For the preparation of ethanolic extract of G. glabra, mature roots of the plant were powdered and mixed with 95% alcohol (1:10 ratio) under continuous agitation at 230 rpm at 40°C for 7 h. The extract was then subjected to vacuum concentration. For HPLC analysis, the extract was dissolved in methanol at a concentration of 1 mg/ml and filtered through a 0.45 μm syringe filter (14).

HPLC analysis was carried out on Waters Alliance 2695 separation module equipped with 2998 photodiode array detector and Phenomenex C18 Luna^® column (5 μm; 250 mm × 4.6 mm). Standards were gifted by Dr. R S Bhakuni. The separation of six markers namely (a) Liquiritigenin (LTG), (b) Isoliquiritigenin (ISL), (c) Formononetin (F), (d) Glycyrhyzzic acid (GA), (e) Glabridin (GLB), and (f) Glycyrrhetinic acid (GTA) was optimized using the mobile phase of (a) water (0.1% formic acid) and (b) acetonitrile (0.1% formic acid) with the given gradient elution using a C18 column (5 μm; 250 mm × 4.6 mm) at a flow rate of 1 ml/min. The column eluent was monitored at 254 nm (14, 15).

Caco-2 cell line was procured from National Centre for Cell Sciences (NCCS), Pune (India). The cell line was routinely maintained in MEM (low glucose) growth medium containing 20% FBS, 1% L-glutamine, 1 mM sodium pyruvate, 1× penicillin-streptomycin, and 1× (2.5 μg/ml) amphotericin B. Cells were grown in T-25 flasks at 37°C, 5% CO₂, 95% humidity with growth medium being changed three times a week and sub-cultured upon reaching 80% confluence with Trypsin EDTA solution (0.25% Trypsin, 53 mM EDTA in Hanks Balanced Salt Solution (HBSS with phenol red, pH 7.4).

For in vitro Vitamin B12 permeability experiments, Caco-2 cells were seeded at a density of 1,00,000 cells in the apical chamber (AP) of Polyethylene Terephthalate (PET) inserts of 12 well transwell plates. A total of 500 and 1,000 μl growth medium was added to the apical (AP) and basolateral (BL) chambers of each transwell, respectively. Growth medium was changed in both AP and BL compartments on alternate days and cells were allowed to grow for 14 days until the formation of tight differentiated monolayers in the AP chambers of inserts. The differentiation status of monolayers was assessed by the measurement of trans-epithelial electrical resistance (TEER) by EVOM² Voltohmeter (WPI, USA) every alternate day. TEER was calculated as Actual TEER = TEER (cell bearing insert) − TEER (blank insert) × area of insert (1.12 cm²). The differentiated Caco-2 monolayers with TEER values ranging from 3,000 to 4,000 Ω.cm² were further considered for the in vitro study (16, 17).

Well-differentiated Caco-2 monolayers with TEER values stated above were employed in in vitro study. On the day of the experiment, the growth medium in Caco-2 monolayer bearing transwells was replaced with HBSS (w/o phenol red, with 25 mM HEPES pH 7.4). Monolayers were washed once with HBSS (500 μl in AP and 1,200 μl in BL compartment) followed by equilibration i.e., incubation in HBSS for 30 min at 37°C in a shaker incubator. After completion of the equilibration step, the in vitro assay was initiated. Stock solutions of Vitamin B12 (Cyanocobalamin) and GgEtOH were prepared in Milli-Q water and DMSO, respectively which were diluted to final working concentrations in HBSS. To the apical chamber bearing Caco-2 monolayers, B12 was added at a concentration of 200 μg/ml (17) and GgEtOH was added at 25, 50, and 100 μg/ml concentration in HBSS (500 μl) whereas basolateral chamber contained only HBSS (1,200 μl). Apical chambers containing only B12 and the combination of B12 + GgEtOH served as control and treatment groups, respectively. The transwell plate was then incubated at 37°C with constant shaking at 50 rpm (Biosan ES-20 orbital shaker incubator). Samples (200 μl) were withdrawn from basolateral chamber after 30 min intervals for 240 min followed by the addition of 200 μl of fresh HBSS to the BL compartment. After assay completion, B12 was quantitated in basolateral samples (collected at various time intervals) spectrophotometrically by measuring absorbance at 360 nm using a microplate reader (FLUOstar Omega). The concentration of Vitamin B12 in samples was deduced from the standard curve followed by the determination of Papp. Two independent experiments were performed with duplicates comprising fully differentiated Caco-2 monolayers treated with B12 only (200 μg/ml) and combination of B12+GgEtOH (25, 50,100 μg/ml).

Apparent permeability coefficient, Papp (cm/s) was calculated from the following equation:

where, dQ/dt (μg/s) is the flux rate, A (cm²) is the surface area of the apical chamber bearing cell monolayer, and C0 (μg/ml) is the initial B12 concentration (apical chamber).

Fold enhancement (FE) was further calculated as the ratio of Papp (B12+GgEtOH) and Papp (B12 alone).

Everted gut sac assay was performed to determine intestinal permeation enhancement of Vitamin B12 in presence of GgEtOH utilizing Swiss albino mice weighing 25–30 g. Everted gut sacs were prepared following the protocol described (18). Mice were sacrificed by cervical dislocation and the small intestine was isolated in oxygenated Krebs Ringer Buffer (KRB) (Glucose 7.78 mM, NaCl 133 mM, KCl 4.56 mM, NaH₂PO4 1.5 mM, MgCl₂ 0.20 mM, NaHCO₃16 mM, CaCl₂3.33 mM, pH 7.4) (19). 10 cm portion from the ileocecal junction (corresponding to the ileum) was separated from the rest of the small intestine. The ileum was flushed with cold normal saline to remove its contents. Ileum was then cut into 4 cm sized segments and gently everted on thin plastic capillary tubing. One end of each everted segment was tied with cotton thread and 500 μl oxygenated KRB was filled through the tubing from another end after which it was tied to create everted gut sacs. Prepared everted gut sacs were initially incubated in oxygenated KRB at 37°C for 10 min. Sacs were then transferred to tubes containing 2 ml oxygenated KRB with added B12 (Cyanocobalamin) at a concentration of 100 μg/ml and GgEtOH extract at 250 and 500 μg/ml concentrations and incubated at 37° for 2 h with constant aeration and shaking at 100 rpm. Sacs incubated with B12 alone served as the control/untreated group whereas sacs incubated with B12+GgEtOH were included in treatment groups. After 30, 60, 90, and 120 min sacs were cut open, serosal fluid collected in 2 ml tubes and short spun for 5 min to separate the debris from the fluid. Hundred microliter of serosal fluid was transferred to 96 wells plate and Vitamin B12 was quantitated from the standard curve by measuring absorbance at 360 nm. Area of each sac was calculated considering it a cylinder (length 4 cm, width/diameter 0.5 cm, radius 0.25 cm) (20). Two experiments were performed, each with two replicates comprising everted gut sacs treated with B12 only (100 μg/ml) and combination of B12+GgEtOH (250 and 500 μg/ml). Apparent permeability coefficient (Papp) was then calculated by the following equation:

where, dQ/dt (μg/s) is the flux rate, A (cm²) is the surface area of the everted gut sac and C0 (μg/ml) is the initial B12 concentration (mucosal compartment).

The pharmacokinetic study of Vitamin B12 was carried out in Swiss albino mice (male), bred and maintained under standard conditions and fed with ATNT 1324-Maintenance Diet pellets. Experiments were conducted under protocol (CIMAP/IAEC/2019-21/06) approved by the Institutional Animal Ethics Committee under the aegis of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.

Swiss albino mice (weighing 25 g) were divided into 12 experimental groups with five mice per group based on different doses (B12 and GgEtOH) and different time intervals (15, 30, 60,120 min). The experimental groups were as follows:

B12 alone (1 mg/kg) (15, 30, 60, 120 min)— 20 animals

B12 (1 mg/kg) + GgEtOH (100 mg/kg) (15, 30, 60, 120 min)—20 animals

B12 (1 mg/kg) + GgEtOH (1000 mg/kg) (15, 30, 60, 120 min)—20 animals

Prior experiment, animals were fasted for 18–24 h with access to water only. Animals were orally administered Cyanocobalamin alone and in combination with GgEtOH extract at doses indicated. For oral administration, GgEtOH extract was initially solubilized in 1% Tween 80 and then the final volume was made up with 0.5% CMC in Milli-Q water. B12 (Cyanocobalamin) solution was also prepared similarly. After defined time intervals (15, 30, 60, and 120 min), 500 μl blood was withdrawn from the orbital plexus of animals and collected in 2 ml tubes containing 50 μl ACD (Acetate Citrate Dextrose) solution (21, 22).

Blood samples were processed for the quantitation of Vitamin B12 by the HPLC method as described elsewhere (21). The processed samples were then transferred to a reversed-phase HPLC system (Waters) equipped with Bridge C18 5 μm column (4.6 mm × 250 mm). The mobile phase consisted of methanol: phosphate buffer (20 mM, pH 3.5) (25:75) with a flow rate of 0.6 ml/min. Vitamin B12 was detected at a wavelength of 360 nm. Pharmacokinetic parameters were evaluated by PK Solver 2.0 software.

Results are expressed as mean ± SD. Statistical analysis was performed on Graph pad prism software (version 5.0). Significance between control (B12 alone) and treatment (B12+GgEtOH) groups was determined by ANOVA followed by Dunnett’s post-hoc test. p < 0.05 was considered significant.

Figure 1A represents the chromatogram comprising standard markers separated by the HPLC protocol described. HPLC fingerprint of GgEtOH has been shown in Figure 1B. The content of marker compounds detected in GgEtOH was LTG—0.65%, ISL—0.17%, F—0.12%, GA—0.69%, and GLB—1.65%.

Vitamin B12 permeation study was carried out in vitro in the Caco-2 model to evaluate the effect of GgEtOH on enhancing intestinal absorption of B12. In presence of GgEtOH, the rate of transport of B12 across Caco-2 monolayers was found to increase gradually with time in a dose-dependent manner (Figure 2A). At 25, 50, and 100 μg/ml GgEtOH concentration, permeation of B12 was significantly enhanced compared to control as indicated by an increase in Papp values of B12 in Caco-2 monolayers treated with B12+GgEtOH (25, 50, 100) combination (Figure 2B). GgEtOH at 25, 50, and 100 μg/ml concentration enhanced B12 absorption by 2.47, 3.68, and 5.9 fold respectively thereby demonstrating an effect on enhancing intestinal membrane permeation (Supplementary Table 1).

Everted gut sac assay was performed with the ileum segments of mouse small intestine to assess the possible effect of GgEtOH on enhancing the permeation of B12 through the intestinal membrane. Sacs incubated with Cyanocobalamin only served as the control group. GgEtOH enhanced absorptive transport of B12 as demonstrated by an increased rate of accumulation of B12 in gut sacs incubated with a combination of B12+GgEtOH than B12 alone (Figure 3A). As shown in Figure 3B, increased apparent permeability (Papp) values of B12 were obtained for sacs incubated with B12+GgEtOH (250 and 500 μg/ml) as compared to B12 alone. GgEtOH extract enhanced intestinal permeation of B12 by 2.6 and 3.7 fold at 250 and 500 μg/ml concentration, respectively during the 2-h experimental duration (Supplementary Table 2). It can thus be considered that the extract is capable of enhancing the absorption of B12 through the small intestine.

The pharmacokinetics study of Vitamin B12 was performed in Swiss albino mice to investigate the effect of GgEtOH on the bioavailability enhancement of B12. Vitamin B12 (Cyanocobalamin) at a dose of 1 mg/kg body weight, alone and in combination with GgEtOH extract (100 and 1,000 mg/kg dose) was administered to animals through oral route. B12 content was determined in blood plasma samples collected at 15, 30, 60, and 120 min post-dosing by HPLC. As shown in Figure 4, the plasma concentration of Vitamin B12 increased in mice treated with B12+GgEtOH 100 mg/kg and B12+GgEtOH 1,000 mg/kg compared to mice treated with B12 only. Further, the pharmacokinetic profile of Vitamin B12 was significantly enhanced in presence of extract. Although, GgEtOH at 100 mg/kg dose significantly increased the Cmax and AUC, it did not contribute to the increment in t_1/2, Tmax, AUC _0–inf and MRT of B12. However, GgEtOH at 1,000 mg/kg dose caused significant enhancement of Cmax and AUC compared to the control group. At 1,000 mg/kg dose of extract, although not significant, but an increase in half-life (t_1/2) and mean residence time (MRT) of B12 was also observed. In B12+GgEtOH 1,000 mg/kg treated group, the Tmax of B12 was significantly improved and was attained in lesser duration (18.75 min) than B12 alone treated group (26.25 min) (Table 1). The results indicated that 1,000 mg/kg dose of the extract led to the enhanced absorption of B12 than 100 mg/kg dose. Overall, GgEtOH improved the bioavailability of Vitamin B12 as evidenced by pharmacokinetic study.