Background/Aim: Vitamin D₃ (VD₃) affects the regulation of the immune system, including the differentiation and function of regulatory T-cells (Tregs). Tregs play an important role in maintaining immune homeostasis in patients with colorectal cancer (CRC). The effects of VD₃ on Treg-associated immune function were investigated in Thai patients in the early stages of CRC. Materials and Methods: Twenty-eight patients were randomized to one of two groups: Untreated or treatment with VD₃ for 3 months. Whole blood samples were collected at baseline, and at 1 and 3 months. Peripheral blood mononuclear cells were isolated and the populations of forkhead box P3-positive Treg cells was analyzed by flow cytometry. The levels of Treg-associated cytokines, interleukin 10 (IL-10) and transforming growth factor beta 1 (TGF-β1), were measured by enzyme-linked immunosorbent assays. Results: Serum VD₃ levels of the VD₃-treated group were significantly increased at 1 (p=0.017) and 3 months (p<0.001) compared to the untreated control group. The mean percentage of Tregs was maintained between 1 and 3 months in the VD₃-treated group. At 3 months, the untreated group had significantly lower Treg levels than the VD₃-treated group (p=0.043). Serum IL-10 levels of the VD₃-treated group were statistically increased at 1 month compared to the control group (p=0.032). No significant difference in serum TGF-β1 levels was observed between the two groups. However, the TGF-β1 level in the VD₃-treated group at 1 month was lower than that of the control. Conclusion: Our findings suggest that VD₃ supplementation can maintain immune responses in the early stages of CRC, helping to control Treg function. Therefore, VD₃ should be supplemented to maintain immune homeostasis, especially in patients with vitamin D deficiency.

Thunbergia laurifolia (TL) has been traditionally used as an antidote and an antipyretic drug by folk healers for centuries in Thailand. Rosmarinic acid (RA) is major compound in TL extract and has attracted great interest due to its potential broad pharmacological effects. Herein, the permeability of RA was investigated in TL extract and as a pure compound in a Caco-2 cell model by using high-performance liquid chromatography with a photodiode array detector (HPLC-PDA). The results reveal that the apparent permeability coefficient (P_app) values of RA in TL extracts and pure RA significantly increased after deconjugation by β-glucuronidase/sulfatase enzymes. Our findings exhibit possible saturable biotransformation of RA and/or membrane transport while penetrated through Caco-2 cells. The cumulative amounts of RA as pure compounds and in TL extracts increased with the exposure time, and the efflux ratio (ER) was 0.27–1.14. RA in the TL extract has a similar absorption in the conjugated form and in the pure compound. The intestinal absorption of them is through passive diffusion. Therefore, our findings conclude that the intestinal transport of RA in TL extracts was mainly penetrated as conjugated forms with glucuronic acid and/or sulfate across Caco-2 cells and transported via passive diffusion.

Fire ant venom is a complex mixture consisting of basic piperidine alkaloids, various biologically active peptides and protein components, including a variety of major allergenic proteins. Tropical fire ant Solenopsis geminata is an important stinging ant species that causes anaphylaxis and serious medical problems. Although the biological activities of allergenic venom proteins that are unique to ant venom, particularly Solenopsis 2 and 4, are still unknown, these proteins are believed to play important roles in mediating the effects of the piperidine derivatives in the venom. In the present study, the cDNA cloning, sequencing and three-dimensional structure of Sol g 4.1 venom protein are described. The recombinant Sol g 4.1 protein (rSol g 4.1) was produced in E. coli, and its possible function as a hydrophobic binding protein was characterized by paralyzing crickets using the 50% piperidine dose (PD50). Moreover, an antiserum was produced in mice to determine the allergenic properties of Sol g 4.1, and the antiserum was capable of binding to Sol g 4.1, as determined by Western blotting. The molecular weight of Sol g 4.1 protein is 16 kDa, as determined by SDS-PAGE. The complete cDNA is 414 bp in length and contains a leader sequence of 19 amino acids. The protein consists of six cysteines that presumably form three disulfide bonds, based on a predicted three-dimensional model, creating the interior hydrophobic pocket and stabilizing the structure. The rSol g 4.1 protein was expressed in inclusion bodies, as determined by SDS-PAGE. Dialysis techniques were used to refold the recombinant protein into the native form. Its secondary structure, which primarily consists of α-helices, was confirmed by circular dichroism analysis, and the three-dimensional model was also verified. The results of allergenic analysis performed on mice showed that the obtained protein was predicted to be allergenically active. Moreover, we report on the possible role of the Sol g 4.1 venom protein, which significantly reduced the PD50 from 0.027 to 0.013% in paralyzed crickets via synergistic effects after interactions with piperidine alkaloids. The primary structure of Sol g 4.1 showed high similarity to that of venom proteins in the Solenopsis 2 and 4 family. Those proteins are life-threatening and produce IgE-mediated anaphylactic reactions in allergic individuals. The possible function of this protein is the binding of the interior hydrophobic pockets with piperidine alkaloids, as determined by the analysis of the structural model and PD50 test.

Thunbergia laurifolia has been a popular herb used in Thai traditional medicine for detoxification and as antipyretic. It contains rosmarinic acid (RA), caffeic acid (CA) and vitexin as major compounds. In order to control the herbal quality, the stability indicating high-performance liquid chromatography (HPLC) was developed and validated. The stability study of compounds in T. laurifolia leaf extract was investigated. The chromatographic separation was performed using a reversed-phase C18 column and mobile phase consisted of 0.5% acetic acid and methanol using a gradient elution with 1.0 mL/min flow rate. The detection wavelength was set at 330 nm. The method was validated for its linearity, precision, accuracy, limit of detection and limit of quantitation. Forced degradation of three compounds in extract showed that they were stable in oxidative condition, but highly labile under alkaline hydrolytic conditions. All three compounds in T. laurifolia leaf extract were stable at room temperature at least 3 months while a remarkable decrease of RA, vitexin and CA in the extract were found in accelerated condition. This finding could be applied for predicting the storage recommendation and expiry of T. laurifolia extract and its related pharmaceutical products.