Isolation, synthesis, and drug interaction potential of secondary metabolites derived from the leaves of miracle tree (Moringa oleifera) against CYP3A4 and CYP2D6 isozymes

Article abstract of DOI:10.1016/j.phymed.2019.153010

Background: Moringa oleifera Lam. is known as a drumstick tree that is widely cultivated in various subtropical and tropical provinces. Previous studies indicated that both aqueous and methanolic extracts of M. oleifera leaves have potent inhibitory effec

Full text of DOI:10.1016/j.phymed.2019.153010

Accepted Manuscript
Isolation, Synthesis, and Drug Interaction Potential of Secondary
Metabolites Derived from the Miracle Tree (Moringa oleifera) Leaves
against CYP3A4 and CYP2D6 Isozymes
Omer I. Fantoukh , Mohamed A. Albadry , Abidah Parveen ,
Mohammed F. Hawwal , Taghreed Majrashi , Zulfiqar Ali ,
Shabana I. Khan , Amar Chittiboyina , Ikhlas A. Khan
PII:
DOI:
Article Number:
Reference:
S0944-7113(19)30176-X
https://doi.org/10.1016/j.phymed.2019.153010
153010
PHYMED 153010
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Phytomedicine
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Revised date:
Accepted date:
24 April 2019
21 June 2019
1 July 2019
Please cite this article as: Omer I. Fantoukh , Mohamed A. Albadry , Abidah Parveen ,
Mohammed F. Hawwal , Taghreed Majrashi , Zulfiqar Ali , Shabana I. Khan , Amar Chittiboyina ,
Ikhlas A. Khan , Isolation, Synthesis, and Drug Interaction Potential of Secondary Metabolites De-
rived from the Miracle Tree (Moringa oleifera) Leaves against CYP3A4 and CYP2D6 Isozymes, Phy-
tomedicine (2019), doi: https://doi.org/10.1016/j.phymed.2019.153010
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ACCEPTED MANUSCRIPT
Isolation, Synthesis, and Drug Interaction Potential of Secondary Metabolites Derived
from the Miracle Tree (Moringa oleifera) Leaves against CYP3A4 and CYP2D6 Isozymes
Omer I. Fantoukha,b,c, Mohamed A. Albadrya, Abidah Parveena,b,d, Mohammed F. Hawwala,b,c, Taghreed
Majrashia,b, Zulfiqar Alia, Shabana I. Khana,b, Amar Chittiboyinaa,b, Ikhlas A. Khana,b,*
Affiliations:
aNational Center for Natural Products Research, University of Mississippi, University, MS 38677, USA
bDivision of Pharmacognosy, Department of BioMolecular Sciences, School of Pharmacy, University of
Mississippi, University, MS 38677, USA
cDepartment of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 4545, Saudi Arabia
dAbbottabad University of Science & Technology, Havelian, KPK, Pakistan
Correspondence:
*Dr. Ikhlas A. Khan, National Center for Natural Products Research, School of Pharmacy, The University of
Mississippi, University, MS 38677, USA E-mail: ikhan@olemiss.edu
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Abstract:
Background: Moringa oleifera Lam. is known as a drumstick tree that is widely cultivated in various
subtropical and tropical provinces. Previous studies indicated that both aqueous and methanolic extracts of M.
oleifera leaves have potent inhibitory effects on two major drug metabolizing Cytochrome P450 enzymes,
namely, CYP3A4 and CYP2D6.
Purpose: The current study was aimed to isolate the secondary metabolites from M. oleifera and investigated
their cytotoxicity and the inhibitory effects on CYP3A4 and CYP2D6 to assess their herb-drug interaction
(HDI) potential.
Methods: Chemical structure elucidation was achieved by interpreting the spectroscopic data (UV, IR, 1D, and
2D NMR experiments), confirming by HR-ESI-MS, and comparing with the previously reported data in the
literature. All the isolates were evaluated for their cytotoxicity against a panel of cell lines (SK-MEL, KB, BT-
549, SK-OV-3, VERO, LLC-PK1, and HepG2) and inhibition of two principal CYP isozymes (CYP3A4 and
CYP2D6).
Results: Phytochemical investigation of M. oleifera leaves resulted in the isolation and characterization of one
new compound, namely omoringone (1), along with twelve known secondary metabolites (2-13) belonging to
several chemical classes including flavonoids, terpenoids, lignans, and phenylalkanoids. A plausible
biosynthetic pathway for compound 1 was provided. Because of the low isolation yield and limited supply,
omoringone (1) and niazirin (12) were successively synthesized. No cytotoxicity was observed on any of the
tested cell lines up to 50 µM. The extract exhibited an inhibitory effect on CYP3A4 isoform (IC50 = 52.5 ± 2.5
µg/mL). Among the isolates, 1-4 and 7-9 inhibited CYP3A4 with the IC50 values ranging from 41.5 to 011 µM
with no remarkable effect on CYP2D6 isozyme.
Conclusion: This work aided in ascertaining components of M. oleifera contributing to CYP3A4 inhibition
exhibited by the extract using an in vitro assay. Nonetheless, further studies are warranted to determine the
bioavailability of the phytochemicals and extrapolate these findings in more physiologically relevant conditions
to further establish the clinical relevance of in vitro observations.
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Keywords:
Moringa olefiera; Moringaceae; rhamnopyranoside synthesis; niazirin; cytotoxicity; CYPs
Abbreviations used
M. olefiera: Moringa oleifera
CC: column chromatography
CYP450: cytochrome P450
MeOH: methanol
HDI: herb-drug interaction
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1 Introduction:
Moringa oleifera Lam. is widely cultivated in various subtropical and tropical areas, and it is known as a
drumstick or miracle tree that belongs to the monogeneric genus Moringa in Moringaceae family. M. oleifera is
a widespread species among 13 others in the genus (Saini et al., 2016). Since 2001, the number of international
congresses and scientific studies increased disseminating information about the incredible properties of this
species. Ayurvedic traditional medicine claims that M. oleifera can prevent 300 diseases, and it has been called
the miracle vegetable because all parts of Moringa such as pods, leaves, roots, and seeds have been exploited to
treat a variety of ailments. Various biological activities such as antioxidant, antihypertensive, antimicrobial,
antidiabetic, anticancer, anti-inflammatory along with immunomodulatory, cardioprotective, and
hepatoprotective effects have been associated with M. oleifera (Leone et al., 2015; Stohs & Hartman, 2015).
Several substrates and xenobiotics are metabolized principally by a superfamily called cytochrome P450
(CYP450). They are heme-containing monooxygenase enzymes responsible for the metabolism of the clinical
drugs and the conversion of the prodrug to the biologically active metabolites in the human body. The catalytic
activity of CYP isozymes can be altered by other medicines or herbs which behave as either CYP inhibitors or
inducers (Denisov et al., 2005). While the former can cause an accumulation of concomitant medicines leading
to acute toxicity profiles, the latter reduce the effectiveness of the concurrent drugs by affecting their
pharmacokinetic properties and eliminating them rapidly from the biological system. Despite the significance of
other CYP isoforms, CYP3A4 and CYP2D6 are accountable in the metabolism of 50% of clinical drugs
(Butterweck et al., 2004). Consequently, the safety and efficacy studies in terms of drug interaction potential
and cytotoxicity of the phytochemicals derived from M. oleifera need to be performed bearing in mind the
popularity and tremendous economic impact of this edible plant species.
Limited preceding studies displayed significant cytotoxicity properties for the extracts of leaves and barks
against breast and colorectal cancer cell lines (Al-Asmari et al., 2015; Jung, 2014; Stohs & Hartman, 2015), and
cytotoxicity against HepG2 cells was demonstrated only with the aqueous extract of M. oleifera leaves (IC50 = 6
mg/mL) (Monera et al., 2008). On the other hand, significant CYP3A4 inhibitory effects were observed, with
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IC50 values of 0.5 and 2.5 mg/mL for methanolic and aqueous extracts of the M. oleifera leaves, respectively
with less inhibitory activities were reported for the root extracts (IC50 > 10 mg/mL) (Monera et al., 2008).
Another study confirmed that the aqueous extract of M. oleifera leaves has a potent inhibition on CYP3A4,
concurring with the previous finding (Awortwe et al., 2014). Both ethanol and aqueous leaves extracts were
found to inhibit human CYP1A2, CYP2D6, CYP2E1, and CYP3A4 activities in vitro in a dose-dependent
manner with the potent inhibition exhibited against CYP1A2 isozyme (IC50 = 13.8 ± 9.8 μg/mL) (Taesotikul et
al., 2010). A fluorescence study revealed that both the extract and chlorogenic acid had some inhibition with
both isozymes CYP3A4 (IC50 = 127.36 ± 2.98 µg/mL and 133.73 ± 1.79 µg/mL) and CYP2D6 (146.50 ± 3.46
µg/mL and 159.22 ± 3.53 µg/mL) respectively, but very less in comparison to their respective positive
inhibitors ketoconazole and quinidine (Ahmmed et al., 2015). The potential of nine selected widely used
tropical medicinal herbs including M. oleifera in inhibiting human cytochrome P450 (CYP) isozymes was
investigated in human liver microsomes. In this study, a twelve probe metabolites of nine probe substrates were
monitored with UPLC/MS-MS, and M. oleifera leaves aqueous extract showed a weak inhibitory effect on
CYP1A2 and CYP2C9 with IC50 > 100 μg/mL (Showande et al., 2018). Owing to its immune booster effect,
nutraceuticals claimed to contain M. oleifera are administered concomitantly with anti-HIV medicines, and co-
administration of M. oleifera at the traditional dose did not alter the steady-state pharmacokinetic of nevirapine
(Monera-Penduka et al., 2017). However, M. oleifera altered the pharmacokinetics of antimalarial treatment,
amodiaquine, in healthy human volunteers showing a significant decrease in the Cmax after concurrent
administration (Olawoye et al., 2018). Similarly, pharmacokinetic studies conducted in Swiss albino mice
indicated that M. oleifera increased the plasma concentration of rifampicin when administered concurrently (Pal
et al., 2011).
However, most of these studies were limited to the solvent extracts level but not conducted on the chemical
constituents. Notably, further studies are vital to determine the phytochemical components of M. oleifera
responsible for the interactions with the metabolism of clinical drugs if consumed simultaneously. Therefore,
the current study aims mainly to isolate the secondary metabolites from M. oleifera and investigate the
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inhibitory effects of the extract and the isolates on two principal drug metabolizing enzymes (CYP3A4 and
CYP2D6) through an in vitro inhibition assay using recombinant CYPs along with the potential of cytotoxicity
effects on tumor and normal cell lines.
2 Materials and methods:
2.1 General experimental procedures:
1
The NMR spectra were obtained on Bruker Avance DRX spectrometer at 400 MHz for H experiments and 100
MHz for 13C experiments. Other 2D experiments (DEPTQ135, COSY, HSQC, HMBC, and NOESY) were
recorded using standard pulse instrument software. Dimethyl sulfoxide-d6 or methanol-d4 were used as the
internal standards and residual solvents to obtain and calibrate the spectrum, respectively. High-resolution
electrospray ionization mass spectra (HR-ESI-MS) were acquired by injection the samples directly to an Agilent
Technologies 6200 series mass spectrometer in the positive and negative ion modes. The specific rotation was
measured on AUTOPOL IV Automatic Polarimeter (Rudolph, Hackettstown, NJ, USA). Agilent Technologies
Cary 630 FTIR IR was used to record the IR spectra. The isolation and fractionation procedures were performed
using column chromatography on silica gel (32-63 μ, Dynamic adsorbents Inc.), Sephadex LH20 (Sigma-
Aldrich), or reversed-phase C-18 (Polar bond, J. T. Baker). The TLC was carried out on silica gel F254
aluminum sheet (20 × 20 cm, Fluka) or silica 60 RP-18 F254S aluminum sheet (20 × 20 cm, Merck). Savant
Speed Vac Plus SC210A Concentrator was employed to concentrate and dry the samples. Visualization of the
spots on TLC was done under UV light at 254 or 365 nm followed by spraying with vanillin-sulphuric acid
reagent (1% vanillin dissolved in 10% H2SO4/ethanol) and then heating via heat gun. The solvents used in the
isolation, fractionation, synthesis, and purification steps were purchased from Fisher Scientific (Fair Lawn, NJ).
2.2 Plant material:
The dried leaves of M. oleifera were obtained from Dr. Bello M. Oluwasesan, Nigeria in August 2016. A
specimen sample (NCNPR # 18332) was prepared and deposited by Dr. Vijayasankar Raman in the herbarium
of NCNPR, School of Pharmacy, the University of Mississippi after authentication. The morphological and
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chemical properties were compared with the reference sample (NCNPR #11695) for the authentication process
at the NCNPR.
2.3 Extraction and isolation of compounds:
The dried leaves of M. oleifera species (671 g) were ground to a fine powder and extracted successively using
continuous extraction method by percolation process with hexanes (4 L × 5 × 12h), acetone (4 L × 5 × 12h), and
methanol (4 L × 5 × 12h), respectively at room temperature. The methanol solution was then evaporated under
reduced pressure at 40 °C to yield a dried methanolic crude extract (73.1 g). The crude extract of the methanol
soluble portion was fractionated by vacuum liquid chromatography (VLC) over reversed phase C-18 column
(25 cm × 10 cm) using water/methanol gradients to provide eleven fractions (Fr. 1-11). Fr. 4 (3.4 g) was further
fractionated over sephadex LH-20 (78 cm × 5.5 cm) with methanol and subsequently subjected on repeated
column chromatography (CC) [silica gel (93 cm × 3 cm), ethyl acetate/chloroform/methanol/water (15:8:4:1
and 6:4:4:1); silica gel (94 cm × 2.5 cm), ethyl acetate/chloroform/methanol/water (15:8:4:1 and 6:4:4:1); and
silica gel (99 cm × 2.5 cm), ethyl acetate/chloroform/methanol/water (10:6:4:1)] followed by preparative TLC
to yield astragalin (4, 5.1 mg), isolariciresinol-3a-O--D-glucopyranoside (9, 5.0 mg), (E)/(Z)-2-hexenyl-β-D-
glucopyranoside (10, 40.1 mg), niazirin (12, 5.6 mg), benzyl--primeveroside (11, 41.8 mg), and methyl-4-(-
L-rhamnopyranosyloxy)benzylcarbamate (13, 4.7 mg). Fr. 5 (4.1 g) was subjected to CC on Sephadex LH-20
(78 cm × 5.5 cm) using methanol as an eluent followed by repeated CC [silica gel (89.5 cm × 4 cm),
chloroform/methanol (9:1, 17:3, and 4:1); silica gel (90 cm × 2.7 cm), chloroform/methanol (9:1 and 4:1); and
silica gel (75 cm × 2 cm), ethyl acetate/chloroform/methanol/water (15:8:4:1)] to afford omoringone (1, 1.5
mg), rutin (2, 117.2 mg), isoquercitrin (3, 792.1 mg), lariciresinol-9-O--D-glucopyranoside (7, 28.9 mg), and
(+)-pinoresinol-4-O--D-glucopyranoside (8, 10.3 mg). Fr. 7 (1.5 g) was applied to CC over Sephadex LH-20
(78 cm × 5.5 cm) eluted with methanol and subsequently applied on normal phase silica gel (92 cm × 3.25 cm)
using ethyl acetate/chloroform/methanol/water gradient (15:8:4:1) to purify (S) linalyl--D-glucopyranoside (5,
5.3 mg) and (S) linalyl--primeveroside (6, 24.2 mg). Chemical Structure elucidation of the isolated compounds
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was performed by analysis of their spectroscopic data including 1D and 2D NMR and confirmed by HR-ESI-
MS spectrometric analysis.
1
13
Omoringone (1): semisolid; UV (CH3OH) λmax nm 313.2, 229.6 nm; H and C NMR (CD3OD), see Table 1;
positive HR-ESI-MS m/z 309.1328 (calcd. for [C16H20O6 +H]+, 309.1338).
2.4 Chemical synthesis of intermediates and compounds:
2.4.1 1, 2, 3, 4-tetra-O-acetate-L-rhamnopyranoside (15):
L-rhamnose monohydrate (14) (2.5 g, 15.23 mmol, 1 equiv.) and a catalytic amount of DMAP (4-dimethyl
aminopyridine) (186 mg, 1.52 mmol, 0.1 equiv.) were dissolved in anhydrous pyridine (8.9 mL). Then, acetic
anhydride (10.4 mL) was added to the solution and stirred for 16 h at room temperature under N2 atmosphere.
After completion of the reaction, the crude mixture was concentrated, and the excess pyridine was co-
evaporated with toluene. The mixture was dissolved in EtOAc, and washed with 1.0 N HCl, NaHCO3, and brine
(aqueous 5% NaCl), respectively. The organic layer was dried over anhydrous Mg2SO4 and co-evaporated with
toluene (2 times).
2.4.2 2, 3, 4-tri-O-acetate-L-rhamnopyranose hemiacetal (16):
Hydrazine acetate (0.27 g, 3.01 mmol, 1.1 equiv.) was added to a solution of 1, 2, 3, 4-tetra-O-acetate-L-
rhamnopyranoside (15) (1 g, 3.01 mmol, 1 equiv.) in DMF (13 mL) at 50°C and stirred the reaction for 2 h
under N2. When the starting material disappears and the product forms, the mixture was diluted with EtOAc and
washed with brine. The crude product was dried over anhydrous Mg2SO4 and concentrated to give the yellow
oily product.
2.4.3 2, 3, 4-tri-O-acetate-α-L-rhamnosyl trichloro acetimidate (17):
2, 3, 4-tri-O-acetate-L-rhamnopyranose hemiacetal (16) (0.87 g, 3 mmol, 1 equiv.) was incubated with
trichloroacetonitrile (2.71 mL, 27 mmol, 9 equiv.) and DBU (0.16 g, 1.05 mmol, 0.35 equiv.) in anhydrous
DCM (10 mL). The mixture was stirred for 2 h at RT and concentrated later using rotary evaporator.
2.4.4 Synthesis of omoringone (1):
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2, 3, 4-tri-O-acetate-α-L-rhamnosyl trichloro acetimidate (17) (0.325 g, 0.75 mmol, 1 equiv.) and 4-hydroxy
benzylidene acetone (18) (0.14 g, 0.90 mmol, 1.2 equiv.) with molecular sieves (3 Å) were taken in freshly
distilled dichloromethane (DCM) (10 mL) at 0°C and stirred for 30 min under N2 atmosphere. Trimethylsilyl
trifluoromethanesulfonate (TMSOTf) (135.11 μL, 0.75 mmol, 1 equiv.) was added dropwise to the reaction
mixture at 0°C and slowly allowed the reaction mixture to RT and stirred for overnight. After completion of all
the starting materials based on TLC, the reaction mixture was filtered and dissolved in CHCl3 (30 mL). The
organic layer was extracted with aq. NaHCO3 solution, dried over anhydrous Mg2SO4 and concentrated under
reduced pressure to provide 4-[(2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl)oxy]-benzylidene acetone (19). This
crude mixture containing the latter was dissolved in anhydrous methanol that contained a catalytic amount of
sodium methoxide (350 μL). The reaction mixture was allowed to stir at ambient temperature for 30 min under
N2. After total consumption of starting material, the reaction mixture was neutralized by the addition of
Amberlite IR-120 (H+) resin. Then, the reaction mixture was filtered and concentrated under reduced pressure
to obtain a crude product containing omoringone (1). The crude product was purified by silica gel
chromatography using a gradient of chloroform: methanol (9: 0, v/v) to obtain omoringone (1) as semisolid (12
mg, 62%).
2.4.5 Synthesis of niazirin (12):
2, 3, 4-tri-O-acetate-α-L-rhamnosyl trichloro acetimidate (17) (0.325 g, 0.75 mmol, 1 equiv.) and 4-hydroxy
benzene acetonitrile (20) (0.12 g, 0.90 mmol, 1.2 equiv.) with molecular sieves (3 Å) were taken in freshly
distilled DCM (10 mL) at 0°C and stirred for 30 min under N2 atmosphere. To this reaction mixture, TMSOTf
(135.11 μL, 0.75 mmol, 1 equiv.) was added dropwise at 0°C and slowly allowed the reaction mixture to RT
and stirred for overnight. After completion of all the starting materials based on TLC, the reaction mixture was
filtered and dissolved in CHCl3 (30 mL). The organic layer was extracted with aq. NaHCO3 solution, dried over
anhydrous Mg2SO4 and concentrated under reduced pressure to provide 4-[(2,3,4-tri-O-acetyl-α-L-
rhamnopyranosyl)oxy]-benzeneacetonitrile (21). This mixture containing compound 21 was dissolved in
anhydrous methanol containing a catalytic amount of sodium methoxide (200 μL). The reaction mixture was
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allowed to stir at ambient temperature for 30 min under N2. After total consumption of starting material, the
reaction mixture was neutralized by the addition of Amberlite IR-120 (H+) resin to neutralize the mixture.
Then, the reaction mixture was filtered and concentrated under reduced pressure to obtain a crude product
containing niazirin (12). The crude product was purified by silica gel chromatography using a gradient of
chloroform: methanol (9: 0, v/v) to obtain niazirin (12) as semisolid (62 mg, 62%).
2.5 Cytotoxicity assay:
The isolated phytochemicals were evaluated for their cytotoxicity towards four human solid tumor cell lines
(melanoma: SK-MEL, epidermal carcinoma: KB, breast ductal carcinoma: BT-549, and ovarian carcinoma: SK-
OV-3), and three kidney and liver cell lines (African green monkey kidney fibroblast: VERO, pig kidney
epithelial: LLC-PK1 and human hepatoma cells: HepG2). DMSO and doxorubicin were included as negative
and positive controls, respectively. The assay was performed in 96-well tissue culture-treated microplates as
described earlier (Zulfiqar et al., 2017). In brief, cells were seeded to the wells of the plate at a density of
10,000 cells/well and incubated for 24 h before treating with various concentrations of the test samples and
incubating for 48 h. Cell viability was determined using the tetrazolium dye (WST-8). IC50 values were
calculated from concentration-response curves of % decrease in cell viability versus test concentrations.
2.6 Inhibition of the catalytic activity of CYP3A4 and CYP2D6 :
The assay to determine the inhibition of the catalytic activity of CYPs was performed using CYP3A4/BFC and
CYP2D6/AMMC inhibitor screening kits (Corning Life Sciences) as reported earlier with some modifications
(Fantoukh et al., 2019). The fluorescent substrates were 7-benzyloxy-trifluoromethyl coumarin (BFC) and 3-[2-
(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (AMMC) for CYP3A4 and CYP2D6,
respectively. The extract of M. oleifera and its constituents were dissolved in methanol and serially diluted in a
solution (100 µL) of cofactors mix, CYP proteins (0.05 mg of protein/mL), and G-6-PDH to achieve six
concentrations ranging from 0.1 to 100 µg/mL for the extract and 0.1 to 100 µM for the pure compounds. The
highest concentration of methanol was 1% (vehicle control). Initial readings were recorded to report the
inherent fluorescence before incubating the plates at 37 °C for 10 min. Initiation of the enzymatic reaction was
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achieved by the addition of the enzyme substrate mixture (100 µL) followed by incubation for 30 min at 370C.
The enzymatic reaction was terminated by the addition of 75 µL of ice cold acetonitrile/0.5 M Tris base (80:20).
Fluorescence was measured on a Spectramax M5 plate reader (Molecular Devices, Sunnyvale, CA, USA) at
excitation/emission wavelengths of 409 / 530 nm to detect 7-hydroxytrifluoromethyl coumarin (HFC) and 390 /
460 nm to detect 3-[2-(N,N-diethylamino)ethyl]-7-hydroxy-4-methyl coumarin hydrochloride (AHMC) as
metabolites of BFC and AMMC, respectively. The inhibitory potential was reported in terms of IC50 values,
obtained from concentration-response curves generated by plotting the tested concentrations against enzyme
inhibition (%) using Microsoft Excel.
3 Results:
3.1 Isolation and Structure elucidation:
All isolated secondary metabolites were obtained from the methanol soluble portion of M. oleifera leaves by
repeated column chromatographic techniques (silica gel, RP-18 silica gel, and Sephadex LH-20) as well as
preparative TLC over silica gel. Phytochemical investigation of MeOH extract for M. oleifera leaves resulted in
the isolation and characterization of one new benzyl acetone rhamnoside, namely omoringone (1), together with
12 known secondary metabolites (2-13). Based on the extensive spectroscopic (1D and 2D NMR) and
spectrometric (HR-ESI-MS) analysis, known compounds were identified as rutin (2), isoquercitrin (3),
astragalin (4), (S) linalyl--D-glucoside (5), (S) linalyl--primeveroside (6), lariciresinol-9-O--D-
glucopyranoside (7), (+)-pinoresinol-4-O--D-glucopyranoside (8), isolariciresinol-3a-O--D-glucopyranoside
(9), (E)/(Z)-2-hexenyl-1-O-β-D-glucopyranoside (10), benzyl--primeveroside (11), niazirin (12), and methyl-4-
(-L-rhamnopyranosyloxy)benzylcarbamate (13) (Figure 1). The NMR data (1H, 13C, DEPT135) for compound
1 and 12 have been shown in Table 1 and Table 2, respectively.
3.2 Synthesis of compounds 1 and 12:
Compounds 1 and 12 were synthesized to obtain sufficient material for performing biological tests. Our
chemical synthesis scheme for compounds 1 and 12 starts with L-rhamnose monohydrate with acetylation step
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followed by regioselective deprotection of the anomeric position and subsequently lead to the
trichloroacetimidate derivative as rhamnosyl donor. The latter reacts with either 4-hydroxy benzylidene acetone
or 4-hydroxy benzene acetonitrile to produce omoringone (1) or niazirin (12), respectively, as described in
Scheme 1.
3.3 Cytotoxicity screening:
All the isolates were evaluated for their cytotoxicity against a panel of cancer and noncancer cell lines (SK-
MEL, KB, BT-549, SK-OV-3, Vero, LLC-PK1, and HepG2). No cytotoxicity effects were observed on any of
the tested cell lines up to a highest concentration of 50 µM (data not shown).
3.4 Inhibition of CYP isoforms by the MeOH extract of M. oleifera and its isolated compounds:
The inhibitory effects of test samples on the catalytic activity of two major CYP isoforms (CYP3A4 and
CYP2D6) were measured. A decrease in the formation of the respective probe metabolites was calculated, and
the IC50 value (concentration responsible for a 50% decrease in activity) was determined from concentration-
response curves. The crude methanolic extract of M. olefiera exhibited a remarkable inhibition (IC50 = 52.50 ±
2.5 μg/mL) against CYP3A4 with no effect on CYP2D6 at 100 μg/mL. It was interesting to note that the activity
of CYP3A4 was inhibited by compounds 1-4 and 7-9 in varying degrees with IC50 values ranging from 41.5 to
011 µM with no remarkable effect observed on the activity of CYP2D6 isozyme making them the potential
major contributors for the inhibitory activity of the extract against CYP3A4 as shown in Table 3.
4 Discussion:
Owing to the ethnobotanical significance of this plant species, the prime research aims to assess the
phytochemical profile, the cytotoxic effect, and drug interaction potential for the extract and isolated secondary
metabolites from M. oleifera. All isolated compounds were obtained from the methanol soluble portion of M.
oleifera leaves by column chromatography using various matrixes (silica gel, RP-18 silica gel, and Sephadex
LH-20) as well as preparative TLC over silica gel. The isolated compounds represent a variety of chemical
classes such as flavonoid, terpenoid, lignan, and other phenolic components (Figure 1). To the best of our
knowledge, compound 1 was found to be a new metabolite. Also, this is the first isolation report for compounds
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5-10 from this species. In addition, compounds 12 and 13 are unique components of M. oleifera, and they could
be employed as analytical markers for the chemical fingerprinting profile and quality control for herbal
supplements claiming to contain this plant species.
The molecular formula of compound 1 was determined as C01H01O1 which was compatible with the protonated
molecule at m/z 309.1328 (calcd. for [C16H20O6 +H]+, 309.1338) in the HRESIMS and displayed seven degrees
1
13
of unsaturation. Investigation of HSQC, COSY, and HMBC spectra allowed assigning the H NMR and C
NMR spectroscopic data. Decoupled 13C NMR spectrum exhibited sixteen resonances which were distinguished
as two methyl, eleven methine, and three non-protonated carbons using DEPTQ135 experiment. Among them,
the compound includes a carbonyl moiety (201.3 ppm) and one deoxyhexose sugar unit which was identified as
3
-L-rhamnopyranose based on coupling constants JH-1'', H-2'' = 1.7 Hz of anomeric proton alongside the other
13
1
characteristic C NMR chemical shift values. Besides, H NMR displayed AB spin system characterized by
roofing effect indicating aromatic ring with symmetry as 1, 4-disubstituted benzene. Two additional ethylenic
carbons were observed (126.3 and 145.4 ppm) which revealed the existence of a double bond. Its geometry was
assigned as (E) configuration based on the high J-value between the vicinal protons (16.3 Hz). A singlet (3H) at
1
2.38 ppm in the H NMR spectrum was indicative of the presence of an acetyl group. The HMBC cross-peak
correlations of the anomeric proton for (H 5.52) with C-4' (C 159.8) indicated that glycone residue was located
at C-4' (Figure 2). All this data was evident to elucidate the structure as (E)-4-[4-[(-L-
rhamnopyranosyl)oxy]phenyl]but-3-en-2-one.
A putative proposed biosynthesis for compound 1 is illustrated in Scheme 2. It is biosynthesized from the
shikimic acid pathway via conversion the phenylalanine moiety to cinnamic acid by phenylalanine ammonia
lyase followed by aromatic hydroxylation via CYP450 to provide p-coumaric acid. The latter was catalyzed by
P-coumarate-CoA ligase to yield p-coumaroyl-CoA. Benzalacetone synthase forms the polyketide intermediate
p-hydroxybenzalacetone through the decarboxylative (Claisen-type) condensation of p-coumaroyl-CoA with
malonyl-CoA, followed by hydrolysis and decarboxylation (Koeduka et al., 2011). In the presence of rhamnosyl
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transferase as tailoring enzyme, the glycone moiety was incorporated into the benzalacetone basic skeleton to
provide compound 1.
Because of the low isolation yields, compounds 1 and 12 were successfully synthesized to obtain sufficient
material for performing biological tests. This work constitutes the first synthesis for compounds 1 and 12.
Comparisons of 1H NMR spectra for natural and synthesized omoringone (1) and niazirin (12) are illustrated in
Figure 3.A, and Figure 4.A, respectively. Comparisons of 13C NMR spectra of natural and synthesized
omoringone (1) and niazirin (12) are shown in Figure 3.B, and Figure 4.B, respectively.
Multiple endogenous substrates and exogenous xenobiotics are predominantly metabolized and biosynthesized
by cytochrome P450. These heme-containing mono-oxygenases are exclusively responsible for the conversion
of the inactive prodrug to the pharmacologically active metabolites in the human body (Denisov et al., 2005).
CYP inhibitors and inducers interact with CYP isozymes in the binding site leading to interference with their
substrates and drugs. Particularly, CYP inhibitors can intensify the toxicity profile while the CYP inducers can
increase the clearance of the co-administered drugs affecting their therapeutic effectiveness. (Butterweck et al.,
2004). Although M. oleifera extracts indicate a high degree of safety, and no adverse effects were reported in
humans so far, some studies highlighted that the chronic administration of M. oleifera extracts might distort the
histoarchitecture of both liver and kidneys which predispose to hepatic and kidney damage and ultimately lead
to hepatotoxicity and nephrotoxicity (Ambi AA, 2011; Awodele et al., 2012; Oyagbemi et al., 2013; Paul CW,
2012). In the current study, we investigated the inhibitory activities of the extract and isolated secondary
metabolites from M. oleifera against two principal CYP450 isozymes (CYP3A4 and CYP2D6) which are
responsible for 50% of clinical drug metabolism. The new compound, namely omoringone (1), in addition to
the flavonols 2-4, and lignans 7-9 exhibited the strongest inhibition against CYP3A4 ranging from 41.5 to 011
µM. Except for astragalin (4) which exhibited a weak inhibitory activity on CYP2D6 (IC50 = 90 ± 5 µM), no
remarkable inhibitory activity was shown against CYP2D6 by the tested samples. On the other hand, the
monoterpenoids 5 and 6 in addition to compounds 10-13 display no effect on both isozymes. Even though the
flavonoids such as rutin and hyperoside show different in vitro IC50 values, they are expected to exert similar
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inhibitory effects in the animal model. This effect could be a consequence for the conversion of the
aforementioned flavonols to flavonol aglycone derivative, quercetin. The latter was extensively evaluated and
found to inhibit several CYP isozymes including CYP3A4, CYP2D6, and CYP2C9 according to prior in vivo
studies (Song et al., 2013; Valentova et al., 2014). Despite the fact that compounds 12 and 13 are unique and
might be considered as analytical markers for M. oleifera, they exhibited no inhibitory effects on both CYP
isozymes tested as mentioned earlier. Although M. oleifera extract and its isolated phytochemicals were tested
against an array of the tumor and normal cell lines, they demonstrated no cytotoxicity effects for the extract and
its constituents at 50 µg/mL and 50 µM, respectively. The results indicate that M. oleifera and its tested
phytoconstituents are less potential to be developed as anticancer agents. In addition, the results suggest that M.
oleifera and its tested ingredients are potentially safe against normal cells such as kidney and liver. This work
aided in ascertaining components in M. oleifera with potential HDI using in vitro human liver microsomes.
Nonetheless, further studies should be conducted to determine the bioavailability of the phytochemicals and
extrapolate the findings to in vivo animal model.
In conclusion, 13 secondary metabolites were isolated from M. oleifera leaves. Omoringone (1) was found to be
a new metabolite. Omoringone (1) and niazirin (12) were successively synthesized to provide sufficient
quantities for conducting the additional bioassays. This work also constitutes the first synthesis for the two
aforementioned compounds. No cytotoxicity effects were observed on any tested cell lines at 50 µg/mL and 50
µM for the extract and phytochemicals, respectively. The extract inhibited CYP3A4 catalytic activity (IC50 =
52.5 ± 2.5 µg/mL) with inhibitory effects observed only with the compounds 1-4 and 7-9 on CYP3A4 with IC50
ranging from 41.5 to 011 µM while no remarkable effect was observed on CYP2D6 isozyme.
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Acknowledgment:
This research was supported by Science-Based Authentication of Dietary Supplements funded by the Food and
Drug Administration grant number [5U01FD004246-07]. Omer and Mohammed extend their appreciation to the
Deanship of Scientific Research and the Research Center, College of Pharmacy, King Saud University for
financial support.
Conflict of interest:
The authors declare that relationships leading to a potential conflict of interest such as commercial or financial
were absent in this research.
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Scheme 1: Synthesis of compounds 1 and 12
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Scheme 2: Plausible biosynthetic pathway for compound 1.
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Table 1: 1H (400 MHz) and 13C (100 MHz) NMR spectroscopic data of compound 1 (in MeOD, δ in ppm, J in
Hz).
Position
Multiplicity
1H (δ)
13C (δ)
27.2
1
2
3
4
CH3
C=O
CH
CH
C
CH
CH
C
CH
CH
CH
CH
CH
CH3
2.38 (3H, s)
201.3
126.3
145.4
129.8
131.2
117.8
159.8
99.6
71.9
72.2
73.7
70.9
6.71 (1H, d, J = 16.3 Hz)
7.64 (1H, d, J = 16.3 Hz)
1'
2', 6'
3', 5'
4'
1''
2''
3''
4''
5''
6''
7.61 (2H, d, J = 8.7 Hz)
7.14 (2H, d, J = 8.7 Hz)
5.52 (1H, d, J = 1.7 Hz)
4.03 (1H, dd, J = 3.4, 1.8 Hz)
3.86 (1H, dd, J = 9.4, 3.5 Hz)
3.49 (1H, t, J = 9.4 Hz)
3.65 – 3.57 (1H, m)
1.24 (3H, d, J = 6.1 Hz)
18.0
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Table 2: 1H (400 MHz) and 13C (100 MHz) NMR spectroscopic data of compound 12 (in MeOD, δ in ppm, J in
Hz).
Position
Multiplicity
1H (δ)
13C (δ)
120.0
22.8
125.6
130.3
118.0
157.2
99.6
71.8
72.1
73.7
70.6
1
2
1'
C≡N
CH2
C
CH
CH
C
CH
CH
CH
CH
CH
CH3
3.81 (2H, s)
2', 6'
3', 5'
4'
1''
2''
3'
4''
5''
6''
7.27 (2H, d, J = 8.6 Hz)
7.07 (2H, d, J = 9.6 Hz)
5.47 (1H, d, J = 1.9 Hz)
4.07 (1H, dd, J = 3.4, 1.9 Hz)
3.91 (1H, dd, J = 9.5, 3.6 Hz)
3.51 (1H, t, J = 9.5 Hz)
3.69 – 3.61 (1H, m)
1.24 (2H, d, J = 6.3 Hz)
18.1
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Table 3: Inhibition of CYP3A4 and CYP2D6 isozymes by M. oleifera extract and isolated compounds
CYP3A4
IC50 µg/mL
52.5 ± 2.5
IC50 µM
CYP2D6
Test sample
M. oleifera MeOH extract
-
41.5 ± 3.5
60 ± 5
65.5 ± 4.5
69 ± 1.5
-
> 100
1
2
3
4
5
6
7
8
9
(E)-4-[4-[(-D-glucopyranosyl)oxy]phenyl]but-3-en-2-one
Rutin
-
Isoquercitrin
> 100
Astragalin
90 ± 5
-
(S) Linalyl--D-glucoside
-
> 100
(S) Linalyl--primeveroside
Lariciresinol-9-O--D-glucopyranoside
(+)-Pinoresinol-4-O--D-glucopyranoside
Isolariciresinol-3a-O--D-glucopyranoside
72.5 ± 7.5
41.5 ± 3.2
011 ± 1
-
-
-
-
-
10 (E)/(Z)-2-Hexenyl-β-D-glucopyranoside
> 100
> 100
-
11
12
13
Benzyl--primeveroside
Niazirin
-
> 100
> 100
Methyl-4-(-L-rhamnopyranosyloxy)benzylcarbamate
KetoconazoleΩ
0.040 ± 0.006
QuinidineΩ
0.050 ± 0.004
(-) indicates the lack of inhibition, (Ω) indicates the positive control for respective CYP450 isozyme. The data are
represented as mean ± SEM of three independent experiments
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Figure legends.
Figure 1: Chemical structures of isolated phytoconstituents 1-13 from M. olefiera
Figure 2: The key HMBC (arrows) and 1H-1H COSY (bold lines) correlations of compounds 1 and 12
Figure 3.A: Comparisons of 1H NMR spectra for natural and synthesized omoringone (1)
Figure 3.B: Comparisons of 13C NMR spectra for natural and synthesized omoringone (1)
Figure 4.A: Comparisons of 1H NMR spectra for natural and synthesized niazirin (12)
Figure 4.B: Comparisons of 13C NMR spectra for natural and synthesized niazirin (12)
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Figure 1: Chemical structures of isolated phytoconstituents 1-13 from M. olefiera
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Figure 2: The key HMBC (arrows) and 1H-1H COSY (bold lines) correlations of compounds 1 and 12
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Figure 3.A: Comparisons of 1H NMR spectra for natural and synthesized omoringone (1)
Figure 3.B: Comparisons of 13C NMR spectra for natural and synthesized omoringone (1)
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Figure 4.A: Comparisons of 1H NMR spectra for natural and synthesized niazirin (12)
Figure 4.B: Comparisons of 13C NMR spectra for natural and synthesized niazirin (12)
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Graphical_abstract
29