Activity-guided Isolation Research Articles

Baicalein isolated from Oroxylum indicum acts as a potent µ- and κ-opioid receptor antagonist agent via the reversal of agonist-mediated cAMP inhibition

The opioid receptors play a pivotal role in the treatment of several neuropsychiatric and neurological disorders. Oroxylum indicum (L.) Kurtz is a very important medicinal plant with several therapeutic applications. It is a main constituent of the Ayurvedic formulation ‘Dashmool’ used for multifaceted disorders by the Indians. However, the constituents of this plant in neurological conditions have not been well studied. Here, we performed activity-guided isolation of compounds for opioid receptor modulator activity. In the study, we found that the isolated compound baicalein (3) has shown the most potent and competitive antagonistic activity at 20 mg/kg dose in vivo experiments. The acute dose of 3 (20 mg/kg) and pan opioid receptor antagonist naloxone (20 mg/kg) block the morphine-induced antinociception and the paw withdrawal latency decreases up to 8.3 s and 9.6 s, respectively. The in silico studies also support our in vitro data that compound 3 binds with MOR and KOR.

Portulaca Oleracea L. Phenolic Amide Methyl (3,4,5-Trimethoxybenzoyl) Valylprolinate Attenuates Diethylhexyl Phthalate-Induced Human Umbilical Vein Endothelial Cells' Inflammation Through NLRP3 and NF-κB Pathways.

Twelve polyphenol derivatives were obtained in a protective activity-guided isolation from the Portulaca oleracea L. extract on a cell model of human umbilical vein endothelial cells (HUVECs) under diethylhexyl phthalate (DEHP) exposure. Among them, methyl (3,4,5-trimethoxybenzoyl) valylprolinate (PP-10) performed the most protective activity and inhibited DEHP exposure-induced HUVECs' apoptosis. PP-10 also inhibited the DEHP-induced inflammatory cytokines (TNF-α, IL-6, IL-1β, and IL-8) and adhesion molecule (ICAM-1 andVCAM-1) overexpression. Furthermore, DEHP-induced NLRP3 inflammasomes' and NF-κB signaling pathway activation was significantly inhibited after the PP-10 treatments. Of note, the current results suggest the potential application of Portulaca oleracea L. and PP-10 in the prevention of DEHP-induced inflammatory damages in HUVECs.

Novel Jatrophane Diterpenoids from Euphorbia esula Promotes Lipid Clearance by Transcriptional Regulation of PCSK9.

PCSK9 has been recognized as an efficient target for hyperlipidemia and related cardiovascular/cerebrovascular diseases. However, PCSK9 inhibitors in the clinic are all biological products, and no small molecules are available yet. In the current work, we discovered that the crude extract of Euphorbia esula (E. esula) promoted LDL uptake in vitro and then obtained 8 new and 12 known jatrophane diterpenoids by activity-guided isolation. After summarized their structure-activity relationship of PCSK9 inhibition, we selected compound 11 (C11) with potent activity and high abundance to investigate its mechanism and in vivo efficacy. Mechanistically, C11 bound with HNF1α to influence its nuclear distribution and subsequently inhibit PCSK9 transcription, thereby enhancing LDLR and promoting LDL uptake. Moreover, C11 demonstrated obvious lipid-lowering activity in HFD mouse model. In conclusion, we first revealed the novel application of E. esula in the discovery of a lipid-lowering candidate and highlighted the potential of C11 in the treatment of hyperlipidemia.

Discovery of procyanidin condensed tannins of (−)-epicatechin from Kratom, Mitragyna speciosa, as virucidal agents against SARS-CoV-2

Kratom, Mitragyna speciosa, is one of the most popular herbs in the West and Southeast Asia. A number of previous works have focused on bioactive alkaloids in this plant; however, non-alkaloids have never been investigated for their biological activities. Antiviral and virucidal assays of a methanol leaf extract of Kratom, M. speciosa, revealed that a crude extract displayed virucidal activity against the SARS-CoV-2. Activity-guided isolation of a methanol leaf extract of Kratom led to the identification of B-type procyanidin condensed tannins of (−)-epicatechin as virucidal compounds against SARS-CoV-2. The fraction containing condensed tannins exhibited virucidal activity with an EC50 value of 8.38 μg/mL and a selectivity index (SI) value >23.86. LC-MS/MS analysis and MALDI-TOF MS identified the structure of the virucidal compounds in Kratom as B-type procyanidin condensed tannins, while gel permeation chromatograph (GPC) revealed weight average molecular weight of 238,946 Da for high molecular-weight condensed tannins. In addition to alkaloids, (−)-epicatechin was found as a major component in the leaves of M. speciosa, but it did not have virucidal activity. Macromolecules of (−)-epicatechin, i.e., procyanidin condensed tannins, showed potent virucidal activity against SARS-CoV-2, suggesting that the high molecular weights of these polyphenols are important for virucidal activity.

Systematic investigation of the material basis, effectiveness and safety of Thesium chinense Turcz. and its preparation Bairui Granules against lung inflammation

BackgroundThesium chinense Turcz. (Named as Bai Rui Cao in Chinese) and its preparations (e.g., Bairui Granules) have been used to treat inflammatory diseases, such as acute mastitis, lobar pneumonia, tonsillitis, coronavirus disease 2019 (COVID-19), and upper respiratory tract infection. However, the material basis, pharmacological efficiency, and safety have not been illustrated.MethodsAnti-inflammatory activity-guided isolation of constituents has been performed using multiple column chromatography, and their structures were elucidated by NMR spectroscopy and ECD calculations. The inhibitory effects on lung inflammation and safety of the crude ethanol extract (CE), Bairui Granules (BG), and the purified active constituents were evaluated using lipopolysaccharide (LPS)-stimulated acute lung inflammation (ALI) mice model or normal mice.ResultsSeven new compounds (1–7) and fifty-six known compounds (8–63) were isolated from T. chinense, and fifty-four were reported from this plant for the first time. The new flavonoid glycosides 1–2, new fatty acids 4–5, new alkaloid 7 as well as the known constituents including flavonoid aglycones 8–11, lignans 46–54, alkaloids 34 and 45, coumarins 57, phenylpropionic acids 27, and simple aromatic compounds 39, 44 and 58 exhibited anti-inflammatory activity. Network pharmacology analysis indicated that anti-inflammation of T. chinense was attributed to flavonoids and alkaloids by regulating inflammation-related proteins (e.g., TNF, NF-κB, TGF-β). Furthermore, constituents of T. chinense including kaempferol-3-O-glucorhamnoside (KN, also named as Bairuisu I, 19), astragalin (AG, Bairuisu II, 12), and kaempferol (KF, Bairuisu III, 8), as well as CE and BG could alleviate lung inflammation caused by LPS in mice by preventing neutrophils infiltration and the expression of the genes for pro-inflammatory cytokines NLRP3, caspase-1, IL-1β, and COX-2. After a 28-day subacute toxicity test, BG at doses of 4.875 g/kg and 9.750 g/kg (equivalent to onefold and twofold the clinically recommended dose) and CE at a dose of 11.138 g/kg (equivalent to fourfold the clinical dose of BG) were found to be safe and non-toxic.ConclusionsThe discovery of sixty-three constituents comprehensively illustrated the material basis of T. chinense. T. chinense and Bairui Granules could alleviate lung inflammation by regulating inflammation-related proteins and no toxicity was observed under the twofold of clinically used doses.

Discovery of New Antimicrobial Metabolites in the Coculture of Medicinal Mushrooms.

Bioactivity screening revealed that the antifungal activities of EtOAc extracts from coculture broths of Trametes versicolor SY630 with either Vanderbylia robiniophila SY341 or Ganoderma gibbosum SY1001 were significantly improved compared to that of monocultures. Activity-guided isolation led to the discovery of five aromatic compounds (1-5) from the coculture broth of T. versicolor SY630 and V. robiniophila SY341 and two sphingolipids (6 and 7) from the coculture broth of T. versicolor SY630 and G. gibbosum SY1001. Tramevandins A-C (1-3) and 17-ene-1-deoxyPS (6) are new compounds, while 1-deoxyPS (7) is a new natural product. Notably, compound 2 represents a novel scaffold, wherein the highly modified p-terphenyl bears a benzyl substituent. The absolute configurations of those new compounds were elucidated by X-ray diffraction, ECD calculations, and analysis of physicochemical constants. Compounds 1, 2, and 5-7 exhibited different degrees of antimicrobial activity, and the antifungal activities of compounds 6 and 7 against Candida albicans and Cryptococcus neoformans are comparable to those of fluconazole, nystatin, and sphingosine, respectively. Transcriptome analysis, propidium iodide staining, ergosterol quantification, and feeding assays showed that the isolated sphingolipids can extensively downregulate the late biosynthetic pathway of ergosterol in C. albicans, representing a promising mechanism to combat antibiotic-resistant fungi.

Molecular docking, molecular dynamics, MM/PBSA approaches and bioactivity studies of nepetanudoside B isolated from endemic Nepeta aristata

Nepetanudoside B (NNB) was isolated from aerial parts of endemic Nepeta aristata crude extract (CH3OH-CHCl3) using silica gel (n-hexane, methanol, ethyl acetate, and dichlorometane, respectively) and sephadex LH-20 (65% Methanol-35% Chloroform) column chromatographies. Preparative-HPLC was used to purify NNB after activity-guided isolation of methanol sub-fractions with enzyme inhibitory and DNA protective properties. The NNB was determined using 1H,13C, COSY, HSQC, HMBC, and LC-MS/MS. The study compared the effects of NNB with conventional drugs in terms of its ability to inhibit enzymes such as urease, α-amylase, carbonic anhydrase (CA), lipase, α-glucosidase, and tyrosinase, as well as its ability to protect DNA. Enzyme kinetic and molecular docking were also used to evaluate this. NNB exhibited the best inhibitory activity on urease (1.28 ± 0.00 µg/mL), lipase (5.83 ± 0.10 µg/mL), BChE (3.73 ± 0.46 µg/mL), tyrosinase (7.39 ± 0.00 µg/mL), α-glucosidase (10.95 ± 0.00 µg/mL), α-amylase (22.11 ± 1.03 µg/mL) and AChE (25.68 ± 3.32 µg/mL), respectively. NNB has higher MolDock scores with binding energy in α-glucosidase (-233) and BChE (-8.90 kcal/mol). In enzyme kinetics studies, it was determined that urease, AChE, α-glucosidase, lipase, and CA were non-competitive , while BChE and tyrosinase were competitive inhibition mechanisms. Their Ki values were calculated as 0.09, 0.24, 0.09, 0.10, 0.08, 0.05, and 0.07 mM, respectively. Molecular dynamics simulation studies were performed for the interactions of NNB-BChE with MM/PBSA binding free energey RMSD, RMSF, Rg, SASA, and also the number of hydrogen bonds was calculated. The suitability and effectiveness of NNB have been proven in the food and pharmaceutical industries. The NNB molecule may lead to development studies as a BChE inhibitor. Communicated by Ramaswamy H. Sarma

Anti-Inflammatory Flavonoids from Agrimonia pilosa Ledeb: Focusing on Activity-Guided Isolation.

To elucidate the anti-inflammatory properties and constituents of Agrimonia pilosa Ledeb. (A. pilosa), a comprehensive investigation was conducted employing activity-guided isolation. The anti-inflammatory effects were evaluated through an in vitro nitric oxide (NO) assay on lipopolysaccharide (LPS)-treated RAW 264.7 macrophage cells. Seven bio-active compounds with anti-inflammatory properties were successfully isolated from the butanol fraction and identified as follows: quercetin-7-O-β-d-rhamnoside (1), apigenin-7-O-β-d-glucopyranoside (2), kaempferol-7-O-β-d-glucopyranoside (3), quercetin (4), kaempferol (5), apigenin (6), and apigenin-7-O-β-d-glucuronide-6″-butylester (7). All isolated compounds showed strong NO inhibitory activity with IC50 values ranging from 1.4 to 31 µM. Compound 6 demonstrated the most potent NO inhibition. Compound 7, a rare flavonoid, was discerned as a novel anti-inflammatory agent, ascertained through its inaugural demonstration of nitric oxide inhibition. Subsequently, a comprehensive structure-activity relationship (SAR) analysis was conducted employing eight flavonoids derived from A. pilosa. The outcomes elucidated that flavones exhibit superior NO inhibitory effects compared to flavonols, and the aglycone form manifests greater potency in NO inhibition than the glycone counterpart. These results highlight A. pilosa as a promising source of effective anti-inflammatory agents and indicate its potential as a health-beneficial dietary supplement and therapeutic material.

Exploring the efficacy of ethnomedicinal plants of Himalayan region against the malaria parasite

Ethnopharmacological relevancePlasmodium falciparum multi-drug resistant (MDR) strains are a great challenge to global health care. This predicament implies the urgent need to discover novel antimalarial drugs candidate from alternative natural sources. The Himalaya constitute a rich repository of medicinal plants which have been used traditionally in the folklore medicine since ages and having no scientific evidence for their activity. Crambe kotschyana Boiss. and Eremurus himalaicus Baker are used for their antipyretic and hepatoprotective properties in Kinnaur district of Himachal Pradesh, India. Aim of the studyThis study would investigate the antiplasmodial efficacy of C. kotschyana and E. himalaicus extracts, their fractions and active components using in vitro, in vivo and in silico approaches to provide a scientific insight into their activity. MethodsThe methanol extracts of C. kotschyana (CKME) and E. himalaicus (EHME) were prepared by maceration followed by fractionation using ethyl acetate. The isolation of flavonoid glycosides isorhamnetin-3, 7-di-O-glucoside from C. kotschyana and luteolin-6-C-glucoside (isoorientin) from E. himalaicus was carried out by antiplasmodial activity-guided isolation. In vitro antimalarial activity was assessed by WHO method while in vitro cytotoxicity was ascertained employing the MTT assay. Molecular docking and molecular dynamics simulation were performed using the Glide module of Schrödinger Software and Gromacs-2022 software package respectively. In vivo curative activity was assessed by Ryley and Peters method. ResultsThe methanol extracts of both the plants illustrated the best antiplasmodial activity followed by the ethyl acetate fractions. Iso-orientin (IC50 6.49 μg/ml) and Isorhamnetin-3,7-di-O-glucoside (IC50 9.22 μg/ml) illustrated considerable in vitro activity even against P. falciparum resistant strain. Extracts/fractions as well as the isolated compounds were found to be non-toxic with CC50 > 640 μg/ml. Molecular docking studies were performed with these 2 O-glucosides against four malaria targets to understand the binding pose of these molecules and the results suggested that these molecules have selectivity for lactate dehydrogenase enzyme. CKME and EHME exhibited curative activity in vivo along with increase in Mean Survival Time of mice. ConclusionThe research delineated the scientific evidence that both the therapeutic herbs possessed antimalarial activity and notably, bioactive compounds responsible to exhibit the antimalarial activity have been isolated, identified and characterized. Further studies are underway to assess the antiplasmodial efficacy of isolated compounds alone and in combination with standard antimalarials.

Activity-guided isolation and identification of antiherpesvirus and antineuroinflammatory active terpenoids from Artemisia vulgaris L. based on the LC-MS/MS molecular network

Seven undescribed terpenoids, comprising two guaiane-type sesquiterpene lactones (1−2), one eucalyptol-type sesquiterpene (3), one monolactone (4), and three triterpenoids (5−7), along with 35 known analogues, were isolated from the leaves of Artemisia vulgaris L. Their structures and configurations were analysed by extensive spectroscopy. Compounds 1, 2, 8−10, 13, 17, 19, and 28 showed antineuroinflammatory activity, and compounds 1 and 2 revealed remarkable antineuroinflammatory effects, with an IC50 value of 2.2 ± 0.1 and 1.6 ± 0.1 μM, more potent than the positive control drug dexamethasone. Furthermore, compounds 1 and 2 could inhibit the expression of BV-2 inflammatory genes (IL-6, TNF-α, IL-1β) induced by LPS, downregulate the critical inflammatory protein production of iNOS and COX-2. The anti-HSV-1 activity screening revealed that compounds 28, 29 and 38 exhibited inhibitory activity against HSV-1 proliferation. Particularly, compound 28 exhibited a significant anti-HSV-1 effect, inhibiting the proliferation of HSV-1 and acyclovir-resistant strains of HSV-1/153 and HSV-1/Blue. Our research identified compounds 1, 2, and 28 from A. vulgaris., which could potentially serve as lead compounds for antineuroinflammatory and anti-HSV-1 activities.

Endophytic Microbes from Medicinal Plants in Fenghuang Mountain as a Source of Antibiotics.

One of the largest concerns with world health today is still antibiotic resistance, which is making it imperative to find efficient alternatives as soon as possible. It has been demonstrated that microbes are reliable sources for the creation of therapeutic antibiotics. This research intends to investigate the endophytic microorganisms from several medicinal plants in Fenghuang Mountain (Jiangsu Province, China) and to discover new antibiotics from their secondary metabolites. A total of 269 endophytic strains were isolated from nine distinct medicinal plants. Taxonomic analysis revealed that there were 20 distinct species among these endophytes, with Streptomyces being the most common genus. Three of the target strains were chosen for scale-up fermentation after preliminary screening of antibacterial activities and the metabolomics investigation using LC-MS. These strains were Penicillium sp. NX-S-6, Streptomyces sp. YHLB-L-2 and Streptomyces sp. ZLBB-S-6. Twenty-three secondary metabolites (1-23), including a new sorbicillin analogue (1), were produced as a result of antibacterial activity-guided isolation. Through spectroscopic analysis using MS and NMR, the structures of yield compounds were clarified. According to antibacterial data, S. aureus or B. subtilis were inhibited to varying degrees by sorrentanone (3), emodic acid (8), GKK1032 B (10), linoleic acid (14), toyocamycin (17) and quinomycin A (21). The most effective antimicrobial agent against S. aureus, B. subtilis, E. coli and A. baumannii was quinomycin A (21). In addition, quinomycin A showed strong antifungal activity against Aspergillus fumigatus, Cryptococcus neoformans, and two clinical isolated strains Aspergillus fumigatus #176 and #339, with MIC as 16, 4, 16 and 16 µg/mL, respectively. This is the first time that bioprospecting of actinobacteria and their secondary metabolites from medicinal plants in Fenghuang Mountain was reported. The finding demonstrates the potential of endophytic microbes in medical plants to produce a variety of natural products. Endophytic microbes will be an important source for new antibiotics.

DNA protection, molecular docking, enzyme inhibition and enzyme kinetic studies of 1,5,9-epideoxyloganic acid isolated from Nepeta aristata with bio-guided fractionation

1,5,9-epideoxyloganic acid (ELA) was isolated from the aerial parts of endemic Nepeta aristata Boiss Et Kotschy Ex Boiss crude extract (methanol:chloroform) using silica gel (hexane, chloroform, ethyl acetate, and methanol) and sephadex LH-20 (65% methanol-35% chloroform) columns. Activity-guided isolation was performed on methanol sub-fractions with DNA protection and enzyme inhibitory activities, and then the ELA was purified by prep-HPLC. The ELA structure, bio-guided isolate, was determined via 1H NMR, 13C NMR, and MS spectrometry. ELA's enzyme inhibition and DNA protection activities were investigated and compared with standard drugs. The inhibition capacity of ELA against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), urease, carbonic anhydrase (CA), α-glucosidase, α-amylase, lipase, and tyrosinase enzymes was evaluated by kinetic and molecular docking results. The ELA displayed the best inhibitory activity on AChE, BChE, α-glucosidase, urease, α-amylase, and tyrosinase with IC50 values of 2.53 ± 0.27, 3.75 ± 0.11, 3.98 ± 0.07, 4.40 ± 0.01, 6.43 ± 0.54 and 7.39 ± 0.00 µg/mL, respectively. ELA acted as a competitive inhibitor against BChE and α-glucosidase and a non-competitive inhibitor against AChE. The ELA’s binding affinity values on AChE, BChE, and α-glucosidase were −7.70, −8.50, and −8.30 kcal/mol, respectively. DNA protection activity of the ELA molecule was determined as 57.53% for form I and 53.57% for form II. In conclusion, the inhibitory activity of ELA demonstrated its effectiveness in terms of its suitability in the pharmaceutical industry. Communicated by Ramaswamy H. Sarma

Discovery and Biosynthesis of Imidazolium Antibiotics from the Probiotic Bacillus licheniformis.

Antibiotic resistance is one of the world's most urgent public health problems, and novel antibiotics to kill drug-resistant bacteria are needed. Natural product-derived small molecules have been the major source of new antibiotics. Here we describe a family of antibacterial metabolites isolated from a probiotic bacterium, Bacillus licheniformis. A cross-streaking assay followed by activity-guided isolation yielded a novel antibacterial metabolite, bacillimidazole G, which possesses a rare imidazolium ring in the structure, showing MIC values of 0.7-2.6 μg/mL against human pathogenic Gram-positive and Gram-negative bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and a lipopolysaccharide (LPS)-lacking Acinetobacter baumannii ΔlpxC. Bacillimidazole G also lowered MICs of colistin, a Gram-negative antibiotic, up to 8-fold against wild-type Escherichia coli MG1655 and A. baumannii. We propose a biosynthetic pathway to the characterized metabolites based on precursor-feeding studies, a chemical biological approach, biomimetic total synthesis, and a biosynthetic gene knockout method.

Secondary metabolites from the underground parts of Valeriana sisymbriifolia Vahl. and their in vitro cytotoxic activities

Cytotoxic activity-guided isolation studies on the underground parts of Valeriana sisymbriifolia Vahl. led to the isolation of 12 secondary metabolites including two undescribed iridoids, sisymbriifolivaltrate and sisymbriifolioside, and two unreported sesquiterpene lactones, sisymbriifolins A and B. Chemical structures of the isolates were established by extensive 1D and 2D NMR analyses as well as HR-ESI-MS. The in vitro cytotoxic activities of the extract, sub-fractions and isolates on lung (A549), breast (MCF7), gastric (HGC27) and prostate (PC3) cancer cell lines were evaluated by MTS assay. Sisymbriifolivaltrate, didrovaltrate, valtrate, 7-homovaltrate and 1-α-acevaltrate exhibited promising cytotoxic activity on MCF7 cell line with IC50 values ranging from 2.5 to 12.3 μM, while valtrate demonstrated the best cytotoxicity against A549 cells with the IC50 value of 7.5 μM. Valtrate and 7-homovaltrate were found to exert noteworthy cytotoxicity towards HGC27 cell line (IC50 values: 2.3 and 3.7 μM, respectively), whereas valtrate, 7-homovaltrate and 1-α-acevaltrate (IC50 values: 2.3–9.7 μM) were found to be potent cytotoxic against PC3 cells. Among the tested compounds, particularly valepotriate-type iridoids were found to be the main cytotoxic principles of V. sisymbriifolia.

Antioxidant Activity-Guided Isolation of Phenolic Compounds from Leaves of Vitex pinnata (Lamiaceae)

The aim of this study is to isolate phenolic compounds from the ethyl acetate fraction of the leaves of V. pinnata Linn, which have antioxidant properties, guided by a TLC-Bioautography (Thin-layer chromatography) with a reagent of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH). The TLC-antioxidant bioautography technique has several advantages. It does not require a long time in testing, the sample used is small, and it can potentially obtain promising antioxidant-positive compounds.The isolation process led to obtaining two phenolic compounds. The UV-Vis, FTIR, and NMR spectrum analysis showed that the compounds were (1) p-hydroxybenzoate and (2) luteolin. Further antioxidant evaluation by DPPH assay showed a potential radical scavenging DPPH of luteolin (2) with an IC50 of 1.56±0.18 µg/mL.

Evaluation of Phytochemical Contents and Biological Activities of Salvia officinalis and Salvia triloba Grown with Organic Farming

Salvia officinalis L., known as medicinal sage, and Salvia triloba L., known as Anatolian sage, belong to the Lamiaceae family and are species that usually grow in the Mediterranean region. In this study, it was aimed to evaluate the in vitro antidiabetic, antiobesity and antioxidant potentials of the extracts prepared by infusion technique from S. officinalis and S. triloba grown by organic farming methods. In addition, the effects of the extracts on the pancreatic cholesterol esterase enzyme were also investigated. Reverse Phase-HPLC technique was used to analyze the phytochemical contents of the extracts. At a concentration of 2 mg/mL, S. officinalis inhibited 64.69% ± 0.23, S. triloba 47.78 ± 2.11% on the α-glucosidase enzyme. Only S. triloba had an inhibitory effect on α-amylase and pancreatic lipase enzyme. On the pancreatic cholesterol esterase enzyme, inhibition values of S. triloba extract at all tested concentrations were found to be higher than S. officinalis extract. When the antioxidant potentials of the extracts were evaluated, the reducing power absorbance values were found to be the highest of the S. officinalis extract. The metal chelating capacity of both extracts at a concentration of 2 mg/mL was calculated as 100%. It was concluded that the ABTS radical scavenging activity of the extracts increased in a dose-dependent manner. With the Reverse Phase-YPSK technique, rosmarinic acid and hesperidin were found to be higher in S. officinalis extract. The presence of hesperidin in S. triloba was detected for the first time in this study. Considering all these findings, it was concluded that activity-guided isolation and in vivo activity studies should be performed because these two species grown by organic farming method have strong α-glucosidase enzyme inhibitory and antioxidant effects.

Discovery of New Triterpenoids Extracted from Camellia oleifera Seed Cake and the Molecular Mechanism Underlying Their Antitumor Activity.

Theasaponin derivatives, which are reported to exert antitumor activity, have been widely reported to exist in edible plants, including in the seed cake of Camellia oleifera (C.), which is extensively grown in south of China. The purpose of this study was to isolate new theasaponin derivatives from C. seed cake and explore their potential antitumor activity and their underlying molecular mechanism. In the present study, we first isolated and identified four theasaponin derivatives (compounds 1, 2, 3, and 4) from the total aglycone extract of the seed cake of Camellia oleifera by utilizing a combination of pre-acid-hydrolysis treatment and activity-guided isolation. Among them, compound 1 (C1) and compound 4 (C4) are newly discovered theasaponins that have not been reported before. The structures of these two new compounds were characterized based on comprehensive 1D and 2D NMR spectroscopy and high-resolution mass spectrometry, as well as data reported in the literature. Secondly, the cytotoxicity and antitumor property of the above four purified compounds were evaluated in selected typical tumor cell lines, Huh-7, HepG2, Hela, A549, and SGC7901, and the results showed that the ED50 value of C4 ranges from 1.5 to 11.3 µM, which is comparable to that of cisplatinum (CDDP) in these five cell lines, indicating that C4 has the most powerful antitumor activity among them. Finally, a preliminary mechanistic investigation was performed to uncover the molecular mechanism underlying the antitumor property of C4, and the results suggested that C4 may trigger apoptosis through the Bcl-2/Caspase-3 and JAK2/STAT3 pathways, and stimulate cell proliferation via the NF-κB/iNOS/COX-2 pathway. Moreover, it was surprising to find that C4 can inhibit the Nrf2/HO-1 pathway, which indicates that C4 has the potency to overcome the resistance to cancer drugs. Therefore, C1 and C4 are two newly identified theasaponin derivatives with antitumor activity from the seed cake of Camellia oleifera, and C4 is a promising antitumor candidate not only for its powerful antitumor activity, but also for its ability to function as an Nrf2 inhibitor to enhance the anticancer drug sensitivity.

Bioactive Saponins of Primula vulgaris Huds. Promote Wound Healing through Inhibition of Collagenase and Elastase Enzymes: in Vivo, in Vitro and in Silico Evaluations.

Primula vulgaris Huds. leaves and roots were used to treat skin damage and inflammation in Anatolian Folk Medicine. This study aimed to assess the ethnopharmacological use of the plant using in vivo, in vitro, and in silico test models. Linear incision and circular excision wound models were used to determine the in vivo wound-healing potential of the plant extracts and fractions. In vitro assays including hyaluronidase, collagenase, and elastase inhibitory activities were carried out for the active compounds to discover their activity pathways. Structure-based molecular modeling was performed to understand inhibitory mechanisms regarding collagenase and elastase at the molecular level. The butanol fraction of the roots of P. vulgaris showed the highest wound-healing activity. Through activity-guided fractionation and isolation techniques, primulasaponin I (1) and primulasaponin I methyl ester (2) were stated as the major active compounds. These compounds exerted their activities through the inhibition of collagenase and elastase enzymes. Primulasaponin I methyl ester isolated from butanol fraction was found to be the strongest agent, especially with the values of 29.65 % on collagenase and 38.92 % on elastase inhibitory activity assays, as well as molecular docking studies. The present study supports scientific data for the traditional use of P. vulgaris and the wound healing properties of the plant can be referred to secondary metabolites as especially saponins found in the roots.

Protective effects of Rubus tereticaulis leaves ethanol extract on rats with ulcerative colitis and bio-guided isolation of its active compounds: A combined in silico, in vitro and in vivo study

The aim of this study was to evaluate the therapeutic effect of active ethanol extract obtained from the leaves of Rubus tereticaulis (RTME) against colitis, and to purify major compounds from this extract by bioassay-directed isolation. Rats with colitis induced via intra-rectal acetic acid administration (5%, v/v) received RTME or sulfasalazine for three consecutive days. On day four, all rats were decapitated, and the colonic tissue samples were collected for macroscopic score, colon weight, reduced glutathione (GSH), myeloperoxidase (MPO), and malondialdehyde (MDA) analyses. The active compounds and chemical composition of RTME were determined by bio-guided isolation and LC-MS/MS, respectively. Compared to the colitis group, the rats treated with RTME displayed significantly lowered macroscopic scores and colon wet weights (p < 0.001). These effects were confirmed biochemically by a decrease in colonic MPO activity (p < 0.001), MDA levels (p < 0.001), and an increase in GSH levels (p < 0.001). Kaempferol-3-O-β-d-glucuronide (RT1) and quercetin-3-O-β-d-glucuronide (RT2) were found to be the major compounds of RTME, as evidenced by in vitro anti-inflammatory and antioxidant activity-guided isolation. Their anti-inflammatory/antioxidant activities were also predicted by docking simulations. Additionally, quinic acid, 5-caffeoylquinic acid, quercetin pentoside, quercetin glucoside, quercetin-3-O-β-d-glucuronide, kaempferol-3-O-β-d-glucuronide, and kaempferol rutinoside were identified in RTME via using LC-MS/MS. RT2, along with other compounds, may be responsible for the observed protective action of RTME against colitis. This study represents the first report on the beneficial effects of RTME in an experimental model of colitis and highlights the potential future use of RTME as a natural alternative to alleviate colitis.

Effects of hydrolysable tannins from Terminalia citrina on type III secretion system (T3SS) and their intestinal metabolite urolithin B represses Salmonella T3SS through Hha–H-NS–HilD–HilC–RtsA–HilA regulatory pathway

Gamma-proteobacteria is a class of gram-negative opportunistic pathogens existing in the intestinal flora, often leading to diarrhea and intestinal infectious diseases, and plays an important role in maintaining intestinal homeostasis. Type III secretion system (T3SS), an important virulence system, is closely related to the adhesion and invasion and pathogenicity to host cells. Therefore, anti-virulence agents targeting T3SS are important strategies for controlling pathogenic infections. In this study, the anti-Salmonella T3SS active compounds neochebulagic acid (1), ellagic acid (2) and urolithin M5 (3) were isolated from seed extract of Terminalia citrina by activity-guided isolation method. Based on the fact that urolithins are the main and stable intestinal microbiota metabolites of hydrolysable tannins, we found that the metabolite urolithin B repressed translation and secretion of SipC through the Hha–H-NS–HilD–HilC–RtsA–HilA regulatory pathway. The results provide evidence for Terminalia seeds and ellagitannin-rich berries and nuts in regulating intestinal homeostasis and treating bacterial infection.