INTRODUCTION:

Amidst the advancement of technology in medicine, the role of traditional phytomedicine could still maintain its importance, even becoming an inspiration for modern drug discoveries^1-5. In this light, our research group investigated several plants with potential medicinal properties, including Annona squamosa^6,7 and Myristica fragrans^8,9. Waste product from cacao (Theobroma cacao L) was found potential in performing antiproliferative activities, especially that of extracted using n-hexane^10-12.

Previously, we have extracted Blumea balsamifera leaves using ethyl acetate solvent, where antioxidant activity of the extract could be used to biosynthesize Cu nanoparticles^13,14. Taken altogether, B. balsamifera is one of the significant phytomedicine, in which its bioactivities required to be explored.

Herein, we intended to screen the bioactivity of B. balsamifera leaves extracted using n-hexane based on its antioxidant and cytotoxic activities. Most of previously reported investigations isolated the essential oils from B. balsamifera¹⁵. Antioxidant activity of B. balsamifera essential oils was found to inhibit nitric oxide (NO) radicals providing protective behavior on neural system against inflammation¹⁶. Anti-inflammatory behavior of essential oils from this plant was also observed in macrophage through the downregulation of proinflammatory cytokines namely tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6¹⁷. Nonetheless, literatures reporting on B. balsamifera leaves extracted using n-hexane solvent by means of partitioning is hardly to be found. Meanwhile, different extraction methods could yield bioactivities and chemical compounds^18-21. Hence, this present work is important in filling the gap of research on providing the phytochemical profile of n-hexane extract from B. balsamifera leaves along with its antioxidant and cytotoxic activities.

MATERIALS AND METHODS:

Materials:

In this study, the followings materials were used: methanol, n-hexane, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and dimethyl sulfoxide. Vitamin C was purchased from the local pharmacy with pharmaceutical grade. Otherwise mentioned, all materials were procured from Merck (Selangor, Malaysia) and analytical grade. Plant specimen was collected from Aceh Selatan Regency, Aceh Province, Indonesia in October 2020. The specimen was brought to the Herbarium Laboratory in the Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Syiah Kuala, Indonesia. The specimen was then identified as B. balsamifera L. Before used, the plant leaves were air-dried and crushed into fine powder.

Extraction of B. balsamifera L leaves:

n-Hexane extract from B. balsamifera L leaves was produced from the partition following the methanolic maceration of the plant simplicia (at room temperature; with four repetitions – 24 h each). The partition itself was performed using separating funnel with n-hexane solvent. The mixture was shaken rigorously, until two separated layer was formed (Figure 1a,b). The lipid layer formed at the bottom of the separating funnel was removed (Figure 1b). The remaining extract was evaporated using rotary evaporator (270 mbar; 50°C; 120rpm).

(A) (b)

Figure 1. Mixture of methanolic maceration product and n-hexane after a rigorous shaking (a) and the two separated layer with lipid residue formed after a few minutes (b).

GC-MS:

Gas chromatography-mass spectroscopy (GC-MS) analysis was carried out to determine the phytoconstituents of the n-hexane extract from B. balsamifera. The GC-MS system utilized was that from Shimadzu, Kyoto, Japan with series no. QP2000A. Firstly, the sample (1μL) was injected into the the GC-MS column (Agilent 19091S-433 HP-5MS; 5% phenyl methyl siloxane). The analysis was performed with increasing temperature of 15°C/min and the flow rate was maintained at 1mL/min. Mass spectrometer acted as a detector for the eluted components of the n-hexane sample, where the data was compared with the reference from the National Institute of Standards and Technology (NIST).

DPPH assay:

Antioxidant properties of the extract was evaluated based on its scavenging activity against DPPH assay following the procedure from previous reports. The n-hexane extract was varied in concentration from 25, 50, to 100ppm, where the extract was dissolved with methanol until the total volume reached 5mL. DPPH 0.4 mM (1mL) was then added to the extract solution and the covered with aluminum foil. Thereafter, the mixture was vortexed until homogenous and incubated (37°C; 30 min). The concentration of DPPH was then measured using UV-vis spectrophotometer (UVmini-1240, Kyoto, Japan) at maximum wavelength of 517nm. Positive control used for the antioxidant properties was vitamin C or ascorbic acid (3—9ppm). The procedure was performed in duplicate.

BSLT assay:

Cytotoxicity of the n-hexane extract was screened using brine shrimp lethality test (BSLT) assay carried out in duplicate. Artemia salina larvae which had been previously hatched and grew for 48 h were placed onto 5 mL containing filled with sea water. The larvae were exposed to the respective n-hexane extract that had been varied in concentrations (1, 10, 100, 500, and 1000ppm) following the dilution with dimethyl sulfoxide. Each group consisted of 10 A. salina larvae. A group which was not exposed with the n-hexane extract was assigned as the control. The container was then stored under a tubular lamp and observed for 24 h.

Data analysis:

Results from DPPH and BSLT assays were presented in averaged values. The minimum concentration required to inhibit a half of the DPPH concentration (IC₅₀) and the minimum concentration required to yield 50% lethality (LC₅₀) were obtained from a linear calibration curve. All data analyses were performed on OriginLab Software (Northampton, MA, USA).

RESULTS AND DISCUSSION:

Antioxidant activity:

Antioxidant activity of the n-hexane extract obtained from B. balsamifera L. leaves was determined based on DPPH inhibition assay, where the data have been presented in Figure 2. The increase on DPPH scavenging activity was found to be concentration-dependent. At highest concentration of the extract sample (100ppm), the inhibition of DPPH was 15.716%. Using the linear fitting curve, IC₅₀ of the n-hexane extract sample was found reaching 281.707ppm. In comparison with the control, vitamin C (IC₅₀=4.326 ppm), the IC₅₀ value of the n-hexane extract was dramatically poorer. Antioxidant value obtained herein could be categorized as weak since the IC₅₀ exceeded 250ppm^22-24.

Figure 2. DPPH inhibition by n-hexane extract from B. balsamifera L. leaves

Low antioxidant activity does not mean that the extract has no active compounds, but it could be ascribed to the presence of inactive or inert compounds unreactive against DPPH. In a study employing Cu nanoparticles synthesized with Polyalthia longifolia roots, the DPPH scavenging activity of the sample was only 509.03ppm but could perform effective antimicrobial activities²⁵. In our previous experiments, we also obtained relatively lower antioxidant activities in n-hexane extract, for example that of from Annona squamosa leaves⁶ and Myristica fragrans Houtt. barks⁹. Ethyl acetate extract from B. balsamifera L. leaves, on the other hand, was found to have IC₅₀ of 64ppm for DPPH inhibition activity¹³. Essential oils extracted using diethyl ether had better DPPH assay-based antioxidant properties with the IC₅₀s ranged from 24 to 29ppm¹⁵.

Cytotoxicity:

Cytotoxicities of the extract and fractions from B. balsamifera L leaves were screened by means of BSLT assay, in which the data have been presented (Figure 3). The number of died A. salina larvae increased as the concentration increased. As in control group, there was no mortality observed. At 100ppm of extract concentration, the mortality percentage was 40%. From the linear equation, the LC₅₀ was found to be 44.47ppm. When the LC₅₀value falls within the range of 30—100 ppm, the phytocompounds could be categorized as moderately active as anticancer²⁶. Hence, the n-hexane extract from B. balsamifera L. leaves was revealed as moderately active anticancer agent.

Figure 3. Mortality of A. salina with respect of n-hexane extract from B. balsamifera L. leaves in different concentrations.

Most of the plant n-hexane extracts are reported to possess high cytotoxicity, such as those from Theobroma cacao L. (0.29—2.37ppm)^10,12. In another study, n-hexane extract from Abelmoschus manihot also possesses cytotoxic activity against breast cancer 4t1 cells line²⁷. However, in a study comparing three extracts from Phyllanthus emblica stem barks obtained using n-hexane, ethyl acetate, and methanol, the n-hexane extract was found to have the lowest cytotoxic activity²⁸. The B. balsamifera L leaves extract is potential to have improved LC₅₀ when used with Cu nanoparticles¹³. Efficacy of B. balsamifera plant have been studied against hepatocellular carcinoma cells²⁹ and cervical cancer cells³⁰. Phytocompounds could facilitate the apoptosis of cells by targeting cancer-related biomolecules, such as glucosaminyl (N‑Acetyl) transferase 3, tyrosine‑protein kinase Met (c‑Met), peroxisome proliferator‑activated receptor γ, sex‑determining region Y and nuclear factor kappa beta ^31-33. In the case B. balsamifera, the mechanism has been proven via APRIL – a member of tumor necrosis factor (TNF)³⁴.

Phytochemical profile:

Chromatogram of the GC-MS analysis performed on n-hexane extract from B. balsamifera L leaves has been presented (Figure 4). The names of compounds along with their retention time and peak area (%) have been presented in Table 1. Two compounds appear to dominate the phytoconstituents of the extract sample, namely borneol L (33.77%) and veridiflorol (22.57%). Other phytocompounds had peak areas of less than 5%, they are: (-)-Caryophyllene oxide (4.95%); bicyclo[2.2.1]heptan-2-one,1,7,7-trimeth (4.01%); trans-caryophyllene (3.44%), beta-selinene (3.41%), and caryophyllene <14-hydroxy-9- (3.12%). In a previous study, the predominant phytocompounds of ethyl acetate extract from B. balsamifera L leaves included naphthalene, cryptomeridiol, eugenol¹³. Further isolation of the ethyl acetate extract, in different previous study, allowed the identification of lutein, beta-carotene, icthyothereol acetate, as well as cryptomeridiol³⁵. Taken altogether, most of the phytocompounds identified herein, are newly reported for B. balsamifera leaves.

Both borneol L and veridiflorol are essential oils which are included as fragrance materials. Borneol has been reported in multiple times for its medicinal benefits including as ischemic stroke therapeutic agent ³⁶ and vasorelaxant agent³⁷. In the case of anticancer properties, borneol could induce cell apoptosis via reactive oxygen species (ROS)^38,39. As for veridiflorol, the compound was found predominantly in Salvia macilenta Boiss. essential oil which had antimicrobial activities⁴⁰. Similarly, methanolic extracts from Syzygium gratum and Melaleuca armillaris which both contained predominant veridiflorol, were reported to possess antimicrobial and antioxidant⁴¹. A plant-derived extract was found cytotoxic against human breast cancer cell line (MCF-7) and colon cancer cell line (HCT116) ⁴². Therefore, phytochemical profile of the n-hexane extract from B. balsamifera L leaves corroborates its potential as anticancer agent.

Table 1. List of compounds identified from the GC-MS system from the B. balsamifera L leaves n-hexane extract.

Compounds	Retention time (min)	Area (%)
Bicyclo[2.2.1]heptan-2-one,1,7,7-trimeth	11.2271	4.01
Borneol L	11.650	33.77
beta-Guaiene	14.234	0.97
trans-Caryophyllene	15.271	3.44
Alloaromadendrene	15.802	2.40
Cycloheptan, 4-metylen-1-me	17.197	0.92
(-)-Caryophyllene oxide	17.341	4.95
Elemol	17.690	0.48
Eudesmol <gamma-> db5-1951	17.875	0.52
Caryophyllene <14-hydroxy-9-	17.966	3.12
beta-Selinene	18.210	3.41
Androstan-17-one, 3-ethyl-3-hydroxy-,	18.311	0.98
Ledene	18.457	1.30
Veridiflorol	19.977	22.57
9-Octadecenoic acid, 12-(acetyloxy)-, met	20.857	1.97
Azuleno[4,5-b]furan-2(3H)-one, decarhydr	22.425	0.91
9,12-Octadecadienoic acid (Z,Z)-, methyl	22.506	0.79
9,12,15-Octadecatrien-1-ol (CAS) OCTA	22.566	1.10
2-Hexadecen-1-ol, 3,7,11,15-tetramethyl-,	22.668	2.94
Tetradecanoic acid, methyl ester (CAS) M	22.791	0.62
S:d15-16-estrone-3-methylethe	23.720	0.66
Isolongifolene	23.819	0.82
Verrucarol	24.049	1.06
5H-Cyclopropa[3,4]benz[1,2-e]azulen-5-o	25.322	0.73
Ergost-25-ene-3,5,6,12-tetrol, (3.beta.,5.al)	25.453	1.44
2,9-Heptadecadiene-4,6-diyn-8-ol, (Z,E)-	25.863	1.89

CONCLUSION:

n-Hexane extract from B. balsamifera L leaves from by means of partitioning has weak antioxidant activity against DPPH radicals and moderate cytotoxicity based on BSLT assay. The presence of borneol L and veridiflorol as major phytoconstituents of the extract further suggest its potential as anticancer agent. Further studies need carried out to confirm the anticancer potential using cancer cell lines. Moreover, the isolation and identification of the pure compound are recommended to unearth the compound responsible for the anticancer drug.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

Authors wish to appreciate the research funding given by Lembaga Penelitian Universitas Syiah Kuala through Lektor Kepala Scheme.

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Received on 08.04.2022 Modified on 20.08.2022

Research J. Pharm. and Tech 2023; 16(4):1664-1668.

DOI: 10.52711/0974-360X.2023.00272