Cold atmospheric plasma-activated medium for potential ovarian cancer therapy.

Cold atmospheric plasma (CAP) has emerged as an innovative tool with broad medical applications, including ovarian cancer (OC) treatment. By bringing CAP in close proximity to liquids such as water or cell culture media, solutions containing reactive oxygen species (ROS) and reactive nitrogen species (RNS) are generated, called plasma-activated media (PAM). In this systematic review, we conduct an in-depth analysis of studies focusing on PAM interactions with biological substrates. We elucidate the diverse mechanisms involved in the activation of different media and the complex network of chemical reactions underlying the generation and consumption of the prominent reactive species. Furthermore, we highlight the promises of PAM in advancing biomedical applications, such as its stability for extended periods under appropriate storage conditions. We also examine the application of PAM as an anti-cancer and anti-metastatic treatment for OC, with a particular emphasis on its ability to induce apoptosis via distinct signaling pathways, inhibit cell growth, suppress cell motility, and enhance the therapeutic effects of chemotherapy. Finally, the future outlook of PAM therapy in biomedical applications is speculated, with emphasis on the safety issues relevant to clinical translation.

Frequency and Types of Anemia in Primary Hypothyroidism Patients: A Prospective Observational Study.

Background Primary hypothyroidism is a common endocrine disorder resulting from inadequate production of thyroid hormones. Anemia is a common condition that can occur in hypothyroidism. Anemia may occur due to nutrient deficiency, such as iron or vitamin B12 deficiency due to chronic disease in hypothyroidism. Therefore, it is important to evaluate the cause of anemia in hypothyroidism.  Objective The aim of this study was to determine the frequency of anemia and its types in patients with primary hypothyroidism. Methods This was a prospective cross-sectional observational study conducted at the Department of Medicine, Jinnah Postgraduate Medical Center, Karachi, Pakistan, using non-probability consecutive sampling. A total of 176 adults aged 18-65 years of either gender, newly diagnosed with primary hypothyroidism, or with any of its symptoms were included in the study. Patients already on anti-thyroid medication and with post-thyroidectomy hypothyroidism were excluded from the study. The duration of the study was 1.5 years, from January 2020 to July 2021. After ethical approval, written informed consent was obtained from each patient. Demographical data along with results of complete blood picture, including Hb and MCV for diagnosing anemia and its types were recorded on a pre-designed proforma. The chi-square test was applied keeping p < 0.05 as statistically significant. Results The mean age of the patients was 42.19 ± 8.43 years, with 59.66% (n = 105) females and 40.34% (n = 71) males. A total of 67% (n =118) patients were found to be anemic. Of these, 38.64% (n = 68) patients had normocytic anemia, 19.32% (n = 34) microcytic anemia, and 9.25% (n = 16) patients had macrocytic anemia; 56.34% (n = 40) males and 74.29% (n = 78) females were reported to be anemic (p = 0.01). Conclusion In our study, the frequency of anemia in patients with hypothyroidism was high, with normocytic anemia being the most common type. It is important to know the type of anemia in hypothyroidism, as normocytic anemia is due to the chronic disease process (anemia of chronic disease) and may not respond to nutrient supplementation. Conversely, microcytic anemia is commonly due to iron deficiency and macrocytic anemia is due to vitamin B12 deficiency and therefore, they require replacement therapy. In any case, it is important to identify and treat the underlying cause of anemia.

Quantitative phase imaging for characterization of single cell growth dynamics.

Quantitative phase imaging (QPI) has emerged as an indispensable tool in the field of biomedicine, offering the ability to obtain quantitative maps of phase changes due to optical path length delays without the need for contrast agents. These maps provide valuable information about cellular morphology and dynamics, unperturbed by the introduction of exogenous substances. In this review, a summary of recent studies that have focused on elucidating the growth dynamics of individual cells using QPI is presented. Specifically, investigations into cellular changes occurring during mitosis, the differentiation of cellular organelles, the assessment of distinct cell death processes (i.e., apoptosis, necrosis, and oncosis) and the precise measurement of live cell temperature are explored. Furthermore, the captivating applications of QPI in theragnostics, where its potential for transformative impact is prominently showcased, are highlighted. Finally, the challenges that need to be overcome for its wider adoption and successful integration into biomedical research are outlined.

Targeted Analysis of Veterinary Drugs in Food Samples by Developing a High-Resolution Tandem Mass Spectral Library.

Veterinary drug residues present in foods can pose severe health threats to the population. The present study aims to develop a high-resolution mass spectral library of 158 veterinary drugs of 16 different classes for their rapid identification in food samples through liquid chromatography-high-resolution electrospray ionization-tandem mass spectrometry (LC-HR-ESI-MS/MS). Standard drugs were pooled according to their log P values and exact masses before analysis. Spectra were collected at system automated collision energy, i.e., of 25-60 eV and four predetermined collision energies (10, 20, 30, and 40 eV) for each compound using a schedule precursor list of [M + H]+, [M + Na]+, and [M + NH4]+ ions. The utility of the developed database was checked by analyzing food samples. A total of 17 veterinary drugs based on the reference standard retention times (RTs), HR-MS spectra, and MS/MS spectra were identified in the analyzed samples. Moreover, five veterinary drugs were selected for quantitative analysis, including doxycycline hyclate, lincomycin, sulfasalazine, moxifloxacin, and diphenoxylate, using liquid chromatography-ion trap mass-spectrometry (LC-IT-MS). Concentrations of the drug were obtained to vary from 0.0805 to 0.9731 mg/kg in food samples and were found to be exceeded in most of the cases as per the maximum residue levels described by Food and Agriculture Organization (FAO)/World Health Organization (WHO). The MS data were submitted to the MetaboLights online database (MTBLS2914). This study will help in the high-throughput screening of multiclass veterinary drugs in foodstuffs.

Photoacoustic imaging for characterization of radiofrequency ablated cardiac tissues.

Photoacoustic (PA) imaging is an emerging technique being explored for various clinical applications. PA imaging offers a portable, inexpensive, stand-alone modality for evaluating optical contrast agents. PA signals are well-correlated with tissue physical parameters and can quantify various physiological variables (e.g., oxygenation of hemoglobin). Moreover, radiofrequency (RF) ablation is a promising treatment for certain cardiac arrhythmias. Assessment of RF-ablated lesions is of clinical importance. The purpose of this study is to elaborate the PA imaging to characterize RF-ablated cardiac tissues. Specifically, we describe the application of PA imaging to identify, characterize, and quantify cardiac RF lesions, highlighting the fundamental principles and unique benefits of this optical imaging technique. Potential future clinical application of PA imaging that reveals additional information about structural damage in RF-treated cardiac tissue are also anticipated.

Variation in Hypothalamic GnIH Expression and Its Association with GnRH and Kiss1 during Pubertal Progression in Male Rhesus Monkeys (Macaca mulatta).

Modulation of pulsatile gonadotropin-releasing hormone (GnRH) secretion across postnatal development in higher primates is not fully understood. While gonadotropin-inhibitory hormone (GnIH) is reported to suppress reproductive axis activity in birds and rodents, little is known about the developmental trajectory of GnIH expression in rhesus monkeys throughout the pubertal transition. This study was aimed at examining the variation in GnIH immunoreactivity (-ir) and associated changes among GnIH, GnRH, and Kiss1 mRNA expression in the hypothalamus of infant, juvenile, prepubertal, and adult male rhesus monkeys. The brains from rhesus macaques were collected from infancy until adulthood and were examined using immunofluorescence and RT-qPCR. The mean GnIH-ir was found to be significantly higher in prepubertal animals (p < 0.01) compared to infants, and significantly reduced in adults (p < 0.001). Significantly higher (p < 0.001) GnRH and Kiss1 mRNA expression was noted in adults while GnIH mRNA expression was the highest at the prepubertal stage (p < 0.001). Significant negative correlations were seen between GnIH-GnRH (p < 0.01) and GnIH-Kiss1 (p < 0.001) expression. Our findings suggest a role for GnIH in the prepubertal suppression of the reproductive axis, with disinhibition of the adult reproductive axis occurring through decreases in GnIH. This pattern of expression suggests that GnIH may be a viable target for the development of novel therapeutics and contraceptives for humans.

Gastric mucosal precancerous lesions in Helicobacter pylori-infected pediatric patients in central China: A single-center, retrospective investigation.

BACKGROUND: Helicobacter pylori (H. pylori) infects about 50% of the world population and is the major cause of chronic gastritis, peptic ulcers, and gastric cancer. Chronic H. pylori infection induces gastric mucosal precancerous lesions mostly in adulthood, and it is debatable whether these pathological conditions can occur in childhood and adolescents as well. Since this is a critical issue to determine if intervention should be offered for this population group, we investigated the gastric mucosal precancerous lesions in pediatric patients in an area in central China with a high prevalence of H. pylori and gastric cancer. AIM: To investigate the relationship of H. pylori infection and gastric mucosal precancerous lesions in children and adolescents in central China. METHODS: We screened 4258 ward-admitted children and adolescent patients with upper gastrointestinal symptoms, and finally enrolled 1015 pediatric patients with H. pylori infection and endoscopic and histological data. H. pylori infection status was determined by rapid urease test and histopathological examination. Both clinical and pathological data were collected and analyzed retrospectively. Occurrence of gastric mucosal precancerous lesions, inflammatory activity and degree of inflammatory cell infiltration between H. pylori-positive and -negative groups were compared. RESULTS: Among the 1015 eligible children and adolescents, the overall H. pylori infection rate was 84.14% (854/1015). The infection rate increased with age. The incidence of gastric mucosal precancerous lesions in H. pylori-infected children was 4.33% (37/854), which included atrophic gastritis (17 cases), intestinal metaplasia (11 cases) and dysplasia (9 cases). In H. pylori-negative patients, only 1 atrophic gastritis case [0.62%, (1/161)] was found (P < 0.05). Active inflammation in H. pylori-infected patients was significantly higher than that in non-infected patients, and the H. pylori-infected group showed more severe lymphocyte and neutrophil granulocyte infiltration (P < 0.001). In addition, endoscopy revealed that the most common findings in H. pylori-positive patients were antral nodularity, but in H. pylori-negative patients only superficial gastritis was observed. CONCLUSION: In children and adolescents, gastric mucosal precancerous lesions occurred in 4.33% of H. pylori-infected patients in central China. These cases included atrophic gastritis, intestinal metaplasia, and dysplasia. The data revealed an obvious critical issue requiring future investigation and intervention for this population group.

Sensitive Detection of Pharmaceutical Drugs and Metabolites in Serum Using Data-Independent Acquisition Mass Spectrometry and Open-Access Data Acquisition Tools.

Data-independent acquisition (DIA) based strategies have been explored in recent years for improving quantitative analysis of metabolites. However, the data analysis is challenging for DIA methods as the resulting spectra are highly multiplexed. Thus, the DIA mode requires advanced software analysis to facilitate the data deconvolution process. We proposed a pipeline for quantitative profiling of pharmaceutical drugs and serum metabolites in DIA mode after comparing the results obtained from full-scan, Data-dependent acquisition (DDA) and DIA modes. using open-access software. Pharmaceutical drugs (10) were pooled in healthy human serum and analysed by LC-ESI-QTOF-MS. MS1 full-scan and Data-dependent (MS2) results were used for identification using MS-DIAL software while deconvolution of MS1/MS2 spectra in DIA mode was achieved by using Skyline software. The results of acquisition methods for quantitative analysis validated the remarkable analytical performance of the constructed workflow, proving it to be a sensitive and reproducible pipeline for biological complex fluids.

In Vitro and In Vivo Studies for the Investigation of γ-Globin Gene Induction by Adhatoda vasica: A Pre-Clinical Study of HbF Inducers for ß-Thalassemia.

Fetal hemoglobin (HbF) is a potent genetic modifier, and the γ-globin gene induction has proven to be a sustainable therapeutic approach for the management of ß-thalassemia. In this study, we have evaluated the HbF induction ability of A. vasica in vitro and in vivo, and the identification of potential therapeutic compounds through a bioassay-guided approach. In vitro benzidine-Hb assay demonstrated strong erythroid differentiation of K562 cells by A. vasica extracts. Subsequently, an in vivo study with an aqueous extract of A. vasica (100 mg/kg) showed significant induction of the γ-globin gene and HbF production. While in the acute study, the hematological and biochemical indices were found to be unaltered at the lower dose of A. vasica. Following the bioassay-guided approach, two isolated compounds, vasicinol (1) and vasicine (2) strongly enhanced HbF levels and showed prominent cellular growth kinetics with ample accumulation of total hemoglobin in K562 cultures. High HbF levels were examined by immunofluorescence and flow cytometry analysis, concomitant with the overexpression in the γ-globin gene level. Compound 1 (0.1 µM) and compound 2 (1 µM) resulted in a greater increase in F-cells (90 and 83%) with marked up (8-fold and 5.1-fold) expression of the γ-globin gene, respectively. Molecular docking studies indicated strong binding affinities of (1) and (2) with HDAC2 and KDM1 protein that predict the possible mechanism of compounds in inhibition of these epigenetic regulators in the γ-globin gene reactivation. Altogether, these observations demonstrated the therapeutic usefulness of A. vasica for fostering HbF production in clinical implications for blood disorders.

Structure-fragmentation study of pentacyclic triterpenoids using electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-QTOFMS/MS).

RATIONALE: Pentacyclic triterpenoids are secondary plant metabolites widespread in fruit peel, leaves, and stem bark. Due to their important biological activities, these compounds are widely screened using advance analytical techniques like electrospray ionization (ESI) mass spectrometry. Over the past few decades, the practice of ESI has been refined into a versatile ionization technique for a wide variety of analytes differing in their chemical makeup, size, complexity, and bimolecular stability. METHODS: The structure-fragmentation relationships (SFRs) of 16 pentacyclic triterpenoids were studied using a positive ion ESI quadrupole time-of-flight mass spectrometry (ESI-QqTOFMS/MS) hybrid instrument. RESULTS: ESI-QqTOFMS (positive ion mode) showed the presence of the protonated molecules [M + H]+ of most of analyzed compounds. Low-energy collision-induced dissociation MS/MS analysis of these molecules indicated multiple losses of water molecules and the loss of the formic acid moiety [M + H - HCOOH]+ and other substituents as the predominant pathway for further fragmentation. Key product ions were identified which resulted from the retro-Diels-Alder cleavage of the ring system. SFRs of all the compounds analyzed were also developed. CONCLUSIONS: We developed a fragmentation pattern of pentacyclic triterpenoids using the ESI-QqTOFMS/MS technique. It was concluded that the formation of key product ions and loss of characteristic neutrals can give detailed insight into structural information about the basic structure and attached substituents.

A UPLC-DAD-Based Bio-Screening Assay for the Evaluation of the Angiotensin Converting Enzyme Inhibitory Potential of Plant Extracts and Compounds: Pyrroquinazoline Alkaloids from Adhatoda vasica as a Case Study.

Angiotensin converting enzyme (ACE) plays a crucial role in regulating blood pressure in the human body. Identification of potential ACE inhibitors from medicinal plants supported the idea of repurposing these medicinal plants against hypertension. A method based on ultra-performance liquid chromatography (UPLC) coupled with a diode array detector (DAD) was used for the rapid screening of plant extracts and purified compounds to determine their ACE inhibitory activity. Hippuryl-histidiyl-leucine (HHL) was used as a substrate, which is converted into hippuric acid (HA) by the action of ACE. A calibration curve of the substrate HHL was developed with the linear regression 0.999. The limits of detection and quantification of this method were found to be 0.134 and 0.4061 mM, respectively. Different parameters of ACE inhibitory assay were optimized, including concentration, incubation time and temperature. The ACE inhibition potential of Adhatoda vasica (methanolic-aqueous extract) and its isolated pyrroquinazoline alkaloids, vasicinol (1), vasicine (2) and vasicinone (3) was evaluated. Compounds 1-3 were characterized by various spectroscopic techniques. The IC50 values of vasicinol (1), vasicine (2) and vasicinone (3) were found to be 6.45, 2.60 and 13.49 mM, respectively. Molecular docking studies of compounds 1-3 were also performed. Among these compounds, vasicinol (1) binds as effectively as captopril, a standard drug of ACE inhibition.

Rapid Identification of Common Secondary Metabolites of Medicinal Herbs Using High-Performance Liquid Chromatography with Evaporative Light Scattering Detector in Extracts.

The discovery and identification of novel natural products of medicinal importance in the herbal medicine industry becomes a challenge. The complexity of this process can be reduced by dereplication strategies. The current study includes a method based on high-performance liquid chromatography (HPLC), using the evaporative light scattering detector (ELSD) to identify the 12 most common secondary metabolites in plant extracts. Twelve compounds including rutin, taxifolin, quercetin, apigenin, kaempferol, betulinic acid, oleanolic acid, betulin, lupeol, stigmasterol, and ß-sitosterol were analyzed simultaneously. The polarity of the compounds varied greatly from highly polar (flavonoids) to non-polar (triterpenes and sterols). This method was also tested for HPLC-DAD and HPLC-ESI-MS/MS analysis. Oleanolic acid and ursolic acid could not be separated in HPLC-ELSD analysis but were differentiated using LC-ESI-MS/MS analysis due to different fragment ions. The regression values (R2 > 0.996) showed good linearity in the range of 50-1000 µg/mL for all compounds. The range of LOD and LOQ values were 7.76-38.30 µg/mL and 23.52-116.06 µg/mL, respectively. %RSD and % trueness values of inter and intraday studies were mostly <10%. This method was applied on 10 species of medicinal plants. The dereplication strategy has the potential to facilitate and shorten the identification process of common secondary metabolites in complex plant extracts.

Management of Fifth Metacarpal Neck Fracture (Boxer's Fracture): A Literature Review.

Boxer's fracture is the fifth metacarpal neck fracture resulting from direct trauma to the clenched fist. Worldwide, this type of fracture is the most typical presentation to emergency departments. The management of fifth metacarpal fractures varies from one setting to another. Conservative management is the preferred option for closed, non-angulated, non-malrotated fractures while open fractures, significant angulation, rotational deformity, and intra-articular extension are recognised indications for surgical intervention. The scope of this article covers the results of a literature review examining the management strategies for such fractures.

Retroperitoneal Ganglioneuroma in a Patient Presenting With Vague Abdominal Pain.

Ganglioneuroblastoma, neuroblastoma, and ganglioneuroma (GN) are the tumors that arise from the neural crest cells. Of these, GN has the most benign origin without metastatic potential. The most common sites of their origin are the posterior mediastinum and retroperitoneum. Although the imaging studies, including CT, are available to detect these tumors, the definitive diagnosis can only be made by histological examination. We present a case of a 40-year-old woman with a retroperitoneal GN causing longstanding, gradually increasing, uncontrolled abdominal pain due to its pressure effect on the pancreas, duodenum, and right kidney with the displacement of the inferior vena cava. An exploratory laparotomy was performed, and the mass was removed. Histopathology confirmed the benign nature of the mass (a GN). These tumors are rarely malignant and mostly asymptomatic. However, in our case, abdominal pain was affecting the patient's life. After a discussion with the patient, an elective surgical procedure was performed, and the patient was symptom-free postoperatively and able to resume her regular routine.

Type I and type II Helicobacter pylori infection status and their impact on gastrin and pepsinogen level in a gastric cancer prevalent area.

BACKGROUND: Type I Helicobacter pylori (H. pylori) infection causes severe gastric inflammation and is a predisposing factor for gastric carcinogenesis. However, its infection status in stepwise gastric disease progression in this gastric cancer prevalent area has not been evaluated; it is also not known its impact on commonly used epidemiological gastric cancer risk markers such as gastrin-17 (G-17) and pepsinogens (PGs) during clinical practice. AIM: To explore the prevalence of type I and type II H. pylori infection status and their impact on G-17 and PG levels in clinical practice. METHODS: Thirty-five hundred and seventy-two hospital admitted patients with upper gastrointestinal symptoms were examined, and 523 patients were enrolled in this study. H. pylori infection was confirmed by both 13C-urea breath test and serological assay. Patients were divided into non-atrophic gastritis (NAG), non-atrophic gastritis with erosion (NAGE), chronic atrophic gastritis (CAG), peptic ulcers (PU) and gastric cancer (GC) groups. Their serological G-17, PG I and PG II values and PG I/PG II ratio were also measured. RESULTS: A total H. pylori infection rate of 3572 examined patients was 75.9%, the infection rate of 523 enrolled patients was 76.9%, among which type I H. pylori infection accounted for 72.4% (291/402) and type II was 27.6%; 88.4% of GC patients were H. pylori positive, and 84.2% of them were type I infection, only 11.6% of GC patients were H. pylori negative. Infection rates of type I H. pylori in NAG, NAGE, CAG, PU and GC groups were 67.9%, 62.7%, 79.7%, 77.6% and 84.2%, respectively. H. pylori infection resulted in significantly higher G-17 and PG II values and decreased PG I/PG II ratio. Both types of H. pylori induced higher G-17 level, but type I strain infection resulted in an increased PG II level and decreased PG I/PG II ratio in NAG, NAGE and CAG groups over uninfected controls. Overall PG I levels showed no difference among all disease groups and in the presence or absence of H. pylori; in stratified analysis, its level was increased in GC and PU patients in H. pylori and type I H. pylori-positive groups. CONCLUSION: Type I H. pylori infection is the major form of infection in this geographic region, and a very low percentage (11.6%) of GC patients are not infected by H. pylori. Both types of H. pylori induce an increase in G-17 level, while type I H. pylori is the major strain that affects PG I and PG IIs level and PG I/PG II ratio in stepwise chronic gastric disease. The data provide insights into H. pylori infection status and indicate the necessity and urgency for bacteria eradication and disease prevention in clinical practice.

A high-throughput method for dereplication and assessment of metabolite distribution in Salvia species using LC-MS/MS.

Dereplication of crude plant extracts through liquid chromatography-mass spectrometry is a powerful technique for the discovery of novel natural products. Unfortunately, this technique is often plagued by a low level of confidence in natural product identification. This is mainly due to the lack of extensive chromatographic and mass spectrometric optimizations that result in improper and incomplete MS/MS fragmentation data. This study proposes a solution to this problem by the optimization of chromatographic separation and mass spectrometry parameters. We report herein a direct and high-throughput strategy for natural product dereplication in five Salvia species using high-resolution ESI-QTOF-MS/MS data. In the present study, we were able to identify a total of forty-seven natural products in crude extracts of five Salvia species using MS/MS fragmentation data. In addition to dereplication of Salvia species, quantitative profiling of twenty-one bioactive constituents of the genus was also performed on an ion trap mass spectrometer. For the quantitation study, method development focused on chromatographic optimizations to achieve maximum sensitivity. The developed dereplication and quantitation strategy can be extended to develop comprehensive metabolic profiles of other plant genera and species and thus can prove useful in the field of drug discovery from plants.

Metabolite distribution and correlation studies of Ziziphus jujuba and Ziziphus nummularia using LC-ESI-MS/MS.

Ziziphus jujuba and Ziziphus nummularia are two important species of the genus Ziziphus. Both plants offer great commercial value and are consumed as food around the world and used for their medicinal values such as anti-inflammatory, antioxidant and hepatoprotective activities. Comprehensive chemical profiling of Z. jujuba and Z. nummularia was done through identification of major metabolites using HPLC-QTOF-MS/MS and quantification of eight analytes using HPLC-IonTrap-MS/MS analysis. A total of 53 compounds were identified from their high-resolution mass spectra in both positive and negative ionization modes. Among these, 52 compounds were found to be present in Z. jujuba, and 45 in Z. nummularia. Chemometric analysis was also performed to assess the distribution of identified compounds and to determine how the obtained data can be used to discriminate between the two species. Moreover, a method for the quantification of eight analytes including, 6″'-feruloylspinosin (1), apigenin (2), apigenin-7-O-glucoside (3), catechin (4), jujuboside A (5), jujuboside B (6), luteolin (7) and quercetin (8) was developed. The method expressed excellent accuracy with less than 3% error and good reproducibility with less than 4% RSD. The limit-of-detection (LOD) was also found to be very low ranging between 0.06 ng/mL to 4.10 ng/mL while limit-of-quantitation (LOQ) values were in the range of 0.17 ng/mL to 12.42 ng/mL. The analyte concentrations were found to be varying from 1.32 mg/kg to 645.76 mg/kg in both species. The developed method was used to identify and quantify marker compounds in fruit and whole plant samples of both species and in their herbal products as well. The present work is unprecedented, as there is no such extensive study targeting fruits, leaves and herbal formulations together using high-resolution techniques. The study will provide great utility in drug discovery, in taxonomical identification of these plants and to develop quality control protocols for their herbal formulations.

Metabolite Profiling and Quantitation of Cucurbitacins in Cucurbitaceae Plants by Liquid Chromatography coupled to Tandem Mass Spectrometry.

Cucurbitaceae is an important plant family because many of its species are consumed as food, and used in herbal medicines, cosmetics, etc. It comprises annual vines and is rich in various bioactive principles which include the cucurbitacins. These steroidal natural products, derived from the triterpene cucurbitane, are mainly the bitter principles of the family Cucurbitaceae. Their biological activities include anti-inflammatory, hepatoprotective, and anti-cancer activities. A total of 10 species belonging to 6 genera of the Cucurbitaceae family along with Cissampelos pareira (Menispermaceae) were included in this study. A comprehensive profiling of certain natural products was developed using HPLC-QTOF-MS/MS analysis and a distribution profile of several major natural products in this family was obtained. A total of 51 natural products were detected in both positive and negative ionization modes, based on accurate masses and fragmentation patterns. Along with this, quantitation of four bioactive cucurbitacins, found in various important plants of the Cucurbitaceae family, was carried out using multiple reaction monitoring (MRM) approach on an ion trap mass spectrometer. Cucurbitacin Q was found to be the most abundant in C. pareira, while Citrullus colocynthis contained all four cucurbitacins in abundant quantities. The developed quantitation method is simple, rapid, and reproducible.

Combining untargeted and targeted metabolomics approaches for the standardization of polyherbal formulations through UPLC-MS/MS.

INTRODUCTION: Polyherbal formulations are an integral part of various indigenous medicinal systems such as Traditional Chinese Medicine (TCM) and Ayurveda. The presence of a very large number of compounds makes the quality control of polyherbal formulations very difficult. OBJECTIVES: To overcome this problem, we have developed a comprehensive strategy for the dereplication of natural products in polyherbal formulations by using Adhatoda vasica as a case study. METHODS: The strategy is based on five major steps: the collection of plant samples from different locations to observe the effects of environmental variables; LC-ESI-MS/MS-based untargeted metabolite profiling of the plant samples to identify marker compounds using extensive chemometric analysis of the obtained data; the identification of marker compounds in polyherbal products; the isolation, purification and characterization of the marker compounds; and MRM-based quantitative analysis of the isolated marker compounds using LC-ESI-MS/MS. RESULTS: Using this strategy, we identified a total of 51 compounds in the methanolic extract of A. vasica plants from 14 accessions. Chemical fingerprinting of the plant led to the identification of characteristic peaks that were used to confirm the presence of A. vasica in complex polyherbal formulations. Four quinazoline alkaloids (marker compounds) were isolated, purified and quantified in various herbal formulations containing A. vasica. CONCLUSION: This method demonstrates a comprehensive strategy based on untargeted and targeted metabolite analysis that can be used for the standardization of complex polyherbal formulations.