{"id":69580,"date":"2025-12-30T11:46:44","date_gmt":"2025-12-30T11:46:44","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=69580"},"modified":"2026-01-03T15:21:05","modified_gmt":"2026-01-03T15:21:05","slug":"comparative-study-on-the-anticancer-potential-of-apigenin-and-syringic-acid-isolated-from-parsley-leaves-against-a375-cell-line","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol18no4\/comparative-study-on-the-anticancer-potential-of-apigenin-and-syringic-acid-isolated-from-parsley-leaves-against-a375-cell-line\/","title":{"rendered":"Comparative Study on the Anticancer Potential of Apigenin and Syringic Acid Isolated from Parsley Leaves Against A375 cell line"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Parsley (<\/span>Petroselinum crispum<\/em> L.<\/em>) is a widely cultivated biennial herb native to the Mediterranean, thriving in rich, well-drained soils under full or partial sunlight, and often grown from seeds planted in early spring.<\/span>1<\/sup> This vibrant green herb is celebrated for its diverse phytochemical profile, including abundant flavonoids like apigenin and luteolin glycosides, volatile oils such as apiol and myristicin, and phenolic acids like syringic and ferulic acids, which together confer potent antioxidant and anti-inflammatory properties.<\/span>2<\/sup><\/p>\n

Traditionally, parsley has been used across cultures as a digestive aid, mild diuretic, breath freshener, and remedy for kidney or bladder issues, while in folk medicine it has also been employed to ease menstrual discomfort and reduce swelling, making it a plant of both culinary and medicinal significance .3<\/sup><\/p>\n

Cancer is a complex disease assigned by the unrestrained growth of abnormal cells, which can infiltrate neighboring tissues and spread to distant sites through metastasis.4<\/sup> Its development is driven by a combination of genetic mutations, epigenetic modifications, and disturbances in key signaling pathways that regulate cellular proliferation, programmed cell death, and differentiation .5.\u00a0\u00a0\u00a0\u00a0 <\/sup>Although conventional treatments like surgery, chemotherapy, and radiation therapy have advanced, they still face significant drawbacks, such as serious side effects, the emergence of treatment resistance, and a lack of selectivity for cancer cells over healthy ones.6<\/sup>,<\/sup>7<\/sup> As a result, there is increasing interest in identifying new therapeutic agents, particularly natural compounds with potential anticancer properties, due to their promise of improved safety and effectiveness.8<\/sup><\/p>\n

Apigenin is a naturally occurring flavonoid found abundantly in many fruits, vegetables, and herbs. It is especially concentrated in parsley, chamomile, celery, and oranges, making these common dietary sources of this bioactive compound .9<\/sup>,<\/sup>10<\/sup> Traditionally, plants rich in apigenin have been used in folk medicine across various cultures to treat conditions such as inflammation, anxiety, insomnia, digestive issues, and skin disorders.8<\/sup> Chamomile tea, for example, is widely consumed as a calming remedy and owes much of its soothing effect to its apigenin content.11<\/sup>,<\/sup>12<\/sup><\/p>\n

Beyond its traditional applications, modern pharmacological research has revealed that apigenin possesses a varied spectrum of biological activities, as antioxidant, anti-inflammatory, antimicrobial, neuroprotective, and notably, anticancer activities.13<\/sup>–<\/sup>15<\/sup> Studies have shown that apigenin can inhibit the growth of cancer cells, induce apoptosis, and suppress tumor angiogenesis and metastasis by modulating various molecular pathways involved in cell cycle regulation and survival .14<\/sup>,<\/sup>15<\/sup><\/p>\n

Because of these promising properties, apigenin is increasingly being explored as a potential therapeutic agent in cancer prevention and treatment strategies.<\/span><\/p>\n

Syringic acid is a naturally occurring phenolic acid that belongs to the class of hydroxybenzoic acids. It is widely distributed in the plant kingdom and can be found in various fruits, vegetables, and medicinal herbs, including grapes, olives, dates, honey, and spices like turmeric and parsley. 1<\/sup>6<\/sup>,<\/sup>17<\/sup> This compound is also abundant in the bark and heartwood of certain trees, such as maple and walnut, as well as in red wine and other plant-derived foods and beverages.16-18<\/sup><\/p>\n

In traditional medicine, syringic acid-rich plants have long been valued for their health-promoting properties. Remedies containing syringic acid sources have been used for centuries to manage inflammation, pain, infections, and digestive problems in different cultures around the world. 19<\/sup>,<\/sup>20<\/sup> These traditional uses laid the foundation for scientific investigations into its potential health benefits.Modern pharmacological research has highlighted syringic acid\u2019s diverse biological activities, including antioxidant, anti-inflammatory, antimicrobial, antidiabetic, neuroprotective, and anticancer effects.21<\/sup>–<\/sup>23<\/sup><\/p>\n

Studies show that syringic acid can neutralize free radicals, reduce inflammatory responses, modulate key metabolic enzymes, and even induce cancer cell apoptosis by influencing signaling pathways related to cell proliferation and survival.22<\/sup>,<\/sup>23<\/sup> These findings support syringic acid\u2019s promise as a natural therapeutic agent for various chronic diseases, including cancer and metabolic disorders. In this study, we hope to isolate two phenolic compounds Apigenin and Syringic acid from ethyl acetate fraction of parsley leaves extract and compared their cytotoxic effects on A375 cell line.<\/p>\n

Materials and Methods<\/strong><\/p>\n

Plant Collection and extraction<\/strong><\/p>\n

Parsley leaves were harvested from a home garden during the summer season and experienced authentication directed by expert Prof. Sukaena Abass. The collected leaves were thoroughly washed, shade-dried, and then mechanically ground into a fine powder. For extraction, 20 g of the powdered parsley leaves were mixed with 150 mL of 90% ethanol and subjected to ultrasonic-assisted extraction using an ultrasonic bath sonicator operating at ( 40 kHz and 45\u202f\u00b0C for 60 minutes) which relies on the principle of acoustic resonance, where small bubbles form and then burst near the plant cell wall, leading to its destruction and the release of active substances into the solution. These resulting cavities are a consequence of the conversion of electrical energy into high-frequency sound waves transmitted through the liquid inside the tank. The resulting mixture was filtered through Whatman filter paper. The filtrate was evaporated to dryness, and the dried extract was subsequently suspended in a biphasic system comprising 150 mL of distilled water and 150 mL of ethyl acetate. The ethyl acetate fraction (EtOAc) was then subjected to Preparative High-performance Liquid Chromatography (PHPLC) for the isolation of phenolic compounds.24<\/sup><\/p>\n

P<\/strong>reparative <\/strong>H<\/strong>igh-performance <\/strong>L<\/strong>iquid <\/strong>C<\/strong>hromatography (<\/strong>P<\/strong>HPLC)<\/strong>Conditions<\/strong><\/p>\n

Quantitative analysis of phenolic compounds in the EtOAc was carried out using PHPLC. The phenolic constituents expected in the fractions were identified by comparing their retention times with those of standard compounds, namely apigenin and syringic acid. The chromatographic separation employed a mobile phase consisting of 1% aqueous acetic acid (solvent A) and acetonitrile (solvent B), with the flow rate set at 3 mL\/min and an injection volume of 300 \u03bcL. A gradient elution was applied, starting with 10% B and increasing linearly to 40% B over 28 min.then from 40% to 60% B by 39 min. and finally from 60% to 90% B by 50 min.After reaching 90% B, the gradient was returned to the initial composition of 10% B (B:A = 10:90) at 55 min.and the system was allowed to equilibrate for an additional 10 min. before concluding the run.25,26<\/sup><\/p>\n

Analysis by Fourier transform infrared (FT-IR)<\/strong><\/p>\n

FT-IR spectra of isolated compounds were subjected in the FTIR spectrometer (Shimadzu)in the range of 500 to 4000 cm<\/span>\u20131<\/sup>wave number<\/span><\/p>\n

Materials<\/strong><\/p>\n

Apigenin and syringic acid St. Were from Sigma-Aldrich, A375 cell lines from Center for Biotechnology Research at Al-Nahrain University.<\/p>\n

Cell Line Maintenance<\/strong><\/p>\n

The procedure of work was conducted for cell line maintenance according toFreshney RI <\/span>(27)<\/sup>.<\/span><\/p>\n

MTT Assay<\/strong><\/p>\n

After treating the cells with apigenin and syringic acid for 72 hours, the culture medium in each well was gently removed. The wells were rinsed twice with phosphate-buffered saline to clear any residual compounds. Next, 100 \u03bcL of fresh cell culture medium and 15 \u03bcL of MTT staining solution were added to each well. Following a 4-hour incubation period, 100 \u03bcL of stop solution was carefully introduced to halt the MTT reaction.<\/span>28<\/sup> The plates were then kept in the incubator overnight to allow complete solubilization of the formazan crystals. Finally, cell viability was assessed by measuring at 570 nm optical density using a spectrophotometer ,Experiment was repeated three times .<\/span><\/p>\n

Statistical analysis<\/strong><\/p>\n

Data analysis was performed with GraphPad Prism version 6, and results were appeared as the mean \u00b1 standard deviation depend on three independent replicates. Statistical comparisons between groups were made using an unpaired t-test, with differences considered statistically significant at P < 0.05.<\/span><\/p>\n

Results<\/strong><\/p>\n

Extraction by Ultrasonic bath sonicator<\/strong><\/p>\n

Ultrasound-assisted extraction was employed as the method of choice for processing the parsley leaves, yielding approximately 11 g of crude extract from 20 g of dried leaf material, representing a notably high extraction efficiency.<\/span><\/p>\n

Isolation of Apigenin and syringic acid by PHPLC<\/strong><\/p>\n

Apigenin and Syringic acid were isolated by PHPLC using the chromatographic settings that cited in the earlier paragraphs, as in Figures. from 1 to 5<\/p>\n\n\n\n
\"\"<\/td>\nFigure 1: HPLC chromatogram for EtOAc<\/strong><\/p>\n

Click here to view Figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 2: HPLC chromatogram of Syringic acid standard.<\/strong>\u00a0<\/strong><\/p>\n

\u00a0\u00a0<\/strong><\/p>\n

Click here to view Figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\u00a0<\/strong>Figure 3: HPLC chromatogram of isolated syringic acid<\/strong><\/p>\n

Click here to view Figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 4: HPLC chromatogram of Apigenin standard.<\/strong><\/p>\n

Click here to view Figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 5: HPLC chromatogram of isolated Apigenin<\/strong><\/p>\n

\u00a0 \u00a0 \u00a0 \u00a0 \u00a0<\/strong><\/p>\n

Click here to view Figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0 \u00a0 \u00a0\u00a0<\/strong><\/p>\n

\u00a0 Table 1:<\/strong>\u00a0R<\/strong>etention time in min. for phenolic compounds in EtOAc fraction<\/strong>\u00a0<\/strong><\/p>\n\n\n\n\n\n
Phenolic compounds<\/strong><\/td>\nRetention time for<\/strong>phenols in EtOAc fraction<\/strong>\u00a0<\/strong><\/td>\nRetention time of phenolic compounds standards<\/strong>\u00a0<\/strong><\/td>\n<\/tr>\n
Syringic acid<\/strong><\/td>\n9.68<\/td>\n9.69<\/td>\n<\/tr>\n
Apigenin<\/strong><\/td>\n3.56<\/td>\n3.58<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Analysis by FT-IR<\/strong><\/p>\n

FT-IR spectroscopy is generally used in phytochemical reports as a fingerprinting technique to compare normal compounds with synthetic standards. The IR spectra of the quarantined compound Apigenin is existing in figure 6. characteristic bands can be seen in the range of 3381-3000cm <\/span>-1<\/sup> (O-H of alcohols stretching vibration),2904-2620 cm<\/span>-1 <\/sup>(C-H stretching vibration for aliphatic ),1650-1512 cm<\/span>-1<\/sup>(C=O)stretching vibration.<\/span>29<\/sup>while the bands present in FT-IR spectrum of isolated syringic acid in figure 7 exhibited distinct peaks at 3361\u00a0cm<\/span>\u20131<\/sup>\u00a0(corresponding to \u2013OH vibrations), 1710\u00a0cm\u2009\u2212\u20091 (representing C\u2009=\u2009O stretching), and 1618\u00a0cm\u2009\u2212\u20091 (indicating aromatic ring group C\u2009=\u2009C stretching). Others peaks were seen at 1367\u00a0cm<\/span>\u20131<\/sup>, 1244\u00a0cm<\/span>\u20131<\/sup>, and 1203\u00a0cm\u2009\u2212\u20091, which corresponded to CH3-, C-O-C, and C-OH groups, respectively as in previous studies.<\/span>30<\/sup><\/p>\n\n\n\n
\"\"<\/td>\nFigure<\/strong> 6: FTIR spectrum of isolated Apigenin compound<\/strong><\/p>\n

Click here to view Figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure<\/strong> 7: FTIR spectrum of isolated Syringic acid compound<\/strong><\/p>\n

Click here to view Figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

MTT Assay<\/strong><\/p>\n

The Cytotoxicity of Apigenin and Syringic acid isolated from plant leaves against A375 cell line compared with (Normal human dermal fibroblast) HdFn cell lines. both cell lines were exposed to serial concentrations from (6.25-400 \u03bcg\/ml) of the Apigenin and Syringic acid to assess their effects on the viability of cell line as shown in figures (8 and 9) respectively. The decrease in A375 cell line viability (%) is represented in tables (2and3) respectively which exhibited a decrease in cell viability (%) with IC<\/span>50<\/sub> values of 40.8 \u03bcg \/ml for apigenin and IC<\/span>50<\/sub> values of 54.5 \u03bcg \/ml for syringic acid. These two compounds induced significant cell death that began at 25 \u03bcg \/mL (P < 0.05, n = 6).<\/span>\u00a0<\/strong><\/p>\n

Table 2:<\/strong>\u00a0Cytotoxicity of Apigenin on A375 cell line and HdFn Cell after 72 hours of Incubation at 37\u00baC<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
Conc. of Apigenin in \u03bcg \/ml<\/strong><\/td>\nHdFn viable cell count<\/strong>Mean \u00b1SD<\/strong><\/td>\nA375<\/strong>cell line <\/strong>count<\/strong>Mean \u00b1SD<\/strong><\/td>\n<\/tr>\n
400<\/strong><\/td>\n62.5386\u00b1 2.7419<\/td>\n22.3753 \u00b12.7365<\/td>\n<\/tr>\n
200<\/strong><\/td>\n74.4986 \u00b12.0764<\/td>\n29.9693\u00b1 2.5524<\/td>\n<\/tr>\n
100<\/strong><\/td>\n88.0846\u00b1 1.0499<\/td>\n39.3180\u00b1 1.5714<\/td>\n<\/tr>\n
50<\/strong><\/td>\n90.8653 \u00b11.1040<\/td>\n51.6603\u00b1 2.8454<\/td>\n<\/tr>\n
25<\/strong><\/td>\n93.6373\u00b1 0.4819<\/td>\n63.0000\u00b1 3.5990<\/td>\n<\/tr>\n
12.5<\/strong><\/td>\n94.0970 \u00b11.2520<\/td>\n76.2653 \u00b14.0336<\/td>\n<\/tr>\n
6.25<\/strong><\/td>\n95.0233 \u00b10.2315<\/td>\n85.5893\u00b1 1.1000<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure<\/strong> 8: IC50<\/sub> of Apigenin on A375 cell line<\/strong><\/p>\n

Click here to view Figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Table 3:<\/strong>\u00a0Cytotoxicity of Syringic acid on A375 cell line and HdFn Cell after 72 hours of Incubation at 37\u00baC<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
Conc. of Syringic acid in \u03bcg \/ml<\/strong><\/td>\nHdFn viable cell count<\/strong>Mean \u00b1SD<\/strong><\/td>\nA375<\/strong>cell line <\/strong>count<\/strong>Mean \u00b1SD<\/strong><\/td>\n<\/tr>\n
400<\/strong><\/td>\n65.5376\u00b1 3.7419<\/td>\n33.3653 \u00b12.7265<\/td>\n<\/tr>\n
200<\/strong><\/td>\n72.4976 \u00b13.07642<\/td>\n40.9593\u00b1 2.5424<\/td>\n<\/tr>\n
100<\/strong><\/td>\n90.0846\u00b1 1.04991<\/td>\n53.3180\u00b1 1.5724<\/td>\n<\/tr>\n
50<\/strong><\/td>\n92.8643 \u00b11.10405<\/td>\n62.6503\u00b1 1.8444<\/td>\n<\/tr>\n
25<\/strong><\/td>\n94.5373\u00b1 0.4819<\/td>\n73.0100\u00b1 3.5980<\/td>\n<\/tr>\n
12.5<\/strong><\/td>\n94.0970 \u00b11.2520<\/td>\n84.2643 \u00b14.0336<\/td>\n<\/tr>\n
6.25<\/strong><\/td>\n95.0433 \u00b10.2315<\/td>\n91.3893\u00b1 1.1000<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure<\/strong> 9: IC50<\/sub> of Syringic acid on A375 cell line<\/strong><\/p>\n

Click here to view Figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Discussion<\/strong><\/p>\n

The results were consistent with previous studies as ultrasonic analysis showed a significant increase in the conc.31<\/sup> of extracted material. Twenty grams of plant leaves yielded 11 grams of crude extract material this is high percentage compared to using the soaking method or the Soxhlet extraction method due to Ultrasound waves made fast formation and collapse of microscopic bubbles in the solvent these bubbles explode the plant cell walls, causing cell disruption and freedom of intracellular phytochemical compounds. Furthermore, using 90% ethanol is an ideal solvent for extracting and isolating the two phenolic compounds, apigenin and syringic acid. The PHPLC results showed high efficiency in isolating the two phenolic compounds, as they had the same retention times compared to the standards compounds. Apigenin had the same retention times was 3.56 as standard apigenin retention time 3.58, and syringic acid showed the same retention time was 9.68 as the standard apigenin retention time 9.69.FT-IR bands gave indications of the presence of functional groups that characterize the apigenin compound, as the peak (3381\u20133000 cm\u207b\u00b9)\u00a0 for O-H group, (2904\u20132620 cm\u207b\u00b9)for C-H group, (1650\u20131512 cm\u207b\u00b9) for C=O group formed the chemical structure of the isolated apigenin compound. While syringic acid FT-IR spectroscopy showed three major band which 3361cm-1<\/sup> for O-H group ,1710 cm-1<\/sup>for C=O group and 1618 cm-1<\/sup> for C=C group the structural components of this compound. As for the MTT test, it showed a direct correlation between increasing concentration and the percentage decrease in live cancer cells. Apigenin had a clear effect, with a IC50 <\/sub>% 40.8 \u03bcg \/ml which higher effect than that of syringic acid with IC50<\/sub> 54.5\u03bcg \/ml %. This is due to the nature of the compound, as it has a more potent effect in inducing apoptosis than syringic acid, which has a simpler phenolic structure than apigenin with flavonoid structure with four hydroxyl group that have effect on PI3K\/Akt and MAPK path way.32<\/sup> The results also showed that both compounds had low activity against normal cells, exhibiting selective properties against cancer cells and being safer against normal cells. There are some limitations to this study. Only one cancer cell line was used, which limits the generalizability of the results to other cancer types. While the two compounds were isolated and characterized, further diagnostic testing is needed to determine their complete structural composition. Therefore, we anticipate conducting more comprehensive future studies to investigate the efficacy of these anticancer compounds.<\/p>\n

Conclusion<\/strong><\/p>\n

The results indicate that two phenolic compounds isolated from parsley leaves Apigenin and Syringic acid exhibited an anti-cancer effect against A375 cell line that depended on increasing concentration of each compound. Apigenin was the more potent cytotoxic compound than Syringic acid. Both compounds demonstrated a safe and harmless effect on normal cells HdFn. Therefore, further preclinical studies to understand their mechanism of action in killing these cancer cells, with the aim of developing natural anti-cancer treatments.<\/p>\n

Acknowledgement<\/strong><\/p>\n

We acknowledge the Department of Pharmacognosy and Plant science in College of Pharmacy, Thi- Qar University to facilitate our work<\/p>\n

Funding Sources<\/strong><\/p>\n

The author(s) received no financial support for the research, authorship, and\/or publication of this article<\/p>\n

Conflict of Interest<\/strong><\/p>\n

The author(s) declares no conflict of interest<\/p>\n

Data Availability Statement<\/strong><\/p>\n

This statement does not apply to this article.<\/p>\n

Ethics Statement<\/strong><\/p>\n

This research did not involve human participants, animal subjects, or any material that requires ethical approval Informed Consent Statement. This study did not involve human participants, and therefore, informed consent was not required.<\/p>\n

Informed Consent Statement<\/strong><\/p>\n

This study did not involve human participants, and therefore, informed consent was not required.\u00a0<\/strong><\/p>\n

Clinical Trial Registration<\/strong><\/p>\n

This research does not involve any clinical trials<\/p>\n

Permission to reproduce material from other sources<\/strong><\/p>\n

Not Applicable\u00a0<\/strong><\/p>\n

Author contributions<\/strong><\/p>\n