Antibacterial and Antioxidant Activities of Triterpenoids Isolated from Endemic Euphorbia arbuscula Stem Latex

This research study aimed to investigate the chemical constituents and evaluate the antibacterial and antioxidant activities of stem latex extracts from the endemic medicinal plant Euphorbia arbuscula found on Socotra Island, Yemen. The study aimed to assess the potential medicinal and veterinary uses of this plant, representing the first evaluation of its properties. The stem latex was extracted using ethanol, and the resulting oil underwent analysis using GC-MS to identify eight compounds. In addition, chromatographic techniques were employed to isolate two triterpenoids, lanosterol and lupeol, from the stem latex. The structures of these compounds were confirmed using IR, MS, and NMR techniques. The antibacterial activity of the extracts and isolated compounds was evaluated against three bacterial strains using the disc diffusion method, revealing only weak antibacterial effects. The study also investigated the antioxidant activity using the DPPH assay, where the ethyl acetate extract exhibited the highest activity with an IC50 value of ±13.55 µg/mL, followed by the chloroform extract with an IC50 of ±21.87 µg/mL. These findings emphasize the potential of Euphorbia arbuscula in the development of new medicines, particularly due to its notable antioxidant activity. The research methodology employed a scientifically rigorous approach, utilizing a comprehensive range of analytical techniques. However, further investigation is required to fully assess the plant's potential as a therapeutic agent.


Introduction
Te Euphorbiaceae plant family holds signifcant importance in Yemen, consisting of 106 species, with 62 species belonging to the genus Euphorbia, out of which 16 are endemic [1,2].Notably, 11 of these endemic species are exclusively found on the island of Socotra.Extensive research has been conducted on various parts of Euphorbia plants, including roots, seeds, latex, lactiferous tubes, stem wood, stem barks, leaves, and whole plants, to explore their chemical compositions and biological activities.
Euphorbia species have a long history of traditional use in various countries worldwide for the treatment of diverse ailments such as cancer, diabetes, heart diseases, cough, earache, and rheumatism [4,9].Te latex of Euphorbia plants contains triterpenoid alcohols, including tigliane diterpenoids, ingenol, phorbol esters, and tirucallol.Tirucallol has demonstrated anti-infammatory and antiparasitic properties, while cycloartenol, another triterpenoid alcohol found in Euphorbia latex, exhibits potential anticancer activity.
Euphorbia arbuscula, commonly known as Amtech, is an endemic plant species found exclusively on the island of Socotra.Te traditional uses of its latex include cauterizing or sealing wounds, alleviating breathing difculties, repelling fies, and providing bandages for sprains or fractures.Furthermore, the latex of Euphorbia arbuscula is utilized as an adhesive and is considered the strongest available on Socotra.
Given the signifcance of the traditional uses of Euphorbia species, particularly Euphorbia arbuscula, in wound healing and fy repellency, this study aims to isolate and identify chemical compounds present in its stem latex.By investigating the chemical constituents of Euphorbia arbuscula, this research seeks to enhance our understanding of its medicinal potential and explore new therapeutic applications.

Plant Material.
Te fresh stem latex of E. arbuscula was collected from Socotra Island, Yemen, in October and December 2014, and the fresh latex was weighed 894 g.Te plant material was identifed by Dr. Abdul Wali Al-Khulaidi (Faculty of Science, Taiz University, the Agricultural Research and Extension Authority, Yemen) and Abdul Habib Al-Qadasi (the Agricultural Research and Extension Authority, Yemen).Its voucher specimen (No.YP2496) was deposited in the Socotra Herbarium.

Plant Preparation and Extraction of Stem Latex.
Te stem latex of E. arbuscula was dried under shade for one week.Te crushed dried stem latex (384 g) was macerated in 90% ethanol (90 ethanol: 10 water; (6 × 1 L)) with shaking for two days at room temperature.Te combined extracts were fltered and evaporated.Te ethanolic dried extract (309.05g) was suspended in 600 mL of warm methanol to give two layers; the lower layer was oily (257 mL, (222.09g)) while the upper layer was methanol.Te methanol layer was frozen for two days (to remove the rest of the oil) to give two layers; the lower layer was separated (48.31 mL, (42.07 g)).Te methanol layer after one weak at room temperature gave a precipitate (4.77 g) which is separated by fltration and then evaporated to give 38.12 g.Te dried methanol extract was dissolved in methanol: water (2 : 1) and then fractionated successively with n-hexane (3 × 500 mL), chloroform (3 × 500 mL), ethyl acetate (6 × 500 mL), and fnally butanol (3 × 500 mL).Each fraction was dried, yielding four fractions of 15.54, 1.85, 1.08 g, and 13.89 g, respectively.At the same time, the frst oily layer was isolated and identifed by GC-MS.Te overall process of stem latex extraction of E. arbuscula is summarized in the fowchart in Figure 1.

Isolation of Constituents.
Compound (1) was isolated for the frst time by sample extraction from the frst oily layer by dissolving 50 g of the oily extract in 30 mL methanol.Dissolved oil was reduced to half by rotary evaporator, n-hexane was added to dissolved oil, and then n-hexane layer gave white powder (5.57g).

Identifcation of Compounds.
All chemicals and solvents were obtained from Sigma-Aldrich (Germany) and Scharlau (Spain).Te Buchi Rotavapor R-200 system with Buchi water bath B-490 and Buchi V-800 vacuum (Germany) was used.TLC spots were visualized under UV light at 254 nm and 365 nm using VIBER-Lourmat equipment (French), and IR spectra were obtained using the potassium bromide disc technique on a JASCO 410 FT-IR.

2.6.
Gas Chromatography-Mass Spectroscopy.Gas chromatography-mass spectrometry (GC-MS) was performed using a Gas Chromatography-Mass Spectrometer GCMS QP2010 Plus (Shimadzu, Japan) equipped with an autosampler.Te sample, dissolved in GC-MS grade chloroform, was injected (1 μL) into a fused silica capillary column HP5-MS (30 m × 0.32 mm, flm thickness 0.25 μm) with a split ratio of 1/50 and helium as the carrier gas.Te oven temperature was initially held at 50 °C for 5 min, then increased at a rate of 5 °C per minute to 240 °C, and maintained at 240 °C for 5 min.Te fnal temperature was 250 °C.Te components of the test oil were identifed by comparing their spectra with those of known compounds stored in the internal library (Wiley; mass spectral library) using a mass spectrometer that was operated in the electron ionization (EI) mode with a beam energy voltage of 70 eV with molecular weight range 30-600 m/z.
Nuclear magnetic resonance (NMR) spectra were recorded using a Bruker spectrometer in CDCl3 as the solvent.Te spectra were obtained at 400 MHz (1H) and 100 MHz ( 13 C).

Biological Activities.
Te extracts of stem latex of E. arbuscula, n-hexane, chloroform, ethyl acetate, butanol, methanol, and ethanol, as well as the isolated pure compounds (1,2), were evaluated for antibacterial and antioxidant activities.

Antibacterial Activity.
Te antibacterial activity was evaluated by disc difusion method [12,13].Bacterial stock cultures were obtained from the Yemeni Pharmacovigilance Center, including Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 8739), and Pseudomonas aeruginosa (ATCC 25619).A stock solution of all samples (500 mg/ml) was prepared in methanol (99.8%).Sterile paper discs (Whatman NO3) with a diameter of 6 mm were impregnated with 15 μl of the samples (500 mg/ml) and air-dried under sterile conditions.Mueller Hinton agar (MHA) (90 mm) was inoculated with the stock bacterial suspension (1-2 × 108 CFU/ml) by streaking the surface in three diferent directions to ensure proper distribution of the inoculum with a sterile cotton swab and incubating of the plates at 37 °C for 24 h.Te antibacterial activities were determined by measuring the diameter of the inhibition zone for each extract and compound against positive controls (30 μg/disc) of gentamicin; 15 μl of the methanol was used as a negative control.Te mean inhibition zone diameter was recorded DPPH were also used as the blank.Te mixture was shaken for 1 min to be homogenous and stored in the dark.After 30 minutes of incubation at 25 °C, the mixture was measured at wavelength 492 nm by using a multiscan spectrum instrument, and the experiment was done in triplicate.Radical scavenging activity was calculated using the following formula: where AC is the absorbance of the negative control, AE is the absorbance of the test sample with DPPH, and AB is the absorbance of the blank.Te correlation between each concentration and its percentage of free radical scavenging was plotted, and the IC 50 was calculated by interpolation.Te activity was expressed as IC 50 , the inhibition concentration of each extract that scavenges 50% of DPPH radicals.

Determination of the Antibacterial Activity.
Te antibacterial activity extracts and pure compounds isolated from the stem latex of E. arbuscula were evaluated against S. aureus, E. coli, and P. aeruginosa using the disc difusion method.Te tested extracts and isolated compounds showed weak and no antibacterial activity.

Determination of Antioxidant Activity.
Te extracts and isolated compounds showed dose-dependent activity, where the % inhibition increased as the concentration of the extract or compounds increased.Te ethyl acetate extract exhibited the highest activity with an IC 50 value of 13.55 μg/mL, which was comparable to ascorbic acid (IC 50 4.09 μg/mL) followed by chloroform extract with an IC 50 value of 21.87 μg/mL, as illustrated in Figure 3.

Discussion
Te GC-MS analysis of the frst oil layer obtained from the latex extract of E. arbuscula revealed the presence of eight compounds, as shown in Table 3. Te major compounds 4 Advances in Pharmacological and Pharmaceutical Sciences identifed were lanosterol (51.98%), ergosta-8,24(28)-dien-3ol-4,14-dimethyl (17.82%), dioctyl phthalate (9.93%), and lupeol (9.86%).Of particular interest was the identifcation of lanosta-8,24-diene-3-one, which has not been previously reported in the Euphorbiacea family.Furthermore, the GC-MS analysis indicated the presence of a rich diversity of triterpenoids in the oil layer.Tese fndings are consistent with previous studies on other Euphorbia species, which have been reported to contain various triterpenoids in their latex and other plant parts.
Compound (1): Te melting point (135-137 °C) was in agreement with the previously published data of Lanosterol [16].Te compound showed a brown spot visible on TLC when it was sprayed with 5% H 2 SO 4 indicating that the compound could be a sterol [17].Te IR spectrum showed

Advances in Pharmacological and Pharmaceutical Sciences
Te identifcation of the compound was performed by comparing its spectral data, including 1 H, 13 C NMR, with Shin et al. [20].Te 1 H NMR spectrum revealed the presence of seven tertiary methyl protons at δ0.75 (H 18 ), 0.95 (H 19 ), 1.68 (H 26 ), 1.60 (H 27 ), 1.00 (H 28 ), 0.80 (H 29 ), and 0.85 (H 30 ).A doublet of three protons at δ0.88 with J � 4 corresponds to (H 21 ), and the triplet of one proton at δ5.11 with J � 8 Hz corresponds to H 24 .Te proton appeared as a doublet of the doublet with J � 4 and 12 Hz at δ3. 25, 3.22.Te identifcation of the compound was performed by comparing its spectral data including 1 H, 13 C NMR with Shin et al., 2000 [20]. 1   ) were due to the presence of a double bond.Te comparison of the spectral data with those reported previously led to the proposal of the lanosterol structure.Lanosterol (lanosta-8,24-dien-3-ol)-type triterpene with a cyclopentanol partial structure in the side chain has not been reported from E. arbuscula.
Compound ( ): Te melting point (215-217 °C) was in agreement with the previously published data of Lupeol [21].Te compound showed a violet spot visible on TLC when sprayed by vanillin sulfuric acid indicating that this compound could be a sterol [17].Te IR spectrum showed a broad strong band at 3397 cm −1 , indicating the presence of OH.Te two bands at 2941 and 2857 cm −1 indicated the CH stretching of CH 3 and CH 2 , respectively.Te C�C vibration was shown around 1638 cm −1 .Bands at 1454 and 1379 (C-H bending) and 1043 cm −1 indicated C-O stretching of OH [21].
Te ), and (C 7 H 11 + ), respectively [22].Te identifcation of this compound was performed by comparing its spectral data including 1 H and 13 C NMR with those published data with Htay et al. [23].Te 1 H NMR spectrum revealed the presence of seven signals singlet for tertiary methyl protons at δ 0.96 (H 23 ), 0.72 (H 24 ), 0.88 (H 25 ), 1.32 (H 26 ), 0.90 (H 27 ), 0.76 (H 28 ), and 1.61 (H 30 ).Multiples of one proton at δ 2.36 correspond to H19.Te H-3 proton appeared as a doublet of the doublet with J � 4.1 and 10.9 Hz at δ3.10, 3.12.Te doublet at δ 4.50 and 4.62 with J � 1.4 Hz due to two methylene protons (H29) is a characteristic of lupeol.) were due to the presence of a double bond.Te compound was identifed as lupeol, and its spectral data showed good agreement with those reported in the literature.Lupeol (lup-20(29)-en-3β-ol) is a pentacyclic triterpene.It has not been reported for E. arbuscula.4.1.Antibacterial Activity.Te antibacterial activity varies with the species of the plant and the plant material.E. thymifolia showed a less potent antibacterial response compared to E. hirta [24].On the other hand, E. cotinifolia was found to be resistant to antibacterial activity against any bacteria tested [25].Recently, researchers shown that the multi-resistant bacterial strains have increased dramatically and thus the treatment of several infections has become very difcult to reduce the therapeutic options [26].

Antioxidant Activity.
Te fndings of our study revealed diferences in the antioxidant activities of plant extracts and isolation compounds.Te highest activity was obtained from the ethyl acetate extract with an IC 50 value of (±13.55 μg/mL), which was comparable to ascorbic acid (IC 50 ± 4.09 μg/mL), followed by the chloroform extract (IC 50 ± 21.87 μg/mL).Te highest activity of the ethyl acetate and chloroform extracts could be due to the synergistic action of the compounds contained, rather than a single chemical substance.Previous studies have reported on the antioxidant activity of various Euphorbia species, including E. acanthamnos, E. macroclada, E. rigida, and E. heyneana [27,28].

Conclusion
Te ethyl acetate extract of Euphorbia arbuscula exhibited the highest antioxidant activity, which was comparable to that of ascorbic acid, followed by the chloroform extract.Antioxidants play a crucial role in protecting health by reducing the risk of chronic diseases, including cancer and heart disease.Further testing of the ethyl acetate extract may yield additional bioactivity, and there is potential for the isolation of bioactive compounds.In addition, more studies are needed to determine the toxicological and pharmacological properties of this plant species.Tis research provides opportunities for future work, as many compounds have not yet been identifed and isolated.

Figure 2 :
Figure 2: GC-MS chromatogram of oil extract of the stem latex E. arbuscula.
[14]h dried stem latex of E. arbuscula (384 g) triplicate tests, and the antibacterial evaluation was defned as strong if the inhibition zone diameter was >15 mm, moderate for diameters between 10 and 15 mm, and weak for diameters >10 mm[14].
Figure 1: Flowchart of extracts and fractionations of stem latex E. arbuscula: methanol (MeOH), n-hexane (n-Hex), chloroform (CHCl 3 ), ethyl acetate (EtOAc), and n-butanol (n-BuOH).fromestimation was carried out in 96 wells plate, and 100 μl of tested samples dissolved were added to each well and mixed with 100 μl of DPPH in methanol (0.1 mM).Te negative control sample containing 100 μL of DPPH was used in the last row of the plate.Te test samples without yield of stem latex of Euphorbia arbuscula extracts is presented in Table1.Te percentage of crude ethanol extract obtained from dry weight of the stem latex of E. arbuscula was 80.48%.Te highest percentage obtained from the crude ethanol extract was the frst extraction of the oil (57.83; 71.86%), while the lowest was obtained from ethylacetate extract (0.28; 0.34%).Te results obtained from the phytochemical screening of stem latex of E. arbuscula extracts are presented in Table2.Phytochemical screening ensured the presence of tanin and phenols in the tested extracts.Steroids and terpenoids were found in the methanol, n-hexane, and chloroform extracts.Te alkaloids, cardiac glycosides, and favonoids were selectively distributed in all latex extracts, while glycosides were absent in all extracts.Te frst oily layer was isolated from methanol identifed by GC-MS.Te phytocomponents were identifed by comparing their mass spectra with those in mass spectral libraries (Wiley; mass spectral library) to ascertain their name, molecular weight, and structure.Figure2shows the total ion chromatogram provided details of peak number, the retention time (RT), name of the compound, molecular formula, molecular weight (MW), and area percentage presented in Table3.At the same time, compound (1) was isolated from the frst oil layer as a white powder dissolved in CHCl 3 ; Rf, 0.73 with EtOAc: n-Hex (7 : 3) and indicated by spray reagent 5% sulfuric acid as a brown zone in visible.Te melting point was 135-137 °C.Te spectral data of the compound were as follows: IR (KBr): ]max cm −1 3440, 2950, 2880, 1680, 1460, 1390, and 1050 cm −1 .Te MS spectra are obtained at 70 eV for C 30 H 50 O m/z relative in- 3 : MeOH (9.5 : 0.5) and indicated as violet zone by vanillin sulfuric acid reagent.Te melting point was 215-217 °C.Te spectral data of the compound were as follows: IR (KBr): ]max cm −1 3397, 2941, 2857, 1638, 1454, 1379, and 1043 cm −1 .Te MS spectra are obtained at 70 eV, for C 30 H 50 O m/z relative intensity of molecular ion (rel.int.%): 426 [M+] (35%), 411 (19.7%), 207 (50.

Table 1 :
Te overall yield and percentage of stem latex extracts of E. arbuscula.: weight of the dried stem latex (384 g) of E. arbuscula and B: weight of the crude ethanol extract (309.05g) stem latex of E. arbuscula. A

Table 2 :
Phytochemical screening of stem latex extracts of E. arbuscula.

Table 3 :
List of chemical composition present in oily layer obtained from extract of stem latex of E. arbuscula by GC-MS.
[18]oad strong band at 3440 cm −1 indicating the presence of OH while two bands at 2950 and 2880 cm −1 (C-H stretching) for CH 3 and CH 2 , respectively.Te C�C vibrations were shown around 1680 cm −1 .Band at 1460 and 1390 cm −1 was assigned for C-H bending and 1050 for C-O stretching of OH[18].Te MS spectrum of the compound (C 30 H 50 O) showed parent molecular ion [M + ] peak at m/z 426 (35%) for (C 30 H 50 O + ).Te ion peak at m/z 411 (100%) corresponds to the molecular formula ion (C 29 H 47 O + ), while the ion peak at m/z 393 (62%) corresponds to the molecular formula ion (C 29 H 45 +