الفهرس 
Literature ReviewOnly 14 pages are availabe for public view
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Abstract
Herbal products have been increasingly used worldwide for ages due to their availability and the familiar concept of their considerable safety. Herbal products are complex multicomponent mixtures that possess a high risk of adulteration and contamination. Despite the continuous use of herbal plants, quality control of these plants remains challenging throughout time. This may be attributed to the complexity of its phytochemical constituents, which requires sophisticated methods for its analysis and hence its quality control. Herbal formulations are not officially recognized due to the lack of an adequate process of standardization and evaluation. Due to the high number of variables affecting the quality control of herbal products, so multivariate analysis is the best tool to find patterns and relationships between these several variables.
Within the Fabaceae family of about 727 genera, fenugreek is one of its members. Concerning the number of species of this family, it comprises approximately 19,325 species. Fenugreek is an angiosperm, herbaceous and annual plant, divided into around 260 species. It is extensively distributed as a food crop throughout India, the Mediterranean region, North Africa, and Yemen. It has many traditional uses in treating digestive disorders, antidiabetic activity, carminative, tonic, aphrodisiac effects, hypocholesterolaemic, and anti-bacterial activities. It has many studied biological activities as anti-inflammatory, antidiabetic, gastroprotective, cardioprotective, and others. It is widely used in Egypt as a spice, flavor, and medicinal herb for different diseases.
This study aimed to develop an accurate model for the quality control of fenugreek using the chromatographic method and spectroscopic methods coupled with chemometrics. The ability of the model to differentiate between fenugreek from different origins was investigated. In addition, we explored the antidiabetic activity of all samples used in the study.
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Part 1: Phytochemical Analysis
Chapter Ⅰ: Ultraviolet Spectroscopic Analysis for Fenugreek Seeds:
UV spectroscopy in combination with PCA was used as an available and easy tool for the discrimination of fenugreek according to geographical origin. PCA results for the samples scanned from (200-400 nm) showed proper discrimination of the samples. The results showed grouping most of the Egyptian samples in one cluster, while Indian samples, IE1 and IE2, were clustered together to the left side with regard to PC1, while samples from Saudi Arabia and Yemen were scattered with Egyptian samples to the right of PC1. A correlation could be confirmed particularly between total polyphenol content and UV spectroscopic analysis from the loading data, which revealed the highest variance level within the wavelengths 200–240 nm, representing mainly the UV absorption by phenolic acids and flavonoidal compounds distributed throughout the plot. Samples characterized by higher levels of polyphenolics were clustered positively along the PC1 axis, as shown in the loading plot. It was noted that samples with the lowest total polyphenol levels also constituted comparatively low absorbance values and were a helpful quality verification tool. This explained the closeness of most Egyptian samples on the right side to PC1.
Chapter Ⅱ: Determination of Polyphenolic and Total Flavonoids content for Fenugreek Seeds
Total phenolic content was calculated according to Folin-Ciocalteu assay using Gallic acid as a standard whose calibration curve and straight-line equation used to calculate TPC among different samples expressed as (mg GAE/ mg dried extract). TPC values were ranging from 14.59 to 33.41 μg/mg. The highest TPC was exhibited by the Yemeni sample YE1, followed by PP3 and EE3. In contrast, the Indian sample IE2 exhibited the lowest value. Whereas Total flavonoid content was calculated according to AlCl3 colorimetric assay using quercetin as a standard whose calibration curve and straight-line equation used to calculate TFC among different samples expressed as (mg QE/ mg dried extract).
On the other hand, the flavonoid content ranged from 5.06 to 20.87 μg/mg. The highest flavonoid content was detected in Egyptian sample EP3 followed by IE3 and EE6, whereas the lowest was assigned to IE1 and IE2. A correlation could be confirmed between the total
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polyphenol content and UV spectroscopic analysis. Samples exhibiting the highest polyphenol levels showed comparatively high absorbance intensity values and served as a useful quality verification tool. This finding is in line with the previously mentioned UV spectroscopic profile PCA combined with TPC and TF content. The loading plot displays TPC and TF in close proximity to wavelengths of the UV scan, which is at the positive side of PC1.
Chapter Ⅲ: High Performance Liquid chromatography (HPLC) Analysis for Fenugreek Seeds
Trigonelline standard was used for the standardization and quantification in different samples. The concentration of trigonelline in each extract was calculated using the regression equation obtained from the trigonelline standard calibration curve. Its concentration was expressed in milligram per gram of fenugreek powder. The obtained trigonelline content showed marked variation among different samples. The results have shown that geographical origin has no great influence on the trigonelline content in fenugreek samples. Trigonelline content ranged from 83.66 and 117.07 mg/g of powdered fenugreek, with the highest content, EP1, and the lowest in PP2. Pharmaceutical preparation samples exhibited the lowest TRG content except for PP1, unlike most Egyptian samples, which showed high TRG content, which implies that pharmaceutical preparation might contain excipients. HPLC metabolic profiling for different samples was constructed utilizing the methanolic extracts. HPLC chromatogram of fenugreek samples from different origins showed remarkable visual similarity regarding different peaks identified. The score plot showed the grouping of most Egyptian samples together on the right side of PC1, while Indian samples were clustered on the lower left side of PC1. Meanwhile, the pharmaceutical preparations were clustered together in close proximity with SE1 on the left of PC1. The Yemeni sample, YE1, was clustered with most Egyptian samples in the upper right of PC1.
Chapter Ⅳ: High Performance Thin Layer chromatography (HPTLC) Analysis for Fenugreek Seeds
The HPTLC profile of fenugreek samples confirmed the presence of trigonelline and 4-Hydroxyisoleucine. Quantification of TRG and 4-HIL in fenugreek samples was achieved via interpolating the peak area in the respective standard calibration curve. Scanning was performed for the methanolic extracts at 265 nm and 395 nm after derivatization with ninhydrin
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reagent. The trigonelline content in fenugreek samples ranged from 19.62 to 67.30 mg/g of dried residue, and that of 4-Hydroxyisoleucine was found to vary from 19.00 to 62.06 mg/g of dry residue. HPTLC was utilized to produce a chemical profile of the methanol extracts of fenugreek samples for all the peak areas detected, and the resulting data were subjected to multivariate analysis. The PCA score plot showed most Egyptian samples (entire and powdered) were clustered together along the right side of PC1 and the Yemeni sample YE1. Pharmaceutical preparation samples were segregated left to PC1, close to the Indian samples clustered lower left to PC1. Whereas the Saudi Arabia sample SE1 was clustered together with Indian samples in the lower left to PC1. The HPTLC model is considered a more powerful tool for discriminating the samples according to their geographical origins.
Part 2: In vitro α-amylase Inhibition Assay for Fenugreek Seeds
Fenugreek extracts were assessed for the α-amylase inhibitory activity to correlate the reported antidiabetic activity to the concentration of trigonelline and 4-Hydroxyisoleucine. The results were expressed as IC50 values, calculated from the regression equations prepared from the concentrations of each extract separately. Acarbose was used as standard, and it showed inhibition of α-amylase enzyme activity with an IC50 value of 34.71 μg/mL. Fenugreek samples showed variable α-amylase enzyme inhibitory activity. The Egyptian sample EE3 showed maximum inhibition with an IC50 value of 40.3 μg/mL followed by the pharmaceutical preparation PP2 (47 μg/mL) and Yemeni sample YE1 (49 μg/mL). In contrast, the Indian sample IE2 showed the lowest inhibitory activity displaying an IC50 value of 814.1 μg/mL. However, no correlation was observed among the results with regard to the contents of trigonelline and 4-hydroxyisoleucine. Undoubtedly, this could be due to the existence of many other antidiabetic phytochemicals predominating in fenugreek that synergistically explain the antidiabetic behavior of the samples and not solely depend on two components.