Extraction of Capsinoid and its Analogs From Pepper Waste of Different Genotypes

Abstract

Pepper, a member of the Capsicum genus of the Solanaceae family, is an annual, cultivated plant that grows in temperate climates. The main analogs of capsinoids, which are the secondary metabolites of peppers, are capsaicin and dihydrocapsaicin. During process, calyxes and peduncles are considered to be waste. Levels of capsinoids in these tissues in not well known. An optimized method was used to extract bioactive materials from the waste products from C. annum L. genotypes. Calyxes and peduncles were collected and dried in the shade. Extractions were in MeOH solutions and the extracts were analyzed using high-performance liquid chromatography (HPLC) to quantify the total amount of capsinoid, capsaicin, and dihydrocapsaicin contents. Capsinoid, and its analogs, were identified in all genotypes at varying concentrations, and their capsaicin and dihydrocapsaicin contents evidenced with liquid chromatography-mass spectrometry (LC-MS). In conclusion, the highest amount of capsinoids was found in chili Samandağ peppers, whereas the lowest amount of capsinoids was found in the red sweet pepper sample. All pepper genotypes, the capsaicin amount was higher comparison to dihydrocapsaicin. Capsanoids and anologues obtained from waste pepper can be used as raw materials in production of value added products.

Capsinoids in the genus Capsicum are secondary metabolites of the plant that belongs to the class of alkaloids. ¹ L-Phenylalanine is the starting material for the synthesis of capsinoids. The mechanism that begins with L-phenylalanine continues with cinnamic acid and its derivatives. The characteristics of capsinoids are composed of properties of different enzymes and control mechanisms. ² This alkaloid has the commonly known analogs capsaicin and dihydrocapsaicin as seen in Figure 1, and dihydrocapsaicin, homocapsaicin, homodihydrocapsaicin, and nonivamide. ^3,4 These capsinoids demonstrate their functional mechanism through the activation of TRPV1 channel, and their most potent analogs are nonivamide and capsaicin, followed by dihydrocapsaicin. ⁵ Capsaicin is the active ingredient that gives pungency to peppers and is the most abundant capsinoid. The taste characteristics of capsaicin were first known in the 1500s, the first extraction was performed in 1846, its structure was determined in 1919, and it was first synthesized in 1930. ⁶ Capsaicin is a valuable bioactive phytochemical used in the pharmaceutical, chemical, and cosmetic industries. ⁷ It is known that phytochemicals are not only used for chemotherapy but also have antioxidant and anticarcinogen effects. As compounds in the class of alkaloids, capsaicins display neurological actions and, therefore, are significant active ingredients used by the pharmaceutical industry. ⁸ Most of these bioactive substances perform their anticarcinogenic action by interrupting the progression of the cell cycle and stimulating tumor cell apoptosis. Many studies have demonstrated that capsaicin has potential for antimutagenic and anticarcinogenic activities. ⁹ It has also been reported that capsaicin induced apoptosis in cancerous cells selectively. ¹⁰ Also, epidemiological studies have reported a positive correlation between the consumption of pepper and the prevention of gastric, pancreatic, and lung cancers. ¹¹

Figure 1

Chemical structure of capsaicin and dihydrocapsaicin.

There are many studies regarding the antioxidant capacity of capsaicin, and capsaicin is known to show very good antioxidant capacity. ¹² In addition, recent studies have reported that capsaicin isolated from hot green pepper had preventive potential against toxic and mutagenic effects of UV-C and H₂O₂ on DNA. ¹³ In terms of nutrition, the consumption of pepper is very common worldwide and many types of hot peppers are more included in the daily diets of many individuals particularly in the Middle East.

Therefore, this study set out to investigate the presence of these active ingredients in the wasted peduncle and calyx parts rather than in the edible parts of peppers. It is estimated that 90% of capsinoids are found in the pericarp (fruit), which constitutes 40% of the pepper, while 10% of capsinoids are found in the seeds. ¹⁴ This study detected capsinoids and its analogs in the calyx and peduncle parts of peppers. For this purpose, the total amount of capsinoids was determined in different hot and sweet pepper types from 4 different genotypes, and the content of capsaicin and dihydrocapsaicin, the most important analogs, was quantified in each genotype.

Materials and Methods

Plant Material

Peduncles and calyxes parts of peppers from 4 different genotypes in C. annum L. species known locally as green hot Apraş pepper, red chili pepper, chili Samandağ pepper, and red sweet pepper were collected in September, during the harvest season from different regions (Gaziantep, Kahramanmaraş, Hatay), and identified at Gaziantep University Department of Biology, Division of Botany.

Purification and Extraction Method for Capsaicin

The industrial-scale purification of capsinoids was introduced and described in the literature by Segi et al in 1995. This method is based on placing capsinoids containing capsaicin in a hydrophilic solvent with a silver compound in an aqueous solution and forming a capsaicin-silver complex, which is soluble in water to provide recovery of highly pure capsaicin. ¹⁵ This method is frequently used in the purification of capsaicin. With an optimized and improved method, this study performed extraction of capsinoids and its analogs with much higher efficiency than the solutions containing low levels of capsinoids. Through this study method, Soxhlet extraction was performed in a 200 mL MeOH solution over 24 hours. Extracts were heated, stirred for 10 minutes with the addition of 1 g activated carbon, and then sieved. The activated carbon was washed a few times with MeOH. It was evaporated until the filtrate obtained had dried. The residue was dissolved in petroleum ether, washed with water in a separatory funnel, and the aqueous layer was discarded. The ether phase was evaporated until dry. The results were samples placed in 10 mL vials as calculated in µg.g⁻¹ as shown in Table 1.

Table 1

Amount of Capsaicin and Dihydrocapsaicin and Total Capsinoids Purified From Pepper Species.

Pepper type	Capsaicin (µg.g−1)	Dihydrocapsaicin (µg.g−1)	Total capsinoids(µg.g−1)
RCP	50.86868	51.86307	102.73175
CSP	95.83977	58.55994	154.39971
RSP	0.38100	0.25983	0.64083
GHAP	8.64513	5.49579	14.14092

RCP, red chili pepper; CSP, chili Samandağ pepper; RSP, red sweet pepper; GHAP, green hot Apraş pepper.

An analysis was performed with 1.00 mL min⁻¹ flow rate on an ODS-3 (4.6 × 250 mm, 5 µm) column in a Shimadzu LC-20AD high-performance liquid chromatography (HPLC) system and acetonitrile/water (70:30%) mobile phase at 230 and 280 nm wavelengths using a UV-Vis detector in isocratic mode. Analysis time was 10 minutes, and the injection volume was 20 µL. Retention time for capsaicin and dihydrocapsaicin was 5.50 and 6.90 minutes, respectively.

Results

Both the peduncle and calyx parts of the peppers were assessed together. The amounts of starting material and dry residue before vial filling are shown in Table 2. Starting materials obtained by drying and grinding the waste material of peppers from 4 different genotypes differed as shown in Table 2. Based on the calculation of each in percentage terms, 0.014 % and 0.016 % of 3.4614 g starting material in sample no. 1 included capsaicin and dihydrocapsaicin, respectively; 0.057 % and 0.033 % of 1.7522 g starting material in sample no. 2 included capsaicin and dihydrocapsaicin, respectively; 0.000112 % and 0.000765 % of 3.3933 g starting material in sample no. 3 included capsaicin and dihydrocapsaicin, respectively; 0.0027 % and 0.0017 % of 3.1713 g starting material in sample no. 4 included capsaicin and dihydrocapsaicin, respectively. Linearity of an analytical procedure is its ability to obtain test results, which are directly proportional to the concentration (amount) of analyte in the sample. The linearity of the method was determined by constructing calibration curve of capsaicin and dihydrocapsaicin at different concentration levels (1, 2.5, 5, 10, 25, 50 µg.g⁻¹) were used. R² = 0.9999864 on standard calibration graph as seen in Figure 2. Pure capsaicin was purchased from Sigma-Aldrich as standard. Quantification was performed by mixing extract samples and chromatograms obtained from standard solutions, and measuring absorbance values at 230 nm wavelength. The results obtained by HPLC were supported by determination of mass on LC-MS. The presence of capsaicin (305.41 g.mol⁻¹) and dihydrocapsaicin (307.43 g.mol⁻¹) was detected in the samples as seen in Figure 3. LC-MS chromotograms of capsaicin and dihydrocapsaicin for each sample are as shown in Supplemental Figures S1-S3. The amount of capsanoids and analogs from RSPs is very low, as shown in Table 1.

Figure 2

Standard calibration curve of capsaicin and dihydrocapsaicin (1, 2.5, 5, 10, 25, 50 µg.g⁻¹).

Figure 3

Chromatogram of positive scan result for capsaicin (306 g.mol⁻¹) and dihydrocapsaicin (308 g.mol⁻¹).

Table 2

Content of Capsaicin and Dihydrocapsaicin in Different Amounts of Dried Pepper Waste.

Pepper type	Starting material (mg)	Dry residue before vial filling(mg)	Capsaicin (mg)	Dihydrocapsaicin(mg)
1	3461.4	7.15 × 10–5	0.5086868	0.5186307
2	1752.2	2.25 × 10–5	0.9583977	0.5855994
3	3393.3	3.97 × 10–5	0.0038100	0.0025983
4	3171.3	0.82 × 10–5	0.064513	0.0549579

Therefore, since LC-MS chromatograms were detected below the detector limit, images were not taken so it could not, therefore, be shown in the attached supplemental information file.

Discussion

Capsinoids distribute unevenly in plants and fruits. Biosynthesis of this secondary metabolite occurs in the placenta of the plant, and then passes to other parts of plant, ie pericarp, seed, and calyces. At this point, an interesting contrast can be seen upon investigation of plants in the Solanaceae family. It was reported that alkaloids were produced in the roots and then translocated to all other parts of the plant. It was reported that alkaloids were produced in roots and then translocated to all other parts of the plant. Detection of capsaicin in other parts of the plant indicated that there could be an anatomical linkage association between the placenta and seeds. ¹⁶ The detection of capsaicin in other parts of the plant suggested that there could be an anatomical leakage association between the placenta and the seeds. ¹⁷ There is a great deal of evidence indicating that the synthesis of capsinoids occurs in the placenta interlocular septum of pepper fruits. Based on the studies conducted, it is known that the biosynthesis of capsinoids occurs in peppers of the Capsicum genus, and is derived from the acylation with branched-chain fatty acid of vanillylamine, an aromatic compound. It was confirmed by HPLC analysis that capsinoids were found in the interlocular septa of pepper fruits. The presence of capsaicinoids is controlled by the Pun1 locus encoding a putative acyltransferase. In the homozygous recessive state, capsaicinoids are not produced by the pepper plants with pun1/pun1 alleles. ¹⁸ Therefore, in the present study, capsanoids were found in the waste parts (peduncle and calyx of peppers) of the different genotypes belonging to the genus Capsicum.

Another study determining the content of capsinoids in peppers quantified capsinoids in the vegetative organs of the Habanero pepper, and evaluated whether pepper plants synthesized or accumulated capsaicins in the vegetative organs as an indirect way to elucidate the systemic regulation of the capsinoid biosynthesis. For this purpose, the habanero pepper, one of the hottest peppers in the world growing in the Yucatan Peninsula, was studied.

The results obtained by chromatographic and enzymatic measurements indicated that habanero pepper plants did not accumulate capsaicinoids in the vegetative organs analyzed under water stress conditions; however, these capsaicinoids were probably found in the reproductive organs. ¹⁹ The extraction of capsinoids from the fruit of peppers of the genus capsicum was first mentioned in the literature by Colins et al in 1995. ²⁰ Later on, Estrada et al extracted capsaicinoids from the vegetative organs of peppers such as from the cells, leaves, and fruit in 2002. ²¹ In our study, purification from the calyx and peduncle parts, which are defined as waste, was performed on peppers from different genotypes for the first time by using a method developed specifically for this study. Within the scope of this reported data, the content of capsinoid and its analogs in the calyx and peduncle parts of peppers was determined and quantification was performed by LC-MS in this study. The highest amount of capsinoids was found in the chili Samandağ pepper, and the amount of capsaicin, which is the active ingredient responsible for pungency, was found to be higher in this genotype compared to other genotypes. Sweet peppers (CH-19 sweet, a cultivar of C. annuum L. with low pungency) contain caspiate, dihydrocaspiate, and nordihydrocaspiate compounds, which have similar characteristics to capsaicin and are known to be found in hot peppers. ²² Therefore, the RSP genotype was chosen for the purpose of this study and found to contain both capsaicin and dihydrocapsaicin in lower quantities than in other genotypes. The contents of caspiate, dihydrocaspiate, and nordihydrocaspiate, which are other analogs of capsinoids found in RSPs in the peppers of this genotype, are planned to be investigated in further studies. A similar study found bioactive capsaicin and carotenoids, which are volatile organic compounds, in the peduncle parts of different particle sizes (0.25, 0.5, and 1 mm) of hot C. annuum L. peppers. Also, the peduncle part was characterized using thermogravimetry, differential scanning calorimetry, and Fourier transform infrared spectroscopy. The highest amount of extract rich in fats was obtained from the peduncle with a particle size of 0.25 mm, by using ethanol. It was also found to be more efficient for the extraction of capsaicin, and Soxhlet extraction was performed by using 200 mL hexane or ethanol solution and later evaporating an aqueous solution. ²³

In this study, a new extraction method, which included the following process steps, was utilized: extracts were heated, stirred for 10 minutes with the addition of 1 g activated carbon, sieved, and the activated carbon was washed a few times with MeOH. Red peppers have commonly been used not only for adding flavor to food but also in the treatment of various diseases since ancient times. Red pepper is used as a drug for cramps, diarrhea, cold, fever, and specific gastric disorders and has therapeutic effects as a stimulant as well as irritant action. ²⁴ These effects are due to capsaicin, the active ingredient that is responsible for the pungency in its content. It is stated that these compounds are detected by taste receptors even in dilutions of 1 part in 15 or 17 million, and have a stimulant effect. ²⁵ Due to their antimicrobial properties, capsinoids are recommended by experts from the Food and Drug Administration to be included in food as an ingredient. ²⁶ Capsaicin demonstrates its main pharmacological action through the peripheral terminal of primary afferent neurons of C-fibers. Capsaicin causes the release of neurotransmitters, in particular substance P, from sensory nerve fibers by exciting the nociceptive C-fiber afferents. Axon-reflex-mediated vasodilation characterized by prolonged cutaneous pain transmission, histamine release, pathological itching, and erythema occurs in consequence of the release of substance P. As a result of its repeated administration, capsaicin inhibits the sense of pain by reducing the release of substance P from the peripheral sensory C-fibers.

Various clinical studies have been reported regarding the use of capsaicin as an active ingredient in various drugs. In particular, creams containing capsaicin are suggested for psoriasis treatment. ²⁷ Therefore, capsaicin is a very important active ingredient in the pharmaceutical industry. These essential alkaloids are used in the pharmaceutical, chemical, cosmetic, and many other industries. Investment of more than USD 500 million is made every year in the United States for both logistic and financial purposes to separate and identify peppers of the genus C. annuum, containing the active ingredient capsaicin, from alkaloids. ²⁸ According to a study, in the agricultural waste from peppers, the peduncle of red peppers was high in phenolics, flavonoids, and capsaicin content, and the wastes were probably used as an additional source of the bioactive products. ²⁹

Conclusion

This study extracted important capsinoids and their analogs from agricultural waste (which do not have any nutritional value) from peppers, which have a significant role in nutrition, and assessed the results. The results we have obtained in terms of evaluating the wastes of peppers, which are continuously produced and consumed in different geographies all over the world, are very important. Capsanoids and the analogs obtained as a result of this study can be used as raw materials in the production of value-added products.

Supplemental Material

Supplementary material - Supplemental material for Extraction of Capsinoid and its Analogs From Pepper Waste of Different Genotypes

Supplemental material, Supplementary material, for Extraction of Capsinoid and its Analogs From Pepper Waste of Different Genotypes by Sibel Bayil Oğuzkan in Natural Product Communications