Development of Methyl Ester Antibody-Based Competitive Indirect ELISA for Quantitative Detection of Mitragynine in Human Urine.

Mitragynine is the main psychoactive compound of Mitragyna speciosa Korth. (kratom). This alkaloid could render psychotropic effects and is often misused as a substitute for commercial drugs. Nowadays, the increasing popularity of kratom has led to the development of a rapid and effective detection method. The detection of mitragynine in a biological sample such as urine requires a highly sensitive and specific method due to the complex nature of mitragynine in urine. Enzyme-linked immunosorbent assay (ELISA) is well known as a rapid screening method for biological samples. In this study, a competitive indirect ELISA was successfully developed using MG-22-OCH3 IgG as a detection antibody for mitragynine in human urine. The mitragynine immunoassay showed a limit of detection and a limit of quantification of 0.412 and 1.25 µg/mL, respectively. The measurement range was between 0.01 and 100.0 µg/mL, with a minimal inhibition (IC50) value of 0.152 µg/mL. The developed ELISA was validated using a gold method such as high-performance liquid chromatography-mass spectrometry (HPLC-MS). The percentage of recovery and the coefficient of variation (CV) for the ELISA and LCMS/MS analyses were 84.0-95.70%, 99.20-112.0%, 7.69-9.78%, and 2.86-6.62%, respectively. This indicates that the developed ELISA is a reliable method that can be used as a rapid approach for quantifying mitragynine content in biological samples.

Methyl ester and aromatic ether modification of mitragynine for generation of mitragynine-specific polyclonal antibodies.

Mitragynine is an alkaloid from Mitragyna speciosa Korth. (kratom), a native tropical plant in Southeast Asia. It could render psychotropic effects and is often misused in substitution for commercial drugs. In recent years, the consumption of kratom has grown rapidly and has led some countries to ban its use. The misuse of kratom can be detected and monitored through the determination of mitragynine from biological samples of the users. Therefore, the development of a rapid and effective detection method is needed. In this study, polyclonal antibodies were produced using mitragynine coupled to a carrier protein (cationic bovine serum albumin, cBSA) as an immunogen, which was prepared with coupling agents (i.e., N, N- dicyclohexylcarbodiimide, DCC and N-hydroxysuccinimide, NHS). It was conjugated to different mitragynine structure, 16-COOCH3 (methyl ester) and 9-OCH3 (aromatic ether). 2,4,6-Trinitrobenzenesulfonic acid (TNBS) method showed that 45 and 46 amino groups were bound to C22-MG-cBSA and C9-MG-cBSA, respectively. Fourier-transform infrared spectroscopy (FTIR) spectral changes at C22- and C9-hydroxymitragynine indicated reduction and demethylation process. In UV-Vis spectra, conjugated mitragynine to cBSA and OVA were displayed at a spectral region at 240-300 nm. For the antibody titre, the C22-MG-cBSA anti-serum showed a significantly higher titre than the C9-MG-cBSA at 1/128000 and 1/32000 dilutions, respectively. The detection range of the developed competitive indirect ELISA (CI-ELISA) was 0.01 to 10.00 µg/mL (R2 = 0.9964). The assay exhibited a limit of detection (LOD) and limit of quantification (LOQ) at 0.041 and 0.124 µg/mL, respectively. The antibody produced is a high-value biorecognition molecule that can be further used in developing immuno-based detection methods such as immunosensors and immunochromatographic lateral flow assays. This will benefit the task force or forensic agencies for toxicological screening with high speed and efficiency.

Preparation, Characterization, and Radiolabeling of [68Ga]Ga-NODAGA-Pamidronic Acid: A Potential PET Bone Imaging Agent.

Early diagnosis of bone metastases is crucial to prevent skeletal-related events, and for that, the non-invasive techniques to diagnose bone metastases that make use of image-guided radiopharmaceuticals are being employed as an alternative to traditional biopsies. Hence, in the present work, we tested the efficacy of a gallium-68 (68Ga)-based compound as a radiopharmaceutical agent towards the bone imaging in positron emitting tomography (PET). For that, we prepared, thoroughly characterized, and radiolabeled [68Ga]Ga-NODAGA-pamidronic acid radiopharmaceutical, a 68Ga precursor for PET bone cancer imaging applications. The preparation of NODAGA-pamidronic acid was performed via the N-Hydroxysuccinimide (NHS) ester strategy and was characterized using liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MSn). The unreacted NODAGA chelator was separated using the ion-suppression reverse phase-high performance liquid chromatography (RP-HPLC) method, and the freeze-dried NODAGA-pamidronic acid was radiolabeled with 68Ga. The radiolabeling condition was found to be most optimum at a pH ranging from 4 to 4.5 and a temperature of above 60 °C. From previous work, we found that the pamidronic acid itself has a good bone binding affinity. Moreover, from the analysis of the results, the ionic structure of radiolabeled [68Ga]Ga-NODAGA-pamidronic acid has the ability to improve the blood clearance and may exert good renal excretion, enhance the bone-to-background ratio, and consequently the final image quality. This was reflected by both the in vitro bone binding assay and in vivo animal biodistribution presented in this research.

A new coumarin from stem bark of Calophyllum wallichianum.

A phytochemical study carried out on the plant, Calophyllum wallichianum has led to the isolation of a new coumarin, wallimarin T (1) and a known coumarin, calanolide E (2) along with two common triterpenes, friedelin (3) and stigmasterol (4). The structures of these compounds were elucidated with the aid of spectroscopic analyses such as FT-IR, GC-MS, and NMR. MIC assay against the Bacillus bacteria were conducted on the extracts and this gave MIC values ranging from 0.313 to 1.25 mg/mL. Compound 2 was weakly inhibitory towards the Bacilli strains with MIC values ranging from 0.25-0.50 mg/mL. Wallimarin T (1) was not active towards all four bacteria. Overall, the extracts exhibited weak bactericidal properties whereas compound 2 was not bactericidal on the tested bacteria. The hexane and chloroform extracts of the plant were found to be inhibitors to the growth of Bacillus megaterium, Bacillus cereus, Bacillus pumilus and Bacillus subtilis.

Isolation and structural modifications of ananixanthone from Calophyllum teysmannii and their cytotoxic activities.

Two naturally occurring xanthones, ananixanthone (1) and ß-mangostin (2), were isolated using column chromatographic method from the n-hexane and methanol extracts of Calophyllum teysmannii, respectively. The major constituent, ananixanthone (1), was subjected to structural modifications via acetylation, methylation and benzylation yielding four new xanthone derivatives, ananixanthone monoacetate (3), ananixanthone diacetate (4), 5-methoxyananixanthone (5) and 5-O-benzylananixanthone (6). Compound 1 together with its four new derivatives were subjected to MTT assay against three cancer cell lines; SNU-1, K562 and LS174T. The results indicated that the parent compound has greater cytotoxicity capabilities against SNU-1 and K562 cell lines with IC50 values of 8.97 ± 0.11 and 2.96 ± 0.06 µg/mL, respectively. Compound 5 on the other hand exhibited better cytotoxicity against LS174T cell line with an IC50 value of 5.76 ± 1.07 µg/mL.

Acetyl- and O-alkyl- derivatives of ß-mangostin from Garcinia mangostana and their anti-inflammatory activities.

Pure ß-mangostin (1) was isolated from the stem bark of Garcinia mangostana L. One monoacetate (2) and five O-alkylated ß-mangostin derivatives (3-7) were synthesised from ß-mangostin. The structures of these compounds were elucidated and determined using spectroscopic techniques such as 1D NMR and MS. The cytotoxicities and anti-inflammatory activities of these five compounds against RAW cell 264.7 were tested. The structural-activity relationship studies indicated that ß-mangostin showed a significant activity against the LPS-induced RAW cell 264.7, while the acetyl- as well as the O-alkyl- ß-mangostin derivatives did not give good activity. Naturally occurring ß-mangostin demonstrated comparatively better anti-inflammatory activity than its synthetic counterparts.

Synthesis of libraries of thiazole, oxazole and imidazole-based cyclic peptides from azole-based amino acids. A new synthetic approach to bistratamides and didmolamides.

Treatment of a 1 : 1 mixture of the thiazole-based amino acids 8a and 8b with FDPP-i-Pr(2)NEt in CH(3)CN gave a mixture of the cyclic trimers 14, 15, 16 and 17 and the cyclic tetramers 19 and 23 in the ratio 2 : 7 : 5 : 8 : 1 : 1 and in a combined yield of 70%. Separate coupling reactions between the bisimidazole amino acid 45 and the thiazole/oxazole amino acids 43a and 42a in the presence of FDPP-i-Pr(2)NEt led to the bisimidazole based cyclic trimers 55 and 57 respectively (54-57%) and to the cyclic tetramer 56 (8-11%). Similar coupling reactions involving the bisthiazole and bisoxazole amino acids 49 and 47 with the imidazole/oxazole/thiazole amino acids 41a, 42a and 43a gave rise to the library of oxazole, thiazole and imidazole-based cyclic peptides 58, 59, 60, 61, 62, 63, 64 and 65. A coupling reaction between the bisthiazole amino acid 49 and the oxazole amino acid 73 led to an efficient (36% overall) synthesis of bistratamide H (67) found in the ascidian Lissoclinum bistratum. Coupling reactions involving oxazolines with thiazole amino acids were less successful. Thus, a coupling reaction between the phenylalanine-based oxazoline amino acid 71a and either the thiazole amino acid 8a or the bisthiazole amino acid 74 gave only a 2% yield of the cyclic hexapeptide didmolamide A (4) found in the ascidian Didemnum molle. Didmolamide B (68) was obtained in 9% yield from a coupling reaction between 74 and the phenylalanine threonine amino acid 72, using either FDPP or DPPA.