Antioxidant and Cytotoxic Properties of Berberis vulgaris (L.) Stem Bark Dry Extract

Berberis vulgaris (L.) has remarkable ethnopharmacological properties and is widely used in traditional medicine. The present study investigated B. vulgaris stem bark (Berberidis cortex) by extraction with 50% ethanol. The main secondary metabolites were quantified, resulting in a polyphenols content of 17.6780 ± 3.9320 mg Eq tannic acid/100 g extract, phenolic acids amount of 3.3886 ± 0.3481 mg Eq chlorogenic acid/100 g extract and 78.95 µg/g berberine. The dried hydro-ethanolic extract (BVE) was thoroughly analyzed using Ultra-High-Performance Liquid Chromatography coupled with High-Resolution Mass Spectrometry (UHPLC–HRMS/MS) and HPLC, and 40 bioactive phenolic constituents were identified. Then, the antioxidant potential of BVE was evaluated using three methods. Our results could explain the protective effects of Berberidis cortex EC50FRAP = 0.1398 mg/mL, IC50ABTS = 0.0442 mg/mL, IC50DPPH = 0.2610 mg/mL compared to ascorbic acid (IC50 = 0.0165 mg/mL). Next, the acute toxicity and teratogenicity of BVE and berberine—berberine sulfate hydrate (BS)—investigated on Daphnia sp. revealed significant BS toxicity after 24 h, while BVE revealed considerable toxicity after 48 h and induced embryonic developmental delays. Finally, the anticancer effects of BVE and BS were evaluated in different tumor cell lines after 24 and 48 h of treatments. The MTS assay evidenced dose- and time-dependent antiproliferative activity, which was higher for BS than BVE. The strongest diminution of tumor cell viability was recorded in the breast (MDA-MB-231), colon (LoVo) cancer, and OSCC (PE/CA-PJ49) cell lines after 48 h of exposure (IC50 < 100 µg/mL). However, no cytotoxicity was reported in the normal epithelial cells (HUVEC) and hepatocellular carcinoma (HT-29) cell lines. Extensive data analysis supports our results, showing a significant correlation between the BVE concentration, phenolic compounds content, antioxidant activity, exposure time, and the viability rate of various normal cells and cancer cell lines.


Introduction
Numerous pharmaceutical companies are focused on researching and developing new formulations based on herbal sources, which can help manage chronic diseases.The World Health Organization also supports conventional plant-based treatments due to their accessibility, safety for long-term uses, and relatively low production costs.This shift toward natural remedies occurred mainly because some synthetic pharmaceutical drugs may have harmful side effects when used for the long-term treatment of chronic diseases [1].Therefore, based on traditional medical systems (Ayurvedic and Chinese), phytotherapy in chronic disorders is currently used as an alternative treatment worldwide.Berberis is a significant plant genus with approximately 500 species worldwide.It belongs to the Berberidaceae family and has considerable potential applications in the food and pharmaceutical industries [2].Berberis species are native to central and southern Europe, Asia (including the northern zones of Pakistan and Iran), and the north-eastern area of the United States.Berberis vulgaris (L.), known as European barberry, common barberry, or Épine-Vinette, has an essential role in herbal therapy; its different parts (fruits, leaves, roots, stem, branches, stem/root bark) have been used in traditional medicine for more than 2500 years.This species can be helpful in various inflammations, high blood pressure, gastrointestinal diseases, hepatic disorders, and diabetes.Numerous studies show that B. vulgaris has valuable pharmacological properties, such as antioxidant, antihyperglycemic, anticholinergic, hypolipidemic, anti-inflammatory, anticancer, and antimicrobial properties.In homeopathy, B. vulgaris is mainly used in urinary lithiasis, dermatology, rheumatism, and liver diseases [3].Berberine, the specific isoquinoline alkaloid mainly extracted from common barberry root and stem barks, is formulated for oral administration alone or in various combinations.The administration of berberine-based phytotherapeutics could have a beneficial impact on lipid and carbohydrate metabolism, particularly on glucose homeostasis, being helpful in weight loss, diabetes mellitus [4] and endocrine disorders, liver diseases [5], cardiovascular diseases, atherosclerosis, neurodegenerative diseases, rheumatic diseases, and infectious diseases [1].Several studies reported berberine-induced toxicity in humans and mice [1].However, toxic phenomena could be diminished through berberine combination with other phytochemicals or plant extracts.Synergistic effects would also be expected in adequate combinations [1].Moreover, B. vulgaris and berberine display anticancer effects through various cell signaling pathways' modulation [6], diminishing tumor cell viability and reducing their multiplication in various neoplasia (lung, breast, ovary, gastric cancer) [1].
In the present study, we aimed to investigate the hydro-ethanolic dry extract of B. vulgaris stem bark (BVE), obtained using a reflux extraction process in 50% ethanol, rotary evaporation, and freeze-drying.Here, 50% ethanol was used as an extraction solvent for obtaining the dry plant extract because of its effectiveness in extracting a broad range of phytochemicals (polar, moderately polar, and some nonpolar compounds); in addition, it has a low toxicity profile compared to other solvents (methanol, acetone, hexane, ethyl acetate or chloroform).Reflux extraction is a low-cost and efficient tool that ensures a high content of bioactive constituents through the consequent rotary evaporation and freeze-drying.The lyophilization (freeze-drying) process provides substantial stability to plant extracts by preserving the secondary metabolites with antioxidant activity.
A complex analysis of BVE's phenolic compounds was performed using ultra-highperformance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS/MS).Berberine was quantified through HPLC-DAD.The BVE's antioxidant potential was in vitro evaluated through the free radical scavenging (DPPH and ABTS) and reducing power (FRAP).The acute toxicity of BVE and berberine-berberine sulfate hydrate (BS)-was assessed in vivo on two Daphnia species.In contrast, their teratogenic potential was evaluated by applying the embryo test to Daphnia magna embryos.Moreover, the antitumor potential of BVE and BS was investigated in several human cancer cell lines: hepatocellular (HEP G2), colon (LoVo and HT-29), breast (MDA-MB-231), ovary (SK-OV-3), and tongue (PE/CA-PJ49), using classical oncolytic drugs as positive controls.Extensive data analyses support our results, showing significant correlations between the BVE concentration, exposure time, phenolic constituent content, antioxidant activity, and cytotoxicity.

Phenolic Compounds (Polyphenols and Phenolic Acids) Quantification
Berberidis cortex dry hydro-ethanolic extract was obtained with a yield of 16.35%.Other studies reported similar yields: 18.7% for roots and 14.7% for leaf extracts in ethanol [6].Ethanol's availability and regulatory approval make it an obvious choice due to its balance of effectiveness, safety, and applicability.Moreover, ethanol's moderate boiling point makes it easy to remove by evaporation, simplifying the process of concentrating the extract and falling within the trend of implementing green technology and using green solvents, which are much safer for the environment.The rotary evaporator could be connected with a vacuum pump, which decreases the boiling point of ethanol (78.2 • C) and facilitates its evaporation.After ethanol collection, it could be subjected to fractional distillation to remove the moisture content and increase its purity.
The standard calibration curves are displayed in Figure S1 in the Supplementary Materials, while the TPC and TPA values are presented in Table 1.BVE is rich in total polyphenols (TPC = 17.6780 ± 3.9320 mg Eq tannic acid/100 g extract); however, it has shown a phenolic acid content (TPA) of only 3.3886 ± 0.3481 mg Eq chlorogenic acid/100 g extract.The literature data show that the TPC in various B. vulgaris extracts is very different.Our hydro-ethanolic extract of Berberidis cortex has a TPC of 1767.80 mg/g, while El-Zahar et al. [7] reported much lower TPC levels in ethanol extracts of roots (147.2 mg/g) and leaves (120.7 mg/g).Och et al. [8] indicated similar TPC values quantified in 80% methanol extracts of various B. vulgaris parts: 58.5 mg/g for the leaf extract, 57.7 mg/g for the stem one, and 52.8 mg/g for the fruit extract.
Figure 1A displays the chromatogram of the primary phytochemicals identified in BVE by UHPLC-MS, and Figure 1B

Antioxidant Activity
Table 2 shows significant differences between the IC 50 /EC 50 values determined by all three methods, IC 50 DPPH = 0.2610 mg/mL, IC 50 ABTS = 0.0442 mg/mL, and EC 50 FRAP = 0.1398 mg/mL, compared to ascorbic acid (IC 50 = 0.0165 mg/mL).Similar values were reported for B. microphylla ethanol extract [9]: ABTS IC 50 = 0.26 mg/mL and DPPH IC 50 = 0.38 mg/mL.The substantial antioxidant potential is underlined by the phenolic constituent content and berberine and other alkaloids, which are known for their protective activity [11]

48-h Acute Toxicity Test Using Daphnia Magna and Daphnia Pulex
After 24 h, D. magna's total lethality was recorded at concentrations ≥ 25 µg/mL BVE and ≥10 µg/mL BS.Similarly, D. pulex's total lethality occurred at concentrations ≥ 50 µg/mL BVE and ≥25 µg/mL BS.Our results revealed that the BS toxicity is higher than BVE for both Daphnia species, with D. magna being more vulnerable than D. pulex.The time-dependent toxicity is more evident in D. magna than in D. pulex.(Figure 2).The lethality curves' analysis revealed that, in the D. magna bioassay (Figure 2A), BS exhibited a lower LC 50 value at 24 h compared to BVE, suggesting the higher toxicity of the pure alkaloid, which was expected.However, at 48 h, the BVE toxicity significantly increases, almost to the same potency as BS.After 48 h of exposure, the concentration-response curves for both tested solutions in D. pulex displayed similar trends to those recorded in D. magna but with differences in the magnitude of lethality (Figure 2B).

Daphnia Magna Embryonic Development Assay
Following the acute toxicity test results, the embryo assay was performed at nonlethal concentrations (2.5 µg/mL BS and 3.125 µg/mL BVE).Minor differences were observed after 24 h (Figure 3a,b).After 48 h, BS stimulated the development of all the embryos (Figure 3c), while BVE exhibited a significant inhibitory effect (Figure 3d), which could be due to the extract s complex composition.Only 20% of the embryos treated with BVE were fully developed, compared to 90% recorded for those exposed to BS.The mobility and viability of neonates developed in BS solution were similar to those of the control.However, they all failed to form the compound eye, even after 48 h of exposure, suggesting a potential developmental risk.

Daphnia Magna Embryonic Development Assay
Following the acute toxicity test results, the embryo assay was performed at non-lethal concentrations (2.5 µg/mL BS and 3.125 µg/mL BVE).Minor differences were observed after 24 h (Figure 3a,b).After 48 h, BS stimulated the development of all the embryos (Figure 3c), while BVE exhibited a significant inhibitory effect (Figure 3d), which could be due to the extract's complex composition.Only 20% of the embryos treated with BVE were fully developed, compared to 90% recorded for those exposed to BS.The mobility and viability of neonates developed in BS solution were similar to those of the control.However, they all failed to form the compound eye, even after 48 h of exposure, suggesting a potential developmental risk.In Daphnia magna, Vesela et al. [12] reported that the berberine chloride toxicity recorded an LC50 of 0.903 µg/mL after 24 h and 0.822 µg/mL at 48 h exposure.Our berberine sulfate hydrate recorded 9.7 µg/mL and 5.3 µg/mL, respectively.In another study on another crustacean [13], 7 µg/mL berberine chloride induced 100% lethality in Artemia salina larvae.These differences could be explained by berberine salt, the animal model species, and the provenance.The D. magna embryos failed to form compound eyes after BS and BVS treatment.Natural berberine also affects cardiovascular system morphogenesis and functionality in Zebrafish embryos [14].Based on these findings, in the Medicinal Plants Monograph Volume 4 [15], the WHO mentions the potential side effects of berberine on humans after consuming more than 500 mg.

In Vitro Anticancer Activity
The antiproliferative activity induced by BVE was evaluated in vitro through several cytotoxic assays by applying different BVE and BS concentrations (6.25-400 µg/mL) to cells derived from six tumor cell lines of different histological origin: HEP G2, LoVo, HT-29, MDA-MB-231, SK-OV-3, and PE/CA-PJ49.Human umbilical vein endothelial cells (HUVECs) were selected as the reference normal cells.
The BVE and BS antiproliferative capacities, as tested on normal human cells and tumor cell lines, are shown in Table 3.
The IC50 values displayed in Table 3 could be interpreted according to Hidayat et al. [16], resulting in an overview of the BVE and BS cytotoxicity in various cell lines.In normal endothelial cells (HUVEC), they have no cytotoxicity after 24 and 48 h (IC50 >> 400 µg/mL).The same interpretation is also available for HT-29 tumor cells, which showed no significant decrease in viability after the BVE/BS treatments.
Generally, BVE exhibited moderate cytotoxicity in the other tumor cells.The most substantial effect, with the lowest IC50 values after 24 and 48 h (IC50 > 100 µg/mL, respectively, IC50 > 50 µg/mL) was seen in breast cancer cells (MDA-MB-231) and OSCC ones (PE/CA-PJ49).In LoVo cells (colon cancer), the cytotoxicity at 24 h was appreciably lower (IC50 > 200 µg/mL) but after 48 h of exposure was moderate, similar to the previous ones (IC50 > 50 µg/mL).BVE exhibited the lowest effect on human ovary cancer (SK-OV-3 cells) and hepatocellular carcinoma (HEP G2 cells) after 24 and 48 h (IC50 > 400 µg/mL and, respectively, >100µg/mL).In Daphnia magna, Vesela et al. [12] reported that the berberine chloride toxicity recorded an LC 50 of 0.903 µg/mL after 24 h and 0.822 µg/mL at 48 h exposure.Our berberine sulfate hydrate recorded 9.7 µg/mL and 5.3 µg/mL, respectively.In another study on another crustacean [13], 7 µg/mL berberine chloride induced 100% lethality in Artemia salina larvae.These differences could be explained by berberine salt, the animal model species, and the provenance.The D. magna embryos failed to form compound eyes after BS and BVS treatment.Natural berberine also affects cardiovascular system morphogenesis and functionality in Zebrafish embryos [14].Based on these findings, in the Medicinal Plants Monograph Volume 4 [15], the WHO mentions the potential side effects of berberine on humans after consuming more than 500 mg.

In Vitro Anticancer Activity
The antiproliferative activity induced by BVE was evaluated in vitro through several cytotoxic assays by applying different BVE and BS concentrations (6.25-400 µg/mL) to cells derived from six tumor cell lines of different histological origin: HEP G2, LoVo, HT-29, MDA-MB-231, SK-OV-3, and PE/CA-PJ49.Human umbilical vein endothelial cells (HUVECs) were selected as the reference normal cells.
The BVE and BS antiproliferative capacities, as tested on normal human cells and tumor cell lines, are shown in Table 3.The IC 50 values displayed in Table 3 could be interpreted according to Hidayat et al. [16], resulting in an overview of the BVE and BS cytotoxicity in various cell lines.In normal endothelial cells (HUVEC), they have no cytotoxicity after 24 and 48 h (IC 50 >> 400 µg/mL).The same interpretation is also available for HT-29 tumor cells, which showed no significant decrease in viability after the BVE/BS treatments.
The results of in vitro studies are detailed and presented in Figure S2 and File S1 in the Supplementary Materials.
After 24 h, BVE cytotoxicity at the selected concentration range (6.25-400 µg/mL) in normal and tumor cell lines showed significant differences (at α < 0.05, p-value was established at 0.0024) between PE/CA-PJ49 and HUVEC, and MDA-MB-231 and HUVEC (p = 0.001, Figure S1a).Substantial differences (p < 0.05) were also observed in the case of HUVEC and all the other tumor cells, except the HT-29 ones, and MDA-MB-231 and HUVEC compared to HT-29 cells (Figure S2a in the Supplementary Materials).
No statistically significant differences were reported between the BVE and BS cytotoxicity for the same exposure period in the same cell line (Figure S2 in the Supplementary Materials).
The cytotoxic activity of BVE was compared to that induced by several drugs (5-Fluorouracil, Cisplatin, and Doxorubicin) [17] that are commonly used in oncological treatments and were applied throughout all the experiments as positive controls.The concentration range used for Cisplatin (CisPt) and 5-Fluorouracil (5-FU) was 3.125-200 µM, while for Doxorubicin (DOX) it was between 0.625 and 40 µM [17], as illustrated in Figures 4 and 5.  Table 1 and Figure 4 show that, in normal cells (HUVEC), both berberine and BVE have no cytotoxic effects (IC50 >> 400 µg/mL).Our results are similar to those from the scientific literature [18].
Figure 5 shows the influence of BVE and BS on cancer cell viability compared to standard oncolytic drugs in the same concentration range (12.5-200 µg/mL for BVE and BS, 12.5-200 µM for 5-FU and CisPt, and 1.25-20 µM for DOX) [17].Table 1 and Figure 4 show that, in normal cells (HUVEC), both berberine and BVE have no cytotoxic effects (IC50 >> 400 µg/mL).Our results are similar to those from the scientific literature [18].
Figure 5 shows the influence of BVE and BS on cancer cell viability compared to standard oncolytic drugs in the same concentration range (12.5-200 µg/mL for BVE and BS, 12.5-200 µM for 5-FU and CisPt, and 1.25-20 µM for DOX) [17].Generally, the tumor cell viability diminution was higher after 48 h than 24 h.In all cases, BS showed a higher cytotoxicity than BVE.In HT-29 and LoVo cells, the BS activity was lower than 5-FU after both exposure times, while in HEP G2, the BS activity was higher than 5-FU after 24 h and lower after 48 h (Figure 5A-F).The PE/CA-PJ49 cell viability decreased in the following order: be > CisPt > BS after 24 and 48 h of treatment (Figure 5K,L).In MDA-MB-231, the cell viability after 24 h decreased in the order of BVE > DOX > BS; after 48 h, BVE acted to a slightly higher extent than DOX (Figure 5G,H).After 24 h, the SK-OV-3 cell viability decreased in the order of DOX > BVE > CisPt > BS, while after 48 h, the previously mentioned order changed: DOX > BVE > BS > CisPt (Figure 5I,J).These effects are due to the potential synergism between the phytochemicals in BVE [19].

Statistical Analysis
The Pearson correlation shows that BVE24 is highly correlated with BS24, BVE48 and BS48 (r = 0.906, r = 0.910, r = 0.866, p < 0.05).BS24 exhibits a strong correlation with 5-FU24, The effects of BVE and BS compared to anticancer drugs on normal endothelial cells' (HUVEC) viability after 24 and 48 h of exposure are displayed in Figure 4.
Table 1 and Figure 4 show that, in normal cells (HUVEC), both berberine and BVE have no cytotoxic effects (IC50 >> 400 µg/mL).Our results are similar to those from the scientific literature [18].
Figure 5 shows the influence of BVE and BS on cancer cell viability compared to standard oncolytic drugs in the same concentration range (12.5-200 µg/mL for BVE and BS, 12.5-200 µM for 5-FU and CisPt, and 1.25-20 µM for DOX) [17].
Generally, the tumor cell viability diminution was higher after 48 h than 24 h.In all cases, BS showed a higher cytotoxicity than BVE.In HT-29 and LoVo cells, the BS activity was lower than 5-FU after both exposure times, while in HEP G2, the BS activity was higher than 5-FU after 24 h and lower after 48 h (Figure 5A-F).The PE/CA-PJ49 cell viability decreased in the following order: be > CisPt > BS after 24 and 48 h of treatment (Figure 5K,L).In MDA-MB-231, the cell viability after 24 h decreased in the order of BVE > DOX > BS; after 48 h, BVE acted to a slightly higher extent than DOX (Figure 5G,H).After 24 h, the SK-OV-3 cell viability decreased in the order of DOX > BVE > CisPt > BS, while after 48 h, the previously mentioned order changed: DOX > BVE > BS > CisPt (Figure 5I,J).These effects are due to the potential synergism between the phytochemicals in BVE [19].

Statistical Analysis
The Pearson correlation shows that BVE24 is highly correlated with BS24, BVE48 and BS48 (r = 0.906, r = 0.910, r = 0.866, p < 0.05).BS24 exhibits a strong correlation with 5-FU24, BVE48 and BS48 (r = 0.813, r = 0.935, r = 0.928, p < 0.05).BVE48 has a considerable correlation with BS48 (r = 0.955) and a moderate one with 5-FU48 (r = 0.788), p < 0.05.Moreover, CisPt24 significantly correlates with DOX24, CisPt48 and DOX48 (r = 0.934, r = 0.997, r = 0.837, p < 0.05), CisPt48 with DOX48 (r = 0.830, p < 0.05), DOX24 with CisPt48 and DOX48 (r = 0.918, r = 0.920, p < 0.05), and 5-FU24 with BVE48 and BS48 (r = 0.866, r = 0.853, p < 0.05); 5FU24 also shows a moderate correlation with 5-FU48 (r = 0.788, p < 0.05).The place of each cytotoxic agent linked to the cell type is shown in Figure 6A, and the similarities between them are displayed in Figure 6B.The correlations between the bioactive phytoconstituents-total phenolic content (TPC) and total phenolic acid (TPA)-and their pharmacological potential are detailed in Table S3 in the Supplementary Material.Their dual redox behavior could explain the antiproliferative effect on tumor cells, leading to decreasing viability; the prooxidant effect of phytochemicals is responsible for the BVE cytotoxicity.The antioxidant effect, measured by three methods, shows a substantial positive correlation with the TPC and TPA (r = 0.972-0.994,p < 0.05).Moreover, the variable parameters determined by all three methods (DPPH, FRAP, ABTS) are intercorrelated (r = 0.997-0.998,p < 0.05) and show a The correlations between the bioactive phytoconstituents-total phenolic content (TPC) and total phenolic acid (TPA)-and their pharmacological potential are detailed in Table S3 in the Supplementary Materials.Their dual redox behavior could explain the antiproliferative effect on tumor cells, leading to decreasing viability; the prooxidant effect of phytochemicals is responsible for the BVE cytotoxicity.The antioxidant effect, measured by three methods, shows a substantial positive correlation with the TPC and TPA (r = 0.972-0.994,p < 0.05).Moreover, the variable parameters determined by all three methods (DPPH, FRAP, ABTS) are intercorrelated (r = 0.997-0.998,p < 0.05) and show a substantial negative correlation with the antiproliferative activity (r = −[0.951-0.999],p < 0.05).Similarly, the TPC and TPA display a significant negative correlation with the cell viability diminution (r = −[0.970-0.997],p < 0.05 (Table S3).The outstanding capacity of B. vulgaris for scavenging ABTS, hydroxyl radicals, and DPPH is due to berberine and phenolic compounds with dual redox behavior that can act synergistically in the extract [2].Recent studies showed that galangin and berberine in a synergic combination might induce esophageal carcinoma cells' apoptosis through cell cycle arrest in the G2/M phase via oxidative stress [19].Moreover, apigenin, gallic acid, and berberine have immunomodulatory potential and could be helpful as immune checkpoint inhibitors and fight cancers via multiple targets [20].
The accessed literature data regarding the cytotoxic effects of berberine evaluated in vitro in various tumor cells, potential mechanisms, and IC 50 values are synthesized in Table 4.  NA-Not available.
In the present study, the BS IC 50 against HEP G2 was slightly over 50 ug/mL, being around that registered in Table 4; the same was true for the colon carcinoma (LoVo), colon cancer (SK-OV-3) and tongue squamous cell carcinoma (PE/CA-PJ49) cell lines.Moreover, Table 4 indicates that the IC 50 of BS against OSCC was 18-136 µM, and our value belongs to this range.For the MDA-MB-231 (breast cancer) cell line, the IC 50 was > 25 µg/mL after 24 h and 12.5 after 24 h.Similar studies investigated the anticancer effects of B. vulgaris extract and berberine chloride in other cancer cell lines, evaluating the cell viability after 24, 48, and even 72 h and reporting various IC 50 values [18,34,35].In HEP G2 (liver cancer), Caco-2 (colon cancer), and MCF-7 (breast cancer), the IC 50 values for barberry extract were 68.02 > 49.96 > 15.61 µg/mL, and for berberine chloride, lower values were recorded: 65.86 > 17.64 > 15.93 µg/mL [34].After 48 h, the IC 50 values drastically decreased: 5.55, 3.84, and 4.44 µg/mL for berberis extract vs. 11.49,5.1, and 4.43 µg/mL for berberine chloride.Moreover, in HEP G2 and CaCo2, the antitumor activity of berberis extract was stronger than that of berberine chloride [34].In our study, both BVE and BS had moderate cytotoxicity.Another research team analyzed the cytotoxicity of B. vulgaris extract in 70% ethanol on breast cancer cell lines (MCF-7) after 24, 48, and 72 h and obtained significantly higher IC50 values, respectively, 4000, 2000 and 1000 µg/mL [35].Och et al. investigated the cytotoxic and proapoptotic properties of B. thunbergii extract and berberine on various hematopoietic cancer cell lines: acute promyelocytic leukemia (HL-60, HL-60/MX1, HL-60/MX2), myeloma (U266B1), acute lymphoblastic leukemia (CCRF/CEM and CEM/C1) and acute T cell leukemia (J45.01)[18].After 24 h, the extract did not show cytotoxic effects in the tested cells, and the IC 50 value of berberine was 80-250 µM [18].However, tumor cells' exposure to a high concentration of B. thunbergii extract influenced the activity of proapoptotic genes (upregulation of B2M, downregulation of BAD and BNIP2, and increased expression of BAX, BAK1, BIK, and CASP9i) in all the leukemia cell lines [18].These phenomena suggest the potential detection of cellular apoptosis after an exposure longer than 24 h, and further experiments in the 72 and 96 h models are requested [18].

Materials and Methods
3.1.Materials 3.1.1.Chemicals All the chemicals were of analytical grade.Analytical standards of 31 compounds were purchased from Sigma-Aldrich, Schnelldorf, Germany.Methanol and ethyl alcohol, HPLC grade, were purchased from Merck, Bucharest, Romania; formic acid (98%) and ultrapure water (LC-MS grade) were also purchased from Merck (Merck Romania, Romania).The Pierce LTQ Velos ESI positive and negative ion calibration solutions (Thermo Fisher Scientific, Dreieich, Germany) calibrated the Orbitrap Mass Spectrometer.
The Daphnia magna Straus for the in vivo studies originated from a culture maintained parthenogenetically at the Department of Pharmaceutical Botany and Cell Biology, Faculty of Pharmacy, "Carol Davila" University of Medicine and Pharmacy Bucharest, since 2012.

B. vulgaris Extract Preparation
B. vulgaris (L.) cortex was harvested in March 2023 from a local ecological crop in Oratia-Lat/Long (in decimal degrees): 45.445199, −27.013190-BuzauCounty, Romania.It was identified by Prof. Octavian Tudorel Olaru, Department of Pharmaceutical Botany and Cell Biology, and Prof. Cerasela Elena Gîrd, Department of Pharmacognosy, Phyto-chemistry and Phytotherapy, Faculty of Pharmacy, "Carol Davila" University of Medicine and Pharmacy, Bucharest.The voucher specimen is also preserved in the Department of Pharmacognosy, Phytochemistry, and Phytotherapy collection.Morphological peculiarities: the vegetable product is presented as flat or slightly recurved fragments; the inner face shows a bright yellow-green fluorescence in UV light (due to berberine).Organoleptic characteristics include a brown-gray color on the outside and a golden-yellow on the inside (due to berberine), which becomes brown through preservation (Figure S4 in the Supplementary Materials), bitter taste, and no smell.As previously described, 50 g of powdered stem bark was subjected to reflux extraction with 50% ethanol (Sigma-Aldrich, Darmstadt, Germany) [37].After filtration, the obtained extract (BVE) was concentrated in a rotary evaporator R100 with a vacuum pump V-700 (BUCHI Corporation, New Castle, DE, USA) and lyophilized (Christ Alpha 1-2/B Braun, BiotechInt, New Delhi, India).

Total Polyphenol Content (TPC)
The Folin-Ciocalteu reagent was used following a spectrophotometric method described extensively in a previously published article [38].The absorbances were measured at 725 nm (Jasco V-530 spectrophotometer, JASCO, Tokyo, Japan), and tannic acid was the standard for the calibration curve in a linear concentration range of 2-9 µg/mL.The TPC is expressed as mg Eq tannic acid/100 g BVE.

Total Phenolic Acid (TPA)
The quantification method was based on the phenolic acids that form nitro derivatives with nitrous acids.Our previously published article detailed it [37].The absorbance was immediately measured at 525 nm (Jasco spectrophotometer, Japan) and compared to a sample that lacked the Arnow reagent.Chlorogenic acid (Sigma-Aldrich, Germany) was used as a standard for the calibration curve in the linear range of 11-53 µg/mL, with R 2 = 0.9998.The total phenolic acid (TPA) content was expressed as mg chlorogenic acid equivalents per gram of extract (mg Eq chlorogenic acid/g BVE).The phenolic profile of BVE was established based on non-targeted tandem mass spectrometry (MS-MS) using the hyphenated technique represented by Ultra-High-Performance Liquid Chromatography (UHPLC) coupled with the Q-Exactive High-Resolution Mass Spectrometer (HRMS).The same method was used to quantify selected phenolic compounds for each available analytical standard (Sigma-Aldrich, Germany).Our previously published study describes all the detailed data [17].

Azinobis-3-Ethylbenzotiazoline-6-Sulfonic Acid Assay (ABTS)
The turquoise-colored ABTS radical resulted from a potent oxidizing agent (potassium persulfate) reaction with the ammonium salt of 2,2 ′ -azino-bis(3-ethylbenzothiazoline-6sulfonic acid).Under the action of the antioxidant, the intensity of the color was reduced to colorless.The absorbance was determined at λ = 734 nm, and the IC 50 value was calculated from the inhibition curves and their linear equations [37].

Ferric-Reducing Antioxidant Power Assay (FRAP)
The antioxidant analyte reacted with Fe 3+ , reducing to Fe 2+ , and imprinting blue.The coloration intensity was directly proportional to the antioxidant activity.The absorbance values were measured at λ = 700 nm (spectrophotometer Jasco V-530) and compared to the control (prepared under the same conditions without sample solution).It was expressed as an EC 50 value; it represented the sample concentration at which the absorbance had a value of 0.5 or half the concentration at which the antioxidant activity was at a maximum, as determined by the trendline equation [40].

48-h Acute Toxicity Test Using Daphnia Magna and Daphnia Pulex
The daphnids belonging to the species Daphnia magna and Daphnia pulex were chosen based on their size from parthenogenetic cultures maintained in an artificial medium for 24 h before testing [41,42].The assay was performed in 24-well culture plates (Greiner Bio-One, Kremsmünster, Austria), with each well containing around 10 organisms.The samples were tested in six concentrations, ranging from 3.125 µg/mL to 100 µg/mL for BVE; as a positive control, BS was used from 0.625 to 20.0 µg/mL.The tests were duplicated, and lethality was assessed at 24 and 48 h.The 50% lethal concentrations (LC 50 ) and the 95% confidence interval (CI95%) of the LC 50 values were determined using GraphPad Prism v 5.1.2008software (GraphPad Software, Boston, MA, USA) [17].

Daphnia Magna Embryonic Development Assay
The following concentrations were chosen for testing: BVE at 3.125 µg/mL and BS at 2.5 µg/mL, based on the results obtained in the viability test.The embryos were exposed to the sample solutions in the dark, maintaining a constant temperature and humidity of 25 • C and 75% RH, respectively.The experiments were carried out on culture plates with 48 wells (Greiner Bio-One, Kremsmünster, Austria).Every 24 h, the embryos were examined at a magnification of 80× under a microscope (bScope ® microscope, Euromex Microscope BV, Arnhem, The Netherlands) to assess the developmental stages and detect abnormalities compared to the untreated control [17].

Cell Cultures and Treatments
The antiproliferative effect of the BVE hydro-ethanolic extract and BS standard was evaluated in vitro in six tumor cell lines (SK-OV-3, LoVo, HEP-G2, HT-29, MDA-MB-231, PE/CA-PJ49), with normal HUVEC cell, used as the control.All the cell lines were cultured in DMEM/F12 medium enriched with 2 mM L-glutamine and 10% fetal calf serum and antibiotics mixture (100 U/mL penicillin and 100 µg/mL streptomycin).They were incubated at 37 • C in a 5% CO 2 humidified atmosphere.For the cytotoxicity assays, the cells were detached from the culture flasks and then cultivated in 96-well flat-bottom plates for 24 h until they reached around 70% confluence.Then, the cells were treated for various periods (24 h and 48 h) with different concentrations of BVE, BS, or oncolytic drugs (5-FU, CisPt, DOX) used as positive controls [43].The BVE and BS stock solutions were prepared by dissolving them in a minimal amount of DMSO and preserved at 4 • C; all the working solutions were prepared from the stocks by serial dilutions with culture medium before each treatment assay [17].

MTS Assay
The cytotoxic potential of BVE and BS was evaluated by a colorimetric cell viability method, the MTS assay, and it was assessed in both tumor and normal cells and compared with the action of oncolytic drugs: DOX, CisPt, and 5-FU [44].
All the assays were performed in triplicate using the CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (MTS) kit (Promega, USA).It contains a reagent mixture of two components: MTS [3-(4,5 dimethylthiazol 2 yl) 5 (3 carboxymethoxy phenyl) 2 (4 sulfophenyl) 2H tetrazolium] and PES (phenazine ethosulphate), a cationic dye with high chemical stability, which may be combined with MTS to form a stable solution [45].The method's principle is based on the ability of metabolically active cells to reduce MTS (a yellow tetrazolium salt) to the colored formazan, which is soluble in the culture medium and can be spectrophotometrically quantified at a 492 nm wavelength.Briefly, 1.5 × 10 4 cells/well were cultured in 100 µL of the medium; after 24 h, the culture supernatants were discarded, and the cells were treated with increasing concentrations of BVE, BS, or reference drug solutions for 24 h or 48 h.At the expiration of the contact time, 20 µL of reagent mixture was added to each well, and the culture dishes were incubated for an additional 4 h at 37 • C, with gentle shaking every 20 min.Absorbance was read at λ = 492 nm with the Dynex ELISA reader (DYNEX Technologies-MRS, Chantilly, VA, USA) [46].
The cell viability was expressed as a percentage, was compared to the untreated cells (considered 100% viable), and was calculated according to the following formula: where T = optical density of treated cells, B = optical density of the blank (culture medium, in the absence of cells), and U = optical density of untreated cells.The obtained results were expressed as the mean values from three different experiments (n = 3) ± standard deviation (SD) [47].For the assessment of the DMSO cytotoxicity, the same experimental determinations were performed as in the MTS assay, and no impairment of cell viability was observed at concentrations lower than 1%.. Also, to observe the possible nonspecific reactions between BVE, BS, or drugs and MTS, their absorbance was determined without cells, and the values were extracted during the calculations.
The correlations between the bioactive constituents of the extracts and their antioxidant activity and cytotoxicity were determined using Principal Component Analysis [49] performed with XLSTAT 2023.1.4.by Lumivero (Denver, CO, USA) through Pearson correlation.A probability value p < 0.05 indicated a statistically significant difference [50].

Conclusions
This research investigated the autochthonous Berberidis cortex, obtaining a dry extract in 50% ethanol through successive reflux extraction, then solvent evaporation and freeze-drying.Through complex UHPLC-HRMS/MS and HPLC-DAD analysis of BVE, 40 phenolic constituents, including berberine, were identified.The main classes of phenolic metabolites (polyphenols and flavonoids) and bioactive representatives were also quantified.BVE's significant antioxidant potential was revealed by in vitro evaluation of the radical scavenging ability and reducing power.Then, the acute toxicity tests highlighted BVE's significant acute toxicity and teratogenicity in Daphnia sp.It also displayed moderate antiproliferative activity in various tumor cell lines and did not affect normal cells.Compared to BVE, berberine showed higher toxicity.It is essential to show that berberine sulfate reduced the viability in several tumor cell lines more than the standard anticancer drugs used as positive controls.

Figure 2 .
Figure 2. The results of the 48 h acute toxicity test using Daphnia sp.Lethality curves obtained after 48 h exposure of Daphnia sp. to BS (a) and BVE (b): D. magna (A) and D. pulex (B).BS-berberine sulfate hydrate; BVE-B.vulgaris stem bark dry hydro-ethanolic extract; NA-the values could not be calculated: * The interval is vast.** The values could not be calculated as the maximum L% was 10%.

Figure 2 .
Figure 2. The results of the 48 h acute toxicity test using Daphnia sp.Lethality curves obtained after 48 h exposure of Daphnia sp. to BS (a) and BVE (b): D. magna (A) and D. pulex (B).BS-berberine sulfate hydrate; BVE-B.vulgaris stem bark dry hydro-ethanolic extract; NA-the values could not be calculated: * The interval is vast.** The values could not be calculated as the maximum L% was 10%.

Table 1 .
Total polyphenols content, total phenolic acids, and antioxidant activity of BVE.

Table 2 .
The phytochemicals identified in B. vulgaris stem bark dry extract (BVE) by UHPLC-HRMS/MS and HPLC-DAD.
shows the HPLC-DAD chromatogram of BVE, where berberine has an RT = 32.513.

Table 4 .
In vitro cytotoxicity of berberine in various tumor cell lines, based on the literature data.