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The clinical treatment of ulcerative colitis (UC) is limited. A traditional Chinese medicinal formula, Huangqin decoction (HQD), is chronicled in Shang Han Lun and is widely used to ameliorate gastrointestinal disorders, such as UC; however, its mechanism is yet to be clarified.The present study aimed to investigate the effect of HQD on 7-day colitis induced by 3% dextran sulfate sodium (DSS) in mice and further explore the inhibitory effect of metabolites on DSS-damaged FHC cells.The therapeutic efficacy of HQD was evaluated in a well-established DSS-induced colitis mice model. The clinical symptoms were analyzed, and biological samples were collected for microscopic examination, metabolomics, metagenomics, and the evaluation of the epithelial barrier function. The mechanism of metabolites regulated by HQD was evaluated in the DSS-induced FHC cell damage model. The samples were collected to detect the physiological functions of the cells.HQD suppressed the inflammation of DSS-induced colitis in vivo, attenuated DSS-induced clinical manifestations, reversed colon length reduction, and reduced histological injury. After HQD treatment, the DSS-induced gut dysbiosis was modulated, and the gut microbiota achieved a new equilibrium state. In addition, HQD activated the mTOR signaling pathway by upregulating amino acid metabolism. Significant phosphorylation of S6 and 4E-BP1 ameliorated intestinal epithelial barrier dysfunction. Moreover, HQD-regulated metabolites protected the epithelial barrier integrity by inhibiting DSS-induced apoptosis of FHC cells and regulating the proteins affecting apoptosis and cell-cell junction.These findings indicated that the mechanism of HQD was related to regulating the gut microbiota and amino acid metabolism, activating the mTOR signaling pathway, and protecting the intestinal mucosal barrier integrity.

Methyl 3,4-dihydroxybenzoate (MDHB) has the potential to prevent neurodegenerative diseases (NDDs). The present work aims to reveal the pharmacokinetics and tissue distribution characteristics of MDHB. The pharmacokinetics and tissue distribution of MDHB were analyzed using LC-MS/MS after a single intragastric administration (50 to 450 mg/kg) in mice, and samples were collected from five animals at specific time points. Pharmacokinetic parameters of MDHB following intragastric administrations were: the time to peak concentration (Tmax) ranged from 0.033 to 0.07 h, the peak concentration (Cmax) ranged from 12,379.158 to 109798.712 μg/l, the elimination half-life (t1/2z) ranged from 0.153 to 1.291 h, the area under the curve (AUC0–∞) ranged from 640.654 to 20,241.081 μg/l × h, the mean residence time (MRT0–∞) ranged from 0.071 to 0.206 h, the apparent volume of distribution (Vz/F) ranged from 17.538 to 45.244 l/kg, and the systemic clearance (Clz/F) ranged from 22.541 to 80.807 l/h/kg. The oral bioavailability of MDHB was 23%. The maximum MDHB content was detected in the stomach, and the minimum content was observed in the testes; the peak content in the brain was 15,666.93 ng/g. The pharmacokinetic characteristics of MDHB include fast absorption, high systemic clearance, a short half-life and an oral bioavailability of 23%. Additionally, MDHB permeates the blood-brain barrier (BBB) and is rapidly distributed to all organs. The identification of the pharmacokinetics of MDHB following its oral administration will contribute to further preclinical and clinical studies of its effects.

Pogostone (PO) is one of the major chemical constituents of the essential oil of Pogostemon cablin (Blanco) Benth. In the present study, the effect of PO on T cell responsiveness was investigated to explore its potential in immunosuppression by a Concanavalin A (ConA)-stimulation model using splenocytes isolated from C57BL/6 mice. Cytotoxicity by PO on normal splenocytes was evaluated by MTS assays. Characteristics of apoptosis, proliferation, and cell cycle were analyzed by flow cytometry. Related expressions of cyclins and cyclin-dependent kinases (CDKs) were also determined by flow cytometry. Inflammatory cytokine profiling was performed emplying cytometric beads assays (CBA). Moreover, the T cell-mediated delayed Type hepersensity (DTH) model was applied to evaluate the immunosuppressive activity of PO. Neither viability reduction in normal splenocytes nor apoptosis in ConA-stimulated splenocytes was observed under PO treatments. Meanwhile, PO remarkably reduced the total population of ConA-stimulated T cell, blocked T cell proliferation induced by Con A, and inhibited the production of IFN-γ and IL-10. This blockade of stimulated T cell proliferation by PO was likely attributed to down-regulation of cyclin E, cyclin B and CDK1 and the subsequent S-phase arrest. Additionally, PO could inhibit the DTH reaction by alleviating ear swelling and inflammatory infiltrations in the DNCB-challenged ear. Taken together, PO exhibited an immunosuppressive property by directly blocking T cell proliferation as well as altering inflammatory cytokine profile, suggesting that PO may have clinical implications for treating autoimmune diseases and other immune-based disorders.

The aim of this study is to evaluate the antibacterial activity and urease inhibitory effects of patchouli alcohol (PA), the bioactive ingredient isolated from Pogostemonis Herba, which has been widely used for the treatment of gastrointestinal disorders. The activities of PA against selected bacteria and fungi were determined by agar dilution method. It was demonstrated that PA exhibited selective antibacterial activity against Helicobacter pylori, without influencing the major normal gastrointestinal bacteria. Noticeably, the antibacterial activity of PA was superior to that of amoxicillin, with minimal inhibition concentration value of 78 µg/mL. On the other hand, PA inhibited ureases from H. pylori and jack bean in concentration-dependent fashion with IC50 values of 2.67 ± 0.79 mM and 2.99 ± 0.41 mM, respectively. Lineweaver-Burk plots indicated that the type of inhibition was non-competitive against H. pylori urease whereas uncompetitive against jack bean urease. Reactivation of PA-inactivated urease assay showed DL-dithiothreitol, the thiol reagent, synergistically inactivated urease with PA instead of enzymatic activity recovery. In conclusion, the selective H. pylori antibacterial activity along with urease inhibitory potential of PA could make it a possible drug candidate for the treatment of H. pylori infection. Copyright © 2014 John Wiley & Sons, Ltd.

In the present study, the inactivation effect of scutellarin (SL) on jack bean urease was investigated to elucidate the inhibitory potency, kinetics and mechanism of inhibition. It was revealed that SL acted as a concentration- and time-dependent inactivator of urease characteristic of slow-binding inhibition with an IC50 of 1.35±0.15 mM. The rapid formation of the initial SL-urease complex with an inhibition constant of Ki=5.37×10(-2) mM was followed by a slow isomerization into the final complex with the overall inhibition constant of Ki*=3.49×10(-3) mM. High effectiveness of thiol protectors, such as L-cysteine (L-cys), 2-mercaptoethanol (2-ME) and dithiothreitol (DTT) significantly slowed down the rate of inactivation, indicating the strategic role of the active site sulfhydryl group in the blocking process. While the insignificant protection by boric acid and fluoride from the inactivation further confirmed that the active site cysteine should be obligatory for urease inhibition, which was also rationalized by the molecular docking study. The inhibition of SL on urease proved to be reversible since SL-blocked urease could be reactivated by DTT application and multidilution. The results obtained indicated that urease inactivation resulted from the reaction between SL and the sulfhydryl group.

The essential oils obtained by hydrodistillation of flowers, young leaves and epicormic branches of Cinnamomum camphora chvar. Borneol were comparatively studied by gas chromatography with flame ionization detector (GC/FID) and GC/mass spectrometry (GC/MS). Among the verified constituents, oxy-terpene accounted for the most abundant constituent in all volatile oils (flowers 50.83%, leaves 70.75%, branches 78.22%). d-Borneol, the characteristic oxy-terpene of the volatile oils of C. camphora chvar. Borneol, was the highest-content compound among all the three parts (flowers 12.13±0.67 g/kg, leaves 17.22±0.85 g/kg and branches 4.47±0.79 g/kg), while camphor was absent in flowers, low in leaves (0.05±0.01 g/kg) but rich in branches (1.09±0.44 g/kg). These findings suggested that volatile oils from the leaves and flowers of C. camphora chvar. Borneol would serve as a better resource for d-borneol preparation, and there might be an α-pinene–borneol–camphor bioconversion during the growth time extension of C. c...

Traditional Chinese medicine (TCM) has been extensively applied as preferable herbal remedy for menopausal symptoms. In the present work, the potential of a TCM recipe named RRF, composed of Radix Astragali, Radix Angelicae Sinensis, and Folium Epimedii, was investigated on a natural aging rat model. After administration of RRF (141, 282, and 564 mg/kg/d), the circulated estradiol (E2) level increased accompanied by a reduction of serum follicle stimulating hormone (FSH). But no significant impact on serum lutenizing hormone (LH) level was observed. As a result of the E2-FSH-LH adjustment, the histomorphology degenerations of ovary, uterus, and vagina of the 11.5-month female rats were alleviated. And lumbar vertebrae trabecular microstructure was also restored under RRF exposure by means of increasing the trabecular area and area rate. Moreover, levels of hypothalamic dopamine (DA) and norepinephrine (NE) rallied significantly after RRF treatment. Results from our studies suggest that RRF possesses a positive regulation on the estrogen imbalance and neurotransmitter disorder, thereby restoring reproductive organ degeneration and skeleton deterioration. The above-mentioned benefits of RRF on the menopause syndromes recommend RRF as a potential candidate for the treatment of perimenopausal syndrome.

A simple and sensitive method was developed and validated for profiling and simultaneous quantitation of seven alkaloids (6-hydroxy-β-carboline-1-carboxylic acid, β-carboline-1-carboxylic acid, β-carboline-1-propanoic acid, 3-methylcanthin-5,6-dione, 5-hydroxy-4-methoxycanthin-6-one, 1-methoxycarbony-β-carboline, and 4,5-dimethoxycanthin-6-one) in Picrasma quassioide grown in different locations by high-performance liquid chromatography with photodiode array detection. The analysis was conducted on a Phenomenex Gemini C(18) column at 35°C with mobile phase of 25 mM aqueous ammonium acetate (pH 4.0, adjusted by glacial acetate acid) and acetonitrile. A common fingerprint chromatograph under 254 nm consisting of 27 peaks was constructed for the evaluation of the similarities among 31 P. quassioide samples. Samples from Guangdong and Guangxi Provinces were found to be within group linkage and showed significant difference from that of Jiangxi Province origin by using principal component analysis and hierarchical clustering analysis. In addition, the seven alkaloids were identified by electrospray ionization mass spectrometry and comparing with reference standards and literature data. All of them were determined simultaneously using the established HPLC method. This rapid and effective analytical method could be employed for quality assessment of P. quassioide, as well as pharmaceutical products containing this herbal material.

Ethnopharmacological relevance Chrysanthemum indicum (Compositae) Linne, Pogostemon cablin (Blanco) Benth and Curcuma wenyujin (Zingiberaceae) Y. H. Chen et C. Ling are three of the extensively used herbal remedies among traditional Chinese medicines for the purpose of anti-inflammation. A traditional Chinese medicine (TCM) recipe named CPZ consisting extracts of the above three herbs, has shown noteworthy anti-influenza activity, which is closely related to its anti-inflammatory feature. Aim of this study To investigated the anti-inflammtory activity of CPZ in vivo for a further exploration of the recipe's anti-inflammatory properties. Materials and methods The anti-inflammatory property of CPZ on acute inflammation was evaluated by inflammatory models of dimethylbenzene (DMB)-induced ear vasodilatation and acetic acid-induced capillary permeability enhancement in mice, as well as the carrageenan-induced paw edema rat model, in which inflammation-related cytokine including prostaglandin E 2 (PGE 2 ), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and nitric oxide (NO) in the edematous paw tissue were determined by enzyme linked immunosorbent assay (ELISA). Moreover, effect of CPZ on chronic inflammation was observed through granuloma formation in rats subjected to cotton pellet implantation. Results CPZ (340, 170, and 85 mg/kg for mice, p.o. ) not only decreased the DMB-induced ear vasodilatation but also attenuated capillary permeability under acetic acid challenge in mice. And the significant inhibition on carrageenan-induced paw edema was observed. Further more, the ELISA results showed that CPZ (170, 85, and 42.5 mg/kg for rats, p.o. ) could up-regulate the level of IL-1β in the edema paw tissue of rats significantly while down-regulate that of PGE 2 , but no apparent effect on TNF-α or NO was observed in the test. Besides, CPZ had a certain degree of restraining effect on the cotton pellet-induced granuloma formation in rats and the highest dose of 170 mg/kg even showed a significant suppression on it. Conclusion The above results indicated that CPZ possessed a potent anti-inflammatory activity, which is indicated to be closely associated with its regulation on IL-1β and PGE 2 thereby mediating the inflammatory response acting at an appropriate level.

Pogostemonis Herba has long been used in traditional Chinese medicine for the treatment of inflammatory disorders. Patchouli alcohol (PA), a tricyclic sesquiterpene isolated from Pogostemonis Herba, is known to possess a variety of pharmacological activities. The present study aimed to investigate the in vivo anti-inflammatory effect of PA using two common inflammatory animal models i.e., xylene-induced ear edema in mice and carrageenan-induced paw edema in rats. The degree of edema in both inflammatory animals, as well as the protein and mRNA expression of some inflammatory mediators including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), prostaglandin E₂ (PGE₂) and nitric oxide (NO) in the hind paw of carrageenan-treated rats were measured. Results showed that PA (10-40 mg/kg) significantly inhibited the ear edema induced by xylene in mice and the paw edema induced by carrageenan in rats. In addition, treatment with PA (10-40 mg/kg) also dose-dependently decreased the production of TNF-α, IL-1β, PGE₂ and NO in the hind paw of carrageenan-treated rats. Furthermore, PA treatment also suppressed the mRNA expression of TNF-α, IL-1β, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the hind paw of carrageenan-treated rats. These results suggest that PA possesses potent anti-inflammatory activity, which may be mediated, at least in part, by down-regulating the mRNA expression of a panel of inflammatory mediators including TNF-α, IL-1β, iNOS and COX-2.

Parkinson's disease is recognized as the second most common neurodegenerative disorder after Alzheimer's disease, characterized by the loss of dopominergic neurons in the substantia nigra pars compacta and can be experimentally mimicked by the use of the neurotoxin, 1‑methyl‑4‑phenylpyridinium ion (MPP(+)), in in vitro models. In this study, we investigated the potential protective effects of apigenin (AP), galangin and genkwanin, naturally occurring plant flavonoids, on the MPP(+)‑induced cytotoxicity in cultured rat adrenal pheochromocytoma cells (PC12 cells). The PC12 cells were pre-treated with various concentrations of the test compounds for 4 h, followed by the challenge with 1,000 µM MPP(+) for 48 h. We found that only pre-treatment with AP (3, 6 and 12 µM) before injury significantly increased cell viability, decreased the release of lactate dehydrogenase, reduced the level of intracellular reactive oxygen species and elevated mitochondrial membrane potential in the MPP(+)‑treated PC12 cells. In addition, AP markedly suppressed the increased rate of apoptosis and the reduced Bcl‑2/Bax ratio induced by MPP(+) in the PC12 cells. Taken together, the findings of this study demonstrate that AP exerts neuroprotective effects against MPP(+)‑induced neurotoxicity in PC12 cells, at least in part, through the inhibition of oxidative damage and the suppression of apoptosis through the mitochondrial pathway.

The aim of this study is to evaluate the antibacterial activity and urease inhibitory effects of patchouli alcohol (PA), the bioactive ingredient isolated from Pogostemonis Herba, which has been widely used for the treatment of gastrointestinal disorders. The activities of PA against selected bacteria and fungi were determined by agar dilution method. It was demonstrated that PA exhibited selective antibacterial activity against Helicobacter pylori, without influencing the major normal gastrointestinal bacteria. Noticeably, the antibacterial activity of PA was superior to that of amoxicillin, with minimal inhibition concentration value of 78 µg/mL. On the other hand, PA inhibited ureases from H.pylori and jack bean in concentration-dependent fashion with IC50 values of 2.67 ± 0.79 mM and 2.99 ± 0.41 mM, respectively. Lineweaver-Burk plots indicated that the type of inhibition was non-competitive against H.pylori urease whereas uncompetitive against jack bean urease. Reactivation of PA-inactivated urease assay showed DL-dithiothreitol, the thiol reagent, synergistically inactivated urease with PA instead of enzymatic activity recovery. In conclusion, the selective H.pylori antibacterial activity along with urease inhibitory potential of PA could make it a possible drug candidate for the treatment of H.pylori infection.

Ischemic stroke has been confirmed to cause neuronal injury due to its insufficient supply of glucose and oxygen to brain tissue. Previous research has shown that oxidative stress, a result of excessive accumulation of reactive oxygen species (ROS), relates to pathophysiology of ischemic stroke, and causes oxidative damage to biomolecules, eventually leading to programmed cell death. Meanwhile, apigenin has been shown to exhibit antioxidant, anti-inflammatory, anti-cancer properties and neuroprotective action. Hence, this study was to investigate the potential mechanisms underlying the neural protection of apigenin on oxygen and glucose deprivation/reperfusion (OGD/R) induced neuronal injury in differentiated PC12 cells. Cells were pretreated with apigenin for 6 h, and then subjected to OGD for 12 h followed by reperfusion for 24 h. The results showed that OGD/R significantly decreased cell viability, mitochondrial membrane potential, mRNA levels of antioxidant and detoxifying enzymes and Nrf2 protein expression, while elevated the release of LDH, cell apoptosis, intracellular ROS level, P53 protein expression and upregulated its downstream genes in PC12 cells. However, apigenin effectively inhibited these undesirable changes induced by OGD/R. Our findings demonstrate that this compound attenuates OGD/R induced neuronal injury mainly by virtue of its anti-apoptosis and antioxidative properties via affecting the expression of Nrf2 and P53, and their downstream target gene transcription.

Baicalin (BA) is the principal component of Radix Scutellariae responsible for its pharmacological activity. In this study, kinetics and mechanism of inhibition by BA against jack-bean urease were investigated for its therapeutic potential. It was revealed that the IC50of BA against jack-bean urease was2.74 ± 0.51 mM, which was proved to be a competitive and concentration-dependent inhibition with slow-binding progress curves. The rapid formation of initial BA-urease complex with an inhibition constant ofKi=3.89 × 10−3 mM was followed by a slow isomerization into the final complex with an overall inhibition constant ofKi*=1.47×10-4 mM. High effectiveness of thiol protectors against BA inhibition indicated that the strategic role of the active-site sulfhydryl group of the urease was involved in the blocking process. Moreover, the inhibition of BA was proved to be reversible due to the fact that urease could be reactivated by dithiothreitol but not reactant dilution. Molecular docking assay suggested that BA made contacts with the important activating sulfhydryl group Cys-592 residues and restricted the mobility of the active-site flap. Taken together, it could be deduced that BA was a competitive inhibitor targeting thiol groups of urease in a slow-binding manner both reversibly and concentration-dependently, serving as a promising urease inhibitor for treatments on urease-related diseases.

Pogostone (PO) is one of the secondary metabolites from Pogostemon cablin (Blanco) Benth. (Lamiaceae), serving as the effective component of the antimicrobial activity. In this study, PO and a series of its analogues were synthesized by the reaction of dehydroacetate and aldehydes in tetrahydrofuran under a nitrogen atmosphere. Their activities against Candida albicans, Gram positive bacteria and Gram negative bacteria were evaluated. The antifungal results demonstrated that PO (MIC ranged from 12 to 97μg/mL against all strains, MFC ranged from 49 to 97μg/mL against all strains) and A3 (MIC ranged from 12 to 49, MFC over 195μg/mL) showed a strong activity against Candida albicans. While A1 (MIC ranged from 49 to 97μg/mL) and A2 (MIC ranged from 24 to 49μg/mL) have only shown effect against Guangzhou clinical isolates, the antibacterial results demonstrated that PO and its analogues showed no effects against the tested bacteria strains. This study suggests that pogostone analogues, with the appropriated structure modification, represented a kind of promising antifungal agents.

Ethnopharmacological relevance: Radix Astragali (root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao, RA), Radix Angelicae sinensis (root of Angelica sinensis (Oliv.) Diels, RAS) and Folium Epimedii (leaves of Epimedium brevicomum Maxim., FE) are three of the extensively applied herbal remedies among traditional Chinese medicines for gynecological disorders and osteoporosis. A derivative herbal recipe—RRF, composed of the three medicines with a weight ratio of 5:1:5, is derived from a famous Chinese herbal formula—Danggui Buxue Tang (DBT). RRF has shown noteworthy protective effect in ovariectomized rats, which might represent a promising candidate for the treatment of perimenopausal disorders. The aim of this study was to investigate the herbal recipe RRF for its efficacy on perimenopausal disorders and the underlying mechanisms via ovariectomy (OVX) models. Materials and methods An experimental model of OVX female rats was applied. Vehicle (Sham and OVX group), RRF (564, 282 and 141 mg/kg/d) and conjugated equine estrogens (CEE, 0.1 mg/kg/d, reference drug) were all administrated orally once daily for 16 weeks post operation. After the treatment, radioimmunoassay for estradiol (E2), lutenizing hormone (LH), follicle stimulating hormone (FSH) and β-endorphin (β--EP), neurotransmitter determination by high-performance liquid chromatography with electrochemical detector (HPLC-ECD) for norepinephrine (NE), dopamine (DA), 5-hydroxytryptamine (5-HT) and 5-HIAA (5-hydroxyindoleacetic acid), bone mineral density (BMD) assay as well as lipid peroxidation assessment, were carried out to probe into the effectiveness of RRF. Results (1) RRF treatment enhanced E2 synthese while diminished the elevated serum FSH and LH levels; in terms of neurotransmitter, β-EP syntheses rallied whereas the hypothalamic NE, DA and 5-HT release experienced varying mitigation in OVX female rats. (2) Repeated administration of RRF was able to attenuate osteoporosis by elevating the BMD levels of total body, and arrest the bone trabeculae degradation. (3) RRF exposure decreased serum levels of constituent MDA and increased endogenous SOD activity. Conclusions Results of the current studies revealed that RRF was capable of acting at multiple targets which presumably underlay its potential protective effect in OVX rats mimicking symptoms as observed in perimenopausal women. Hence, RRF might represent a promising candidate in the treatment of perimenopausal disorders in midlife women.

A simple and sensitive method was developed and validated for profiling and simultaneous quantitation of seven alkaloids (6-hydroxy-β-carboline-1-carboxylic acid, β-carboline-1-carboxylic acid, β-carboline-1-propanoic acid, 3-methylcanthin-5,6-dione, 5-hydroxy-4-methoxycanthin-6-one, 1-methoxycarbony-β-carboline, and 4,5-dimethoxycanthin-6-one) in Picrasma quassioide grown in different locations by high-performance liquid chromatography with photodiode array detection. The analysis was conducted on a Phenomenex Gemini C(18) column at 35°C with mobile phase of 25 mM aqueous ammonium acetate (pH 4.0, adjusted by glacial acetate acid) and acetonitrile. A common fingerprint chromatograph under 254 nm consisting of 27 peaks was constructed for the evaluation of the similarities among 31 P. quassioide samples. Samples from Guangdong and Guangxi Provinces were found to be within group linkage and showed significant difference from that of Jiangxi Province origin by using principal component analysis and hierarchical clustering analysis. In addition, the seven alkaloids were identified by electrospray ionization mass spectrometry and comparing with reference standards and literature data. All of them were determined simultaneously using the established HPLC method. This rapid and effective analytical method could be employed for quality assessment of P. quassioide, as well as pharmaceutical products containing this herbal material.

OBJECTIVE To establish the HPLC chromatographic fingerprint of Kumu injection and to simultaneously determine the contents of three beta-carboline alkaloids, comprehensively evaluating the immanent quality of Kumu injection. METHOD The chromatographic analysis was performed on a Phenomenex Gemini C18 ( 4.6 mm x 250 mm, 5 microm) column with the gradient elution solvent system composed of methanol and 30 mmol x L(-1) aqueous ammonium acetate (adjusted with glacial acetic acid to pH 4.5). Similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine (2004 A) was used in data analysis. RESULT Sixteen co-possessing peaks were selected as the fingerprints of Kumu injection, and 7 peaks were identified by chemical reference substances. There were good similarities between the standard fingerprint chromatogram and each fingerprint chromatogram from the eleven samples for their similarity coefficients were not less than 0.9. Three kinds of beta-carboline alkaloids were separated well. The correlation coefficients were 0.999 9. The linear ranges of three components were 0.020 0-0.300 0, 0.102 0-1.530 0, 0.015 2-0. 228 0 microg, respectively, and the average recoveries ranged were from 99.5% to 102%. CONCLUSION The method of fingerprint combined with quantitative analysis is sensitive, selective, and provide scientific basis for quality control of Kumu Injection.

Purpose: To develop and validate a simple and selective high performance liquid chromatography photo diode array mass spectrometry (HPLC-PDA-MS/MS) method for simultaneous determination and confirmation of seven major active alkaloids (6-Hydroxy-ß-Carboline-1-carboxylic acid, ß-Carboline-1- carboxylic acid, ß-Carboline-1-propanoic acid, 3-Methylcanthin-5,6-dione, 4-Methoxy-3-methylcanthine- 5,6-dione, 5-Hydroxy-4-methoxycanthin-6-one, 4,5-Dimethoxycanthin-6-one) in Kumu injections (KMIs)Methods: For the analysis of the preparation, the optimal chromatographic condition was achieved on a Phenomenex Gemini C18 column with gradient elution of 25 mM aqueous ammonium acetate (pH = 4.0 adjusted by glacial acetate acid) and acetonitrile with flow rate at 1.0 mL/min, column temperature at 35oC and detection wavelengths at 245, 260 and 271 nm.Results: Excellent linear behavior over the investigated concentration ranges was observed with regression coeffcient (R2) > 0.9997 for all analytes. Intra- and inter-day precisions for all studied constituents ranged from 0.20 to 1.80 %. Recoveries of the assayed constituents were in the range of 98.73 to 100.34 %. The results showed the contents of these seven marker compounds differed significantly among different batches of KMIs both from the same and different manufacturers.Conclusion: The validated method was reliable, accurate, repeatable and can be applied to routine quality assessment of these active components in KMIs.Keywords: Alkaloids, High performance liquid chromatography, Photo diode array, Mass spectrometry, Kumu injection, Quality control

The inhibitory effect of andrographolide sodium bisulphite (ASB) on jack bean urease (JBU) and Helicobacter pylori urease (HPU) was performed to elucidate the inhibitory potency, kinetics and mechanism of inhibition in 20 mM phosphate buffer, pH 7.0, 2 mM EDTA, 25 °C. The ammonia formations, indicator of urease activity, were examined using modified spectrophotometric Berthelot (phenol-hypochlorite) method. The inhibitory effect of ASB was characterized with IC50 values. Lineweaver-Burk and Dixon plots for JBU inhibition of ASB was constructed from the kinetic data. SH-blocking reagents and competitive active site Ni2+ binding inhibitors were employed for mechanism study. Molecular docking technique was used to provide some information on binding conformations as well as confirm the inhibition mode. The IC50 of ASB against JBU and HPU was 3.28 ± 0.13 mM and 3.17 ± 0.34 mM, respectively. The inhibition proved to be competitive and concentration- dependent in a slow-binding progress. The rapid formation of initial ASB-JBU complex with an inhibition constant of K i = 2.86 × 10−3 mM was followed by a slow isomerization into the final complex with an overall inhibition constant of K i * = 1.33 × 10−4 mM. The protective experiment proved that the urease active site is involved in the binding of ASB. Thiol reagents (L-cysteine and dithiothreithol) strongly protect the enzyme from the loss of enzymatic activity, while boric acid and fluoride show weaker protection, indicating that the active-site sulfhydryl group of JBU was potentially involved in the blocking process. Moreover, inhibition of ASB proved to be reversible since ASB-inactivated JBU could be reactivated by dithiothreitol application. Molecular docking assay suggested that ASB made contacts with the important sulfhydryl group Cys-592 residue and restricted the mobility of the active-site flap. ASB was a competitive inhibitor targeting thiol groups of urease in a slow-binding manner both reversibly and concentration-dependently, serving as a promising urease inhibitor for the treatment of urease-related diseases.

Background A traditional Chinese Medicine (TCM) formula, HZJW, has been applied in clinics in China for gastrointestinal disorders. However, the therapeutic mechanism underlying its efficacy and safety remained to be defined. The present investigation was undertaken to evaluate the formula HZJW for its gastroprotective potential, possible effect on Helicobacter pylori along with safety to justify its anti-ulcer action and safe clinical application. Methods The gastroduodenal cytoprotective potential was evaluated in rodent experimental models (HCl/Ethanol and NSAID-induced ulcer protocols). The anti-H. pylori property was assessed by agar dilution assay in vitro and analysis in vivo including rapid urease test, immunogold test and histopathology. For toxicity assessment, acute toxicity study was performed according to fixed dose procedure with a single oral administration of HZJW to mice. In the oral chronic toxicity, rats (80 males, 80 females) were administrated HZJW orally in 0, 1000, 2500, or 5000 mg/kg/day doses for 26 weeks (n = 40/group of each sex). Clinical signs, mortality, body weights, feed consumption, ophthalmology, hematology, serum biochemistry, gross findings, organ weights and histopathology were examined at the end of the 13- and 26-week dosing period, as well as after the 4-week recovery period. Results In the HCl/Ethanol-induced ulcer model, it was observed that oral administration with HZJW (260, 520 and 1040 mg/kg) and ranitidine (250 mg/kg) significantly reduced the ulcerative lesion index (116.70 ± 36.4, 102.20 ± 18.20, 84.10 ± 12.1 and 73.70 ± 16.70) in a dose-dependent manner, respectively, with respect to control group (134.10 ± 31.69). Significant inhibition was also observed in ulcerative index from aspirin-induced ulcer model, with decreases of 35.40 ± 5.93, 31.30 ± 8.08, 26.80 ± 8.27and 20.40 ± 6.93 for the groups treated with HZJW and ranitidine, in parallel to controls (41.60 ± 10.80). On the other hand, treatment with HZJW efficaciously eradicated H. pylori in infected mice in rapid urease test (RUT) and immunogold antibody assay, as further confirmed by reduction of H. pylori presence in histopathological analysis. In the in vitro assay, MICs for HZJW and amoxicillin (positive control) were 125 and 0.12 μg/mL respectively. The LD50 of HZJW was over 18.0 g/kg for mice. No drug-induced abnormalities were found as clinical signs, body weight, food consumption, hematology, blood biochemistry, ophthalmology and histopathology results across three doses. No target organ was identified. The No Observed Adverse Effect Level (NOAEL) of HZJW was determined to be 5,000 mg/kg/day for both sexes, a dose that was equivalent to 50 times of human dose. Conclusions These results suggested the efficacy and safety of HZJW in healing peptic ulcer and combating H. pylori, which corroborated their conventional indications and contributed to their antiulcer pharmacological validation, lending more credence to its clinical application for the traditional treatment of stomach complaints symptomatic of peptic ulcer disease (PUD). HZJW might have the potential for further development as a safe and effective alternative/complementary to conventional medication in treating gastrointestinal (GI) disorders.