Influence of process parameters on the extraction of capsaicinoids from various Capsicum species cultivated in Romania

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1 Romanian Biotechnological Letters Vol. 20, No. 5, 2015 Copyright 2015 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER Influence of process parameters on the extraction of capsaicinoids from various Capsicum species cultivated in Romania Received for publication, May 10, 2015 Accepted, August 6, 2015 Abstract ROXANA-MĂDĂLINA STOICA 1, OVIDIU POPA 1, SULTANA NIȚĂ 2, NARCISA BĂBEANU 1 1 University of Agronomic Sciences and Veterinary Medicine of Bucharest, Faculty of Biotechnologies, 59 Mărăşti Blvd, District 1, , Bucharest, Romania, 2 National Institutes for Chemical Pharmaceutical Research and Development, 112 Vitan Av., District 3, Bucharest, Romania Corresponding author narcisa.babeanu@gmail.com The chili pepper is a very important plant used worldwide as a vegetable, a spice, and an external medicine. Pepper fruits are important sources of vitamins C and E, provitamin A, capsaicinoids and carotenoids. Capsaicin is one of the five chemical compounds forming the class of capsaicinoids (capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homodihydrocapsaicin and homocapsaicin) and the predominant compound in peppers. The extraction of the oleoresin from the fruit of hot pepper can be made using organic solvents but the yield varies with peppers variety and the conditions of extraction processing. In this work, three different varieties of Capsicum sp. have been characterized by their capsaicinoids content. Capsaicinoids were identified in all extracts with a concentration ranging from 0.6 to 0.8% (dry weight) and the best results were obtained with 96% ethanol as solvent. Keywords: capsaicin analysis, extraction, organic solvents, chili fruits 1. Introduction A traditional plant with many pharmacological effects is chilifruit (Capsicum species). Around the world there are known five varieties of Capsicum sp., namely C.annuum, C.frutescens, C.chinense, C.baccatum, C.pubescens (MUSFIROH & al.,[1]). Chili fruits have various uses as a pungent flavor in food, natural coloring, pharmaceutical ingredient and as sprays for riot control and self-defense. The pungent flavor of chilies is due to a group of closely related alkaloids called capsaicinoids (SANATOMBI & SHARMA, [2]). Capsaicin and dihydrocapsaicin together account for about 90% of total pungency (ISLAM & al., [3]; AMRUTHRAJ & al., [4]; ANWAR & al., [5]). In addition to capsaicin and dihydrocapsaicin, many less abundant capsaicinoids have been detected in Capsicum extracts, including nordihydrocapsaicin, homocapsaicin and homodihydrocapsaicin (MOZSIKSI & al., [6]). The amount of capsaicinoids in peppers is dependent on the genetic makeup of the plant, the developmental stage, and the environment where it is grown, such as geographical origin and temperature, soil nutrients, light, water stress (ZEWDIE & BOSLAND, [7]; BOSLAND & BARAL, [8]; GONZALEZ-ZAMORA & al., [9]; SGANZERLA & al., [10]). The capsaicinoids begin to accumulate in the early stages of fruit development and they reach a maximum rate as the fruit matures. Romanian Biotechnological Letters, Vol. 20, No. 5,

2 ROXANA-MĂDĂLINA STOICA, OVIDIU POPA, SULTANA NIȚĂ, NARCISA BĂBEANU Capsaicin is the active ingredient in topical analgesics used against pain, anti-arthritic and anti-inflammatory ointments, and also possess powerful antioxidant, anti-mutagenic and antitumoral properties. As well, there are many patents on insecticides, insect or animal repellents and pesticides containing capsaicinoids (DANG& al., [11]). The major focus of the present study was to compare and to select a suitable solvent for the extraction of capsaicinoids from three varieties of Capsicum cultivated in Romania, using different organic solvents (methanol, acetone, acetonitrile, ethanol). 2. Materials and methods Standards and Chemical Reagents The solvents, such as methanol, acetonitrile, acetone and ethanol, used for extraction of capsaicinoids were purchased from Merck (Germany). Pure capsaicin obtained from Sigma Aldrich was used as reference standard. HPLC grade chemicals were used for the mobile phase and all the other reagents used in the analysis were analytical grade. Plant material Dried fruits of three varieties of hot peppers, such as Fresno, Hot cherry and Congo Trinidad (CT) were used for extraction of capsaicinoids. Peppers used in this study were harvested from a local farm in September 2014 at maturity stage and at full fruit size. Capsaicinoids extraction Capsaicinoids are soluble in various solvents such as chloroform, acetone, ethyl ether, ethyl acetate, methyl chloride, ethanol, and 2-propanol, methanol, acetonitrile, among others (Santamaria R.I. et al., 2000). First fully ripe fruits samples were allowed to dry at 50 C for hours, to a moisture content ranging between 3-4%, and crushed into powder before extraction. For grinding it was used a Retsch GM200 laboratory grinder. The amount of milled vegetal material was about 50 g and the grinding duration varied between 30 secs and 60 secs. Every time, 25 g powder was extracted with 300 ml organic solvents, using Soxhlet extraction (Buchi B-811), for 5 hours. The temperature was adjusted to obtain at least 40 cycles. After extraction, the solvent was removed using a rotary evaporator (Buchi R215), and the vacuum level was adjusted so the temperature to be less than 40 degrees Celsius. The total capsaicinoids content (capsaicin, dihydrocapsaicin and nordihydrocapsaicin) was determined using High Performance Liquid Chromatography (Elite La Chrom chromatograph).as standards we used methanol solution of capsaicin (0.05%), nordihydrocapsaicin (0.025%) and dihydrocapsaicin (0.025%). For separation it was used a phenylsilicagel column (5μm),L= m, Ø=4.6 mm, t = 30 C. Mobile phase was acetonitrile :1g/L solution of phosphoric acid = 40:62 (V/V), flow rate: 1.0 ml/min, detection: λ=225 nm, injection:10µl, the elution order being: nordihydrocaspsaicin, nonivamide, capsaicin, dihydrocapsaicin Romanian Biotechnological Letters, Vol. 20, No. 5, 2015

3 Influence of process parameters on the extraction of capsaicinoids from various Capsicum species cultivated in Romania 3. Results and discussion Granulometric distribution of pepper powder resulted after grinding is presented in Figures 1-3. Figure 1. Influence of grinding duration on granulometric distribution of pepper powder (7000 rpm, CT variety). Determination of average diameter was calculated in the assumption of a log-normal distribution of particles size. As can be seen from Fig. 2 and 3, data fitted reasonable well, considering this type of distribution. Figure 2. Cumulative undersize mass fraction (7000 rpm, 30 sec, CT variety). Figure 3. Cumulative undersize mass fraction (7000 rpm, 60 sec, CT variety). Romanian Biotechnological Letters, Vol. 20, No. 5,

ROXANA-MĂDĂLINA STOICA, OVIDIU POPA, SULTANA NIȚĂ, NARCISA BĂBEANU It can be also observed that by increasing the grinding time, the average diameter of particles decreases from 419 μm to 300 μm.

4 ROXANA-MĂDĂLINA STOICA, OVIDIU POPA, SULTANA NIȚĂ, NARCISA BĂBEANU It can be also observed that by increasing the grinding time, the average diameter of particles decreases from 419 μm to 300 μm. Following grinding, the resulted material was subject to solvent extraction experiments. From all three Capsicum varieties, the best results were obtained with CT variety, while the lowest results were obtained with Hot cherry variety. Our finding are similar to other reports (ANTONIOUS & JARRET, [12] GNAYFEED & al., [13]) which showed that Capsicum chinense species showed higher capsaicin content than the Capsicum annum accession/varieties. The sum of capsaicin and dihydrocapsaicin reported to total capsacinoids concentration in the tested cultivars ranges between 75%-85%. (Figure 4). Comparing the efficiency of capsaicinoids extraction from the three varieties of Capsicum sp. with the four solvents tested, it can be observed that ethanol extract shows the higher content of capsaicinoids (0.872%), while the methanol extract shows the lowest content of capsaicinoids (0.643%) (Table 1). As it can be observed from Figure 5, the organic solvent influences the total amount of the extracted capsaicinoids, but not their ratio in the extract. The data from Table 2 suggest that the granulometric distribution of Capsicum powder has no significant effect on the extraction yield. Figure 4. Comparative chart on the capsaicinoids concentrations of Capsicum sp.(etoh). Table 1. The percentage of capsaicinoids content* obtained with organic solvents from Capsicum sp./dry weight Solvents EtOH MeOH Me 2 CO Acetonitrile Capsaicinoids Nordihydrocapsaicin (%) 0.101± ± ± ±0.020 Capsaicin (%) 0.507± ± ± ±0.029 Dihydrocapsaicin (%) 0.264± ± ± ±0.021 Total (%) 0.872± ± ± ±0.025 (*values are the mean ± s for 3 repetitions) Romanian Biotechnological Letters, Vol. 20, No. 5, 2015

Influence of process parameters on the extraction of capsaicinoids from various Capsicum species cultivated in Romania 1.0 Capsaicin; DCH % 0.8 0.6 0.4 0.2 0.

Capsaicin/dihydrocapsaicin ratio obtained by EtOH extraction is significantly higher than that obtained with other solvents. Similar results were obtained for extraction with methanol (JUANGSAMOOT J.

5 Influence of process parameters on the extraction of capsaicinoids from various Capsicum species cultivated in Romania 1.0 Capsaicin; DCH % EtOH C MeOH C-DCH Me2CO Acetonitrile Figure 5. Comparative chart on the influence of solvents on concentrations of capsaicinoids. Capsaicin/dihydrocapsaicin ratio obtained by EtOH extraction is significantly higher than that obtained with other solvents. Similar results were obtained for extraction with methanol (JUANGSAMOOT J. & al., [14]. AL OTHMAN & al.[15] tained a capsaicin/ dihydrocapsaicin ratio close to value 1 with EtOH, but this value can be a characteristic of the varieties tested. Table 2. The influence of granulometric distribution on the amount of most important capsaicinoids (EtOH) Grinding time Capsaicinoids* 30 sec 2 30 sec Capsaicin (%) C (%) + DHC(%) 75, *-capsaicin; DHC- dihydrocapsaicin 4. Conclusions This study proposed to investigate the influence of particle size on the extraction efficiency and also, to determine the best organic solvents for the extraction of capsaicinoids from the tested pepper varieties. Our results shows that the best values were obtained with ethanol, the highest capsaicinoids content extracted, comparative with methanol, the smallest amount of capsaicinoids extracted. The experiments also proved that in our conditions the particles size distribution did not significantly influence the extraction yield. 5. Acknowledgements This paper was published under the frame of European Social Fund, Human Resources Development Operational Programme , project POSDRU/159/1.5/S/ Romanian Biotechnological Letters, Vol. 20, No. 5,

6 ROXANA-MĂDĂLINA STOICA, OVIDIU POPA, SULTANA NIȚĂ, NARCISA BĂBEANU References 1. MUSFIROH I., TREESYEANGELINA M., MUCHTARIDI M., Capsaicin level of various Capsicum fruits. International Journal of Pharmacy and Pharmaceutical Sciences, 5(1), 248, 251,2013; 2. SANATOMBI K., SHARMA G.J., Capsaicin content and pungency of different Capsicum spp. cultivars. Not. Bot. Hort. Agrobot., 36(2), 89, 90, 2008; 3. ISLAM MD.A., SHARMA SHYAM S., SINHA P., NEGI MADAN S., NEOG B., TRIPATHI SH.BH., Variability in capsaicinoid content in different landraces of Capsicum cultivated in north-eastern India. ScientiaHorticulturae, 183, 66, 71, 2015; 4. AMRUTHRAJ N.J. PREETAM RAJ J.P., LEBEL L.A., Polar aprotic extraction of capsaicinoids from Capsicum Chinense Bhut Jolokia for Antimicrobial activity, International Journal of Biological & Pharmaceutical Research, 4(12), 959, 964, 2013; 5. ANWAR E.F., RAMADON D.H., Formulation and evaluation of gel and emulgel of chili extract (Capsicum frutescens L.) as topical dosage forms. International Journal of Pharmacy and Pharmaceutical Sciences, 6(3), 13, 16, 2014; 6. MOZSIKSI G., PAST T., SALAM O., ABDEL M.E., KUZMA M., PERJESI P., Interdisciplinary review for correlation between the plant origin capsaicinoids, non-steroidal antiinflammatory drugs, gastrointestinal mucosal damage and prevention in animals and human beings. Inflammopharmacol., 17, 113,150, 2009; 7. ZEWDIE Y., BOSLAND P.W., Evaluation of genotype, environment, and genotype by environment interaction for capsaicinoids in Capsicum annuum L..Euphytica, 111, 185, 190, 2000; 8. BOSLAND P.W., BARAL J.B., Bhut Jolokia - The World s Hottest Known Chile Pepper is a Putative Naturally Occurring Interspecific Hybrid HortScience, 42(2), 222, 224, 2007; 9. GONZALEZ-Z.A., SIERRA-CAMPOS E., LUNA-ORTEGA J.G., PEREZ-MORALES R., ORTIZ- JUAN C.R., GARCIA-HERNANDEZ J.L., Characterization of Different Capsicum Varieties by Evaluation of Their Capsaicinoids Content by High Performance Liquid Chromatography, Determination of Pungency and Effect of High Temperature. Molecules, 18, 13471,13486, 2013; 10. SGANZERLA M., JANCLEI P.C., TAVARES DE MELO A.M., GODOY H.T., Fast method for capsaicinoids analysis from Capsicum chinense fruits. Food Research International, 64,718, 725,2014; 11. DANG Y.Y., ZHANG H., XIU Z.L., Three-liquid-phase Extraction and Separation of Capsanthin and Capsaicin from Capsicum annum L. Czech J. Food Sci., 32, (1), , 2014; 12. ANTONIOUS, G.F., Jarret R.T., Screening Capsicum accessions for capsaicinoids content. J. Environ. Sci. Health, B 41(5),717, 729, 2006; 13. GNAYFEED, M. H., DAOOD H. G., BIACS P. A., ALCARAZ C. F., Content of bioactive compounds in pungent spice red pepper (paprika) as affected by ripening and genotype. J. Sci. Food Agric., 81(15), 1580,1585, 2001; 14. JUANGSAMOOT J., RUANGVIRIYACHAI C., TECHAWONGSTIEN S., CHANTHAI S., Determination of capsaicin and dihydrocapsaicin in some hot chilli varieties by RP-HPLC-PDA after magnetic stirring extraction and clean up with C cartridge, International Food Research Journal,19(3), 1217,1226, 2012; 15. AL OTHMAN Z.A., AHMED Y.B.H., HABILA M.A., GHAFAR A.A., Determination of Capsaicin and Dihydrocapsaicin in Capsicum Fruit Samples using High Performance Liquid Chromatography, Molecules, 16,8919, 8929, Romanian Biotechnological Letters, Vol. 20, No. 5, 2015

Determination of Capsaicin Content and Pungency Level of Five Different Peppers Grown in Nigeria.

Determination of Capsaicin Content and Pungency Level of Five Different Peppers Grown in Nigeria. 1 C.O. Nwokem *, 2 E.B. Agbaji, 2 J.A. Kagbu, 2 E.J. Ekanem 1 National Research Institute for Chemical