Two xanthones and two rotameric (3⟶8) biflavonoids from the Cameroonian medicinal plant Allanblackia floribunda Oliv. (Guttiferae)

1 Two xanthones and two rotameric (3⟶8) biflavonoids from the Cameroonian medicinal plant Allanblackia floribunda Oliv. (Guttiferae) Bel Youssouf G. Mountessoua,b, Joseph Tchamgouea, Jean Paul Dzoyemc, Roland T. Tchuenguemc, Frank Surupd, Muhammad I. Choudharyb, Ivan R. Greene, Simeon F. Kouama,* aDepartment of Chemistry, Higher Teacher Training College, University of Yaoundé I, P.O. Box 47, Yaoundé, Cameroon bH.E.J. Research Institute of Chemistry, International Centre for Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi-75270, Pakistan cDepartment of Biochemistry, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon dDepartment of Microbial Drugs, Helmholtz Centre for Infection Research and German Centre for Infection Research, partner site Hannover/Braunschweig, Inhoffenstrasse 7, D-31824 Braunschweig, Germany eDepartment of Chemistry and Polymer Science, University of Stellenbosch, P/Bag X1, Matieland, Stellenbosch, 7602, South Africa *Corresponding author: Tel.: +237 694 464 535; e-mail address: [email protected] (S. F. Kouam). 2 Abstract Two xanthones, 2-(3-hydroxy-3,3-dimethyldihydroallyl)-dihydro-6-deoxyisojacareubin (1) and dihydro-6-deoxyjacareubin (2), and two 3⟶8 rotameric biflavonoids, (2R,3S)- volkensiflavone-7-O-𝛽-acetylglucopyranoside (3) and (2S,3S)-morelloflavone-7-O-𝛽acetylglucopyranoside (4), together with fifteen known compounds, were isolated from a dichloromethane/methanol (1/1, v/v) extract of the bark of the plant Allanblackia floribunda. The structures of the new compounds were elucidated by NMR spectroscopy and mass spectroscopic techniques and those of the known ones were deduced by comparison with data reported in the literature. The isolated biflavonoids were obtained as mixtures of conformers exhibiting duplicate NMR signals in solution at 25 °C and their respective absolute configurations were assigned using circular dichroism spectroscopy. Some of the isolated compounds were assessed for their antibacterial and antioxidant properties. 3 1. Introduction Allanblackia floribunda Oliver belongs to the Guttiferae family and is widely distributed along the coastal regions of West Africa. In folk medicine, different parts of this plant are either used alone or in combination with other plants for the treatment of several human ailments including upper respiratory tract infections, dysentery, diarrhoea, and toothache.1 Biological and pharmacological investigations of extracts of this plant have enabled a better understanding of their use in traditional medicine. For instance, the antitumor, radical scavenging, antimycobacterial, antibacterial and antifungal activities of the root bark extract of A. floribunda have been reported.2 Investigations of this plant have led to the isolation of a series of secondary metabolites belonging to different classes including triterpenoids,3 benzophenones,4 xanthones5 and biflavonoids.2,4 Some of these compounds exhibit a wide range of biological and pharmacological activities including antioxidant, antitumor,2 cytotoxic,5 anti-inflammatory, antimicrobial and antifungal activities,6,7 as well as HIV inhibitory activity.8 As part of our continuing search for biologically active compounds from Cameroonian medicinal plants,9–12 we have investigated the bark of A. floribunda for its minor secondary metabolites and herein report the isolation, structural elucidation and biological activities of two new xanthones (1 and 2) and two new rotameric biflavonoids (3 and 4). 2. Results and Discussion The dichloromethane/methanol extract of the stem bark of A. floribunda was subjected to repeated column chromatography to give several fractions, which were further purified over sephadex LH–20 and preparative HPLC to yield a total of nineteen compounds of which compounds 1–4 were unknown. 4 3 OO O OH OH 2 R1R2 H 6OH 7H OH O OH O O O 16 O OH O O OH OH 1 12 3 4 4a 5 6 7 8 4b 8a 9 1' 2' 3' 4'' 5' 9a 1'' 2'' 3'' 5'' 4' TFA/ O O OH O O OH HO OH I-A I-B I-C II-D II-E II-F 1 2 3 5 6 4 71' 8 9 10 2' 3' 4' 5' 6' 1 2 3 4 5 6 78 1'' 2'' 3'' 4'' 5'' 6'' 9 10 OH OH R2 O O OH O O OH HO OH I-A I-B I-C II-D II-E II-F OH R1 O OH 2'' 4'' 5'' 6'' 8'' 7'' 1'' 3'' 4 5H OH 8 O OH OH OH OH HO 9 O O OH O O OH HO OH OH OH O HO HO OAc O OH O HO HO OH O OH O HO HO OH O OH O HO HO O O Figure 1. Chemical structures of compounds 1−9 and 16. Compound 1 was obtained as a yellow solid (m.p. 186–187 °C), which reacted positively to FeCl3 reagent. Its molecular formula C23H26O6 was established from the positive ion mode HRESIMS, which showed ion clusters [M+H]+ at m/z 399.1802 (calcd. for C23H27O6: 399.1808). The IR spectrum showed strong absorptions for hydroxyl and conjugated carbonyl groups at 3400 and 1640 cm–1 respectively, while the UV spectrum exhibited characteristic absorption bands of xanthones at 𝜆max 234 and 250 nm.13 The 1H NMR spectrum of compound 1 displayed an ABC spin system at 𝛿H 7.73 (dd, 1.8; 7.9 Hz), 7.27 (dd, 1.8; 7.9 Hz) and 7.22 (pseudo t, 7.9 Hz) assignable to a 1,2,3-trisubstituted benzene ring. The 2,2-dimethyldihydropyran moiety was deduced from the signals at 𝛿H 2.93 and 1.76 (2H each, t, 7.9 Hz) and 𝛿H 1.37 (6H, s) which was further confirmed by the 13C NMR spectrum with resonances at 𝛿C 72.7, 41.3, 29.8 and 16.4. This 2,2-dimethyldihydropyran group was attached at C-3 and C-4 of the xanthone skeleton of compound 1 as illustrated by HMBC correlations (Fig. 2) observed between the methylene protons at 𝛿H 2.93 (H-1ꞌ) with the carbon signals at 𝛿C 158.8 (C-3); 152.0 (C-4a); 107.9 (C-4); 72.7 (C-3′) and 41.3 (C-2′). On the other hand, the presence of a 3-hydroxy-3-methylbutyl group was also deduced by the presence of signals at 𝛿H 2.77 and 1.77 (2H each, t, 7.0 Hz) and 1.40 5 (6H, s) which were further confirmed in the 13C NMR spectrum with resonances at 𝛿C 72.7, 41.3, 29.8 and 16.4 respectively. Thus, HMBC correlations between the methylene protons at 𝛿H 2.77 (H-1ꞌꞌ) and the carbon signals at 𝛿C 158.8 (C-3); 103.7 (C-2); 76.2 (C-3′′) and 31.7 (C-2′′) clearly identified the point of attachment at C-2 of the xanthone skeleton. Based on the above investigations, structure 1 was named 2-(3-hydroxy-3,3-dimethyldihydroallyl)-dihydro-6deoxyisojacareubin (Fig. 1). The proposed structure was fully supported by HMBC, DEPT and COSY spectra. Key HMBC correlations of compounds 1 are illustrated in Fig. 2. Furthermore, compound 1 was heated at reflux in TFA for 30 min. The resulting product, bispyranoxanthone (16) allowed additional confirmation of the proposed structure for 1. 3 O O HO HO OH O O H3C O O OH O O OH HO OH OH H H O O O OH OH 2 OH O O O OH OH 1 Figure 2. Key HMBC correlations of compounds 1–3. 6 Table 1. 13C and 1H NMR data of compounds 1 and 16 (CDCl3) at 25 °C. C and H no. 1 16 13C (176 MHz) 1H (500 MHz) 13C (176 MHz) 1H (700 MHz) 𝛿C (𝑚) 𝛿H (𝑛H, J in Hz) 𝛿C (m) 𝛿H (𝑛H, J in Hz) 1 158.2 (C) - 154.2 (C) - 2 103.7 (C) 6.19 (1H, s) 105.8 (C) 6.19 (1H, s) 3 158.8 (C) - 157.8 (C) - 4 107.9 (C) - 99.6 (C) - 4a 152.0 (C) - 153.9 (C) - 4b 144.6 (C) - 143.5 (C) - 5 145.5 (C) - 144.1 (C) - 6 119.9 (CH) 7.27 (dd, 1.8; 7.9) 119.0 (CH) 7.28 (1H, dd, 1.5, 8.0) 7 123.6 (CH) 7.22 (pseudo t, 7.9) 123.8 (CH) 7.23 (1H, pseudo t, 8.0) 8 116.0 (CH) 7.73 (dd, 1.8; 7.9) 117.9 (CH) 7.64 (1H, dd, 1.5, 8.0) 8a 121.0 (C) - 106.2 (C) - 9 181.3 (C) - 176.1 (C) - 9a 102.6 (C) - 102.0 (C) - 1 16.4 (CH2) 2.93 (2H, t, 7.9) 16.7 (CH2) 2.93 (2H, t, 6.7) 2 41.3 (CH2) 1.76 (2H, t, 7.9) 31.7 (CH2) 1.93 (2H, t, 6.7) 3 72.7 (C) - 76.3 (C) - 4 29.8 (CH3) 1.37 (3H, s) 26.9 (CH3) 1.42 (3H, s) 5 29.8 (CH3) 1.37 (3H, s) 26.9 (CH3) 1.42 (3H, s) 1 16.2 (CH2) 2.77 (2H, t, 7.0) 17.0 (CH2) 2.65 (2H, t, 6.7) 2 31.7 (CH2) 1.87 (2H, t, 7.0) 31.5 (CH2) 1.84 (2H, t, 6.7) 3 76.2 (C) - 76.0 (C) - 4 26.9 (CH3) 1.40 (3H, s) 26.6 (CH3) 1.45 (3H, s) 5 26.9 (CH3) 1.40 (3H, s) 26.6 (CH3) 1.45 (3H, s) 7 Compound 2, named dihydro-6-deoxyjacareubin, was obtained as an orange solid (m.p. 190–192 °C) which also reacted positively to FeCl3 reagent. Its molecular formula C18H16O5 was established from the positive ion mode HRESIMS, which showed ion clusters [M+H]+ at m/z 313.1072 (calcd. for C18H17O5: 313.1076). The IR spectrum showed strong absorptions for hydroxyl and conjugated carbonyl groups at 3400 and 1640 cm–1 respectively, while the UV spectrum exhibited absorption maxima characteristic of xanthones at 𝜆max 234 and 250 nm.13 The 1H and 13C NMR spectra of compound 2 showed similarities with those of compound 1 and additionally displayed two methylene signals at 𝛿H 2.75 and 1.87 (2H each, t, 6.7 Hz) together with a 6-proton singlet at 𝛿H 1.40 suggesting the presence of a 2,2-dimethyldihydropyran moiety. Furthermore, an ABC spin system at 𝛿H 7.78 (1H, dd, 1.5; 8.0 Hz), 7.32 (dd, 1.5; 8.0 Hz) and 7.25 (pseudo t, 8.0 Hz) was suggestive of a 1,2,3-trisubstituted benzene ring. The 13C NMR spectrum of 2 displayed 18 carbon signals which were assigned in combination with DEPT and HSQC experiments to two methyls, two methylenes, four methines and ten quaternary carbons, including a carbonyl signal at 𝛿C 180.7 ppm. The presence of a 2,2-dimethyldihydropyran moiety was further confirmed by signals in the 13C NMR spectrum at 𝛿C 76.6 (C-3), 31.7 (C-2), 26.8 (C-4/C-5) and 16.0 (C-1). The HMBC spectrum proved pivotal in order to attach this 2,2dimethyldihydropyran moiety to the xanthone skeleton. Thus, correlations between the methylene protons at 𝛿H 2.75 (H-1′) with the carbon signals at 𝛿C 160.9 (C-1); 102.6 (C-9a); 76.6 (C-3′) and 31.7 (C-2′) suggested that C-1′ (𝛿C 16.0) was attached to C-2 (𝛿C 104.6) of the xanthone skeleton. In addition, correlations of the aromatic proton signal at 𝛿H 6.38 (1H, s, H-4) with carbon signals at 𝛿C 161.8 (C-3); 154.7 (C-4a); 104.6 (C-2) and 102.6 (C-9a) were also crucial in confirming the 2,2-dimethyldihydropyran moiety being attached at positions 2 and 3 of the xanthone skeleton. Moreover, the position of the chelated hydroxyl (𝛿H 13.22) group at C-5 was assigned based on the correlations observed between the aromatic proton at 𝛿H 7.78 (1H, dd, 1.5; 8.0, H-8) with the carbonyl group signal at 𝛿C 180.7. The proposed structure of compound 2 (Fig. 2) was fully supported by HMBC, DEPT and COSY spectra. Its physical and spectroscopic data were consistent with those of a known synthetic compound.14 To the best of our knowledge, compound 2 is herein reported for the first time from a natural source. 8 Table 2. 13C and 1H NMR data of compound 2 (CDCl3) at 25 °C. C and H no. 13C (176 MHz) 1H (500 MHz) 𝛿C (m) 𝛿H (𝑛H, J in Hz) 1 160.9 (C) - 2 104.6 (C) - 3 161.8 (C) - 4 94.9 (CH) 6.38 (1H, s) 4a 154.7 (C) - 4b 144.1 (C) - 5 144.2 (C) - 6 119.9 (CH) 7.32 (1H, dd, 1.5, 8.0) 7 123.7 (CH) 7.25 (1H, pseudo t, 8.0) 8 116.9 (CH) 7.78 (1H, dd, 1.5, 8.0) 8a 121.1 (C) - 9 180.7 (C) - 9a 102.6 (C) - 1′ 16.0 (CH2) 2.75 (2H, t, 6.7) 2′ 31.7 (CH2) 1.87 (2H, t, 6.7) 3′ 79.6 (C) - 4′ 26.8 (CH3) 1.40 (3H, s) 5′ 26.8 (CH3) 1.40 (3H, s) Compound 3 was obtained as a yellow solid, and had the molecular formula C38H32O16 as established from low resolution FAB-MS (negative mode) and HRFAB-MS (positive mode) techniques in which the molecular ions appeared at m/z 743.3 [M–H]+ and 745.1781 [M+H]+ (calcd. for C38H33O16: 745.1769), respectively. The IR spectrum of compound 3 showed strong absorption bands around 3400 and 1700 cm–1 indicating the presence of hydroxyl and carbonyl groups, respectively. Although the prep. HPLC and LC-MS chromatograms indicated this compound to be pure, both its 1H and 13C NMR spectra exhibited doubled sets of signals, suggesting the presence of two conformers. The 1H NMR spectrum displayed characteristic signals of a sugar moiety (acetylglucopyranosyl) with the anomeric proton signal at 𝛿H 5.24 (H1‴) and a multiplet of non-anomeric protons in the up field region (𝛿H 3.70–3.48) along with a two-proton triplet of an oxymethylene entity at 𝛿H 4.36 ppm. The 13C NMR spectrum displayed signals for 38 carbons, which were sorted by DEPT and HSQC techniques into one methyl, one methylene, nineteen methines and seventeen quaternary carbons, including signals for three carbonyls at 𝛿C 172.6, 184.0 and 197.3 ppm (Table 4). Compounds with similar spectral data 9 have been previously reported from the plants of the genus Garcinia of the Guttiferae family.15– 18 Therefore, comparison of the spectroscopic data of compound 3 with those described in the literature enabled us to deduce that it was a monoglycosylated biflavonoid derivative presenting doubled NMR signals. It is noteworthy that the doubled signals in the NMR spectra of biflavonoids at room temperature may be due to the fact that the molecules adopt different conformations16 which arise from the restricted free rotation of the 3→8 interflavanyl bond.17 Nevertheless, these NMR signals can be merged into a single set of signals at higher temperatures because under these conditions, the molecules do not exist in a preferred stable conformation.19 However, the structural elucidation of compound 3 based on its NMR data recorded at ambient temperature was not straightforward. In the 1H NMR spectrum, except for a pair of doublets at 𝛿H 5.96 and 5.97 (1H each, d, 1.6 Hz, H-6, H-8) assignable to two meta-coupled protons of ring I-A of the flavanone moiety of both the major and minor conformers, the 1H NMR resonances of the major conformer were mostly observed in the deshielded aromatic region (𝛿H 6.40–7.80) whereas, signals appearing in the up-field regions were assigned to the sugar moiety. The 1H NMR spectrum of compound 3 also displayed characteristic signals of flavones at 𝛿H 6.45 (1H, s, H-II-3) and 𝛿H 5.71 and 5.33 (1H each, d, 12.0 Hz, H-I-2, H-I-3) indicating the presence of both moieties in the molecule. The coupling constant of 12.0 Hz indicates a biaxial (trans) configuration of the two protons of ring I-C. In addition, characteristic signals for two separate AA'BB' systems were observed at 𝛿H 7.73 and 6.91 (2H each, d, 8.8 Hz, H-3″/5″, H-2″/6″) for one system and 𝛿H 7.02 and 6.34 (2H each, d, 8.4 Hz, H-2′/6′, H-3′/5′) assignable to the two 1,4-disubstituted aromatic ring systems of the flavone (ring II-E) and flavanone (ring I-B) moiety, respectively. The correlations of these sets of protons viz. H-2″/6″ with H-3″/5″and H-2′/6′with H-3′/5′ were fully supported by their 1H–1H COSY correlations (see ESI) both for the major and the minor conformers. A one proton singlet assignable to H-6 of each of the flavone units was evident in the 1H NMR spectrum. Interestingly, signals attributable to an acetyl group were observed in the 1H and 13C NMR spectra at 𝛿H/C 1.95/20.7 (CH3) and 172.7 (CH3C=O). The complete assignment of the two conformers of compound 3, as presented in Tables 3 and 4 respectively, was facilitated by the HSQC and HMBC spectra. The acetylglucopyranosyl moiety was linked at position C-7 of the flavone unit and deduced by correlations observed in the HMBC spectrum between the anomeric proton at 𝛿H 5.24 with carbon signals at 𝛿C 161.6 (C-7) and 𝛿C 100.0 (C-6) of ring D (Fig. 2). 16 Silica gel 60 (0.230–0.400 mm) and (0.040–0.063 mm) was used as adsorbents for flash and column chromatography respectively. The semi-pure compounds were finally purified successively over Sephadex LH-20 (bead size 25–100 μm, Sigma-Aldrich) with CH2Cl2–MeOH (1/1, v/v), reverse (JAIGEL–ODS H80, serial no. 209963) and normal (JAIGEL–SIL, D-60-10, serial no. 051300228) phase preparative HPLC. The specifications of these columns were 250 mm length×20 mm inner diameter; 4𝜇m particle size and 80 Å pore size. Merck TLC plates (silica gel 60 F254) were used for the detection of the purity of compounds. Melting points were determined with Gallekamp apparatus. UV spectra were recorded using a PerkinElmer Lambda 25 UV/Vis spectrometer. IR spectra were recorded in KBr on a PerkinElmer 2000 FT-IR spectrophotometer. EI/ESI mass spectra were recorded on an Agilent 5975C MSD and Thermo Finnigan MAT95XL mass spectrometers at the Hussein Ebrahim Jamal Research Institute of Chemistry (HEJ–RIC) of the International Centre for Chemical and Biological Sciences (ICCBS), University of Karachi (UOK), Karachi Pakistan. The NMR experiments of pure compounds were performed in different deuterated solvents depending on their solubility, using Bruker Ascend 400, 500 and 600 MHz (1H and 13C) spectrometers equipped with a Bruker 5 mm Broadband probe (depending on the amount). Chemical shifts (𝛿) in ppm are referenced to tetramethylsilane (TMS) at 0.00 ppm for 1H and 13C. Coupling constants are expressed in hertz (Hz). 4.2. Plant material The stem bark of A. floribunda Oliver was collected in November 2014 in the Kye-Ossi, Ntem valley Division in the South region of Cameroon. The sample was identified by Mr. Victor Nana, a retired botanist at the National Herbarium of Cameroun, Centre region, where a voucher specimen was deposited as voucher No: 52904/HNC. 4.3. Extraction and isolation The air-dried and ground stem bark (2.2 kg) of A. floribunda was extracted at room temperature with a mixture of CH2Cl2/MeOH (1/1, v/v) for 48 h and concentrated to a viscous black residue (556 g). A part of this residue (550.0 g) was then subjected to flash chromatographic separation over a silica gel (230–400 mesh) column using a stepwise gradient of 𝑛-Hex/EtOAc (ranging from 0 to 100% of EtOAc, v/v), followed by a gradient of EtOAc/MeOH (ranging from 9/1 to 8/2, v/v), to afford a total of 58 fractions (𝑓r1–𝑓r58) of ca. 17 1000 mL per fraction. Fraction 𝑓r15 (eluted with 𝑛-Hex/EtOAc 9/1) precipitated to give compound 10 (100 mg, yellow solid). Further precipitation of the remaining aliquot of this same fraction gave an inseparable mixture of phytosterols (180 mg, white powder). Fraction 𝑓r22 (𝑛Hex/EtOAc 8/2, v/v) precipitated to afford compound 12 (6 mg, yellow solid). Fractions 𝑓r48, 𝑓r50, 𝑓r51 and 𝑓r53 obtained with pure EtOAc, were mass-precipitated to give four different yellow amorphous powders labelled Y5 (0.5 g), Y6 (3.4 g), Y7 (2.1 g) and Y8 (0.4 g) respectively. All four impure powders were subsequently separately purified over reverse phase preparative HPLC (column JAIGEL–ODS H80) using a mixture of acetonitrile/water: 1/1 + 0.08% of TFA as eluent, with the following settings: UV sensitivity 0.05; RI sensitivity 50; initial & final flow rates: 3 & 4 mL/min; initial & final pressures: 47 & 64 psi. Consequently, compounds 5 (75 mg), 6 (100 mg), 7 (25 mg) and 8 (105 mg) were eluted with retention times of 22, 18, 14 and 12 min respectively. All 58 fractions (𝑓r1–𝑓r58) from the flash chromatography were combined into 4 main fractions (A–D) on the basis of their TLC analyses. Fraction A (𝑓r1–𝑓r7: 850 mg) obtained with pure 𝑛-Hex as eluent, consisted of fatty acids and was not investigated further. Fraction B (𝑓r8–𝑓r33: 38.7 g) obtained with 𝑛-Hex/EtOAc (8/2–4/6, v/v), was subjected to further column chromatography over silica gel (0.040–0.063 mm) and eluted with a gradient of 𝑛-Hex/EtOAc (9.5/0.5–0/10, v/v) to produce 100 fractions (B1–B100) of ca. 500 mL each which were combined on the basis of TLC analysis. The first 28 fractions from fraction B (B1–B28), were eluted with a mixture of 𝑛-Hex/EtOAc (9.5/0.5, v/v) and combined on the basis of their TLC profiles into two sub-fractions. The first sub-fraction b15–b20 (200 mg) was rechromatographed over Sephadex LH-20 (CH2Cl2/MeOH (1/1, v/v) and then over normal phase prep. HPLC, using an isocratic mode solvent of 𝑛-Hex/EtOAc (8.8/1.2, v/v), to afford compound 14 (2.5 mg, orange oil) with a retention time of 44 min. The second sub-fraction b21–b28 (150 mg) showed a complex mixture of oils and was not further investigated. The second series of fractions from fraction B (B29-B40) was eluted with 𝑛-Hex/EtOAc (9/1, v/v) and combined, then was further chromatographed and eluted on normal phase preparative HPLC, using an isocratic mode of solvent 𝑛-Hex/EtOAc (8.5/1.5, v/v) to afford compound 2 (0.7 mg, orange solid), at a retention time of 20 min. The third series of fractions from fraction B (B41-B43) (30 mg) was eluted with 𝑛-Hex/EtOAc (8/2, v/v), then rechromatographed and eluted on normal phase preparative HPLC, using an isocratic mode of solvent 𝑛-Hex/EtOAc (7/3, v/v) to afford compound 1 (12 mg, yellow solid), at a retention time of 25 min. The fourth series of fractions from fraction B (B44-B45) (88 mg) was 18 rechromatographed over Sephadex LH-20 (CH2Cl2/MeOH (1/1, v/v), to afford compound 11 (18 mg, yellow solid). The fifth series of fractions from fraction B (B52–B59) (10 mg) was further chromatographed and eluted on normal phase preparative HPLC, using an isocratic mode of solvent 𝑛-Hex/EtOAc (7.5/2.5, v/v) to afford compound 13 (1.8 mg, yellow solid), at a retention time of 44 min. From the sixth series of fractions from fraction B (B60–B77), a white solid compound 15 (60 mg, white neat solid) was obtained. The last series of fractions obtained from fraction B (B78–B100) eluted with 𝑛-Hex/EtOAc (7/3–2/8, v/v) was found to be a complex mixture of compounds and was not further investigated. Fraction C (𝑓r34–𝑓r57: 137 g), eluted with 𝑛-Hex/EtOAc 3/7–1/9, v/v) from the flash chromatography was shown to contain the already purified compounds 5; 6 and 8 and therefore was not further investigated. A part of fraction D (𝑓r58: 80 g; pure EtOAc) was subjected to further chromatographic separations over sephadex LH-20 using CH2Cl2/MeOH (1/1) as eluent to afford eighteen subfractions (D1–D18). Sub-fraction D9 (1.8 g) was subjected to silica gel column chromatography and eluted with an isocratic system of 𝑛-Hex/EtOAc (6/4, v/v) to afford a mixture of glycosylated sterols (105 mg). Sub-fractions D5 (150 mg), D16 (200 mg) and D17 (300 mg) were separately purified on a reverse phase prep. HPLC using isocratic solvents to afford compounds 9 (5 mg, orange oil), 3 (15 mg, yellow solid), and 4 (18.8 mg, yellow solid) respectively. 4.3.1. 2-(3-hydroxy-3,3-dimethyldihydroallyl)-dihydro-6-deoxyisojacareubin (1). Yellow solid from Hex/EtOAc 7/3, m.p. 186–187 °C; UV (MeOH) – 𝜆max nm (PDA): 234, 250, 269, 330 nm; HRESIMS m/z 399.1802 (calcd. for C23H27O6: 399.1808). For 1H and 13C NMR data, see Table 1. 4.3.2. dihydro-6-deoxyjacareubin (2). Orange solid from Hex/EtOAc 8.5/1.5, m.p. 190–192 °C; UV (MeOH) – 𝜆max nm (PDA): 234, 250, 269, 330 nm; HRESIMS [M+H]+ at m/z 313.1072 (calcd. for C18H17O5: 313.1076). For 1H and 13C NMR data, see Table 2. 4.3.3. (2R,3S)-volkensiflavone-7-O-𝛽-acetylglucopyranoside (3). Yellow solid from EtOAc fraction; m.p. 241–243 °C; [𝛼]25D = 0° (c = 0.067, MeOH); UV (MeOH) – 𝜆max nm (log ℰ): 229 (3.91), 292 (3.80), 324 (3.73); IR (KBr): 𝜈max = 3418, 2927, 1728, 1645, 1604, 1510, 1451, 1370, 1243, 1172, 1082, 834, 742, 622, 526 cm–1; HRFESIMS m/z 745.1781 [M+H]+ (calcd. for C38H33O16: 745.1769). For 1H and 13C NMR data, see Tables 3 & 4. 19 4.3.4. (2S,3S)-morelloflavone-7-O-𝛽-acetylglucopyranoside (4). Yellow solid from EtOAc; m.p. 245–247 °C; [𝛼]20D = 0° (c = 0.046, MeOH); UV (MeOH) – 𝜆max nm (log ℰ): 222 (4.31), 230 (4.39), 287 (4.36), 291 (4.36), 344 (4.18); IR (KBr): 𝜈max = 3384, 1726, 1643, 1603, 1515, 1452, 1369, 1262, 1169, 1082, 834, 741, 630, 559, 526 cm–1; HRESIMS m/z 761.1732 [M+H]+ (calcd. for C38H33O17: 761.1718). For 1H and 13C NMR data, see Tables 3 and 4. 4.3.5. Conversion of (1) to (16) Compound 1 (10 mg) was dissolved in trifluoroacetic acid (2 mL) and was heated under reflux for 30 min. The resulting mixture was separated and purified by prep. HPLC with 𝑛Hex/EtOAc (7/3, v/v) to afford compound 16 (7.4 mg, 74%), at a retention time of 22 min. Compound 16 was obtained as a pale yellow oil and identified as 1,5-dihydroxy-1,2,3,4-bis(2,2dimethyldihydropyrano)xanthone. HRESIMS [M+H]+ at m/z 381.1702 (calcd. for C23H25O5: 381.1702). For 1H and 13C NMR data, see Table 1. 4.3.6. Assays for antibacterial and antioxidant activities The antibacterial activity was evaluated using the broth microdilution method by determining the minimum inhibitory concentration (MIC) values against the five bacteria strains viz., Escherichia coli (ATCC 25922); Pseudomonas aeruginosa (ATCC 27853); Staphylococcus aureus (ATCC BAA1026); Enterococcus faecalis (ATCC 29212); Proteus mirabilis (isolate). The antioxidant capacity of the compounds was evaluated based on the principle of scavenging the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical. Acknowledgements BYGM is grateful to The World Academy of Sciences (TWAS) for an eight-month fellowship under the auspices of the ICCBS-TWAS programme to work at the H.E.J. Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan. This study was also supported by the Alexander von Humboldt Foundation and the German Academic Exchange Service (DAAD) through the equipment subsidies to SFK. 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