Development of an RP-HPLC Method for Quantifying Diclofenac Diethylamine, Methyl Salicylate, and Capsaicin in Pharmaceutical Formulation and Skin Samples.

An RP-HPLC method with a UV detector was developed for the simultaneous quantification of diclofenac diethylamine, methyl salicylate, and capsaicin in a pharmaceutical formulation and rabbit skin samples. The separation was achieved using a Thermo Scientific ACCLAIMTM 120 C18 column (Waltham, MA, USA, 4.6 mm × 150 mm, 5 µm). The optimized elution phase consisted of deionized water adjusted to pH = 3 using phosphoric acid mixed with acetonitrile in a 35:65% (v/v) ratio with isocratic elution. The flow rate was set at 0.7 mL/min, and the detection was performed at 205 nm and 25 °C. The method exhibits good linearity for capsaicin (0.05-70.0 µg/mL), methyl salicylate (0.05-100.0 µg/mL), and diclofenac diethylamine (0.05-100.0 µg/mL), with low LOD values (0.0249, 0.0271, and 0.0038 for capsaicin, methyl salicylate, and diclofenac diethylamine, respectively). The RSD% values were below 3.0%, indicating good precision. The overall greenness score of the method was 0.61, reflecting its environmentally friendly nature. The developed RP-HPLC method was successfully applied to analyze Omni Hot Gel® pharmaceutical formulation and rabbit skin permeation samples.

Genetic analysis of pungency deficiency in Japanese chili pepper 'Shishito' (Capsicum annuum) revealed its unique heredity and brought the discovery of two genetic loci involved with the reduction of pungency.

The sensation of pungency generated by capsaicinoids is a characteristic trait of chili peppers (Capsicum spp.), and the presence or absence of pungency is central in determining its usage as a spice or a vegetable. In the present study, we aimed to clarify the heredity and genetic factors involved in the deficiency of pungency (quite low pungency) that is uniquely observed in the Japanese chili pepper 'Shishito' (Capsicum annuum). First, the F2 population ('Shishito' × pungent variety 'Takanotsume') was used for segregation analysis, and pungency level was investigated using capsaicinoid quantification with high-performance liquid chromatography. Also, restriction site associated DNA sequencing of the F2 population was performed, and genetic map construction and quantitative trait locus (QTL) mapping were implemented. The results indicated that the F2 population showed varying capsaicinoid content and two major QTLs were detected, Shql3 and Shql7, which explained 39.8 and 19.7% of the genetic variance, respectively. According to these results, the quite low pungency of 'Shishito' was a quantitative trait that involved at least the two loci. Further, this trait was completely separate from general non-pungent traits controlled by individual recessive genes, as described in previous studies. The present study is the first report to investigate the genetic mechanism of pungency deficiency in Japanese chili peppers, and our results provide new insights into the genetic regulation of pungency in chili pepper.

Capsaicin derived from endemic chili landraces combats Shigella pathogen: Insights on intracellular inhibition mechanism.

Ethnic tribals in northeast India have been growing and maintaining local chili landraces for ages. These chilies are known for their characteristic pungency and immense therapeutic properties. Capsaicin, a significant chili metabolite, is recognized as a natural drug for pain relief, diabetic neuropathy, psoriasis, arthritis, etc. In this study, we tried to observe the influence of locality factors on the pungency and bioactive features of Capsicum annuum L. landraces. We also checked the gastro-protective ability of these chilies, especially in the cure of shigellosis. Phytometabolite characterization and estimation were done through spectrophotometric methods. Preparative and analytical HPLC techniques were employed for extracting and purifying capsaicin-enriched fractions. Shigella flexneri growth retardation was determined through the broth dilution method. Gentamicin protection assay and ELISA were done to assess the intracellular invasion and IL-1ß inflammasome production by S.flexneri. The correlation analyses postulated that phenols, flavonoids, chlorophylls, ß-carotene, and capsaicin synthase upregulation strongly influenced capsaicin biosynthesis in chili cultivars. Correspondingly, the inhibitory efficacy of the HPLC-purified Balijuri-derived capsaicin was more effective than the Raja-derived capsaicin in inhibiting intracellular Shigella growth. Reduced levels of pro-inflammatory cytokine (IL1ß) in capsaicin-treated Shigella-infected cells probably reduced inflammation-mediated intestinal damage, limiting bacterial spread. This investigation advocates the unique potential of local chilies in curing deadly 'shigellosis' with mechanistic evidence. Our observation justifies the traditional healing practices of the ethnic people of NE India.

Biosynthesis of capsaicinoids in pungent peppers under salinity stress.

The synthesis of capsaicinoids occurs in the placenta of the fruits of pungent peppers. However, the mechanism of capsaicinoids' biosynthesis in pungent peppers under salinity stress conditions is unknown. The Habanero and Maras genotypes, the hottest peppers in the world, were chosen as plant material for this study, and they were grown under normal and salinity (5 dS m-1 ) conditions. The results showed that salinity stress harmed plant growth but increased the capsaicin content by 35.11% and 37.00%, as well as the dihydrocapsaicin content by 30.82% and 72.89% in the fruits of the Maras and Habanero genotypes, respectively, at 30 days after planting. The expression analysis of key genes in capsaicinoids biosynthesis revealed that the PAL1, pAMT, KAS, and PUN1 genes were overexpressed in the vegetative and reproductive organs of pungent peppers under normal conditions. However, under salinity stress, overexpression of PAL1, pAMT, and PUN1 genes was identified in the roots of both genotypes, which was accompanied by an increase in capsaicin and dihydrocapsaicin content. The findings showed that salinity stress caused an enhancement in the capsaicin and dihydrocapsaicin contents in the roots, leaves, and fruits of pungent peppers. Nonetheless, it was found that the production of capsaicinoids is generally not restricted to the fruits of pungent peppers.

Identification of novel SNPs in Pun1 locus for pungency in Capsicum species.

BACKGROUND: Capsaicin and its analogues known as capsaicinoids are the principal sources of pungency in Capsicum spp. In this study, characterization of North-West Himalayan chilli germplasm and commercial landraces of different Indian states known for different pungency-color combinations was done based on capsaicin concentration. Moreover, molecular variation in pungency among high, medium and mild/not pungent Capsicum spp., especially those adapted to North-West Himalayas were elucidated. METHODS AND RESULTS: Forty-nine genotypes of chilli comprising breeding lines of Kashmiri origin, commercial landraces of Southern Indian origin and one of the world's hottest chilli Bhut Jolokia from Nagaland state of India were used as an experimental material. Wide variation in capsaicin content was observed among the genotypes, wherein, Bhut Jolokia (Capsicum chinense) expressed the highest capsaicin content (10,500.75 µg/g). Further, molecular analysis of PunI gene was done for discovering SNPs responsible for variations in pungency. In the non-pungent Nishat-1 (Capsicum annuum var. grossum), the 650 bp DNA fragment was not amplified due to 2.5 kb deletion spanning the putative promoter and first exon of AT3. The amplified DNA product for high and medium pungent was sequencing. Sequence alignment among revealed SNPs which were further observed responsible for variations in amino acid sequence and protein structure. CONCLUSION: The observed variation in protein structure might be responsible for high capsaicin production in one genotype as compared to the other and hence the protein conformation determines its interaction with the substrate.

A Little Pepper-Upper? Systematic Review of Randomized Controlled Studies on Capsaicinoids, Capsinoids, and Exercise Performance.

Capsaicinoids and capsinoids are bioactive compounds mostly found in peppers. Although preclinical studies have reported that these compounds can improve exercise performance due to transient receptor potential vanilloid subtype 1 (TRPV1)-mediated thermogenesis, sympathetic modulation, and releasing calcium, it is still unclear how they affect exercise performance in humans as ergogenic supplements. Conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses reporting guide 2020, this systematic review examined the ergogenic effect of capsaicinoids and capsinoids on exercise performance in healthy adults. A total of 19 randomized placebo-controlled trials were included in the study. Studies were accessed by searching five databases (PubMed, Scopus, SPORTDiscus, Web of Science, and Cochrane Library). The quality of the studies was evaluated using the Cochrane risk-of-bias assessment tool. According to the study results, 10 studies examining the effect of capsaicinoid and capsinoid supplements on exercise performance reported positive effects. Also, the effect of capsaicinoids and capsinoids on exercise performance is more pronounced in resistance training. This difference, which varies according to the type of exercise, may be due to the correlation between capsaicin transient receptor potential vanilloid subtype 1 and insulin-like growth factor-1.

Phenolic Compounds and Capsaicinoids in Three Capsicum annuum Varieties: From Analytical Characterization to In Silico Hypotheses on Biological Activity.

The affinity of specific phenolic compounds (PCs) and capsaicinoids (CAPs) present in three Capsicum annuum varieties (Friariello, Cayenne and Dzuljunska Sipka) to the transient receptor potential vanilloid member 1 (TRPV1) was investigated by integrating an analytic approach for the simultaneous extraction and analysis through high-performance liquid chromatography coupled with ion trap mass spectrometry (HPLC/ITMS) and UV detection (HPLC-UV) of PCs and CAPs and structural bioinformatics based on the protein modelling and molecular simulations of protein-ligand docking. Overall, a total of 35 compounds were identified in the different samples and CAPs were quantified. The highest content of total polyphenols was recorded in the pungent Dzuljunska Sipka variety (8.91 ± 0.05 gGAE/Kg DW) while the lowest was found in the non-pungent variety Friariello (3.58 ± 0.02 gGAE/Kg DW). Protein modelling generated for the first time a complete model of the homotetrameric human TRPV1, and it was used for docking simulations with the compounds detected via the analytic approach, as well as with other compounds, as an inhibitor reference. The simulations indicate that different capsaicinoids can interact with the receptor, providing details on the molecular interaction, with similar predicted binding energy values. These results offer new insights into the interaction of capsaicinoids with TRPV1 and their possible actions.

Protective effects of Capsicum fruits and their constituents on damage in TNF-α-stimulated human dermal fibroblasts.

BACKGROUND: Antioxidant and anti-inflammatory effects of natural products on skin cells have been proved to be effective in improving skin damage. Capsicum species contain capsaicinoids that have antioxidant and anti-inflammatory properties, and various subspecies are cultivated. In this study, the effects of four Capsicum fruits and major constituents on oxidative stress and inflammatory reactions were measured using human dermal fibroblasts (HDFs) to verify their effects on skin damage. RESULTS: The inhibitory effects of nitric oxide (NO), reactive oxygen species (ROS), and prostaglandin E2 (PGE2 ) by cucumber hot pepper, red pepper (RDP), Shishito pepper (SSP), and Cheongyang pepper were determined in HDFs. RDP and SSP inhibited the production of NO, ROS, and PGE2 in tumor necrosis factor-alpha-stimulated HDFs. Additionally, SSP seeds restored tumor necrosis factor-alpha-induced increase in matrix metalloproteinase-1 and decreased procollagen I α1 (COLIA1). In high-performance liquid chromatography analysis of the capsaicinoids capsaicin (CAP) and dihydrocapsaicin (DHC), CAP was detected at a higher level than DHC in the peel and seeds of all four types of Capsicum fruits, and the total amount of capsaicinoids was the highest in SSP. CAP and DHC, which are major constituents of Capsicum fruits, also inhibited NO, ROS, and PGE2 and restored matrix metalloproteinase-1 and procollagen I α1. CONCLUSION: RDP and SSP were shown to have a significant protective effect on skin damage, including oxidative stress, inflammatory reactions, and reduction of collagens. Capsaicinoids CAP and DHC were proved as active constituents. This research may provide basic data for developing Capsicum fruits as ingredients to improve skin damage, such as inflammation and skin aging. © 2022 Society of Chemical Industry.

Peppermint Essential Oil and Its Major Volatiles as Protective Agents against Soft Rot Caused by Fusarium sambucinum in Cera Pepper (Capsicum pubescens).

Cera pepper (Capsicum pubescens) is an exotic fruit considered as a rich source of nutraceuticals with known benefits for human health and also an economic resource for local producers in Mexico. The present investigation reports on the in vitro and in situ antifungal activity of the essential oil from Mentha piperita and its two major volatiles (menthol and menthone) against Fusarium sambucinum, which is a causal agent of soft rot in cera pepper. The application of these components in pepper fruits previously infected with F. sambucinum caused a significant delay (p<0.05) in the emergence of soft rot symptoms. This effect was reflected in the maintenance of pH and fruit firmness during a period of 10 days. The nutrimental content of the fruits (protein, fiber, fat and other proximate parameters) was conserved in the same period of time. The nutraceutical content of these fruits was estimated by the quantification of seven carotenoids (violaxanthin, cis-violaxanthin, luteoxanthin, antheraxanthin, lutein, zeaxanthin and ß-carotene), ascorbic acid and capsaicinoids (capsaicin and dihydrocapsaicin). According to our results, the essential oil from M. Piperita and its major volatiles exerted a preservative effect on these metabolites. Our findings demonstrated that the essential oil of M. Piperita and its major volatiles represent an ecological alternative for the control of fusariosis caused by F. sambucinum in cera peppers under postharvest conditions.

Development and Validation of an HPLC Method for Simultaneous Determination of Capsaicinoids and Camphor in Over-the-Counter Medication for Topical Use.

A reverse-phase high-performance liquid chromatography method was developed to determine and quantify capsaicin (trans-8-methyl-N-vanillyl-6- nonenamid), dihydrocapsaicin (8-methyl-N-vanillylnonanamide), and camphor (trimethylbicyclo[2.2.1]heptan-2-one). It is applicable in analyses of over-the-counter (OTC) medications for topical use and raw materials such as chili pepper oleoresin. Chromatographic separation was carried out on a C18 column using an isocratic flow of the mobile phase containing acetonitrile and ultrapure water in a ratio of 2:3, with pH adjusted to 3.2 using glacial acetic acid, and a flow rate of 1.5 mL/min. The concentration of the eluting compounds was monitored by a diode-array detector at a wavelength of 281 nm. The method was evaluated for several validation parameters, including selectivity, accuracy (confidence intervals < 0.05%), repeatability, and intermediate precision. The limit of detection (LOD) was determined to be 0.070 µg/mL for capsaicin, 0.211 µg/mL for dihydrocapsaicin, and 0.060 µg/mL for camphor. The limit of quantification (LOQ) was determined to be 0.212 µg/mL for capsaicin, 0.640 µg/mL for dihydrocapsaicin, and 0.320 µg/mL for camphor. Linearity was set in the range of 2.5-200 µg/mL for capsaicin and dihydrocapsaicin and 25-2000 µg/mL for camphor. The suggested analytical method can be used for quality control of formulated pharmaceutical products containing capsaicinoids, camphor, and propolis.

Factors affecting the capsaicinoid profile of hot peppers and biological activity of their non-pungent analogs (Capsinoids) present in sweet peppers.

Capsaicinoids are acid amides of C9-C11 branched-chain fatty acids and vanillylamine and constitute important chemical compounds of Capsicum annuum together with their non-pungent analogs (capsinoids) which have an impressive list of health benefit properties (i.e., analgesia, anti-obesity, thermogenic, cardiovascular, gastrointestinal, antioxidant, anti-bacterial, anti-virulence, anti-inflamatory, anti-diabetic, inhibits angiogenesis, and improves glucose metabolism) . In this review, the state of art on how capsaicinoids are affected by different pre- and postharvest factors is discussed together with their biological activity. For instance, high light intensity and heat treatments may reduce capsaicinoid content in fruits probably due to the loss of activity of capsaicin synthase (CS) and phenylalanine ammonia lyase (PAL). The pungency in peppers varies also with environment, genotype or cultivar, node position, fruiting and maturity stages, nitrogen and potassium contents. As the fruit mature, capsaicinoid levels increase. Fruits from the second node tend to have higher accumulation of pungency than those of other positions and the pungency decreases linearly as the node position increase. Sodium hydroxide treatment reduces the pungency of pepper fruit as it hydrolyzes and modifies one of the features (vanillyl group, the acid-amide linkage and alkyl side chain) of capsaicin molecule. Salt and water stress increase PAL and capsaicin synthase activity and increase the capsaicinoid accumulation in fruit, by negatively regulating peroxidase activity at appropriate levels. Future research must be directed in better understanding the changes of capsinoids during pre and post-harvest management, the causal drivers of the loss of activity of the aminotransferase gene (pAMT) and if possible, studies with genetically modified sweet peppers with functional pAMT. Available data provided in this review can be used in different agricultural programs related to developing new cultivars with specific pungency levels. The contents of capsaicinoids and capsinoids in both fresh fruits and marketed products are also of remarkable importance considering the preferences of certain niches in market where higher added-value products might be commercialized.

Electroanalytical overview: the pungency of chile and chilli products determined via the sensing of capsaicinoids.

When you bite into a chile pepper or eat food containing chile (chilli), one might feel heat, or other associated feelings, some good such as the release of endorphins, and some bad. The heat, or pungency, and related feelings from eating chile peppers are the result of their chemical composition, i.e. the concentrations of capsaicinoids. The major components are capsaicin and dihydrocapsaicin, which occur in chiles in the ratio of 6 : 4. Other capsaicinoids occur in smaller concentrations and are known as the "minor" capsaicinoids. Wilbur L. Scoville in 1912 created an organoleptic test, now known as the Scoville scale, which asked a panel of tasters to state when an increasingly dilute solution of the chile pepper in alcohol no longer burned the mouth. Following the Scoville scale, a plethora of analytical techniques later followed. In this overview, we explore the endeavours directed to the development of electrochemical-based sensors for the determination of capsaicin and related compounds, starting from their use in hyphenated laboratory set-ups to their modern use as stand-alone electroanalytical sensors. The latter have the advantage of providing a rapid and sensitive methodology that has the potential to be translated in the field; future trends and issues to be overcome are consequently suggested.

Patterns and compound specific stable carbon isotope analysis (δ13 C) of capsaicinoids in Cayenne chilli fruits of different ripening stages.

INTRODUCTION: Capsaicinoids are alkaloids of high pungency which are exclusively formed by fruits of the genus Capsicum. Capsaicinoid content and composition of Capsicum fruits are influenced by ripening. OBJECTIVE: Determination of changes in content and pattern of individual capsaicinoids in chilli pods with fruit ripening. Compound specific stable carbon isotope analysis (δ13 C values (‰), CSIA) was used for a better understanding of capsaicinoid development during fruit ripening. METHODOLOGY: Cayenne chillies (Capsicum annuum) were grown in a glasshouse and harvested from different plants at four ripening stages (unripe, semi-ripe, ripe, overripe). Nine capsaicinoids (one verified by synthesis) were quantified by gas chromatography with mass spectrometry (GC-MS). For CSIA, an acetylation method for derivatising capsaicinoids was developed. RESULTS: Variations in the patterns of the nine capsaicinoids were not relatable to a distinct ripening stage and also total contents varied extensively from plant to plant. However, changes in total capsaicinoid concentrations were systematic. In almost all plants, maximum values were reached in unripe fruits, then decreased to semi-ripe samples and increased again in the following ripening process. Likewise, δ13 C values of individual capsaicinoids were always by ~2‰ heavier in unripe than in semi-ripe or ripe fruits. However, direction of changes in sum-δ13 C values (‰) (taking contributions of all capsaicinoids together) could not be explained by corresponding variations in capsaicinoid concentrations. CONCLUSIONS: Both quantification and δ13 C values (‰) verified the presence of ripening-related changes in the capsaicinoid content which may be caused by simultaneously proceeding reactions like synthesis, storage and degradation of capsaicinoids.

A sensitive 1 H NMR spectroscopic method for the quantification of capsaicin and capsaicinoid: morpho-chemical characterisation of chili land races from northeast India.

INTRODUCTION: Proton (1 H) nuclear magnetic resonance (NMR) spectroscopy based analytical method for the quantification of capsaicin (major pungent principle of chili) has certain advantages including short data acquisition time and better structural authentication. Earlier NMR methods are associated with either of the bottlenecks such as low or lack of information on the sensitivity and scope for the quantification of total capsaicinoid. OBJECTIVE: To develop a sensitive 1 H quantitative NMR (qNMR) technique for capsaicin and total capsaicinoid in dry chili and chili oleoresin and to demonstrate its applicability in a real sample set. METHOD: A 1 H qNMR method was developed using benzene as the internal standard for the quantification of capsaicin (terminal methyl signal) as well as total capsaicinoid (benzyl methylene signal) in dry chili and oleoresin and validated in terms of specificity, linearity, sensitivity, accuracy and precision. RESULTS: The developed 1 H qNMR method was specific, sensitive (limit of detection 4.4 µg/mL and limit of quantitation 14.8 µg/mL), linear in the range 0.083-8.33 mg/mL of capsaicin, accurate and precise. The credibility of the developed method was showcased in the morpho-chemical characterisation of commercially available 15 chili land races from northeast India. The analysis identified the land races with a wide range of capsaicin (trace to 1.49% in the dry fruit and trace to 6.21% in the oleoresin w/w) and oleoresin content (3.35-26.78% w/w). CONCLUSION: The standardized 1 H qNMR method facilitated the findings of chemical basis for the selection of chili land races from this region, capable of producing high-yielding oleoresin with intended degree of pungency.

Characterization of newly developed pepper cultivars (Capsicum chinense) 'Dieta0011-0301', 'Dieta0011-0602', 'Dieta0041-0401', and 'Dieta0041-0601' containing high capsinoid concentrations and a strong fruity aroma.

Capsaicinoids are responsible for the pungent flavor of peppers (Capsicum sp.). The cultivar CH-19 Sweet is a non-pungent pepper mutant that biosynthesizes the low-pungent capsaicinoid analogs, capsinoids. Capsinoids possess important pharmaceutical properties. However, capsinoid concentrations are very low in CH-19 Sweet, and Capsicum cultivars with high content capsinoids are desirable for industrial applications of capsinoids. Habanero, Bhut Jolokia, and Infinity are species of Capsicum chinense, and have strong pungency and intense fruity flavors. In the present study, we report new cultivars with high concentrations of capsinoids (more than ten-fold higher than in CH-19 Sweet), and showed that these cultivars (Dieta0011-0301 and Dieta0011-0602 from Bhut Jolokia, Dieta0041-0401 and Dieta0041-0601 from Infinity) are of nutritional and medicinal value and have fruity aromas. We also obtained a vanilla bean flavor, vanillyl alcohol, and vanillyl ethyl ether from capsinoids in the fruit of these cultivars following the addition of ethanol at room temperature. ABBREVIATIONS: p-AMT: putative aminotransferase; C. annuum: Capsicum annuum; C. chinense: Capsicum chinense; dCAPS: derived Cleaved Amplified Polymorphic Sequences.

Mutation in the putative ketoacyl-ACP reductase CaKR1 induces loss of pungency in Capsicum.

KEY MESSAGE: A putative ketoacyl-ACP reductase (CaKR1) that was not previously known to be associated with pungency of Capsicum was identified from map-based cloning and functional characterization. The pungency of chili pepper fruits is due to the presence of capsaicinoids, which are synthesized through the convergence of the phenylpropanoid and branched-chain fatty acid pathways. The extensive, global use of pungent and non-pungent peppers underlines the importance of understanding the genetic mechanism underlying capsaicinoid biosynthesis for breeding pepper cultivars. Although Capsicum is one of the earliest domesticated plant genera, the only reported genetic causes of its loss of pungency are mutations in acyltransferase (Pun1) and putative aminotransferase (pAMT). In this study, a single recessive gene responsible for the non-pungency of pepper No.3341 (C. chinense) was identified on chromosome 10 using an F2 population derived from a cross between Habanero and No.3341. Five candidate genes were identified in the target region, within a distance of 220 kb. A candidate gene, a putative ketoacyl-ACP reductase (CaKR1), of No.3341 had an insertion of a 4.5-kb transposable element (TE) sequence in the first intron, resulting in the production of a truncated transcript missing the region coding the catalytic domain. Virus-induced gene silencing of CaKR1 in pungent peppers resulted in the decreased accumulation of capsaicinoids, a phenotype consistent with No.3341. Moreover, GC-MS analysis of 8-methyl-6-nonenoic acid, which is predicted to be synthesized during the elongation cycle of branched-chain fatty acid biosynthesis, revealed that its deficiency in No.3341. Genetic, genomic, transcriptional, silencing, and biochemical precursor analyses performed in combination provide a solid ground for the conclusion that CaKR1 is involved in capsaicinoid biosynthesis and that its disruption results in a loss of pungency.

Alternative mass spectrometry techniques for the validation of the fragmentation pattern of capsaicin and dihydrocapsaicin.

RATIONALE: Capsaicinoids are prevalent secondary metabolites in many natural and synthetic pharmacological compounds. To date, several soft ionization studies related to capsaicinoids have been reported; they all proposed a common fragmentation pattern based on a rearrangement of the aromatic double bonds and the fragmentation of the various positional acyl chains. However, the mechanism has never been validated by high-resolution analyses. Consequently, in this work, a validated fragmentation mechanism of the main capsaicinoids, capsaicin (1) and dihydrocapsaicin (2), is offered. METHODS: In order to propose and validate a common electron ionization (EI) fragmentation mechanism for the target analytes, the following mass spectrometric methods were employed: collision-induced dissociation (CID) by means of linked scans (LS), reinforcing the methodology by high-resolution mass spectrometry (HRMS), in addition to appropriate deuterium-labeled experiments performed using gas chromatography/mass spectrometry (GC/MS) and direct analysis in real time (DART). RESULTS: In a first stage, a common EI fragmentation pattern comprising two pathways was proposed for compounds 1 and 2; then, the suggested mechanism was validated by CID-LS together with HRMS complemented by DART-deuterium-labeling studies. The obtained results are indicative that the corresponding molecular ions were conveniently observed, m/z 305 and m/z 307; it is worth noting that the common base peak is in correspondence with a tropylium ion derivative (m/z 137), as a consequence of a McLafferty rearrangement. In addition to these highlighted fragments, other common ions, m/z 122 and m/z 94, and their corresponding trajectory, were confirmed using the same approach. Finally, the proposed mechanism was complementarily validated by deuterium-labeling studies, taking into account the two exchangeable hydrogens present in the phenolic and the amidic moieties. CONCLUSIONS: A common validated EI fragmentation pattern for both capsaicin and dihydrocapsaicin was established using appropriated mass spectrometric methods together with convenient hydrogen/deuterium labeling. This study provides a new alternative to validate mechanisms of fragmentation of important natural products.

A simple 1 H NMR based assay of total capsaicinoid levels in Capsicum using signal suppression in non-deuterated solvent.

BACKGROUND: The heat of Capsicum fruits is routinely assayed using high-performance liquid chromatography (HPLC) to determine capsaicin (CA) and dihydrocapsaicin (DHC) levels. The assay can be time consuming, with each HPLC run typically lasting 10 min. Nuclear magnetic resonance (NMR) is eminently suitable for quantification of fruit extracts, although it has been largely ignored for quantitative chilli analysis. The present study describes a novel approach using solvent suppression in protic solvent (i.e. non-deuterated) to quantify total capsaicinoid levels in chilli extracts. RESULTS: Using solvent suppression techniques and maleic acid as an internal standard, capsaicinoid content in a series of accurately weighed standard samples was determined over a range between 40 and 720 ppm (0.13-2.35 mmolar) with high accuracy and precision. The measurement was linear over the entire range. This method was subsequently used with ten authentic Capsicum samples (seven chinense, two annuum and one baccatum) and showed an excellent correlation with the HPLC data. CONCLUSION: The results of the present study confirm that NMR in non-deuterated solvent can provide a rapid and robust assessment of the pungency of capsicum fruits. © 2018 Society of Chemical Industry.

Genetic diversity analysis of selected Capsicum annuum genotypes based on morphophysiological, yield characteristics and their biochemical properties.

BACKGROUND: Assessment of the different desirable characters among chili genotypes has expanded the effective selection for crop improvement. Identification of genetically superior parents is important in assortment of the best parents to develop new chili hybrids. RESULTS: This study was done to assess the hereditary assorted variety of selected genotypes of Capsicum annuum based on their morphophysiological and yield traits in two planting seasons. The biochemical properties, capsaicinoid content (capsaicin and dihydrocapsaicin), total phenolics content and antioxidant action determination of unripe and ripe chili pepper fruits were carried out in dry fruits. AVPP9813 and Kulai 907 were observed to have high fruit yields, with 541.39 and 502.64 g per plant, respectively. The most increased genotypic coefficient of variation (GCV) and phenotypic coefficient of variation (PCV) were shown by the fruit number per plant (49.71% and 66.04%, respectively). High heritability was observed in yield characters viz-à-viz fruit weight, length and girth and indicated high genetic advance. Eight groups were obtained from the cluster analysis. For the biochemical analysis, the capsaicinoid content and total phenolic content were high in Chili Bangi 3 at unripe and ripe fruit stages, while for antioxidant activity SDP203 was the highest in ripe dry fruit. CONCLUSION: Higher GCV and PCV, combined with moderate to high heritability and high hereditary progress, were seen in number of fruit per plant, fruit yield per plant and fruit weight per fruit. These findings are beneficial for chili pepper breeders to select desirable quantitative characters in C. annuum in their breeding program. © 2018 Society of Chemical Industry.

Preservation of red pepper flakes using microwave-combined cold plasma treatment.

BACKGROUND: Red pepper flakes are often contaminated with various microorganisms; however, any technologies aiming to decontaminate the flakes should also maintain their quality properties. This study investigated the effect of microwave-combined cold plasma treatment (MCPT) at different microwave power densities on microbial inactivation and preservation of red pepper flakes. Red pepper flake samples inoculated with spores of Bacillus cereus or Aspergillus flavus and without inoculation were subjected to MCPT at 900 W for 20 min at either low microwave power density (LMCPT, 0.17 W m-2 ) or high microwave power density (HMCPT, 0.25 W m-2 ). RESULTS: The numbers of B. cereus and A. flavus spores on red pepper flakes after LMCPT and HMCPT were initially reduced by 0.7 ± 0.1 and 1.4 ± 0.3 log spores cm-2 and by 1.5 ± 0.3 and 1.5 ± 0.2 log spores cm-2 respectively and remained constant for 150 days at 25 °C. Immediately after HMCPT, the concentrations of capsaicin and ascorbic acid in the flakes were significantly lower than in untreated samples; however, no difference in concentration was detected during storage. Neither LMCPT nor HMCPT affected the antioxidant activity or color of the flakes during storage. LMCPT also did not affect the sensory properties and the concentrations of capsaicin and dihydrocapsaicin of the flakes, indicating its suitability in preserving their quality properties. CONCLUSION: MCPT may provide an effective non-thermal treatment for food preservation which can improve the microbial safety and stability of red pepper flakes while maintaining intact their qualitative properties. © 2018 Society of Chemical Industry.