Phytochemical Analysis, Antioxidant Activities In Vitro and In Vivo, and Theoretical Calculation of Different Extracts of Euphorbia fischeriana

Euphorbia fischeriana has a long-standing history of use in traditional medicine for the treatment of tuberculosis diseases. However, the plant’s therapeutic potential extends beyond this specific ailment. The present study aimed to investigate the antioxidant properties of Euphorbia fischeriana and lay the groundwork for further research on its potential therapeutic applications. Phytochemical tests were performed on the plant, and 11 types of phytochemicals were identified. Ultraviolet–visible spectrophotometry was used to evaluate the active components and antioxidant properties of eight different solvent extracts, ultimately selecting acetone extract for further research. UHPLC-ESI-Q-TOF-MS identified 43 compounds in the acetone extract, and chemical calculations were used to isolate those with high content and antioxidant activity. Three stability experiments confirmed the extract’s stability, while cell viability and oral acute toxicity studies demonstrated its relatively low toxicity. In rats, the acetone extract showed significant protective effects against D-galactosamine-induced liver damage through histopathological examination and biochemical analysis. These results suggest that Euphorbia fischeriana’s acetone extract has potential in treating diseases related to oxidative imbalances. Therefore, this study highlights the plant’s potential therapeutic applications while providing insight into its antioxidant properties.


Introduction
Euphorbia is a diverse genus comprising around 2000 species, making it the largest in the spurge family and one of the largest among angiosperms. This genus has a global distribution, with a higher concentration found in Africa and Central and South America; however, 80 species are also found in China, distributed in both the northern and southern regions [1]. Genus Euphorbia has a long history of medicinal use, particularly in traditional medicine, whereby it has been used for treating respiratory tract infections, digestive discomfort, microbial infections, body and skin irritation, snake and scorpion bites, and body pain, among other conditions [2]. In a systematic review of Euphorbia plants, researchers analyzed both the chemical composition and pharmacological properties of the genus. The review revealed that Euphorbia plants contain a variety of valuable compounds such as diterpenoids, triterpenoids, sesquiterpenoids, steroids, and flavonoids. In addition, in the Over 200 chemical constituents, including diterpenoids, triterpenoids, cyclic terpenoids, acetophenones, flavonoids, coumarins, steroids, phenolic acids, and tannins, have been identified from the roots of E. fischeriana [18]. Given the structure and type of compounds present in the plant, it is reasonable to infer that E. fischeriana possesses antioxidant properties. However, to date, only one report has been published on the antioxidant activity of the plant, demonstrating that the volatile oil extracted from its roots has a certain scavenging effect on DPPH [19]. Hence, the present study aims to investigate various solvent extracts of E. fischeriana and determine the most suitable solvent for extracting its antioxidant components using the content of active components Over 200 chemical constituents, including diterpenoids, triterpenoids, cyclic terpenoids, acetophenones, flavonoids, coumarins, steroids, phenolic acids, and tannins, have been identified from the roots of E. fischeriana [18]. Given the structure and type of compounds present in the plant, it is reasonable to infer that E. fischeriana possesses antioxidant properties. However, to date, only one report has been published on the antioxidant activity of the plant, demonstrating that the volatile oil extracted from its roots has a certain scavenging effect on DPPH [19]. Hence, the present study aims to investigate various solvent extracts of E. fischeriana and determine the most suitable solvent for extracting its antioxidant components using the content of active components and antioxidant capacity as evaluation criteria. Additionally, the study seeks to utilize UHPLC-MS technology to identify the components present in the solvent extract and calculate the antioxidant capacity of the principal components, thereby laying a foundation for the clinical application of E. fischeriana. Importantly, this study will significantly expand our knowledge on the potential of E. fischeriana as an antioxidant agent and, therefore, contribute to the development of new and improved therapeutic interventions against oxidative-stress-related diseases. Table 1 shows the identification of eleven classes of phytochemicals in Euphorbia fischeriana. Saponins, anthraquinones, cardiac glycosides, and cyanogenic glycosides were not detected. Two interesting phenomena were observed in the conducted experiments. The first phenomenon involves the detection of flavonoids in which two results were positive and the other two were negative. This contradiction might be attributed to the low flavonoid content in plants. It may also be due to the high sensitivity and low detection limit of AlCl 3 and lead acetate tests, but with poor sensitivity in Shinoda and alkaline reagent tests. However, this speculation needs verification through total flavonoid content determination. The second phenomenon is the positive results in all three alkaloid experiments, suggesting the presence of alkaloids in Euphorbia fischeriana. This new finding is novel and has not been reported in previous studies. The content of total alkaloids must still be determined.

Yields
Eight solvents with distinct polarities were utilized to extract Euphorbia fischeriana powder. Extraction yields ranged from 8.0 ± 0.1% to 33.4 ± 0.4% (w/w) ( Table 2). The aqueous extract had the highest yield owing to the abundance of water-soluble constituents such as polyphenols, proteins, and carbohydrates, followed by methanol, ethanol, and acetone extracts. The dichloromethane and hexane extracts had the lowest yield. While the aqueous extract had the highest yield, it generally contains ineffective ingredients, such as pigments and pectins. Additionally, if carbohydrates are not the research object, the significant amount of carbohydrates present in the aqueous extract can interfere with the study of other components, affecting the outcomes of compound identification and biological activity. Consequently, this study did not investigate the aqueous extract, and further research on the selection of solvent extract should consider the active ingredient content and antioxidant activity outcomes. Carbohydrates are vital components of animal and plant cells, with crucial physiological functions such as storing and supplying energy, saving protein, and preventing the production of ketone bodies [20]. Glycosides, in particular, have various pharmacological effects, as evidenced by acetophenone glycosides from Euphorbia fischeriana demonstrating inhibitory effects against Mycobacterium smegmatis [21]. This study evaluated the carbohydrate content of various solvent extracts of Euphorbia fischeriana, and as indicated in Table 3, the total carbohydrate content (TCC) ranged from 0.0 to 537.8 ± 19.6 mg glucose equivalents (GE)/g extract. Significant differences were observed among the groups, with the aqueous extract having the highest TCC, followed by the ethanol extract. Notably, no TCC was detected in the dichloromethane extract. This finding may be attributed to the presence of carbohydrates and glycoside components in the aqueous extract. Table 3. Total carbohydrate content (TCC), total protein content (TP ro C), total triterpenoid content (TT ri C), total phenolic content (TP he C), total flavonoid content (TFC), total tannin content (TT an C), gallotannin content (GC), condensed tannin content (CTC), and total alkaloid content (TAC) of Euphorbia fischeriana extracted with different solvents. Furthermore, plant protein possesses diverse properties and nutritional value, and is readily digested and absorbed by the human body. Plant protein has been associated with a range of health benefits, including immune regulation, antioxidant, and anti-fatigue effects [22]. In the present study, the aqueous extract of Euphorbia fischeriana showed the highest TP ro C, which was 643.2 ± 12.5 mg bovine serum albumin equivalents (BSAE)/g extract ( Table 3). The methanol, ethanol, and acetone extracts also showed higher TP ro C levels. Previous studies have shown that higher TP ro C values indicate the presence of more antioxidant components in the extract [23], suggesting that the ethanol and acetone extracts of Euphorbia fischeriana contain antioxidants.

Total Triterpenoid Content (TT ri C)
Triterpenoids are important bioactive compounds that exhibit diverse biological activities, including anticancer, antiallergy, antiatherosclerosis, and antiulcer properties [24]. In the present study, the TT ri C was determined in eight different solvent extracts of Euphorbia fischeriana. As shown in Table 3, the acetone extract exhibited the highest TT ri C, which was 870.5 ± 49.8 mg ginsenoside Re equivalents (GRE)/g extract, followed by the ethyl acetate extract. These results of this study are consistent with a previous report indicating that Euphorbia fischeriana is abundant in triterpenoids [18].

Total Phenolic Content (TP he C)
Plant polyphenols, such as flavonoids, are another group of bioactive substances that have excellent antioxidant capacity and display antitumor and antiviral activities. They also play a critical role in the prevention of cardiovascular disease and dementia [25] and are widely used in the cosmetic, food, and medical industries [26]. The TP he C in the eight solvent extracts of Euphorbia fischeriana was also measured in this study ( Table 3). The TP he C values ranged from 0.0 to 38.6 ± 2.2 mg gallic acid equivalents (GAE)/g extract, with the acetone extract showing the highest TP he C. These results further highlight the therapeutic potential of Euphorbia fischeriana as a rich source of polyphenols with a wide range of biological activities.

Total Flavonoid Content (TFC)
Flavonoids are a subclass of polyphenols that are known to prevent cell degeneration and aging, inhibit the growth of cancer cells, regulate blood pressure and cholesterol, and exhibit preventive effects against cardiovascular and cerebrovascular diseases [27]. Unfortunately, the total flavonoid content (TFC) in the different solvent extracts of Euphorbia fischeriana was found to be very low, ranging from 1.4 ± 0.0 to 3.2 ± 0.1 mg quercetin equivalents (QE)/g extract (Table 3). This finding can explain the contradictory results of the four qualitative experimental analyses conducted earlier, as the low content of flavonoids might have resulted in different experimental sensitivities.

Total Tannin Content (TT an C), Gallotannin Content (GC), and Condensed Tannin Content (CTC)
Tannins are bioactive compounds that possess antibacterial and antiviral properties. They are also capable of removing superoxide free radicals from the body and delaying aging [28]. To evaluate the tannin content in various solvent extracts of Euphorbia fischeriana, the GC, CTC, and TT an C were determined ( Table 3). The acetone extract exhibited the highest GC and CTC. The aqueous extract had the highest TT an C and the acetone extract had the second-highest TT an C. These results demonstrate the potential of Euphorbia fischeriana as a rich source of tannin and further support the therapeutic benefits of this plant.

Total Alkaloid Content (TAC)
Alkaloids are a group of bioactive compounds that have a wide range of pharmacological activities, including the protection of the cardiovascular, nervous, and immune Molecules 2023, 28, 5172 7 of 33 systems, and anti-cancer properties [29]. While the results of qualitative experiments on three alkaloids tested in this study were all positive, the TAC of different extracts was found to be very small, ranging from 1.6 ± 0.0 to 2.4 ± 0.0 mg berberine hydrochloride equivalents (BHE)/g extract (Table 3). This finding indicates that the detection limits of the three qualitative experiments were low and sensitive.
Overall, this study provides valuable insights into the potential of Euphorbia fischeriana as a source of bioactive compounds, including carbohydrates, glycosides, plant protein, phenolics, triterpenoids, tannins, and alkaloids, with various physiological and pharmacological functions. Further research is required to explore the mechanisms of action and the potential applications of these compounds in health and medical research. This could significantly contribute to our understanding of the therapeutic potential of Euphorbia fischeriana and the development of new treatments for various diseases.

DPPH and ABTS
The assessment of the free radical scavenging ability of different extracts is essential to understand the antioxidant potential of natural products. DPPH and ABTS scavenging assays are widely utilized to evaluate the free radical scavenging ability in vitro [30]. DPPH is a fat-soluble free radical while ABTS is water-soluble, and each assay targets different types of free radicals, allowing for the identification of specific antioxidants.
In our experiment, we investigated the free radical scavenging ability of various extracts of Euphorbia fischeriana. Our results are consistent with the existing literature. Specifically, the methanol, ethanol, and acetone extracts showed robust antioxidant capacity in DPPH assays, while the acetone and ethanol extracts also displayed strong ABTS scavenging activity (Table 4). Among the tested Euphorbia species, Euphorbia ebracteolata, Euphorbia tirucalli, and Euphorbia heyneana exhibited potent DPPH scavenging ability while Euphorbia tirucalli and Euphorbia Atlantic demonstrated strong ABTS scavenging activity [31][32][33][34].

Hydroxyl Radicals and Superoxide Radicals
The study of hydroxyl radicals and superoxide radicals produced in the body is also crucial to evaluate the antioxidant capacity of a compound. The scavenging of these radicals showcases the ability of antioxidants to combat oxidative toxicity in cells [35].
Our findings indicated that the acetone extract of Euphorbia fischeriana exhibited the most robust hydroxyl radical scavenging activity, followed by the ethanol extract. Additionally, in experiments conducted with superoxide radicals, the dichloromethane extract was the most potent scavenger using curcumin as a positive control (Table 4). Previous data also indicated that Euphorbia heyneana possesses the ability to scavenge both hydroxyl radicals and superoxide radicals [33].

FRAP and CUPRAC
Assessing the antioxidant capacity of samples is an important step in identifying potential therapeutic agents. One method of measuring this capacity is by evaluating the sample's ability to reduce iron and copper ions [36]. Two commonly used methods for this evaluation are FRAP and CUPRAC assays. FRAP experiments are carried out in acidic conditions, while CUPRAC experiments are conducted in neutral conditions, closer to the physiological environment, making the latter method more reliable for assessing therapeutic potential.
In our study, we used FRAP and CUPRAC assays to evaluate the antioxidant capacity of various Euphorbia fischeriana extracts. The methanol, ethanol, and acetone extracts exhibited strong antioxidant capacity in FRAP assays, and these results were confirmed in CUPRAC experiments (Table 5). Our findings support existing literature, which identified Euphorbia hirta, Euphorbia heterophylla, and Euphorbia convolvuloides as having good antioxidant performance in FRAP and CUPRAC assays [37].

Metal Chelating
The presence of iron and copper ions can accelerate the Fenton reaction, leading to increased oxidative stress in vivo [38]. Therefore, identifying effective metal-ion chelators is critical in the search for potential therapeutic agents.
Our research showed that the ethyl acetate extract of Euphorbia fischeriana exhibited the highest chelating activity toward ferrous and copper ions. The chelating activity of the other solvent extracts was lower in comparison (Table 5). Additionally, Euphorbia neriifolia showed strong chelating activity toward ferrous ions [39].
Overall, these results suggest the potential of Euphorbia species as a rich source of natural antioxidant and chelating compounds that could be developed into powerful therapeutic agents. Further research is necessary to identify the active compounds responsible for these properties and to assess their bioavailability and pharmacological effects.

Hydrogen Peroxide (H 2 O 2 )
H 2 O 2 is a potent oxidizing agent and a byproduct of human metabolism. In excess, it can lead to cell and tissue damage, making its direct removal essential for maintaining the body's health [40]. Our results showed that the acetone extract of Euphorbia fischeriana had the highest H 2 O 2 scavenging activity ( Table 6). Another Euphorbia species, Euphorbia neriifolia, also showed significant H 2 O 2 scavenging ability [39], suggesting that Euphorbia species, in general, may possess potent H 2 O 2 scavenging capabilities. 2.4.6. β-Carotene Bleaching β-Carotene is a widely used polyene colorant that is prone to oxidation, causing it to lose its yellow color [41]. Antioxidants can slow down the bleaching rate of β-carotene by inhibiting its oxidation process. The strength of an antioxidant can be determined by the degree of decrease in β-carotene absorbance over time. In our research, we evaluated the antioxidant capacity of different extracts of Euphorbia fischeriana using the β-carotene bleaching assay. Our results showed that the methanol, ethanol, and acetone extracts exhibited strong antioxidant capacity (Table 6), comparable to that of synthetic antioxidants such as BHT and BHA ( Figure 2). These findings suggest that Euphorbia fischeriana may be a potential source of natural antioxidants that have the ability to inhibit oxidation processes and preserve food quality during storage. Moreover, literature reports show that six different Euphorbia species also display β-carotene bleaching inhibition activity. These findings support the potential of the Euphorbia genus to provide natural antioxidants [42,43]. The results suggest that the Euphorbia species could be a promising source for developing natural antioxidants with potential for various food and medicinal applications. Further research is warranted to identify the active compounds in Euphorbia fischeriana and other Euphorbia species that contribute to the antioxidant activity.

Singlet Oxygen
Singlet oxygen is another reactive oxygen species that can lead to cellular damage [44]. In our experiment, we found that the methanol extract of Euphorbia fischeriana had the best singlet oxygen scavenging activity, followed by the dichloromethane and acetone extracts ( Table 6). This result suggests that Euphorbia fischeriana may be a valuable source

Singlet Oxygen
Singlet oxygen is another reactive oxygen species that can lead to cellular damage [44]. In our experiment, we found that the methanol extract of Euphorbia fischeriana had the best singlet oxygen scavenging activity, followed by the dichloromethane and acetone extracts ( Table 6). This result suggests that Euphorbia fischeriana may be a valuable source of natural compounds with singlet oxygen scavenging properties.

Hypochlorous Acid (HClO)
HClO is a potent oxidant that plays a crucial role in defending against pathogen invasion, but excessive HClO can disrupt the organism's oxidative balance and lead to disease [45]. We found that among the Euphorbia fischeriana extracts, the aqueous extract had the best HClO scavenging ability, followed by the acetone extract (Table 6). This finding suggests that Euphorbia fischeriana extracts may contain natural compounds that could be useful in the modulation of HClO levels in the body.

Nitric Oxide (NO)
NO is a gas that can pass freely through biofilms in the living body and participate in many biological processes, but it can also cause cellular damage by modifying protein function. Our results showed that both the aqueous and acetone extracts of Euphorbia fischeriana exhibited strong NO scavenging ability ( Figure 3). These findings suggest that Euphorbia fischeriana extracts may contain natural compounds that could be useful in the regulation of NO levels in the body. The potential of Euphorbia species in scavenging NO is further supported by research on Euphorbia neriifolia and Euphorbia tirucalli, which also displayed robust NO scavenging ability [39,46]. exhibited the highest TTriC, suggesting that acetone is a more effective solvent for extracting terpenoids than ethanol. Based on this result, we will proceed with further research using the acetone extract. Further studies on the chemical composition of this extract will enable the identification of specific compounds responsible for its antioxidant activity. We also plan to conduct more in vivo experiments to further substantiate its antioxidant properties and assess its safety for potential use in therapeutic applications.

UHPLC-MS Analysis
In this study, the chemical composition of the acetone extract of Euphorbia fischeriana was analyzed using UHPLC-ESI-Q-TOF-MS. Molecular ions and fragment ions were matched with reference data, resulting in the identification of 39 bioactive substances ( Table 7). The structures of these compounds are presented in Figure 4. The UHPLC-MS results obtained in positive-ion mode are shown in Figure 5   Our research results demonstrate that Euphorbia fischeriana could emerge as a valuable source of natural compounds with potent antioxidant activity, with its acetone extract showing particularly promising results. The acetone extract displayed strong TT ri C, TP he C, GC, and CTC values. Additionally, the acetone extract also exhibited robust ABTS, hydroxyl radicals, and H 2 O 2 scavenging abilities, together with highest FRAP and CUPRAC values. The antioxidant activity of the ethanol extract was found to be comparable to that of the acetone extract, indicating its potential for further study. However, given that terpenoids make up 70% of the more than 200 compounds isolated from Euphorbia fischeriana, they are considered the primary active components and, therefore, an important focus of research. Notably, while the ethanol extract failed to detect triterpenoids in the determination of total triterpenoids, the acetone extract exhibited the highest TTriC, suggesting that acetone is a more effective solvent for extracting terpenoids than ethanol. Based on this result, we will proceed with further research using the acetone extract. Further studies on the chemical composition of this extract will enable the identification of specific compounds responsible for its antioxidant activity. We also plan to conduct more in vivo experiments to further substantiate its antioxidant properties and assess its safety for potential use in therapeutic applications.

UHPLC-MS Analysis
In this study, the chemical composition of the acetone extract of Euphorbia fischeriana was analyzed using UHPLC-ESI-Q-TOF-MS. Molecular ions and fragment ions were matched with reference data, resulting in the identification of 39 bioactive substances ( Table 7). The structures of these compounds are presented in Figure 4. The UHPLC-MS results obtained in positive-ion mode are shown in Figure 5           In the UHPLC-MS results, the glycosides contained in Euphorbia fischeriana are vulnerable to neutral loss of different glycosyls upon collision dissociation, such as glucose (m/z 162), arabinose (m/z 132), and xylose (m/z 132). Upon analysis, peak 1 in the mass spectrum showed an ion at m/z 365.1062. The MS 2 spectrum of this ion exhibited a fragment at m/z 162.1125 that resulted from the loss of hexose (162 Da) from the deprotonated ion at m/z 365.1062. Thus, peak 1 was identified as sucrose [47].

Molecular Electrostatic Potential (MEP) Surface Map
In this study, ten large peaks were selected based on their peak area from large to small, and their corresponding compounds were numbered as compounds 1-15. A detailed overview of the compounds and their corresponding numbers can be found in Table 8 and Figure 6.

Frontier Molecular Orbital
The frontier molecular orbital theory is a powerful tool in predicting the chemical reactivity of a given system. Investigations into the energy levels of frontier molecular orbitals offer valuable insights into the reactivity of bioactive molecules [76]. This theory is based on studying the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of molecules. The energies associated with the Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO) play a crucial role in determining the reactivity of molecules, as they typically participate in chemical reactions [77]. The HOMO of a molecule determines its antioxidant capacity, while the strength of its oxidation capacity depends on the energy of the LUMO. Therefore, the lower the energy of the LUMO, the stronger the oxidation capacity, and the higher the energy of the HOMO, the stronger the antioxidant capacity of the molecule. The energy difference between the HOMO and LUMO is referred to as the "band gap." To determine the band gap-the energy difference between the HOMO and the LUMO-an optimized geometry was utilized, and calculations were performed using density functional theory [78]. A smaller band gap indicates that the molecule is easier to excite.
This study aimed to explore the antioxidant activities of the fifteen compounds corresponding to the top ten peaks. To compare their antioxidant capacity, we selected two positive controls, trolox and gallic acid, which were previously used in the experiments. According to theoretical calculations, all fifteen compounds had a HOMO The principle of an antioxidant is to reduce the concentration of oxygen, and the activated site for antioxidant activity is expected to be the region of positive electrostatic potential. Darker areas of blue signify a more potent antioxidant activity since they exhibit higher positive electrostatic potential. As illustrated in Figure 7, compounds 9, 10, 14, and 15 possessed a more positive electrostatic potential than the other compounds, suggesting that they might be better antioxidants.

Frontier Molecular Orbital
The frontier molecular orbital theory is a powerful tool in predicting the chemical reactivity of a given system. Investigations into the energy levels of frontier molecular orbitals offer valuable insights into the reactivity of bioactive molecules [76]. This theory is based on studying the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of molecules. The energies associated with the Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO) play a crucial role in determining the reactivity of molecules, as they typically participate in chemical reactions [77]. The HOMO of a molecule determines its antioxidant capacity, while the strength of its oxidation capacity depends on the energy of the LUMO. Therefore, the lower the energy of the LUMO, the stronger the oxidation capacity, and the higher the energy of the HOMO, the stronger the antioxidant capacity of the molecule. The energy difference between the HOMO and LUMO is referred to as the "band gap." To determine the band gap-the energy difference between the HOMO and the LUMO-an optimized geometry was utilized, and calculations were performed using density functional theory [78]. A smaller band gap indicates that the molecule is easier to excite.
This study aimed to explore the antioxidant activities of the fifteen compounds corresponding to the top ten peaks. To compare their antioxidant capacity, we selected two positive controls, trolox and gallic acid, which were previously used in the experiments. According to theoretical calculations, all fifteen compounds had a HOMO distribution ranging from −5.98 eV to −7.93 eV, indicating that they exhibit a certain antioxidant capacity. Among them, compounds 9 (−6.07 eV) and 14 (−5.98 eV) displayed superior HOMO performance compared to gallic acid (−6.12 eV) but were weaker than trolox (−5.42 eV). The findings of this study indicate that the presence of phenolic hydroxyl groups and glycoside groups in the compound structure plays a crucial role in increasing the energy levels of HOMO orbitals, thereby enhancing the antioxidant potential of these compounds. The band gap, which determines the chemical reactivity, is closely associated with the ease of excitation of the investigated molecules. Analysis of Figure 8  In conclusion, based on these calculations, compounds 9 and 14 demonstrate strong antioxidant capacity and are more likely to engage in chemical reactions, suggesting that they may serve as primary antioxidant components in Euphorbia fischeriana.

Stability Studies of Acetone Extract
Our study explored the stability of the acetone extract of Euphorbia fischeriana and its antioxidant properties through a series of experiments. Figures 9-11 depict the outcomes of these experiments. We discovered that the extract's TPheC value and ABTS scavenging activity were generally stable when subjected to changes in pH. The highest value for TPheC of the extract was noted at pH 7, and it decreased slightly with an increase or decrease in pH. In comparison, the ABTS scavenging activity showed a gradual decrease with increasing pH. The stronger the alkalinity, the greater was the impact on the acidic system of the ABTS experiment, which likely accounted for the observed decrease. Regarding heating time, we observed a slight reduction in the TPheC value and ABTS scavenging activity of the extract. However, the impact was minimal. Concerning stability experiments using an in vitro simulation of the human digestive system, the TPheC value of the acetone extract decreased gradually with time. We concluded that gastric acid, pepsin, trypsin, pancreatin, and bile may all have affected the extract, leading to the gradual decrease in the TPheC value. The ABTS scavenging activity of the extract followed a similar trend as the TPheC. Nevertheless, our stability studies revealed that the antioxidant components of the acetone extract of Euphorbia fischeriana were stable, which is promising for maintaining its efficacy under various physiological conditions. In conclusion, based on these calculations, compounds 9 and 14 demonstrate strong antioxidant capacity and are more likely to engage in chemical reactions, suggesting that they may serve as primary antioxidant components in Euphorbia fischeriana.

Stability Studies of Acetone Extract
Our study explored the stability of the acetone extract of Euphorbia fischeriana and its antioxidant properties through a series of experiments. Figures 9-11 depict the outcomes of these experiments. We discovered that the extract's TP he C value and ABTS scavenging activity were generally stable when subjected to changes in pH. The highest value for TP he C of the extract was noted at pH 7, and it decreased slightly with an increase or decrease in pH. In comparison, the ABTS scavenging activity showed a gradual decrease with increasing pH. The stronger the alkalinity, the greater was the impact on the acidic system of the ABTS experiment, which likely accounted for the observed decrease. Regarding heating time, we observed a slight reduction in the TP he C value and ABTS scavenging activity of the extract. However, the impact was minimal. Concerning stability experiments using an in vitro simulation of the human digestive system, the TP he C value of the acetone extract decreased gradually with time. We concluded that gastric acid, pepsin, trypsin, pancreatin, and bile may all have affected the extract, leading to the gradual decrease in the TP he C value. The ABTS scavenging activity of the extract followed a similar trend as the TP he C. Nevertheless, our stability studies revealed that the antioxidant components of the acetone extract of Euphorbia fischeriana were stable, which is promising for maintaining its efficacy under various physiological conditions.

Cell Viability
Our experiment examined the effects of the acetone extract of Euphorbia fischeriana on the cellular morphology of TM 3 mouse Leydig cells after 24 and 48 h of treatment, as shown in Figure 12. The results indicated that at low doses (25 and 50 µg/mL), the acetone extract did not exhibit any cytotoxicity. In fact, it even promoted cell proliferation to a certain extent with increasing incubation time. However, at moderate doses (100 µg/mL), the extract induced some cytotoxicity, but this did not increase with the extension of incubation time. On the other hand, at high doses (200 µg/mL), the extract demonstrated clear cytotoxicity, and its toxic effects increased significantly with the extension of incubation time (Table 9). These results emphasize the importance of dosage when using Euphorbia fischeriana as a therapeutic agent. Our findings suggest that Euphorbia fischeriana extract has the potential to be developed into an effective drug for certain health conditions. However, it is necessary to pay careful attention to the dose administered to avoid possible toxic effects. It is worth noting that different Euphorbia species have varying degrees of cytotoxicity that can affect different types of cells. For instance, Euphorbia vajraveleu has been found to be non-toxic to normal cells, H9C2, and has negligible toxic effects on cervical cancer cells, Hela [79]. Conversely, Euphorbia lathyris, another species of this genus, has been found to show strong cytotoxicity against a variety of cancer cells [80]. These differences in cytotoxicity among Euphorbia species may be attributed to the varying chemical compositions and concentrations of their extracts.

Cell Viability
Our experiment examined the effects of the acetone extract of Euphorbia fischeria the cellular morphology of TM3 mouse Leydig cells after 24 and 48 h of treatme shown in Figure 12. The results indicated that at low doses (25 and 50 μg/mL acetone extract did not exhibit any cytotoxicity. In fact, it even promoted cell prolife to a certain extent with increasing incubation time. However, at moderate dose μg/mL), the extract induced some cytotoxicity, but this did not increase wit extension of incubation time. On the other hand, at high doses (200 μg/mL), the e demonstrated clear cytotoxicity, and its toxic effects increased significantly wit extension of incubation time (Table 9). These results emphasize the importance of d when using Euphorbia fischeriana as a therapeutic agent. Our findings sugges Euphorbia fischeriana extract has the potential to be developed into an effective dru certain health conditions. However, it is necessary to pay careful attention to the administered to avoid possible toxic effects. It is worth noting that different Eup species have varying degrees of cytotoxicity that can affect different types of cell instance, Euphorbia vajraveleu has been found to be non-toxic to normal cells, H9C2 has negligible toxic effects on cervical cancer cells, Hela [79]. Conversely, Eup lathyris, another species of this genus, has been found to show strong cytotoxicity a a variety of cancer cells [80]. These differences in cytotoxicity among Euphorbia s may be attributed to the varying chemical compositions and concentrations of extracts.

Oral Acute Toxicity Study
In our study, we administered a single dose of 2000 mg/kg of Euphorbia fischeriana acetone extract orally to a group of mice, and noted that none of the 20 tested mice died within 24 h. This finding suggests that the toxicity associated with acetone extract is relatively low. Similarly, a study on Euphorbia fusiformis found that mice tolerated up to 5000 mg/kg bw without any incidence of mortality. The single dose LD 50 was determined to be 10,000 mg/kg bw in this study [81]. Another study evaluated the safety of Euphorbia hirta extract in rats and found that a single dose of 5000 mg/kg did not induce any significant side effects or mortalities during the 14-day observation period [82]. Taken together, these studies provide compelling evidence indicating that Euphorbia plants are relatively safe for use in rats and mice. However, it should be noted that these findings do not necessarily imply that Euphorbia plants are safe for human consumption. To date, only a limited number of clinical studies have been conducted on Euphorbia extracts. More research, including preclinical and clinical studies, is needed to evaluate their safety and efficacy in humans.

Hepatoprotective Activity
The liver is a critical metabolic organ in vertebrates that plays a vital role in processing nutrients, storing energy, and eliminating waste substances. When the liver is damaged, it can lead to a decrease in the fluidity of the liver cell membrane, which causes an increase in cell permeability. As a result, enzymes such as aspartate aminotransferase, alanine aminotransferase, and γ-glutamyl transpeptidase are released into the bloodstream. Liver damage can also impair the binding and excretion of bilirubin in bile, which increases the concentration of total bilirubin in the blood [83].
To examine the effectiveness of Euphorbia fischeriana acetone extract in managing liver damage, we conducted experiments on rats. Forty rats were randomly divided into five groups (n = 8). Each group received a different treatment orally. The control group (group I) was given 0.5% carboxymethylcellulose sodium. The negative control group (group II) was administered D-galactosamine and 0.5% carboxymethylcellulose sodium. The high-dose group (group III) received Euphorbia fischeriana acetone extract at 300 mg/kg BW. The low-dose group (group IV) received Euphorbia fischeriana acetone extract at 150 mg/kg BW. Finally, the comparison group (group V) was given silymarin at 100 mg/kg BW.
We fed the rats with Euphorbia fischeriana acetone extract every day for seven days by gavage, followed by D-galactosamine injection. The results showed that pretreatment with the acetone extract (high and low dose groups) significantly reduced the viscera index in the rats compared to group II, indicating an improvement in liver condition (p < 0.001, Figure 13). Moreover, the high-dose group had a significantly better effect on liver function than silymarin (p < 0.001, Figure 13).
Our results revealed that compared with group II, the activity levels of liver enzymes including aspartate aminotransferase, alanine aminotransferase, and γ-glutamyl transpeptidase were significantly reduced in the groups treated with Euphorbia fischeriana acetone extract (p < 0.001, shown in Figure 14). In particular, compared to group II, the reduced activity of alanine aminotransferase was 63.57% and 68.17% in groups III and IV, respectively, while aspartate aminotransferase was reduced by 61.54% and 62.72%, respectively. These findings suggest that the low-dose acetone extract of Euphorbia fischeriana has a potent curative effect on liver damage caused by D-galactosamine. Our results revealed that compared with group II, the activity levels of liver enzymes including aspartate aminotransferase, alanine aminotransferase, and γ-glutamyl transpeptidase were significantly reduced in the groups treated with Euphorbia fischeriana acetone extract (p < 0.001, shown in Figure 14). In particular, compared to group II, the reduced activity of alanine aminotransferase was 63.57% and 68.17% in groups III and IV, respectively, while aspartate aminotransferase was reduced by 61.54% and 62.72%, respectively. These findings suggest that the low-dose acetone extract of Euphorbia fischeriana has a potent curative effect on liver damage caused by D-galactosamine.
The reduction in the activity of liver enzymes observed in our study is indicative of the amelioration of liver damage. Increased activity levels of these enzymes in the liver are an indication of liver inflammation and injury. Therefore, the significant reduction in the activity of aspartate aminotransferase, alanine aminotransferase, and γ-glutamyl transpeptidase in the treatment groups supports the use of Euphorbia fischeriana acetone extract as a potential therapeutic agent for liver-related diseases and disorders.   Silymarin. Significantly different from the control group at ** p < 0.01 and *** p < 0.001. Significantly different from the D-GalN group at ## p < 0.01 and ### p < 0.001. Significantly different from the D-GalN + SMN group at $$ p < 0.01 and $$$ p < 0.001.
Our results revealed that compared with group II, the activity levels of liver enzymes including aspartate aminotransferase, alanine aminotransferase, and γ-glutamyl transpeptidase were significantly reduced in the groups treated with Euphorbia fischeriana acetone extract (p < 0.001, shown in Figure 14). In particular, compared to group II, the reduced activity of alanine aminotransferase was 63.57% and 68.17% in groups III and IV, respectively, while aspartate aminotransferase was reduced by 61.54% and 62.72%, respectively. These findings suggest that the low-dose acetone extract of Euphorbia fischeriana has a potent curative effect on liver damage caused by D-galactosamine.
The reduction in the activity of liver enzymes observed in our study is indicative of the amelioration of liver damage. Increased activity levels of these enzymes in the liver are an indication of liver inflammation and injury. Therefore, the significant reduction in the activity of aspartate aminotransferase, alanine aminotransferase, and γ-glutamyl transpeptidase in the treatment groups supports the use of Euphorbia fischeriana acetone extract as a potential therapeutic agent for liver-related diseases and disorders. The reduction in the activity of liver enzymes observed in our study is indicative of the amelioration of liver damage. Increased activity levels of these enzymes in the liver are an indication of liver inflammation and injury. Therefore, the significant reduction in the activity of aspartate aminotransferase, alanine aminotransferase, and γ-glutamyl transpeptidase in the treatment groups supports the use of Euphorbia fischeriana acetone extract as a potential therapeutic agent for liver-related diseases and disorders.
Moreover, compared with group II, the albumin level was significantly reduced in the rats, while treatment with both silymarin and the acetone extract significantly increased the albumin level after 24 h of modeling (p < 0.001). The low-dose group was better than the high-dose group in improving albumin levels. Additionally, D-galactosamine injection increased the concentration of total bilirubin in the blood of rats in group II, while the concentration of total bilirubin in groups III-V decreased significantly compared to group II, with the low-dose group performing better than the high-dose group and being comparable to silymarin. Furthermore, D-galactosamine-induced liver injury increased the production of reactive oxygen species and reduced the efficacy of antioxidants in vivo. This led to an increase in the level of malondialdehyde and a decrease in the level of glutathione in the liver of rats. Compared with group II, the effect of the acetone extract on enhancing glutathione levels was not ideal, but it significantly reduced malondialdehyde levels (p < 0.001, shown in Figure 15). The low-dose group performed better than the high-dose group and was comparable to silymarin in reducing malondialdehyde levels. rats in group II, while the concentration of total bilirubin in groups III-V decreased significantly compared to group II, with the low-dose group performing better than the high-dose group and being comparable to silymarin. Furthermore, D-galactosamine-induced liver injury increased the production of reactive oxygen species and reduced the efficacy of antioxidants in vivo. This led to an increase in the level of malondialdehyde and a decrease in the level of glutathione in the liver of rats. Compared with group II, the effect of the acetone extract on enhancing glutathione levels was not ideal, but it significantly reduced malondialdehyde levels (p < 0.001, shown in Figure 15). The low-dose group performed better than the high-dose group and was comparable to silymarin in reducing malondialdehyde levels.
In summary, our findings indicate that the low-dose acetone extract of Euphorbia fischeriana has a good liver-protective effect through antioxidant activity. The overall protective effect of this extract is comparable to that of silymarin, suggesting its potential as a therapeutic agent for liver-related disorders and diseases. Significantly different from the control group at * p < 0.05 and *** p < 0.001. Significantly different from the D-GalN group at # p < 0.05 ## p < 0.01 and ### p < 0.001. Significantly different from the D-GalN + SMN group at $$ p < 0.01 and $$$ p < 0.001.
In summary, our findings indicate that the low-dose acetone extract of Euphorbia fischeriana has a good liver-protective effect through antioxidant activity. The overall protective effect of this extract is comparable to that of silymarin, suggesting its potential as a therapeutic agent for liver-related disorders and diseases.
The results of the histopathological examination are presented in Figure 15. In the control group, hepatocytes were arranged in a normal pattern, and no inflammatory cell infiltration was observed around the portal area ( Figure 16A). However, in group II, the liver's histological structure was disordered, the hepatic cord was absent, single-cell necrosis was visible (no-tailed arrow), and a large number of inflammatory cells were present (long-tailed arrow) ( Figure 16B). The magnified view of group II at 400× further revealed the presence of increased inflammatory cell infiltration and necrosis ( Figure 16C). Remarkably, the high-dose group showed significant improvement in hepatocyte injury with fewer inflammatory and necrotic cells ( Figure 16D). Similarly, the low-dose group demonstrated better results compared to the high-dose group, with fewer inflammatory and necrotic cells ( Figure 16E). It was interesting to note that the positive group had the best curative effect, which was evident by the significant reduction in inflammatory and necrotic cells and the relatively complete morphology of hepatocytes ( Figure 16F). necrosis was visible (no-tailed arrow), and a large number of inflammatory cells were present (long-tailed arrow) ( Figure 16B). The magnified view of group II at 400× further revealed the presence of increased inflammatory cell infiltration and necrosis ( Figure  16C). Remarkably, the high-dose group showed significant improvement in hepatocyte injury with fewer inflammatory and necrotic cells ( Figure 16D). Similarly, the low-dose group demonstrated better results compared to the high-dose group, with fewer inflammatory and necrotic cells ( Figure 16E). It was interesting to note that the positive group had the best curative effect, which was evident by the significant reduction in inflammatory and necrotic cells and the relatively complete morphology of hepatocytes ( Figure 16F). It is essential to understand the significance of the findings observed in this study. The histopathological examination reveals the extent of damage to the liver caused by D-galactosamine. The presence of inflammatory cells, necrotic cells, and a disordered histological structure in group II demonstrates the severity of liver damage induced by D-galactosamine. The reduction in these parameters in the positive, high-dose, and low-dose groups indicates the hepatoprotective effect of the Euphorbia fischeriana acetone extract. These findings support the potential use of Euphorbia fischeriana extract in the treatment of liver disorders.
Recent research has shown that different species of Euphorbia plants have significant hepatoprotective effects. For instance, Euphorbia fusiformis has been found to decrease elevated biochemical parameters to a level comparable to that of the control group [78]. Another study conducted on Euphorbia antiquorum demonstrated that it significantly increased the level of reduced glutathione in tissues by reducing the activities of serum It is essential to understand the significance of the findings observed in this study. The histopathological examination reveals the extent of damage to the liver caused by D-galactosamine. The presence of inflammatory cells, necrotic cells, and a disordered histological structure in group II demonstrates the severity of liver damage induced by Dgalactosamine. The reduction in these parameters in the positive, high-dose, and low-dose groups indicates the hepatoprotective effect of the Euphorbia fischeriana acetone extract. These findings support the potential use of Euphorbia fischeriana extract in the treatment of liver disorders.
Recent research has shown that different species of Euphorbia plants have significant hepatoprotective effects. For instance, Euphorbia fusiformis has been found to decrease elevated biochemical parameters to a level comparable to that of the control group [78]. Another study conducted on Euphorbia antiquorum demonstrated that it significantly increased the level of reduced glutathione in tissues by reducing the activities of serum enzymes, bilirubin, triglycerides, and lipid peroxidation in rats. Furthermore, its hepatoprotective and antioxidant activities were comparable to those of silymarin [84].
These observations suggest that Euphorbia plants could be potential sources of natural liver protective agents and antioxidants. The hepatoprotective properties of these plants are likely due to the antioxidant principle and potential of the ingredients they contain. Our study found that Euphorbia fischeriana acetone extract has a beneficial effect on liver health, which suggests its potential therapeutic applications in managing liver-related disorders. However, further research is necessary to determine the bioactive components responsible for these beneficial effects, as well as the optimal dosage and course of treatment. It is essential to identify the optimal dosage and timing to ensure the extract's maximum therapeutic benefits while minimizing any potential side-effects. Clinical studies are also necessary to validate the results of our study and provide more precise recommendations for using the extract in human patients. In conclusion, our results highlight the potential of Euphorbia plants as a natural source of liver-protective agents and antioxidants. Our findings indicate that Euphorbia fischeriana acetone extract has a beneficial effect on liver health and supports further exploration of its therapeutic potential in managing liverrelated diseases and disorders. We hope this study will inspire more research and clinical studies on Euphorbia plants as natural therapeutic agents for liver-related ailments.

Qualitative Phytochemical Analysis
Qualitative phytochemical analysis was performed on 15 types of chemical components, following a previously established method [23]. The detailed experimental procedure is described in the Supplementary Materials.

Quantitative Phytochemical Analysis
Quantitative phytochemical analysis was conducted to determine the concentration of various compounds such as TCC, TP ro C, TT ri C, TP he C, TFC, TT an C, CTC, GC, and TAC, using methods previously described in reference [23]. The detailed experimental procedure is described in the Supplementary Material.

Antioxidant Activity Assays
Antioxidant activity assays were performed using a range of different methods, including DPPH, ABTS, hydroxyl radicals, superoxide radicals, FRAP, CUPRAC, metal chelating, H 2 O 2 , HClO, β-carotene bleaching, and NO. These assays were conducted following previously established protocols [23]. The detailed experimental procedure is described in the Supplementary Materials.

Computational Methods
All calculations were performed using the Gaussian 09 program package at the B3LYP-D3/6-311G (d, p) level [85][86][87][88][89]. Following the optimization of the molecular structures, frequency calculations were performed to ensure that the optimized structures corresponded to minimum energy points with no virtual frequencies. Furthermore, the MEP surface of compounds 1-15 were analyzed using MULTIWFN software and the VMD program, while the frontier molecular orbitals (HOMO and LUMO) were analyzed using GaussView [90][91][92].

Cell-Viability Assay
To evaluate the cell viability, the TM 3 mouse cell line was used in the MTT assay, which was carried out as described in reference [23].

Oral Acute Toxicity Study
An oral acute toxicity study was conducted according to previously established methods [93].

Hepatoprotective Experiments
The hepatoprotective experiments performed included animal selection, experimental protocols, histopathological examination, and biochemical analyses, following the procedures outlined in reference [93].

Statistical Analysis
Statistical analysis was performed to assess the significance of the data. The data were presented as means with the standard error of the mean. One-way analysis of variance with post hoc least significant difference tests was used to test for significant correlations between groups. Pearson's correlation analysis was used to investigate the relationship between antioxidant activity and total active constituents. p-values of 0.05, 0.01, and 0.001 were considered significant, highly significant, and very highly significant, respectively.

Conclusions
Euphorbia fischeriana is a well-known medicinal plant that has been used in traditional medicine for many years. Despite the numerous reports on the components of this plant, its antioxidant activity in vivo and in vitro and the identification of compounds that have antioxidant activities are still unknown. Therefore, this study aimed to fill this knowledge gap by investigating the antioxidant properties of Euphorbia fischeriana. The results of phytochemical analysis showed that Euphorbia fischeriana contains 11 types of phytochemicals. The contents of active components and antioxidant properties were evaluated in eight different solvent extracts of Euphorbia fischeriana using ultraviolet-visible spectrophotometry. Among these, the acetone extract exhibited the highest contents of active components and antioxidant activity and was selected as the object of further study. Further analysis of the acetone extract led to the identification of 43 specific compounds. To determine which compounds have antioxidant activities, the top ten peaks were selected for theoretical calculations of antioxidant capacity. The results confirmed the presence of antioxidant components in Euphorbia fischeriana acetone extract and helped clarify the antioxidant mechanism of this plant. To evaluate the effectiveness of Euphorbia fischeriana acetone extract as a potential antioxidant agent, its stability and antioxidant capacity were evaluated during heating, at different pH values, and after in vitro digestion. The results showed that the acetone extract exhibited excellent stability and antioxidant capacity even in adverse conditions, indicating its potential therapeutic applications. Finally, in vivo antioxidant experiments were conducted, and the results showed that the low-dose acetone extract displayed a significantly better protective effect on liver injury in rats.
In conclusion, our findings demonstrate that Euphorbia fischeriana acetone extract contains compounds with potent antioxidant properties. The identification of these active components provides a foundation for further exploration of this plant for its therapeutic potential in treating diseases related to oxidative stress. However, given the potential cytotoxicity of Euphorbia species, more in-depth research is needed to ensure their safe and effective use. We hope that our study will inspire further research on the antioxidant properties of Euphorbia fischeriana and contribute to the development of novel natural antioxidants for human health and wellbeing.