Abstract
The chemical compositions were determined by the leaf essential oils of 2 Vietnamese plants, Piper betle f. densum and Disepalum plagioneurum. The main chemical classes in the P densum leaf essential oil were sesquiterpene hydrocarbons (30.1%) and oxygenated sesquiterpenes (60.1%), with γ-elemene (12.7%), valeranone (9.3%), and ishwarone (6.0%) being the principal compounds. Disepalum plagioneurum leaf essential oil was dominated by monoterpene hydrocarbons (23.9%) and sesquiterpene hydrocarbons (59.8%). Bicyclogermacrene (26.8%), (E)-caryophyllene (12.7%), (E)-β-ocimene (8.4%), and (Z)-β-ocimene (6.0%) were the major compounds of this essential oil. With the same MIC of 64 µg/mL, the leaf essential oils of P betle f. densum and D plagioneurum strongly controlled the growth of the Gram (+) bacterium Clostridium sporogenes NCTC 12935 and the fungus Aspergillus niger ATCC 1015, respectively. It was also found that D plagioneurum leaf essential oil was cytotoxic to cancer cell lines MCF7 and HeLa with IC50 values of 63.2 and 41.4 µg/mL, respectively.
Introduction
Piperaceae (or pepper) is a large family of small trees, shrubs, and herbs. This family is divided into 5 genera, namely Macropiper, Zippelia, Piper, Peperomia, and Manekia, with about 3600 accepted species. 1 These are now widely distributed in both pantropical and neotropical zones. 1 Piperaceae species, especially Piper sp., are aromatic and can produce essential oils. 1 Essential oils extracted from different organs of many Piper species have generally been constituted of terpenoids, phenylpropanoids, and alkaloids. 1 Of the Piper species, P betle is among the most attractive for essential oil studies. Phytol, carvacrol, chavicol, chavibetol, and especially eugenol, are the major compounds in P betle leaf oils. 2 For instance, eugenol constituted 14% to 64% of the leaf oil from India.3,4 Eugenol (22.7%) was also one of the major compounds in Vietnamese P betle leaf essential oil. 5
The Annonaceae is the largest family in the Magnoliales order. 2 About 112 genera and 2440 species have been recorded, which are concentrated in the tropics, with few species found in temperate areas.6,7 Numerous Annonaceae species are of economic value since their extracts and metabolites, especially essential oils, are used as raw materials in the cosmetic and perfumery industries, and for medicinal use. 8 For instance, Ylang-ylang (Cananga odorata) essential oil is one of the most extensively used natural ingredients in the perfume industry, earning the name “Queen of Perfumes.” 9
As can be seen, the plants of these 2 families are thought to be rich resources of essential oils. In Vietnam, there have been plenty of phytochemical investigations using GC-MS (gas chromatography-mass spectrometry) analyses of Piperaceae and Annonaceae essential oils. Piper nigrum (black pepper) seed essential oil, collected from Gialai was dominated by 3-carene (29.2%), δ-limonene (20.9%), caryophyllene (15.0%), and β-pinene (9.8%). 10 The main compounds in Piper laosanum fresh leaf essential oil were α-curcumene (12.0%), germacrene D (6.3%), and sabinene (6.1%). 11 Piper albispicum leaf oil, gathered from Hatinh, showed the best antimicrobial activity against Pseudomonas aeruginosa with a MIC of 5.82 µg/mL due to the role of chavicol acetate (16.3%) and bicyclogermacrene (13.8%). 12 Our previous report indicated that Polyalthia viridis leaf and stem essential oils (Annonaceae), collected from Quangtri, have cytotoxic, antimicrobial, and anti-inflammatory effects. 13
Piper betle f. densum (Blume) Fosberg (synonyms: Piper betle var. densum (Blume) C. DC., or Piper densum Blume), locally named Tieu day, is an endemic species found in Kontum and Lamdong provinces of Vietnam. 14 Disepalum plagioneurum (Diels) D. M. Johnson (synonym: Polyalthia pingpienensis), locally named Nhoc trai khop la thuon, is an Annonaceae species found in China, Laos, and Vietnam. 15 To date, only one phytochemical report has dealt with the isolation of acetogenin derivatives from the leaves of D plagioneurum. 15 The current study aimed to describe the chemical composition of the essential oils from these 2 plants. These oils have been further taken into consideration for their antimicrobial and cytotoxic properties.
Results and Discussion
The yellow essential oil from P betle f. densum fresh leaves was obtained with a yield of 0.15% (v/w). A total of 45 compounds were identified, which represented 92.5% of the composition (Table 1). Sesquiterpene hydrocarbons (30.1%) and their oxygenated derivatives (60.3%) were the primary chemical classes. Other types were present in trace amounts, including monoterpene hydrocarbons (0.7%), oxygenated monoterpenes (0.8%), diterpene hydrocarbons (0.4%), and nonterpenic compounds (0.2%). γ-Elemene (12.7%), valeranone (9.3%), and ishwarone (6.0%) were the principal compounds. Piper betle f. densum leaf essential oil was further characterized by the appearance of other compounds with an amount greater than 1.0%, consisting of trans-cycloisolongifol-5-ol (4.7%), trans-calamenene (4.7%), cis-sesquisabinene hydrate (4.7%), epi-cedrol (4.6%), cis-cadinene ether (4.4%), occidentalol (3.5%), p-menth-1-ene-7,8-diol (3.3%), germacrene B (3.2%), α-amorphene (2.9%), (Z)-α-santalol acetate (2.5%), β-bisabolenal (2.4%), eudesm-7(11)-en-4-ol acetate (1.8%), 14-hydroxy-α-humulene (1.7%), longipinanol (1.6%), cedr-8-en-13-ol (1.5%), β-vetivenene (1.3%), epi-cubebol (1.2%), β-(Z)-curcumen-12-ol (1.2%), (E)-caryophyllene (1.1%), and germacra-4(15),5,10(14)-trien-1-α-ol (1.1%). As mentioned above, the chemical compositions of essential oils of P betle f. densum and P betle are quite different.2-5
Chemical Compounds in Piper betle f. densum Leaf Oil.a
Abbreviations: Rt, retention time; RIE, retention indices relative to n-alkanes (C7-C40) on Equity-5 column; RIL, retention indices from Adams 16 and the NIST standard database. 17
Bold: major compounds.
Powdered D plagioneurum leaves were hydrodistilled to give a yellow essential oil with a yield of 0.17% v/w. A total of 42 compounds were identified, which accounted for 99.9% of the composition (Table 2). This essential oil was predominated by sesquiterpene hydrocarbons (59.8%) and monoterpene hydrocarbons (23.9%). Oxygenated monoterpenes, oxygenated sesquiterpenes, and nonterpenic compounds formed 9.6%, 6.3%, and 0.3%, respectively. Bicyclogermacrene (26.8%), (E)-caryophyllene (12.7%), (E)-β-ocimene (8.4%), and (Z)-β-ocimene (6.0%) were the major compounds in this essential oil. Some compounds reached more than 1.0%, including germacrene D (4.3%), 1,8-cineole (3.7%), (E)-nerolidol (3.7%), bicycloelemene (2.9%), β-myrcene (3.6%), α-pinene (2.3%), perillene (2.3%), α-copaene (2.3%), α-humulene (2.0%), sabinene (1.8%), α-terpinyl acetate (1.7%), β-elemene (1.4%), β-selinene (1.2%), aromadendrene (1.1%), lianalool acetate (1.0%), δ-cadinene (1.0%), and spathulenol (1.0%). This result matches well with previous reports since bicyclogermacrene can be seen as the major compound of the essential oils of Vietnamese Polyalthia species, for example, P viridis leaf (17.1%), 13 P harmandii leaf (20.9%), 18 P harmandii stem (27.9%), 18 and Polyalthia suberosa leaf (26.3%). 19
Chemical Compounds in Disepalum plagioneurum Leaf Oil.a
Abbreviations: Rt, retention time; RIE, retention indices relative to n-alkanes (C7-C40) on Equity-5 column; RIL, retention indices from Adams 16 and the NIST standard database. 17
Bold: major compounds.
In the antimicrobial assay (Table 3), P betle f. densum leaf essential oil showed strong activity against the Gram (+) bacterium Clostridium sporogenes with a MIC of 64 µg/mL, whereas D plagioneurum leaf oil controlled the growth of Bacillus subtilis and Staphylococcus aureus with the same MIC of 128 µg/mL. Both oils suppressed the growth of 2 Gram (–) bacteria Escherichia coli and Pseudomonas aeruginosa with the same MIC of 128 µg/mL. The 2 studied oils were inactive against the fungus Aspergillus brasiliensis (MIC > 256 µg/mL) and moderately active against the fungus Fusarium oxysporum with the same MIC of 128 µg/mL. In contrast to P betle f. densum leaf oil (MIC > 256 µg/mL), D plagioneurum leaf oil successfully inhibited the growth of the fungus Aspergillus niger with a MIC of 64 µg/mL. In the last case, these 2 essential oils failed against 2 yeasts Candida albicans and Saccharomyces cerevisiae (MIC > 256 µg/mL).
Antimicrobial Activity of the Studied Essential Oils.
Huong et al reported that P albispicum leaf and stem essential oil possessed MIC values of 9.07 to 10.91 µg/mL against Enterococcus faecalis and C albicans. 12 Piper pendulispicum leaf and stem essential oils (MIC 16-32 µg/mL) were equivalent to the positive control cycloheximide (MIC 32 µg/mL) against C albicans. 20 Polyalthia suberosa twig essential oil successfully controlled P aeruginosa, A niger, and C albicans with the same MIC of 50 μg/mL. 18 Likewise, P viridis stem essential oil produced the same MIC of 50 µg/mL against A niger and C albicans. 13 Collectively, it is expected to use Vietnamese Piperaceae and Annonaceae essential oils in antimicrobial treatments.
Both essential oil samples were submitted to cytotoxic assay against the growth of HepG2, MCF7, and HeLa cells, with ellipticine used as a positive control (HepG2: IC50 = 0.69 µg/mL, MCF7; IC50 = 0.81 µg/mL, and HeLa: IC50 = 0.78 µg/mL). Disepalum plagioneurum leaf essential oil showed cytotoxicity towards cancer cells MCF7 and HeLa with IC50 values of 63.2 and 41.4 µg/mL, respectively, but was inactive towards HepG2 (IC50 > 256 µg/mL). Piper betle f. densum leaf essential oil did not inhibit any of these 3 cancer cell lines (IC50 > 256 µg/mL). In line with previous results, Vietnamese Polyalthia essential oils showed potential for cytotoxic treatments. Polyalthia viridis stem oil and P suberosa leaf essential oil prevented the proliferation of HepG2, MCF7, and A549 cells with IC50 values of 56.7 to 69.9 μg/mL.13,19 Considering the role of the major compounds, bicyclogermacrence was mainly responsible for the cytotoxicity of Nectandra leucantha leaf essential oil against MCF7, HCT, U-87, and B16F10-Nex2 cancer cells. 21 The cytotoxicity of Annona muricata leaf essential oil against MCF7 cells (99.2% kill at 100 μg/mL) is likely due to the relatively high concentration of (E)-caryophyllene (38.9%). 22 Hence, our current data provide new information for further research.
Conclusion
For the first time, the current research reports the chemical compositions of the essential oils of 2 Vietnamese plants, in which γ-elemene (12.7%), valeranone (9.3%), and ishwarone (6.0%) were the predominant compounds in P betle f. densum leaf essential oil, while D plagioneurum leaf essential oil was associated with the presence of the major compounds bicyclogermacrene (26.8%), (E)-caryophyllene (12.7%), (E)-β-ocimene (8.4%), and (Z)-β-ocimene (6.0%). The leaf essential oils of P densum and D plagioneurum exhibited strong antimicrobial activity against the Gram (+) bacterium C sporogenes and fungus A niger with the same MIC of 64 µg/mL, respectively. In addition, D plagioneurum leaf essential oil showed cytotoxicity toward cancer cells MCF7 and HeLa with IC50 values of 63.2 and 41.4 µg/mL, respectively.
Materials and Methods
Plant Materials
The fresh leaves of Piper betle f. densum (Blume) Fosberg and Disepalum plagioneurum (Diels) D. M. Johnson were collected from Pu Hoat and Pu Huong Natural Reserves, Nghean, Vietnam in March-2023, respectively. The plants were identified by our co-author Prof. Le Thi Huong. Two voucher specimens PD-2023 (P betle f. densum leaves) and DP-2023 (D plagioneurum leaves) were deposited at the faculty of Agriculture, Forestry and Fishery, Nghe An University of Economics.
Distillation
Each fresh powdered sample (1.5 kg) was submitted to hydrodistillation for 3.0 h. The extraction was performed using a Clevenger apparatus. The essential oils obtained by decantation were dried over Na2SO4 and then kept in sealed vials at −5 °C for further analysis.
GC-MS Analysis
GC-MS analysis was performed using a Shimadzu Technologies GCMS-QP2010 Plus (Shimadzu) chromatograph equipped with a fused silica Equity-5 capillary column (30 m 0.25 mm, film thickness 0.25 µm, Supelco). 23 The analytical settings were as follows: 1.5 mL/min of carrier helium, 280 °C injector and interface temperatures, and a temperature ramp from 60 °C (2 min hold) to 240 °C (10 min hold) at 3 °C/min, then to 280 °C at 5 °C/min for the column (40-min hold). A split ratio of 10:1 was used to inject the samples. The inlet pressure was 93.2 kPa, and the injection volume was 1.0 µL. The MS settings were ionization voltage 70 eV, detector voltage 0.82 kV, and acquisition scan mass range of 40 to 500 amu at a sampling rate of 0.5 scan/s. By co-injecting the constituents and comparing the results to a homologous series of n-alkanes (C7-C40), the retention indices (RI) of chemical constituents were calculated. Quantification was carried out on the basis of the relative area of the total ion chromatogram (TIC) peaks of volatile compounds. Chemical identification was carried out by comparison of RI values with those reported by Adams, 16 and the NIST standard databases. 17
Antimicrobial Assay
The pathogenic ATCC (American Type Culture Collection) strains, including 3 Gram (+) bacteria, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, and C sporogenes NCTC 12935, 2 Gram (–) bacteria, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 15442, 3 fungi, Aspergillus brasiliensis ATCC 16404, A niger ATCC 1015 and Fusarium oxysporum ATCC 46591, and 2 yeasts, Candida albicans ATCC 10231 and Saccharomyces cerevisiae ATCC 4098, were used in this study. All strains were cultured on Muller Hilton Agar (MHA, Merck) plates for one day at 37 °C.
The assay was described in previous work.24-26 Briefly, the essential oil samples were dissolved in DMSO (5%) to reach concentrations of 4 to 256 µg/mL. A total of 180 µL of bacterial suspension with 106 CFU mL−1 in Muller Hilton Broth and 20 µL of essential oil were placed in each well (MHB, Merck). The mixture was incubated at 37 °C, and the OD (optical density) was determined at 600 nm using an Elisa reader (RNE-9002, USA). The lowest concentration that showed no growth was identified as the MIC. The assays were performed 3 times. The same procedures were used for the negative control, which contained MHB and Tween, and the positive controls, which contained MHB and bacterial suspension without the tested sample. Streptomycin and tetracycline were used as reference compounds for the respective Gram (+) and Gram (–) bacteria, whereas nystatin was used for fungi and yeasts.
Cytotoxic Assay
The cytotoxicity of the 2 oil samples was tested on the proliferation of A549 (human lung cancer), HepG2 (human hepatocellular cancer), and MCF-7 (human breast cancer) cells using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay.13,19 The cancer cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (100 U/mL) and penicillin/streptomycin (100 g/mL) at 37 °C in a humidified 5% CO2 atmosphere. The MTT assay was carried out as follows: human cancer cells (2.0 × 105 cells/mL) were treated for 72 h with either 256-32 µg/mL of the oil samples or the standard compound ellipticine. After incubation, MTT (0.1 mg) was added to each well. Cells were then incubated at 37 °C for 4 h. The plates were centrifuged at 1.000 rpm for 6 min at 25 °C, and the media was then aspirated. Continuously, DMSO (150 µL) was added to each well to dissolve the formazan crystals. The OD (optical density) was measured at 540 nm using an Accu-Tell Elisa Reader (ABER-2, inhibitory percentage that caused a reduction in the absorbance, compared with the untreated controls. The IC50 (50% inhibitory concentration) values were calculated using dose–response curves.
Supplemental Material
sj-docx-1-npx-10.1177_1934578X231190689 - Supplemental material for Essential Oils of Two Vietnamese Plants Piper betle f. densum (Piperaceae) and Disepalum plagioneurum (Annonaceae): Chemical Composition, Antimicrobial, and Cytotoxic Activities
Supplemental material, sj-docx-1-npx-10.1177_1934578X231190689 for Essential Oils of Two Vietnamese Plants Piper betle f. densum (Piperaceae) and Disepalum plagioneurum (Annonaceae): Chemical Composition, Antimicrobial, and Cytotoxic Activities by Do Ngoc Dai, Ty Viet Pham, Nguyen Dinh Luyen, Vo Thi Dung, Le Thi Huong and Ninh The Son in Natural Product Communications
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.
Supplemental Material
Supplemental material for this article is available online.
References
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
