Hypericum alpestre extract exhibits in vitro and in vivo anticancer properties by regulating the cellular antioxidant system and metabolic pathway of L‐arginine

Conventional treatment methods are not effective enough to fight the rapid increase in cancer cases. The interest is increasing in the investigation of herbal sources for the development of new anticancer therapeutics. This study aims to investigate the antitumor capacity of Hypericum alpestre (H. alpestre) extract in vitro and in vivo, either alone or in combination with the inhibitors of the l‐arginine/polyamine/nitric oxide (NO) pathway, and to characterize its active phytochemicals using advanced chromatographic techniques. Our previous reports suggest beneficial effects of the arginase inhibitor NG‐hydroxy‐nor‐ l‐arginine and NO inhibitor NG‐nitro‐Larginine methyl ester in the treatment of breast cancer via downregulation of polyamine and NO synthesis. Here, the antitumor properties of H. alpestre and its combinations were explored in vivo, in a rat model of mammary gland carcinogenesis induced by subcutaneous injection of 7,12‐dimethylbenz[a]anthracene. The study revealed strong antiradical activity of H. alpestre aerial part extract in chemical (DPPH/ABTS) tests. In the in vitro antioxidant activity test, the H. alpestre extract demonstrated pro‐oxidant characteristics in human colorectal (HT29) cells, which were contingent upon the hemostatic condition of the cells. The H. alpestre extract expressed a cytotoxic effect on HT29 and breast cancer (MCF‐7) cells measured by the MTT test. According to comet assay results, H. alpestre extract did not exhibit genotoxic activity nor possessed antigenotoxic properties in HT29 cells. Overall, 233 substances have been identified and annotated in H. alpestre extract using the LC‐Q‐Orbitrap HRMS system. In vivo experiments using rat breast cancer models revealed that the H. alpestre extract activated the antioxidant enzymes in the liver, brain, and tumors. H. alpestre combined with chemotherapeutic agents attenuated cancer‐like histological alterations and showed significant reductions in tumor blood vessel area. Thus, either alone or in combination with Nω‐OH‐nor‐ l‐arginine and Nω‐nitro‐ l‐arginine methyl ester, H. alpestre extract exhibits pro‐ and antioxidant, antiangiogenic, and cytotoxic effects.


| INTRODUCTION
Based on World Health Organization (WHO) reports, cancer is a prominent global cause of death, responsible for approximately 10 million fatalities in 2020.This accounts for nearly one in six deaths caused by cancer worldwide. 1Based on future prognosis, it is anticipated that the annual number of cancer-related deaths will reach 13 million by the year 2030. 2 In terms of prevalence, the most frequently observed types of cancer include breast cancer (with an annual incidence of 2.26 million cases), lung cancer (2.21 million), colon cancer (1.93 million), and prostate cancer (1.41 million).
Conventional cancer treatment methods such as chemotherapy, radiation therapy, immunotherapy, and so forth, have several limitations, including recurrence, side effects (e.g., stomatitis, fatigue, cognitive dysfunction, bowel and bladder dysfunction, mental health issues, etc.), development of multidrug resistance, and high cost. 3,4ere is growing interest in reaffirming the chemopreventive properties of plant-borne bioactive substances. 5Several bioactive phytochemicals are effective in the therapy of various diseases including cancer.Around 70% of the anticancer medications that have received approval from the FDA in the past few decades are estimated to have originated from natural sources or are synthetic substances that mimic natural substances. 2 Studies have shown that natural products exhibit potential antitumor properties through their ability to target disrupted signaling mechanisms that underlie various cancer hallmarks, such as drug resistance, metabolic reprogramming, stemness features, Epithelial-Mesenchymal Transition (EMT), and the maintenance of the tumor microenvironment. 2 Phytochemicals have been shown to exhibit promising clinical outcomes through their redox-active, anti-inflammatory, and anticarcinogenic activities by modulating abnormal signaling associated with tumor formation and progression. 6,7e suppression of cancer cell proliferation by phytochemicals is frequently associated with perturbations in cellular redox balance. 6ncer cells possess a significant antioxidant potency, enabling them to sustain the redox balance at nontoxic levels.In contrast, cancer cells exhibit higher basal levels of reactive oxygen species (ROS) compared to normal cells, rendering them more susceptible when subjected to further elevation of ROS levels. 8Therefore, targeting intracellular redox systems with phytochemicals with pro-oxidant properties may induce an excessive burden of oxidative stress within cancer cells.This, in turn, triggers various cellular mechanisms leading to cell death, thereby impeding cancer progression and activating the intrinsic pathway for apoptosis. 8,9Emerging research indicates that pro-oxidant agents have the potential to function as a novel class of anticancer drugs that specifically target tumor cells. 10In numerous clinical trials and studies, anticancer properties of various phytochemicals or herbal extracts have been investigated for their ability to modulate oxidative stress within the tumor environment.
Those substances include berberine, piperine, curcumin, artemisinin, resveratrol, paclitaxel, isothiocyanates, green tea, mistletoe extract, noscapine, and so forth. 11On the other hand, substances of natural origin in combination with conventional chemotherapeutic agents may act synergistically by enhancing the anticancer effect of the drugs and simultaneously reducing their adverse side effects. 2,6In addition to their direct anticancer effects, natural products have also been found to enhance the susceptibility of cancer cells toward other anticancer drugs, leading to better therapeutic outcomes with fewer side effects.Combining natural products with other drugs or inhibitors can further enhance their therapeutic benefits by inducing apoptosis and reducing drug resistance through the modulation of diverse underlying mechanisms.For instance, studies have demonstrated that combining pharmacologically active phytochemicals with arginase and NOS inhibitors can be a promising strategy for treating cancer. 6We have recently reported that ethanol extract of Rumex obtusifolius in combination with arginase (nor-NOHA-N ω -OH-nor-Larginine) and NOS (L-NAME-Nω-nitro-L-arginine) inhibitors exhibited beneficial therapeutic outcomes in vivo, in the rat model of breast cancer. 6e modulation of amino acid metabolism has emerged as a promising strategy in diverse forms of anticancer therapy due to its crucial role in regulating the immune response. 12Arginine, an essential amino acid, plays a crucial role in various cellular mechanisms, such as the production of nitric oxide (NO) and polyamines.It is also a direct activator for different kinases. 13NO and polyamines are key participants in proliferation, angiogenesis, and metastasis.Regulation of these substances is essential for the processes of cancer suppression. 14It is possible to regulate the

Significance Statement
Our HPLC/MS/MS data shows that Hypericum alpestre (HA) contains a variety and abundance of natural antioxidants.amount of these substances by inhibiting the Arginase and NO synthase (NOS). 15The high activity of NOS, an enzyme that produces NO, has been detected in cancer cells from different types of tissues and has been linked to tumor grade, rate of proliferation, and the expression of signaling molecules. 16Our previous study demonstrated the anticancer effect of nor-NOHA and L-NAME substances in experimental models of breast cancer. 17,18The use of plant extract compounds to simultaneously target various cellular aspects of cancer while also regulating the L-arginine pathway through the administration of inhibitors is an innovative approach and holds great promise in the field of cancer treatment.
Hypericum (family Hypericaceae) is a genus of plants that includes 500 species of herbs, shrubs, and trees well known for their healing properties, and widely used in the folk medicine of different countries. 19,20Particularly, the extracts of these plants are used to treat wounds and bruises, dysenteries, acute mastitis, jaundices, hepatitis, sore furuncles, skin inflammation, nerve pain hemoptysis, epistaxis, metrorrhagia, irregular menstruations, burns, and hemorrhages. 19,21In traditional medicine in some countries, it has also been used for the treatment of various tumors. 224][25][26][27][28] Upon screening of ethanolic extracts of 11 wild plant species for their cytotoxic properties, H. alpestre extract showed one of the most potent in inhibiting the growth of A549 (lung adenocarcinoma) and HeLa (cervical carcinoma) cancer cells. 29Therefore, it was selected for further comprehensive analyses as a potential source of anticancer substances or preparations.
The main goal of this study was to evaluate the effectiveness of a new cancer treatment method proposed by our research group.This method involves administering plant extracts in combination with arginase and NOS inhibitors to a rat breast cancer model in vivo.

| Chemicals and reagents
All chemicals used in the research were purchased from Sigma-Aldrich.The OxiSelect™ Cellular Antioxidant Assay Kit originated from Cell Biolabs, Inc.

| Collection and extraction of plant material
Hypericum alpestre subsp.Polygonifolium (Rupr.)Avet.& Takht.(HA) aerial parts were collected from the Tavush region of the Republic of Armenia, at elevations ranging from 1500 to 2500 meters above sea level.The plant was submitted to the Herbarium of YSU, where they were it was assigned a Voucher specimen serial number (ERCB 13206).The grounded HA aerial parts were macerated with 96% ethanol at a 10:1 solvent-tosample ratio (v/w) as described before. 30For in vivo experiments, stock solutions were prepared, in which dried HA extracts were dissolved in DMSO.These solutions were subsequently diluted in saline, resulting in a final concentration of 1% v/v of DMSO. 30A crude alcoholic extract was prepared as previously described, with a concentration of 50 mg dry weight (DW)/mL, for the cellular antioxidant activity (CAA) and 3-(4,5dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) tests. 29The percent yield of substances in the 50 mg DW/mL extract was quantified by the following procedure performed in triplicates: first, 500 µL of the extract was dried, and then the dry weight was determined by weighing each sample.The percent yield was calculated to be 17.33 ± 2.31%.

| Phytochemical characterization and antioxidant profiling of H. alpestre extract
Metabolomic characterization of H. alpestre extract was done with an advanced high-resolution mass spectrometry technique.Dionex Ultimate 3000 UHPLC chromatographic system (Thermo Fisher, Dionex) fitted with SynergyTM Hydro-RP A (150 × 4.5 mm, 4 µm, Phenomenex) column was used, maintained at a temperature of 30°C, as previously described. 31A Q ExactiveTM Focus quadrupole-Orbitrap mass spectrometer (Thermo Fisher) equipped with a heated electrospray ionization source (HESI II) was connected to the advanced chromatographic system.
The profiling of H. alpestre antioxidants was performed with the use of the HPLC-DAD system (Agilent) coupled with a Pinnacle PCX Derivatization Instrument (Pickering Laboratories Inc.) and UV-VIS detector (Agilent). 62 mmol/L), and antibiotics (100 µg/mL streptomycin, 100 U/mL penicillin, and 0.25 µg/mL of Amphotericin B).The cells were grown in a Smart Cell Incubator (Heal Force) at 37°C with 5% CO 2 and a humidified atmosphere, following the previously described protocol.32 Potential contamination of cultured cells with mycoplasma was routinely assessed using the Universal Mycoplasma Detection Kit from ATCC (30-1012 K™).The experiments were performed using cell lines from passages 7 to 20.

| Antioxidant potential of H. alpestre extract determined by chemical tests and cellular antioxidant activity assay
HA extract's antioxidant capacity was determined by standard spectrophotometric methods, using 1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals according to the procedure described before. 33The cellular antioxidant activity of H. alpestre extract was evaluated in HT29 cells using the OxiSelect Assay Kit. 346 | Cell growth-inhibiting (MTT assay), genotoxic and DNA-protecting properties (Comet assay) of H. alpestre extract The impact of different concentrations of HA extract on the growth of HT29 and MCF-7 exposed for 3, 6, 24, or 72 h was evaluated using the MTT test, following the methodology described previously. 35The genotoxic properties of the HA extract, as well as its potential to protect DNA against the damaging effects of hydrogen peroxide, were assessed with comet assay 36 following the established procedure described. 6

| Animals and experimental groups
The experimental procedures involving animals were conducted following the guidelines outlined in Directive 2010/63/EU. 37hical approval was obtained from the Armenian National Center of Bioethics.Female albino rats weighing between 120 and 150 g were randomly distributed into 10 groups, with each group comprising eight animals (except for the DMBA group, which had 10 animals as detailed in Tables 1 and 2. Following 28 weeks of DMBA administration, the rats were euthanized by isoflurane using a precision vaporizer with an induction chamber and waste gas scavenger (RWD Life Science).Isoflurane was administered slowly up to 5% until respiratory arrest occurred.Animals were monitored for the cessation of respiration and remained in the euthanasia chamber for an additional 60 s after respiration had ceased. 38e animals underwent a 1-week acclimatization period before the experiment.They were housed in cages with a total area of 3500 cm 2 , in a temperature-controlled room set at 25°C, with a 12-h light/12-h dark cycle and relative humidity of 50−55%.The animals were maintained under constant environmental and nutritional conditions (Animal Care house, YSU, Faculty of Biology).
4][45][46] The tumor was first detected by touch and its size was subsequently measured and monitored regularly.At the end of the experiment, the type of breast cancer was confirmed through a histological analysis.
Blood was obtained from the lateral tail vein as described. 40,43jections (i.p.) of extract and chemotherapeutic agents were performed following "The Laboratory Rat" protocol. 44The laboratory personnel conducted daily health monitoring and tumor measurements.The body weights of the animals were monitored weekly throughout the entire experiment.
The selection of plant extract concentrations was based on in vitro findings and information available in the literature from similar studies involving other plant extracts. 45Tumor volume (V, cm3) was assessed through caliper measurements and computed using the equation V = π × (4/3) × (l/2) × (w/2) × (h/2), where l, w, and h correspond to the length, width, and height of tumor, respectively.The tumor height was determined using the formula h = 2/3 l.
No differences were observed between the following groups: control (healthy rats) group, control + saline, and control + DMSO/saline/HA groups.The toxicity profile was evaluated in the control group showing a lack of toxicity at 2.5 mg/kg of H. alpestre extract.

| NO quantity measurement
The levels of NO in the blood plasma were quantified by measuring nitrite ions using the Griess assay. 46Briefly, 100 μL of plasma samples were combined with 100 μL of Griess reactant.Subsequently, the supernatant obtained was transferred to tubes with cadmium pellets and left to incubate at room (25°C) for 12 h, facilitating the conversion of nitrate to nitrite.The absorbance was measured at a wavelength of 550 nm (a standard curve constructed using NaNO 2 ).
T A B L E 1 Quantity of rats in experimental groups during distinct weeks of experiments.a The total number of rats examined during those weeks is presented.
b There were six rats in the DMBA group at Week 24, and mortality reached 100% from Week 25 to 28. c Breast cancer.

| Antioxidant enzyme systems
The catalase activity in different tissues was measured by the titration method 47 and expressed as the μmol of hydrogen peroxide broken down in 1 mg of protein. 48Peroxidase activity was determined according to the spectroscopic method, 49 and the activity of peroxidase was expressed in μmol/min/mg protein.
Superoxide dismutase (SOD) activity of different tissues was measured according to the degree of inhibition of the adrenaline self-oxidation reaction rate as shown before. 50SOD activity was expressed in conventional units of activity; 50% inhibition of the reaction was taken as 1 conventional unit of enzyme activity.

| Arginase activity
The modified Diacetyl Monoxime colorimetric method was employed to assess the arginase activity in blood plasma. 51The enzyme activity was quantified as the amount of urea formed per second, measured in micromoles.

| Polyamines
Plasma polyamine levels were assessed with a Seiler Thin-Layer Chromatographic assay modified for blood analysis as described before. 51About 50 μL of the dansylated sample was applied to the pre-absorbing area of silica gel plates.The plates were developed for 2 h using chloroform/triethylamine (25:2 v/v) mobile phase.The lanes were carefully scraped off.The collected polyamines were eluted with 2 mL of ethyl acetate, and the resulting solution was measured for absorption at 505 nm.

| Lipid peroxidation assay
The lipid peroxidation in blood plasma was assessed by measuring malondialdehyde (MDA) with the thiobarbituric acid method. 51

| Quantity of ammonia
The indophenol method, as previously described, 51 was used to measure the concentration of ammonia in the blood plasma.

| Interleukin-2 (IL-2) level by ELISA
The concentrations of IL-2 were quantified in both blood plasma and tumor samples using an ELISA kit (RAB0288, Sigma-Aldrich) following the manufacturer's instructions. 6 A B L E 2 Experimental design.

| Histological analysis
After DMBA administration, palpable mammary tumors were surgically removed at specific time points.The excised tumors were then fixed (10% buffered formalin) and prepared for histological analysis using H&E staining. 51Light microscopy (BM-190/T/SP Trinocular, Boeco) was used to visualize the stained tissue sections, and images were captured with a B-CAM10 CCD camera (Boeco).To assess the histological characteristics of breast cancer, the Nottingham Histologic Score system 51   human digestive tract, which is directly exposed to ingested food constituents, including plant compounds, while the MCF-7 cells were selected as an in vitro equivalent and thus justification for the use of the rat breast cancer model. 47,52 found that the ethanolic extract of HA inhibited the growth of both HT29 and MCF-7 cells in a time-and dosedependent manner (Figure 1A,B).In both cell lines, considerable cell growth inhibition was seen after  53 Genotoxic and anti-genotoxic effects of HA extract in cellular models were investigated using the comet assay.This method allows for assessing the genotoxicity of a substance by measuring the fragmentation of cellular DNA in response to exposure to genotoxins.
The comet assay can also be employed to assess the DNA protection effect of tested compounds against the genotoxic impact of ROS.
Neither genotoxic nor DNA protective effects were observed in HT29 cells (Figure 1C,D).

| H. alpestre combined with chemotherapeutic agents increases survivability and decreases tumor multiplicity
In vivo experiments were carried out using a rat mammary carcinogenesis model induced by DMBA.2C).These findings suggest that combining H. alpestre extracts with L-NAME or nor-NOHA could be a potent therapeutical approach to prevent cancer development or its treatment.

| Correlation between arginase activity and polyamine quantity
We  Nitrite anions levels decreased by Week 28 but remained higher than in the Control group (p < .0001, Figure 3B).The BC + HA + L-NAME group exhibited the most beneficial effect in terms of the nitrite levels, displaying a 20−50% reduction compared to groups where these agents were administered separately (p < .0001).
DMBA induction significantly increased blood NH 4 + and MDA quantity versus the Control group (p < .0001, Figure 3C,D).All experimental groups except for BC + nor-NOHA demonstrated reduced ammonia levels at Week 13 (Figure 3C).The HA and HA + L-NAME groups presented the levels of ammonia similar to the Control group.Ammonium concentrations remained low in all treatment groups by Week 28, with no significant differences observed, except for the BC + nor-NOHA group, where ammonia levels increased by 25% compared to the Control group.
The HA + nor-NOHA and HA + L-NAME groups did not display an elevation in MDA levels between Weeks 13 and 28 compared to the Control group (Figure 3D).At Week 13, MDA levels in the BC + nor-NOHA and BC + L-NAME groups resembled those in the BC group.Therefore, the combinations of HA with L-NAME and nor-NOHA demonstrated a strong inhibition of lipid peroxidation during the post-treatment period and did not induce hyperammonemia.

| H. alpestre regulates antioxidant enzymatic system work
Further, the level of lipid peroxidation in the tumor, liver, kidneys, and brain of rats was explored by determining the amount of MDA.As shown in Figure 5, the processes of lipid peroxidation were more intense in the kidneys while it was relatively low in the brain.
Specifically, the levels of MDA were 1.19 µmol/g in the kidneys and 0.147 µmol/g in the brain.In the liver, the concentrations of MDA were decreased by 88.22% in the HA-treated versus control groups (Figure 5A).Furthermore, the levels of MDA in the kidneys and brain were decreased by 69.16% and 18.37%, respectively in the HA-treated versus control groups.We also found that the level of MDA in the liver and kidney of the cancer group decreased by 32.65%, and 48.57%, respectively, compared with the nontumor group, meanwhile, but the level of lipid peroxidation in the brain did not change significantly compared to control animals.The results show that there is no increase in MDA in the tissues in the BC group compared to the Control group, but it is markedly increased in the blood (Figure 3D).Despite all this, the enzymes of the antioxidant system in tissues are more sensitive to treatment.The catalase activity in the brains was 2.1 and 1.9 times lower than in the liver and kidneys.This low catalase activity explains the low antioxidant capacity 54 and a high level of lipid peroxidation in brain tissue.
Stimulation of the activity of catalase and peroxidase enzymes was observed in the studied organs of healthy rats treated with HA extract (Figure 5B,D).The increase in catalase activity was observed in all tested organs, with the maximum stimulation in the brain (by 16.47%).Regarding peroxidase activity, HA extract contributed to the stimulation by 50% in the liver.The activity of catalase and peroxidase enzymes in the liver of animals with breast cancer (BC group) increased by 39.3% and 58.53%, respectively (Figure 5D).
Peroxidase activity was significantly stimulated in the kidneys as well.
The catalase activity in the brain of animals with breast cancer increased by 25% versus controls, whereas peroxidase activity decreased by 78.54%.Interestingly, the combination of HA extract and L-NAME caused a total inhibition of the activity of catalase and peroxidase enzymes in the brain tissue (Figure 5B,D).
It was found that in healthy rats, SOD is the most active in the liver and brain (Figure 5C) which is probably the reason for the decrease in the number of free radicals and, consequently, the decrease in the level of MDA compared to other organs.In the liver of healthy animals that received HA extract, SOD activity decreased by 23.47% compared to the Control group but increased by 37.25% in the kidneys, in contrast to the activity of catalase and peroxidase under similar conditions.In the liver and brain of the BC group, SOD activity decreased by 48.98% and 34.69%, respectively, compared to healthy animals, and significantly increased in the kidneys (Figure 5C).
Significant stimulation of SOD activity in the liver and brain of animals treated with HA extract was detected.
In the tumors, our data revealed that the level of MDA in animals treated with HA extract was increased by 34.7% compared to the BC  1 and 2 for information on the sample size.
group.In the BC + HA + nor-NOHA and BC + HA + L-NAME groups, the amount of MDA is reduced by 2 and 3 times, respectively.SOD activity in the tumors was increased in the BC + HA, BC + HA+nor-NOHA, and BC + HA + L-NAME groups compared to the BC group.
The activities of catalase and peroxidase were decreased by 22.92% and 58.69%, respectively, in the BC + HA group compared to the BC group (Figure 5E).In the case of combinations, the values obtained are close to the results obtained during exposure to the plant alone.

| The change of IL-2 under the influence of H. alpestre
IL-2 is a crucial cytokine for the regulation of T-cell activation and measuring this cytokine can be a treatment indicator for cancer. 55ood levels of IL-2 were 2 times higher than in the BC + HA group compared to the BC group (Figure 6A).IL-2 production in the tumor and blood of rats was observed to be 1.5-fold higher in the BC + HA + L-NAME group compared to the BC group (Figure 6B).
However, the quantity of IL-2 in the blood of the BC + HA + nor-NOHA/L-NAME group did not differ significantly from the BC group and was 1.5 times lower compared to the Control group.

| H. alpestre combined with chemotherapeutic agents attenuate cancer-like histological alterations
The histopathological findings are displayed in The CAA test is specifically designed to replicate biological conditions, including a pH of 7.4 and a temperature of 37°C.
Furthermore, it incorporates the factors of bioavailability, distribution, and cellular metabolism of the antioxidants being tested, so it is more biologically relevant than chemical tests.
Based on the CAA assay the reproducibility was not achieved (variations from prooxidant activity to antioxidant activity were found in independent repetitions).Nonetheless, we can state that although H. alpestre ethanol extract showed considerable antioxidant properties during chemical tests it acted as a pro-oxidant in cellular models depending on the homeostasis of these particular cells.
3.9 | The characterization of phenolic substances in H. alpestre extract The Supplementary Table presents a  in the literature as well (Table 3).7,12-Dimethylbenz(a)anthracene (DMBA), a PAH, is a widely studied carcinogen used to induce tumors in rodents. 84Human exposure to DMBA can occur from cigarette smoke, car exhaust, and the burning of organic substances such as coal, oil, wood, and garbage, as well as smoked and burned foods. 18Along with the development of civilization, the influence of all this is factors is increasing.Now it is more than necessary to find means to neutralize and prevent the pathological effects of these compounds.That 'saviors' belong to natural compounds, especially those that can be, or already are, used in food.The herb H. alpestre can be endowed with just such a property.This study aimed to determine the anticancer effects of HA.
Considering the high biological and cytotoxic properties of H. alpestre antioxidant/pro-oxidant and anticancer properties of ethanol extract of H. alpestre (HA) aerial parts were explored in the study.We have previously shown a strong cytotoxic activity of HA extracts toward two other cancerous cell lines: HeLa and A549. 85The strong antioxidant potential of the HA extract in different chemical tests has been also shown before. 86In a previous study, we showed a high extracts. 23The presence of certain substances found in HA extract significantly contributes to its overall antioxidant activity. 87This is due to the strong association between the phenolic content and antioxidant capacity.However, in our previous research, we also found that the H. alpestre extract can also present a prooxidant activity. 28Specifically, it caused downregulation of catalase activity and an upregulation of SOD activity in BV-2 cell lines, which indicates that exposure to the extract can induce oxidative stress in these cells. 88Prooxidant properties of H. perforatum extract and the other well-explored species within the same genus were also reported. 89,90e authors state that it could be due to the substance hypericin, which is known for its pro-oxidant properties.The use of prooxidants to modulate ROS has emerged as a promising approach to selectively target malignant cells during cancer treatment with chemotherapy agents. 91Therefore, the investigation of anticancer properties of HA extract can have much interest.

| The relationship between arginase and NOS is an important factor in anticancer therapy
The excessive expression of arginase and iNOS has been previously linked to inflammatory disorders. 88Therefore, it was proposed to utilize the interaction between arginase and NOS in cancer as a potential strategy for practical anticancer approaches.Through the use of combined treatments, it became possible to prevent inflammation caused by ROS while effectively suppressing the activities of arginase and NOS. 88,92As a result, this promotes antiinflammatory processes.
It is important to note that inflammation plays a significant role in the development of drug resistance in cancer, 93 so we hypothesize that the HA extract could potentially prevent the development of drug resistance.Specifically, it is known that during inflammation, there is an increase in the activity of COX-2, 94

| HA exhibits high antioxidant activity in an in vivo model
In vivo, data using rat breast cancer model revealed that the HA extract activated SOD and CAT enzymes in the liver, brain, and tumors of rats in experimental but not control groups, thereby enhancing antioxidant protection.The latter was accompanied by a decrease in the concentration of MDA in the blood and tumors, which also confirmed the antioxidant protective effect of the extract.
From the published literature, antioxidant protection leads to antiinflammatory, antiproliferative, antimetastatic, and antiangiogenic effects. 95The above-mentioned processes were studied by evaluat- The activity of arginase and NOS enzymes may be accompanied by an increase in the activity of COX-2, which is a key process in inflammation. 97Further studies will be needed to determine the role of HA in immune responses in the settings of breast cancer.
The observed impact of HA extract on the antioxidant enzymatic system of the animals in both control and experimental groups is likely a result of its high phenolic content, which was reported earlier. 86Phenolic substances and terpenoids are involved in redox reactions and act as neutralizing agents for ROS and the effectiveness of the latter in reducing the risk of cardiovascular diseases and diabetes mellitus has been proven. 98We assumed that after the injection of animals with HA extract, their body received additional  Dihydromyricetin -Antioxidant, anticancer, anti-inflamatory 67,68 Myricetin-arabinoside + Antioxidant 69 Myricetin -Antioxidant, anticancer, anti-inflamatory 70,71 Flavonols Quercetin-hexoside + Anticancer, Antioxidant, and anti-inflammatory 72,73 Quercetin-pentoside isomer + Anticancer, antioxidant, and anti-inflammatory 72,73 Quercetin + Antioxidant, anti-inflammatory, and anticancer 60,70,[73][74][75] Quercetin-acetyl-glucoside + Antioxidants  substances remaining unknown (Table 1).The metabolomic analysis of HA aerial part extracts unveiled the presence of several major phenolic constituents, suggesting that these substances might contribute to its cytotoxic effects (Table 2).Considering the strong in vitro growth inhibiting properties of HA extract against different cancerous cell lines including the breast cancer model, its anticancer properties were further explored in in vivo rat mammary carcinogenic model.

| Genotoxicity of H. alpestre
According to literature data, plant extracts containing antioxidant substances are able to protect cellular DNA from H 2 O 2 -induced damage.However, antioxidant substances may sometimes increase H 2 O 2 -induced DNA damage through the stimulation of prooxidant cellular mechanisms . 101Considering that HA extracts showed prooxidant properties in CAA assay in this study, which was also observed in other research, we assumed that HA extract could cause cellular DNA damage in HT29 cells as well.It was important to show that no mutagenic and carcinogenic risks are to be expected before proposing medical applications.The extract did not possess anti-genotoxic properties as well.Our results do not confirm reports suggesting genotoxic or anti-genotoxic properties of species within the Hypericum genus.For example, Sarimahmut et al. 102 showed that H. adenotrichum extract possessed genotoxic effects on human lymphocytes demonstrated by different methods including comet assay.In contrast, another study showed that H. brasiliense extract did not exhibit a genotoxic effect in in vivo rat models. 103In several studies, anti-genotoxic effects of Hypericum spp extracts were found.
For instance, high protection by H. retusum flower, fruit, and seed extracts was shown. 104Thus, literature data suggest that genotoxic or anti-genotoxic activities of plant extracts within the Hypericum genus may differ.

H
. alpestre possessed a remarkable cytotoxic effect on HT29 and MCF-7 cancer cell cultures and acted as a prooxidant in cellular models.The combination of H. alpestre extract with inhibitors of the L-arginine metabolic pathway presents synergistic Anticancer effects against the induced mammary tumor.The H. alpestre extract alone or combined with L-arginine metabolic regulatory compounds, demonstrates significant potential for the development of novel therapeutic models.

3 | RESULTS 3 . 1 |
Cytotoxicity and genotoxicity of H. alpestre extract MTT assay was used to evaluate the inhibitory activity of H. alpestre (HA) extract in two cancer cell lines.The colon adenocarcinoma (HT29) and breast cancer (MCF-7) cell lines were employed in the study.The HT29 cell line is widely used as a cancer model of the next measured the impact of HA extract on the activity of arginase, and the level of polyamines in the rat breast cancer model, either alone or in combination with L-NAME or nor-NOHA.Arginase activity was significantly increased in the blood of the BC group at Weeks 8, 13, and 28, versus the Control group (p < .0001,Figure3A).However, the combinations of HA with nor-NOHA orL-NAME    prevented this elevation at Weeks 13 and 28.During these specific time points, the arginase activity in the BC + HA + nor-NOHA group gradually reverted to levels comparable to the control group.The increase in arginase activity was accompanied by changes in blood polyamine levels.These levels were notably elevated in the BC relative to the Control group at all examined time points post-DMBA exposure (p < .0001,Figure4A,B,C).However, a decline in polyamine levels was observed in the BC + HA+nor-NOHA and BC + HA + L-NAME groups at Weeks 13 and 28.The most effective reduction in polyamines was recorded in the BC + HA + nor-NOHA group.Specifically, in the BC + HA + nor-NOHA group, the levels of putrescine, spermidine, and spermine remained unchanged at Week 13 and similar to the control group (Figure4A,B,C).At Week 28, the BC + HA + nor-NOHA group maintained approximately 18% lower levels of polyamines than the BC group.In BC + HA + nor-NOHA and BC + HA + L-NAME groups, the reduction in polyamine levels was about 1.5−2 times greater than in the groups treated with nor-NOHA, L-NAME, and HA alone.

F I G U R E 1
Inhibition of cell growth determined by MTT test in (A) HT29 and (B) MCF-7 cell cultures treated by Hypericum alpestre extract for 3, 6, 24, and 72 h versus untreated control.Genotoxic effect of H. alpestre expressed as % DNA in comet tail evaluated in HT29 cells (C), assessing the ability of H. alpestre to protect DNA of HT29 cells from H 2 O 2 -induced oxidative damage expressed as % DNA in comet tail evaluated in HT29 cells-(D).Results are expressed as mean ± SD from three independent experiments.C− (Negative control), nontreated cells; C+ (Positive control), cells treated with 150 μM H 2 O 2 .3.4 | H. alpestre downregulates NO generation, lipid peroxidation, and ammonia levelDuring Weeks 13−28, an increase in blood nitrite levels was observed in the BC group (p < .0001,Figure3B).However, the administration of nor-NOHA, L-NAME, and HA extract and their combinations decreased the nitrite levels at Weeks 13 and 28 after DMBA injection (p < .0001,Figure3B).Nitrite levels in the BC + HA, BC + HA + nor-NOHA, and BC + HA + L-NAME groups approached levels similar to the Control group at Week 13.

F I G U R E 2
Kaplan−Meier survival curve (A), tumor multiplicity (B), and volumes (cm 3 ) (C) in all experimental groups.Significance (p < .05)was calculated by Log-rank (Mantel-Cox) (p = .0306)and Logrank (test for trend: p = .0120)tests in (A), Dunnett's multiple comparisons test in (B) and two-way ANOVA and Tukey's multiple comparisons test in C. Results are represented as mean ± SD. *p < .05,**p < .01,***p < .001,****p < .0001(n = 10 for the DMBA group, n = 8 for the other groups).Survivability (a), tumor multiplicity (B), and volume (cm 3 ) (C) were monitored over a 28-week study period.Tumor volume was measured by summing the volumes of all tumors within one rat in each experimental group.

Figure 7 .
In the BC group (Figure7A,B), spindle cell carcinoma, invasive lobular carcinoma, grade 1 papillary carcinoma, and sarcomatoid carcinoma were identified.Invasive lobular carcinoma was observed in the BC+nor-NOHA, BC + L-NAME, and BC + HA groups (Figure7C−E).The spindle cell carcinoma displayed round spindle cells with pleomorphic nuclei arranged in an uncertain pattern (indicated by black arrows).In invasive lobular carcinoma, the cancerous cells exhibit a distinctive pattern where they arrange themselves in single-file lines and display vacuoles within their cytoplasm.Grade I papillary carcinoma showed numerous papillary ledges supported by thin connective tissue cores (indicated by black arrows).Sarcomatoid carcinoma displayed spindle cells without visible epithelial separation.In the BC + HA + L-NAME group, histopathological examination revealed lobular carcinoma in approximately 50% of cases (poor prognosis) (Figure7F), and 50% of rats exhibited ductal in situ proliferation defined as Grade I (Figure7G).BC + HA + nor-NOHA rats demonstrated sarcomatoid carcinoma (60% with poor prognosis) (Figure7H), and 40% of rats displayed ductal in situ proliferation defined as Grade I (Figure7I).Additionally, Figure7Jpresents the assessment of the vasculature in breast tumors.The BC group exhibited a threefold increase in vascular surface area compared to the Control group (p < .0001).The groups treated with BC + HA+nor-NOHA and BC + HA + L-NAME both showed significant reductions in tumor blood vessel area.3.8 | Antioxidant capacity of H. alpestre extractSince we observed the beneficial effects of H. alpestre in vitro and in vivo, we then measured the antioxidant activity of the HA extract using DPPH/ABTS assays (Figure8A,B) and the CAA tests F I G U R E 4 The change in rat blood of polyamines levels (PUT: A, SPD: B, SPM: C) at various key weeks.The results are presented as mean ± SEM. *p < .05,**p < .01,***p < .001,****p < .0001,NS: not significant.See Tables 1 and 2 for the sample size.HA, Hypericum alpestre; PUT, putrescine; SPD, spermine; SPM, spermine.F I G U R E 5 Regulation of activity of antioxidant enzymes by the herbal extract and L-arginine pathway inhibitors.Results are presented as mean ± SEM. *p < .05,**p < .01,***p < .001,****p < .0001.n = 5. (Figure 8C).For both DPPH and ABTS tests, the slope of the line that describes the relationship between extract concentration and the number of scavenged radicals was calculated to find the regression coefficient.This coefficient, which represents stoichiometric value, was determined after 10 min of reaction (n 10 ).According to data obtained, DPPH/ABTS stoichiometry values of the H. alpestre were 2․ 129 and 3․174, respectively, indicating that a 1 µg of H. alpestre extract reduced 10 −3 µg of DPPH/ABTS radicals, resulting in the respective stoichiometry values (Figure 8A,B).
comprehensive list of compounds detected in the HA extract, including the molecular formulas, and theoretical and experimental mass.The categorized substances include condensed tannins and their derivatives (21 substances), hydroxycinnamic acids and their derivatives (18 substances), flavonols (17 substances), hydroxybenzoic acids and their derivatives (14 substances), flavones (12 substances), flavanones (11 substances), flavan-3-ols (7 substances), hydrolyzable tannins (7 substances), anthraquinones (7 substances), lignans (6 substances), fatty acids (5 substances), isoflavones (4 substances), naphthols (4 substances), dihydrochalcones and their derivatives (3 substances), coumarins and their derivatives (2 substances), tocopherols (2 substances) triterpenoids (1 substance), and flavanonols (1 substance) (See Supporting Information: Table and Figure 9A for further details).The substances were carefully examined based on their UV-visible spectrum and assigned to specific groups.The identification of the substances involved analyzing full scan MS and MS2 spectra acquired for prominent m/z signals detected in negative ion mode, their retention time, and information from literature data.Major phenolic constituents identified in HA extract with possible bioactive properties are presented in Table 3. Online post-column derivatization of analytes using the ABTS reagent was carried out following the HPLC analysis of the sample.This process involved the reduction reaction during standard ABTS colorimetric tests, which caused notable alteration in the UV-visible spectrum and resulted in a change in the absorption of the ABTS reagent (leading to discoloration).The chromatogram recorded after derivatization at 734 nm showed negative peaks, indicating the presence of redox-active substances (Figure 9C).The DAD detector recorded approximately 30 major constituents in the chromatograms at 270 nm before derivatization (Figure 9B).Out of these substances, 16 were found to have antioxidant activity after the derivatization.Substances 25, 28, 48, 70, 76, 78, 108, 124, and 139, which are coumarins and derivatives, had the highest impact on the overall antioxidant capacity.Following them were substances 6, 19, 35, 60, and 138, which are flavan-3-ols, as well as substances 7 and 20, which are hydroxycinnamates.Antioxidant properties of several identified major H. alpestre components were reported

F I G U R E 7
Histopathological alterations in the mammary glands at Week 28 (H&E staining x400).(A, B) Breast cancer; (C) BC + nor-NOHA (DN); (D) BC + L-NAME (DL); (E) BC + HA (DH); (F, G) BC + HA + L-NAME (DHL), (H, I) BC + HA + nor-NOHA (DHN).The histological scores were assessed using the Nottingham Score system (k, Grade I [scores 3−5], Grade II [scores 6−7], and Grade III [scores 8−9]), and the breast tumor vasculature was evaluated (J, 10 nonoverlapping micrographs were captured at 1000-fold magnification from each animal, covering the entire tissue section).Statistical significance (p < .05)was assessed using a two-tailed unpaired T-test.The results are presented as mean ± SEM. *p < .05,**p < .01,***p < .001,****p < .0001.content of total phenolic compounds and flavonoids in H. alpestre which regulates changes in arginase and NOS activity.The inhibition of arginase and NOS enzymes in cancer also resulted in the dysregulation of COX-2 activity.We found that combined treatment with HA extract and nor-NOHA effectively suppressed the production of polyamines, leading to a decrease of polyamine levels in the blood to levels similar to the control group.Considering the crucial role of polyamines in the abnormal growth of cells and cancer metastasis, it can be speculated that one of the mechanisms of the anticancer effect of the proposed herb-drug combination may be attributed to the disruption of polyamine synthesis.Notably, our data show that the HA+nor-NOHA treatment showed greater effectiveness compared to using the individual components alone or the combination of HA + L-NAME.
ing the changes in the activity of arginase, the quantity of polyamines, NO, and IL-2, and breast histological analysis.Inhibition of the arginase by nor-NOHA reduced the quantity of polyamines that otherwise would stimulate cell proliferation and migration.Cancer cells thus lose regulation of growth and proliferation and become liable to undergo apoptosis.Inhibition of NOS by L-NAME reduced the amount of NO in cancer, leading to antiangiogenic effects.We confirmed the latter by showing the reduction of the blood vessel volume in the tumor.Inhibition of NOS, especially in combination with the plant extract, reduced the level of peroxynitrite thereby attenuating its aggressive oxidative effects.96The latter allowed us to increase the efficiency of our treatment model.The HA extract F I G U R E 8 Antioxidant capacity of aerial part extract of Hypericum alpestre evaluated by spectrophotometric tests: (A)-ABTS and (B)-DPPH or in vitro CAA test-(C).CAA was tested by exposing HT29 cells to HA extract for 1 h.The data are expressed as means ± SD from three independent experiments, p < .05for spectrophotometric tests.inhibitedarginase and NOS activities to the same levels as specific inhibitors alone.The reason for this effect may be the improvement of the condition of sick rats by the effect of the HA, or a direct effect on the activity of these enzymes.Inhibition of arginase and NOS enzymes leads to an increase in the amount of L-arginine amino acid, which is an initiator of anticancer response by CD4+ T cells and CD8+ T cells.This can lead to an increment in IL-2 levels and NK-cell and T-killer cell activation, which promotes antitumor cytotoxicity.

F I G U R E 9
HPLC-MS (A), HPLC-DAD 270 nm (B) chromatograms of phenolic constituents of Hypericum alpestre aerial part extract and the antioxidant profile (C) after (734 nm) post-column derivatization with ABTS reagent, for the identity of peaks, see Spp.

4. 3 |
The characterization and cytotoxicity of H. alpestre Phenolic compounds, as the most prevalent plant secondary metabolites, have attracted considerable attention due to their antioxidant/prooxidant properties and their potential role in the mitigation of diverse diseases associated with oxidative stress, including cancer.99,100Considering the high content of phenolics including flavonoids, the strong antioxidant properties of the H. alpestre extract can be attributed to the presence of redox-active constituents within the metabolome of this herb.The characterization of HA extracts' phenolic constituents was done using the LC-Q-Orbitrap HRMS technique.A total of 244 constituents were identified in H. alpestre extracts, with 233 constituents annotated and 11

Table .
T A B L E 3 Major identified phytochemicals in the extract Hypericum alpestre with potent anticancer, antioxidant, and anti-inflammatory properties.