Phenolic composition, antioxidant, and cytotoxic effects on HeLa and HepG2 cancer cell lines of Mespilus germanica grown in Turkey

Abstract This study screened flavonoids and phenolic acids, antioxidant and cytotoxic effects of Mespilus germanica leaf and fruit samples. The RP-HPLC-DAD analysis allowed the identification of hesperidin, epicatechin, epigallocatechin, benzoic, p-hydroxybenzoic, vanillic, protocatechuic, syringic, caffeic, ferulic, sinapic and p-coumaric acids in various extracts. Fruit alkaline-hydrolysable phenolic acids extract (BHPA), leaf bound phenolic acids from basic hydrolysis-2 extract (BPBH2) and leaf free flavan-3-ol extract exhibited the largest DPPH, OH and NO radicals scavenging activity, respectively. Leaf flavone extract showed strong cytotoxicity on the HepG2 cell line (IC50 = 36.49 ± 1.12 μg/mL) as well as good •OH scavenging and Fe2+ chelation activities. Additionally, leaf bound phenolic acids from acid hydrolysis-1 extract (BPAH1) demonstrated strong cytotoxicity on the HeLa cell line (IC50 = 36.24 ± 1.89 μg/mL). This study suggests Turkish medlar as a natural source of phenolic compounds with potential application in food and pharmaceutical industries as anticancer/antioxidant agents. Graphical Abstract

1. Introduction natural compounds, for example, flavonoids and phenolic acids, that could be found in different plant species play a significant role in the discovery and development of new drugs for the treatment of various diseases (safari and ahmady-asbchin 2019).these bioactive compounds have also shown promising therapeutic effects for various types of cancer, including cervical and liver cancers.despite decades of scientific research and enormous progress in the diagnosis, prognosis, and therapy of cancers, the disease is still among the leading causes of death worldwide (allegra et al. 2022).to contribute to this field, the current study investigated phenolic compounds in turkish medlar that could have cytotoxic effects against cervical and liver cancers, employing HeLa and HepG2 cells.Mespilus germanica (medlar) has flavor and aroma properties due to the lipids and phenols found in it, thus promoting its industrial application.medlar is rich in organic acids, mineral elements, carbohydrates, proteins, vitamins, fatty acids, carotenoids, and phenolic compounds, with antibacterial, antioxidant and anticancer effects (Voaides et al. 2021;sadeghinejad et al. 2022).
although there are published data on the phenolic compounds isolated from turkish medlar, reports on the plant's cytotoxicity on HeLa and HepG2 cancer cell lines were not found in the literature, while the antioxidant evaluations were primarily limited to the dppH • scavenging activity, metal chelation, reducing power, and FRap assays.as a result, the goal of this work was to apply different extraction procedures to produce phenolic extracts from medlar leaf and fruit samples, and profile flavonoids and phenolic acids via Rp-HpLC-dad.the study further investigated the antioxidant activity of the twelve extracts using six different methods, and the cytotoxic effect of the extracts on cancer cell lines HeLa and HepG2 by mtt assay.

Results and discussion
phenolics derived from plant materials are gaining popularity in the cosmetic, food and pharmaceutical industries due to their beneficial properties.the extraction of phenolic compounds from the samples was accomplished and the yield depends upon the method and solvent used (table s2). the fruit sample showed higher yields in all extractions except Fpa and BpBH 2 .

Phenolic profile using RP-HPLC-DAD
seven phenolic acids comprising three benzoic acid derivatives (benzoic, p-hydroxybenzoic and vanillic acids) and four cinnamic acid derivatives (caffeic, ferulic, sinapic and p-coumaric acids) were identified using Rp-HpLC-dad in six leaf extracts, and none in BpaH 2 extract (table s3).Benzoic acid (43.83 ± 1.85 µg/g dWe ) in BpBH 1 and sinapic acid (0.64 ± 0.05 µg/g dWe ) in aHpa had the highest and lowest concentration, respectively.sadeghinejad et al. ( 2022) reported caffeic, ferulic and p-coumaric acids in iranian medlar leaf.table s4 displays the phenolic acid profile of fruit extracts.protocatechuic, syringic, vanillic and ferulic acids were identified in four fruit extracts: Fpa, BHpa, BpBH 1 and BpBH 2 .Ferulic acid came to the fore in terms of concentration (9.30 ± 0.58 µg/g dWe ) and was found in all the four extracts.Rop et al. ( 2010) identified ferulic, protocatechuic and vanillic acids in common medlar fruit collected from Czech Republic.Ferulic and protocatechuic acids were also determined in polish medlar fruit extracts (Żołnierczyk et al. 2021). in this study, base hydrolysis is a successful approach for phenolic acid extraction from the fruit of turkish medlar, but acid hydrolysis is not.Hot acid hydrolysis can destroy phenolic acids, making identification difficult (Robbins 2003).plants use phenolic acids to defend themselves against biotic and abiotic stressors.
Hesperidin in flavanone extracts of leaf and fruit, epicatechin and epigallocatechin in fruit free flavan-3-ol extract, are the three flavonoid compounds identified in this study (table s5).Żołnierczyk et al. ( 2021) also reported a substantial amount of epicatechin in crude, water and methanolic extracts of polish medlar fruit.Hesperidin, epicatechin and epigallocatechin are promising compounds with medicinal properties such as antioxidant, anticancer, anti-ageing and antibacterial effects (Chen et al. 2022).the biological properties of flavonoids are accredited to their configurational structure, the position of functional groups, and the total number of hydroxyl groups attached to the structure (Chen et al. 2022).

Antioxidant activity
the results of antioxidant activity are presented in table s6. the dppH • scavenging activity of the medlar extracts was concentration dependent.Fruit BHpa extract (iC 50 = 47.87 ± 0.61 µg/mL) and leaf free flavan-3-ol extract (iC 50 = 48.60 ± 0.59 µg/mL) showed the highest and statistically similar (p > 0.05) dppH • scavenging activity.phenolic compounds present in plants are known to scavenge dppH • via proton donation.Ferulic and syringic acids were determined in considerable amounts in these extracts.medlar extracts showed a strong • oH scavenging effect in the range of 8.60 ± 0.06 to 14.49 ± 0.18 µg/mL.specifically, leaf BpBH 2 extract was the largest scavenger of • oH, and HpLC analysis revealed antioxidant compounds, ferulic and p-hydroxybenzoic acids, in the extract. in general, it can be said that leaf extracts were more effective than fruit extracts in quenching no • radicals.Flavanone, free flavan-3-ol, Fpa, BHpa and aHpa extracts of the leaf showed strong no • scavenging properties (iC 50 < 30 µg/ mL).some of the phenolic compounds found in these extracts have numerous pharmacological effects (antimicrobial, anticancer and antioxidant activities).scavenging of dppH • , • oH and no • is an important parameter that may be used to evaluate the antioxidant activity of any food sample.
Fe 2+ -chelation is crucial in avoiding free radicals related to Fe 2+ .Fruit Fpa and BpBH 1 , and leaf BpBH 1 and BpBH 2 extracts showed the highest and statistically similar results (p > 0.05).the chelation capacity of medlar may relate to phenolic compounds identified in its extracts.the elimination of Fe 2+ from the circulation by chelation may be a viable strategy for preventing oxidative stress-induced diseases.Higher ferric-reducing powers were demonstrated by leaf free flavan-3-ol, Fpa and BpaH 2 extracts, and fruit BHpa extract (>380 µg Vit-C eq /g dWe ). the observed reduction might be attributed to the flavonoids and phenolic acids found in the extracts, which reduce Fe 3+ to Fe 2+ , which is then chelated by the antioxidant molecules.the reducing power of all extracts measured using the FRap approach was concentration dependent.the leaf BpBH 1 extract produced good results (811.00 ± 3.01 µg Vit-C eq /g dWe ). the FRap measures the reduction of Fe 3+ to Fe 2+ by the electron donor in the sample.Gülçin et al. (2011) simultaneously evaluated dppH • scavenging activity, Fe 2+ -chelation, Fe 3+ to Fe 2+ reduction power, and FRap of turkish medlar aqueous lyophilized extract.

Cytotoxic activity
this research studied the cytotoxicity of medlar extracts on HeLa or HepG2 cells at 24 and 48 h after incubation.as demonstrated in Figure s1, the leaf BpaH 1 extract showed the strongest cytotoxic effects on HeLa 24 h after incubation (iC 50 = 36.24± 1.89 μg/mL), which is substantially close to the american national Cancer institute's defined threshold (iC 50 ≤ 30 μg/mL) (suffness 1990).HpLC examination indicated the presence of p-coumaric acid in this extract.the cytotoxic properties of p-coumaric acid on HeLa cell lines were reported (malik and dhiman 2022).Furthermore, the leaf flavone extract exhibited a substantial effect on HeLa 48 h after incubation (iC 50 = 40.00± 2.32 μg/mL).as shown in Figure s2, leaf aHpa, Fpa and flavone extracts, as well as fruit BpBH 2 and aHpa extracts, displayed significant cytotoxicity on HepG2 cancer cell lines 24 h post incubation (p > 0.05).Leaf flavone extract also showed strong cytotoxicity (iC 50 = 36.49± 1.12 μg/mL) against HepG2 cancer cell lines 48 h after incubation.the phenolic compounds found in the extracts may be responsible for the cytotoxic action.

Conclusions
the phenolic yields, antioxidant, and cytotoxic properties of the two samples are greatly influenced by the chemicals employed in the extraction operations.the extraction of bound phenolic acid from the samples was more effective through base hydrolysis.this study found medlar samples to be a rich source of flavonoids, free and conjugated phenolic acids with antioxidant activity, and cytotoxic effect on HeLa and HepG2 cell lines.

Disclosure statement
no potential conflict of interest was reported by the authors.

Funding
the author(s) reported there is no funding associated with the work featured in this article.