Pterostilbene

Hippocampal neuronal HT22 and astrocytoma U251 cells were cultured in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) (37°C, 5% CO₂), which was replaced by FBS-free DMEM 24 h before the experiment. Culture media were changed every 2 days. Cell culture reagents were obtained from Gibco (Grand Island, NY, USA). PTE dissolved with DMSO was diluted in DMEM in advance (DMSO ≤ 0.1%). Before OGD/R, U251 cells were treated with PTE (2.5 or 5 μM) or vehicle for 4 h.

This trial was a prospective, randomized, double-blind placebo-controlled intervention trial. This trial was approved by the University of Mississippi Medical Center Institutional Review Board. The target population was patients with hypercholesterolemia, defined as a baseline total cholesterol ≥200 mg/dL and/or, baseline low-density lipoprotein cholesterol ≥100 mg/dL. Patients were included if they were ≥18 years of age and on either no cholesterol therapy or cholesterol medication at a stable dose for at least 2 months prior to baseline laboratory. Patients were excluded if they had significant hepatic, renal, or gastrointestinal tract disease, were receiving thiazolidinediones or fibric acid derivatives, had current overt cardiovascular disease, were women of reproductive potential not receiving birth control, or were pregnant/nursing women.
The trial planned for an enrollment of 80. Subjects were divided equally into one of four groups: pterostilbene 50 mg twice daily (low dose), pterostilbene 125 mg twice daily (high dose), pterostilbene 50 mg/grape extract 100 mg twice daily (low dose + grape extract), or matching placebo twice daily for 6–8 weeks. Ciprofibrate is a peroxisome proliferator-activated receptor-α (PPAR-α) agonist [8 (link)]. Pterostilbene has demonstrated similar PPAR-α activation at approximately equimolar concentrations in animal models [4 (link)]. Since a standard human dose of ciprofibrate is 100 mg/day, this dose was selected for the lowest effective pterostilbene dose in monotherapy and combination. The higher daily dose was evaluated to assess for potential dose-related efficacy or adverse effect. All patients received identical counseling on lifestyle intervention and compliance with currently prescribed medication regimens.
The manufacturer of the pterostilbene and placebo products was deemed in compliance by the FDA current good manufacturing practices prior to the initiation of this trial. The process of pterostilbene synthesis for this trial is described elsewhere [13 ].
The safety markers included biochemical and subjective measures collected at two visits (baseline and final). Donated blood was analyzed for all biochemical measures at the same laboratory values. Primary safety measures included Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), serum creatinine, and blood glucose. Other safety markers include blood electrolytes and symptomatic subjective adverse drug reactions (ADRs) collected during patient interviews at baseline and final visits. Blood pressure, cholesterol, and weight were collected and reported separately as efficacy endpoints. Pill counts were utilized to assess for compliance.
Linear mixed models were used to estimate primary safety measure treatment effects in order to account for intra subject associations arising from the repeated measures before and after longitudinal design. The underlying missing-at-random architecture implicit in mixed models was assumed. Various models were fit to examine potential baseline effects including as appropriate the following:

3-way interaction models of final outcome × treatment group × baseline value;

2-way interaction models including all 2-way terms from (1) but excluding the 3-way term;

models assuming baseline value affected change similarly across treatment groups;

models assuming change in outcome were independent of baseline value.

Each model was examined in unadjusted and adjusted form (adjusting for age, gender and race). Final reported treatment effects were obtained from the simplest appropriate adjusted model for each outcome. For secondary measures compared to baseline and/or placebo, a t-test was performed for continuous data and a Fisher's exact test was performed for dichotomous data.

Octacalcium phosphate crystals
(OCP), a type of basic calcium phosphate (BCP) often deposited in
joints, causing acute inflammatory arthritis and joint degeneration,^{29 (link)} were used to induce OA in this study. OCP precipitate
was collected from dicalcium phosphate (Sigma-Aldrich, Darmstadt,
Germany) and 0.05 M diammonium phosphate solution (Sigma-Aldrich,
Darmstadt, Germany) according to a previous study.^{30 (link)} The OCP powder collected after precipitation was washed
with distilled water and dried at 37 °C. Pterostilbene (PT) with
a purity of 98% was purchased from Combi Blocks (San Diego, USA).
LPS and ATP were purchased from Sigma-Aldrich (Darmstadt, Germany).

Twenty-five-day-old stylo plants (genotype ‘2001-71’) were used. The stylo seeds were provided by the Tropical Crop Germplasm Research Institute, Haikou, Hainan province, China. Stylo seeds were soaked in hot water (80 °C) for 3 min and germinated for 3 days. After germination, the seeds were transplanted into seedling boxes filled with equal volumes of vermiculite and nutrient soil. The seedlings were grown in a greenhouse with an average temperature of 28 °C for approximately 3 weeks [9 (link)]. Colletotrichum gloeosporioides strain WC-02 was used in this study and described previously [39 (link)]. The inoculation of C. gloeosporioides was performed according to the previous study [9 (link),25 (link)]. The fungus was grown on potato dextrose agar for 3 days, and the spore was produced in complete medium for 3 days. The spores were harvested and diluted with sterile water. The concentration of spore suspension was determined using a hemocytometer under a light microscope and adjusted to 10⁶ spores mL⁻¹. The stylo plants were spray-inoculated with a spore suspension containing 0.02% silweet L-77 with 500 μmol L⁻¹ phloretin (Sigma-Aldrich, St. Louis, MO, USA) and 500 μmol L⁻¹ pterostilbene (Sigma-Aldrich, St. Louis, MO, USA) (Cg + PP treatment) or an equal amount of ethanol solvent (Cg treatment) [26 (link)]. The inoculated plants were placed in a dark room at 28 °C and 90% humidity for 12 h. Then, the plants were transferred to a growth chamber at 28 °C, 90% humidity and 16 h photoperiod with a light intensity of 800 μmol m⁻² s⁻¹. Stylo plants sprayed with 500 μmol L⁻¹ phloretin and 500 μmol L⁻¹ pterostilbene containing 0.02% Silweet L-77 (PP treatment) or an equal amount of ethanol solvent (CK) were used as physiological controls.