Evaluation of phytochemical composition, antioxidant, cytotoxic and in silico studies of ethyl acetate fractions of Tristaniopsis merguensis leaves

Abstract

Tristaniopsis merguensis is a member of the Myrtaceae family. The leaves of T. merguensis are frequently utilized as herbal tea. The study aimed to evaluate the polyphenolic content, antioxidant, and cytotoxic properties of ethyl acetate fractions derived from Tristaniopsis merguensis (ETM). In addition, it also aimed to conduct an LC-HRMS analysis of a potential fraction to ascertain its suitability as an antioxidant and anticancer active ingredient. An antioxidant evaluation was carried out using the ABTS and DPPH methods. Meanwhile, an in silico study was performed using the target protein elastase (HER-2). A total of twenty metabolites of T. merguensis were identified in the potential fraction ETM.05. In antioxidant testing, the T. merguensis fraction, with code ETM.05, demonstrated potent antioxidant activity based on the ABTS and DPPH methods, with IC₅₀ values of 64.830 ± 2.803 µg/mL and 40.252 ± 0.032 µg/mL, respectively. This resulted in the categorization of the fraction as a strong antioxidant. In the anticancer testing, ETM.05 also demonstrated potent anticancer activity, with an IC₅₀ value of 10.66 µg/mL, which was categorized as a very strong activity in comparison to the positive control (cisplatin), which exhibited an IC₅₀ value of 12.24 µg/mL. The in silico study indicates that the identified metabolites have the potential to bind to the target protein HER-2, which plays a role in remodeling the breast cancer process. This research suggests that the fraction ETM.05 from the ethyl acetate leaves fraction of T. merguensis has the potential to serve as an active antioxidant and anticancer source.

Graphical Abstract

Highlights

• •A total of twenty metabolites of T. merguensis were identified in the potential fraction ETM.05.
• •The T. merguensis fraction, with code ETM.05, demonstrated potent antioxidant activity based on the ABTS and DPPH methods, with IC₅₀ values of 64.830 ± 2.803 µg/mL and 40.252 ± 0.032 µg/mL, respectively.
• •ETM.05 also demonstrated potent anticancer activity, with an IC₅₀ value of 10.66 µg/mL.

1. Introductions

Natural plant extracts have been studied as herbal medicine, aiming to utilize their activities as anticancer and antioxidant ingredient agents [1]. Plant extracts can be incorporated into the formulations used in the medicine. Given their simple cultivation, some plant species in Indonesia have great potential to be used as extracts. The use of medicinal plants remains a tradition among the ethnic communities living in the undulating planes and foothills of major forests [2], [3]. Numerous secondary metabolites produced by plants, such as flavonoids and phenolics, have numerous therapeutic and health benefits [4].

Natural antioxidants are considered safer than synthetic antioxidants. These antioxidants, rich in phenol and flavonoids, can effectively scavenge free radicals and check progressive oxidative damage [5]. By fighting against free radicals, antioxidants offer protection against various diseases [6]. Some of the medicinal properties of plants might be attributed to their scavenging activity [7].

Cancer is regarded as one of the world’s most prevalent diseases and is one of the world’s leading causes of mortality, with its incidence continuing to rise [8]. A statistical analysis of mortality rates indicates that one in six women and one in five men will develop a lethal tumor at some point in their lives [9]. Natural products, particularly those exhibiting antioxidant activity, have been demonstrated to be an effective alternative for reducing tumor cell mass or preventing tumor formation at the early stages [10], [11]. A multitude of phytochemical classes of compounds have been observed to exert antioxidant activity, including flavonoids, polyphenols, alkaloids, terpenes, and other compounds [12].

Tristaniopsis merguensis, locally known as pelawan, belongs to the family of Myrtaceae. This plant is well renownedin west Indonesia and throughout the Bangka Belitung Islands, where it has a significant impact on health as herbal tea [13]. T. merguensis occurs in many different types of lowland to lower montane forest up to 1300 m altitude, often along the rivers or near the coast [14]. The leaves of plant-based natural products have long been a primary source of drug discoveries. Scientific literature supports the antioxidant properties of the leaf extract. The antioxidant activity of acetone extract obtained through the microwave-assisted extraction (MAE) method has been demonstrated to be more potent than that of ethanol extract, with an IC₅₀ value of 9.501 μg/mL [15]. The total phenolic content of the acetone extract derived from T. merguensis leaves was determined to be 215.22 mg GAE/g [16].

Our previous study demonstrated that ethyl acetate fraction exhibited the highest cytotoxic activity compared to n-heksane and ethyl acetate extract of T. merguensis leaves [17]. In this research, different fractions of Triptaniopsis merguensis from ethyl acetate leaf extracts were used to evaluate the phytochemical composition, antioxidant, in vitro cytotoxic activities, LC-HRMS analyses of potential fraction and an in silico study against HER-2 breast cancer protein. Antioxidant activity was assessed using DPPH and ABTS assay. Both methods were used for the pre-evaluation of the possible antioxidant activity, serving as an indicator of the cytotoxic activity. In vitro cytotoxic test was utilized to evaluate the toxicity capacity against breast cancer MCF-7 cell lines, while LC-HRMS was employed to analyze the chemical compounds in the potent fraction. However, no reports have detailed the phytochemical composition, antioxidant, and cytotoxic potential of fractions from the ethyl acetate of T. merguensis leaf extract. Lastly, active compounds from potential fractions were applied to an in silico study using moleculer docking against HER-2 breast cancer proteins.

2. Materials and methods

2.1. Chemicals

Unless otherwise specified, the chemicals and standards utilized in this study were of analytical grade. α-tocopherol (Sigma-Aldrich), 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) were purchased from Sigma-Aldrich. 96 well plate (Merck NEST 701001), Dimethyl sulfoxide (DMSO) (Merck D1435), Chemicals included phosphate-buffered saline (PBS), antibiotic (Sigma Aldrich P4333), and cisplatin (EDQM C2210000) were used. Additional materials needed included Trypan Blue (Sigma Aldrich T-8154), FBS (Gibco 10270–106), Trypsin-EDTA (Gibco 25200–056), and RPMI (Gibco 11875–093).

2.2. Plant materials

The leaves of Tristaniopsis merguensis were collected in February 2023 from Namang City, Bangka Island, Bangka Belitung Province, Indonesia. The specimens were then air-dried for 14 days at room temperature. The plant was identified and deposited by the Laboratory of Plant Systematics, Universitas Gadjah Mada, Yogyakarta, Indonesia, with a voucher specimen (No. 0262/S. Tb/II/2023).

2.3. T. merguensis extract preparation

The leaf powder of T. merguensis underwent a solid-liquid extraction as demonstrated by Musa et al. (2023) with some modifications [18]. Dried Leaves of Tristaniopsis merguensis were macerated at room temperature for 48 h in ethyl acetate at a solid-liquid ratio of 1/10 (w/v). The dried leaves were macerated in 250 g of dried leaves with 2.5 L of ethyl acetate solvent. The ethyl acetate macerates were filtered using a Whatman No. 2 filter paper. The extracts were then concentrated using a vacuum rotary evaporator at 45°C. After evaporation, the extracts were introduced into a Memmert-type oven (Model, Memmert) at 30°C until their weight stabilized to provide ethyl acetate crude extract. The yield of the extracts was measured [19].

2.4. Fractionated ethyl acetate extract

The ethyl acetate extract of T. merguensis was fractionated using column chromatography. The ethyl acetate and n-hexane were used as a solvent combination. About 50 g of ethyl acetate extract was subjected to column chromatography. The column was prepared by utilizing a gradient of 10 % to obtain a total of 10 fractions (ETM.01-ETM.10). Briefly described, ETM.01 solution was made with a combination of n-hexane (180 mL)/ethyl acetate (20 mL). ETM.02 solution was made with a combination of n-hexane (160 mL)/ethyl acetate (40 mL). ETM.03 solution was made with a combination of n-hexane (140 mL)/ethyl acetate (60 mL). ETM.04 solution was made with a combination of n-hexane (120 mL)/ethyl acetate (80 mL). ETM.05 solution was made with a combination of n-hexane (100 mL)/ethyl acetate (100 mL). ETM.06 solution was made with a combination of n-hexane (80 mL)/ethyl acetate (120 mL). ETM.07 solution was made with a combination of n-hexane (60 mL)/ethyl acetate (140 mL). ETM.08 solution was made with a combination of n-hexane (40 mL)/ethyl acetate (160 mL). ETM.09 solution was made with a combination of n-hexane (20 mL)/ethyl acetate (180 mL). ETM.08 solution was made with ethyl acetate (200 mL). All solvents were subjected to the column chromatography sequentially. All fractions were evaporated to determine the % yield. The phytochemical screening approach was employed in a qualitative test to identify metabolites that were secondary in ETM.01- ETM,10 samples. Subsequently, several experiments were conducted using phytochemical testing methods to identify alkaloids, phenols, tannins, flavonoids, steroids, and terpenoids

2.5. Total phenolics content

The TPC was determined using the Folin-Ciocalteu method previously described by Kabri et al. [20], with certain modifications [20]. The ETM.01-ETM.10 were prepared at a concentration of 1000 µg/mL. In summary, approximately 1 mL of Folin-Ciocalteu reagent was combined with 200 μL of fractions and 0.8 mL of a 7.5 % (m/v) Na₂CO₃ solution. Following a 60-minute incubation period at room temperature, the absorbance was measured at a wavelength of 765 nm. A calibration curve was prepared using a series of gallic acid solutions with varying concentrations. The results of the total phenolic content (TPC) were expressed as mg GAE/g of the dried extract. All tests were conducted in triplicate [21].

2.6. Total flavonoids content

The TFC was determined according to the methods proposed by Alkowni et al., (2023) with some modifications [22]. Briefly, 0.5 mL of the ETM.01-ETM.10 with a concentration of 1000 µg/mL was mixed with 0.1 mL of 1 % AlCl₂ (1 %), 0.1 mL of natrium acetate (1 M), and 2.8 mL of aquadest. Standard solutions of quercetin were prepared at concentrations of 80, 70, 60, 50, and 40 µg/mL. Then, shake and incubate them for 30 minutes at room temperature. The color intensity was then measured at 510 nm using a UV–VIS spectrophotometer. The total flavonoid content was determined as quercetin equivalent (QE) (40, 50, 60, 70, and 80 μg/mL, R² 0.982) and was expressed as mg of QE/g [23].

2.7. DPPH radical scavenging activity

The DPPH scavenging activity of the ETM.01-ETM.10 was determined in accordance with the methodology outlined by Tjammal et al. (2022), with certain modifications [24]. The fractions and the positive control α-tocopherol were placed in separate tubes. Subsequently, 0.6 mL of DPPH was added to each tube, resulting in the desired final concentrations. The experiments were conducted in triplicate. The decrease in absorbance at 517 nm was determined for all samples following a 30-minute incubation period. The complete assay procedure is provided in the supplementary material.

2.8. ABTS radical scavenging activity

The free radical scavenging activity of ETM.01-ETM.10 was determined using an ABTS radical cation (ABTS•+) decolorization assay by Jiangseubchatveera et al., (2023) with certain modifications [7]. The samples were prepared in methanol at various concentrations (0, 20, 40, 60, 80, and 100 µg/mL). Subsequently, 200 µL of each sample was added to 1.8 mL of the ABTS•+ solution in tubes and allowed to stand at room temperature for 15 minutes. Subsequently, the absorbance at 734 nm was quantified. The complete assay procedure is provided in the supplementary material.

2.9. Cytotoxic activity determination

The in vitro cytotoxicity of ETM.01-ETM.10 was studied in MCF-7 breast cancer cell lines. PrestoBlue® assay was used to assess the cytotoxicity for all fractions using a cell viability test. Cells were cultured in a medium obtained from RPMI. The samples with varying doses of 3.91, 7.81, 15.63, 31.25, 62.50, 125.00, 250.00, and 500.00 µg/mL were added to the medium, followed by the addition of a control. Thereafter, DMSO was added, and the sample absorbance (A) was read at 570 nm. The cell viability percentage was determined as follows: [Absorbance of treated sample/ Absorbance of control sample] x 100 [25], [26].

2.10. Liquid chromatography high-resolution mass spectrometry (LC-HRMS/MS) analysis

The ETM.05 potential fraction was subjected to analysis via ultra-high-performance liquid chromatography coupled with targeted HRMS. This involved the use of a Thermo Scientific Dionex Ultimate 3000 RSLC Nano UHPLC, in conjunction with a Thermo Scientific Q Extractive (Thermo Fisher Scientific, Massachusetts, USA). An LC-HRMS analysis was conducted using a mobile phase comprising A [27]. Mobile phase A and B consisted of 0.1 percent formic acid in water and formic acid in methanol, respectively. The analysis was conducted on a column AcclaimTM Vanquish C18 Dim. (mm) 150 × 2.1 with a flow rate of 40 μL/min, an injection volume of 5 μL, and a gradient over a 25-minute analysis time. The experiments were conducted in parallel reaction monitoring with a resolution of 35,000 full width at half maximum (FWHM), heated electrospray ionization, and positive ionization, while the data processing was conducted with the Thermo Scientific software [28], [29].

2.11. In silico study by molecular docking

The chemical structure of twenty compounds tentatively identified from the LC-HRMS analysis were subjected to molecular docking analysis in accordance with the procedure described by Wulan et al. (2023) [30]. An in silico analysis of the enzyme structure was conducted using the Protein Data Bank (https://www.rcsb.org/structure/HER-2). Initially, non-standard residues such as water molecules and native ligands were removed from the PDB (3RCD) file. The results of the molecular docking analysis were evaluated based on a number of parameters, including the orientation of the ligand structure, the presence and strength of hydrophobic interactions, the formation of hydrogen bonds, and the value of the binding energy for each ligand.

2.12. Statistical analysis

The data were reported as the mean ± standard deviation. The data were subjected to statistical evaluation using a one-way analysis of variance (ANOVA). The significance level was set at p < 0.05).

3. Results

3.1. Percent yield

T. merguensis leaves were identified and deposited in the Laboratory of Plant Systematics, Universitas Gadjah Mada, Indonesia, with a voucher specimen (No. 0262/S. Tb/II/2023). In the current study, 1000 g of powdered leaves were extracted using ethyl acetate. The percent yield of ethyl acetate extracts of T. merguensis leaves was 18 %.

3.2. Fractionated ethyl acetate extract

Fractions were prepared using column chromatography method and ten fractions (ETM.01-TMF.10) were obtained. The profile of Thin Layer Chromatography (TLC) is shown in Fig. 1. N-hexane and ethyl acetate were used as the eluents in a 3:2 ratio. The weights of the fractions were as follows: ETM.01 was 5.7488 g, ETM.02 was 14.6574 g, ETM.03 was 1.1982 g, ETM.04 was 3.4001, ETM.05 was 3.2182 g, ETM.06 was 3.6141 g, ETM.07 was 2.9321, ETM.08 was 8.3965 g, ETM.09 was 2.1753 g, and ETM.10 was 1.9205 g.

Fig. 1.

Thin Layer Chromatography (TLC) of ETM.01-ETM.10. 1.a in UV λ 365 nm, 1.b in UV λ 254 nm. The eluent used was n-hexane/ethyl acetate with a ratio of 3:2 mL.

3.3. Phytochemical screening of ethyl acetate fractions

The phytochemical evaluation of T. merguensis ethyl acetate leaf fractions is displayed in Table 1. Based on the results, HPF was found to contain alkaloids, flavonoids, triterpenoids, and steroids. EPF contained alkaloids, flavonoids, triterpenoids, steroids, and phenolic/tannins, while MPF had alkaloids, flavonoids, triterpenoids, and phenolic/tannins. These findings, which demonstrated the presence of secondary metabolites in leaves, were in conformity with other studies.

Table 1.

Phytochemical screening of T. merguensis.

Fractions	Alkaloids	Flavonoids	Phenolics	Triterpenoids	Saponins
ETM.01	-	+	+	+	-
ETM.02	-	+	+	+	-
ETM.03	-	+	+	+	-
ETM.04	-	+	+	+	-
ETM.05	+	+	+	+	+
ETM.06	+	+	+	+	+
ETM.07	+	+	+	+	+
ETM.08	+	+	+	+	+
ETM.09	+	+	+	+	-
ETM.10	+	+	+	+	-

+ = present, - = absent

3.4. Total phenolic and flavonoid content

The TPC and TFC of ethyl acetate fractions is shown in Table 2. The phenolic content of ETM fractions was measured from the gallic acid calibration curve, where y = 0.0002x + 0.0186 (R² value = 0.9878). ETM.05 exhibited a high phenolic content of 17.534 ± 0.716 mg GAE/g (Fig. 2). The results for the TFC were derived from the quercetin calibration curve where y = 0.00026x + 0.01320 (R² value = 0.9826). In addition, ETM.05 also contained a total flavonoid content, measuring 17.732 ± 0.157 mg QE/g (Fig. 3).

Table 2.

The total phenolic and flavonoid content of ethyl acetate fractions. The data represent mean ± SD. The tests were performed in triplicate with two different extract preparations.

Fractions	% yield	TFC (mg QE/g)	TPC (mg GAE/g)
ETM.01	5.75	5.838 ± 0.115	5.993 ± 0.082
ETM.02	14.66	6.823 ± 0.087	7.552 ± 0.123
ETM.03	1.19	8.439 ± 0.087	8.573 ± 0.172
ETM.04	3.40	14.778 ± 0.043	15.742 ± 0.335
ETM.05	3.22	17.732 ± 0.157	17.534 ± 0.716
ETM.06	3.61	11.394 ± 0.190	14.971 ± 0.112
ETM.07	2.93	6.192 ± 0.075	8.717 ± 0.163
ETM.08	8.39	7.151 ± 0.087	8.412 ± 0.081
ETM.09	2.17	8.692 ± 0.075	9.165 ± 0.082
ETM.10	1.92	13.944 ± 0.157	15.079 ± 0.135

Fig. 2.

The total phenolic content of ETM.01- ETM.10.

Fig. 3.

The total flavonoid content of ETM.01- ETM.10.

3.5. Antioxidant activity of ETM.01-ETM.10 by DPPH and ABTS methods

ETM.05 demonstrated a good DPPH radical scavenging potential, having an IC₅₀ value of 40.252 ± 0.032 µg/mL, compared to α-tocopherol, which had an IC₅₀ value of 23.887 ± 1.13 µg/mL. The antioxidant activity quantified of ETM.05, measured by ABTS assay, showed an IC₅₀ value of 64.830 ± 2.803 µg/mL, while that of α-tocopherol was found to be 29.647 ± 1.113 µg/mL. The data revealed that ETM.05 had good antioxidant activity, comparable to that of α-tocopherol (Table 3). The percentage inhibition of DPPH free radical scavenging is shown in Fig. 4 and the percentage inhibition of ABTS free radical scavenging is presented in Fig. 5. A lower IC₅₀ value indicates a more potent inhibitor, as it requires a lower concentration to achieve the same level of inhibition Table 4.

Table 3.

Antioxidant activity of ETM.01-ETM.10. The data represent mean ± SD. The tests were performed in triplicate with two different extract preparations.

Fractions	ABTS (X ± SD)	DPPH (X ± SD)
ETM.01	200.655 ± 3.889	204.399 ± 2.363
ETM.02	217.345 ± 1.749	193.827 ± 5.048
ETM.03	181.811 ± 2.004	150.542 ± 3.917
ETM.04	102.408 ± 0.513	97.285 ± 2.548
ETM.05	64.830 ± 2.803	40.252 ± 0.032
ETM.06	192.024 ± 6.778	137.427 ± 0.600
ETM.07	215.273 ± 8.391	227.642 ± 1.222
ETM.08	203.824 ± 4.095	184.626 ± 2.170
ETM.09	166.590 ± 2.151	157.236 ± 0.858
ETM.10	210.336 ± 1.281	131.028 ± 1.167
α-tocopherol	29.647 ± 1.113	23.887 ± 1.13

Fig. 4.

The DPPH free radical scavenging activity of ETM.01-ETM.10. The data represent mean ± SD. The tests were performed in triplicate.

Fig. 5.

The ABTS free radical scavenging activity of ETM.01-ETM.10. The data represent mean ± SD. The tests were performed in triplicate.

Table 4.

Cytotoxic activity of ETM.01-ETM.10 in breast cancer cells line MCF-7.

Fractions	IC50 (µg/mL)	Category
ETM.01	154.90	Moderate
ETM.02	152.00	Moderate
ETM.03	99.55	Strong
ETM.04	21.33	Very strong
ETM.05	10.66	Very strong
ETM.06	51.92	Strong
ETM.07	56.33	Strong
ETM.08	71.60	Strong
ETM.09	36.68	Very strong
ETM.10	377.10	Weak
Cisplatin	12.24	Very strong

3.6. Cytotoxic activity of ETM.01-ETM.10 in breast cancer cells line MCF-7

The cytotoxicity of ETM.01-ETM.10 was evaluated against MCF-7 breast cancer cells using the MTT assay. The concentrations ranged from 3.91, 7.81, 15.63, 31.25, 62.5, 125.0, 250.0 and 500.0 µg/mL. ETM.05 demonstrated the highest cytotoxic activity with an IC₅₀ value of 10.66 µg/mL, suggesting strong anti-proliferative potential. This was followed by ETM.04 and ETM.09, with IC₅₀ values of 21.334 µg/mL and 36.68 µg/mL, respectively, indicating that ETM.05 possesses the most potent anti-proliferative activity of all the fractions tested. The cell viability is shown in Fig. 6.

Fig. 6.

The viability was evaluated using the MTT assay after a one-day treatment period. The data were shown as mean ± SD and the significance threshold was chosen at *p < 0.05.

3.7. LC-HRMS analysis of potent fraction

Based on the antioxidant and cytotoxic activity test, ETM.05 showed the most potent antioxidant and cytotoxic activity. The total ion chromatograms (TIC) from ETM.05 fraction of T. merguensis, detected using LC-HRMS, are shown in Fig. 7. LC-HRMS analysis of ETM.05 revealed the presence of 20 phenolic and flavonoid compounds (Fig. 9).

Fig. 7.

The morphology of the MCF-7 cell line of ETM.05 with a scale bar indicating 20 times magnification.

Fig. 9.

(2 R)-5-hydroxy-7-methoxy-2-phenyl-3,4-dihydro-2H-1-benzopyran-4-one (1), 5,6-dimethoxy-2-(2-methoxyphenyl)-4H-chromen-4-one (2), Osthol (3), Yangonin (4), Diphenolic acid (5), 4-methoxy-9-(2-methylbut-3-en-2-yl)-7H-furo [3,2-g] chromen-7-one (6), Phloretin (7), Pinocembrin (8), 1a,7a-Dihydro-3,6-dihydroxy-1a-(3-methyl-2-buten-1-yl) naphtha [2,3-b]oxirene-2,7-dione (9), Caffeic acid phenethyl ester (10), Formononetin (11), Zingerol (12), senkyunolide B (13), 1-(2,4-Dihydroxy-5-methoxyphenyl)-2-(4-methoxyphenyl)-1-propanone (14), 2,6-di-tert-butyl-4-ethylphenol (15), Glycitein (16), 5,7-dihydroxy-6-methoxy-2-phenyl-3,4-dihydro-2H-1-benzopyran-4-one (17), Equol (18), 3′,4′-Dihydroxyphenylacetone (19), Acetylshikonin (20).

3.8. Molecular docking study

The parameters utilized in this molecular docking study were amino acid residues, hydrogen bonds, and binding energy. Fig. 8 depicts the compound (ligand) structure of ETM.05, derived from the predicted LC-HRMS/MS Fig. 9. The visualization results indicate that all compounds from the ETM.05 fraction were capable of interacting with the active site of HER-2 (Table 5, Table 6). The data indicates that the compounds in the ETM.05 of T. merguensis leaves exhibit binding energies ranging from 4.94 to 7.62 kcal/mol (Table 6). Of these, twenty form bonds with the amino acids in breast cancer HER-2 target proteins, as evidenced by the reported interactions with native ligands (Figs. 10 and 11).

Fig. 8.

Total ion chromatograms (TIC) from ETM.05 fraction of T. merguensis detected using LC-HRMS.

Table 5.

The main constituents detected from ETM.05 fractions of T. merguensis using LC-HRMS.

Name	Formula	Molecule Weight	RT (min)	Absolute Error (ppm)	Classes
1.(2 R)−5-hydroxy−7-methoxy−2-phenyl−3,4-dihydro-2H−1-benzopyran−4-one	C16H14O4	270.0888	11.214	4.8382	Flavonoid
2.5,6-dimethoxy−2-(2-methoxyphenyl)-4H-chromen−4-one	C18H16O5	312.09895	12.768	4.3283	Flavonoid
3.Osthol	C15H16O3	244.10951	10.168	3.7046	Phenolic
4.Yangonin	C15H14O4	258.08896	9.756	2.3828	Phenolic
5.Diphenolic acid	C17H18O4	286.12017	12.592	3.2088	Phenolic
6.4-methoxy−9-(2-methylbut−3-en−2-yl)-7H-furo[3,2-g] chromen−7-one	C17H16O4	284.10468	11.837	−1.5393	Phenolic
7.Phloretin	C15H14O5	274.08397	8.721	2.4092	Phenolic
8.Pinocembrin	C15H12O4	256.07347	10.411	2.2666	Flavonoid
9.1a,7a-Dihydro−3,6-dihydroxy−1a-(3-methyl−2-buten−1-yl)naphth [2,3-b]oxirene−2,7-dione	C15H14O5	274.08388	10.413	1.0644	Phenolic
10.Caffeic acid phenethyl ester	C17H16O4	284.10469	10.972	1.9878	Phenolic
11.Formononetin	C16H12O4	268.07345	10.767	3.2344	Phenolic
12.Zingerol	C11H16O3	196.10974	6.118	4.2123	Flavonoid
13.Senkyunolide B	C12H12O3	204.07819	13.617	1.7887	Phenolic
14.1-(2,4-Dihydroxy−5-methoxyphenyl)−2-(4-methoxyphenyl)−1-propanone	C17H18O5	302.11507	10.633	1.9245	Phenolic
15.2,6-di-tert-butyl−4-ethylphenol	C16H26O	234.19814	11.608	2.1214	Phenolic
16.Glycitein	C16H12O5	284.06836	7.783	3.5116	Flavonoid
17.5,7-dihydroxy−6-methoxy−2-phenyl−3,4-dihydro-2H−1-benzopyran−4-one	C16H14O5	286.08387	10.271	1.6572	Flavonoid
18.Equol	C15H14O3	242.09401	10.518	2.8677	Flavonoid
19.3',4′-Dihydroxyphenylacetone	C9H10O3	166.06274	6.631	1.6267	Phenolic
20.Acetylshikonin	C18H18O6	330.1097	11.258	1.7022	Phenolic

Table 6.

The value of binding energy and amino acid residues that bind to the HER-2 breast cancer protein.

Name	Binding energy (kcal/mol)	Inhibition constant (Ki. × 10−6 µmol	H-bond (Å)	Hydrophobic
TAK−285 (native ligand)	−5.08	187.48	Asp863 (2.77), Met801(1.74)	Phe864, Ala751, Met801, Leu852, Leu800, Lys753, Leu796, Leu785
1.(2 R)−5-hydroxy−7-methoxy−2-phenyl−3,4-dihydro-2H−1-benzopyran−4-one	−7.14	5.81	Asp863 (2.10), Phe864 (3.07), Leu785 (2.31)	Ala751, Val734, Lys753, Leu785
2.5,6-dimethoxy−2-(2-methoxyphenyl)-4H-chromen−4-one	−7.27	4.72	Lys753 (2.67), Thr862 (2.66)	Ala751, Val734, Lys753, Leu785
3.Osthol	−6.84	9.61	Asp863 (2.09)	Phe864, Leu796, Lys753, Leu785, Asp863
4.Yangonin	−6.81	10.26	Lys753 (1.88), Leu785 (2.08)	Leu785
5.Diphenolic acid	−7.58	2.78	Met801 (2.07), Asp863 (2.17), Lys753 (1.80), Leu796 (2.06), Ala751 (2.15)	Val734, Thr798, Ala751, Leu852, Thr862
6.4-methoxy−9-(2-methylbut−3-en−2-yl)-7H-furo[3,2-g]chromen−7-one	−7.38	3.9	-	Lys753, Leu796, Phe864, Met774, Leu785
7.Phloretin	−6.59	14.88	Asp863 (2.11), Ser783 (1.94), Leu796 (1.87)	Lys753, Ala751, Val734, Met774, Leu785
8.Pinocembrin	−6.73	11.68	Ser783 (2.01, 2.35), Thr862 (2.98)	Leu785, Leu796
9.1a,7a-Dihydro−3,6-dihydroxy−1a-(3-methyl−2-buten−1-yl)naphth[2,3-b]oxirene−2,7-dione	−6.94	8.25	Asp863 (1.93, 2.59), Lys753 (3.05), Thr862 (2.91), Leu796 (2.18)	Phe864, Leu785, Met774, Val734, Ala751, Lys753
10.Caffeic acid phenethyl ester	−6.61	14.26	Met801 (2.03, 2.47), Lys753 (2.01)	Leu852, Ala751, Leu796, Thr798, Lys753
11.Formononetin	−7.62	2.59	Asp863 (2.67), Leu785 (2.12), Ser783 (1.79)	Leu785, Leu796, Lys753
12.Zingerol	−5.16	164.02	Asp863 (1.87), Lys753 (2.30), Thr862 (2.17)	Lys753
13.Senkyunolide B	−6.14	31.77	Met801 (2.03)	Ala751, Lys753, Leu796, Leu852
14.1-(2,4-Dihydroxy−5-methoxyphenyl)−2-(4-methoxyphenyl)−1-propanone	−6.29	24.48	Met801 (1.97), Lys753 (2.40)	Leu852, Ala751, Val734, Lys753
15.2,6-di-tert-butyl−4-ethylphenol	−7.14	5.79	Asp863 (1.97)	Phe864, Leu785, Ala751, Leu796, Lys753
16.Glycitein	−6.63	13.7	Met801 (2.09), Ser783 (2.16), Thr862 (2.05)	Thr798, Leu862, Ala751, Lys753
17.5,7-dihydroxy−6-methoxy−2-phenyl−3,4-dihydro-2H−1-benzopyran−4-one	−6.46	18.49	Asp863 (2.10), Leu796 (2.31)
18.Equol	−7.51	3.15	Gly865 (2.05), Lys753 (2.41), Thr798 (1.66)	Leu755, Leu796, Lys753, Leu785
19.3',4′-Dihydroxyphenylacetone	−4.94	238.21	Ser783 (2.09), Thr798 (1.81)	Leu785
20.Acetylshikonin	−7.29	4.5	Asp863 (2.09), Thr798 (2.11), Ser783 (2.66)	Leu785, Ala751, Leu852, Val734

Fig. 11.

Structure of the breast cancer receptor (HER-2) (a) and re-docking of native ligand (co-crystal) results into the breast cancer pocket, validating the method with an RMSD value of 1.05 Å (b).

4. Discussion

Studies on the biological activity of Tristaniopsis merguensis leaves and their relationship with chemical constituents, both in vitro and in silico, are still very limited. The common phytochemistry content from ethyl acetate fraction of the T. merguensis leaves includes alkaloids, flavonoids, tannins, phenolics, terpenoids, steroids, and saponins. The ETM.05, ETM.06, ETM.07 and ETM.08 contained alkaloids, flavonoids, phenolics, saponins and triterpenoids. The ETM,01, ETM.02 ETM.03, and ETM.04 only contained flavonoids, phenolics, and triterpenoids. ETM.09 and ETM.10 did not contain saponins. Flavonoids and phenolics constituent the major classes of phytoconstituents found in Triptaniopsis merguensis leaves and the family of Myrtaceae [31], [32].

The thin-layer chromatography results for fractions ETM.01-ETM.10, as illustrated in Fig. 1, indicate that ETM.04, ETM.05, and ETM.06 contain higher concentrations of phenolic and flavonoid compounds. This is corroborated by the presence of numerous compounds that fluoresce under ultraviolet light at 365 and 254 nanometers. Flavonoids and phenolic compounds possess conjugated double bonds in their aromatic rings, which enable them to fluoresce under ultraviolet light at wavelengths of 254 and 365 nanometers [33], [34].

The TPC was investigated using the Folin–Ciocalteu reagent. The statistical analysis of these results exhibited significant differences in TPC (p < 0.05). The ETM.05 fraction showed the highest TPC at 17.534 ± 0.716 mg GAE/g, followed by ETM.04 with 15.742 ± 0.335 mg GAE/g. Meanwhile, the ETM.01 fraction contained the lowest TPC at 5.993 ± 0.082 mg GAE/g. Previously, the TPC of ethyl acetate fraction from the acetone extract of Triptaniopsis merguensis was estimated to be 86.724 ± 1.83 mg GAE/g [35], while the acetone extract itself was reported to contain 215.22 mg GAE/g [35]. The highest TFC content was found in ETM.05 (17.732 ± 0.157 mg QE/g), followed by ETM.04 fraction (14.778 ± 0.043 mg QE/g), and ETM.10 (13.944 ± 0.157 mg QE/g). Moreover, the ETM.01 fraction had the lowest TFC content (5.838 ± 0.115 mg QE/g). The eluent utilized for eluting ETM.05 was a 5:5 ratio of n-hexane and ethyl acetate. This ratio has been demonstrated to be effective for eluting phenolic and flavonoid compounds in column chromatography due to its semipolar nature [36].

Results of the antioxidant activity analysis based on IC₅₀ values using the DPPH method and ABTS method are shown in Table 3 and Table 4, respectively. The analysis was compared with α-tocopherol as a positive control for DPPH and ABTS. The DPPH and ABTS radical scavenging assays are straightforward and widely utilized techniques for investigating the antioxidant capacity of diverse plant extracts and fractions. The strength of the antioxidant activity was determined based on the IC₅₀ value, as referenced by Nurhasnawati et al. (2019) [37]. The following scale was used to categorize the strength of the activity: values below 50 ppm were considered very strong, 50–100 ppm as strong, 100–250 ppm as moderate, 250–500 ppm as weak, and above 500 ppm as inactive. As illustrated in Fig. 3, Fig. 4, an increase in the percentage of inhibition is accompanied by a corresponding decrease in the IC₅₀ value. A lower IC₅₀ value indicates that a low concentration is sufficient to inhibit free radicals, thereby demonstrating strong antioxidant activity. Based on IC₅₀ of DPPH radical scavenging activity for the ETM.01-ETM.10, the highest IC₅₀ value was observed in ETM.05 with IC₅₀ of 64.830 ± 2.803 µg/mL in ABTS and 40.252 ± 0.032 µg/mL in DPPH.

α-tocopherol as a positive control with IC₅₀ value of 29.647 ± 1.113 µg/mL in ABTS and 23.887 ± 1.13 µg/mL in DPPH has very strong antioxidant activity compared to ETM.05. We hypothesized that the ETM.05 had the highest antioxidants because it contained the highest amount of TPC (Table 2). Differences in chemical constituents in the ETM.05-ETM.10 might play an important role in this phenomenon. Moreover, the ETM.05 had higher antioxidant activity than other fractions. Several previous studies have demonstrated that the antioxidant activity of T. merguensis leaf extract falls into the “very strong” category when evaluated using the DPPH method. Enggiwanto et al., (2018) reported that the ethanol extract of Triptaniopsis merguensis leaves exhibited the strongest antioxidant (DPPH) activity with IC₅₀ of 18.277 µg/mL, while aseton extract, obtained through Microwave-Assisted Extraction (MAE), had IC₅₀ of 13.312 µg/mL, both classified in the “very strong” category. Roanisca et al., (2019) revealed that the antioxidant capacity of the acetone extract had an IC₅₀ value of 22,1454 µg/mL (very strong category) through the DPPH method. In their study, the total phenolic content and DPPH radical scavenging activity of the extract and all its fractions suggested a positive correlation. According to the results of total phenolic and flavonoid contents (Table 2), the ETM.05 exhibited high TPC and TFC which were very proportional to the high antioxidant activities in DPPH and ABTS. This agrees with another study indicating that the ethyl acetate and methanol fractions contain TPC and TFC which are responsible for the antioxidant activity. To the best of our knowledge, this is the first study to report the radical scavenging activity of ethyl acetate fraction from T. merguensis leaves using both DPPH and ABTS assays.

The difference in the antioxidant capacity is influenced by multiple factors, with the type of solvent used for fractionation being the most significant [38], [39]. In addition, plants contain a mixture of polar and non-polar secondary metabolites compounds, which can be dissolved in solvents of varying polarities [40]. The empirical principle, “like dissolves like”, means that polar compounds dissolve in polar solvents [41]. The ETM.01 recorded the lowest antioxidant capacity in both methods (DPPH and ABTS). The study reported that the compounds dissolved in n-hexane were less polar, such as lipids, which exhibit weak radical scavenging capacity [42], [43]. Cascaes et al. [36] and Silva et al. [44] revealed that the family of Myrtaceae contained high levels of phenolic and flavonoid content. The study proved its antioxidant, antihyperglycemic, and antibacterial effects [45].

The in vitro anticancer activity of ETM.01-ETM.10 fraction from ethyl acetate extract of T. merguensis leaves was evaluated through the MTT assay against MCF-7. In vitro results demonstrated that the ETM.01-ETM.10 fractions revealed mild to significant inhibitory potential, as indicated by the percentage of cell viability (Fig. 6). At concentrations of 3.91 and 7.81 µg/mL, the viability of MCF-7 cells remains relatively high, exceeding 80 %. This implies that at low concentrations, the compounds have not yet exhibited significant toxic effects on the cells. The high viability observed at these concentrations suggests that the majority of cells remain viable and functional. An increase in the fractions concentration to 15.63 µg/mL was observed to result in a 20 % reduction in cell viability in ETM.05. This reduction signifies that at a concentration of 15.63 µg/mL, the compound exerts cytotoxic effects, inhibiting cancer cell growth and triggering cell death. At the highest concentration of 500 µg/mL, there is a significant reduction in cell viability, with values dropping to approximately 50 % across all fractions. This precipitous decline suggests that all fractions demonstrate robust anticancer efficacy at elevated concentrations, resulting in the demise of the majority of MCF-7 cancer cells. Of the fractions tested, ETM.05 exhibited the most pronounced reduction in cell viability, followed by ETM.04 and ETM.09.

The lowest IC₅₀ value was observed in the ETM.05 fraction, followed by ETM.04, ETM.09, ETM.06, ETM.07, ETM.08, and ETM.03, with respective IC₅₀ values of 10.66, 21.33, 36.68, 51.92, 56.33, 71.60, and 99.55 µg/mL. These findings were based on the dose-response curve obtained from the MTT assay. A low IC₅₀ value (≤ 100 µg/mL) implies that the tested compounds exhibit robust anticancer activity against MCF-7 cells [46]. These findings indicate that the fractions ETM.05, ETM.04, ETM.09, ETM.06, ETM.07, ETM.08, and ETM.03 may possess considerable potential as anticancer agents against MCF-7 cells. The results of the cytotoxicity test in this study demonstrate a positive correlation with antioxidant activity and the total content of phenolic and flavonoid compounds. The results suggest that the compounds in the ETM.05 fraction exert their effects through a cytotoxic mechanism, specifically by inducing apoptosis (programmed cell death) in MCF-7 cells. This may be attributed to oxidative stress, cell cycle inhibition, or interaction with cellular receptors involved in cancer cell proliferation [47]. These results clearly establish the benefit of the ethyl acetate fraction from T. merguensis leaves. The toxicity and specificity studies demonstrated that the plants can be allowed for further application [48]. In order to identify the compound present in ETM.05, an LC-HRMS analysis was conducted.

LC-HRMS analyses revealed the presence of 20 major compounds comprising 7 flavonoid groups and 13 phenolic groups. The results of the LC-HRMS analysis indicate a positive correlation with the phenolic and flavonoid content. The phenolic compounds group consisted of osthol (3), yangonin (4), diphenolic acid (5), 4-methoxy-9-(2-methylbut-3-en-2-yl)-7H-furo [3,2-g] chromen-7-one (6), phloretin (7), 1a,7a-dihydro-3,6-dihydroxy-1a-(3-methyl-2-buten-1-yl) naphtha [2,3-b]oxirene-2,7-dione (9), caffeic acid phenethyl ester (10), formononetin (11), senkyunolide B (13), 1-(2,4-dihydroxy-5-methoxyphenyl)-2-(4-methoxyphenyl)-1-propanone (14), 2,6-di-tert-butyl-4-ethylphenol (15), 3′,4′-dihydroxyphenylacetone (19), and acetylshikonin (20). Meanwhile, the flavonoid compound group consisted of 2 R)-5-hydroxy-7-methoxy-2-phenyl-3,4-dihydro-2H-1-benzopyran-4-one (1), 5,6-dimethoxy-2-(2-methoxyphenyl)-4H-chromen-4-one (2), pinocembrin (8), zingerol (12), glycitein (16), 5,7-dihydroxy-6-methoxy-2-phenyl-3,4-dihydro-2H-1-benzopyran-4-one (17), and equol (18).

In the present study, the mechanism of interaction and correlation of the phenolic and flavonoid compounds of ETM.05 and their bioactivity against breast cancer were analyzed in silico. The in silico breast cancer activity of compounds from the ETM.05 fraction (Fig. 10) was assessed through molecular docking to predict the interactions between ligands (compounds in ETM.05) and the target proteins of breast cancer enzymes (HER-2). HER-2 is overexpressed in several cancers, such as breast cancer, and is associated with aggressive tumor growth and poor prognosis. Studying HER2 can help design therapies targeting its overexpression or activation. The molecular structure of the breast cancer’s receptor protein, native ligan (co-crystal), and overlay of co-crystal re-docking result can be seen in Fig. 10. To validate the docking method, the re-docking of the native ligand into the target protein pocket was carried out, resulting in the calculation of the Root Mean Square Deviation (RMSD) value. The results of the validation process indicate that the RMSD value of 1.05 Å is within the acceptable range, thereby demonstrating that the parameters utilized in the molecular docking method are in compliance with the established requirements. A smaller RMSD value indicates a greater degree of alignment between the crystallographic ligand position and the re-bonded ligand position.

Fig. 10.

2D interactions of co-crystals TAK-285 (native ligands) and compounds (2 R)-5-hydroxy-7-methoxy-2-phenyl-3,4-dihydro-2H-1-benzopyran-4-one (1), 5,6-dimethoxy-2-(2-methoxyphenyl)-4H-chromen-4-one (2), Osthol (3), Yangonin (4), Diphenolic acid (5), 4-methoxy-9-(2-methylbut-3-en-2-yl)-7H-furo [3,2-g] chromen-7-one (6), Phloretin (7), Pinocembrin (8), 1a,7a-Dihydro-3,6-dihydroxy-1a-(3-methyl-2-buten-1-yl) naphtha [2,3-b]oxirene-2,7-dione (9), Caffeic acid phenethyl ester (10), Formononetin (11), Zingerol (12), senkyunolide B (13), 1-(2,4-Dihydroxy-5-methoxyphenyl)-2-(4-methoxyphenyl)-1-propanone (14), 2,6-di-tert-butyl-4-ethylphenol (15), Glycitein (16), 5,7-dihydroxy-6-methoxy-2-phenyl-3,4-dihydro-2H-1-benzopyran-4-one (17), Equol (18), 3′,4′-Dihydroxyphenylacetone (19), Acetylshikonin (20).

The compounds that exhibited the strongest interactions were compounds 11, 5, 18, 6, 20, 2, 1, 15, 9, 3, 4, 8, 10, 16, 7, 17, 14, 13, and 12, with bond-free energy values of −7.62, −7.58, −7.51, −7.38, −7.29, −7.27, −7.14, −7.14, −6.94, −6.84, −6.81, −6.73, −6.61, −6.63, −6.59, −6.46, −6.29, −6.14 and −5.16 kcal/mol, respectively (Table 6). The binding energy of TAK-285 (native ligand) was −5.08 kcal/mol. It was found that the 19 compounds interact lower than the native ligand (Fig. 10). However, the bond-free energies of compound 19 are higher than that of the native ligand. Nevertheless, the binding pattern to key amino acids differs from that observed in co-crystals. A more negative bond free energy (DG) value indicates enhanced stability between the ligand and the target protein (receptor), resulting in a more robust bond.

As illustrated in Fig. 10, the compound that exerts the most pronounced influence on the receptor is the hydrogen bonding of the hydroxyl group (O–H). Furthermore, the nucleophilic water molecule participates in a reaction with the hydroxyl group attached to the aromatics [49]. In essence, this impedes the substrate's functionality by hindering the hydrolysis reaction. Hydrogen bonding represents a type of interaction that can facilitate the stabilization of the binding of a ligand to its receptors [50]. Furthermore, van der Waals interactions occur between the ETM.05 compound and the amino acid residue of the elastase, which can enhance conformational stability. The 19 most promising compounds from ETM.05 are phenolic and flavonoid derivatives with a planar aromatic ring.

The results of the in silico studies indicate that the majority of the phenolic and flavonoid compounds identified in the ETM.05 fraction are well accommodated at the binding site of the target enzyme, with a relatively large free binding energy, averaging −7 kcal/mol. The in silico molecular docking findings are in accordance with the in vitro results, which demonstrate a cytotoxic effect, particularly in the ETM.05 of the ethyl acetate fraction of T. merguensis leaves [51].

5. Conclusion

A total of twenty metabolites, predominantly phenolic compounds, were identified in the ETM.05 fraction. The antioxidant activity of the compound was evaluated using the ABTS and DPPH methods, which yielded IC₅₀ values of 64.830 µg/mL and 40.252 µg/mL, respectively. Anticancer testing against MCF-7 breast cancer cells demonstrated remarkable cytotoxicity (IC₅₀ of 10.66 µg/mL), exceeding the efficacy of cisplatin (IC₅₀ of 12.24 µg/mL). Molecular docking in silico validated the capacity of the metabolites to bind to HER-2, a protein implicated in the pathogenesis of breast cancer. The ETM.05 fraction derived from T. merguensis exhibits considerable promise as an antioxidant and anticancer agent.

Funding

The study was funded under the Indonesia Endowment Funds for Education Agency (LPDP) and Center for Higher Education Funding (BPPT).

CRediT authorship contribution statement

Respati Tri Swasono: Validation, Methodology, Formal analysis, Conceptualization. Tri Raharjo: Writing – original draft, Validation, Software, Conceptualization. Boima Situmeang: Methodology, Funding acquisition, Conceptualization.

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Boima Situmeang reports financial support was provided by Indonesia Endowment Funds for Education (LPDP) and Center for Higher Education Funding (BPPT). Boima Situmeang reports a relationship with Indonesia Endowment Funds for Education (LPDP) and Center for Higher Education Funding (BPPT) that includes: funding grants. Boima Situmeang has patent pending to 02012/J5.2.3./BPI.06/9/2022.

Handling Editor: Prof. L.H. Lash

Appendix A. Supplementary material

Supplementary material

Data Availability

Data will be made available on request.