Article

Introduction

Helianthus annuus, commonly known as the sunflower, is a widely recognized flowering plant valued for its distinctive large, bright yellow blooms and significant economic and ecological roles. Belonging to the Asteraceae family, sunflowers are native to North America and have been cultivated globally for centuries for their seeds, oil, and ornamental beauty. The plant thrives in diverse climatic conditions, particularly in well-drained soils under full sunlight, making it adaptable to various agricultural settings [1]. Known for its high oil content, sunflower oil is rich in unsaturated fatty acids, particularly linoleic and oleic acids, which have numerous applications in food, cosmetics, and biodiesel production. Beyond its agricultural and industrial uses, Helianthus annuus has garnered attention in traditional and modern medicine due to its rich phytochemical profile. Sunflower seeds and other plant parts contain bioactive compounds, including flavonoids, phenolic acids, tannins, alkaloids, and saponins, which exhibit antioxidant, anti-inflammatory, and antimicrobial properties. Historically, sunflower preparations have been used in folk medicine to treat ailments such as respiratory infections, gastrointestinal issues, and inflammatory conditions [2], the petals, leaves, and roots have been utilized in various traditional remedies to promote wound healing, alleviate muscle pain, and support immune health.

Recent studies have explored the potential health benefits of sunflower-derived compounds, particularly their role in mitigating oxidative stress, which is linked to chronic diseases such as cardiovascular conditions, cancer, and neurodegenerative disorders. In the current study, the phytochemical profile of Helianthus annuus flowers is investigated through a systematic extraction process using a range of solvents [3]. This analysis aims to identify and quantify key bioactive compounds, providing insights into the therapeutic potential of sunflower extracts and supporting the broader application of this plant in nutraceuticals and pharmaceuticals.

Sunflower (Helianthus annuus L.) is an annual flowering plant highly valued for its economic, nutritional, and therapeutic properties. As a member of the Asteraceae family, the sunflower is native to North America and has spread globally due to its versatility and adaptability. Known for its large, bright yellow blooms, sunflower cultivation primarily focuses on its seeds, which are a rich source of oil and protein [4]. Sunflower oil, extracted from the seeds, is widely used in cooking and is prized for its high content of unsaturated fatty acids, particularly linoleic and oleic acids, which support cardiovascular health. Additionally, sunflower seeds contain various bioactive compounds, including phenolic acids, flavonoids, tannins, and alkaloids, which contribute to the plant’s medicinal benefits. Beyond its nutritional and industrial applications, Helianthus annuus has garnered attention in ethnomedicine and modern herbal practices. The seeds and other parts of the plant have been traditionally used to alleviate respiratory disorders, support immune health, and reduce inflammation [5]. Sunflower extracts are recognized for their antioxidant properties, which help mitigate oxidative stress and may protect against chronic diseases such as heart disease, cancer, and neurodegenerative disorders. The adaptability of sunflower plants to a wide range of environmental conditions, from temperate to subtropical climates, makes them resilient crops for diverse agricultural systems. This study investigates the phytochemical profile of Helianthus annuus flowers through a systematic extraction and analysis process, aiming to reveal the therapeutic potential of sunflower compounds and their possible applications in pharmaceuticals, nutraceuticals, and cosmetics.

The demand for sunflower (Helianthus annuus L.) extracts and bioactive compounds has surged, particularly with the increasing recognition of their therapeutic properties. Sunflower extracts have shown potential benefits in addressing various health issues, such as cancer, cardiovascular disease, hypertension, and psychological conditions, gaining further relevance in light of the global health challenges posed by the COVID-19 pandemic. Sunflower seeds and flowers are rich sources of phytochemicals, including phenolic acids, flavonoids (notably glycosidic derivatives of quercetin and kaempferol), phytosterols, monounsaturated and polyunsaturated fatty acids, vitamins (such as tocopherols and thiamine), amino acids, and essential minerals [6]. These bioactive components contribute to sunflower’s antioxidant, anti-inflammatory, and cardioprotective effects. Among sunflower’s most studied compounds are chlorogenic acid and tocopherols, which exhibit robust antioxidant properties and play key roles in sunflower’s therapeutic profile. Despite the primary focus on seeds for oil production, other parts of the sunflower, such as petals and leaves, contain valuable phytochemicals and are often discarded as agricultural waste. Considering the substantial biomass produced alongside seed harvesting, which could reach several hundred thousand tons annually, there is a growing interest in utilizing sunflower waste to develop sustainable, health-promoting products. This emerging research highlights the potential of sunflower as a valuable source of bioactive compounds, supporting its use in pharmaceuticals, nutraceuticals, and functional foods.

Chemical Composition / Phytochemistry of Sunflower (Helianthus annuus)

The primary composition of sunflower includes approximately 4–8% water, 15–20% proteins, 20–30% lipids, 10–15% carbohydrates, and 8–10% fiber. Sunflower seeds are particularly valued for their high oil content, with oil making up around 20–50% of the seed, depending on the cultivar. The oil is rich in essential fatty acids, predominantly linoleic acid (48–74%) and oleic acid (14–40%), making it beneficial for cardiovascular health. Sunflower oil also contains minor quantities of saturated fatty acids, such as palmitic and stearic acids, in lower concentrations. Sunflower seeds are a good source of vitamins, particularly vitamin E (tocopherols), which acts as a powerful antioxidant, helping to protect cells from oxidative damage. They also contain B vitamins, including thiamine (vitamin B1), niacin, and folic acid. In addition, sunflower seeds have a variety of minerals, such as magnesium, potassium, phosphorus, selenium, and zinc, which are important for maintaining healthy bodily functions.

Phytochemical analysis of sunflower has revealed the presence of phenolic compounds, flavonoids, saponins, alkaloids, and sterols, which contribute to its medicinal properties. The major phenolic acids identified include chlorogenic, caffeic, and ferulic acids, which exhibit strong antioxidant activities [7]. These compounds are primarily found in the hull and the seed coat, making sunflower an excellent source of dietary antioxidants. Carotenoids, mainly β-carotene and lutein, are present in sunflower petals and contribute to its vibrant yellow color. These carotenoids not only play a role in photosynthesis but also have potential health benefits for human consumption, such as supporting eye health. Additionally, phytosterols, such as campesterol, stigmasterol, and beta-sitosterol, are abundant in sunflower oil, contributing to its cholesterol-lowering properties. Sunflower seeds are increasingly recognized for their bioactive components, including the antioxidant activity of tocopherols and polyphenols, making them valuable in both culinary and nutraceutical applications. The high content of linoleic and oleic acids also makes sunflower oil a popular choice in food and cosmetic products for its moisturizing and anti-inflammatory properties.

Collection and Authentication of Plant Material
The flowers of Helianthus annuus (sunflower) were collected from local farms in the Bhubaneswar region of Odisha, India, during the month of September in the year 2023. The plant material was authenticated by Head, College of Agriculture, Dhule. Mahatma Phule Krishi Vidhyapeeth, Rahuri. Maharashtra. India – 424004 (voucher specimen number: SF2023/01/Students).

Drying
Following collection, the sunflower flowers were thoroughly cleaned, and the petals were separated from the disc florets. The petals were then spread out on clean newspapers in a shaded area to dry for ten days. Subsequently, the dried petals were transferred to a hot air oven set at 40 °C and maintained for one hour to ensure the complete removal of moisture content prior to the extraction process.

Successive Extraction Using Soxhlet Apparatus
To prepare the extracts of Helianthus annuus (sunflower) petals, fresh petals were collected and thoroughly washed with flowing water to eliminate any dirt or debris. The petals were then subjected to extraction using various solvents, including hexane, chloroform, ethyl acetate, and methanol. After washing, the petals were rinsed again under flowing distilled water to remove any residual contaminants. Dried petal powder was obtained by grinding the dried petals using a mechanical grinder and sieving to achieve a uniform particle size.

The petal powder was then extracted successively in a Soxhlet apparatus with hexane at 60 °C, ethyl acetate at 77 °C, chloroform at 61 °C, and methanol at 65 °C. The extraction temperatures were set to the boiling points of the solvents to facilitate a faster cycling rate of fresh solvent. A duration of six hours was allocated to each solvent for hot continuous and successive extraction. All extracts obtained were concentrated and dried in an oven at 45 °C. The dried extracts were subsequently used for phytochemical screening.

Screening of Phytochemicals
The petal extract of Helianthus annuus (sunflower) was subjected to preliminary phytochemical screening to assess the presence of secondary metabolites. Phytochemical tests were conducted following standard procedures, and tests were performed for alkaloids, flavonoids, saponins, steroids and terpenoids, phenolic compounds, tannins, cardiac glycosides, glycosides, coumarins, anthraquinones, quinones, and resins. Various qualitative tests were employed to determine the presence or absence of bioactive compounds.

Detection of Alkaloids
For the detection of alkaloids, Mayer’s test was performed. Samples of the petal extract were dissolved in a diluted hydrochloric acid solution and subsequently filtered. Two to three drops of Mayer’s reagent were added to 2 ml of the filtrate. A positive reaction was indicated by the formation of a creamy white precipitate.

Test for Flavonoids
To test for flavonoids, a small amount of concentrated hydrochloric acid and magnesium turnings were added to the test solution, which was then heated gently for five minutes. The appearance of a reddish tint in the mixture indicated the presence of flavonoids.

Test for Saponins
A small amount of the powdered sunflower petals was boiled with 20 mL of water at a low temperature for two minutes. The mixture was then filtered through a fine sieve to remove contaminants. The resulting filtrate was diluted with water to a final volume of 5 mL and vigorously stirred. The presence of saponins was indicated by the formation of a stable foam.

Detection of Steroids and Terpenoids
For the Liebermann–Burchardt test, a mixture of 1 mL of the petal extract, 1 mL of chloroform, 2 mL of acetic anhydride, and 1-2 drops of concentrated sulfuric acid was prepared. The formation of a dark green color in the solution indicated the presence of steroids.

Test for Phenolic Compounds
The presence of phenolic compounds was assessed using two reagents. A small amount of the powdered sample was treated with a 5% ferric chloride solution, which produced a deep violet-black coloration, confirming the presence of phenolic compounds. Additionally, the presence of phenolics was indicated by the formation of a precipitate when treated with lead acetate solution.

Test for Tannins
A small quantity of the powdered petal sample was dissolved in water, and the aqueous extract was treated with a few drops of ferric chloride solution. The development of a bluish-black color indicated the presence of tannins.

Detection of Glycosides
To test for glycosides, the extract was heated in either an alcohol or hydroalcoholic solution. For Baljet’s test, the mixture was treated with a 2% sodium picrate solution. The appearance of a golden orange color in the mixture indicated the presence of glycosides.

Detection of Glycosides
b) Legal’s Test
To identify glycosides, the test solution was alkalized with pyridine, and the addition of 2% sodium nitroprusside resulted in a color change from pink to red, indicating the presence of glycosides.

c) Keller-Killiani Test
In this test, 100 mg of the sunflower petal extract was mixed with 1 mL of ferric chloride solution and 1 drop of glacial acetic acid. Following this, 1 mL of concentrated sulfuric acid was carefully added to the mixture. The appearance of a brown band at the interface indicated the presence of glycosides in the sample.

Detection of Coumarins
For the detection of coumarins, 2 mL of the aqueous extract was diluted and then treated with 3 mL of a 10% sodium hydroxide solution. The development of a golden color suggested the presence of coumarins.

Test for Phytosterols
To test for phytosterols, the extract solution was mixed vigorously before the addition of strong sulfuric acid. The mixture was allowed to settle, and if phytosterols were present, the chloroform layer at the bottom of the solution would turn crimson.

Detection of Quinones
The presence of quinones was assessed by treating 1 mL of crude extract with diluted sodium hydroxide. The appearance of a red or blue-green color indicated the presence of quinones.

Detection of Resins
To test for resins, 2 mL of extract was mixed with 5 to 10 drops of acetic anhydride in a saucepan over medium heat. Following this, 0.5 mL of sulfuric acid was added. A deep purple color formation indicated the likely presence of resins.

Detection of Cardiac Glycosides
For the Keller-Kiliani test specific to cardiac glycosides, 2 mL of the sunflower extract was mixed with 1 mL each of ferrous chloride, concentrated sulfuric acid, and glacial acetic acid. The solution turned emerald green upon illumination, suggesting the presence of cardiac glycosides.

Detection of Leucoanthocyanins
To detect leucoanthocyanins, an equal volume of the sunflower extract and isoamyl alcohol was mixed. The formation of a red color in the upper layer indicated the presence of leucoanthocyanins.

Detection of Anthraquinones
For the detection of anthraquinones, a small amount of finely powdered sunflower petals was added to a mixture stirred with chloroform for five minutes. After filtering the contents, 5 mL of ammonia solution was quickly added to the mixture. The appearance of a vivid pink color in the aqueous layer suggested the presence of anthraquinones.

Detection of Fixed Oils
To assess the presence of fixed oils, a small sample of the powdered sunflower petals was compressed between two filter sheets. The presence of fixed oils was indicated by any observable oil residue left on the filter paper.

Quantification of Total Alkaloid Content
To quantify the total alkaloid content, 1 mg of the sunflower extract was dissolved in dimethyl sulfoxide and treated with 1 mL of 2N hydrochloric acid, followed by filtration. The resulting solution was transferred to a separating funnel, and 5 mL of bromocresol green solution and 5 mL of phosphate buffer were added. The mixture was vigorously shaken with varying volumes (1, 2, 3, and 4 mL) of chloroform, and the combined organic phases were collected in a 10 mL volumetric flask and diluted to the mark with chloroform. A series of reference standard solutions of atropine (20, 40, 60, 80, and 100 μg/mL) were prepared in the same manner. The absorbance of both the standard and test solutions was measured against a reagent blank at 470 nm using a UV/Visible spectrophotometer. The alkaloid content was expressed as mg of alkaloids equivalent per gram of the plant extract.

Quantification of Total Flavonoid Content
The total flavonoid content was determined using a colorimetric assay involving aluminum chloride. In a 10 mL flask, 1 mL of the sunflower extract was mixed with 4 mL of distilled water. After adding 0.30 mL of 5% sodium nitrite and allowing it to react for 5 minutes, 0.3 mL of 10% aluminum chloride was added. After another 5 minutes, 2 mL of 1M sodium hydroxide was introduced, and the solution was diluted to 10 mL with distilled water. A series of standard solutions of quercetin (20, 40, 60, 80, and 100 μg/mL) were prepared similarly. The absorbance for both test and standard solutions was measured using a reagent blank at 510 nm with a UV/Visible spectrophotometer. The total flavonoid content was expressed as mg of quercetin equivalent per gram of extract.

Quantification of Total Tannin Content
The total tannin content was quantified using the Folin-Ciocalteu method. In a 10 mL volumetric flask, 0.1 mL of sunflower extract was combined with 7.5 mL of distilled water, 0.5 mL of Folin-Ciocalteu phenol reagent, and 1 mL of 35% sodium carbonate (Na2CO3) solution, then diluted to 10 mL with distilled water. The reagent mixture was thoroughly shaken and allowed to incubate at 30°C for 30 minutes. A series of gallic acid standard solutions (20, 40, 60, 80, and 100 μg/mL) were prepared in the same manner. The absorbance of both standard and test solutions was measured against a blank at 725 nm using a UV/Visible spectrophotometer. The total tannin content was expressed as mg of gallic acid equivalent (GAE) per gram of extract.

Quantification of Total Phenolic Compound Content
The concentration of phenolic compounds in the sunflower extract was quantified using the Folin-Ciocalteu method. The reaction mixture contained 1 mL of sunflower extract and 9 mL of distilled water. Following this, 1 mL of Folin-Ciocalteu phenol reagent was added and the mixture was shaken well. After allowing the mixture to react for 5 minutes, 10 mL of 7% sodium carbonate (Na₂CO₃) solution was added to the mixture, bringing the total volume to 25 mL. A series of gallic acid standard solutions (20, 40, 60, 80, and 100 μg/mL) were prepared similarly. The mixture was incubated for 90 minutes at 30°C, after which the absorbance of both test and standard solutions was measured against a reagent blank at 550 nm using a UV/Visible spectrophotometer. The total phenolic content was denoted as mg of gallic acid equivalent (GAE) per gram of extract.

Morphological Description
Helianthus annuus L. (Sunflower) is a tall, annual flowering plant known for its large, vibrant flower heads, which are among the most recognizable symbols of summer. Native to the Americas, sunflowers are cultivated worldwide for their seeds and oil. The plant typically grows between 1.5 to 3.5 meters in height, with a sturdy, hairy stem that can support its large flower heads.

The leaves are broad, heart-shaped, and have a rough texture, measuring up to 30 cm in length and arranged in a spiral pattern around the stem. The sunflower’s distinctive flower heads are made up of numerous tiny disc florets at the center, surrounded by bright yellow ray florets that attract pollinators. These flower heads can measure up to 30 cm in diameter and follow the sun’s movement across the sky, a behavior known as heliotropism. The sunflower’s fruit is a large, edible seed that develops after the flowers have been pollinated. Each seed is flat, oval, and can vary in color from black to striped or solid gray. The seeds are encased in a hard outer shell, which provides protection. The roots of the sunflower are fibrous and spread widely, allowing the plant to access moisture and nutrients from the soil. Sunflowers thrive in well-drained soil and require full sunlight for optimal growth. They are known for their drought resistance and can adapt to various soil types. Sunflowers are typically grown as annuals, with their life cycle completing within one growing season, and they produce a high yield of seeds, making them an essential crop for both food and oil production.

Common Names of Helianthus annuus L. (Sunflower)

• Common Name: Sunflower
• Hindi: सूरजमुखी (Surajmukhi)
• Kannada: ಸೂರ್ಯಕಾಂತಿ (Suryakanti)
• Malayalam: സൂര്യകാന്തി (Suryakanthi)
• Marathi: सूर्यमुखी (Suryamukhi)
• Sanskrit: सूर्यमुखी (Suryamukhi)
• Urdu: سورج مکھی (Sooraj Mukhi)
• Telugu: సూర్యఫలము (Suryaphalamu)

Qualitative Phytochemical Analysis of Helianthus annuus L. (Sunflower)

Phytochemicals, primarily responsible for the formation of secondary metabolites in plants, play a crucial role in plant defense while also exhibiting therapeutic effects that can improve human health. As a result, these compounds are the focus of extensive research. This study aimed to analyze the phytochemistry of the sunflower extracts to identify the secondary metabolites present. The extracts were prepared using hexane, chloroform, ethyl acetate, and methanol to assess the presence of various compounds, including alkaloids, flavonoids, saponins, steroids, terpenoids, tannins, glycosides, coumarins, phytosterols, quinones, anthraquinones, cardiac glycosides, leucoanthocyanins, fixed oils, and resins.

The qualitative analysis revealed that the stigma of Helianthus annuus can be classified into multiple phytochemical categories. According to the results provided (Table-1 and Fig-1), a comparison of the solvents employed in the extraction process was made.

The phytochemical investigation showed that the methanol extract was the only extract containing alkaloids, glycosides, and saponins. Flavonoids and coumarins were detected in all extracts except for the petroleum ether extract. The chloroform and methanol extracts were found to contain steroids and terpenoids. Both chloroform and ethyl acetate extracts contained phenols and tannins, while phytosterols were present in all extracts except for the chloroform extract. Cardiac glycosides were identified in all extracts except for the ethyl acetate extract. The chloroform extract uniquely contained quinones and anthraquinones, whereas the ethyl acetate extract revealed the presence of resins. Additionally, both the petroleum ether and chloroform extracts showed a favorable reaction for fixed oils. Notably, none of the extracts contained leucoanthocyanins (Table-2).

To produce crude extracts, the following solvents were utilized, resulting in different percentages of yield: hexane (6.2% yield), chloroform (24.16% yield), ethyl acetate (38.2% yield), and methanol (52.5%).

To determine the distribution and concentration of distinct bioactive components, the phytochemical content of sunflower (Helianthus annuus) extracts was assessed using a variety of solvents. The analysis revealed no detectable alkaloids in any of the sunflower extracts, indicating that this plant primarily stores beneficial compounds in other phytochemical classes. In terms of flavonoids, the ethyl acetate extract exhibited the highest content, measured at 650 mg/g Quercetin equivalent, followed by the methanol extract at 570 mg/g and the chloroform extract at 440 mg/g, while no flavonoids were found in the petroleum ether extract. The total phenolic content was similarly evaluated, with the ethyl acetate extract yielding the highest concentration of phenols at 580 mg/g, comparable to gallic acid. The chloroform extract contained 440 mg/g of phenolic compounds, whereas the petroleum ether and methanol extracts showed no detectable phenolic content. Tannin concentrations followed a similar pattern, with the ethyl acetate extract recording the highest at 486 mg/g, and the chloroform extract providing a lower concentration of 360 mg/g. No tannins were found in the petroleum ether and methanol extracts, highlighting the selective nature of these solvents in extracting phytochemicals. Overall, the significant variations observed in phytochemical extraction emphasize the importance of choosing the right solvent and underscore Helianthus annuus as a valuable source of bioactive compounds for various applications, including nutrition and health.

Table 3. Helianthus annuus (sunflower) stigma extract and quantifying the phytochemical content in different solvents

Discussion

Plant-derived phytochemicals are considered potential therapeutic agents with minimal side effects compared to chemically manufactured medications. Several studies have explored the therapeutic applications of sunflower (Helianthus annuus). Beyond its popularity as an oilseed and ornamental plant, sunflower has been found to be significant in treating various diseases, including cardiovascular conditions, digestive ailments, and inflammatory disorders. The presence of phytochemicals such as flavonoids, phenolic acids, and carotenoids in sunflower contributes to its therapeutic benefits. Sunflower extracts have demonstrated antioxidant properties that can help mitigate oxidative stress, making them valuable in promoting heart health. Additionally, sunflower oil is known for its high content of unsaturated fats, which can aid in lowering cholesterol levels and supporting cardiovascular health. Some compounds derived from sunflower have shown promise in alleviating symptoms associated with respiratory conditions due to their anti-inflammatory properties. For example, certain flavonoids may help reduce inflammation in the airways. Furthermore, the antioxidant effects of sunflower extracts can support overall health by combating free radicals and enhancing immune function. Present qualitative phytochemical studies reveal the presence of various compounds, including flavonoids, saponins, terpenoids, and phenolic compounds. In crude extract preparation, hexane (6.2% yield), chloroform (24.16% yield), ethyl acetate (38.2% yield), and methanol (52.5% yield) were utilized. The study assessed the phytochemical content of sunflower extracts across different solvents, revealing the highest concentrations of flavonoids in the ethyl acetate extract, followed by methanol and chloroform. Additionally, the largest concentrations of phenols and tannins were found in the ethyl acetate extract, followed by the chloroform extract.

Conclusion

Phytochemical screening is crucial for discovering diverse phytoconstituents in plant extracts. Helianthus annuus L. (sunflower) is an important medicinal plant extensively cultivated for its nutritional and economic value. Sunflower and its components possess significant pharmacological properties, including anti-inflammatory and antioxidant effects. Further clinical trials will undoubtedly yield fresh insights into the unexplored qualities and biological constituents of sunflower, aimed at treating or preventing various diseases and disorders. This research establishes a significant foundation for subsequent studies on the separation and characterization of phytoconstituents from sunflower for potential therapeutic applications. While this work primarily focused on qualitative analysis and screening, a quantitative analysis of the bioactivity of its compounds, along with infrared (IR) spectral analysis of the various phytochemicals, would be advantageous. The investigation would be further enhanced by developing methods for the detection, analysis, and separation of phytoconstituents in sunflower extracts.

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