L tartaric acid

To perform H&E and tartrate-resistant acid phosphatase (TRAP) staining, tissues fixed in 4% paraformaldehyde underwent decalcification in 10% ethylenediaminetetraacetic acid at 4 °C for 3 weeks. They were then embedded in paraffin after dehydration, and 4-µm-thick sections were created. Sectioning of the paraffin-embedded samples was performed along the sagittal plane.
H&E staining was performed using haematoxylin (Vector Laboratories, Inc., Burlingame, CA) for 3 minutes, followed by 10 seconds of eosin (Wako). TRAP staining was performed with Naphthol AS-BI phosphate (Sigma), sodium nitrite (Wako), 0.08 M L(+)-tartaric acid (Wako), and pararosaniline hydrochloride (Sigma) in 0.1 M of sodium acetate buffer (pH 5.0) at 37 °C in an incubator for 20 minutes, followed by nuclear counterstaining with haematoxylin. Under an Olympus light microscope (Olympus, Tokyo, Japan), TRAP-positive multinuclear giant cells adjacent to the collagen fibres were identified as osteoclasts. We counted the osteoclasts that were present in regions of bone regeneration and measured the area that consisted of new collagen (Olympus cellSens standard version 1.13; Olympus). The number of osteoclasts in the decalcified bone area (mm⁻²) was represented by the number of osteoclasts in the new collagen area.

L-(+)-Tartaric acid (1.00 g, 6.66 mmol, 203-00052, Fujifilm-Wako, Japan) was dissolved in MeOH (22.2 mL) and cooled to −80 °C. SOCl₂ (1.5 mL, 20.7 mmol, 206-01103, Fujifilm-Wako, Japan) was added dropwise to the solution. The mixture was stirred for 3 h and was allowed to warm to room temperature. After 12.5 h, the reaction solution was concentrated in vacuo to obtain a colorless oil, which was dissolved in CH₂Cl₂ (22.2 mL). 2.2-Dimethoxypropane (5.4 mL, 43.8 mmol, 042-06963, Fujifilm-Wako, Japan) and p-TsOH·H₂O (634 mg, 3.33 mmol, 34208-05, Nacalai tesque, Japan) were added and the mixture was stirred with refluxing (45 °C). After 4 h, the mixture was warmed to room temperature and saturated acrylamide (10 mL, 00807-05, Nacalai tesque, Japan) was added slowly. AcOEt and H₂O were added, the mixture was extracted with AcOEt, the organic layer was washed with brine, then it was dried over anhydrous MgSO₄ and filtered and the solvent was evaporated under reduced pressure. The residue was purified by silica gel chromatography (hexane-AcOEt = 8:1 v/v) to give (R,R)-diethyl 2,3-O-isopropylidentartrate (1.11 g, 5.07 mmol, 76%, 2 steps) as a yellow oil. ¹H-NMR (600 MHz, CDCl₃) δ 4.73 (s, 2H), 3.75 (s, 6H), 1.41 (s, 6H). The spectral data are in perfect agreement with the report by Hilpert et al.

All crude drugs adhered to the quality control of the 18th Edition of the Japanese Pharmacopoeia [1 ]. The dried sliced form (approximately 2 mm wide) of Pinellia tuber (lot number, #009120002) and cut dried ginger (lot number, 8L25M) were purchased from Tochimoto Tenkaido (Osaka, Japan) and Daiko Shoyaku (Nagoya, Japan), respectively. Oxalic acid and L-( +)-tartaric acid were purchased from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). L-(–)-malic acid and citric acid monohydrate were purchased from Sigma Aldrich (St. Louis, MO, USA) and Nacalai Tesque (Kyoto, Japan), respectively.