Euglenatides, Potent Antiproliferative Cyclic Peptides Isolated from the Freshwater Photosynthetic Microalga Euglena gracilis.

By limiting the nitrogen source to glutamic acid, we isolated cyclic peptides from Euglena gracilis containing asparagine and non-proteinogenic amino acids. Structure elucidation was accomplished through spectroscopic methods, mass spectrometry and chemical degradation. The euglenatides potently inhibit pathogenic fungi and cancer cell lines e.g., euglenatide B exhibiting IC50 values of 4.3 µM in Aspergillus fumigatus and 0.29 µM in MCF-7 breast cancer cells. In an unprecedented convergence of non-ribosomal peptide synthetase and polyketide synthase assembly-line biosynthesis between unicellular species and the metazoan kingdom, euglenatides bear resemblance to nemamides from Caenorhabditis elegans and inhibited both producing organisms E. gracilis and C. elegans. By molecular network analysis, we detected over forty euglenatide-like metabolites in E. gracilis, E. sanguinea and E. mutabilis, suggesting an important biological role for these natural products.

A comprehensive LCMS/MS characterization for the green extracted cucurbitane-triterpenoid glycosides from bitter melon (Momordica charantia) fruit.

A first-time green extraction and LCMSMS analysis for karavilosides (KVs) VIII, X, and XI in different parts (skin, pith, and seed) of the fresh and dried fruit of bitter melon (BM) is reported herein. Ultrasonication for green extraction whereas, LCMS/MS for KVs quantification were used. More extract yield (675.80 ± 163.57 mg/g) was observed for the dried fruit parts compared to the fresh BM-fruit parts (513.20 ± 75.42 mg/g). The fresh skin (343.40 ± 54.07 mg/4g) and dried seeds (311.80 and 77.95 ± 38.98) exhibited more yield whereas, the solvent yield (mg/4mg) observed was; H2O (651.70) > EtOH (227.20) > EtAC (163.30) > ACT (146.80). The LCMS/MS yield for the KVs revealed a descending order; KVXI (2376.44 ppb) > KVX (639.17 ppb) > KVVIII (599.83 ppb). More correlation was seen for the solvent Vs extract yield whereas, the KVs revealed more correlation for the BM-fruit part (P = 0.05). The study comprehensively characterized the parts of fresh and dried BM-fruits in terms of extract yield and KVs amount.

Anti-Infective Activity of Momordica charantia Extract with Molecular Docking of Its Triterpenoid Glycosides.

Momordica charantia, commonly known as bitter melon, is a fruiting plant that has been used for several diseases including infectious diseases. In this study, we report the antibacterial, antifungal, and antiviral activity of different bitter melon fruit parts originating from India and Saudi Arabia. The in vitro experiments are supported by the molecular docking of karavilosides to verify their role in the bioactivity. The antimicrobial assays revealed activity against Candida albicans, Escherichia coli, and Staphylococcus aureus. The extracts exhibited the potent inhibition of HIV-I reverse transcriptase, with an IC50 of 0.125 mg/mL observed for the pith extract originating from Saudi Arabia and the standard drug doxorubicin. The molecular docking of karavilosides exhibited a significant affinity to reverse transcriptase comparable to Rilpivirine and higher than that of doxorubicin. These outcomes encourage the precious bioactive components of the seed and pith of the Saudi bitter melon fruits to be further studied for isolation and structure elucidation.

Solanum pseudocapsicum vs Capsicum annum; comparative phenolics profiling using green ultrasonic extraction and UHPLC analysis.

BACKGROUND: Solanum pseudocapsicum (PC) and Capsicum annum (CA) belongs to the family of Solanaceae. CA have been reported a rich source of phenolics whereas, the phenolics content of GA (gallic acid), SC (scopoletin), RA (rosmarinic acid), and RV (resveratrol) are yet to be reported for the PC-fruit. This study comparatively evaluates the phenolics profile for different parts (seeds and skin) and colors (green and red) of the PC- and CA-fruits using the green solvents of ethanol (ET), acetone (AC), water (H2O), and different combinations of these solvents. METHODOLOGY: Ultrasonics extraction (US) and UHPLC analysis were employed for phenolics evaluation. RESULTS: The USMD (method development) revealed the highest extract yield of 62 mg/100 mg for the PC-skin in ET:AC (70:30) solvent whereas, more phenolics (ppm) were observed for PC-seeds in ET:AC (50:50) solvent, particularly the SC (29.46) and GA (16.92). The UHPLCMDMV exhibited significant accuracies (100.70-114.14 %) with r2-values (0.9993-0.9997) in the linearity range of 1-200 ppm. The USMV (method validation) in PC- and CA-fruit parts and colors revealed more extract yields for the red skin part of the PC- (180.5 mg) and CA-fruit (126.2 mg). The phenolics were seen more in the green seeds of the PC-fruit (ppm); SC (276), GA (147.36), RV (28.54), and RA (23.87) followed by the green PC-skin, and red/green CA-seeds. The statistical models of mean differences, ANOVA, and Pearson's correlation showed significant differences for the PC-fruit parts (seeds and skin) and colors (red and green) vs extract yield and phenolics content (P = 0.05). CONCLUSION: PC-and CA-fruits were successfully evaluated where the seeds for the green fruits exhibited more phenolics amount.

The effect of natural antioxidants, pH, and green solvents upon catechins stability during ultrasonic extraction from green tea leaves (Camellia sinensis).

BACKGROUND: This is a first-time report to evaluate the effect of natural antioxidants, pH, and green solvents upon catechins yield and stability during the active process of extraction from green tea leaves. METHODOLOGY: Green solvents (model-A) augmented with piperine (PPN) and quercetin (QT) as natural antioxidants (model-B) at different pH 2-6 (model-C) were used to extract catechins from green tea leaves using an ultrasonic extraction process (USE). For quantification of catechins (EC; epicatechins, ECG; epicatechin gallate, and EGCG; epigallocatechin gallate), a green and sensitive UHPLC-MS/MS method was developed and validated. RESULTS: The UHPLC-MS/MS method showed an accuracy of 98.3-102.6 % within the linearity range of 1-500 ppb for EC (m/z) 289 â†’ 245 â†’ 109, ECG (m/z) 441.2 â†’ 169 â†’ 289, and EGCG (m/z) 457.1 â†’ 169 â†’ 125.1. The general yield (ppb) for EC, ECG, and EGCG was observed with the ranges and sum of (N = 180) 0.06-157.80 and 6696.83, 0.04-316.93 and 12632.60 and, 0.12-584.11 and 26144.83, respectively. Model-C revealed the highest yield for catechins at the lowest pH-2 with an individual catechin yield of EGCG (584.11) > ECG (316.93) > EC (157.80) in CW2. In terms of stability, EGCG was the most unstable catechin whereas, catechins extracted in model-B exhibited more stability (%recovery of 14.70 for EC, 10.55 for ECG, and 5.36 for EGCG in BEP). Moreover, model-B showed the minimal degradation for catechins within the range of 11.81-94.64 (BEP); even the most degradable EGCG was seen with the smallest %loss of 11.81-94.64 at time 24-70 h, as compared to the loss of > 95 % in model-A and C. The ANOVA score for catechins yield was; F11,168 = 61.06 (EC), F11,168 = 66.53 (ECG), and F11,168 = 48.92 (EGCG) (P = 0.00) with mean scores of (M = 94.63, SD = 25.46) for EC, (M = 194.87, SD = 51.41) ECG, and (M = 357.57, SD = 96.80) EGCG in CE2. CONCLUSION: A significant effect on catechins yield and stability was observed with the use of natural antioxidants and lowest pH-2.

Ultrasonic-assisted extraction of fenugreek flavonoids and its geographical-based comparative evaluation using green UHPLC-DAD analysis.

BACKGROUND: This study, for the first time, reports a simultaneous determination of flavonoids; rutin (RT), quercetin (QT), luteolin (LT), and kaempferol (KF) in different origins of fenugreek seeds samples (N = 45) using a green UHPLC-DAD analysis METHODOLOGY: Ultrasound-assisted extraction (UAE) was employed to extract fenugreek flavonoids using different polarity solvents of n-hexane (n-hex), dichloromethane (DCM), and methanol (MeOH) RESULTS: The extract yield on an individual basis was observed in the range of 1.03-17.29 mg, with the highest yield (mg/sample) for the Egyptian sample (17.29 mg). The highest total extract yield (mg/origin) was observed for the Iranian sample (82.28 ±â€¯5.38). The solvent with the highest extract yield (mg) was n-hex 169.35 ±â€¯13.47, followed by MeOH 114.39 ±â€¯12.27. The validated green UHPLC-DAD method resulted in a short runtime (9 min) with an accuracy of 97.86(±12.32)-101.37(±5.91), r2-values = 0.993-0.999, LOD = 2.09-4.48 ppm, and LOQ = 6.33-13.57 ppm for flavonoids analysis within the linearity range of 1-500 ppm. The general yield for flavonoids exhibited a descending order (ppm): RT (2924.55 ±â€¯143.84) > QT (457.05 ±â€¯34.07) > LT (82.37 ±â€¯3.27) > KF (4.54 ±â€¯0.00). The yield (ppm) for the flavonoids was more in MeOH solvent (3424.81 ±â€¯235.44) constructing a descending order of MeOH > n-hex > DCM. For an individual flavonoid yield; MeOH was seen with an order of RT > QT > LT, n-hex (LT > QT), and DCM (RT > LT > QT). The statistical analysis of PCA (principle component analysis) revealed a widespread distribution of flavonoids in fenugreek seeds with a variance of 35.93% (PC1). Moreover, flavonoids extraction was prone to the nature and specificity of the solvent used (PC2: 33.34%) rather than the amount of the extract yield (P = 0.00). The K-mean cluster analysis showed the origins with higher flavonoids yield in appropriate solvent as I3M (Indian accession # 3 MeOH extract) with more QT amount, IR2M (Iranian accession # 2 MeOH extract) with more LT amount along with I2M (Indian accession # 2 MeOH extract) and Q2M (Qassim Saudi Arabia accession # 2 MeOH extract) containing high amount of RT. The outcomes are supported by KMO (Kaiser-Meyer-Olkin) and Bartlett's test value of 0.56 with X2-value of 191.87 (P = 0.00) CONCLUSION: The samples were effectively evaluated and standardized in terms of flavonoid amount suggesting a significant variation in fenugreek quality.

Antidiabetic and Anticancer Potentials of Mangifera indica L. from Different Geographical Origins.

Mango fruit is well known for its nutritional and health benefits due to the presence of a plethora of phytochemical classes. The quality of mango fruit and its biological activities may change depending upon the variation in geographical factors. For the first time, this study comprehensively screened the biological activities of all four parts of the mango fruit from twelve different origins. Various cell lines (MCF7, HCT116, HepG2, MRC5) were used to screen the extracts for their cytotoxicity, glucose uptake, glutathione peroxidase activity, and α-amylase inhibition. MTT assays were carried out to calculate the IC50 values for the most effective extracts. The seed part from Kenya and Sri Lanka origins exhibited an IC50 value of 14.44 ± 3.61 (HCT116) and 17.19 ± 1.60 (MCF7). The seed part for Yemen Badami (119 ± 0.08) and epicarp part of Thailand (119 ± 0.11) mango fruit showed a significant increase in glucose utilization (50 µg/mL) as compared to the standard drug metformin (123 ± 0.07). The seed extracts of Yemen Taimoor seed (0.46 ± 0.05) and Yemen Badami (0.62 ± 0.13) produced a significant reduction in GPx activity (50 µg/mL) compared to the control cells (100 µg/mL). For α-amylase inhibition, the lowest IC50 value was observed for the endocarp part of Yemen Kalabathoor (108.8 ± 0.70 µg/mL). PCA, ANOVA, and Pearson's statistical models revealed a significant correlation for the fruit part vs. biological activities, and seed part vs. cytotoxicity and α-amylase activity (p = 0.05). The seed of mango fruit exhibited significant biological activities; hence, further in-depth metabolomic and in vivo studies are essential to effectively utilize the seed part for the treatment of various diseases.

Gas Chromatography-Mass Spectrometry (GC-MS) Metabolites Profiling and Biological Activities of Various Capsicum annum cultivars.

This study evaluates the quality variation for twenty-seven capsicum fruit (CF) samples, in terms of their volatile oil composition and biological activities. The GCMS analysis revealed the presence of seventy one chemical compounds from different chemical classes with an average (%) composition of: 26.13 (alcohols) > 18.82 (hydrocarbons) > 14.97 (esters) > 3.08 (ketones) > 1.14 (others) > 1.07 (acids) > 0.72 (sugar) > 0.42 (aldehydes) > 0.15 (amino compounds). Alcohols and hydrocarbons were the most abundant in these CF samples with 1-Decanol, 2-octyl- and docosanoic acid, docosyl ester as the major components, respectively. The % inhibition in cytotoxicity assays was observed in the range of 9−47 (MCF7) and 4−41 (HCT116) whereas, the zone of inhibition (mm) for the antimicrobial activity was found to be 0.0−17 (P. aeruginosa) > 0.0−13 (E. coli and S. aureus). Moreover, the samples with the largest zone of inhibition in the agar-well-diffusion method (C16, C19, and C26) upon further evaluation presented the least MIC and MBC values against P. aeruginosa with an MIC and MBC (µg/mL) of 6.3 and 12.5, respectively. The outcome for GCMS and biological activities were further supported by statistical tools of PCA and K-mean cluster analysis which confirmed the C16 CF sample with the best activity followed by C5, C13 (the best cytotoxic), and C19, C26 (the best antimicrobial). The statistical analysis exhibited a high Chi-square value of 5931.68 (GCMS) and 32.19 (biological activities) with p = 0.00 for KMO and Bartlett's Test of Sphericity. The 27-CF samples were effectively distinguished based on quality variation, and the C16 CF sample exhibited significant potential for further study.

ICP-MS determination of elemental abundance in traditional medicinal plants commonly used in the Kingdom of Saudi Arabia.

Medicinal plants are widely used in the Kingdom of Saudi Arabia to treat various ailments in the form of folk medicine. Forty four such medicinal plant samples were collected from local markets and evaluated for the presence of 14 elements (Mn, Cr, Co, Ni, Cu, Mo, Al, Pb, Ba, Zn, Ag, Hg, Bi, Cd). Microwave-assisted digestion with inductively coupled plasma-mass spectrometry (ICP-MS) was applied to determine the elemental composition in these medicinal plants. Widespread occurrence of these elements was observed in all plant samples, except for Bi and Co where the lowest mean values of 0.03 ± 0.04 and 0.03 ± 0.15 were observed, respectively. The descending order for mean (µg/g) elemental occurrence observed was as follows: Ba > Al > Zn > Ni > Mn > Ba > Hg > Mo > Cu > Cr > Ag > Cd > Co > Bi, whereas the range for these elements in the 44-medicinal plants was as follows: Pb > Al > Zn > Ni > Mn > Cu > Mo > Ag > Ba > Hg > Co > Cd > Cr > Bi. Pb, Hg and Cd were found beyond the maximum limits in these medicinal plants, while the remaining elements were found well within the range of maximum limits. A number of medicinal plants showed high amounts of these elements. Some plants contained more than one element, such as Foeniculum vulgare Mill (Pb, Hg, Cd), Ricinus communis (Pb, Cd), Vigna radiata (Pb, Cd) and Sesamum indicum (Pb, Hg). The data matrix was validated through the statistical tools of principal component analysis (X2 = 160.44, P = .00), Pearson's correlation (P = .01 and 0.05), and K-mean cluster analysis (F = 104.55, P = .00). The findings of the study provide baseline data for the comparative analysis of these medicinal plants, which may help select safe medicinal plants in terms of consumer-based use and its utilisation for the treatment of various ailments.

Biological Screening of Glycyrrhiza glabra L. from Different Origins for Antidiabetic and Anticancer Activity.

BACKGROUND: Geographical variation may affect the phytochemistry as well as the biological activities of Glycyrrhiza glabra (licorice) root. Herein, a series of biological activities were performed to evaluate the impact of geographical origin on the biological potential of eight different licorice samples. METHODOLOGY: Cell culture studies were performed for cytotoxicity (MCF7, HCT116, HepG2, and MRC5), glucose uptake assay (HepG2), and glutathione peroxidase activity (HepG2), whereas α-amylase inhibition activity was tested for antidiabetic potential. RESULTS: The Indian sample was observed to be more cytotoxic against MCF7 (22%) and HCT116 (43%) with an IC50 value of 56.10 (±2.38) µg/mL against the MCF7 cell line. The glucose uptake was seen with a mean value of 96 (±2.82) and a range of 92-101%. For glutathione peroxidase activity (GPx), the Syrian (0.31 ± 0.11) and Pakistani samples (0.21 ± 0.08) revealed a significant activity, whereas the Palestinian (70 ± 0.09) and Indian samples (68±0.06) effectively inhibited the α-amylase activity, with the lowest IC50 value (67.11 ± 0.97) µg/mL for the Palestinian sample. The statistical models of PCA (principal component analysis) and K-mean cluster analysis were performed to correlate the geographical origin, extract yield, and biological activities for the eight licorice samples of different origins. CONCLUSION: The licorice samples exhibited significant cytotoxic, GPx, and α-amylase inhibitory activity. The samples with higher extract yield showed more potential in these biological activities.

Development of a Critical Appraisal Tool (AIMRDA) for the Peer-Review of Studies Assessing the Anticancer Activity of Natural Products: A Step towards Reproducibility.

The journal of APJCP (Asian Pacific Journal of Cancer Prevention) focuses to gather relevant and up-to-date novel information's related to cancer sciences. The research methodologies and approaches adopted by the researcher are prone to variation which may be desirable in the context of novel scientific findings however, the reproducibility for these studies needs to be unified and assured. The reproducibility issues are highly concerned when preclinical studies are reported in cancer, for natural products in particular. The natural products and medicinal plants are prone to a wide variation in terms of phytochemistry and phyto-pharmacology, ultimately affecting the end results for cancer studies. Hence the need for specific guidelines to adopt a best-practice in cancer research are utmost essential. The current AIMRDA guidelines aims to develop a consensus-based tool in order to enhance the quality and assure the reproducibility of studies reporting natural products in cancer prevention. A core working committee of the experts developed an initial draft for the guidelines where more focus was kept for the inclusion of specific items not covered in previous published tools. The initial draft was peer-reviewed, experts-views provided, and improved by a scientific committee comprising of field research experts, editorial experts of different journals, and academics working in different organization worldwide. The feedback from continuous online meetings, mail communications, and webinars resulted a final draft in the shape of a checklist tool, covering the best practices related to the field of natural products research in cancer prevention and treatment. It is mandatory for the authors to read and follow the AIMRDA tool, and be aware of the good-practices to be followed in cancer research prior to any submission to APJCP. Though the tool is developed based on experts in the field, it needs to be further updated and validated in practice via implementation in the field.

Green and novel ultrasonic extraction with UHPLC-MSMS analysis of natural sweetener (Glycyrrhizic acid) from Glycyrrhiza glabra; a multifactorial mechanistic evaluation based on statistical analysis.

A novel, green and eco-friendly, cost-effective, fast, and reliable high energy ultrasonication (US) extraction with UHPLC-MSMS (Ultrahigh performance liquid chromatography with mass spectrometry) quantification of Glycyrrhizic acid (GZA) is reported herein for the first time. The study provides useful insights regarding the effect of US-factors with statistical analysis and mechanisms, involved in GZA-extraction and analysis. An US-extraction method (US-MD) was developed using three levels of US factors: solvents (AC (acetone), EtOH (ethanol), H2O (water)), time (1, 2, 3 min), amplitudes (30, 40, 50%), pulse (10/0.5, 20/0.5, 30/0.5 sec), particle sizes (0.5, 1, 1.4 mm), and temperatures (20, 30, 40 °C). The US-MD was further validated with high accuracy 98.96 ± 6.82 and r2 = 0.995 whereas, an in-house analytical method (UHPLC-MSMS) was developed and validated to quantify the GZAamount. UHPLCMSMS-MD resulted in a retention time of 0.31 min with MSMS (821.400 > 351.200) in a 1 min run time whereas, UHPLCMSMS-MV showed high accuracy and precision with r2 = 0.998 for GZA. Statistical analysis of K-mean clustering finalized US-set-of-factors showing optimum extract yield (mg/1mg) of 0.48 with sum (2.41 ± 014) and mean (0.27) along with a high GZA-amount (µg/mg) of 8.23 with sum (43.31 ± 2.07) and mean (4.81) for H2O in 3 min at 40 °C using particle size (1.4 mm), amplitude (50%), and pulse (30/0.5). Large scale application of US-UHPLCMSMS confirmed the evaluation power of the method showing the order for GZA amount; Egypt > Pakistan > Syria > India > Palestine > America > Georgia > Morocco. A significant effect for US factors Vs extract yield and GZA amount was observed however, solvent*GZA-amount and extract yield*particle size were more significantly correlated compared to time*temperature*amplitude*pulse analyzed via PCA, GLM-UniANOVA, K-mean, and Pearson's correlation (P ≤ 0.05). A combined mechanism of shear stress, macroturbulence due to acoustic cavitation and implosions, sonochemical, and sonocapillary effect were noted for the US technique producing higher extract yield and GZA amount from licorice.

Epigenetic modulation of secondary metabolite profiles in Aspergillus calidoustus and Aspergillus westerdijkiae through histone deacetylase (HDAC) inhibition by vorinostat.

The effects of epigenetic modulation on secondary metabolite biosynthesis were investigated with five Aspergillus species cultured in the presence of either the DNA methyltransferase inhibitor 5-azacitidine or the histone deacetylase inhibitor vorinostat. With Aspergillus calidoustus and Aspergillus westerdijkiae, fermentation in the presence of vorinostat (100 µM) induced significant changes in secondary metabolite profile with examples of both induction and repression. We identified putative biosynthetic gene clusters for emericellamide in A. calidoustus and ochratoxin in A. westerdijkiae. A substantial induction in production levels was observed for two secondary metabolites: the diketopiperazine alkaloid phenylahistin in A. calidoustus and the polyketide penicillic acid in A. westerdijkiae, indicating the potential of epigenetic regulation for the activation of silent fungal biosynthetic pathways.

A Decade of Antifungal Leads from Natural Products: 2010-2019.

In this review, we discuss novel natural products discovered within the last decade that are reported to have antifungal activity against pathogenic species. Nearly a hundred natural products were identified that originate from bacteria, algae, fungi, sponges, and plants. Fungi were the most prolific source of antifungal compounds discovered during the period of review. The structural diversity of these antifungal leads encompasses all the major classes of natural products including polyketides, shikimate metabolites, terpenoids, alkaloids, and peptides.