Madhugrit (batch #1MDT-210081) was sourced from Divya Pharmacy, India. The standards for HPLC analysis namely gallic acid, magnoflorine, piperine, rutin, ellagic acid, coumarin, cinnamic acid, and palmatine were obtained from Sigma-Aldrich, USA; protocatechuic acid, corilagin from Natural remedies, India, and methyl gallate from TCI chemicals, India. Reagents namely RPMI 1640 (no glucose), DMEM (low glucose), DPBS, antibiotic-antimycotic solution, starch, Malondialdehyde (MDA), Trichloroacetic acid (TCA), LPS, and 2′,7′-Dichlorofluorescin diacetate (H₂DCFDA) were procured from Sigma-Aldrich, USA. Heat-inactivated FBS, TPVG, Bovine serum albumin (BSA), Nile red, and Alamar blue were obtained from HiMedia, India. Horse serum was bought from Gibco, USA. Chemicals, 2-thiobarbituric acid (TBA), D-glucose, Metformin, and dexamethasone were obtained from TCI chemicals, India. Phorbol 12-myristate 13-acetate (PMA) was purchased from Alfa Aesar, UK. Mitomycin C was obtained from Roche, Germany. Pierce BCA protein assay kit and sodium azide were obtained from Thermo Fisher Scientific, USA. Human TNF-α and IL-6 ELISA kits were obtained from BD Biosciences, USA. QUANTI-Blue reagent was purchased from InvivoGen, USA.

Madhugrit powder(500 mg) was diluted with 10 ml water: methanol (50:50), sonicated for 30 min, centrifuged at 10,000×g for 5 min, and filtered by a 0.45 µm nylon filter. This solution was further used for the metabolite analysis. Prominence-XR UHPLC system (Shimadzu, Japan) equipped with a Quaternary pump (Nexera XR LC-20AD XR), DAD detector (SPD-M20 A), auto-sampler (Nexera XR SIL-20 AC XR), degassing unit (DGU-20A 5R) and column oven (CTO-10 AS VP) were used for analysis. Separation was achieved using a Shodex C18-4E (5µm, 4.6 X 250 mm) column subjected to binary gradient elution. The two solvents used for the analysis were: (A) water containing 0.1% orthophosphoric acid (pH 2.5) and (B) 0.1% orthophosphoric acid in a mixture of acetonitrile and water (88:12). Gradient programming of the solvent system: 5% B for 0-5 min, 5-10% B from 5-20 min, 10% B from 20-30 min, 10-20% B from 30-40 min, 20-35% B from 40-50 min, 35-60% B from 50-65 min, 60-85% B from 65-70 min, 85-5% B from 70-71 min and 5% B from 71-75 min with a flow rate of 1 ml/min. A 10 µl of standard and test solution was injected and the column temperature was maintained at 30°C. Wavelength of 270 nm was used for gallic acid, methyl gallate, protocatechuic acid, magnoflorine, corilagin, coumarin, cinnamic acid, piperine, and palmatine; 250 nm for ellagic acid and 350 nm for rutin. The obtained chromatograms of standards and sample were overlayed and the phytometabolites were quantified respectively at different retention times (RT).

The rat pancreatic cell line AR42J; rat myoblast cells L6; human monocytic cells THP-1 were procured from the ATCC licensed repository, National Centre for Cell Science, India. The human keratinocyte cells HaCaT were purchased from Krishgen Biosystems, India. The reporter cell lines namely HEK-Blue TNF-α, HEK-Blue IL-1β, and THP1-Blue NF-κB were obtained from In vivoGen, USA. AR42J, L6, and HaCaT were propagated in normal glucose (NG, 5.5 mM) DMEM supplemented with 10% FBS and 1% antibiotic-antimycotic solution. THP-1 cells were cultured in normal NG (5.5 mM) RPMI 1640 supplemented with 10% FBS and 1% antibiotic-antimycotic solution. The reporter cell lines were cultured as per the manufacturer’s instructions. The cultured cells were maintained at 37°C and 5% CO₂ in a humidified incubator and used within 5 passages after revival.

The effect of Madhugrit on the viability of AR42J, L6, THP-1 monocytes, THP1 macrophages, and HaCaT, were evaluated by Alamar blue dye (15 µg/ml). Madhugrit (10, 30, 100 µg/ml) was added during differentiation of AR42J into amylase-secreting exocrine cells by 100 nM dexamethasone and incubated for 72 hr (31–33). The L6 cells were differentiated with 2% Horse serum containing media for 72 hr post which the cells were incubated with Madhugrit (10-100 µg/ml) for 24 hr. THP1 monocytes and macrophages were incubated with Madhugrit (10-100 µg/ml) for 48 hr. HaCaT cells were also treated for 48 hr with Madhugrit (10-100 µg/ml). Following incubation with Alamar blue dye, the plates were read at Ex. 560/Em.590 nm by Envision multimode plate reader (PerkinElmer, USA). All treatments were performed with NG (5.5 mM) culture media. Data were presented as mean ± SEM (n=3).

AR42J cells were plated at a density of 1×10⁶ cells per well in a 6-well plate. Post 72 hr treatment, as described earlier, cells were washed with DPBS and induced with 1% (w/v) starch for 1 hr after which the supernatant was collected and α-amylase (U/L) levels were analyzed by Architect ci8200 biochemical analyzer (Abbott Diagnostics, USA), at an external laboratory. Undifferentiated cells (UDC) were used as normal control. Data were presented as mean ± SEM (n=3).

L6 cells were seeded at a density of 2×10⁵ cells per well in 12-well plate. After differentiation, cells were treated with Madhugrit (10, 30, and 100 µg/ml) or Metformin (2 mM) in serum-free NG (5.5 mM) culture media. After 24 hr the cells were washed with DPBS and stimulated with 100 µM 2-NBDG dye for 1 hr in DPBS, thereafter cells were washed twice with DPBS and lysed by 2 phased freeze-thaw process. The cell lysate was collected, centrifuged at 12,000×g for 10 min and 100 µl of the supernatant was pipetted in 96-well black plate and read at Ex.475/Em.529 nm by Envision multimode plate reader (PerkinElmer, USA). Data were presented as mean ± SEM (n=3).

The evaluation of the anti-glycation activity of Madhugrit was done as per Starowicz et al. (34) with slight modifications. Briefly, glucose (90 mg/ml) and BSA (10 mg/ml) were separately dissolved in DPBS. Then, Madhugrit (0-100 µg/ml) or Metformin (2 mM) were mixed with a BSA-glucose solution. The use of 0.01% sodium azide was done to prevent microbe development. After 1 week of incubation at 37°C, the fluorescence of AGEs was measured at Ex.350/Em.420 nm and the inhibition of AGEs formation was evaluated. Data were presented as mean ± SEM (n=3).

THP-1 monocytes (5×10⁵ cells) were pre-treated with Madhugrit (10, 30, and 100 µg/ml) or Metformin (2 mM) in T-25 flasks for 24 hr. Afterward, the cells were centrifuged, washed with DPBS, and plated at a density of 3×10⁴ cells/well in a 96-well plate. The cells were again treated with Madhugrit or Metformin in presence of 20 ng/ml PMA for 24 hr. All treatments were performed with NG (5.5 mM) culture media. Post incubation the cells were washed and Alamar blue assay was performed. Data were presented as mean ± SEM (n=3).

THP-1 monocytes were plated at a density of 1×10⁵ cells per well. The cells were differentiated to macrophages and pre-treated with Madhugrit (10, 30, and 100 µg/ml) or Metformin (2 mM) for 24 hr. Following incubation, the cells were washed and stimulated with 100 ng/ml LPS in presence of Madhugrit (10, 30, and 100 µg/ml) or Metformin (2 mM) for 24 hr. Untreated control (UC) group was taken as normal control. All treatments were performed with NG (5.5 mM) culture media. The cell supernatant was then collected and assessed for TNF-α and IL-6 release by sandwich ELISA as per the manufacturer’s instructions. Data were presented as mean ± SEM (n=4).

THP-1 macrophages were pre-treated with Madhugrit (10, 30, and 100 µg/ml) or Metformin (2 mM) for 24 hr. Later on, the cells were stimulated for 24 hr by LPS (500 ng/ml) and divided into 5 groups: (1) UC NG (5.5 mM), (2) LPS NG (5.5 mM), (3) UC HG (25 mM), (4) LPS HG (25 mM) and (5) LPS HG (25 mM) with Madhugrit (10, 30, 100 µg/ml) or Metformin (2 mM). After 24 hr the cell supernatant was collected and incubated with the NF-κB, TNF-α, and IL-1β reporter cells. Subsequently, based on the secreted embryonic alkaline phosphatase (SEAP) levels released by the reporter cells the activity of NF-κB, TNF-α and IL-1β was evaluated by QUANTI-Blue reagent as per the manufacturer’s instructions. The plates were read at an optical density of 630 nm by Envision multimode plate reader. Data were presented as mean ± SEM (n=3).

HaCaT cells (2×10⁵/well) were cultured in 12 well plates and pre-treated with Madhugrit (10, 30, 100 µg/ml) or Metformin (2 mM) for 24 hr. Later on, the cells were washed with DPBS and induced with mitomycin C (5 µg/ml) to inhibit proliferation. The cells were washed and wounded by a 10 µl plastic pipette tip and further rinsed with DPBS to remove cell debris. The cells were treated and divided into 5 groups: (1) UC NG (5.5 mM), (2) UC HG (25 mM), and (3) HG (25 mM) with Madhugrit (10, 30, 100 µg/ml) or Metformin (2 mM). Images were acquired at 0 hr and 24 hr. Analysis of % wound closure and rate of migration (µm/hr) were done by Fiji, ImageJ (NIH, USA) (13, 35). Data were presented as mean ± SEM (n=3).

The N2 (wild type) C. elegans strain was procured from the Caenorhabditis Genetics Center (CGC) and maintained in NGM (nematode growth medium) seeded with E. coli OP50 at 20°C. A synchronization technique was used to separate the eggs from the worms by treatment with an alkaline hypochlorite solution (25, 36). The hatched eggs released the L1 larvae, used for exposure to various treatments. The L1 nematodes were exposed to different concentrations of Madhugrit (3, 10, and 30 μg/ml) or Metformin (2 mM) for 24 hr. After incubation, the nematodes were transferred to an NGM plate seeded with E. coli OP50 with or without high glucose (HG, 100 mM) supplementation along with Madhugrit (3, 10, and 30 μg/ml) or Metformin (2 mM). Treatment scheme and parameters analyzed on C. elegans experimental model are depicted in Figure 1 .

The nematodes were treated as described above and were maintained on NGM/E. coli OP50 plates until they reached larval stage L4. To evaluate progeny size, one nematode from each treated group was transferred to a new NGM plate seeded with E. coli OP50 with or without 100 mM glucose supplementation along with Madhugrit (3, 10, and 30 μg/ml) or Metformin (2 mM). The total number of progenies were counted by ZEISS Stemi 305 stereo microscope (Carl Zeiss, Germany). Data were presented as mean ± SEM (n=5).

After pre-treatment, 30 worms of the L4 stage were transferred to new NGM plates seeded with E. coli OP50 with or without 100 mM glucose supplementation along with Madhugrit (3, 10, and 30 μg/ml) or Metformin (2 mM). The worms that displayed a curling behavior (37) were counted by ZEISS Stemi 305 stereo microscope (Carl Zeiss, Germany). Data were presented as mean ± SEM (n=5).

The pre-treated L4 stage nematodes were transferred to a new NGM plate seeded with E. coli OP50 with or without 100 mM glucose supplementation along with Madhugrit (3, 10, and 30 μg/ml) or Metformin (2 mM). On Day 5, worms were washed off from the plates and centrifuged at 600×g for 1 min. Worms were washed rigorously and placed in lysis buffer (Tris 100 mM, NaCl 150 mM, EGTA 1 mM, EDTA 1 mM, Triton X-100 1%, and sodium deoxycholate 0.5%) supplemented with protease inhibitor cocktail (Thermo Fisher Scientific, USA) and passed through three cycles of freeze-thaw and centrifuged at 14000×g for 10 min. The supernatant was collected and analyzed for glucose and triglyceride levels by Erba 200 biochemical analyzer (Erba Mannheim, Germany). Protein concentration was determined using the Pierce BCA protein assay kit. The obtained values were further normalized with protein concentration. Data were presented as mean ± SEM (n=3).

After treatment, worms were washed off from the plates and centrifuged at 600×g for 1 min. The worms were washed thrice and fixed with 40% isopropanol for 3 min, centrifuged at 600×g for 1 min, and stained with Nile red as per the protocol of Escorcia et al. (38). The images were acquired using a FITC filter on an Olympus BX43 microscope equipped with a Mantra imaging platform (PerkinElmer, USA) and further processed on Inform 2.2 software suite (PerkinElmer, USA).

ROS levels in C. elegans were detected by the H₂DCFDA dye as mentioned by Zhou et al. (39) with slight modifications. Briefly, worms were lysed, centrifuged and the supernatant was incubated with H₂DCFDA (200 µM) at 37°C for 1 hr. The plates were read at Ex. 495/Em.523 nm by Envision multimode plate reader and the fluorescence values were normalized with protein concentration (40). Data were presented as mean ± SEM (n=3).

The MDA content from the lysate of treated nematodes was measured by the TBA-TCA method (41). The optical density was read at 532 nm by Envision multimode plate reader and the amount of MDA present in the samples was determined from the standard curve. The data was further normalized with protein concentration. Data were presented as mean ± SEM (n=3).

Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons post-hoc test. Data were analyzed using GraphPad Prism 7 (GraphPad Software, USA). Results were considered to be statistically significant at a probability level of p < 0.05.

The quantitative analysis of phytometabolites using standard marker compounds ( Figure 2 ) confirmed the presence of rutin, ellagic acid, gallic acid, protocatechuic acid, methyl gallate, magnoflorine, corilagin, palmatine, coumarin, cinnamic acid and piperine in Madhugrit. The quantitative analysis of phytochemicals using reference standards is mentioned in Table 1 .

The effect of Madhugrit on the viability of cells was assessed on rat pancreatic exocrine like AR42J cells ( Figures 3A, F ), rat L6 myotubes ( Figures 3B, G ), human monocytic THP1 cells ( Figures 3C, H ), human THP1 macrophages ( Figures 3D, I ) and human HaCaT epidermal keratinocytes ( Figures 3E, J ). The cell lines were subjected to various concentrations of Madhugrit (10, 30, and 100 µg/ml) and no significant decrease in cell viability was observed even at 100 µg/ml concentration in all the cell lines. These preliminary results indicated that Madhugrit is non-toxic at all physiologically relevant concentrations and has no effect on the metabolic functionality of cells.

The AR42J cells was differentiated into an exocrine acinar-like pancreatic cell line (31) as the undifferentiated cells secrete less α-amylase in response to starch ( Figure 4A ). The bioactivity of Madhugrit in presence of starch load which mimics the physiological postprandial condition was determined on the differentiated cells. It was observed that Madhugrit (10, 30, and 100 µg/ml) treated cells released significantly (p <0.01) less α-amylase compared to control ( Figure 4A ) in response to 1% (w/v) starch. Next, to confirm the hypoglycemic activity of Madhugrit we evaluated its effect on glucose uptake levels in L6 cells (42). When compared with the control group, Madhugrit treatment with 10, 30, and 100 µg/ml significantly (p <0.05) promoted the uptake of glucose by 1.64, 2.48, and 2.84-fold respectively, in a concentration-dependent manner ( Figure 4B ). The results were at par with the positive control drug Metformin (2 mM) which also significantly (p <0.05) enhanced the glucose uptake levels by 2.53-fold compared to normal control.

Advanced glycation end products (AGEs) are formed in high amounts during diabetes which get accumulated and develop oxidative stress (43). It was observed that Madhugrit (3-100 µg/ml) inhibited the in vitro formation of AGEs in a concentration-dependent manner ( Figure 4C ). An inhibition of more than 15% was observed in the Madhugrit (100 µg/ml) treated group. Hence, it was established that Madhugrit possesses a property to inhibit AGEs formation.

The differentiation of monocytes to macrophages is a critical event that activates various pro-inflammatory signaling networks. The anti-inflammatory properties of Madhugrit were confirmed by the use of the in vitro model of THP1 monocyte to macrophage differentiation by PMA. It was observed that compared to control, Madhugrit at the concentration of 100 µg/ml significantly (p <0.01) decreased the conversion of monocytes to macrophages in presence of PMA ( Figure 5A ) as depicted by Alamar blue assay. Similarly, a significant (p <0.05) decrease was also observed in Metformin (2 mM) treated cells which was also previously reported by Vasamsetti et al. (28). Furthermore, the levels of LPS induced pro-inflammatory cytokines namely TNF-α and IL-6 were also evaluated post-Madhugrit treatment. An increase in the levels of LPS is observed due to an imbalance of gut microbiota during diabetes. LPS upon binding to toll-like receptor 4, found on immune cells activates a series of pro-inflammatory cascades, generally accompanied by an increase in levels of TNF-α and IL-6 (14, 44, 45). Madhugrit (10, 30, and 100 µg/ml) treated THP1 macrophages inhibited the release of LPS (100 ng/ml) induced TNF-α and IL-6 release, in a concentration-dependent manner ( Figures 5B, C ). The inhibitory effect of Madhugrit at 100 µg/ml concentration was more pronounced than Metformin (2 mM) in the case of TNF-α ( Figure 5B ) and IL-6 release ( Figure 5C ). Taken together, Madhugrit has anti-inflammatory properties, evident by its potential to inhibit macrophage differentiation and release of pro-inflammatory cytokines which is helpful to control the low-grade inflammation during diabetes.

Macrophages under hyperglycemic state constantly express a pro-inflammatory phenotype which is more prominent in presence of exogenous stimulants (endotoxins) (3). In high glucose (25 mM) conditions, it was observed that THP1 macrophages without any LPS (500 ng/ml) challenge, compared to normal glucose (5.5 mM) conditions, showed an increase in their pro-inflammatory phenotype which became more pronounced upon LPS stimulation ( Figures 5D–F ). It was observed that Madhugrit (10, 30, and 100 µg/ml) treated macrophages, in a concentration-dependent manner, were able to decrease their pro-inflammatory phenotype when exposed to high glucose and LPS, evident from the significantly decreased activity of NF-κB, TNF-α, and IL-1β ( Figures 5D–F ). Similar findings were observed in the Metformin (2 mM) treated group as well ( Figures 5D–F ). Taken together, these results suggest that Madhugrit has potentials to reduce persistent inflammatory phenotype of macrophages exposed to high glucose and bacterial endotoxins, in chronic diabetic conditions.

One of the common complication of diabetes is delayed and impaired wound healing process (46). A controlled inflammation and re-epithelization of the wound are paramount for effective wound healing. Keratinocyte migration, which is an integral part of re-epithelization is inhibited in high glucose conditions (13). Based on Madhugrit’s ability to resolve inflammation, we sought to examine its activity on wound healing under hyperglycemic conditions. As the HaCaT cells were induced with mitomycin C, the cell proliferation process was inhibited to remove any confounding observations related to the rate of migration. The HaCaT cells under high glucose (25 mM) condition displayed a visible defect in the wound healing process. In comparison, the cells treated with Madhugrit (10, 30, and 100 µg/ml) showed a prominent ability for wound healing, but it was not apparent in the Metformin (2 mM) treated group ( Figure 6A ). Based on the image analysis a significant (p <0.05) decrease in the percentage of wound closure and rate of cell migration was observed when HaCaT cells were kept in high glucose conditions ( Figures 6B, C ). Upon Madhugrit treatment, the cells started to display effective wound healing in a concentration-dependent manner. Notably, at 100 µg/ml concentration of Madhugrit the rate of migration and percentage of wound closure was equivalent to that of HaCaT cells kept in normal glucose (5.5 mM) condition ( Figures 6B, C ). These results suggest that Madhugrit has a potential to normalize the wound healing process in a high glucose condition.

A constant level of high glucose leads to multi-organ dysfunction which underlies the complications of diabetes (47). To confirm the potency of Madhugrit in vivo, the non-mammalian model of C. elegans was utilized as the effects of glucose toxicity have been previously reported on the nematode (25). The effect of standalone Madhugrit (3-30 µg/ml) treatment on Brood size was evaluated which showed that it had no toxic effect (data not shown). Further experiments were performed using the same concentrations of Madhugrit. The worms exposed to high glucose (100 mM) showed a significant (p <0.001) reduction in their brood size but the worms exposed to 30 µg/ml concentration of Madhugrit were able to significantly (p <0.001) counteract the negative effects of glucose on their egg laying capacity. Similar effects were observed in the case of Metformin ( Figure 7A ). Under high glucose exposure, C. elegans displayed aberrant behavior like curling. Nearly, 80% of the worms were observed to have curling behavior due to glucose-induced stress. Madhugrit (3, 10, and 30 µg/ml) treated worms showed a decrease in the incidences of curling behavior in a concentration-dependent manner ( Figure 7B ). The positive control drug Metformin (2 mM) also reduced % curling in the high glucose exposed worms. The observed increase in brood size and a decrease in % curling suggests that Madhugrit has a strong potential to negate the effects of glucose toxicity on normal physiology.

The qualitative assessment of the lipid content post glucose exposure and Madhugrit treatment was evaluated by Nile red staining. The worms exposed to 100 mM glucose clearly showed increased lipid accumulation compared to worms grown without added glucose. Madhugrit (3, 10, and 30 µg/ml) treated worms showed a visible reduction in lipid content in a dose-dependent manner. Worms with Metformin (2 mM) treatment also displayed less lipid deposition ( Figure 7C ). Furthermore, the worms exposed to high glucose (100 mM) showed a significant (p <0.05) increase in their glucose (0.92 mM/mg protein) and triglyceride (137.8 mg/mg protein) levels compared to worms grown without added glucose ( Figures 7D, E ). Such biochemical alterations were significantly reduced in the Madhugrit (3, 10, and 30 µg/ml) treated nematodes. Metformin (2 mM) treated worms also efficiently resisted the biochemical changes in response to high glucose exposure. The worms treated with 30 µg/ml of Madhugrit had nearly normalized levels of glucose and triglyceride levels ( Figures 7D, E ).

The evaluation of changes in ROS levels was done by the normalized fluorescence intensity of dichlorofluorescein (DCF) (48). High glucose exposure significantly (p <0.001) increased the DCF fluorescence compared to worms grown without added glucose ( Figure 7F ). Madhugrit (3, 10, 30 µg/ml) treated nematodes were able to counteract the development of ROS in response to high glucose as observed by the dose-dependent decrease in fluorescence ( Figure 7F ). An increase in ROS causes the overproduction of MDA, an indicator of oxidative stress (49). The worms exposed to 100 mM glucose displayed a significant (p <0.001) increase in MDA (404.9 nM/mg protein) levels ( Figure 7G ). Whereas, Madhugrit (3, 10, 30 µg/ml) treated worms were able to offset the generation of oxidative stress in a dose-dependent fashion ( Figure 7G ). The 30 µg/ml Madhugrit treated worms had MDA (121.4 nM/mg protein) levels equivalent to the worms grown without added glucose (121.3 nM/mg protein). Collectively, these results implied that Madhugrit possesses robust anti-oxidant properties.