Progress in the analysis of phytocannabinoids by HPLC and UPLC (or UHPLC) during 2020-2023.

INTRODUCTION: Organic molecules that bind to cannabinoid receptors are known as cannabinoids. These molecules possess pharmacological properties similar to those produced by Cannabis sativa L. High-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC, also known as ultra-high-performance liquid chromatography, UHPLC) have become the most widely used analytical tools for detection and quantification of phytocannabinoids in various matrices. HPLC and UPLC (or UHPLC) are usually coupled to an ultraviolet (UV), photodiode array (PDA), or mass spectrometric (MS) detector. OBJECTIVE: To critically appraise the literature on the application of HPLC and UPLC (or UHPLC) methods for the analysis of phytocannabinoids published from January 2020 to December 2023. METHODOLOGY: An extensive literature search was conducted using Web of Science, PubMed, and Google Scholar and published materials including relevant books. In various combinations, using cannabinoid in all combinations, cannabis, hemp, hashish, C. sativa, marijuana, analysis, HPLC, UHPLC, UPLC, and quantitative, qualitative, and quality control were used as the keywords for the literature search. RESULTS: Several HPLC- and UPLC (or UHPLC)-based methods for the analysis of phytocannabinoids were reported. While simple HPLC-UV or HPLC-PDA-based methods were common, the use of HPLC-MS, HPLC-MS/MS, UPLC (or UHPLC)-PDA, UPLC (or UHPLC)-MS, and UPLC (or UHPLC)-MS/MS was also reported. Applications of mathematical and computational models for optimization of protocols were noted. Pre-analyses included various environmentally friendly extraction protocols. CONCLUSION: During the last 4 years, HPLC and UPLC (or UHPLC) remained the main analytical tools for phytocannabinoid analysis in different matrices.

The Identification and Cytotoxic Evaluation of Nutmeg (Myristica fragrans Houtt.) and Its Substituents.

The aril and seed of nutmeg, Myristica fragrans Houtt. (Myristicaceae), hold significant value in various industries globally. Our preliminary research found two morphological variations: a globose shape and an oval shape. Due to these different characteristics, the safety of consumers is of primary concern. Thus, authentication and comparative pharmacological and toxicity analyses are necessary. In this study, pharmacognostic and advanced phytochemical analyses, DNA barcoding, cytotoxicity, and the anti-nitric oxide production of commercial Thai nutmeg were examined. Via morphologic examinations and TLC fingerprinting, all the sampled aril and seed were categorized into globose and oval-shaped groups. The results of HPLC, GC-MS, and LC-MS/MS experiments revealed distinct differences between these groups. The DNA barcoding of the trnH-psbA region using the BLAST method and neighbor-joining tree analyses confirmed the globose nutmeg as M. fragrans and the oval-shaped variant as M. argentea. A comparison was then carried out between the potential toxicity and anti-inflammatory capabilities of M. fragrans and M. argentea. Cytotoxicity tests on HaCaT, 3T3-L1, Caco-2, HEK293, and RAW264.7 were performed using both methanolic extracts and volatile oil from the arils and seeds of both species. This study concludes that blending or substituting these two species maintains their therapeutic integrity without posing safety concerns.

Novel non-cytotoxic antimicrobial peptides WSKK11 and WSRR11 with potent activity against Cutibacterium acnes.

OBJECTIVES: Cutibacterium acnes is one of the common multifactorial causes that play an important role in the pathophysiology of acne vulgaris. We aimed to develop novel antimicrobial peptides for reduction of the hypercolonization. METHODS: Six cationic peptides were derived by de novo designation. The antimicrobial and cytotoxic activities of peptides were investigated. The peptide conformation was determined by circular dichroism spectrometry. The antimicrobial effects of peptides were evaluated using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and DNA-binding ability assay. RESULTS: Among designed peptides, WSKK11 and WSRR11 were effective antimicrobials against C. acnes at MICs of 128 and 64 mg/L, respectively. The MICs of WSKK11 against Staphylococcus epidermidis, Staphylococcus aureus and Candida albicans were 8, 8 and 32 mg/L, while those of WSRR11 were 64, 32 and 32 mg/L, respectively. WSKK11 and WSRR11 were less toxic to human erythrocytes (<2%) and not toxic to macrophages, keratinocytes and fibroblasts up to 512 mg/L. WSKK11 and WSRR11 mostly revealed the conformation of the undefined or random coil structures under mimicked environmental conditions. The peptides affected cell surfaces and cell membranes of C. acnes as well as possibly translocating through the cell membrane, observed by a combination of SEM and TEM, respectively. WSKK11 and WSRR11 had the ability to bind bacterial DNA. CONCLUSIONS: The two novel antimicrobial peptides WSKK11 and WSRR11 are members of a new class of antimicrobial agents that could deal with acne problems. Therefore, the antimicrobial peptides may be promising novel active agents for dermatological, beauty and cosmeceutical applications.

Novel antioxidant and anti-inflammatory peptides from the Siamese crocodile (Crocodylus siamensis) hemoglobin hydrolysate.

Novel antioxidant and anti-inflammatory peptides were isolated from hydrolysates of Siamese crocodile (Crocodylus siamensis) hemoglobin. C. siamensis hemoglobin hydrolysates (CHHs) were obtained by pepsin digestion at different incubation times (2, 4, 6, and 8 H) at 37 °C and subjected to antioxidant and anti-inflammatory activity assessment. CHH obtained by 2-H hydrolysis (2H-CHH) showed the highest anti-inflammatory activity with respect to decreasing nitric oxide (NO) production, whereas the strongest antioxidant activity was found for 6-H hydrolysis (6H-CHH) against nitric oxide radicals. To evaluate the anti-inflammatory and antioxidant activity of individual peptide components, 2H-CHH and 6H-CHH were purified by semipreparative HPLC. Peptide fraction P57 isolated from 6H-CHH was found to exhibit the highest nitric oxide radical inhibition activity (32.0%). Moreover, purification of 2H-CHH yielded peptide fraction P16, which displayed a high efficacy in decreasing NO production of macrophage RAW 264.7 cells (83.2%) and significantly reduced proinflammatory cytokines and inflammatory mediators interleukin-6 (IL-6), interleukin-1 beta (IL-1ß), and prostaglandin-E2 (PGE2 ) production to about 2.0, 0.3, and 1.9 ng/mL, respectively. Using LTQ orbitrap XL mass spectrometry, active peptide sequences were identified as antioxidant KIYFPHF (KF7), anti-inflammatory SAFNPHEKQ (SQ9), and IIHNEKVQAHGKKVL (IL15). Additionally, CHHs simulated gastric and intestinal in vitro digestion positively contributed to antioxidant and anti-inflammatory activity. Taken collectively, the results of this work demonstrate that CHHs contain several peptides with anti-inflammatory and antioxidant properties, which may prove valuable as treatment or supplement against diseases associated with inflammation and oxidative stress.

Cationic Antimicrobial Peptides Derived from Crocodylus siamensis Leukocyte Extract, Revealing Anticancer Activity and Apoptotic Induction on Human Cervical Cancer Cells.

Known antimicrobial peptides KT2 and RT2 as well as the novel RP9 derived from the leukocyte extract of the freshwater crocodile (Crocodylus siamensis) were used to evaluate the ability in killing human cervical cancer cells. RP9 in the extract was purified by a combination of anion exchange column and reversed-phase HPLC, and its sequence was analyzed by mass spectrometry. The novel peptide could inhibit Gram-negative Vibrio cholerae (clinical isolation) and Gram-positive Bacillus pumilus TISTR 905, and its MIC values were 61.2 µM. From scanning electron microscopy, the peptide was seen to affect bacterial surfaces directly. KT2 and RT2, which are designed antimicrobial peptides using the C. siamensis Leucrocin I template, as well as RP9 were chemically synthesized for investigation of anticancer activity. By Sulforhodamine B colorimetric assay, these antimicrobial peptides could inhibit both HeLa and CaSki cancer cell lines. The IC50 values of KT2 and RT2 for HeLa and CaSki cells showed 28.7-53.4 and 17.3-30.8 µM, while those of RP9 were 126.2 and 168.3 µM, respectively. Additionally, the best candidate peptides KT2 and RT2 were used to determine the apoptotic induction on cancer cells by human apoptosis array assay. As a result, KT2 and RT2 were observed to induce apoptotic cell death in HeLa cells. Therefore, these results indicate that KT2 and RT2 with antimicrobial activity have a highly potent ability to kill human cervical cancer cells.

Design and synthesis of cationic antibacterial peptide based on Leucrocin I sequence, antibacterial peptide from crocodile (Crocodylus siamensis) white blood cell extracts.

Leucrocin I is an antibacterial peptide isolated from crocodile (Crocodylus siamensis) white blood cell extracts. Based on Leucrocin I sequence, cationic peptide, NY15, was designed, synthesized and evaluated for antibacterial activity against Bacillus sphaericus TISTR 678, Bacillus megaterium (clinical isolate), Vibrio cholerae (clinical isolate), Salmonella typhi (clinical isolate), Salmonella typhi ATCC 5784 and Escherichia coli 0157:H7. The efficacy of the peptide made from all L-amino acids was also compared with all D-amino acids. The peptide made from all D-amino acids was more active than the corresponding L-enantiomer. In our detailed study, the interaction between peptides and the cell membrane of Vibrio cholerae as part of their killing mechanism was studied by fluorescence and electron microscopy. The results show that the membrane was the target of action of the peptides. Finally, the cytotoxicity assays revealed that both L-NY15 and D-NY15 peptides are non-toxic to mammalian cells at bacteriolytic concentrations.

Purification and characterization of antioxidant peptides from leukocyte extract of Crocodylus siamensis.

Antioxidant peptides were isolated from the leukocyte extract of the Siamese crocodile, Crocodylus siamensis. Crocodile leukocyte was extracted by a combination of methods including freeze-thawing, acetic acid extraction and homogenization. The peptides in the leukocyte extract were purified by anion exchange chromatography and reversed phase-high performance liquid chromatography. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay was used to evaluate the antioxidant activity of the elution peaks at each purification step. As a result, there were two purified peptides exhibiting strong antioxidant activity in reducing free radicals on DPPH molecules. The amino acid sequences of these peptides were determined by LC-MS/MS as TDVLGLPAK (912.5 Da) and DPNAALPAGPR (1,148.6 Da), and their IC50 values were 153.4 and 95.7 µM, respectively. The results of this study therefore indicate that leukocyte extract of C. siamensis contains peptides with antioxidant activity which could be used as a novel antioxidant.

Improving the antibacterial activity and selectivity of an ultra short peptide by hydrophobic and hydrophilic amino acid stretches.

The principle of amino acid stretches tagged at the C terminal of Luecrocin I, which is an ultra-short antibacterial peptide, by tryptophan and arginine or lysine has been reported. The choice of amino acid type at each stretch position depends on the hydrophobic and hydrophilic regions visualized in the helical wheel pattern of Luecrocin I. Oligopeptide tagging should also consider the properties such as positive charge, hydrophobicity, the content of hydrophobic amino acids, polar angle, the properly hydrophilic and hydrophobic facets. Amidation at C terminal and lysine substitute for arginine can increase selectivity between mammalian cells (hemolytic and MTT assay) and bacterial cells tested. KT2 and RT2 which have 53% hydrophobic residues, 7 positive charges, 160° polar angle, -0.02 (KT2) and -0.04 (RT2) hydrophobicity were effective against S. typhi DMST 22842, S. epidermidis ATCC 12228, E. coli ATCC 25922 and V. cholerae non-O1, non-O139. The SEM images implied that the antibacterial mechanism of RT2 and KT2 may depend on concentration rather than time. Finally, RT2 and KT2 can be new antibacterial agents or may be further developed for alternative antibiotics.