Flavonoids of Lonchocarpus montanus A . M . G . Azevedo and biological activity

The analysis of root extracts from Lonchocarpus montanus A.M.G. Azevedo resulted in the isolation of twenty three compounds chiefly flavonoids of which five (four flavonoids and one benzophenone) are described for the first time. The molecular structures of the new compounds (1-5) were determined through spectral analysis (UV, IR, MS and NMR) as being: 2′-hydroxy-8-(α,α-dimethylallyl)-2′′, 2′′dimethylpyrano-(5′′,6′′:3′,4′)-dibenzoylmethane (1), 2′-methoxy-8-(α, α-dimethylallyl)-2′′, 2′′-dimethylpyrano-(5′′,6′′:3′,4′)-dibenzoylmethane (2), 4′-methoxy-2′′,2′′-dimethylpyrano-(5′′,6′′:8,7)-flavone (3), 2′′(1-hydroxy-1-methylethyl)-furano-(4′′,5′′:8,7)-flavone (4) and [2′-methoxy-furano-(4′′,5′′:3′,4′)-phenyl]phenylmethanone (5). Additionally, fifteen fatty acids were detected through GC-MS analysis of the corresponding methyl esters [(CH3)2CH(CH2)8COOH and CH3(CH2)nCOOH (n = 6, 12-24)]. Quantitative RP-HPLC showed that the most abundant flavonoids in the petroleum ether and dichloromethane extracts were pongamol (19%) and lanceolatine B (8.0%), respectively. In the bioautography assay, the extracts, pongamol (9), lanceolatine B (10), isolonchocarpin (14), derriobtusone A (17) and medicarpine (18) were active against Staphilococus aureus whereas 9 also against Bacillus subtilis and Cladosporium cladosporioides. Compound 1, 2′′,2′′-dimethylpyrano-(5′′,6′′:8,7)-flavone (11) and furano-(12′′,13′′:7,8)4′-methoxy flavone (12) were active against Fusarium oxysporium whereas 11 also against Rhizopus orizae. The extracts, compounds 9, 10, 17 and (E)-7-O-methoxypongamol (23) displayed high toxicity in the brine shrimp lethality assay.


INTRODUCTION
The genus Lonchocarpus encompasses 150 species, including 24 native to Brazil.L. montanus is a new native species (A.M.G.A. Tozzi, unpublished data).The botanical classification was based on morphology of the flowers and fruits and the inflorescence structure, which has been considered an outstanding character.Accordingly L. montanus and L. obtusus Benth revealed a strong similarity and were allocated to the Unguiflora section in Lonchocarpus subgenus Lonchocarpus (A.M.G.A. Tozzi, unpublished data).L. obtusus has furnished flavonoids, including the new auronol derriobtusone A, (Do Nascimento et al. 1976).The natural aurones and auronols comprise a very small group of flavonoids.L. montanus is an ornamental tree popularly known as "cabelouro" or "carrancudo".It is widely distributed through the states of Bahia, Goias, Minas Gerais and Tocantins.
This paper describes the isolation and characterization of four new flavonoids (1-4) and one new benzophenone derivative (5) together with eighteen known compounds (6-23) and a mixture of fatty acids (24-38), from root extracts of L. montanus.The results obtained after submitting extracts (petroleum ether and dichloromethane) and compounds 1, 6, 9-12, 14, 17, 18 to bioautography (against bacteria and fungi) as well as extracts (petroleum ether, dichloromethane and methanol) and compounds 9, 10, 17, to brine shrimp lethality are also included.The analysis of the extracts was performed through chromatography (CC, TLC, GC-MS and RP-HPLC).

GENERAL PROCEDURES
Melting points were determined using a Mettler FP5 apparatus.The optical rotations were measured on a Carl Zeiss Jena Polamat A polarimeter or a Jasco J-720 spectropolarimeter.IR spectra (film or KBr) were recorded using a Perkin-Elmer Model 1600 FT-IR 1600 instrument.UV spectra were recorded on a HP Diode Array Spectrophotometer 8452 A spectrometer using MeOH as solvent.NMR spectra were recorded on Varian INOVA-500 (500 MHz for 1 H and 125 MHz for 13 C), Gemini 300 (300 MHz for 1 H and 75 MHz for 13 C) or Brucker AC 300 P (300 MHz for 1 H and 75 MHz for 13 C) spectrometers using CDCl 3 as solvent and TMS as internal standard.The DEPT experiments were performed using polarization transfer pulses of 90 • and 135 • .EIMS, direct probe, HREIMS and MS/MS experiments were performed on a VG Auto Spec-Fisions Instrument by using electron ionization at 70 eV.GC-MS was performed on a Hewlett Packard (Model HP 5890 B SERIES II) instrument.Column chromatography (CC) separations were on silica gel 60 (70-230 mesh, Merck).TLC was performed on commercial plates (silica gel G and GF 254 , Merck) while preparative TLC used precoated 1000 m thick Merck silica gel 60 F 254 glass plates.Compounds were detected by UV (λ = 254 and 366 nm) irradiation and/or with an ethanolic solution of anisaldehyde, sulfuric acid and acetic acid (90 mL:5mL:1mL), followed by heating.The solvent mixtures were prepared a active compounds (LD 50 ≤ 1000 µg mL -1 ) 48 .b made using concentrations of 50, 5.0 and 0.5 µg mL -1 . in volume ratios.Reversed-phase HPLC analyses were performed using a C-18 Nova Pak column, with diode-array detection.Identification of the peaks in the chromatogram of each extract (petroleum ether and dichloromethane) was carried out by co-injection with five flavonoids, 9, 10, 14, 17, 18 (one each time) isolated from the extracts of L. montanus roots and further purified by preparative HPLC.

GC-MS ANALYSIS OF METHYL ESTERS
The methyl fatty acids esters were analyzed by GC-MS using a HP 5970 mass selective detector coupled to a HP-gas chromatograph having a HP5 fused silica capillary column (30 m × 0.25 mm i.d., 0.25 µm film).The instrument was operated with injector and detector temperatures of 260 • C and 280 • C, respectively, in the splitless (2 µL injection) and constant flow mode.The temperature of the GC column was increased from 50 to 300 • C at a rate of 10 • C min -1 and held at 300 • C for 5 min.The carrier gas was helium at a flow rate of 1.0 mL min -1 .Mass spectra were taken over the m/z 40-600 range with an ionizing voltage of 70 eV.Linear retention time indices (RI) for the methyl fatty acids esters 25-39 were determined by comparing their retention times with those of n-paraffin standards and mass spectral data (Adams 1995).All mass spectra were identified by using an on-line library (Wiley 275) or authentic compounds (Table I).

QUANTITATIVE HPLC ANALYSIS
Light petroleum and dichloromethane root extracts from L. montanus were dissolved in acetonitrile.An aliquot (2 µL) of each extract was analyzed on a Hewlett Packard model 1090, series II/M HPLC with a Waters Nova-Pak C-18, 3 µm, column, with mobile phase gradient of CH 3 CN-H 2 O (48-52) for 25 min and then to CH 3  during 20 min at a flow rate of 0.8 mL min -1 , keeping the column at room temperature.Detection was with a HP photodiode array detector.The identification of the peaks in the chromatograms of each extract (petroleum ether and dichloromethane) was carried out by individual co-injections with each one of the most abundant flavonoids (9, 10, 14, 17 and 18) isolated from the extracts of L. montanus, which were first purified by semi-preparative HPLC.Each compound was dissolved in acetonitrile and purified with a semi-preparative HPLC (Waters) system, consisting of an universal liquid chromatograph injector, a model 484 variable-wavelength detector (adjusted to 230 nm), a model 740 data module and a model 600E system controller and pump, with a Regis ODS, 5 µm, 250 × 10 mm i.d.column, and a CH 3 CN-H 2 O gradient of 20-100% CH 3 CN in 45 min at a flow rate of 2.0 mLmin -1 .The corresponding retention times were: 9 (7.8 min), 10 (8.5 min), 14 (29 min), 17 (13 min) and 18 (4.2min).

CALIBRATION CURVES
Standard solutions of each quantified flavonoid (9, 10, 17) were prepared by serial dilutions in acetonitrile.Calibration curves were obtained by plotting the integrated peak areas at the maximum UV absorption for each compound versus concentration by performing linear regression analysis with correlation coefficients of 0.9999 for each analyzed compound.Quantification was made by external calibration in the petroleum ether extract the concentrations were found to be 9 (19%), 10 (10.6%) and 17 (13.6%)while in the dichloromethane extract they were: 9 (6.7%), 10 (8.0%) and 17 (6.4%).
The Brine shrimp lethality assay was performed according to McLaughlin (McLaughlin 1995).The extracts (petroleum ether, dichloromethane and methanol) and compounds 9, 10, 17 displayed high toxicity as revealed by very low LC 50 values (Table II).

EXTRACTION AND ISOLATION
Dried and pulverized roots (259 g) of L. montanus were successively extracted with petroleum ether (30-60 • C), CH 2 Cl 2 and MeOH in a Soxhlet apparatus.After solvent evaporation, the petrol ether extract gave a viscous yellow oil (5.1 g), while the dichloromethane (3.5 g) and the methanol (6.0 g) extracts gave brown oils.Part of the petroleum ether extract (4.32 g) was fractionated by silica gel CC eluted first with petroleum ether/CHCl 3 (1:1).The eluent polarity was gradually increased by addition of CHCl 3 and then MeOH to furnish 295 fractions (30 mL each) which were reduced to 28 groups after TLC analysis.Most of the compounds were found in ten groups ranging from fractions 34 to 240.A sample of each was further fractionated by successive preparative TLC (silica gel) as described below and recovered from TLC plates by extraction with mixtures of CH 2 Cl 2 and MeOH.
Compound 2 exhibited a molecular ion [M] + at m/z 404.198792 in HREIMS, which is fourteen mass units higher than 1, suggesting that the hydroxyl group was replaced by a methoxy group, for a molecular formula of C 26 H 28 O 4 (calc. 404.198760).In the low resolution mass spectrum the base peak at m/z 217 [C 13 H 13 O 3 ] + originates from C-8/C-9 bond cleavage proving that the methoxy group is on B ring.The fragmentaion pathway (Fig. 2) is analogous to that of 1. NMR data (Table III) were also very similar those of 1 except for the lack of a signal corresponding to a hydrogen bonded hydroxy group at C-2 in the 1 H NMR spectrum and the presence of a methoxy group (δ 3.51, 3H, s, 2 -OCH 3 ) that was confirmed in the 13 C NMR spectrum (δ 66.0, 2 -OCH 3 ).The unusual chemical shift value observed for the methoxy group in the 1 H NMR spectrum evidenced that the methyl group stays out of the aromatic ring plane in order to relieve sterical hindrance with the α,α-dimethylallyl group at C-8, as can be demonstrated through the corresponding molecular model.
A minor compound together with 9 was detected through GC-MS.The corresponding mass spectrum (t R = 23.3min) displayed a molecular ion [M] + of m/z 280 (23%) which is fourteen mass units lower than that of 9, according to the molecular formula C 17 H 12 O 4 .The peaks at m/z 161 (16%) and m/z 105 (100%) correspond to the fragments from the cleavage of C8-C9 and C7-C8 bonds, respectively, leading to the suggestion that this compound can be demethylpongamol, a new compound that is probably the biogenetic precursor of 1. Alternative modes of furano fusion with ring A however cannot be discarded.
The known flavonoids (6-20, 23; see Table IV), β-sitosterol (21) and stigmasterol ( 22) were characterized by comparison of the respective spectral data with those found in the literature.
The petroleum ether, dichloromethane and methanol extracts and the flavonoids 9, 10, 17 and 23 were also submitted to the brine shrimp lethality bioassay (McLaughlin 1995) and displayed high toxicity as revealed by very low LC 50 values (Table II).
Derriobtusone A ( 17) is a very rare auronol which was isolated only from L. obtusus.The occurrence of Derriobtusone A (17), among the most abundant compounds in L. montanus reinforces its allocation together with L. obtusus at Unguiflora section in Lonchocarpus subgenus Lonchocarpus.The structures of the most abundant flavonoids (9, 10, 17) furnished by L. montanus are closely related.These findings suggest a biosynthetic pathway consisted by alternative oxidative steps starting from the same chalcone (Fig. 3).However no aurone was found in L. montanus.Collins for critical reading and English revision.The authors are also very grateful to Dr. Luzia Koike for supervising the analysis of the methyl esters mixture.

Fig. 3 -
Fig. 3 -Rationalization of a biosynthetic pathway leading to the most abundant flavonoids isolated from Lonchocarpus montanus A.M.G.Azevedo.

TABLE II Brine shrimp lethality test results for extracts and some flavonoids from Lonchocarpus montanus. a
b made using concentrations of 50, 5.0 and 0.5 µg mL -1 .c made using concentrations of 1000, 100 and 10 µg mL -1 .PLANT MATERIAL Roots of L. montanus were collected in Taquatinga do Tocantins, Tocantins State, Brazil in March, 1998.The plant was identified by Dr. A.M.G.Azevedo from the Biology Institute of Campinas State University (UNICAMP), Campinas, SP, Brazil.A voucher specimen (B.A.S. Pereira and D. Alvarenga 1717 -UEC) is deposited at the herbarium of Campinas State University (UNICAMP).