Microbial metabolism of cannflavin A (1) and B (2), two biologically active flavonoids isolated from Cannabis sativa L., produced five metabolites (3–7). Incubation of 1 and 2 with Mucor ramannianus (ATCC 9628) and Beauveria bassiana (ATCC 13144), respectively, yielded 6″S,7″-dihydroxycannflavin A (3), 6″S,7″-dihydroxycannflavin A 7-sulfate (4) and 6″S,7″-dihydroxycannflavin A 4′-O-α-L-rhamnopyranoside (5), and cannflavin B 7-O-β-D-4‴-O-methylglucopyranoside (6) and cannflavin B 7-sulfate (7), respectively. All compounds were evaluated for antimicrobial and antiprotozoal activity.
Cannabis sativa L. (Family: Cannabaceae), a plant originating in Central Asia, is cultivated worldwide as a source of fiber, energy, food and medicinal or narcotic preparations (Flores-Sanchez and Verpoorte, 2008). Recently, a number of new cannabis constituents were identified, increasing the total from 489 in 2005 (ElSohly and Slade, 2005) to 537 in 2009 (Ahmed et al., 2008a,b; Appendino et al., 2008; Radwan et al., 2009, 2008a,b), while the number of cannabinoids increased from 70 to 109. The cannabinoids are the most studied cannabis constituents, in particular Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive component (Galal et al., 2009); however, research has shown that some of the other cannabinoids also exhibit pharmacological activities, e.g., the nonpsychotropic cannabinoid cannabidiol (CBD) displays antihyperalgesic, antipsychotic, anticonvulsant, neuroprotective and antiemetic properties (Galal et al., 2009). Other classes of compounds reported for cannabis include terpenes, sugars, hydrocarbons, steroids, flavonoids, nitrogenous compounds, noncannabinoid phenols and amino acids (ElSohly and Slade, 2005).
More than 4000 flavonoids have been identified and numerous beneficial health effects have been reported, e.g., antiinflammatory (Gomes et al., 2008), antiviral (Naithani et al., 2008), anticancer (Mojzisova and Mojzis, 2008), cardioprotective (Mojzisova and Mojzis, 2008), antioxidant (Ibrahim et al., 2008), antiprotozoal (Fotie, 2008) and antimicrobial (Heinonen, 2007) activities.
Twenty-six flavonoids have been isolated from cannabis (ElSohly and Slade, 2005; Flores-Sanchez and Verpoorte, 2008; Radwan et al., 2008a), representing seven chemical structures (vitexin, isovitexin, apigenin, luteolin, kaempferol, orientin and quercetin) with different glycosylation, prenylation, geranylation and methylation patterns. Cannflavin A and B, two methylated isoprenoid flavones, represent the first aglycone flavonoids uniquely isolated from cannabis. The antileishmanial activity for cannflavin A and B was reported as strong (IC50 10.3 μM) (Radwan et al., 2008a) and moderate (IC50 13.6 μM) (Radwan et al., 2008b), respectively.
Microorganisms are used as predictive models for mammalian drug metabolism to establish the metabolic fate of biologically active compounds, while providing sufficient amounts of metabolite for structure elucidation and pharmacological evaluation. These models are also employed to obtain more active or less toxic substances and selective derivatives (Venisetty and Ciddi, 2003).
We herein report the microbial metabolism of 1 and 2 via a panel of 41 microorganisms, as well as the isolation, structure elucidation and activity (antimicrobial and antiprotozoal) of the resultant metabolites.
2. Results and discussion
Initial screening of 1 and 2 was carried out using a standard two-stage procedure (Ibrahim et al., 2008), with three and five of the 41 microorganisms showing the ability to transform 1 (three polar metabolites) and 2 (two polar metabolites), respectively. TLC analysis indicated that Mucor ramannianus (ATCC 9628) and Beauveria bassiana (ATCC 13144) had the highest transformational efficiencies for 1 and 2, respectively, and was thus chosen for scaleup fermentation. Preparative scale fermentation of 1 with M. ramannianus yielded three new metabolites [6″S,7″-dihydroxycannflavin A (3), 6″S,7″-dihydroxycannflavin A 7-sulfate (4) and 6″S,7″-dihydroxycannflavin A 4′-O-α-L-rhamnopyranoside (5)], while preparative scale fermentation of 2 with B. bassiana yielded two new metabolites [cannflavin B 7-O-β-D-4‴-O-methylglucopyranoside (6) and cannflavin B 7-sulfate (7)].
Metabolite 3 was isolated as a light yellow powder with molecular formula C26H30O8 based on HRESIMS and 13C NMR spectroscopic data (12° of unsaturation). The UV λmax at 275 and 350 nm indicated a flavonoid structure (Avula et al., 2009). The 13C and DEPT-135 NMR spectroscopic data displayed an sp3 oxymethine (δC 72.3) and an sp3 geminal dimethyl oxygenated quaternary carbon (δC 77.7), in addition to eight oxygenated carbon resonances similar to those found in 1 (Choi et al., 2004). A comparison of the 1H (Table 1) and 13C (Table 2) NMR data of 1 and 3 indicated almost identical resonances for both compounds, except for the replacement of the C-6″/C-7″ olefinic resonances in 1 [δH 5.04 (t, J = 7.0, H-6″); δC 125.2 (C-6″), 131.6 (C-7″)] by oxymethine [δH 3.80 (m); δC 77.7 (C-6″)] and oxygenated quaternary carbon [δC72.3 (C-7″)] resonances in 3. A series of HMBC (H-6″/C-4″, C-8″, C-9″; C-6″/H2-4″, H3-8″, H3-9″; C-7″/H3-8″, H3-9″; H2-5″/C-9″) and COSY (H-6″/H2-5″/H2-4″) correlations confirmed the assignment of hydroxy groups at C-6″ and C-7″, establishing 3 as 6″,7″-dihydroxycannflavin A.
Metabolite 4, a yellow amorphous powder, was shown to have a molecular formula C26H30O11S (HRESIMS). The IR spectroscopy displayed characteristic sulfate bands at νmax 1050 (C–O–S) and 1250 (S=O) cm−1 (Ibrahim, 2005). The 1H (Table 1) and 13C (Table 2) NMR chemical shifts of 4 and 1 were similar, with significant differences being the downfield shift of H-8, C-6, C-8 and C-10 (0.55, 2.5, 4.0 and 0.6 ppm, respectively) and the upfield shift of C-7 (1.9 ppm), supporting the sulfation of the C-7 hydroxy (Barron and Ibrahim, 1987). The C-6 geranyl group in 4 was 6″,7″-dihydroxylated as in 3 based on HMBC (H-6″/C-4″, C-8″, C-9″; C-6″/H3-8″; C-7″/H3-8″, H3-9″) and COSY (H-6″/H2-5″/H2-4″) correlations and the absence of C-6″/C-7″ olefinic NMR resonances, establishing 4 as 6″,7″-dihydroxycannflavin A 7-sulfate.
Metabolite 5 (HRESIMS: C32H40O12), isolated as a light yellow powder, displayed similar 1H (Table 1) and 13C (Table 2) NMR resonances to 3 and 4, with additional resonances indicating a pyranose moiety [1H NMR: δH 5.50 (1H, br s, H-1‴), 1.04 (3H, d, J = 6.0, H3-6‴); 13C NMR: δC98.4 (C-1‴), 16.1 (C-6‴)]. The pyranoside location was determined at C-4′ via HMBC correlation of the anomeric proton and C-4′ (δC 149.0), while its identity was determined through GC–MS analysis. The acetylated thiazolidine derivative of the hydrolysis product of 5 and of authentic L-rhamnose displayed the same GC–MS retention time (15.64 min) (Shukla et al., 2009). The chemical shift of the anomeric carbon [δC 98.4 (C-1‴)] suggested an α-O-glycosidic linkage (Herath et al., 2008; Ibrahim et al., 2008), establishing 5 as 6″,7″-dihydroxycannflavin A 4′-O-α-L-rhamnopyranoside.
The absolute configuration at C-6″ was determined through application of the modified Mosher’s method (Δδ = δS − δR) for 5 (Ahmed et al., 2008a; Murata et al., 2008; Nakamura et al., 2009; Zhang et al., 2009). The 1H NMR resonances for the H3-8″ and H3-9″ protons of the S-MTPA ester (5a) were observed downfield compared to those of the R-MTPA ester (5b) [Δδ(H3-8″) = +0.02 and Δδ(H3-9″) = +0.03], indicating S absolute configuration at C-6″. The absolute configurations at C-6″ for 3 and 4 were subsequently determined as S by comparison of their NMR data with that of 5 (Tables 1 and and22).
Metabolite 6, a yellow amorphous solid with molecular formula C28H32O11 (HRESIMS), displayed similar 1H (Table 1) and 13C (Table 2) NMR resonances to 2 (Choi et al., 2004), with additional resonances indicating a 4‴-O-methylglucopyranoside unit [δH 5.04 (1H, d, J = 7.6, H-1‴), 3.54 (3H, s, 4‴-OCH3)]. NMR spectroscopic data (13C, DEPT-135 and HMQC) confirmed the presence of the methylated glycoside moiety via an anomeric (δC 100.1), two hydroxymethine (δC73.6, 76.4), one methoxymethine (δC 79.0), one oxygenated methine (δC 76.4), one hydroxymethylene (δC 60.2) and one O-methyl (δC 59.5) carbon resonance, in addition to the resonances for the cannflavin B structure. The anomeric proton coupling constant (J = 7.6 Hz) indicated β-O-glycosidic linkage, while HMBC correlation (H-1‴/C-7) revealed the location at C-7 (Herath et al., 2008, 2006; Ibrahim et al., 2008). Consequently, 6 was identified as cannflavin B 7-O-β-D-4‴-O-methylglucopyranoside. B. bassiana has previously been shown to produce 4-O-methyl-D-glucopyranoside-phenol adducts (Kikuchi et al., 2004; Zhan and Gunatilaka, 2005, 2008).
Metabolite 7, a yellow solid, was shown to have a molecular formula C21H20O9S (HRESIMS), while IR spectroscopy indicated characteristic sulfate bands at νmax 1050 (C–O–S) and 1250 (S=O) cm−1 (Ibrahim, 2005). The 1H (Table 1) and 13C (Table 2) NMR chemical shifts of 7 and 2were similar, with significant differences in the downfield shift of H-8, C-6, C-8 and C-10 (0.57, 2.2, 3.8 and 0.4 ppm, respectively) and the upfield shift of C-7 (2.9 ppm), supporting the sulfation of the C-7 hydroxy (Barron and Ibrahim, 1987). HMBC correlations (H-8/C-6, C-7) confirmed the sulfation position and established 7 as cannflavin B 7-sulfate.
3. Concluding remarks
In conclusion, in the present study, three metabolites of cannflavin A (1), produced by M. ramannianus, and two metabolites of cannflavin B (2), produced by B. bassiana, were isolated, with B. bassiana producing higher transformation yields than M. ramannianus. The enzymatic reactions included hydroxylation, O-glycosylation and sulfate conjugation, as well as combinations of these transformations. O-Glycosylation occurred at the C-4′ and C-7 hydroxy groups of 1 and 2, respectively, while sulfate conjugation occurred exclusively at the C-7 hydroxy. This study demonstrates the use of microbial transformation techniques to prepare new derivatives of flavonoids.
4.1. General experimental procedures
NMR spectra were recorded in DMSO-d6 on Bruker Avance DPX-500 (1H: 500 MHz; 13C: 125 MHz) and Varian AS400 (1H: 400 MHz; 13C: 100 MHz) spectrometers. IR spectra were recorded on a Bruker Tensor 27 spectrophotometer. UV spectra were obtained on a Varian Cary 50 Bio UV–visible spectrophotometer. Optical rotations were measured at ambient temperature using a Rudolph Research Analytical Autopol IV automatic polarimeter. HRESIMS was obtained using a Bruker BioApex FTMS. GC–MS analysis was carried out on a HP 6890 series GC, equipped with a split/splitless capillary injector, a HP 6890 Series injector autosampler, and a DB-5 ms column (30 m × 0.25 mm × 0.25 μm, Agilent). The GC was interfaced to a HP 5973 quadrupole mass selective detector through a transfer line set at 280 °C. The injector temperature was 250 °C, and 1 μL injections were performed in the split (1:10) mode. Column flow was set at a constant pressure of 20 psi, giving an initial flow of 2.2 mL/min, using helium as carrier gas. The oven temperature was raised from 150 (hold 1 min) to 250 °C (hold 30 min) at a rate of 10 °C/min. The filament was operated at 70 eV, with an emission current of 35 μA. The multiplier voltage was automatically set to 2247 V. The ion source and quadrupole temperatures were 230 and 150 °C, respectively. The acquisition range was m/z 30–800 at 1.95 scans per second, starting 3.5 min after injection. TLC was carried out on aluminum-backed plates pre-coated with silica gel F254 (20 × 20 cm, 200 μm, 60 Å, Merck) and on glass-backed plates pre-coated with silica gel F254 (10 × 20 cm, 200 μm, 60 Å, Analtech). Visualization was accomplished by spraying with p-anisaldehyde [0.5 mL in glacial acetic acid (50 mL) and sulfuric acid (97%, 1 mL)] spray reagent followed by heating. Flash silica gel (40–63 μm, 60 Å, Silicycle) and Sephadex LH-20 (25–100 μm, lipophilic, Sigma–Aldrich) were used for column chromatography. High-performance flash chromatography (HPFC) was performed using a Horizon Biotage system (Biotage, Inc., Charlottesvillle, VA) with normal phase silica gel cartridges (KP-SIL, 10 g, 40–63 μm, 60 Å, Biotage).
Cannflavin A (1) and B (2) were isolated from C. sativa L. (Radwan et al., 2008a, 2008b) grown at the University of Mississippi. Whole buds of mature female plants were harvested, air-dried and stored in barrels at low temperature (−24 °C).
Microorganisms (41) housed at the National Center for Natural Products Research, University of Mississippi, were used in preliminary screening experiments to identify organisms capable of metabolizing 1 and 2.
4.4. Analytical scale fermentation
Medium-α, consisting of dextrose (20 g), NaCl (5 g), K2HPO4 (5 g), bacto-peptone (5 g) (Difco Labs, Detroit, MI) and yeast extract (5 g) (Difco Labs) in distilled H2O (1000 mL), was used in the twostage fermentation experiments. Initial screening was performed in Erlenmeyer flasks (125 mL) containing medium-α (25 mL). The media were autoclaved (20 min, 121 °C, 15 psi) before inoculation with each of the 41 microorganisms. The substrates were separately dissolved in dimethylformamide (DMF) (5 mg/500 μL) and added to 24 h old stage II cultures. Incubation (room temperature, 14 days, 100 rpm) was achieved on a rotary shaker (New Brunswick Model G10-21), while samples were monitored at 7 day intervals. Samples (5 mL) were extracted (5 mL, EtOAc/iso-PrOH, 8:2) and centrifuged (5 min, 3000 rpm), and the organic layers analyzed by TLC (CHCl3/MeOH solvent systems). Culture and substrate controls were simultaneously maintained with the above fermentation experiments (Herath et al., 2006, 2008; Ibrahim et al., 2008).
4.5. Preparative scale fermentation
Preparative scale fermentations of 1 and 2 with M. ramannianus (ATCC 9628) and B. bassiana(ATCC 13144), respectively, were performed (vide supra) in six flasks (2 L each) containing medium-α (500 mL each). Each flask was inoculated with the relevant microorganism and the substrate (1: 5 × 100 mg; 2: 1 × 100 mg), dissolved in DMF (10 mL), added. Incubation (room temperature, 14 days, 100 rpm) was followed by extraction (3 × 500 mL, EtOAc/iso-PrOH 8:2), evaporation of the solvent and dehydration over anhydrous Na2SO4 to provide the final extract (860 and 220 mg, respectively).
4.6. Isolation and purification of the metabolites
Sephadex LH-20 chromatography (MeOH) of the resulting extracts from 1 (860 mg) and 2 (220 mg) yielded 3 (5.2 mg) and 4 (6.5 mg), and 6 (31.0 mg) and 7 (7.0 mg), respectively, while 5 (10.0 mg) was subsequently purified through preparative thin layer chromatography (EtOAc/MeOH/H2O, 30:5:4).
4.6.1. 6″S,7″-Dihydroxycannflavin A (3)
Light yellow powder (5.2 mg, 1.04% yield); [α]25D −55 (c 0.2, MeOH); UV (MeOH) λmax (log ε): 275 (4.1), 350 (4.02), +NaOMe 270, 410, +NaOAc 270, 405, +AlCl3 280 nm; IR (neat) νmax: 3406, 1657, 1360, 1210, 1073 cm−1; for 1H and 13C NMR spectroscopic data, see Tables 1 and and2,2, respectively; HRESIMS m/z 469.1929 [M−H]−(calcd for C26H29O8, 469.1862).
4.6.2. 6″S,7″-Dihydroxycannflavin A 7-sulfate (4)
Yellow amorphous solid (6.5 mg, 1.3% yield); [α]25D −60 (c 0.2, MeOH); UV (MeOH) λmax (log ε): 275 (4.1), 340 (4.02), +NaOMe 270, 410, +NaOAc 270, 405, +AlCl3 280 nm; IR (neat) νmax: 3400, 1625, 1600, 1250 (S=O), 1050 (C–O–S) cm−1; for 1H and 13C NMR spectroscopic data, see Tables 1 and and2,2, respectively; HRESIMS m/z 551.1450 [M+H]+ (calcd for C26H31O11S, 551.1587).
4.6.3. 6″S,7″-Dihydroxycannflavin A 4′-O-α-L-rhamnopyranoside (5)
Light yellow powder (10.0 mg, 2.0% yield); [α]25D −122 (c 0.5, MeOH); UV (MeOH) λmax (log ε): 275 (4.01), 365 (4.04), +NaOMe 275, 430, +NaOAc 290, 335, +AlCl3 280, 360 nm; IR (neat) νmax: 3398, 1677, 1290, 1190, 1069 cm−1; for 1H and 13C NMR spectroscopic data, see Tables 1 and and2,2, respectively; HRESIMS m/z 639.2425 [M+Na]+ (calcd for C32H40NaO12, 639.2417).
4.6.4. Cannflavin B 7-O-β-D-4‴-O-methylglucopyranoside (6)
Yellow amorphous solid (31.0 mg, 31.0% yield); [α]25D −30 (c 0.5, MeOH); UV (MeOH) λmax (log ε): 275 (4.01), 340 (4.03), +NaOMe 264, 395, 405, +NaOAc 270, 345, 405, +AlCl3 280, 355 nm; IR (neat) νmax: 3398, 1677, 1290, 1190, 1069 cm−1; for 1H and 13C NMR spectroscopic data, see Tables 1 and and2,2, respectively; HRESIMS m/z 545.2133 [M+H]+ (calcd for C28H33O11, 545.2023).
4.6.5. Cannflavin B 7-sulfate (7)
Yellow solid (7.0 mg, 7.0% yield); UV (MeOH) λmax (log ε): 275 (4.01), 340 (4.02), +NaOMe 264, 395, 410, +NaOAc 270, 404, 445, +AlCl3 280, 355 nm; IR (neat) νmax: 3400, 1625, 1600, 1250 (S=O), 1050 (C–O–S) cm−1; for 1H and 13C NMR spectroscopic data, see Tables 1 and and2,2, respectively; HRESIMS m/z 471.0811 [M+Na]+ (calcd for C21H20NaO9S, 471.0726).
4.7. Acid hydrolysis and identification of the sugar units
Compounds 5 and 6 (2 mg) were refluxed separately with HCl (0.5 N, 3 mL, 2 h), followed by addition of water (10 mL) and extraction with CHCl3 (3 × 5 mL). The aqueous layers were dried to give the hexoses, which were identified through NMR spectroscopy, GC–MS analysis of their acetylated thiazolidine derivatives and through comparison of their specific rotation with authentic samples.
4.8. GC–MS analysis of the sugar units
The sugars obtained through acid hydrolysis of 5 and 6 were separately dissolved in pyridine (1 mL), added to L-cysteine methyl ester hydrochloride (0.1 M) in pyridine (2 mL), and the mixture heated (60 °C, 1 h). An equal volume of acetic anhydride (ca. 3 mL) was added and the heating continued (1 h). The acetylated thiazolidine sugar derivatives were subjected to GC–MS analysis and their retention times compared with acetylated thiazolidine derivatives of authentic sugars (Sigma–Aldrich): D-glucose, 21.32 min; L-glucose, 22.68 min; D-glactose, 22.12 min; L-rhamnose, 15.64 min; L-xylose, 16.90 min; L-arabinose, 16.11 min (Shukla et al., 2009).
4.9. Modified Mosher’s method
The Mosher esters were directly prepared in two NMR tubes by reacting 5 (2 mg) in pyridine-d5(100 μL) with (R)-(−)- (30 μL) and (S)-(+)-MTPA-Cl (30 μL), respectively, at room temperature (2 h) to afford the S– (5a) and R-MRPA (5b) esters, respectively. 1H NMR data were measured directly from the reaction mixtures (Ahmed et al., 2008a).
4.10. Biological activities
The isolated metabolites were evaluated for in vitro antifungal [Candida albicans (ATCC 90028), Candida glabrata (ATCC 90030), Candida krusei (ATCC 6258) and Aspergillus fumigat (ATCC 90906)], antibacterial [Escherichia coli (ATCC 35218), Pseudomonas aeruginosa (ATCC 27853), Mycobacterium intracellulare (ATCC 23068) and Staphylococcus aureus (ATCC 29213)], antileishmanial (culture of Leishmania donovani) and antimalarial [Plasmodium falciparum (D6 and W2 clones)] activity (Babu et al., 2006; Bharate et al., 2007; Radwan et al., 2008b).
The project described was supported in part by the National Institute on Drug Abuse, Contract No. N01DA-5-7746, and in part by the National Center for Research Resources, Grant No. 5P20RR021929-02. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the National Center for Research Resources. We are grateful to Dr. Bharathi Avula (University of Mississippi) for assistance with the HRESIMS, and to Dr. Melissa Jacob, Ms. Marsha Wright, Dr. Babu Tekwani and Dr. Shabana Khan (University of Mississippi) for conducting the antimicrobial and antiprotozoal testing.
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