Diaion hp 20

Optical rotation was measured with a Rudolph Research Analytical AutoPol IV Automatic Polarimeter. UV and IR spectra were obtained with Shimadzu UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan) and Thermo scientific Nicolet iS50FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively. CD spectra were recorded on Jasco J-815 circular dichroism spectrophotometer (Jasco Products Company, Oklahoma City, OK, USA) in methanol at the concentration of 0.01 mg/mL (the length of the cell path was 1 cm). NMR spectra including 1D and 2D experiments were recorded on a Bruker 400 MHz NMR; HPLC was carried out on Thermo scientific Ultimate 3000 LC system using a Phenomenex Luna phenyl-hexyl column (100 mm × 21.2 mm, 5 μm particle size, Phenomenex, Torrance, CA, USA) and a Phenomenex Luna C18 HPLC column (250 mm × 10 mm, 5 μm particle size, Phenomenex, Torrance, CA, USA). All solvents were HPLC grade. Column chromatography was performed using Diaion HP-20 (Sigma, St. Louis, MO, USA).

For pre-concentration, column material of 95 g Diaion^® HP-20 (Sigma-Aldrich, Steinheim, Germany) was prepared by incubation in MeOH for 15 min followed by incubation and equilibration in water for 15 min before use. Thereafter, a column was packed with the material and loaded with 5 L of culture supernatant. After washing the column with 150 mL of water, the elution was carried out using 620 mL of acetonitrile (ACN)+0.1% FA. The yellow eluate was collected in a round flask and the organic solvent removed. A total of 50 L of culture supernatant was pre-concentrated to 60 mL of eluate.

Five kilograms fresh leaves of Uncaria gambir Roxb. harvested in Kebun Tumbuhan Obat Farmasi (KTOF) UPT, Sumatran Biota Laboratory, Andalas University. The sample specimen was identified and deposited in the herbarium of Andalas University (No: 343/K-ID/ANDA/2019). The leaves were laid at oven (50 ^oC, 24 h). All of the sample were pulvered by a grinder.
The powdered leaves of Uncaria gambir Roxb., were then soaked in methanol 70% for 9 days at room temperature with frequent agitation for a period of 3 days. The methanol extract was subsequently filtered through Whatman No. 1 filter paper. Further step, to promote the rapid removal of excess solvent, the samples were put on flask of rotary evaporator to obtained extraction yields 18.65%.
Liquid-liquid extraction was done against 500 g of methanol extracts with hexane, ethyl acetate, and butanol to obtained hexane fraction (0%), ethyl acetate fraction (40.6%), butanol fraction (10%). 10 g of butanol fraction was directly subjected to a Diaion HP-20 (Sigma Aldrich, Singapore) column (9 cm i.d. x 60 cm) with H₂O by increasing amounts of MeOH in stepwise gradient elution to obtain 10 subfractions SA01–SA10, respectively. SA03 was a proanthocyanidin fraction, a brown powder 31.2 mg (0.32%). A brown bottle storage was required for all of the Diaion HP-20 fractions until further use.

The cellular Pol stimulated compound was purified from the screened medicinal plant using various column chromatography, consisting of Diaion HP-20 (Sigma Aldrich, St. Louis, MO, USA) and silica gel 60 (Merck Millipore, Darmstadt, Germany), and HPLC, consisting of Chromatorex ODS DM1020T (Fuji Silysia Ltd., Durham, NC, USA). The purified compound was identified using high-resolution mass spectrometry (Xevo G2 Tof: Waters; Milford, MA, USA) and nuclear magnetic resonance equipment (JNM-ECS400: JEOL RESONANCE; Tokyo, Japan).

Ptp1b (human, recombinant), p-nitrophenyl phosphate, EDTA, citric acid, dithiothreitol, gallic acid (≥98·0 %) and ursolic acid (≥98·0 %) were obtained from Sigma Aldrich. Materials for chromatographic studies included pre-coated silica plates (60 GF 254; 20 × 20 cm) (Fluka for TLC, Diaion HP 20; Sigma-Aldrich Co.). Silica gel H (Merck) and Lichroprep RP-18 silica gel (15–25 µm; Merck) were used for vacuum liquid chromatography, and silica gel 60 (70–230 mesh ASTM; Fluka) and Sephadex LH-20 (Sigma-Aldrich Co.) for column chromatography. The following solvent systems were used for developing the chromatograms. S1: n-hexane–ethyl acetate (9:1, v/v); S2: methylene chloride–methanol (9·5:0·5, v/v); S3: ethylacetate–methanol–water (10:1·6:1·2, by vol.). Spots were visualised by spraying with p-anisaldehyde–sulfuric acid or natural products–polyethyleneglycol reagent (NP/PEG). ¹H-NMR and ¹³C-NMR spectra were recorded on a Bruker high-performance digital Fourier transform-NMR spectrophotometer operating at 400 (¹H) and 100 (¹³C) MHz in DMSO-d6 as a solvent and chemical shift were given in δ (parts per million) relative to solvent as an internal standard. The ¹H-NMR was run for 1 h and ¹³C-NMR was run for 6 h at room temperature. Mass spectra were measured on a MS QQQ mass spectrometer equipped with an electrospray ion source in negative ion mode.

Strain IE-3 was grown anaerobically in serum vials at 30°C for 48 h for the maximum production of a LMW peptide. Antimicrobial compound was extracted from CFB using 2% activated Diaion HP20 (Sigma, USA) hydrophobic resin. The crude extract obtained was further purified through cation exchange (Capto S, GE Healthcare, USA) chromatography column linked to an AKTA prime plus (GE healthcare, USA), in 20 mM sodium acetate buffer (pH 4.6) and eluted with NaCl gradient (50 to 1000 mM) in binding buffer. The peptide was desalted using dialysis tube (molecular cutoff 0.5 kDa, Spectrum, USA). Approximate molecular mass of peptide was determined by gel filtration column (Sodex KW-802.5) using standard molecular weight markers as described earlier [31 (link)]. Purity was confirmed by reversed phase HPLC (10 mm × 250 mm × 150 Å) C-18 column (venusil, Agela Technologies) under isocratic flow (1.5 ml/min) of acetonitrile (20%) along with 0.1% TFA. Elution was monitored at 200–340 nm wavelength range on PDA detector and peaks were collected by fraction collector (1260 Infinity, Agilent technology, USA).