HPTLC Stability Indicating Analytical Method of Andrographolide and 5-fluorouracil with Network Pharmacology Analysis against Cancer.

BACKGROUND: Herbal drugs when used in combination with chemotherapeutic drugs can reduce the side effects and increase the efficacy by acting on multiple targets. Andrographolide (AG), a diterpene lactone isolated from Andrographis paniculata Nees, is a bioactive compound with anticancer potential, and 5-fluorouracil (FU), a pyrimidine analogue, is used in the treatment of cancer. Both drugs are used to formulate combination nanoformulation to increase absorption, thereby increasing their oral bioavailability. OBJECTIVE: The study aimed to develop and validate stability indicating simultaneous HPTLC method for quantification of FU and AG in combination nanoformulation along with in silico docking and network pharmacology analysis to understand the interaction between the drugs and cancer targets. METHODS: Chromatographic separation was performed using mobile phase chloroform: methanol: formic acid (9: 0.5: 0.5, v/v/v) on HPTLC silica plates 60 F254 as a stationary phase using UV-Vis detector and HPTLC scanner at 254 nm. Further, in silico docking analysis was performed to predict the binding affinity of AG and FU with different proteins and network pharmacology to find out the exact biomolecular relationship of AG and FU in alleviating cancer. RESULTS: The data from the calibration curve showed a good linear regression relationship with r² = 0.9981 (FU) and r² = 0.9977 (AG) in the concentration range of 0.1-2.0 µg/mL. The developed method was validated according to the ICH guidelines. Stability studies showed changes in peak patterns and areas. Bioinformatic and network pharmacology analyses of AG and FU with target proteins and genes associated with cancer play a multimechanistic role in alleviating cancer. CONCLUSION: The developed method has been concluded to be robust, simple, precise, reproducible, accurate, and stability indicating for simultaneous quantification of AG and FU, and the molecular interaction studies have further indicated that the combination nanoformulation of AG and FU could be effective against cancer.

Validation and Standardization of Gallic Acid and Ellagic Acid in Quercus Infectoria, Terminalia Chebula, and Pistacia Integerrima.

BACKGROUND: Due to its medicinal properties, Pistacia integerrima is in high demand and is extensively used as a key ingredient in various formulations. However, its popularity has led to its inclusion on the International Union for Conservation of Nature threatened category list. In Ayurvedic texts, such as Bhaishajaya Ratnavali, Quercus infectoria is recommended as a substitute for P. integerrima in different formulations. Additionally, Yogratnakar highlights that Terminalia chebula shares similar therapeutic properties with P. integerrima. OBJECTIVE: The objective of the current study was to gather scientific data on metabolite profiling and marker-based comparative analysis of Q. infectoria, T. chebula, and P. integerrima. METHODS: In present study, hydroalcoholic and aqueous extracts of all three plants were prepared and standardized for the comparative evaluation of secondary metabolites. TLC was carried out for the comparative fingerprinting of the extracts using chloroform-methanol-glacial acetic acid-water (60 + 8 + 32 + 10, by volume) as a solvent system. A fast, sensitive, selective, and robust HPLC method was developed to determine gallic acid and ellagic acid from both extracts of all three plants. The method was validated for precision, robustness, accuracy, LOD and LOQ as per the International Conference on Harmonization guidelines. RESULTS: The TLC analysis revealed the presence of several metabolites, and the pattern of metabolites in the plants exhibited a certain degree of similarity. A highly precise and reliable quantification technique was created for gallic acid and ellagic acid, operating within a linear concentration range of 81.18-288.22 µg/mL and 3.83-13.66 µg/mL, respectively. The correlation coefficients for gallic acid and ellagic acid were 0.997 and 0.996, indicating good linear relationships. The gallic acid content in all three plants ranged from 3.74 to 10.16% w/w, while the ellagic acid content ranged from 0.10 to 1.24% w/w. CONCLUSION: The study contributes to the scientific understanding of the metabolite profiles and comparative analysis of Q. infectoria, T. chebula, and P. integerrima. The findings provide valuable insights into the chemical composition of these plants and can be used for various applications in herbal medicine. HIGHLIGHTS: This pioneering scientific approach highlights the phytochemical similarities between Q. infectoria, T. chebula and P. integerrima.

Multi-Mechanistic and Therapeutic Exploration of Nephroprotective Effect of Traditional Ayurvedic Polyherbal Formulation Using In Silico, In Vitro and In Vivo Approaches.

Based on traditional therapeutic claims, NEERI KFT (a traditional Ayurvedic polyherbal preparation) has been innovatively developed in recent time on the decades of experience for treating kidney dysfunction. Due to the lack of scientific evidence, the present investigations are needed to support the rationale use of NEERI KFT. Considering the facts, the study investigated the nephroprotective effect of NEERI KFT against kidney dysfunction using in silico, in vitro and in vivo approaches. In this study, phytochemical and network pharmacology studies were performed for the developed formulation to evaluate the molecular mechanism of NEERI KFT in the amelioration of kidney disease. In vitro nephroprotective and antioxidant effect of NEERI KFT was determined on HEK 293 cells against cisplatin-induced cytotoxicity and oxidative stress. In vivo nephroprotective effect of NEERI KFT was determined against cisplatin-induced nephrotoxicity in Wistar rats, via assessing biochemical markers, antioxidant enzymes and inflammatory cytokines such as TNF-α, IL-1ß, CASP-3, etc. The results showed that the compounds such as gallic acid, caffeic acid and ferulic acid are the major constituents of NEERI KFT, while network pharmacology analysis indicated a strong interaction between polyphenols and several genes (CASPs, ILs, AGTR1, AKT, ACE2, SOD1, etc.) involved in the pathophysiology of kidney disease. In vivo studies showed a significant (p < 0.05) ameliorative effect on biochemical markers and antioxidant enzymes (SOD, CAT, GSH, etc.), and regulates inflammatory cytokine (TNF-α, IL-1ß, CASP-3) expression in kidney tissue. Hence, it can be concluded that NEERI KFT subsequently alleviates renal dysfunction mediated by cisplatin via attenuating oxidative and inflammatory stress, thus preserving the normalcy of kidney function.

A Comprehensive Study to Explore Tyrosinase Inhibitory Medicinal Plants and Respective Phytochemicals for Hyperpigmentation; Molecular Approach and Future Perspectives.

Tyrosinase is a copper-containing key substance in the pigmentation of mammalian hair and skin. Melanin synthesis is influenced by a variety of extrinsic and internal variables, including hormone fluctuations, inflammation, ageing, and subsequent ultraviolet light exposure. Melasma, senile lentigines, freckles, and diminished colour are all undesirable side effects of excessive melanin production. The current review provides the pursuit of effective and safe tyrosinase inhibitors derived from medicinal plants and ascribes updated inferences on current practices. Commercially available tyrosinase inhibitors provide an even skin tone and are used clinically to treat hyperpigmentation and related disorders. This review focuses on the mechanism of melanogenesis and on experimentally verified potent and natural tyrosinase inhibitors. Bioactive compounds such as phenols, flavonoids, stilbenes, and few traditional herbal formulations from the Indian system of medicine, have been used for long in India and subcontinents for the effective management of melanogenesis and related problems. Scientific information was gathered from different sources of databases such as PubMed, Google Scholar, Springer, Scopus, and Science Direct, as well as the literature found in medicinal plant books. This critically summarized review ensures to aid researchers and enterprises working on tyrosinase inhibitors and on conditions associated with melanogenesis, to get one-step solutions for identifying more safe and effective natural remedies.

Metabolite Profiling, Stability Testing, Pharmacokinetics, and In Vivo Pattern Recognition Analysis of Arq-e-Keora: A Traditional Unani Formulation.

BACKGROUND: Arq-e-Keora is a liquid formulation of the Unani system of medicine and used since long for the management of weakness of heart, palpitations, etc. OBJECTIVE: The study was carried out to generate a scientific data for its metabolite profiling, stability testing, pharmacokinetics, and pattern recognition analysis of Arq-e-Keora. METHOD: Arq-e-Keora has been prepared as water distillate of male wpadix of Pandanus odoratissimus L.f. TLC profiling of Arq-e-Keora was performed using hexane and acetone (7:3, v/v) as a solvent system. The metabolic profiling of volatile compounds was carried out using GC-MS. Pharmacokinetic analysis was performed through GC-MS to evaluate how quickly it absorbs and distributes in plasma. The pattern recognition analysis was performed in order to recognize the pattern and fate of metabolites in rat plasma up to 24 h after single oral administration of Arq-e-Keora. RESULTS: TLC and GC-MS analysis resulted in profiling of 11 and 21 metabolites, respectively. GC-MS analysis revealed that phenethyl alcohol, alpha-terpinolene, beta terpinene, alpha terpinene, beta fenchyl alcohol, hexadecanoic acid, and octadecanoic acid are the major metabolites found in Arq-e-Keora. The stability analysis showed that most of the compounds are stable at refrigerator temperature during their consumption. Pharmacokinetics data of phenethyl alcohol showed its absorption was rapid, with Tmax occurring within 1 h after oral administration of Arq-e-Keora. In vivo pattern recognition analysis suggests that some metabolite expression was altered after its oral administration. CONCLUSIONS: As a result, our model could be used for quality, stability, and pharmacokinetic evaluation of various Unani formulations mentioned in Unani Pharmacopoeia of India. HIGHLIGHTS: This is the first study of pharmacokinetic analysis and metabolite pattern of traditional Unani formulation after its oral administration in Wistar rats.

Metabolomic Profiling and Identification of Antioxidant and Antidiabetic Compounds from Leaves of Different Varieties of Morus alba Linn Grown in Kashmir.

Mulberry (Morus alba L.) is commonly cultivated in Asian countries as a traditional medicine and food supplement. Four Kashmiri Morus alba varieties (Zagtul, Chtattatual, Chattatual Zaingir, and Brentul Kashmir) were evaluated for their proximate composition, mineral content, total phenolic and flavonoid content, antioxidant potential, and antihyperglycemic activity. Furthermore, TLC-MS-bioautography was used for the identification of antioxidant and antidiabetic compounds in the best active extract. Lastly, UPLC-MS was employed for metabolomic profiling of the best variety of M. alba. Among all the varieties, the Zagtul variety was found to have the highest phenolic (71.10 ± 0.44 mg GAE/g DW) and flavonoid (53.22 ± 0.69 mg rutin/g DW) content. The highest antioxidant potential (DPPH) with an IC50 value of 107.88 ± 3.8 µg/mL was recorded for the Zagtul variety. Similarly, α-amylase and α-glucosidase inhibition for antidiabetic potential with IC50 74.76 ± 6.76 and 109.19 ± 5.78 µg/mL, respectively, was recorded in Zagtul variety. TLC-MS-bioautography for identification of bioactive compounds revealed the presence of chlorogenic acid for antioxidant potential and 1-deoxynojirimycin (DNJ) and syringic acid for antidiabetic potential. Further, bioactive compounds responsible for diverse functions of M. alba were confirmed by UPLC-MS in both negative and positive modes. However, major compounds in the Zagtul variety were identified as chlorogenic acid, moracin N, gallic acid, ferulic acid, morin, 1-deoxynojirimycin, and syringic acid. Hence, based on our findings, it can be concluded that M. alba leaves can be consumed as a promising dietary supplement and can be formulated as phytopharmaceutical for the management of various metabolic disorders.

Development of Synergy-Based Combination of Methanolic Extract of Andrographis paniculata and Berberis aristata Against E. coli and S. aureus.

This study evaluates the antibacterial activity and phytochemical characterizations of Andrographis paniculata extract (APE) and Berberis aristata extract (BAE). The stem of Andrographis paniculata (AP) and root of Berberis aristata (BA) were extracted with methanol. The results confirmed that APE and BAE possess high phenolic and flavonoid content. The antioxidant activity of the APE and BAE showed an elevated potential to scavenge DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals with IC50 of 95.03 µg/mL and 256.26 µg/mL, respectively. A total of 35 and 32 metabolites in APE and BAE, respectively, were identified through mass spectrometry analysis, whereas 17 and 12 metabolites in APE and BAE, respectively, were detected through high-performance thin-layer chromatography (HPTLC) fingerprinting profiling. Antibacterial activity of the extracts was performed by the well diffusion and microdilution method, and the findings showed that APE and BAE had antibacterial activities against E. coli and S. aureus. The growth curve and time-kill study showed that the extracts had a bacteriostatic effect. A combination study with the standard drug was carried out using the microdilution checkerboard method in which most of the combinations showed synergistic interactions. The findings of this study have shown that APE and BAE are good sources of antibacterial compounds and can be used for treating infectious diseases caused by E. coli and S. aureus.

Benefits of Black Cohosh (Cimicifuga racemosa) for Women Health: An Up-Close and In-Depth Review.

Women's health is an imminent concern worldwide, but it remains an ignored segment of research in most developing countries, and is yet to take the center stage in even developed nations. Some exclusive female health concerns revolve around both pathological and physiological aspects. These gender-specific maladies include breast, cervical, and ovarian cancers, and physiological concerns such as menopause and osteoporosis, which are often coexistent. Recently, women's health issues, including postmenopausal syndrome, have attracted the attention of researchers and practitioners alike, opening newer pharmaceutical research and clinical avenues. Although not counted as a disease, postmenopausal syndrome (PMS) is a female health phenomenon underpinned by hormonal depletion. Enhanced life expectancy in women has added to their suffering, and pharmacological interventions are needed. Amongst the available treatment modalities, the use of numerous botanicals has emerged as an efficient health management tool for women. Cimicifuga racemosa (CR or Black Cohosh) is a plant/herb which has been traditionally exploited and extensively used by women. This review is an attempt to compile and provide a summary of the importance of CR in complementary and alternative therapies for the improvement of various disorders related to women, such as menopausal syndrome, mammary cancer, and osteoporosis. It aims to systematically highlight the bioactive constituents, pharmacology, pharmacokinetics, therapeutic potentials, quality control processes, chromatographic techniques, and possible mechanisms of action of clinically effective phytomedicine for women's health. Various clinical trials and patents relating to CR and women's health have been collated. Furthermore, the plant and its related products have been considered from a regulatory perspective to reveal its commercial feasibility. The present review summarizes the existing data on CR focusing on women's health, which can help to introduce this traditional phytomedicine to the world and provide some reference for future drug development.

Potential nephroprotective phytochemicals: Mechanism and future prospects.

ETHNOPHARMACOLOGICAL RELEVANCE: Kidney disease (KD) is one of the serious health issues, which causes worrisome morbidity and economic burden. Therapeutic strategies are available however majority of them are associated with severe adverse effects and poor patient compliance and adherence. This explorative article was undertaken to provide a holistic review of known nephroprotective (NP) phytoconstituents along with their research-based evidences on mechanism, sources, and clinical trials that may play essential role in prevention and cure of KD. AIM OF THE STUDY: The present systematic review aimed to provide in-depth and better evidences of the global burden of KD, phytoconstituents as NP with emphasis on mechanism of action both in vitro and in vivo, their wide biological sources as well as their clinical efficacy in management of kidney disease and its related disorders. MATERIAL AND METHODS: Comprehensive information was searched systematically from electronic databases, namely, PubMed, Sciencedirect, Wiley, Scopus, Google scholar and Springer until February 2021 to find relevant data for publication on phytoconstituents with nephroprotective potential. RESULTS: In total, 24,327 articles were screened in first search for "phytoconstituents and medicinal plants for nephroprotection and kidney disorder". On the basis of exclusion and inclusion criteria, 24,091 were excluded. Only 236 papers were spotted to have superlative quality data, which is appropriate under titles and sub-titles of the present review. The phytoconstituents having multiple research evidence along with wide number of medicinal plants sources and mechanism reported for nephroprotection have been selected and reviewed. CONCLUSION: This review, based on pre-clinical and clinical data of NP phytoconstituents, provides scientific-basis for the rational discovery, development and utilization of these upcoming treatment practices. Further,-more clinical studies are warranted to improve the pharmacodynamic and pharmacokinetic understanding of phytoconstituents. Also, more specific evaluation for natural sources is needed.

Analysis of polyphenols in Aegle marmelos leaf and ameliorative efficacy against diabetic mice through restoration of antioxidant and anti-inflammatory status.

The biomedical survey reports edible plant Aegle marmelos has been utilized for centuries by tribal communities in India as a dietary supplement for the management of diabetes. Herein, we have investigated cytotoxicity, cytoprotective and antidiabetic activity of characterized alkaloid-free hydroalcoholic extract of A. marmelos (AFEAM; 200 and 400 mg/kg). Identification of polyphenols and quantification of major compounds were done using UPLC-MS and HPTLC, respectively. AFEAM showed good cytocompatibility and cytoprotective potential against oxidative stress induced by hyperglycemia in HepG2 cells. The AFEAM intake had significantly ameliorated the serum blood glucose level, state of dyslipidemia, level of pro-inflammatory markers (tumor necrosis factor-α, interleukin-6, and interleukin-1ß), and antioxidant (superoxide dismutase, catalase, glutathione peroxidase, and malondialdehyde) status in diabetic mice. Histological examination of the treated groups showed amelioration of damaged pancreas, liver, and kidney tissues. Conclusively, AFEAM intake might be promising dietary supplements for prediabetics as well as an adjuvant to modern treatment in diabetics. PRACTICAL APPLICATIONS: Different reports have been published on Aegle marmelos but as per our understanding till date, no study has been reported on the amelioration of diabetes due to alkaloid free hydroalcoholic extract of A. marmelos /polyphenolic content in the animal model. The result of this study indicated that A. marmelos supplementation effectively ameliorates diabetes through the restoration of antioxidant and anti-inflammatory status. This study has collated sufficient scientific evidence for the dietary application of A. marmelos in society especially for prediabetics, however, it can also be used as an adjuvant to modern treatments in diabetics.

Modulating effects of the synergistic combination of extracts of herbal drugs on cyclophosphamide-induced immunosuppressed mice.

OBJECTIVE: Taking leads from the available research, we aimed to develop a synergy-based herbal combination of Tinospora cordifolia (TC), Phyllanthus emblica (PE), and Piper nigrum (PN). Also, evaluating their synergistic effect on CP-induced immunosuppression in mice model and exploring the possible mechanisms involved in reversing the damage. METHODOLOGY: The immunomodulatory activity of combination, of TC stem, PE fruits, and PN dried fruits, was determined by in vitro assays (splenocyte proliferation and pinocytic activity of peritoneal macrophages of mice) and in vivo study using CP-induced immunosuppression model in Swiss Albino mice. The ratio was optimized for combining three by in vitro MTT assay. The combination was further evaluated for anti-oxidant activity by DPPH scavenging method and quantified for its bioactive metabolites by HPTLC. Serum collected on day 0, 4, 7 and 14 was employed for estimation of haematogram (haematocrit, TLC, DLC, and haemoglobin, etc) and immune parameters (IL-10, IL-6 and TNF-α) by ELISA. RESULTS: The study demonstrated, that combination of herbal extracts at an intermediate dose could inhibit the proliferation of spleen cells and peritoneal macrophages (P ≤ 0.0001) and induce suppression of pro-inflammatory mediators, and also certified that combination exerts synergized effects. The results showed that the combination possess potential antioxidant activity by DPPH scavenging method (IC50-113.5 µg/ml). It was identified that combination significantly (P ≤ 0.0001) improved the immune markers, haematogram parameters, and histological parameters, with maximum protection offered by an intermediate dose. CONCLUSION: The results suggested that present combination could be further explored clinically as potent synergy-based therapeutic approach for immune modulation.

Development of Synergy-Based Combination for Learning and Memory Using in vitro, in vivo and TLC-MS-Bioautographic Studies.

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 ß-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.

Nutritional constituents of mulberry and their potential applications in food and pharmaceuticals: A review.

Mulberry is a fast growing deciduous plant found in wide variety of climatic, topographical and soil conditions, and is widely distributed from temperate to subtropical regions. Due to presence of valuable phytochemical constituents, mulberry as a whole plant has been utilized as a functional food since long time. Mulberry fruits are difficult to preserve as they have relatively high water content. Therefore for proper utilization, different value-added products like syrups, squashes, teas, pestil sand köme, pekmez (turkuish by-products), yogurts, jams, jellies, wines, vinegar, breads, biscuits, parathas, and many more are made. In overseas, these value-added products are commercially sold and easily available, though in India, this versatile medicinal plant is still missing its identity at commercial and industrial scale. Leaves of mulberry are economically viable due to their important role in the sericulture industry since ancient times. Mulberries or its extracts exhibit excellent anti-microbial, anti-hyperglycaemic, anti-hyperlipidemic, anti-inflammatory, anti-cancer effects and is used to combat different acute and chronic diseases. Different parts of Morus species like fruits, leaves, twigs, and bark exhibit strong anti-tyrosinase inhibition activity that makes it a suitable candidate in cosmetic industries as a whitening agent. The current review provides a comprehensive discussion concerning the phytochemical constituents, functionality and nutraceutical potential of mulberry and as a common ingredient in various cosmetic products.

Anticancer potential of andrographolide from Andrographis paniculata (Burm.f.) Nees and its mechanisms of action.

ETHNOPHARMACOLOGICAL RELEVANCE: Synthetic drugs used for cancer treatment have side effects that may be immunosupressive, can cause liver, kidney and cardiac toxicity, and infertility and ovarian failure, among others. Thus, herbal drugs could be used in the cancer treatment as an adjuvant therapy. Andrographis paniculata (Burm.f.) Nees (AP) is one of the traditional herbs used in different alternative medicinal systems such as Ayurveda, Unani, Chinese, Malayi, Siddha, etc. for the treatment of various disorders and diseases including cancer. AIM OF THE STUDY: The aim of writing this review is to highlight the medicinal importance of AP and its main phytoconstituent andrographolide (AG). The main emphasis was given on the anticancer activity of AG, its proposed mechanisms of action, novel approaches used to improve its biopharmaceutical properties with the perspective of evidence-based research, and its development as an adjuvant therapy for cancer treatment in future. MATERIALS AND METHODS: Literature survey was conducted and research papers were retrieved from different databases such as Pubmed, Google Scholar, ACS, Wiley online library, ScienceDirect, Springer, and Scopus during 1970-2020. Research articles, review articles, and short communications, etc. were used for this purpose. The papers were selected on the basis of exclusion and inclusion criteria. RESULTS: Different anticancer mechanisms of AG have been reportedly proven such as cell cycle arrest, apoptosis, NF-κß inhibition, antiangiogenesis, cytokine inhibition, etc. whereas its pharmacokinetic properties showed its highly protein bound nature, Cyt P400 (CYP) inhibition, low aqueous solubility, poor oral bioavailability, etc. Different novel formulations of AG have been investigated to increase its bioavailability for better efficacy. CONCLUSION: This review can provide knowledge about the potential applicability of AP or AG as an adjuvant therapy in cancer treatment. Further research is needed before making any conclusion about the efficacy in humans as an adjuvant therapy in cancer.

Metabolomic Profiling and Immunomodulatory Activity of a Polyherbal Combination in Cyclophosphamide-Induced Immunosuppressed Mice.

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.

TLC-Based Metabolite Profiling and Bioactivity-Based Scientific Validation for Use of Water Extracts in AYUSH Formulations.

We aimed to develop a chromatographic method for scientific validation of water extract of some important Indian traditional plants used in AYUSH-based formulation as immunomodulator and to evaluate their bioactive potential. Fruits of Phyllanthus emblica L. and Piper nigrum L., stem of Tinospora cordifolia (Willd.) Miers, rhizome of Curcuma longa L., leaves of Ocimum sanctum L. and Achillea millefolium L., roots of Withania somnifera L., and stem bark of Azadirachta indica A. Juss. were coarsely powdered and extracted in three different solvents (water, ethanol, and hydroethanol). The antioxidant potential was determined through 1, 1-diphenyl-2-picrylhydrazyl and ferric reducing capacity methods. Thin-layer chromatography (TLC) was carried out for the comparative metabolite profiling of the extracts using toluene, ethyl acetate, and formic acid (5 : 4 : 1, v/v/v) as a solvent system. In vitro immunomodulatory activity of the extracts has been tested on splenocyte proliferation and pinocytic assay. Hydroethanolic extract (HEE) of most of the plant materials has the highest phenolic and flavonoid contents, followed by water extract (WE) and ethanolic extract (EE), whereas the water extracts of most of the plant material showed better antioxidant activity. Almost all extract exhibited splenocyte proliferation and pinocytic activity in a dose-dependent manner. But water extract showed significantly higher splenocyte proliferation and pinocytic activity as compared to the other two extracts. TLC analysis resulted in detection of totally 63 and 56 metabolites at 254 nm and 366 nm, respectively. Through principal component analysis (PCA), it was observed that metabolite pattern of different extracts from same plant materials may be different or similar. This preliminary result can be used for quality evaluation and to develop a synergy-based polyherbal combination of water extracts of selected plant materials.

Ashwagandha in brain disorders: A review of recent developments.

ETHNOPHARMACOLOGICAL RELEVANCE: Withania somnifera (Family: Solanaceae), commonly known as Ashwagandha or Indian ginseng is distributed widely in India, Nepal, China and Yemen. The roots of plant consist of active phytoconstituents mainly withanolides, alkaloids and sitoindosides and are conventionally used for the treatment of multiple brain disorders. AIM OF THE REVIEW: This review aims to critically assess and summarize the current state and implication of Ashwagandha in brain disorders. We have mainly focussed on the reported neuroactive phytoconstituents, available marketed products, pharmacological studies, mechanism of action and recent patents published related to neuroprotective effects of Ashwagandha in brain disorders. MATERIALS AND METHODS: All the information and data was collected on Ashwagandha using keywords "Ashwagandha" along with "Phytoconstituents", "Ayurvedic, Unani and Homeopathy marketed formulation", "Brain disorders", "Mechanism" and "Patents". Following sources were searched for data collection: electronic scientific databases such as Science Direct, Google Scholar, Elsevier, PubMed, Wiley On-line Library, Taylor and Francis, Springer; books such as AYUSH Pharmacopoeia; authentic textbooks and formularies. RESULTS: Identified neuroprotective phytoconstituents of Ashwagandha are sitoindosides VII-X, withaferin A, withanosides IV, withanols, withanolide A, withanolide B, anaferine, beta-sitosterol, withanolide D with key pharmacological effects in brain disorders mainly anxiety, Alzheimer's, Parkinson's, Schizophrenia, Huntington's disease, dyslexia, depression, autism, addiction, amyotrophic lateral sclerosis, attention deficit hyperactivity disorder and bipolar disorders. The literature survey does not highlight any toxic effects of Ashwagandha. Further, multiple available marketed products and patents recognized its beneficial role in various brain disorders; however, very few data is available on mechanistic pathway and clinical studies of Ashwagandha for various brain disorders is scarce and not promising. CONCLUSION: The review concludes the results of recent studies on Ashwagandha suggesting its extensive potential as neuroprotective in various brain disorders as supported by preclinical studies, clinical trials and published patents. However vague understanding of the mechanistic pathways involved in imparting the neuroprotective effect of Ashwagandha warrants further study to promote it as a promising drug candidate.

Quality Control and Stability Testing of Arq Formulations of Unani Pharmacopeia of India Using HPTLC and GC-MS.

BACKGROUND: There is an increasing global demand for traditional medicines because of their efficacy, ease of availability, and few or no side effects. The Unani medicines have long been used in India and abroad for various disorders and diseases. Arqiyat (Arq; distillate) is one of the most widely used Unani formulation prepared by simple distillation from single or combination drugs. OBJECTIVE: In the present investigation, an attempt has been made to generate data for quality control and stability testing of Arq formulations of the Unani Pharmacopeia of India that includes only three Arq formulations. METHOD: Arq-e-Nana (AeN), Arq-e-Gazar (AeG), and Arq-e-Brinjasif (AeB) were prepared through hydrodistillation. Quality control and stability analysis were performed by using HPTLC and GC-MS. RESULTS: Dichloromethane extract of AeB showed maximum of 10 bands at different retardation factor values; however, there are 7 bands in AeN as well as in AeG. GC-MS analysis showed presence of 10 metabolites in AeG, 8 in AeN, and 9 in AeB. Stability studies showed that the shelf life of Arq formulations would be enhanced at refrigeration (5 ± 2°C) conditions. CONCLUSIONS: The present study highlights quality control and stability analysis of Arq formulations of Unani Pharmacopeia of India using HPTLC and GC-MS.

HPTLC and UPLC-MS/MS Methods for Quality Control Analysis of Itrifal Formulations of Unani System of Medicine.

BACKGROUND: Ultra-performance LC (UPLC)-tandem MS (MS/MS) and high-performance TLC (HPTLC) assay methods were developed for chemical fingerprinting and quantitative analysis of bioactive constituents of a certain "Itrifal formulation," a traditional kind of Unani medicine. OBJECTIVE: In the present investigation, HPTLC and UPLC-MS/MS methods were developed and validated for the detection and quantification of major metabolites present in itrifal formulation. METHODS: The metabolites present in the formulation were separated using modern chromatographic techniques, and a quantitative analysis was performed. Analytical performance of the proposed HPTLC and UPLC-MS/MS methods was validated as per the defined guidelines with respect to linearity, accuracy, precision, robustness, and specificity. RESULTS: The developed UPLC-MS/MS and HPTLC methods were used for quantification of gallic acid, tannic acid, catechin, and quercetin. All four constituents were quantified by UPLC-MS/MS, while two constituents were quantified by HPTLC in the commercial itrifal formulation. The calibration plot was found to be linear, accurate, precise, robust, and specific for both HPTLC and UPLC-MS/MS. CONCLUSIONS: The present methods were successfully applied for analysis of the given markers in itrifal formulations. The same can be used for QC and stability testing of itrifal formulations.

Chromatographic Profiling of Rose Petals in Unani Formulations (Gulkand, Arq-e-Gulab, and Rose Sharbat) Using HPTLC and GC-MS.

BACKGROUND: One of the most precious systems of traditional medicine is the Unani system of medicine. A wide variety of formulations indigenous to the Unani medicine have been preserved over the years. These formulations are potent and effective even after ages of postformulation. Rose petals are an example of such an herb, which is extensively and popularly used in Unani formulations for edible and cosmetic purposes. Rose petals are rich in terpenes, glycosides, flavonoids, and anthocyanins. OBJECTIVE: The aim of this study was to characterize the phytochemical profiling of different rose varieties and their marketed formulations. METHOD: HPTLC method was developed for detecting overall profile and assessing variations among the rose varieties available in market along with the popular formulations of rose such as gulkand (Brand A and Brand B), Arq-e-gulab or Gulab Jal (Brand C), and sharbat (Brand D). GC-MS analysis was also carried out for fingerprinting of rose varieties and formulations. RESULTS: HPTLC and GC-MS fingerprinting showed some common peaks in rose samples as well as in the formulation samples. The methods also gave different peaks for the adulterant that might be used in place of rose. CONCLUSIONS: Both the methods could be used for standardization of herbal products containing rose as one of the ingredients and also used to check for the adulteration. HIGHLIGHTS: The current advanced chromatographic analysis is a valuable tool to determine the quality of the formulation.