Article http://dx.doi.org/10.21577/1984-6835.20210066 a Fundação Oswaldo Cruz, Farmanguinhos, Programa de Pós-Graduação em Pesquisa Translacional em Fármacos e Medicamentos, Rua Sizenando Nabuco 100, Manguinhos, CEP 21021-250, Rio de Janeiro-RJ, Brazil. b Universidade do Estado do Rio de Janeiro, Instituto de Biologia, Programa de Pósgraduação em Biologia Vegetal, Maracanã, CEP 20550-013, Rio de Janeiro-RJ, Brazil. c Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rua Pacheco Leão 915, Jardim Botânico, CEP 22460-030, Rio de Janeiro-RJ, Brazil; d Universidade Federal do Rio de Janeiro, Campus Macaé, Laboratório de Produtos Bioativos, Av. Aluizio da Silva Gomes 50, Novo Cavaleiros, CEP 27930-560, MacaéRJ, Brazil; e Universidade Estadual do Norte Fluminense Darcy Ribeiro, Centro de Biociências e Biotecnologia, Laboratório de Biologia do Reconhecer, Av. Alberto Lamego 2000, Parque Califórnia, CEP 28013-602, Campos dos Goytacazes-RJ, Brazil; f Universidade Castelo Branco, Faculdade de Farmácia, Av. de Santa Cruz 1631, Realengo, CEP 21710-255, Rio de Janeiro-RJ, Brazil. Antimycobacterial Activity and Chemical Characterization of the Essential Oils from Reproductive Organs of Piper lhotzkyanum Kunth (Piperaceae) Atividade Antimicobacteriana e Caracterização Química dos Óleos Essenciais dos Órgãos Reprodutivos de Piper lhotzkyanum Kunth (Piperaceae) Claudete Costa-Oliveira,a Ygor J. Ramos,a,b,c George A. Queiroz,c Elsie F. Guimarães,c Marlon Heggdorne-Araujo,d Elena B. Lassounskaia,e Michelle F. Muzitano,d Diego S. Marcelino,f Davyson de L. Moreiraa,b,* Piper lhotzkyanum Kunth belongs to the Piperaceae family, is a medicinal plant also known as “Aperta-ruão” and “Beque-cheiroso”. This species is used in folk medicine to treat rheumatism, sore throats, gastrointestinal problems, and bronchial conditions. The present study aimed to analyze the chemical composition and biological activity of the essential oils (EOs) from reproductive organs of P. lhotzkyanum against strains of Mycobacterium tuberculosis H37Rv (ATCC, 25618). The EOs were obtained by hydrodistillation and characterized by GC-MS and GC-FID. Chemical composition of the volatile mixture showed to be rich in monoterpenes for both inflorescences (IFl) and infructescences (IFr). These monoterpenes included α-phellandrene, β-phellandrene, α-pinene, and β-pinene for both samples. The antimycobacterial activity showed minimum inhibitory concentration (MIC) of 76.51 µg/mL and 128 µg/mL for IFr and IFl, respectively. By these results, the volatile fraction of IFr showed promising activity against M. tuberculosis. The chemical composition and antimicrobial properties of IFl and IFr EOs have been reported for the first time. Keywords: Inflorescence; infructescence; Mycobacterium tuberculosis; monoterpenes, phellandrene. 1. Introduction *E-mail: davysonmoreira@hotmail.com Recebido: 22 de Março de 2021 Aceito: 22 de Março de 2021 Publicado online: 6 de Outubro de 2021 Rev. Virtual Quim., 2021, 13 (5), 1196-1202 ©2021 Sociedade Brasileira de Química Piper genus (Piperaceae) has commercial, ecological, and medicinal importance.1 Biological properties of essential oils (EOs) from plants of this genus have been reported. 2-8 The most popular species is P. nigrum L., known as black pepper, widely used as a condiment and as medicine, mainly in Asia.9 Piper lhotzkyanum Kunt is a perennial shrub from 1 to 2 m high and popularly known as “beque-cheiroso”,10-11 common in the Brazilian Amazon rainforest and the Brazilian Atlantic forest.12 This species is used in folk medicine in infusion preparation for the treatment of rheumatism, sore throats, gastrointestinal problems, bronchial conditions, among others. The infructescences of P. lhotzkyanum have a strong flavor and produce a burning sensation and analgesia when chewed.10,13-14 Tuberculosis (TB) is a neglected disease caused by Mycobacterium tuberculosis and is one of the most significant motive of death since 19th century.8 According to WHO 230,000 children died from one million infected with TB.15 In this scenario, it is worth noting the increase in multidrug-resistant TB to drugs such as fluoroquinolones and injectables such as amikacin, kanamycin, capreomycin, further reducing the range of treatment options. There is an urgency regarding the discovery of new drugs and an alternative would be the combination of commercial drugs with natural compounds that could be tested to enhancer antibiotic activity.15-19 It is reported that natural products, including EOs, and their isolated compounds have inhibitory activity against the growth of M. tuberculosis, while some have been selected as prototype molecules for the development of new anti-tuberculosis agents.20-22 Despite being well reported that EOs of the Piper genus have demonstrated biological activities,2-8 such as antibacterial, antiproliferative,23-26 antileishmanial,16 insecticidal, fungicidal,25 antioxidant, and cytotoxic, 26 to date there is nothing published demonstrating the EO composition of P. lhotzkyanum with biological properties, besides a study showing great ovicidal activity against Anticarsia gemmatalis (soybean caterpillar) (LC50 = 1.6%).27 This is an open-access article distributed under the terms of the Creative Commons Attribution License. 1196 Costa-Oliveira Also, the chemical composition of EOs from inflorescences (IFl) and infructescences (IFr) (reproductive organs) for P. lhotzkyanum has never been described. Therefore, the aim of this study was to perform an analysis of the EO chemical composition of reproductive organs of P. lhotzkyanum from altitude in addition to reporting its antimycobacterial activity. 2. Material and Methods 2.1. Plant material and essential oil extraction Inflorescences and infructescences of Piper lhotzkyanum Kunth were collected in a region of altitude in the Atlantic Forest at Serra dos Órgãos National Park, near the city of Teresópolis, Rio de Janeiro (Altitude: 1,144.69 m and GPS: 12°11′45′S; 38°58”05’W) in 2019. This study was registered in the Genetic Heritage Management Council (CGEN n. AE4E953) and in the Biodiversity Authorization and Information System (SISBIO n. 57296-1). The botanical identification was made by Dr. Elsie Franklin Guimarães and Msc. George Azevedo Queiroz at Rio de Janeiro Botanical Garden Research Institute (JBRJ). Herborized samples were deposited at the Herbarium RB (01426181). The fresh plant material (100 g, 700 mL of distilled water) was subjected to hydrodistillation for 2 h in a Clevenger-type apparatus for EOs extracting. The obtained samples were drying over anhydrous sodium sulfate (Na2SO4, Sigma-Aldrich, Brazil), kept in sealed amber vials, and stored at -20 °C for five days until gas chromatography (GC) analysis. The total yield of EO was registered as a percentage value, considering weight of EO (g)/ 100 g of fresh plant material.28-30 2.2. Essential oils analysis The obtained EOs were diluted in dichloromethane (HPLC grade, Tedia, Brazil) until 1.000 ppm. All samples were injected 1 µL, splitless,28-29 for chemical identification by gas chromatography coupled to mass spectrometry (GC-MS) and for quantification by GC coupled to Flame Ionization Detection (GC-FID). GC-MS analysis was performed using a gas chromatograph 6890 GC coupled to an Agilent MS 5973N mass spectrometer (Hewlett-Packard, Brazil), operating at 70 eV of ionization energy, in positive mode, and mass range of m/z 40 – 600 atomic mass units (u). The GC conditions were an HP-5MS capillary column (30 m x 0.25 mm id x 0.25 μm film thickness), temperature programming from 60 °C to 240 °C with an increase of 3 °C/min, using helium (99.99%) as carrier gas at a constant flow rate of 1.0 mL/min. The injector and detector were set at 270 °C, the transfer line was set at 280 °C and samples were injected 1 μL splitless. GC-FID analysis was achieved in an HP-Agilent 6890 gas chromatograph (Hewlett-Packard, Brazil) equipped with an HP-5MS capillary column (30 m x 0.25 mm id x 0.25 μm Vol. 13, No. 5, 2021 film thickness), temperature setting from 60 °C to 240 °C, with an increase of 3 °C/min, using hydrogen as carrier gas at a constant flow rate of 1.0 mL/min. The injector and detector temperatures were set at 270 °C. Samples were injected 1 μL splitless. Retention indices (RI) as well as the quantification of the peak area were achieved based on the results of the GC-FID. Relative percentage of individual components was calculated based on the peak areas of the GC without correction of the FID response factor. The compounds were identified based on the fragmentation pattern of the mass spectrum compared with literature records (National Institute of Standards and Technology – NIST, 2010; Wiley7n), as well as calculated RI referring to a homologous series of n-alkanes (C8 -C28, Sigma -Aldrich, Brazil).31-32 2.3. Antibacterial activity The standard virulent strain of Mycobacterium tuberculosis H37Rv (ATCC, 25618) was grown in 7H9 (BACTO) culture medium, supplemented with 10% albumin, dextrose, catalase (ADC) (BC®), 0.05% of tween 80, and kept in an incubator (Scientific – Water-Jacketed incubator) at 37 °C and 5% CO2, until the beginning of the growth phase. Samples were evaluated for their antimycobacterial activity using the tetrazole salt assay in a 96-well microplate at concentrations of 16, 32, 64 and 128 μg/mL. For this test, a suspension was prepared with M. tuberculosis H37Rv (300 µL of mycobacteria in 7.2 of 7H9 culture medium supplemented with 10% ADC, approximately 3 x107 Colony Forming Units – CFU/ mL) and kept in an incubator at 37 °C and 5% CO2 until the beginning of the log phase (exponential growth phase). The CFU dosage for turbidity was standardized and monitored in a spectrophotometer (Hitachi – Model U-1100) at an optical density (O.D.) of 600 nm. Subsequently, in the logarithmic growth phase, 50 µL of this suspension were plated in a 96-well microplate (1x106 CFU/ well). The EO samples (50µL / well) were previously diluted in 7H9 supplemented with ADC in a concentration 2 times higher than the desired final concentration and added to the microplate where the mycobacteria already contained. The sealed plate was incubated at 37 °C and 5% CO2 for 5 days. After this period, 10 µL per well of a 5 mg/ mL solution of tetrazole 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyl-tetrazole (MTT) in saline phosphate buffer (PBS) was added sterile. Three hours later, 100 µL of the lysis buffer was added (20% w/ v sodium dodecyl sulfate (SDS)/ 50% dimethylformamide (DMF) in distilled water – pH 4.7). The microplate reading was performed on a spectrophotometer at 570 nm (Hitachi – Model U-1100).33 Treatment with rifampicin (0.032; 0.08; 0.2 and 1 µg/ mL) was used as a positive control of antimycobacterial activity in the wells containing only the bacilli. Negative control was set in wells containing bacilli and without treatment. To calculate the percentage of inhibition of mycobacterial growth, equation (1) was used. 1197 Antimycobacterial Activity and Chemical Characterization of the Essential Oils from Reproductive Organs of Piper lhotzkyanum Kunth (Piperaceae) (1) 2.4. Statistical analysis The statistical analysis to show differences in the antimicrobial activity of the analyzed EOs was performed by the ANOVA test, using Statistica® software. The value of p < 0.05 was considered statistically significant. 3. Results Strongly yellowish essential oils were obtained from fresh IFr and IFl of P. lhotzkyanum. The yield of EOs was at 2.4% for IFl and 2.5% for IFr (w/ w). It was possible to identify a total of 39 and 20 compounds that correspond to 93.9% and 97.1% of total chemical composition, respectively, which are shown in Table 1. A total of 29 compounds for IFl and 12 for IFr was identified in less than 1.0% of relative percentage, and corresponding to 11.3% and 3.2%, respectively. The relative percentage of monoterpenes in the EOs of the reproductive organs was quite pronounced (IFl – 86.16%; IFr – 92.34%). The major constituents were identified as non-oxygenated monoterpenes α-phellandrene (IFr – 56.4%; IFl – 48.52%), β-phellandrene (IFr – 14.5%; IFl – 8.49%), α-pinene (IFr – 6.8%; IFl – 11.25) and β-pinene (IFr – 6.9%; IFl – 8.35) (Figure 1). The mass spectra of the main components from the reproductive organs of P. lhotzkyanum (70 eV, positive mode, m/z 40 – 600 u) is shown on the supplementary material (Figures S1-4). The antimicrobial activity of the EOs is shown in Figure 2. The sample IFr showed greater activity than IFl (MIC of 76 and 128 µg/mL, respectively). Figure 1. Chemical structures of the main identified constituents in the essential oils from inflorescences and infructescences of Piper lhotzkyanum Kunth Figure 2. Growth inhibition of Mycobacterium tuberculosis H37Rv after treatment with Piper lhotzkyanum Kunth essential oils Legend. Results of MTT assay after 5 days of incubation in the presence of samples at concentrations of 16, 32, 64 and 128 µg/mL. Positive control = M. tuberculosis H37Rv treated with rifampicin (reference drug); negative control = M. tuberculosis H37Rv without treatment. Statistical analysis: Oneway ANOVA followed by the Tukey test *** p < 0.001, ** p < 0.01 and * p < 0.05 compared to the negative control (Mtb H37Rv 1x106 CFU / mL). Triplicate results represented as mean ± standard error. IFl - Essential oil from inflorescences; IFr - Essential oil from infructescences. 1198 Rev. Virtual Quim. Costa-Oliveira Table 1. Chemical composition of the essential oil from reproductive organs of Piper lhotzkyanum Kunth. Compoundsa RI calb RI litc α-thujene 923 Relative percentage (%) IFl IFr 924 0.1 tr 6.8 α-pinene 931 932 11.2 camphene 944 946 0.3 - β-pinene 970 974 8.3 6.9 6-methyl-5-hepten-2-one 988 989 tr tr myrcene 995 988 1.1 0.7 α–phellandrene 1009 1002 48.5 56.4 δ-3-carene 1010 1008 3.5 3.7 α–terpinene 1012 1014 1.3 - p-cymene 1022 1020 tr tr o-cymene 1023 1022 - tr limonene 1025 1024 tr 1.9 β-phellandrene 1028 1025 8.5 14.5 cis-β-ocimene 1036 1032 2.6 0.6 γ-terpinene 1057 1054 0.2 0.3 0.5 terpinolene 1085 1086 0.4 linalool 1103 1095 1.7 - unidentified (monoterpene) - - - 1.8 trans-sabinene hydrate 1104 1098 tr - menth-2-en-1-ol 1116 1118 tr - terpinen 4-ol 1178 1174 tr - α-terpineol 1189 1186 0.2 - cis-piperitol 1205 1195 tr - α-copaene 1381 1374 0.2 - β-elemene 1407 1389 0.0 - α-gurjunene 1411 1409 0.1 - cis-caryophyllene 1425 1417 2.4 2.0 - γ-elemene 1433 1434 0.5 aromadendrene 1440 1439 0.1 - α-humulene 1456 1452 0.3 - β-selinene 1480 1489 0.5 0.5 α-selinene 1494 1498 0.6 0.1 0.3 β-curcumene 1496 1514 - γ-cadinene 1509 1513 0.1 - δ-cadinene 1518 1522 0.5 0.1 zonarene 1524 1528 0.1 - selina-3,7(11)-diene 1547 1545 tr - germacrene B 1553 1559 0.1 - caryophyllene oxide 1581 1582 tr - guaiol 1596 1600 tr - α-epi-muurolol 1645 1640 0.1 - Non-Oxygenated Monoterpenes 86.16 92.34 Oxygenated Monoterpenes 1.99 1.77 Non-Oxygenated Sesquiterpenes 5.50 3.03 Oxygenated Sesquiterpenes 0.18 0.00 Total 93.85 98.91 a All compounds were identified by MS and RI in accordance with experimental. aCompounds are listed in order of elution. bRetention indices calculated from retention times in relation to those of the n-alkanes series on a HP-5MS analytical column (see experimental). cRetention indices from the literature. IFl = Inflorescences, IFr = Infructescences. tr = compound < 0.05%. Vol. 13, No. 5, 2021 1199 Antimycobacterial Activity and Chemical Characterization of the Essential Oils from Reproductive Organs of Piper lhotzkyanum Kunth (Piperaceae) 4. Discussion 5. Conclusions In this article we analyzed for the first time the chemical composition and the antimycobacterial potential of reproductive organs of the species P. lhotzkyanum. This work represents the first biological activity described for the inflorescences and infructescences of this species. Studies referring to the leaf EO of P. lhotzkyanum was published before by Krinski (2018),27 and showed ovicidal activity. The EO yields for the reproductive organs (~ 2.5%, w/w) were considered high, a fact that is not common for species of Piper, with some exceptions, for example, Piper cubeba L. (4.4%, w/w). However more studies is necessary to evaluate this yield during different phenological periods.34-39 This fact is crucial in natural products, mainly, to be exploited commercially or in the production of pharmacological assets. Besides, this high EO yield may have an importance role in the ecological issues related to P. lhotzkyanum, such as pollinators attraction or herbivore repellency. Considering herbivore repellency, the identified compounds in the volatile mixture may present important biological properties such as antimicrobial. This study reported a predominance of non-oxygenated monoterpenes in the reproductive organs which differs from some results found in the literature for other Piper species,39-40. The high relative percentage of α- and β-phellandrenes, that are used in the food and perfume industries are very interesting.41-42 Compound α-phellandrene, a cyclic monoterpene, is also found in the EOs from plants such as Schinus terebinthifolius Raddi (15.7%), Solanum erianthum D.Don (17.5%), Thymus kotschyanus Boiss and Hohen (10.8%), Cupressus atlantica Gaussen (5.5%), Anethum graveolens L. (32.0%), Myrica gale L. (8.0%) and Piper mullesua D. Don (22.8%). Literature records showed antinociceptive, anti-inflammatory, 43 antimicrobial,44 anticancer,45 and hyperalgesic activities46 related to this monoterpene. The evaluation of antimycobacterial activity for both EOs against strains of M. tuberculosis showed promising activity for IFl and IFr. MIC values < 100 µg/mL have been found to be ideal candidates against M. tuberculosis, while values of 100 – 200 µg/mL are considered moderate candidates. 8,47 Therefore, EO from IFr (MIC = 76 mg/ mL) could be a promising candidate to proceed in a further study since synergism of EOs with standard drugs used to treat TB may be an option in the treatment of this condition.17 Some studies relate the antimicrobial activity of EOs, mainly, to their monoterpenoid constituents. Mechanism of action proposed that monoterpenes act on the disruption and dysregulation of the bacterial membrane function.48-51 Interestingly to note that the richest EO in monoterpenes from infructescences showed greater activity, strengthening the hypothesis that they may interact with the phospholipid membranes of M. tuberculosis. The chemical composition of the essential oil from reproductive organs of Piper lhotzkyanum is described here for the first time. The studied essential oils and their volatile components can provide an important source of new antimycobacterial agents. In addition, the high essential oils´ yield and the great relative percentage of phellandrene may represent an important source of this monoterpene for the industry. The essential oil from infructescences was the most active against M. tuberculosis. These findings contribute with new data on the chemical constitution and antimycobacterial potential of the essential oils from reproductive organs of P. lhotzkyanum collected at high altitude site in the Atlantic Forest. 1200 Acknowledgment This work was supported by CAPES, FAPERJ and PROEP-CNPq (407845/2017-8). References 1. Parrini, R.; Pardo, C. S.; Pacheco, J. F.; Conhecendo as plantas cujos frutos e recursos florais são consumidos pelas aves na Mata Atlântica do Parque Nacional da Serra dos Órgãos. Atualidades Ornitologicas 2017, 199, 38. [Link] 2. Martins, A. 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