Institute of Biosciences and Technology, MGM University, Chatrapati Sambhaji Nagar-431003
Flavonoids are polyphenolic compounds known for their antioxidant properties and various health benefits. The current study focused on the evaluation and quantification of flavonoids Morin, Naringin, Quercetin, and Rutin in blackberry (Rubus fruticosus) leaves. High-Performance Liquid Chromatography (HPLC) is employed for the qualitative analysis of these compounds using a reverse-phase column with 0.1% phosphoric acid and acetonitrile in a 25:75 ratio. The standard retention times for Quercetin, Morin, Naringin, and Rutin were 2.513 min, 3.887 min, 7.130 min, and 4.780 min, respectively. In blackberry leaf extracts, Quercetin, Morin, and Naringin are identified at retention times of 2.567 min, 3.640 min, and 5.673 min, respectively, these results confirm the presence of key flavonoids with potential health benefits in blackberry leaves
Blackberries (Rubus fruticosus) belong to the Rosaceae family, one of the largest and most economically important plant families, commonly known as the rose family. It comprises over 90 genera and 2,500 species, including various fruits, ornamentals, and shrubs. This family includes other well-known fruits such as apples, pears, strawberries, raspberries, cherries, peaches, and almonds [1&2].
Blackberries contain numerous phytochemicals and include powerful antioxidants such as polyphenols, flavonoids, anthocyanins, tannins, and phenolic acids. The flavonoids present include quercetin, kaempferol, and myricetin. Blackberries are also rich in vitamin C, fiber, and folate, which contribute to their overall health benefits [4]. In terms of flavonoids, blackberries are packed with several types, most notably quercetin and anthocyanins like cyanidin-3-glucoside. These compounds possess potent anti-inflammatory, antioxidant, and antimicrobial properties. Anthocyanins, which belong to the flavonoid group, help reduce oxidative stress by neutralizing harmful free radicals, thereby protecting the body from various chronic diseases.[5] Morin is a natural flavonoid found in various plants, including members of the Moraceae family such as mulberries. Chemically, it is known as 2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one. As a flavonoid, it has a characteristic structure that includes a three-ring system with multiple hydroxyl groups. Naringin is a flavonoid glycoside commonly found in citrus fruits, particularly in grapefruits and bitter oranges. Chemically, it is classified as a flavanone glycoside, specifically the 7-O-neohesperidoside of naringenin. Quercetin’s chemical structure consists of a flavonol backbone, with hydroxyl groups (-OH) attached to positions 3, 5, 7, 3', and 4' of the flavonoid nucleus. Health Benefits of quercetin has been studied for various potential health benefits. Rutin, also known as quercetin-3-rutinoside, is a flavonol glycoside composed of quercetin (aglycone) and the disaccharide rutinose (rhamnose-glucose). Rutin is commonly found in foods such as buckwheat, citrus fruits (especially citrus peels), apples, berries, and some medicinal herbs [6]. The health benefits of blackberries are numerous, thanks to their high content of vitamins, minerals, fiber, and phytochemicals. Regular consumption of blackberries can help improve heart health by reducing cholesterol levels and improving blood pressure regulation due to their high antioxidant content. The fiber and polyphenols in blackberries promote digestive health and prevent constipation. The antioxidants, including vitamin C and anthocyanins, may also reduce the risk of certain cancers by preventing oxidative damage to cells [6]. Additionally, blackberries support brain health, as the antioxidants may help prevent neurodegenerative conditions, while vitamin K and manganese in blackberries promote healthy bones. Their low glycemic index makes them an excellent choice for blood sugar control, making them beneficial for people with diabetes. Overall, blackberries are a versatile and nutritionally dense fruit with multiple health-promoting benefits.[7]
The aim was to evaluate and determine the presence of flavonoids Morin, Naringenin, Quercetin, and Rutin is done using the HPLC method. The rationale behind choosing leaves over fruits is their constant availability making more sustainable and accessible resources for further research and practical application in medicinal biotechnology.
Methodology
Preparation and extraction of plant extract:
Collect the leaf samples. Dry the leaves using the shed dry method. Crush the leaves using molten and pistil until they're a fine powder. The powder will be stored in a dark drawer until used.
Soxhlation
Samples are prepared and weighed 20 grams. The apparatus is cleaned using ethanol. The sample and solvent system are loaded into the apparatus. The apparatus is kept in the heating mantel at 30?. The eight cycles of Soxhlation have been taken. The extract is stored at room temperature for further analysis [16].
HPLC (High-performance liquid chromatography)
High-performance liquid Chromatography (HPLC) is a technique used to separate, identify, and quantify compounds in a mixture. The mobile phase typically consists of HPLC water, organic solvents, and buffers at a specific pH. The sample is prepared by dissolving it in a suitable solvent and then filtered through a 0.22 or 0.45 µm filter to remove particulates. The HPLC system consists of a pump, injector, column, and detector. The mobile phase is pumped through the column at a constant flow rate, and the sample is injected into the system using an autosampler or manual injector. The column separates the compounds based on their interactions with the stationary phase. A UV-Vis detector or other suitable detectors detect the separated components [17].
The elution is monitored, and the retention times of the compounds are recorded. The data are analyzed to determine the concentration and purity of the analytes based on the peak areas or heights. After the analysis, the column is typically flushed with a high proportion of organic solvent to remove residual compounds and maintain column performance. The obtained result will be compared against the standard for further evaluation.
RESULTS
The HPLC analysis of the standard is performed using an RP column, with a mobile phase consisting of 0.1% of H3PO4 + Acetonitrile in a ratio of 25:75 at a flow rate of 1 mL/min. The detection is carried out at a wavelength of 350 nm, and the column temperature is maintained at 37 °C. The injection volume is 20 µL. The retention time for Quercetin, Morin, Naringin, and Rutin are 2.513 min, 3.887 min, 7.130 min, and 4.780 min, respectively, depicted in Fig.1- 4.
The HPLC analysis of the sample also followed the same protocol as the standard performed. The retention times of Morin, Naringin, Quercetin, and Rutin are observed at 3.640, 7.840, and 2.567 min, and 4.480min with a peak area of 1.7572, 189.5961, 48.1537, And 12.0042 mAU*min. observe in Fig.5.
Fig:1 Standard Chromatogram of Morin
Fig.2 Standard Chromatogram of Naringin
Fig.3 Standard Chromatogram of Quercetin
Fig.4 Chromatogram of Standard rutin
Fig.5 Chromatogram of blackberry
DISCUSSION
The evaluation and quantification of flavonoids from blackberry involved Soxhlet extraction followed by High-Performance Liquid Chromatography (HPLC) analysis. Soxhlet extraction efficiently isolated flavonoids, while HPLC provided precise identification and quantification of morin, rutin, quercetin, and naringin. The standard retention times for the four flavonoids were 2.513 min for Quercetin, 3.887 min for Morin, 7.130 min for Naringin, and 4.780 min for Rutin. In blackberry leaf extracts Quercetin, Morin, Naringin, and Rutin are identified at retention times of 2.567 min, 3.640 min, 5.673 min, and 4.480 min respectively. These results confirm the presence of key flavonoids with potential health benefits in blackberry leaves. Previous studies have also reported the presence of flavonoids in blackberry leaves and their potential antioxidant and antimicrobial properties. The current study provides a detailed analysis of the flavonoid composition in blackberry leaves, Results indicated that blackberry leaves contain significant amounts of morin, rutin, quercetin, and naringin. HPLC demonstrated high selectivity and sensitivity, effectively separating the flavonoid compounds. The detection wavelength used is 350 nm to capture optimal absorbance
REFERENCES
HOW TO CITE: Dr. Raja Kumar Parabathina*, Sunil Kothargasti, Vishal Lolge, Nidhi Dubey, Sanika Girgaonkar, Studies on the Evaluation of Flavonoids (Morin, Naringin, Quercetin & Rutin) in Blackberry Leaves By HPLC, Int. J. of Pharm. Sci., 2024, Vol 2, Issue 11, 904-909. https://doi.org/10.5281/zenodo.14202760
10.5281/zenodo.14202760
