A New Macrolactone,Racemolide Along With Seven Known Compounds With Biological Activities From Mangrove Plant,Lumnitzera racemosa

Abstract

One new macrolide, racemolide (1), was isolated from an MeOH extract of the leaves of Lumnitzera racemosa with 7 known compounds (2-8) by various chromatographic techniques via silica gel, octadecylsilyl column chromatography and high-performance liquid chromatography. The structure of the new compound was determined through a combination of spectroscopic and chemical analyses. The isolated compounds were evaluated for their antileishmanial, hepatoprotective, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities. Compound 1 showed potent antileishmanial activity at inhibition % value of 67.6 ± 1.24% compared with the clinical drug miltefosine (93.3 ± 4.39%). Compounds 1 and 5 possessed moderate hepatoprotective activity against acetaminophen (APAP)-induced hepatotoxicity using human HepG2 cells at protection % value of 21.70 ± 1.97% and 27.5 ± 1.41%, respectively, compared with the standard glycyrrhizin (69.50 ± 3.53%). Compounds 4, 6, 7, and 8 had high DPPH radical scavenging activity (91.10 ± 4.25, 91.53 ± 0.05, 91.91 ± 0.09, and 86.48 ± 2.87%, respectively), while compounds 3 and 5 showed moderate (45.38 ± 3.62% and 62.20 ± 1.47%, respectively), comparable with the standard trolox (89.21 ± 5.88%). These results suggest that the new compound, racemolide (1), and known compounds (3-8) are valuable as promising antileishmanial, hepatoprotective, and anti-oxidant agents.

Lumnitzera racemosa Willd. (Family: Combretaceae) is a small tree that is cultivated in the mangrove swamps of Australia and Asia. A fluid obtained from slits made in the stem has been used conventionally to treat itches. ¹ Antihypertensive activity has been newly stated for the aqueous acetone extract of the plant ² Marine halophytes, such as mangroves and related species have many and various metabolites exhibiting antibacterial, antifungal, antiviral, antidiarrheal, hepatoprotective, antifeedant, insecticidal, cytotoxic, and antiplasmodial activities. ^{3 -12} The presence of saponins, triterpenes, sterols, hydroquinones, flavonoids, tannins, aliphatic alcohols, and phenolics ^{13 -16} has been previously reported from this plant. The metabolites that were isolated from mangroves often possess unique structural features and incorporate new or unusual assemblages of functional groups. ¹⁷ The present study deals with the isolation and structural elucidation of a new macrolactone, named racemolide (1) along with 7 known compounds (2-8) from the leaves of L. racemosa collected from subtropical marine regions in Japan, Okinawa (Figure 1). The isolated compounds were examined for their antileishmanial, hepatoprotective, and DPPH radical scavenging activities. From the EtOAc fraction of a MeOH extract of the leaves of L. racemosa, 8 compounds were isolated and identified; 1 new compound namely racemolide (1) and 7 known compounds (2-8). The structures of known compounds were elucidated to be loliolide (2), ¹⁸ protocatechuic acid (3), ¹⁹ methyl gallate (4), ²⁰ (+)-lyoniresinol (5), ²¹ myricetin 3-O-(2″-O-galloyl)-α-rhamnopyranoside (6), ²² quercetin 3-O- (2″-O-galloyl)-α-rhamnopyranoside (7), and quercetin (8) ²³ by comparing their physical characters and spectroscopic data with those reported in literature.

Figure 1

Structure of the isolated compounds from L. racemosa.

Compound (1) was obtained as colorless amorphous powder, having the molecular formula C₁₈H₁₈O₄ as determined by positive-ion mode high-resolution (HR)-electrospray ionization (ESI)-mass spectrometry (MS). The IR spectrum of 1 showed the bands at 3390 cm⁻¹ and 1714 cm⁻¹ corresponding to hydroxy and carbonyl group absorptions, respectively. The ¹H nuclear magnetic resonance (NMR) spectrum of 1 displayed the presence of 2 sets of trisubstitued benzene rings at δ_H 6.77 (1H, d, J = 8 Hz), 6.83 (1H, d, J = 8 Hz), 7.00 (1H, d, J = 2 Hz), 7.01 (1H, dd, J = 8, 2 Hz), 7.04 (1H, dd, J = 8, 2 Hz), and 7.16 (1H, d, J = 2 Hz), 3 triplet-like methylene protons at δ_H 2.61, 2.83, and 3.00 (each with J = 6 Hz), 1 multiplet methylene protons at δ_H 2.27 and 1 triplet-like oxymethylene proton at δ_H 4.30 with J = 6 Hz. The ¹³C NMR spectrum of 1 showed the presence of 18 carbon signals including; 4 methylenes, 1 oxymethylene, 1 carbonyl, 6 methines, and 6 quaternaryy carbons. The further NMR analyses were performed with the aid of ¹H-¹H correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC) and heteronuclear multiple-bound correlation (HMBC) spectroscopies. The HMBC spectrum displayed a strong correlation between the methylene protons (H₂-9′) at δ_H 4.30 and the carbonyl carbon (C-9) at δ_C 177.7 indicating the lactone formation between C-9 and C-9′ (Figure 2). The connections between C-7 and C-8 and from C-7′ to C-9′ were confirmed from the ¹H-¹H COSY correlations between H-7/H-8 , H-7′/H-8′, and H-8′/H-9′, respectively. From these data, the structure of compound 1 was elucidated to be a new macrolactone, designated as racemolide as shown in Figure 2, which is probably biosynthesized through intra-molecular lactonization of magnolol like 3,3'-neolignane precursor. In conclusion, chemical investigation of the leaves of L. racemosa resulted in the isolation and identification of 8 compounds, including a new macrolactone, named racemolide. The new macrolide (1) showed potent antileishmanial activity at inhibition % value of 67.6 ± 1.24%, compared to the positive control miltefosine (93.3 ± 4.39%) at the same concentration (50 µM). None of the remaining compounds (2-8) exhibited any significant antileishmanial activity. Compounds 1 and 5 had weak hepatoprotective activity against APAP-induced hepatotoxicity using human HepG2 cells at protection % value of 21.70 ± 1.97%, 27.5 ± 1.41%, respectively, compared to glycyrrhizin as a positive control (69.50 ± 3.53%) at the same concentration (50 µM). Compounds 4, 6, 7, and 8 possessed high DPPH radical scavenging activity at inhibition % value of 91.10 ± 4.25%, 91.53 ± 0.05%, 91.91 ± 0.09%, and 86.48 ± 2.87%, respectively, while compounds 3 and 5 exhibited moderate DPPH radical scavenging activity (45.38 ± 3.62% and 62.20 ± 1.47%, respectively), on the other hand, compounds 1 and 2 had no DPPH radical scavenging activity (4.29 ± 1.31% and 2.30 ± 0.72%, respectively) compared to the standard trolox (89.21 ± 5.88) at the same concentration (25 µM) (Table 1).The results of this study provide a scientific rationale for the therapeutic use of L. racemosa and set a basis for the further development of antioxidant, antileishmanial, and hepatoprotective products for the medical uses.

Figure 2

Selected COSY and HMBC correlations of 1.

Table 1

Antileishmanial, Hepatoprotective, and DPPH Radical Scavenging Activities of the Isolated Compounds (1-8) From L. Rascemosa.

Compounds	% Inhibition (50 µM) leishmania	IC50 (µM)	% Inhibition (50 µM)APAP induced toxicity	IC50 (µM)	% Inhibition (25 µM)DPPH radical	IC50 (µM)
1	67.6 ± 1.24	37.97 ± 1.32	21.7 ± 1.97	>100	NA	>100
2	NA	>100	NA	>100	NA	>100
3	NA	>100	NA	>100	45.4 ± 3.62	28.9 ± 3.56
4	NA	>100	NA	>100	91.1 ± 4.25	14.5 ± 5.66
5	NA	>100	27.5 ± 1.41	88.3 ± 4.51	62.2 ± 1.47	15.7 ± 1.18
6	NA	>100	NA	>100	91.5 ± 0.05	7.40 ± 1.03
7	NA	>100	NA	>100	91.9 ± 0.09	7.20 ± 0.42
8	NA	>100	NA	>100	86.5 ± 2.87	9.33 ± 0.99
Miltefosine	93.3 ± 4.39	22.49 ± 2.10	NA	>100	-	-
Glycyrrhizin	-	-	69.5 ± 3.53	11.74 ± 5.72	-	-
Trolox	-	-	-	-	89.2 ± 5.88	5.9 ± 2.19

NA: not active (<20% for antileishmanial, <10% for hepatoprotection, <20% for DPPH).

Experimental

General Experimental Procedures

IR spectrum was obtained on a Horiba FT-710 Fourier transform infrared spectrophotometer. ¹H and ¹³C NMR spectra were recorded on a JEOL JNM α-600 spectrometer with tetramethylsilane as an internal standard. HR-ESI mass spectrum was taken on a LTQ Orbitrap XL mass spectrometer. Silica gel column chromatography (CC) was performed on silica gel 60 [(E. Merck, Darmstadt, Germany), 70–230 mesh]. Reversed-phase [octadecylsilanized silica gel (ODS)] open CC (RPCC) was performed on Cosmosil 75C18-OPN (Nacalai Tesque, Kyoto, Japan) (Φ = 2 cm, L = 40 cm, 10 g fractions being collected). High-performance liquid chromatography (HPLC) was performed on an ODS column [Inertsil ODS-3; GL Science, Tokyo, Japan; (Φ = 10 mm, L = 25 cm, flow rate: 2.0 mL/min), using a refractive index and/or a UV detector. Precoated silica gel 60 F254 plates (E. Merck; 0.25 mm in thickness) were used for TLC analyses, visualized by spraying with a 10% H₂SO₄ solution in EtOH and heating to around 150°C on a hotplate. The microorganisms used in this study, Leishmania major is provided from Institute of Tropical Medicine, Nagasaki University.

Plant Material

The leaves of L. racemosa were collected in August 2002 in Okinawa, Japan. It was kindly identified by Prof. M. Aramoto. A voucher specimen of the plant is deposited in the Herbarium of Hiroshima University (02-LR-Okinawa-0825).

Extraction and Isolation

The air-dried powdered leaves of L. racemosa (5.27 kg) were extracted with 100% methanol till exhaustion and then concentrated under reduced pressure to yield a viscous gummy material. This residue was dissolved in water and defatted with n-hexane to yield hexane layer (58.8 g). The aqueous layer was evaporated to remove a trace amount of organic solvent and then extracted with EtOAc and n-BuOH. The EtOAc and n-BuOH fractions were concentrated under reduced pressure to give 139.1 g and 89.9 g of residues, respectively. The remaining aqueous layer was concentrated to furnish a water-soluble fraction.

The EtOAc fraction (139.1 g) was fractionated by CC on a highly porous synthetic resin, Diaion HP-20 (ɸ = 60 mm, L = 70 cm). The column was eluted initially with 100% MeOH (6 L) then 100% acetone (6 L). The MeOH fraction (94.30 g) was subjected to silica gel CC (2.8 kg), (ɸ = 80, L = 96 cm). The column was eluted initially with CHCl₃, then with gradient of CHCl₃–MeOH (98 : 2, 96 : 4, 94 : 6, 90 : 10, 85 : 15, 80 : 20, 75 : 25, 70 : 30, 60 : 40, 40 : 60), and finally with 100% MeOH, 500 mL fractions being collected. Similar fractions have been combined, affording 16 fractions (Fr.1 ~ 16).

The fifth fraction Fr.5 (5.60 g) was purified on RPCC, affording 5 fractions. The first fraction Fr.5-1 (196.2 mg) was purified by HPLC (30% MeOH) to produce compound 2 (2.0 mg). The third fraction Fr.5–3 (280.3 mg) was purified by HPLC (35% MeOH) to give compound 1 (10 mg).

The tenth fraction Fr.10 (5.0 g) was separated on RPCC, yielding 2 fractions. Fraction Fr.10-1(337.3 mg) was purified by HPLC (30% MeOH) to afford compounds 3 (67.4 mg) and 4 (31.5 mg). Fraction Fr.10-2 (8.4 mg) was purified by HPLC (40% MeOH) to give compound 5 (5.0 mg).

The thirteenth fraction Fr.13 (10.9 g) was purified on RPCC, producing 5 fractions. The fifth fraction Fr.13-5 (0.30 g) was purified by HPLC (60% MeOH) to furnish compounds 6 (15.3 mg), 7 (19.6 mg), and 8 (8.3 mg).

Racemolide (1)

Colorless amorphous powder.

IR (film) ν _max 3390, 2963, 1714, 1594, 1518, 1427, 1271, 1218 cm⁻¹.

¹H NMR (600 MHz, CD₃OD): Table 2.

Table 2

: ¹³C and ¹H NMR Spectral Data for 1 (CD₃OD, 150 and 600 MHz, Respectively).

Position	δ C	δ H, multiplicity, J in Hz
1	134.4	-
2	134.4	7.00, d, 2
3	128.4	-
4	153.3	-
5	116.9	6.77, d, 8
6	129.5	7.01, dd, 8, 2
7	31.1	3.00, 2 H, t, 6
8	37.1	2.61, 2 H, t, 6
9	177.7	-
1'	133.1	-
2'	134.6	7.16, d, 2
3'	127.7	-
4'	152.6	-
5'	117.4	6.83, d, 8
6'	130.4	7.04, dd, 8, 2
7'	32.0	2.83, 2 H, t, 6
8'	26.5	2.27, 2 H, m
9'	67.4	4.30, 2 H, t, 6

m: multiplet signal.

¹³C NMR (150 MHz, CD₃OD): Table 2.

HR-ESI-MS (positive-ion mode) m/z: 321.1635 [M + Na]⁺ (calcd. for C₁₈H₁₈O₄Na: 321.1633).

Biological Assay

Antileishmanial Assay

The leishmanicidal activities of isolated compounds were performed using the colorimetric MTT assay. Medium 199 supplemented with 10% heat-inactivated fetal bovine serum and 100 µg/mL of kanamycin was used as the cell culture medium. The test compounds were dissolved in dimethyl sulfoxide (DMSO) and added to the each well of the 96-well microtitration plates at 1% as the final concentration. The compounds were tested at final concentrations of 100, 50 and 25 µM. Leishmania major cells (2 × 10⁵ cells/well) were cultured in an incubator at 25^°C for 72 hours and then MTT solution was added to each well and the plates were incubated overnight at 25^°C. The absorbance was measured at 540 nm using a Molecular Device Versamex tunable microplate reader. miltefosine was used as a positive control. The inhibition % was calculated using the following equation:

$${\displaystyle \mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}(\mathrm{\%})=[1-({\mathrm{A}}_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}-{\mathrm{A}}_{\mathrm{b}\mathrm{a}\mathrm{c}\mathrm{k}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}}/{\mathrm{A}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}-{\mathrm{A}}_{\mathrm{b}\mathrm{a}\mathrm{c}\mathrm{k}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}})]\times 100}$$

where A _control is the absorbance of the control reaction mixture (containing DMSO and all reagents except for the test compounds). ²⁴

Hepatoprotection Assay

After 3 day subculture, 100 µL of diluted cell suspension was added to the wells of 96-well microtiter plates (3 × 10⁴ cells/well), then cultured in a 5% CO₂ incubator at 37^°C for 24 H. The supernatant was aspirated off and then 100 µL of test compounds (50 µM) dissolved in DMEM in the presence of acetaminophen (20 mM) were added to the each well of the 96-well microtiter plate, and then incubated at 37^°C for 36 H. After that a MTT solution was added to each well and the plates were incubated for 1.5 H. After removal of the supernatant, the formazan precipitates were dissolved in 100 µL of DMSO. The absorbance was measured using a Molecular Device Versamax tunable microplate reader at 570 nm. The concentration of vehicle (DMSO) in each well is adjusted to contain 1%. The inhibition % was calculated using the following equation:

$${\displaystyle \mathrm{\%}\mathrm{V}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}=[({\mathrm{A}}_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}-{\mathrm{A}}_{\mathrm{b}\mathrm{a}\mathrm{c}\mathrm{k}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}})/({\mathrm{A}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}-{\mathrm{A}}_{\mathrm{b}\mathrm{a}\mathrm{c}\mathrm{k}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}})]\times 100}$$

where A _control is the absorbance of the control reaction mixture (containing DMSO and all reagents except for the test compounds). ²⁵

DPPH Radical Scavenging Assay

The absorbance with various concentrations of the tested compounds dissolved in MeOH (100 µL) in a 96-well microtiter plate was measured at 515 nm as A_blank. Then, 200 µM DPPH solution (100 µL) was added to each well, followed by incubation at room temperature for 30 minutes. The absorbance was measured again as A_sample. The % inhibition was calculated using the following equation:

$${\displaystyle \mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}(\mathrm{\%})=[1-({\mathrm{A}}_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}-{\mathrm{A}}_{\mathrm{b}\mathrm{a}\mathrm{c}\mathrm{k}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}})/({\mathrm{A}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}-{\mathrm{A}}_{\mathrm{b}\mathrm{a}\mathrm{c}\mathrm{k}\mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}})]\times 100}$$

where A_control is the absorbance of the control reaction mixture (containing DMSO and all reagents except for the test compounds). ²⁶