretation, editing and finalizing the manuscript. Satwinderjeet Kaur—study conception, design, investigation, data curation, methodology, validation. Zakir Showkat Khan, Gurjeet Kaur, Gholamreza Abdi—investigation, editing, finalizing the manuscript.

Funding

No funding was provided.

Data availability

Data related to this research is included in the article.

Competing interests

The authors declare no competing interests.