PMMB 2023, 6, 1; a0000331. doi: 10.36877/pmmb.0000331 http://journals.hh-publisher.com/index.php/pmmb Original Research Article HPLC-UV-MS/MS Profiling of Phenolics from Euphorbia nicaeensis (All.) Leaf and Stem and Its Antioxidant and Anti-Protein Denaturation Activities Khawla Bouaouda1, Chaimae Elagdi2, Naoufal El Hachlafi3, Karima Mohtadi1, Mohammed Hsaine2, Anass Kettani1, Rachid Flouchi3, Khang Wen Goh4, Abdelhakim Bouyahya5, Hanae Naceiri Mrabti6, Rachid Saile1, Hassan Taki1* Article History 1Laboratory of Biology and Health, University Hassan II of Casablanca, Faculty of Sciences Ben M’Sik, Casablanca, Morocco, P.O.Box 7955; khawla.bouaouda-etu@etu.univh2c.ma (KB); karima.mohtadi@univh2c.ma (KM); anass.kettani@univh2c.ma (AK); rachid.saile@univh2c.ma (RS) 2 Laboratory of Ecology and Environment, University Hassan II of Casablanca, Faculty of Sciences Ben M’Sik, Casablanca, Morocco, P.O.Box 7955; chaimaa.elagdi-etu@etu.univh2c.ma (CE); mohammed.hsaine@univh2c.ma (MH) 3Laboratory of Microbial Biotechnology and Bioactive Molecules, Sciences and Technologies Faculty, Sidi Mohamed Ben Abdellah University, P.O. Box 2202, Imouzzer Road, Fez, Morocco; naoufal.elhachlafi@usmba.ac.ma (NEH), racinf@gmail.com (RF) 4Faculty of Data Science and Information Technology, INTI International University, 71800 Nilai, Malaysia; khangwen.goh@newinti.edu.my (KWG) 5Laboratory of Human Pathologies Biology, Department of Biology, Faculty of Sciences, Mohammed V University in Rabat, Rabat 10106, Morocco; a.bouyahya@um5r.ac.ma (AB) 6High Institute of Nursing Professions and Health Techniques Casablanca, Morocco; naceiri.mrabti.hanae@gmail.com (HNM) *Corresponding author: Hassan Taki, University Hassan II of Casablanca, Faculty of Sciences Ben M’Sik, Casablanca, Morocco; hassan.taki@univh2c.ma (HT) Received: 19 March 2023; Received in Revised Form: 21 April 2023; Accepted: 10 May 2023; Available Online: 05 June 2023 Abstract: This work focuses on the leaves and stems of Euphorbia nicaeensis All. to confirm its historical use by the Moroccan population. The phytochemical profile of the plant by HPLC-UV-MS/MS was identified for the first time, and the biological activities of each plant organ was evaluated separately by maceration and ultrasonic-assisted extraction. The evaluation of antioxidant activity based on DPPH assay and hydrogen peroxide scavenging assay showed that leaves could be used as a natural source of antioxidants as they provide a potent antioxidant effect (DPPH IC50 = 23.47±0.62 µg/ml), (H2O2 IC50 = 110.27 ± 3.59 µg/ml) compared to stems. These results were proven by HPLC-UV-MS/MS analysis and revealed that E. nicaeensis leaves are richer in phenolic compounds, especially quercetin and derivatives, known for their antioxidant properties. In contrast, the stems could be considered a potential anti-inflammatory agent considering their solid anti-inflammatory activity. The PMMB 2023, 6, 1; a0000331 2 of 20 most potent effects were obtained at a concentration of 2 mg/ml, which induced 83.98% and 82.04% inhibition against bovine albumin and egg albumin denaturation, respectively, compared to the control. In addition, the stem phytochemical profile indicated the presence of some compounds with anti-inflammatory effects, such as fargesin and nuciferine. Likewise, the findings showed that ultrasound-assisted extraction was more effective than maceration. Keywords: Euphorbia nicaeensis All., Maceration, Ultrasound extraction, Antioxidant activity, Anti-inflammatory activity, HPLC-UV-MS/MS 1. Introduction Aromatic and medicinal plants have long been regarded as a vital source of therapeutics and curative remedies due to the presence of bioactive compounds and phytochemical constituents. Medicinal herbs have proven to be the basis of traditional medicine worldwide. Curing and healing human diseases has always been related to using entire plants or parts of them in remedy preparation [1]. Moroccan population has traditionally employed aromatic and medicinal plants; over time, people have gained knowledge of old cosmetics and pharmacopoeia. Oral communication is still used to pass on essential information about these activities from generation to generation [2]. However, several medicinal plants are currently underexploited in Morocco despite their ancestral use in treating human diseases. Euphorbia nicaeensis All. is one of these plants on which this investigation focuses. It is a species of Euphorbiaceae family, a perennial spurge herbaceous plant that prefers calcareous soils. It grows in sunny and dry places and is distributed in the Mediterranean and central Europe with a strong morphological variability in leaves and bracts [3]. E. nicaeensis is an abundant species in the rocky lawns of Morocco [4], latex is the most traditionally used part of the plant, and it has been used in the past to attack warts and erase dead flesh. The population was aware of the vesicant properties of latex on the skin, eyes, and mucous membranes [5]. There are also other uses of leaves and stems against bacterial infections, hepatitis, tuberculosis, typhoid, and other diseases [6–8]. Numerous studies have proven the traditional use of E. nicaeensis and its anthelmintic, antifungal, and anticancer activities due to the jatrophane diterpenoid isolated from the root extracts and latex [9–11]. Previous studies have also evaluated some biological activities of E. nicaeensis aerial parts and detected its anti-inflammatory properties [12,13]. This study aims to assess the phytochemical profile of Euphorbia nicaeensis by HPLC-UV- MS/MS analysis for the first time and to evaluate the anti-inflammatory properties and antioxidant activity of leaves and stems. Therefore, two extraction methods were compared, probe ultrasonic-assisted extraction and maceration. PMMB 2023, 6, 1; a0000331 3 of 20 2. Materials and Methods 2.1. Plant material The plant was harvested from the Ifrane region (Figure 1). The Lambert coordinates: 33° 32′ 44.4″ N, 5° 19′ 17.89″ W. and the plant have undergone drying after separation of the leaves. Figure 1. Photo of the species Euphorbia nicaeensis All. 2.2. Extraction procedure 2.2.1. Maceration An amount of 2.5 g of ground plant powder (leaves and stems) was extracted by 50 ml (methanol - water) (80:20) v/v. The maceration extracts were kept under stirring for 24 hours, then filtered under a Buchner funnel and concentrated by rotary evaporation. The residues were collected and stored until further analysis. 2.2.2. Probe ultrasonic-assisted extraction (PUAE) Probe ultrasonic-assisted extraction (PUAE) is one of the main extraction methods used for plant materials. Sonication causes cavitation and implosion, which causes cell-wall rupture and increases the number of disturbed cells. When disturbed, the solvent penetrates the cell, and the intracellular plant material is absorbed into the solvent [14]. The extraction was done using an ultrasonic probe system (Bioblock scientific, Vibra Cell 75042). The probe was submerged 1.5 cm under the surface of the mixture (methanol- water) (80:20) v/v and 2.5 g of plant powder. The extraction was performed at the maximum power settings of the transducer (100%, 400 W), at 24 kHz, for 15 min, with a 20 s pulse. Following extraction, the extract was filtered and concentrated by rotary evaporation until dryness and was stored for further use. 2.3. Phytochemical screening Phytochemical screening of Euphorbia nicaeensis All. leaves and stems were performed using standardized laboratory protocols. The presence or absence of phenols, tannins [15], flavonoids [16], saponins [17], flavonol, carbohydrates, alkaloids, gums, amino acids [18], sterols and polyterpenes [19], was analyzed accordingly. PMMB 2023, 6, 1; a0000331 4 of 20 2.4. Total phenolic content (TPC) The phenolic compounds were measured using Folin-Ciocalteu method [20]. In brief, 50 µl of Folin-Ciocalteu reagent was added to the sample, followed by 150 µl of Na2CO3 after 10 min, and the volume was made up to 1 ml with water. The absorbance was measured in a spectrophotometer reader at 760 nm after 2 hours of incubation and compared to the Gallic acid calibration curve (5 – 100 µg/ml), R2= 0.998. The results were expressed as mg Gallic acid equivalents /g dry weight (mg GAE/g DW). 2.5. Total flavonoid content (TFC) According to [21], 250 µl of the extract solution was mixed with 1 ml of distilled water and 75 µl of NaNO2 solution (5 %). After 6 min, 75 µl of Alcl3 solution (10 %) was added. The mixture was allowed to stand for 6 min before adding 1 ml of NaOH solution (4 %) and bringing the final volume to 2.5 ml with distilled water. The mixture was properly vortexed and allowed to stand for 15 min in darkness. The absorbance was measured at 510 nm and compared to the catechin standard curve (5 – 400 µg/ml) R2= 0.997. The results were expressed as mg of catechin equivalent per g dry weight (mg CE/g DW). 2.6. Flavonol content (FC) The content of flavonols was determined according to the method described by [22]. An aliquot of 500 µl of extract was added to 500 µl of Aluminium chloride (20 mg/ml) and 1.5 ml of sodium acetate (50 mg/ml). The mixture was properly vortexed and left to stand in darkness for 2.5 h. The absorbance was read at 440 nm, and the result was expressed referring to the absorbance of standard quercetin solution prepared in the same conditions (5 – 200 µg/ml), R2= 0,995. The flavonol content is expressed in mg Quercetin equivalents per g dry weight (mg QE/g DW). 2.7. Condensed tannin content (CTC) The assay was executed as reported previously [23]. An aliquot of 100 µl of each extract was added to 1.5 ml of Vanillin (4 %) and 750 µl of concentrated hydrochloric acid (HCL). The mixture was vortexed, left to stand in the dark for 20 min, and the absorbance was recorded at 500 nm. The condensed tannin content was counted related to the Catechin calibration curve (5 – 400 µg/ml) R2= 0.985, elaborated in the same manner. The results are expressed as mg of Catechin equivalent per 100 g dry weight (mg CE/100g DW). 2.8. Lipid-soluble pigment content According to [21], 150 mg of vegetal powder (leaves and stems) was mixed for 1 min with 10 ml of Acetone/Hexane (4:6). The mixture was filtered through Whatman filter paper grade 4. The absorbance was recorded at 453,505,645, and 663 nm. The content of β-carotene, lycopene, chlorophyll a, and chlorophyll b was calculated according to the following equations, expressed in µg per g dry weight (DW): PMMB 2023, 6, 1; a0000331 5 of 20 β-carotene = 0.216 × 𝐴663 − 1.220 × 𝐴645 − 0.304 × 𝐴505 + 0.452 × 𝐴453 Lycopene = −0.0458 × 𝐴663 + 0.204 × 𝐴645 − 0.304 × 𝐴505 + 0.452 × 𝐴453 Chlorophyll a = 0.999 × 𝐴663 − 0.0989 × 𝐴645 Chlorophyll b = 0.328 × 𝐴663 + 1.77 × 𝐴645 A453, A505, A645, and A663 are the absorbance measured at 453,505,645, and 663 nm, respectively. 2.9. In vitro antioxidant activity 2.9.1. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay The radical scavenging ability of the extract was carried out as described by [24–26]. 500 µl of extract solution was mixed with 1 ml of DPPH solution (0.1 mM in methanol) freshly prepared. The mixture was vortexed properly and left to stand in the dark for 60 min. The reduction of DPPH was measured at 515 nm, and the scavenging effect was estimated based on the percentage of scavenging DPPH radicals using the following equation: % 𝐃𝐏𝐏𝐇 𝐬𝐜𝐚𝐯𝐞𝐧𝐠𝐢𝐧𝐠 𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲 = 𝑨𝐃𝐏𝐏𝐇 − 𝑨𝐬 𝑨𝐃𝐏𝐏𝐇 × 𝟏𝟎𝟎 ADPPH is the absorbance of DPPH and AS is the absorbance of DPPH when the sample has been added at different concentrations. The IC50 value is the concentration that scavenges 50 % of DPPH radicals, and it is calculated from the scavenging effect percentage graph. Ascorbic acid was used as a standard. 2.9.2. Scavenging of hydrogen peroxide assay The performance of hydrogen peroxide (H2O2) scavenging was investigated as follows [27]. The phosphate buffer (50 mM, 7.4 pH) received a 40 mM H2O2 solution. In order to quantify the mixture's absorbance spectrophotometrically at 230 nm, all experimental samples were combined with 0.6 ml of H2O2 solution. The mixture was then incubated for 10 minutes. Ascorbic acid served as the standard, while phosphate buffer functioned as the control. Hydrogen peroxide scavenging (%) was calculated using the formula below. % 𝐇𝐲𝐝𝐫𝐨𝐠𝐞𝐧 𝐩𝐞𝐫𝐨𝐱𝐢𝐝𝐞 𝐬𝐜𝐚𝐯𝐞𝐧𝐠𝐢𝐧𝐠 𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲 = 𝑨𝟎 − 𝑨𝐬 𝑨𝟎 × 𝟏𝟎𝟎 Where As is the absorbance in the presence of the ascorbic acid standard or samples, and Ao is the absorbance of the blank. Ascorbic acid was used as a standard. PMMB 2023, 6, 1; a0000331 6 of 20 2.10. In vitro anti-inflammatory activity 2.10.1. Bovine Serum Albumin Assay (BSA) Protein denaturation is one of the causes of inflammatory and rheumatic diseases, as reported by a previous study [28]. Any compound that provides greater than 20% inhibition of protein denaturation is considered a potential anti-inflammatory agent and could be useful for treating several diseases [29]. BSA assay of leaf and stem extracts was determined using the method reported by [27]. 0.45 ml of bovine serum albumin was mixed with 0.05 ml of samples at various concentrations (200 - 2000 μg/ml). The mixture was incubated for 25 min at 40°C, and phosphate buffer saline (2.5 ml; pH 6.3) was added to tubes. The control received phosphate buffer solution (0.05 ml) instead of extract. The absorbance was measured using a spectrophotometer at 660 nm, and the percentage inhibition of BSA denaturation was calculated using the following equation: % 𝐢𝐧𝐡𝐢𝐛𝐢𝐭𝐢𝐨𝐧 𝐨𝐟 𝐁𝐒𝐀 𝐝𝐞𝐧𝐚𝐭𝐮𝐫𝐚𝐭𝐢𝐨𝐧 = 𝑨𝟏 − 𝑨𝟐 𝑨𝟏 × 𝟏𝟎𝟎 Where A1 = absorbance of the control and A2 = absorbance of the test sample. 2.10.2. Chicken Egg albumin assay (CEA) Using the technique described by [30], the ability of our extracts to inhibit protein denaturation was examined. To 2.8 ml of PBS solution, 2 ml of extract and 0.2 ml of chicken egg albumin were added. All samples were kept at room temperature for 15 minutes before being heated to 70 °C for 10 minutes. Diclofenac was employed as standard, and the absorbance was measured at 660 nm. The equation below was used to determine the inhibition of egg albumin denaturation: % 𝐢𝐧𝐡𝐢𝐛𝐢𝐭𝐢𝐨𝐧 𝐨𝐟 𝐞𝐠𝐠 𝐚𝐥𝐛𝐮𝐦𝐢𝐧 𝐝𝐞𝐧𝐚𝐭𝐮𝐫𝐚𝐭𝐢𝐨𝐧 = 𝑨𝟏 − 𝑨𝟐 𝑨𝟏 × 𝟏𝟎𝟎 A1 is the control's absorbance and A2 is the test sample's absorbance. 2.11. Phenolic profile analysis by HPLC-UV-MS/MS Phenolic compounds were qualitatively analyzed using a liquid chromatography system coupled with a triple quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). The suggested technique was carried out using a Kinetex C18 reversed- phase column (100 x 4.6 mm, 2.6 m particles). From solvent A (0.1% formic acid aqueous solution) and solvent B (methanol), gradient separation was created following the method described by [31]. Retention time and spectrum matching with nine standards were used to identify the phenolic compounds, along with the NIST-MS/MS library. PMMB 2023, 6, 1; a0000331 7 of 20 2.12. Statistical analysis T-test was used to evaluate the statistical differences between extraction methods (maceration and probe-assisted ultrasound extraction) and organs (leaves and stems). Experiments were run in triplicate, and the results are expressed as mean values of three analyses. Data are statistically significant at p<0.05 and highly significant at p<0.001. Pearson correlation coefficient (r), DPPH (IC50), and H2O2 (IC50) were determined using GraphPad prism 8.0.2. 3. Results 3.1. Phytochemical screening The results of phytochemical screening of Euphorbia nicaeensis All. leaves and stems are shown in Table 1. Phenols, tannins, saponins, and carbohydrates are strongly present in all plant organs. Flavonoids, flavonol glycosides, sterols, and polyterpenes are highly present in leaves and small amounts in stems. Alkaloids and amino acids are present in tiny quantities, quinones and mucilage are absent in all plant organs. Table 1. Phytochemical screening results of leaves and stems of Euphorbia nicaeensis All. Phytochemical compounds Leaves Stems Phenols / Tanins (++) (++) Flavonoids (++) (+) Flavonol glycosides (+) (+/-) Alkaloids (+/-) (+/-) Saponins (++) (++) Carbohydrates (++) (++) Quinones (-) (-) Gums and mucilages (-) (-) Amino acids (+) (+) Sterols and Polyterpenes (++) (+) (+): Presence of phytochemicals, (-): Absence of phytochemicals, (++): Presence in significant amounts, (+/-): Presence in small amounts. PMMB 2023, 6, 1; a0000331 8 of 20 3.2. Total phenol (TPC), Total flavonoid (TFC), Flavonol content (FC), and Condensed tannin content (CTC) The carried-out tests indicated that the leaves and stems of Euphorbia nicaeensis are rich in phenolic compounds. The percentage yield of leaf and stem extracts by maceration (3.32%, 1.68%), and ultrasonic-assisted extraction (12.69 ± 0.17%, 4.62 ± 1.55%), respectively, showed that the highest extraction yield of both leaves and stems was obtained with probe ultrasonic-assisted extraction (PUAE). The (TPC) values of PUAE extract of leaves and stems (80.13 ± 1.61 mg GAE/g DW, 77.79 ± 2.29mg GAE/g DW), respectively, were statically higher (t-test, p<0.05) than that of maceration extracts (68.69 ± 0.91mg GAE/g DW, 68.31 ± 1.83 mg GAE/g DW). This data also reveals that the leaves are richer in flavonoids, condensed tannins, and flavonols than the stems as presented in Table 2. Table 2. Total phenol, flavonoids, condensed tannins, flavonols contents and extraction yield of maceration extracts and probe ultrasonic-assisted extract of Euphorbia nicaeensis All. leaves and stems. ELM* ESM* ELU* ESU* p1 p2 p3 p4 TPC (mg GAE/g DW) 68.69±0.91 68.31± 1.83 80.13± 1.61 77.79±2.29 0.574 0.042 <0.001 <0.001 TFC (mg CE/g DW) 32.83 ±3.84 21.83 ±9.93 49.22 ±0.192 45.27 ±2.5 0.047 0.031 0.004 0.010 CTC (mg CE/100g DW) 1.33±1.1 1.88±1.92 7.61±1.92 1.22±0.92 0.450 0.002 0.001 0.361 FC (mg QE/g DW) 4.80±0.08 1.23 ±0.27 36.14±0.08 5.88±0.33 <0.001 <0.001 <0.001 <0.001 Extraction yield (%) 3.32±0.59 1.68±0.28 12.69±0.17 4.62±1.55 0.024 0.011 <0.001 0.077 ELM: Euphorbia leaf maceration, ESM: Euphorbia stem maceration, ELU: Euphorbia leaves ultrasonic- assisted extraction, ESU: Euphorbia stems ultrasonic-assisted extraction, TPC: Total flavonoid content, TFC: Total flavonoid content, CTC: Condensed tannin contents, FC: Flavonols content, p1: p-value (ELM – ESM), p2: p-value (ELU – ESU), p3: p-value (ELM – ELU), p4: p-value (ESM – ESU). *Means ± SD from triplicate determinations. p-value is considered significant at p<0.05 and highly significant at p<0.001. PMMB 2023, 6, 1; a0000331 9 of 20 3.3. Lipid-soluble pigment content As shown in Figure 2, this study revealed that leaves and stems differed significantly (p<0.05) in the content of chlorophyll pigment. The leaves are richer in chlorophyll a and b (0.167±0.001 µg/g DW, 0.108± 0.002 µg/g DW) than the stems (0.016 µg/g DW, 0.049± 0.001 µg/g DW). These findings also showed that leaves have significantly higher (p<0.05) β-carotene content (0.035 ± 0.001 µg/g DW) compared to stems (0.006 ± 0.001 µg/g DW). In addition, low lycopene concentration was detected in both leaves and stems (0.033 ±0.001 µg/g DW, 0.036 µg/g DW), respectively, with no significant difference (p>0.05). Figure 2. Lipid-soluble pigment content of leaves and stems of Euphorbia nicaeensis. Values are means ± S.D of three independent measurements. EL: Euphorbia leaves, ES: Euphorbia stems. The values with the same superscript letters are not significantly different (p>0.05). 3.4. In vitro antioxidant activity The evaluation of the antioxidant activity of E. nicaeensis leaf and stem extracts was screened by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay and scavenging of hydrogen peroxide (H2O2) assay. All extracts showed antioxidant activity in Figure 3. The results were positively correlated to the concentration, and the maximum scavenging activity was recorded at the highest concentration for all extracts. Furthermore, it was observed that PUAE provided a strong significant DPPH scavenging effect (p<0.01), translated by a low IC50 value of leaf extracts (IC50 = 23.47±0.62 µg/ml) closely compared to Ascorbic Acid (IC50 EL ES β-carotene 0.035 0.006 Lycopene 0.033 0.036 Chlorophyll a 0.167 0.016 Chlorophyll b 0.108 0.049 0.000 0.020 0.040 0.060 0.080 0.100 0.120 0.140 0.160 0.180 Li p id -s o lu b le p ig m e n ts c o n te n t µ g /g ± 0.001 ±0.000a ±0.000 ±0.002 ± 0.001 ±0.001a ±0.001 ±0.002 PMMB 2023, 6, 1; a0000331 10 of 20 =19.89±0.14 µg/ml). While the maceration leaf extract showed a low antioxidant effect (IC50 = 79.76±1.93 µg/ml). Figure 3. DPPH radical scavenging activity (A) and H2O2 scavenging activity (B) versus concentration (μg/mL). ELM: Euphorbia leaf maceration, ESM: Euphorbia stem maceration, ELU: Euphorbia leaf ultrasonic- assisted extraction, ESU: Euphorbia stem ultrasonic-assisted extraction. The results are expressed as means ± SD of three independent measurements. The stem extract of PUAE also exhibited a moderate antioxidant effect (IC50 = 42.45 ± 2.30µg/ml) compared to the maceration extract (IC50 = 61.16 ± 1.53µg/ml). However, it was observed that stem maceration extract has a significantly (p<0.01) lower IC50 value than leaf maceration extract, which implies a higher antioxidant capacity, as shown in Table 3, similarly, for H2O2 scavenging results. The lowest IC50 was observed in PUAE leaf extract (IC50=110.27 ± 3.59 µg/ml) followed by stem extract (IC50 = 131.51 ± 1.80 µg/ml) in comparison to maceration (IC50 = 678.32 ± 7.22 µg/ml, IC50 = 1073.63 ± 1.08 µg/ml, respectively). Table 3. Scavenging activity of leaf and stem extracts of Euphorbia nicaeensis All. Scavenging activity (IC50 µg/ml) ESM* DPPH assay H2O2 assay 61.16 ± 1.53 1073.63 ± 1.08 ELM* 79.76 ± 1.93 678.32 ± 7.22 ESU* 42.46 ± 2.30 131.51 ± 1.80 ELU* 23.48 ± 0.62 110.27 ± 3.59 AA* 19.90 ± 0.14 35.41 ± 0.23 *Means ± SD from triplicate determinations, p < 0.001. (A) (B) PMMB 2023, 6, 1; a0000331 11 of 20 The correlation analysis between antioxidant activity and phenolic content of the extracts is presented in Table 4. Pearson's coefficient revealed a positive relationship between antioxidant activity and total phenolics and flavonoids' plant content. The DPPH assay of antioxidant activity and TPC showed a Pearson coefficient of r=0.8827 and r=0.7768 for total flavonoids content with no significant correlation (p>0.05). Moreover, a highly significant correlation was observed between the H2O2 assay and the composition of the plant in both TPC (p < 0.001) and TFC (p < 0.05). Table 4. Pearson correlation test between total phenolic content, total flavonoids content, and antioxidant activity. *Significant at p <0.05; **Significant at p<0.001; DPPH: DPPH scavenging activity; H2O2: hydrogen peroxide scavenging activity; TPC: Total flavonoid content; TFC: Total flavonoid content. 3.5. In vitro anti-inflammatory activity According to the results presented in Table 5, all extracts showed an inhibition of Bovine Serum Albumin (BSA) and chicken egg albumin denaturation (CEA), which increased with the sample concentration. The highest inhibitions were observed at the 2000 µg/ml dose for all extracts. The findings revealed a notable inhibition of serum albumin denaturation (83.98% and 75.39 %, p<0.001) and egg albumin denaturation (82.04% and 78.87%, p<0.001) induced by PUAE extracts of stems and leaves, respectively, at the dose of (2000 µg/ml). The maceration extracts of leaves and stems showed a lower inhibition of bovine serum albumin (38.28% and 43.36%, p<0.001) at the same concentration. The inhibition of egg albumin revealed similar results. Maceration extracts from stems and leaves showed a weak inhibition (49.85%, 62.52%, respectively). While the standard drug diclofenac sodium exhibited potent inhibition (97.73%, p<0.001) at the same concentration when compared to the control. Pearson's correlation (r) DPPH (1/IC50) H2O2 (1/IC50) TPC 0.8827 0.9995** TFC 0.7768 0.9505* PMMB 2023, 6, 1; a0000331 12 of 20 Stem extracts from maceration and ultrasound-assisted extraction showed a significantly higher level of protein denaturation inhibition (p<0.05) than leaf extracts at the concentration range of (2000- 500 µg/ml). Table 5. In vitro anti-inflammatory activity of Euphorbia nicaeensis All. Treatment Dosage (µg.ml-1) Bovine serum albumin denaturation Chicken Egg albumin denaturation Absorbance % Inhibition Absorbance % Inhibition Control - 0.917 ± 0.015 - 0.913 ± 0.015 _ ELM 200 0.652 ± 0.0006 28.90 0.286 ± 0.003 72.48 300 0.591 ± 0.002 a 35.54 0.221 ± 0.001 72.08 500 0.571 ± 0.0026 37.77 0.218 ± 0.003 70.95 1000 0.570 ± 0.0021 37.89 0.197 ± 0.001 68.80 2000 0.566 ± 0.001 38.28 0.193 ± 0.001 62.52 ESM 200 0.677 ± 0.0072 26.17 0.279 ± 0.001 73.32 300 0.570 ± 0.006 37.89 0.223 ± 0.001 63.94 500 0.541 ± 0.0066 41.02 0.206 ± 0.004 59.82 1000 0.536 ± 0.0061 42.19 0.192 ± 0.001 52.04 2000 0.519 ± 0.009 43.36 0.164 ± 0.004 49.85 ELU 200 0.638 ± 0.003 30.47 0.342 ± 0.003 68.72 300 0.605 ± 0.0096 a 33.98 0.285 ± 0.004 b 75.77 500 0.390 ± 0.0046 57.42 0.265 ± 0.003 76.13 1000 0.276 ± 0.0053 69.92 0.255 ± 0.004 78.43 2000 0.226 ± 0.001 75.39 0.251 ± 0.001 78.87 ESU 200 0.613 ± 0.0044 33.20 0.458 ± 0.006 69.49 300 0.537 ± 0.001 41.41 0.438 ± 0.012 b 75.62 500 0.308 ± 0.0053 66.41 0.367 ± 0.002 77.48 1000 0.208 ± 0.000 77.34 0.329 ± 0.003 78.98 2000 0.147 ± 0.0035 83.98 0.244 ± 0.002 82.04 Diclofenac sodium 200 0.079 ± 0.0026 91.41 0.054 ± 0.004 94.12 300 0.062 ± 0.001 93.23 0.047 ± 0.002 94.89 500 0.057 ± 0.0036 93.75 0.041 ± 0.002 95.47 1000 0.050 ± 0.0017 94.53 0.035 ± 0.004 96.17 2000 0.021 ± 0.0044 97.73 0.022 ± 0.004 97.59 The results are expressed as means ± SD of three independent measurements. ELM: Euphorbia leaf maceration, ESM: Euphorbia stem maceration. ELU: Euphorbia leaf ultrasonic-assisted extraction, ESU: Euphorbia stem ultrasonic-assisted extraction. the values with the same superscript letters are not significantly different (p>0.05). PMMB 2023, 6, 1; a0000331 13 of 20 3.6. Phenolic profile analysis by HPLC-MS Figure 4. HPLC-MS/MS chromatogram of UAE stem extract. Figure 5. HPLC-MS/MS chromatogram of UAE leave extract. Based on the previous results of the phenolic compounds assay, UAE leaf, and stem extracts were retained for HPLC-UV-MS/MS analysis. The presence of the compounds identified by HPLC-UV analysis was confirmed using HPLC-MS/MS. Comparisons with reference standards, NIST library, and earlier literature reports helped identify. The retention times of the standards tested did not match the peaks detected in the chromatograms of the leaf and stem extracts (Figures 4 and 5). This is because many are in the form of sugar derivatives that are rarely available. Therefore, mass spectrometry was utilized to characterize the peaks and allowed further identification of the compounds. Comparing the MS/MS data with those in the NIST library and other references revealed several phenolic compounds. PMMB 2023, 6, 1; a0000331 14 of 20 Table 6 demonstrates the abundance of different phytochemicals present in both extracts, with a slight difference in composition, including flavonols, phenolic acids, lignan, calchone, carotenoids, and alkaloids. Fargesin, 2’-hydroxy-3,4,4’,6’- tetramethoxychalcone, nuciferin, quercetin 3'-methyl ether, and isoquercetin are detected in both extracts with different peak airs. Isoquercetin was detected as the predominant compound in the leaf extract, followed by quercitrin hydrate and guaiaverin. The stem extract is rich in 2’-hydroxy- 3,4,4’,6’- tetramethoxychalcone, representing the highest peak (22.04%), followed by Quercetin 3'-methyl ether. Table 6. HPLC-MS/MS tentative identification of phenolics and derivatives in E. nicaeensis leaf and stem extracts. Leave Stem N° RT Tentative identification Air % Ref N° RT Tentative identification Air% Ref 1 1.70 Fargesin 5.72 N 1 1.70 Fargesin 3.34 N 2 1.92 2’-hydroxy-3,4,4’,6’- tetramethoxychalcone 4.51 N 2 1.96 2’-hydroxy-3,4,4’,6’- tetramethoxychalcone 22.04 N 3 3.18 Nuciferine 2.08 N 3 3.18 Nuciferine 8.14 N 4 4.86 Quercetin 3’-methyl ether 3.90 N 4 4.78 Quercetin 3’-methyl ether 10.02 N 5 10.08 5,7,3’,4’- Tetramethoxyisoflavone 2.11 N 5 10.38 Lutein 3.65 N 6 10.97 Galloylquinic acid 1.79 [32] 6 17.77 Dilinolenin 3.11 N 7 19.90 Quercetin-galactoside- gallate 8.70 [33] 7 21.11 Isoquercetine 0.94 N 8 20.47 Myricitrin 6.59 N 8 21.52 2'-Hydroxy-2,4,4'- trimethoxychalcone 9.59 N 9 21.11 Isoquercetin 27.49 N 10 22.57 Guaiaverin 11.78 N 11 22.99 Quercitrin hydrate 14.49 [31] RT: retention time, N: NIST-MS/MS library, Air %: Relative peak area PMMB 2023, 6, 1; a0000331 15 of 20 4. Discussion Most of the phytochemical screening findings align with a previous investigation conducted in Serbia for Euphorbia nicaeensis ssp. glareosa [34]. However, some differences were observed, namely, the presence of alkaloids and the absence of quinones in E. nicaeensis All. leaves and stems, compared to this subspecies. This could be due to several parameters, including genetics, differences in harvesting site, rainfall, light, season, harvesting period, topography, soil type, and extraction method [35]. The phytochemical assay showed that the leaves and stems of E. nicaeensis are rich in total phenolic, flavonoids, and flavonols and contain a moderate amount of condensed tannins. Flavonoids, particularly flavonols, are effective antifungal agents against various pathogens, including Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, Trichophyton rubrum and Trichophyton beigelii.[36–40]. Condensed tannins have also been demonstrated to have potent anthelmintic properties against worm infections. These findings support the traditional usage of Euphorbia nicaeensis to treat fungal infections and parasite disorders [41]. By comparing the results of total phenolic content (TPC), total flavonoid content (TFC), condensed tannin content (CTC), flavonol content (FC), and the yield of the two extraction methods, it can be concluded that probe ultrasound-assisted extraction (PUAE) is more efficient than maceration. These might be linked to acoustic cavitation processes, which could cause a forceful impact on the solid surface, resulting in an enhanced extraction rate, as already reported [42]. It is therefore inferred that PUAE could be a convenient technique for phenolic compound extraction. However, the maceration extract of stems proved to have a higher antioxidant effect than leaf maceration extract. These results can be interpreted by the variation of the nature and chemical structure of the phenolic compounds present in each sample. In some cases, the highest antioxidant activity may be observed in extracts with a low phenolic content. Synergy may occur between the major antioxidants (phenolic compounds) and other minor constituents of the plant, so this could significantly impact the differences in their antioxidant activity [43]. This study revealed that E. nicaeensis All. leaves possess a potent antioxidant effect close to ascorbic acid activity. A higher free radical scavenging and hydrogen peroxide scavenging effect were observed in probe ultrasound-assisted extraction leaf extracts, compared to stems. The difference between leaves and stems in extraction yield and total phenolic contents could partially explain this. Studies have reported that leaves contain a high amount of phenolic compounds than stems [44]. A potent correlation was observed between phenolic compounds and H2O2 scavenging activity, as reported by reports [45,46]. However, the inclusion of other substances, particularly sugars, which, according to [47], can interfere with quantification and antioxidant activity assays, may cause a non-significant correlation between TPC and DPPH. HPLC-UV-MS/MS analysis showed that the tested extracts are rich in phenolic compounds, especially flavonoids (flavonol glycosides). Quercetin and its derivatives are the PMMB 2023, 6, 1; a0000331 16 of 20 most abandoned compounds in the tested extracts, especially in the leaves, and studies have reported their different biological virtues [48,49]. The presence of isoquercetin and quercitrin hydrate represented by the highest peaks, may be among the leading causes of the significant antioxidant properties of the leaves. Fargesin, myricitrin, and guaiaverin also play a crucial role in plant health and display several therapeutic values due to their antioxidant or/and anti- inflammatory properties [50,51]. From this, we can infer that the phenolic compounds of the plant are the major cause of the scavenging effect of the extracts, supporting the results obtained and explaining why the leaves have a higher antioxidant power than the stems. Therefore, leaves are a potential source of natural antioxidants and bioactive compounds. The results of previous research on different species and subspecies of the Euphorbiacea family have demonstrated that E. nicaeensis All. has a higher DPPH radical inhibition potential than E. retusa Forssk. (IC50 leaf = 287.52±2.92 µg/ml, IC50 stem=225.87±3.88 µg/ml [52] and E. hirta L. (IC50 leaf = 803 µg/ml, IC50 stem= 1358 µg/ml) [53], as well as E. heterophylla (IC50 = 194.28±0.22 µg/ml) [54]. The subspecies Euphorbia nicaeensis ssp. glareosa showed more than 50% inhibition of DPPH (56.5%), which qualifies the plant as moderately active [34]. Yet no previous study on the in-vitro antioxidant activity of E. nicaeensis All. has been published. The aerial part of E. nicaeensis All. is recognized as having anti-inflammatory virtue as it contains glyceroglycolipids tested for their anti-inflammatory activity [55]. This study's results agree with what has been previously published. In vitro tests showed that the plant has a strong anti-inflammatory effect, inhibiting the degradation of bovine serum albumin (BSA) and chicken egg albumin (CEA), observed mainly in the stem extracts. Nuciferine (alkaloid) and fargesin (lignan) are among the main compounds identified in the stem extract according to chromatograms and MS/MS identification; they are phytochemical compounds known for their anti-inflammatory virtues [50,56] which proves the results of the anti- inflammatory activities performed. The inhibition of thermal degradation of proteins by extract can be explained by several mechanisms, depending on the type of plant extract and the protein studied [57]: Proteins can be protected from thermal degradation by antioxidants because they can scavenge reactive oxygen species (ROS) and reduce oxidative degradation [58]. When phenolic chemicals interact with proteins, their properties, such as solubility, digestibility, and thermal stability, may alter. However, extracts rich in phenolic compounds have been employed to inhibit protein denaturation and improve thermal stability [59]. The antioxidant effect of E. nicaeensis extracts resulting from the presence of phenolic compounds, specifically quercetin, and derivatives, play an essential role in the stability of proteins and inhibit denaturation caused by heat treatment, as already reported. Based on the results of anti-inflammatory activity and those reported by a previous study, we can deduce that the aerial part of E. nicaeensis, especially the stems, provides a PMMB 2023, 6, 1; a0000331 17 of 20 potent anti-inflammatory activity and could be considered a potential anti- inflammatory agent. 5. Conclusion This study aimed to investigate the phytochemical profile and determine the antioxidant and anti-inflammatory activities of leaf and stem extracts of Euphorbia nicaeensis All. It has been observed that ultrasound-assisted extraction is an efficient and recommended method for phenolic compound extraction. This work also showed that all extracts are rich in phenolic compounds and exhibited efficient antioxidant and anti- inflammatory properties with significant differences between organs. HPLC-UV-MS/MS confirmed the presence of phenolic compounds, particularly quercetin and its derivatives, present mainly in the leaf extracts. The leaves showed the highest antioxidant activity, while the most potent anti-inflammatory effect was observed in the stem extracts. The phenolic composition indicated the presence of several compounds with anti-inflammatory properties. These findings confirmed E. nicaeensis historical medicinal uses and opened up new ways and possibilities of developing this plant in the pharmaceutical and food fields as a new source of bioactive compounds unaffected by commercial breeding. However, it is evident that additional research, such as the molecular identification of the bioactive substances responsible for these biological activities, is required to offer a more satisfactory and clearer perspective to this study. Author Contributions: Conceptualization, KB and HT.; methodology, KB, HT, CE, RS; software, NEH, CE.; validation, X.X., Y.Y. and Z.Z.; formal analysis, NEH, HNM, KB, KM, MH, RF; investigation KB, HT, CE; resources KWG, AB, HNM; data curation, AK and KB.; writing—original draft preparation, KB and CE; writing—review and editing AB, NEH, HT, KWG. Funding: No external funding was provided for this research. Acknowledgments: Authors of this work express their sincere thanks to everyone who contributed to the realization of this study, especially Abderrahmane Belhouari, Professor of statistics at the Faculty of Science BEN MSIK and Said Zaidoune, Professor of English at the Faculty of Letters BEN MSIK. 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