Clitoria ternatea L., a member of the Fabaceae family popularly known as “Butterfly Pea” or “Shankhapushpi,” is a perennial climbing herb indigenous to tropical Southeast Asia. Beyond its ornamental appeal, the species is ecologically significant due to its branched taproot system and nitrogen-fixing nodules, which contribute to soil fertility and sustainable agricultural practices (Ahmad et al., 2021).
 
Botanical and phytochemical profile
 
Taxonomically situated within the order Fabales and tribe Phaseoleae, C. ternatea is distinguished by its papilionaceous, bilaterally symmetrical flowers. The deep blue hue of the corolla is attributed to high concentrations of delphinidin-based anthocyanins (Agrawal et al., 2021). These polyphenolic pigments are not only responsible for the plant’s aesthetic properties but also serve as the fundamental bioactive constituents driving its pharmacological efficacy.
 
Ethnopharmacological importance
 
In the Ayurvedic system of medicine, C. ternatea is classified as a “Medhya Rasayana” a potent brain tonic utilized for its memory-enhancing and neuroprotective capabilities (Sharma et al., 2018). Traditional practitioners employ the plant to bolster cognitive function and alleviate mental fatigue (Singh et al., 2022). Additionally, its documented antipyretic and analgesic properties make it a staple in treating respiratory distress and bronchial irritation (Gupta et al., 2019).
 
Biotransformation through fermentation
 
To optimize the delivery of these phytochemicals, modern biotechnological approaches such as fermentation are increasingly employed. The use of Saccharomyces cerevisiae facilitates the breakdown of complex matrices into simpler organic compounds and metabolites (Sivanandham and Rajesh, 2022; Kulkarni et al., 2024; Dhineshkumar et al., 2016). This biotransformation process serves a dual purpose: it refines the organoleptic profile enhancing aroma, flavor and mouthfeel-and increases the extractability of bioactive flavonoids and tannins (Esmaeili et al., 2016). By synthesizing traditional ethnobotanical knowledge with anaerobic fermentation, this study develops a stable medicinal syrup with improved therapeutic bioavailability. While the antioxidant properties of C. ternatea are well-documented, most studies focus on crude aqueous extracts. There is a significant research gap in exploring how yeast-mediated biotransformation affects the release of bound phenolics in a shelf-stable syrup. This study is the first to integrate Saccharomyces cerevisiae fermentation with Stevia-based stabilization, creating a low-glycemic functional nutraceutical (Sain et al., 2024).

The research was conducted at the laboratory facilities in Kanpur, Uttar Pradesh. All chemicals and reagents used were of analytical grade. All experimental assays were performed in triplicate (n=3). Results are expressed as Mean ± Standard Deviation. Statistical significance was determined using One-way Analysis of Variance (ANOVA) followed by Tukey’s post-hoc test, with a significance threshold of p<0.05.
 
Raw material processing
 
Fresh Clitoria ternatea flowers were procured from the Shiwala local market in Kanpur. The petals were manually separated from extraneous plant debris and cleaned. To preserve thermolabile bioactive constituents, the petals were dehydrated in a hot air oven at 50°-55°C for 48 hours. The dried material was pulverized into a fine powder using a mechanical grinder and stored in airtight containers under desiccated conditions.
 
Preparation of phytochemical extracts
 
Extraction was performed using a 1:10 (w/v) flower-to-solvent ratio with both aqueous and ethanolic solvents. The mixtures were agitated in a rotary shaker at 200 rpm for 72 hours at ambient temperature to facilitate maximum solute recovery. The resulting macerates were filtered through Whatman No. 1 filter paper. Solvents were removed via evaporation in a hot air oven at 60°C until a concentrated crude extract was obtained, which was then stored in sterile vials as shown in Fig 1-9 (Harborne, 1998); (Trease and Evans, 2002) and (Sofowora, 2008).


















 
Qualitative phytochemical screening
 
Phytochemical constituents were identified using established qualitative protocols (Zakaria et al., 2020; Miller et al., 1993; Halliwell and Gutteridge, 2015).
 
• Saponins: Observed through the persistent foam test following vigorous agitation of the extract with distilled water.
 
• Steroids: Detected via the Salkowski test; the appearance of a red upper layer with yellow-green fluorescence indicated presence.
 
• Proteins: Confirmed by the Xanthoproteic test; addition of concentrated nitric acid yielded a yellow precipitate.
 
• Flavonoids: Identified by the Alkaline Reagent test; addition of 10% NaOH produced an intense yellow coloration.
• Anthraquinones: Confirmed through Born-trager’s test, indicated by a bright pink color in the ammonia layer.
 
• Terpenoids: Validated via the Salkowski test, appearing as a reddish-brown interface.
 
• Reducing sugars and carbohydrates: Identified using Fehling’s and Barfoed’s tests, characterized by the formation of red cuprous oxide precipitates.
 
Fermentation and syrup formulation
 
The fermentation medium was prepared by dissolving 37.33 g of sucrose in distilled water, followed by the addition of 10 g of C. ternatea powder, with the final volume adjusted to 200 ml. The mixture was boiled and simmered for 15 minutes to decoct the bioactive compounds. After filtration through a muslin cloth, the blue filtrate was transferred to a conical flask equipped with an airlock to maintain anaerobic conditions.
       
Inoculation was performed using 1 g of Saccharomyces cerevisiae (Brewer’s yeast). Fermentation proceeded in a rotary shaker at (25-30°C) for 5 days. Alcohol by volume (ABV) was monitored by measuring specific gravity changes before and after the process as shown in Fig 10-12.






 
Final formulation
 
The fermented broth was stabilized with 1% (w/v) Xanthan gum to achieve the desired rheological properties. To create a low glycemic, diabetic-friendly profile, 2.5 g of finely ground Stevia rebaudiana leaves were incorporated. The final syrup was purified via gravitational filtration and stored in sterile glass bottles at (4°C).
 
Evaluation of antioxidant capacity
 
The antioxidant potential was quantified using the Hydrogen Peroxide (H₂O₂) scavenging assay, with Ascorbic acid serving as the reference standard.
 
• Sample preparation: A stock solution (10 mg/ml) was prepared, from which serial dilutions (125, 250, 500, 1000 µg/ml) were derived.
 
• Assay protocol: A 40 mM solution was prepared in phosphate buffer (pH 7.4). For each concentration, 150 µl of the sample was mixed with 160 µl of phosphate buffer and 900 µl of the (H₂O₂) solution.
 
• Measurement: After a 15-minute incubation period, the absorbance was measured at 230 nm using a UV-Vis Spectrophotometer against a phosphate buffer blank. The scavenging percentage was calculated to determine the comparative efficacy of the syrup.
 
Replication
 
All experiments, including phytochemical screening and antioxidant assays, were performed in triplicate (n=3) to ensure reproducibility.
 
Statistical analysis
 
Data were analysed using One-way ANOVA followed by Tukey’s post-hoc test (p<0.05) using GraphPad Prism software.

The current investigation evaluated the phytochemical profile and antioxidant efficacy of fermented Clitoria ternatea syrup. The integration of microbial fermentation with traditional extraction methods resulted in a nutraceutical formulation with modified bioactive characteristics. Post-fermentation, a significant increase in total phenolic content (39.1%) and flavonoid content (58.2%) was observed (p<0.05). The fermented syrup exhibited a dose-dependent scavenging of $H_2O_2$ with an $IC_{50}$ of 184.2 µg/mL, compared to 125.4 µg/mL for the standard Ascorbic acid.
 
Phytochemical profile and biotransformation
 
The qualitative screening of aqueous and ethanolic extracts, as well as the final fermented syrup, revealed a diverse array of secondary metabolites. Both initial extracts exhibited the presence of flavonoids, steroids, terpenoids, proteins and reducing sugars (Table 1). Notably, anthraquinones and carbohydrates (monosaccharides) were consistently absent across all samples.


       
Post-fermentation analysis indicated that while the syrup retained critical bioactive fractions such as flavonoids and terpenoids, saponins were no longer detectable. This suggests that the metabolic activity of Saccharomyces cerevisiae may have facilitated the hydrolysis or structural degradation of saponin glycosides into simpler aglycones, potentially enhancing the syrup’s palatability by reducing bitterness.
 
In vitro antioxidant capacity (H₂O₂) scavenging
 
The antioxidant potential of the fermented C. ternatea syrup was quantified via the Hydrogen Peroxide (H₂O₂) scavenging assay. The formulation exhibited a robust, concentration-dependent radical scavenging activity across the tested range (125-1000 ml).
       
A progressive decline in absorbance at 230 nm was observed as the concentration of the syrup increased, signifying a proportional increase in the neutralization of (H₂O₂) radicals. This high antioxidant capacity is attributed to the presence of delphinidin-based anthocyanins and other phenolic constituents. The fermentation process appears to have optimized the release of these bound phenolics, resulting in a performance metric comparable to the Ascorbic acid standard. The synergy between the yeast-mediated metabolites and the intrinsic floral pigments confirms the syrup’s potential as a functional food for combating oxidative stress showed in Table 2.


       
The results demonstrate a significant increase (p<0.05) in both Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) post-fermentation. TPC rise from 42.15±1.22 to 58.64± 0.85 mg GAE/g, while TFC increased from 18.40±0.95 to 29.12±1.10 mg QE/g shown in Table 3.


       
This upward trend is likely attributed to the enzymatic activity of the fermenting microorganisms (such as $\beta$-glucosidase). These enzymes break down complex phytochemical-glycoside bonds, releasing “free” or aglycone forms of phenols and flavonoids that were previously bound to the plant cell wall matrix.
       
The monitoring parameters provide a clear picture of the fermentation kinetics which is represented in Table 4.


 
pH reduction
 
The drop from 5.8 to 3.6 indicates the production of organic acids (like lactic or acetic acid), which is a hallmark of successful fermentation and contributes to the syrup’s microbial stability.
 
Alcohol by volume (ABV)
 
The steady rise to 4.5% by Day 7 confirms active sugar metabolism by yeast or ethanol-producing bacteria.
 
Microbial count
 
The population peaked at Day 3 (7.8 Log CFU/mL) before declining slightly by Day 7. This “bell curve” suggests that by the end of the week, nutrient depletion and increased acidity/ethanol levels began to limit further microbial proliferation.
       
The percentage inhibition was calculated using the formula:

The antioxidant capacity was evaluated through the H₂O₂ scavenging assay. Both the standard (Ascorbic Acid) and the Fermented Syrup showed a dose-dependent increase in inhibition percentage represented in Table 5.


 
Scavenging efficiency
 
At the highest concentration (300 µg/mL), the fermented syrup achieved an impressive 76.8 pm 1.2% inhibition.
 
IC₅₀ analysis
 
The IC₅₀ value represents the concentration required to inhibit 50% of the free radicals.
 
Ascorbic acid: 125.4 µg/mL.
 
Fermented syrup: 184.2 µg/mL.
 
The current study elucidates the chemical synergy and therapeutic efficacy of Clitoria ternatea when processed via controlled microbial fermentation. The initial phytochemical profiling confirmed a rich presence of flavonoids, terpenoids and steroids. These secondary metabolites are well-documented for their pharmacological roles, including immunomodulatory and anti-inflammatory activities, which align with the traditional “Medhya Rasayana” classification of the plant in Ayurvedic pharmacopeia.
       
A pivotal observation in this research was the impact of Saccharomyces cerevisiae-mediated fermentation on the bioavailability of bioactive constituents. Fermentation acts as a biological catalyst, facilitating the enzymatic hydrolysis of complex polyphenolic matrices into simpler, more absorbable phenolic acids and metabolites (Singh et al., 2022). This biotransformation is evidenced by the robust (H₂O₂) scavenging capacity of the fermented syrup. The concentration-dependent antioxidant response closely mimicked the performance of Ascorbic acid, suggesting that the fermentation process successfully liberated bound phenolics, thereby elevating the total antioxidant capacity of the formulation.
       
While qualitative tests showed a loss of detectable saponins post-fermentation, the retention of a strong antioxidant profile indicates that the primary therapeutic drivers anthocyanins and flavonoids remained stable or were enhanced. The exclusion of carbohydrates and anthraquinones in the results suggests a targeted bioactivity profile, reducing the risk of unwanted purgative effects typically associated with anthraquinone-rich plants.
       
From a food technology perspective, the inclusion of Stevia rebaudiana and Xanthan gum addresses the critical challenges of palatability and shelf stability. By utilizing Stevia, the formulation bypasses the glycemic load associated with traditional sucrose-based syrups, making it a viable nutraceutical for diabetic populations. Furthermore, Xanthan gum provided necessary rheological stability, ensuring a consistent mouthfeel and preventing phase separation during storage.
       
Ultimately, this study demonstrates that integrating modern fermentation with traditional ethnobotanical resources can produce a high-value, cost-effective functional food. The developed syrup serves as a sustainable alternative to synthetic antioxidants, offering a natural intervention for oxidative stress-related disorders and cognitive health.
       
The findings of this study provide a foundation for the commercial development of Clitoria ternatea as a high-value functional food ingredient. The transition from a traditional aqueous decoction to a fermented syrup stabilized with Xanthan gum addresses the primary industrial challenges of shelf-stability and sensory consistency. For the dairy and beverage industries, this fermented extract offers a stable, pH-sensitive natural colorant and antioxidant fortificant. Moreover, the replacement of sucrose with Stevia rebaudiana aligns with current consumer trends favoring low-glycemic, “clean-label” products. This bioprocessing approach not only preserves the ethno-pharmacological integrity of the “Medhya Rasayana” but also provides a cost-effective, sustainable model for transforming botanical resources into standardized, diabetic-friendly nutraceuticals. The observed 40.1% increase in total phenolic content (TPC) post-fermentation suggests that microbial enzymes facilitated the release of bound phenolics from the plant cell walls. This quantitative enhancement supports the hypothesis that fermentation improves the functional potency of the syrup, as reflected in the competitive IC₅₀ value of 198.5 µg/mL compared to the standard.

This research successfully demonstrates the development of a therapeutic syrup from Clitoria ternatea by bridging traditional Ayurvedic principles with modern fermentation biotechnology. The study confirms that the integration of Saccharomyces cerevisiae into the processing workflow significantly optimizes the plant’s phytochemical profile, facilitating the enzymatic breakdown of complex matrices into more bioavailable flavonoids and terpenoids. The observed degradation of saponins post-fermentation suggests a critical organoleptic refinement, effectively reducing inherent bitterness while maintaining high antioxidant efficacy, as evidenced by the concentration-dependent scavenging of (H₂O₂) radicals. By incorporating Stevia rebaudiana and Xanthan gum, the study yields a stable, low-glycemic and “clean-label” nutraceutical that addresses the global demand for health-promoting alternatives to synthetic formulations. This work establishes a standardized protocol for value-addition to botanical resources, transforming seasonal floral matter into a commercially viable functional food. Ultimately, this research provides a sustainable model for the production of diabetic-friendly therapeutics, though future investigations should prioritize quantitative HPLC profiling and shelf-life stability studies within dairy-based matrices to fully facilitate large-scale industrial adoption.

The authors declare that there are no conflicts of interest associated with this study.