Chemical composition, anti-inflammatory and cytotoxic activity of essential oils from two Luvunga species (L. scandens and L. hongiaoensis) from Vietnam

Abstract New essential oils (EOs) extracted from different parts of two Luvunga species (L. scandens and L. hongiaoensis) from Vietnam were investigated for their chemical composition, anti-inflammatory and cytotoxic activity. Sixty-nine total compounds were identified in the EOs by GC/MS. The major constituent of the leaf, fruit, and root EOs from L. scandens was β-caryophyllene (71.5%, 63.0%, and 31.5% respectively). The main compounds in L. hongiaoensis EOs were β-elemene (34.3% in leaf oil) and caryophyllene oxide (21.2% in root oil, 19.4% in stem oil). The EO from L. scandens fruits significantly inhibited nitric oxide production on LPS-induced RAW264.7 cells (IC50 = 37.95 ± 2.76 µg/mL). The EOs from L. hongiaoensis roots and L. scandens leaves and fruits exhibited cytotoxic activity against MCF-7, SK-LU-1, and HepG2 (IC50 from 49.74 ± 3.36 to 97.82 ± 8.61 µg/mL). This is the first report on L. hongiaoensis EOs and significantly complements the composition and bioactivity of L. scandens EOs. Graphical Abstract


Introduction
The genus Luvunga Buch.-Ham.ex Wight & Arn.belongs to the family Rutaceae and comprises about 15 species found in tropical evergreen forests in Southeast Asia, India, Sri Lanka, Australia, and New Guinea (Mazo and Tahil 2021).Luvunga species are climbing woody lianas with inverted or sometimes strongly recurved thorns that act as anchors to the branches of trees (Mazo and Tahil 2021).Among the Luvunga species, L. scandens roots, stems, and fruits have been used in the traditional medicine of India, Vietnam, Thailand, Myanmar, Malaysia, and China to treat kidney diseases, fatigue, malaria, cirrhosis, ascites, rheumatism, muscle pain, and scorpion stings (Mishra and Agrawal 1988;Ong et al. 2011;Sirinut et al. 2017;Li et al. 2018).Since the 1980s, the EO of L. scandens pulp in India had been studied, showing that it contained mainly cineol (32.3%) and methyl cinnamate (14.5%) and had antifungal, antibacterial, antiaflatoxigenic, antioxidant activity (Mishra and Chauhan 1983;Garg and Jain 1999;Dubey et al. 2017), CNS depressant, anticonvulsant effects in albino rats, reduced blood pressure, slightly reduced rectal temperature in rats, and inhibited acetylcholine activity on isolated ileum and uterus of guinea pigs (Mishra and Agrawal 1988).Plant extracts of L. scandens have been shown to have anti-proliferative effects against the human malignant melanoma cell line (Sama Naziyah et al. 2018), squamous carcinoma cell line (Shaban et al. 2017) and cytotoxic effects against the MCF-7 cancer cell line (Al-Zikri et al. 2016).A variety of triterpenoids, phytosterols, limonoids, coumarins, and alkaloids were isolated and identified from different parts of this plant (Al-Zikri et al. 2014;Taher et al. 2016;Nguyen et al. 2017;Sirinut et al. 2017;Tran et al. 2019;Ichwan et al. 2021), among which two triterpenoids, flindissol and 3-oxotirucalla-7,24-dien-21-oic-acid isolated from L. scandens, showed remarkable inhibitory effect on the cancer cell lines MCF-7 and HSC-3 with IC 50 value in µM region (Al-Zikri et al. 2014;Ichwan et al. 2021).
In Vietnam, four species of Luvunga have been recorded: L. sarmentosa, L. nitida, L. scandens, and L. hongiaoensis, of which the last two species are distributed in Bidoup-Nui Ba National Park, Lam Dong province (Ho 2003;Tagane et al. 2020).While L. scandens is a well-known medicinal plant species, L. hongiaoensis is a newly discovered species, recently described by Tagane et al. (2020).This species is endemic to Vietnam, only found in Hon Giao area, Bidoup-Nui Ba National Park, and has never been studied for its chemical composition and biological activity.The chemical composition of EO from L. scandens pulp and leaves were investigated by Mishra and Chauhan (1983) and Van et al. (2021), respectively.However, the chemical composition and bioactivity of EOs from other plant parts of L. scandens have not been reported.Therefore, this study was carried out to clarify and compare the chemical composition of the EOs from the roots, stems, leaves, and fruits of L. scandens and L. hongiaoensis.The EOs obtained from the plant parts with higher EO content were also selected to evaluate their potential anti-inflammatory activity through inhibition of nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells and cytotoxic activity against three cancer lines MCF-7, SK-LU-1, and HepG2.

Chemical composition of L. scandens and L. hongiaoensis oils
The composition of investigated EOs from roots, leaves, and fruits of L. scandens and roots, leaves, and stems of L. hongiaoensis is presented in the Supplemental material (Table S2).The GC chromatograms of the EOs and some identified names of the major peaks are given in the Supplemental material (Figure S1-S6).A total of sixty-nine compounds were identified in the six investigated EOs.Sesquiterpene hydrocarbons and their oxygenated derivatives were the major constituents of EOs from all investigated parts of the two Luvunga species, accounting for 93.6%, 99.9%, and 99.9% of the L. scandens root, leaf and fruit EOs and 94.3%, 99.5% and 92.0% of the root, leaf and stem EOs of L. hongiaoensis, respectively.β-Elemene, β-caryophyllene, spathulenol, and caryophyllene oxide were the only components present in all six EOs and were also the most abundant with a large variation between these oils.All three EOs from L. scandens contained β-caryophyllene as the main constituent, with the highest content in the leaf oil (71.5%), the fruit oil (63.0%), and the root oil (31.5%), while in the EOs from L. hongiaoensis, β-elemene was most abundant in the leaf oil (34.3%), caryophyllene oxide was predominant in the root oil (21.2%) and the stem oil (19.4%).
The root EO of L. scandens contained forty-one compounds, of which β-caryophyllene (31.5%) and caryophyllene oxide (28.6%) were found as the major constituents.Eight compounds were characterized in L. scandens leaf EO, including β-caryophyllene (71.5%) and valencene (17.8%) as the most abundant components.The predominant constituents in the ten identified compounds of L. scandens fruit EO were β-caryophyllene (63.0%), followed by germacrene D (8.5%), α-humulene (8.2%), caryophyllene oxide (8.2%), and valencene (8.1%).The chemical composition of EO from L. scandens leaves collected in a sandy forest in Vietnam was identified in a previous study (Van et al. 2021), showing notable differences when, in addition to the main constituent β-caryophyllene (34.7%), there were also the presence of limonene (21.3%) and α-humulene (10.9%).Similarly, cineol and methyl cinnamate, the main constituents of the EO from L. scandens pulp in India (Garg and Jain 1999), were not found in the same species in this study.These differences may be due to geographical differences and natural conditions that affect the accumulation of substances in plants (Chrystal et al. 2020).In general, the composition of EOs from L. scandens was previously described only in one paper on leaf oil and one article on pulp oil, with no publications on the composition of EOs from the roots.

NO production inhibitory activity of LSL, LSFR, and LHR
Some essential oils, especially those from plants in the Rutaceae family, have anti-inflammatory properties (Oliveira et al. 2022).They can modulate inflammation by inhibiting enzymes, blocking mediators or reducing oxidative stress (Lucca et al. 2022).Nitric oxide (NO) is an important molecule of the host defense mechanism and participates in maintaining hemostasis, regulating vascular integrity, or regulating nerve activity (Hirst and Robson 2011).Overproduction of NO causes pathological problems associated with acute and chronic inflammation, neurodegeneration, apoptosis, and necrosis (Whittle 1995;Nagai et al. 2003).A large amount of NO is produced when RAW 264.7 cells are activated by LPS.Therefore, NO production in LPS-induced mouse macrophage RAW 264.7 cells was used to evaluate the anti-inflammatory potential of EOs.Of the three EOs tested, only the EO from L. scandens fruits (LSFR) showed significant activity.It showed a moderate ability to inhibit NO production with an IC 50 value of 37.95 ± 2.76 µg/mL, while the IC 50 value of positive control L-NMMA was 7.90 ± 0.63 µg/mL (Table S3).It was also not toxic to macrophage cells (91.76% cell survival at the concentration of 100 μg/mL).The EOs from L. scandens leaves (LSL) and L. hongiaosensis roots (LHR) were not considered notable samples for anti-inflammatory activity as they showed cytotoxicity to macrophage cells with 69.39 and 65.53% cell survival, respectively at the concentration of 100 μg/mL.Some of the main components of the EOs from L. scandens fruits which are known to possess anti-inflammatory activity such as β-caryophyllene (Fidyt et al. 2016;Francomano et al. 2019), caryophyllene oxide (Fidyt et al. 2016), α-humulene (Rogerio et al. 2009), spathulenol (Dos Santos et al. 2022) and valencene (Tsoyi et al. 2011), might have contributed to the general NO production inhibitory effect of LSFR.However, each EO was a mixture of many components that could have a synergistic or inhibitory effect on each other.So the presence of those compounds in LSL and LHR was not enough to bring about an inhibitory effect on NO production without causing macrophage cell death.Therefore, further studies on the activity of each EO component and its anti-inflammatory mechanism are needed.

Cytotoxic activity of LSL, LSFR, and LHR on cancer cell lines
To investigate the antitumor potential of EO samples, three cell lines belonging to the most common cancer types, such as breast, lung, and liver cancers, were selected for screening.The EOs obtained from the two Luvunga species with the higher yield among the six EOs, including LSL, LSFR, and LHR, were selected for in vitro cytotoxicity evaluation.The cytotoxicity of EOs LSL, LSFR, and LHR on the human breast cancer cell line (MCF-7), human lung cancer cell line (SK-LU-1), and human hepatocellular cancer cell line (HepG2) was evaluated using SRB assay.All three EOs showed moderate cytotoxic effects on all three tested cell lines with the IC 50 value ranging from 49.74 ± 3.36 to 97.82 ± 8.61 µg/mL (Table S4).Of the three tested EO samples, LHR exhibited the best cytotoxic effect on all three cancer cell lines MCF-7, SK-LU-1, and HepG2, with the corresponding IC 50 values of 64.99 ± 4.06, 49.74 ± 3.36, and 61.67 ± 5.97 µg/mL, while the IC 50 values of the positive control ellipticine were 0.43 ± 0.02, 0.35 ± 0.02, and 0.44 ± 0.03 µg/mL, respectively.
In general, the EOs from two Luvunga species in the present study contained major components with a variety of known scientifically proven biological effects.Their anti-inflammatory effects through inhibition of NO production in RAW 264.7 cells and cytotoxic activity against three human carcinoma cell lines may provide more basis for further research into their medicinal application.

Plant materials
The roots, stems, and leaves of Luvunga hongiaoensis Tagane were collected on March 4 th 2022 at coordinates 12 °17'60"N, 108 °70'00"E, while the roots, leaves, and fruits of Luvunga scandens (Roxb.)Buch.-Ham. in Wight & Arn. were collected on May 6 th 2022 at coordinates 11 °46'69"N, 108 °06'80"E, from Bidoup-Nui Ba National Park, Lam Dong province, Vietnam.The plants were taxonomically identified by our research team member Dr Luong Van Dung, Faculty of Biology, Dalat University, Vietnam.Voucher specimens (DL220401 and DL220402 for L. hongiaoensis and L. scandens, respectively) have been deposited in the Herbarium of Dalat University.The leaf samples were fully expanded leaves; the fruit samples were mature green whole fruits; the stem samples included both stems and small branches; the root samples included both main roots and lateral roots, washed clean of soil, drained of water.

Obtaining of EOs
The fresh materials were cut and ground in an electric grinder, then hydrodistilled using a Clevenger apparatus, according to the Vietnamese Pharmacopoeia (2017), for three and a half hours.The experiment was repeated three times.The obtained EOs were dehydrated by sodium sulfate and stored in sealed glass vials at 4 °C before being used for chemical analysis or bioassay.The EO samples obtained from roots, stems, and leaves of L. hongiaoensis, and roots, leaves, and fruits of L. scandens were designated as LHR, LHS, LHL, LSR, LSL, and LSFR, respectively.Their yields are shown in Table S1.

Analysis of EOs
The analysis of the EOs was performed using gas chromatography combined with mass spectrometry on a SCION 456-GC/SCION SQ (SCION Instruments, USA), equipped with fused silica capillary Rxi-5MS column (30 m × 0.25 mm, 0.25 μm coating thickness; crossbond 5% diphenyl/95% dimethyl polysiloxane; Restek, USA).Helium was used as the carrier gas at 1.0 mL/min; the oven temperature programme was gradually increased from 60 °C to 246 °C at 3 °C/min, and increased from 246 °C to 280 °C at 20 °C/min; the injector was operating in the split ratio at 1:20, injector temperature 250 °C; the injected sample was 0.1 μL of 10% (v/v) solution of the EOs in GC grade n-hexane.MSD was operated in the EI mode (70 eV), scanning mass range of 50-500 amu, ion source temperature of 250 °C at a sampling rate of 1.0 scan/sec.Linear retention indices (RIs) of the compounds were determined relative to homologous series of n-alkanes C 9 -C 28 under the same experimental conditions.The identification of the compounds was carried out based on a comparison of their mass spectra and RI to those from the NIST mass spectra library (version 2.4, 2020) and the literature (Adams 2017;Linstrom and Mallard 2022).The relative percentages of the compounds in the EOs were calculated on the basis of the peak area by GC software.

Cell culture
All cell lines were cultured in DMEM supplemented with 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, and 10% FBS.The cells were subcultured every three days at a 1:3 ratio and maintained in a humidified chamber at 37 °C, 5% CO 2 (Tsai et al. 2007;Joo et al. 2014).

NO inhibition assay
The inhibitory effect of the EO samples on NO production was evaluated in LPS-stimulated RAW 264.7 cells.RAW 274.7 cells were seeded in 96-well plates at a concentration of 2 x 10 5 cells/well and incubated at 37 °C in a 5% CO 2 humidified atmosphere for 24 h.Then, the culture media were aspirated and replaced with FBS-free DMEM media for 3 h.Test samples at different concentrations (100, 20, 4, and 0.8 μg/mL) were added to each well of 96-well plates, and the cultivation was continued under identical conditions for 2 h.The cells were then stimulated to produce NO by LPS (10 μg/mL) for 24 h.The presence of nitrite was determined using the NO detection kit Griess Reagent System (Promega Cooperation, WI, USA).Briefly, a mixture of 100 μL of cell culture medium and an equal volume of Griess reagent: 50 μL of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid, and 50 μL 0.1% (w/v) N-1-naphthylethylenediamine dihydrochloride in water, was incubated at room temperature for 10 min.Then the absorbance was recorded in a microplate reader at 540 nm.The nitrite content of each sample was determined using the NaNO 2 standard content curve and compared with the negative control (LPS).FBS-free DMEM medium was used as a blank sample, while N G -Methyl-L-arginine acetate (L-NMMA) (Sigma) was used as the positive control.The experiment was performed in triplicate.The inhibitory potency of NO was measured at doses of 100, 20, 4, and 0.8 μg/mL, respectively, and was estimated as the half-maximum inhibitory concentration (IC 50 ), calculated by the Table Curve Version 4.0 program (Systat Software Inc., San Jose, CA, USA) (Tsai et al. 2007;Joo et al. 2014).

MTT cell viability assay
EO samples were added to the wells of a 96-well plate seeded with RAW 264.7 cells to obtain concentrations similar to those of the NO experiment.After 24 h of incubation, MTT was added to a final concentration of 5 mg/mL, and the cells were further incubated at 37 °C in a 5% CO 2 humidified atmosphere for the next 24 h.Finally, after removing the supernatant, the formazan crystals were dissolved in 50 µL DMSO, and the absorbance was measured at 540 nm by using a microplate reader.The percentage of surviving cells was calculated relative to the blank control group (Tsai et al. 2007;Joo et al. 2014).

Cytotoxicity assay
The cytotoxic effect of the EOs on the cancer cell lines SK-LU-1, MCF-7, and HepG2 was evaluated by using SRB colorimetric assay, developed by Skehan et al. and used commonly in the National Cancer Institue (USA) as the accepted method for evaluating the cytotoxic potential of compounds or extracts through a panel of human cancer cell lines (Skehan et al. 1990;Hughes et al. 2011).Cell density was determined based on measuring the optical density (OD) of total cellular proteins stained with SRB.Briefly, the cells were grown in 96-well microtiter plates with 190 µL culture medium per well.After 24 h, 10 µL of the test samples dissolved in 10% DMSO was added to the wells, and incubation was continued at 37 °C for 48 h with 5% CO 2 .After that, TCA was added to fix the cells for one hour, and then SRB was added to stain cell proteins for 30 min.The nonstaining dye was washed off with acetic acid three times.SRB staining protein was dissolved in 10 mM unbuffered Tris base.The OD at 540 nm was measured by using an ELISA Plate Reader.Ellipticine was used as the positive control.The inhibitory percentage of cell growth was calculated by the following formula: (%) inhibition = 100% -[(OD sample -OD day 0 )/(OD blank control -OD day 0 )] x 100.Experiments were carried out in triplicate for the accuracy of data.The TableCurve 2Dv4 computer software was used for data analysis and IC 50 calculation.