1. INTRODUCTION:

Litsea cubeba (Lour.) Pers has several anonyms, including Litsea citrata Blume.; Tethrantera citrata (Blume) Nees; Tethrantera polyantha Wall¹ is included in the Lauraceae family^2,3. In Indonesia, this plant is called Attarasa or Krangean. In Indonesia, krangean grows wild in groups on mountain slopes in Sumatra, Kalimantan, and Java at an altitude of 700 - 2300 m above sea level.

Krangean is a woody plant that produces triacylglycerol (oil), and its seeds contain up to 49.1% oil⁴. Litsea. cubeba (LC) is of high economic value as all plant parts contain essential oil, usually a light yellow or yellow-clear oily liquid with a lemon like aroma and pungent taste⁵. According to data published in 2011, essential oil of LC was ranked in the top 20 essential oils priced at 17–20 euros/kg. China is the world's largest producer and exporter of LC essential oil, with an estimated production of 1500–2000 t per annum⁶. The dominant essential oil content of LC is monoterpenes (94.4-98.4%), especially neral and geranial (78.7-87.4%). D-Limonene (0.7-5.3%). Krangean bark identity compounds: Lupeol⁷, Sineol⁸.

LC also claimed have diversity of functional properties, such as antimicrobial, antioxidant, anticancer, anti-inflammatory, insect repellent, and insecticidal activity, Litsea. cubeba essential oil has wide use in perfumes, food additives, medicines, botanical insecticides and cosmetics⁹. The research is expected to determine the most potential and safe extracts to be used as cosmetic agents, especially whitening from the Litsea cubeba (Lour.) Pers plant. The antioxidant activities by means of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and β-carotene scavenging activity (BCB) were determined. The cytotoxicity was performed against human fibroblast cell lines (3T3) and melanoma cell lines (and B16F10).

Litsea cubeba (Lour.) Pers (8)

2. MATERIALS AND METHODS:

2.1 Material:

Bark, fruit and leaves of Litsea cubeba (LC) were collected, identified, and prepared in accordance with recognized techniques

2.2 Preparation of LC ethanol extracts (LCEE):

2.2.a Maceration (8,10):

The cleaned LC bark, fruit, and leaves samples were dried in an oven at no more than 50°C. The dried powdered simplicia was extracted by maceration with a composition of powder: 96% ethanol solvent (1:10 v/v). The total macerate results were collected and evaporated using a rotary evaporator at a temperature of 50°C until a thick extract was obtained”

2.3 Antioxidant Assay:

2.3.a. DPPH (1,1-diphenyl-2-picrylhydrazyl) (11,12).

The test was carried out by inserting 2.0ml of extract and 0.5ml of 1mM DPPH solution and adding ethanol up to 5.0ml into the tube. The absorbance was read at λmax DPPH. The control was made by mixing 0.5ml of DPPH and ethanol up to 5.0ml.

2.3.b. ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid) assay (13,14).

Determination of antioxidant activity was carried out by putting 4.0mL of ABTS solution into a test tube, adding 0.2mL of sample solution for each concentration. Furthermore, the mixture was homogenized by vortexing for 1 minute and left to stand according to the operating time of each test solution in a dark place. The absorbance of the solution was read at the maximum wavelength. The same steps were taken in measuring the concentration of the quercetin standard series in reading the absorbance. The absorbance of the fractions of LC obtained was compared with the ABTS absorbance to obtain the % antioxidant activity. The calculation of the percentage of antioxidant activity can use the following formula equation:

Abs control – Abs sample

% Antioxidant activity = ------------------------- x 100%

Abs contro

A linear regression equation calculates the sample concentration that will reduce free radicals by 50% (IC₅₀) using sample concentration data with percentage antioxidant activity. As a positive control or comparison, epigallocatechin gallate (EGCG) and trolox are employed

2.3.c. BCB (β-carotene scavenging activity) (15):

A mixture of β-carotene and linoleic acid was prepared by dissolving 0.5mg β-carotene in 1mL chloroform and 25µL linoleic acid in 200mg Tween 20. The chloroform was then completely evaporated under a vacuum, and 100mL of distilled water was subsequently added to the residue. Then the mixture was shaken vigorously to form an emulsion. From this emulsion, 2.5mL was transferred into different test tubes containing 350µL of ALE at different concentrations. All samples were vortexed for 1 min and placed at 50°C in a water bath for 2h with a negative control (blank), which contained the same volume of ethanol instead of the samples. The absorbance of samples was measured at 470nm using a spectrophotometer at the initial time (t = 0) against a blank (emulsion without β-carotene). A standard epigallocatechin gallate (EGCG) and trolox were used as a positive control. Antioxidant activities (inhibitions percentage, I%) of the samples were calculated using the following Equation (2):

Where A _{sample t=0} and A _sample
t=2h are the absorbance values for the test sample at the beginning of the experiments and after 2 h assay, respectively. A _{control t=0} and A _control
t=2h are the absorbance values for the control at the beginning of the experiments and after 2 h assay, respectively.

2.4 Cell Culture (16):

Vero and B16F10 mouse skin melanoma cells have been collected from the UGM Faculty of Medicine, Parasitology lab. “Vero cells were cultured in RPMI-1640 (Roswell Park Memorial Institute) with a mixture of 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, and 10% heat-inactivated fetal bovine serum (FBS). In contrast, B16F10 cells were cultivated at 37°C in DMEM (Dulbecco's modified Eagle). The cells were incubated in 5% CO₂ at 37°C.”

2.5 Antitoxicity Assay against B16F10 and Vero Cells:

In the mitochondria of live cells, yellow MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole]” is transformed into purple formazan. The absorbance of this coloured solution can be determined at a particular wavelength (500-600nm) using a spectrophotometer. As a result, the application may analyze the extract's antitoxicity based on cell survival or viability. When the quantity of purple formazan generated by agent-treated cells is contrasted with that of untreated control cells, a substance's effectiveness in causing cell death can be evaluated. In 96-well microplates, 100µl of B16F10 and/or Vero cell suspension was added “at a density of 1.5 x 10⁴ cells.” Cell cultures were treated with extract concentrations that ranged “from 15.63 to 1000µg/ml and doxorubicin concentrations that ranged from 1.5 to 50µg/ml, respectively. Three evaluations were conducted for each therapy group. The treated cell cultures were incubated at 37°C with 5% CO₂ for a whole day. Following incubation, the cells were washed with PBS, and the culture media was removed. 100µl of MTT solution was applied to each well, and the wells were incubated for four hours at 37°C with 5% CO₂.”Following the incubation period, a stopper of 100µl of 10% SDS reagent was added, and the mixture was left overnight. The ELISA reader was used to measure the cell absorbance at 595nm.” A curve was created to calculate the test drug's IC₅₀ value against the T47D cell line between the concentration and the percentage of living cells. The following formula was used to calculate the significant percentage test of cell viability

TC – CM

% Cell viability = ------------------ x 100

CC - CM

TC is the absorbance of treatment cells, CC is the absorbance of control cells and CM is the absorbance of control media.”

2.6 Selectivity Index (SI) (17):

Selectivity index was determined using following formula:

IC50 on cell vero

SI = --------------------------------

IC50 on tumor cell lines

2.7 Statistical Analysis:

Three independent replications of each experiment were conducted. Data was analyzed using GraphPad Prism 5.03 (GraphPad Software, Inc.; USA) to obtain mean values and standard deviations. Data were statistically processed. The "R Project for Statistical Computing version R3.1.0" program “(The R Foundation of Statistical Computing, Vienna, Austria)” was used to evaluate the reliability of the Student T test results.

2.8 Analysis of Bioactive Compound Content from extract with LC-HRMS:

The compound content in the skin, fruit and leaves extracts of Litsea cubeba was analyzed using LC-HRMS (Liquid Chromatography-High Resolution Mass Spectrometry) at the Gunung Kidul National Research and Innovation Agency. Compounds that were successfully analyzed will be matched with reference compounds in the library. The instrument uses liquid chromatography (Thermo Scientific™ Vanquish™ UHPLC Binary Pump) and Orbitrap high-resolution mass spectrometry (Thermo Scientific™ Q Exactive™ Hybrid Quadrupole-Orbitrap™ High Resolution Mass Spectrometer). Liquid chromatography was performed using a Thermo Scientific™ Accucore™ Phenyl-Hexyl 100mm × 2.1mm ID × 2.6µm analytical column.

RESULT AND DISCUSSION:

2.9 Effect of Ethanol Extracts of Litsea cubeba (LCEE) on Antioxidant Activity:

Table 1. The IC₅₀ values of LCEE by using DPPH, ABTS, and BCB methods. IC₅₀ was reported as mean values ± SEM of three independent assays.

Sampel	IC₅₀(µg/mL)^*
Sampel	DPPH	ABTS	β-carotene bleaching (BCB)
Bark of LCEE	87.82±1.05	89.95±1.98	179.78±2.31
Fruit of LCEE	159.56±1.06	51.28±1.88	681.52±4.10
Leaves of LCEE	74.707±1.1	117.02±1.85	153.70±1.56
Positive control
EGCG		1.85±1.05	9.78±1.40
Trolox		4.78±2.11	1.13±4.12

Antioxidant activity categories with IC₅₀^* values (µg/mL): very strong <20, strong <100, moderate 100-500, weak >500.

According to the antioxidant test results, LCEE leaves had a small quantity of antioxidant activity with the DPPH IC₅₀ method compared to other LCEE parts of 74.707 ± 1.1 µg/ml with a strong category. In the ABTS method test, the smallest IC₅₀ result was fruit of LC (51.28 ± 1.88 µg/ml). What is surprising is that, although DPPH and ABTS are methods based on radical scavengers, the IC₅₀ of leaves of LC extract gave the most significant results (117.02 ± 1.85 µg/ml). BCB method can estimate the ability of an antioxidant to inhibit lipid peroxidation. Using the BCB technique, the antioxidant activity test, once again, leaves of LC gave the smallest results (153.70 ± 1.56 µg/ml).

2.2 Effect of Etthanol Extracts of Litsea cubeba (LCEE) on Cytotoxic Activity:

Table 2. The IC₅₀ values of LCEE against B16F10, 3T3, and Vero cells. IC₅₀ was reported as mean values ± SEM of three independent assays.

Sample	IC₅₀(µg/ml)^*			Selectivity Index (SI)^**
Sample	B16F10	VERO	3T3	VERO/ B16F10	3T3/B16F10
Bark of LCEE	401.93± 1.33	473.66± 3.76	376.80±2.06	1.18	0.94
Fruit of LCEE	49.33± 5.92	251.28± 1.55	67.09± 1.82	5.09	1.36
Leaves of LCEE	35.81± 1.85	303.80± 2.98	455.77± 2.25	8.48	12.73

IC₅₀^* values (µg/ml) generated three cytotoxic categories: Potential <100, Moderate 100-1000, and Non-toxic >1000. SI^** scores were used to classify three selectivity categories: Non-selective action is indicated by SI values ≤1, moderate selectivity is shown by SI values <5, and SI values indicate selective action > 5.

1.3 Cytotoxic activity of ethanol extracts of Litsea cubeba (LCEE) against B16F10 and Vero cells:

An MTT test assessed the cytotoxicity of B16F10 melanoma cells, 3T3 fibroblasts, and Vero normal cells. The examined sample was used to conduct a 24-hour treatment of 1x10⁴ cells. The IC₅₀value determined the cytotoxic effect of the sample on the test cells, while the cytotoxicity relationship between test cells was determined by the selectivity index (SI) values. A positive value indicates higher selectivity against B16F10 melanoma cells, while a negative value or less than one (≤1) indicates higher toxicity against Vero and 3T3 cells and lower against B16F10 melanoma cells (18-21)

The IC₅₀ value of the LC fruit and leaves extracts showed potential cytotoxic activity with a positive SI (selectivity index) value for all concentrations that suppressed B16F10 melanoma cell proliferation. The 5.09 and 8.48 SI values indicated that the fruit and leaves of LC extract had more selective cytotoxic activity against B16F10 melanoma cells than Vero cells, as indicated by the SI values of ≤1. These results suggest that fruit and leaves of LCEE can be developed as antiproliferative agents against cancer cells. These findings are consistent with prior studies showing that a substance could serve as an anticancer agent if it is the capacity to accurately target cancer cells without endangering healthy cells ^21,22

2.2. Chemical composition of extract from different parts of Litsea cubeba (LC) by HRMS:

Max Area in HRMS (High-Resolution Mass Spectrometry) analysis refers to the area's maximum value under the peak curve representing a particular ion in the mass spectrum. The peak area measures the number of ions detected in a certain period, which also reflects the number of compounds represented by the ions in the sample ^19,24,25,26. The 20 most significant composition of compounds found in the fruit, leaves, and bark parts of LC is in Table 4.

Table 4. Composition of the 10 largest chemical bioactive compounds in fruit, skin and leaves extracts of LC

S. No	Compound name (bark)	% Area	Compound name (fruit)	% Area	Compound name (leaves)	% Area
1	“(1E)-7-(3,4-Dihydroxyphenyl) -1-phenyl-1-heptene-3,5-dione”	13.25	“2-Hydroxy-3-isobutyl-9,10-dimethoxy-1,2,3,4, 6,7-hexahydro-11bH-benzo(a)quinolizine”	3.89	17beta-Trenbolone	8.27
2	Sinomenine	11.32	ethylestrenol	3.80	“4-[3-(benzylamino) butyl]-2-ethoxyphenol”	4.23
3	N-Methylhernagine	8.69	Anhydrovitamin A	3.36	“(1E)-7-(3,4-Dihydroxy phenyl)-1-phenyl-1-heptene-3,5-dione”	2.90
4	Galaxolidone	7.78	ethylestrenol	3.0	N-Methylhernagine	2.78
5	Ethylmorphine	5.32	paspaline	2.95	Choline	2.29
6	ethylestrenol	2,38	Vitamin A	2,72	“Ethylmorphine”	2,16
7	Isococculidine	2,25	Monolaurin	2,56	“1-[4-(1-adamantyl) phenoxy]-3-piperidinopropan -2-ol hydrochloride”	2,13
8	“2-Aminoestra -1,3,5 (10) -triene-3,17β-diol”	2,04	Anhydrovitamin A	2,31	Warfarin	2,11
9	Choline	1,82	Naltrexone	1,72	6-Acetylmorphine	2,04
10	Ethylmorphine	1,73	p-cymene	1,72	“4-Ethyl-7-hydroxy-3-(p-methoxyphenyl) coumarin”	1,76
11	Codeine	1,65	1,2,3,4-Tetramethyl-1,3-cyclopentadiene	1,71	"1-[4-(1-adamantyl) phenoxy]-3-piperidinopropan -2-ol hydrochloride”	1,71
12	“9-Oxo-10(E), 12(E)-octadecadienoic acid”	1,44	ethylestrenol	1,59	Warfarin	1,59
13	Idrocilamide	1,34	“IPMP”	1,33	6-Acetylmorphine	1,33
14	ethylestrenol	1,26	“9,17-dioxo-1,2,3,4,10, 19-hexanorandrostan-5-oic acid”	1,32	“4-Ethyl-7-hydroxy-3- (p-methoxyphenyl) coumarin”	1,32
15	“α-Eleostearic acid”	1,26	abietatriene	1,26	“1-[4-(1-adamantyl)phenoxy]-3-piperidinopropan-2-ol hydrochloride”	1,26
16	“syn-labda-8(17),12E,14-triene”	1,19	Ginkgoneolic acid	1,21	Warfarin	1,21
17	Hernagine	1,04	“2-Hydroxy-3-isobutyl-9,10-dimethoxy- 1,2,3, 4,6,7- hexahydro-11bH-benzo(a)quinolizine”	1,08	6-Acetylmorphine	1,08
18	(±)-Eucalyptol	0,88	ethylestrenol	1,06	“4-Ethyl-7-hydroxy-3- (p-methoxyphenyl) coumarin”	1,06
19	Ethylmorphine	0,87	(9cis)-Retinal	1,04	1-[4-(1-adamantyl)phenoxy]-3-piperidinopropan-2-ol hydrochloride	1,04
20	Betaine	0,79	“1-[4-(1-adamantyl)phenoxy] -3-piperidinopropan -2-ol hydrochloride”	1,02	Warfarin	1,02

Table 4 shows the 20 largest compound contents of LC bark, leaves and fruit. Each extract contains more than 600 compounds detected by HRMS. From the data above, there is no similarity in the compounds produced, but if you look at all the data there are many similarities in the same data, including 9-Oxo-10(E),12(E)-octadecadienoic acid and (-)-Caryophyllene oxide which are in the range of 0.5-1.4%. The similarity of several compounds may indicate the same activity. However, if you look at the results of the selectivity index (SI) in the LC bark extract (table 4), bark has an SI value of less than 1 indicating that the sample cannot be used because it is toxic not only to test cells but also to normal cells. This may be due to the presence of gaxolidine compounds with high concentrations (7.73%) where this compound has toxic activity^27,28. In everyday use, this compound is an environmental contaminant and affects the nerves. This still needs to be studied further because it is possible that LC bark is contaminated after post-harvest.

Figure 2. Gaxolidine (hexamethylidanopyran) C18 H24 O2, MW: 272,178

CONCLUSIONS:

The results of antioxidant activity showed that LC leaves extract gave the strongest IC₅₀ results in the DPPH and BCB methods. The strongest results were obtained from LC fruit extract in the ABTS method. Compared to extracts of other parts of LC, the leaves of LCEE have the highest potential cytotoxic activity, with an IC₅₀ value of 35.81±1.85µg/ml. Their SI values of 8.48 and 12.73 indicate that they are the most selective against B16F10 melanoma cells than normal Vero and 3T3 cells. HRMS results showed that the extracts of the three parts of LC contained between 600-650 compounds. There were similarities in the content of compounds, including “9-Oxo-10 (E), 12 (E) -octadecadienoic acid,” and (-) -Caryophyllene oxide, which are in the range of 0.5-1.4%. The bark extract of LC gave a selectivity index result of <1, possibly because it contains a large amount of the toxic compound gaxolidine (7.78%).

AUTHOR CONTRIBUTIONS:

Erna Prawita Setyowati: Writing, Methodology, Conceptualization, Diah Tri Utami: Writing, Methodology, Rony Abdi Syahputra, Yahdiana Harahap, Aminah Dalimunthe, Bonglee Kim, Sari Haryanti: Methodology.

ACKNOWLEDGMENTS:

The research, writing, and/or publication of this work were made possible by financial support received by the author(s). We thank Universitas Gadjah Mada (UGM) for funding this study under Riset Kolaborasi Indonesia (RKI Scheme C) 2024 and BRIN for making HRMS use easier.

DATA AVAILABILITY STATEMENT:

The data is contained within the article.

CONFLICT OF INTEREST:

The authors declare that they have no conflicts of interest.

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Received on 06.03.2025 Revised on 22.07.2025

Accepted on 26.10.2025 Published on 10.02.2026

Available online from February 16, 2026

Research J. Pharmacy and Technology. 2026;19(2):651-656.

DOI: 10.52711/0974-360X.2026.00095

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