Cascara Sagrada

The bark of Magnolia officinalis Rehd. et Wils. was dried in the shade at room temperature and stored in a dark, cold room until use. The air-dried bark of Magnolia officinalis Rehd. et Wils. (3 kg) was cut into pieces and extracted twice with 95% (v/v) ethanol (four times as much as the weight of the dried plants) for 3 days at room temperature. After filtration through the 400-mesh filter cloth, the filtrate was filtered again through filter paper (Whatman Grade No. 5) and concentrated under reduced pressure. The combined extract (450 g) was suspended in H₂O and the aqueous suspension was extracted with n-hexane, ethyl acetate, and n-BuOH, respectively. The n-hexane layer was evaporated to dryness to give a residue (70 g), which was chromatographed on silica gel with n-hexane:ethyl acetate (9:1) gradient to yield a crude fraction that included 4-O-methylhonokiol. This fraction was repeatedly purified by silica gel chromatography using n-hexane:ethyl acetate as the eluent to obtain pure 4-O-methylhonokiol (Fig. 1a). 4-O-methylhonokiol was identified by ¹H-NMR and ¹³C-NMR. The results of the NMR data are as follows and are in agreement with previously published data [24 (link)]. ¹H-NMR (400 MHz, CDCl₃): δ 3.36 (2H, d, J = 7 Hz, H-7), 3.44 (2H, d, J = 7 Hz, 7′-H), 3.89 (3H, s, OMe), 5.05–5.14 (5H, m, H-9, H-9′, OH), 5.93–6.07 (2H, m, H-8, H-8′), 6.92 (1H, d, J = 7 Hz, Ar-H), 6.97 (1H, d, J = 8 Hz, Ar-H), 7.04–7.08 (2H, m, Ar-H), 7.24–7.31 (2H, m, Ar-H). ¹³C-NMR (100 MHz, CDCl₃): δ 34.5 (C-7), 39.6 (C-7′), 55.8 (OMe), 111.2 (C-3′), 115.7 (C-4′), 115.8 (C-9), 116.1 (C-9′), 128.0 (C-1′), 128.1 (C-6), 129.0 (C-3), 129.2 (C-1), 130.0 (C-5), 130.4 (C-6′), 130.7 (C-2), 132.4 (C-5′), 136.7 (C-8), 138.0 (C-8′), 151.0 (C-2′), 157.2 (C-4). The ethanol extract of Magnolia officinalis contained 16.6% 4-O-methylhonokiol, followed by 16.5% honokiol and 12.9% magnolol, and 42–45% others.Fig. 1

Chemical structure of 4-O-methylhonokiol (a) and experimental scheme (b)

Onshore, the carbonates, sediments, and nodules were separately ground into powder with a sterile porcelain mortar and pestle. The nodules, which were only loosely consolidated and thus could have contained sediment-phase contamination, were preprocessed in order to thoroughly remove sediment as previously described (19 (link)), with the exception of nodule 5118N (see Table S1 in the supplemental material). Genomic DNA was extracted following the general procedure of the MoBio PowerSoil kit (MoBio, St. Louis, MO) (see reference 5 (link) for variations from default protocol), using ~400 mg powder. For wood samples, a sterile razor blade was used to collect shavings from the exterior, avoiding the bark and any observed animals (e.g., shipworms) whenever possible. DNA from wood samples was extracted using the MoBio PowerPlantPro kit’s recommended protocol, with 40 µl of phenolic separation solution and ~70 mg wood shavings. Bottom water samples from nearby station HR-9 (see Fig. S1 in the supplemental material) were collected on a 0.2-µm filter and extracted by phenol-chloroform followed by CsCl density gradient centrifugation (66 (link)).
Preparation for sequencing of the V4 region of the 16S rRNA gene was performed with universal primers according to the protocol recommended by the EMP (iTag sequencing [67 ]) (http://www.earthmicrobiome.org/emp-standard-protocols/16s/) (68 (link), 69 (link)), with minor modifications as previously described (19). Raw sequences were generated on an Illumina MiSeq platform at Laragen, Inc. (Los Angeles, CA). In-house data processing was completed in QIIME1.8.0 and included joining paired ends, quality trimming, chimera checking, 97% OTU clustering, singleton removal, PCR contaminant removal, 0.01% relative abundance threshold removal, and rarefaction to 16,051 sequences per sample (see Text S1 in the supplemental material). Taxonomic assignments were generated according to an appended version of the Silva 115 database (for details, see reference 19 (link)).