INTRODUCTION:

The various phytogeographical areas of Jordan are rich in a variety of wild plants¹. Twenty percent of all Jordan’s plant species are medicinal plants that are used by local inhabitants and in pharmaceutical industry². There are more than 2500 plant species, 868 genera, and 142 families^1,3-5. Medicinal plants that are rich in bioactive compounds are abundant in Jordan’s geographic regions. In Jordan, more than 485 species of medicinal plants and 99 families have been identified as therapeutic agents⁶.

Medicinal plants in Jordan can be exemplified by Varthemia iphinoides, Bongardia chrysogonum, Ajuga chia, Salvia palaestina, Micromeria nervosa and Rubus sanguineus and many others ^2,3,7,8.

There has been increasing interest in the extracts of medicinal plants because they have potential of numerous benefits as a source of drugs to all mankind. Nowadays, the discovery of natural and plant -based products that can prevent the growth of cancer cells with minimum side effects, is urgent and essential⁵³. The medicinal value of these plants lies on bioactive phytochemical constituents that produce definite physiological action on the human body. Examples on the most important bioactive phytochemical constituents are alkaloids, essential oils, flavonoids, tannins, terpenoids, saponins, phenolic compounds and many more⁹. A wide variety of free radical scavenging molecules are found in plants, such as flavonoids, anthocyanins, carotenoids, vitamins, dietary glutathione, and endogenous metabolites are rich in antioxidant activities⁴⁶.

A variety of plant families in Jordan has been investigated for their anti-tumoral effect^10-14. In Jordan, cancer is the second leading cause of death after heart disease, and breast and colon cancers are among the most prevalent types¹⁵. The genus Alcea includes more than 40 subspecies¹⁶. Some of them are not studied for their health-related properties, and the investigated ones were not given enough attention. A. setosa is prevalent in east Mediterranean area¹⁶. It is a perennial herb that is rich of pollen and nectar and characterized with pink flowers and 1–2 m tall stalks with a diameter of 8–13 cm^17,41.

The plant is traditionally orally consumed in some countries as a diuretic, expectorant, and emollient, to treat inflammation and asthma, and to relieve stomach and intestine pain^18,19. The roots are used for cough and tooth inflammation³. Methanolic extract from flowers of A. setosa was found to be compromised mainly of polyphenols and flavonoids²⁰. Leaves methanolic extract from A. setosa has been studied for its anti-tumor potential against mouse fibrosarcoma cells, and no cytotoxic activity was found²¹. A. setosa has been evaluated for its antioxidant activity only twice earlier. In either study, it did not show any significant effect^20,22. With regards to the phytochemical analysis of essential oil and methanolic extract, no previous studies in the available literature were found to be performed on A. setosa.

MATERIAL AND METHODS:

Plant material:

A. setosa flowers were collected from Mahis and Wadi Al- Seer (31°57'26.2"N 35°50'46.1"E 31°57'35.4"N+ 35°50'17.6"E) in North western Jordan in May, 2020. The plant was taxonomically and authentically identified by Professor Sawsan Oran; a plant taxonomist, (Department of Biological Sciences, the University of Jordan). Voucher specimens have been deposited at the herbarium of the Department of Biological Sciences- University of Jordan [specimen 4].

Plant Extraction:

The flowers of Alcea setosa were dried at room temperature (23-25 °C) for (2-3) weeks at botany research lab at the department of biological sciences at the University of Jordan; dried plants were powdered using an electrical blender. The powdered plant then exposed to methanolic extraction. 100 gm of each powdered plant was dissolved in 950 ml methanol and stirred using hotplate magnetic stirrer. The solvent left for seven days then the extraction filtered by Whitman no.1 filtered paper. The filtrated solvents were then evaporated using rotary evaporator at 60 °C. The evaporated extract was kept in refrigerator for further study (stock extracts)²³.

Identification of extract components:

For stock solution preparation, an appropriate amount of Alcea setosa methanolic extract was dissolved with 2.0 ml of Dimethyl sulfoxide-DMSO (analytical grade), then completed to 50 ml by Acetonitrile. The sample was centrifuged at 4000 rpm for 2.0 min, 1.0 ml was taken and transferred to auto-sampler and 3.0 µl was injected. for identification of exact MS and retention time. All the other reagents, Acetonitrile, methanol, water, and formic acid used were LC/MS grade. All standards used for identification of ms/z and the retention time.

A Bruker Daltonik (Bremen, Germany) Impact II ESI-Q-TOF System equipped with Bruker Dalotonik Elute UPLC system (Bremen, Germany) was used for screening compounds of interest. Standards for identification of m/z with high resolution Bruker TOF MS and exact retention time of each analyte after chromatographic separation were used.

Cell culture:

Human colorectal adenocarcinoma cells (CACO2), breast cancer cells (T47D), and normal human fibroblasts (MRC5) were purchased from American Type Culture Collection Company (ATCC). The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) mixed with 10% fetal bovine serum (FBS), 1% L-glutamine and penicillin (100 U/ml) and incubated in a humidiﬁed atmosphere of 5% CO₂ at 37°C. After three days of incubation the cells were harvested by trypsin-EDTA solution and incubated for 10 minutes. 3 ml of culture media was added to the flask, and then the cells were transferred to a tube and vortexed. Finally, the cells were counted, and viability test was performed by trypan blue dye test. The cells were seeded in multi-well plates at the appropriate density recommended for each test^35,37.

Cytotoxicity Assays

Trypan exclusion assay:

Trypan blue assay is one of the earliest and most common methods for counting cells and determining increase or decrease in number of viable cells to indicate proliferation activity³⁸. Viable cells with intact plasma membranes that are not destructed by the extract remain clear and exclude the blue dye, while nonviable cells with ruptured membrane, regardless of death mechanism, are stained blue under the light microscope. Cells were seeded at high concentration (1.0 x10⁴ cells/ml) in 96-well plates and incubated at 37°C to allow for cell attachment. Then 100 µl of drug mixed with media was added to each well and allowed to incubate for 72 hours. After 72 hours, the contents of the wells were emptied by trypsinization and 25 μl of the cell suspension and 25 µl of trypan blue dye (sigma) were mixed in a mixing well. After 30 seconds, 25 µl of the mixture was added to a hemocytometer and observed under microscope. The number of stained cells and the total number of cells were counted. The IC50 indicating a net loss of cells following treatment was calculated from:

[(Ti-Tz)/Tz] x 100 = – 50, where Ti is the initial cell count and Tz is the final cell count³⁸.

Neutral Red Assay:

Neutral red assay is one the most common employed tests for the detection of cell viability following exposure to anticancer substances. The assay is based on detecting the accumulation of the neutral red dye in the lysosomes of viable, uninjured cells³⁹. According to previous procedure⁴⁰, about 10,000 cells from each cell line were seeded in each well of 96-well plates and incubated overnight at 37°C and 5% CO₂. The extract of A. setosa was prepared by mixing 16 µl of the extracts stock (25 mg/ml) with 984 µl media, and using the equation: C1*V1 = C2*V2. A serial dilution of (800, 400, 200, 100, 50) µg/mL were prepared, and incubated again for 24 hours. Doxorubicin was used as positive control. Then, the cells were washed with phosphate-buffered saline PBS (1X) and the supernatant was discarded. A total of 100 μl neutral red (NR) solution (50 μg/ml) was added and incubated at 37°C for 2 hours. NR then was removed, and wells were washed with PBS, and after 10 minutes, absorbance was detected by a dual-wavelength UV spectrometer at 520 nm with a 650 nm reference wavelength. The percentage of antiproliferative activity compared to the untreated cells was determined as:

% Antiproliferative activity = [100 × (Absorbance of untreated group−Absorbance of treated group)]/Absorbance of untreated group.

MTT cytotoxicity assay:

The diluted extract of A. setosa (25 mg/ml) was added to about 10,000 cells from each cell line (T47D, CACO2, and MRC5) at final concentrations of 800, 400, 200, 100, 50, 25, 12.5, 6.25 and 3.125 µg/ml, and the cells were incubated for 72hrs. After exposure, 3-(4,5-dimethylthiazol-2w-yl)-2,5-diphenyl tetrazolium bromide (MTT) was added to the wells and incubated for another 4 hours. 100 ul of DMSO solution was then added to solubilize the MTT crystals before reading optical density (OD) by multi-well plate reader (Bio-Tek Instrument, USA) at 570 nm using a reference wavelength of 630 nm^35,37,50,51. Additionally, doxorubicin at final concentrations of 0.1, 0.5, 1 ,5, 10, 25, 50, 100, 200 µg/mL was used as positive control. The percentage of viability was calculated as the absorbance ratio between treated and untreated (control) wells; hence the absorbance of untreated cells was considered as 100%. Percentage of growth inhibition of cells was calculated as follows: % Inhibition = 100 − (Treated OD/Non-treated OD) × 100)²¹. IC50 values were calculated as concentrations that exhibited 50% inhibition of proliferation on the tested cell line.

Antioxidant activity:

Following a published protocol³³, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity assay was employed to measure the antioxidant activity. DPPH is useful to assess antioxidant activity of specific compounds of extracts, and is a stable free radical reactive with substance able to donate a hydrogen atom⁴⁹. Briefly, a volume of 200 mL of 400, 600, 800, and 1000 µg/ml of the extract or 100, 50, 25, 12.5, 6.25, 3.125, and 1.56 ug/ml ascorbic acid (standard) were prepared in methanol and added to 2 mL DPPH (0.21 mM in 95% ethanol). The mixture was shaken, left for 60 min at room temperature in the dark, and the absorbance was detected at 517 nm in a spectrophotometer. The percentage of DPPH inhibition were calculated using the following equation: percentage of inhibition = [(Ac -As)/(Ac)] x 100, where Ac is the absorbance of the control reaction and As is the absorbance of the sample reaction. The sample concentration (in 1 mL reaction mixture) providing 50% inhibition (IC50) was estimated by plotting percentages of inhibition against concentrations of sample.

Statistical analysis:

All assays were conducted in triplicate. The results were expressed as mean ± SD, and the differences between the means were tested for significance in the statistical analysis software IBM SPSS Statistics Version 23 (Armonk, New York, USA) using one-way analysis of variance (ANOVA) followed by Tukey post-hoc test. The significance level was set at P < 0.05.

RESULTS AND DISCUSSION:

A. setosa composition:

The methanolic extract of A. setosa flowers gave an amount of (~ 5g) of paste. The extract was analyzed by means of Liquid Chromatography–Mass Spectrometry which combined the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. The separated compounds were successfully identified by applying MS data library matching coupled with comparison standards for identification of m/z with high resolution and exact retention time of each analyte after chromatographic separation. Results shown in table 1 represents the components of methanolic extract from A. setosa.

Table 1: Chemical composition of Alcea setosa methanolic extract analyzed by LC-MS.

No.	Name	m/z meas.	RT [min]	Molecular Formula	Area of peaks
1	3,5-Dimethoxy-4-hydroxyacetophenone (ketone)	195.0613	4.92	C10H12O4	25302
2	3-Rha-7-Rha Quercetin (NMR)( flavonol derivative)	593.1463	5.55	C27H30O15	1570084
3	Apigenin-7-O-glucoside (Apigetrin)( Flavone derivatives)	431.0941	7.54	C21H20O10	41321
4	FAHFA 18:1; FAHFA 2:0/16:1; [M-H]- (Fatty acids)	311.2183	15.94	C18H32O4	175079
5	FAHFA 18:2; FAHFA 2:0/16:2; [M-H]- (fatty acids)	309.203	14.78	C18H30O4	125602
6	Genistein (isoflavones derivative)	269.0425	9.94	C15H10O5	413195
7	Hispidulin (flavone)	299.0527	10.79	C16H12O6	146875
8	Isorhoifolin (flavone)	577.1516	6.33	C27H30O14	2035325
9	Kaempferol-3-O-glucoside (Flavone derivatives)	447.0889	6.56	C21H20O11	744671
10	Salicylic acid (organic acid)	137.0224	5.75	C7H6O3	60954
11	Scopoletin (coumarin derivative)	191.0327	5.07	C10H8O4	22262
12	Tiliroside (flavone derivative)	593.1254	9.06	C30H26O13	339072

Fig. 1: LC-MS chromatogram of Alcea setosa methanolic extract

Table 2: IC₅₀ values (mean ± SD µg/ml) of A. setosa extract from three cytotoxicity assays.

Cytotoxicity assay	Treatment	Cell line
Cytotoxicity assay	Treatment	Caco II	T47D	Fibroblasts (MRC5)
Trypan blue	ME	74.14 ± 0.42	110.3 ± 0.36	162.4 ± 0.26
Neutral Red	ME	134.4 ± 0.26	164 ± 0.21	200 ± 0.36
MTT assay	ME	138.4 ± 0.32	142.6 ± 0.23	446.3 ± 0.42
MTT assay	Doxorubicin	4.5 ± 0.36	2.66 ± 0.44	6.3 ± 0.09

The difference in IC50 values between tumoral and normal cells suggests the potential of extract to be used in treatment of cancer. Moreover, according to the classification of natural constituents cytotoxicity, that describes ingredients with IC50 values of 100 – 1000 µg/ml as potentially harmful to cancerous cells³⁴, our cytotoxicity results indicate the possibility of considering A. setosa methanolic extract as a therapeutic suggestion in cancer treatment. The significance of the differences between the mean percentages of the viability according to extract concentration is illustrated in Fig. 2 and Fig. 3 for the results of MTT and neutral red assays, respectively. For all the studied cell lines, Fig. 2 indicates significantly lower viability mean values recorded for the highest concentration of extract. Similarly, as shown in Fig. 3, neutral red results illustrate that the extract inhibited the growth of the three cell lines on a dose dependent manner; compared to the control group, treated cells showed significant decrease in viability as the dose of extract increased.

The cytotoxicity of A. setosa could be explained by the high content of flavon derivatives such as Kaempferol-3-O-glucoside as explained by Al-Qudah and his team^25,26. Interestingly, Tin and his colleagues have reported cytotoxic activities by Isorhoifolin of A. setosa crude ethanol extraction against HepG2 cancer cell with IC50 of 47.03 µg/mL²⁷.

In addition, the presence of bioactive phenolics such as Apigenin, Kaempferol-3-O-rutinoside and Quercetin 3-O-glucoside supports the belief in Kurdistan folk medicine that the root of Euphorbia condylocarpa plant is an effective treatment for cancer as suggested by Hassan and his team²⁸. Cytotoxicity of A. setosa extract maybe attributed to the presence of Genistein; an Isoflavon derivative, and is a natural tyrosine kinase inhibitor which exhibited protection from cancers. Genistein was shown to be anti- proliferative and an inducer of cell cycle arrest at the G₂-M phase in prostate, breast, and jurkat T cell leukemia cell lines^29,32.

Another study has investigated the polyphenol extract of Hibiscus sabdariffa on some cancer cell lines, one was estrogen receptor-expressing breast cancer cell line (T47D); results have showed 50% growth inhibition in a dose dependent manner⁵². Those findings suggest that A. setosa phenolic compounds played an important role in inducing cytotoxicity in our experiments. According to Azab’s discussion of the modern research reports of the biological activities of the various species of Alcea genus, Alcea rosea is the most investigated subspecies, whereas, the very common subspecies A. setosa, have not been studied yet¹⁶. With regards to A. setosa in Jordan no cytotoxicity data were found for the comparison purpose.

Antioxidant activity:

The antioxidant activity of A. setosa extract was assessed using DPPH assay. The concentrations ranged from 200 ug/ml to 1000 µg/ml. The scavenging ability of different compounds is concentration dependent and increases with concentration gradient. Ascorbic acid (100 µg/ml) was used as a positive control. The IC50 of ascorbic acid and A. setosa extract was 4.1ug/ml, 30.0 µg/ml respectively. The antioxidant activity of A.setosa extract is expressed in Table 3 in terms of IC50 values. The IC50 value for the DPPH radical scavenging activity was 30.0 ± 0.26 µg/ml.

Table 3: Antioxidant activity of J. phoenicea EO (Mean ± SD µg/ml).

Sample	IC50
A.setosa	30.0 ± 0.26
Ascorbic acid	4.1 ± 0.15

Several studies have described the antioxidant activity of A. setosa extract; a study concerning A. setosa, revealed the studied extracts exhibited very weak antioxidant activity. The leaf extract was more potent which displayed an inhibition of 72% of the radical at 1 mg/mL ²². Our results indicated that A. setosa flower extract exhibited a moderate scavenging activity with IC50 of 30 µg/ml.

In another study, the IC50 for the DPPH radical scavenging activity was above 1000 µg/ml²⁰.

The moderate antioxidant activity shown in this study is potentially attributed to the high content of Salicylic acid in the extract³⁰; Peak area for Salicylic acid in the extract was 60954. High phenolic content, such as flavons and flavanols in the A. setosa extract may be the contributor to the moderate scavenging activity exhibited²⁹. A similar study on ethanolic extract of Euphorbia hirta showed a dose dependent antioxidant activity due to the presence of phenol and flavonoid which exhibit high radicle scavenging ability⁴⁷. These phytochemicals can scavenge the reactive oxygen species (ROS) in the body, such as hydroxyl radical, hydrogen peroxide superoxide anion, and singlet oxygen. The oxidative damage initiated by these species may lead to DNA damage⁵⁵.

Phenolic and flavonoid compounds are essential secondary metabolites that contain an aromatic ring with at least one hydroxyl group. Phenolic compounds donate electrons and mainly is the hydroxyl groups which contribute to antioxidant activity. Alkaloids, flavanoids, tannins, and steroids are common phytochemicals present in plant methanolic extracts⁵⁴. Phenolic compounds prevent oxidative stress, inhibit free radicles, decompose peroxide, and scavenge oxygen species in biological systems. Flavonoids are polyphenolic compounds with low molecular weight, and are good antioxidant source that increase the overall antioxidant power of an organism and protect it against lipid peroxidation⁴². In fact, the isoflavones, have exhibited to inhibit NF-κB activation stimulated by ROS suggesting their ability to scavenge free radicles. Isoflavones could have inhibited cancer growth by inducing apoptosis and the modulating expression of the genes related to the cell growth and apoptotic processes³¹.

CONCLUSION:

To our knowledge, this is the first study for the chemical analysis for the extract contents of A. setosa and the evaluation the antiproliferative of antioxidant potential for the Jordanian species A. setosa. The methanolic extract of A. setosa from Jordan was found to be rich in flavons, fatty acids flavons derivatives, ketone derivative, flavonol derivative, organic acid, and coumarin derivative. The biological active compounds explain preferential in-vitro antiproliferative and antioxidant potential of the extract on the tested cancer cell lines. A. setosa is recommended for further studies and identification of the main phytochemical components and their anti-proliferative mechanism.

FUNDING INFORMATION:

This project was funded by the Deanship of academic research of the University of Jordan- Amman.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 27.02.2021 Modified on 25.03.2021

Research J. Pharm.and Tech 2021; 14(12):6447-6454.

DOI: 10.52711/0974-360X.2021.01115

Hana R. Bajes1*, Sawsan A. Oran2, Yasser K. Bustanji3

Hana R. Bajes¹*, Sawsan A. Oran², Yasser K. Bustanji³