Ward Taifi (Taif rose) is considered one of the most important economic products of Taif, Saudi Arabia. In this study both fresh and dry Taif rose were biologically and phytochemically investigated. The 80% methanol extracts and
The use of natural resources especially plants increases daily for the discovery of new therapeutic agents. Medicinal plants are the richest bioresource of drugs in traditional medicines, modern medicines, nutraceuticals, food supplements, folk medicines, pharmaceutical intermediates, and chemical entities for synthetic drugs. Natural products from plants continue to be used in pharmaceutical preparations as crude extracts, fractions, or pure compounds. Several active compounds have been discovered in plants and used directly as patented drugs like Taxol, Artemisinin, and Maprouneacin [
Taif rose, Ward Taifi (
All solvents, standards, and reagents are analytical and HPLC grade. 1,1-diphenyl picrylhydrazyl (DPPH)• free radical and Folin-Ciocalteu’s reagent (FCR) are from Fluka Chemicals. Aluminum chloride, sodium carbonate, sodium phosphate, ammonium molybdate, ascorbic acid, petroleum ether, ethyl acetate, methanol, ethanol, acetic acid, trichloroacetic acid, formic acid, sulphuric acid, sulphorhodamine-B (SRB), catechin, taxifolin, rutin, quercetin 3-O-
During harvest season (March–April 2012), Taif roses were collected from rose farms in the El-Hadda region, Taif governorate, Saudi Arabia. After removing the green parts of roses, part of them was used fresh, and another part was dried on shade air-dried place and powdered using an electric mill. Known weights of cut fresh, and dry powdered rose (900 and 300 g, resp.) were extracted three times by 80% methanol: 4 L for fresh rose and 2 L for dry rose. The solvent was removed under vacuum using rotary evaporator affording known weight of each 80% methanol extract (112 and 97 g). Thirty grams of each 80% methanol extract were dissolved in 150 mL distilled water and partitioned with
Three different chemical methods were used for evaluating the antioxidant activity of crude methanol extracts,
This method depends on the reduction of purple DPPH radicals to a yellow coloured diphenyl-picrylhydrazine, and the remaining DPPH radicals which show maximum absorption at 517 nm were measured using UV-Vis spectrophotometer (Jenway 6405). Two mL of different concentrations of each sample were added to 2 mL solution of 0.1 mM DPPH. An equal amount of methanol and DPPH served as control. After 20 min of incubation at 37°C in the dark, the absorbance was recorded at 517 nm. The experiment was performed in triplicates. The DPPH radical scavenging activity was calculated according to the following equation:
The assay is based on the reduction of Mo (VI) to Mo (V) by the antioxidants and subsequent formation of a green phosphate/Mo (V) complex at acid pH. 300
Two mL of each sample and ascorbic acid in methanol (200
The crude methanol extracts,
The experiment was repeated 3 times. The percentage of cell survival was calculated according to the following equation:
In this study, the total phenolic, flavonoid, and flavonol contents of crude methanol extracts,
The total phenolic content of plant extracts was determined using Folin-Ciocalteu’s reagent (FCR). 100
The flavonoid content was determined by aluminium chloride method using rutin as a reference compound. 100
The content of flavonols was determined by using quercetin as a reference compound. 1 mL of each sample solution (0.001 g/mL) was mixed with 1 mL aluminium trichloride (20 mg/mL) and 3 mL sodium acetate (50 mg/mL). The absorbance at 440 nm was read after 2.5 h. The absorption of the standard quercetin solution (0.5 mg/mL) in methanol was measured under the same conditions. All determinations were carried out in triplicates. The amount of flavonols in plant extracts in quercetin equivalents (QE) was calculated by the same formula used in flavonoids (
Ten standard stock solutions, catechin (500
Direct infusion method using ESI-MS (Waters 3100) in negative ion mode was performed to get ESI(−ve)-MS fingerprinting for fresh and dry Taif roses. The analytical conditions for injection include the injection of all samples (5 mg/mL) directly to the ion source by means of a syringe pump at flow rate (0.02 mL/min) for ten minutes. The analytical conditions for mass spectrophotometer were capillary voltage (3 kV), cone voltage (30 and 70 eV), desolvation temperature (350°C), desolvation gas flow (700 L/h), cone gas flow (50 L/h), and source temperature (150°C). Mass spectra were scanned in ESI negative mode in the range between
LC-ESI-MS analysis system consists of HPLC (Waters Alliance 2695) and mass detector (Waters 3100). The mobile phases were prepared daily by filtering through 0.45
Each standard compound was chromatography using the previous analytical conditions. For each standard, the retention time and mass spectrum were determined. From each individual standard stock solution, a mixed stock solution containing ten analytes were prepared and diluted to appropriate different concentrations for establishing calibration curves. For quantitative analysis, six different concentrations of a mixed stock solution containing ten analytes were injected. A calibration curve was obtained by plotting the peak area versus the concentration of each standard. Chromatograms of samples obtained were analyzed using Maslynx 4.1 software based on the comparison of retention times of the samples with those of the standards for qualitative analysis and calibration curve for quantitative analysis.
All determinations in Tables
DPPH free radical scavenging activity, total antioxidant capacity, and reducing power activity of 80% methanol extracts,
Extract | DPPH free radical scavenging activity | Total antioxidant capacity |
Reducing power activity (mg equivalent to ascorbic acid/g extract)4 | |
---|---|---|---|---|
SC50 ( |
(mg ascorbic acid equivalent/g extract)2 | |||
Fresh roses | ||||
80% MeOH |
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Aqueous fraction | >100d | — |
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Dry roses | ||||
80% MeOH |
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Aqueous fraction | >100d | — |
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Ascorbic acid |
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— | — | — |
Values of SC50, total antioxidant capacity reducing power activity, were expressed as mean of triplicate determinations ± standard deviation (
1SC50: concentration in
2Radical scavenging activity expressed by mg ascorbic acid equivalent/g extract.
3Antioxidant capacity monitored by the phosphomolybdenum method expressed by mg ascorbic acid equivalent/g extract.
4Reducing power activity expressed by mg ascorbic acid equivalent/g extract.
Total amount of phenolic, flavonoid, and flavonol compounds of 80% methanol extracts,
Extract | Total phenolics |
Total flavonoids |
Total flavonols |
---|---|---|---|
Fresh roses | |||
80% MeOH | 61.54 ± 3.88c | 30.94 ± 0.39c | 21.01 ± 0.55c |
|
186.84 ± 6.94e | 63.18 ± 0.76d | 34.46 ± 0.58d |
Aqueous fraction | 7.73 ± 2.21a | 3.61 ± 0.34a | 3.08 ± 0.10a |
Dry roses | |||
80% MeOH | 49.38 ± 1.27b | 24.94 ± 1.25b | 14.20 ± 0.59b |
|
177.99 ± 7.25d | 65.59 ± 0.82e | 40.51 ± 0.85e |
Aqueous fraction | 7.74 ± 1.91a | 3.74 ± 0.17a | 2.81 ± 0.13a |
Values of SC50, total antioxidant capacity reducing power activity, were expressed as mean of triplicate determinations ± standard deviation (
1Total phenolics expressed by mg gallic acid equivalent/g extract.
2Total flavonoids expressed by mg quercetin equivalent/g extract.
3Total flavonols expressed by mg quercetin equivalent/g extract.
Cytotoxic activity expressed by IC50 (
The total phenolic contents and biological activity were reported in some
Free radicals and reactive oxygen species have been proposed to induce cellular damage and to be involved in several human diseases such as cancer, arteriosclerosis, and inflammatory disorders as well as in aging processes [
The antioxidant activity of 80% methanol extracts,
Cancer is a complicated group of diseases characterized by the uncontrolled growth and spread of abnormal cells, and the mortality that results from the common forms of cancer is still unacceptably high [
Figure
Many reports attributed the biological properties of roses to its high contents of phenolic compounds [
The correlation coefficient between the total antioxidant capacity monitored by phosphomolybdenum method and the total phenolic, flavonoid, and flavonol contents of 80% methanol extracts,
Hyphenated HPLC-MS technique is an important method used for identifying complex mixtures, especially the phenolics in the crude extract or its fraction found in the plant, either by using standard compounds (target identification) or by comparing mass spectrum obtained with literatures (tentative identification) [
After several trials to obtain good separation of the ten standard phenolic compounds mixture by LC-MS (Figure
Method validation data for ten phenolic compounds by RP-HPLC-ESI-MS.
Peak number | Compounds | Sample loading linearity range ( |
Regression equation | Correlation coefficient ( |
---|---|---|---|---|
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Catechin | 0.1–0.8 |
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0.878 |
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Taxifolin | 0.1–0.8 |
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0.998 |
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Quercetin-3-glucose-( |
0.05–0.4 |
|
0.990 |
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Rutin | 0.05–0.4 |
|
0.997 |
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Quercetin 3-O- |
0.05–0.4 |
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0.995 |
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Quercetin 3-O- |
0.1–0.8 |
|
0.998 |
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Kaempferol 3-O- |
0.05–0.4 |
|
0.997 |
|
Quercetin | 0.025–0.2 |
|
0.998 |
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Apigenin | 0.025–0.2 |
|
0.996 |
Total ion chromatogram of ten phenolic compound standards using LC-ESI negative mass spectrometry: Catechin
The mass spectrum for both methanol and aqueous fractions obtained by direct infusion of samples into the negative ion mode ESI-MS showed the presence of a major peak at
After optimizing the LC separation method of standards, the samples were injected in LC-MS under the same conditions. Figures
Total ion chromatograms (TIC) of 80% methanol extract (a),
Total ion chromatograms (TIC) of 80% methanol extract (a),
Chemical structures of compounds identified and tentatively identified from fresh and dry Taif rose.
The molecular mass of sugar units in glycosides was calculated from the difference mass of molecular ion peaks, such that a difference of 132 indicates pentose (xylose/arabinose); a difference of 146 indicates deoxyhexose (rhamnose); a difference of 162 mass units indicates a hexose (glucose/galactose); a difference of 176 indicates glucuronic acid. Known peaks were identified by comparing their
Peak assignment, molecular weight (MW), molecular ion (M−), mass ion fragments, and tentative identification of compounds detected in 80% methanol extract,
Peak number | MW |
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Tentative identification | |
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M− | Fragments | |||
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192 | 191 | 127, 93, 85 | Quinic acid |
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170 | 169 | 125, 79 | Gallic acid |
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484 | 483 | 331, 313, 169, 125 | Digalloyl hexose |
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184 | 183 | 169, 147, 124, 78 | Methyl gallic acid derivative |
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786 | 785 | 633, 615, 483, 301, 169, 125 | Digalloyl DHHP hexose |
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466 | 465 | 313, 301, 169, 147, 125 | Digalloyl deoxyhexose |
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786 | 785 | 633, 615, 483, 331, 313, 301, 169, 125 | Digalloyl DHHP hexose |
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968 | 967 | 785, 765, 667, 505, 301, 183, 169 | Unknown ellagitannin |
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968 | 967 | 785, 765, 633, 615, 483, 451, 301, 182, 169, 125 | Unknown ellagitannin |
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938 | 937 | 783, 657, 465, 301, 169, 125 | Unknown ellagitannin |
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968 | 967 | 785, 765, 639, 450, 314, 301, 169, 147, 124 | Unknown ellagitannin |
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616 | 615 | 463, 313, 301, 169 | Quercetin-3-glucose-( |
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938 | 937 | 785, 766, 615, 313, 301, 183, 169, 125 | Unknown ellagitannin |
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610 | 609 | 463, 301 | Rutina |
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464 | 463 | 301, 229, 179, 150 | Quercetin 3-O- |
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600 | 599 | 463, 300, 179, 169, 151 | Quercetin-hexose-protocatechuic acid |
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434 | 433 | 301, 151, 179 | Quercetin-O-pentose |
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600 | 609 | 447, 435, 284, 169, 151 | Kaempferol-hexose-gallic acid |
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610/594 | 609/593 | 435, 433, 301, 285, 169, 151 | Quercetin/Kaempferol derivatives |
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448 | 447 | 284, 179, 151 | Kaempferol 3-O- |
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600 | 599 | 447, 285, 197, 169, 151 | Kaempferol-O-hexose-O-gallic acid |
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418 | 417 | 284, 197, 227 | Kaempferol-O-pentose |
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594 | 593 | 417, 285, 197, 151, 147 | Kaempferol-O-pentose-O-glucuronic acid |
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432 | 431 | 284, 255, 227 | Kaempferol 3-O- |
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652 | 651 | 609, 447, 301, 147 | Quercetin acetyldisaccharides |
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610 | 609 | 463, 447, 301, 147 | Quercetin-O-hexose-O-deoxyhexose |
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636 | 635 | 487, 285 | Kaempferol acetyldisaccharides |
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594 | 593 | 447, 430, 285, 151 | Kaempferol-O-hexose-O-deoxyhexose |
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605 | 604 | 582, 462, 342 | Unknown non-phenolic compound |
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Standard compounds | ||||
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290 | 289 | 244, 221, 150, 136, 123 | Catechin |
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304 | 303 | 284, 274, 217, 179, 151 | Taxifolin |
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616 | 615 | 463, 313, 301, 271, 169, 151, 147 | Quercetin-3-glucose-6-gallic acid |
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610 | 609 | 463, 301, 179, 151, 147 | Rutin |
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464 | 463 | 300, 271, 254, 179, 151 | Quercetin 3-O- |
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448 | 447 | 300, 270, 179, 151 | Quercetin 3-O- |
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448 | 447 | 284, 179, 151 | Kaempferol 3-O- |
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432 | 431 | 248, 254, 227, 198, 147 | Kaempferol 3-O- |
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302 | 301 | 179, 151 | Quercetin |
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270 | 269 | 225, 199, 159, 151, 117 | Apigenin |
Quantity of compounds detected in 80% methanol extract,
Peak number | Compound | Fresh rose (mg/g extract) | Dry rose (mg/g extract) | ||||
---|---|---|---|---|---|---|---|
80% MeOH |
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Aqueous | 80% MeOH |
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Aqueous | ||
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Quinic acida | 1546329 | 351441 | 1749393 | 2076361 | 731132 | 26452471 |
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Gallic acid | 3.22 | 6.31 | — | 2.84 | 5.39 | — |
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Digalloyl hexose1 | 0.09 | 0.31 | — | 0.04 | 0.16 | — |
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Methyl gallic acid derivative2 | 3.57 | 7.6 | — | 2.1 | 5.3 | — |
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Digalloyl HHDP hexose1 | 1.03 | 2.84 | — | 1.17 | 2.80 | — |
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Digalloyl deoxyhexose1 | 0.09 | 0.22 | — | 0.11 | 0.19 | — |
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Digalloyl HHDP hexose1 | 2.18 | 5.35 | — | 2.60 | 5.32 | — |
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Unknown ellagitannin1 | 1.57 | 4.06 | — | 1.12 | 2.66 | — |
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Unknown ellagitannin1 | 2.52 | 6.30 | — | 1.72 | 3.85 | — |
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Unknown ellagitannin1 | 0.22 | 0.80 | — | 0.49 | 0.40 | — |
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Unknown ellagitannin1 | 0.75 | 1.56 | — | 0.87 | 1.44 | — |
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Quercetin-3-glucose-6-gallic acid | 1.33 | 1.38 | — | 0.64 | 1.43 | — |
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Unknown ellagitannin1 | 2.06 | 4.07 | — | 1.61 | 3.50 | — |
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Rutin | 0.10 | 0.17 | — | 0.10 | 0.24 | — |
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Quercetin 3-O- |
1.90 | 3.93 | — | 2.28 | 4.72 | — |
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Quercetin-glucose-protocatechuic acid1 | 1.46 | 3.62 | — | 1.93 | 3.64 | — |
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Quercetin-pentoside3 | 0.21 | 0.65 | — | 0.29 | 0.78 | — |
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Kaempferol-hexose-gallic acid1 | 1.27 | 4.77 | — | 1.70 | 3.33 | — |
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Quercetin/Kaempferol derivatives4 | 2.81 | 5.88 | — | 3.43 | 6.66 | — |
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Kaempferol 3-O- |
7.50 | 16.21 | — | 9.15 | 17.60 | — |
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Kaempferol-hexose-gallic acid1 | 0.14 | 0.31 | — | 0.22 | 0.43 | — |
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Kaempferol-pentoside5 | 0.53 | 1.30 | — | 0.87 | 1.50 | — |
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Kaempferol-pentoside glucuronic acid4 | 1.95 | 5.21 | — | 2.65 | 5.71 | — |
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Kaempferol 3-O- |
0.54 | 1.31 | — | 0.88 | 1.50 | — |
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Quercetin acetyl disaccharides4 | 0.23 | 0.50 | — | 0.21 | 0.58 | — |
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Quercetin disaccharides4 | 0.26 | 0.65 | — | 0.19 | 0.66 | — |
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Kaempferol acetyl disaccharides4 | 1.27 | 3.28 | — | 1.50 | 3.44 | — |
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Kaempferol hexoside-deoxyhexose4 | 1.46 | 3.94 | — | 1.59 | 3.74 | — |
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Unknown non-phenolic compounda | 2776578 | 6225284 | — | 541747 | 7069559 |
Quantified as 1quercetin-3-glucose-6-gallic acid; 2gallic acid; 3quercetin 3-O-
aDue to lake of similar or related standard compounds, the quantity is represented by area under curve.
The results of this study provide evidence that the 80% methanol extracts,
Authors have declared that there is no conflict of interests in this paper.
The authors are very grateful to Taif University, Saudi Arabia, for supporting this work (Project no. 2-433-1871) and deeply grateful to the supporter of Chair of Research and Development Studied for Taif rose, Taif University, Saudi Arabia.