New Flavanol and Cycloartane Glucosides from Landoltia punctata

Chemical investigation on the constituents of Landoltia punctata led to the isolation and identification of 17 compounds, four of which were new and identified as (3β,24S)-9,19-cycloartane-3,22,24,25-tetraol 3-O-[β-d-glucopyranosyl-(1→2)]-[β-d-glucopyranosyl-(1→6)]-β-d-glucopyranoside (1), (3β,24S)-9,19-cycloartane-3,24,25-triol 3-O-[β-d-glucopyranosyl-(1→2)]-[β-d-glucopyranosyl-(1→6)]-β-d-glucopyranoside (2), 3,4'-dihydroxy-7,3'-dimethoxyflavan-5-O-β-d-glucopyranoside (3) and 3,4'-dihydroxy-4,7,3'-trimethoxyflavan-5-O-β-d-glucopyranoside (4). Their structures were elucidated by spectroscopic, chemical, and biochemical methods. Thus, cycloartane triterpenoids were discovered in the Lemnaceae family for the first time. Compound 3 showed antioxidant capacity in the positively charged 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical (ABTS+•) and superoxide anion radical scavenging assays.


Introduction
The plant of the Lemnaceae, commonly known as duckweed, is a kind of aquatic monocot angiosperm and the smallest with flowers, which is widely distributed on the surface of still, slowly flowing, and polluted waters around the World. In this family, there are 37 species, representing five genera: Lemna, Landoltia, Spirodela, Wolffia and Wolffiella [1,2]. Duckweeds grow very fast and proliferate quickly by budding, allowing them to colonize freshwater habitats rapidly and produce 13

OPEN ACCESS
to 38 metric tons/hectare/year dry weight of plant mass [3,4]. Since duckweeds can absorb pollutants (eg. N, P and heavy metals) from wastewater, they have been commonly used in the treatment of domestic and animal wastewater streams for many years [5][6][7]. Meanwhile, because of the relatively high starch and low lignin content in this plant, it has been proved to be an ideal bioresource for bioethanol production [4,8]. Supported by the Minister of Science and Technology and the Major Projects of Knowledge Innovation Program of Chinese Academy of Sciences, our group have been focused on the exploitation of duckweed in the fields of biofuels and natural products.
In our previous study, Landoltia punctata demonstrated obvious advantages in the biofuel aspect for its higher starch content and easier acquisition among the five genera in Lemnaceae [9]. Large scale cultivation of L. punctata has been carried out by our group, and tons of L. punctata biomass can be obtained in a very short time. It is no doubt that L. punctata has become a new resource for natural products such as proteins, polysaccharides, amino acids, and other small molecules.

O-[β-D-glucopyranosyl-(1→2)]-[β-D-glucopyranosyl-(1→6)]-β-D-glucopyranoside.
The HR-ESI-MS spectrum of 2 (m/z 969.5406 [M + Na] + , calc. for C 48 H 82 O 18 Na) supported a molecular formula of C 48 H 82 O 18 . The NMR spectra of 2 were very similar to those of 1, except for one less oxygenated methine at δ C 71.3. Detailed inspection of the HMBC spectrum led to a conclusion that 2 had one less OH on C-22 than 1, on the basis of the key HMBC correlation of 21-CH 3 (δ H 1.01) with C-22 (δ C 23.2). The linkage of three glucoses on the triterpenoid aglycon was determined to be the same way as 1 on the basis of HMBC and 1 H, 1 H-COSY signals. The stereochemistry of 2 was determined on the basis of NOESY cross peaks of H-8 and H-19a/18-CH 3 , 18-CH 3 and H-12a, H-19a and 29-CH 3 , H-16b and 30-CH 3 , 21-CH 3 and H-17, H-17 and 30-CH 3 . Acid hydrolysis of 2 gave D-glucoses, which was identified by comparing the optical rotation value with an authentic sample. Finally the structure of 2 was determined to be (3β,24S)-9,19-cycloartane-3,24,25-  group. Enzymatic hydrolysis of 3 with β-D-glucosidase afforded D-glucose, which was identified by direct comparison with the authentic sample by TLC [19]. In view of above evidences, it was concluded that 3 was a flavanol glucoside.
In the combination of the 13 C-NMR and HSQC spectra of 3, the 23 carbon resonances could be easily attributed to a flavanol moiety, a glucopyranose unit (δ C 62.7, 71.6, 75.1, 78.2, 78.4, 102.8), and two methoxyls (δ C 56.0, 56.6). In order to determine the location of substituent groups on the flavanol moiety, HMBC and NOESY experiments were performed. As a result, the NOESY correlations of the methoxyl H-atoms at δ H 3.82 with H-6 and H-8, H-6 with H-1', and the other methoxyl H-atoms at δ H 3.95 with H-2', together with the HMBC correlations of 7-OCH 3 to C-7, 3'-OCH 3 to C-3', indicated the glucose and two methoxyls were situated at C-5, C-7, and C-3' respectively ( Figures 4 and 5).
Compound 4 was isolated as amorphous powder. The molecular formula was C 24 H 30 O 12 , determined by negative-ion at m/z 509.1662 in the HR-ESI-MS. Enzymatic hydrolysis of compound 4 with β-D-glucosidase gave D-glucose. Comparing its NMR spectra with those of 3, it was evident that 4 contained one more methoxyl at δ H 3.65/δ C 56.6 and one oxygenated CH (δ C 73.5) than 3.
The location of one more methoxyl at C-4 was determined by the HMBC correlation of 4-OCH 3 at δ H 3.65 with C-4 (δ C 73.5). The other substitutions of 4 were confirmed by the same way as 3.  The configuration of 4 was elucidated on the basis of NOESY correlations and coupling constants.

Biological Activity Assay
According to previous bioactivity reports on flavonoids [29][30][31], antimicrobial and antioxidant activities of compound 3 were evaluated. Two pathogenic bacteria (Bacillus subtilis, Escherichia coli) and two fungi (Aspergillus niger, Saccharomyces cerevisiae) were selected for antifungal and antibacterial assays, respectively. The compound 3 showed no antimicrobial activity (C > 50 μg/mL). In the ABTS cation radical scavenging assay, the inhibition rate of 3 was 82.0% at 1 mg/mL (Positive control vitamin C, 75.2%, C = 0.1 mg/mL). The superoxide anion radical scavenging assay suggested that 3 showed 53.5% superoxide anion radical scavenging capacity at 2 mg/mL. While the positive control luteolin was 53.8% at 0.625 mg/mL. Unfortunately, the low available amount of compounds 1, 2, and 4 precluded the antimicrobial and antioxidant assays.

Material
The duckweed Landoltia punctata (G. Meyer) Les & Crawford was grown under natural conditions and collected in Kunming, Yunnan province, P. R. China. The duckweed was collected in May, washed with water, and then dried at 60 °C. The test strains Aspergillus niger, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis were obtained from Chengdu Institute of Biology, Chinese Academy of Sciences (CAS), P. R. China.
The same CC of the n-BuOH extract (60 g) on MCI gel as that of EtOAc extract gave four fractions (I-IV). Fraction II was subjected to preparative thin layer chromatography (TLC) and Sephadex LH-20 CC to afford compounds 1 (13 mg) and 2 (10 mg). Faction III was separated over silica gel with CHCl 3 /MeOH (7:1-0:1, v/v) as eluent to generate 4 fractions (IIIa-IIId). Fr. IIIb was separated by HPLC using MeOH/H 2 O (45:55, v/v) as the eluent to yield compounds 13 and 14. Fr. IIIc and IIId were further purified by TLC and then Sephadex LH-20 CC to yield compounds 3 (18 mg) and 4 (8 mg) respectively.

Hydrolysis
Acid hydrolysis. Compounds 1 and 2 (10 mg) were independently dissolved in aqueous solution of 2 N HCl (6 mL) and stirred at 85 °C overnight. After cooled down, the reaction mixture was neutralized and then extracted with CHCl 3 . The water layer were concentrated to dryness, dissolved in water to constant volume, and analyzed by comparing their TLC profile and optical rotation values with the standard sample of D-glucose [21,22], respectively.

Biological Activity Assay
According to the reported [32], antifungal and antibacterial activities were determined using the Oxford cup method with Methicillin sodium as the positive control. The antioxidant activity assay was performed on the DPPH, ABTS +• , and superoxide anion radical scavenging models complying with the previously published literature [33].

Conclusions
In this study, we identified 12 flavonoids including two new flavanol glucosides from L. punctata. The result showed that apigenin or luteolin flavonoids are the main constituents of this species, which is in good agreement with the previous reports and our previous study results that the transcripts for key enzymes of flavonoid biosynthesis in L. punctata expressed in high abundance at the transcriptional level [9,10,34]. Flavonoids are generally biosynthesized to cope with environmental stressors such as ultraviolet radiation, ozone, heavy metals, nutrient limitation, herbivores, and so on. The high content of flavonoids in L. punctata could be related to environmental stressors. Meanwhile cycloartane triterpenoids were discovered in Lemnaceae family for the first time in this study. Many cycloartane triterpenoids possessed diverse bioactivities such as anti-inflammatory, anti-tumor, anti-viral, immuno-regulatory, hypoglycemic, cardiovascular system, nervous system and hepatoprotective effects [35,36], some of which offer good prospectives in medical applications. This study demonstrates that L. punctata is a new source for cycloartane triterpenoids.