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Background: Presently, varied case reports demonstrated an increase or decrease in blood concentration of diverse conventional drugs, often co-administered with edible fruits, spices, or vegetables. The overarching aim of this research is to elucidate the fluctuations in tacrolimus (TAC) blood concentration on the consumption of pomegranate rind extract (PRE).Methods: A pharmacokinetic (PK) study was conducted with two groups, vis-a-vis PRE + TAC (3 mg/kg) and TAC (3 mg/kg) alone groups. An experimental study was conducted in three different manners: Single-dose (S) PRE (200 mg/kg), 7-day repetitive (7-R) PRE (200 mg/kg) dosing, and multiple (M) PRE doses (100, 200, 400, and 800 mg/kg). All the blood samples (approximately 300 μl) were drawn at different time intervals, i.e., 30 min, 1, 2, 4, 8, and 12 h after oral administration of TAC (3 mg/kg). The estimation of TAC in rat plasma was done using the hyphenated technique LC-MS/MS where the mass spectrometer used was a triple-stage quadrupole in multiple-reaction monitoring (MRM) mode.Results: The findings depict that in comparison with the TAC (3 mg/kg) alone group with the 7-day repetitive (7-R) PRE (200 mg/kg) dosing, the Cmax was found to be 9.03 ± 1.21 ng/ml; AUC from time zero to infinity (AUC0-∞), 61.91 ± 17.37 ngh/ml, while the TAC (3 mg/kg) + PRE group exhibited an increase in PK parameters of TAC (Cmax 22.48 ± 3.07 ng/ml; AUC0-∞ 153.08 ± 13.24 ng h/ml). The authors further investigated in what manner the PRE affects the PK of TAC in animals. For this, docking studies with major phytoconstituents present in the PRE with CYP3A4 isoenzyme were carried out. Ellagitannins (dock score, −11.64) and punicalagin (dock score, −10.68) were again used for molecular simulation studies with TAC. To validate our findings, a CYP3A4 inhibitory in vitro assay was conducted.Conclusion: Based on the integrated in vivo and in silico studies, we concluded that pomegranate rind extract interacts strongly with CYP isoenzyme and is therefore responsible for the altered PK profile of TAC.

Background: The identification of genoprotectants is a promising strategy for improving human health. Piper longum has drawn scientific attention because of its diverse biological effects and traditional utilization. The current investigation aims to evaluate the genome-stabilizing potential of Piper longum against cyclophosphamide-associated genotoxicity.Methods: We adopted a funnel screening with a three-tier evaluation approach, where Piper longum was investigated in an acellular medium, peripheral blood lymphocytes, and a rodent model. The genoprotective action of the Piper longum extract was initially performed with plasmid pBluescript SK(-) DNA. Furthermore, the extract and various fractions were screened against cyclophosphamide-induced genotoxicity using a cytokinesis-block micronucleus assay and a chromosomal aberration assay in human peripheral blood lymphocytes. The genome-stabilizing action of the extract and potent (hexane) fraction was further confirmed in vivo in Wistar albino rats by evaluating them using mammalian erythrocyte micronucleus tests, DNA fragmentation, oxidative stress markers, 8-hydroxy-2-deoxyguanosine (8-OHdG), γH2AX, and histopathological lesions in the liver and hippocampus. Additionally, acute and sub-acute toxicity studies were conducted following the Organization for Economic Co-operation and Development (OECD) guidelines for rats. Furthermore, the extract was quantified and characterized by high-performance thin-layer chromatography (HPTLC), ultra-high performance liquid chromatography–mass spectroscopy (UPLC-MS), and gas chromatography–mass spectrometry (GC-MS).Results: The Piper longum ethanol extract was shown to protect plasmid pBluescript SK(-) DNA against H2O2-induced strand breaks. In human lymphocytes, the extract and hexane fraction showed a reduction in micronucleus formation (p < 0.001) and chromosomal aberrations (p < 0.01) against cyclophosphamide. Furthermore, the extract and fraction treatment, when administered at 200 mg/kg for 28 days in Wistar rats, restored cyclophosphamide-induced genomic instability by reducing micronucleus formation and DNA fragmentation; restoring redox homeostasis; decreasing 8-OHdG, a hallmark of oxidative DNA damage; reducing γH2AX, a DNA double-strand break (DSB) marker; and preserving the liver and hippocampus against histopathological lesions. The extract and fraction revealed no signs of systemic toxicity at the used doses. Piperine and piperlongumine are the major alkaloids quantified along with the presence of flavonoids in the ethanol extract and the presence of fatty acids and terpenoids in the hexane fraction of Piper longum.Conclusion: Our investigation confirms the genoprotective action of Piper longum by reducing cyclophosphamide-associated cytogenotoxicity, oxidative stress, hepato- and neurotoxicity, oxidative DNA damage, and DNA double-strand breaks. The outcomes are critical for mitigating the genotoxic effects of chemotherapy recipients, requiring further attention.

The study was aimed to develop a characterized polyherbal combination as an immunomodulator containing Phyllanthus emblica L., Piper nigrum L., Withania somnifera (L.) Dunal, and Tinospora cordifolia (Willd.) Miers. Through response surface methodology (RSM), the ratio of aqueous extracts of four plant materials was optimized and comprised 49.76% of P. emblica, 1.35% of P. nigrum, 5.41% of W. somnifera, and 43.43% of T. cordifolia for optimum immunomodulatory activity. The optimized combination showed antioxidant potential and contains more than 180 metabolites, out of which gallic acid, quercetin, ellagic acid, caffeic acid, kaempferitrin, and p-coumaric acid are some common and significant metabolites found in plant extracts and in polyherbal combination. Treatment with the polyherbal combination of different doses in cyclophosphamide-induced immunosuppressed mice significantly (p < 0.01) enhanced the subsets of immune cells such as natural killer (NK) cells (60%), B cells (18%), CD4 cells (14%), and CD8 cells (7%). The characterized polyherbal combination exhibited potent immunomodulatory activity, which can be further explored clinically for its therapeutic applicability.

The toxicity and disposal concerns of organic solvents used in conventional extraction purposes has entailed the need for greener alternatives. Among such techniques, supercritical fluid extraction (SFE) has gained popularity by yielding extracts of high purity in a much faster manner. Carbon dioxide (CO2) is generally preferred as a supercritical solvent because of its lower temperature requirements, better diffusivity and easy removal. The present study describes the characterization of supercritical CO2 extracts of Indian variety of Cordyceps sinensis (CS)- a high-altitude medicinal mushroom widely revered in traditional medicine for its extensive anti-hypercholesterolemic, anti-inflammatory, anti-proliferative and energy-enhancing properties. Experimental parameters viz. 300 and 350 bar of extraction pressure, 60°C of temperature, 0.4°L/h CO2 of flow rate and use of 1% (v/v) of ethanol as entrainer were optimized to prepare three different extracts namely, CSF1, CSF2 and CSF3. High-performance thin-layer chromatography (HPTLC) was used for assessing the quality of all the extracts in terms of cordycepin, the pivot biomarker compound in CS. Characterization by HPTLC and GC-MS confirmed the presence of flavonoids and nucleobases and, volatile organic compounds (VOCs), respectively. The chromatographic data acquired from metabolite profiling were subjected to chemometric analysis in an open source R studio which illustrated interrelatedness between CSF1 and CSF2 in terms of two major principal components. i.e. Dim 1 and Dim 2 whose values were 40.33 and 30.52% in variables factor map plotted using the HPTLC-generated retardation factor values. The factor maps based on retention times of the VOCs exhibited a variance of Dim 1 = 43.95% and Dim 2 = 24.85%. Furthermore, the extracts demonstrated appreciable antibacterial activity against Escherichia coli and Salmonella typhi by generation of reactive oxygen species (ROS), protein leakage and efflux pump inhibition within bacterial pathogens. CSFs were elucidated to be significantly cytoprotective (p < 0.05) in a simulated hypobaric hypoxia milieu (0.5% oxygen). CSF2 showed the best results by effectively improving the viability of human embryonic kidney (HEK 293) cells to 82.36 ± 1.76% at an optimum dose of 100 µg/ml. Levels of hypoxia inducible factor-1 alpha (HIF-1α) were modulated four-fold upon supplementation with CSF2. The results collectively evinced that the CSF extracts are substantially bioactive and could be effectively utilized as mycotherapeutics for multiple bioeffects.

The present study is aimed at developing a synergistic combination to enhance learning and memory in Alzheimer’s patients with the help of eight common medicinal plants used in the AYUSH system. Aqueous and hydroalcoholic extracts of eight medicinal plants from the AYUSH system of medicine were prepared. These were subjected to in vitro anticholinesterase activity, to find out the combination index of synergistic combination. The synergistic combination and their individual extracts were subjected to total phenol, flavonoid and antioxidant activity estimation. Further, in vivo neurobehavioral studies in rats were carried out followed by TLC-MS-bioautographic identification of bioactive metabolites. Out of the sixteen extracts, aqueous extracts of Withania somnifera (L.) Dunal (WSA) and Myristica fragrans (L.) Dunal (MFA) were selected for the development of synergistic combination based on their IC50 value in vitro anticholinesterase assay. The synergistic combination inhibited the anticholinesterase activity significantly as compared to the individual extracts of WSA and MFA. The synergistic combination also showed more phenolic and flavonoid contents with potential antioxidant activity. The TLC-bioautography showed four white spots in WSA, signifying sitoindosides VII, VIII, quercetin, isopelletierine and Withanolide S as AChE inhibitory compounds while showing five white spots of anti-cholinesterase active metabolites identified as eugenol, methyl eugenol, myristic acid, galbacin and β-sitosterol in MFA. The observation of neurocognitive behavior in amnesia induced subjects manifested that both the synergistic combinations showed comparable results to that of standard piracetam, though the synergistic combination containing a higher concentration of WSA showed more appreciable results in ameliorating dementia in rats. The study suggests that the synergy based combination successfully enhanced memory and learning by abating free radical and acetylcholine levels, and increased learning and memory in rats, providing a strong rationale for its use in the treatment of dementia and Alzheimer’s disease.

Safoof-e-Pathar phori (SPP) is an Unani poly-herbomineral formulation, which has for a long time been used as a medicine due to its antiurolithiatic activity, as per the Unani Pharmacopoeia. This powder formulation is prepared using six different plant/mineral constituents. In this study, we explored the antiurolithiatic and antioxidant potentials of SPP (at 700 and 1,000 mg/kg) in albino Wistar rats with urolithiasis induced by 0.75% ethylene glycol (EG) and 1% ammonium chloride (AC). Long-term oral toxicity studies were performed according to the Organization for Economic Co-operation and Development (OECD) guidelines for 90 days at an oral dose of 700 mg/kg of SPP. The EG urolithiatic toxicant group had significantly higher levels of urinary calcium, serum creatinine, blood urea, and tissue lipid peroxidation and significantly (p < 0.001 vs control) lower levels of urinary sodium and potassium than the normal control group. Histopathological examination revealed the presence of refractile crystals in the tubular epithelial cell and damage to proximal tubular epithelium in the toxicant group but not in the SPP treatment groups. Treatment of SPP at 700 and 1,000 mg/kg significantly (p < 0.001 vs toxicant) lowered urinary calcium, serum creatinine, blood urea, and lipid peroxidation in urolithiatic rats, 21 days after induction of urolithiasis compared to the toxicant group. A long-term oral toxicity study revealed the normal growth of animals without any significant change in hematological, hepatic, and renal parameters; there was no evidence of abnormal histology of the heart, kidney, liver, spleen, or stomach tissues. These results suggest the usefulness of SPP as an antiurolithiatic and an antioxidant agent, and long-term daily oral consumption of SPP was found to be safe in albino Wistar rats for up to 3 months. Thus, SPP may be safe for clinical use as an antiurolithiatic formulation.

The cases of COVID-19 are still increasing day-by-day worldwide, even after a year of its first occurrence in Wuhan city of China. The spreading of SARS-CoV-2 infection is very fast and different from other SARS-CoV infections possibly due to structural differences in S proteins. The patients with severe diseases may die due to acute respiratory distress syndrome (ARDS) caused by systemic inflammatory reactions due to the excessive release of pro-inflammatory cytokines and chemokines by the immune effector cells. In India too, it is spreading very rapidly, although the case fatality rate is below 1.50% (https://www.statista.com), which is markedly less than in other countries, despite the dense population and minimal health infrastructure in rural areas. This may be due to the routine use of many immunomodulator medicinal plants and traditional AYUSH formulations by the Indian people. This communication reviews the AYUSH recommended formulations and their ingredients, routinely used medicinal plants and formulations by Indian population as well as other promising Indian medicinal plants, which can be tested against COVID-19. Special emphasis is placed on Indian medicinal plants reported for antiviral, immunomodulatory and anti-allergic/anti-inflammatory activities and they are categorized for prioritization in research on the basis of earlier reports. The traditional AYUSH medicines currently under clinical trials against COVID-19 are also discussed as well as furtherance of pre-clinical and clinical testing of the potential traditional medicines against COVID-19 and SARS-CoV-2. The results of the clinical studies on AYUSH drugs will guide the policymakers from the AYUSH systems of medicines to maneuver their policies for public health, provide information to the global scientific community and could form a platform for collaborative studies at national and global levels. It is thereby suggested that promising AYUSH formulations and Indian medicinal plants must be investigated on a priority basis to solve the current crisis.

Aqueous extract of the bark of Terminalia arjuna (TA) is used by a large population in the Indian subcontinent for treating various cardiovascular conditions. Animal experiments have shown its anti-atherogenic, anti-hypertensive, and anti-inflammatory effects. It has several bioactive ingredients with hemodynamic, ROS scavenging, and anti-inflammatory properties. Earlier we have done limited proteomic and transcriptomic analysis to show its efficacy in ameliorating cardiac hypertrophy induced by isoproterenol (ISO) in rats. In the present study we have used high-throughput sequencing of the mRNA from control and treated rat heart to further establish its efficacy. ISO (5 mg/kg/day s.c.) was administered in male adult rats for 14 days to induce cardiac hypertrophy. Standardized aqueous extract TA bark extract was administered orally. Total RNA were isolated from control, ISO, ISO + TA, and TA treated rat hearts and subjected to high throughput sequence analysis. The modulations of the transcript levels were then subjected to bio-informatics analyses using established software. Treatment with ISO downregulated 1,129 genes and upregulated 204 others. Pre-treatment with the TA bark extracts markedly restored that expression pattern with only 97 genes upregulated and 85 genes downregulated. The TA alone group had only 88 upregulated and 26 downregulated genes. The overall profile of expression in ISO + TA and TA alone groups closely matched with the control group. The genes that were modulated included those involved in metabolism, activation of receptors and cell signaling, and cardiovascular and other diseases. Networks associated with those genes included those involved in angiogenesis, extracellular matrix organization, integrin binding, inflammation, drug metabolism, redox metabolism, oxidative phosphorylation, and organization of myofibril. Overlaying of the networks in ISO and ISO_TA group showed that those activated in ISO group were mostly absent in ISO_TA and TA group, suggesting a global effect of the TA extracts. This study for the first time reveals that TA partially or completely restores the gene regulatory network perturbed by ISO treatment in rat heart; signifying its efficacy in checking ISO-induced cardiac hypertrophy.

Moringa oleifera Lam. (family; Moringaceae), commonly known as drumstick, have been used for centuries as a part of the Ayurvedic system for several diseases without having any scientific data. Demineralized water was used to prepare aqueous extract by maceration for 24 h and complete metabolic profiling was performed using GC-MS and HPLC. Hypoglycemic properties of extract have been tested on carbohydrate digesting enzyme activity, yeast cell uptake, muscle glucose uptake, and intestinal glucose absorption. Type 2 diabetes was induced by feeding high-fat diet (HFD) for 8 weeks and a single injection of streptozotocin (STZ, 45 mg/kg body weight, intraperitoneally) was used for the induction of type 1 diabetes. Aqueous extract of M. oleifera leaf was given orally at a dose of 100 mg/kg to STZ-induced rats and 200 mg/kg in HFD mice for 3 weeks after diabetes induction. Aqueous extract remarkably inhibited the activity of α-amylase and α-glucosidase and it displayed improved antioxidant capacity, glucose tolerance and rate of glucose uptake in yeast cell. In STZ-induced diabetic rats, it produces a maximum fall up to 47.86% in acute effect whereas, in chronic effect, it was 44.5% as compared to control. The fasting blood glucose, lipid profile, liver marker enzyme level were significantly (p < 0.05) restored in both HFD and STZ experimental model. Multivariate principal component analysis on polar and lipophilic metabolites revealed clear distinctions in the metabolite pattern in extract and in blood after its oral administration. Thus, the aqueous extract can be used as phytopharmaceuticals for the management of diabetes by using as adjuvants or alone.

Solanum nigrum L., is traditionally used for the management of the various liver disorders. Investigating the effect of polarity based fractionation of S. nigrum for its hepatoprotective effect on Hep G2 cells in vitro to provide base of its activity by quantifying in steroidal glycosides responsible for hepatoprotective potential. A new UPLC-ESI-MS/MS method following a high performance thin layer chromatography (HPTLC) has been developed and validated for quantification of steroidal glycosides and aglycone (solasonine, solamargine, and solasodine, respectively). The in vitro antioxidant potential, total phenolics, and flavonoid content were also determined in different fractions. The newly developed UPLC-ESI-MS/MS and HPTLC methods were linear (r2 ≥ 0.99), precise, accurate, and showing recovery more than 97%. The n-butanol enriched fraction of S. nigrum berries was found to be the most potent hepatoprotective fraction against all other fractions as it showed significantly (p < 0.01) better in vitro anti-oxidant potential than other fractions. Quantification by both methods revealed that, content of steroidal glycosides and aglycones are more than 20% in n-butanol fraction as compared to other fractions. The screened steroidal glycoside n-butanol enriched fraction underwent bioefficacy studies against D-galactosamine and H2O2 induced toxicity in HepG2 cell line showing significant (p < 0.05) liver protection. However, developed method can be used for the quality control analysis with respect to targeted metabolites and it can be explored for the pharmacokinetic and pharmacodynamic analysis in future.

Purpose. The intention of the proposed work is to study the presence of the aflatoxins B1, B2, G1 and G2 in medicinal plants, namely Mucuna pruriens, Delphinium denudatum and Portulaca oleraceae. Methodology. The aflatoxins were extracted, purified by immunoaffinity column chromatography and analysed by high-performance liquid chromatography–tandem quadrupole mass spectrometry with electrospray ionisation (HPLC–MS/MS). Fungal count was carried out in PDA media. Results. A good linear relationship was found for AFB1, AFB2, AFG1 and AFG2 at 1–10 ppb (r>0.9995). The analyte accuracy under three different spiking levels was 86.7–108.1 %, with low per cent relative standard deviations in each case. The aflatoxins can be separated within 5 to7 min using an Agilent XDB C18-column. We found that AFB1 and AFB2 were in trace amounts below the detection limit in M. pruriens whilst they were not detected in D. denudatum. P. oleraceae was found to be contaminated with AFB1 and AFB2. AFG1 and AFG2 were not detected in M. pruriens, P. oleraceae and were below the detection limit in D. denudatum. This was consistent with very low numbers of fungal colonies observed after 6 hr of incubation. Conclusion. The analytical method developed is simple, precise, accurate, economical and can be effectively used to determine the aflatoxins in medicinal plants and therefore to control the quality of products. The aflatoxin levels in the plant extracts examined were related to the minimal fungal load in the medicinal plants examined. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

Type 2 diabetes mellitus (T2DM) is a growing global public health issue, and dipeptidyl peptidase-4 (DPP-4) is a potential therapeutic target in T2DM. Several synthetic anti-DPP-4 medications can be used to treat T2DM. However, because of adverse effects, there is an unmet demand for the development of safe and effective medications. Natural medicines are receiving greater interest due to the inherent safety of natural compounds. Glycyrrhiza uralensis (licorice) is widely consumed and used as medicine. In this study, we investigated the abilities of a crude water extract (CWE) of G. uralensis and two of its constituents (licochalcone A (LicA) and licochalcone B (LicB)) to inhibit the enzymatic activity of DPP-4 in silico and in vitro. In silico studies showed that LicA and LicB bind tightly to the catalytic site of DPP-4 and have 11 amino acid residue interactions in common with the control inhibitor sitagliptin. Protein-protein interactions studies of LicA-DPP4 and LicB-DPP4 complexes with GLP1 and GIP reduced the DPP-4 to GLP1 and GIP interactions, indicated that these constituents might reduce the degradations of GLP1 and GIP. In addition, molecular dynamics simulations revealed that LicA and LicB stably bound to DPP-4 enzyme. Furthermore, DPP-4 enzyme assay showed the CWE of G. uralensis, LicA, and LicB concentration-dependently inhibited DPP-4; LicA and LicB had an estimated IC50 values of 347.93 and 797.84 μM, respectively. LicA and LicB inhibited DPP-4 at high concentrations, suggesting that these compounds could be used as functional food ingredients to manage T2DM.

Myostatin (MSTN) is a well-reported negative regulator of muscle growth and a member of the transforming growth factor (TGF) family. MSTN has important functions in skeletal muscle (SM), and its crucial involvement in several disorders has made it an important therapeutic target. Several strategies based on the use of natural compounds to inhibitory peptides are being used to inhibit the activity of MSTN. This review delivers an overview of the current state of knowledge about SM and myogenesis with particular emphasis on the structural characteristics and regulatory functions of MSTN during myogenesis and its involvements in various muscle related disorders. In addition, we review the diverse approaches used to inhibit the activity of MSTN, especially in silico approaches to the screening of natural compounds and the design of novel short peptides derived from proteins that typically interact with MSTN.

Skeletal muscle (SM) comprises around 40% of total body weight and is among the most important plastic tissues, as it supports skeletal development, controls body temperature, and manages glucose levels. Extracellular matrix (ECM) maintains the integrity of SM, enables biochemical signaling, provides structural support, and plays a vital role during myogenesis. Several human diseases are coupled with dysfunctions of the ECM, and several ECM components are involved in disease pathologies that affect almost all organ systems. Thus, mutations in ECM genes that encode proteins and their transmembrane receptors can result in diverse SM diseases, a large proportion of which are types of fibrosis and muscular dystrophy. In this review, we present major ECM components of SMs related to muscle-associated diseases, and discuss two major ECM myopathies, namely, collagen myopathy and laminin myopathies, and their therapeutic managements. A comprehensive understanding of the mechanisms underlying these ECM-related myopathies would undoubtedly aid the discovery of novel treatments for these devastating diseases.