Anti-oral Microbial Flavanes from Broussonetia papyrifera Under the Guidance of Bioassay

Abstract A new flavane, bropapyriferol (1), and eleven known ones were isolated from the EtOAc part of Broussonetia papyrifera under the guidance of bioassay. The structure of compound 1 was determined by extensive 1D and 2D NMR, [α]D spectroscopic data and quantum computation. Daphnegiravan F (2) and 5,7,3′,4′-tetrahydroxy-3-methoxy-8,5′-diprenylflavone (3) showed significantly anti-oral microbial activity against five Gram-positive strains and three Gram-negative strains in vitro. Especially, compound 3 was more potent in suppressing Actinomyces naeslundii and Porphyromonas gingivalis (MIC = 1.95 ppm) than the positive control, triclosan. Graphical Abstract Electronic supplementary material The online version of this article (doi:10.1007/s13659-019-0197-y) contains supplementary material, which is available to authorized users.


Introduction
Broussonetia papyrifera, a deciduous broadleaf plant in the family Moraceae, is native to Asia, and mainly distributed in China, Japan, Vietnam and India. B. papyrifera also known as paper mulberry is a highly valued plant in China, whose leaves, roots, barks and fruits are used in traditional Chinese medicines (TCMs) for diuretic, anti-rheumatic, anti-bacterial, anti-inflammatory purposes. Its bark is a good source for papermaking and leaves are high quality feed. Now, this plant widely grows in Europe, the United States and Africa as introduced a species [1,2]. The main constituents of B. papyrifera are flavones, lignans, diterpenes and triterpenes 1 3 [3][4][5][6][7][8]. In the previous investigation, three flavanes, kazinol B, 7,4′-dihydroxy-3′-prenylflavan and 3′-(3-methylbut-2enyl)-3′,4′,7-trihydroxyflavane, and one triterpene, oleanolic acid, from the cortex were revealed with anti-oral microbial activity [9]. In order to clarify the anti-oral microbial effects of B. papyrifera and the active constituents, the preliminary bioassay revealed that the EtOAc part showed increased activity comparing to the total extraction. Thus, detailed investigation was performed on the active EtOAc part and yielded one new flavane, bropapyriferol, and eleven known ones. Herein, we report their isolation, structural elucidation and anti-oral microbial activities. In the 13 C NMR spectrum, one methine (δ C 75.7) and two methylenes (δ C 30.7 and 25.8) were characteristic for a flavane skeleton when taking 12 aromatic carbons into consideration. One prenyl group was deduced from the methylene (δ H 3.83, d, J = 6.6 Hz; δ C 25.4), tri-substituted double bond (δ H 5.49, t, J = 6.6 Hz; δ C 124.8 and 130.5) and two tertiary methyls (δ H 1.75 and 1.63, both s; δ C 18.0 and 25.8). From the above analyses, this compound was proposed to be a prenylated flavane [6].

Structure Elucidation
The NMR data of 1 showed similarity with those of daphnegiravan F [10], and the main difference was that compound 1 had an addition 2-hydroxypropan-2-yl group (two methyls: δ H 1.41 and 1.49, δ C 26.4 and 25.8; one oxygenated quaternary carbon: δ C 70.8) and two methines (δ H 6.07 and 4.94, δ C 74.3 and 99.1) instead of a double bond (Table 1). Detailed analysis of the 2D NMR spectra well established its planar structure: the 1 H-1 H COSY correlation of H-5 with H-6, and HMBC correlations from H-6 and H-8 to C-4a, and from H-5 to C-7 verified a hydroxyl group at C-7; the HMBC correlations from H-8′ to C-2′, and from H-7′ to C-1′ and C-3′ suggested the prenyl group at C-2′; the 1 H-1 H COSY correlation of H-12′ with H-13′, and HMBC correlations from H-15′ and H-16′ to C-13′ indicated a hydroxyl group at C-12′ and 2-hydroxypropan-2-yl group at C-13′ (Fig. 1).
The stereochemistry at C-12′ and C-13′ was deduced as trans-form by the coupling constant (J H-12′,H-13′ = 4.4 Hz) and comparing with that of 16-hydroxycudratrixanthone Q [11]. In order to determine the stereochemistry of 1, the two possible configurations, 2S*,12′S*,13′S*-1 (1a) and 2S*,12′R*,13′R*-1 (1b), were applied to quantum computation at the B3LYP/6-311 + g(d,2p) level for the 13 C NMR data. As shown in Fig. 2, the calculated NMR data for 1b matched well with the experimented data, with the correlation coefficient (R 2 ) of 0.9979, when comparing with that of the calculated 1a. In addition, the correlation coefficient (R 2 ) was up to 0.9997 when only the carbons (C-4′, 5′, 6′, 12′, 13′ and 14′) near C-12′ and C-13′ takes into consideration. The absolute stereochemistry was determined to be 2R, 12′S, 13′S by the positive [α] D value which is consistent with the previous report and quantum calculation [12]. Thus, the structure of 1 was determined and named to be bropapyriferol.
T h e k n o w n c o m p o u n d s w e r e d e t e rm i n e d a s d a p h n e g i r a v a n F ( 2 ) [ 1 0 ] ,

Anti-oral Microbial Activity
The total extraction and each fraction were assayed for the anti-oral microbial activity against two Gram-positive and two Gram-positive strains. As shown in Table 2, the total extraction showed activity against the four assayed   Table 3, compound 2 exhibited significant activity against all the strains except for S. sorbrinus, with MIC values of 7.8, 7.8, 3.9, 15.6, 62.5, 3.9, 3.9 and 7.8 ppm, which is comparable to the positive control, triclosan. Compound 3 could obviously inhibit five Gram-positive strains and one Gram-negative strain (P. gingivalis), with MIC values between 1.95 and 15.6 ppm. For A. naeslundii and P. gingivalis, compound 3 showed an MIC value of 1.95 ppm, even higher than triclosan (MIC = 3.9 and 7.8 ppm, repectively). In addition, compound 10 also showed comparable inhibition with triclosan on two Gram-negative strains, F. nucleatum (MIC = 7.8 ppm) and P. gingivalis (MIC = 7.8 ppm).

General Procedures
Optical rotations were collected on a Jasco model 1020 polarimeter (Horiba, Tokyo, Japan). UV spectra were determined on a Shimadzu UV-2401A spectrophotometer (Shimadzu, Kyoto, Japan). IR (KBr) spectra were recorded on a Bio-Rad FTS-135 spectrometer (Bio-Rad, Hercules, California, USA). 1D and 2D NMR spectra were recorded on a Bruker AM-400 NMR or DRX-500 spectrometer with TMS as the internal standard (Bruker, Bremerhaven, Germany). MS data were collected on a VG Auto Spec-3000 spectrometer (VG, Manchester, UK) and an API Qstar Pulsar hybrid Q-TOF mass spectrometer (AB-Sciex, Framingham, MA, USA).

Plant Materials
The

Extraction and Isolation
The air-dried aerial part (5 kg) of B. papyrifera was powdered and extracted with EtOH (90%) at room temperature for two times. The combined extract was solved in water and partitioned with petroleum ether (PE), EtOAc and n-butanol, successively. Based on the preliminary bioassay, the EtOAc part showed the highest anti-oral microbial activity, and was thus applied for the following investigation. The EtOAc part was separated by silica gel CC using CHCl 3 -MeOH gradient (from 9:1 to 5:5) to yield six fractions, Frs. A-F. Fr. D was fractionated by MPLC system on a MCI gel CHP20P column with MeOH-H

Spectroscopic Data of Bropapyriferol (1)
Colorless   [22]. In brief, a serial doubling dilution of each sample in the range 0.12-250 ppm was prepared in a 96-well plate. 100 μL of bacterial suspension was added into the 96-well plate, and the final concentration in each well was adjusted to 5 × 10 7 CFU/mL. After incubated aerobically or anaerobically at 37 °C for about 20 h, the plate was determined by a BioTek EL × 808 Microplate Reader. The bacteria growth was indicated by the turbidity determined at OD 630 , which was performed in duplicate. The minimum inhibitory concentration (MIC) is defined as the lowest concentration of the sample at which the microorganism tested does not demonstrate visible growth. Triclosan (Purity > 99%, Ciba Specialty Chemicals) was used as the positive control.

Quantum Computation
The 13 C NMR and [α] D calculations for compound 1 were carried out using Gaussian 09 program. Conformational search was achieved by Spartan '14 in MMFF94 s force field, and the lowest conformer was further optimized with the hf/3-21 g and DFT b3lyp/6-311 + g(d,p) methods in Gaussian 09 program package. 13 C NMR shielding constants were calculated with the GIAO method at b3lyp/6-311 + g(d,2p) level in pyridine with PCM, which were converted into chemical shifts by referencing to TMS.
[α] D values were calculated at b3lyp/6-31 g(d,p) level based on the above DFT optimized geometries.