NVEO 2020, Volume 7, Issue 4

Volume 7, Issue 4, 2020 e-ISSN: 2148-9637

NATURAL VOLATILES & ESSENTIAL OILS A Quarterly Open Access Scientific Journal

NVEO Publisher: BADEBIO Ltd.

NATURAL VOLATILES & ESSENTIAL OILS Volume 7, Issue 4, 2020

RESEARCH ARTICLES

Pages 1-7 Essential oil composition and antioxidant activity of Reinwardtiodendron cinereum (Hiern) Mabb. (Meliaceae); Wan Mohd Nuzul Hakimi Wan Salleh, Shamsul Khamis and Muhammad Helmi Nadri,

8-13 Chemical composition and acetylcholinesterase inhibition of the essential oil of Cyathocalyx pruniferus (Maingay ex Hook.f. & Thomson) J. Sinclair; Wan Mohd Nuzul Hakimi Wan Salleh, Shamsul Khamis and Muhammad Helmi Nadri

14-25 Bioactivity evaluation of the native Amazonian species of Ecuador: Piper lineatum Ruiz & Pav. essential oil; Eduardo Valarezo, Gabriela Merino, Claudia Cruz- Erazo and Luis Cartuche

26-33 Extraction process optimization and characterization of the Pomelo (Citrus grandis L.) peel essential oils grown in Tien Giang Province, Vietnam; Dao Tan Phat, Kha Chan Tuyen, Xuan Phong Huynh, Tran Thanh Truc

34-40 Characterization of Dysphania ambrosioides (L.) Mosyakin & Clemants essential oil from Vietnam; Tran Thi Kim Ngan, Pham Minh Quan and Tran Quoc Toan

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Nat. Volatiles & Essent. Oils, 2020; 7(4): 1-7 Salleh et al. DOI: 10.37929/nveo.770245

RESEARCH ARTICLE Essential oil composition and antioxidant activity of Reinwardtiodendron cinereum (Hiern) Mabb. (Meliaceae)

Wan Mohd Nuzul Hakimi Wan Salleh1,*, Shamsul Khamis2, Muhammad Helmi Nadri3, Hakimi Kassim4 and Alene Tawang4

1Department of Chemistry, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris, 35900 Tanjong Malim, Perak, MALAYSIA 2School of Environmental and Natural Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, MALAYSIA 3Innovation Centre in Agritechnology (ICA), Universiti Teknologi Malaysia, 84600 Pagoh, Muar, Johor, MALAYSIA 4Department of Biology, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris, 35900 Tanjong Malim, Perak, MALAYSIA

*Corresponding author. Email: [email protected] Submitted: 16.07.2020; Accepted: 02.10.2020

Abstract This study was designed to investigate the chemical composition and antioxidant activity of the essential oil from Reinwardtiodendron cinereum (Hiern) Mabb. growing in Malaysia. The essential oil was obtained by hydrodistillation and fully analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The analysis led to the identification of thirty-five components representing 83.6% of the total oil. The most abundant components were α-zingiberene (15.4%), pimaradiene (10.1%), (E)-caryophyllene (7.5%), and β-elemene (6.3%). The antioxidant activity was determined by DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging, total phenolic content, and β-carotene linoleic acid bleaching assays. The essential oil showed weak activity in

DPPH radical scavenging (IC50 value of 105.2 µg/mL), phenolic content (45.5 ± 0.2 mg GA/g), and β-carotene linoleic acid bleaching (52.5 ± 0.2%) assays. To the best of our knowledge, this is the first study of the chemical composition and antioxidant activities of the essential oil report concerning the genus Reinwardtiodendron.

Keywords: Antioxidant, essential oil, Meliaceae, Reinwardtiodendron cinereum, α-Zingiberene

Introduction Essential oils are chemically characterized as complex mixtures of low molecular weight compounds and some of them are highly volatile and capable of generating flavors (Trombetta et al., 2005). Scientific studies have shown the role of essential oils in biological interactions among plants and they are potential therapeutic including anti-inflammatory, analgesic, anti-tumor, antifungal and antibacterial activities (Osei- Safo et al., 2010; Salleh et al., 2014a, 2016a). Meliaceae, also called Mahogany family is a flowering plant family of mostly trees and shrubs in the order Sapindales. It is composed of 50 genera and 1400 species. In Southeast Asia, species of Meliaceae are widely found from lowlands to higher elevation highlands, and are one of important components in tropical and subtropical evergreen forests (Perez-Flores et al., 2012). A great variety of plants belonging to the Meliaceae family have been phytochemically investigated and several compounds have been isolated and identified. They were limonoids, triterpenes, steroids, diterpenes, sesquiterpenes, and coumarins (Soares et al., 2012; Scur et al., 2016). Reinwardtiodendron is one of the genus of Meliaceae, distributed mainly in Peninsular Malaysia, Borneo, and Sumatra. It is composed of only seven species, which are R. anamalaiense, R. celebicum, R. cinereum, R. humile, R. kinabaluense, R. kostermansii, and R. merrillii (Mabberley, 2011).

Nat. Volatiles & Essent. Oils, 2020; 7(4): 1-7 Salleh et al. DOI: 10.37929/nveo.770245

Reinwardtiodendron cinereum (Hiern) Mabb. is a tree of up to 27 m height. The leaf is 10-15 cm long, while the bark is smooth with scaly patches and conspicuous knobbly tubercles. The colour for the inner bark is white, while the fruits are globose or glabrous with a yellow colour (Mabberley, 2011). There has been no information of this plant in traditional or folk medicine practice. Most of the species of genus are unexplored, both pharmacologically and phytochemically. Previous phytochemical investigation of R. cinereum has resulted in the isolation of onocerane triterpenoids (Nugroho et al., 2018). Besides, the bark extract of R. cinereum was found to be cytotoxic against human promyelocytic leukaemia HL-60 cells (Nugroho et al., 2018). Meanwhile, the literature search did not reveal any report on the essential oil composition of the genus Reinwardtiodendron. As a continuation part of our systematic evaluation of the aromatic flora of Malaysia (Salleh et al., 2014b, 2015a, 2015b, 2015c), this study reports the chemical composition and antioxidant activities of the essential oil from the leaves of R. cinereum. Materials and Methods Plant material Sample of Reinwardtiodendron cinereum was collected from Behrang, Perak in January 2019, and identified by Dr. Shamsul Khamis from University Kebangsaan Malaysia (UKM). The voucher specimen (SK165/19) was deposited at UKMB Herbarium University Kebangsaan Malaysia. Solvents and chemicals β-Carotene, linoleic acid, DPPH•, gallic acid and butylated hydroxytoluene (BHT) were obtained from Sigma- Aldrich Chemie (Steinheim, Germany). Analytical grade methanol, ethanol, and dimethylsulfoxide (DMSO), HPLC grade chloroform, Folin-Ciocalteu’s reagent, anhydrous sodium sulfate, sodium carbonate, polyoxyethylene sorbitan monopalmitate (Tween-40) were purchased from Merck (Darmstadt, Germany). Extraction of the essential oil The fresh leaf (500 g) was subjected to hydrodistillation in Clevenger-type apparatus for 4 hours. The essential oil obtained and was dried over anhydrous magnesium sulfate then stored at 4-6 °C. The yield (v/w) of the obtained essential oil was 1.8%, expressed as a percentage of absolute dry weight. Gas chromatography (GC) analysis GC analysis were performed on an Agilent Technologies 7890B and an Agilent 7890B FID equipped with DB- 5 column. Helium was used as a carrier gas at a flow rate of 0.7 mL/min. Injector and detector temperature were set at 250 and 280 °C, respectively. The oven temperature was kept at 50 °C, then gradually raised to 280 °C at 5 °C/min and finally held isothermally for 15 min. Diluted samples (1/100 in diethyl ether, v/v) of 1.0 μL were injected manually (split ratio 50:1). The injection was repeated three times and the peak area percents were reported as means ± SD of triplicates. Calculation of peak area percentage was carried out by using the GC HP Chemstation software (Agilent Technologies). Gas chromatography-mass spectrometry analysis GC-MS chromatograms were recorded using Agilent Technologies 7890A and Agilent 5975 GC MSD equipped. The GC was equipped with HP-5MS column (30 m × 0.25 mm × 0.25 μm film thickness). Helium was used as carrier gas at a flow rate of 1 mL/min. Injector temperature was 250 °C. The oven temperature was programmed from 50 °C (5 min hold) to 250 °C at 10 °C/min and finally held isothermally for 15 min. For GC- MS detection, an electron ionization system, with ionization energy of 70 eV was used. A scan rate of 0.5 s (cycle time: 0.2 s) was applied, covering mass range was from 40–400 amu.

Nat. Volatiles & Essent. Oils, 2020; 7(4): 1-7 Salleh et al. DOI: 10.37929/nveo.770245

Identification of chemical components For the identification of essential oil components, co-injection with the above standards were used, together with correspondence of retention indices and mass spectra with respect to those occurring in ADAMS, NIST 08 and FFNSC2 libraries (Adams, 2007). Semi-quantification of essential oil components was made by peak area normalization considering the same response factor for all volatile components. Percentages values were the mean of three chromatographic analyses. Antioxidant activity DPPH radical scavenging The free radical scavenging activity was measured by the DPPH method with minor modifications (Salleh et al., 2014c). Each sample of stock solution (1.0 mg/mL) was diluted to a final concentration of 1000−7.8 μg/mL. Then, a total of 3.8 mL of 50 μM DPPH• methanolic solution (1 mg/50 mL) was added to 0.2 mL of each sample solution and allowed to react at room temperature for 30 min. The absorbance of the mixtures was measured at 517 nm. A control was prepared without a sample or standard and measured immediately at 0 min. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity and vice versa. The percent inhibitions (I %) of DPPH radical were calculated as follow: I % = [ A blank – A sample / A blank ] ×100; where

A blank is the absorbance value of the control reaction and A sample is the absorbance values of the test samples.

The sample concentration providing 50% inhibition (IC50) was calculated by plotting inhibition percentages against concentrations of the sample. All tests were carried out in triplicate and IC50 values were reported as means ± SD of triplicate. Total phenolic content (TPC) Total phenolic contents of the essential oil were determined as described previously (Salleh et al., 2014c). A sample of stock solution (1.0 mg/mL) was diluted in methanol to final concentrations of 1000 μg/mL. A 0.1 mL aliquot of sample was pipetted into a test tube containing 0.9 mL of methanol, then 0.05 mL Folin-

Ciocalteu’s reagent was added, and the flask was thoroughly shaken. After 3 min, 0.5 mL of 5 % Na2CO3 solution was added and the mixture was allowed to stand for 2 h with intermittent shaking. Then, 2.5 mL of methanol was added and left to stand in the dark for 1 h. The absorbance measurements were recorded at 765 nm. The same procedure was repeated for the standard gallic acid solutions. The concentration of total phenolic compounds in the oil was expressed as mg of gallic acid equivalent per gram of sample. Tests were carried out in triplicate and the gallic acid equivalent value was reported as mean ± SD of triplicate. β-Carotene-linoleic acid bleaching assay The β-carotene-linoleic acid bleaching assay was determined as described previously with minor modifications (Salleh et al., 2016b). A mixture of β-carotene and linoleic acid was prepared by adding together of 0.5 mg β-carotene in 1 mL chloroform 25 μL linoleic acid and 200 mg Tween 40. The chloroform was then completely evaporated and 100 mL of oxygenated distilled water was subsequently added to the residue and mixed gently to form a clear yellowish emulsion. The essential oil (2 g/L) was in methanol and 350 μL of each sample solution was added to 2.5 mL of the above mixture in test tubes and mixed thoroughly. The test tubes were incubated in a water bath at 50 °C for 2 h together with two blanks, one contained BHT (positive control) and the other contained the same volume of methanol. The absorbance was measured at 470 nm on an ultraviolet-visible (UV–Vis) spectrometer. Antioxidant activities (inhibitions percentage, I %) of the samples were calculated using the following equation: I % = [A β-carotene after 2 h / A initial β-carotene] × 100; where

A β-carotene after 2 h assay is the absorbance value of β-carotene after 2 h assay remaining in the samples and Ainitial

Nat. Volatiles & Essent. Oils, 2020; 7(4): 1-7 Salleh et al. DOI: 10.37929/nveo.770245

β-carotene is the absorbance value of β-carotene at the beginning of the experiment. All tests were carried out in triplicate and inhibition percentages were reported as means ± SD of triplicate. Statistical analysis Data obtained from essential oil analysis and antioxidant were expressed as mean values. The statistical analyses were carried out by employing one way ANOVA (p<0.05). A statistical package (SPSS version 11.0) was used for the data analysis. Results and Discussion The essential oil was obtained by hydrodistillation as a pale yellow colour from the fresh leaves of R. cinereum. The chemical compositions of the essential oil were analysed by GC, GC-MS and Kovats Indices (KI). The identified components are listed in Table 1 according to their elution on a DB-5 column in parallel with their KI and their relative’s percentage.

Table 1. Chemical composition of Reinwardtiodendron cinereum essential oil

Percentage Identification No. Components KIa KIb (%)c methods 1 α-Patchoulene 1455 1457 0.2 RI, MS 2 α-Cubebene 1458 1460 0.3 RI, MS 3 4,5-di-epi-Aristolochene 1468 1467 0.3 RI, MS 4 δ-Elemene 1470 1468 0.5 RI, MS 5 α-Copaene 1490 1491 1.7 RI, MS 6 α-Gurjunene 1530 1529 0.2 RI, MS 7 trans-Cadina-1,4-diene 1535 1532 0.5 RI, MS 8 α-cis-Bergamotene 1562 1559 0.2 RI, MS 9 β-Copaene 1580 1579 0.3 RI, MS 10 α-Cedrene 1585 1582 0.5 RI, MS 11 (Z)-Caryophyllene 1589 1588 4.6 RI, MS 12 β-Elemene 1590 1590 6.3 RI, MS, Std 13 (E)-Caryophyllene 1595 1598 7.5 RI, MS, Std 14 Aromadendrene 1625 1620 0.5 RI, MS 15 Pogostol 1650 1651 0.5 RI, MS 16 α-Humulene 1665 1666 2.1 RI, MS 17 γ-Muurolene 1690 1689 1.8 RI, MS 18 Germacrene D 1705 1708 4.0 RI, MS 19 β-Selinene 1715 1716 2.7 RI, MS 20 α-Zingiberene 1720 1720 15.4 RI, MS, Std 21 α-Muurolene 1725 1723 1.0 RI, MS 22 (E, E)-α-Farnesene 1745 1743 2.4 RI, MS 23 Xanthorrhizol 1751 1751 1.3 RI, MS 24 Geranyl acetate 1752 1752 0.7 RI, MS 25 δ-Cadinene 1756 1755 5.9 RI, MS, Std 26 γ-Cadinene 1765 1763 2.4 RI, MS 27 (E)-α-Bisabolene 1775 1775 0.4 RI, MS 28 Germacrene B 1825 1823 0.5 RI, MS

Nat. Volatiles & Essent. Oils, 2020; 7(4): 1-7 Salleh et al. DOI: 10.37929/nveo.770245

29 Pimaradiene 1910 1909 10.1 RI, MS 30 Elemol 2075 2078 0.3 RI, MS 31 Globulol 2080 2082 2.7 RI, MS 32 1-epi-Cubenol 2086 2088 1.9 RI, MS 33 Spathulenol 2020 2126 0.8 RI, MS 34 α-Cadinol 2222 2227 3.0 RI, MS 35 Manool oxide 2075 2376 0.3 RI, MS Sesquiterpene hydrocarbon 72.6 Oxygenated sesquiterpenes 11.0 Total (%) 83.6 aLinear retention index experimentally determined using homologous series of C6-C30 alkanes. bLinear retention index taken from Adams (2007) or NIST 08 (2008) and literature. cRelative percentage values are means of three determinations ± SD. Identification methods: Std, based on comparison with authentic compounds; MS, based on comparison with Wıley, Adams, FFNSC2 and NIST 08 MS databases; RI, based on comparison of calculated RI with those reported in Adams, FFNSC 2 and NIST 08.

Analysis of the essential oil had successfully characterized thirty-five components, accounting for 83.57 % of the total composition. They were grouped into sesquiterpene hydrocarbons and oxygenated sesquiterpenes. Sesquiterpene hydrocarbons were the most dominant components which constituted 27 components, accounting for 72.6% of the total composition. Meanwhile, oxygenated sesquiterpenes were present in appreciable amounts which comprised 8 components, accounting for 11 % of the total composition. The most abundant components in the essential oil were α-zingiberene (15.4%), pimaradiene (10.1%), (E)- caryophyllene (7.5%), β-element (6.3 %), δ-cadinene (5.9%), (Z)-caryophyllene (4.6%), germacrene D (4.0%), and α-cadinol (3.4%). In addition, the other minor components detected in the essential oil in more than 2 % were β-selinene (2.7%), globulol (2.7%), (E,E)-α-farnesene (2.4%), γ-cadinene (2.4%), and α-humulene (2.1%). The essential oil was screened for their possible antioxidant activity by DPPH radical scavenging, total phenolic contents, and β-carotene/linoleic acid bleaching assays. The results are shown in Table 2. The essential oil exhibited weak DPPH radical scavenging activity (IC50 of 105.2 µg/mL) compared to standard antioxidant, BHT (IC50 of 18.5 µg/mL). The low activity of the essential oil was attributed to the low phenolic content of the essential oil (25.5 ± 0.2 mg GA/g) which is responsible for antioxidant activity (Salleh et al., 2016a). This was supported by the results of the Folin-Ciocalteu assay on the essential oil, which showed a low amount of oxygenated sesquiterpenes. In addition, β-carotene/linoleic acid bleaching assay was evaluated by measuring the inhibition of conjugated diene hydroperoxides starting from linoleic acid oxidation. The essential oil gave 52.5 ± 0.2% which were lower compared to BHT, 95.5 ± 0.1%.

Table 2. Antioxidant activity of the Reinwardtiodendron cinereum essential oil

β-carotene/linoleic acid DPPH IC50 TPC, Gallic acid equivalent Samples (%) (μg/mL) (mg GA/g) Essential oil 52.5 ± 0.2 105.2 ± 0.2 25.5 ± 0.2 BHT 95.5 ± 0.1 18.5 ± 0.1 ND Data represent mean±standard deviation of three independent experiments; ND – not determined α-Zingiberene is a monocyclic sesquiterpene that is the main flavour component of ginger. Previous findings showed that α-zingiberene exhibits apoptotic effects on SiHa cells (Lee, 2016). In addition, the purified α- zingiberene from the essential oil of Casearia sylvestris showed a cytotoxic activity against HeLa, U-87, SiHa, and HL60 cell lines, with IC50 values of 63.2, 153.0, 48.0, and 98.7 μg/mL, respectively (Bou et al., 2013). Similar to our results, α-zingiberene has been reported to be the major component from various species of

Nat. Volatiles & Essent. Oils, 2020; 7(4): 1-7 Salleh et al. DOI: 10.37929/nveo.770245

Zingiberaceae family, including Zingiber officinale (India: rhizomes 46.71%) (Sharma et al., 2016), Curcuma longa (China: rhizomes 25.05%) (Zhang et al., 2017) and Amomum muricarpum (Vietnam: fruits 6.3%) (Huong et al., 2015). In addition, α-zingiberene was found to be rich in the essential oils of Senecio selloi (aerial parts 54.0%) (Silva et al., 2013), Casearia sylvestris (leaves 48.31%) (Bou et al., 2013), Guarea kunthiana (aerial parts 34.48%) (Pandini et al., 2018), Piper lucaeanum (leaves 30.4%) (Marques et al., 2015), and Polyalthia longifolia (leaves20.0%) (Ouattara et al., 2014). Thus, these components are considered to be worth furthermore study to develop it as chemotherapeutics. In conclusion, the genus Reinwardtiodendron is still poorly discovered an unidentified as far as its essential oil composition is concerned. This research is the first report on the essential oil composition of R. cinereum growing in Malaysia. The high concentration of α-zingiberene makes it a good candidate for a chemical marker for Reinwardtiodendron species. In addition, the preliminary evaluation of its antioxidant activity is the first step described in the literature for this species and taken together, the data obtained here inspire more studies supporting the possibility of linking the chemical contents with particular biological properties.

ACKNOWLEDGMENT

The author would like to thank Department of Chemistry, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris (UPSI) for research facilities. REFERENCES

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Nat. Volatiles & Essent. Oils, 2020; 7(4): 8-13 Salleh et al. DOI: 10.37929/nveo.770303

RESEARCH ARTICLE Chemical composition and acetylcholinesterase inhibition of the essential oil of Cyathocalyx pruniferus (Maingay ex Hook.f. & Thomson) J. Sinclair Wan Mohd Nuzul Hakimi Wan Salleh1,*, Shamsul Khamis2, Muhammad Helmi Nadri3, Hakimi Kassim4 and Alene Tawang4

1Department of Chemistry, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris, 35900 Tanjong Malim, Perak, MALAYSIA 2School of Environmental and Natural Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, MALAYSIA 3Innovation Centre in Agritechnology (ICA), Universiti Teknologi Malaysia, Hub Pendidikan Tinggi Pagoh, 84600 Pagoh, Muar, Johor, MALAYSIA 4Department of Biology, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris, 35900 Tanjong Malim, Perak, MALAYSIA

*Corresponding author. Email: [email protected] Submitted: 16.07.2020; Accepted: 24.11.2020

Abstract The chemical composition and acetylcholinesterase inhibitory activity of the essential oil from the leaf of Cyathocalyx pruniferus growing in Pahang, Malaysia was investigated for the first time. The essential oil was obtained by hydrodistillation and fully characterized by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). A total of thirty chemical components (95.5%) were successfully identified in the essential oil which was characterized by high proportions of α-pinene (25.4%), germacrene D (20.2%), β-caryophyllene (10.8%), and δ-cadinene (6.4%). Acetylcholinesterase inhibitory activity was evaluated against the Ellman method, and the essential oil showed good inhibition against acetylcholinesterase (Percentage inhibition: 75.5%) assay.

Keywords: Essential oil, Cyathocalyx pruniferus, Annonaceae, α-pinene, germacrene D, acetylcholinesterase

Introduction The genus Cyathocalyx belonging to the Annonaceae family consists of approximately 36 species of monopodial trees. It is widely distributed in primary and secondary tropical lowland forests of Southeast Asia, with a center of diversity in western Malaysia (Peninsular Malaysia, Sumatra, and Borneo) (Wang and Saunders, 2006). Cyathocalyx pruniferus (Maingay ex Hook.f. & Thomson) J. Sinclair. is locally known as Antoi beludu in Malaysia (Burkill, 1966). International Plant Names Index reported that the plant is a synonym of Drepananthus pruniferus Maingay ex Hook.f. & Thomson (IPNI, 2020). Literature reviews indicated that only a small number of species in the genus Cyathocalyx have been investigated for its chemical compounds and biological activities. Previous phytochemical investigation on Cyathocalyx species led to the isolation of alkaloids and diterpenoids (Wijeratne et al., 1995a, 1995b). However, there is little information about the chemical composition and the biological properties of the essential oil of the genus Cyathocalyx. Previous study on the essential oil of Cyathocalyx have been reported on Cyathocalyx zeylanicus collected from India (Hisham et al., 2012). In continuation of our systematic studies on pharmacologically active volatiles from Malaysian plants (Salleh et al.,2014, 2015a, 2015b, 2016a, 2016b), we describe in this paper an evaluation of the chemical composition and acetylcholinesterase inhibitory activity of the essential oil from the leaf of C. pruniferus.

Nat. Volatiles & Essent. Oils, 2020; 7(4): 8-13 Salleh et al. DOI: 10.37929/nveo.770303

Materials and Methods Plant material Sample of Cyathocalyx pruniferus was collected from Gambang, Pahang in September 2019, and identified by Dr. Shamsul Khamis from Universiti Kebangsaan Malaysia (UKM). The voucher specimen (SK242/19) was deposited at UKMB Herbarium, Faculty of Science and Technology UKM. Extraction of essential oil The fresh leaf (300 g) was subjected to hydrodistillation in Clevenger-type apparatus for 4 hours. The essential oil obtained was dried over anhydrous magnesium sulphate and stored at 4-6°C. Gas chromatography (GC) analysis GC analysis were performed on an Agilent Technologies 7890B and an Agilent 7890B FID equipped with DB- 5 column. Helium was used as a carrier gas at a flow rate of 0.7 mL/min. Injector and detector temperature were set at 250 and 280 °C, respectively. The oven temperature was kept at 50 °C, then gradually raised to 280 °C at 5 °C/min and finally held isothermally for 15 min. Diluted samples (1/100 in diethyl ether, v/v) of 1.0 μL were injected manually (split ratio 50:1). The injection was repeated three times and the peak area percentages were reported as means ± SD of triplicates. Calculation of peak area percentage was carried out by using the GC HP Chemstation software (Agilent Technologies). Gas chromatography-mass spectrometry (GC-MS) analysis GC-MS chromatograms were recorded using Agilent Technologies 7890A and Agilent 5975 GC MSD equipped. The GC was equipped with HP-5MS column. Helium was used as carrier gas at a flow rate of 1 mL/min. Injector temperature was 250 °C. The oven temperature was programmed from 50 °C (5 min hold) to 250 °C at 10 °C/min and finally held isothermally for 15 min. For GC-MS detection, an electron ionization system, with ionization energy of 70 eV was used. A scan rate of 0.5 s (cycle time: 0.2 s) was applied, covering a mass range from 40–400 amu. Identification of chemical components For identification of essential oil components, co-injection with the standards (major components) were used, together with correspondence of retention indices and mass spectra with respect to those occurring in Adams, NIST 08 and FFNSC2 libraries (Adams, 2007). Semi-quantification of essential oil components was made by peak area normalization considering the same response factor for all volatile components. Percentages values were the mean of three chromatographic analyses. Acetylcholinesterase inhibitory activity AChE inhibitory activity of the essential oil was measured by slightly modifying the spectrophotometric method developed by Salleh et al. (2014b). Electric eel AChE was used, while acetylthiocholine iodide was employed as substrates of the reaction. DTNB acid was used for the measurement of the acetylcholinesterase activity. Briefly, in this method, 140 μL of sodium phosphate buffer (pH 8.0), 20 μL of DTNB, 20 μL of essential oil and 20 μL of AChE solution were added by multichannel automatic pipette in a 96-well microplate and incubated for 15 min at 25 °C. The reaction was then initiated with the addition of 10 μL of acetylthiocholine iodide. Hydrolysis of acetylthiocholine iodide was monitored by the formation of the yellow 5-thio-2- nitrobenzoate anion as a result of the reaction of DTNB with thiocholines, catalysed by enzymes at 412 nm utilizing a 96-well microplate reader (Epoch Micro-Volume Spectrophotometer, USA). Percentage of

Nat. Volatiles & Essent. Oils, 2020; 7(4): 8-13 Salleh et al. DOI: 10.37929/nveo.770303

inhibition (%I) of AChE was determined by comparison of rates of reaction of samples relative to blank sample (EtOH in phosphate buffer pH 8) using the formula: %I = [ E - S / E ] × 100; where E is the activity of enzyme without test sample and S is the activity of enzyme with test sample. The experiments were done in triplicate. Galantamine was used as a reference. Statistical analysis Data obtained from essential oil analysis and bioactivity were expressed as mean values. The statistical analyses were carried out by employing one-way ANOVA (p<0.05). A statistical package (SPSS version 11.0) was used for the data analysis. Results and Discussion The essential oil was isolated by hydrodistillation from the fresh leaf of C. pruniferus was analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The yield of the essential oil obtained was 0.13% (w/w). The percentage composition of the essential oil, retention time and retention indices of the components are shown in Table 1.

Table 1. Chemical composition of C. pruniferus essential oil

No. Components KIa KIb Percentage (%)

1 α-Pinene 930 0932 25.4 ± 0.3 2 Camphene 945 946 0.4 ± 0.1 3 Sabinene 970 0969 0.4 ± 0.1 4 β-Pinene 972 0974 1.1 ± 0.1 5 α-Terpinene 1015 1014 0.4 ± 0.2 6 Limonene 1025 1024 0.3 ± 0.1 7 β-Elemene 1336 1335 0.5 ± 0.1 8 α-Cubebene 1346 1345 0.9 ± 0.1 9 α-Copaene 1376 1374 1.1 ± 0.1 10 β-Caryophyllene 1415 1417 10.8 ± 0.3 11 Aromadendrene 1440 1439 1.3 ± 0.1 12 α-Humulene 1450 1452 0.3 ± 0.2 13 Alloaromadendrene 1460 1458 0.5 ± 0.2 14 α-Amorphene 1485 1483 1.1 ± 0.1 15 Germacrene D 1486 1484 20.2 ± 0.2 16 β-Selinene 1490 1489 0.3 ± 0.2 17 α-Muurolene 1505 1500 0.5 ± 0.2 18 β-Bisabolene 1502 1505 0.8 ± 0.1 19 (E,E)-α-Farenesene 1505 1505 2.6 ± 0.2 20 -Cadinene 1510 1513 0.5 ± 0.1 21 δ-Cadinene 1520 1522 6.4 ± 0.1 22 Elemol 1550 1548 0.7 ± 0.1 23 Germacrene B 1560 1559 2.4 ± 0.3 24 (E)-Nerolidol 1560 1561 1.2 ± 0.1

Nat. Volatiles & Essent. Oils, 2020; 7(4): 8-13 Salleh et al. DOI: 10.37929/nveo.770303

25 Spathulenol 1575 1577 2.8 ± 0.2 26 Caryophyllene oxide 1580 1582 2.9 ± 0.2 27 Globulol 1590 1590 3.0 ± 0.2 28 Guaiol 1600 1600 2.9 ± 0.2 29 t-Muurolol 1645 1644 1.3 ± 0.1 30 α-Cadinol 1650 1652 2.7 ± 0.2 Monoterpene hydrocarbons 28.0 ± 0.1 Sesquiterpene hydrocarbons 50.2 ± 0.2 Oxygenated sesquiterpenes 17.4 ± 0.2 Total (%) 95.53 ± 0.2 aLinear retention index, experimentally determined using homologous series of C6-C30 alkanes; bLinear retention index taken from Adams (2007) or NIST 08 (2008) and literature; cRelative percentage values are means of three determinations ± SD

The chemical components identified from the essential oil were thirty, forming 95.5% of the total oil composition. The essential oil consisted mainly of sesquiterpenes hydrocarbon (50.2%), monoterpenes hydrocarbon (28.0%), and oxygenated sesquiterpenes (17.4%). The most abundant components of the essential oil were α-pinene (25.4%), germacrene D (20.2%), β-caryophyllene (10.8%), and δ-cadinene (6.4%). The other minor components detected in the essential oil in more than 2% were globulol (3.0%), caryophyllene oxide (2.9%), guaiol (2.9%), spathulenol (2.8%), α-cadinol (2.7%). (E,E)-α-farnesene (2.6%), and germacrene B (2.4%). In comparison to the previous study (Hisham et al., 2012), analyses of the leaf oil of Cyathocalyx zeylanicus has successfully identified thirty-two compounds, comprising 98.0% of total oil. The essential oil was reported to show high amounts of β-caryophyllene (21.6%), α-pinene (20.4%) and (E)-β- ocimene (11.8%). Chemical differences in the essential oil composition of plant species concerning their geographical origins and harvesting season have been reported showing that the chemical and biological diversity of aromatic and medicinal plants depend on factors such as cultivation area, climatic conditions, vegetation phase, and genetic modifications. In fact, these factors influence the plant’s biosynthetic pathways and consequently, the relative proportion of the main characteristic components (Salleh et al., 2016b). Acetylcholinesterase inhibitory activity (I%) was tested against acetylcholinesterase (AChE) enzyme. It was compared with that of galantamine, as a standard drug against Alzheimer’s disease. The essential oil indicated good AChE (I%: 75.5%) inhibitory activity at 1,000 mg/mL concentration, compared to galantamine which gave 85.6% inhibition. In previous reports, AChE inhibition can be explained by the high content of α- pinene and β-pinene have anticholinesterase activity (Picollo et al., 2008). This study shows that α-pinene as the major component in this oil hence may contribute to the AChE inhibition. α-Pinene is a bicyclic monoterpene widely found in nature, acting as an insect-repellent agent in plant defence (Huang et al., 2013). A variety of interesting pharmacological properties have been attributed to α- pinene, including anti-inflammatory, bronchodilator, hypoglycemic, sedative, antioxidant, and broad- spectrum antibiotic activities (Mercier et al., 2009; Violante et al., 2012; Da Silva et al., 2012). Previous studies have reported the presence of α-pinene in the essential oils obtained from Annonaceae family, such as Polyalthia korintii (leaf oil: 43.2%) (Sherin et al., 2018), Guatteria costaricensis (leaf oil: 36.3%) (Palazzo et al., 2009), and Xylopia langsdorffiana (fruit oil: 34.6%) (Moura et al., 2016). Meanwhile, germacrene D was also present as the predominant component in the oil. This component is one of the most common plant volatiles considered to be a biogenetic precursor of many sesquiterpenes such as cadinane, muurolane, and

Nat. Volatiles & Essent. Oils, 2020; 7(4): 8-13 Salleh et al. DOI: 10.37929/nveo.770303

amorphane derivatives (Fujita, 1990). This metabolite is involved in plant-insect interaction acting as a pheromone on receptor neurons (Stranden et al., 2002). Germacrene D was also shown as an important deterrent and insecticidal agent against different parasites such as mosquitos, aphids, and ticks (Bruce et al., 2005). Previously, germacrene D was found abundant in Cardiopetalum calophyllum (flower oil: 37.03%) (Xavier et al., 2016), Uvaria rufa (stem oil: 38.4%) (Thang et al., 2014) and Fissistigma pallens (leaf oil: 30.2%) (Hoferl et al., 2013), another member of the Annonaceae family.

ACKNOWLEDGMENT

The author would like to thank Department of Chemistry, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris (UPSI) for research facilities. REFERENCES

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RESEARCH ARTICLE Bioactivity evaluation of the native Amazonian species of Ecuador: Piper lineatum Ruiz & Pav. essential oil Eduardo Valarezo*, Gabriela Merino, Claudia Cruz-Erazo and Luis Cartuche

Departamento de Química y Ciencias Exactas, Universidad Técnica Particular de Loja, Loja 110150, ECUADOR

*Corresponding author. Email: [email protected] Submitted: 18.08.2020; Accepted: 07.012.2020

Abstract In the present research, the essential oil from Piper lineatum Ruiz & Pav. was analysed by GC/MS and GC/FID, respectively. A total of thirty-seven chemical compounds were identified, which represented 98.9% of the essential oil composition. The main compounds were apiole (21.5%), safrole (19.2%), and myristicin (13.8%), respectively. The in vitro antimicrobial activity and antifungal activity of the oil was assayed against two Gram positive bacteria, five Gram negative bacteria and two fungi. The essential oil from P. lineatum showed an inhibitory activity against Gram-positive bacterium Klebsiella pneumoniae (ATCC 9997), and against dermatophytic fungus Trichophyton rubrum (ATCC 28188) with a MIC of 500 μg/mL in both cases. The antioxidant activity of essential oil was explored using DPPH and ABTS radical scavenging method, by means of both assays the essential oil showed a weak antioxidant activity. To the best of our knowledge, this is the first report on the chemical composition and biological activity of essential oil from this species.

Keywords: Essential oil, Piper lineatum, Apiole, Safrole, Antimicrobial activity, Antioxidant activity

Introduction The Piperaceae is a large family of flowering plants widely distributed throughout the tropical and subtropical regions (Yuncker, 1973). The Piperaceae also known as the pepper family belongs to order Piperales. The family comprises about 13 genera (The Plant List, 2013), of which the genera Piper and Peperomia contain more than 98% of the species (about 2618 species), and the other three genera contain less than 2% (about 40 species) (Missouri Botanical Garden, 2017). The genus Piper is the largest in the Piperaceae family and is made up of around 1457 species (Missouri Botanical Garden, 2017), many of which have been used as culinary spices and recognized by their medicinal properties in traditional medicine background. Worldwide Piper species are widely used for their antibacterial, antifungal, anti-inflammatory and disinfectant effects and to prevent pains, stomach-ache, and as remedies against parasites, fever, gastritis, flu, rheumatism, cough, headache, skin, prostate problems, etc. (Salehi et al., 2019). In South America, a large number of species of this genus have been reported with uses in traditional medicine (Salehi et al., 2019). As an example, Piper umbellatum L. is traditionally used as an anti-inflammatory in Brazil and to treat fever in Peru (Duke, Bogenschutz-Godwin, & Ottesen, 2009). Piper marginatum Jacq. leaves and stems are used in Brazil, especially in Paraíba State, against snake bites and as a sedative, while in north-eastern Brazil and in the northern region, especially in the Amazon, P. marginatum is commonly used for the treatment of inflammatory diseases, snake bites, as well as the liver and bile duct diseases (Brú & Guzman, 2016). Durant-Archibold et al. have reported on the folklore uses of P. hispidum in various South American countries, a leaf infusion is drunk for its antihemorrhagic and diuretic effects in Brazil while in Ecuador it is applied to kill head lice and a leaf decoction is used in Colombia to treat malaria (Durant-Archibold, Santana, & Gupta, 2018). Piper genus is a well-known and widely distributed pantropical taxon of aromatic plants, rich in essential oils (EOs), which can be found in many tissues and organs: fruits, seeds, leaves, branches, roots and stems (da Silva et al., 2017; Naim & Mahboob, 2020). Piper essential oils have insecticidal, acaricidal, repellent and antifeedant effects and some are nematicidal and phytotoxic (Jaramillo-Colorado, Pino-Benitez, & González-

Nat. Volatiles & Essent. Oils, 2020; 7(4): 14-25 Valarezo et al. DOI: 10.37929/nveo.782412

Coloma, 2019). Piper species EOs are rich in all classes of volatile chemical compounds, but the composition is highly variable and these differences seem to depend on polymorphism, plant part, geographical differences, environmental conditions and chemotypes (Thin et al., 2018). According to Mgbeahuruike, Yrjönen, Vuorela and Holm (2017) there are more than 270 identified compounds in Piper spp. EOs. Amongst the most important compounds found in the Piper EOs are monoterpene hydrocarbons, oxygenated monoterpenoids, sesquiterpene hydrocarbons and oxygenated sesquiterpenoids, also in these oils you can find the phenylpropanoids such as safrole, dillapiole, myristicin, elemicin, (Z)-asarone, eugenol, apiole, and sarisan (da Silva et al., 2017). Safrole is abundant in nature among diverse plant genera such as Sassafras, Ocotea, Cinnamomum, Myristica, and Piper (Kemprai et al., 2020), in EOs of some species like Piper hispidinervum collected in southern Brazil this compound is even found in percentages of around 80% (Andrés et al., 2017). Safrol is recognized as a weak hepatocarcinogen with demonstrated genotoxicity in rodents led to strict restrictions on its use in food by various regulatory bodies globally. Which has caused that many government authorities have enforced laws to restrict the production and harvesting of safrole‐ bearing plants (Kemprai et al., 2020). However, the essential oils that contain safrole show biological activity, especially repellent activity, for this reason some scientists suggest the use of these oils as a potential natural insecticide, for example for stored-product insect pests (Zapata & Smagghe, 2010). For Ecuador, 450 species of the Piperaceae family belonging to four genera have been registered, of which the most diverse is the genus Peperomia with 180 native and 50 endemic species, followed by the genus Piper with 157 native and 61 endemic species (Jørgesen & León-Yáñez, 1999; León-Yánez et al., 2019). Ecuadorian native communities in the Amazonian region use different species of Piper. For example, the stems of P. augustum and P. conejosense are broken off and used as toothbrushes (Davis & Yost, 1983; Evans Schultes, 1985). Piper ecuadorense, known as matico de monte, is a plant used by many indigenous communities in Loja and Zamora provinces to treat hangover, as a disinfectant and in wound healing (Ramírez, Cartuche, Morocho, Aguilar, & Malagon, 2013). The Leaf and flower of Piper aduncum L. are used as disinfectant, gastritis, influenza, rheumatism and cough. The whole plant of Piper barbatum Kunth, named as Cordoncillo is used to treat headache, stomach pain, dermatitis, disinfectant, the leaf of Piper bogotense C. DC. called Sacha guando is used in hepatic pain, the stem and leaf of Piper crassinervium Kunth (Guabiduca) is used to treat diabetes, gastritis, prostate problems and the leaf of Piper ecuadorense Sodiro (matico del monte) is used as hangover, disinfectant, healing of wounds (Tene et al., 2007). Piper lineatum Ruiz & Pav. is a native aromatic shrub of Ecuador and Peru. In Ecuador is widely distributed between 0-2500 m a.s.l in the Amazonian provinces of Morona Santiago, Napo, Pastaza, Pichincha, Tungurahua and Zamora Chinchipe, and in the Andean province of Pichincha (Tropicos, 2020). Ecuador is considered a biodiversity hotspot with many vegetal species per unit surface area, which locates this country in the sixth position in the world. Piper aromatic species in Ecuador have been used as a condiment or in traditional medicine, for that reason, the aim of this research was to determine the chemical composition and antifungal and antibacterial activity of the essential oil of Piper lineatum, as well as, to value its antioxidant activity. Materials and Methods Materials Dichloromethane, sodium sulfate anhydrous, 2,2-diphenyl-1-picrylhydryl (DPPH•) and 2,2’-azinobis-3- ethylbenzothiazoline-6-sulfonic acid (ABTS) were purchased from Sigma-Aldrich. Alliphatic hydrocarbons standard was purchased from CHEM SERVICE under the name of Diesel Range Organics Mixture #2-GRO/DRO

Nat. Volatiles & Essent. Oils, 2020; 7(4): 14-25 Valarezo et al. DOI: 10.37929/nveo.782412

and with the code M-TPH6X4-1ML. Helium was purchased from INDURA Ecuador. Microbiological media as Sabourad and Mueller Himton Broth were purchased from DIPCO. Dimethylsulfoxide (DMSO) was obtained from Fisher. All chemicals were of analytical grade and used without further purifications. Plant material The collection of the species, authorized by the Ministerio del Ambiente de Ecuador (MAE), with authorization N° 001-IC-FLO-DBAP-VS-DRLZCH-MA, was performed in Zamora canton, province of Zamora Chinchipe, Ecuadorian Amazon, at a latitude of 4°08'25" S and a longitude of 78°55'51" W. The collection was performed at an altitude of 1075 m a.s.l. The storage and transfer of the plant material was carried out in airtight plastic containers until they were hydrodistilled. Temperature collection was at room temperature (26 °C) and the transfer temperature was 24 °C, the pressure was approximately 88 KPa (room pressure). The botanical specimens were identified by Dr. Bolivar Merino, at the herbarium of the “Universidad Nacional de Loja”. A voucher specimen is preserved in the Herbarium of the Universidad Técnica Particular de Loja under code PPN-809.

Moisture of plant material The moisture of plant material was determined using test method AOAC 930.04-1930, Loss on drying (Moisture) in plants. For which, an analytical balance (model Mettler AC 100, ±0.0001) and an oven (Memmert Universal Oven UN260) were used. The loss on drying (LOD) as estimate of moisture content was calculated as follows: % (w/w) LOD = % (w/w) moisture = 100 x (weight loss on drying, g/weight test portion, g). All the experiments were performed in triplicated and the results are expressed as mean values. Essential oil isolation The material was processed fresh, immediately after arriving at the laboratory, between 2 and 4 hours after being collected. The plant material was submitted to hydrodistillation by four hours in a Clevenger-type apparatus. Subsequently, the collected essential oil was subjected to removal of moisture by addition of anhydrous sodium sulphate and finally, it was stored in amber sealed vials at 4 °C to protect it from light until being used in the subsequent analysis (Valarezo, Guamán, Paguay, & Meneses, 2019). Determination of physical properties of essential oil The density and refraction index of essential oils were determined according to the standard AFNOR NF T 75- 111 and AFNOR NF T 75-112, respectively. For density, a pycnometer (1 mL) and an analytical balance (model Mettler AC 100, ±0.0001) were used, whereas for refraction index a refractometer (model ABBE) was used. The procedures were repeated three times and all measurements were performed at 20°C. Gas chromatography/Flame ionization detector (GC/FID) The analyses of the chemical composition of the essential oils were performed on an Agilent gas chromatograph (model 6890N series) equipped with a flame ionization detector (FID). A nonpolar column DB-5ms (5%-phenyl-methylpolyxilosane) 30 m x 0.25 mm, thickness 0.25 µm was used. An automatic injector (series 7683) in split mode was used. The samples, 1 µL of solution (1/100, v/v, essential oil/dichloromethane), were injected with a split ratio of 1:50. Helium was used as a carrier gas at 0.9 mL/min in constant flow mode. The initial oven temperature was held at 50 °C for 3 min and then it was heated to 210 °C with a ramp of 2.5 °C/min, and the temperature was maintained for 3 min until the end. The injector and detector temperatures were 210 °C, and 250 °C; respectively. Retention index (IR) of the compounds was

Nat. Volatiles & Essent. Oils, 2020; 7(4): 14-25 Valarezo et al. DOI: 10.37929/nveo.782412

determined based on the standard of aliphatic hydrocarbons, which were injected after the oils at the same conditions. Gas chromatography/Mass spectrometry (GC/MS) The GC/MS analyses were performed using an Agilent chromatograph coupled to a mass spectrometer (quadrupole) detector (model Agilent series 5973 inert). The spectrometer was operated at 70 eV, electron multiplier 1600 eV, scan rate: 2 scan/s and mass range: 40-350 m/z. It was provided with a computerized system MSD-Chemstation D.01.00 SP1. The same columns described in GC/FID section were used. The ion source temperature was set at 250 °C. The identification of the oil components was based on a comparison of both mass spectrum data and relative retention indices with the published literature (Adams, 2007; NIST 05, 2005; NIST, 2020). The relative amounts of individual components were calculated based on the GC peak area (FID response) without using a correction factor. Biological activity Evaluation of antibacterial activity Antibacterial activity was evaluated against five Gram-negative and two Gram-positive bacteria, as shown in Table 2. The bacterial strains were incubated overnight at 37°C in Mueller-Hinton (MH) broth; subsequently, the cell suspension was adjusted with a sterile physiological solution to obtain an equivalent concentration of 0.5 in the McFarland scale (1.5 x 108 cells/mL). Another dilution was done in MH broth to adjust the concentration of inoculum to 2 x 106 CFU/mL. A volume of 100 µL from the inoculum were transferred into wells containing 100 µL of MH broth with the higher concentration of the essential oil as described below, giving a final concentration of 5 x 105 CFU/mL. Minimum inhibitory concentration (MIC) was determined by two fold serial dilution method using 96-well microtiter plates (Clinical and Laboratory Standards Institute, 2012).Twenty microliters of a solution of each essential oil [20 µL/mL of DMSO (v/v)] was dissolved in 180 µL of MH broth; subsequently, 100 µL of this solution was transferred to a next well containing 100 µL of MH broth. The procedure was done several times to achieve concentrations ranging from 1000 to 8 µg/mL. Gentamicin (1 mg/mL) was used as the positive control for Gram-positive and Gram-negative bacteria. The microplates were sealed and incubated at 37 °C for 24 hr. Evaluation of antifungal activity The antifungal activity of the essential oils against two fungal organisms was determined by the microdilution method (Clinical and Laboratory Standards Institute, 2008; NCCLS, 2002) as described for the antibacterial activity. A reserve solution of spore fungi was diluted with Sabouraud dextrose broth (7 mL) and stored at 4°C until analysis. Minimum inhibitory concentration was determined using a final concentration of 5 x 104 spores/mL in 96- well microtitre plates. Essential oil was dissolved in Sabouraud dextrose broth with fungal inoculum to achieve the required concentrations from 1000 – 0.5 µg/mL. Itraconazole was used as the positive antimycotic control. The microplates were sealed and incubated at 28 °C for 72 hr. In the antibacterial and antifungal activity tests, a negative control with DMSO was also considered (positive growth with 5% DMSO as final concentration). MIC was the lowest concentration of essential oil that prevented visible bacterial or fungal growth, respectively. All the experiments were performed in triplicated and the results are expressed as mean values.

Nat. Volatiles & Essent. Oils, 2020; 7(4): 14-25 Valarezo et al. DOI: 10.37929/nveo.782412

Antioxidant capacity DPPH radical scavenging activity The DPPH free radical scavenging activity of oils was measured based on the scavenging activity of the stabilized 2,2-diphenyl-1-picrylhydryl radical. This assay was performed based on the technique of Brand Williams et al. (Brand-Williams, Cuvelier, & Berset, 1995) with some modifications described by Thaipong et al. (Thaipong, Boonprakob, Crosby, Cisneros-Zevallos, & Hawkins Byrne, 2006). Briefly, to prepare the radical stock solution, 24 mg of DPPH• was dissolved in 100 mL of methanol. The working solution was prepared by diluting 10 mL of the radical stock solution with 45 mL of methanol to obtain a reading of 1.1 ± 0.02 absorbance units at a wavelength of 515 nm in a UV spectrophotometer (Genesys 10S UV-Vis Spectrophotometer, Thermo Scientific). The working solution was prepared fresh daily. Take 150 µL of the sample solution with different concentrations of P. lineatum essential oil (0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5 mg/mL) and add 2850 µL of DPPH• working solution in an amber vial. Vigorously shake for three minutes and allowed to react for 24 hours at room temperature protected from light. The final absorbance was measured at a wavelength of 515 nm. The same amount of methanol was added instead of the sample solution as a blank control, and BHT and Trolox were used as positive controls. The percentage of free radical-scavenging capacity was calculated as follows: R (%) = [(As-Ai)/As] x 100%, where Ai is the absorbance of DPPH• mixed with essential oil and as is the sample blank absorbance of DPPH• in which sample has been replaced with methanol The oil concentration providing 50% inhibition (IC50) was calculated from the graph by plotting inhibition % against oil concentration. All measurements were performed in triplicate and reported as the average value. ABTS radical cation scavenging activity The ABTS assay was performed using the procedure described by Arnao et al. (Arnao, Cano, & Acosta, 2001) with some modifications by Thaipong et al. (Thaipong et al., 2006). Two stock solutions were prepared: 7.4 µM ABTS•+ and 2.6 µM potassium persulfate. ABTS radical cation (ABTS•+) was produced after mix both solutions in equal quantities and allowed to react protected from light during 12 hours to obtain the ABTS standard solution. For the study of essential oil, 1 mL of the ABTS standard solution was diluted with 60 mL of methanol to obtain an absorbance of 1.1 ± 0.02 in a spectrophotometer UV to 734 nm. From each concentration (0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5 mg/mL) as well with each essential oil, 150 µL were added to 2850 µL of the ABTS•+ working solution, then was allowed to react by 2 hours protected from light, after this the absorbance was measured at 734 nm. The same amount of deionized water was added instead of the sample solution as a blank control, BHT and Trolox were used as positive controls. The sample's removal rate of ABTS radicals can be expressed as: SA (%) = (Ao-Ai)/Ao x 100%; Ao: Absorbance of ABTS•+ mixed with the sample; Ai: absorbance after reaction of ABTS•+ with the sample. The oil concentration providing 50% inhibition (IC50) was calculated from the graph by plotting inhibition % against oil concentration. All measurements were performed in triplicate and reported as the average value. Results and Discussion

Through hydrodistillation in a Clevenger-type apparatus, was obtained 5 mL of essential oil from 1500 g of Piper lineatum, what represents a yield of 0.33 ± 0.02% (v/w) or 3.3 mL/kg, referred to fresh plant material (60 ± 1% of moisture), equivalent to 0.83% on moisture free basis, which is considered as a low yield (Molares, González, Ladio, & Agueda Castro, 2009). To our knowledge, this is the first report of the study of essential oil from Piper lineatum. The essential oil (EO) presented as a viscous liquid of subjective color light-yellow. The essential oil relative density was d20 =0.8792 ± 0.0011 and refraction index was n20 =1.6918 ± 0.0017.

Nat. Volatiles & Essent. Oils, 2020; 7(4): 14-25 Valarezo et al. DOI: 10.37929/nveo.782412

Essential oil compounds identification The identification of volatile compounds present in Piper ecuadorense of Ecuador was carried out by means of gas chromatography equipped with a flame ionization detector (GC/FID) and gas chromatography coupled to a mass spectrometer detector (GC/MS) using capillary nonpolar column DB-5Ms. The results obtained are summarized in Table 1. Thirty-seven individual compounds were detected in the EO of P. lineatum, which represent a 98.87% of the total essential oil. Most essential oils are mainly composed of terpenes and sesquiterpenes, however, in this study was determined that the EO of P. lineatum has a higher percentage (40%) of compounds classified as OTC (other compounds). In percentage, the compounds classified as oxygenated monoterpenes (OXM) ranked second and the aliphatic monoterpene hydrocarbons (ALM) the third. The largest number (ten) of compounds belonged to the group at ALM.

Table 1. Chemical composition of essential oil from Piper lineatum.

P. lineatum MM Peak # Compounda,b RI RIref Type CF %c SD (Da)

1 α-Pinene 932 932 1.91 0.15 ALM C10H16 136.13

2 Camphene 947 946 tr - ALM C10H16 136.13

3 β-Pinene 973 974 1.81 0.16 ALM C10H16 136.13

4 Myrcene 986 988 0.36 0.01 ALM C10H16 136.13

5 α-Phellandrene 1005 1002 0.39 0.04 ALM C10H16 136.13

6 α-Terpinene 1013 1014 2.02 0.27 ALM C10H16 136.13

7 ρ-Cymene 1021 1020 0.95 0.11 ARM C10H14 134.10

8 1,8-Cineole 1029 1026 8.91 0.76 OXM C10H18O 154.14

9 (Z)-β-Ocimene 1034 1032 1.17 0.12 ALM C10H16 136.13

10 (E)-β-Ocimene 1045 1044 3.19 0.32 ALM C10H16 136.13

11 γ-Terpinene 1055 1054 4.23 0.34 ALM C10H16 136.13

12 Terpinolene 1082 1086 0.89 0.03 ALM C10H16 136.13

13 Linalool 1098 1095 0.86 0.02 OXM C10H18O 154.14

14 Terpinen-4-ol 1170 1174 0.07 0.00 OXM C10H18O 154.14

15 α-Terpineol 1191 1186 0.58 0.02 OXM C10H18O 154.13

16 Safrole 1291 1285 19.16 1.72 OXM C10H10O2 162.07

17 δ-Elemene 1338 1335 0.19 0.02 ALS C15H24 204.19

18 α-Cubebene 1347 1345 tr - ALS C15H24 204.19

19 α-Copaene 1376 1374 0.38 0.05 ALS C15H24 204.19

20 (E)-Isosafrole 1375 1373 0.05 0.01 OXM C10H10O2 162.07

21 β-Cubebene 1388 1387 0.07 0.01 ALS C15H24 204.19

22 β-Elemene 1390 1389 tr - ALS C15H24 204.19

23 Methyl eugenol 1405 1403 3.09 0.10 OTC C11H14O2 178.10

24 trans-Caryophyllene 1415 1417 1.90 0.21 ALS C15H24 204.19

25 β-Gurjunene 1435 1431 tr - ALS C15H24 204.19

26 α-Humulene 1451 1452 0.69 0.16 ALS C15H24 204.19

27 trans-Cadina-1(6),4-diene 1478 1475 1.55 0.26 ALS C15H24 204.19

28 Germacrene D 1476 1480 1.67 0.27 ALS C15H24 204.19

29 Viridiflorene (=Ledene) 1498 1496 0.11 0.01 ALS C15H24 204.19

30 δ-Amorphene 1515 1511 1.96 0.02 ALS C15H24 204.19

31 Myristicin 1519 1517 13.78 0.49 OTC C11H12O3 192.08

Nat. Volatiles & Essent. Oils, 2020; 7(4): 14-25 Valarezo et al. DOI: 10.37929/nveo.782412

P. lineatum MM Peak # Compounda,b RI RIref Type CF %c SD (Da)

32 δ-Cadinene 1524 1522 1.55 0.03 ALS C15H24 204.19

33 Elemicin 1558 1555 1.25 0.05 OTC C12H16O3 208.11

34 Viridiflorol 1595 1592 0.48 0.01 OXS C15H26O 222.20

35 epi-α-Cadinol 1639 1638 0.17 0.01 OXS C15H26O 222.20

36 α-Eudesmol 1647 1652 1.96 0.02 OXS C15H26O 222.20

37 Apiole 1673 1677 21.48 1.68 OTC C12H14O4 222.09 Aliphatic monoterpene hydrocarbons (ALM) 15.98 Aromatic monoterpene hydrocarbons (ARM) 0.95 Oxygenated monoterpenes (OXM) 29.63 Aliphatic sesquiterpene hydrocarbons (ALS) 10.11 Oxygenated Sesquiterpene (OXS) 2.61 Other compounds (OTC) 39.60 Total identified 98.87 aCompounds ordered according to the elution order in the column DB5-Ms. bAll compounds were identified by MS and RI: MS. by comparison of the mass spectrum with those of the computer mass libraries Wiley 7. Adams 2007 and NIST 05 2005; RI. by comparison of RI with those reported in literature; Adams 2007. NIST 05 2005 and NIST 2020. cPercentage values are means of nine determinations. tr. trace (< 0.05%); -. not detected; RI. retention indices in the a-polar column (DB5-Ms); RIref. references: Adams 2007. NIST 05 2005 and NIST 2020. SD. standard deviation; CF. Chemical Formula; MM. Monoisotopic mass.

The main constituent of the essential oil was apiole (OTC, CAS 523-80-8) with 21.48 ± 1.68%, safrole (OXM, CAS 94-59-7) was the second compound with the highest percentage with the 19.16 ± 1.72%. Other main compounds were myristicin (OTC, 13.78 ± 0.49%) and 1,8-cineole (OXM, 8.91 ± 0.76%). Representative amounts of γ-terpinene 4.23 ± 0.34% and (E)-β-ocimene 3.19 ± 0.32% were also identified. Regarding previous studies about essential oil from species belonging to genus piper, apiol (64.24%) and safrole (64.54%) were the major constituents of the EO from Colombian species P. holtonii and P. auritum respectively (Pineda M., Vizcaíno P., García P., Gil G., & Durango R., 2012), 1,8-cineole (13.0%) and safrole (14.9%) were identified as major compounds in a study carried out on essential oil from leaves of species P. carpunya grown in the Peruvian Amazon (Vargas, Velasco-Negueruela, Pérez-Alonso, Palá-Paúl, & Vallejo, 2004), and apiole (28.62%) was identified as one of the main components in the P. aduncum essential oil (Santana et al., 2015).

With regard to toxicity of the apiol (CF: C12H14O4, MM: 222.09), it has been reported that parsley apiole has acute toxicity to humans, the lowest total dose of parsley apiole causing death is 4.2 g (2.1 g/day for two days) the lowest fatal daily dose is 770 mg, which was taken for 14 days; the lowest single fatal dose is 8 g. At least 19 g has been survived. Parsley apiole is hepatotoxic and nephrotoxic (Tisserand & Young, 2014). In scientific reports, safrole (CF: C10H10O2, MM: 162.07) is considered a human carcinogen based on sufficient evidence of hepatocarcinogenic in rats and mice. The EPA has evaluated the carcinogenicity of safrole and classifies it as possibly carcinogenic to humans (Group B2: the agent (mixture) is possibly carcinogenic to humans). Safrole cannot be used as fragrance ingredient, the International Fragrance Association (IFRA) recommend that the essential oils containing safrole should not be used at levels in which the total concentration exceeds 0.01% in consumer products (Clarke, 2008; Tisserand & Young, 2014). However, P. lineatum essential oil also contains 1,8-cineole (eucalyptol, CF: C10H18O, MM: 154.14) which is used in the treatment of respiratory tract diseases due to its antimicrobial, mucolytic, broncholytic, and antiinflammatory properties (Aprotosoaie, Luca, Trifan, & Miron, 2019), Santos in the 2004 (Santos et al., 2004) stated that 1,8-cineole reduce colon inflammation in rats.

Nat. Volatiles & Essent. Oils, 2020; 7(4): 14-25 Valarezo et al. DOI: 10.37929/nveo.782412

Biological activity Essential oils are complex mixtures of chemical compounds, which may have biological synergism or antagonistic properties. When two or more carcinogenic constituents are present in an essential oil or in a mixture of essential oils, it has been have assumed that such actions are additive. When an essential oil or a mixture of oils contains a minority of carcinogenic, and a majority of anticarcinogenic constituents, it is assumed that the principle of antagonism applies (Tisserand & Young, 2014). To estimate the net activities (including synergism and antagonism) of the compounds present in the essential oil, the biological activity was determined based on their antibacterial, antifungal and antioxidant activities. Essential oils obtained from aerial parts of P. lineatum were assessed by microdilution broth method and the values of minimum inhibitory concentration (MIC, expressed in μg/ml) are shown in Table 2.

Table 2. Antibacterial and antifungal activity of essential oil from Piper lineatum, given as minimal inhibitory concentration (MIC, g/mL).

Piper lineatum Positive controlb Microorganism MIC (g/mL)a Gram-negative bacteria Pseudomonas aeruginosa (ATCC 27853) >1000 0.39 Klebsiella pneumoniae (ATCC 9997) 500 0.39 Proteus vulgaris (ATCC 8427) >1000 0.39 Escherichia coli (ATCC 25922) >1000 0.39 Salmonella typhimurium (LT2) >1000 1.95 Gram-positive bacteria Enterococcus faecalis (ATCC 29212) >1000 1.95 Staphylococcus aureus (ATCC 25923) >1000 0.39 Dermatophytes Fungi Trichophyton rubrum (ATCC 28188) 500 0.39 Trichophyton mentagrophytes (ATCC 28185) >1000 0.39 a Mean of nine determinations. b Gentamicine for Gram-negative and Gram-positive bacteria and Itraconazole for fungi.

Currently, there are no accepted standard criteria for defining the in vitro antimicrobial activity of natural products. However, from an extensive review about this issue, Van Vuuren and Holl (2017) suggested a more detailed classification scale for extracts and essential oils that resulted more relevant to characterize the activity of our samples. For an essential oil, a moderate activity is reported when a MIC value lies between 500 to 1000 µg/mL, from 101 to 500 µg/mL the activity is strong, meanwhile, an activity ≤ 100 µg/mL is considered to be very strong. On basis, the essential oil from P. lineatum presented a strong activity against Klebsiella pneumoniae (ATCC 9997) and Trichophyton rubrum (ATCC 28188) with a MIC of 500 μg/mL in both cases. The antibacterial capacity of the EO of P. carpunya may be due to its high concentration in 1,8-cineole, which has been reported as a compound with antibacterial capacity (Hendry, Worthington, Conway, & Lambert, 2009). Further, 1,8-cineole showed significant levels of synergistic interaction combined with antimicrobial compounds like chlorhexidine gluconate (CHG) against Staphylococcus aureus, S. aureus, Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis and Candida albicans (Şimşek & Duman, 2017). According to Stefano et al. apiol and myristicin resulted inactive against bacterial strains Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa, and antifungal strains Candida albicans and Candida tropicalis, at the maximum tested concentration of 200 μg/mL (Stefano, Pitonzo, & Schillaci, 2011). However, the essential oil extracted from the seeds of dill (Anethum graveolens L.), which contains 16.8% of apiole, showed antifungal capacity against Aspergillus flavus, the antifungal

Nat. Volatiles & Essent. Oils, 2020; 7(4): 14-25 Valarezo et al. DOI: 10.37929/nveo.782412

activity of dill oil results from its ability to disrupt the permeability barrier of the plasma membrane and from the mitochondrial dysfunction-induced reactive oxygen species accumulation (Tian et al., 2012). Additionally, the essential oil of P. lineatum contains safrole which is considered a hepatotoxin. Using Escherichia coli- expressed human P450, recombinant cytochrome P450 (CYP, P450) and human liver microsomes, was demonstrated that safrole is a potent inhibitor of human CYP1A2, CYP2A6, and CYP2E1, also inhibits with relatively less potency CYP2D6 and CYP3A4 (Ueng, Hsieh, & Don, 2005). Antioxidant capacity The essential oil of P. lineatum was explored for antioxidant activity using DPPH radical and ABTS radical cation scavenging activity, IC50 was used as a measure value of inhibition concentration of 50% of the activity, and BTH and Trolox as positive control. Through DPPH• method, the essential oils studied showed little antioxidant activity (25% of inhibition to 2500 ppm), but did not provide IC50 values (>2500 g/mL) in the concentration ranges tested. Among standards tested Trolox (IC50 = 580 ± 40 g/mL) was the most efficient.

Employing the ABTS technique, with 2500 ppm an inhibition of 21% was obtained, but did not provide IC50 values in the concentration ranges tested (Table 3).

Table 3. Antioxidant activity of essential oils of Piper lineatum DPPH• ABTS•+ Sample IC50* (µg/mL) Piper lineatum EO >2500 >2500 BHT 980 ± 50 290 ± 20 Trolox 580 ± 40 380 ± 30 * IC50 = Inhibition Concentration of 50%

The absence of antioxidant activity, for the essential oil of this study, in the DPPH assay can be explained by the fact that terpene compounds are not capable of donating a hydrogen atom and the low solubility provided by them in the reaction medium of the assay, because of this test utilizes methanol as solvent (Mata et al., 2007). However, the ABTS method is especially useful for investigations of lipophilic antioxidants and is appropriate for the investigation of antioxidant activity of essential oils (Andrade et al., 2013), the absence of antioxidant activity in essential oils of P. lineatum mainly consisting of monoterpene and sesquiterpene with antioxidant activity may be due to antagonism with carcinogenic compounds like safrole. In this regard, it should be considered that the apiole, the main compound in the EO of P. lineatum, has been shown to inhibit human colon cancer cell (COLO 205 cells) growth through induction of G0/G1 cell cycle arrest and apoptotic cell death (Wu et al., 2019). Therefore, this compound could be isolated from the essential oil and used as an active principle or as a raw material to obtain derivatives, since apiol derivatives have also shown induced anti-proliferative effects on colon cancer (Wu et al., 2019). Conclusion In Ecuador, many species belonging to the genus piper are used as culinary spices or in traditional medicine especially in the Amazon, without making an exact distinction by species, which causes that medicinal properties are attributed to most species of the genus Piper, however many of these species possess compounds that can be harmful to human health. In this research a complete characterization of the chemical composition, physical properties, and antimicrobial and antioxidant activity was conducted over the essential oil of Piper lineatum. A total thirty-seven individual compounds were detected in the essential oil, being the apiole and safrole the main compounds. The essential oil showed to be active against Klebsiella pneumoniae and Trichophyton rubrum.

Nat. Volatiles & Essent. Oils, 2020; 7(4): 14-25 Valarezo et al. DOI: 10.37929/nveo.782412

ACKNOWLEDGMENT

Financial support for this study was granted by “Universidad Técnica Particular de Loja” through PROY_QUI_0010. The authors thank to Ministerio del Ambiente- Ecuador, for providing the plant collection and research permissions through “Autorización de Investigación Científica” N° 001-IC-FLO-DBAP-VS-DRLZCH-MA”. REFERENCES

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Nat. Volatiles & Essent. Oils, 2020; 7(4): 26-33 Phat et al. DOI: 10.37929/nveo.780505

RESEARCH ARTICLE Extraction process optimization and characterization of the Pomelo (Citrus grandis L.) peel essential oils grown in Tien Giang Province, Vietnam Dao Tan Phat1,2,3, Kha Chan Tuyen4, Xuan Phong Huynh5 and Tran Thanh Truc6,*

1Graduate University of Science and Technology, Vietnam Academy of Science and Technology, Ha Noi, VIETNAM 2Center of Excellence for Biochemistry and Natural Products, Nguyen Tat Thanh University, Ho Chi Minh City, VIETNAM 3NTT Hi-Tech Institute, Nguyen Tat Thanh University, Ho Chi Minh City, VIETNAM 4Faculty of Food Science and Technology, Nong Lam University, Ho Chi Minh City, VIETNAM 5Biotechnology Research and Development Institute, Can Tho City, VIETNAM 6College of Agriculture, Can Tho University, Can Tho City, VIETNAM

*Corresponding author. Email: [email protected] Submitted: 14.08.2020; Accepted: 19.12.2020

Abstract Hydrodistillation was used to obtain the essential oil from pomelo (Citrus grandis L.) peels collected in Vietnam. The effects of extraction variables including size of material, temperature, extraction time, and water-to-material ratio on the yield of essential oil were investigated. In addition, to assess the quality of pomelo essential oil, the chemical composition was compared. The optimized conditions were as follows: material size of grind, temperature 120 °C, water to material ratio 5 mL/g, and extraction time 105 min., respectively. The chemical composition of the pomelo oil was then determined by GC-MS, where 5 components were identified, of which, limonene was the highest (97.1%). This method can be considered as a green method of extraction method as it is less energy intensive process and offers high performance. The results are expected to aid in justification of hydrodistillation in industrial applications and in refining of extraction parameters.

Keywords: Pomelo peels, Citrus grandis L., hydro-distillation, GC-MS analysis

Introduction Essential oils are widely applied in various industries such as cosmetics, food and pharmaceuticals and carry wide clinical implications (Dao et al., 2019a; Thuong Nhan et al., 2020; Thuy et al., 2019). To obtain essential oils from raw materials, a wide range of methods could be adopted. While old, traditional methods, such as hydrodistillation and expression, only require simple instruments to produce essential oils with adequate quality, newly developed extraction techniques (instant controlled pressure drop technique, supercritical fluids, liquefied gas technology and ultrasound extraction) allow for better solvent and energy saving and capability to afford products with better quality and sustainability (Chemat et al., 2020). However, newer technologies are often more complex and require costly instruments such as microwave irradiators, steam generators and vacuum pumps. Therefore, intensification of established processes plays a key role in improving essential oil-derived products and have been attracting scientific attention recently. Essential oils isolated from pomelo (Citrus grandis L.) fruits are a widely adopted agent with a wide range of applications and have been known as alternative for antimicrobial and antioxidant agents (Mokbel & Suganuma, 2006). It has been shown that bioactivities of the essential oils are dependent on their chemical compositions (Uysal et al., 2011). Therefore, chemical composition of pomelo essential oil from different countries is also a subject of extensive research. One important component of pomelo essential oil is limonene, accounting for around 85 to 96% of total content while myrcene, sabinene, α-pinene and α- terpinene have been reported at less than 2.5% (Tsai et al., 2017; Shaw, 1979). Other minor components

Nat. Volatiles & Essent. Oils, 2020; 7(4): 26-33 Phat et al. DOI: 10.37929/nveo.780505

including nootkatone, octanal, octyl acetate, citronellyl acetate, citral and carvone have been found at trace levels in the essential oil (<1.0%) and have been designated as the main agents of scents (Ortuno et al., 1995; Sawamura & Kuriyama, 1988). Such variance in pomelo essential oil composition could be attributable to the variety of the pomelo plant, the plant part, used extraction technique and process parameters. To date, numerous extraction methods including microwave-assisted hydrodistillation and supercritical CO2 extraction have been attempted to isolate essential oil bearing extracts from pomelo materials (Hien et al., 2018; Gyawali et al., 2012). However, such processes lack applicability in producing essential oil at commercial scale due to their high operational costs, complexity and limited scalability. In this study, pomelo essential oils were extracted from peels of pomelo collected from Tien Giang province, Vietnam. Although this region is known as a major producer of pomelo, essential oil of the fruit originating from this region was not extensively studied. Hydrodistillation was selected as the extraction method in this study due to its ease of operation, low cost and the ability to perform exhaustive extraction from raw materials. The extraction parameters were also optimized with respect to maximal essential oil yield and the obtained product was characterized by gas chromatography-mass spectrometry (GC-MS) to determine the volatile composition. The results are expected to contribute to enhanced valorization of pomelo fruits and justify the use of hydrodistillation in larger scale manufacturing processes. Materials and Methods Materials and chemical Fresh pomelo (Citrus grandis L.) fruits were selected from Tien Giang province (latitudes 10°25’13’’N and longitudes 106o17’49’’E) of Vietnam in March 2020, then washed and peeled manually. The peels were cut into pieces of approximately 2x2 cm2 and kept in a plastic bag at low temperatures (4°C). Prior to distillation, the sample was exactly weighted, treated and used immediately to avoid the loss of essential oil. The water content of fresh pomelo peels was 73.76±0.19, which was determined following the Association of Official Analytical Chemists (AOAC) method 925.10. Hydro-distillation process Hydro-distillation technique was used to extract pomelo peel essential oil. The procedure is as follows. First, 100g of peels were placed in a 1L round-bottom flask with 500 mL of distilled, deionized water and connected to a Clevenger’s distillation unit. The essential oil was extracted by hydro-distillation, which was carried out for 120 min. The extraction duration that allows exhaustive oil extraction from peel tissues was justified by previous studies (Dao et al., 2019; Thuy et al., 2019). The obtained essential oil, which was collected in Clevenger’s receiver, was separated and anhydrous with Na2SO4. The extracted essential oil was stored in a glass amber bottle and refrigerated at 4 °C until further analysis. Each extraction was performed three times under same conditions. Yield of pomelo peels oil (Y) obtained for every run that was calculated as follow (1):

푉표푙푢푚푒 표푓 푒푠푠푒푛푡푖푎푙 표푖푙(푚푙) Y (%) = 100% (1) 퐴푚표푢푛푡 표푓 푟푎푤 푚푎푡푒푟푖푎푙푠 (푔) Single factor investigation was used to survey the optimum extraction conditions of the hydrodistillation process. Four factors including material size, water-material ratio, temperature and extraction time were consequently allowed to vary. The yield of essential oil was selected as the outcome of the optimization. The survey is performed to estimate the best conditions for the extraction process, and it was repeated to avoid errors.

Nat. Volatiles & Essent. Oils, 2020; 7(4): 26-33 Phat et al. DOI: 10.37929/nveo.780505

Gas chromatography–mass spectrometry analysis A Gas Chromatography-Mass Spectrometry (GC-MS) is used to analyze the composition of the essential oils of all extraction methods. 25 µL sample of essential oil in 1.0 mL n-hexane. Name of the equipment: GC Agilent 6890N, MS 5973 inert with HP5-MS column, head column pressure 9.3 psi. GC-MS system was performed hold under the following conditions: carrier gas He; flow rate 1.0 mL/min; split 1:100; injection volume 1.0 µL; injection temperature 250 °C; oven temperature progress included an initial hold at 50 °C for 2 min, and a rise to 80 °C at 2 °C /min, and them to 150 °C at 5 °C /min, continue rising to 200 °C at 10 °C /min and rise to 300 °C at 20 °C /min for 5 min. Results and Discussion Single factor investigation Effects of four experimental parameters on the yield of pomelo essential oil are shown in Figure 1.A. refers to the change in obtained essential oil yield when changing the size of materials. Generally, the performance of pomelo essential oil extraction improved as the size of the material decreased. Specifically, the efficiency increased from 1% to 1.8% when changing the size from the original size to puree. This can be explained as follows. The smaller the material size, the faster the water diffuses into the essential oil sacs, promoting the release of essential oils under thermal exposure and in turn causing better extraction yield. On the contrary, the large peel sizes will yield lesser oil due to small surface area in contact with the solvent. Therefore, grind peels have been chosen in subsequent investigations (Dao et al., 2019b; Tran et al., 2019). One of the main factors influencing the extraction process is the extraction temperature. It is observed from Figure 1b that when the temperature increases from 100 to 120 °C, the yield of the extracted essential oil gradually increases from 1.5 to 1.8%. However, the yield of essential oil ceased to increase at 1.8% and tended to decrease when rising temperature to 130 °C and to 140 °C. This observation can be explained from the fact that the heat and the steam generated at 120 °C exerts stronger rupture on the plant cells, therefore accentuating the pushing of essential oil to the surrounding media. This is consistent with result of Tran et al. [2018] where the efficiency of the process of extracting plants was found to be greatly affected by temperature. Therefore, for pomelo peel material, the temperature of 120°C is the optimal for hydrodistillation and beyond this temperature, the essential oil might degrade thus impairing extraction yield. Similarly, the effect of extraction time on yield of pomelo essential oil can be seen in Figure 1c. At the first stage, extraction time and process yield were directly proportional to each other. At the 105-minute mark, yield reached its highest value at 1.85% and then decreased to 1.75% and to 1.6 % at 120 min and at 135 min, respectively. It is regarded that the longer the extraction time, the more oil is obtained. As distillation time extends to a certain limit, the amount of essential oil obtained reaches a state of saturation and is likely to be denatured due to prolonged heat exposure (Boukhatem et al., 2014). The determination of extraction time is related to energy consumption and production cost of the process. Therefore, 105 min is selected as appropriate duration to conduct the next experiment. During distillation, at the boiling point, the essential oils in the plant cells diffuse out the surface of the material and are swept away by the steam. Simultaneously, water penetrates the material in the opposite direction and the oil continues to be swept into the water. The process occurs until the essential oil in the tissues completely escapes. Therefore, determining the ratio of water to raw materials is necessary for the

Nat. Volatiles & Essent. Oils, 2020; 7(4): 26-33 Phat et al. DOI: 10.37929/nveo.780505

extraction process to attain maximum yield. The effect of ratio water and material can be seen in Figure 1d. As the ratio changed, the process efficiency also changed, and the highest yield was achieved at 5: 1 mL/g. At a low water ratio of 2-3 mL/g, the low amount of collected essential oil is due to the insufficient amount of water required dissolve the colloidal wrappers surrounding the essential oil bag. This leaves a large quantity of residual essential oil in the material, impairing the efficiency of the process. On the other hand, at higher ratios such as 6-7:1 mL/g, the amount of essential oil that is obtained is also not high, possibly due to slow water evaporation rate and oil losses in the collection tube, effectively reducing the efficiency of the distillation process. In practical terms, large amounts of water also lead to prolonged distillation times and higher operation costs. The highest amount of essential oil obtained (1.9%) at the water-material ratio of 5:1 mL/g.

Figure 1. Effects of four experimental parameters on the yield of pomelo essential oil

(C ) (D)

(A) Size of the materials, (B) Temperature, (C) Extraction time, (D) Water-to-material ratio to the yield of the essential oil

The results of GC-MS The compositions of essential oil from pomelo peels was determined by comparing the retention time and mass spectra of the compounds in the essential oil with the spectral data library. Citrus essential oils are usually characterized by the abundance of one or two main compounds. Essential oils from grapefruit which was reported to be strong antibacterial activity against the bacterial strains (Gram-negative and Gram-positive bacteria), yeast and mold tested by Chanthaphon et al. (2008). The mechanism of this

Nat. Volatiles & Essent. Oils, 2020; 7(4): 26-33 Phat et al. DOI: 10.37929/nveo.780505

bioactivity was elaborated by Lis-Balchin (1996) maintaining that limonene, α-pinene, and sabinene might disrupt membrane of microorganisms and inhibit ion transport processes and respiration. In this study, around 100g of pomelo fruit peel yielded around 1.9 mL of essential oil using Clevenger’s apparatus at optimal conditions. The composition of the volatile oil was analyzed by GC-MS (Table 1 and Figure 2). Five constituents were detected from peels, accounting for almost 100% essential oil content. limonene, α-pinene and β-myrcene were the main components in the essential oil. The most abundant component was limonene, a monoterpene hydrocarbon, at 97.1%. Other important constituents were β- myrcene (1.3%) and α-Pinene (0.7%). These results are similar with the other literature data for Citrus genus (Uysal et al., 2011; Chen & Viljoen, 2010). Limonene is a well-studied compound with diverse bioactivities and finds numerous uses in cosmetic, food and beverage industries. In agri-food applications, limonene is a common ingredient in pesticide, herbicide and anti-spoilage products. In medicinal field, limonene also finds clinical relevance due to its antioxidant activity. However, limonene instability and susceptibility against external conditions have called for development of preservation processes such as microencapsulation, coacervation and nano-emulsification and urged for further innovations in enhancing biocompatibility of resulting products (Ibanez et al., 2020; Mahato et al., 2019). Citrus species have been well known as an abundance source for limonene, whose content has been reported to range from 30.1 to 75.5% (Patil et al., 2009; Kamal et al., 2011). The content of limonene in pomelo peel oil was also greatly varied depending on the origin of the material and the extraction technique. Our result indicates that hydrodistillation might be justifiable when it comes to extraction performance with regard to limonene content in the resulting pomelo peel oil. Khan et al. (2007) indicated that limonene in the essential oil hydrodistilled from Pakistani pomelo peels contributed to around 72.3% of total content. Meanwhile, essential oils obtained by pressing from Japanese and Kenyan peels showed limonene content of 87.1% and 94.8%, respectively (Sawamura et al., 2001; Njoroge et al., 2005). On the contrary, very low limonene content (3.7%) was determined in Korean pomelo volatiles extracted by supercritical fluid, which could be explained by more complex composition of supercritical extracts (Gyawali et al., 2012). These discrepancies suggest that both material origin and extraction technique could largely determine the composition of the essential oil products and that rationalization for extraction technique should be individually made for each purpose of use.

Table1. Chemical constituents of pomelo oil

Peak R.T min Compounds % 1 7.261 α-Pinene 0.7 2 8.997 Sabinene 0.2 3 9.938 β-Myrcene 1.3 4 10.482 α-Phellandrene 0.7 5 11.936 Limonene 97.1

Nat. Volatiles & Essent. Oils, 2020; 7(4): 26-33 Phat et al. DOI: 10.37929/nveo.780505

Figure 2. The GC-MS chromatogram of essential oil from pomelo peels

Conclusion The extraction of essential oil from pomelo peels was attempted using hydro-distillation method. The process was optimized by varying several parameters including water-to-materials ratio, temperature, and extraction time. The optimum conditions for extraction process are as follows: temperature of 120°C, ratio of 5:1 mL/g, and extraction time of 105 minutes. Pomelo essential oil yield was 1.9% under these conditions. Besides, GC-MS analysis indicates that the composition of pomelo oil in Vietnam obtained using hydro-distillation method contains approximately 97% limonene. Therefore, hydro-distillation can be considered a green extraction method because it is a low energy process and does not require a subsequent solvent separation stage. Further studies should contemplate assessment of bioactivities of pomelo essential oil with respect to varying volatile compositions.

ACKNOWLEDGMENT

This research was funded by the Science and Technology Program of Ministry of Education and Training (Vietnam): "Application and Development of Advanced Technologies in Preservation and Processing of Aquatics and Agricultural Products in the Mekong Delta" (Code: CT2020.01.TCT-07). REFERENCES

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RESEARCH ARTICLE Characterization of Dysphania ambrosioides (L.) Mosyakin & Clemants essential oil from Vietnam

Tran Thi Kim Ngan1,2, Pham Minh Quan3,4 and Tran Quoc Toan3,4

1Center of Excellence for Biochemistry and Natural Products, Nguyen Tat Thanh University, Ho Chi Minh City 700000, VIETNAM 2NTT Hi-Tech Institute, Nguyen Tat Thanh University, Ho Chi Minh City 700000, VIETNAM 3Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, Hanoi 100000, VIETNAM 4Graduate University of Science and Technology, Vietnam Academy of Science and Technology, Hanoi 100000, VIETNAM

*Corresponding author. Email: [email protected] Submitted: 14.08.2020; Accepted: 27.12.2020

Abstract Dysphania ambrosioides (L.) Mosyakin & Clemants is a fragrant herb widely distributed from warm temperate regions to subtropical and tropical regions of the Northern Hemisphere. The essential oil from D. ambrosioides is the main ingredient used in traditional medicine due to its anti-malarial, anti-cancer, and anti-helminthic properties, among others. The chemical composition of D. ambrosioides essential oil from the aerial part was extracted by hydrodistillation and analysis by gas chromatography-mass spectrometry, resulting the yield of 0.8%. The main components were characterized as 2,3-dehydro-1,4-cineole (55%), α-terpinene (15.2%), iso-ascaridole (15.3%), and p-cymene (9.8%), respectively.

Keywords: D.ambrosioides, Essential oil, GC-MS, Hydrodistillation

Introduction Dysphania ambrosioides (synonym Chenopodium ambrosioides L.), known as wormseed, is a herbaceous shrub belonging to the family Amaranthaceae. It is native to Central and South America and distributed mainly from warm temperate to subtropical and tropical climates in the Northern Hemisphere. It finds useful culinary and agricultural uses. In folk medicine, D. ambrosioides and its essential oils were used to treat gut worm, limit the growth of some fungi, treat parasite infections in non-ruminant livestock, cats and dogs, and humans for many years (Soares et al., 2017). This herb is widely known in popular medicine as anthelmintic, vermifuge, and emmenagogue and used in the treatment of digestive, respiratory, urogenital, vascular, and nervous disorders (Ávila-Blanco et al., 2014). From 1916 to 1921, in Brazil, more than one million people received the essential oil of D. ambrosioides for the control of intestinal worms (Barros et al., 2019). Many studies have reported effects of D. ambrosioides essential oil in activities against intestinal worms, leishmaniasis, schistosomiasis and pest insects, which may be attributable to ascaridol (Monzote et al., 2014; Lohani et al., 2012; Kau, 2000; Harraz et al., 2015). In addition, it was shown that D. ambrosioides essential oil contained carvacrol, α-pinene, p-cymene, α-terpinene, α-terpinyl acetate, and limonene (Harraz et al., 2015; Zhu et al., 2012; Jardim et al., 2008; Sá et al., 2016; Owolabi et al., 2009; Onocha et al., 1999). Ávila-Blanco et al. (2014) evaluated the in vitro and in vivo antiamoebic activity of the essential oil of D. ambrosioides in an amoebic liver abscess hamster model. The result showed that the oral administration of essential oil (8 mg/kg and 80 mg/kg) to hamster infected with Entamoeba histolytica reverted the infection. In the study of Soares et al. (2017), authors assessed the in vitro schistosomicidal effects of D. ambrosioides essential oil on Schistosoma mansoni and results demonstrated the promising schistosomicidal potential of this essential oil, which had the monoterpenes cis-piperitone oxide (35.2%), p- cymene (14.5%) isoascaridole (14.1%), and α-terpinene (11.6%) as the major constituents. Result in

Nat. Volatiles & Essent. Oils, 2020; 7(4): 34-40 Ngan et al. DOI: 10.37929/nveo.780483

research of Ketzis et al. (2002) showed that the essential oil did reduce viability of eggs of Haemonchus contortus in vitro, although, short-term treatment of individual goats with the oil were not effective in reducing the number of nematode adults or eggs. Monzote et al. (2014) examined antileishmanial activity of essential oil and its main components against experimental cutaneous leishmaniasis in BALB/c mice. Results demonstrated that the D. ambrosioides essential oil showed a better efficacy against experimental cutaneous leishmaniasis caused by L. amazonensis in comparison with its pure major compounds. Futhermore, essential oil of D. ambrosioides has been studied as a nature insecticide. Pavela et al. (2018) investigated effect of D. ambrosioides essential oil on the housefly, Musca domestica. And the results showed that this essential was more toxic to adults of M. domestica showing a LD50 of 51.7 μg/ adult. Also, the LC50 values of the essential oil from Chinese D. ambrosioides and its active compound (Z) ascaridole against maize weevil adults, Sitophilus zeamais, were 3.08 and 0.84 mg/L air respectively (Chu et al., 2011). And they possessed nematicidal activity against the root-knot nematodes, Meloidogyne incognita (Bai et al., 2011). In this study, we aimed to compare the chemical compositions of essential oil of D. ambrosioides obtained from the mountainous area of northern Vietnam, where the altitude is from 1200-1500 m above sea level with special weather, with some other studies in the world. According to our knowledge, this is the first study in Vietnam with D. ambrosioides essential oil in this region. Materials and Methods Plant materials D. ambrosioides was collected in Lung Phin commune, Bac Ha district, Lao Cai Province, Vietnam (22° 36′ 15″ N, 104° 20′ 55″E). The plant was collected during flowering season in May-June 2019 and was identified by Assoc. Prof. Tran Huy Thai, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology. A voucher specimen was deposited under the code DG-01 at Organic Biochemistry Laboratory, Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology. After harvesting, the materials were cleaned and had damaged or waterlogged parts removed. Then aerial parts of the plant were dried and stored for extraction. Extraction of the volatile essential oil 200 g of dried D. ambrosioides aerial parts were coarsely ground. Afterwards, the powder was placed in the flask of the Clevenger type hydrodistillation system. The material to water ratio was 1:3 (w/w) and the temperature of the heating mantle was set to 110 °C. The hydrodistillation process took place for 3 hours to afford the mixture consisting of volatiles and water. To separate water from essential oils, anhydrous

Na2SO4 was used. The essential oil was stored in dark glass bottles for compositional analysis. Gas chromatography–mass spectrometry analysis An Agilent Technologies HP7890A GC, coupled with a mass spectrum detector (MSD) Agilent Technologies HP5975C and a DB-XLB column (60 m x 0.25 mm, film thickness 0.25 µm, Agilent Technologies), was utilized to perform GC-MS analysis of the essential oils. The temperature of the injector and detector was initiated at 250 oC and 280 oC respectively. The temperature progress of the column began at 40 °C, increased to 140 °C at 20 °C/min, and then to 270 °C at 4 °C/min. Helium was used as carrier gas and the flow rate was set at 1 mL/min. Injection in the distillation apparatus was performed by splitting with the ratio was 100:1. The volume injected was 1 µL of essential oils. GC-MS was performed under following MSD conditions:

Nat. Volatiles & Essent. Oils, 2020; 7(4): 34-40 Ngan et al. DOI: 10.37929/nveo.780483

ionization voltage of 70 eV, the emission current 40 mA, acquisitions scan mass range 35-450 amu under full scan. A homologous n-alkane series was used as a standard for the determination of retention time indices (RI) of each component in the essential oil sample. The relative percentages (%) of individual components were calculated based on the GC peak area (MSD response) without correction. Results and Discussion The yield of D. ambrosioides essential oil obtained by hydrodistillation was calculated to be 0.8435%. The afforded essential oil exhibited a pungent odor, bitter taste and pale yellow color. The density of the essential oil ranged from 0.965 to 0.990. Current yield performance is higher than the results obtained with Brazilian and Yemenian D. ambrosioides, at 0.3 and 0.52% respectively (dry mass basis) (Jardim et al., 2008; Al-badani et al., 2017). However, much higher extraction yield was reported in a previous study using aerial parts of Chinese D. ambrosioides, at 2.12% (Zhu et al., 2012). The yield difference could be attributable on the climatic conditions of the ecological source, the region, the fertility, the status of the material used and the extraction process of the essential oil. Volatile composition of D. ambrosioides essential oil were characterized by GC-MS and results are presented in Table 1. The chromatogram, which signals the presence of constituents through the appearance of peaks, was shown in Figure 1. On the chromatogram, a total of 11 peaks corresponding to 11 active ingredients were identified. By measuring the corresponding peak areas, it was revealed that 11 identified components accounted for 99.13% of the total amount of essential oils and unidentified compounds attributed to 1.17% of total volatile content. The main constituents of essential oils were defined as 2,3-dehydro-1,4-cineole (55%), α-terpinene (15.2%), iso-ascaridole (15.3%), and p-cymene (9.8%). Other compounds all have content of less than 1%. The chemical composition of the essential oil in the present study is completely different from other previous reports. Specifically, in Owolabi et al. (2009) isolated and identified the main compounds in Nigerian D. ambrosioides essential oils, showing that α- terpinene (63.1%) and p-cymene (26.4%) were two major components together with less abundant compounds including ascaridole (3.9%), 1.8-cineole (1.3%), isorebidole (3.6%), trans-verbenyl acetate (0.5%) and γ-terpinene (0.5%). Similarly, Gupta et al. (2002) showed that India D. ambrosioides oil contained α-terpinene (63.6%), p-cymene (19.5%) and ascaridole (6.2%) as the main components. Another study on compositional variability in volatiles from different pretreatment of D. ambrosioides cultivated in Cuba reported that the major components were α-terpinene (17.0-20.7%), p-cymene (20.2-21.1%) and ascaridole (30.5-47.1%) (Monzote et al., 2011). On the other hand, ascaridole has been identified as the main compound with largest percentage in D. ambrosioides essential oil isolated from Yemen and Madagascar materials. Accordingly, Ascaridole (54.2%), iso-ascaridole (27.7%), and p-cymene (8.1%) are the main compounds in essential oil in Yemen and p-cymene (16.2%), ascaridole (41.8%), iso-ascaridole (18, 1%) from Madagascar material (Al-badani et al., 2017; Cavalli et al., 2004). While Ascaridole was absent in the Vietnamese essential oil sample; 2,3-dehydro-1,4-cineole, which is the most abundant compound in this study was absent in Yemen, Madagascar, Nigeria and India samples (Table 2) (Figure 2). Compositions of essential oils are generally complex and tend to vary with developmental stage of the plant or the climatic conditions in which they are harvested (Zhang et al., 2017). Based on current results and the comparison with the literature, it is suggested that difference in extraction technique may give volatile compositions having contrasting major compounds. In addition, as the ascaridole compound could be easily denatured under elevated temperature (150 °C) to form isoascaridole, the discrepancies between reported ascaridole contents could also be attributed measurement errors due to formation of isomers (Cavalli et al., 2004). Another explanation is that interaction between plants and the environment via

Nat. Volatiles & Essent. Oils, 2020; 7(4): 34-40 Ngan et al. DOI: 10.37929/nveo.780483

secretion and biosynthesis of secondary metabolites may alter essential oil composition and compound quantities (Sá et al., 2016). The diverse biological activity of D. ambrosioides essential oils is due to its chemical composition. The highly variable chemical composition of D. ambrosioides essential oils with respect to plant part, enviroment, harvesting time, geographical cultivate, weather, soil, manure sources (Barros et al., 2019; Bibiano et al., 2019).

Table 1. Chemical components of D. ambrosioides essential oil

No RI Component % 1 1022 α-Terpinene 15.2 2 1029 p-Cymene 9.8 3 1063 -Terpinene 0.2 4 1094 m-Cymene 0.2 5 1125 4-Hydroxy-4-methylcyclohex-2-enone 0.5 6 1251 2,3-Dehydro-1,4-cineole 55.0 7 1263 Piperitone epoxide 0.6 8 1266 unknown (69, 168, ) 1.2 9 1294 Menth-2-en-1,4-diol 0.8 10 1304 Carvacrol 0.4 11 1316 iso-Ascaridole 15.3 Total 99.1

Figure 1. Chromatogram of D. ambrosioides essential oil.

Peak numbers (1-11) are in parentheses: (1): α-terpinene, (2): p-cymene,(3): -terpinene (4): m-cymene, (5): 4-hydroxy-4- methylcyclohex-2-enone, (6): 2,3-dehydro-1,4-cineole, (7): piperitone epoxide, (8): unknown (69, 168, RI 1266), (9): menth-2-en- 1,4-diol, (10): carvacrol, (11): iso-ascaridole

Nat. Volatiles & Essent. Oils, 2020; 7(4): 34-40 Ngan et al. DOI: 10.37929/nveo.780483

Table 2. D. ambrosioides essential oil composition from different origins

Percentage( %)

Compounds Vietnam Yemen Madagascar Nigeria India (Present study) (Al-badani et (Cavalli et al., (Owolabi et (Gupta et al., al., 2017) 2004) al., 2009) 2002) α-Terpinene 15.19 0.70 9.70 63.10 63.60 p-Cymene 9.80 8.10 16.20 26.40 19.50 Ascaridole - 54.20 41.80 3.90 6.20 2,3-Dehydro-1,4-cineole 54.99 - - - - iso-Ascaridole 15.31 27.70 18.10 3.60

Figure 2: Structures of 2,3-Dehydro-1,4-cineole and Ascaridole

Conclusion In this study, the chemical composition of the essential oil isolated from aerial parts of Dysphania ambrosioides collected in the Lao Cai Province, Vietnam, was determined. To the best of our knowledge, this is the first study on the characterization of the essential oil composition of this species from Vietnam. Essential oil was isolated by hydro-distillation method. The yield of essential oil obtained was 0.8%. GC-MS analysis was used to identify the chemical compounds of obtained essential oil.

ACKNOWLEDGMENT

This research is funded by Vietnam Academy of Science and Technology with Project code NG 0001/19-20.

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