Ashwagandha supplementation has been associated with reduced self-reported stress and anxiety, although most trials have used doses of 250–600 mg daily for 6–8 weeks. This study examined the effects of a low-dose ashwagandha extract (Ashwa.30™) on perceived stress, mood, fatigue, and stress reactivity in stressed adults. In this 28-day, randomised, double-blind, placebo-controlled trial, 60 adults aged 18–65 years with self-reported stress were randomly allocated to receive ashwagandha 30 mg/day or placebo. Outcomes included the Depression, Anxiety, and Stress Scale, daily mood ratings, and the Chalder Fatigue Scale. The Socially Evaluated Cold-Pressor Test (SECPT) was conducted on Days 7 and 28, with subjective stress and fatigue, cardiovascular and electrodermal measures, skin temperature, and salivary cortisol and chromogranin A assessed. Compared to the placebo, ashwagandha intake was associated with greater reductions in self-reported stress in the full analysis set (primary analysis; p = 0.046). The per protocol set analysis was directionally consistent but not statistically significant. Mean daily stress ratings were also lower in the ashwagandha group (p = 0.043). During the Day 28 SECPT, the ashwagandha group reported lower stress ratings (p = 0.044), and salivary cortisol concentrations remained relatively stable in the ashwagandha group but increased in the placebo group, suggesting attenuation of cortisol reactivity (p = 0.047). However, no statistically significant between-group differences were detected for other secondary and physiological outcomes. An ashwagandha extract (Ashwa.30™) administered at 30 mg daily was associated with reduced self-reported stress in the Full Analysis Set and attenuation of SECPT-related salivary cortisol reactivity. These preliminary findings support further investigation in larger, adequately powered trials. Australian and New Zealand Clinical Trials Registry: (ACTRN12625000129482) https://www.anzctr.org.au/ACTRN12625000129482p.aspx .

Asparagus racemosus, commonly known as Shatavari, is an established Ayurvedic plant whose bioactivity is credited to its steroidal saponins, generally referred to as shatavarins, exerting adaptogenic and estrogenic effects. However, traditional methods of isolation often produce products with limited purity and poor reproducibility, which seriously limit scalability in pharmacological applications. This study describes an effective, scalable, flash chromatography-based approach toward the isolation and purification of six bioactive steroidal saponins, AR-1 to AR-6, from AR root material. The methanolic extract was subjected to successive liquid-liquid partitioning and multistep chromatographic purification to give high-purity compounds identified as spirostan derivatives (AR-1 to AR-4), shatavarins, and aglycone derivatives (AR-5 and AR-6). The structural identities and chemical homogeneity were clearly supported by various spectroscopic analyses: HPLC-ELSD, FTIR, NMR, and LC-MS. The in silico ADME predictions performed by SwissADME were used as a prioritization strategy to inform compound selection for biological testing, providing relative comparisons of absorption potential rather than validating oral bioavailability. Derivatives of aglycone compounds AR-5 and AR-6 showed greater predicted absorption potential, while highly glycosylated saponins AR-2 and AR-4 showed low permeability, as expected for compounds of their size and polarity. While steroidal saponins are not typical of oral drug chemical space, these models correlate well with permeability and metabolic properties. Molecular docking studies of GPER and estrogen receptors (ERα, ERβ, and ERRγ) showed binding affinities of -7.7 to -12.4 kcal/mol, with AR-1, AR-2, and AR-4 showing a preference for hydrophobic and hydrogen-bonding interactions in GPER and ERα binding pockets typical of estrogen ligands. The developed flash chromatography workflow represents an efficient, scalable route to pharmacologically relevant standards of shatavarins. Combined with chromatographic and in silico results, the findings reveal the translational potential of AR saponins as multitarget phytoestrogens for hormone-modulatory and adaptogenic therapeutic applications.

[This corrects the article DOI: 10.1371/journal.pone.0285012.].

Background The immune system plays a crucial role in protecting the host by regulating biological activities in multiple organs and circulatory fluids. The phytoextracts are evident to modulate a series of biological activities and play a protective role in managing healthy conditions through interaction with the immune system and exhibit antioxidant, antitumor, anti-inflammatory, antimicrobial, and immunomodulatory activities. Objectives Understanding the function and regulation of immunomodulation may provide potential strategies for the development of natural extracts for the management of adverse conditions. Materials and Methods In the present study, we have investigated the immunomodulatory activity of proprietary polyherbal formulation (Phytocee™) containing Emblica officinalis, Ocimum sanctum , and Withania somnifera using in vitro macrophage phagocytosis and ex vivo natural killer (NK) cell activity. The effect of water and methanolic extracts of Phytocee on NK cell activity was performed using mice splenocytes as effector cells and YAC-1 fibroblast as target cells. The effect of Phytocee on phagocytosis in murine macrophage (J774A.1) cells against fluorescein-labeled bioparticles was also investigated. Results The significant increase in NK cell activity of 35% at 200 µg/mL of water extract and dose-dependent increase with methanol extract confirmed the NK cell-mediated immunomodulation of Phytocee. A phagocytic accumulation of bioparticles within macrophages at 100 and 200 µg/mL of extract group suggests a macrophage activating role and immuno-effectiveness of Phytocee herbal formulation. Conclusion Based on the current data and the historical use of ingredients in the polyherbal formulation support the immunoprotective role of Phytocee. Keywords Immunomodulation , natural killer cells , macrophages , , , , , ,

Background Andrographis paniculata (Burm. f.) Nees is a well-recognized medicinal plant known for its benefits against a multitude of ailments. The intent of the present study was to evaluate the effect of a standardized extract of A. paniculata on innate immunity. Materials and Methods Phagocytic activity was measured in terms of phagocytic index using carbon clearance methodology in mice. In another experiment, NK cells (effector cells) were isolated from the mouse spleen after the dosing period, and NK cells-mediated lysis of target cells (YAC-1 cells) were determined using an MTT assay. Results The phagocytic index and NK cell activity were found to be significantly ( p < 0.05) different in the treated group compared to the control group in the tested animal models at a dose of 41 mg/kg. Conclusion The results suggest the potential of A. paniculata to enhance innate immunity. Keywords Andrographis , carbon clearance , immunity , NK cells , phagocytes

Panchatikta is an Ayurvedic drug combination of 5 bitter-tasting drugs, namely Azadirachta indica, Tinospora cordifolia, Justicia adhatoda, Solanum virginianum and Trichosanthes dioica. In this study, estimations of the phenolic, flavonoids and tannin contents, HPTLC profile and in vitro anti-oxidant and anti-microbial potential of 3 extracts of the Panchatikta were carried out. The total phenolic content of the Panchatikta combination was almost the same in ethanol, water and hydroalcoholic extracts. Flavonoid content and tannin content were highest in ethanol extract. HPTLC profiling of the 3 extracts of Panchatikta showed the maximum number of peaks for the ethanolic extract. Water extract showed a comparatively smaller number of peaks. The maximum number of peaks was seen after derivatisation. In the DPPH assay, gallic acid has IC50 value of 5.50 ± 0.38 µg/mL; ethanol and hydroalcoholic extracts have IC50 close to 60 µg/mL and water extract has around 90 µg/mL. The ABTS assay showed standard ascorbic acid has a IC50 value of 67.33 ± 0.58 µg/mL while Panchatikta extracts have a much higher IC50. In nitric oxide assay also, tested samples have a higher IC50 when compared with that of ascorbic acid (177.33 ± 5.09). In well diffusion assay for antibacterial screening, all 3 extracts have a significant zone of inhibition at concentrations of 500 µg and 1000 µg. Pseudomonas aeruginosa was found to be resistant to all 3 test samples and standard antibiotic discs. Minimum activity was obtained against Klebsiella pneumonia. Panchatikta extracts at concentrations of 500 µg and 1000 µg showed significant activity against the fungal strains Aspergillus niger and Fusarium oxysporum. In conclusion, the results of the study can provide scientific support for the traditional usage of the Ayurvedic drug combination Panchatikta for various ailments.

Background: Andrographis paniculata (Burm. f.) Nees is a well-recognized medicinal plant known for its benefits against a multitude of ailments. The intent of the present study was to evaluate the effect of a standardized extract of A. paniculata on innate immunity.Materials and Methods: Phagocytic activity was measured in terms of phagocytic index using carbon clearance methodology in mice. In another experiment, NK cells (effector cells) were isolated from the mouse spleen after the dosing period, and NK cells-mediated lysis of target cells (YAC-1 cells) were determined using an MTT assay.Results: The phagocytic index and NK cell activity were found to be significantly (p < 0.05) different in the treated group compared to the control group in the tested animal models at a dose of 41 mg/kg.Conclusion: The results suggest the potential of A. paniculata to enhance innate immunity.

Ocimum tenuiflorum, commonly referred to as holy basil or Tulsi, has a long history of use in traditional medicine systems, particularly Ayurveda, due to its various health benefits, including anti-inflammatory, antioxidant, and adaptogenic properties. In contemporary contexts, this plant is progressively incorporated into dietary supplements and nutraceuticals. Given its widespread use and potential health beneficial properties, it is imperative to scientifically evaluate the safety of Ocimum tenuiflorum. This study presents comprehensive safety assessments of a standardized extract of Ocimum tenuiflorum. We conducted a series of genotoxicity studies, including the bacterial reverse mutation test (BRMT), in-vitro mammalian chromosomal aberration (CA) test, and in-vivo mammalian erythrocyte micronucleus (MN) test in Swiss Albino mice. Additionally, an acute oral toxicity study was performed using Sprague Dawley rats, adhering to OECD guidelines in a GLP-compliant laboratory. The results showed no mutagenic effect with O. tenuiflorum extract up to a dose of 5000 µg/plate in BRMT. The results of CA test revealed the non clastogenic activity of O. tenuiflorum extract up to a dose of 500 µg/mL with and without metabolic activation (S9). Ocimum tenuiflorum extract was found to be non-clastogenic at the highest tested dose of 2000 mg/kg bodyweight in invivo MN test. In acute oral toxicity study, O. tenuiflorum extract was found to be safe up to 5 g/kg bodyweight in Wistar rats. Collectively, these findings suggest that Ocimum tenuiflorum extract is non-genotoxic and safe for oral consumption up to 5000 mg/kg body weight in Sprague Dawley rats.

Panchatikta is an Ayurvedic multiherbal combination containing five bitter-tasting drugs-Azadirachta indica, Tinospora cordifolia, Justicia adhatoda, Solanum virginianum, and Trichosanthes dioica. The objective of this study is to develop standards for Panchatikta ingredients using pharmacognostic parameters and to analyze the chemical composition. The plant materials were collected from different natural habitats, authenticated, dried, ground, and the combination was prepared by mixing the ingredients in equal amounts followed by macroscopic, organoleptic, microscopic, histochemical, and physicochemical characterization according to the standard protocol of the Ayurvedic Pharmacopoeia of India. Special morphological characteristics of the A. indica (stem bark), T. cordifolia (stem), J. adhatoda (roots), S. virginainum (roots), and T. dioica (aerial parts) were identified. The anatomical structure of each drug was observed, and microscopic markers were developed for identification. Histochemical deposits such as starch, oil, lignin, and tannin were located in each plant material. Powder characters such as fibers from phloem with pointed ends and narrow lumen, parenchyma cells containing starch grains, crystal fibers, fragments of bordered pitted vessels, fragments of pitted vessels, cells containing black powdery masses, and epidermal cells from leaves in surface view with an anomocytic stomata were identified as the key features for the Panchatikta combination. The organoleptic characterization of component drugs and combinations was performed. The physicochemical evaluation of parameters such as ash content (5.53 +/- 0.29), water-insoluble ash (4.57 +/- 0.32), acid-insoluble ash (1.55 +/- 0.31), alcohol-soluble extract (5.9 +/- 1.21), water-soluble extract (9.19 +/- 0.21), fiber content (33.27 +/- 1.37), total sugar (1.83 +/- 0.65), and reducing sugar (1.73 +/- 0.40) were evaluated for the Panchatikta combination. Preliminary phytochemical tests were carried out to verify the presence of alkaloids, flavonoids, saponins, tannins, phenols, glycosides, and steroids. The HPTLC profile comparison of ethanolic, hydroalcoholic, and aqueous extracts of Panchatikta and single drugs showed some type of synergy and antagonistic activities among the chemical principles present. Marker-based screening of azadirachtin, betulinic acid, and lupeol was done in Panchatikta ethanol using HPTLC. As Panchatikta is prescribed for various life-threatening diseases including cancer, the standards developed here ensure the authenticity of the ingredients, which in turn ensures the quality of the prepared medicines.

Background: The immune system plays a crucial role in protecting the host by regulating biological activities in multiple organs and circulatory fluids. The phytoextracts are evident to modulate a series of biological activities and play a protective role in managing healthy conditions through interaction with the immune system and exhibit antioxidant, antitumor, anti-inflammatory, antimicrobial, and immunomodulatory activities. Purpose: Understanding the function and regulation of immunomodulation may provide potential strategies for the development of natural extracts for the management of adverse conditions. Methods: In the present study, we have investigated the immunomodulatory activity of proprietary polyherbal formulation (Phytocee (TM)) containing Emblica officinalis, Ocimum sanctum, and Withania somnifera using in vitro macrophage phagocytosis and ex vivo natural killer (NK) cell activity. The effect of water and methanolic extracts of Phytocee on NK cell activity was performed using mice splenocytes as effector cells and YAC-1 fibroblast as target cells. The effect of Phytocee on phagocytosis in murine macrophage (J774A.1) cells against fluorescein-labeled bioparticles was also investigated. Results; The significant increase in NK cell activity of 35% at 200 mu g/mL of water extract and dose-dependent increase with methanol extract confirmed the NK cell-mediated immunomodulation of Phytocee. A phagocytic accumulation of bioparticles within macrophages at 100 and 200 mu g/mL of extract group suggests a macrophage activating role and immuno-effectiveness of Phytocee herbal formulation. Conclusion: Based on the current data and the historical use of ingredients in the polyherbal formulation support the immunoprotective role of Phytocee.

BACKGROUND:Bacopa monnieri (BM) is traditionally used in human diseases for its antioxidant, anti-inflammatory and neuroprotective effects. However, its anticancer potential has been poorly understood.AIM:The aim of this study was to explore the detailed anticancer mechanism of BM against oral cancer and to identify the bioactive BM fraction for possible cancer therapeutics.RESULTS:We performed bioactivity-guided fractionation and identified that the aqueous fraction of the ethanolic extract of BM (BM-AF) had a potent anticancer potential in both in vitro and in vivo oral cancer models. BM-AF inhibited cell viability, colony formation, cell migration and induced apoptotic cell death in Cal33 and FaDu cells. BM-AF at low doses promoted mitophagy and BM-AF mediated mitophagy was PARKIN dependent. In addition, BM-AF inhibited arecoline induced reactive oxygen species production in Cal33 cells. Moreover, BM-AF supressed arecoline-induced NLR family pyrin domain containing 3 (NLRP3) inflammasome activation through mitophagy in Cal33 cells. The in vivo antitumor effect of BM-AF was further validated in C57BL/6J mice through a 4-nitroquinolin-1-oxide and arecoline-induced oral cancer model. The tumor incidence was significantly reduced in the BM-AF treated group. Further, data obtained from western blot and immunohistochemistry analysis showed increased expression of apoptotic markers and decreased expression of inflammasome markers in the tongue tissue obtained from BM-AF treated mice in comparison with the non-treated tumor bearing mice.CONCLUSION:In conclusion, BM-AF exhibited potent anticancer activity through apoptosis induction and mitophagy-dependent inhibition of NLRP3 inflammasome activation in both in vitro and in vivo oral cancer models. Moreover, we have investigated apoptosis and mitophagy-inducing compounds from this plant extract having anticancer activity against oral cancer cells.

Ocimum tenuiflorum is a sacred medicinal plant bestowed with multiple health benefits. This plant is traditionally considered an adaptogen. Many scientific studies have indicated the anti-stress potential of Ocimum tenuiflorum but with higher doses. The present study investigated the effects of Holixer (TM) (a clinically studied standardized Ocimum tenuiflorum extract) on modulating stress using two in vivo models, namely the swim endurance study in mice and forced swim test in rats. In addition, we explored the mechanism of action of Holixer (TM) on the HPA axis using two in vitro cell-based assays to check for its inhibitory effect on cortisol release and CRF1 receptor antagonistic activity. Ocimum tenuiflorum extract enhanced the swimming time in mice, reduced the stress-induced increase in immobility time, and prevented the increase in corticosterone in rats subjected to the forced swim test. Further, Ocimum tenuiflorum extract inhibited cortisol release and exhibited a significant CRF1 receptor antagonist activity. Thus, Ocimum tenuiflorum extract was found effective in managing stress, and the effect could be due to the inhibition of cortisol release and the antagonistic effect on the CRF1 receptors.

Terminalia arjuna, is an important medicinal plant mainly used for cardiovascular ailments in the traditional Indian system of medicine Ayurveda. The stem bark of T. arjuna is generally employed in Ayurvedic formulations. The present study undertakes the possibilities of judicious utilization of stem bark and inner wood combinations of T. arjuna instead of bark alone in herbal medicines. Different combinations of bark and inner wood portions were prepared and extracted. Preliminary phytochemical analyses by qualitative tests, and quantitative estimations of total phenolics, and flavonoid contents revealed predominant results in bark extract. Anti-oxidant activities like DPPH, and ABTS radical scavenging assays were carried out and bark extract showed leading results and the combination sample also showed promising values. Comparative chemical profiling of the samples was done using High-performance thin layer chromatography to identify and quantify major chemical constituents. Flavonoids such as catechin, epicatechin, and phenolic acid, gallic acid were identified by HPTLC profiling, in all samples. On quantification of gallic acid, the bark and sample BS1 showed promising results when compared with other combination samples. The identified compounds were proven with medicinal activities against cardiovascular-related ailments. The present study concluded that BS1 (75:25) of bark and wood combination could be used instead of bark (B) for medicinal preparations without affecting the quality so the 25% usage of bark can be reduced and 25% of the stem can be saved from waste. The study will be beneficial for Ayurvedic industries using the bark T. arjuna for medicinal preparation since the wastage of wood can be reduced without affecting the quality of medicine.

•Euphorbia thymifolia and E. hirta are two medicinally important species used in Ayurveda.•Metabolite profiling by Q-TOF-LC-MS/MS led to the identification of many active compounds from the selected species.•Both species showed anti-cancer activity against ascites carcinoma in mice model.

Andrographis paniculata , commonly known as green chiretta, is a traditionally used plant in India, China, and Southeast Asian countries for its varied health benefits including immune health. The objective of the present study was to assess the safety of AP-Bio®, a standardized A. paniculata extract in Sprague Dawley rats by following the Organisation for Economic Cooperation and Development (OECD) test guidelines of acute and 90-day repeated dose sub-chronic toxicity studies. AP-Bio® did not show any treatment-related clinical signs of toxicity or mortality during the 14-day observation period in animals tested in the single-dose acute oral toxicity study up to a dose of 5000 mg/kg body weight. In the 90-day repeated dose sub-chronic oral toxicity study, no treatment-related adverse clinical signs were observed in any of the treated groups (300, 600, and 900 mg/kg). All treated animals showed usual weight gain and comparable feed intake. The ophthalmoscope examination did not reveal any abnormalities. Also, no toxicologically significant changes were observed in urinalysis, hematology, and blood chemistry parameters. Absolute organ weights and relative organ weights of vital organs did not differ significantly compared to control. Gross and histopathological findings did not show any remarkable and treatment-related changes. Results of the safety evaluation showed the median lethal dose (LD 50 ) of AP-Bio® was found to be more than 5000 mg/kg rat body weight and the no observed adverse effect level (NOAEL) of AP-Bio® was found to be 900 mg/kg rat body weight.

History of safe use, HoSU preparations are formulations mentioned in ancient literature and are according to the Ayurvedic system of medicine. Due to the advent of new technologies, there is a shift in the manufacturing methodologies of Ayurvedic medicines involving extractions and fractionations. This shift, while has helped manufacturers improve their productivity, raises a question on the equivalence to chemistry, and toxicity when compared to traditional products. As a case study, an attempt is made to establish equivalence between HoSU preparation of Terminalia chebula with commercially available aqueous and alcoholic extracts and one of its fractions. Chemical analysis of the products using principal component analysis, revealed similar chemistry whereas the LC50 values of HoSU preparation were safest followed by commercial preparations and the reference marker compound when tested on zebrafish. The generated data indicated that chemical composition of all 4 samples tested were similar qualitatively, though variations were observed quantitatively. PCA plot indicated that the HoSU extract, commercial alcoholic extract, and commercial water extract were similar whereas water acetone extract was dissimilar. This emphasizes the need for conducting an equivalence study in terms of chemistry and toxicity between HoSU products and commercially available products.

CONTEXT:Turmeric (Curcuma longa) is a common medicinal plant used in traditional medicine that also has been scientifically validated for its antioxidant, anti-arthritic, anticancer, analgesic, and anti-inflammatory properties. Researchers have still not much explored the beneficial effects of the curcuminoid-free portion of turmeric. NR-INF-02 is a proprietary, patented aqueous extract of Curcuma longa comprising turmerosaccharides with a novel phytochemical composition.OBJECTIVE:The study intended to evaluate the safety and tolerability of NR-INF-02 in healthy adult volunteers at doses of 1000 and 2000 mg, administered for 84 days.DESIGN:The study employed a randomized, open label, two-arm, parallel-group design.SETTING:The trial was carried at 2 sites, the Meenakshi Multispecialty Hospital in Chennai, Tamil Nadu, India and the Vijaya Super Specialty Hospital in Nellore, Andhra Pradesh, India.PARTICIPANTS:Participants were healthy adult, male or female volunteers, aged 18-65 years with a body mass index of ≥18.5 kg/m2 and ≤ 24.9 kg/m2 and a body weight of at least 55 kg for men and 48 kg for women.INTERVENTION:Participants were randomly divided into 2 groups with 24 participants each for a total of 48 participants. They received either 1000 or 2000 mg of NR-INF-02 for 84 days.OUTCOME MEASURES:The incidence of adverse events and the changes from baseline in clinical laboratory parameters-including hematological, biochemical, and urinalysis parameters-were assessed at baseline, at day 42, and postintervention at day 84 as primary endpoints for safety. Secondary endpoints were the changes in vital signs and the difference in the results of an electrocardiogram (ECG) between baseline and days 42 and 84.RESULTS:The NR-INF-02 at doses of 1000 and 2000 mg demonstrated a 4.17% and 20.83% incidence of adverse events (AEs), respectively. The AEs were mild to moderate and were either probably or possibly related, but not definitively, related to treatment. A detailed examination of hematological, biochemical, and urological parameters and of ECG results and vital signs didn't indicate any untoward effects for any participant.CONCLUSION:The study found NR-INF-02 to be safe and tolerable at both tested doses for the given duration of the trial for healthy adult volunteers.

Abstract Background Intestinal epithelial barrier dysfunction predisposes to many gastrointestinal, metabolic, and psychological disorders. A flavonoid rich extract of Glycyrrhiza glabra (FREG) has previously been reported to possess anti-inflammatory, antioxidant, and antiulcer properties. Aim To investigate the effect of FREG (GutGard®) on restoring intestinal barrier function in tumor necrosis factor-alpha (TNF-α) stimulated human colonic adenocarcinoma cell monolayer (Caco-2) and 2,4,6-Trinitrobenzenesulfonic acid (TNBS) induced ulcerative colitis in rats. Methods In in vitro, human intestinal Caco-2 cell monolayers were treated with TNF-α in the presence or absence of FREG and the paracellular permeability to FITC-conjugated 4-kD dextran (FD4) was measured to evaluate protection against the barrier dysfunction. In in vivo, intestinal barrier dysfunction was induced in male albino Wistar rats via intrarectal instillation of TNBS. Subsequently, the rats were treated orally with either FREG at 6.25, 12.5, and 25 mg/kg body weight, or Mesacol (250 mg/kg) for 5 days. On day 5, intestinal epithelial permeability was assessed with FD4 leakage into the serum. Also, colonic inflammation, colon morphology, histology and macroscopic score, weight to length ratio were evaluated. The activity of myeloperoxidase (MPO), TNF- α, secretory IgA levels and tight junction proteins expression were evaluated in rat’s colon. Results FREG protected the intestinal epithelial barrier integrity in human intestinal Caco-2 cells in vitro. FREG administration significantly improved the intestinal epithelial barrier function as evident from significant reduction in FD4 leakage. The colon morphology, histology score, macroscopic score, colon weight to length ratio also indicates beneficial effects of FREG on barrier function. In addition, FREG regulated the tight junction proteins, and markedly decreased TNF-α, MPO levels and significantly increased the secretory IgA levels in TNBS induced colitis rats. Conclusion The study findings support the protective action of FREG on intestinal epithelial barrier integrity indicating its potential in protecting from implications of leaky gut.

Glycyrrhiza glabra (G. glabra) is well known for its health benefits based on the traditional and current scientific evidence. The aim of the present study was to evaluate the safety of GutGard, a standardised-flavonoid rich extract of G. glabra. The study was designed to evaluate the acute and subchronic oral toxicity of GutGard in Sprague Dawley rats according to the procedures and methods of Organisation for Economic Cooperation and Development (OECD) test guidelines for acute and subchronic toxicity. A single dose of GutGard at 5000 mg/kg body weight did not produce treatment related clinical signs of toxicity or mortality in any of the animals tested during the 14-day observation period. Therefore, the median lethal dose was estimated to be more than 5000 mg/kg. A subchronic oral toxicity study for 90 days in rats at the dose levels of 250, 500, and 1000 mg/kg did not show any treatment related adverse clinical signs. The treated animals exhibited normal weight gain and comparable feed intake. Ophthalmoscope examination did not reveal any abnormalities. Further, GutGard administration in rats did not show any clinical evidence of toxicity with respect to urinalysis, haematology, and blood chemistry parameters. The relative organ weight of vital organs did not differ significantly as compared to control. Gross and histopathological findings did not show any remarkable and treatment related changes. Based on the current experimental study findings, the median lethal dose (LD50) of GutGard was found to be >5000 mg/kg b.wt and the no observed adverse effect level (NOAEL) was found to be 1000 mg/kg rat b.wt.