TWO NEW METABOLITES; 7β-OH-UZARIGENIN AND 7β-OH-ODOROSIDE-H; THROUGH BIOTRANSFORMATION OF UZARIGENIN AND ODOROSIDE-H

TWO NEW METABOLITES; 7β-OH-UZARIGENIN AND 7β-OH-ODOROSIDE-H; THROUGH BIOTRANSFORMATION OF UZARIGENIN AND ODOROSIDE-H

Ahmed A. Zaki ^1*, Nadia E. Hashish ^{1, 2}, Mohamed A. Amer ¹ and M.-F. Lahloub¹

Pharmacognosy Department, Faculty of Pharmacy ¹, Mansoura University, Mansoura 35516, Egypt

Faculty of Pharmacy ², Delta University for Science and Technology. Daqahlia, Egypt

ABSTRACT: Two new cardenolide metabolites, 7β-OH-uzarigenin (1M) and 7β-OH-odoroside-H (2M) were obtained through the biotransformation of uzarigenin (1) and odoroside-H (2). Compounds 1 and 2 were isolated from the air-dried roots of Nerium oleander L. The plant, belonging to family Apocynaceae, was collected in October, 2010 from Mansoura university garden in Egypt. The methylene chloride extract of the N. Oleander roots was chromatographed using silica gel column, gradually eluted by petroleum ether: Ethyl acetate with increasing proportions of EtOAc. Further purification by repeated crystallization from hot methanol afforded both compounds. The biological transformation was acheived by Cunninghamella echinulata NRRL 1382 with different incubation periods. The structures of the isolated compounds and the newly assigned metabolites were identified using extensive studies; (¹H, ¹³C, HSQC, HMQC, ¹H-¹H COSY and ¹H-¹³C HMBC), mass spectral analysis (EI and ESI /MS) and IR.

Uzarigenin, Odoroside-H, Cunninghamella, Nerium oleander

INTRODUCTION: Nerium oleander L. (Apocynaceae) plant is widely distributed through the Mediterranean region and sub-tropical Asia and is indigenous to the Indo-Pakistan subcontinent. The plant is widely cultivated as ornamental plant/shrub in many countries including Egypt. N. oleander is poisonous to human, animal and certain insects mainly due to the presence of cardio-tonic compounds; namely cardenolides. Different parts of the plant were studied chemically but to a lesser extent its roots. Some of the previously isolated cardenolides such as digioxin and digitoxigenin were subjected to biotransformation by microorganisms and/ or plant cell suspensions.^1-6

Uzarigenin (1) and odoroside-H (2) were isolated during the present study from the methylene chloride extract of N. oleander roots. Reviewing the literature revealed no reports discusses the biotransformation of both compounds, hence they were subjected to microbial metabolism followed by identification of the chemical structures of the resulted metabolites by analyzing their spectroscopic data including 1D, 2D NMR as well as high resolution mass spectrometry.

MATERIALS AND METHODS:

General procedures

Melting points were measured with Fisher-Johns Scientific melting point apparatus and uncorrected. IR spectra were recorded on Mattson 5000 FT-IR spectrophotometer in KBr pellets. 1D and 2D NMR spectra were run on Jeol spectrophotometers (400 MHz for 1H-NMR and 100 MHz for 13C-NMR) CD₃OD with TMS as an internal standard. The Chemical shift values (δ) were given in parts per million (ppm), and the coupling constants were in hertz (Hz). HRESI Mass spectra were performed on Bruker MicrOTOF Mass Spectrometer. All the solvents used for extraction and isolation were of analytical grade. TLC was performed on silica gel G. Separation and purification were carried out by column chromatography on silica gel (200–300 mesh). Compounds were detected by spraying with vanillin/ H₂SO₄ and heating at 105 ^oC for 2 min.

Isolation of compounds 1 and 2

Nerium oleander roots were air dried at ambient temperature and grinded just prior to being used for the present investigation. A voucher specimen has been deposited at the department of pharmacognosy department, University of Mansoura (NO-023). The grinded roots (13.0 kg) were extracted exhaustively by maceration with methanol (15 L X 5). Extract was collected by filteration and the solvent was evaporated. The resulted dry extract was suspended in hydro-alcohol, and then partitioned with petroleum ether, methylene chloride and EtOAc, respectively, to afford dried petroleum ether- (123.0 g), methylene chloride- (100.0 g) and EtOAc-soluble (100.0 g) residues.

Methylene chloride extract  was applied on to the top of a silica gel column (500 g, 60 x 5 cm) previously packed in petroleum ether. Gradient elution was performed using pet. ether: EtOAc with increasing proportions of EtOAc. The effluent was collected in 250 ml fractions. Each fraction was monitored by TLC using EtOAc in pet. ether (different proportions) as a developing system and vanillin / H₂SO₄ acid as a spray reagent. Fraction A eluted with pet. ether: EtOAc (6.5: 3.5) revealed the presence of one major spot with R_f  0.61 and two minor spots with R_f  0.38 and 0.23; using pet. ether: EtOAc;  4 : 6  as developing system. On evaporation of the solvent, white amorphous powder of compound 1 was deposited.

It was further purified by repeated crystallization from hot methanol to afford 1 (500 mg), on TLC it turns turquoise after spraying with vanillin / H₂SO₄ and heating. Fractions B eluted with pet. ether: EtOAc (4: 6). TLC of collected fractions revealed the presence of one major brown spot with R_f 0.48, after spraying with vanillin/ sulfuric and heating, using EtOAc 100% as developing system. The residue after drying of combined fractions washed several times with ethyl acetate to afford yellow powder. When the yellow powder was allowed to crystallize from methanol it gave yellow rosette, which was designated as compound 2 (1000 mg).

Biotransformation of compound 1 and 2

Medium:All biotransformation experiments were performed in a medium consists of: 2% glucose, 0.5% yeast extract, 0.5 % peptone, 0.5% NaCl and 0.5% K₂HPO₄. The PH of the medium was adjusted to 7.0 using 6 N HCl before autoclaving for 20 min at 121 ^oC and 15 psi.

 Screening:Forty microorganisms were grown according to the standard two-stage fermentation protocol ⁷. Stage I culture was started by suspending spores and mycelia from filamentous fungal culture slants in 2 ml sterile medium and transferring the suspension to 25 ml medium contained in 125 ml flasks fifth of their volume of the prepared medium. After incubation for 72 hours, stage II cultures (two sets) were initiated by transferring 2 ml of stage I cultures to 125 ml-flasks containing 25 ml medium. After 24 hours, compounds 1 and 2 (0.2 mg/ml medium in 1:1 dimethylformamide (DMF) – methanol (MeOH)) were added to the flasks. Culture controls consisted of fermentation blanks in which the organisms were grown under identical conditions but without substrate addition. Substrate controls were composed of a sterile medium to which the substrate was added and incubated without microorganisms. The screening revealed that Cunninghamella echinulata NRRL 1382 is able to metabolize inoculated compounds.

 Scaling up and isolation of metabolites

Compounds 1 and 2 (0.4 g each) was evenly distributed among to two sets of Cunninghamella echinulata NRRL 1382 culture; each set consists of twenty five flasks 250 ml, each containing 50 ml medium of 24 hours-old C. echinulata stage II cultures. The cultures were incubated for two days in case of 1 and for five days in case of 2 (200 rpm, 27 ^oC) and extracted with EtOAc (1L x 3). The combined extracts were dried and evaporated to yield 340 mg (for 2) and 300 mg (for 1) viscous residues. EtOAc extracts were separately column chromatographed on silica gel (60 x 1.2 cm) and gradiently eluted using 60 – 80 % EtOAc in pet. ether. These columns afforded compound 1 metabolite (1M), 11 mg with R^f 0.52 using   EtOAc - Pet. ether (70 %). Compound 2 metabolite (2M), 8 mg with R^f 0.22 using   EtOAc - Pet. ether (80 %). Both metabolites gave turquoise in color after sparying with vanillin/ H₂SO₄ and heating for 2 min.

FIGURE 1. CHEMICAL STRUCTURES OF 1 (UZARIGENIN), 2 (ODOROSIDE-H) AND THEIR BIOTRANSFORMATION PRODUCTS BY C. ECHINULATA NRRL 1382; 1M (7β-HYDROXY-UZARGENIN) AND 2M (7β-HYDROXY-ODOROSIDEH)

RESULTS AND DISCUSSION:

Compounds 1 and 2 were isolated from the methylene chloride extract of Nerium oleander roots by column chromatographic methods. Their structures were identified as uzarigenin and odoroside-H (figure 1), respectively, on the basis of their IR, 1D NMR, 2D NMR and mass spectroscopic data ^8-10.

Compound 1 isolated as colorless needles, its molecular formula is C₂₃H₃₄O₄ deduced from EI-MS peaks; m/z 375 [M+H], m/z 356 [M-H₂O], m/z 338 [M-2H₂O] and mass fragment at m/z 85 that supported the butenolide side chain. APT experiment spectrum (Table 1) revealed 23 carbon signals, one carbonyl carbon at δ 175.8, one

quaternary olefinic carbon at δ 176.5, one olefinic methine at δ 116.8 and one oxygenated methylene carbon at δ 73.8 were characteristic for five-membered lactone ring of cardenolides. One carbinylic at δ 84.8, one oxygenated methine at δ 70.4 and two angular methyl at δ 11.8 and 15.4 were characteristic for the steroid nucleus with trans fusion of ring A and B which resulted in α-orientation of H-5¹¹. ¹H-NMR spectrum showed two double doublets of one proton each at δ 4.6 and 4.7 (J = 18.3, 3.6 Hz), and one proton broad singlet at δ 5.6, characteristic for α,β- unsaturated γ-lactone of cardenolides ^{12, 13}.

They were assigned for H-21a, H-21b and H-22, respectively. HMQC spectrum connected them to carbons at δ 73.8 (C-21) and 116.8 (C-22). Two doublets at δ 0.55 and 0.62 (J = 3.2)  three proton each were attributed to H-18 and H-19, respectively, linked to carbons at 11.8 and 15.4 ppm, respectively, in HMQC spectrum, and their upfield shift indicated this aglycone part is included in H-5α series ¹³. One proton double doublet at δ 2.6 (J = 9.2, 5.5) was assigned for C-17 (δ_C 50.8) and this confirmed the β-orientation of butenolide side.  One proton multiplet at δ 3.3 was assigned for normally oxygenated methine during biosynthesis (C-3, δ_C 70.4), chemical shift of C-3 and H-3 chemical shift and multiplicity indicated the configuration of hydroxyl group as β ¹⁴. It was concluded that compound 1 is uzarigenin (14β-hydroxy-5-α-card-20(22)-enolide).

Compound 2 isolated as yellow rosettes. EI-MS showed peak [M⁺] at m/z 534 which coincident with the molecular formula C₃₀H₄₆O₈ and peak at m/z 161 and 178 indicated the sugar composition as C₇H₁₃O₄. APT experiment exhibited (Table 1), one carbinylic at δ 85.2 and two methyl at δ 16.3 and 23.5 were characteristic for the steroid nucleus with Cis fusion of ring A and B which resulted in β-orientation of H-5 ¹⁵. In addition to presence of six oxygenated methine groups at δ 101.4, 83, 74.2, 70.3, 70.1 and 67.8; one of them is anomeric carbon of 6-deoxy sugar due to presence of additional methyl group at 15.7 ppm. The sugar is attached to C-3 of aglycone on the basis of the downfield chemical shift of C-3 (δ_C 74.2) as well as biogenetic consideration that steroids are normally oxygenated at C-3 during biosynthesis. Its axial orientation was decided due to the broad singlet at δ 3.99 attributable for equatorial carbinylic H-3 ¹⁴.  One proton doublet at δ 4.2 (J= 7.8 Hz) was assigned for anomeric proton for anomeric carbon 101.4 ppm. The chemical shift and coupling constant of anomeric proton suggested β-linkage of the sugar. Proton signals at δ 3.55 (m), 3.2 (b, dd, J=9.6, 3.2), 3.8 (br d, J= 3.2 Hz), 3.4 (m), and 1.28 (d, J= 5.96 Hz) were assigned for C-2’(δ_c 70.3), C-3’(δ_c 83), C-4’(δ_c 67.8), C-5’ (δ_c 70.1) and C-6` (δ_c 15.7), respectively. In addition to presence of singlet at 3.5 ppm, was assigned for OCH₃ (δ_c 57.2). From the chemical shifts of sugar carbons and proton, we concluded the sugar is β-D-digitalose ^16-18.It was concluded that compound 2 is odoroside-H (3β-O-(β-D-digitalosyl)-14β-hydroxy-5-β-card-20(22)-enolide).

Compounds 1 and 2 were successfully metabolized by Cunninghamella echinulata NRRL 1382. The substrate was added into a 2-day-old cultivation of the microorganisms and one product was resulted from each compound. The products were isolated by chromatographic methods after an additional incubation period of two days in case of 1 and five days in case of 2. Their structures were identified as: 7β-OH-uzarigenin (1M) and 7β-OH-odoroside-H (2M) (Figure 1), respectively, on the basis of their IR, 1D NMR, 2D NMR and mass spectroscopic data.

Compound 1M was isolated as white needles, its positive mode HR ESI/MS showed  ion peak [M+Na]⁺ at m/z 413.22890 consistent with the molecular formula C₂₃H₃₄O₅ Na; ion peak [2M+Na] ⁺ at m/z 803.46793 and ion peak [3M+Na] ⁺  at m/z  1193.70429. Negative mode of HR ESI/MS revealed ion peak [M-H]^- at m/z 389.23428; ion peak[M+HCOO]^- at m/z 435.23946 and peak [2M-H]^- at m/z 779.46931. ¹³C-NMR spectrum showed one additional methine signal at δ 75.4 accompanied by the disappearance of carbon signal with chemical shift typical of C-7 (δ_C 27.2). Besides, downfield shift of C-6 from 28.4 to 37.9 ppm and of C-8 from δ 41.0 to 47.1. ¹H-¹H COSY spectrum revealed the correlation between H-7 (3.7 ppm, J = 4.9, 10.6) and proton signal at δ 1.66 (d, J = 10.6, C-8, δ_C 47.1). Correlation was proved between H-7 and proton signal at 1.51 (C-6, δ_C 37.9 from HSQC). Coupling constants of H-7 (J_{7α, 6α}= 4.9 and J_{7α, 6β}= 10.6) and coupling constant of H-8 (J_{7α, 8β}= 10.6) confirm the position of 7β-OH. Compound 1M was concluded to be the new compound 3β, 7β, 14β-trihydroxy-5-α-card-20(22)-enolide.

Compound 2M was isolated as yellow rosettes, its positive mode HR ESI/MS showed  ion peak [M+H]⁺ at m/z 551.35087 consistent with the molecular formula C₃₀H₄₆O₉; ion peak [2M+Na] ⁺ at m/z 1123.60610; ion peak [M-sugar+Na] ⁺ at m/z 413.26328 and ion peak [3M+Na] ⁺  at m/z  1674.91577. Negative mode HR ESI/MS showed ion peak [M-H] at m/z 549.31173; ion peak [M+HCOO] at m/z 595.31797 and peak [2M+HCOO] at m/z 1145.62234. ¹³C-NMR (Table 1) showed one additional methine signal at 71.2 ppm accompanied by the disappearance of carbon signal with chemical shift typical of C-7 (δ_C 21.3). Besides, downfield shift of C-6 from 26.9 to 37.8 ppm and of C-8 from δ 41.5 to 47.4. All of these conclusions suggested the hydroxylation of C-7, because other assumptions like 12β-OH with chemical shift about 75 ppm would cause C-13 to appear at about 55 ppm, and this does not occur. ¹H-¹H COSY spectrum revealed the correlation between H-7 (3.88 ppm, m) and proton signal at δ 1.7 (C-8, δ_C 47.4). Correlation between H-7 and proton signal at δ 1.51 (C-6, δ_C 37.8). Coupling constants of H-17 (J_{17α, 16α}= 5.2 and J_{17α, 16β}= 9.44) confirming the 17α- orientation. Compound 2M was concluded to be 3β-O-(β-D-digitalosyl)-7β, 14β-dihydroxy-5-β-card-20(22)-enolide. To the best of our knowledge this is a new compound.

 Product 1M (7β-Hydroxy-uzarigenin), white needles; mp 256–260 ^oC; IR (KBr) υ_max 3495, 2990, 2857, 1796, 1740, 1620 cm^-1; PI-HR ESI/MS m/z 413.22890 [M+Na]⁺, 803.46793 [2M+Na]⁺,1193.70429 [3M+Na]⁺; NI-HR ESI/MS m/z 389.23428 [M-H], 435.23946 [M+HCOO], 779.46931 [2M-H]. ¹H-NMR (CD3OD , 400 MHz): 1.51 (m, H-1), 3.8 (m, 3.51,  J = 3.8, H-3), 3.7 (dt , J = 4.9, 10.6, H-7), 1.66 (d , J = 10.6, H-8), 1.45 (H-12), 1.88 (H-15), 2.1 (H-16), 2.8 (dd, J = 8.96, 4.76, H-17), 0.90(s, H-18), 0.86 ( s, H-19),  4.9 (dd, J = 18.4, 1.32, H-21a), 5.1(dd, J = 18.4, 1.32, H-21b), 5.9 (s, H-22). ¹³C NMR (CD3OD, 100 MHz) spectral data are summarized in Table 1.

 Product 2M (7β-Hydroxy-odoroside-H), yellow rosettes; mp 241–246 ^oC; IR (KBr) υ_max3475, 2933, 2860, 1791, 1736, 1626 cm^-1; PI-HR ESI/MS m/z 551.35087 [M+H]⁺, 1123.60610 [2M+Na]⁺, 413.26328 [M-sugar+Na]⁺; 1674.91577 [3M+Na]⁺; NI- HR ESI/MS m/z 549.31173 [M-H]^-, 595.31797 [M+HCOO]^-, 1145.62234 [2M+HCOO]^-. ¹H-NMR (CD3OD , 400 MHz): 1.7 (m, H-1), 2.16, 1.95 (H-2), 3.98 (br s, H-3), 2.23, 1..85 (H-4), 1.48 (H-5), 1.85, 1.45 (H-6), 3.88 (m, H-7), 1.7 (H-8), 1.41, 1.43 (H-11), 1.7 (H-12), 1.37 (H-15), 1.56, 1.62 (H-16), 2.8 (dd, J = 9.44, 5.2, H-17), 0.90(s, H-18), 0.98 (s, H-19),  4.9 (dd, J = 18.4, 1.44, H-21a),  5.1 (dd , J = 18.4, 1.44, H-21b), 5.9 (s, H-22), 4.23(d,  J = 7.8, H-1`), 3.52 (m, H-2`), 3.2 (br dd, J =9.6, 3.2, H-3`), 3.8 (br d, J =3.2, H-4`), 3.52(m, H-5`), 1.26 (d,  J = 6.48, H-6`), 3.45 (s, OCH₃). ¹³C NMR (CD3OD, 100 MHz) spectral data are summarized in Table 1.

TABLE 1: NMR SPECTROSCOPIC (δ) DATA OF ISOLATED COMPOUNDS 1, 2, 1M AND 2M (δ VALUES IN ppm). SPECTRA WERE RECORDED IN CD₃OD, 400 MHz

CONCLUSION: In conclusion, for the first time, compounds uzarigenin and odoroside-H (1 &2) were isolated during this study from Nerium oleander roots. The biotransformation of both compounds by Cunninghamella echinulata NRRL 1382, resulted in the formation of two new metabolites; 7β-OH derivatives of both.

ACKNOWLEDGEMENT: Authors acknowledge Dr. Shinnosuke Uno, Laboratory of Chemical Biology and Molecular Imaging, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Japan, for carrying out NMR spectra of compounds.

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    How to cite this article:

    Zaki AA, Hashish NE, Md. Amer A and Lahloub MF: Two New Metabolites; 7β-OH-uzarigenin and 7β-OH-odoroside-H; through Biotransformation of Uzarigenin and Odoroside-H.Int J Pharm Sci Res2014; 5(11): 4987-92.doi: 10.13040/IJPSR.0975-8232.5 (11).4987-92.

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