الفهرس 
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Abstract
Rosaceae (rose family) is an ancient plant family representing 19th largest family of plants, and the third most economically important plant family in the temperate regions and containing many genera ranged from 95 to more than 100 genera and 2830−3100 species. Rosaceae members afford high-value nutritive crops and contribute favorable aesthetic and industrial products.
Fragaria L. is a genus of strawberry fruit which is a unique within the Rosaceae that has (basic chromosome of x = 7) with a small basic (x=7) genome size and is comprised of 20-30 species, only 23 species of them are wild including 13 diploids (2n = 2x = 14), four tetraploids, one hexaploid, and four octoploids, Fragaria × ananassa is an octoploid species with 56 chromosomes (2x = 8n = 56). This species called (pineapple or pine strawberry) where its smelling like pinapple (Ananas). Strawberries are perennial, herbaceous, low-growing plants. Strawberries are capable of vegetative propagation via the production of runners (stolons), which are trailing above ground stems that can take root at their nodes to establish new, clonal daughter plants.
Strawberry is a very important crop all over the world due to its nutritional value and organoleptic characteristics and its gainfulness. Egypt is an important country in the production of strawberries and its industry in Egypt increased very fast, because this country can export fresh fruits to Europe during the cold season from mid-November to mid-February and the main production areas of Egypt found in the Nile Delta (parallel 30/32◦ N) including Noubaria (1,875 ha), Qalubia (1,093 ha), Beheira (444 ha), Ismailia (1,049 ha), Sharkia (1,067 ha) and Monoufia (172 ha).
The present study work includes the following:
Part I: DNA fingerprinting of five cultivars of strawberry (Fragaria × ananassa Duch.) & Botanical study of strawberry.
Part II: Phytochemical study of the leaves and fruits of five cultivars of strawberry (Fragaria × ananassa Duch.).
Part III: Biological study of the leaves and fruits of two cultivars (Festival & Red Merlin) of strawberry (Fragaria × ananassa Duch.).
Part I: DNA fingerprinting of five cultivars of strawberry (Fragaria × ananassa Duch.) & Botanical study of strawberry.
Chapter 1: DNA fingerprinting of five cultivars of strawberry (Fragaria × ananassa Duch.)
The five cultivars of strawberry were subjected to random amplified polymorphic DNA (RAPD) analysis to assess the genetic variability and similarity at the molecular level. Eleven oligonucleotide primers were amplified successfully to evaluate the degree of diversity and similarity between the five studied cultivars.
RAPD bands were treated as presence or absence without considering their percentage. For estimating genetic distance among the investigated samples, each of DNA bands was treated as a unit character, and then genetic similarity was analyzed.
All the eleven random primers produced distinguishable polymorphic bands in each of the DNA sample. The RAPD experiments produced a total of 151 bands of which 52 bands were polymorphic. The percentage of total polymorphic loci was (34.4%) indicating a lower level of polymorphism which partially due to the strict genetic relationships of some cultivars analysed in this study and due to the low number of cultivars under investigation.
Primers A14, A17, A18, A19 and G10 were found to be the most effective in producing unique bands, and those five primers of eleven used, were produced a total of 10 unique bands in three cultivars (Suzana, Tamar and Winter Dawn), so primer-A14 can be used as specific genetic marker for authentication of Tamar cultivar where it generated three unique bands for this cultivar only.
Results revealed that the five examined strawberry cultivars were segregated according to the data obtained by the RAPD-PCR technique into two main clusters, and the first one cluster contains only one cultivar [Winter Dawn (4)*], that mean this cultivar showed the lowest similarities with the others four cultivars in percentages (80.8, 81.6, 85.3 and 90%) which referred to cultivars Suzana, Tamar, Festival and Red Merlin, respectively. But on the other hand the second cluster was divided into three subclusters showing the most similar cultivars (Festival and Tamar) in subcluster 3, and the second similar cultivar (Red Merlin) in subcluster 2, and the third one (Suzana) cultivar in subcluster 1.
Chapter 2: Botanical study of leaves of two cultivars of strawberry (Festival & Red Merlin):
A. Macroscopical examination:
Average height and width for mature plants is 23 cm and 30 cm respectively, there are about five to six sizes of strawberry leaves arranged from outside to inside of the canopy were arranged in spiral manner around the crown, it is evergreen stemless plant having stipulate trifoliate glossy dark green leaf on the upper surface with hirsute pubescent surface, and greyish green from lower surface and less pubescent than the upper except in the midrib and lateral veins regions is more pubescent than the upper. The terminal leaflet: is obovate in Red Merlin cultivar and is obovate to slightly round in Festival cultivar, the lateral leaflets: are ovate and nearly identical to each other and identical in both varieties.
The lamina of leaflet has crenate margin, toothed apex and with symmetric base of terminal one and asymmetrical base for both lateral leaflets. The midrib and lateral veins are prominent on the lower surface, and the venation is pinnate reticulate.
The petiole is long yellowish green in color and in some cases in reddish tinge color, grooved from one side and solid, its length and diameter average is about 120 mm and 3.5 mm respectively, and petioles have a medium pubescence.
B. Microscopical examination:
1. Leaves:
The transverse section through the lamina and midrib region of the terminal leaflet of adult leaves shows dorsiventral mesophyll, the midrib is more prominent on the lower surface and extra broad prominence observed at the 1/3 basal part of leaflet, but depressed on the upper surface. The palisade mesophyll usually consist of two rows of columnar cells which being interrupted in the midrib region and replaced by two rows of collenchymatous cells beneath each epidermal layers (upper & lower). A central crescent shape collateral vascular bundle (open vascular bundle) is present in the midrib region with pericycle of parenchymatous cells, also cluster crystals of calcium oxalate are scattered in the cortical region.
Upper epidermis: showed large polygonal tabular cells with straight thick cellulosic anticlinal wall, covered with smooth waxy cuticle, and not showed any type of stomata and lower epidermis: showed similar cells to the upper epidermis but smaller in size, the cells showing thick cellulosic wavey anticlinal wall, and mainly anomocytic stomata and scarcely anisocytic type stomata were present, but neural epidermis: showed polygonal axially elongated epidermal cells, with straight thick cellulosic anticlinal wall and the stomata are absent in the neural region, and the two types of hairs were more abundant on these cells especially on the lower surface.
The mesophyll is dorsiventral, being differentiated into disconnected upper palisade mesophyll and lower spongy mesophyll tissues. The transcurrent palisade mesophyll consists of two rows of cylindrical columnar cells, but spongy mesophyll consists of 3-4 layers of irregular shaped parenchymatous cells with large intercellular spaces.
The midrib consists of 4-5 rows of collenchymatous cells just beneath the upper epidermis and 2-3 rows just above the lower epidermis, any collenchymatous cells followed by 4-5 rows and 3-4 rows of thin walled parenchymatous cells showing narrow intercellular spaces for lower and upper epidermis respectively. The cortical tissues contain scattered cluster crystal of calcium oxalate, tannin cells, and the endodermis is undifferentiated.
There are two vascular bundles appeared in the midrib region one is large and the second adjacent one is smaller, each one of them is collateral and crescent in shape, consisting of radiating wholly lignified xylem that located up toward the upper epidermis compensating (vessels, tracheids, wood fibers and wood parenchyma) and phloem is located downward consisting of small thin cellulosic walled parenchymatous cells, the medullary rays are uniserriate consisting of radially elongated non-lignified parenchymatous cells and the pericycle consists of thin parenchymatous cells.
2. Petiole:
The transverse section through the petiole showing nearly cordate outline diagram, it is convex on one side and slightly curved to nearly straight on the other side. It shows an epidermis followed by a relatively wide cortex showing many scattered cluster crystals of calcium oxalate, this cortex interrupted with central large arc-shaped vascular bundle and two smaller vascular bundles in the both upper sides. The epidermis is formed of axially elongated polygonal thick cellulosic parenchymatous cells with straight anticlinal wall and showing few numbers of anomocytic stomata. The cortical cells are nearly similar in the shape, arrangement and contents to those of the midrib of the leaf, and there are 2-4 rows of collenchymatous cells just beneath the epidermis followed by thin walled parenchymatous cells which contain tannin cells and scattered cluster crystals of calcium oxalate. The three vascular bundles (one is central large crescent shaped and the other ones are lateral small on the both upside direction of the middle one) are collateral and the pericycle fibers as interrupted arc shape below the phloem in the central one and continuous arc shape in the both lateral vascular bundles.
Surface scanning of strawberry leaves of Festival and Red Merlin cultivars using scanning electron microscopy (SEM):
Upper surface (Festival cultivar): epicuticular wax is less denser and more thicker than of Red Merlin, and this surface showed many carvings by (SEM), but Red Merlin cultivar: epicuticular wax is very fine and thinner (membranous platelets) than of Festival but denser and thinner than of lower surface of the same cultivar, and this surface showed less carvings than of Festival by (SEM).
Lower surface (Festival cultivar): epicuticular wax is very fine and denser than of upper one of the same cultivar and the lower of Red Merlin were appeared as interwoven threads, epicuticular wax is denser and thicker than of upper one of the same cultivar and the lower of Festival cultivar were appeared as dense threads and protrusion.
Part II: Phytochemical study of the leaves and fruits of five cultivars of strawberry (Fragaria × ananassa Duch.)
Chapter 1: Preliminary phytochemical screening:
The results of the phytochemical screening show the presence of sterols and/or triterpenes in low concentration, free and combined flavonoids, tannins in the leaves and fruits of strawberry, while steam volatiles are present as traces in both organs.
Crystalline sublimate, alkaloids and/ or nitrogenous bases, anthraquinones, cardiac glycosides are absent in both organs.
Chapter 2: Volatile constituents and lipid contents:
A. Investigation and comparative study of volatile constituents of the fresh and frozen fruits of strawberry fruits (Fragaria × ananassa Duch.) along with their antimicrobial activity:
The volatile constituents of each of the fresh and frozen fruits of strawberry were prepared using solvent-solvent extraction method using n-hexane and yielded 0.05%, 0.049%, 0.039 %, 0.041 % and 0.042 % v/w % for fresh fruits of Festival, Red Merlin, Suzana, Tamar and Winter-Dawn, respectively. But frozen fruits yielded 0.04 %, 0.041 %, 0.035 %, 0.038 % and 0.033 % v/w % for Festival, Red Merlin, Suzana, Tamar and Winter-Dawn, respectively.
The GC/MS analysis of the volatile constituents of the fresh and frozen fruits under investigation revealed the presence of 38 different aroma compounds, distributed in different manner between the five cultivars of frozen and fresh forms.
The identification of those compounds were based on the comparing their retention time and mass fragmentation pattern with those of the reviewed literature along with other published data (Adams book) as well as through the predicted GC/MS library search (NIST).
The most abundant compounds determined in the volatile constituents are ethyl octadecanoate 15.52% in Tamar fresh aroma, E-14-hexadecenal 17.27% and 13.69% in Festival and Red Merlin fresh aroma respectively, octadecanol 17.65% and 15.71% in Festival and Red Merlin fresh aroma respectively, nerolidol 11.37% in Winter Dawn fresh aroma, geraniol 9.64% in Suzana frozen aroma, and tetradecene 15.41% and 10.36% in Festival and Red Merlin fresh aroma respectively.
The total identified volatile constituents in all five cultivars showed different pattern upon freezing from the fresh fruits, where there is a notable increase in total percentage of alcohols and aldehydes upon freezing and a notable decrease in the total percentage of terpenes, alkenes, lactones, ionones and ketones upon freezing, but esters percentage not changed upon freezing, finally the most stable cultivars against freezing are Festival and Red Merlin.
The most effective cultivars more than the standard control against Staphylococcus aureus, Klebseilla pneumoniae and Candida Albicans are Festival and Winter Dawn. But Festival and Red Merlin more effective than the standard control against Bacillus subtilis.
B. Study of the lipid content of leaves and fruits of two cultivars of strawberry (Festival & Red Merlin):
GLC analysis of the unsaponifiable matter:
The GLC analysis of the unsaponifiable matter of the leaves and fruits of both Festival and Red Merlin cultivars (leaves & fruits) revealed identification of 57 different compounds, and not all of them represented in only one sample and distributed between all samples under investigation.
The percentage of total hydrocarbons in the unsaponifiable fractions of leaves ranged from 8.19%-9.91% for Red Merlin and Festival, respectively and ranged from 9.27%-13.87% for Festival and Red Merlin fruits, respectively. Results showed that the fruits are richer than leaves in total percentage of hydrocarbons and the richest unsaponifiable fraction is Red Merlin fruit.
GLC analysis of fatty acid methyl ester (FAME):
The GLC analysis of the saponifiable matter of the leaves and fruits of both Festival and Red Merlin cultivars (leaves & fruits) revealed identification of 26 different saturated and unsaturated fatty acids ester derivatives, and not all of them represented in only one sample and distributed between all samples under investigation.
The percentage of total identified saturated fatty acids (69.2%) is higher than the percentage of total identified unsaturated fatty acids (30.8%). The results showed that the Methyl linolenate is most abundant unsaturated fatty acid derivative identified in Red Merlin fruits 23.39% followed by Festival fruit 16.22%, also Methyl oleate is the second unsaturated fatty acid detected in the four samples under investigation, where about 14.52%, 11.21% for Red Merlin and Festival fruits, respectively.
Chapter 3: Nutritional evaluation:
Determination of vitamin C, minrals and proximates contents of five different cultivars of strawberry (Fragaria × ananassa Duch.) fruits:
A. Determination of minerals and vitamin C content in strawberry (Fragaria × ananassa Duch.) fruits:
The assessment of minerals (K, P, Mg and Ca) and vitamin C were carried out according to the AOAC procedures.
The result revealed that vitamin C content differently varied among the five strawberry genotypes was approximately a 2-fold difference between the lowest (Suzana 22.7mg/100gm FW) and highest contents (Tamar 47.7 mg/100gm FW), where the vitamin C content was strongly genotype dependent. About 300 gram of fresh strawberry satisfied to supply the normal person with the daily demands of vitamin C, where the RDA (recommended daily allowance) of vitamin C is 75-90 mg per day so all the cultivars under investigation considered a good source of vitamin C especially Tamar cultivar.
Phosphorous and potassium represented the most significant minerals found in strawberry and also varied in wide range between cultivars, so affected by the genotype but the remain assessed minerals not largely affected with the genotype, Suzana cultivar considered the most rich cultivar with minerals among the studied cultivars, so consumption of about 300 gm of strawberry fruits per day will provide the normal person with about 12%, 6% and 15% of daily demand of phosphorous and magnesium, calcium and potassium respectively.
B. Determination of proximates content of five cultivars of strawberry (Fragaria × ananassa Duch.) Fruits:
All the proximates (water, carbohydrates, protein, fats and total ash) of the five cultivars of strawberry fruits were measured according to the method of (Cunniff, 1995), and the results revealed that the total carbohydrates content ranged from (7.45-9.46 gm %) for Red Merlin and Suzana cultivar respectively. The protein content varied from (0.59 -1.04 gm %), and the highest content of protein was found in Festival cultivar (1.04 gm %) and all cultivars showed high moisture content. The highest content was in Red Merlin, Winter Dawn and Tamar cultivars (91.49, 91.4, 91.18 gm %) respectively. Festival and Suzana cultivars had a lower moisture content of (89.96, 89.03 gm %) respectively.
In all proximates assessment, we found that the protein and the total carbohydrates contents were affected by the cultivar type. Other than the remained factors, and there was a difference approximately a 1.7- fold and 1.3-fold between the highest and lowest values, respectively.
Chapter 4: HPLC/MS analysis and colorimetric assays.
A. Non-targeted identification of active constituents of five cultivars of strawberry (Fragaria × ananassa Duch.) leaves and fruits using HPLC/DAD/ESI-HRMS:
Thirty three 33 metabolites had been putatively identified by LC-HRMS from the alcoholic extracts of five cultivars of strawberry leaves and fruits including twenty one polyphenolic, three sterols, one monoterpene and eight triterpenes compounds.
Most of the terpenes were present in leaves, very few in fruits and represented in low concentration. On the other hand, flavonoids and phenolic metabolites were presented in higher number but lower concentration in leaves.
B. Qualitative and quantitative determination of total flavonoids and phenolics of five cultivars of strawberry (Fragaria × ananassa Duch.) leaves and fruits using colorimetric assay and HPLC/MS:
Quantitative determination of total flavonoids and phenolics of five cultivars of strawberry (Fragaria × ananassa Duch.) leaves and fruits using colorimetric assay:
This study was performed in order to estimate the total flavonoids and phenolic contents in leaves and fruits of five cultivars of strawberry. This method depending on measuring the intensity of the color developed when flavonoids were complexed with aluminium chloride and the percentage was calculated as rutin according to previous standard calibration curve, or when phenolics complexed with Folin-Ciocalteu reagent and the percentage was calculated as gallic acid according to previous standard calibration curve.
The total flavonoid content of fruits and leaves in this study varied from 0.09 to 0.22 as g rutin/100 g FW for fresh fruits and from 2.01 to 2.55 as g rutin/100g DW for dried leaves. There was about 1.2-2.5 fold difference between the lowest and the highest values of total flavonoids for leaves and fruits respectively.
The total phenolic content of fruits and leaves in this study varied from 0.21to 0.37 as g gallic/100g FW for fresh fruits and from 1.62 to 2.76 as g gallic/100g DW for dried leaves. There was about 1.7-1.8 fold difference between the lowest and the highest values of total phenolics for leaves and fruits respectively.
Qualitative and quantitative determination of flavonoids and phenolics of five cultivars of strawberry (Fragaria × ananassa Duch.) leaves and fruits using HPLC Analysis:
HPLC analysis of alcoholic extracts of five strawberry cultivars leaves and fruits revealed the identification and quantification of thirty five polyphenolic compounds according to authentics (12 flavonoids and 23 phenolic acids and its derivatives).
Red Merlin leaves, Winter Dawn leaves, Festival fruits and Red Merlin fruits were showed the highest total identified flavonoids, but Winter Dawn leaves, Festival leaves, Red Merlin fruits and Festival fruits were showed the highest total identified content of phenolic acids and its derivatives, and the most abundant flavonoids identified were hesperidin followed by naringin, rutin and quercetrin. The total flavonoids ranged from 6.1778- 2639.17 mg/100g for Winter Dawn and Suzana leaves respectively and ranged from 494.36 – 277.32 mg/100g for Red Merlin and Suzana fruits respectively.
The most abundant phenolic acids identified were E-vanillic followed by pyrogallol, ellagic acid, benzoic, catechol, protocatecheuic acid, chlorogenic acid, vanillic and 3-OH tyrosol. The total phenolics ranged from 7136.8-1051.94 mg/100g for Red Merlin and Suzana leaves respectively, and ranged from 179.85-62.5 mg/100g for Festival and Tamar fruits respectively.
Chapter 5: Extraction, isolation and identification of the major compounds of the ethyl acetate extract of strawberry (Fragaria × ananassa Duch.) leaves of Festival cultivar:
30 gm ethyl acetate fraction of strawberry leaves (Festival cultivar) was fractionated on a silica gel column. Gradient elution was carried out using 100% dichloromethane (DCM) and increasing the polarity by 10% stepwise with methanol till 100% methanol. The obtained fractions were separately subjected to re-chromatographed on silica gel and Sephadex LH-20 (using methanol and methanol- water mixtures of increasing polarity) for further purification. Fractionation and purification of the main ethyl acetate extract subfractions of strawberry leaves (Festival cultivar) revealed isolation of seven compounds (C1, C2, C3, C4, C5, C6 and C7).
Compound C1: Kaempferol (yellow powder, 100 mg).
Compound C2: trans-p-Coumaric acid (colorless needles, 200 mg).
Compound C3: Quercetin (yellow powder, 100 mg).
Compound C4: p-Tyrosol (colorless needle crystals, 40 mg).
Compound C5: Methyl gallate (white needle crystals, 100 mg).
Compound C6: Eutigoside A (white powder, 70 mg).
Compound C7: trans-tiliroside (yellow powder, 150 mg).
Part III: Biological study of the leaves and fruits of two cultivars (Festival & Red Merlin) of strawberry (Fragaria × ananassa Duch.)
Chapter 1: Pharmacological screening (in vivo & in vitro study).
A. A Study of the Pharmacological Activity of the leaves and fruits of strawberry (Fragaria × ananassa Duch.) Festival and Red Merlin cultivars:
1. Acute oral toxicity study of the leaves and fruits of strawberry (Fragaria × ananassa Duch.) Festival and Red Merlin cultivars different extracts:
There was no mortality in animals at all doses of the extracts up to 5000 mg/kg b.wt., so the oral LD50 of the extracts is more than 5000 mg/kg.
2. Induction of diabetes mellitus:
Female albino rats of the Sprague Dawely Strain (150-170g) were injected intra-peritoneal (i.p.) by a single injection of (50 mg/kg body weight) of freshly prepared streptozotocin (STZ) dissolved in 0.1M citrate buffer (pH 4.5) to induce diabetes mellitus type 2, then after seven days the rats were screened for fasting blood glucose level and the rats with blood glucose level of 250-300 mg/dl were taken for study. Streptozotocin (STZ) was used as a drug induces diabetes mellitus, dyslipidemia, oxidative stress and dysfunction of many organs as (liver, kidney) in the experimental studies.
2.1. Antihyperglycemic activity:
All the tested treatments (alcoholic extract & aqueous infusion) of leaves and (alcoholic extract, aqueous infusion and juice) for fruits, ameliorated the blood glucose level and glycosylated haemoglobin (HbA1C %) in diabetic rats in comparable to the non-treated diabetic rats specially alcoholic extracts of leaves and fruits followed by aqueous infusion and finally juice, and the most effective one is Festival leaf alcoholic extract were decreased the blood glucose level to the half in comparable to the non-treated diabetic rats from 276.6 to 142.9 mg/dl and also decreased HbA1C % from 9.617 to 7.215.
2.2. Anti-hepatotoxicity:
All the liver function parameters ameliorated upon treating the STZ-diabetic rats with alcoholic extracts, aqueous infusions and juices of both leaves and fruits of (Fragaria × ananassa Duch.) Festival and Red Merlin cultivars, and serum level of ALT showed a significant decrease in all experimental rats treated with the all treatments even juices, where decreased from147.17 to 50.82 U/ml by alcoholic extract of Festival leaves.
2.3. Anti-hyperlipidemic activity:
The tested (alcoholic extracts, aqueous infusions) for leaves and (alcoholic extracts, aqueous infusions and juices) for fruits of (Fragaria × ananassa. Duch) Festival and Red Merlin cultivars ameliorated the lipid profile of diabetic rats in comparable to the non-treated diabetic rats at the tested dose (100 mg/kg b.wt), so they considered as a moderate antihyperlipidemic drugs, and serum lipids and lipoproteins (T.C, T.G, HDL-c, VLDL-c) improved by all the tested treatments but there was a significant decrease in LDL-c serum level by the alcoholic extracts of leaves and fruits followed by the aqueous infusions of leaves and fruits in comparable to the non-treated diabetic rats. Also, There was a significant decrease in risk 1 ratio (T.C/HDL-c) in diabetic rats treated with alcoholic extracts of both leaves followed by alcoholic extracts of both fruits, and all of them more effective than the standard treatment, also there was a notable decrease in risk 2 ratio (LDL-c/HDL-c) by all the tested treatments, while risk 1 ratio (T.C/HDL-c) decreased from 3.12 to 0.68 and risk 2 ratio (LDL-c/HDL-c) decreased from 4.9 to 2.08 by alcoholic extract of Festival leaves cultivar.
2.4. Nephroprotective activity:
All the tested treatments ameliorated all the kidney function parameters (serum urea, creatinine and uric acid), but serum creatinine level significantly decreased in diabetic rats treated with the alcoholic extracts of both leaves under investigation in comparable to the non-treated diabetic rats and their effect nearly equal to gliclazide followed by the rats treated with alcoholic extracts of both fruits, and its level decreased from 1.62 to 0.895 mg/dl by the alcoholic extract of leaves of Festival cultivar, and from 1.62 to 0.85 mg/dl by the alcoholic extract of leaves of Red Merlin cultivar. Both alcoholic extracts of leaves decreased the serum urea with about 35% from the non-treated diabetic rats.
2.5. Antioxidant activity:
All the tested treatments ameliorated the oxidative stress conditions in the diabetic treated rats in comparable to the diabetic non-treated rats, and the total antioxidant capacity (TAC) significantly increased in diabetic rats treated with alcoholic extracts of both leaves. Its level was increased from 0.26 to 0.48 and to 0.45 mmole/l for Festival and Red Merlin cultivars respectively followed by that treated with alcoholic extracts of fruits then the aqueous infusions of both leaves. While reduced glutathione level (GSH) significantly increased in the diabetic rats treated with alcoholic extract of Festival leaf in comparable to diabetic non-treated rats, and increased in duplicate form from 25.96 to 51.66 mg/dl.
B. In vitro assessment of the cytotoxic activity of strawberry (Fragaria × ananassa Duch.) leaves and fruits of Festival and Red Merlin cultivars using MTT assay:
The alcoholic extracts of the leaves and fruits of strawberry Festival and Red Merlin cultivars were tested against three human cell lines: HePG2 (Hepatocellular carcinoma cell line), MCF7 (Breast carcinoma cell line) and HCT (colorectal carcinoma cell line) as well as normal cell line as control using MTT reduction assay method.
The results revealed that all treatments reduced cell viability in a dose-dependent manner, and the alcoholic extract of Festival leaves is the most effective one against all the tested cell lines which having IC50 920 µg/ml, 263 µg/ml, 298 µg/ml against liver, breast and colorectal carcinoma cell lines respectively, followed by the alcoholic extract of Red Merlin leaves and having IC50 954 µg/ml, 467 µg/ml, 378 µg/ml against liver, breast and colorectal carcinoma cell lines respectively. Festival cultivar more effective than Red Merlin cultivar, and also the most sensitive cell lines to the alcoholic extracts of strawberry even leaves or fruits are breast carcinoma cell line and colorectal carcinoma cell line.
C. In vitro assessment of the antioxidant activity of strawberry (Fragaria × ananassa Duch.) leaves and fruits of Festival and Red Merlin cultivars using DPPH assay:
The radical scavenging activity of strawberry leaves (alcoholic and aqueous) extracts, and fruits (alcoholic, aqueous and juice) extracts of only two cultivars (Festival and Red Merlin) were assessed using DPPH assay. The degree of discoloration of DPPH indicates the free-radical scavenging efficiency of the extract. The extract concentrations providing 50% inhibition (IC50) was calculated from the graph of % DPPH inhibition against extract concentration. The antioxidant power of different extracts expressed by IC50 so all the different extracts of Festival cultivar showed better results than the extracts of Red Merlin cultivar and also extracts of leaves better than the fruits
Chapter 2: Antimicrobial study (in vitro study).
Antimicrobial Screening of Five cultivars of Strawberry (Fragaria × ananassa Duch.) Alcoholic extracts of Leaves and Fruits:
The antimicrobial activity of the five alcoholic extracts of leaves and five alcoholic extracts of fruits were determined using agar well diffusion method. All the extracts were tested in vitro for their antibacterial activity against four gram positive bacteria and four gram negative bacteria. Antifungal activity determined against four fungi microorganisms using Sabouraud Dextrose agar medium, using ampicillin, vancomycin, gentamicin and amphotericin B as standards for gram positive bacteria, MRSA, gram negative bacteria and fungi, respectively.
Results revealed that the most potent leaves cultivars were Red Merlin and Tamar against all micro-organisms except Aspergillus niger and all the leaves under investigation were more effective against gram negative bacteria than the gram positive bacteria and showed a moderate antifungal activity but the most potent from fruits were Red Merlin and Festival against gram negative bacteria, MRSA and fungi except Aspergillus niger, Festival cultivar was the most potent one against gram positive followed by Winter Dawn cultivar.
On general the alcoholic extracts of fruits were more effective than the alcoholic extracts of leaves.
Table (44): A list of the isolated compounds from strawberry (Fragaria × ananassa Duch.) leaves of Festival cultivar:
Compound Structure Remarks
Kaempferol
Firstly reported
p-coumaric acid
Previously reported
Quercetin
Firstly reported
p-Tyrosol
Secondly reported
Methyl gallate
Firstly reported
Eutigoside A
Secondly reported
Trans-Tiliroside
Previously reported
Table (45): A list of the identified compounds from five cultivars of strawberry (Fragaria × ananassa Duch.) leaves and fruits using HPLC/DAD/ESI-HRMS:
Compound Structure Organ Remarks
Benzaldehyde
Leaf,
Fruit Firstly reported
4-methoxy-benzaldehyde
Leaf,
Fruit Firstly reported
p-coumaric acid
Leaf,
Fruit Previously reported
Methyl salicylate
Fruit Firstly reported
Ellagic acid
Leaf,
Fruit Previously reported
Methyl trans-p-coumarate-4-glucoside
Leaf,
Fruit Firstly reported
O-trans-cinnamoyl-β-D-glucopyranose
Leaf Firstly reported in leaf
Lysidiside A
Leaf Firstly reported in leaf
Kaempferol
Leaf,
Fruit Firstly reported in leaf
Catechin
Leaf,
Fruit Previously reported
5,7-dihydroxy-2-phenyl-chromen-4-one (Chrysin)
Leaf Previously reported
Taxifolin
Leaf,
Fruit Previously reported
Procyanidin B3
Leaf,
Fruit Previously reported
trans-tiliroside [Kaempferol-3-β-D-(6-O-trans-p-coumaroyl) glucopyranoside]
Leaf,
Fruit Previously reported
Taxifolin 3-O-α-L-arabinofuranoside
Leaf,
Fruit Previously reported
7-O-trans-cinnamoylchrysin
Leaf,
Fruit Previously reported
Phloridzin (phloretin 2’-O-β-D-glucoside)
Fruit Previously reported
Afzelechin (4α →8) pelargonidin 3-O-β-glucopyranoside
Fruit Previously reported
Catechin (4α →8) pelargonidin 3-O-β-glucopyranoside
Fruit Previously reported
Pelargonidin-3-glucoside chloride (Callistephinchlorid)
Fruit Previously reported
Cyanidin 3-O-β-D-glucoside
Fruit Previously reported
p-mentha-1,8-diene (limonene)
Fruit Firstly reported
Suavissimoside R1
Leaf,
Fruit Firstly reported in leaf
(6S, 9R)-roseoside
Fruit Previously reported
Sericic acid
Leaf Firstly reported in leaf
Citroside A
Fruit Previously reported
Ursolic acid
Leaf Previously reported
Polnolic acid
Leaf Firstly reported in leaf
Tormentic acid
Leaf Firstly reported in leaf
Fupenzic acid Leaf Firstly reported in leaf
Campesterol
Leaf Firstly reported in leaf
Stigmasterol Leaf Firstly reported in leaf