1. Introduction
Compounds known as capsaicinoids cause the spicy flavor (pungency) of chili pepper fruit. The primary capsaicinoid in chili pepper is capsaicin, followed by dihydrocapsaicin, nordihydrocapsaicin, homodihydrocapsaicin and homocapsaicin. Capsaicin and dihydrocapsaicin account for approximately 90% of capsaicinoids in chili pepper fruit, are the two most potent capsaicinoids and their molecules differ only in the saturation of the acyl group [
1,
2,
3]. Capsaicin (trans-8-methyl-
N-vanillyl-6-nonenamide) is a crystalline, lipophilic, colorless and odorless alkaloid with the molecular formula C
18H
27NO
3. Its molecular weight is 305.40 g/mol, and it is fat-, alcohol- and oil-soluble. First crystallized in 1876 by Tresh, who named it, capsaicin’s molecular structure was resolved by Nelson and Dawson in 1919 [
4]. Capsaicin displays
cis/trans isomerism because the double bond prevents internal rotation. Capsaicin is always found as the
trans isomer because in the
cis form, the -CH(CH
3)
2 and the longer chain on the other side of the double bond will be close together, causing them to repel each other slightly; this steric hindrance does not exist in the
trans isomer. This additional strain imposed causes the
cis isomer to be a less stable arrangement than the
trans isomer (
Figure 1) [
5].
Figure 1.
Regions of the molecule of capsaicin. A (aromatic ring); B (amide bond); and C (hydrophobic side chain).
Figure 1.
Regions of the molecule of capsaicin. A (aromatic ring); B (amide bond); and C (hydrophobic side chain).
Structure-activity relationships (SAR) for capsaicin agonists (a substance that is capable of binding to a receptor and elicit a response in the cell), have previously been rationalized by dividing the capsaicin molecule into three regions: A (aromatic ring), B (amide bond) and C (hydrophobic side chain) (
Figure 1). It is known that the substituents in the positions 3 and 4 of the A-ring are essential for potent agonist activity, and the phenolic 4-OH group in capsaicin analogues is of particular importance, H-bond donor/acceptor properties of the phenol group are key for agonist activity [
6].C-Region SAR in capsaicin analogues have been discussed in detail in some reports. In summary, a hydrophobic group, e.g., an octyl chain or substituted benzyl or group, is required for high potency. Optimally, such aralkyl groups are substituted in the
para position by small hydrophobic moieties. [
2], by other way Barbero
et al., [
7] reported the importance of lateral chain lengths for the bioactivity of capsaicinoids, which was higher between 8 and 9 carbons atoms.
The more than twenty known capsaicinoids are all amides formed from condensation of vanillylamine and fatty acids of different chain lengths. The forms of different natural capsaicinoids depend on the number of lateral chain carbons (R) or the presence or absence of unsaturations (
Figure 2). Capsaicinoids are synthesized naturally in the placenta of chili fruits from enzymatic condensation of vanillylamine and different-sized fatty acid chains which are elongated by a fatty acid synthase. This condensation is caused by the capsaicin synthase enzyme (CS) acting specifically on fatty acid chain length, which requires Mg
2+, ATP and coenzyme A (CoA) and vanillylamine, the phenolic portion, formed from phenylalanine as a product in the phenylpropanoid pathway, while the fatty acid is formed from the amino acids valine or leucine. Structural differences among the capsaicinoids are defined by the nature of the lateral chain, which ranges from 9 to 11 carbons long with a variable number of double bonds located in different positions along the chain (
Figure 2).
Figure 2.
Chemical structure of different capsaicinoids and their analogues. R Capsaicinoids and analogues.
Figure 2.
Chemical structure of different capsaicinoids and their analogues. R Capsaicinoids and analogues.
Capsaicinoids are important in the food and pharmaceutical industries. For this reason, a number of researchers are engaged in improving their production be it by manipulating chili plant cultivation conditions [
8], chemical synthesis [
9], enzymatic synthesis [
10,
11] or alternative methods such as cell or tissue culture [
12]. To date, research has shown that capsaicinoids, and capsaicin in particular, have a wide variety of biological and physiological activities which provide them functions such as antioxidants [
13], anticarcinogenics [
14], promotion of energy metabolism and suppression of fat accumulation [
15], and anti-inflammatories [
16]. However, the potential applications of these molecules are limited by the irritation caused by their pungency; this has driven the search for and characterization of analogous molecules without inherent and undesirable effects [
14,
17,
18].
3. Capsaicin Characterization
Growing interest in capsaicin has led to its characterization with methods such as spectrophotometry UV-VIS and chromatography. These have been modified over time to develop more sensitive, faster capsaicin characterization techniques.
Thin-layer chromatography has been used to detect capsaicin on ground pepper [
52], and multi-band thin-layer chromatography has been used to evaluate the R
f of capsaicin in different adsorbents and the same mobile phase under identical conditions (
i.e. time, temperature, solvent vapor equilibrium,
etc.) [
53]. High-performance liquid chromatography (HPLC) has been widely used for characterization of capsaicin and it analogues [
54,
55,
56,
57,
58,
59]. Using different detectors, such as fluorescence, HPLC has been used to study capsaicinoids [
60]. For example, this method has been applied to detect capsaicin, zucapsaicin and civamide (a capsaicin
cis-isomer) within a 1 to 100 ng/mL concentration interval [
61], and to characterize six capsaicin analogues up to a 3 ng concentration in animal tissues and blood [
62]. This method was also used to quantify air-borne capsaicin and dihydrocapsaicin at a pickling and pepper manufacturing plant. Capsaicin concentration was 0.015 and dihydrocapsaicin concentration was 0.02 μg per sample; the capsaicin:dihydrocapsaicin ratio remained within a 0.3:1 to 0.5:1 range, not coinciding with the higher capsaicin content in
Capsicum [
63]. Storage stability in capsaicin solutions has been monitored using HPLC during a one year period, in a concentration range of 0.5 to 128 µM and four temperature/light combinations: 4 °C in darkness; room temperature in darkness; room temperature in light; and −20 °C in darkness. Under the tested conditions, the 4 µM treatment exhibited optimum performance, remaining stable for one year when stored at 4 °C in darkness [
64]. In another study, HPLC with a diode array detector allowed quantification of capsaicin at levels below 100 Scoville units (the Scoville unit indicates the amount of capsaicin present) when mixed with nordihydrocapsaicin and dihydrocapsaicin, and at levels below 1000 Scoville units when mixed with spices such as cumin and pepper [
65]. Reverse-phase HPLC was applied to identify and quantify four lipidic hydroperoxide isomers produced by oxidation of linoleic acid to evaluate capsaicin’s antioxidant activity [
66].
Technological advances in the use of HPLC for organic compound analysis have been applied to capsaicin characterization, specifically liquid chromatography-mass spectrometry (LC-MS) [
67,
68,
69] and liquid chromatography-tandem mass spectrometry (LC-MS-MS) [
70]. The latter method was used to analyze capsaicinoid content in commercial pepper sprays with a standard quantification curve within a 10 to 1,000 ng/mL concentration interval. Capsaicin, dihydrocapsaicin and nonivamide were identified in the sprays within a 0.7 to 40.5 mg/mL interval [
71]. In other applications, LC-MS-MS has been used to quantify capsaicin, dihydrocapsaicin and nonivamide in the blood and tissues of rats exposed to inhalations of these three compounds with resulting capsaicin concentrations of 1.0 to 90.4 ng/mL in blood, 5.0 to 167 pg/mg in lungs, and 2.0 to 3.4 pg/mg in liver [
72]. Simultaneous quantification of capsaicin and dihydrocapsaicin in extracts from different
Capsicum genotypes has been accomplished using liquid chromatography-electrospray/time-of-flight mass spectrometry with detection limits of 6 ng for capsaicin and 1.2 ng for dihydrocapsaicin [
73,
74]. Liquid chromatography quadrupole ion trap mass spectrometry (LC-ESI/MS/MS) has been successfully applied to quantify capsaicin’s
in vitro half-life (2.3 to 4.1 min) [
75], as well as blood capsaicin content in rats subcutaneously administered capsaicin and dihydrocapsaicin [
76].
In a same way gas chromatography (GC) has been used in the study of capsaicin [
77]. In a comparison of capsaicin detection using GC and HPLC, the detection limit was 1.0 µg/g with a linear capsaicin calibration curve of 1 to 250 µg/mL with GC and 0.5 to 50 µg/mL with HPLC [
78]. The two methods have also been used in tandem (GCL) for determining the pungency in raw spices, extracts, food and pharmaceutical products. The pungency data obtained by both methods, the sensory test and CGL, ranged from 50,000 to 2,000,000 Scoville units with a correlation coefficient of 0.95 by a period of one year [
79,
80].
Solid phase microextraction-gas chromatography-mass spectrometry is another method used to study capsaicinoids [
81]. In an analysis of eleven pepper varieties and four pepper-based sauces, use of this method did not require derivatization, which is required with GC, and it effectively identified and quantified capsaicin (55–25,459 µg/g) and dihydrocapsaicin (93–1,130 µg/g) in the peppers (4.3–717.3 µg/g) and sauces (1.0–134.8 µg/g) [
82].
A modification of capillary chromatography that has been applied to capsaicin and dihydrocapsaicin in different varieties of
Capsicum frutescens, is the micellar electrokinetic capillary chromatography. In this method, samples are separated by differential partition between micelles (pseudo stationary stage) and the mobile phase (aqueous buffer at pH 9). Both capsaicin and dihydrocapsaicin were detected within 11 min with an excellent resolution [
83].
Capsaicin’s hydrophobic nature suggests that it may influence membrane structure, which was studied using the dipalmitoylphosphatidylcholine and dielaidoylphosphatidylethanolamine membrane models and the differential scanning calorimetry, fluorescent probe spectroscopy and
31P-nuclear magnetic resonance techniques. The results were used to propose the possible implications of capsaicin’s effects in biological membranes [
84].
Proton nuclear magnetic resonance (
1H-NMR) and carbon nuclear magnetic resonance (
13C-NMR) were used in a comparative analysis of capsaicinoids purity and structure [
85].
1H-NMR has also been used in quantitative analysis of capsaicin in
Capsicum frutescens L. without previous derivatization [
86].
13C-NMR,
1H-NMR and α- and β-glucosydase hydrolysis were used to identify the structure of two capsaicin glucosides: capsaicin-β-
D-glucopyranoside and dihydrocapsaicin-β-
D-glucopyranoside. In this study, the glucosides were not detected in non-pungent
C. annum crops and a positive correlation was observed between capsaicin and dihydrocapsaicin levels and their glucosides [
87].
The UV-VIS spectrophotometric method is one of the most inexpensive and accessible for capsaicin quantification; indeed, most laboratories have a UV-VIS spectrophotometer. However, analysis is restricted to capsaicin solutions with microgram-level concentrations [
88].
Kachoosangi
et al. [
89] discuss capsaicin detection methods other than chromatography and spectroscopy with detection ranges from 0.5 to 35 µM, while capsaicin’s molecular and electronic structure can be analyzed using the density functional theory [
90].