Prebiotic Potential of Gymnema sylvestre Leaf Extract: An In-Vitro Evaluation Using Selected Probiotic Strains

The gastrointestinal tract contains a complex microbial community that contributes to digestion, metabolic processes, immune regulation and maintenance of intestinal homeostasis. Growing interest in the gut microbiota has increased research into probiotics, prebiotics, postbiotics and synbiotics as functional approaches for supporting host health. Prebiotics are substrates that are selectively utilized by host microorganisms and can produce beneficial changes in the composition or activity of the microbiota. Examples include inulin, fructo-oligosaccharides and galacto-oligosaccharides, recent research has expanded toward resistant starches, plant polysaccharides and selected plant-derived compounds^{. [1,2,3]}

Plant materials are attractive sources for prebiotic research because they contain dietary carbohydrates, fibers, polyphenols and other phytochemicals. Several plant, fruit, vegetable and herbal extracts have been reported to stimulate beneficial bacterial growth or demonstrate resistance to simulated gastrointestinal digestion. Such properties may allow plant-derived materials to reach the intestinal environment in a form that remains available for microbial utilization^{. [4,5,6]}

Gymnema sylvestre is a perennial woody climber widely distributed in tropical regions and is traditionally known as Gurmar. Leaves contain triterpenoid saponins, gymnemic acids, flavonoids, alkaloids, tannins and phenolic constituents. Previous research has extensively investigated its antidiabetic, antioxidant, antimicrobial and other pharmacological properties. However, the prebiotic potential of its aqueous leaf extract remains comparatively less explored. ^[7,8,9,10]

2. MATERIALS AND METHODS

2.1 PLANT MATERIAL AND EXTRACTION

Fresh leaves of Gymnema sylvestre were collected in the month of January ,2026 and authenticated by the Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pradesh (Plant Code No. SVUH: 2610). The leaves were washed, shade-dried and powdered. 50g of powder were mixed with 500 mL distilled water (1:10 w/v) and subjected to maceration for 48 h with intermittent stirring. The mixture was filtered through Whatman No. 1 filter paper. The filtrate was concentrated at 35–40°C by using rotary vacuum evaporator and the dried extract was stored at 4°C in amber colored glass bottles for future use.  ^[11,12,13]

Percentage yield: Percentage yield (%) = (weight of dried extract / weight of plant powder) × 100.

2.2 QUALITATIVE PHYTOCHEMICAL SCREENING

The obtained extract was designated as GSAE (Gymnema sylvestre Aqueous Extract). The aqueous extract was subjected to preliminary qualitative phytochemical screening. Carbohydrates were assessed by Molisch's test, reducing sugars by Benedict's test, proteins by the Biuret test, amino acids by the Ninhydrin test, alkaloids by Dragendorff's test, flavonoids by the Shinoda test, tannins and phenolic compounds by ferric chloride tests, saponins by the foam test, terpenoids by the Salkowski test, steroids by the Liebermann–Burchard test and glycosides by the Keller–Killiani test^{. [14,15]}

2.3 FTIR CHARACTERIZATION

The dried aqueous extract was finely powdered. Approximately 1–2 mg of extract was mixed thoroughly with 100–200 mg spectroscopic-grade potassium bromide (KBr). The mixture was compressed under high pressure using a hydraulic press to prepare a transparent KBr pellet. The pellet was immediately placed in the FTIR spectrophotometer, and the spectrum was recorded over 4000–400 cm⁻¹. Characteristic absorption bands were interpreted with respect to their corresponding functional groups^{. [16, 17, 18]}

2.4 PROBIOTIC STRAINS AND CULTURE CONDITIONS

The study used Lactobacillus sp. strain YVU and Bacillus stercoris strain VFT. Lactobacillus sp. strain YVU was identified by 16S rRNA gene sequencing and reported with GenBank accession PP587743.1; its isolation source was Bajra crop root soil. Bacillus stercoris strain VFT was identified by 16S rRNA gene sequencing and reported with GenBank accession PX992070.1; its isolation source was fermented tomatoes.

De Man–Rogosa–Sharpe (MRS) broth and MRS agar were used as culture media. Culture media were sterilized by autoclaving at 121°C and 15 psi for 15–20 min. Active cultures were prepared in sterile MRS broth and incubated at 37°C for 24 h. ^[19,20]

2.5 GROWTH STIMULATION ASSAY

The growth stimulation assay was performed to evaluate the effect of GSAE on viable bacterial growth. MRS broth containing the bacterial culture without extract served as the control. Treatment groups contained GSAE at 0.25% or 0.50% (w/v). For Bacillus stercoris, 0.025 g and 0.05 g extract were added to 10 mL broth for the 0.25% and 0.50% treatments, respectively. The corresponding quantities were used for Lactobacillus sp. cultures. Cultures were incubated at 37°C for 24 h.

Following incubation, cultures were centrifuged at 3800 rpm for 10 min, the supernatant was discarded, and the bacterial pellet was washed with sterile PBS. The pellet was resuspended in PBS and serially diluted up to 10⁻⁷. Appropriate dilutions were spread on MRS agar and incubated at 37°C for 24 h. Colonies were counted and bacterial population was expressed as CFU/mL^{. [21-26]}

2.6 SIMULATED GASTRIC ACID HYDROLYSIS

Artificial gastric fluid was prepared using NaCl (8 g/L), Na₂HPO₄·2H₂O (8.25 g/L), KCl (0.2 g/L), NaH₂PO₄ (14.35 g/L), MgCl₂·6H₂O (0.18 g/L) and CaCl₂·2H₂O (0.1 g/L). The pH was adjusted to 1.0, 2.0, 3.0 and 4.0 using HCl, followed by addition of pepsin to a final concentration of 1000 U/mL. GSAE was prepared at 0.5% (w/v) and mixed with 2 mL artificial gastric fluid. The reaction mixtures were incubated at 37°C. Measurements were recorded after 24h of incubation. Reducing sugar and total carbohydrate contents were estimated and hydrolysis was calculated as:

Hydrolysis (%) = (reducing sugar released / total carbohydrate content) × 100^{. [27,28]}

2.7 SIMULATED INTESTINAL FLUID HYDROLYSIS

For simulated intestinal digestibility, the thesis describes artificial intestinal fluid consisting of 1× PBS containing 0.5% (w/v) bile salts and trypsin at a final concentration of 1000 U/mL. The methodology section reports adjustment to pH 8.0, whereas the Results section reports measurements at pH 6.8 and 7.4. Measurements were recorded after 24h of incubation. Reducing sugar and total carbohydrate contents were estimated and hydrolysis was calculated. ^[29,30]

3. RESULTS & DISCUSSION

3.1 EXTRACTION YIELD

From 50 g of Gymnema sylvestre leaf powder, 4.7 g of dried aqueous extract was obtained. The calculated extraction yield was 9.4%.

Table 1: Extraction yield of GSAE

3.2 PHYTOCHEMICAL PROFILE

Table 2: Phytochemical screening of GSAE

3.3 FTIR CHARACTERIZATION OF GSAE

Figure 1: FTIR Spectrum of Gymnema sylvestre leaves aqueous extract (GSAE)

Table 3: FTIR Interpretation of GSAE

Peak (cm⁻¹)	Observation	Significance
3395	Broad O–H stretching band	Indicates hydroxyl groups from phenols and polysaccharides.
2924	C–H stretching	Corresponds to aliphatic C–H stretching
1734	C=O stretching	Indicates the presence of carbonyl groups.
1632	C=C stretching	Confirm aromatic compounds and flavonoids.
1246–1074	C–O and C–O–C stretching	Indicates glycosides and carbohydrate-rich polysaccharides are associated with prebiotic properties.

Figure 2. Growth of Bacillus stercoris in control & treatment groups

Figure 3. Growth of Lactobacillus sp. in control & treatment groups

Table 6: Effect of gastric pH on hydrolysis of G. sylvestre leaves aqueous extract

Sr. No	pH of Gastric Juice	Incubation time (h)	% Hydrolysis	Observation
1.	1.0	2	13.8 ± 0.5	Maximum hydrolysis observed under highly acidic condition
2.	2.0	2	10.4±0.4	Moderate
3.	3.0	2	7.2±0.3	Lower
4.	4.0	2	3.9±0.2	Minimum hydrolysis, better stability

Figure 4: Effect of gastric pH on hydrolysis of GSAE

Table 7: Effect of intestinal pH on hydrolysis of GSAE

Sr. No	pH of Intestinal Juice	Incubation time (h)	% Hydrolysis	Observation
1.	6.8	2	5.6 ± 0.3	Slight hydrolysis observed
2.	7.4	2	3.8 ± 0.2	Low hydrolysis and better stability

Figure 5. Effect of intestinal pH on hydrolysis of GSAE

From Table 7, Figure 5: The sample exhibited low hydrolysis under simulated intestinal conditions. At pH 6.8, the percentage hydrolysis was 5.6 ± 0.3%, indicating slight degradation. At pH 7.4, hydrolysis decreased to 3.8 ± 0.2%, demonstrating greater stability under near-neutral intestinal conditions. The low degree of hydrolysis suggests that the sample remained relatively resistant to intestinal digestion, supporting its potential prebiotic activity.

4. CONCLUSION

The present study evaluated the in-vitro prebiotic potential of Gymnema sylvestre leaves aqueous extract on probiotic strains (Lactobacillus spp. and Bacillus stercoris). The extract demonstrated the ability to promote the growth of probiotic bacteria, indicating its potential as a natural prebiotic. The gastric acid and intestinal fluid resistance studies suggested that the extract remained relatively stable under simulated gastrointestinal conditions, which is an important characteristic of an effective prebiotic.

FTIR analysis confirmed the presence of functional groups associated with carbohydrates and other bioactive compounds that may contribute to its prebiotic efficacy. Overall, the study suggests that Gymnema sylvestre leaves aqueous extract has promising potential as a natural prebiotic ingredient and may be explored further for its application in functional foods and nutraceutical formulations.

ACKNOWLEDGEMENTS

The authors sincerely acknowledge RIPER College, Anantapur, for providing the permission for doing FTIR analysis, Fourth Apple Biosciences, Chandragiri Road, Tirupati and YV University, Kadapa for performing growth assay.

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