Phytochemical analysis, antioxidant, α-glucosidase inhibitory activity, and toxicity evaluation of Orthosiphon stamineus leaf extract.

Ocimum aristatum, commonly known as O. stamineus, has been widely studied for its potential as an herbal medicine candidate. This research aims to compare the efficacy of water and 100% ethanolic extracts of O. stamineus as α-glucosidase inhibitors and antioxidants, as well as toxicity against zebrafish embryos. Based on the study findings, water extract of O. stamineus leaves exhibited superior inhibition activity against α-glucosidase, ABTS, and DPPH, with IC50 values of approximately 43.623 ± 0.039 µg/mL, 27.556 ± 0.125 µg/mL, and 95.047 ± 1.587 µg/mL, respectively. The major active compounds identified in the extract include fatty acid groups and their derivates such as linoleic acid, α-eleostearic acid, stearic acid, oleanolic acid, and corchorifatty acid F. Phenolic groups such as caffeic acid, rosmarinic acid, 3,4-Dihydroxybenzaldehyde, norfenefrine, caftaric acid, and 2-hydroxyphenylalanine and flavonoids and their derivates including 5,7-Dihydroxychromone, 5,7-Dihydroxy-2,6-dimethyl-4H-chromen-4-one, eupatorin, and others were also identified in the extract. Carboxylic acid groups and triterpenoids such as azelaic acid and asiatic acid were also present. This study found that the water extract of O. stamineus is non-toxic to zebrafish embryos and does not affect the development of zebrafish larvae at concentrations lower than 500 µg/mL. These findings highlight the potential of the water extract of O. stamineus as a valuable herbal medicine candidate, particularly for its potent α-glucosidase inhibition and antioxidant properties, and affirm its safety in zebrafish embryos at tested concentrations.

Study on the mechanism of Orthosiphon aristatus (Blume) Miq. in the treatment of hyperuricemia by microbiome combined with metabonomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Growing evidence indicates that hyperuricemia is closely associated with gut microbiota dysbiosis. Orthosiphon aristatus (Blume) Miq. (O. aristatus), as a traditional Chinese medicine, has been widely used to treat hyperuricemia in China. However, the mechanism by which O. aristatus treats hyperuricemia has not been clarified. AIM OF THE STUDY: In this study, we investigated whether the molecular mechanism underlying the anti-hyperuricemia effect of O. aristatus is related to the regulation of gut microbiota by 16S rDNA gene sequencing combined with widely targeted metabolomics. MATERIALS AND METHODS: Hyperuricemia was induced in rats by administration of 10% fructose and 20% yeast, and the uricosuric effect was assessed by measuring the uric acid (UA) levels in serum and cecal contents. Intestinal morphology was observed by hematoxylin and eosin (HE) staining. To explore the effects of O. aristatus on the gut microbiota and its metabolites, we utilized 16S rDNA gene sequencing combined with widely targeted metabolomics. Furthermore, metabolic pathway enrichment analysis was performed on the screened differential metabolites. The real time quantitative polymerase chain reaction (RT-PCR) and western blotting (WB) were used to detect the expression of relevant proteins in the key pathway. RESULTS: Our results indicated that O. aristatus intervention decreased serum UA levels and increased the UA levels in cecal contents in hyperuricemic rats. Additionally, O. aristatus improved intestinal morphology and altered the composition of the gut microbiota and its metabolites. Specifically, 16S rDNA revealed that O. aristatus treatment significantly reduced the abundance of unidentified-Ruminococcaceae and Lachnospiraceae-NK4A136-group. Meanwhile, widely targeted metabolomics showed that 17 metabolites, including lactose, 4-oxopentanoate and butyrate, were elevated, while 55 metabolites, such as flavin adenine dinucleotide and xanthine, were reduced. Metabolic pathway enrichment analysis found that O. aristatus was mainly involved in purine metabolism. Moreover, RT-PCR and WB suggested that O. aristatus could significantly up-regulate the expression of UA excretion transporter ATP-binding cassette subfamily G member 2 (ABCG2) in the intestine. CONCLUSION: O. aristatus exerts UA-lowering effect by regulating the gut microbiota and ABCG2 expression, indicating that this herb holds great promise in the treatment of hyperuricemia.

An integrative analysis to predict the active compounds and explore polypharmacological mechanisms of Orthosiphon stamineus Benth.

BACKGROUND: Orthosiphon stamineus Benth is a dietary supplement and traditional Chinese herb with widespread clinical applications, but a comprehensive understanding of its active compounds and polypharmacological mechanisms is lacking. This study aimed to systematically investigate the natural compounds and molecular mechanisms of O. stamineus via network pharmacology. METHODS: Information on compounds from O. stamineus was collected via literature retrieval, while physicochemical properties and drug-likeness were evaluated using SwissADME. Protein targets were screened using SwissTargetPrediction, while the compound-target networks were constructed and analyzed via Cytoscape with CytoHubba for seed compounds and core targets. Enrichment analysis and disease ontology analysis were then carried out, generating target-function and compound-target-disease networks to intuitively explore potential pharmacological mechanisms. Lastly, the relationship between active compounds and targets was confirmed via molecular docking and dynamics simulation. RESULTS: A total of 22 key active compounds and 65 targets were identified and the main polypharmacological mechanisms of O. stamineus were addressed. The molecular docking results suggested that nearly all core compounds and their targets possess good binding affinity. In addition, the separation of receptor and ligands was not observed in all dynamics simulation processes, whereas complexes of orthosiphol Z-AR and Y-AR performed best in simulations of molecular dynamics. CONCLUSION: This study successfully identified the polypharmacological mechanisms of the main compounds in O. stamineus, and predicted five seed compounds along with 10 core targets. Moreover, orthosiphol Z, orthosiphol Y, and their derivatives can be utilized as lead compounds for further research and development. The findings here provide improved guidance for subsequent experiments, and we identified potential active compounds for drug discovery or health promotion.

Molecular docking and dynamics simulation of Orthosiphon stamineus against SGLT1 and SGLT2.

Orthosiphon stamineus Benth a traditional medicine used in the treatment of diabetes and kidney diseases. Sodium-glucose co-transporter (SGLT1 and SGLT2) inhibitors are the novel group of drugs used to treat patients with type 2 diabetes mellitus. In this study 20 phytochemical compounds from Orthosiphon stamineus Benth were obtained from 3 databases viz Dr.Duke's phytochemical, Ethno botanical database and IMPPAT. They were subjected to physiochemical, drug likeliness, and ADMET and toxicity predictions. Homology modeling and molecular docking against SGLT1 and SGLT2 were performed and the stability of the selected drug molecule was validated by molecular dynamic (MD) simulation for 200 ns. Among the 20 compounds, 14-Dexo-14-O-acetylorthosiphol Y alone showed higher binding affinity with SGLT1 and SGLT2 protein with the binding energy of -9.6 and -11.4 Kcal/mol respectively and had highest affinity towards SGLT2 inhibitor. This compound also satisfied Lipinski rule of 5 and had a good ADMET profile. The compound is non-toxic to marine organisms and to normal cell lines and non-mutagenic. The RMSD value attained equilibrium at 150 ns with the stability around 4.8 Å and no significant deviation was reported from 160 to 200 ns for SGLT2. Our study suggests that 14-Dexo-14-O-acetylorthosiphol Y showed promising results against the SGLT2 and could be considered as a potent anti-diabetic drug.Communicated by Ramaswamy H. Sarma.

Seven-day Oral Intake of Orthosiphon stamineus Leaves Infusion Exerts Antiadhesive Ex Vivo Activity Against Uropathogenic E. coli in Urine Samples.

Orthosiphon stamineus leaves (Java tea) extract is traditionally used for the treatment of urinary tract infections. According to recent in vitro data, animal infection studies, and transcriptomic investigations, polymethoxylated flavones from Java tea exert antiadhesive activity against uropathogenic Escherichia coli (UPEC). This antiadhesive activity has been shown to reduce bladder and kidney lesion in a mice infection model. As no data on the antivirulent activity of Java tea intake on humans are available, a biomedical study was performed on 20 healthy volunteers who self-administered Orthosiphon infusion (4 × 3 g per day, orally) for 7 days. The herbal material used for the study conformed to the specification of the European Pharmacopoeia, and ultra high-performance liquid chromatography (UHPLC) of the infusion showed rosmarinic acid, caffeic acid, and cichoric acid to be the main compounds aside from polymethoxylated flavones. Rosmarinic acid was quantified in the tea preparations with 243 ± 22 µg/mL, indicating sufficient reproducibility of the preparation of the infusion. Urine samples were obtained during the biomedical study on day 1 (control urine, prior to Java tea intake), 3, 6 and 8. Antiadhesive activity of the urine samples was quantified by flowcytometric assay using pre-treated UPEC NU14 and human T24 bladder cells. Pooled urine samples indicated significant inhibition of bacterial adhesion on day 3, 6 and 8. The urine samples had no influence on the invasion of UPEC into host cells. Bacterial proliferation was slightly reduced after 24 h incubation with the urine samples. Gene expression analysis (qPCR) revealed strong induction of fitness and motility gene fliC and downregulation of hemin uptake system chuT. These data correlate with previously reported datasets from in vitro transcriptomic analysis. Increased bacterial motility was monitored using a motility assay in soft agar with UPEC UTI89. The intake of Java tea had no effect on the concentration of Tamm-Horsfall Protein in the urine samples. The present study explains the antiadhesive and anti-infective effect of the plant extract by triggering UPEC from a sessile lifestyle into a motile bacterial form, with reduced adhesive capacity.

Suppression of Melanoma Growth in a Murine Tumour Model Using Orthosiphon stamineus Benth. Extract Loaded in Ethanolic Phospholipid Vesicles (Spherosome).

BACKGROUND: Orthosiphon stamineus Benth (O.S) is a traditional south-east Asian herb. The extract of O.S is used in the formulation of ethanolic nanolipid vesicle system to have considerable potential for tumour therapeutics. METHODS: The research objective is to develop and characterise the anticancer and antiangiogenic effect of O.S extract in the form of nano-ethanolic spherosomes (ESP) using phospholipids in melanoma. Spherosomes formulation of O.S was developed using the thin-film re-hydration method and converted to gel using Acrypol 1%. The formulations were subjected to optimisation and physical-chemical characterisations like particle size, surface charge, DSC, FTIR, and TEM. Cytotoxicity of O.S and ESP was studied using an endothelial cell line (EA. hy926). Furthermore, anti-melanoma effect of O.S spherosome gel was studied in albino mice after topical administration. RESULTS: ESP-6 with the ratio of extract (O.S): cholesterol: phospholipid (1: 6: 0.5) showed the highest entrapment efficiency (80.56 ± 0.84%) using ultraviolet spectroscopy. In-vivo permeation/penetration studies revealed deeper absorption of ESP-6 compared to a hydroethanolic gel of O.S. In-vitro and in vivo anti-melanoma studies demonstrated the significant tumour-suppressing effect of ESP-6 on murine melanoma. Percentage inhibition of tumour growth by O.S and ESP-6 at 3000 mg/kg showed to be 63.98 ± 7.86% and 87.76 ± 7.90%, respectively. CONCLUSION: Spherosome vesicles were developed with a smooth surface. The results demonstrated that O.S extract showed no toxicity when tested on the endothelial cell line. O.S loaded in spherosomes has the potential to lower the growth of melanoma in mice. The spherosomes loaded with O.S do not promote tumour growth or act as antiangiogenetic in melanoma.

A Systematic Review of Orthosiphon stamineus Benth. in the Treatment of Diabetes and Its Complications.

(1) Background: Orthosiphon stamineus Benth. is a traditional medicine used in the treatment of diabetes and chronic renal failure in southern China, Malaysia, and Thailand. Diabetes is a chronic metabolic disease and the number of diabetic patients in the world is increasing. This review aimed to systematically review the effects of O. stamineus in the treatment of diabetes and its complications and the pharmacodynamic material basis. (2) Methods: This systematic review was conducted following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA), using the databases ScienceDirect, PubMed, and Web of Science. (3) Results: Thirty-one articles related to O. stamineus and diabetes were included. The mechanisms of O. stamineus in the treatment of diabetes and its complications mainly included inhibiting α-amylase and α-glucosidase activities, antioxidant and anti-inflammatory activities, regulating lipid metabolism, promoting insulin secretion, ameliorating insulin resistance, increasing glucose uptake, promoting glycolysis, inhibiting gluconeogenesis, promoting glucagon-likepeptide-1 (GLP-1) secretion and antiglycation activity. Phenolic acids, flavonoids and triterpenoids might be the main components for hypoglycemia effects in O. stamineus. (4) Conclusion: O. stamineus could be an antidiabetic agent to treat diabetes and its complications. However, it needs further study on a pharmacodynamic substance basis and the mechanisms of effective constituents.

Gut Microbiota of Pigs Metabolizes Extracts of Filipendula ulmaria and Orthosiphon aristatus-Herbal Remedies Used in Urinary Tract Disorders.

Urinary tract infections influence the mortality rate in pigs and are linked to extensive antibiotic usage in the farm industry. Filipendula ulmaria (L.) Maxim. and Orthosiphon aristatus (Blume) Miq. are widespread medicinal plants traditionally used to treat urinary tract disorders. As their preparations are orally administered, the metabolism of their constituents by gut microbiota before absorption should be considered. Until now, no experiments had been performed to describe the biotransformation of tthose plants' extracts by animal gut microbiota. The study evaluates the influence of pig intestinal microbiota on the structure of active compounds in flowers of F. ulmaria and leaves of O. aristatus. The incubations of the extracts with piglet gut microbiota were performed in anaerobic conditions, and the samples of the batch culture were collected for 24 h. In F. ulmaria, the main metabolites were quercetin and kaempferol, which were products of the deglycosylation of flavonoids. After 24 h incubation of O. aristatus extract with the piglet gut microbiota, 2 main metabolites were observed. One, tentatively identified as 3-(3-dihydroxyphenyl)propionic acid, is likely the primary metabolite of the most abundant depsides and phenolic acids. The results confirm the formation of the compounds with anti-inflammatory and diuretic activity in the microbiota cultures, which might suggest F. ulmaria and O. aristatus for treating urinary tract disorders in piglets. Based on the similarities of human and pig gut microbiota, the pig model can help estimate the metabolic pathways of natural products in humans.

Micropropagation and Secondary Metabolites Content of White-Purple Varieties of Orthosiphon aristatus Blume Miq.

<b>Background and Objective:</b> The cat whiskers plant (<i>Orthosiphon aristatus</i> Blume Miq) is a plant that has been widely used as raw material for traditional medicine. The population of white-purple varieties of <i>O. aristatus</i> is decreasing efforts to maintain the white-purple <i>O. aristatus</i> need to be done keeping in mind its potential as raw material for traditional medicine. This study aims to determine the composition of a suitable medium in growing plantlet <i>O. aristatus</i> white-purple varieties and the content of its secondary metabolites. <b>Materials and Methods:</b> The internode explants were induced on MS medium added by various combinations of zeatin and 2,4-Dichlorophenoxyacetic acid (2,4-D). Root induction was carried out on shoots formed on MS medium with Indole-3-Butyric Acid (IBA). The acclimatization process was carried out using soil media. Determination of secondary metabolite levels was carried out on <i>O. aristatus</i> (<i>in vitro</i> culture) and wild-type plants aged ten months using high-performance liquid chromatography (HPLC). <b>Results:</b> MS+BAP 2ppm+NAA3 ppm media was the optimal medium for growing shoots in leaf explants. Media MS+zeatin 3 ppm+2,4-D 2 ppm produced good shoot growth on internode explants. The best root induction occurred in MS+IBA media of 0.75 ppm. The acclimatization process was successful on shoots originating from the internode, while those from leaf explants had not succeeded in growing and developing. <b>Conclusion:</b> The levels of rosmarinic acid and sinensetin in the white-purple variety <i>O. aristatus</i> (<i>in vitro</i> culture) were 1.08 and 1.62% w/w and higher than those of wild varieties.

Complementary effects of Orthosiphon stamineus standardized ethanolic extract and rosmarinic acid in combination with gemcitabine on pancreatic cancer.

BACKGROUND: Pancreatic cancer is one of the most notorious cancers and is known for its highly invasive characteristics, drug resistance, and metastatic progression. Unfortunately, many patients with advanced pancreatic cancer become insensitive towards gemcitabine treatment. Orthosiphon stamineus (O.s) is used widely as a traditional medicine for the treatment of multiple ailments, including cancer in South East Asia. The present in vitro study was designed to investigate the complementary effects of an ethanolic extract of O.s (Et. O.s) or rosmarinic acid in combination with gemcitabine on Panc-1 pancreatic cancer cells. METHOD: Cell viability and colony formation assays were used to determine the 50% inhibitory concentration (IC50) of Et. O.s, rosmarinic acid, and gemcitabine. Different doses of gemcitabine in combination with Et. O.s or rosmarinic acid were tested against Panc-1 to select the best concentrations which possessed synergistic effects. Elucidation of molecular mechanisms responsible for mediating chemo-sensitivity in Panc-1 was performed using Quantitative Real-time PCR (QPCR), flow cytometry and immunohistochemistry. RESULTS: Et. O.s was found to significantly sensitise Panc-1 towards gemcitabine by reducing the gene expression of multidrug-resistant protein family (MDR) (MDR-1, MRP-4, and MRP-5) and molecules related to epithelial-mesenchymal transition (ZEB-1 and Snail-1). An induction of the human equilibrate nucleoside transporter-1 (hENT-1) gene was also found in cells treated with Et. O.s-gemcitabine. The Et. O.s-gemcitabine combination induced cellular senescence, cell death and cell cycle arrest in Panc-1. In addition, the inhibition of Notch signalling was demonstrated through the downregulation of Notch 1 intracellular domain in this treatment group. In contrast, rosmarinic acid-gemcitabine combination showed no additional effects on cellular senescence, apoptosis, epithelial mesenchymal transition (EMT) markers, the MRP-4 and MRP-5 multi-drug resistance protein family, hENT-1, and the Notch pathway through Notch 1 intracellular domain. CONCLUSION: This study provides valuable insights on the use of Et. O.s to complement gemcitabine in targeting pancreatic cancer in vitro, suggesting its potential use as a novel complementary treatment in pancreatic cancer patients.

"Kidney Tea" and Its Bioactive Secondary Metabolites for Treatment of Gout.

Clerodendranthus spicatus, popularly known as "kidney tea" in China, is consumed traditionally as a functional food for treatment of renal inflammation, dysuria, and gout. We evaluated the effects of C. spicatus on gout by assessing activities of antihyperuricemia, anti-gouty arthritis, and analgesia in vivo, and the results indicated that the ethyl acetate fraction shows potential activities. Subsequent phytochemical investigation of this fraction led to the isolation of 32 compounds, consisting of 20 diterpenoids (including the new orthosiphonones E and F), 2 triterpenoids, 6 flavonoids, 2 lignanoids, and 2 phenolic acid derivatives. Pharmacological investigation of the pure compounds in the cellular model of hyperuricemia indicated that 12 compounds could promote the excretion of uric acid at 10 µg/mL, and compounds 3, 4, 5, and 21 had better effects than that of benzbromarone, a famous uricosuric drug. Furthermore, compounds 4, 6, 7, 9, 14, 15, 23, 26, and 31 showed significant anti-gouty arthritis activity in monosodium urate (MSU)-induced joint swelling at the dose of 50 mg/kg, while compounds 4, 5, 7, 9, and 26 exhibited significant inhibition of pain induced by acetic acid. Our findings provided scientific justification to support the traditional application of "kidney tea" for treating gout and suggested its good application prospects in the future.

Antihyperuricemic and nephroprotective effects of extracts from Orthosiphon stamineus in hyperuricemic mice.

OBJECTIVES: To investigate the antihyperuricemia and nephroprotective effects of Orthosiphon stamineus extracts on hyperuricemia (HUA) mice and explore the potential mechanisms. METHODS: Orthosiphon stamineus extracts were extracted using 50% ethanol and enriched using ethyl acetate, and characterised utilising UPLC/ESI-MS. A potassium oxonate (PO) induced hyperuricemic mouse model was used to evaluate antihyperuricemia and nephroprotective effects of O. stamineus ethyl acetate extracts (OSE). KEY FINDINGS: Eight constituents from OSE were identified and OSE treatment ameliorated HUA by regulating key indicators of kidney dysfunction and xanthine oxidase, adenosine deaminase activity and urate transporters in hyperuricemic mice. Moreover, in renal histopathology analysis, OSE significantly alleviated kidney injury. CONCLUSIONS: These findings demonstrate that OSE has antihyperuricemic and nephroprotective effects on PO-induced HUA mice and those results indicate that OSE could be a safe and effective agent or functional ingredient for treating HUA.

Optimization of Ultrasound-Assisted Extraction Conditions Followed by Solid Phase Extraction Fractionation from Orthosiphon stamineus Benth (Lamiace) Leaves for Antiproliferative Effect on Prostate Cancer Cells.

Primarily, optimization of ultrasonic-assisted extraction (UAE) conditions of Orthospihon stamineus was evaluated and verified using a central composite design (CCD) based on three factors including extraction time (minutes), ultrasound amplitude (A), and solvent concentration (%). The response surface methodology (RSM) was performed to develop an extraction method with maximum yield and high rosmarinic acid content. The optimal UAE conditions were as follows: extraction time 21 min, ultrasound amplitudes 62 A, and solvent composition 70% ethanol in water. The crude extract was further fractionated using solid-phase extraction (SPE), where six sequential fractions that varied in polarity (0-100% Acetonitrile in water) were obtained. Next, the six fractions were evaluated for their antioxidant and anti-cancer properties. This study found that Fraction 2 (F2) contained the highest rosmarinic acid content and showed the strongest antioxidant activity. Additionally, F2 showed an anti-proliferative effect against prostate cancer (DU145) with no harmful effect on normal cells.

Polymethoxylated flavones from Orthosiphon stamineus leaves as antiadhesive compounds against uropathogenic E. coli.

Aqueous and acetone extracts of O. stamineus leaves reduce the adhesion of uropathogenic E. coli (UPEC, strain UTI89) to T24 bladder cells significantly (IC25 ~ 524 mg/mL, resp. 40 µg/mL). The acteonic extract had no cytotoxic effects against UPEC in concentrations that inhibited the bacterial adhesion. The extract significantly reduced the gene expression of fimH, fimC, fimD, csgA and focG, which are strongly involved in the formation of bacterial adhesins. The antiadhesive effect was due to the presence of polymethoxylated flavones, enriched in the acetonic extract. Five flavones have been isolated by fast centrifugal partition chromatography, followed by preparative HPLC. Eupatorin, ladanein, salvigenin, sinensetin, 5,6,7,4'-tetramethoxyflavone and 5-hydroxy-6,7,3',4'-tetramethoxyflavone were identified as the main polymethoxylated flavones. With the exception of eupatorin, all of these flavones reduced the bacterial adhesion in a concentration depending manner, indicating that B-ring hydroxylation and methoxylation seems to have a major impact on the antiadhesive activity. In addition, this was confirmed by investigation of the flavones chrysoeriol and diosmetin, which had only very weak antiadhesive activity. From these data, Orthosiphon extracts can be assessed to have a pronounced antiadhesive activity against UPEC, based on a variety of polymethoxylated flavones.

Orthosipon stamineus extract exerts inhibition of bacterial adhesion and chaperon-usher system of uropathogenic Escherichia coli-a transcriptomic study.

Specific recognition and bacterial adhesion to host cells by uropathogenic Escherichia coli (UPEC) are the first steps towards infection of epithelial tissue of the human urogenital system. Therefore, targeting of UPEC virulence factors, relevant for adhesion, is a promising approach for prevention of recurrent urinary tract infections (UTI). A fully characterized plant-derived aqueous extract from the leaves of Orthosiphon stamineus (OWE), a plant traditionally used in clinical practice in Europe and Asia for UTI, has been shown to exert strong antiadhesive effects under in vitro and in vivo conditions. For improved understanding of the underlying mechanisms, transcriptome analysis of OWE-treated UPEC strain UTI89 by Illumina sequencing and cross-validation of these data by qPCR indicated significant downregulation of bacterial adhesins (curli, type 1-, F1C-, and P fimbriae) and of the chaperone-mediated protein folding/unfolding and pilus assembly process; in contrast, flagellar and motility-related genes were upregulated. We conclude that OWE transforms the sessile lifestyle of bacteria into a motile one and therefore disables bacterial attachment to the host cell. Additionally, the extract inhibited gene expression of multiple iron-acquisition systems (ent, fep, feo, fhu, chu, sit, ybt). The present study explains the antiadhesive and anti-infective effect of the plant extract by pinpointing specific biochemical and molecular targets.

Simultaneous Quantification and Pharmacokinetic Study of Nine Bioactive Components of Orthosiphon stamineus Benth. Extract in Rat Plasma by UHPLC-MS/MS.

Orthosiphon stamineus Benth. (OS) is a traditional folk medicine for the treatment of kidney stones and other urinary tract diseases. In this study, a rapid and sensitive Ultra high-performance liquid chromatography (UHPLC)-MS/MS approach was established and validated for the simultaneous quantification of nine bioactive components in rat plasma. The nine components from OS extract detected in rat plasma were danshensu, protocatechuic acid, caffeic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B, cichoric acid, sinensetin and eupatorin. After liquid-liquid extraction with ethyl acetate, the plasma samples were subjected to a triple quadrupole mass spectrometer employing electrospray ionization (ESI) technique and operating in multiple reaction monitoring (MRM) with both positive and negative ion modes. The standard curves showed good linear regression (r > 0.9915) over the concentration range for the nine analytes. The inter-day and intra-day precision and accuracy were found to be within 15% of the nominal concentration. The recovery and stability of nine compounds were all demonstrated to be within acceptable limits. The approach was successfully applied to investigate the pharmacokinetic analysis of the nine bioactive components after oral administration of OS extract in rats.

Identification of Metabolites of Eupatorin in Vivo and in Vitro Based on UHPLC-Q-TOF-MS/MS.

Eupatorin is the major bioactive component of Java tea (Orthosiphon stamineus), exhibiting strong anticancer and anti-inflammatory activities. However, no research on the metabolism of eupatorin has been reported to date. In the present study, ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) combined with an efficient online data acquisition and a multiple data processing method were developed for metabolite identification in vivo (rat plasma, bile, urine and feces) and in vitro (rat liver microsomes and intestinal flora). A total of 51 metabolites in vivo, 60 metabolites in vitro were structurally characterized. The loss of CH2, CH2O, O, CO, oxidation, methylation, glucuronidation, sulfate conjugation, N-acetylation, hydrogenation, ketone formation, glycine conjugation, glutamine conjugation and glucose conjugation were the main metabolic pathways of eupatorin. This was the first identification of metabolites of eupatorin in vivo and in vitro and it will provide reference and valuable evidence for further development of new pharmaceuticals and pharmacological mechanisms.

Changes and degradation kinetics of some bioactive compounds in dried Orthosiphon aristatus (Java tea) leaves during elevated temperature storage.

BACKGROUND: Orthosiphon aristatus is a traditional medicinal herb mostly used in Southeast Asia and which has many health benefits. Packaging types and storage temperatures were investigated in order to select the best conditions for producing high bioactive compounds (BC) from two kinds of dried O. aristatus leaves. RESULTS: Blanched leaves were vacuum packed in polypropylene (PP) and aluminum foil laminated with polyethylene terephthalate and polyethylene (PET/Al/PE) and dried in a freeze dryer (B_FD) or heat pump-assisted dehumidified dryer (B_HPD60) at 60 °C prior to storage at 15, 25 and 35 °C for 6 months. Leaves in PET/Al/PE bags had higher total phenolic content (TPC), antioxidant activity (AOA) and BC than in PP bags (P ≤ 0.05). Storage at 15 °C retained the highest TPC and AOA in PET/Al/PE bags (P ≤ 0.05). The degradation kinetics for BC, sinensetin and eupatorin followed first-order kinetics. Half-lives (t1/2 ) for BC in PET/Al/PE were higher than in PP and were the highest at 15 °C for both packaging types. CONCLUSIONS: Low temperature and PET/Al/PE bags provided the highest bioactive compound retention. The dried leaves from B_HPD60 and packed in PET/Al/PE bags had higher resistance to degradation of sinensetin than B_FD in PP bags. © 2018 Society of Chemical Industry.

Molecular Modeling and Simulation of Transketolase from Orthosiphon stamineus.

BACKGROUND: Orthosiphon stamineus is a traditional medicinal plant in Southeast Asia countries with various well-known pharmacological activities such as antidiabetic, diuretics and antitumor activities. Transketolase is one of the proteins identified in the leaves of the plant and transketolase is believed able to lower blood sugar level in human through non-pancreatic mechanism. In order to understand the protein behavioral properties, 3D model of transketolase and analysis of protein structure are of obvious interest. METHODS: In the present study, 3D model of transketolase was constructed and its atomic characteristics revealed. Besides, molecular dynamic simulation of the protein at 310 K and 368 K deciphered transketolase may be a thermophilic protein as the structure does not distort even at elevated temperature. This study also used the protein at 310 K and 368 K resimulated back at 310 K environment. RESULTS: The results revealed that the protein is stable at all condition which suggest that it has high capacity to adapt at different environment not only at high temperature but also from high temperature condition to low temperature where the structure remains unchanged while retaining protein function. CONCLUSION: The thermostability properties of transketolase is beneficial for pharmaceutical industries as most of the drug making processes are at high temperature condition.

Qualitative and quantitative analysis on the chemical constituents in Orthosiphon stamineus Benth. using ultra high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

A sensitive and efficient method was established and validated for qualitative and quantitative analysis on the chemical constituents in Orthosiphon stamineus Benth. (O. stamineus) using ultra high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Based on the retention time and MS spectra, 61 compounds were detected by using ultra high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry. 52 chemical structures in the O. stamineus extracts including 26 phenolic acids, 11 flavonoids, 6 diterpenoids, 4 fatty acids and 5 tanshinones were tentatively identified without the time-consuming process of isolation. Moreover, five chemical constituents (Danshensu, Caffeic acid, Rosmarinic acid, Sinensetin and Eupatorin) were quantified in three different batches of O. stamineus samples by the developed ultra high-performance liquid chromatography coupled with electrospray ionization triple-quadrupole mass spectrometry method in 10 min. The method validation of the five compounds was performed with acceptable linearity (R2, 0.9930-0.9997), precision (RSD, 1.87-10.36%), repeatability (RSD, 0.59-4.87%) and recovery (105.30-110.53%, RSD ≤ 13.90%).