INTRODUCTION:

Lung cancer is a disease that can cause death in both men and women, with an estimated 1.3 million deaths each year. In addition, treatment options available for advanced-stage disease are limited¹^,². One factor that causes lung cancer is smoking.

Air pollution, alcohol intake, nutrition, genetic predisposition, and occupational exposure are other factors. A high risk of lung cancer is caused by air pollution, both indoors and outdoors. Small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) are the two primary subtypes of lung cancer. It is estimated that 80% of all lung cancer cases are NSCLC. Usually, this kind of lung cancer is discovered when the metastases have progressed. By now, there is very little chance that surgery will have a curative impact. Additionally, a primary factor in NSCLC treatment failure that contributes to tumor recurrence and disease progression is medication resistance. Natural goods contain various anticancer substances that have been studied and turned into potent chemotherapeutic cancer medications. However, many of these chemotherapeutic drugs have been shown to produce significant toxic side effects and drug resistance. Therefore, continued research should be conducted to find more effective natural products with fewer side effects³. One promising strategy is to utilize natural ingredients. In today's era, natural ingredient-based treatments are increasingly popular. Most people feel safe in their use due to fewer side effects. A study conducted by the WHO gives credit to herbal remedies due to the therapeutic effects of natural ingredients. The increasing use of natural ingredients in medicine encourages researchers to identify chemical compounds that can provide pharmacological potential¹. One of the plants that can be used as a natural herbal medicine is the Chromolaena odorata L. weed plant. The C. odorata plant is part of the Compositae family. It is one type of weed that grows in tropical and humid sub-tropical regions worldwide. It has allelochemical compounds that can inhibit the growth of surrounding plants, both similar and different types⁴. C. odorata is thought to have a reasonably high defense because it is easy to grow even though it has been cut down and is often found on vacant land with dense and clumpy growth⁵. Environmentally friendly C. odorata weed control is one of the alternatives that can be done by utilizing the potential of its chemical compounds because C. odorata is also an herbal plant that contains vital chemical compounds⁶. Indonesian people, especially in the South Sulawesi region, use it as a wound medicine, dysentery, malaria, diarrhea, toothache, cough medicine, and laryngitis in traditional medicine⁷. The ability of C. odorata to cure various diseases cannot be separated from the active compounds contained in it, including alkaloids, flavonoids, steroids, terpenoids, phenolics, quinone saponins, and tannins⁸. Some studies show that C. odorata has bioactivity as antibacterial, antifungal, anti–inflammatory, anticancer, antiplasmodial, antidiabetic, and antioxidant⁹^,¹⁰. Therefore, this study characterizes the chemical content profile of C. odorata weed plants, in this case the leaves (LOD), stems (SOD), and roots (ROD) of C. odorata, and tests the antioxidant activity with the Nitric Oxide (NO), BCB and CUPRAC methods. As one of the test parameters for medicinal raw materials, especially as an alternative anti–lung cancer drug, the quality of the drug can be known by ensuring the authenticity of the ingredients used. Each medicinal plant contains certain chemical elements. A set of chemicals from medicinal plants can be analyzed and identified to ensure their authenticity. The chemical content in C. odorata weed plants can be known qualitatively and quantitatively using various UV–Vis spectrophotometric techniques, FT–IR, and GC–MS chromatography. One method of separating the components of the mixture used in this study is extraction¹¹. Extraction of C. odorata with 70% ethanol also has high antioxidant activity, so it is suitable for anticancer, antidiabetes⁴^,¹², anti–inflammatory¹³, and provide antigenic effects or stimulate the immune response, especially in producing antibodies and also conducted in silico tests through molecular docking simulations and predictions of absorption, distribution, bioavailability and toxicity, as anticancer guide compounds that have the potential to be used as anti–lung cancer candidates through EGFR inhibition.

MATERIALS AND METHODS:

The collection and extraction of Chromolaena odorata L. weed:

C. odorata plants were obtained from Panaikang Village, Pangkep Regency, South Sulawesi, Indonesia, and were determined with specimen number 0027/C/UD–FF/UMI/VIII/2022. In the Pharmacognosy–Phytochemistry Laboratory/VII/2021 at the Pharmacognosy–Phytochemistry Laboratory, Faculty of Pharmacy, Muslim University of Indonesia. C. odorata plants were separated from the attached dirt and then washed thoroughly and drained. Clean parts of the C. odorata plant leaves (LOD) stems (SOD), and roots (ROD) were placed on a tray and then dried in an oven at 40⁰C. The dried samples were then powdered and extracted using 70% ethanol; after extracting, each sample was evaporated with a rotary evaporator until the ethanol extract of C. odorata LOD, SOD, ROD was obtained.

Phytochemical screening:

This study conducted the qualitative screening of chemical components in the ethanol extract¹⁴^,¹⁵ of C. odorata weeds. The aim was to determine the presence of alkaloids, flavonoids, steroids, terpenoids, saponins, and phenols¹⁶.

Total phenolic content (TPC) and total flavonoid content (TFC):

Total phenolic content (TPC): Using the Folin–Ciocalteu reagent, TPC was evaluated. First, 0.5mL of Folin–Ciocalteu reagent, 7.5mL of ionized water, and 2 mg of dry mass of crude extract were combined. After 15minutes at room temperature, 1.5mL of 20% (b/v) sodium carbonate was added to the mixture. After 20 minutes of heating in water and cooling in an ice bath, the combination was tested for absorbance at its maximum wavelength using a Shimadzu U-1900 spectrophotometer. The quantity of TPC was determined using the calibration curve for gallic acid as a comparator. Gallic acid equivalents (GAE) for dry plant material were used to express the results.

Total flavonoid contents (TFC):

The quantification of total flavonoids was performed through a colorimetric method employing the AlCl₃ reagent. The dissolution of 10grams of extract in 10mL of ethanol yielded a concentration of 1000 parts per million (ppm). In the experimental procedure, Pipetting the volume of extract 1mL. Subsequently, this volume was combined with 1mL of a solution containing 2% AlCl₃ and 0.1mL of a sodium acetate solution with a concentration of 1M. The specimen was subjected to incubation for a duration of one hour under ambient conditions. The determination of absorbance was conducted using the UV–Vis spectrophotometric technique, specifically at a wavelength of 432nm. The samples were prepared in triplicate for each analysis, and the quantification of total flavonoid levels was expressed as milligrams of quercetin equivalent (GAE) per gram of extract¹⁷.

Chemical characterization using FT–IR analysis (Fourier Transform Infrared Spectroscopy Spectrum):

Using an FT–IR spectrometer outfitted with an infrared source¹⁸, a potassium bromide beam splitter, and a susceptible DigiTectTM Detector system, vibrational spectra for LOD, SOD, and ROD odorata extracts were acquired. To reduce the amount of water absorbed, a disk with dimensions of 13 mm in diameter and 1-2mm thick, containing roughly 1 mg of sample evenly distributed in 150mg of KBr, was gently heated to 130°C. The disk was then scanned immediately and thoroughly in the 4000–400cm^–1 wave number region in transmission mode with a resolution level of 8 cm^–1 with 135 consecutive scans. Before collecting each spectrum, the ATR crystal was cleaned with absolute ethanol to remove any residue.

Chemical characterization using GC–MS analysis:

The GC–MS analysis was conducted in accordance with the established methodology outlined by Jana et al¹⁹. The Thermo Scientific, Trace GC Ultra/ISQ Single Quadrupole MS instrument was utilized, along with a TG–5MS fused silica capillary column measuring 30 m in length, 0.251mm in diameter, and featuring a 0.1 mm film thickness. A gas chromatography mass spectrometry (GC–MS) identification technique employed an electron ionization system characterized by an ionization energy of 70 electron volts (eV). The carrier gas employed in this experiment was helium gas, which was utilized at a flow rate of 1ml/min. The temperature of the injector and mass spectrometer (MS) transfer line was adjusted to 280°C. The initial temperature of the oven was set to 50°C, and it was maintained at this temperature for a duration of 2 minutes. Subsequently, the temperature was increased to 150°C at a rate of 7°C per minute. Following this, the temperature was further raised to 270°C at a rate of 5°C per minute, and it was held at this temperature for 2 minutes. Finally, the temperature was increased to the definitive value of 310°C at a rate of 3.5°C per minute, and it was maintained at this temperature for a duration of 10 minutes. The relative peak area percent was utilized to quantitatively determine all identified compounds. The components were provisionally recognized by comparing their non-relative retention times and mass spectra with those available in the National Institute of Standards and Technology (NIST) and Wiley data library of GC–MS techniques²⁰^,²¹.

Antioxidant analysis by NO (Nitric Oxide), BCB, and CUPRAC methods.:

NO (Nitric Oxide) radical scavenging assay:

The free radical scavenging²²^,²³ activity was measured using the Nitric Oxide Assay, following the methodology described by (Boora et al., 2014). Concisely, different sequential dilutions of the tested sample (1.5mL) were introduced into a solution (1.5 mL) containing 0.1mmol of NO. Control groups consisting of ethanol and NO were utilized, with the volumes of these substances equal. Following a 60–minute incubation period, the sample was treated with Griess reagent based on manual instruction. The solution was diluted to a final volume of 5mL using distilled water and subsequently subjected to incubation for 30 minutes. Subsequently, the mixture was quantified using a UV–Vis spectrophotometer set at 542nm. The IC₅₀ value, which signifies the percentage inhibition, was determined using the formula that calculates the concentration of the tested sample needed to eliminate 50% of the radicals. The IC₅₀ value, which represents the percentage inhibition, was calculated using the following formula²⁴.

(Abs Blank – Abs Sample)

Free Radical Scavenging = ------------------------- x100 %

Absorbansi Blank

Inhibition of lipid peroxidation by beta carotene bleaching (BCB):

The BCB assay test was run with a few minor adjustments. In order to prepare beta carotene emulsion (BCE), 20mg of beta carotene powder was dissolved in 0.2mL of chloroform, then 0.2mL of linoleic acid, and 2 mL of tween 20. The residual chloroform was evaporated after thorough mixing. Up to 100 milliliters of distilled water were added to the solution to dilute it and create a translucent emulsion. Each extract solution's serial volume was created by combining 1mL of BCE with 5mL of ethanol. A spectrophotometer (Shimadzu UV–1900) was used to measure the absorbance at 467nm following 20 and 120 minutes of incubation at 50C. The absorbance was measured using the same approach after producing a blank solution (1 mL BCE in 5mL ethanol). As comparators, quercetin and butyl hydroxytoluene (BHT) were employed. The antioxidant activity was determined by comparing the sample's degradation to the blank solutions. The absorbance data were then processed using the formula (ln(a/b)1/t), where t is the incubation time (120 minutes) and is the absorbance value after 0 minutes of incubation. b is the absorbance value after 120 minutes. Ln represents the relationship between natural logarithms.

CUPRAC (Cupric et al. Capacity) radical scavenging assay:

CUPRAC solution was done by mixing 5mL CuCl₂.2H₂O 0.01M; 5mL Neocupproine Ethanolic 0.0075 M; 5mL NH₄Ac Buffer 1 M, and 1mL of distilled water, stirring was done. Samples and cupric reagent were incubated for 30 minutes with 1mL ethanol p.a in a vial, and the absorbance was measured at 450 nm using a UV– Vis Spectrophotometer²⁵. GAEAC (Galate et al. Capacity) of the sample was obtained by calculating the absorbance of the sample against the concentration series of quinine standard solution and recorded as equivalent to µmol/g sample obtained from the calibration curve results with the equation y = bx + a, where the antioxidant activity profile was obtained by interpolating the absorbance value of the sample as value (y) so that the antioxidant activity profile as value (x) in the sample can be known. The value is used to calculate the reducing power of the sample with the formula:

GAEAC =

In silico assay with molecular docking:

Molecular docking analysis of the eight compounds generated through GC–MS analysis First, the 5UG8 protein complex was separated between macromolecules and Gefitinib ligand using the Discovery Studio Visualizer® program²⁶, then optimized by adding hydrogen atoms and energy minimization. The ligand was redrawn using the ChemDraw® Ultra 14.0 program, and energy was minimized using the Chem3D® Pro 12.0 program and then saved in .pdb format. After the preparation, the determination of the physicochemical properties of the compound based on Lipinski's Rule of Five. ²⁷

RESULT:

Extraction, Percent yield, Total phenolic contents (TPC) and Total flavonoid contents (TFC):

Maceration was the method used to extract the three plant organs of C. odorata. 70% concentration of ethanol was used as the solvent for the extraction. The yield percentage achieved can be used to gauge the solvent's ability to extract chemicals. Alkaloid, terpenoid, phenolic, flavonoid, and saponin content in C. odorata leaf, stem, and root extracts were found using phytochemical experiments with chemical reagents (Table 1). In addition, the outcomes of contrasting total flavonoid content (TFC) ²⁸ utilizing quercetin with total phenolic content (TPC) using gallic acid. The percentage yield, TPC, and TFC of the three C. odorata weed parts that were extracted using 70% ethanol are displayed in Table 2.

Results of antioxidant activity testing by NO, BCB, and CUPRAC method:

Antioxidant activity testing on ethanol extracts³¹ of C. odorata LOD, SOD, and ROD (as depicted in Figure 3) showed that all extracts exhibited antioxidant properties ³², through their ability to counteract free radicals³³.

NO (Nitrat Oksida) assay:

The antioxidant activity with the same concentration was tested in vitro using nitric oxide (NO) (as depicted in Figure a), with quercetin control (Figure d). Nitric oxide reacts with O₂ and H⁺/H^- to form peroxy nitrite and hydrogen radicals, damaging tissues and cells through free radical mechanisms³⁴. NO from sodium nitroprusside forms nitrite with oxygen. Nitrite ions undergo diazotization with sulfanilamide acid and pair with naphthyl ethylenediamine, forming a pink color at a wavelength of 546 nm. Leaf ethanol extract showed the highest antioxidant activity with an IC₅₀ value of 28,641 µgmL^–1, classified as a powerful antioxidant (IC₅₀ ≤ 50 µg mL^–1). The stem ethanol extract showed vigorous antioxidant activity (IC₅₀ = 50–100 µg mL^–1) with an IC₅₀ value of 54.04µg mL^–1. In contrast, the root ethanol extract showed weak antioxidant activity with an IC₅₀ value of 146.147µg mL^–1 (IC₅₀ = 150–200µg mL^–1).

BCB (Beta carotene bleaching) assay:

The C. odorata extract's antioxidant capacity was expressed as an IC₅₀ value (Figure b). A higher sample concentration is necessary to impede the amount of beta-carotene degradation. Each C. odorata extract had a significantly different antioxidant capacity among the samples, according to the test results for antioxidant ability in reducing the rate of beta-carotene degradation by lipid peroxidation (P<0.0001, n=3).

Figure 3. Nitric oxide (NO) radical scavenging activity (a), ABTS radical scavenging activity (b), and cupric reducing antioxidant capacity (CUPRAC) (c) of the three organs of leaf (LOD), stem (SOD) and root (ROD) ethanol extracts of C. odorata plants (all performed with three repetitions), Quercetin positive control (d), Ascorbic acid positive control (e), Beta Carotene Bleaching positive control (f).

The LOD sample (IC₅₀<50µg mL^-1) showed high antioxidant properties, with quercetin as positive control and BHT (IC₅₀ = 2.536µg mL^-1) as positive control; from the results of antioxidant activity with BHT, it was found that the ethanol extract of LOD had highly active activity with IC₅₀ value of 13.227µg mL^–1, IC₅₀ of SOD = 55.353, and IC₅₀ value of ROD extract = 145.089µg mL^-1. The ROD extract of BHT was employed as a reference because BHT is known to have particular antioxidant characteristics in avoiding lipid peroxidation, and the SOD sample demonstrated strong antioxidant properties (IC₅₀ = 100–150µg mL^–1).

CUPRAC (Cupric Ion Reducing Antioxidant Capacity) essay:

In this test, the CUPRAC (Cupric Ion Reducing Antioxidant Capacity)³⁵ method was used using extracts of C. odorata leaf, steam, and root samples. The principle of the CUPRAC method is that the blue Cu(Nc)₂²⁺ reagent will be reduced to yellow Cu(Nc)²⁺ ³⁶. From the results of antioxidant activity testing of several parts of the C. odorata plant using the CUPRAC method, the results were declared equivalent to querceti (Figure d) as a comparison can be seen in Figure c, where the LOD extract has a significantly higher reducing ability (p = 0.05) with a value of 782.215 ± 2.872 l MQEV/g among the samples, followed by SOD (600.404 ± 2.111), and ROD (397.823±2.131).

Correlation between total phenolic content (TPC) and total flavonoid content (TFC) and various antioxidant activities.:

To ascertain the correlation between TPC, TFC³⁷, and other antioxidant activity tests of C. odorata extracts, principal component analysis (PCA) was conducted. According to Figure 4 and Table 4, the initial principal components, PC1 and PC2, accounted for 96.78% and 3.22% of the total variance in the data set, respectively. According to the results, NO was on PC2. At the same time, CUPRAC, TPC, and TFC were situated on the right side of PC1 and BCB (Figure a). Table 4 displays the Pearson correlation coefficient, which indicates a robust negative association between TPC and several antioxidant activities of C. odorata extract (TPC–NO: r = - 0.-0.945; TPC–BCB: r = - 0.979; TFC–NO: r = - 0.985); TFC–BCB: r = - 0.934). Positive linear associations, on the other hand, were noted. According to the findings, PC1 and BCB were situated on the right side, where CUPRAC, TPC, and TFC were. PC2 has NO (Figure a). Table 3 displays the Pearson correlation coefficient, which indicates a robust negative association between TPC and several antioxidant activities of C. odorata extract (TPC–NO: r = - 0.-0.945; TPC–BCB: r = - 0.979; TFC–NO: r = - 0.985); TFC–BCB: r = - 0.934)—on the other hand, using several C. odorata extracts, favorable linear correlations were found between TPC and TFC (r = 0.987), TPC and CUPRAC (r = 0.999), and TFC and TPC (r = 0.987).

Table 4. Chemical composition of ethanol extracts of three organs of C. odorata analyzed by GC-MS and values of binding energy and amino acid residues that bind to Gefitinib (5GU8).

RT	MF	Exctracts	Main Fragments	Moleculer Ion	Identified Compounds	Bond-free energy (kcal/ mol)	H-bond/ vander waals Interaction*	Bond Distance
10.49	C₉H₈O₃	LOD, SOD	163, 147, 126, 124	[M⁺H]⁺	3-(4-hydroxyphenyl) prop-2-enoic acid	-7.25	Met(793), Thr(854)	2.2, 1.78
12.62	C₁₁H₁₄N_2O	LOD, SOD	195, 171, 126	[M⁺ Na] ⁺; [M⁺H]	7,11-diazatricyclo [7.3.1.02,7] trideca-2,4-dien-6-one	-6.88	Met(793), Thr(854), Gln (791)	2.00; 1.65, 3.0
15.03	C₁₅H₁₂O5	LOD, SOD	272, 225, 181, 138, 123, 97,83	[M⁺Na] ⁺ ; [M⁺H]	(2S)-5,7-dihydroxy-2-(4-ydroxyphenyl)-2,3-ihydrochromen-4-one	-9.15	Met(793), Gln(791), Thr(854), Asp(855), Lys(745), Leu(718)	2.24, 2.49, 2.07, 3.02, 1.99, 1.85
20.58	C₁₆H₁₂O₅	LOD, SOD, ROD	284, 225, 180, 56	[M⁺H]⁺	2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one	-9.08	Met(793), Lys(745), Leu (718)\	2.10, 5.3, 2.02
22.05	C₁₆H₃₂O₂	LOD, SOD, ROD	255, 196, 151, 95,94	[M⁺H^]+	n-Hexadecanoic acid	-7.17	Lys(745), Thr(854)	1.77, 2.05
28.57	C₂₀H₃₆O₂	LOD, ROD	284, 225, 180, 56	[M⁺H]⁺	(1r,2r,4as,8as)-1-[(3r)-3-hydroxy-3-methylpent-4-enyl]-2,5,5,8a-tetramethyl-3,4,4a,6,7,8-hexahydro-1h-naphthalen-2-ol	-9.27	Lys(745), Thr(854)	4.95, 1.95
30.3	C₁₅H₁₂O₅	LOD, SOD, ROD	272, 225, 181, 138, 123, 97,83	[M⁺H]⁺	5,7-dihydroxy-2-(4-hydroxyphenyl) chromen-4-one	-9.84	Met(793), Gln(791), Thr(854), Asp (855), Lys(745), Leu(854)	2.29, 2.69, 2.07, 2.99, 1.95, 1.93
34.44	C₃₂H₅₂O₂	LOD	463, 393, 301, 109, 95, 81, 69	[M⁺Na] ⁺	Lanosta-8,24-dien-3-ol, acetate, (3 beta.)	-9.11	Cys (797); Ala (743); Val (726); Phe (723)	4.66. 3.15, 4.08

Note: RT =Retention time, MF = Molecular Formula

DISCUSSION:

The plant Chromolaena odorata L. is a weed that gets less attention even though it has potential as a material that can be used in various traditional medicine systems because it can produce various secondary metabolites that offer a variety of biological activities³⁹. This research focuses on identifying chemical compounds as anti-cancer, especially lung anti-cancer, from C. odorata plant organ extracts based on antioxidant activity and in vitro and molecular docking approaches. In this study, the extraction method separated mixed materials using 70% ethanol solvent. The maceration procedure was carried out by soaking each powder of LOD, SOD, and ROD of C. odorata in 70% ethanol solvent three times 24 hours while occasionally stirring. This choice was made because ethanol has a lower dielectric constant than water, which has a dielectric constant of 80. The dielectric constant measures polarity, with higher values indicating greater solvent polarity. Based on their respective dielectric constants, ethanol and water have a lower polarity⁴⁰. Moreover, compared to the use of other solvents like ethyl acetate and n-hexane, which have low polarity, the use of ethanol solvent has been shown to draw carbohydrate group components, phenolic flavonoids, alkaloids, steroids, and terpenoids to permit large percentage yield values⁴¹. This is also supported by the analysis using FT–IR and GC–MS⁴², three dominant compounds were obtained from the three extracts of LOD, SOD, and ROD C. odorata, including: diazatricyclo [7.3.1.02.7] trideca-2,4-dien-6-one; n–hexadecanoic acid and 5,7-dihydroxy-2-(4-hydroxyphenyl) chrome-4-one, this shows that from the three extracts of C. odorata in general, three compounds were obtained. Data in general, two groups of phenolic compounds and one phthalic acid were obtained, while from the FT–IR results, the composition of the functional groups of compounds that characterize the ethanol extracts of LOD, SOD, and ROD C. odorata lies in the difference in the composition of the functional groups of the compounds present. These compounds are characterized by O–H groups and aromatic rings containing C=C functional groups. In addition, the extract showed the presence of C=O carbonyl groups, particularly conjugated aldehydes. Furthermore, typical absorption patterns of C=C functional groups, namely conjugated alkenes, were observed⁴³^,⁴⁴. Based on the results of GC-MS and FT-IR identification, flavonoid, phenolic, alkaloid, terpenoid, and palmitic acid compounds can be identified. It has pharmacological and antioxidant effects from the compounds obtained, primarily on phenolic and flavonoid compounds.³⁷ and at the same time as an anticancer. In ethanol extracts of LOD, SOD, and ROD C.odorata using 70% ethanol solvent, the LOD extract yielded the highest extract yield at 10.27%, followed by the SOD extract at 7.05% and the ROD at 3.78%.. This study obtained the highest TPC content in the C.odorata LOD extract compared to other extracts. However, the SOD C.odorata extract showed higher TPC than the ROD C.odorata extract and followed by a relatively higher amount of TFC in the SOD C.odorata extract compared to the other extracts (Table 5). This may be due to the higher amount of bioactive compounds in LOD extract compared to SOD and ROD C.odorata, thus affecting the total TPC and TFC. The TFC found in this study indicates that flavonoids from C.odorata's three organs may be a vital source of antioxidant compounds.

Antioxidant assays in the in vitro study⁴⁵, including NO, BCB, and CUPRAC, were analyzed to determine the antioxidant potential of crude extracts from three plant organs of C. odorata weed (Figure 3). NO, BCB and CUPRAC methods showed extreme activity in the ethanol extract of LOD C. odorata. The radical scavenging activity of the ethanol extract of LOD C. odorata is stronger than the other extracts, indicating that the number of phytochemical compounds in the ethanol extract of LOD C. odorata is higher, especially for phenol and flavonoid compounds and n-hexene acid compounds (Figure 2). Where flavonoids have a hydroxyl group (–OH) attached to the aromatic carbon ring. Resonantly stabilized free radical derivatives of these compounds show relatively low reactivity compared to most other free radicals, so their role as antioxidants is relatively high⁴⁶^,⁴⁷. And n–hexadecanoic acid is a long–chain saturated fatty acid compound that has potential as an antioxidant⁴⁸ due to the formation of free radicals R* by releasing H atoms from fatty acid molecules and when fatty acid radicals (R*) from the initiation stage meet with oxygen will form peroxide radicals (ROO*). Peroxide radicals formed with other fats (R1H) form hydroperoxides (ROOH) and new fatty acid radical molecules (R1*), and the last stage is the termination stage, where highly unstable hydroperoxides are broken down into short-chain organic compounds by reacting with free radicals (R*). The TPC value results⁴⁹, are highly correlated with the antioxidant activity found in this investigation. Similar to this conclusion⁵⁰, it has been previously reported that antioxidant activity is connected to phenolic content, and the higher the phenolic concentration, the better the antioxidant potential. The results of this investigation indicated a robust negative association between TPC and different antioxidant activities of C. odorata extracts, as indicated by the Pearson correlation coefficient and PCA (Table 4 and Figure 4). The inverse association between the antioxidant activity values accounts for this negative correlation. In contrast, a positive linear association (r = 0.987) was discovered between TPC and TFC. Thus, total phenolic and flavonoid levels in seaweed crude extracts are essential indicators of antioxidant efficacy. According to the loaded values and factor maps, the primary contributors to PC1 were TPC, NO, BCB, and CUPRAC. TFC was the primary cause of PC2 during the same period (Figure 4). These findings suggest that TPC and TFC significantly impact the antioxidant activity of C. odorata extracts in all three organs.

The molecular tethering process is carried out using the Auto Dock Tools application. Parameters of molecular tethering results can be seen from the value of Gibbs free energy or binding energy (ΔG). The lower the bond energy value, the stronger the compound-complex bond with the receptor. The first stage is validation by re-docking between standard ligands and macromolecules. The re-docking result of EGFR macromolecule and its natural ligand N-[(3R,4R)-4fluoro-1-{6-[(1-methyl-1H-pyrazole-4-yl) amino] -9-(propane-2-yl) -9H-purin-2-yl} pyrrolidin-3-yl] propenamide which has been previously optimized with Auto dock Tools® application (The Scripps Research Institute, America) and then docked again. Based on the validation results, the position was obtained x = –13.156 y = 15.093 z = –25.719 with RMSD 1.899 Å, and ΔG value –7.72kcal/mol inhibition constant 495.16 nM. Visualization of re-docking results can be seen in Figure 6. The re-docking results obtained an RMSD value of 1.899 Å, which means the receptor is qualified because of the RMSD value of 1.899 Å. Qualified because the RMSD value <2 Å can be used to tether the test compound⁵¹. Furthermore, molecular tethering simulations were carried out with the same application, using eight compounds and Gefitinib as a ligand against EGFR in oriented docking. Of the eight tethered compounds, we obtained that the best compound was 5,7-dihydroxy-2-(4-hydroxyphenyl) chrom-4-one with ΔG –9.84 kcal/mol, even better than Gefitinib which was the positive control (ΔG –7.72 kcal/mol) - by what has been done by llll stating that the compounds [7. 3.1.02.7] trideca-2,4-dien-6-one and 5,7-dihydroxy-2-(4-hydroxyphenyl) chrom-4-one have been shown to exhibit anticancer effects, primarily on lung cancer ⁵²^, ⁵³. The compound can trigger cell apoptosis by inhibiting cancer cell proliferation, inducing autophagy, and regulating the cell cycle. In addition, they decrease cancer cell motility and inhibit cancer cell migration and invasion. Recently, the compound 5,7-dihydroxy-2-(4-hydroxyphenyl) chrom-4-one showed anticancer activity by stimulating immune responses⁴⁷. During the process, several signaling pathways and protein kinases are modulated by compound 5,7-dihydroxy-2-(4-hydroxyphenyl) chrom-4-one. This suggests that EGFR will have a more stable binding with the C. odorata compound than Gefitinib (Table 3). The re-docking results of natural ligand 5UG8 with EGFR were visualized with the help of Discovery Studio Visualizer®. Hydrogen bonds and hydrophobic interactions formed between natural ligands and EGFR were analyzed ⁵⁴. Based on the optimization results, the number of hydrogen bonds between natural ligands and EGFR is two hydrogen bonds involving one amino acid, Met793. In addition to hydrogen bonds, there are interactions involving Gln791, Leu718, Thr854, Asp855, and Lys745. Based on the results of hydrogen bond analysis of test compounds with EGFR, some test compounds have bonded to identical amino acid residues as natural ligands and Gefitinib on the receptor. Test compounds that bind to identical amino acid residues as natural ligands can have the same biological activity as natural ligands. The hydrogen bond formed at the interaction of EGFR and its natural ligand involves the amino acid residue Met793. The comparator compound Gefitinib also involves the amino acid residue Met793. Met793 is an amino acid residue involved in the interaction of EGFR inhibitors. On EGFR this test compound has more hydrogen bonds, namely at Met793, Gln791, Leu718, Thr854, Asp855 and Lys745 (Figure 7). The bond distance influences the bond interactions that occur. Weak interactions are only possible if the molecular surfaces are nearby and complementary. Therefore, the bond strength is highly dependent on the distance. The bond distance formed from the hydrogen bond interaction of the compound against EGFR can be observed (Table 4). This indicates that the bond formed between the compounds of C. odorata extract and EGFR is strong. According to the In Silico study, the majority of the phenolic group chemicals found in C. odorata LOD, SOD, and ROD extracts have potential applications as anti-cancer drugs, particularly for lung cancer.

CONCLUSION:

Based on the results of the study, ethanol extracts of the three organs of leaves (LOD), stems (SOD), and roots (ROD) of Chromolaena odorata L. plants from phytochemical identification identified eight secondary metabolites, and the majority were phenolic compounds. Each C. odorata extract contains high antioxidant potential and has a considerable correlation with TPC and TFC in C. odorata extracts. The in vitro and silico analysis of C. odorata plants with 5GUB target protein showed that most of the phenolic group compounds identified in ethanol extracts of C. odorata LOD, SOD, and ROD can be used as a potential source of antioxidants and as a traditional medicine, especially as a candidate for lung cancer.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

ACKNOWLEDGMENTS:

This research is a National Competitive Research (Research Cooperation - Domestic) funded by the Ministry of Education, Culture, Research and Technology, Republic of Indonesia.

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Received on 11.01.2024 Revised on 08.05.2024

Accepted on 19.08.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):6020-6031.

DOI: 10.52711/0974-360X.2024.00914

Reagen	Compound	Colour	Organ C. odorata
Reagen	Compound	Colour	Leaf	Stem	Root
Dragendrof	Alkaloid	red precipitate	+	+	-
Diluted NaOH Reagent	Flavonoids	fading yellow	+	+	+
Libermann-Burchard	Steroids	brown color ring circle	+	+	+
Salkowski’s	Triterpenoid	golden yellow	+	+	-
Liebermann-Burchard	Saponin	creating foam	+	-	-
Folin-ciocalteu	Phenol	blue	+	+	+

Ekstrak C. odorata	% Yield of extracts	TPC (GAE mg/g) of dry plant matter	TFC (CE mg/g) of dry plant matter
LOD	10.27 ± 0.07	7.518 ± 0.03	2.152 ± 0.04
SOD	7.05 ± 0.05	4.922 ± 0.04	1.055 ± 0.022
ROD	3.78 ± 0.0235	2.319 ± 0.023	0.434 ± 0.41

Correlation	TPC	TFC	NO	BCB	CUPRAC
TPC	1	0.98732	-0.9459	-0.9791	0.99954
TFC	0.98732	1	-0.9854	-0.9344	0.98204
NO	-0.94593	-0.9854	1	0.86019	-0.9356
ABTS	-0.9791	-0.9344	0.86019	1	-0.9848
CUPRAC	0.99954	0.98204	-0.9356	-0.9848	1