Polyoxypregnane Ester Derivatives and Lignans from Euphorbia gossypina var. coccinea Pax.

Abstract

From the aerial parts of Euphorbia gossypina var. coccinea Pax., eight new pregnane glycosides (euphogossypins A–H, 1–8) of the cynanforidine and deacetylmetaplexigenin aglycons, two new lignans (gossypilignans A and B, 9 and 10), and four known compounds, namely, the pregnane 12-O-benzoyldeaxcylmetaplexigenin (11), the lignan 9α-hydroxypinoresinol (12), and the flavonoids naringenin (13) and quercitrin (14) were isolated. The structure elucidation of the new compounds was carried out by a spectroscopic analysis, including HRMS, 1D (¹H, ¹³C JMOD), and 2D NMR (HSQC, ¹H–¹H COSY, HMBC, and NOESY) experiments. The obtained pregnane glycosides were substituted with acetyl and benzoyl ester moieties, and sugar chains containing thevetose, cymarose, digitoxose, and glucose monosaccharides. All of the compounds are described for the first time from E. gossypina var. coccinea. The isolated pregnanes and lignans were tested for their antiproliferative activity on HeLa cells using the MTT assay; the compounds exerted no significant effect against the tumor cells.

1. Introduction

Plants belonging to the genus Euphorbia are known to possess considerable chemical, medicinal, and economic importance [1]. Terpenes, including diterpenes and triterpenes, steroids, cerebrosides, glycerols, and phenolic compounds are characteristic constituents of Euphorbia species [2]. In the course of our work focusing on the isolation of special metabolites from various Euphorbia species, the chemical composition of Euphorbia gossypina var. coccinea Pax. (Euphorbiaceae) was thoroughly investigated [3]. The aim of our work was the identification of the special metabolites of the plant. This plant is a perennial, much-branched, succulent, herbaceous, evergreen shrub native to Kenya and Tanzania. Preparations of E. gossypina have long been used in traditional medicine for the treatment of swollen legs and general body pain. Moreover, it is applied as eye drops in the treatment of conjunctivitis and warts [4]. The diluted latex of small twigs is taken to treat laryngitis [5]. In Somalia, the latex is applied to treat mange in livestock [6]. There is no literature data on the phytochemistry and pharmacology of E. gossypina var. coccinea.

Herein, we report the isolation and structure elucidation of fourteen compounds, among them being eight new polyoxypregnane ester derivatives (euphogossypins A–H, 1–8), two new lignans (gossypilignans A and B, 9 and 10), and four known compounds, including the pregnane 12-O-benzoyldeacylmetaplexigenin (11), the lignan 9α-hydroxypinoresinol (12), and the flavonoids naringenin (13) and querctirin (14) from E. gossypina var. coccinea.

Pregnane glycosides are C₂₁ steroidal natural compounds, in which the pregnane part is attached to different sugars. These compounds demonstrate a fair degree of diversity in their aglycone part with different numbers and types of sugar units being attached to the aglycone at position C-3 [7]. The sugar part of these compounds can be a mono- or disaccharide, or an oligosaccharide chain arranged mainly linearly to the pregnane skeleton through an acetal linkage. The characteristic monosaccharides of pregnane glycosides are d-glucose, l-rhamnose, d-cymarose, d-oleandrose, d-allose, and d-digitoxose. The occurrence of pregnane glyosides is characteristic of the Asclepiadaceae, Apocynaceae, Malpighiaceae, Ranunculaceae, and Zygophyllaceae families [8]. Pregnane glycosides are reported to possess noteworthy pharmacological properties, such as immunosuppressant, cytotoxic, antidepressant, anti-inflammatory, antioxidant, antibacterial, antifungal, and antiproliferative activities [8]. Therefore, the isolated compounds were tested for their antiproliferative activity against the HeLa cell line using the MTT assay.

2. Results and Discussion

2.1. Isolation of Compounds

The dried and ground aerial parts of the plant were extracted with methanol. After evaporation of the methanol, the extract was dissolved in 50% aqueous methanol, and a solvent–solvent partition was performed with n-hexane, CHCl₃, and EtOAc. The CHCl₃ extract was separated by normal (NP) and reversed-phase (RP) vacuum liquid chromatography (VLC), and then it was purified by preparative thin layer chromatography (prep. TLC) and high-performance liquid chromatography (HPLC) to yield eight new pregnane glycosides (1–8), two new lignans (9, 10), one known pregnane and one lignan, and two known flavonoids (Figure 1).

2.2. Structure Elucidation of the Compounds

The structure determination of the isolated compounds was carried out by an extensive spectroscopic analysis, including one- and two-dimensional NMR and HRMS measurements.

Compound 1 was obtained as a white amorphous powder. Its molecular formula was determined as C₄₉H₇₂O₁₇ by the HRESIMS ion at m/z 955.4660 [M + Na]⁺ (calcd for C₄₉H₇₂O₁₇Na, 955.4662). The ¹H NMR spectrum of 1 showed the resonances of three anomeric protons at δ_H 4.85 (dd, J = 2.0 and 9.0 Hz), 4.76 (dd, J = 1.8 and 9.5 Hz), and 4.30 (d, J = 7.8 Hz), three methoxy groups at δ_H 3.42, 3.44, and 3.65 (each 3H, s), and three secondary methyl groups at δ_H 1.22 (d, J = 6.3 Hz), 1.27 (d, J = 6.2 Hz), and 1.31 (d, J = 6.2 Hz), suggesting the presence of a trisaccharide unit in 1 composed of deoxymethyl sugars. Moreover, the ¹H NMR spectrum contained signals ascribable to three methyl groups displayed at δ_H 1.12 (3H, s), 1.54 (3H, s), and 2.06 (3H, s), and to an olefinic proton at δ_H 5.38 (1H, br s). The proton signals at δ_H 7.93 (2H, dd), 7.43 (2H, t), and 7.55 (1H, t) showed the presence of a benzoyl group in the molecule. The JMOD spectrum indicated that compound 1 contained two carbonyls, seven non-protonated carbons (of which three were oxygenated), twenty-two methines (of which eleven were oxygenated), nine methylenes, and nine methyl carbons (of which three were methoxy). The ¹H and ¹³C JMOD NMR data dictated that 1 was a pregnane glycoside. Of these, 21 carbons were assigned to a pregnane skeleton, 7 to a benzoyl function, and 19 to a trisaccharide moiety. The HMBC correlations between H-19 (δ_H 1.12) and C-1 (δ_C 38.9), C-5 (δ_C 140.8), C-9 (δ_C 43.8), and C-10 (δ_C 37.3) suggested the position of a double bond at C-5/C-6. The HMBC correlations between H-18 (δ_H 1.54) and C-12 (δ_C 73.3), C-14 (δ_C 88.1), and C-17 (δ_C 91.6), between H-21 (δ_H 2.06) and C-17 (δ_C 91.6), and H-6 (δ_H 5.38) and C-8 (δ_C 74.4) demonstrated the positions of hydroxy groups at C-8, C-14, and C-17 (Figure 2). The aglycone moiety of 1 was, therefore, determined to be 12β-benzoyloxy-3β,8β,14β,17β-tetrahydroxypregn-5-ene (cynanforidine), a C/D-cis-polyoxypregnane ester [9]. The relative configuration of the molecule was determined by the analysis of NOESY correlations and literature data of similar structures reported previously.

The aglycone of 1 was supposed to have the same configuration as those of pregnanes isolated from Gymnema sylvestre [10]. The multiplicity of H-12 (δ_H 4.83 (dd, J = 4.2, 11.9 Hz)) implied that the configuration of H-12 was axial (α-configuration, Figure 2). The NOESY correlations between H-3 (δ_H 3.57), H_α-1 (δ_H 1.89), and H_α-4 (δ_H 2.40), and between H-12 (δ_H 4.83) and H-9 (δ_H 1.59) determined the configurations of the oxygenated groups at C-3 and C-12 to be β. NOESY correlations detected between Hβ-1 (δ_H 1.13) and Hβ-4 (δ_H 2.29) showed the α-position of these protons. The C-12 benzoyl group was confirmed by HMBC correlation from H-12 (δ_H 4.83) to the benzoyl carbonyl C-1′ (δ_C 165.5). The large coupling constants between the H-1 and H-2 of monosaccharide moieties, and the HMBC correlations between Thv H-1 (δ_H 4.30) and Cym II C-4 (δ_C 82.8), Cym II H-1 (δ_H 4.76) and Cym I C-4 (δ_C 82.7), and between Cym I H-1 (δ_H 4.85) and aglycone C-3 (δ_C 78.0) indicated the sugar linkages as β-d-thevetopyranosyl-(1→4)-β-d-cymaropyranosyl-(1→4)-β-d-cymaropyranoside and at C-3 of aglycone. Based on the above evidence, the structure of 1 was elucidated as cynanforidine 3-O-β-d-thevetopyranosyl-(1→4)-β-d-cymaropyranosyl-(1→4)-β-d-cymaropyranoside, a new compound named euphogossypin A.

Compound 2 was isolated as a white amorphous powder. Its ¹H and JMOD spectra exhibited the characteristic signals of a pregnane aglycone, one benzoyl unit, and three sugar moieties (Table 1). In addition, NMR data of 2 were similar to those of euphogossypin A (1), except for the difference in sugar units at C-3. A careful analysis of NMR data led to the conclusion that two cymarose units were replaced by two digitoxose monosaccharides. The HMBC correlations between Thv H-1 (δ_H 4.82) and Dig II C-4 (δ_C 84.1), Dig II H- 1 (δ_H 5.41) and Dig I C-4 (δ_C 83.9), and between Dig I H-1 (δ_H 5.48) and aglycone C-3 (δ_C 78.1) confirmed the sugar linkages to be 3-O-β-d-thevetopyranosyl-(1→4)-β-d-digitoxopyranosyl-(1→4)-β-d-digitoxopyranoside. Consequently, compound 2 was identified as a new compound, cynanforidine 3-O-β-d-thevetopyranosyl-(1→4)-β-d-digitoxopyranosyl-(1→4)-β-d-digitoxopyranoside, named euphogossypin B.

The molecular formula of 3 was determined as C₄₈H₇₀O₁₇ by the HRESIMS molecular ion at m/z 941.4508 [M + Na]⁺ (calcd for C₄₈H₇₀O₁₇Na 941.4510). The ¹H and JMOD data (Table 1) suggested that the structure of 3 was similar to those of 1 and 2, except for the difference in monosaccharide units in the C-3 sugar chain. The sugar moieties were found to be d-cymarose, d-digitoxose, and d-thevetose. The sugar linkages (β-d-thevetopyranosyl-(1→4)-β-d-digitoxopyranosyl-(1→4)-β-d-cymaropyranoside) to each other and to the pregnane skeleton were confirmed by the HMBC correlations from Thv H-1 (δ_H 4.35) to Dig C-4 (δ_C 83.8), from Dig H-1 (δ_H 4.89) to Cym C-4 (δ_C 83.9), and from Cym H-4 (δ_H 3.24) to C-3 (δ_C 79.3). Similarly to those of 1 and 2, the aglycone was found to be 12β-benzoyloxy-3β,8β,14β,17β-tetrahydroxypregn-5-ene (cynanforidine). Consequently, the structure of 3 was determined as cynanforidine 3-O-β-d-thevetopyranosyl-(1→4)-β-d-digitoxopyranosyl-(1→4)-β-d-cymaropyranoside and named euphogossypin C.

HRESIMS and NMR data of compounds 4–6 suggested that they were tetrasaccharide derivatives due to the presence of four anomeric carbon and proton signals, one more than observed for compounds 1–3. The additional sugar unit was identified as d-glucopyranose (Table 2). The polyoxypregnane ester aglycone was the same as in compounds 1–3. The large coupling constant between H-1 and H-2 of the glucose unit, and the HMBC correlations between Glc H-1 (δ_H 4.43) and Thv C-4 (δ_C 82.8) in 4–6, indicated the sugar linkages as β-d-glucopyranosyl-(1→4)-β-d-thevetopyranosyl-(1→4)-β-d-cymaropyranosyl-(1→4)-β-d-cymaropyranoside in 4, β-d-glucopyranosyl-(1→4)-β-d-thevetopyranosyl-(1→4)-β-d-digitoxopyranosyl-(1→4)-β-d-digitoxopyranoside in 5, and β-d-glucopyranosyl-(1→4)-β-d-thevetopyranosyl-(1→4)-β-d-digitoxopyranosyl-(1→4)-β-d-cymaropyranoside in 6. Based on the above evidence, the structure of 4 was deduced to be cynanforidine β-d-glucopyranosyl-(1→4)-β-d-thevetopyranosyl-(1→4)-β-d-cymaropyranosyl-(1→4)-β-d-cymaropyranoside, and was named euphogossypin D, 5 was determined as cynanforidine β-d-glucopyranosyl-(1→4)-β-d-thevetopyranosyl-(1→4)-β-d-digitoxopyranosyl-(1→4)-β-d-digitoxopyranoside and named euphogossypin E, and 6 was characterized as cynanforidine β-d-glucopyranosyl-(1→4)-β-d-thevetopyranosyl-(1→4)-β-d-digitoxopyranosyl-(1→4)-β-d-cymaropyranoside and named euphogossypin F.

The NMR data of compound 7 were very similar to that of 2, with the only difference between them being the replacement of the benzoyl moiety into an acetyl substituent at C-12. Therefore, the aglycone of 7 was identified as metaplexigenin (Table 3) [11]. The connecting sugar chain was determined as β-d-thevetopyranosyl-(1→4)-β-d-digitoxopyranosyl-(1→4)-β-d-digitoxopyranoside based on the 1D and 2D NMR spectral data. Compound 7 was, thus, characterized as metaplexigenin β-d-thevetopyranosyl-(1→4)-β-d-digitoxopyranosyl-(1→4)-β-d-digitoxopyranoside and named euphogossypin G.

Compound 8 wore the same C-3 trisaccharide sugar chain, β-d-thevetopyranosyl-(1→4)-β-d-digitoxopyranosyl-(1→4)-β-d-cymaropyranoside, as 3 (Table 3). By the NMR data, it was determined to be a deacetylmetaplexigenin derivative and named euphogossypin H.

Gossypilignan A (9) was obtained as a pale yellow, amorphous solid. The molecular formula of 9 was determined as C₂₂H₃₀O₇ from its HRESIMS ion observed at m/z 429.1889 [M + Na]⁺ (calcd for C₂₂H₃₀O₇Na, 429.1884). The ¹H and ¹³C JMOD NMR data indicated the presence of four methoxy (δ_H 2 × 3.82, s, and 2 × 3.83, s; δ_C 4 × 56.8), two methyl (δ_H 0.68, d, J = 6.9 Hz, and 0.76, d, J = 7.0 Hz; δ_C 10.0 and 12.1), one methylene (δ_H 3.35 and 3.45; δ_C 67.3), and three methine groups (δ_H 1.77, m, 2.62, m, 3.52, d; δ_C 37.0, 37.2, and 57.9) (Table 4). Additionally, the ¹H NMR spectrum of 9 showed aromatic protons at δ_H 6.64, s and 6.66, s (2H each), implying that the aromatic rings were tetrasubstituted. The ¹³C NMR spectrum of 9 also supported the presence of six oxygenated aromatic carbons at δ_C 149.1, 149.2, and 134.7 (each 2C). According to the HMBC correlations from H-2, H-6, H-2′, and H-6′ to C-7, both aromatic rings were attached to C-7. The ¹H-¹H COSY correlations of H-7/H-8/H-8′/H-7′, H-8/H-9, and H-8′/H-9′, as well as the HMBC correlations from H-7 to C-8, C-8′, and C-9, indicated the presence of a 2,3-dimethylbutane moiety (Figure 3). By comparison, the skeleton of 9 was found to be the same as that of kadangustin J [12]. According to the chemical shift of δ_C 67.3 (C-7′), a hydroxy group should be placed at C-7′. Four methoxy groups were located at C-3, C-5, C-3′, and C-5, respectively, while C-4 and C-4′ were substituted with hydroxy groups. Thus, the structure of compound 9 was determined to be 4,4-di-(4-hydroxy-3,5-dimethoxyphenyl)-2,3-dimethylbutanol and named gossypilignan A. As compound 9 was a 7,7-diaryl-8,8′-dimethylbutan-1-ol lignan, it had two chiral centers. Based on the investigation of Davidson et al., syn- and anti-isomers of such compounds can be distinguished based on the significant differences between their ¹H NMR data [13]; therefore, as in the case of 9, both methyl groups were β-oriented, proving that this compound was a syn-isomer.

The molecular formula of 10 was determined as C₂₂H₂₈O₇ by the HRESIMS pseudo-ion at m/z 427.1734 [M + Na]⁺. The ¹H NMR spectrum of 10 showed four aromatic hydrogens as two similar systems, one at δ_H 6.69 (2H, br s), and another at 6.63 (2H, br s), indicating the presence of two 1,3,4,5-tetrasubstituted benzene rings (Table 4). The chemical shifts observed for aromatic hydrogens along with the presence of four singlets corresponding to methoxy hydrogens at δ_H 2 × 3.84 and 2 × 3.86 (s, 3H each) indicated the presence of 3,5-dimethoxy-4-hydroxyphenyl groups in this compound. The ¹³C NMR data corroborated the structural determination of these aromatic rings. Moreover, ¹H NMR spectral data suggested a nonsymmetric tetrahydrofuran lignan, through signals corresponding to two methyl groups at δ_H 1.00 (d, J = 6.4 Hz) and 0.63 (d, J = 7.0 Hz) in addition to two oxybenzyl methines at δ_H 4.64 (d, J = 9.3) and 5.47 (d, J = 4.4 Hz). The attachment of aromatic rings to the tetrahydrofuran ring was determined by an HMBC experiment. In the HMBC spectrum, interactions were observed between H-2′ and C-7′ and C-4′, between H-6′/C-4′ and C-2′, and between H-2 and C-7. These data indicated the presence of the two 3,5-dimethoxy-4-hydroxyphenyl structural parts at C-7 and C-7′, respectively. Based on previous literature data [14,15], the coupling constant of 9.3 Hz for the doublet at δ_H 4.64 (H-7) indicated that this hydrogen was in a trans configuration with the adjacent H-8, while the coupling constant of 4.4 Hz of H-7′ demonstrated its cis relationship with H-8′. NOE correlations confirmed the relative stereochemistry at the tetrahydrofurane ring as trans (C-7/C-8), cis (C-8/C-8′), and cis (C-8′/C-7′). Moreover, NOE effects proved the cis configuration of the aromatic group at C-7′ and methyl groups at C-9 and C-9′. These data permitted the establishment of the structure of the new tetrahydrofuran lignan 10 as rel-(7S,8R,7′S,8′S)-3,5,3′,5′-tetramethoxy-4,4′-dihydroxy-7,7′-epoxylignan and named gossypilignan B.

The known compounds were identified as 12-O-benzoyldeacylmetaplexigenin, 9α-hydroxypinoresinol [16], naringenin [17], and quercitrin [18]. All compounds were isolated for the first time from the plant. Such polyoxypregnane ester derivatives were isolated previously, mainly from Asclepiadaceae species (e.g., Cynanchum wilfordi and Leptadenia hastata) [9]. Though Euphorbia species are frequently characterized by the abundant presence of various terpenoids, this is only the second report of pregnanes from a plant belonging to the Euphorbiaceae family. Previously, two pregnane glycosides were identified from the aerial parts of Croton ruizianus. Their aglycon was 3β,14β,15β,16α-tetrahydroxypregnan-20-one, while the connecting sugar parts were either 3-O-β-d-Glu-(1→4)-β-d-Ole-(1→4)-β-d-Ole-(1→4)-β-d-Dig-(1→4)-β-d-Ole or 3-O-β-d-Ole-(1→4)-β-d-Ole-(1→4)-β-d-Dig-(1→4)-β-d-Ole (Ole—oleandrose; Dig—digitoxose) [19]. Pregnane glycosides isolated from E. gossypina var. coccinea were substituted with thevetose, cymarose, digitoxose, and glucose-containing linear sugar chains.

The 7,7-diarylbutanol seco-lignans are an interesting class of lignans with both chemical and pharmacological importance. Cytotoxic, anti-HIV-1, and antioxidant activities have been previously reported for this class of natural products [12,20,21].

Pregnane glycosides are reported to possess various biological activities, an antiproliferative effect being one of the most important ones [22]. Amplexoeside A, isolated from an Asclepiadaceae species Asclepias amplexcicaulis, showed antiproliferative activity in the KB assay. Condurangoglycoides identified from Marsdenia congurango were proved to be active against Sarcoma 180 and Ehrlich sarcoma in mice [23,24]. Pregnane glycosides (Ap and Ao) from Dregea uolubilis showed activity against Ehrlich carcinoma [25]. Moreover, compounds with an antitumor effect were isolated from Aspidopterys obcordata, Caralluma dalzielii, C. negevensis, C. quadrangular, Cynanchum wilfordi, Desmidorchis flava, Gelsemium sempervirens, Marsdenia tenacissima, Periploca sepium, Solenostemma argel, and Stizophyllum riparium [8,26,27,28]. Although the molecular backbone of lignans consists of only two phenylpropane (C6–C3) units, they show an enormous structural diversity, and promising pharmacological effects. A considerable number of plant lignans (e.g., podophyllotoxin, sesamin, diphyllin, enterolactone, arctiin, matairesinol, and taxiresinol) were identified to possess antiproliferative activity [29]. Since, previously, several pregnane glycosides and lignans were reported to display strong antiproliferative activities, all isolated compounds belonging to these groups of special metabolites were tested for their antiproliferative properties against the HeLa cell line using the MTT assay. Doxorubicin and cisplatin were used as positive controls (IC₅₀s 0.02 ± 0.003 μM and 2.07 ± 0.07 μM, respectively). Among the tested compounds, only euphogossypin A (1) exerted weak antiproliferative activity (IC₅₀ 52.4 ± 0.23 µM), while the others proved to be inactive (Table S1).

3. Materials and Methods

3.1. General Experimental Procedures

NMR spectra were recorded in CDCl₃, CD₃OD, and C₅D₅N on a Bruker Avance DRX 500 spectrometer (Billerica, MA, USA) at 500 MHz (¹H) and 125 MHz (¹³C). The signals of the deuterated solvents were taken as reference. The chemical shift values (δ) were given in ppm and coupling constants (J) were in Hz. Two-dimensional (2D) experiments were performed with standard Bruker software. In the COSY, HSQC, and HMBC experiments, gradient-enhanced versions were used. High-resolution MS spectra were acquired on a Thermo Scientific Q-Exactive Plus Orbitrap mass spectrometer (Waltham, MA, USA) equipped with ESI ion source in positive ionization mode. The data were acquired and processed with MassLynx software.

Column chromatography (CC) was performed on polyamide (MP Biomedicals, Irvine, CA, USA). Normal-phase vacuum liquid chromatography (VLC) was carried out on silica gel (Kieselgel 60 GF₂₅₄, 15 μm, Merck, Darmstadt, Germany). Thin-layer chromatography was performed on Kieselgel 60 RP-18 F₂₅₄ and Kieselgel 60 F₂₅₄ (Merck, Darmstadt, Germany). Spots were detected under UV light (245 nm and 336 nm) and made visible with vanillin sulfuric acid reagent and heating at 105 °C for 1 min. The high-performance liquid chromatography (HPLC) separation was carried out on a Waters HPLC (Waters 600 controller, Waters 600 pump, and Waters 2998 photodiode array detector), using RP (LiChrospher RP-18, 5 μm, 250 × 4 mm, Merck, Darmstadt, Germany) column. In the case of the RP-HPLC separation, the mobile phase consisted of MeOH (solvent A) and H₂O (solvent B). The flow rate was 1 mL/min. The data were acquired and processed with the Empower software.

All solvents used for CC were of at least analytical grade (VWR Ltd., Szeged, Hungary). Ultra-pure water was prepared with a Milli-Q water purification system (Millipore, France).

3.2. Plant Material

Aerial parts of Euphorbia gossypina var. coccinea Pax. were collected in Kenya (GPS coordinates 1°24′24.31777″ S, 36°42′53.86125″ E), Africa, in July 2018, and identified by Patrick Chalo Mutiso, a taxonomist (Department of Biological Sciences, Faculty of Science and Technology, University of Nairobi). A voucher specimen (no. UON 2018/249) was deposited at the Herbarium of the School of Biological Sciences, University of Nairobi, Kenya.

3.3. Isolation of Compounds

The dried and ground aerial parts of E. gossypina var. coccinea (1 kg) were extracted with methanol at room temperature. After concentration, the extract (45 g) was dissolved in 50% aqueous methanol, and solvent–solvent partitions were performed with hexane, CHCl_3, and EtOAc. The CHCl₃ fraction (28.6 g) was purified by polyamide column chromatography using MeOH–H₂O (4:1) to remove chlorophyll. Thereafter, the yielded fraction (22.8 g) was purified by VLC on silica gel with a gradient system of cyclohexane–EtOAc–EtOH (from 8:2:0 to 6:3:1) and CHCl₃–MeOH (from 9:1 to 1:1). The TLC determination and combination of the fractions afforded 20 main fractions (Fr. 1–20). Fraction 9 (1.4 g) was further chromatographed by RP-VLC using MeOH–H₂O gradient solvent system (8:2–2:8), and 9 fractions (Fr. 9/1–9) were obtained. Fraction 9/3 (87.9 mg) was subjected to preparative TLC using CHCl₃–MeOH (8:2) as eluent, and was, subsequently, purified by RP-HPLC. Gradient elution was applied, starting at 10% A (methanol) and 90% B (H₂O) for 1 min, then linearly increased to 50% A (in 10 min) to yield compound 10 (t_R = 3.1 min, 2.6 mg). Fraction 10 (1.6 g) was separated by NP-VLC using a toluene–acetone gradient system (from 9:1 to 6:4) to obtain 9 subfractions (Fr. 10/1–9). Fr. 10/4 (371 mg) was chromatographed by NP-VLC using cyclohexane–EtOAc–EtOH gradient system (from 9:1:0 to 6:3:1) to yield 6 fractions (Fr. 10/4/1–6). 9α-hydroxypinoresinol (5.8 mg) was isolated from Fr. 10/4/3 (87 mg) by preparative TLC using CHCl₃–MeOH (95:5) as mobile phase. Fr. 10/5 (188 mg) was purified by RP-TLC using MeOH–H₂O (8:2) as eluent, and further purified by RP-HPLC by using gradient elution, started at 10% A (methanol) and 90% B (H₂O) for 1 min, then linearly increased to 50% A (in 10 min) to yield compound 9 (t_R = 7.3 min, 12.1 mg). Fr. 10/6 (123 mg) was separated by RP-TLC using MeOH–H₂O (8:2) as solvent system and compound 1 (11.2 mg) was obtained. Fraction 11 (4.9 g) was chromatographed by NP-VLC using the gradient system of toluene–acetone (from 9:1 to 1:1) to yield 11 subfractions (Fr. 11/1–11). Fraction 11/9 (193.5 mg) was purified by RP-TLC using MeOH–H₂O (8:2) as an eluent to yield compound 2 (19.7 mg) and two subfractions (Fr. 11/9/1 (8.1 mg) and 2 (6.9 mg)). Both fractions were purified by RP-HPLC (the gradient started at MeOH–H₂O (2:8) for 1 min, then linearly ramped up to 1:1 in 10 min and held 0.5 min, then returned to the initial conditions within 1 min, and kept for 2 min) to yield compounds 8 (t_R = 5.2 min, 6.4 mg) and 7 (t_R = 6.1 min, 5.1 mg). Fr. 13 (165 mg) was purified by NP-TLC using CHCl₃–MeOH (85:15) as mobile phase to isolate compound 4 (12.7 mg). Fraction 15 (5.9 g) was separated by RP-VLC using a gradient system of MeOH–H₂O (from 1:9 to 9:1) to yield 7 subfractions (Fr. 15/1–7). Fraction 15/3 (50 mg) was subjected to an NP-TLC using toluene–acetone (1:1) as an eluent to obtain naringenin (5 mg). Fraction 15/6 (75 mg) was subjected to RP-TLC using MeOH–H₂O (7:3) to yield quercitrin (8.9 mg). Fraction 15/7 (329 mg) was purified by RP-HPLC using MeOH–H₂O (8:2) isocratic solvent system to obtain 12-O-benzoyldeacylmetaplexigenin (t_R = 11 min, 47.1 mg). Fraction 15/8 (75 mg) was purified by RP-HPLC (the gradient started at MeOH–H₂O (2:8) for 1 min, then linearly ramped up to 1:1 in 10 min and held 0.5 min, then returned to the initial conditions within 1 min and kept for 2 min) to yield compound 6 (t_R = 5.5 min, 13.9 mg). Fr. 16 (1.6 g) was subjected to an NP-VLC using CHCl₃–MeOH gradient system (from 98:2 to 1:1) to yield 9 subfractions (Fr. 16/1–9). Fr. 16/6 (106 mg) was further purified by RP-HPLC using MeOH–H₂O (7:3) isocratic solvent system to obtain compound 5 (t_R = 7.2 min, 24.3 mg).

3.3.1. Euphogossypin A (1)

White amorphous powder; [α${]}_{\mathrm{D}}^{25}$ +12 (c 0.1, MeOH); HRESIMS m/z: 955.4660 [M + Na]⁺ (calcd for C₄₉H₇₂O₁₇Na, 955.4662); ¹H (CDCl₃, 500 MHz) and ¹³C NMR (CDCl₃, 125 MHz) data, see Table 1.

3.3.2. Euphogossypin B (2)

White amorphous powder; [α${]}_{\mathrm{D}}^{25}$ +24 (c 0.2, MeOH); HRESIMS m/z: 927.4352 [M + Na]⁺ (calcd for C₄₇H₆₈O₁₇Na, 927.4349); ¹H (C₅D₅N, 500 MHz) and ¹³C NMR ((C₅D₅N, 125 MHz) data, see Table 1.

3.3.3. Euphogossypin C (3)

White amorphous powder; [α${]}_{\mathrm{D}}^{25}$ +5 (c 0.1, MeOH); HRESIMS m/z: 941.4508 [M + Na]⁺ (calcd for C₄₈H₇₀O₁₇Na 941.4510); ¹H (CD₃OD, 500 MHz) and ¹³C NMR (CD₃OD, 125 MHz) data, see Table 1

3.3.4. Euphogossypin D (4)

White amorphous powder; [α${]}_{\mathrm{D}}^{25}$ +63 (c 0.15, MeOH); HRESIMS m/z: 1103.5029 [M + Na]⁺ (calcd for C₅₄H₈₀O₂₂Na, 1103.5033); ¹H (CD₃OD, 500 MHz) and ¹³C NMR (CD₃OD, 125 MHz) data, see Table 2.

3.3.5. Euphogossypin E (5)

White amorphous powder; [α${]}_{\mathrm{D}}^{25}$ 0 (c 0.15, MeOH); HRESIMS m/z: 1089.4869 [M + Na]⁺ (calcd for C₅₃H₇₈O₂₂Na, 1089.4877); ¹H (CD₃OD, 500 MHz) and ¹³C NMR (CD₃OD, 125 MHz) data, see Table 2.

3.3.6. Euphogossypin F (6)

White amorphous powder; [α${]}_{\mathrm{D}}^{25}$ +3 (c 0.15, MeOH); HRESIMS m/z: 1103.5029 [M + Na]⁺ (calcd for C₅₄H₈₀O₂₂Na, 1103.5033); ¹H (CD₃OD, 500 MHz) and ¹³C-NMR (CD₃OD, 125 MHz) data, see Table 2.

3.3.7. Euphogossypin G (7)

White amorphous powder; [α${]}_{\mathrm{D}}^{25}$ +12 (c 0.05, MeOH); HRESIMS m/z: 865.4188 [M + Na]⁺ (calcd for C₄₂H₆₆O₁₇Na, 865.4192); ¹H (CDCl₃, 500 MHz) and ¹³C NMR (CDCl₃, 125 MHz) data, see Table 3.

3.3.8. Euphogossypin H (8)

White amorphous powder; [α${]}_{\mathrm{D}}^{25}$ +33 (c 0.15, MeOH); HRESIMS m/z: 837.4238 [M + Na]⁺ (calcd for C₄₁H₆₆O₁₆Na, 837.4243); ¹H (CDCl₃, 500 MHz) and ¹³C NMR (CDCl₃, 125 MHz) data, see Table 3.

3.3.9. Gossypilignan A (9)

Pale yellow amorphous solid; [α${]}_{\mathrm{D}}^{25}$ −13 (c 0.1, MeOH); HRESIMS m/z: 429.1889 [M + Na]⁺ (calcd for C₂₂H₃₀O₇Na, 429.1884); ¹H (CD₃OD, 500 MHz) and ¹³C NMR (CD₃OD, 125 MHz) data, see Table 4.

3.3.10. Gossypilignan B (10)

White amorphous powder; [α${]}_{\mathrm{D}}^{25}$ −5 (c 0.05, MeOH); HRESIMS m/z: 427.1734 [M + Na]⁺ (calcd for C₂₂H₂₈O₇Na, 427.1727); ¹H (CD₃OD, 500 MHz) and ¹³C NMR (CD₃OD, 125 MHz) data, see Table 4.

3.3.11. 12-O-Benzoyldeacylmetaplexigenin

¹H NMR (in CDCl₃) δ_H 1.15 (3H, s, Me-19), 1.15, 1.90 (2H, m, H-1), 1.55 (3H, s. Me-18), 1.56, 1.83 (2H, m, H-2), 1.60 (1H, m H-9), 1.96 (2H, m, H-11), 2.03 (2H, m, H-15), 2.07 (3H, s, Me-21), 2.25 (2H, m, H-7), 2.30–2.39 (2H, m, H-4), 1.92, 2.86 (2H, m, H-16), 3.57 (1H, m, H-3), 4.86 (1H, dd, J = 10.2, 5.6 Hz, H-12), and 5.39 (1H, br s, H-6); ¹³C NMR see Table 1.

3.4. Antiproliferative Assays

3.4.1. Cell Line

Human cervix carcinoma (HeLa) cells were cultured in Eagle’s Minimal Essential Medium (EMEM, containing 4.5 g/L glucose) supplemented with a non-essential amino acid mixture, a selection of vitamins, and 10% heat-inactivated fetal bovine serum. The cell line was detached with 0.25% trypsin and 0.02% EDTA for 5 min at 37 °C. The cell line was purchased from LGC Promochem, Teddington, England.

3.4.2. Antiproliferative Assay

The antiproliferative assay of the isolated compounds (1–3, 5–12) against the human cervix carcinoma (HeLa) cell line was performed by MTT, using cisplatin and doxorubicin as positive controls. This assay was carried out as previously described [30,31].

4. Conclusions

In this article, nine polyoxypregnane ester derivatives, including euphogossypins A–H (1–8) as new natural products, two new lignans (gossypilignans A (9) and B (10)), and 12-O-benzoyldeaxcylmetaplexigenin (11), 9α-hydroxypinoresinol (12), naringenin (13), and quercitrin (14) as known compounds, were characterized from the aerial parts of E. gossypina var. coccinea. Their planar structures were elucidated by comprehensive spectroscopic data. All compounds were isolated for the first time from the plant. Although previously several pregnane glycosides and lignans have been found to be active as antiproliferative agents, none of the compounds isolated from E. gossypina var. coccinea showed remarkable antiproliferative activity against the Hela cells. Our findings enrich the knowledge of special metabolites of Euphorbia species.

Author Contributions

Conceptualization, A.V.; methodology, A.V.; software, N.S. and G.S.; investigation, R.H., N.K., R.B., N.S., C.Z.D., P.W.M. and G.S.; resources, J.H.; writing—original draft preparation, R.H. and A.V.; writing—review and editing, A.V., N.K. and J.H.; project administration, J.H.; funding acquisition, J.H. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article and Supplementary Material.

Acknowledgments

The authors acknowledge Patrick Chalo Mutiso, a taxonomist (Department of Biological Sciences, Faculty of Science and Technology, University of Nairobi), for the collection and identification of plant material.

Conflicts of Interest

The authors declare no conflict of interest.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures and Tables

Figure 1. New isolated compounds (1–10) from E. gossypina var. coccinea.

Figure 1. New isolated compounds (1–10) from E. gossypina var. coccinea.

Figure 2. The 1H-1H COSY (—) and key HMBC (→) correlations of 1.

Figure 3. Selected 1H-1H COSY (—) and HMBC (→) correlations of 9.

Table 1

The ¹H and ¹³C NMR data for compounds 1–3.

Atom	1 *		2 *		3 *
	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)	δC
1	1.13, m, α/1.89, m, β	38.9, CH2	1.14, m, α/1.83, m, β	39.4, CH2	1.14, m, α/1.83 m, β	39.8, CH2
2	1.62, m, β/1.92, m, α	29.1, CH2	1.82, m, β/2.11, m, α	30.3, CH2	1.58, m, β/1.87 m, α	30.2, CH2
3	3.57, m	78.0, CH	3.89, m	78.1, CH	3.54, m	79.3, CH
4	2.29, m, β/2.40, m, α	38.9, CH2	2.43, m, * β/2.55, m, α	39.7, CH2	2.22, m; 2.38 dd (3.4, 12.7)	39.8, CH2
5	-	140.8, C	-	139.9, C	-	140.3, C
6	5.38, br s	117.8, CH	5.31, br s	119.6, CH	5.36, br d (4.7)	119.7, CH
7	2.22, m	34.4, CH2	2.38, m/2.50, m	35.2, CH2	2.12–2.22 m	35.2, CH2
8	-	74.4, C	-	74.8, C	-	75.2, C
9	1.59, m	43.8, CH	1.80, m	44.9, CH	1.59 m	45.1, CH
10	-	37.3, C	-	37.9, C	-	38.2, C
11	1.94, m	24.3, CH2	2.21, m, α/2.36, m, β	25.5, CH2	1.81, m, α/2.00, m, β	25.5, CH2
12	4.83, dd (4.2, 11.9)	73.3, CH	5.36, dd (4.3, 11.5)	77.5, CH	4.83 dd (4.3, 11.9)	74.7, CH
13	-	58.5, C	-	58.8, C	-	59.1, C
14	-	88.1, C	-	90.0, C	-	90.0, C
15	2.03, m	33.4, CH2	2.14–2.21, m	34.3, CH2	1.92, m; 2.06, * m	34.3, CH2
16	1.92, m, β/2.85, m, α	32.1, CH2	2.07, m/3.27, m	33.7, CH2	1.73, m, β/2.87, m, α	33.5, CH2
17	-	91.6, C	-	93.0, C	-	93.1, C
18	1.54, s	9.7, CH3	2.10, s	11.3, CH3	1.67, s	10.6, CH3
19	1.12, s	18.7, CH3	1.34, s	18.6, CH3	1.16, s	18.6, CH3
20	-	209.5, C	-	210.6, C	-	212.2, C
21	2.06, s	27.5, CH3	2.37, s	28.2, CH3	2.05, s	27.8, CH3
Bz
1′	-	165.5, C	-	165.8, C	-	166.7, C
2′	-	130.1, C	-	131.8, C	-	131.6, C
3′,7′	7.93, dd (1.1, 8.2)	129.7, CH	8.31, d (7.8)	130.4, CH	7.95, d (7.9)	130.5, CH
4′,6′	7.43, t (8.0)	128.6, CH	7.49, t (7.7)	129.4, CH	7.48, dd (7.9, 7.4)	129.6, CH
5′	7.55, t (8.1)	133.3, CH	7.59, t (7.7)	133.7, CH	7.61, t (7.4)	134.3, CH
	Cym I		Dig I		Cym
1	4.85, dd (2.0, 9.0)	96.2, CH	5.48, d (9.4)	96.9, CH	4.87, dd (1.9, 8.9)	97.2, CH
2	1.58, m, a/2.08, m, e	35.7, CH2	2.05, m; 2.43, m *	39.5, CH2	1.54, m, a/2.06, m, * e	36.7, CH2
3	3.80, m	77.2, * CH	4.65, m	68.0, CH	3.85, m	78.6, CH
4	3.21, dd (3.0, 9.6)	82.7, CH	3.53, m	83.9, CH	3.24, m *	83.9, CH
5	3.84, m	68.7, CH	4.31, m	69.1, CH	3.81, m	70.0, CH
6	1.22, d (6.3)	18.3, CH3	1.45, d (6.0)	19.1, CH3	1.20, d (6.2)	18.5, CH3
3-OMe	3.42, s *	58.1, # CH3			3.44, s	58.5, CH3
	Cym II		Dig II		Dig
1	4.76, dd (1.8, 9.5)	99.7, CH	5.41, d (9.6)	100.3, CH	4.89, dd (1.7, 9.1)	101.0, CH
2	1.65, m, a/2.16, m, e	35.3, CH2	2.00, m, a/2.43, m, * e	39.4, CH2	1.71, m, a/2.02, m, e	38.8, CH2
3	3.78, m	77.1,* CH	4.70, m	68.3, CH	4.21, m	68.6, CH
4	3.26, dd (2.9, 9.6)	82.8, CH	3.60, m	84.1, CH	3.24, * m	83.8, CH
5	3.90, m	68.4, CH	4.37, m	69.5, CH	3.87, m	69.5, CH
6	1.27, d (6.2)	18.6, CH3	1.59, d (6.1)	19.0, CH3	1.31, d (6.2)	18.6, CH3
3-OMe	3.44, s *	58.2, # CH3
	Thv		Thv		Thv
	4.30, d (7.8)	104.5, CH	4.82, d (7.9)	106.3, CH	4.35, d (7.9)	105.5, CH
	3.51, m	74.8, CH	3.85, m	75.3, CH	3.28, m	75.2, CH
	3.10, t (9.0)	85.4, CH	3.61, m	88.3, CH	3.01, m	87.5, CH
	3.18, t (9.2)	74.8, CH	3.58, m	76.3, CH	3.04, m	76.5, CH
	3.36, dd (6.2, 9.2)	71.8, CH	3.73, m	73.2, CH	3.31, + m	73.2, CH
	1.31, d (6.2)	17.9, CH3	1.53, d (6.0)	18.9, CH3	1.25, d (6.1)	18.1, CH3
3-OMe	3.65, s	60.8, CH3	3.91, s	61.4, CH3	3.63, s	61.1, CH3

* in CDCl₃ (1), pyridine-d₅ (2), methanol-d₄ (3), ^# interchangeable signals; ⁺ overlaps with solvent signal.

Table 2

The ¹H and ¹³C NMR data for compounds 4–6 in CD₃OD.

Atom	4		5		6
	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)	δC
1	1.14, m, α/1.85, m, β	39.8, * CH2	1.16, m, α/1.85 m, β	39.8, CH2	1.16, m, α/1.84, m, β	39.8, CH2
2	1.60, m, * β/1.88, m, α	30.2, CH2	1.61, m, * β/1.88 m, α	30.1, CH2	1.59, m, β/1.87, m, α	30.2, CH2
3	3.55, m	79.3, CH	3.56, m	79.3, CH	3.54, m	79.3, CH
4	2.23, m, β/2.38, m, α	39.8, * CH2	2.23, m; 2.40 dd (3.5, 12.7)	39.8, CH2	2.22, m, β/2.38, m, α	39.8, CH2
5	-	140.2, C	-	140.3, C	-	140.3, C
6	5.37, br d (4.6)	119.7, CH	5.37, br d (4.6)	119.6, CH	5.36, br s	119.7, CH
7	2.13–2.22, m	35.2, CH2	2.14–2.22 m	35.2, CH2	2.14–2.21, m	35.2, CH2
8	-	75.0, C	-	75.0, C	-	75.0, C
9	1.61, m *	45.1, CH	1.61 m *	45.1, CH	1.60, m	45.1, CH
10	-	38.2, C	-	38.2, C	-	38.2, C
11	1.83, m, α/2.02, m, β	25.5, CH2	1.83, m, α/2.02, m, β	25.4, CH2	1.82, m, α/2.01, m, β	25.4 CH2
12	4.83, dd +	74.7, CH	4.82 dd (4.3, 11.9)	74.7, CH	4.82, dd (overlaps)	73.3, CH
13	-	59.1, C	-	59.1, C	-	59.1, C
14	-	90.0, C	-	90.0, C	-	90.0, C
15	1.94, m; 2.07, m	34.3, CH2	1.94, m; 2.08, m	34.3, CH2	1.93, m; 2.07, m	34.3, CH2
16	1.75, m, β/2.87, m, α	33.5, CH2	1.75, m, β/2.87, m, α	33.5, CH2	1.74, m, β/2.87, m, α	33.5, CH2
17	-	93.1, C	-	93.1, C	-	93.1, C
18	1.67, s	10.6, CH3	1.67, s	10.6, CH3	1.67, s	10.6, CH3
19	1.16, s	18.6, CH3	1.16, s	18.6, CH3	1.16, s	18.6, CH3
20	-	212.3, C	-	212.2, C	-	212.2, C
21	2.06, s	27.8, CH3	2.06, s	27.8, CH3	2.05, s	27.8, CH3
Bz
1′	-	166.7, C	-	166.7, C	-	166.7, C
2′	-	131.5, C	-	131.5, C	-	131.5, C
3′,7′	7.95, d (7.9)	130.5, CH	7.95, d (7.9)	130.5, CH	7.95, d (8.2)	130.5, CH
4′,6′	7.48, t (7.8)	129.6, CH	7.48, t (7.9)	129.5, CH	7.48, t (7.9)	129.6, CH
5′	7.60, t (7.5)	134.3, CH	7.61, t (7.8)	134.3, CH	7.60, t (7.9)	134.3, CH
	Cym I		Dig I		Cym
1	4.87, dd (1.6, 9.6)	97.2, CH	4.96, dd (1.7, 9.7)	97.0, CH	4.87, dd (1.9, 8.9)	97.2, CH
2	1.55, m, a/2.07, m, * e	36.6, CH2	1.68, m, a/1.96, m, e	38.9, CH2	1.54, m, a/2.06, m, e	36.7, CH2
3	3.85, m #	78.6, CH	4.24, m *	68.4, CH	3.85, m	78.6, CH
4	3.24, m *	83.9, CH	3.23, m *	83.7, CH	3.26, m *	83.8, * CH
5	3.81, m	70.0, CH	3.81, m	69.5, CH	3.82, m	70.0, CH
6	1.19, d (6.3)	18.5,# CH3	1.21, d (6.2)	18.5, * CH3	1.20, d (6.1)	18.5, CH3
3-OMe	3.43, s #	58.4, CH3			3.44, s	58.5, CH3
	Cym II		Dig II		Dig
1	4.80, m +	101.1, CH	4.93, dd (1.6, 9.7)	100.4, CH	4.89, dd (1.7, 9.1)	101.0, CH
2	1,59, m, a/2.14, m, * e	36.4, CH2	1.76, m, a/2.03, m, e	38.7, CH2	1.71, m, a/2.02, m, e	38.8, CH2
3	3.84, m *	78.6, CH	4.22, m *	68.5, CH	4.22, m	65.6, CH
4	3.28, m	84.1, CH	3.27, m	83.8, CH	3.26, m *	83.9, * CH
5	3.88, m *	70.1, CH	3.91, m	69.7, CH	3.88, m	69.5, CH
6	1.30, d (6.3)	18.7, CH3	1.31, d (6.2)	18.5, CH3	1.31, d (6.2)	18.6, CH3
3-OMe	3.44, s #	58.6, CH3
	Thv		Thv		Thv
	4.34, d (7.8)	106.1, CH	4.37, d (7.8)	105.5, CH	4.37, d (7.8)	105.5, CH
	3.30, m	75.0, CH	3.34, m	74.7, CH	3.33, m	74.8, CH
	3.19, m *	86.1, CH	3.20, m	86.0, CH	3.19, m	86.0, CH
	3.37, m	82.8, CH	3.38, m	82.8, CH	3.37, m	82.8, CH
	3.47, m	72.5, CH	3.48, m	72.6, CH	3.48, m	72.6, CH
	1.37, d (6.1)	18.5, # CH3	1.36, d (6.2)	18.6, CH3	1.36, d (6.2)	18.5, CH3
3-OMe	3.63, s	61.2, CH3	3.63, s	61.3, CH3	3.62, s	61.3, CH3
	Glc		Glc		Glc
1	4.43, d (7.7)	104.3, CH	4.43, d (7.8)	104.3, CH	4.43 d (7.8)	104.3, CH
2	3.18, m *	75.7, CH	3.18, m	75.7, CH	3.18, m	75.7, CH
3	3.35, m	78.0, CH	3.35, m	78.0, CH	3.35, m	78.0, CH
4	3.23, m *	71.9, CH	3.23, m *	71.9, CH	3.22, m	71.9, CH
5	3.26, m	78.4, CH	3.26, m	78.4, CH	3.26, m *	78.4, CH
6	3.64, dd (6.4, 12.0); 3.87, m	63.2, CH2	3.64, dd (6.4, 12.0); 3.87, dd (2.0, 12.0)	63.2, CH2	3.64, m; 3.87, m	63.2, CH2

*^,# interchangeable signals; ⁺ overlaps with residual water.

Table 3

The ¹H and ¹³C NMR data for compounds 7 and 8 in CDCl₃.

Atom	7		8
	δH (J in Hz)	δC	δH (J in Hz)	δC
1	1.10, m, α/1.86, m, β	39.0, * CH2	1.08, m, α/1.87, m, β	39.0, * CH2
2	1.64, m, β/1.93, m, * α	29.0, CH2	1.66, m, β/1.94, m, α	29.2, CH2
3	3.57, m	78.1, CH	3.55, m	78.1, CH
4	2.29, m, β/2.41, m, α	39.0, * CH2	2.30, m, β/2.38, m, α	39.1, * CH2
5	-	141.4, C	-	140.8, C
6	5.35, br s	117.5, CH	5.36, br s	117.9, CH
7	2.20, m	34.3, CH2	2.18, m	34.5, CH2
8	-	74.7, C	-	74.5, C
9	1.52, m	43.9, CH	1.46, dd (3.2, 13.1)	44.4, CH
10	-	37.4, C	-	37.3, C
11	1.78, m	24.4, CH2	1.60, m, α/1.90, m, β	28.2, CH2
12	4.51,dd (5.8, 10.3)	72.7, CH	5.68, m	69.7, CH
13	-	57.8, C	-	61.1, C
14	-	88.3, C	-	88.0, C
15	1.93, m *	32.8, CH2	1.94, m *	34.3, CH2
16	1.83, m, β/2.87, m, α	32.4, CH2	1.92, m, β/2.75, m, α	33.7, CH2
17	-	91.9, C	-	92.1, C
18	1.42, s	9.4, CH3	1.27, s	7.9, CH3
19	1.12, s	18.9, CH3	1.16, s	18.9, CH3
20	-	209.4, C	-	213.9, C
21	2.24, s	27.4, CH3	2.34, s	28.4, CH3
12-OAc		170.0, C
	1.95, s	20.8, CH3
14-OH	3.94, s		4.12, br s
17-OH	4.42, s		4.61, br s
	Dig I		Cym
1	4.92, dd (1.7, 9.3)	96.1, CH	4.85, br d (9.5) *	96.3, CH
2	1.72, m, a/2.08, m, e	37.3, CH2	1.59, m; 2.09, m	35.9, CH2
3	4.24, m *	66.7, CH	3.81, m	77.3, CH
4	3.23, dd (3.0, 9.4)	82.8, CH	3.24, dd (2.9, 9.6)	82.9, CH
5	3.79, dq (6.3, 9.4)	68.3, CH	3.85, m #	68.7, CH
6	1.23, d (6.3)	18.4, CH3	1.22, d (6.2)	18.4, CH3
3-OMe			3.45, s	58.2, CH3
	Dig II		Dig
1	4.91, dd (1.7, 9.3)	98.5, CH	4.85, br d (9.5) *	99.6, CH
2	1.75, m, a/2.14, m, e	37.0, CH2	1.77, m, a/2.15, m, * e	37.1, CH2
3	4.23, m *	66.8, CH	4.21, m	66.9, CH
4	3.26, dd (2.9, 9.4)	83.2, CH	3.27, dd (3.0, 9.4)	83.4, CH
5	3.90, dq (6.2, 9.4)	68.2, CH	3.86, m #	67.9, CH
6	1.29, d (6.2)	18.5, CH3	1.29, d (6.2)	18.5, CH3
3-OMe
	Thv		Thv
	4.35, d (7.7)	103.5, CH	4.34, d (7.7)	103.5, CH
	3.47, m	74.7, CH	3.45, m	74.7, CH
	3.11, t (9.0)	85.4, CH	3.10, t (9.0)	88.3, CH
	3.19, t (9.0)	74.8, CH	3.19, t (9.0)	74.8, CH
	3.40, m	72.2, CH	3.39, dd (6.1, 9.0)	72.2, CH
	1.32, d (6.1)	17.9, CH3	1.31, d (6.1)	17.9, CH3
3-OMe	3.66, s	60.9, CH3	3.66, s	60.8, CH3

*^,# interchangeable signals.

Table 4

The ¹H and ¹³C NMR data for compounds 9 and 10 in CD₃OD.

Atom	9		10
	δH (J in Hz)	δC	δH (J in Hz)	δC
1	-	137.0, C	-	136.2, C
2,6	6.66, s	106.4, CH	6.69, br s	104.7, CH
3,5	-	149.1/149.2, C	-	149.3, CH
4	-	134.7, C	-	134.7, C
7	3.52, d (11.8)	57.9, CH	4.64, d (9.3)	87.6, CH
8	2.62, m	37.2, C	2.49, m *	48.6, C
9	0.68, d (6.9)	12.1, CH	1.00, d (6.4)	12.1, CH
3,5-OMe	3.82, s/3.83, s	56.8, CH3	3.86, s	56.7/56.8, CH3
1′	-	137.8, C	-	132.5, C
2′,6′	6.64, s	106.3, CH	6.63, br s	104.3, CH
3′,5′	-	149.1/149.2, C	-	149.1, C
4′	-	134.7, C	-	135.4, C
7′	3.35, dd (6.6, 10.7)3.45, dd (8.3, 10.7)	67.3, C	5.47, d (4.4)	86.5, CH
8′	1.77, m	37.0, CH3	2.48, m *	44.6, CH
9′	0.76, d (7.0) s	10.0, CH3	0.63, d (7.0)	9.8, CH3
3′,5′-OMe	3.82, s/3.83, s	56.8, C	3.84, s	56.7/56.8, CH3

* interchangeable signals.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants11101299/s1, Figures S1–S58: One-dimensional and two-dimensional NMR spectra of isolated compounds (1–10); Table S1: Antiproliferative activity of the isolated compounds (1–3, 5–12) in HeLa cell line.

References

1. Vasas, A.; Hohmann, J. Euphorbia Diterpenes: Isolation, Structure, Biological Activity, and Synthesis (2008−2012). Chem. Rev.; 2014; 114, pp. 8579-8612. [DOI: https://dx.doi.org/10.1021/cr400541j] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25036812]

2. Shi, Q.W.; Su, X.H.; Kiyota, H. Chemical and pharmacological research of the plants in genus Euphorbia. Chem. Rev.; 2008; 108, pp. 4295-4327. [DOI: https://dx.doi.org/10.1021/cr078350s] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/18817355]

3. Euphorbia gossypina; Available online: https://powo.science.kew.org/taxon/urn:lsid:ipni.org:names:346654-1 (accessed on 31 March 2022).

4. Sharma, P.; Singh, G. A review of plant species used to treat conjunctivitis. Phytother Res.; 2002; 16, pp. 1-22. [DOI: https://dx.doi.org/10.1002/ptr.1076] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/11807959]

5. Schmelzer, G.H.; Gurib-Fakim, A. Plant Resources of Tropical Africa 11 (1), Medicinal Plants1; PROTA Foundation/Backhuys Publisher/CTA: Wageningen, The Netherlands, 2008; Volume 11, 300.

6. Catley, A.; Mohammed, A.A. Ethnoveterinary knowledge in Sanaag region, Somaliland (Part II): Notes on local methods of treating and preventing livestock disease. Nomadic Peoples; 1996; 39, pp. 135-145.

7. Araya, J.J.; Binns, F.; Kindscher, K.; Timmermann, B.N. Verticillosides A–M: Polyoxygenated pregnane glycosides from Asclepias verticillata L. Phytochemistry; 2012; 78, pp. 179-189. [DOI: https://dx.doi.org/10.1016/j.phytochem.2012.02.019]

8. Si, Y.; Sha, X.S.; Shi, L.L.; Wei, H.Y.; Jin, Y.X.; Ma, G.X. Review on pregnane glycosides and their biological activities. Phytochem. Lett.; 2022; 47, pp. 1-17. [DOI: https://dx.doi.org/10.1016/j.phytol.2021.10.007]

9. Tsukamoto, S.; Hayashi, K.; Mitsuhashi, H. Studies on the constituents of Asclepiadaceae plants. LX. Further studies on glycosides with a novel sugar chain containing a pair of optically isomeric sugars, d- and l-cymarose, from Cynanchum wilfordi. Chem. Pharm. Bull.; 1985; 33, pp. 2294-2304. [DOI: https://dx.doi.org/10.1248/cpb.33.2294]

10. Kiem, P.V.; Yen, D.T.H.; Hung, N.V.; Nhiem, N.X.; Tai, B.H.; Trang, D.T.; Yen, P.H.; Ngoc, T.M.; Minh, C.V.; Park, S. et al. Five new pregnane glycosides from Gymnema sylvestre and their α-glucosidase and α-amylase inhibitory acitivities. Molecules; 2020; 25, 2525. [DOI: https://dx.doi.org/10.3390/molecules25112525]

11. Aquino, R. New polyoxypregnane ester derivatives from Leptadenia hastata. J. Nat. Prod.; 1996; 59, pp. 555-564. [DOI: https://dx.doi.org/10.1021/np960251e]

12. Gao, X.M.; Pu, J.X.; Huang, S.X.; Yang, L.M.; Huang, H.; Xiao, W.L.; Zheng, Y.T.; Sun, H.D. Lignans from Kadsura angustifolia. J. Nat. Prod.; 2008; 71, pp. 558-563. [DOI: https://dx.doi.org/10.1021/np0705108]

13. Davidson, S.J.; Rye, C.E.; Barker, D. Using NMR to determine the relative stereochemistry of 7,7-diaryl-8,8′-dimethylbutan-1-ol lignans. Phytochem. Lett.; 2015; 14, pp. 138-142. [DOI: https://dx.doi.org/10.1016/j.phytol.2015.09.014]

14. Yue, J.M.; Chen, Y.Z.; Hua, S.M.; Cheng, J.L.; Cui, Y.X. Ganschisandrine, a lignan from Schisandra sphenanthera. Phytochemistry; 1989; 28, pp. 1774-1776.

15. Da Silva Filho, A.A.; Albuquerque, S.; e Silva, M.L.A.; Eberlin, M.N.; Tomazela, D.M.; Bastos, J.K. Tetrahydrofuran lignans from Nectandra megapotamica with trypanocidal activity. J. Nat. Prod.; 2004; 67, pp. 42-45. [DOI: https://dx.doi.org/10.1021/np0302697] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/14738383]

16. Abe, F.; Yamauchi, T. 9α-hydroxypinoresinol, 9α-hydroxymedioresinol and related lignans from Allamanda neriifolia. Phytochemistry; 1988; 27, pp. 575-577. [DOI: https://dx.doi.org/10.1016/0031-9422(88)83144-3]

17. Cordenonsi, L.M.; Sponchiado, R.M.; Campanharo, S.C.; Garcia, C.V.; Raffin, R.P.; Schapoval, E.E.S. Study of flavonoids presente in Pomelo (Citrus máxima) by DSC, UV-VIS, IR, 1H and 13C NMR and MS. Drug Anal. Res.; 2017; 1, pp. 31-37. [DOI: https://dx.doi.org/10.22456/2527-2616.74097]

18. Zhong, X.N.; Otsuka, H.; Ide, T.; Hirata, E.; Takushi, A.; Takeda, Y. Three flavonol glycosides from leaves of Myrsine seguinii. Phytochemistry; 1997; 46, pp. 943-946. [DOI: https://dx.doi.org/10.1016/S0031-9422(97)00366-X]

19. Piacente, S.; Belisario, A.B.; Castillo, H.D.; Pizza, C.; Feo, V.D. Croton ruizianus: Platelet proaggregating activity of two new pregnane glycosides. J. Nat. Prod.; 1998; 61, pp. 318-322. [DOI: https://dx.doi.org/10.1021/np970399d]

20. Xu, L.J.; Huang, F.; Chen, S.B.; Li, L.N.; Chen, S.L.; Xiao, P.G. A cytotoxic neolignan from Schisandra propinqua (Wall.) Baill. J. Integr. Plant Biol.; 2008; 48, pp. 1493-1497. [DOI: https://dx.doi.org/10.1111/j.1744-7909.2006.00323.x]

21. Huang, F.; Xu, L.; Shi, G. Antioxidant isolated from Schisandra propinqua (Wall.) Baill. Biol. Res.; 2009; 42, pp. 351-356. [DOI: https://dx.doi.org/10.4067/S0716-97602009000300009]

22. Deepak, D.; Srivastav, S.; Khare, A. Pregnane Glycosides. Fortschritte der Chemie Organischer Naturstoffe/Progress in the Chemistry of Organic Natural Products; Springer: Vienna, Austria, 1997; Volume 71, [DOI: https://dx.doi.org/10.1007/978-3-7091-6529-4_3]

23. Hayashi, K.; Wada, K.; Mitsuhashi, H.; Bando, H.; Takase, M.; Terada, S.; Koide, Y.; Aiba, T.; Narita, T.; Mizuno, D. Antitumor active glycosides from Condurango cortex. Chem. Pharm. Bull.; 1980; 28, pp. 1954-1958. [DOI: https://dx.doi.org/10.1248/cpb.28.1954]

24. Hayashi, K.; Wada, K.; Mitsuhashi, H.; Bando, H.; Takase, M.; Terada, S.; Koide, Y.; Aiba, T.; Narita, T.; Mizuno, D. Further investigation of antitumor condurangoglycosides with C-18 oxygenated aglycone. Chem. Pharm. Bull.; 1981; 29, pp. 2725-2730. [DOI: https://dx.doi.org/10.1248/cpb.29.2725] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/7349290]

25. Yoshimura, S.I.; Narita, H.; Hayashi, K.; Mitsuhashi, H. Studies on the constituents of Asclepiadaceae plants. LVI. Isolation of new antitumor-active glycosides from Dregea volubilis (L.) Benth. Chem. Pharm. Bull.; 1983; 31, 3971. [DOI: https://dx.doi.org/10.1248/cpb.31.3971] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/6671238]

26. Panda, N.; Banerjee, S.; Mandal, N.B.; Sahu, N.P. Pregnane glycosides. Nat. Prod. Commun.; 2006; 1, pp. 665-695. [DOI: https://dx.doi.org/10.1177/1934578X0600100813]

27. Plaza, A.; Perrone, A.; Balestrieri, M.L.; Felice, F.; Balestrieri, C.; Hamed, A.I.; Pizza, C.; Piacente, S. New unusual pregnane glycosides with antiproliferative activity from Solenostemma argel. Steroids; 2005; 70, pp. 594-603. [DOI: https://dx.doi.org/10.1016/j.steroids.2005.02.019]

28. Hunag, L.J.; Wang, B.; Zhang, J.X.; Yan, C.; Mu, S.Z.; Hao, X.J. Studies on cytotoxic pregnane sapogenins from Cynanchum wilfordii. Fitoterapia; 2015; 101, pp. 107-116. [DOI: https://dx.doi.org/10.1016/j.fitote.2014.10.014]

29. Saleem, M.; Kim, H.J.; Ali, M.S.; Lee, Y.S. An update on bioactive plant lignans. Nat. Prod. Rep.; 2005; 22, pp. 696-716. [DOI: https://dx.doi.org/10.1039/b514045p]

30. Stefkó, D.; Kúsz, N.; Szemerédi, N.; Barta, A.; Spengler, G.; Berkecz, R.; Hohmann, J.; Vasas, A. Unique phenanthrenes from Juncus ensifolius and their anti-proliferative and synergistic effects with the conventional anticancer agent doxorubicin against human cancer cell lines. Pharmaceutics; 2022; 14, 608. [DOI: https://dx.doi.org/10.3390/pharmaceutics14030608]

31. Bacher, F.; Wittmann, C.; Nové, M.; Spengler, G.; Marć, M.A.; Enyedy, E.A.; Darvasiová, D.; Rapta, P.; Reinere, T.; Arion, V.B. Novel latonduine derived proligands and their copper(II) complexes show cytotoxicity in the nanomolar range in human colon adenocarcinoma cells and in vitro cancer selectivity. Dalton Trans.; 2019; 48, 10464. [DOI: https://dx.doi.org/10.1039/C9DT01238A]