Pharmacognostical And Phytochemical Studies of Aparajita (Clitoria Ternatea Linn.)

Clitoria ternatea Linn. Commonly known as Aparajita in traditional Indian medicine, holds a revered place in the history of ancient healthcare systems, particularly Ayurveda. This plant has been celebrated for centuries not only for its vibrant beauty but also for its remarkable therapeutic applications.¹ In classical Ayurvedic texts, Aparajita is classified under Medhya Rasayana, a group of herbs known to enhance memory, intellect, and longevity. Historically, it has been used by traditional healers to treat a wide range of ailments including epilepsy, anxiety, bronchitis, fever, and urinary tract infections. The roots were often used as a purgative and brain tonic,² while the flowers were employed to calm the nervous system and cleanse the blood. In various regions of India and Southeast Asia, the plant has also been used in religious rituals, where its deep blue flowers symbolize purity and protection. This longstanding traditional use reflects a deep cultural belief in the plant’s healing powers, passed down through generations.³ As modern science increasingly turns toward nature for solutions to complex health challenges, the importance of validating traditional medicinal plants has gained prominence. With the rise in demand for safe, effective, and standardized herbal remedies, a comprehensive understanding of a plant’s pharmacognostical and phytochemical characteristics is essential.⁴ Pharmacognostical studies, which include macroscopic and microscopic evaluations of plant materials, help in the accurate identification and authentication of medicinal plants. These studies are vital in preventing adulteration and ensuring quality control in herbal preparations. Alongside this, phytochemical investigations are crucial for detecting the presence of bioactive compounds—such as alkaloids, flavonoids, saponins, glycosides, and tannins—that are responsible for the plant’s medicinal effects. Together, these scientific approaches provide a foundation for transforming traditional knowledge into evidence-based herbal therapies.⁵ Clitoria ternatea Linn. is a fast-growing perennial climber belonging to the family Fabaceae. It is native to tropical and subtropical regions, particularly in India, Thailand, Indonesia, and Africa, where it grows wild or is cultivated as an ornamental or medicinal plant. The plant is easily recognized by its strikingly vivid blue or white papilionaceous flowers, compound leaves, and slender twining stems. All parts of the plant—roots, stems, leaves, seeds, and flowers—are used in traditional medicine and are known to contain a wide spectrum of phytoconstituents. Scientific studies have identified several important chemical compounds in Clitoria ternatea, including anthocyanins (especially ternatins, responsible for the blue color), flavonoids like kaempferol and quercetin, triterpenoids, saponins, and phenolic compounds. These constituents contribute to its wide range of reported pharmacological activities, including antioxidant, anti-inflammatory, antistress, nootropic, antimicrobial, and antidiabetic effects.^6-8

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 Figure 1: Clitoria ternatea Plant

Clitoria ternatea is extensively used in traditional medicine. Its roots and seeds, known as "Aparajita" in Ayurveda, act as nerve tonics, laxatives, and alteratives. The plant treats body aches, infections, urogenital disorders, intestinal worms, and animal stings. Roots are purgative, laxative, and diuretic, addressing issues like indigestion, arthritis, skin diseases, and abdominal enlargement. Root juice of the white-flowered variety is used for migraines, while root decoctions aid in rheumatism and ear diseases. Powdered seeds mixed with ginger act as laxatives and are used for colic and swollen joints. The plant is also employed for snakebite and scorpion stings.^9-10 Given the traditional importance and growing scientific interest in Clitoria ternatea, the present study was undertaken to carry out a detailed pharmacognostical and phytochemical investigation of this plant. The study aims to document the macroscopic and microscopic features of the plant to support its identification and standardization, and to screen for the presence of key phytochemicals that support its therapeutic claims. By combining traditional knowledge with modern scientific methods, this research seeks to contribute to the growing body of evidence that supports the safe and effective use of Clitoria ternatea in herbal medicine, ultimately promoting its potential for integration into contemporary healthcare systems.

MATERIAL AND METHODS:

Macroscopic Characteristics

The leaves, flowers, roots, and seeds of Clitoria  ternatea were analyzed for their macroscopic and organoleptic features. These included size, shape, color, surface texture, fracture characteristics, odor, and taste, aiding in plant identity and purity assessment. Visual inspection helped screen out adulterated or low-quality samples.^11-13

Size, Color & Texture Analysis

Samples were measured using rulers and graph paper, while colors were observed in diffuse daylight. Surface and fracture characteristics were studied using magnifying lenses and tactile methods. Odor was evaluated by inhalation or crushing, and taste tests were conducted only when necessary.^14-20

Microscopic Characteristics

Microscopic analysis followed macroscopic evaluation to confirm plant identity. Standard optical microscopes and botanical tools were used to study cellular details. Preparation involved staining, mounting, and observation of plant tissues.^21-25

Section Cutting Techniques

a. Leaf: Midrib sections were cut using pith block method and examined for vascular and epidermal structures.

 b. Flower: Sections revealed number of floral whorls, ovary chambers, ovules, placentation, and stigma features.

c. Root: Softened root pieces were sectioned, stained, and analyzed for primary and secondary tissues.

d. Seed: The seed coat was examined after alkali treatment, and sections of boiled seeds were prepared using pith or wax embedding.

Standardization of Clitoria ternatea

Plant Collection and Authentication

Clitoria ternatea whole plant was collected from Hi-tech college of Pharmacy campus and authenticated. Leaves, flowers, roots, and seeds were air-dried and powdered (sieve no. 40) for analysis.^26-29

Extractive Values

Different solvent extractive values (e.g., methanol, ethanol, chloroform, etc.) were determined by macerating 5g of each plant part in 100 ml solvent, filtering, evaporating, drying at 105°C, and calculating the % extractive value.

Loss on Drying (LOD)

LOD was calculated by heating 2–3 g of plant powder at 110°C until constant weight. It indicates moisture and volatile content.

Ash Values

Total, acid-insoluble, and water-soluble ash values were estimated by incinerating samples and treating with HCl or water. These assess inorganic residues and contamination.

Foreign Matter

50 g of plant sample was examined visually and with a sieve to separate and quantify any non-plant material, dust, or mineral matter.

Extraction of Leaves, Flowers, Roots, and Seeds of Clitoria ternatea Linn. with Different Solvents

The extractive values of Clitoria ternatea (Aparajita) for its various parts—leaves, flowers, roots, and seeds—were determined by extracting each with different solvents to assess the amount of soluble constituents. For the leaves, the highest extractive value was obtained using acetic acid (12.17%), followed by n-butanol (11.79%) and petroleum ether (3.19%). Other solvents yielded lower values, with methanol at 3.05%, aqueous extract at 2.49%, and hexane at 0.40%. The least effective solvent was butyl acetate, with an extractive value of 0.11%.^29-32 For the flowers, methanol provided the highest extractive value (20.07%), closely followed by acetic acid (19.25%). Acetone extracted 2.13%, while the aqueous extract resulted in 5.01%. Other solvents, including petroleum ether (1.90%), butyl acetate (1.47%), and ethyl acetate (1.02%), showed relatively lower extractive values. The remaining solvents, such as chloroform (0.85%), n-butanol (0.80%), ethanol (0.80%), and hexane (0.45%), exhibited minimal extraction efficiency. In the case of the roots, n-butanol again yielded the highest extractive value at 7.12%, followed by methanol (6.85%) and acetic acid (6.65%). Aqueous extraction resulted in 4.91%, and butyl acetate extracted 1.78%. Other solvents, including chloroform (1.52%), petroleum ether (1.11%), acetone (0.65%), ethanol (0.40%), ethyl acetate (0.35%), and hexane (0.34%), provided lower extractive values. For the seeds, acetic acid provided the highest extractive value at 7.95%, with petroleum ether extracting 7.13%. The aqueous extract yielded 5.56%, and ethanol resulted in 1.82%. Methanol (1.65%), acetone (1.22%), and chloroform (1.10%) also showed moderate extraction efficiency. Other solvents, including hexane (1.00%), ethyl acetate (0.95%), butyl acetate (0.68%), and n-butanol (0.59%), were less effective in extracting the soluble components.^33-35

Extraction of Plant Parts Using Different Solvents

The leaves, flowers, roots, and seeds of Clitoria ternatea Linn. (Aparajita) were shade-dried, powdered (40# mesh), and subjected to successive hot continuous extraction using a Soxhlet apparatus. Eleven solvents were selected based on polarity—petroleum ether, hexane, n-butanol, butyl acetate, chloroform, ethyl acetate, acetone, methanol, ethanol, acetic acid, and water. After each extraction, the residue was dried and the next solvent was used. The extracts were concentrated on a water bath, and the yield, color, and consistency were recorded. This method ensures maximum recovery of active phytochemicals for pharmacological evaluation.^36-37

Phytochemical Screening of Extracts

Qualitative phytochemical analysis was performed on the extracts to identify major constituents. Tests confirmed the presence of carbohydrates (Molisch’s, Fehling’s, Benedict’s), proteins (Biuret, Millon’s, Xanthoprotein), amino acids (Ninhydrin, Millon’s), steroids (Salkowski, Liebermann-Burchard), glycosides (Baljet’s, Legal’s, Keller-Killani), flavonoids (Shinoda, Lead acetate, Ferric chloride), alkaloids (Dragendorff’s, Mayer’s, Hager’s, Wagner’s), and tannins/phenolic compounds (FeCl?, lead acetate, gelatin). These results suggest a rich phytochemical profile, supporting the plant’s traditional medicinal use.^38-40

RESULT AND DISCUSSIONS:

Preliminary Pharmacognostic Characteristics of Clitoria ternatea Linn.

Leaves

The leaves are pinnate with 5–7 ovate leaflets (3–5 cm × 2–3 cm), glabrous on the upper side with short appressed hairs and pubescent underneath. They are arranged alternately with an imparipinnate type. Morphologically, the leaves possess an obtuse apex and entire margins.

Table 1: Organoleptic Characteristics of C. ternatea Leaves

                   
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Figure 2: Plants of Clitoria ternatea Linn. (Aparajita)

Flowers

The fresh flowers are blue with a characteristic odor and soft texture. Upon drying, they turn whitish-blue and become fibrous, while the powdered form is light blue to yellowish brown, with no odor and a coarse texture.

Table 2: Organoleptic Characteristics of C. ternatea Flowers

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Figure 3: Fresh (A), and dried flowers (B) of Clitoria ternatea Linn. (Aparajita) Roots

The roots are primary, fibrous in type, with a stout taproot and tortuous lateral branches. They are cylindrical (1–5 mm thick), smooth to fibrous in texture, with occasional cracks due to lenticels.

Table 3: Organoleptic Characteristics of C. ternatea Roots

Seeds

The pods are linear-oblong, flattened (4–13 cm × 0.8–1.2 cm), with thickened margins and persistent styles. Mature pods are pale brown, dehiscent, and sparsely pubescent. Each pod contains 8–11 oblong, somewhat flattened seeds (4.5–7 mm × 3–4 mm), olive brown to chocolate in color, shiny, mottled, and minutely pitted. Seed morphology may vary with environmental conditions.

Table 4: Organoleptic Characteristics of C. ternatea Seeds

Leaves

The leaf features a single-layered epidermis with a thick cuticle and unicellular to tricellular hooked trichomes on both surfaces. The vascular bundle is crescent-shaped, with pericycle forming a broken ring. Mesophyll consists of oval to polygonal parenchymatous cells. The leaf exhibits a dorsiventral structure, with palisade cells beneath the upper epidermis, and sclerenchyma at the midrib. Calcium oxalate crystals are present in the vascular bundles.

Flowers

The flower's epidermis is a single layer of wavy cells with simple trichomes and a thick cuticle. The cortex contains radially arranged parenchymatous cells, oil glands, and a ring of bicollateral vascular bundles. Parenchyma is rich in calcium oxalate crystals, and fibers are pink-stained.

Roots

The root’s epidermis consists of 10-20 layers of thin-walled, tangential cork cells. The cortex has large parenchymatous cells filled with starch and occasional calcium oxalate crystals. The vascular bundle is encircled by lignified cortical fibers. Xylem vessels are pitted with bordered pits, and fibers are slit-pitted.

Seeds

The seed coat consists of an epidermis with transparent, thick-walled polygonal cells, containing mucilage. The sub-epidermis includes sclerenchyma, parenchyma, and pigment layers. The endosperm and cotyledon cells are polyhedral with calcium oxalate crystals.

Standardization of C. ternatea

Extractive Values of C. ternatea (Leaves, Flowers, Roots, and Seeds)

The extractive values for different solvents used to extract C. ternatea plant parts are shown in Table 18. The highest extractive value for leaves was observed with n-butanol (11.79 g/100 g), while methanol yielded the highest extractive value for flowers (20.07 g/100 g). For roots, n-butanol was the most effective solvent (7.12 g/100 g), and for seeds, petroleum ether showed the highest value (7.13 g/100 g).

Table 5: Extractive Values (g/100 g) for Different Solvents and Plant Parts of C. ternatea

Moisture Content of C. ternatea

The moisture content of the different parts of C. ternatea, determined by loss in weight on drying, is summarized in Table 19. Seeds had the highest moisture content at 5%, followed by flowers (4%) and roots (2.5%). Leaves had the lowest moisture content at 2%.

Table 6: Moisture Content (Loss on Drying) in Different Parts of C. ternatea

Ash and Foreign Matter Content of C. ternatea

The ash and foreign matter content for various parts of C. ternatea are presented in Table 20. The highest total ash content was found in leaves (11%), while flowers showed the lowest value (8.5%). Roots and seeds had relatively low ash contents (4% and 3.5%, respectively). Additionally, the water-soluble ash content in leaves was 6%, and the acid-insoluble ash content was 4%.

Table 7: Ash and Foreign Matter Content in Different Parts of C. ternatea

Microbial Contamination in C. ternatea Plant Parts

Microbial contamination in the crude plant materials was assessed and is shown in Table 21. All plant parts—leaves, flowers, roots, and seeds—had low bacterial and fungal contamination. Notably, no Enterobacteria, E. coli, Salmonella spp., Staphylococcus, or Pseudomonas aeruginosa were detected in any of the plant parts.

Table 8: Microbial Contamination in Crude Drugs of C. ternatea

Phytochemical Screening of Leaves, Flowers, Roots, and Seeds Extracts of Clitoria ternatea Linn.

Phytochemical screening of Clitoria ternatea (Aparajita) was conducted on extracts obtained from the leaves, flowers, roots, and seeds using eleven different solvents: petroleum ether, hexane, n-butanol, butyl acetate, chloroform, ethyl acetate, acetone, methanol, ethanol, acetic acid, and aqueous. The analysis tested for the presence of various chemical groups including carbohydrates, reducing sugars, alkaloids, volatile oils, proteins, amino acids, fats, oils, steroids, glycosides (cardiac, anthraquinone, saponin, cyanogenic, and coumarin), flavonoids, tannins, and phenolic compounds. For the leaves (Table 9), carbohydrates were found in petroleum ether, hexane, methanol, and acetic acid extracts. Reducing sugars were present in methanol and acetic acid extracts, while non-reducing polysaccharides were detected in acetone and methanol extracts. Alkaloids were identified in n-butanol, butyl acetate, chloroform, acetone, methanol, and ethanol extracts. Proteins were found in ethyl acetate, acetone, and ethanol, while amino acids were observed in ethyl acetate and ethanol. Steroids were present in petroleum ether, hexane, and chloroform extracts. Glycosides such as cardiac glycosides appeared in the hexane extract, anthraquinone glycosides in n-butanol and acetone extracts, and saponin glycosides in chloroform, ethyl acetate, and aqueous extracts. Flavonoids were observed in n-butanol, acetone, and methanol extracts, and tannins and phenolic compounds were found in n-butanol and methanol extracts. However, oils, fats, and volatile oils were not detected in the leaves. For the flowers (Table 10), carbohydrates were present in petroleum ether, hexane, n-butanol, butyl acetate, chloroform, ethyl acetate, and ethanol extracts. Reducing sugars were detected in petroleum ether, methanol, and acetic acid extracts, while monosaccharides were found in acetone and methanol extracts. Non-reducing polysaccharides were present in all extracts except petroleum ether and methanol. Alkaloids were identified in petroleum ether, acetone, methanol, ethanol, acetic acid, and aqueous extracts. Fats, oils, and volatile oils were detected in petroleum ether, hexane, n-butanol, butyl acetate, and chloroform, with volatile oils also found in chloroform. Proteins were observed in acetone, methanol, ethanol, and aqueous extracts, but amino acids were not present. Steroids were found in petroleum ether, hexane, n-butanol, butyl acetate, chloroform, ethyl acetate, and acetone extracts. Cardiac glycosides were present in petroleum ether, methanol, and aqueous extracts, while saponin glycosides were only detected in aqueous extracts. Flavonoids were recorded in acetone and aqueous extracts, and tannins and phenolic compounds were identified in acetone, methanol, ethanol, and aqueous extracts. In the roots (Table 11), carbohydrates and reducing sugars were found in all eleven extracts, though monosaccharides and non-reducing polysaccharides were absent. Alkaloids were present in petroleum ether, hexane, ethyl acetate, acetone, methanol, ethanol, acetic acid, and aqueous extracts. Fats, oils, and volatile oils were recorded in petroleum ether, hexane, n-butanol, butyl acetate, chloroform, and ethyl acetate, with volatile oils also detected in acetone. Proteins were present in all extracts except ethyl acetate, and xanthoproteic tests were positive only in acetone, methanol, ethanol, acetic acid, and aqueous extracts. Amino acids were found in all extracts except ethyl acetate, with tyrosine detected in petroleum ether, hexane, n-butanol, and butyl acetate, and tyrosine also identified in chloroform extract. Steroids were present in petroleum ether, hexane, n-butanol, butyl acetate, chloroform, and ethyl acetate. Cardiac glycosides were found in butyl acetate, ethyl acetate, and saponin glycosides in petroleum ether, hexane, chloroform, methanol, and acetic acid. Flavonoids were detected in methanol and ethanol, and tannins and phenolic compounds were present in petroleum ether and aqueous extracts. For the seeds (Table 12), carbohydrates and reducing sugars were present in all extracts except ethyl acetate, while non-reducing polysaccharides were detected only in the ethyl acetate extract. Alkaloids were found in petroleum ether, hexane, acetone, methanol, ethanol, acetic acid, and aqueous extracts. Fats and oils, as well as volatile oils, were detected in petroleum ether, hexane, n-butanol, butyl acetate, chloroform, ethyl acetate, and acetone. Proteins were observed in ethyl acetate, acetone, methanol, ethanol, acetic acid, and aqueous extracts. Steroids were recorded in petroleum ether, hexane, n-butanol, butyl acetate, and chloroform. Cardiac glycosides were found in n-butanol, butyl acetate, ethyl acetate, acetone, methanol, ethanol, acetic acid, and aqueous extracts. Saponin glycosides were detected in petroleum ether, hexane, n-butanol, ethyl acetate, ethanol, and aqueous extracts. Flavonoids were present in methanol, ethanol, acetic acid, and aqueous extracts, while tannins and phenolic compounds were found in acetone, methanol, ethanol, and acetic acid extracts. However, monosaccharides, amino acids, anthraquinone, cyanogenic, and coumarin glycosides were not detected in any of the eleven extracts.

Table 9: Phytochemical analysis for C. ternatea leaf extracts

Table 10: Phytochemical Analysis For C. Ternatea Flower Extracts