Assessing the cytotoxicity of phenolic and terpene fractions extracted from Iraqi Prunus arabica on AMJ13 and SK-GT-4 human cancer cell lines [version 2; peer review: 1 approved with reservations

Background: Breast and esophageal cancer are the most aggressive and prominent causes of death worldwide. In addition, these cancers showed resistance to current chemotherapy regimens with limited success rates and fatal outcomes. Recently many studies reported the significant cytotoxic effects of phenolic and terpene fractions extracted from various Prunus species against different cancer cell lines. As a result, it has a good chance to be tested as a complement or replacement for standard chemotherapies. Methods: The study aimed to evaluate the cytotoxicity of phenolic and terpene fractions extracted from Iraqi Prunus arabica on breast (AMJ13) and esophageal (SK-GT-4) cancer cell lines by using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide). Analysis using the Chou-Talalay method was performed to assess the synergistic effect between the extracted fractions and chemotherapeutic agent (docetaxel). Moreover, high-performance


Introduction
A critical global health issue is breast cancer, the most prevalent cancer diagnosed worldwide, with 2.26 million incidents expected and it is the major cause of cancer-related deaths among women. Although breast cancer is considered a common disease in developed societies, in 2020, the world's less developed regions accounted for two-thirds of breast cancer-related deaths and more than 50% of all breast cancer diagnoses. 1 In terms of cancer-related deaths, esophageal cancer (EC) occurs sixth on the list of the deadliest diseases (436,000 fatalities). Meanwhile, 473,000 cases have been recorded worldwide. 2 Even though chemotherapeutic regimens and radiation therapy are more effective methods for treating cancer, they are nonselective, have substantial side effects, and can harm normal healthy tissues, leading to severe unanticipated and undesirable side effects. 3 Initially, several tumors appeared amenable to treatment. However, with time, resistance might develop for a number of reasons, such as mutations in DNA and metabolic changes that cause the drug to be inhibited and degraded. 4 The occurrence of natural compounds as anticancer agents is estimated to exceed 60% of the current anticancer drugs. 5 These drugs from natural origins can be used for both cancer prevention and treatment due to their pharmacological safety and can be used independently or in conjunction with existing chemotherapeutic treatments to improve the therapeutic efficacy or to reduce chemotherapy-induced toxic effects. 6 The constant change and injury that occurs in the human body over decades requires a development of revolutionary and highly precise "arms" capable of successfully combating malignant cells. Natural products are a powerful and endless source for identifying the finest anticancer prospects. 7 Prunus arabica is recognized as a distinct species from the farmed almond P. dulcis. Both, however, are members of the Prunus genus and the Rosacea family. This species was given its scientific name based on its geographical location where it first appeared. This taxon is indigenous to mild climate Asia regions, including the Fertile Crescent Mountains, as well as Turkey, Iran, and Iraq. 8 Prunus arabica is a thick undomesticated almond species with an unbarked stem that remains green even during dormancy. 9 A wide range of biological and pharmacological effects from different Prunus species show great promise for the treatment of various cancers. 10 Flavonoids, steroids, terpenoids, poly phenols, and other chemicals have all been identified from various Prunus species. 11 Polyphenols and terpenoids which are found plentifully in plants, show anticancer effects, via inhibiting cancer cell growth, blood vessel formation, metastasis, inflammation, and inducing cell death. 12 For this purpose, the anticancer effect of phenolic and terpene fractions from Prunus arabica extract was investigated using the AMJ13 and SK-GT-4 cancer cell lines and normal human fibroblasts (NHF).

Preparation and separation of plant extract
The full protocol can be found in the Extended data. 45 Approximately 500 gm of granular powdered plant that had been shade-dried for 12 days then defatted for 24 hours with hexane (BDH chemicals, England cat-no. BDH24575.100E) in a ratio of 1:3 W/V before being dried at room temperature. In a Soxhlet apparatus (BOECO, Germany) the defatted plant components were separated using two liters of 80% ethanol (Sigma-Aldrich, Germany cat-no1070172511) until completely depleted. A thick, dark-greenish-yellow residual (known as the crude fraction A) was obtained by drying the alcoholic extract by evaporation at low pressure and temperatures below 40°C by using IKA RV 10 Rotary Evaporator (Germany). This fraction was acidified using 300 ml of 5% HCl (Sigma-Aldrich cat-no. 1009861000) to pH 2 and then split with ethyl acetate (Sigma-Aldrich, Germany cat-no. 319902-1L) to acquire two distinct layers (the acidic aqueous layer and ethyl acetate layer-crude fraction). The crude fraction was dried out using low-pressure evaporation in an IKA RV 10 Rotary Evaporator (Germany) then basified with 300 ml of 5% NaOH (Honeywell, USA cat-no. 30620) to pH

REVISED Amendments from Version 1
Notable addition based on the reviewer's suggestion is the inclusion of selectivity index analysis. This addition enhances the robustness and relevance of our research. Furthermore, Standard deviation (SD) values for the data points in Figures 1-3 (that represent the percentage of cytotoxicity) have been added to provide a better understanding of the variability in the results and improve the overall quality of the figures. Alongside the reported IC50 values, the corresponding (SD) values are included. This will give readers a more comprehensive view of the precision and reliability of the IC50 measurements. More papers and some recent reviews are included to provide a more in-depth comparison of the results of this study to those of other studies with similar plant extracts. The references in the results and discussion sections have been updated and revised.
Any further responses from the reviewers can be found at the end of the article 10 and extracted by adding chloroform (Honeywell, USA cat-no. C2432) to the separatory funnel in order to obtain two layers; the aqueous basic layer and the and chloroform layer. The basic water layer evaporated to the point of dryness and acidified with 5% HCl to reach pH 2, then extracted with ethyl acetate to get another fraction designated as fraction (F-B). The chloroform layer which was separated by the same steps and partitioned with 80% methanol (Biochem Chemopharma, France cat-no. 213032500) and petroleum ether (Sigma-Aldrich, Germany cat-no. 32299) to obtain another two layers; fraction (C) the petroleum ether and methanol fraction which was considered as fraction D. 13 Preliminary qualitative phytochemical analysis of fractionated extract of Prunus arabica The full protocol can be found in the Extended data. 46 Standard protocols were used in chemical testing to identify the active components using ethanolic extracts from various plant fractions. 14 I. Before usage, the solvents were processed using 0.45 mm pore size (Millipore) membrane filters.

Instrumentation and analytical conditions
Individual phenolic components identification was conducted by reversed-phase high-performance liquid chromatography utilizing a Sykam HPLC chromatographic system (Germany) integrated with ultraviolet detection (Sykam S 3240 UV/Vis Multichannel Detector, Germany) and sample delivery system (Sykam S1122 Solvent deliver system, Germany). The column temperature was maintained at 30°C (Sykam S 4011 column thermo controller, Germany). The gradient elution method, with eluent A and eluent B (methanol and 1% (v/v) formic acid in water respectively), was carried out as follows: initial 0-4 min, 40% B; 4-10 min, 50% B; and flow-rate of 0.7 ml/min. Approximately 100 μL of the samples were injected. An autosampler (Skynm S5200 sample injector, Germany) analyzed the standards automatically. Spectral data was recorded at a 280 nm. 15 The following conditions were used for the terpene fraction; mobile phase acetonitrile: distilled water: acetic acid (60:25:5), column = C18-ODS (25 cm * 4.6 mm), detector = UV-220 nm, and the flow rate was 1 ml/min.

Cytotoxicity assays
The full protocol can be found in the Extended data. 47 The MTT assay for cell viability 19 was performed to measure the cytotoxic effect of the extracted fractions. Cell lines were planted into 96-well plates (Santa Cruz Biotechnology, USA cat-no. sc-204447) at 1 Â 10 4 cells/well. After 24 hrs, or until a confluent monolayer was achieved, cells were treated with the tested compound. After 72 hours of exposure, cell viability was assessed; the medium was removed by aspiration and 28 μL of 2 mg/ml MTT (MTT stain obtained from Bio-World, USA cat-no. 42000092-1) was added and incubated at 37°C for 1.5 hours.
After removing the MTT solution, the crystals remaining in the wells were solubilized by the addition of 130 μL of dimethyl sulfoxide (DMSO) (Santacruz Biotechnology, USA cat-no. sc-202581) was used to solubilize the residual crystals in the wells, proceeded by incubation for 15 min at 37°C with shaking. 20 Using a microplate reader (Genex-lab, USA cat-no. MR-100) the absorbance was measured at 492 nm; the experiment was carried out in triplicate. Untreated cells, MTT solution, and a solubilizing buffer (DMSO) will be placed in the wells designated as a control. Due to its proven lack of cytotoxicity in cell culture, a final concentration of 0.5% DMSO was used for this study. The following formula was applied to determine the percent of cytotoxicity 21 : %Cytotoxicity ¼ 100 À cell viability AT: Absorbance of treated cells with tested compounds, ANT: absorbance of untreated cells.

Chou-Talalay analysis
Synergism or interaction of phenolic and terpene fractions with docetaxel was investigated using a non-constant ratio. Analysis of the combination was performed by the Chou-Talalay method. CompuSyn was used to derive the corresponding combination indices (CI) (CompuSyn, Inc., Paramus, NJ, USA). The combination indices were calculated using nonconstant phenolic, terpene, and docetaxel ratios and mutually exclusive formulations. A CI value between 0.9 and 1.1 indicates an additive effect, a CI value below 0.9 denotes synergy, and a CI above 1.1 denotes antagonism. 22 Selectivity Index analysis The Selectivity Index (SI) is calculated by dividing a compound's IC 50 value against normal cells by its IC 50 value against cancer cells. 23,24 High-selectivity compounds have a SI value greater than 3, while low-selectivity compounds have a SI value of 3. 24

Statistical analysis
The MTT assay results were analyzed statistically with an ANOVA test in GraphPad V 7.00 (for windows), an openaccess alternative that can perform an equivalent function is R. The unpaired t-test was used to compare groups; P-values <0.05 were considered statistically significant.

Qualitative phytochemical analysis
The phytochemical screening results are given in Table 1.  To discover individual compounds of extracted fractions, HPLC analysis was used. Seven phenolic acids (p-coumaric acid, ferulic acid, gallic acid, caffeic acid, quercetin, rutin, catechin) and three terpenes (B sitosterol, campesterol, stigmasterol) were detected from extracted fractions of Prunus arabica, as shown in Tables 2 and 3.

In vitro cytotoxic activity
The MTT assay was used to evaluate the cytotoxicity and therapeutic efficacy of the isolated fractions in the cancer and normal cell lines (AMJ13, SK-GT-4 and NHF). As shown in Figures 1-3, the results showed that the treatments significantly decreased the viability of the cancer cells (>50% at higher concentrations) with minimal cytotoxic effects on the normal cells (<50% cytotoxicity). On the AMJ13 cell line, phenolic, terpene fractions, and docetaxel had a half maximal inhibitory concentration (IC 50 ) of 29.34AE2.37, 8.455AE3.02, and 14.51AE0.77 μg/ml, respectively, as shown in

Extract-docetaxel potential interaction (Chou-Talalay analysis)
The possible interactions between extracted fractions (phenolic, terpene) and docetaxel therapy on the AMJ13 and SK-GT-4 cell lines were analyzed. The quantification of synergism or antagonism is defined as a mass-action law issue (determined by the combination index CI values) and cannot be determined by the statistical p values. 22 Chou-Talalay equations were used to calculate the combination's value. A CI value less than 0.9, the effects were assumed to be synergistic; a CI value between 0.9 and 1.1, the effects were considered as additive; while a value larger than 1.1, the effects were assumed to be antagonistic. 25 After an exposure period of 72 hours, the phenolic fraction of P. arabica with docetaxel produced a strong to very strong synergic cytotoxic effect against AMJ13 and SK-GT-4 cancer cell lines in comparison with a single treatment. The terpene fraction showed almost the same synergism effect when combined with docetaxel ( Figure 5).

Morphological analysis (crystal violet staining)
Morphological alterations were examined with crystal violet staining of the cell lines after 72 hr. Exposure to the IC 50 of the phenolic fraction, terpene, docetaxel, and their combinations with chemotherapy as shown in Figures 6, 7 and 8. The captured images are for cells treated with (100 μg/ml) of the tested fractions or their combinations with docetaxel on cancer or normal cell lines.

Selectivity index
The provided Table 4 illustrates the selectivity index for the tested compound in this study.

Discussion
Polyphenols and terpenes are the most abundant and widely distributed compound in the plant kingdoms and groups. 26 Prunus species have been found to be a potential dietary supplement and a good source of phenolic and terpene bioactive chemicals. 27 In the current study, lead acetate and NaOH tests for polyphenols gave a positive result, meaning the presence of phenolic compounds in P. arabica extract. The dark color might indicate the presence of large quantities of polyphenols and flavonoids. 28 The H 2 SO 4 test gave a dark pink or red color and greenish color, respectively, as an indication of the presence of steroids, while the chloroform and sulphuric acid test produced a greyish color which was considered an indication of the presence of terpenes.
The data for the HPLC analysis showed that the phenolic fraction of the extracted P. arabica contains eight phenolic compounds (p-Coumaric acid, ferulic acid, gallic acid, caffeic acid, quercetin, rutin, catechin, and chlorogenic acid), the terpene fraction contained three major terpenes (β-sitosterol, stigmasterol, and campesterol) as well as some non-phenolic organic and inorganic components at deceptive values.   From the eight phenolic compounds found in our phenolic fraction of the P. arabica extract, the highest component concentration was ferulic acid (3.992 mg/gm) with a retention time of 3.22 min, which may contribute to the high efficacy of the phenolic extract as a cytotoxic biological chemical on different cancer cell lines. 29 Research has indicated that ferulic acid induces cell death by decreasing the Bcl-2 and increasing the BAX gene expression or by upregulation of caspase-3 and cleaved caspase-9. 30 The highest component of the terpene fraction was campesterol (4.358 mg/gm), with a retention time of 11.5 min. A study by Hyocheol B. et al. confirmed that campesterol could inhibit both cellular growth and cell cycle progression by regulating the PCNA (proliferating cell nuclear antigen) and PI3K/MAPK (phosphatidylinositol-3-kinase/mitogenactivated protein kinase) signal pathways. Moreover, their results also showed that campesterol could prevent the clustering of ovarian cancer cells. 30 Some undiscovered phenolic and terpene compounds may be presented by peaks on the chromatograms, hydroperoxides or peroxides produced from terpenes are likely responsible for these found but unidentified peaks. 31 The present work studied the cytotoxic effects of the extracted fractions (phenolic and terpene) of P. arabica alone and in combination with docetaxel and compared their novel effects with the single chemotherapeutic agent (docetaxel) on AMJ13, SK-GT-4, and NHF cell lines. Breast cancer and esophageal carcinoma are considered highly malignant tumors, which lead to poor prognoses. 32 The low efficacy of currently available breast and esophageal cancer chemotherapeutics and radiation moreover these therapies are associated with severe adverse effects and patients can develop resistance to these agents. 33,34 In vitro, the results of this study appear that the treatment with the phenolic and terpene extract of P. arabica significantly reduced cell viability and triggered apoptosis when compared to the control group in both AMJ13 and SK-GT-4 cell lines (Figures 1-2). During this study against the AMJ13 cell line, the terpene fraction showed comparable cytotoxic effects to docetaxel even in concentrations as low as 25 μg/ml, the IC 50 for phenolic terpene and docetaxel against AMJ13 cell line was 29.34AE2.37, 8.455AE3.02 and 14.51AE0.77 μg/ml respectively ( Figure 4).
The phenolic fraction showed almost an equal cytotoxicity to docetaxel against the SK-GT-4 cell line, while terpene showed less significant cytotoxicity in comparison with other tested treatments; the IC 50 for phenolic terpene and docetaxel against SK-GT-4 was 21.97AE3.56, 15.14AE3.26, 0.7125AE0.084 μg/ml respectively as shown in Figure 4. All treatment concentrations for phenolic and terpene fractions failed to show significant cytotoxicity on the NHF cell line (less than 50%).
Many unique chemical components, including polyphenols, flavonoids, alkaloids, and terpenes, have been identified from Prunus species. The great structural variety of these compounds underlies their unique biological properties, which include bioavailability, antioxidant activity, and specific interactions with cell receptors and enzymes. 35 Researchers have found that flavonoids have a wide range of biological effects in mammals, including antimicrobial, antiviral, analgesic, anti-allergic, hepatoprotective, cytostatic, and apoptotic properties. 36 The current study confirms the findings of previous studies which showed that phytosterols, such as quercetin and β-sitosterol, protect against a wide variety of diseases and exhibit selective cytotoxicity towards cancer cells, as evidenced by high apoptosis indices in cells exposed to quercetin's anticancer activity. 37 Many studies have demonstrated that flavonoids have cytotoxic effects, including modulating ROS-scavenging enzyme activities, cell cycle arrest, induction of apoptosis and autophagy, and suppression of cancer cell proliferation and invasiveness. In healthy cells, flavonoids function as antioxidants, but in cancer cells, they become strong prooxidants that induce apoptotic pathways and downregulate pro-inflammatory signals. 38 Stigmasterol's cytotoxic actions come from its ability to induce autophagy in tumor cells, decrease their proliferation and spread, and promote their apoptosis. 39 The increased sensitivity of cancer cells to cytotoxicity was another goal of this investigation by using phenolic or terpene fractions with docetaxel and the cumulative effects of many dosages. The MTT assay performed with docetaxel in the presence of varying amounts of phenolic or terpene fractions. According to the findings, the phenolic, terpene, and docetaxel combination substantially decreased cancer cell viability without causing appreciable damage to normal cells. The Chou-Talalay equation was used to evaluate the combinations.
The degree of synergy or antagonism cannot be assessed by p value in a statistical manner but can be quantified using CI values (combination index values). 40 Nearly all of the doses examined showed synergistic cytotoxicity against the cancer cell lines. To demonstrate their safety, testing on a normal human fibroblast cell line showed no effect at any dose of the combination of phenolic and terpene fractions with docetaxel. There have been a number of studies suggesting that phenolic acids and terpenes may boost the effect of other chemotherapies on breast cancer. 41 However, this is the first study to provide empirical evidence of synergy between phenolic and terpene fractions with docetaxel on AMJ13 and SK-GT-4 cell line ( Figure 5 and Table 5). As seen in the current study and after crystal violet staining, the AMJ13, SK-GT-4, and NHF cells that were exposed to 72h of extracted fractions and docetaxel and the combination therapy revealed cell shrinkage, cytoplasm and cell membrane disappearance, stromal edema, nucleus shrinkage and a marked decrease in the number of cells compared with control (untreated) cells. The effect of combination therapy was more prominent than single therapy as the cells showed more shrinkage, extensive cell damage, and necrosis.
As a result of their specificity, chemopreventive medicines only target cancer cells. A compound's ability to selectively destroy cancer cells while having a minimal effect on healthy cells is measured by its "selectivity index." The compound's low selectivity index suggests that it is less hazardous to healthy cells than cancerous ones. Compounds having high SI values may provide a safer and more effective cancer treatment option. 43 Based on the test results the phenolic fraction is found to be less selective for all tested cells (SI<3). Meanwhile, terpene fraction is selective for AMJ13 cancer cells with (SI>3) and less selective for SK-GT-4 cancer cells. The Docetaxel treatment (positive control) showed an excellent SI toward SK-GT-4 cell line (SI>3). The results of this study need a further evaluation to determine the potential cytotoxicity in animal models.

Conclusion
It was observed from the results that the P. arabica phenolic and terpene extracts have significant cytotoxic activity on breast cancer and esophageal cancer cell lines with minimal effect on normal cells, due to the presence of effective compounds in this extract. Moreover, these active compounds increased the cytotoxic activity of docetaxel on cancer cell  The project contains the following underlying data: • Figure 1  • Figure 5 -B -Terpene plus docetaxel AMJ13.html (Cytotoxicity of Prunus arabica terpene fraction combination with docetaxel on AMJ13 cancer cell line. I-Dose-Effect Curve at 50% cytotoxicity, II-Isobologram analysis displays synergism rate between terpene fraction and docetaxel at all points of the combination as they located at the lower left of the hypotenuse, demonstrating the effect is synergistic at a 50% cytotoxicity dose, III-showing the combination index data location for each dose.) • Figure 5 -D -Terpene plus docetaxel SK-GT-4.html (Cytotoxicity of Prunus arabica terpene fraction combination with docetaxel on SK-GT-4 cancer cell line. I-Dose-Effect Curve at 50% cytotoxicity, II-Isobologram analysis displays synergism rate between terpene fraction and docetaxel at all points of the combination as they located at the lower left of the hypotenuse, demonstrating the effect is synergistic at a 50% cytotoxicity dose, III-showing the combination index data location for each dose.) • Page 7; '...while a CI value larger than 1.1, the effects were assumed to be antagonistic...' and Figure 5 legends page 9; '...CI value less than 1 indicates an antagonism...'' CI value less than or larger tahan 1 assumed antagonistic? Please correct.
Please write axis name in Figure 4. 10.
The selectivity index is an important parameter for indicates the selectivity of a given samples between normal and cancer cells. Samples are expected less cytotoxic towards the healthy cells according to the cancer cells. Please calculate the selectivity indexes of samples.

11.
Authors should compare the results of this study some other similar plant extracts with more details. References should be improved and updated in the results and discussion section. Indeed, the field of similar samples with regard to both their chemistry and biological relevance, include a significant number of articles and some recent reviews 12.
Please use only one of the terms antiproliferative activity, cytotoxic activity, or anticancer activity in all manuscripts.

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate? Yes I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Author Response 05 Jun 2023 matin mahmood Dear Assoc. Prof. SERAP ŞAHİN BÖLÜKBAŞI, Thank you for your thoughtful review of our manuscript. We greatly appreciate your feedback and suggestions for improvement. In our experiments, we used untreated cells, MTT solution, and a solubilizing buffer (DMSO) as controls. To ensure the integrity of our experimental results and minimize any potential confounding effects, we carefully selected a final concentration of 0.5% DMSO for our study. This concentration has been widely reported in the literature as non-toxic and compatible with various cell types. Regarding the morphological analysis section of this study, it is worth noting that the analysis was carried out using only a concentration of 100 ug/ml, and it is important to clarify that no analysis was conducted specifically to IC 50 values.

Sincerely, Matin A. Mahmood
Competing Interests: No competing interests were disclosed.
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