Abstract
In this study, we analyzed the volatile constituents of the n-hexane extract from the rhizomes of Homalomena cochinchinensis Engl. by gas chromatography-mass spectrometry (GC-MS). We also investigated the anti-inflammatory, acetylcholinesterase (AChE) inhibitory, and cytotoxic activities of this extract. GC-MS analysis revealed the presence of 25 compounds, of which linalool was the primary component (17.6%). The extract significantly inhibited lipopolysaccharide-induced production of nitric oxide in RAW264.7 cells (IC50 = 21.08 ± 1.80 µg/mL); displayed weak cytotoxic activity against SK-LU-1, HepG2, and MCF7 cancer cell lines (IC50 values ranged from 154.60 to 184.25 µg/mL); and exhibited a weak capacity for AChE inhibition (IC50 = 131.10 ± 9.76 µg/mL). This report is the first to examine the volatile components and biological activities of the n-hexane extract from rhizomes of Homalomena cochinchinensis.
Introduction
Given the increasing demand for herbal drugs, natural health products, and secondary metabolites of medicinal plants, global medicinal plant use is growing rapidly.1,2 Homalomena is a large genus in the Araceae family with approximately 250 species that are primarily distributed in tropical and subtropical regions. 3 Some species play important roles in traditional medicine in countries such as Vietnam, China, India, and Malaysia. In Vietnam, for example, H occulta and H cochinchinensis are used to treat rheumatism, joint pain, paralysis, and inflammation. 4 In addition, in northeastern India, the leaves and rhizomes of H aromatica are used to treat joint pain and skin infections. 5 Recent reports have also described the antibacterial activity of rhizome essential oil and constituents in the acetone extract of H cochinchinensis.6,7 Investigating the composition and biological activities of H cochinchinensis rhizomes is important for clarifying the role of this plant in folk medicine. As part of an ongoing investigation of bioactive metabolites from plants of the Homalomena genus collected in Vietnam,8-10 we report herein the composition of volatile components from H cochinchinensis rhizomes. We also investigated the extract's cytotoxic activity against SK-LU-1 (human lung adenocarcinoma), HepG2 (human hepatocellular carcinoma), and MCF7 (human breast cancer) cell lines; its inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells; and its capacity for acetylcholinesterase (AChE) inhibition.
Results and Discussion
Extraction and Profiling
Gas chromatography-mass spectrometry (GC-MS) analysis of the n-hexane extract (Supplemental Figure S2) revealed the phytochemical diversity of its volatile constituents (Table 1). The components of this extract could be classified into monoterpene hydrocarbons, sesquiterpene hydrocarbons, fatty acids, and fatty acid esters.
Volatile Components of the n-Hexane Extract From the Rhizomes of Homalomena cochinchinensis.
Abbreviations: MS, mass spectrum; Co-GC, co-injection with authentic compounds; RI*, retention index on Equity-5 column; RIa, retention index literature comparison.
The volatile profile of the n-hexane fraction was characterized by high proportions of oplopanone (16.7%), khusinol acetate (10.5%), elemol acetate (4.4%), teucmosin (3.3%), and spathulenol (2.5%). Therefore, sesquiterpenoids were dominant (53.9%) in this species. Linalool (17.6%) was the most abundant component in the n-hexane-soluble portion of H cochinchinensis rhizomes. Interestingly, the chemical composition of the n-hexane extract observed in the present study was very different from that of H. cochinchinensis rhizome essential oil reported in the literature. In particular, Van et al 6 reported that the primary components of H cochinchinensis rhizome essential oil are monoterpenoids (89.6%), whereas our work identified sesquiterpenoids (53.9%) as the main constituents of the n-hexane extract. α-Sabinene (4.2%) is also a major component of rhizome essential oil but was not detected in the n-hexane extract. Nevertheless, linalool was found to be the most abundant component in both the essential oil and n-hexane extract of H cochinchinensis rhizomes (57.4% and 17.6%, respectively). Previous studies have reported high linalool content in the acetone extract of both rhizomes and the aerial parts of H cochinchinenesis. 7 Linalool was also reported as the major constituent of many Homalomena essential oils such as H aromatica rhizome (62.5%), 11 H aromatica root (58.3%), 12 H occulta rhizome (36.9%), 13 and H sagittifolia rhizome (61.9%). 14 Collectively, linalool is considered one of the chemical markers of the Homalomena genus.
Anti-Inflammatory Activity
The anti-inflammatory activity of H cochinchinensis rhizome n-hexane extract is presented in Table 2. The n-hexane extract possessed significant NO-inhibitory activity, with an IC50 value of 21.08 ± 1.80 µg/mL. Its potency was 2 times lower than the positive control, L-NMMA (N(G)-monomethyl L-arginine) (IC50 = 8.90 ± 0.48 µg/mL). Moreover, this extract showed no cytotoxicity against RAW264.7 cells. Thus, the n-hexane extract of H cochinchinensis rhizomes could be considered a natural source of a novel anti-inflammatory agent.
In Vitro Anti-Inflammatory Activity of n-Hexane Extract of HCH.
Abbreviations: HCH, Homalomena cochinchinensis; L-NMMA, N(G)-monomethyl L-arginine; NO, nitric oxide; IC50, concentration that inhibits 50% of cell growth.
Cytotoxicity and Anti-AChE Activities
The cytotoxicity and AChE inhibitory capacity of the n-hexane extract are presented in Table 3. The n-hexane extract showed weak inhibitory effects on the examined cell lines, with IC50 values ranging from 154.60 to 184.25 µg/mL. In addition, this extract demonstrated weak AChE-inhibitory ability (IC50 = 131.10 ± 9.76 µg/mL). It is possible that low-polarity fractions could be contained with low-molecular-weight compounds expected to easily pass through the blood–brain barrier. 15
Cytotoxicity and Anti-Acetylcholinesterase Activity of n-Hexane Extract From Homalomena cochinchinensis rhizomes.
Abbreviations: HCH, Homalomena cochinchinensis; SK-LU-1, human lung adenocarcinoma; HepG2, human hepatocellular carcinoma; MCF7, human breast cancer; Anti-AChE, anti-acetylcholinesterase.
Ellipticine and berberine were used as positive control; IC50 (concentration that inhibits 50% of cell growth).
Previous studies have demonstrated that linalool shows anticancer, AChE-inhibitory, and neuroprotective activities.16,17 Moreover, sesquiterpenes reportedly have potent AChE inhibitory activities and may be considered natural cognitive enhancers for patients with Alzheimer's disease. 18 Recently, we observed that teucmosin, a rare sesquiterpene lactone from H pendula, exhibited significant NO and AChE inhibitory capacities. 10 Thus, the biological activities of the n-hexane extract of H cochinchinensis rhizomes in this work could be explained by the presence of high linalool (17.6%), teucmosin (3.3%), and sesquiterpene hydrocarbon (53.9%) contents.
Notably, the n-hexane fraction likely contains nonpolar, high-molecular-weight components that are not detectable by GC-MS but may contribute to the biological activities of H cochinchinensis rhizomes. Therefore, further phytochemical and biological evaluation of H cochinchinensis rhizome n-hexane extract is necessary to fully characterize its active constituents.
Conclusion
Twenty-five compounds in the n-hexane extract of H cochinchinensis rhizomes were identified via GC-MS analysis. Linalool was identified as the primary component. The extract displayed significant NO-inhibitory effects; weak cytotoxic activity against SK-LU-1, Hep G2, and MCF7 cancer cell lines; and weak AChE-inhibitory capacity. This study is the first to investigate the volatile components and biological activities of n-hexane extract from H cochinchinensis rhizomes. The study improves our understanding of H cochinchinensis and indicates that its rhizomes have the potential for the development of new anti-inflammatory agents.
Materials and Methods
Preparation of Plant Extract
H cochinchinensis rhizomes were collected in Dong Nai province, Vietnam in May 2022 (Supplemental Figure S1). The plant was authenticated by Dr Anh Tuan Le (Mientrung Institute for Scientific Research, Vietnam National Museum of Nature, VAST, Vietnam). A voucher specimen (TNK-DN-02) has been deposited at the Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Vietnam (Supplemental Figure S10). The rhizomes were washed and dried to remove any dust. The dried rhizomes of H cochinchinensis were cut into small pieces and extracted with methanol (MeOH) (each, 7 days × 3 times) at room temperature. The residue was filtered, and the solvents were removed to yield the crude extract. The methanolic extract was suspended in water and partitioned with n-hexane (50 mL × 3 times). The n-hexane layer was concentrated in vacuo before analyzing the chemical composition and evaluating the biological activities.
Analysis of the Volatile Compounds
GC-MS was used to determine the volatile components using a Shimadzu GCMS-QP2010 Plus system (Kyoto, Japan) with an Equity-5 capillary column (30 m × 0.25 mm, 0.25 m film thickness), and a mass spectrometer (MSD QP2010 Plus). The n-hexane extract (1 mg) and dichloromethane were diluted in a ratio of 1:100, and 1 µL was used for analysis. After 2 min of preheating the oven to 60 °C, the temperature was scheduled from 240 °C at a rate of 3 °C/min (10 min hold) and increased to 280 °C at a rate of 5 °C/min (40 min hold). The carrier gas was helium at a flow rate of 1.5 mL/min. The following conditions are applied to mass detectors: mass acquisition range of 40 to 500 and interface temperature of 280 °C. The samples were injected using splitless injection mode. By comparing the mass spectra to the Wiley 7 and NIST 11 libraries, the constituents were identified and the retention index was calculated using a standard solution of C8 – C38 alkanes and comparing them with literature data. 19 The GC peak area was used to calculate the relative quantities of each component, without correction.
Anti-Inflammatory Assay
The n-hexane extract was evaluated for its inhibitory activity against the LPS-induced production of NO in RAW 264.7 cells. The nitrite concentration, as an indicator of the presence of NO in the culture medium, was measured by the Griess reaction. The detailed protocols have been carefully described in our previous reports.20,21
AChE Inhibition Assay
AChE inhibitory activity of the n-hexane extract was performed in 96-well microtiter plates and measured via an assay described by Ellman et al 22 with slight modification. The detailed protocols have been described in our previous publication. 23
Sulforhodamine B (SRB) Assay for Evaluating Cytotoxic Activity
The cytotoxic activity of the extract was determined by SRB assay, as described by Monks et al. 24 The detailed protocols were given in our earlier publication. 25
Supplemental Material
sj-docx-1-npx-10.1177_1934578X231168481 - Supplemental material for Volatile Components and Biological Activities of n-Hexane Extract From Rhizomes of Homalomena cochinchinensis
Supplemental material, sj-docx-1-npx-10.1177_1934578X231168481 for Volatile Components and Biological Activities of n-Hexane Extract From Rhizomes of Homalomena cochinchinensis by Linh Thuy Khanh Nguyen, Phu Quynh Dinh Nguyen, Nghia Ai Thi Doan, Chau Bao Hoai Nguyen, Tuan Quoc Doan, Linh Thuy Thi Tran, Hoai Thi Nguyen and Duc Viet Ho in Natural Product Communications
Footnotes
Acknowledgments
This research was supported by Hue University (ID No. DHH2022-04-165) and in part by the Strong Research Group Program of Hue University (ID No. NCM.DHH.2023.02).
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) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Hue University (ID No. DHH2022-04-165), and in part by the Strong Research Group Program of Hue University (ID No. NCM.DHH.2023.02).
Ethical Approval
Not applicable, because this article does not contain any studies with human or animal subjects.
Statement of Human and Animal Rights
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Informed Consent
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Trial Registration
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References
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