Psidium guajava Linn. (P. guajava), commonly known as guava and referred to as Jewafa in Arabic, is a medicinal plant belonging to the Myrtaceae family, which includes about 75 genera and nearly 3,000 species, primarily evergreen trees and shrubs found in tropical and subtropical regions. The P. guajava tree can grow up to 12 meters tall, with smooth brown bark and green, elliptical to oval leaves with an acute tip. Its tropical tree is cultivated globally and recognized for its nutritional and medicinal values [1, 2].

Guava trees produce nutrient-rich fruits high in vitamins, fiber, and essential nutrients. Originally native to Latin America, P. guajava is now grown across various tropical and subtropical regions and has long been used in traditional medicine to treat numerous ailments. The tree, often reaching up to 35 feet, also has leaves and bark with a rich history of medicinal applications that continue to be valued in modern herbal medicine [3, 4].

Psidium guajava Linn. (P. guajava), is known for its rich history of medicinal use, particularly in traditional practices for treating diverse health conditions. The roots, bark, leaves, and immature fruits of P. guajava have been traditionally utilized to address ailments such as inflammation, diabetes, hypertension, dental caries, wounds, pain, and fever [5]. Renowned for its antispasmodic and antimicrobial properties, P. guajava is also valued as a hypoglycemic agent, particularly for managing diarrheal diseases and dysentery, with studies supporting its efficacy in treating diabetes and gastrointestinal conditions [6, 7]. The pharmacological potential of guava has been substantiated through numerous studies, revealing activities such as antioxidant, hepatoprotective, anti-allergic, antimicrobial, anti-plasmodial, cytotoxic, cardioprotective, anti-cough, anti-inflammatory, and analgesic effects. These findings align with its traditional applications and support its potential in clinical settings for conditions ranging from rotaviral enteritis to diabetes [7]. Additionally, ethnobotanical and clinical research highlights guava’s broad efficacy against gastrointestinal, respiratory, and oral infections, as well as skin issues, cancer, cardiovascular diseases, and malnutrition. This versatile plant is even explored for animal health and various commercial uses [8].

Recent studies have extended the clinical relevance of P. guajava to viral infections, including COVID-19. Research on guava leaf extract has shown positive effects on inflammatory markers in asymptomatic and mild COVID-19 cases, where supplementation contributed to improved recovery times and reduced inflammation without adverse effects. These results suggest P. guajava extract may support inflammation management and expedite recovery in mild COVID-19, though further studies are needed to confirm its potential in more severe cases [9]. Psidium guajava Linn, is a nutritionally and medicinally valuable plant rich in diverse phytochemicals, including phenolics, flavonoids, triterpenoids, tannins, vitamins, essential oils, and sesquiterpene alcohols. Guava leaves contain notable compounds such as rutin, naringenin, gallic acid, catechin, epicatechin, kaempferol, quercetin, and guaijaverin, which contribute to its well-documented antimicrobial, antioxidant, and anti-inflammatory properties. The guava fruit, high in vitamin C (80 mg per 100 g) and vitamin A, also serves as a good source of pectin and dietary fibre. Due to these extensive benefits, guava is cultivated worldwide, with India leading production at approximately 4,054,000 metric tons from 265,000 hectares annually [6]. Guava’s pharmacological value extends across a range of bioactive metabolites, particularly phenolics, flavonoids, carotenoids, terpenoids, and triterpenes, which exhibit antibacterial, antidiarrheal, antioxidant, and anti-inflammatory effects. Flavonoids like quercetin are noted for their antidiarrheal action, relaxing intestinal smooth muscles and inhibiting bowel contractions, while triterpenes and polyphenols in guava leaves enhance its antispasmodic and antioxidant properties [7]. Research has also highlighted P. guajava’s anticancer potential, with various in vitro and in vivo studies documenting its chemo preventive and chemotherapeutic effects against cancers such as breast, gastrointestinal, genitourinary, and skin cancers. Compounds isolated from guava, including 3,5-dihydroxy-2,4-dimethyl1-O-(60-O-galloyl-b-D-glucopyranosyl)-benzophenone, guajadial, guajavadial A-C, and psiguajadial A-B, along with guava extracts, demonstrate antiproliferative, pro-apoptotic, anti-inflammatory, antioxidant, and antiangiogenic actions, all of which contribute to its antineoplastic effects [3]. With a rich array of additional phytochemicals such as polysaccharides, proteins, sesquiterpenoid alcohols, triterpenoid acids, alkaloids, glycosides, steroids, and saponins, guava also supports traditional medicinal uses for treating conditions like diarrhoea, infections, and inflammation. This extensive phytochemical composition, along with its flavonoid and triterpene content, underpins guava’s broad therapeutic potential and pharmacological relevance [4]. Morin is a natural flavonoid found in various plants, particularly in the Moraceae family. Its chemical structure is 2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one, featuring a three-ring system with multiple hydroxyl groups. Naringin, a flavonoid glycoside, is mainly present in citrus fruits, particularly grapefruits and bitter oranges. It is chemically defined as the 7-O-neohesperidoside of naringenin. Quercetin, a flavanol, has hydroxyl groups attached at positions 3, 5, 7, 3', and 4', and is well-known for its potential health benefits. Rutin, or quercetin-3-rutinoside, is a flavanol glycoside composed of quercetin and the disaccharide rutinose, and can be found in foods like buckwheat, citrus fruits, apples, and berries [10-11]. The purpose of the study was to assess and identify the presence of these flavonoids—Morin, Naringenin, Quercetin, and Rutin in Psidium guajava Linn leaves using the HPLC method. Leaves were chosen over fruits due to their consistent availability, making them a more sustainable and accessible resource for ongoing research and practical applications in medicinal biotechnology.

Methodology

Preparation And Extraction Of Plant Extract:

Collected the leaf samples and dried them using the shed drying method. Crushed the dried leaves with a mortar and pestle until they formed a fine powder. Stored the powdered leaves in a dark, cool place until ready for use.

Soxhlation

The samples were prepared and weighed to 20 grams. The apparatus was cleaned using ethanol. The sample and solvent system were loaded into the apparatus, which was then placed in a heating mantle at 30°C. Eight cycles of Soxhlet extraction were performed. The extract was stored at room temperature for further analysis [12].

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 generally consists of HPLC-grade water, organic solvents, and buffers at a specific pH. The sample is prepared by dissolving it in an appropriate solvent and filtering it through a 0.22 or 0.45 µm filter to eliminate particulates.

The HPLC system includes 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 either an autosampler or manual injector. The column separates the compounds based on their interactions with the stationary phase. A UV-Vis detector or other appropriate detectors identify the separated components [13]. 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 ensure column performance. The obtained results are compared with the standard for further evaluation.

RESULTS

The HPLC analysis of the standard was carried out using an RP column, with the mobile phase consisting of 0.1% H?PO? and acetonitrile in a 25:75 ratio at a flow rate of 1 mL/min. Detection was performed at a wavelength of 350 nm, and the column temperature was maintained at 37°C. The injection volume was 20 µL. The retention times for Quercetin, Morin, Naringin, and Rutin were 2.513 min, 3.887 min, 7.130 min, and 4.780 min, respectively, as shown in Fig. 1-4.

For the sample analysis, the same protocol as the standard was followed. The retention times for Naringin, and Quercetin were observed at 7.13 min, and 2.527 min, respectively, with peak areas of 1.7572, 189.5961, 48.1537, and 12.0042 mAU*min, as shown in Fig. 5.

       
           
       
   Fig.4 Chromatogram of Standard rutin

           
       
    Fig.5 Chromatogram of Psidium guajava Linn