Onosma mutabilis: Phytochemical composition, antioxidant, cytotoxicity, and acute oral toxicity

Abstract The traditional use of Onosma L. species as a remedy motivated scientists to discover great biological/pharmacological potentials in this plant. In the current study, in addition to the phytochemical composition of methanol (MeOH), water, and ethyl acetate extract of aerial parts of Onosma mutabilis Boiss., an endemic plant species in the flora of Kurdistan, Iraq, in vitro antioxidant, cytotoxicity, and oral toxicity activity were investigated. Results of total phenolic and total flavonoid tests show the MeOH extract superiority, and the results of Gas chromatography–mass spectrophotometer(GS/GS‐MS) show 18 chemical compounds in the MeOH extract, and the majority of the detected compounds were alkaloids (78.77%) and steroids (11.48%), namely as 5,8‐dihydroxy‐2‐(4‐methylpent‐3‐enyl) naphthalene‐1,4‐dione (48.60%), 3‐O‐Methyl‐d‐glucose (27.49%), β‐Sitosterol (6.81%), Phenol, 2,4‐bis (1,1‐dimethyl ethyl)‐, phosphite (3.46%), and 24,25‐Dihydroxycholecalciferol (3.14%). Results of the antioxidant tests show the MeOH extract superiority in the phosphomolybdenum assay, radical scavenging [on 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) and 2,2′‐azino‐bis (3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS)] assays, and reducing power [cupric reducing antioxidant capacity (CUPRAC) and ferric reducing antioxidant power (FRAP)] assays (1.45, 3.54, 2.33, 1.12, 1.62, mg/ml, respectively). The cytotoxicity results of the plant extract are presented as IC50 (inhibitory concentration at 50%) on the prostate cancer cells (DU‐145), mammary cancer cells (MCF‐7), and human cervix carcinoma (Hep2c), at which values ranged from 28.79 to 41.83 μg/ml. Results of the acute toxicity in the dose‐dependent trail (100, 200, 300, 600 mg/kg of MeOH) show the absence of the behavior and appearance changes of female Wister rats. Overall, O. mutabilis extract exhibited significant natural potentials probably because of its polar phytochemicals, which could be an alternative source for remedial, nutrient, and cosmetic manufacture.


| INTRODUC TI ON
The plant is one of the 150 Onosma species known to exist in the Asia continents. The Onosma L. genus belongs to the family Boraginaceae that has been known to exist in the tropical, subtropical, and temperate areas of the world. Turkey is the richest Asian country in terms of Onosma species (95 species) followed by China and Pakistan with 29 and 8 species, respectively. Although earlier reports show about 200 species worldwide, continued systematic botanical studies increased the number of Onosma species, and it is estimated now to reach approximately 230 species (Özgen et al., 2004;El-Shazly et al., 2003;Kumar et al., 2013). The medicinal and industrial importance of Onosma species attracted researchers to explore more about their phytochemical and biological activities in recent years (Pal & Chaudhury, 2010).
Natural products have gained more attention in recent years because of the drawback related to chemically synthetic drugs.
Growing plants rich in certain phytochemicals provide new scope to the cosmetic pharma and agro-industry. A natural metabolically inactive glucose analog called 3-O-Methyl-D-Glucose offers significant protection for dried mouse sperm at above freezing temperatures without the need for poration of the cell membrane and improves desiccation tolerance of keratinocytes (Liu et al., 2012); (Norris et al., 2006). Furthermore, 3-O-Methyl-d-glucose (methyl glucose) is often used to study blood-brain barrier transport and the distribution spaces of hexoses in the brain (Jay et al., 1990). Continued exploration of the genus Onosma species revealed important phytochemicals including flavonoids (hesperidin, apigenin, luteolin), phenolics (rosmarinic acid, ferulic acid, vanillic acid), alkannin, and shikanon also known as isohexenyl-naphthazarins in O. pulchra, O. ambigens, O. gigantea, and O. heterophyllum, each possessing several biological and pharmacological activities Sarikurkcu et al., 2018Sarikurkcu et al., , 2020aSarikurkcu et al., , 2020b. The researchers also suggested O. bracteatum as a drug candidate for the cure of prostate cancer, lung cancer, and breast cancer, as a result of its cytotoxic effect against different cancer cells (Imran et al., 2018). The previous study also shown O. arenaria as rich source of different alkaloid classes (El-Shazly et al., 2003). Alkaloids are organic compounds with significant biological and pharmaceutical potentials (Huizing & Malingré, 1981); (Souza et al., 2020).
The search for plant phytochemicals with greater antioxidant activity has doubled in the last decades. This rising interest in this biological activity of the plant is started when people start to realize the harmful effect of food preserves, which are used to delay exploiting of food, on human health (Menghani et al., 2011). The health damage of food additives was mainly linked to metabolic disorders such as gastrointestinal disorders, cancer, and arthritis, which results from oxidative stress, a condition when the antioxidant defense system cannot neutralize the number of reactive oxygen species and free radicals that are produced by or enter the body. Antioxidant defense system includes innate biological produced elements such as superoxide dismutase, catalase, and hydro peroxidase, and acquired antioxidant molecules from plants. Therefore, consuming plants enriched with antioxidant molecules will empower the antioxidant defense system in fighting free radicals and minimize oxidative damage (Shanmugam et al., 2018). Plant antioxidants mainly include phenolics, flavonoids, and alkaloids, and choosing a suitable extraction method necessary to maximize gained plant antioxidants because of phytochemical or solvent polarity (Wangensteen et al., 2004).
Cancer is a major health problem that includes almost 200 types of cell lines, resulting in uncontrolled cell proliferation and differentiation as spreading into surrounding tissues and organs. The curing process of the cancer patients usually detained by many obstacles, mainly drug resistance, toxicity, and decreased specification (Vukic et al., 2017). Thus, the search for new natural curative agent for controlling those health problems has been doubled in recent years.
Naphthoquinones from O. Visianii Clem and O. paniculata were reported as a strong antiproliferative compound against breast carcinoma, colon cancer, and melanoma cell lines (Kretschmer et al., 2012;Vukic et al., 2018). Furthermore, the phenolic compounds from O.
The medicinal plant's increasing demand by consumers led to the development of multiform supplements from a single plant by various methods. As a result, natural compound studies majorly focused on the identification and maximizing gained isolates, ignoring the chemical profile changes that could threaten a consumer's health. (Sellami et al., 2011;Wojdyło et al., 2014). Hence, toxicity test is considered as a valuable mandatory safety measure for any medicinal plant that claimed traditionally to have curative effects (Newman & Cragg, 2012;Pariyani et al., 2015). The current work inspiration is taken from the traditional usage of onsoma species and considered as the first record on the chemical profile and biological activity of O. mutabilis.

| Plant collection
| 5757 JABBAR in an ultrasonic bath at room temperature for two hours. The solvents drained out by a rotary evaporator in a water bath at 40℃ to obtain the solid crude extract; then, freeze-drying was performed to complete solvent removal. The obtained dry extract was 23.67, 9.42, and 2.34% (w/w) for the MeOH, water, and ethyl acetate extracts, respectively. Then, they were stored at +4℃ until analyzed (Vardanega et al., 2014).

| Phytochemical analysis
The MeOH, water, and ethyl acetate extract of O. mutabilis were investigated qualitatively for total phenolic and total flavonoid by using the spectrophotometer technique (Kähkönen et al., 1999). Then, the MeOH extract was examined qualitatively by using the GC/GC-MS technique (Vardanega et al., 2014); (McLafferty et al., 1989). The analysis detail of both techniques is given in the Appendix S1.

| Statistical analysis
The statistical analysis of the biological activity was applied in triplicate to ensure the accuracy, and results are presented as mean and standard deviation (mean ± SD). The differences in the test activity between the extracts were found by Student's t test (α = 0.05) using SPSS v. 14.0 program. The obtained data were analyzed by using one-way analysis (ANOVA) of the SPSS statistical software package, version 24.0 for Windows. The values of the significance were set at p < .05.  Tables 3 and 4, respectively. Finally, the oral toxicity activity is presented in the Table 5. The data of the following sections are handled in a systematic order.

| Phytochemical profile
As declared by now, the current work investigates the chemical profile and the biological activity of O. mutabilis extract. Thus, the chemical profile results are discussed at first. The chemical constituents analyzed by two different methods, qualitative method applied for the total phenolic and total flavonoid estimation by the spectrophotometer (

| Antioxidant activity
The investigation of the antioxidant activity of O. mutabilis was performed by using different assays. The phosphomolybdenum assay was applied to test total antioxidant activity. The DPPH and ABTS assays were applied to test the free radical scavenging activity of the extracts. Finally, the CUPRAC and FRAP assays were applied to evaluate the reducing power activity. The data results are presented in the (Table 3).
In the phosphomolybdenum assay, the phosphatemolybdenum is incubated with the extract, and the conversion of phophatemolybdenum is estimated to reveal the total antioxidant activity of the extract. The IC 50 value of the methanolic extract in the antioxidant activity was 1.45 ± 0.05 mg/ml and was found to be very close to the Trolox value (1.03 ± 0.02 mg/ml) ( Table 3). The antioxidant activity of the water extract and the ethyl acetate extract was 2.14 ± 0.09 and 3.91 ± 0.26 mg/ml, respectively, and was found to be lower than that of the MeOH extract. The results presented here are good compatible with the chemical profile of the extracts. Based on the Tukey's test results, the total antioxidant activity of the MeOH extract and the Trolox was significantly differed at %5 ( Table 3).
The radical scavenging ABTS assay showed higher scavenging activity by the extracts than that from the DPPH assay. The MeOH extract showed to be more influential in the scavenging DPPH and ABTS radicals that could be linked with its higher phenolic and flavonoid contents (   (El-Shazly et al., 2003;Yıldız et al., 2020;Damianakos et al., 2014a).
The earlier in vivo investigation showed significant antioxidant activity of Onosma armeniacum K. extract and have correlated this activity to the plant's naphthoquinone contents (Cadirci et al., 2007). In the current study, phytochemical profiling showed alkaloids as the main constituents (78.77%) which may be responsible for their efficient antioxidant potentials. The previous study has shown a positive correlation between the increased antioxidant potential of maca extract with its high alkaloids contents 62.4% (Gan et al., 2017). The current study also detected a significant amount of β-sitosterol, a herbal nutraceutical that are recently highlighted as antioxidant and possibly futuristic remedy for numerous health problems (Babu & Jayaraman, 2020). Furthermore, 2,4-bis (1,1-dimethyl ethyl)-, phosphite (alkanox240) was found in significant amount (3.45%) that has been known as an organo-phosphite antioxidant exhibiting excellent hydrolytic stability and reduces peroxide-induced oxidative degradation of most polymeric substances (Johnson et al., 2005). The 24,25-Dihydroxycholecalciferol was another significantly detected compound in the current study, a well-known antioxidant rolling as an antiaging agent by reducing oxidative stress and DNA damages, lowering cell senescence, and deactivating p53-p21signaling pathways (Richardson, 2001). The literature data cited above are considered as reliable confirmation on the contribution of detected phytochemicals to the antioxidant activity of O. mutabilis extracts.

| Cytotoxicity activity
Medicinal herbs have been used therapeutically for ages and are still considered as raw materials for modern remedies. According to the previous estimation, natural products contribute to 60% of today's anticancer drug production (Gordaliza, 2007). Another study reported that herbal medicine is used as a curative agent by 50% of breast cancer and by 37% of prostate cancer patients (Richardson, 2001). Previous preclinical studies showed the efficacy of natural compounds to inhibit human cervical carcinoma and other cancers (Park et al., 2021); (Aung et al., 2017 Liparis Nervosa extract with its pyrrolizidine alkaloid contents . The current study showed an increased percentage of alkaloids in the methanolic extract of O. mutabilis, which could be correlated with its significantly cytotoxicity activity. Previously, the anticancer potentials of alkaloids from traditional medicinal plants have been well screened by highlighting the molecular mechanism of their actions (Mondal et al., 2019). The earlier study also correlated the anticancer activity of O. nigricaule extract with its alkaloid contents, namely deoxyshikonin, b,b-dimethylacryl shikonin, and acetyl shikonin (Ozgen et al., 2010). Additionally, previous researchers reported triterpenoid, bauerenone, and β-sitosterol from Onosma limitaneum extract as effective antiproliferative agent against various tumor cells (Ahmad et al., 2005). Furthermore, several studies have reported shikonin as anticancer agent because of its ability to induce apoptosis in different human tumor cells from gastric cancer, prostate cancer, and breast cancer (Liang et al., 2016;Gara et al., 2015;Lin et al., 2018). Different mechanisms proposed on how shikonin induce apoptosis, and a particular study detected that shikonin stimulates p53-mediated cell cycle arrest and apoptosis in A375-S2 melanoma cells by caspase 9-dependent mechanism (Wu et al., 2004), while another study reported that shikonin induces apoptosis by  (Awad et al., 2001). Furthermore, earlier study reported that β-sitosterol detained colonic epithelial cell proliferation because of its decreased absorption property (Baskar et al., 2012). The literature referenced above was considered as the reliable data on the involvement of detected phytochemicals in the cytotoxicity activity of O. mutabilis extract against tested cell lines.

| Oral toxicity test
The obtained information on the oral toxicity test showed a lack of undesired changes in the appearance, behavior, and life status of rats under treatment of 100, 200, 300, and 600 mg/kg of the MeOH extract of O. mutabilis for 7 days (Table 5).
Exploring the acute toxicity of various doses of the MeOH extract in the current study can be considered a useful safety control for the potentiality of consuming it in repeated administrations.
Mortality, appearance, and behavioral changes are considered as the starting signs of toxicity usually noticed in acute toxicity studies (Lee et al., 2014). Interestingly, our subacute toxicity study showed no significant sign of toxicity. As the rats were safe even after 600 mg/ kg administration for 7 repeated days, the predicted LD 50 of the  (Khalili et al., 2010); (Redzić et al., 2009). Another in vivo toxicity study on rats showed the safety of O. echioides extract in subacute toxicity test and dosedependent increased mortality of rats in acute toxicity test with the LD 50 claimed to be 1,000 mg/kg body weight (Shoaib et al., 2020).
The data provided here can be considered as the first step toward the identification of the phytochemical and biocompatibility of O. mutabilis. However, further studies are required to explore the mechanism of the actions possessed by the phytochemicals and to investigate the toxicity effect of O. mutabilis extract at higher doses for longer periods.

| CON CLUS ION
In the current study, the phytochemical profile of O. mutabilis was provided as useful data behind its biological potentials, namely antioxidant and cytotoxicity activity. Antioxidant activity test results showed the methanolic extract as the strongest antioxidant agent that could serve as food additive to delay expiration.
Phenolic, flavonoid, and alkaloid contents, especially Deoxy shikonin, β-sitosterol, Phenol, 2,4-bis (1,1-dimethyl ethyl)-, phosphite, and 24,25-Dihydroxycholecalciferol, are thought to be involved in the antioxidant activity of the extracts. The cytotoxicity potentials significantly exhibited by the methanolic extract, which may be correlated with its alkaloid and β-sitosterol contents. Hence, encouraging by their natural potentials, the plant species can be an alternative and valuable source for remedial, nutrient, and cosmetic manufacture.

ACK N OWLED G M ENT
The author acknowledged to the Salahaddin University to provide their facility for the current study.

CO N FLI C T O F I NTE R E S T
The author declares no conflict of interest.

E TH I C A L A PPROVA L
The study followed the Iraqi ethical guidelines for the care and use of laboratory animals, and the study protocol was approved by the ECETHC (Ethical Committee of Erbil Technical Health College) (Reference No. 34 in 25-06-2021).

DATA AVA I L A B I L I T Y S TAT E M E N T
The materials related to this study are available in the Appendix S1, in the online version, at doi: