Some pungent principles of spices cause the adrenal medulla to secrete catecholamine in anesthetized rats

We recently reported that capsaicin, a pungent principle of hot red pepper, evokes catecholamine secretion from the rat adrenal medulla. In this study, the effects of some pungent principles of spices on adrenal catecholamine secretion were investigated as compared with that of capsaicin. An increase in catecholamine, especially epinephrine, secretion was observed not only on capsaicin infusion but also on piperine (a pungent principle of pepper) and zingerone (ginger) infusion. Even on infusion of the same amount (650 nmol/kg, i.v.), the order of potency as to catecholamine secretion was capsaicin much greater than piperine greater than or equal to zingerone. While, sulfur-containing and volatile pungent principles, allylisothiocyanate (mustard, etc.) and diallyldisulfide (garlic, etc.), did not even cause slight catecholamine secretion. Furthermore, these adrenergic secretagogues were readily transported via the gut into the body. These results indicate that some pungent principles of dietary spices can induce a warming action via adrenal catecholamine secretion.     

Pungent qualities of sanshool-related compounds evaluated by a sensory test and activation of rat TRPV1

The detection threshold and taste characteristics of sanshools were examined by sensory evaluation, after isolating four sanshools (alpha-, beta-, gamma-, and delta-), and two hydroxy sanshools (alpha- and beta-) from the pericarp of Japanese pepper. The Scoville unit (SU) values of the four sanshools were in the range of 80,000-110,000, while those of hydroxy sanshools were 3-5 fold lower than corresponding sanshools. The pungent qualities of each sanshool were different. Burning and tingling were predominantly perceived and lasted for the longest time with alpha-sanshool. Burning and fresh for gamma-sanshool, and tingling and numbing for hydroxy alpha-sanshool were perceived. Tests on the activation of rat TRPV1 were also performed. All of them were weak agonists. Among them, gamma-sanshool was the most potent agonist, although its EC50 value of 5.3 microM was 230 fold higher than that of capsaicin. These results indicate that it would be difficult to explain the pungent quality of each sanshool simply in terms of TRPV1 activation.     

Studies on spice principles as antioxidants in the inhibition of lipid peroxidation of rat liver microsomes

Polyunsaturated fatty acids (PUFA) are vulnerable to peroxidative attack. Protecting PUFA from peroxidation is essential to utilize their beneficial effects in health and in preventing disease. The antioxidants vitamin E, t-butylhydroxy toluene (BHT) and t-butylhydroxy anisole (BHA) inhibited ascorbate/Fe²⁺-induced lipid peroxidation in rat liver microsomes. In addition, a number of spice principles, for example, curcumin (5–50 µM) from turmeric, eugenol (25–150 µM) from cloves and capsaicin (25–150 µM) from red chillies inhibited lipid peroxidation in a dose-dependent manner. Zingerone from ginger inhibited lipid peroxidation at high concentrations (> 150 µM) whereas linalool (coriander), piperine (black pepper) and cuminaldehyde (cumin) had only marginal inhibitory effects even at high concentrations (600 µM). The inhibition of lipid peroxidation by curcumin and eugenol was reversed by adding high concentrations of Fe²⁺.     

TIME-INTENSITY EVALUATION OF ORAL BURN1

Temporal oral burn profiles for cinnamaldehyde, piperine and capsaicin were quantified using time-intensity (TI) parameters. Despite large judge-to-judge variation, TI profiles for eaters and noneaters of chile peppers differed significantly. Eaters had lower maximum intensities, shorter total times, and slower maximum rates of onset and decay. Judges with substantial desensitization also had shorter time-to-maximum intensities. Desensitized judges not only scored the burn of capsaicin as less intense, but also the burn of cinnamaldehyde and piperine. The burn of cinnamaldehyde solutions increased and decayed quickly; whereas, burns due to piperine and capsaicin increased and decayed slowly. Capsaicin compared to cinnamaldehyde, had time-to-max, plateau time, and total time that were 1.5, 2.0 and 3.0 fold larger; whereas, the maximum rates of onset and decay were 0.7 and 0.2 fold smaller, respectively. Temporal differences are believed to reflect differences in neural pathways for the irritant species.     

Simultaneous determination of cinnamaldehyde, eugenol and piperine by HPTLC densitometric method

An HPTLC densitometric method for the simultaneous determination of cinnamaldehyde and eugenol as well as trace amounts of piperine in pepper-contaminated cinnamon was developed. The applicability of the method was tested with cinnamon bark powder adulterated with pepper powder, cinnamon oil, clove powder, clove oil and a commercial preparation containing cinnamaldehyde and eugenol. The method was validated for specificity, precision, accuracy and robustness. The method was found to be precise for different concentrations of cinnamaldehyde, eugenol and piperine. The accuracy of the method was checked by conducting a recovery study at three different levels. The linearity was found to be in the ranges 52.54-735.56, 533.2-8531.2 and 50-300 ng/spot, respectively, with correlation coefficients of 0.9985 +/- 0.04, 0.9982 +/- 0.06 and 0.9937 +/- 0.11 for cinnamaldehyde, eugenol and piperine.     

Quantitative analysis of sanshool compounds in Japanese pepper (Xanthoxylum piperitum DC.) and their pungent characteristics

The distributions of each sanshool in the Japanese pepper plant grown in various regions and the change in composition of sanshools during maturation of the fruit were investigated. The degree of pungency, defined as the amount of a sanshool/the threshold value, was calculated, and the pungent qualities of the products were evaluated and compared. The degree of pungency and amount of a sanshool showed a positive correlation. In young leaves and flowers, the degree of pungency was less than that in the fruits, the main compound being alpha-sanshool, while the two hydroxy sanshools were detected only in trace amounts. The main compound in fruits was hydroxy alpha-sanshool, whose threshold value was higher than that of alpha-sanshool. It is concluded that the pungency of Japanese pepper should be evaluated not only by the threshold values, but also by the pungent qualities, the composition of sanshools, and the usage of each product of Japanese pepper.     

Spice phenolics inhibit human PMNL 5-lipoxygenase

A wide variety of phenolic compounds and flavonoids present in spices possess potent antioxidant, antimutagenic and anticarcinogenic activities. We examined whether 5-lipoxygenase (5-LO), the key enzyme involved in biosynthesis of leukotrienes is a possible target for the spices. Effect of aqueous extracts of turmeric, cloves, pepper, chili, cinnamon, onion and also their respective active principles viz., curcumin, eugenol, piperine, capsaicin, cinnamaldehyde, quercetin, and allyl sulfide were tested on human PMNL 5-LO activity by spectrophotomeric and HPLC methods. The formation of 5-LO product 5-HETE was significantly inhibited in a concentration-dependent manner with IC(50) values of 0.122-1.44 mg for aqueous extracts of spices and 25-83 microM for active principles, respectively. The order of inhibitory activity was of quercetin>eugenol>curcumin>cinnamaldehyde>piperine>capsaicin>allyl sulfide. Quercetin, eugenol and curcumin with one or more phenolic ring and methoxy groups in their structure showed high inhibitory effect, while the non-phenolic spice principle allyl sulfide showed least inhibitory effect on 5-LO. The inhibitory effect of quercetin, curcumin and eugenol was similar to that of synthetic 5-LO inhibitors-phenidone and NDGA. Moreover, the inhibitory potency of aqueous extracts of spice correlated with the active principles of their respective spices. The synergistic or antagonistic effect of mixtures of spice active principles and spice extracts were investigated and all the combinations of spice active principles/extracts exerted synergistic effect in inhibiting 5-LO activity. These findings clearly suggest that phenolic compounds present in spices might have physiological role in modulating 5-LO pathway.     

Enhanced intestinal uptake of iron,zinc and calcium in rats fed pungent spice principles – Piperine,capsaicin and ginger (Zingiber officinale)

In view of the wide-spread deficiency of iron and zinc in populations dependent on plant foods, it is desirable to improve the bioavailability of the same. Specific dietary spices may alter the ultrastructure and permeability characteristics of intestines. Groups of Wistar rats were fed piperine, capsaicin and ginger containing diets for 8 weeks in order to examine their possible influence on intestinal absorption of iron, zinc and calcium. Everted segments of duodenum, jejunum and ileum portions of small intestines isolated from these rats were examined for ex vivo uptake of iron, zinc and calcium from incubations containing digesta of finger millet. Higher uptake of iron, zinc and calcium by the intestinal segments from spice-fed animals was observed. The increase in the mineral uptake was the highest for calcium with >100% in some cases. The positive influence of dietary capsaicin was more pronounced on zinc uptake as compared to that of iron. Uptake of the glutamic acid standard was 87% and 62% higher in the case of jejunal segments of rats fed piperine and ginger. The higher intestinal uptake of iron and zinc as a result of consumption of pungent spices could encourage a strategy to reduce deficiency of these trace elements prevalent in population dependent on plant based foods.     

Isolation of cDNA clones differentially accumulated in the placenta of pungent pepper by suppression subtractive hybridization

Capsaicinoids responsible for pungency of chili pepper are synthesized exclusively in the placenta tissue of the fruit. As an elementary step in the molecular genetics study of capsaicinoid biosynthesis, a cDNA library was constructed from the placenta of a highly pungent pepper, Capsicum chinense cv. Habanero using the suppression subtractive hybridization (SSH). Thirty-nine cDNA clones from about 400 subtracted clones were selected through dot blot analysis and according to their nucleotides sequence. Sequence information of the chosen clones was evaluated by comparing it with DNA and protein databases. Results showed that the cDNA clones could be divided into 4 groups; cDNAs with similarities in genes encoding metabolic enzymes including acyl transferase and fatty acid alcohol oxidase (Group I), putative cell wall proteins (Group II), biotic and abiotic stress-inducible proteins (Group III), and lastly, cDNAs with no similarity (Group IV). Northern blot analysis was performed to confirm that these clones are differentially expressed in pungent pepper. The results revealed that all cDNA clones were differentially expressed in pungent pepper. In addition, the cDNA clones of Groups I and IV were differentially or preferentially expressed in the placenta of pungent pepper.     

Responses of the ciliates tetrahymena and paramecium to vertebrate odorants and tastants

The ciliates Tetrahymena and Paramecium respond to strong depolarizing stimuli with Ca(2+)-based action potentials, ciliary reversals, and consequent bouts of backward and forward swimming called "avoidance reactions" (ARs). We found that several representative tastants and odorants cause repetitive ARs in Tetrahymena and Paramecium at low (nM to microM) concentrations. Tetrahymena responded well to capsaicin, quinine, quinacrine, denatonium benzoate, eugenol, piperine, chloroquine, carvacrol, allyl isothiocyanate (AITC), and menthol. Chemosensory adaptation was seen with carvacrol, eugenol, quinacrine, and capsaicin. Cross-adaptation was seen between some of these compounds, suggesting possible similarities in their chemosensory transduction or adaptation pathways. Paramecium only responded well to AITC, quinacrine, piperine, and eugenol (with the effective concentration for 50% response [EC(50)] values in the microM range) while chemosensory adaptation was only seen to eugenol in Paramecium, suggesting possible species differences. Tetrahymena and Paramecium may have primitive receptors that can recognize these and other compounds or some of these compounds can act independently of specific receptors.     

Individual differences in perception of bitterness from capsaicin, piperine and zingerone

It was recently shown that in some subjects capsaicin can evoke bitterness as well as burning and stinging, particularly in the circumvallate (CV) region of the tongue. Because perception of bitterness from capsaicin is characterized by large individual differences, the main goal of the present study was to learn whether people who taste capsaicin as bitter also report bitterness from structurally similar sensory irritants that are known to stimulate capsaicin-sensitive neurons. The irritancy and taste of capsaicin and two of its most commonly studied congeners, piperine and zingerone, were measured in individuals who had been screened for visibility of, and reliable access to, the CV papillae. Approximately half of these individuals reported tasting bitterness from all three irritants when the stimuli were swabbed directly onto the CV papillae. Concentrations that produced similar levels of burning sensation across subjects also produced similar (though lower) levels of bitter taste. These results are consistent with the hypothesis that capsaicin and its congeners stimulate bitterness via a common sensory receptor that is distributed differentially among individuals. Additionally, bitter tasters rated gustatory qualities (but not burning and stinging) slightly but significantly higher than did bitter non-tasters, which suggests that perception of capsaicin bitterness is associated with a higher overall taste responsiveness (but not chemesthetic responsiveness) in the CV region.     

TRPA1 Agonists—Allyl Isothiocyanate and Cinnamaldehyde—Induce Adrenaline Secretion

Thermosensitive transient receptor potential (TRP) channels, especially TRPV1 and TRPA1, are activated by the pungent compounds present in spices. TRPV1 activation by the intake of capsaicin, the irritant in hot pepper, induces adrenaline secretion and increases energy consumption. TRPV1 is mainly expressed in the sensory neurons and coexpressed with TRPA1 at a high frequency. However, the mechanism underlying adrenaline secretion by TRPA1 agonists such as allyl isothiocyanate (AITC) and cinnamaldehyde (CNA), the pungent ingredients in mustard and cinnamon, is not known. We examined whether AITC and CNA could induce adrenaline secretion in anesthetized rats. An intravenous injection of AITC or CNA (10 mg/kg) increased adrenaline secretion. These responses disappeared completely in capsaicin-treated rats with an impaired sensory nerve function. Moreover, pretreatment with cholinergic blockers (hexamethonium and atropine) attenuated the AITC- or CNA-induced adrenaline secretion. These results suggest that TRPA1 agonists activate the sensory nerves and induce adrenaline secretion via the central nervous system.     

Metabolite biodiversity in pepper (Capsicum) fruits of thirty-two diverse accessions: variation in health-related compounds and implications for breeding

A comprehensive study on morphology and biochemical compounds of 32 Capsicum spp. accessions has been performed. Accessions represented four pepper species, Capsicum annuum, Capsicum frutescens, Capsicum chinense and Capsicum baccatum which were selected by their variation in morphological characters such as fruit color, pungency and origin. Major metabolites in fruits of pepper, carotenoids, capsaicinoids (pungency), flavonoid glycosides, and vitamins C and E were analyzed and quantified by high performance liquid chromatography. The results showed that composition and level of metabolites in fruits varied greatly between accessions and was independent of species and geographical location. Fruit color was determined by the accumulation of specific carotenoids leading to salmon, yellow, orange, red and brown colored fruits. Levels of both O- and C-glycosides of quercetin, luteolin and apigenin varied strongly between accessions. All non-pungent accessions were devoid of capsaicins, whereas capsaicinoid levels ranged from 0.07 up to 80 mg/100g fr. wt. in fruit pericarp. In general, pungent accessions accumulated the highest capsaicinoid levels in placenta plus seed tissue compared to pericarp. The non-pungent capsaicinoid analogs, capsiates, could be detected at low levels in some pungent accessions. All accessions accumulated high levels of vitamin C, up to 200 mg/100g fr. wt. The highest vitamin E concentration found was 16 mg/100g fr. wt. Based on these metabolic data, five accessions were selected for further metabolic and molecular analysis, in order to isolate key genes involved in the production of these compounds and to assist future breeding programs aimed at optimizing the levels of health-related compounds in pepper fruit.     

TRPA1 agonists--allyl isothiocyanate and cinnamaldehyde--induce adrenaline secretion

Thermosensitive transient receptor potential (TRP) channels, especially TRPV1 and TRPA1, are activated by the pungent compounds present in spices. TRPV1 activation by the intake of capsaicin, the irritant in hot pepper, induces adrenaline secretion and increases energy consumption. TRPV1 is mainly expressed in the sensory neurons and coexpressed with TRPA1 at a high frequency. However, the mechanism underlying adrenaline secretion by TRPA1 agonists such as allyl isothiocyanate (AITC) and cinnamaldehyde (CNA), the pungent ingredients in mustard and cinnamon, is not known. We examined whether AITC and CNA could induce adrenaline secretion in anesthetized rats. An intravenous injection of AITC or CNA (10 mg/kg) increased adrenaline secretion. These responses disappeared completely in capsaicin-treated rats with an impaired sensory nerve function. Moreover, pretreatment with cholinergic blockers (hexamethonium and atropine) attenuated the AITC- or CNA-induced adrenaline secretion. These results suggest that TRPA1 agonists activate the sensory nerves and induce adrenaline secretion via the central nervous system.     

Capsaicin cross-desensitization on the tongue: psychophysical evidence that oral chemical irritation is mediated by more than one sensory pathway

Psychophysical measurements were made of the perceived intensityand quality of sensations of chemical irritation before andafter the tip of the tongue had been desensitized to capsaicin(10 ppm). The results of the first experiment showed that capsaicindesensitization tended to reduce the perceived intensity ofirritation produced by approximately equipotent concentrationsof capsaicin (3 ppm), ethanol (30%), cinnamic aldehyde (2.5%)and NaCl (5M) applied to the tongue on filter paper disks; however,the reduction in irritation was less for the latter three compoundsthan for capsaicin and failed to reach statistical significancefor ethanol. Ratings of sensation quality suggested that thefour irritants produced different quality ‘profiles’,and that ethanol and cinnamic aldehyde were characterized bysensations of numbness as well as by sensations of burning andstinging/pricking. Follow-up experiments in which subjects ratedthe perceived intensity of individual sensation qualities showedthat desensitization dramatically reduced the burning and stinging/prickingcomponents of irritation, but left the sensations of numbnessand chemogenic warmth unchanged. It is concluded that lingualchemesthetic sensations are multidimensional, and mediated byboth capsaicinsensitive and capsaicin-insensitive sensory pathways.     

Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin

Six members of the mammalian transient receptor potential (TRP) ion channels respond to varied temperature thresholds. The natural compounds capsaicin and menthol activate noxious heat-sensitive TRPV1 and cold-sensitive TRPM8, respectively. The burning and cooling perception of capsaicin and menthol demonstrate that these ion channels mediate thermosensation. We show that, in addition to noxious cold, pungent natural compounds present in cinnamon oil, wintergreen oil, clove oil, mustard oil, and ginger all activate TRPA1 (ANKTM1). Bradykinin, an inflammatory peptide acting through its G protein-coupled receptor, also activates TRPA1. We further show that phospholipase C is an important signaling component for TRPA1 activation. Cinnamaldehyde, the most specific TRPA1 activator, excites a subset of sensory neurons highly enriched in cold-sensitive neurons and elicits nociceptive behavior in mice. Collectively, these data demonstrate that TRPA1 activation elicits a painful sensation and provide a potential molecular model for why noxious cold can paradoxically be perceived as burning pain.     

Spice active principles as the inhibitors of human platelet aggregation and thromboxane biosynthesis

Spice active principles are reported to have anti-diabetic, anti-hypercholesterolemic, antilithogenic, anti-inflammatory, anti-microbial and anti-cancer properties. In our previous report we have shown that spices and their active principles inhibit 5-lipoxygenase and also formation of leukotriene C4. In this study, we report the modulatory effect of spice active principles viz., eugenol, capsaicin, piperine, quercetin, curcumin, cinnamaldehyde and allyl sulphide on in vitro human platelet aggregation. We have demonstrated that spice active principles inhibit platelet aggregation induced by different agonists, namely ADP (50 μM), collagen (500 μg/ml), arachidonic acid (AA) (1.0 mM) and calcium ionophore A-23187 (20 μM). Spice active principles showed preferential inhibition of arachidonic acid-induced platelet aggregation compared to other agonists. Among the spice active principles tested, eugenol and capsaicin are found to be most potent inhibitors of AA-induced platelet aggregation with IC50 values of 0.5 and 14.6 μM, respectively. The order of potency of spice principles in inhibiting AA-induced platelet aggregation is eugenol>capsaicin>curcumin>cinnamaldehyde>piperine>allyl sulphide>quercetin. Eugenol is found to be 29-fold more potent than aspirin in inhibiting AA-induced human platelet aggregation. Eugenol and capsaicin inhibited thromboxane B2 (TXB2) formation in platelets in a dose-dependent manner challenged with AA apparently by the inhibition of the cyclooxygenase (COX-1). Eugenol-mediated inhibition of platelet aggregation is further confirmed by dose-dependent decrease in malondialdehyde (MDA) in platelets. Further, eugenol and capsaicin inhibited platelet aggregation induced by agonists—collagen, ADP and calcium ionophore but to a lesser degree compared to AA. These results clearly suggest that spice principles have beneficial effects in modulating human platelet aggregation.     

A versatile PCR marker for pungency in Capsicum spp.

Pungency in Capsicum spp. is an important quality trait for pepper breeding. The perception of pungency in pepper is due to the presence of a group of compounds named capsaicinoids, only found within the Capsicum genus. How pungency is controlled at genetic and molecular levels has not been completely elucidated. The use of molecular markers to assess pungency trait is required for molecular breeding, despite the difficulty of development of universal markers for this trait. In this work, a DNA sequence possibly related to pungency with a high similarity to Pun1 locus was studied, and sequence analysis of this homolog revealed a 15?bp deletion in non-pungent pepper accessions. An allele-specific pair of primers was designed and specific fragments of 479?bp from non-pungent and 494?bp from pungent accessions were obtained. Polymorphism of this marker, named MAP1, was tested in a wide range of accessions, belonging to several Capsicum species, including pungent and non-pungent accessions of C. annuum L., and pungent accessions of C. chinense, C. baccatum, C. frutescens, C. pubescens, C. galapagoense, C. eximium, C. tovarii, C. cardenasii, and C. chacoense. All these Capsicum accessions were correctly discriminated. The marker suitability to assess pungency in domesticated and wild Capsicum species was demonstrated, and therefore it will be very useful in marker assisted selection (MAS). Moreover, MAP1 was located in a saturated pepper linkage map and its possible relationship with the Pun1 locus has been discussed. Among the available markers for this complex quality trait, the marker developed in this study is the most universal so far.     

Non-pungent Capsicum contains a deletion in the capsaicinoid synthetase gene, which allows early detection of pungency with SCAR markers

The capsaicinoid synthetase (CS) gene cosegregated perfectly with the C locus, which controls the presence of pungency, in 121 F2 individuals from a cross between 'ECW123R' and 'CM334', both of Capsicum annuum. We concluded that CS and C are tightly linked. Sequence analysis of the genes of four pungent and four non-pungent pepper lines showed that the non-pungent peppers had a 2,529 bp-deletion in the 5' upstream region of CS. We have developed molecular markers of the C locus to detect pungency at the seedling stage. Based on the deleted sequence, we developed five SCAR markers, two of them being codominant. These SCAR markers will be useful for easy, accurate, and early detection of non-pungent individuals in breeding programs.