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Two novel phytotoxic substances from Leucas aspera
Journal of Plant Physiology 171 (2014) 877–883
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Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Physiology
Two novel phytotoxic substances from Leucas aspera A.K.M. Mominul Islam a,∗ , Osamu Ohno b , Kiyotake Suenaga b , Hisashi Kato-Noguchi a a b
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki, Kagawa 761-0795, Japan Department of Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku, Yokohama 223-8522, Japan
a r t i c l e
i n f o
Article history: Received 7 January 2014 Received in revised form 19 February 2014 Accepted 6 March 2014 Available online 15 March 2014 Keywords: Bioactive compounds Diterpene Lamiaceae Medicinal plant Plant growth inhibitor
a b s t r a c t Leucas aspera (Lamiaceae), an aromatic herbaceous plant, is well known for many medicinal properties and a number of bioactive compounds against animal cells have been isolated. However, phytotoxic substances from L. aspera have not yet been documented in the literature. Therefore, current research was conducted to explore the phytotoxic properties and substances in L. aspera. Aqueous methanol extracts of L. aspera inhibited the germination and growth of garden cress (Lepidum sativum) and barnyard grass (Echinochloa crus-galli), and the inhibitory activities were concentration dependent. These results suggest that the plant may have phytotoxic substances. The extracts were then purified by several chromatographic runs. The final purification was achieved by reversed-phase HPLC to give an equilibrium (or inseparable) 3:2 mixture of two labdane type diterpenes (compounds 1 and 2). These compounds were characterized as (rel 5S,6R,8R,9R,10S,13S,15S,16R)-6-acetoxy-9,13;15,16-diepoxy-15-hydroxy-16-methoxylabdane (1) and (rel 5S,6R,8R,9R,10S,13S,15R,16R)-6-acetoxy-9,13;15,16-diepoxy-15-hydroxy-16-methoxylabdane (2) by spectroscopic analyses. A mixture of the two compounds inhibits the germination and seedling growth of garden cress and barnyard grass at concentrations greater than 30 and 3 M, respectively. The concentration required for 50% growth inhibition (I50 ) of the test species ranges from 31 to 80 M, which suggests that the mixture of these compounds, are responsible for the phytotoxic activity of L. aspera plant extract. © 2014 Elsevier GmbH. All rights reserved.
Introduction Phytotoxic substances are released into the surrounding environment from both above and below ground parts of phytotoxic plants through a number of processes (Rice, 1984; Weir et al., 2004). Upon release, these substances inhibited the germination and growth of other adjacent and/or succeeding plants, or even the secreting plant itself by affecting their many physiological properties (Weir et al., 2004; Yu et al., 2003). These phytotoxic substances also have the potential to modify rhizosphere soil properties including microbial biomass carbon and microbial community (Zhou et al., 2013), which may affect the properties of adjacent and/or succeeding plant species (Callaway and Aschehoug, 2000). Therefore, those phytotoxic plants especially their phytotoxic substances
Abbreviations: DW, dry weight; I50 , concentration required for 50% growth inhibition. ∗ Corresponding author. Tel.: +81 09028961982. E-mail address: [email protected] (A.K.M.M. Islam). http://dx.doi.org/10.1016/j.jplph.2014.03.003 0176-1617/© 2014 Elsevier GmbH. All rights reserved.
have received special attention due to their agricultural potential to develop natural herbicides for eco-friendly weed management strategies (Duke et al., 2000; Macías et al., 2007). Leucas aspera is a medicinally important herbaceous plant belonging to Lamiaceae (Labiatae) family. It grows as a competitive weed in high land crop fields, homesteads, fallow lands, and along the roadsides of both tropical and temperate Asia, and Africa. The plant is also well-known to traditional healers due to its antioxidant, analgesic-antipyretic, anti-rheumatic, anti-inflammatory, anti-bacterial, anti-fungal, anti-venom, larvicidal and many other medicinal properties (Prajapati et al., 2010; Srinivasan et al., 2011). A number of chemical constituents have been isolated and characterized from the different parts of L. aspera (Prajapati et al., 2010, Srinivasan et al., 2011). Although the phytochemical study of L. aspera was started many years ago (Shirazi, 1947), no phytotoxic substance has been reported except for a few preliminary phytotoxicity bioassay studies (Islam and Kato-Noguchi, 2012, 2013a; Roy et al., 2006). In the present work, we isolated and characterized two novel phytotoxic compounds from the aqueous methanol extract of L. aspera.
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Materials and methods Plant materials Plants of Leucas aspera (Willd.) Link were collected from the field laboratory of Bangladesh Agricultural University, Mymensingh2202, Bangladesh during July–August 2011. After collection, plants were washed with tap water; sun dried and then kept in a refrigerator at 2 ◦ C until extraction. Garden cress (Lepidum sativum L.) and barnyard grass (Echinochloa crus-galli (L.) Beauv.) seeds/seedlings were used for bioassays. Garden cress was chosen due to its known seedling growth pattern and higher sensitivity to phytotoxic substances (Xuan et al., 2005), and barnyard grass was chosen as it is the most common paddy weed throughout the world and has developed high resistance against many synthetic herbicides (Heap, 2013). Extraction and bioassay Whole plants (leaves, stem and roots) of dried L. aspera (1.6 kg) were cut into small pieces and extracted with 9 L of 70% (v/v) aqueous methanol for 48 h. After filtration using one layer of filter paper (No. 2; Advantec, Toyo Roshi Kaisha Ltd., Tokyo, Japan), the residue was re-extracted with same volume of methanol for another 48 h and filtered, and two filtrates were combined and evaporated. An aliquot of the extract (final assay concentration was 3, 10, 30 and 100 mg dry weight [DW] equivalent extract/mL) was evaporated to dryness at 40 ◦ C, dissolved in methanol and added to a sheet of filter paper (No. 2) in 28 mm Petri dishes. The methanol was evaporated in a draft chamber then the filter paper was moistened with 0.6 mL of 0.05% (v/v) aqueous solution of Tween 20 (polyoxyethylene sorbitan monolaurate; Nacalai Tesque, Inc., Kyoto, Japan). Ten seeds of garden cress or barnyard grass were placed on the filter paper in Petri dishes. The Petri dishes were then incubated in a dark chamber at 25 ◦ C. Germination was considered when the radical emerge by rupturing the seed coat as per Faria et al. (2005), and was measured at every 12 h intervals up to 48 h (the time when germination became constant). The germination percentage to controls (without extracts) was determined as per Islam and Kato-Noguchi (2013b). For growth bioassay, 10 seeds of garden cress or 10 seedlings of barnyard grass (germinated in the darkness at 25 ◦ C for 24 h) were sown in the Petri dishes. The shoot and root lengths of the seedlings were calculated at 48 h after incubation in darkness at 25 ◦ C. The percentage length of seedlings was then determined by reference to the length of control (without extracts) seedlings.
(50 g, GE Healthcare Bio-Sciences AB, Uppsala, Sweden), and eluted with 20, 40, 50, 60, 70 and 80% (v/v) of aqueous methanol and methanol (300 mL per step). The most active fraction eluted at 50% aqueous methanol was dissolved in 20% (v/v) aqueous methanol (1.0 mL) and loaded onto reverse-phase C18 cartridges (YMC Co. Ltd., Kyoto, Japan). The cartridge was eluted with 20, 40, 50, 60, 70 and 80% (v/v) aqueous methanol and methanol (30 mL per step). The active fraction obtained from 70% aqueous methanol was finally purified by reverse phase HPLC (HP 3 m, 4.6 × 250 mm I.D., Inertsil ODS-3, GL Science Inc., Tokyo, Japan) eluted at a flow rate of 0.5 mL/min with 60% (v/v) aqueous methanol and detected at 220 nm with 40 ◦ C oven temperature. Inhibitory activity was found in a peak fraction eluted between 175 and 200 min as a colorless substance. The fraction gave an equilibrium (or inseparable) 3:2 mixture of compounds 1 and 2. The compounds were characterized by high-resolution ESI mass data, and 1D and 2D NMR spectra. Compounds 1 and 2: HRESIMS m/z 433.2558 [M+Na]+ , = −0.8 mmu (calcd for C23 H38 O6 Na, 433.2566); [˛]D 18 −28.4◦ (c 0.2, acetone) for a mixture with the ratio of 3:2; 1 H NMR of 1 (600 MHz, CD3 OD) ıH 5.39 (ddd, J = 5.5, 5.5, 2.8 Hz, H6), 5.29 (dd, J = 5.8, 6.9 Hz, H15), 4.26 (s, H16), 3.41 (s, 3H, 16OCH3 ), 2.26 (dd, J = 6.9, 12.4 Hz, H14b), 2.19 (m, H11b), 2.14 (dd, J = 5.5, 12.4 Hz, H14a), 2.08 (m, H8), 2.02 (s, 3H, H2 ), 1.91 (m, H12a), 1.91 (m, H12b), 1.73 (m, H11a), 1.72 (m, H2b), 1.71 (m, H1b), 1.70 (m, H5), 1.66 (m, H7b), 1.51 (m, H2a), 1.45 (ddd, J = 3.1, 6.2, 14.4 Hz, H7a), 1.37 (m, H1a), 1.35 (m, H3b), 1.25 (d, J = 3.4 Hz, 3H, H20), 1.20 (m, H3a), 1.00 (s, 3H, H19), 0.94 (s, 3H, H18), 0.86 (d, J = 6.5 Hz, 3H, H17); 13 C NMR of 1 (150 MHz, CD OD) ␦ 172.5 (C1 ), 106.4 (C16), 97.8 3 C (C15), 94.6 (C9), 90.6 (C13), 72.1 (C6), 54.9 (16OCH3 ), 49.7 (C5), 45.2 (C3), 44.1 (C10), 44.0 (C14), 37.9 (C12), 37.7 (C7), 35.1 (C4), 33.6 (C1), 33.4 (C18), 32.9 (C8), 30.0 (C11), 24.2 (C19), 21.9 (C2 ), 20.2 (C20), 20.0 (C2), 17.5 (C17); 1 H NMR of 2 (600 MHz, CD3 OD) ıH 5.45 (d, J = 5.8 Hz, H15), 5.39 (ddd, J = 5.5, 5.5, 2.8 Hz, H6), 4.37 (s, H16), 3.37 (s, 3H, 16OCH3 ), 2.54 (dd, J = 6.5, 12.7 Hz, H14b), 2.19 (m, H11b), 2.10 (m, H12a), 2.10 (m, H12b), 2.08 (m, H8), 2.02 (s, 3H, H2 ), 1.76 (m, H14a), 1.73 (m, H11a), 1.72 (m, H2b), 1.71 (m, H1b), 1.70 (m, H5), 1.66 (m, H7b), 1.51 (m, H2a), 1.45 (ddd, J = 3.1, 6.2, 14.4 Hz, H7a), 1.37 (m, H1a), 1.35 (m, H3b), 1.25 (d, J = 3.4 Hz, 3H, H20), 1.20 (m, H3a), 1.00 (s, 3H, H19), 0.94 (s, 3H, H18), 0.86 (d, J = 6.5 Hz, 3H, H17); 13 C NMR of 2 (150 MHz, CD3 OD) ıC 172.5 (C1 ), 108.0 (C16), 97.3 (C15), 94.4 (C9), 89.7 (C13), 72.1 (C6), 54.8 (16OCH3 ), 49.7 (C5), 45.2 (C3), 44.1 (C10), 44.1 (C14), 39.3 (C12), 37.7 (C7), 35.1 (C4), 33.6 (C1), 33.4 (C18), 32.9 (C8), 30.0 (C11), 24.2 (C19), 21.9 (C2 ), 20.2 (C20), 20.0 (C2), 17.5 (C17).
Bioassay of the isolated compounds Purification of active substance Whole plants of dried L. aspera were extracted as stated above and evaporated with a rotary evaporator at 40 ◦ C to produce an aqueous residue. The aqueous residue was then divided into two equal parts, and each part was adjusted to pH 7.0 with 1 M phosphate buffer and partitioned three times against an equal volume of ethyl acetate to yield aqueous and ethyl acetate fractions. The biological activity of these two fractions was measured by garden cress and barnyard grass growth bioassay. The ethyl acetate fraction was then separated by silica gel column (60 g of silica gel 60, spherical, 70–230 mesh, Nacalai Tesque, Inc.), eluted stepwise with n-hexane containing increasing amounts of ethyl acetate (10% per step, v/v), ethyl acetate, acetone and methanol (300 mL per step). The biological activity of the fractions was determined using garden cress bioassay according to the aforesaid procedure, and inhibitory activity was found in the fraction obtained with 60% n-hexane in ethyl acetate. After evaporation, the active residue was applied to Sephadex LH-20
The mixture of these compounds was dissolved in 0.2 mL of methanol to prepare assay concentrations, added to a sheet of filter paper (No. 2) in 28 mm Petri dishes. Ten seeds of garden cress, or 10 seeds (for germination bioassay) or seedlings (for growth bioassay) of barnyard grass were sown on the Petri dishes and biological activity was determined by the aforementioned procedure.
Statistical analysis All the bioassays were conducted with three replications and repeated twice using a completely randomized design with 10 seeds/seedlings for each determination. Significant differences between treatment and controls were examined by Student’s ttest for each test plant species. The concentration required for 50% growth inhibition (I50 ) of the test species in the assay was determined from the regression equation of the concentration response curves.
A.K.M.M. Islam et al. / Journal of Plant Physiology 171 (2014) 877–883
Fig. 1. Effects of aqueous methanol extract of L. aspera on the germination of garden cress and barnyard grass. Concentrations of tested samples corresponded to the extracts obtained from 3, 10, 30 and 100 mg dry weight of L. aspera. Vertical bars represent error bars with standard deviations. Means ± SE from three independent experiments with 10 seeds for each determination are shown. Asterisks indicate a significant difference between control and treatment *p < 0.05 and **p < 0.01.
Results Phytotoxicity of L. aspera plant extracts Aqueous methanol extract of L. aspera inhibited the germination and growth of garden cress and barnyard grass, and the inhibitory activity was concentration dependent. The inhibitory activity was more prominent on garden cress than barnyard grass. At 100 mg DW equivalent extract/mL, the germination of garden cress was completely inhibited, whereas that of barnyard grass was 31% of control at 48 h after incubation (Fig. 1). At the same concentration the shoot and root growth of garden cress were inhibited by 15% and 4% of control, whereas those of the barnyard grass were 28% and 0% of control, respectively (data not shown). These results indicate that L. aspera plant extracts have phytotoxic potential and may possess phytotoxic substances. As seedling growth of the test species was more sensitive than germination, throughout the whole purification steps the biological activity of the extract was measured by growth bioassay. Concentration and test-plant-species dependent inhibitory activities on the shoot and root growth of garden cress and barnyard grass were also observed on both ethyl acetate and aqueous fractions of the plant extract (Figs. 2 and 3). At 100 mg DW equivalent extract/mL, ethyl acetate fraction completely inhibited the shoot and root growth of garden cress (Fig. 2), and inhibited the shoot and root growth of barnyard grass to 58 and 2% of control (Fig. 3), respectively. At the same concentration, aqueous fraction inhibited the shoot growth of garden cress and barnyard grass to 18 and 45% of control, respectively; and inhibited the root growth of garden cress and barnyard grass to 9 and 14% of control (Figs. 2 and 3),
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Fig. 2. Effects of ethyl acetate and aqueous fractions isolated from an aqueous methanol extract of L. aspera on growth of garden cress. Concentrations of tested samples corresponded to the extracts obtained from 3, 10, 30 and 100 mg dry weight of L. aspera. Vertical bars represent error bars with standard deviations. Means ± SE from three independent experiments with 10 seeds for each determination are shown. Asterisks indicate a significant difference between control and treatment *p < 0.05, **p < 0.01 and ***p < 0.001.
respectively. As the inhibitory activity of the ethyl acetate fraction was greater than that of the aqueous fraction, the purification process was further continued with the ethyl acetate fraction. Structure determination of compounds 1 and 2 The ethyl acetate fraction was then purified through silica gel column, Sephadex LH 20 column and reverse phase C18 cartridges. The biological activity of all fractions was determined using garden cress growth bioassay. Final purification of the compounds was achieved by reversed-phase HPLC (ODS, MeOH–H2 O) to give an equilibrium (or inseparable) 3:2 mixture of compounds 1 and 2 (Fig. 4). The molecular formula of both 1 and 2 was determined to be C23 H38 O6 by HRESIMS (m/z 433.2558, calcd for C23 H38 O6 Na [M+Na]+ , 433.2566). The 1 H NMR spectrum showed duplicate signals (3:2) for H12ab, H14ab, H15, H16 and 16OCH3 suggesting the presence of epimeric isomers. An NMR study (1 H and 13 C NMR, DEPT, COSY, and HSQC) and considering the molecular formula it is apparent that both 1 and 2 contained six methyl groups including one methoxy, seven methylenes, five methines including three oxymethines, and five quaternary carbons. The 1 H and 13 C NMR signals (Table 1) were assigned from these observations. A detailed analysis of the COSY, HSQC and HMBC spectra of 1 and 2 in CD3 OD allowed us to elucidate the following four partial structures: C1-C3, C5-C17, C11-C12, and C14-C15. The connectivity of these fragments was elucidated based on the HMBC techniques (Fig. 5). HMBC crosspeaks of H3, H5, H18, and H19 to quaternary carbon C4 (ıC 35.1), H18 and H19 to C5 (ıC 49.7), and H18 and H19 to C3 (ıC 45.2) established the connection among C3, C4, C5, C18, and C19. HMBC correlations from H5, H6, and H20 to quaternary carbon C10
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Fig. 5. COSY and HMBC correlations of compounds 1 and 2.
Fig. 3. Effects of ethyl acetate and aqueous fractions isolated from an aqueous methanol extract of L. aspera on growth of barnyard grass. Concentrations of tested samples corresponded to the extracts obtained from 3, 10, 30 and 100 mg dry weight of L. aspera. Vertical bars represent error bars with standard deviations. Means ± SE from three independent experiments with 10 seeds for each determination are shown. Asterisks indicate a significant difference between control and treatment *p < 0.05, **p < 0.01 and ***p < 0.001.
(ıC 44.1) and C9 (ıC 51.7), and of H20 to C1 (ıC 33.6) constructed the connection of C1, C5, and C20 to C10. Meanwhile, the connectivity among C8, C9, C10, and C11 was elucidated based on HMBC correlations of H7a, H12ab, H17, H20 to quaternary carbon C9 (ıC 94.6 for 1, 94.4 for 2). The connectivity among C12, C13, C14, and C16 was also elucidated based on HMBC correlations of H14ab and H16 to quaternary carbon C13 (ıC 90.6 for 1, 89.7 for 2), H12ab, H14a and H15 to C16 (ıC 106.4 for 1, 108.0 for 2), and H14ab and H16 to
C12 (␦C 37.9 for 1, 39.3 for 2). The HMBC crosspeaks of H6 and H2 to the carbonyl carbon C1 (ıC 172.5) revealed an acetoxy group at C6. Furthermore, the methoxy group was determined to be located at C16 based on the HMBC correlation between them. Thus, the planar structure of 1 and 2 could be determined to be a labdane-type diterpene possessing two spiro-tetrahydrofuran rings. The relative stereostructures of 1 and 2 were determined based on the NOE correlations observed in the NOESY spectrum (Fig. 6). The NOE correlations at H1a/H5, H2a/H19, H2a/H20, H5/H6, H5/H7b, H7a/H17, H6/H18, and H8/H20 revealed that the 6, 6-bicyclic ring system was present in a chair–chair conformation. The NOE correlations of H11a/H20, H14a/H17, H12ab/H16, and H16/H14a confirmed the relative stereochemistries at C9 and C13. The NOE correlation at H14a/H15 of 1 and the correlation at H14b/H15 of 2 revealed their relative stereochemistries at C15, and thus, 2 was concluded to be the 15-epimer of 1. As shown above, the structures of 1 and 2 were determined to be (rel 5S,6R,8R,9R,10S,13S,15S,16R)-6acetoxy-9,13;15,16-diepoxy-15-hydroxy-16-methoxylabdane (1) and (rel 5S,6R,8R,9R,10S,13S,15R,16R)-6-acetoxy-9,13;15,16diepoxy-15-hydroxy-16-methoxylabdane (2) (Fig. 4). They were identified as the analogs of (rel 5S,6R,8R,9R,10S,13S,15S,16R)-6acetoxy-9,13;15,16-diepoxy-15,16-dimethoxylabdane and (rel 5S,6R,8R,9R,10S,13S,15R,16R)-6-acetoxy-9,13;15,16-diepoxy-15,
Fig. 4. Structures of the compounds.
A.K.M.M. Islam et al. / Journal of Plant Physiology 171 (2014) 877–883
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Table 1 NMR data for compounds 1 and 2 in CD3 OD. Position
1
2
ıH /ppm (mult., J in Hz)a 1a 1b 2a 2b 3a 3b 4 5 6 7a 7b 8 9 10 11a 11b 12a 12b 13 14a 14b 15 16 17 18 19 20 1 2 16OCH3 a b
ıC /ppmb
1.37 (m) 1.71 (m) 1.51 (m) 1.72 (m) 1.20 (m) 1.35 (m)
33.6
1.37 (m) 1.71 (m) 1.51 (m) 1.72 (m) 1.20 (m) 1.35 (m)
20.0 45.2 35.1 49.7 72.1 37.7
1.70 (m) 5.39 (ddd, 5.5, 5.5, 2.8) 1.45 (ddd, 3.1, 6.2, 14.4) 1.66 (m) 2.08 (m)
1.70 (m) 5.39 (ddd, 5.5, 5.5, 2.8) 1.45 (ddd, 3.1, 6.2, 14.4) 1.66 (m) 2.08 (m)
32.9 94.6 44.1 30.0
1.73 (m) 2.19 (m) 1.91 (m) 1.91 (m)
1.73 (m) 2.19 (m) 2.10 (m) 2.10 (m)
37.9 90.6 44.0
2.14 (dd, 5.5, 12.4) 2.26 (dd, 6.9, 12.4) 5.29 (dd, 5.8, 6.9) 4.26 (s) 0.86 (d, 6.5, 3H) 0.94 (s, 3H) 1.00 (s, 3H) 1.25 (d, 3.4, 3H)
1.76 (m) 2.54 (dd, 6.5, 12.7) 5.45 (d, 5.8) 4.37 (s) 0.86 (d, 6.5, 3H) 0.94 (s, 3H) 1.00 (s, 3H) 1.25 (d, 3.4, 3H)
97.8 106.4 17.5 33.4 24.2 20.2 172.5 21.9 54.9
2.02 (s, 3H) 3.41 (s, 3H)
ıH /ppm (mult., J in Hz)a
2.02 (s, 3H) 3.37 (s, 3H)
ıC /ppmb 33.6 20.0 45.2 35.1 49.7 72.1 37.7 32.9 94.4 44.1 30.0 39.3 89.7 44.1 97.3 108.0 17.5 33.4 24.2 20.2 172.5 21.9 54.8
Recorded at 600 MHz. Recorded at 150 MHz.
16-dimethoxylabdane, which were isolated from the fruit of Vitex rotundifolia (Ono et al., 1999). Effects of the isolated compounds on seed germination and seedling growth The compounds mixture was evaluated for their germination and growth inhibitory activities using seeds of garden cress and barnyard grass (Figs. 7 and 8). At concentrations greater than 30 M the mixture significantly inhibited the germination of both test species and a complete inhibition was observed at 300 M or greater than that concentration (Fig. 7). The shoot and root growth of garden cress and barnyard grass were significantly inhibited by the mixture at concentrations greater than 3 M (Fig. 8). The inhibition increased with increasing concentrations. The concentrations required for 50% growth
inhibition (I50 ) of garden cress shoots and roots were 31.1 and 56.9 M, respectively, whereas for barnyard grass they were 79.8 and 33.0 M, respectively. The I50 values for the shoots and roots of garden cress were 2.6-fold lower, and 1.7-fold higher than that of the barnyard grass, respectively. The results indicate that the mixture of these compounds (compounds 1 and 2) are phytotoxic, and are responsible for the phytotoxic activity of L. aspera extract.
Discussion The aqueous methanol extract of L. aspera showed a concentration dependent inhibitory activity on the seed germination and seedling growth of garden cress and barnyard grass. The seedling growth is more sensitive to the extract than seed germination. The higher sensitivity of seedling growth to phytotoxic substances was
Fig. 6. NOESY correlations of compounds 1 and 2.
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Fig. 7. Effects of compounds 1 and 2 mixtures on the germination of garden cress and barnyard grass. Means ± SE from three independent experiments with 10 seedlings for each determination are shown.
also reported by Rasmussen and Einhellig (1977), and Williams and Hoagland (1982). The results suggest that the aqueous methanol extract of L. aspera is phytotoxic and may possess phytotoxic substances. Two novel phytotoxic substances from the plant extracts were isolated and characterized as an inseparable mixture of C15 epimeric diterpenes in the proportion of 3:2 (compound 1:compound 2). Those compounds were unable to separate, even though different solvent compositions plus a number of columns have been used. This could be due to: (i) either those two compounds may co-exist in nature as equilibrium, or (ii) their fast inter-conversion reactions. An inseparable mixture of C15 epimeric diterpenes was also reported from L. neufliseana, another species of the same genus by Khalil et al. (1996). Labdane type diterpenes are reported to have a broad spectrum of biological activities such as antimycobacterial (Kulkarni et al., 2013), antimicrobial (Souza et al., 2011), antifeedant (Bohlmann et al., 1982), cytotoxic activities (Zani et al., 2000) and prostaglandin-induced contractions inhibitors (Sadhu et al., 2006). However, report about their phytotoxic activities is very infrequent (DellaGreca et al., 2000). Besides, a number of compounds had been isolated from the different parts of L. aspera (Chouhan and Singh, 2011; Gerige et al., 2007; Mangathayaru et al., 2006; Pradhan et al., 1990; Sadhu et al., 2006), none of them are reported to have phytotoxic potential. Therefore, the findings of this research could explore new dimension for the phytotoxic activity of labdane type diterpenes, more specifically the phytotoxic activity of L. aspera with other adjacent plant species under natural settings. This is the first report of the phytotoxic activities of compounds 1 and 2 mixtures, and their presence in L. aspera. In summary, the aqueous methanol extracts of L. aspera are phytotoxic, and thus contain phytotoxic substances. An equilibrium (or inseparable) 3:2 mixture of two labdane type diterpenes, (rel 5S,6R,8R,9R,10S,13S,15S,16R)-6-acetoxy9,13;15,16-diepoxy-15-hydroxy-16-methoxylabdane (1) and (rel 5S,6R,8R,9R,10S,13S,15R,16R)-6-acetoxy-9,13;15,16-diepoxy-15hydroxy-16-methoxylabdane (2) were isolated and characterized from the plant extracts. The data generated in this study showed that the isolated compounds mixture have inhibitory activity on germination and seedling growth of both target species at concentrations greater than 30 and 3 M, respectively. The I50 values of these two compounds for garden cress and barnyard grass lies in between 31 and 80 M. The shoot growth of garden cress was more sensitive to the compounds mixture than that of barnyard grass. On the other hand, root growth of barnyard grass was more sensitive than that of garden cress. Therefore, we conclude that the mixtures of compounds 1 and 2 are phytotoxic and might play an important role in the phytotoxic activity of L. aspera under natural settings.
References
Fig. 8. Effects of compounds 1 and 2 mixtures on the shoot and root growth of garden cress and barnyard grass seedlings. Means ± SE from three independent experiments with 10 seedlings for each determination are shown.
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Contents lists available at ScienceDirect
Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph
Physiology
Two novel phytotoxic substances from Leucas aspera A.K.M. Mominul Islam a,∗ , Osamu Ohno b , Kiyotake Suenaga b , Hisashi Kato-Noguchi a a b
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki, Kagawa 761-0795, Japan Department of Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku, Yokohama 223-8522, Japan
a r t i c l e
i n f o
Article history: Received 7 January 2014 Received in revised form 19 February 2014 Accepted 6 March 2014 Available online 15 March 2014 Keywords: Bioactive compounds Diterpene Lamiaceae Medicinal plant Plant growth inhibitor
a b s t r a c t Leucas aspera (Lamiaceae), an aromatic herbaceous plant, is well known for many medicinal properties and a number of bioactive compounds against animal cells have been isolated. However, phytotoxic substances from L. aspera have not yet been documented in the literature. Therefore, current research was conducted to explore the phytotoxic properties and substances in L. aspera. Aqueous methanol extracts of L. aspera inhibited the germination and growth of garden cress (Lepidum sativum) and barnyard grass (Echinochloa crus-galli), and the inhibitory activities were concentration dependent. These results suggest that the plant may have phytotoxic substances. The extracts were then purified by several chromatographic runs. The final purification was achieved by reversed-phase HPLC to give an equilibrium (or inseparable) 3:2 mixture of two labdane type diterpenes (compounds 1 and 2). These compounds were characterized as (rel 5S,6R,8R,9R,10S,13S,15S,16R)-6-acetoxy-9,13;15,16-diepoxy-15-hydroxy-16-methoxylabdane (1) and (rel 5S,6R,8R,9R,10S,13S,15R,16R)-6-acetoxy-9,13;15,16-diepoxy-15-hydroxy-16-methoxylabdane (2) by spectroscopic analyses. A mixture of the two compounds inhibits the germination and seedling growth of garden cress and barnyard grass at concentrations greater than 30 and 3 M, respectively. The concentration required for 50% growth inhibition (I50 ) of the test species ranges from 31 to 80 M, which suggests that the mixture of these compounds, are responsible for the phytotoxic activity of L. aspera plant extract. © 2014 Elsevier GmbH. All rights reserved.
Introduction Phytotoxic substances are released into the surrounding environment from both above and below ground parts of phytotoxic plants through a number of processes (Rice, 1984; Weir et al., 2004). Upon release, these substances inhibited the germination and growth of other adjacent and/or succeeding plants, or even the secreting plant itself by affecting their many physiological properties (Weir et al., 2004; Yu et al., 2003). These phytotoxic substances also have the potential to modify rhizosphere soil properties including microbial biomass carbon and microbial community (Zhou et al., 2013), which may affect the properties of adjacent and/or succeeding plant species (Callaway and Aschehoug, 2000). Therefore, those phytotoxic plants especially their phytotoxic substances
Abbreviations: DW, dry weight; I50 , concentration required for 50% growth inhibition. ∗ Corresponding author. Tel.: +81 09028961982. E-mail address: [email protected] (A.K.M.M. Islam). http://dx.doi.org/10.1016/j.jplph.2014.03.003 0176-1617/© 2014 Elsevier GmbH. All rights reserved.
have received special attention due to their agricultural potential to develop natural herbicides for eco-friendly weed management strategies (Duke et al., 2000; Macías et al., 2007). Leucas aspera is a medicinally important herbaceous plant belonging to Lamiaceae (Labiatae) family. It grows as a competitive weed in high land crop fields, homesteads, fallow lands, and along the roadsides of both tropical and temperate Asia, and Africa. The plant is also well-known to traditional healers due to its antioxidant, analgesic-antipyretic, anti-rheumatic, anti-inflammatory, anti-bacterial, anti-fungal, anti-venom, larvicidal and many other medicinal properties (Prajapati et al., 2010; Srinivasan et al., 2011). A number of chemical constituents have been isolated and characterized from the different parts of L. aspera (Prajapati et al., 2010, Srinivasan et al., 2011). Although the phytochemical study of L. aspera was started many years ago (Shirazi, 1947), no phytotoxic substance has been reported except for a few preliminary phytotoxicity bioassay studies (Islam and Kato-Noguchi, 2012, 2013a; Roy et al., 2006). In the present work, we isolated and characterized two novel phytotoxic compounds from the aqueous methanol extract of L. aspera.
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Materials and methods Plant materials Plants of Leucas aspera (Willd.) Link were collected from the field laboratory of Bangladesh Agricultural University, Mymensingh2202, Bangladesh during July–August 2011. After collection, plants were washed with tap water; sun dried and then kept in a refrigerator at 2 ◦ C until extraction. Garden cress (Lepidum sativum L.) and barnyard grass (Echinochloa crus-galli (L.) Beauv.) seeds/seedlings were used for bioassays. Garden cress was chosen due to its known seedling growth pattern and higher sensitivity to phytotoxic substances (Xuan et al., 2005), and barnyard grass was chosen as it is the most common paddy weed throughout the world and has developed high resistance against many synthetic herbicides (Heap, 2013). Extraction and bioassay Whole plants (leaves, stem and roots) of dried L. aspera (1.6 kg) were cut into small pieces and extracted with 9 L of 70% (v/v) aqueous methanol for 48 h. After filtration using one layer of filter paper (No. 2; Advantec, Toyo Roshi Kaisha Ltd., Tokyo, Japan), the residue was re-extracted with same volume of methanol for another 48 h and filtered, and two filtrates were combined and evaporated. An aliquot of the extract (final assay concentration was 3, 10, 30 and 100 mg dry weight [DW] equivalent extract/mL) was evaporated to dryness at 40 ◦ C, dissolved in methanol and added to a sheet of filter paper (No. 2) in 28 mm Petri dishes. The methanol was evaporated in a draft chamber then the filter paper was moistened with 0.6 mL of 0.05% (v/v) aqueous solution of Tween 20 (polyoxyethylene sorbitan monolaurate; Nacalai Tesque, Inc., Kyoto, Japan). Ten seeds of garden cress or barnyard grass were placed on the filter paper in Petri dishes. The Petri dishes were then incubated in a dark chamber at 25 ◦ C. Germination was considered when the radical emerge by rupturing the seed coat as per Faria et al. (2005), and was measured at every 12 h intervals up to 48 h (the time when germination became constant). The germination percentage to controls (without extracts) was determined as per Islam and Kato-Noguchi (2013b). For growth bioassay, 10 seeds of garden cress or 10 seedlings of barnyard grass (germinated in the darkness at 25 ◦ C for 24 h) were sown in the Petri dishes. The shoot and root lengths of the seedlings were calculated at 48 h after incubation in darkness at 25 ◦ C. The percentage length of seedlings was then determined by reference to the length of control (without extracts) seedlings.
(50 g, GE Healthcare Bio-Sciences AB, Uppsala, Sweden), and eluted with 20, 40, 50, 60, 70 and 80% (v/v) of aqueous methanol and methanol (300 mL per step). The most active fraction eluted at 50% aqueous methanol was dissolved in 20% (v/v) aqueous methanol (1.0 mL) and loaded onto reverse-phase C18 cartridges (YMC Co. Ltd., Kyoto, Japan). The cartridge was eluted with 20, 40, 50, 60, 70 and 80% (v/v) aqueous methanol and methanol (30 mL per step). The active fraction obtained from 70% aqueous methanol was finally purified by reverse phase HPLC (HP 3 m, 4.6 × 250 mm I.D., Inertsil ODS-3, GL Science Inc., Tokyo, Japan) eluted at a flow rate of 0.5 mL/min with 60% (v/v) aqueous methanol and detected at 220 nm with 40 ◦ C oven temperature. Inhibitory activity was found in a peak fraction eluted between 175 and 200 min as a colorless substance. The fraction gave an equilibrium (or inseparable) 3:2 mixture of compounds 1 and 2. The compounds were characterized by high-resolution ESI mass data, and 1D and 2D NMR spectra. Compounds 1 and 2: HRESIMS m/z 433.2558 [M+Na]+ , = −0.8 mmu (calcd for C23 H38 O6 Na, 433.2566); [˛]D 18 −28.4◦ (c 0.2, acetone) for a mixture with the ratio of 3:2; 1 H NMR of 1 (600 MHz, CD3 OD) ıH 5.39 (ddd, J = 5.5, 5.5, 2.8 Hz, H6), 5.29 (dd, J = 5.8, 6.9 Hz, H15), 4.26 (s, H16), 3.41 (s, 3H, 16OCH3 ), 2.26 (dd, J = 6.9, 12.4 Hz, H14b), 2.19 (m, H11b), 2.14 (dd, J = 5.5, 12.4 Hz, H14a), 2.08 (m, H8), 2.02 (s, 3H, H2 ), 1.91 (m, H12a), 1.91 (m, H12b), 1.73 (m, H11a), 1.72 (m, H2b), 1.71 (m, H1b), 1.70 (m, H5), 1.66 (m, H7b), 1.51 (m, H2a), 1.45 (ddd, J = 3.1, 6.2, 14.4 Hz, H7a), 1.37 (m, H1a), 1.35 (m, H3b), 1.25 (d, J = 3.4 Hz, 3H, H20), 1.20 (m, H3a), 1.00 (s, 3H, H19), 0.94 (s, 3H, H18), 0.86 (d, J = 6.5 Hz, 3H, H17); 13 C NMR of 1 (150 MHz, CD OD) ␦ 172.5 (C1 ), 106.4 (C16), 97.8 3 C (C15), 94.6 (C9), 90.6 (C13), 72.1 (C6), 54.9 (16OCH3 ), 49.7 (C5), 45.2 (C3), 44.1 (C10), 44.0 (C14), 37.9 (C12), 37.7 (C7), 35.1 (C4), 33.6 (C1), 33.4 (C18), 32.9 (C8), 30.0 (C11), 24.2 (C19), 21.9 (C2 ), 20.2 (C20), 20.0 (C2), 17.5 (C17); 1 H NMR of 2 (600 MHz, CD3 OD) ıH 5.45 (d, J = 5.8 Hz, H15), 5.39 (ddd, J = 5.5, 5.5, 2.8 Hz, H6), 4.37 (s, H16), 3.37 (s, 3H, 16OCH3 ), 2.54 (dd, J = 6.5, 12.7 Hz, H14b), 2.19 (m, H11b), 2.10 (m, H12a), 2.10 (m, H12b), 2.08 (m, H8), 2.02 (s, 3H, H2 ), 1.76 (m, H14a), 1.73 (m, H11a), 1.72 (m, H2b), 1.71 (m, H1b), 1.70 (m, H5), 1.66 (m, H7b), 1.51 (m, H2a), 1.45 (ddd, J = 3.1, 6.2, 14.4 Hz, H7a), 1.37 (m, H1a), 1.35 (m, H3b), 1.25 (d, J = 3.4 Hz, 3H, H20), 1.20 (m, H3a), 1.00 (s, 3H, H19), 0.94 (s, 3H, H18), 0.86 (d, J = 6.5 Hz, 3H, H17); 13 C NMR of 2 (150 MHz, CD3 OD) ıC 172.5 (C1 ), 108.0 (C16), 97.3 (C15), 94.4 (C9), 89.7 (C13), 72.1 (C6), 54.8 (16OCH3 ), 49.7 (C5), 45.2 (C3), 44.1 (C10), 44.1 (C14), 39.3 (C12), 37.7 (C7), 35.1 (C4), 33.6 (C1), 33.4 (C18), 32.9 (C8), 30.0 (C11), 24.2 (C19), 21.9 (C2 ), 20.2 (C20), 20.0 (C2), 17.5 (C17).
Bioassay of the isolated compounds Purification of active substance Whole plants of dried L. aspera were extracted as stated above and evaporated with a rotary evaporator at 40 ◦ C to produce an aqueous residue. The aqueous residue was then divided into two equal parts, and each part was adjusted to pH 7.0 with 1 M phosphate buffer and partitioned three times against an equal volume of ethyl acetate to yield aqueous and ethyl acetate fractions. The biological activity of these two fractions was measured by garden cress and barnyard grass growth bioassay. The ethyl acetate fraction was then separated by silica gel column (60 g of silica gel 60, spherical, 70–230 mesh, Nacalai Tesque, Inc.), eluted stepwise with n-hexane containing increasing amounts of ethyl acetate (10% per step, v/v), ethyl acetate, acetone and methanol (300 mL per step). The biological activity of the fractions was determined using garden cress bioassay according to the aforesaid procedure, and inhibitory activity was found in the fraction obtained with 60% n-hexane in ethyl acetate. After evaporation, the active residue was applied to Sephadex LH-20
The mixture of these compounds was dissolved in 0.2 mL of methanol to prepare assay concentrations, added to a sheet of filter paper (No. 2) in 28 mm Petri dishes. Ten seeds of garden cress, or 10 seeds (for germination bioassay) or seedlings (for growth bioassay) of barnyard grass were sown on the Petri dishes and biological activity was determined by the aforementioned procedure.
Statistical analysis All the bioassays were conducted with three replications and repeated twice using a completely randomized design with 10 seeds/seedlings for each determination. Significant differences between treatment and controls were examined by Student’s ttest for each test plant species. The concentration required for 50% growth inhibition (I50 ) of the test species in the assay was determined from the regression equation of the concentration response curves.
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Fig. 1. Effects of aqueous methanol extract of L. aspera on the germination of garden cress and barnyard grass. Concentrations of tested samples corresponded to the extracts obtained from 3, 10, 30 and 100 mg dry weight of L. aspera. Vertical bars represent error bars with standard deviations. Means ± SE from three independent experiments with 10 seeds for each determination are shown. Asterisks indicate a significant difference between control and treatment *p < 0.05 and **p < 0.01.
Results Phytotoxicity of L. aspera plant extracts Aqueous methanol extract of L. aspera inhibited the germination and growth of garden cress and barnyard grass, and the inhibitory activity was concentration dependent. The inhibitory activity was more prominent on garden cress than barnyard grass. At 100 mg DW equivalent extract/mL, the germination of garden cress was completely inhibited, whereas that of barnyard grass was 31% of control at 48 h after incubation (Fig. 1). At the same concentration the shoot and root growth of garden cress were inhibited by 15% and 4% of control, whereas those of the barnyard grass were 28% and 0% of control, respectively (data not shown). These results indicate that L. aspera plant extracts have phytotoxic potential and may possess phytotoxic substances. As seedling growth of the test species was more sensitive than germination, throughout the whole purification steps the biological activity of the extract was measured by growth bioassay. Concentration and test-plant-species dependent inhibitory activities on the shoot and root growth of garden cress and barnyard grass were also observed on both ethyl acetate and aqueous fractions of the plant extract (Figs. 2 and 3). At 100 mg DW equivalent extract/mL, ethyl acetate fraction completely inhibited the shoot and root growth of garden cress (Fig. 2), and inhibited the shoot and root growth of barnyard grass to 58 and 2% of control (Fig. 3), respectively. At the same concentration, aqueous fraction inhibited the shoot growth of garden cress and barnyard grass to 18 and 45% of control, respectively; and inhibited the root growth of garden cress and barnyard grass to 9 and 14% of control (Figs. 2 and 3),
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Fig. 2. Effects of ethyl acetate and aqueous fractions isolated from an aqueous methanol extract of L. aspera on growth of garden cress. Concentrations of tested samples corresponded to the extracts obtained from 3, 10, 30 and 100 mg dry weight of L. aspera. Vertical bars represent error bars with standard deviations. Means ± SE from three independent experiments with 10 seeds for each determination are shown. Asterisks indicate a significant difference between control and treatment *p < 0.05, **p < 0.01 and ***p < 0.001.
respectively. As the inhibitory activity of the ethyl acetate fraction was greater than that of the aqueous fraction, the purification process was further continued with the ethyl acetate fraction. Structure determination of compounds 1 and 2 The ethyl acetate fraction was then purified through silica gel column, Sephadex LH 20 column and reverse phase C18 cartridges. The biological activity of all fractions was determined using garden cress growth bioassay. Final purification of the compounds was achieved by reversed-phase HPLC (ODS, MeOH–H2 O) to give an equilibrium (or inseparable) 3:2 mixture of compounds 1 and 2 (Fig. 4). The molecular formula of both 1 and 2 was determined to be C23 H38 O6 by HRESIMS (m/z 433.2558, calcd for C23 H38 O6 Na [M+Na]+ , 433.2566). The 1 H NMR spectrum showed duplicate signals (3:2) for H12ab, H14ab, H15, H16 and 16OCH3 suggesting the presence of epimeric isomers. An NMR study (1 H and 13 C NMR, DEPT, COSY, and HSQC) and considering the molecular formula it is apparent that both 1 and 2 contained six methyl groups including one methoxy, seven methylenes, five methines including three oxymethines, and five quaternary carbons. The 1 H and 13 C NMR signals (Table 1) were assigned from these observations. A detailed analysis of the COSY, HSQC and HMBC spectra of 1 and 2 in CD3 OD allowed us to elucidate the following four partial structures: C1-C3, C5-C17, C11-C12, and C14-C15. The connectivity of these fragments was elucidated based on the HMBC techniques (Fig. 5). HMBC crosspeaks of H3, H5, H18, and H19 to quaternary carbon C4 (ıC 35.1), H18 and H19 to C5 (ıC 49.7), and H18 and H19 to C3 (ıC 45.2) established the connection among C3, C4, C5, C18, and C19. HMBC correlations from H5, H6, and H20 to quaternary carbon C10
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Fig. 5. COSY and HMBC correlations of compounds 1 and 2.
Fig. 3. Effects of ethyl acetate and aqueous fractions isolated from an aqueous methanol extract of L. aspera on growth of barnyard grass. Concentrations of tested samples corresponded to the extracts obtained from 3, 10, 30 and 100 mg dry weight of L. aspera. Vertical bars represent error bars with standard deviations. Means ± SE from three independent experiments with 10 seeds for each determination are shown. Asterisks indicate a significant difference between control and treatment *p < 0.05, **p < 0.01 and ***p < 0.001.
(ıC 44.1) and C9 (ıC 51.7), and of H20 to C1 (ıC 33.6) constructed the connection of C1, C5, and C20 to C10. Meanwhile, the connectivity among C8, C9, C10, and C11 was elucidated based on HMBC correlations of H7a, H12ab, H17, H20 to quaternary carbon C9 (ıC 94.6 for 1, 94.4 for 2). The connectivity among C12, C13, C14, and C16 was also elucidated based on HMBC correlations of H14ab and H16 to quaternary carbon C13 (ıC 90.6 for 1, 89.7 for 2), H12ab, H14a and H15 to C16 (ıC 106.4 for 1, 108.0 for 2), and H14ab and H16 to
C12 (␦C 37.9 for 1, 39.3 for 2). The HMBC crosspeaks of H6 and H2 to the carbonyl carbon C1 (ıC 172.5) revealed an acetoxy group at C6. Furthermore, the methoxy group was determined to be located at C16 based on the HMBC correlation between them. Thus, the planar structure of 1 and 2 could be determined to be a labdane-type diterpene possessing two spiro-tetrahydrofuran rings. The relative stereostructures of 1 and 2 were determined based on the NOE correlations observed in the NOESY spectrum (Fig. 6). The NOE correlations at H1a/H5, H2a/H19, H2a/H20, H5/H6, H5/H7b, H7a/H17, H6/H18, and H8/H20 revealed that the 6, 6-bicyclic ring system was present in a chair–chair conformation. The NOE correlations of H11a/H20, H14a/H17, H12ab/H16, and H16/H14a confirmed the relative stereochemistries at C9 and C13. The NOE correlation at H14a/H15 of 1 and the correlation at H14b/H15 of 2 revealed their relative stereochemistries at C15, and thus, 2 was concluded to be the 15-epimer of 1. As shown above, the structures of 1 and 2 were determined to be (rel 5S,6R,8R,9R,10S,13S,15S,16R)-6acetoxy-9,13;15,16-diepoxy-15-hydroxy-16-methoxylabdane (1) and (rel 5S,6R,8R,9R,10S,13S,15R,16R)-6-acetoxy-9,13;15,16diepoxy-15-hydroxy-16-methoxylabdane (2) (Fig. 4). They were identified as the analogs of (rel 5S,6R,8R,9R,10S,13S,15S,16R)-6acetoxy-9,13;15,16-diepoxy-15,16-dimethoxylabdane and (rel 5S,6R,8R,9R,10S,13S,15R,16R)-6-acetoxy-9,13;15,16-diepoxy-15,
Fig. 4. Structures of the compounds.
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Table 1 NMR data for compounds 1 and 2 in CD3 OD. Position
1
2
ıH /ppm (mult., J in Hz)a 1a 1b 2a 2b 3a 3b 4 5 6 7a 7b 8 9 10 11a 11b 12a 12b 13 14a 14b 15 16 17 18 19 20 1 2 16OCH3 a b
ıC /ppmb
1.37 (m) 1.71 (m) 1.51 (m) 1.72 (m) 1.20 (m) 1.35 (m)
33.6
1.37 (m) 1.71 (m) 1.51 (m) 1.72 (m) 1.20 (m) 1.35 (m)
20.0 45.2 35.1 49.7 72.1 37.7
1.70 (m) 5.39 (ddd, 5.5, 5.5, 2.8) 1.45 (ddd, 3.1, 6.2, 14.4) 1.66 (m) 2.08 (m)
1.70 (m) 5.39 (ddd, 5.5, 5.5, 2.8) 1.45 (ddd, 3.1, 6.2, 14.4) 1.66 (m) 2.08 (m)
32.9 94.6 44.1 30.0
1.73 (m) 2.19 (m) 1.91 (m) 1.91 (m)
1.73 (m) 2.19 (m) 2.10 (m) 2.10 (m)
37.9 90.6 44.0
2.14 (dd, 5.5, 12.4) 2.26 (dd, 6.9, 12.4) 5.29 (dd, 5.8, 6.9) 4.26 (s) 0.86 (d, 6.5, 3H) 0.94 (s, 3H) 1.00 (s, 3H) 1.25 (d, 3.4, 3H)
1.76 (m) 2.54 (dd, 6.5, 12.7) 5.45 (d, 5.8) 4.37 (s) 0.86 (d, 6.5, 3H) 0.94 (s, 3H) 1.00 (s, 3H) 1.25 (d, 3.4, 3H)
97.8 106.4 17.5 33.4 24.2 20.2 172.5 21.9 54.9
2.02 (s, 3H) 3.41 (s, 3H)
ıH /ppm (mult., J in Hz)a
2.02 (s, 3H) 3.37 (s, 3H)
ıC /ppmb 33.6 20.0 45.2 35.1 49.7 72.1 37.7 32.9 94.4 44.1 30.0 39.3 89.7 44.1 97.3 108.0 17.5 33.4 24.2 20.2 172.5 21.9 54.8
Recorded at 600 MHz. Recorded at 150 MHz.
16-dimethoxylabdane, which were isolated from the fruit of Vitex rotundifolia (Ono et al., 1999). Effects of the isolated compounds on seed germination and seedling growth The compounds mixture was evaluated for their germination and growth inhibitory activities using seeds of garden cress and barnyard grass (Figs. 7 and 8). At concentrations greater than 30 M the mixture significantly inhibited the germination of both test species and a complete inhibition was observed at 300 M or greater than that concentration (Fig. 7). The shoot and root growth of garden cress and barnyard grass were significantly inhibited by the mixture at concentrations greater than 3 M (Fig. 8). The inhibition increased with increasing concentrations. The concentrations required for 50% growth
inhibition (I50 ) of garden cress shoots and roots were 31.1 and 56.9 M, respectively, whereas for barnyard grass they were 79.8 and 33.0 M, respectively. The I50 values for the shoots and roots of garden cress were 2.6-fold lower, and 1.7-fold higher than that of the barnyard grass, respectively. The results indicate that the mixture of these compounds (compounds 1 and 2) are phytotoxic, and are responsible for the phytotoxic activity of L. aspera extract.
Discussion The aqueous methanol extract of L. aspera showed a concentration dependent inhibitory activity on the seed germination and seedling growth of garden cress and barnyard grass. The seedling growth is more sensitive to the extract than seed germination. The higher sensitivity of seedling growth to phytotoxic substances was
Fig. 6. NOESY correlations of compounds 1 and 2.
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Fig. 7. Effects of compounds 1 and 2 mixtures on the germination of garden cress and barnyard grass. Means ± SE from three independent experiments with 10 seedlings for each determination are shown.
also reported by Rasmussen and Einhellig (1977), and Williams and Hoagland (1982). The results suggest that the aqueous methanol extract of L. aspera is phytotoxic and may possess phytotoxic substances. Two novel phytotoxic substances from the plant extracts were isolated and characterized as an inseparable mixture of C15 epimeric diterpenes in the proportion of 3:2 (compound 1:compound 2). Those compounds were unable to separate, even though different solvent compositions plus a number of columns have been used. This could be due to: (i) either those two compounds may co-exist in nature as equilibrium, or (ii) their fast inter-conversion reactions. An inseparable mixture of C15 epimeric diterpenes was also reported from L. neufliseana, another species of the same genus by Khalil et al. (1996). Labdane type diterpenes are reported to have a broad spectrum of biological activities such as antimycobacterial (Kulkarni et al., 2013), antimicrobial (Souza et al., 2011), antifeedant (Bohlmann et al., 1982), cytotoxic activities (Zani et al., 2000) and prostaglandin-induced contractions inhibitors (Sadhu et al., 2006). However, report about their phytotoxic activities is very infrequent (DellaGreca et al., 2000). Besides, a number of compounds had been isolated from the different parts of L. aspera (Chouhan and Singh, 2011; Gerige et al., 2007; Mangathayaru et al., 2006; Pradhan et al., 1990; Sadhu et al., 2006), none of them are reported to have phytotoxic potential. Therefore, the findings of this research could explore new dimension for the phytotoxic activity of labdane type diterpenes, more specifically the phytotoxic activity of L. aspera with other adjacent plant species under natural settings. This is the first report of the phytotoxic activities of compounds 1 and 2 mixtures, and their presence in L. aspera. In summary, the aqueous methanol extracts of L. aspera are phytotoxic, and thus contain phytotoxic substances. An equilibrium (or inseparable) 3:2 mixture of two labdane type diterpenes, (rel 5S,6R,8R,9R,10S,13S,15S,16R)-6-acetoxy9,13;15,16-diepoxy-15-hydroxy-16-methoxylabdane (1) and (rel 5S,6R,8R,9R,10S,13S,15R,16R)-6-acetoxy-9,13;15,16-diepoxy-15hydroxy-16-methoxylabdane (2) were isolated and characterized from the plant extracts. The data generated in this study showed that the isolated compounds mixture have inhibitory activity on germination and seedling growth of both target species at concentrations greater than 30 and 3 M, respectively. The I50 values of these two compounds for garden cress and barnyard grass lies in between 31 and 80 M. The shoot growth of garden cress was more sensitive to the compounds mixture than that of barnyard grass. On the other hand, root growth of barnyard grass was more sensitive than that of garden cress. Therefore, we conclude that the mixtures of compounds 1 and 2 are phytotoxic and might play an important role in the phytotoxic activity of L. aspera under natural settings.
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Fig. 8. Effects of compounds 1 and 2 mixtures on the shoot and root growth of garden cress and barnyard grass seedlings. Means ± SE from three independent experiments with 10 seedlings for each determination are shown.
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