Cve 3100

Mealworms were washed, sterilized, freeze-dried, and pulverized using a sway-type pulverizer (KSP-35, Korea medi Co., LTD, Daegu, Korea). The freeze-dried powder was mixed with 70% ethanol, sonicated (350 J, 10 s, twice) using an ultrasonic processor (VCX500, Sonics & Materials, Inc., Newtown, CT, USA), and kept at 25 °C for 30 min. The extract was then centrifuged at 3500 rpm for 10 min. This was followed by the supernatant being separated and collected. The supernatant was filtered using a 0.45 μm PVDF syringe filter (GE Healthcare, Little Chalfont, UK) before being transferred into pre-weighed containers. The filtrate was then concentrated using a vacuum rotary evaporator (CVE-3100; EYELA, Tokyo, Japan). TME was stored at −70 °C until further use.

Free amino acids were extracted based on the previously established method (Tsuchiya et al., 2013 (link)). The amount of 1 g of soil was extracted three times with 80% (v/v) aqueous ethanol (pH adjusted to 3.0 with HCl) at 60°C. This step was to remove most of the extractable amino acid, which is easily available for plant root and other organisms. Combined fractions were evaporated to dryness using a vacuum centrifugal evaporator (CVE-3100; EYELA, Tokyo, Japan), equipped with a cold glass trap (Uni trap UT-1000; EYELA).

The obtained PEG-block-PCys (P1(SH)) was solubilized in pyridine (100 mL) and butyric anhydride (150 mL), and the solution was purged with N₂ for 15 min. The solution was stirred at 50 °C for 3 d. The acylation reaction solution was directly dialyzed against water for preparing Nano^Cys(Bu)(P1) (MWCO = 1000), followed by concentrating the obtained solution using a centrifugal evaporator (EYELA, CVE-3100). The concentration of the resulting solution was determined by weighing the obtained polymer after lyophilizing 100 μL of the solution. Finally, the solution was diluted with MilliQ water to achieve the target concentration. GPC (DMF, PEO standard): M_n(GPC) = 7600, Đ(GPC) = 1.36. ¹H NMR (600 MHz, CDCl₃/TFA = 15/1 (v/v), r.t., δ = 7.26 ppm (CHCl₃)): δ 8.32–7.45 (brs, –COCH(CH₂SCO(CH₂)₂CH₃)NH–), 4.89–4.60 (brs, –COCH(CH₂SCO(CH₂)₂CH₃)NH–), 3.96–3.58 (brs, –OCH₂CH₂–), 3.5 (3H, s, –OCH₃), 3.44–3.10 (brs, –CH₂ SCO(CH₂)₂CH₃), 2.68–2.46 (brs, –SCOCH₂CH₂CH₃), 1.80–1.53 (brs, –SCOCH₂CH₂CH₃), 1.10–0.74 (brs, –SCOCH₂CH₂CH₃).
Nano^Cys(Bu)(P2) and Nano^Cys(Bu)(P3) were prepared in the same manner, after the protection reactions of P2(SH) and P3(SH).

For fractionation of LMW-PPE, we employed an HPLC gradient system equipped with a syringe-loading sample injector, dual pumps, an MD-2010 multi-UV detector (Jasco, Tokyo, Japan) and a C 18 reversed-phase column (Inertsil ODS-3, 5 μm, 4.6 × 250 mm, GL Science, Tokyo, Japan). For high reproducibility, an analytical column was used for fractionation rather than a preparative one. The HPLC solvents were 0.05% trifluoroacetic acid in water (A) and HPLC-grade acetonitrile (B). One hundred microliters of the LMW-PPE was applied to the HPLC column and separated using the following gradient at a flow rate of 1 mL/min: hold at 100% solvent A from 0 to 10 min, then linear gradient from 0% B in 10 min to 95% B in 30 min. The eluents were collected in sample tubes every 2 min and dried using a centrifugal evaporator CVE-3100 (EYELA, Tokyo, Japan). The residues were reconstituted with 100 μL of PBS and filtered through a 0.22 μm membrane before using the NAD recovery assay.

After cardiac blood collection, blood was collected in ethylenediaminetetraacetic acid-containing tube (BD Microtainer^® MAP; Becton-Dickinson, Franklin Lakes, NJ, USA) under anesthesia with pentobarbital (50 mg/kg, intraperitoneal administration). The samples were centrifuged at 3500× g for 10 min at room temperature. The obtained supernatant was used as plasma sample. Apart from these plasma collection experiments, the oral cavities of rats were cleaned by small cotton balls soaked in saline under anesthesia with pentobarbital (50 mg/kg, intraperitoneal administration). The rats were then injected with pilocarpine (1.0 mg/kg, subcutaneous administration) to stimulate salivary secretion. The saliva was subsequently collected from the oral cavity using a micropipette and placed in tubes in an ice bath. These samples of plasma and saliva were stored at −80 °C until use. Equal volume of 10% trichloroacetic acid (TCA) was mixed with the samples of plasma or saliva, followed by centrifuging at 10,000× g for 10 min at 4 °C. To remove TCA, the supernatant was washed with water-saturated diethyl ether. Thirty µL of the aqueous layer was dried under reduced pressure with a rotary evaporator (CVE-3100; Eyela, Tokyo, Japan) at 40 °C.