Comparative analysis of chemical composition and antioxidant activity of essential oil and hydrolate from black pepper fruit (Piper nigrum L.)




black pepper essential oil, hydrolate, aqueous phase of suspension, chemical composition, antioxidant activity


The objective of this study was to analyze the chemical composition of the essential oil (EO) and hydrolate of black pepper fruit, as well as their antioxidant activity. The EO was obtained by Clevenger-type hydrodistillation with hydromodule 1:10 m/v for 240 minutes, and the hydrolate was collected after the hydrodistillation process. The qualitative composition of EO was determined by GC/MS and quantitative by GC/FID method, while the qualitative composition of a hydrolate was determined by HS-SPME-GC/MS and quantitative composition by HS-SPME-GC/FID method. The antioxidant activity was investigated by DPPH assay. Fifty-five compounds were identified from black pepper EO, where the most abundant compounds were (E)-caryophyllene (41.6 %), limonene (9.7 %), and sabinene (8.6 %). Twelve compounds were identified from black pepper hydrolate, where the most abundant compounds were α-terpineol (34.7 %), borneol (17.3 %), and terpinen-4-ol (13.9 %). The hydrolate showed higher antioxidant activity after 20 minutes of incubation with an EC50 value of 0.993 ± 0.011 mg/cm3 compared to the EO with an EC50 value of 67.72 ± 1.871 mg/cm3.

According to the results obtained in this study, both the EO and hydrolate are good sources of natural antioxidants with potential uses in the food, organic agriculture, pharmaceutical, and cosmetic industries as a safer alternative to synthetic additives. Furthermore, the possibility of wider uses of the hydrolate should be investigated in more detail. Although researchers interested in hydrolate investigation focus mostly on their antioxidant activity, these "aromatic wastes" could also present promising cosmetic activities.


(1) Parmar, V. S.; Jain, S. C.; Bisht, K. S.; Jain, R.; Taneja, P.; Jha, A.; Tyagi O. D.; Prasad A. K.; Wengel J.; Olsen C. E.; Boll, P. M., Phytochemistry of the genus Piper. Phytochemistry 1997, 46 (4), 597–673.

(2) Salehi, B.; Zakaria, Z. A.; Gyawali, R.; Ibrahim, S. A.; Rajkovic, J.; Shinwari, Z. K..; Khan, T.; Sharifi-Rad, J.; Ozleyen, A.; Turkdonmez, E.; Valussi, M.; Tumer, T. B.; Fidalgo, L.M.; Martorell, M.; Setzer, W. N., Piper species: A comprehensive review on their phyto-chemistry, biological activities and applications. Molecules 2019, 24 (7), 1364.

(3) Milenković, A. N.; Stanojević, L. P., Black pepper: chemical composition and biological activities. Adv.Technol. 2021, 10 (2), 40–50.

(4) Ashokkumar, K.; Murugan, M.; Dhanya, M. K.; Pandian, A.; Warkentin, T. D., Phytochemistry and therapeutic potential of black pepper [Piper nigrum (L.)] essential oil and piperine: a review. Clin. Phytoscience. 2021, 7 (1), 1–11.

(5) Dosoky, N. S.; Satyal, P.; Barata, L. M.; Da Silva, J. K. R.; Setzer, W. N., Volatiles of black pepper fruits (Piper nigrum L.). Molecules 2019, 24 (23), 4244: 1–13.

(6) Kapoor, I. P. S.; Singh, B.; Singh, G.; De Heluani, C. S.; De Lampasona, M. P.; Catalan, C. A., Chemistry and in vitro antioxidant activity of volatile oil and oleoresins of black pepper (Piper nigrum). J. Agric. Food Chem. 2009, 57 (12), 5358–5364.

(7) Politeo, O.; Jukić, M.; Miloš M., Chemical composition and antioxidant activity of essential oils of twelve spice plants. Croat. Chem. Acta 2006, 79 (4), 545–552.

(8) Orav, A.; Stulova, I.; Kailas, T.; Mürisepp, M., Effect of storage on the essential oil composition of Piper nigrum L. fruits of different ripening states. J. Agric. Food Chem. 2004, 52 (9), 2582–2586.

(9) Aćimović, M. G.; Tešević, V. V.; Smiljanić, K. T.; Cvetković, M. T.; Stanković, J. M.; Kiprovski, B. M.; Sikora, V. S., Hydrolates: By-products of essential oil distillation: chemical composition, biological activity and potential uses. Adv. Technol. 2020, 9 (2), 54–70.

(10) Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th ed. Adams, R. P., (Eds.). Allured Publishing Co., Illinois, 2007.

ISBN 978-1-932633-21-4

(11) Gas Chromatography and Mass Spectrometry: a Practical Guide, 2nd ed. Sparkman, D. O.; Penton, Z. E.; Fulton, K. G. (Eds).; Elsevier Inc., Oxford, USA, 2011.

(12) Stanojević, Lj. P.; Stanojević, J. S.; Savić, V. Lj.; Cvetković, D. J.; Kolarević, A.; Marjanović-Balaban, Ž.; Nikolić, Lj. B., Peppermint and basil essential oils: chemical composition, in vitro antioxidant activity and in vivo estimation of skin irritation. J. Essent. Oil-Bear Plants 2019, 22, 979–993.

(13) Abera, W. A. Optimization and characterization of essential oil from black pepper (Nigrum) seeds using the distillation extraction method for perfume additives. Am. J. Chem. Eng. 2022, 10 (3), 53–62.

(14) Wang, Y. L.; Wang, L., Tan, J.; Li, R.; Jiang, Z. T.; Tang, S. H., Comparative analysis of intracellular and in vitro antioxidant activities of essential oil from white and black pepper (Piper nigrum L.). Front. Pharmacol. 2021, 12, 1344.

(15) Dinh, P. N.; Cam, H. D. T.; Quoc, T. P., Comparison of essential oil extracted from black pepper by using various distillation methods in laboratory scale. IOP Conf. Ser.: Mater. Sci. Eng. 2020, 991 (1), 012050.

(16) Babushok, V. I.; Linstrom, P. J.; Zenkevich, I. G., Retention indices for frequently reported compounds of plant essential oils. J. Phys. Chem. Ref. Data. 2011, 40 (4), 043101–47.

(17) Sruthi, D.; Zachariah, T. J.; Leela, N. K.; Jayarajan, K. Correlation between chemical profiles of black pepper (Piper nigrum L.) var. Panniyur-1 collected from different locations. J. Med. Plants Res. 2013, 7, 2349–57.

(18) Kapoor, I. P. S.; Singh, B.; Singh, G.; De Heluani, C. S.; De Lampasona, M. P.; Catalan, C. A., Chemistry and in vitro antioxidant activity of volatile oil and oleoresins of black pepper (Piper nigrum). J. Agric. Food Chem. 2009, 57 (12), 5358–5364.

(19) Jirovetz, L.; Buchbauer, G., Ngassoum, M. B.; Geissler, M., Aroma compound analysis of Piper nigrum and Piper guineense essential oils from Cameroon using solidphase microextraction–gas chromatography, solid-phase microextraction–gas chromatography–mass spec-trometry and olfactometry. J. Chromatogr. A. 2002, 976 (1–2), 265–275.

(20) Tran, T. H.; Le Ha, K.; Nguyen, D. C.; Dao, T. P.; Le Nhan, T. H.; Nguyen, D. H.; Nguyen, T. D.; Vo, D. N.; Tran, Q. T.; Bach, L. G., The study on extraction process and analysis of components in essential oils of black pepper (Piper nigrum L.) seeds harvested in Gia Lai Province, Vietnam. Processes 2019, 7 (2), 56.

(21) Morshed, S.; Hossain, M. D.; Ahmad, M.; Junayed, M., Physicochemical characteristics of essential oil of black pepper (Piper nigrum) cultivated in Chittagong, Bangladesh. J. Food Qual. Hazards Control. 2017, 4 (3), 66–69.

(22) Tchoumbougnang, F.; Jazet, D. P. M.; Sameza, M. L.; Fombotioh, N.; Vyry, W. N. A.; Henri, A. Z. P.; Menut, C., Comparative essential oils composition and insecticidal effect of different tissues of Piper capense L., Piper guineense Schum. et Thonn., Piper nigrum L. and Piper umbellatum L. grown in Cameroon. Afr. J. Biotechnol. 2009, 8 (3), 424–431.

(23) Jeena, K.; Liju, V. B.; Umadevi, N. P.; Kuttan, R., Antioxidant, anti-inflammatory and antinociceptive properties of black pepper essential oil (Piper nigrum Linn). J. Essent. Oil-Bear. Plants. 2014, 17 (1), 1–12.

(24) Vinturelle, R.; Mattos, C.; Meloni, J.; Nogueira, J.; Nunes, M. J.; Vaz, I. S.; Rocha, L.; Lione, V.; Castro, H. C.; Chagas, E. F. D., In vitro evaluation of essential oils derived from Piper nigrum (Piperaceae) and Citrus limonum (Rutaceae) against the tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Biochem. Res. Int. 2017, (2017), 5342947: 1–9.

(25) Collin, G.; Gagnon, H., Chemical composition and stability of the hydrosol obtained during the production of essential oils. III. The case of Myrica gale L., Comptonia peregrine (L.) Coulter and Ledum groenlandicum Retzius. Am. J. Essent. Oil. 2016, 4, 07–19.

(26) Zekri, N.; Amalich, S.; Elbelghiti, M. A.; Zair, T., Phytochemical screening and chemical composition of essential oils and hydrosols of Mentha species from Morocco. Adv. Environ. Biol. 2014, 8, 10–18.

(27) Turek, C.; Stintzing, F. C., Stability of essential oils: a review. Compr. Rev. Food Sci. Food Saf. 2013, 12 (1), 40–53.

(28) Zengin, H.; Baysal, A. H., Antibacterial and antioxidant activity of essential oil terpenes against pathogenic and spoilage-forming bacteria and cell structure-activity relationships evaluated by SEM microscopy. Molecules 2014, 19 (11), 17773–17798.

(29) Lei, C.; Jianyu, S.; Lin, L.; Bing, L.; Wang, L., A new source of natural D-borneol and its characteristic. J. Med. Plants Res. 2011, 205 (15), 3440–3447.

(30) Misharina, T. A., Antiradical properties of essential oils and extracts from coriander, cardamom, white, red, and black peppers. Appl. Biochem. Microbiol. 2016, 52 (1), 79–86.



2022-12-28 — Updated on 2022-12-30


How to Cite

Milenković, A., Stanojević, J. ., & Stanojević, L. . (2022). Comparative analysis of chemical composition and antioxidant activity of essential oil and hydrolate from black pepper fruit (Piper nigrum L.). Macedonian Journal of Chemistry and Chemical Engineering, 41(2), 209–220. (Original work published December 28, 2022)



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