GC-MS Characterization,in Vitro Antioxidant and Anti-Inflammatory Activities of Essential oil from the Leaves of Litsea balansae Lecomte

Abstract

Objective: This study aimed to analyze the essential oil obtained from Litsea balansae Lecomte leaves and investigate its potential as an anti-inflammatory and antioxidant agent. Methods: Fresh L balansae leaves were collected in Quangtri, Vietnam, and subjected to hydro-distillation using a Clevenger-type apparatus to extract the essential oil. The essential oil composition was analyzed using gas chromatography–mass spectrometry (GC-MS). Antioxidant capacity was evaluated through the 2,2-Diphenyl-1-picrylhydrazyl assay, while the inhibition of nitric oxide production was assessed in lipopolysaccharide-activated RAW264.7 cells. Results: GC-MS analysis identified 71 compounds, constituting 99.3% of the essential oil. Key constituents included carvacrol (20.8%), caryophyllene oxide (11.0%), cis-β-guaiene (6.4%), khusimone (3.9%), bicycloelemene (3.7%), and spathulenol (3.7%). The essential oil exhibited a moderate antioxidant effect (half maximal inhibitory concentration [IC₅₀]= 208.37 ± 10.67 µg/mL) and significant anti-inflammatory activity (IC₅₀= 28.09 ± 1.53 µg/mL). Conclusion: The essential oil derived from L balansae leaves showcases potential as an anti-inflammatory and antioxidant agent, as indicated by its chemical composition and biological activities. These findings suggest promising avenues for further research in the development of therapeutic agents from natural sources.

Introduction

Lauraceae, also known as the laurel family, is a diverse and widespread group of flowering plants with more than 2500 species of trees and shrubs. Among the 45 genera of this family, Litsea is the most diverse genera which comprises roughly 400 species known for their aromatic properties and various uses.^1–3 These plants typically thrive in tropical and subtropical climates, including Asia, North America, and Australia, where they are appreciated for their cultural, culinary, and medicinal significance. ⁴ In traditional medicine, the leaves, fruits, or essential oils derived from Litsea plants have been employed to treat various ailments, including digestive issues, respiratory conditions, and skin disorders. ⁴ These plants contain terpenes and phenolic compounds with broad-spectrum activity such as anti-inflammatory,^2,4 antioxidant,^5,6 antimicrobial, ⁷ and anticancer^8,9 properties.

Litsea balansae, commonly known as “Bời lời balansa” in VietNam, is a medium-sized evergreen tree with a dense and rounded crown. This species can be found in various regions throughout the country, particularly in mountainous areas and forests.^10,11 In Vietnamese traditional medicine, infusions or decoctions made from parts of L balansae plant are often used to treat ailments such as fever, respiratory infections, digestive disorders, and skin conditions.^12,13 To the best of our knowledge, no information could be seen on either chemical constituents or biological activities of L balansae essential oil. Therefore, this study aims to provide the chemical composition, anti-inflammatory and antioxidant activities of the leaf essential oil derived from L balansae plant for the first time.

Results and Discussion

The fresh leaves of L balansae plant have been collected from Quangtri, Vietnam. They were hydro-distilled using a Clevenger-type apparatus to afford the essential oil. After that, the essential oil was analyzed by gas chromatography–mass spectrometry (GC-MS), and the outcomes are outlined in Table 1. The yield of the yellow essential oil was found to be 0.1% (v/w) when calculated based on the fresh weight. In total, we identified 71 compounds, which accounted for 99.3% of the oil's composition. These compounds were categorized into 3 main chemical groups, namely oxygenated sesquiterpenoids (45.2%), sesquiterpene hydrocarbons (26.9%), and oxygenated monoterpenoids (23.7%). Additionally, nonterpenic compounds were present in smaller amounts (3.5%) (Table 1, Figure 1). Among oxygenated sesquiterpenoids, caryophyllene oxide presented with a dominant proportion (11.0%), followed by khusimone (3.9%), spathulenol (3.7%), vulgarone B (2.8%), α-cadinol (2.6%), γ-eudesmol (1.9%), mustakone (1.7%), and several others. Trace amounts of compounds such as 1-epi-cubenol, α-muurolol, carotol, and viridiflorol were also detected. Sesquiterpene hydrocarbons constituted the second most abundant group, representing 26.9% of the essential oil. Prominent constituents in this class included cis-β-guaiene (6.4%), α-amorphene (2.5%), β-elemene (1.4%), and δ-cadinene (1.4%). Other compounds, including aromadendrene, α-copaene, allo-aromadendrene, and α-trans-bergamotene, were present in smaller amounts (Table 1). Oxygenated monoterpenoids comprised 23.7% of the essential oil composition, with carvacrol being the most abundant compound at 20.8%. Additionally, α-terpineol, borneol, endo-fenchol, iso-dihydro carveol acetate, neoiso-3-thujanol, and santalone were identified in this group. Surprisingly, monoterpene hydrocarbons were not detected in essential oil. Nonterpenic compounds were identified in the essential oil at a relatively low content of 3.5%. These compounds included n-decanal, 2-undecanone, dodecanal, n-nonanol, thymohydroquinone dimethyl ether, n-nonanal, and 2-dodecanone.

Figure 1.

The major chemical constituents of L balansae leaf essential oil: Carvacrol (A), caryophyllene oxide (B), cis-β-guaiene (C), khusimone (D), bicycloelemene (E) and spathulenol (F).

Table 1.

Chemical Compounds in L balansae Leaf Essential oil.

No	RT	Compounds	RIE	RIL	Concentration (%)
1	13.14	n-Nonanal	1105	1100	0.2
2	13.53	endo-Fenchol	1114	1114	0.6
3	15.06	neoiso-3-Thujanol	1148	1147	0.1
4	15.83	Borneol	1166	1165	0.7
5	16.11	n-Nonanol	1172	1165	0.3
6	16.35	Santalone	1178	1177	0.1
7	16.95	α-Terpineol	1191	1186	1.0
8	17.61	n-Decanal	1206	1201	1.6
9	21.54	2-Undecanone	1295	1293	0.5
10	21.93	Carvacrol	1304	1298	20.8
11	22.99	iso-Dihydro carveol acetate	1328	1326	0.4
12	23.41	Bicycloelemene	1338	1338	3.7
13	25.07	α-Copaene	1377	1374	0.7
14	25.77	β-Elemene	1393	1389	1.4
15	25.87	2-Dodecanone	1395	1389	0.2
16	26.45	Dodecanal	1409	1408	0.4
17	26.60	α-Cedrene	1413	1410	0.3
18	26.74	cis-α-Bergamotene	1416	1411	0.4
19	26.91	(E)-Caryophyllene	1420	1417	1.2
20	27.09	Thymohydroquinone dimethyl ether	1425	1424	0.3
21	27.25	(E)-α-Ionone	1429	1428	0.4
22	27.50	γ-Elemene	1435	1434	0.3
23	27.58	trans-α-Bergamotene	1437	1432	0.6
24	27.72	Aromadendrene	1440	1439	0.9
25	27.90	6,9-Guaiadiene	1445	1442	0.2
26	28.32	α-Humulene	1455	1452	1.2
27	28.57	allo-Aromadendrene	1461	1458	0.7
28	29.22	β-Chamigrene	1477	1476	1.3
29	29.52	ar-Curcumene	1484	1479	0.6
30	29.66	α-Amorphene	1488	1483	2.5
31	29.87	δ-Selinene	1493	1492	0.3
32	30.09	cis-β-Guaiene	1498	1492	6.4
33	30.23	epi-Cubebol	1501	1493	1.0
34	30.56	(Z)-α-Bisabolene	1510	1506	0.4
35	31.15	δ-Cadinene	1525	1525	1.4
36	31.32	Kessane	1529	1529	0.1
37	31.58	trans-Cadina-1,4-diene	1536	1533	0.8
38	31.72	α-Cadinene	1539	1537	0.3
39	31.88	Selina-3,7(11)-diene	1544	1545	0.5
40	32.17	Elemol	1551	1548	0.4
41	32.48	trans-Dauca-4(11),7-diene	1559	1556	0.2
42	32.58	Germacrene B	1561	1559	0.6
43	32.73	(E)-Nerolidol	1565	1561	0.4
44	32.98	Caryophyllenyl alcohol	1572	1570	1.0
45	33.30	Spathulenol	1580	1577	3.7
46	33.52	Caryophyllene oxide	1586	1582	11.0
47	33.84	Viridiflorol	1594	1592	0.7
48	33.93	Carotol	1596	1594	0.7
49	34.08	Guaiol	1600	1600	0.3
50	34.24	Khusimone	1604	1604	3.9
51	34.51	Humulene epoxide II	1611	1608	1.1
52	34.66	cis-Isolongifolanone	1615	1612	1.0
53	35.02	Isolongifolan-7-α-ol	1621	1618	0.6
54	35.02	10-epi-γ-Eudesmol	1625	1622	0.6
55	35.25	1-epi-Cubenol	1631	1627	0.8
56	35.36	γ-Eudesmol	1634	1630	1.9
57	35.53	Caryophylla-4(12),8(13)-dien-5α-ol	1638	1639	1.2
58	35.53	allo-Aromadendrene epoxide	1640	1639	1.2
59	35.76	α-Muurolol	1644	1644	0.7
60	36.05	Vulgarone B	1652	1649	2.8
61	36.20	α-Cadinol	1657	1652	2.6
62	36.32	α-Bisabolol oxide B	1660	1656	1.2
63	36.64	14-hydroxy-(Z)-Caryophyllene	1668	1666	0.6
64	36.82	14-hydroxy-9-epi-(E)-Caryophyllene	1673	1668	1.6
65	37.03	Mustakone	1679	1676	1.7
66	39.08	Eremophilone	1736	1734	0.6
67	39.67	Xanthorrhizol	1752	1751	0.3
68	40.22	14-oxy-α-Muurolene	1768	1767	0.4
69	40.77	(E)-α-Atlantone	1783	1777	0.3
70	40.95	γ-Eudesmol acetate	1788	1783	0.2
71	42.91	(2E,6E)-Farnesyl acetate	1846	1845	0.2
		Total			99.3
		Oxygenated monoterpenes			23.7
		Sesquiterpene hydrocarbons			26.9
		Oxygenated sesquiterpenes			45.2
		Non-terpenic compounds			3.5

Abbreviations: RT, retention time; RI_E, retention indices relative to n-alkanes (C₇-C₄₀) on equity-5 column; RI_L, retention indices from the Adams book.¹⁴

According to reported data, there are some differences between the major chemical composition of L balansae leaf oil and other Litsea species collected in Vietnam such as L cambodiana, L viridis, L cubeba, L glutinosa, L helferi, L ferruginea, L verticillata. For instance, some of monoterpene hydrocarbons included α-pinene, camphene, sabinene, β-pinene, myrcene, α-phellandrene, p-cymene, limonene presented in leaf essential oil of Litsea species as a major component. However, these compounds were not identified in L balansae leaf essential oil. Similarly, 1,8-cineole and linalool, 2 major chemotypes of L. cubeba leaf essential oil, were conspicuously absent in L balansae. γ-Terpinene, a significant constituent of L ferruginea leaf essential oil, is also identified with minor quantities in other Litsea species such as L viridis, L cubeba, L glutinosa, L verticillata. However, γ-terpinene is not identified in L balansae leaf essential oil. Surprisingly, the abundance carvacrol (20.8%), cis-β-guaiene (6.4%) and khusimone (3.9%) presented in L balansae essential oil were conspicuously absent in all of the other oil samples.^15,16,17,18 The differences observed in the chemical classes and composition of Litsea essential oils can be attributed to a range of factors. These include the nature of the plant, its geographical location, the methods used for extraction, the specific parts of the plant utilized, the conditions under which it was cultivated, and the stage of maturity at which it was harvested. In summary, it is these variations in chemical composition that ultimately contribute to the distinctive biological characteristics of L balansae essential oil.

In addition, the antioxidant capacity of the essential oil was assessed using the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay, as detailed in Table 2. The essential oil displayed moderate activity, with an half maximal inhibitory concentration (IC₅₀) value of 208.37 ± 10.67 µg/mL. Interestingly, this IC₅₀ value is comparable to that of the positive control, L-ascorbic acid, which had an IC₅₀ value of 7.60 ± 0.31 µg/mL. It's worth noting that the essential oil from L balansae leaves exhibited only moderate antioxidant efficacy, which could be attributed to the relatively low concentrations of total phenolic compounds in this oil. In contrast, previous studies have reported significant antioxidant activity in the essential oils of Litsea cubeba and Litsea pungens.^19,20

Table 2.

IC₅₀ Values of DPPH Scavenging of Leaf Essential oil of L balansae.

Concentration (µg/mL)	% DPPH scavenging
	Leaf essential oil of L balansae		L-Ascorbic acidb
	Average	SD	Average	SD
500	86.61	1.88	-	-
100	32.80	1.52	95.56	1.18
20	6.10	1.11	72.04	1.29
4	3.58	0.29	34.25	0.79
0.8	-	-	12.34	0.34
IC50a	208.37 ± 10.67		7.60 ± 0.31

Abbreviations: SD: standard deviation; IC₅₀, half maximal inhibitory concentration.

^a

IC₅₀: scavenging concentration at 50%—concentration that neutralizes 50% of DPPH-free radicals.

^b

Positive control;

Nitric oxide (NO) plays a crucial role in the development of inflammatory responses, and excessive production of NO can lead to various pathological issues, including acute and chronic inflammation, apoptosis, necrosis, and neurodegenerative diseases. Inhibiting the accumulation of NO presents a promising therapeutic strategy.^21,22 To explore this further, we evaluated inhibitory activity on NO production in LPS-induced RAW264.7 macrophage cells of the essential oil, and the outcomes are presented in Table 3. Notably, the essential oil of L balansae exhibited significant inhibition of NO production with an IC₅₀ value of 28.09 ± 1.53 µg/mL. As a reference point, the positive control dexamethasone displayed an IC₅₀ value of 15.95 ± 1.05 µg/mL. A literature survey has shown that carvacrol exerts anti-inflammatory effects through the modulation of inflammatory mediators, including IL-1β and prostanoids while caryophyllene oxide, isolated from the bark of Annona squamosa, has demonstrated noteworthy and quantifiable anti-inflammatory activity in empirical studies.^23,24 These components were found in the essential oil of L balansae with high amounts, 20.8 and 11.0%, respectively. Therefore, it is reasonable to believe that these compounds contribute significantly to the anti-inflammatory activity of L balansae essential oil. These findings strongly suggest that the essential oil from L balansae plant will be a source for discovering new anti-inflammatory agents.

Table 3.

In Vitro Anti-Inflammatory Activity of Leaf Essential oil of L balansae.

Concentration (µg/mL)	Leaf essential oil of L balansae		Dexamethasonea
	NO inhibition rate (%)	Viability rate (%)	NO inhibition rate (%)	Viability rate (%)
100	90.04	95.79	81.36	95.18
20	42.81	97.86	52.27	98.56
4	19.97	-	40.39	-
0.8	9.50	-	34.73	-
IC50b	28.09 ± 1.53		15.95 ± 1.05

^a

Positive control.

^b

IC₅₀: concentration that inhibits 50% of cell growth.

Conclusion

In this research, we present, for the first time, a comprehensive analysis of the phytochemical composition, anti-inflammatory, and antioxidant properties of L balansae leaf essential oil. The oil itself is a yellow liquid with a distinct aromatic fragrance. Through our analysis, we identified a total of 71 compounds, with carvacrol (20.8%), caryophyllene oxide (11.0%), cis-β-guaiene (6.4%), khusimone (3.9%), bicycloelemene (3.7%), and spathulenol (3.7%) being the predominant components. Furthermore, the essential oil exhibited notable DPPH radical scavenging activity, as well as the ability to inhibit LPS-induced NO production in RAW 264.7 cells. The calculated IC₅₀ values were 208.37 ± 10.67 and 28.09 ± 1.53 µg/mL, respectively. These findings represent a significant contribution to the understanding of the chemical composition of L balansae. Moreover, they underscore the potential of this plant as a valuable resource for the discovery of new anti-inflammatory and antioxidant agents.

Materials and Methods

Plant Materials

On Monday, June 12, 2023, fresh leaves of L balansae were collected from Quangtri, Vietnam, located at N 17° 03.291’, E 107° 04.344’. The botanical name of the plant was identified by Dr Anh Tuan Le, who is one of the co-authors of our research. To establish a reference for future studies, a voucher specimen labeled LBTB-2023 (L balansae leaves) was deposited at the Faculty of Chemistry, University of Education, Hue University in Vietnam.

Distillation

Each fresh powder sample weighing 1.0 kg was subjected to hydro-distillation using a Clevenger apparatus. The extraction process lasted for 4.0 h, during which the essential oils were released from the plant material. The oils obtained by decantation after hydro-distillation were dried over Na₂SO₄ to remove any residual water content. Subsequently, the dried essential oils were stored in sealed vials maintained at a temperature of −5 °C to ensure their stability and preserve their chemical composition for further analysis. ²⁵

The GC-MS Analysis

The GC-MS analysis was conducted using a Shimadzu Technologies GCMS-QP2010 Plus chromatograph (Shimadzu, Kyoto, Japan) equipped with a fused silica Equity-5 capillary column (30 m × 0.25 mm, film thickness 0.25 µm, Supelco).^26,27,28,29 The analytical parameters were set as follows: Helium was used as the carrier gas at a flow rate of 1.5 mL/min, with injector and interface temperatures set at 280 °C. The temperature program involved a ramp from 60 °C (held for 2 min) to 240 °C (held for 10 min) at a rate of 3 °C/min, followed by a final ramp to 280 °C (held for 40 min) at a rate of 5 °C/min for the column. A split ratio of 10:1 was used for sample injection, with an inlet pressure of 93.2 kPa and an injection volume of 1.0 µL. The mass spectrometer settings consisted of an ionization voltage of 70 eV, a detector voltage of 0.82 kV, and data acquisition in the scan mass range of 40 to 500 amu at a sampling rate of 0.5 scan/s. The identification of chemical constituents was accomplished by co-injecting the samples and comparing the retention indices (RI) to a homologous series of n-alkanes (C₇-C₄₀), as well as referencing the Adams book.¹⁴ Quantification was carried out on the basis of the relative area of the total ion chromatogram peaks of volatile compounds. The comprehensive analysis of the obtained GC-MS data allows for the identification and characterization of the chemical compounds present in the L balansae essential oils, serving as a foundation for further investigations and potential applications.

Measurement of Nitric Oxide Production of the Essential Oil

A total of 274.7 cells from the RAW cell line were distributed into a 96-well plate, with each well containing a density of 5.104 cells. The plate was then placed in a 5% CO₂ incubator at a temperature of 37 °C for 24 h. Following this incubation period, the medium in each well was removed, and a fresh FBS-free DMEM medium was added for a 3-h period. After pretreatment with different concentrations of the essential oil for 2 h, the cells were further stimulated with LPS (1 g/mL) for an additional 24 h. To assess nitric oxide (NO) production, the accumulation of nitrite (NO2⁻) in the culture medium was measured using a colorimetric assay based on the Griess Reagent System (Promega Corporation). Briefly, 100 μL of cell culture medium was combined with an equal volume of Griess Reagent, which consists of 50 μL of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid and 50 μL of 0.1% (w/v) N-1-naphthylethylenediamine dihydrochloride in water. This mixture was then incubated for 10 min at room temperature, and the nitrite concentration was determined by measuring the absorbance at 540 nm using a microplate reader. For reference, a fresh culture medium was utilized as a blank in all experiments. Dexamethasone served as the positive control in this study. To quantify the amount of nitrite in the samples, a NaNO₂ serial dilution concentration standard curve was used, and nitrite production was subsequently measured. The NO inhibition capacity of the sample was calculated using the following formula: ³⁰

$$\begin{array}{rl}(\text{\%})\mathrm{inhibition}& =100\text{\%}\\ & -{\displaystyle \frac{\mathrm{concentration}\mathrm{of}\mathrm{NO}\mathrm{sample}}{\mathrm{concentration}\mathrm{of}\mathrm{NO}\mathrm{negative}\mathrm{control}}\times 100\text{\%}}\end{array}$$

The IC₅₀ value was calculated using Table Curve 2Dv4 software (Systat Software Inc.). The data obtained from 3 independent experiments were statistically analyzed and presented as the mean ± standard deviation (SD).

DPPH Radical Scavenging Activity

The DPPH radical scavenging tests were conducted following Abramovič et al's method with some modifications.^31,32 The sample was diluted with a methanol stock solution and then further diluted with double distilled water to prepare a range of solutions with different concentrations. _L-Ascorbic acid from Sigma was used as a reference control, and its aqueous solutions at various concentrations were also prepared using double distilled water. To create the DPPH solution, DPPH from Sigma was dissolved in 100% methanol to make a 0.25 M solution. The experimental procedure involved adding 100 µL of the research sample in methanol at different concentrations to separate wells in a 96-well plate. Then, 100 µL of the prepared DPPH solution was added in a 1:1 ratio to each well. The control well (blank well) contained 100 µL of water and 100 µL of DPPH. After incubating the plate at room temperature for 30 min, the absorbance of the solutions (OD) was measured at 517 nm. The ability of the test sample to neutralize the free radicals generated from DPPH was determined using the following equations:

$$\text{\%}\mathrm{Scavenging}\mathrm{Activities}=[(\mathrm{ODc}\text{–}\mathrm{ODs})/\mathrm{ODc}]\mathrm{x}100$$

$$\mathrm{Where}:\mathrm{ODc}=\mathrm{OD}(\mathrm{well}\mathrm{without}\mathrm{reagent})\text{-}\mathrm{OD}(\mathrm{blank}\mathrm{well})$$

$$\mathrm{ODs}=\mathrm{OD}(\mathrm{reagent}\mathrm{well})-\mathrm{OD}(\mathrm{blank}\mathrm{well})$$

The IC₅₀ value (scavenging concentration at 50%), representing the concentration at which 50% of DPPH-free radicals are neutralized, was determined using TableCurve 2Dv4 software.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231214159 - Supplemental material for GC-MS Characterization, in Vitro Antioxidant and Anti-Inflammatory Activities of Essential oil from the Leaves of Litsea balansae Lecomte

Supplemental material, sj-docx-1-npx-10.1177_1934578X231214159 for GC-MS Characterization, in Vitro Antioxidant and Anti-Inflammatory Activities of Essential oil from the Leaves of Litsea balansae Lecomte by Duc V. Ho, Hanh N. T. Hoang, Nguyen H. Nguyen, Hang B. Do, Hung Q. Vo, Anh T. Le, Thang Q. Le and Ty V. Pham in Natural Product Communications