Chemo-profiling and bioactivities of Taif rose ( Rosa damascena Mill.) industrial by-products after hydrodistillation

This study investigated the chemical composition of Rosa damascena Mill. (grown in the Taif region of Saudi Arabia), in relation to their potential as antioxidants and anticancer agents. The rose flowers are used to produce essential oils and hydrosol, but the process generates a significant amount of —solid and liquid. They contain biologically active compounds and can be treated as by-products. This study aimed to characterize these materials and analyze their extracts for biological activity. The total phenolic, flavonol, and flavonoid contents were quantified, and their chemical compositions were determined using liquid chromatography-mass spectrometry analysis. The antioxidant properties were estimated in vitro using three methods [1,1-diphenyl picrylhydrazyl (DPPH) radical, phosphomolybdenum, and ferric reducing power assays], while an anticancer test was carried out using the liver carcinoma cell line (HepG2). The findings showed that the texture’s n -butanol ( n -BuOH) fraction had the highest overall phenolic, flavonoid, and flavonol contents (230.69 ± 10.50 mg gallic acid equivalents /g ext., 71.66 ± 3.15 mg RE/g ext., and 51.70 ± 0.61 mg QE/g ext., respectively). The liquid chromatography-electrospray ionization-mass spectrometry analysis of the by-product extracts led to identifying 55 polyphenolic compounds, including organic acids, flavonoids, phenolic acids, and tannins, of which some constituents were described in Rosa extracts for the first time. In addition, the n -BuOH fraction of the texture and wastewater had a significant DPPH radical scavenging activity with SC 50s values of 7.57 ± 0.01 and 12.78 ± 0.13 µg/ml, respectively. Furthermore, the cytotoxic activity of the n -BuOH fraction of texture showed the highest activity on HepG2 (IC 50 = 12.1 ± 0.85 μg/ml) followed by wastewater n -BuOH fr. (IC 50 = 14.00 ± 1.05 μg/ml) and the methanolic extract of texture (IC 50 = 20.5 ± 1.41 μg/ml). Our findings indicated that rose industrial by-products are enriched with polyphenolic compounds with promising antioxidants and anticancer activities. Thus, they could be used as a food supplement.


INTRODUCTION
The Taif region in Saudi Arabia is famous for the cultivation of a kind of Damask rose known as Rosa damascena Mill.var.trigintipetala Dieck or Taif rose.The plant is considered an important economical source of essential oil and floral water (Dobreva et al., 2020).Furthermore, local rose oil is recognized as one of the most distinguished and reputational perfumes around the globe (Abdel-Hameed et al., 2016).After the production of essential oil with hydrodistillation technology, a huge amount of waste materials (by-products), including texture and wastewater, is produced.They are usually discarded as industrial waste without effective use and cause environmental pollution (Gerasimova et al., 2022).However, the by-products can also be used in the production of methane biogas, phenyl ethyl alcohol, compost, residue concrete, health sludge, cosmetics, and natural antioxidant supplement (Erbaş and Baydar, 2016).Bulgarian and Greece R. damascena wastewater extracts after oil production are rich in polyphenols, which hardly degrade and have remarkable bioactivities (Ilieva et al., 2022).Other studies reported the existence of various secondary metabolites, including quercetin, kaempferol, ellagic acid, and their glycoside derivatives, as well as observing some pharmacological properties (Dina et al., 2021;Kumar et al., 2008;Rusanov et al., 2014;Schiber et al., 2006;Sabahi et al., 2020).Our previous studies were focused on the chemical profiling of the nonpolar constituents of R. damascena and the evaluation of their cytogenetic and cytotoxic activities in which both absolute rose oil and concrete were safe at a concentration of 10 μg/ml on normal human blood lymphocytes.Moreover, the concrete exerted cytotoxic activity against HepG2 and MCF7 cell lines with IC 50 = 16.28 and 18.09 μg/ml, and the absolute oil had IC 50 of 24.94 and 19.69 μg/ml, respectively.The nonpolar constituents were investigated using gas chromatography-mass spectrometry, which led to the phenyl ethanol, β-citronellol, geraniol, eugenol, and methyl eugenol being the major constituents (Hagag et al., 2014).On the other hand, the liquid chromatography-electrospray ionizationmass spectrometry (LC-ESI-MS) was used to characterize the polar constituents, and the results revealed the existence of 28 compounds categorized as phenolic acids, ellagitannins, and flavonoids (Abdel-Hameed et al., 2013).To the best of our knowledge, an excellent safety effect of R. damascena wastewater extracts on chronic, subchronic, and acute toxicity in adult albino mice was observed.In addition, the total phenolic, flavonoid, and flavonol contents as well as their antioxidant potentials have been estimated (Abdel-Hameed et al., 2015).
Hepatic cells are important in the drug development process because the liver is the major detoxification organ of the human body and is primarily involved in drug metabolism and interactions.Hence, HepG2 cells are the most commonly used in vitro models, particularly in the study of liver physiology and cancer (Harjumäki et al., 2019).
Herein, the industrial by-products (wastewater and texture) after the hydrodistillation method were phytochemically investigated by determining their phenolic, flavonol, and flavonoid contents.Afterward, the polar active ingredients were characterized using LC-ESI-MS analysis.Furthermore, the anticancer effects using HepG2 cells of Rosa by-product extracts and the antioxidant potentials were evaluated.

Collection of plant materials
Rosa damascena flowers were collected during the harvest season (March-April) from the Taif region for the production of essential oil.A fresh sample of the plant was verified by Prof. Mohamed Fadle, Professor of Plant Taxonomy, Faculty of Science, Taif University, KSA.A voucher sample (no.1532018) was preserved at the Department of Medicinal Chemistry, Theodor Bilharz Research Institute, Giza, Egypt.In rose oil production factories, the freshly collected flowers were mixed with water in a tin-lined copper boiler (10,000 roses × 50 l water), then simmered gently by the gas heater for about 6-10 hours according to the boiler size.After the essential oil production, a huge amount of by-products (wastewater and texture) was collected in the distillation container.

Preparation of extracts
Four liters of R. damascena industrial by-products were collected from one of Taif's rose oil factories, then filtrated by cleaned cotton bags by squeezing the texture separated from the wastewater.Three hundred grams of texture by-product was extracted by methanol (MeOH) for one week at room temperature.A rotary evaporator (Buchi, Switzerland) was used to remove the solvent under pressure to afford 74.63 g of MeOH extract from the texture (MT); this step was repeated two times.In parallel, the wastewater obtained from squeezing the original by-product was centrifuged and filtered by Whatman No. 1 filter paper, then the filtrate was evaporated to yield 54.31 g of dried wastewater extract (W).Moreover, 20 g of each of the MT and W extracts were dissolved in 120 ml of distilled water, then partitioned with n-BuOH (3 × 120 ml).The organic and aqueous layers were separated and dried to give 7.62 g of n-BuOH fr.from texture MeOH extract (BT), 11.80 g of aqueous fraction from texture MeOH extract (AT), 5.71 g n-BuOH fraction from wastewater extract (BW), and 13.82 g aqueous fraction from wastewater extract (AW), respectively, as shown in Figure 1.All extracts were stored in brown glass bottles at room temperature for biological and chemical studies.

Quantification of total phenolic content
FCR assay was used to determine the total phenolic content tested extracts as described previously by Abdel-Hameed et al. (2009).Briefly, the tested extracts (100 µl) at a concentration of 100 µg/ml were mixed with FCR (500 µl) and neutralized with 20% Na 2 CO 3 solution (1.5 ml).The reaction mixture was carefully stirred, diluted with distilled water to a volume of 10 ml, and incubated for 2 hours at room temperature to allow for color development.At 765 nm, the absorbance was recorded in comparison to a blank that had all the reagents without samples at the same conditions using a UV-visible spectrophotometer (Jenway 6405, USA).The total phenolic content was measured using gallic acid as a reference and expressed as milligram gallic acid equivalents (GAE) per gram of extract.

Quantification of total flavonoid content
The flavonoid content was analyzed by the AlCl 3 colorimetric method (Abdel-Hameed et al., 2009).Briefly, each extract (100 µl) at a concentration of 100 mg/ml was added to 2% AlCl 3 (100 µl) dissolved in ethanol, acetic acid one drop, and the mixture was diluted up to 5 ml.After 40 minutes at ambient temperature, the reaction mixture's absorbance was measured at 415 nm against a blank containing all reagents without the samples using a UV-visible spectrophotometer (Jenway 6405, USA).All determinations were carried out in triplicate, and rutin was used as a reference.The total phenolic content was calculated as milligram rutin equivalents (REs) per gram of extract.

Quantification of total flavonol content
The content of flavonols was examined using an AlCl 3 assay (Abdel-Hameed et al., 2009).Briefly, 1 ml of each sample solution (100 mg/ml) was mixed with 1 ml of AlCl 3 (20 mg/ml) and 3 ml of sodium acetate (50 mg/ml).The reaction mixture was shaken well, and the absorbance was recorded at 440 nm after 2.5 hours.Quercetin was used as standard, and the absorption of a quercetin solution in MeOH (0.5 mg/ml) was determined.The experiments were performed in triplicate, and the amount of total flavonols was measured in milligram of quercetin equivalents (QEs) per gram of extract.

LC-ESI-MS analysis
A hybrid 3100 mass detector system (Waters, Alliance, USA) was conducted in the high performance liquid chromatography system (2695-series) and coupled with an electrospray ionization (ESI) source.A membrane disc filter with a 0.45 m pore size was used to filter the mobile phases, then sonicated to remove any gases.After several optimization attempts, two mobile phases were used: mobile phase A consisted of distilled water acidified with 0.1% formic acid (FA), and mobile phase B consisted of acetonitrile/MeOH [1:1 v/v] acidified with 0.1% FA.Gradient elution was applied, using 5% B for 5 minutes, 10% B for 5 minutes, 50% B for 45 minutes, 95% B for 10 minutes, and finally 5% B for 5 minutes.The injection volume of the sample was 10 µl and the mobile phase flow rate (0.6 ml/minute) split 1/1 before the mass interface.With the following settings, the analysis was carried out in negative ion mode within the m/z 50-1,000 range and other parameters, including capillary voltage (3 kV), cone voltage (50 eV), ion source temperature (150°C), desolvation temperature (350°C), and cone gas flow (50 l/hours), and desolvation gas flow (600 l/hour).Polyphenols were identified based on their mass spectrum and retention time (tR) and information from the literature after the obtained MS data were processed using Maslynx 4.1 software.

1, 1-Diphenyl-2-picrylhydrazyl radical assay
This technique relies on reducing the DPPH radical (purple color) to a diphenyl-picrylhydrazine stable molecule (yellow color).The DPPH radical scavenging activity was described by Osman et al. (2022).Briefly, 2 ml of R. damascena by-product extracts at different concentrations dissolved in MeOH was mixed with a 2 ml solution of 0.1 mM DPPH radical (dissolved in MeOH).The control contained 2 ml MeOH and 2 ml DPPH radical.The mixture was left to stand for 20 minutes at 37°C in the dark and a UV/Vis spectrophotometer (Jenway 6405, USA) was used to determine the absorbance at 517 nm.The experiment was performed in triplicate and ascorbic acid was used as a positive reference.The following equation was used to determine the DPPH radical scavenging rate: where A S and A C are the absorbances of the sample and control, respectively.The half-maximal scavenging concentration (SC 50 ) values were determined and expressed as the mean ± standard deviation (SD) in μg/ml.

Phosphomolybdenum assay
The phosphomolybdenum assay was carried out by the method of Prieto et al. (1999).It is predicated on the antioxidants' reduction of Mo (VI) to Mo (V) and the following formation of a green phosphate/Mo (V) complex.Briefly, 3 ml of the phosphomolybdenum reagent (28 mM sodium phosphate, 0.6 M sulfuric acid, and 4 mM ammonium molybdate) was added to 300 µl of the extract solution (100 μg/ml) in sealed test tubes.In a water bath set at 95°C, the reaction mixture was incubated for 90 minutes.The absorbance of the examined by-product extracts was measured at 695 nm using a UV/Vis spectrophotometer (Jenway 6405, USA) after cooling to room temperature in comparison with a blank sample containing 0.3 ml pure MeOH without extract.The experiment was performed in triplicate and the results were given in terms of milligram of ascorbic acid equivalents per gram of dried extracts (mg AAE/ g ext.).

Ferric reducing power (FRP) assay
This method was described by Abdel-Hameed et al. (2015).In total, 2 ml of by-product extracts (200 µg/ml) dissolved in MeOH was added to 2 ml of sodium phosphate buffer (0.2M, pH 6.6) and 2 ml of potassium ferricyanide (1%).The mixture was heated to 50°C in a water bath for 20 minutes.The mixture was then centrifuged at 3,000 rpm for 10 minutes at room temperature, with 2 ml of trichloroacetic acid (TCA) added.Immediately, 2 ml of the residual solution was added to 0.5 ml of 0.1% ferric chloride and 2 ml of MeOH.The absorbance of the samples, the blank, and the ascorbic acid reference were recorded at 700 nm using a UV/Vis spectrophotometer (Jenway 6405, USA).All experiments were done in triplicate and ascorbic acid equivalents in milligram/ gram of extract represented the FRP activity.

Determination of the anticancer activity
This assay was performed at the National Cancer Institute (NCI) in Egypt.Rosa damascena by-product extracts were tested in vitro using a HepG2 cell line (Obtained frozen in liquid nitrogen at −180°C from the American Type Culture Collection and were handled in NCI, by serial subculturing).Using SRB dye to stain the total amount of cellular protein, this assay indirectly calculates the number of cells.The test was described by Skehan et al. (1990).Briefly, at a concentration of 5 × 10 4 −10 5 cells/well in a medium, the cells were seeded in 96-well microtiter plates and allowed to attach to the plates for 24 hours.Each individual sample was made in wells, which were then incubated for 48 hours at 37°C in 5% CO 2 .After 24 hours, cells were treated with the relevant samples at different concentrations (0, 5, 12.5, 25, and 50 mg/ml).Then, the volume per well was increased to 200 ml using a fresh medium, and the incubation time was extended for another 24, 48, and 72 hours.After that, the cells were fixed with 50 μl of cold TCA (50%) for 1 hour at 4°C.Following the 30 minutes incubation, the cells were washed four times with 1% acetic acid.The plates were then desiccated and 100 μl/well of 10 mM Tris base (pH 10.5) was used to solubilize the dye for 5 minutes at 1,600 rpm on a shaker (Orbital shaker OS 20, Boeco, Germany).Using a spectrophotometer at 564 nm with an ELIZA microplate reader (Meter tech.Σ 960, USA), the optical density (OD) of each well was calculated.
The mean values of each drug concentration were computed after the mean background absorbances were automatically removed.For each cell line, the experiment was carried out three times.The following equation was used to determine the percentage of cell survival fraction: Survival fraction (%) = (OD treated cells / OD control cells ) × 100.

Statistical analysis
All determinations were carried out in triplicate and presented as mean ± SD using SPSS 13.0 program.The differences between each group were analyzed by one-way analysis of variance (ANOVA) with Tukey's posthoc test.Statistical significance was set to p < 0.05.

Total phenolic, flavonoid, and flavonol contents
The waste materials of R. damascena were examined for their total phenolic, flavonoid, and flavonol contents.According to the findings shown in Table 1, the BT fraction represented the highest total phenolic, flavonoid, and flavonol contents (230.69 ± 10.50 mg GAE/g ext., 71.66 ± 3.15 mg RE/g ext., and 51.70 ± 0.61 mg QE/g ext., respectively) followed by the BW fraction (118.29 ± 2.92 mg GAE/g ext., 40.16 ± 1.05 mg RE/g ext., and 24.61 ± 0.75 mg QE/g ext., respectively).The MT extract (92.13 ± 4.18 mg GAE/g ext., 28.42 ± 0.63 mg RE/g ext., and 18.16 ± 0.81 mg QE/g ext., respectively) and the W extract (48.27 ± 1.27 mg GAE/g ext., 16.68 ± 0.73 mg RE/g ext., and 8.47 ± 0.44 mg QE/g ext.), respectively, had reduced amounts of the phenolic, flavonoid, and flavonol contents.The AW and AT fractions contained the lowest amounts.In comparison to published data, these results are in agreement with those of Baydar and Baydar (2013), who reported high phenolic content of Turkish R. damascena MeOH extract from spent flowers.Dina et al. (2021) reported the high phenolic and flavonoid contents of the aqueous extract of hydro-distilled petals of R. damascena growing in Greece.In contrast, other studies reported that the total phenolic and flavonoid contents in the wastewater of R. damascene were 7.2 ± 0.2 mg GAE/ml and 1.14 ± 0.01 mg/ml, respectively (Gateva et al., 2022).Thus, these results revealed that the geographical origin, extraction conditions, and solvent have significant effects on phenolic and flavonoid contents.

LC-ESI-MS analysis
The liquid chromatography coupled with mass spectrometer experiments were performed in order to investigate the phytoconstituents of R. damascena extracts.In total, 55 polyphenolic compounds, including organic acids, phenolic acids, tannins, and flavonoids, have been detected in the MT, W extracts, and their   2.The structures of the proposed identified compounds are shown in Figure 2; Figures 3 and 4 illustrate the LC-ESI-MS total ion chromatograms of Taif rose wastewater and texture extracts.In the present study, the compound number has been attributed to its elution time in all extracts.
Tannins were represented as one of the major constituents of R. damascena by-product extracts, including compounds 3, 6, 7, 12, and 14, which had a precursor ion peak at m/z 483 and characteristic product ions at m/z 331, 169 No.   damascena (Elkhateeb et al., 2007;Dina et al., 2021).Compound 53 exhibited an [M-H] − at m/z 582 and different fragments at m/z 462 and 342 due to the liberation of a coumaric acid.Therefore, it was characterized as N', N", N'''-tris-p-coumaroyl spermidine, which is classified as hydroxycinnamic acid amide (Negri et al., 2018).

Antioxidant activity
In this study, three methods were carried out, including the FRP and phosphomolybdenum assays which are based on a single-electron transfer mechanism, while DPPH radical assay is based on radical scavenging (H transfer).The FRP mechanism cannot detect the antioxidants that can be detected by DPPH radical.Thus, the reduction of Fe 3+ and Mo (VI), as well as the formation of their complexes, is really important since when they are in a "free" state, they can catalyze the production of hydroxyl radicals which consider highly toxic radicals (Zhu et al., 2011).

DPPH radical scavenging activity
The stable free radical DPPH has an absorption band at 517 nm.It can lose this absorption by taking in an electron or a certain type of free radical, which results in a visual shift from purple to yellow color.One of its advantages is that it can accommodate many samples in a short time with high sensitivity to record the active ingredients at low concentrations (Qingming et al., 2010).In the present study, all of the by-product extracts exhibited DPPH radical scavenging properties.This was observed by the low SC 50 value, which indicates a higher antioxidant activity (Table 3).The BT and BW fractions had significant SC 50 values of 7.57 ± 0.01 and 12.78 ± 0.13 µg/ml, respectively.The MT and W extracts showed higher SC 50 values (SC 50 = 17.79 ± 0.10 and 34.25 ± 0.49 µg/ml, respectively), while the AT and AW fractions had a low antiradical scavenging activity with a value of >100 µg/ml.In addition, ascorbic acid was used as standard and represented a powerful antiradical activity with an SC 50 value of 5.83 ± 0.24 µg/ml.It was reported that the DPPH assay is applicable to hydrophobic systems like highly pigmented plants, such as roses, cherries, and red cabbage (Floegel et al., 2011).Therefore, the results of tested extracts of R. damascene showed a significant DPPH radical scavenging activity, especially BT and BW fractions.

FRP assay
This assay monitoring the reduction of Fe 3+ to Fe 2+ was used to determine the extracts' FRP.Depending on the FRP of the tested samples, the test solution's yellow hue changes to green or blue in this assay.Antioxidant-containing materials result in the reduction of the Fe 3+/ ferricyanide complex to the Fe 2+ form, which can be observed by measuring the appearance of Perl's Prussian blue at 700 nm (Georgieva et al., 2021).The results in Table 3 showed that the BT and BW fractions had a good metal-reducing capacity (513.63 ± 4.25 and 359.23 ± 3.28 mg AAE /g ext., respectively), followed by the MT and W extracts (254.26 ± 5.81 and 207.24 ± 1.87 mg AAE /g ext., respectively).However, the aqueous fractions of both AT and AW showed the lowest metalreducing power with a value of 107.02 ± 1.56 and 66.46 ± 2.70 mg AAE /g ext., respectively).Therefore, the tested extracts and fractions afforded some degree of electron donating capacity in a concentration-dependent manner.It was reported that an FRP of the aqueous extract of R. damascena had 5.08 ± 0.07 mmol Fe 2+/ g (Dudonné et al., 2009).On the other hand, the hydroethanolic extract of the Moroccan R. damascena flowers displayed 0.20 ± 0.1 mg/ml (Chroho et al., 2022).

Phosphomolybdenum assay
The phosphomolybdenum assay or total antioxidant capacity (TAC) method is based on the reduction of Mo (VI) of ammonium molybdate to Mo (V) to form a green subsequent of phosphate/Mo (V) complex at acidic pH (Abdel-Hameed et al., 2009).In this study, the reduction effect of examined extracts and fractions were arranged in the following order BT fraction with the value of 518.37 ± 25.31 mg AAE /g ext.˃ BW fraction (392.90 ± 9.56 mg AAE /g ext.) ˃ MT extract (380.22 ± 13.17 mg AAE /g ext.) ˃ W extract (309.21 ± 9.05 mg AAE /g ext.) ˃ AT fraction (138.15 ± 11.61mg AAE /g ext.) ˃ AW fraction (60.83 ± 10.06 mg AAE /g ext.) as shown in Table 3.These results suggest the powerful electron donating properties of BT and BW fractions with a large metal reduction capacity.Moreover, our results demonstrated a close correlation exists between the polarity of the extracting solvent and the content of active ingredients.
The previous studies reported that the TAC of the hydroethanolic extract from R. damascene flowers was 213.22 mg AAE/ g extract (Chroho et al., 2022).In addition, the ethanolic extract of fresh and spent flowers of R. damascena showed TAC with values of 372.26 ± 0.96 to 351.36 ± 0.84 mg/g extract (Ozkan et al., 2004).Indeed, our study showed that the extract from R. damascena flowers exhibited a powerful TAC, which may be due to the presence of phenolic compounds.

The anticancer activity
Environmental, chemical, physical, metabolic, and genetic variables all play a part in the development and progression of cancer in a multi-step process (Gomaa, 2013).There are so many kinds of cancers that affect the human body, among them hepatocellular carcinoma, which is considered the fourth leading cause of all deaths worldwide.For a variety of causes, including alcoholism, hepatitis B virus, hepatitis C virus, hemochromatosis, type 2 diabetes, hemophilia, and oxidative stress, it typically develops in the backdrop of cirrhosis (Barmoudeh et al., 2022;Ly et al., 2021).Cancer therapies frequently employ natural ingredients, particularly those derived from plants.The newly proposed idea of a broad-spectrum integrative approach to cancer prevention and therapy is based on the fact that they contain a wide range of bioactive secondary metabolites (Al-Dabbagh et al., 2018).Herein, the effects of R. damascena by-products were investigated against the HepG2 cell line and doxorubicin was used as a positive control (Fig. 5).The results revealed that the BT fraction showed the highest potent anticancer activity (IC 50 = 12.1 ± 0.85 μg/ml), followed by the W extract (14.00 ± 1.05 μg/ml), MT extract (20.5 ± 1.41 μg/ml), and AT fraction (21.1 ± 1.6441 μg/ml).The BW and AW fractions exhibited the lowest IC 50 value of 23.2 ± 1.57 and 34.7 ± 1.18 μg/ml, respectively.In contrast, doxorubicin had IC 50 = 3.73 ± 0.09 μg/ml.A previous study carried out by Gateva et al. (2022) reported that R. damascena wastewater fraction has a cytoprotective effect and is toxic at concentrations of 100 μg/ ml and higher in HepG2 cells using the (3-[4,5-dimethylthiazol-2yl]-2,5 diphenyl tetrazolium assay (Gateva et al., 2022).Thus, the potent anticancer activity of R. damascena industrial by-product extracts may be due to the presence of high levels of polyphenolic compounds.

CONCLUSION
In summary, the current study demonstrated that the industrial by-product extracts of R. damascena contain high levels of phenolic, flavonoids, and flavonol contents, especially for BT fr., BW fr., and MT extract, respectively.The LC-ESI-MS analysis led to identifying of 55 polyphenolic compounds in the tested extracts classified as organic acids, phenolic acids, tannins, and flavonoids.Furthermore, the BT and BW fractions had a high DPPH radical scavenging activity (SC 50 = 7.57 ± 0.01 and 12.78 ± 0.13 µg/ml, respectively), which is near to the value of ascorbic acid (SC 50 = 5.83 ± 0.24 µg/ml) as well as in the two other in vitro assays.Also, they exhibited a high inhibition activity of HepG2 cells (IC 50 = 12.1 ± 0.85 and 14.00 ± 1.05 μg/ ml, respectively).Thus, the reuse of R. damascena by-products will not only provide significant environmental benefits but also will contribute to evaluating their extracts with potential applications in pharmaceutical industries.

AUTHORS CONTRIBUTIONS
Ezzat E. A. Osman designed and performed the experiments analyzed and interpreted the data and results, the original manuscript.Salih A. Bazaid provided instrument facilities and revised the manuscript.El-Sayed S. Abdel-Hameed designed the experiments and wrote and revised the original manuscript.

This study does not involve experiments on animals or
The datasets generated during and/or analyzed during

Figure 1 .
Figure 1.A scheme of extraction and fractionation process of R. damascena industrial by-products.

Figure 2 .
Figure 2. Chemical structures of compounds of interest in R. damascena wastewater and texture by-products.

Figure 3 .
Figure 3. LC-ESI-MS total ion chromatograms of R. damascena wastewater and texture extracts; (A) MT extract, (B) BT fraction, (C) BW fraction, and (D) W extract at the negative ion mode.

Figure 4 .
Figure 4. LC-ESI-MS total ion chromatograms of R. damascena wastewater and texture extracts: (A) AW fraction and (B) AT fraction at the negative ion mode.

Table 1 .
Total phenolic, flavonoids, and flavonol contents of rose wastewater and texture by-product extracts of R. damascena.
The results were expressed as mean ± standard deviation (SD) (n = 3); the lettering ( a, b, c ) indicated the significant difference (p < 0.05) in each group utilizing one-way analysis of variance (ANOVA, Tukey's post hoc test).In group A: a Significant difference between W ext. and BW fr.& AW fr.; b Significant difference between BW fr. and W ext. & AW fr.; c Significant difference between AW fr. and W ext. & BW fr.In group B: a Significant difference between MT ext.and BT fr.& AT fr.; b Significant difference between BT fr. and MT ext.& AT fr.; c Significant difference between AT fr. and MT ext.& BT fr.

Table 2 .
Assigned identification of chemical constituents of R. damascena wastewater and texture by-products by LC-ESI-MS in negative ion mode.

Table 3 .
Total antioxidant capacity, FRP activity, and DPPH free radical scavenging activity of R. damascena wastewater and texture by-product extracts.Tukey , s post hoc test).In group A: a significant difference between W ext. and BW fr.& AW fr.; b significant difference between BW fr. and W ext. & AW fr.; c significant difference between AW fr. and W ext. & BW fr.In group B: a significant difference between MT ext.and BT fr.& AT fr.; b significant difference between BT fr. and MT ext.& AT fr.; c significant difference between AT fr. and MT ext.& BT fr.