Advances in Research on Bioactivity, Toxicity, Metabolism, and Pharmacokinetics of Usnic Acid In Vitro and In Vivo.

Lichens are among the most widely distributed plants on earth and have the longest growth cycle. Usnic acid is an abundant characteristic secondary metabolite of lichens and the earliest lichen compound used commercially. It has diverse pharmacological activities, such as anti-inflammatory, antibacterial, antiviral, anticancer, antioxidant, and photoprotective effects, and promotes wound healing. It is widely used in dietary supplements, daily chemical products (fodder, dyes, food, perfumery, and cosmetics), and medicine. However, some studies have found that usnic acid can cause allergic dermatitis and drug-induced liver injury. In this paper, the bioactivity, toxicity, in vivo and in vitro metabolism, and pharmacokinetics of usnic acid were summarized. The aims were to develop and utilize usnic acid and provide reference for its future research.

The antinociceptive and anti-inflammatory potential and pharmacokinetic study of significant alkamides ingredients from Asarum Linn.

ETHNOPHARMACOLOGICAL RELEVANCE: Asari Radix et Rhizoma (ARR), including 3 major plants of genus Asarum Linn, A. heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag., A. sieboldii Miq. f. sieboldii and A. sieboldii Miq f. seoulense (Nakai) C. Y. Cheng et C. S. Yang, is one of the most important traditional herbal medicine in Asia with tremendous pharmacological activities. For a long time, researchers focus attention on studing asarinin and essential oils, the indicating ingredients of ARR, but paid less attention to another characteristic component, alkamides. The role of alkamides in the major efficacy of ARR medication remains to be elucidated. AIM OF THE STUDY: This study aims to investigate the contribution of alkamides in the efficacy of ARR according to the evaluation of antinociceptive and anti-inflammatory effects and in vivo pharmacokinetics processes. MATERIALS AND METHODS: For pharmacodynamic study, the analgesic and anti-inflammatory effects of alkamides-enriched fraction (ARRA) were comparatively evaluated by writhing test, hot plate test, and ear swelling test in mice after oral administration. For pharmacokinetic study, an UHPLC-MS/MS method was developed for the simultaneous determination of N-isobutyl-2E,4E,8Z,10Z/E-dodecatetraenamide (DDA) and other 6 major characteristic ingredients of ARR in rat plasma. The analytical method was validated and successfully applied to the pharmacokinetic study of ARR extract and DDA. RESULTS: Pharmacodynamic study show that the ARR and ARRA can significantly inhibit the writhing times of mice caused by acetic acid administration, increase the pain threshold of thermal stimulation, and inhibit xylene treated ear swelling degree by reduce PGE2 and TNF-α levels in the inflamed tissue. For pharmacokinetic study, the pharmacokinetic parameters of Vd/F and CL/F after intravenous administration in rats of DDA are 63.94 ± 32.12 L/kg and 0.33 ± 0.06 L/min/kg, respectively. The plasma drug concentration declined with the T1/2 value of 2.25 ± 0.96 h, and the MRT0-∞ was 2.23 ± 1.02 h. The absolute bioavailability of DDA after oral administration was calculated as 10.73%. DDA, methyleugenol, and asarinin have relatively high AUC0-∞ values when the ethanol and water extract of ARR is orally administered. CONCLUSIONS: ARRA is a kind of active ingredients with potential analgesic and anti-inflammatory effects that played a significant role in the major efficacy of ARR. DDA, the major compound of ARRA, has a high level of exposure in vivo, which could be is suitable for the pharmacokinetic marker or new quality marker of ARR.

Simultaneous LC-MS/MS bioanalysis of alkaloids, terpenoids, and flavonoids in rat plasma through salting-out-assisted liquid-liquid extraction after oral administration of extract from Tetradium ruticarpum and Glycyrrhiza uralensis: a sample preparation strategy to broaden analyte coverage of herbal medicines.

Herbal medicines have historically been practiced in combinatorial way, which achieves therapeutic efficacy by integrative effects of multi-components. Thus, the accurate and precise measurement of multi bioactive components in matrices is inalienable to understanding the metabolism and disposition of herbal medicines. In this study, aiming to provide a strategy that improves analyte coverage, evaluation of six protocols employing sample pretreatment methods- protein precipitation (PPT), liquid-liquid extraction (LLE), sugaring-out-assisted liquid-liquid extraction (SULLE), and salting-out-assisted liquid-liquid extraction (SALLE)- was performed by LC-MS/MS using rat plasma and a mixture of alkaloid (evodiamine, rutaecarpine, dehydroevodiamine), terpenoid (limonin, rutaevin, obacunone), and flavonoid (liquiritin, isoliquiritin, liquiritigenin) standards isolated from Tetradium ruticarpum and Glycyrrhiza uralensis. These protocols were as follows: (1) PPT with methanol, (2) PPT with acetonitrile, (3) LLE with methyl tertiary-butyl ether-dichloromethane, (4) LLE with ethyl acetate-n-butanol, (5) SALLE with ammonium acetate, (6) SULLE with glucose. The results suggested that SALLE produced broader analyte coverage with satisfactory reproducibility, acceptable recovery, and low matrix interference. Then, sample preparation procedure of SALLE, chromatographic conditions, and mass spectrometric parameters were optimized, followed by method validation, showing that good sensitivity (LLOQ ≤ 1 ng mL-1), linearity (r ≥ 0.9933), precision (RSD ≤ 14.45%), accuracy (89.54~110.87%), and stability could be achieved. Next, the developed method was applied successfully to determine the pharmacokinetic behavior of the nine compounds in rat plasma after intragastric administration with an extract from Tetradium ruticarpum and Glycyrrhiza uralensis (Wuzhuyu-Gancao pair). Based on an extensive review and experiments, a sample preparation procedure that matches with LC-MS/MS technique and can get wider analyte coverage was outlined. The developed SALLE method is rapid, reliable, and suitable for bioanalysis of analytes with diverse polarity, which was expected to be a promising strategy for the pharmacokinetic studies of herbal medicines. Graphical abstract.

Potential mechanisms of tremor tolerance induced in rats by the repeated administration of total alkaloid extracts from the seeds of Peganum harmala Linn.

ETHNOPHARMACOLOGICAL RELEVANCE: The seeds of Peganum harmala Linn have been widely used for the treatment of nervous, cardiovascular, gastrointestinal, respiratory, and endocrine diseases and many other human ailments. However, tremor toxicity occurs after overdose and is tolerated following multiple dosing. Thus far, little is known about the underlying mechanisms of tremors and tremor tolerance. AIM OF THE STUDY: To investigate the potential mechanisms of tremors and tremor tolerance induced in rats by the repeated administration of total alkaloid extracts from the seeds of P. harmala (TAEP). MATERIALS AND METHODS: A tremor model was induced in male Wistar rats by administering TAEP at a dose of 150 mg/kg/day. To evaluate tremor action, behavioral assessment was conducted by using a custom-built tremor acquisition and analysis system. To investigate the relationships between tremors and neurotransmitter levels in the brain, various neurotransmitters were simultaneously quantified by an ultra-performance liquid chromatography combined with electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) system, and the association between these two parameters was analyzed using Pearson correlation coefficients. To further elucidate the potential mechanisms of the alterations of neurotransmitter levels in cortical tissues, the protein expression levels of several important enzymes and transporters that are closely related to neurotransmitter levels were investigated. In addition, neuropathological analysis was conducted to assess the effect of TAEP on neurons in the brain. To further clarify the potential mechanisms of TAEP-induced neurodegeneration in the brain, c-fos was subjected to immunohistochemical analysis, and oxidative stress markers were examined. RESULTS: Tremors initially occurred in rats after the oral administration of TAEP at a dose of 150 mg/kg/day. However, they were tolerated following repeated dosing. The levels of 5-hydroxytryptamine (5-HT) and glycine (Gly) in cortical tissues were most likely associated with the tremor response. Tremor tolerance also likely resulted from the degeneration of cerebellar Purkinje cells. Furthermore, the alteration of 5-HT levels was mainly attributed to the downregulated expression of monoamine oxidase A (MAO-A). The degeneration of Purkinje neurons might have resulted from the overexpression of c-fos and increased oxidative stress in the cerebellum after the multiple dosing of TAEP. CONCLUSION: The tremor response induced by TAEP at high doses is closely related to the concentrations of 5-HT and Gly in cortical tissues. Tremor tolerance may also be attributed to the degeneration of cerebellar Purkinje cells after the repeated dosing of TAEP. Further studies should be conducted to elucidate the interaction of the alkaloids on the neurotransmitter receptors, the expression of related neurotransmitter receptors, the specific signaling pathway involved in regulating MAO-A, and the mechanism of the loss and functional recovery of cerebellar Purkinje neurons.

Identification of photodegraded derivatives of usnic acid with improved toxicity profile and UVA/UVB protection in normal human L02 hepatocytes and epidermal melanocytes.

This study focused on the investigation of photodegradation of usnic acid (UA) which is a natural UV absorbing agent in lichens. Despite years of historical use in food supplement, traditional medicine or cosmetic products in many countries, liver toxicity has been found to be one of the severe and life threatening adverse effects in early 2000's. Such severe side effect has limited UA or its synthesized derivatives for further use clinically or commercially. In this study, extracted UA from Usnea longissima in methanol was exposed to natural sunlight for 21 days. Five photodegraded derivatives (1 to 5) with two new and three previously explored compounds were isolated and purified by column chromatography and preparative liquid chromatography. The structures of these derivatives were identified based on the data of nuclear magnetic resonance spectrum, mass spectrum, optical rotation, infrared spectrum, X-ray crystallography and/or electronic circulation dichroism. The cytotoxicity of (+)-UA and 2 to 5 in liver L02 cells and melanocytes were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Experimental results indicated that IC50 of (+)-UA in liver L02 cells and melanocytes were 24.4 and 6.9 µM respectively, while compound 2 to 5 have lower cytotoxicity with IC50 of 326.7, 1085.0, 62.7 and 152.4 µM in L02 cells and 87.7, 297.7, 60.2 and 85.0 µM in melanocytes respectively. Besides, (+)-UA and these derivatives were exposed to fix dosed of UVA or UVB. The anti-UVA/UVB activity was determined via Hoechst33342/propidium iodide double staining method, and quantified by computer linked fluorescence microscope equipped with CellsSense Dimension system. Based on analysis, Compound 2 to 5 captured prominent UVA/UVB protection capacity in both hepatocytes and melanocytes (p < .001). In addition, the effects of chemicals on tyrosinase were evaluated via Western Blot analysis. In terms of tyrosinase expression, only 2 showed significant stimulating effect (p < .05). However, the safe use of these derivatives cutaneously should be further studied. In conclusion, the photodegraded derivatives (2 to 5) of extracted UA have lower hepatotoxicity than (+)-UA and captured significant UV protection activities.

Simultaneous determination of polyphenols and triterpenes in pomegranate peel based on high-performance liquid chromatography fingerprint by solvent extraction and ratio blending method in tandem with wavelength switching.

Traditionally, pomegranate (Punica granatum L.) has been consumed as fresh fruit or as pomegranate juice. Pomegranate peel, the dried husk of P· granatum, is an important herbal medicine for treating diarrhea, hemostasis and insect-induced abdominal pain in China. However, the quality control methods for pomegranate peel remain unsatisfactory. In this work, a new HPLC-based qualitative and quantitative method for quality control of pomegranate peel was developed and validated for the simultaneous determination of polyphenols and triterpenes (including punicalagins A and B, ellagic acid, oleanolic acid and ursolic acid) by solvent extraction and ratio blending method in tandem with wavelength switching. The average recoveries were 98.07-100.61% with relative standard deviation no more than 4.27%. In addition, the fingerprint analysis was conducted to interpret the consistency of the quality test. Thirteen characteristic peaks were selected to evaluate the similarities of 16 batches of pomegranate peel. The similarities of samples were all more than 0.80, indicating that the samples from different areas of China were consistent. The results demonstrated that quantitative analysis and the HPLC fingerprint as a characteristic distinguishing method combining similarity evaluation can be successfully used to assess the quality and to identify the authenticity of pomegranate peel.

Mechanism-based pharmacokinetics-pharmacodynamics studies of harmine and harmaline on neurotransmitters regulatory effects in healthy rats: Challenge on monoamine oxidase and acetylcholinesterase inhibition.

BACKGROUND: ß-Carboline alkaloid harmine (HAR) and harmaline (HAL) are monoamine oxidase (MAO) and acetylcholinesterase (AChE) inhibitors. However, whether HAR and HAL inhibit MAO or AChE selectively and competitively is unclear. PURPOSE: The purpose of this study was to investigate the potential competition inhibition of HAR and HAL on MAO and AChE in brain endothelial cells (RBE4) and in healthy rats to provide a basis for the application of the inhibitors in the treatment of patients with depression and with Parkinson's disease or Alzheimer's disease. STUDY DESIGN/METHODS: The transport properties of HAR and HAL by using blood-brain barrier models constructed with RBE4 were systematically investigated. Then, the modulation effects of HAR and HAL on CNS neurotransmitters (NTs) in healthy rat brains were determined by a microdialysis method coupled with LC-MS/MS. The competition inhibition of HAR and HAL on MAO and AChE was evaluated through real time-PCR, Western blot analysis, and molecular docking experiments. RESULTS: Results showed that HAL and HAR can be detected in the blood and striatum 300 min after intravenous injection (1 mg/kg). Choline (Ch), gamma-aminobutyric acid (GABA), glutamate (Glu), and phenylalanine (Phe) levels in the striatum decreased in a time-dependent manner after the HAL treatment, with average velocities of 1.41, 0.73, 3.86, and 1.10 (ng/ml)/min, respectively. The Ch and GABA levels in the striatum decreased after the HAR treatment, with average velocities of 1.16 and 0.22 ng/ml/min, respectively. The results of the cocktail experiment using the human liver enzyme indicated that the IC50 value of HAL on MAO-A was 0.10 ± 0.08 µm and that of HAR was 0.38 ± 0.21 µm. Their IC50 values on AChE were not obtained. These findings indicated that HAL and HAR selectively acted on MAO in vitro. However, RT-PCR and Western blot analysis results showed that the AChE mRNA and protein expression decreased in a time-dependent manner in RBE4 cells after the HAR and HAL treatments. CONCLUSION: NT analysis results showed that HAL and HAR selectively affect AChE in vivo. HAL and HAR may be highly and suitably developed for the treatment of Alzheimer's disease.

Subchronic toxicity and concomitant toxicokinetics of long-term oral administration of total alkaloid extracts from seeds of Peganum harmala Linn: A 28-day study in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The seeds of Peganum harmala Linn, in which the most abundant active compounds are harmaline and harmine, have been widely used as a traditional medicine in various countries to treat a broad spectrum of diseases including asthma, cough, depression, Parkinson's and Alzheimer's diseases. However, few studies on long-term or subchronic toxicity of seeds of P. harmala were reported after overdose. AIM OF THE STUDY: To investigate the subchronic toxicity and concomitant toxicokinetics of total alkaloid extracts from seeds of P. harmala (TAEP) after oral administration for four weeks in rats. MATERIALS AND METHODS: The subchronic toxicity and concomitant toxicokinetics of TAEP were evaluated after 28-day oral administration in rats at daily dose levels of 15, 45, and 150 mg/kg. The signs of toxicity and mortality were monitored and recorded daily. The body weight and average food consumption were measured weekly. The analyses of hematology, biochemistry, urine, relative organ weights and histopathology were conducted at the termination of treatment and recovery phase. For concomitant toxicokinetics study, the plasma toxicokinetic parameters, tissue distribution, and excretion of predominant ingredients harmaline and harmine in TAEP and metabolites harmalol and harmol were tested. RESULTS: Following initial repeated exposure to high-dose (150 mg/kg/day) of TAEP excitotoxic reaction, such as tremor, was observed, but tolerated on the fourth day after multiple dosing. The significant alterations in blood glucose and lipid metabolism in liver were observed, but recovered after four weeks of drug withdrawal. The no-observed-adverse-effect level (NOAEL) of TAEP was considered to be 45 mg/kg/day under the present study conditions. There were no significant gender differences in most indexes of subchronic toxicity throughout the experimental period with the exception of food consumption and body weight. In concomitant toxicokinetics study, the alterations of dynamic characteristic for harmaline, harmine and metabolite harmol after multiple oral administration at three doses had been observed. Harmaline, harmine and metabolites harmalol and harmol were widely distributed in organs and there was no accumulation in the tissues examined. The reduction of harmaline and metabolite harmalol in brain after multiple dosing at dose of 150 mg/kg might be closely related to the tremor tolerance. The main excretory pathway for metabolites harmalol and harmol was urinary excretion via kidney. CONCLUSIONS: The results revealed that TAEP at doses of 15 and 45 mg/kg/day in rats might be safe. Excitotoxic reaction such as tremor occurred initially at dose of 150 mg/kg/day, however, the toxicity was tolerant and reversible. In addition, harmaline and harmine in TAEP had a quick absorption into blood and metabolized to harmalol and harmol, and there was no drug accumulation in the detected tissues. Further studies should be investigated to clarify the mechanisms of tremor tolerance and neurotoxicity of TAEP.

Establishment and application of a new HPLC qualitative and quantitative assay for Gentiana Macrophyllae Radix based on characteristic constituents of anofinic acid and its derivatives.

Gentiana Macrophylla Radix (GMR) is officially used as traditional Chinese medicine, but easily confused with Gentianae Radix et Rhizoma (GRR) and adulterants. This study aimed to establish an HPLC method for qualitative and quantitative analysis of GMR based on characteristic components, anofinic acid and its derivatives. HPLC analysis was performed on a C18 column with gradient elution using acetonitrile and 0.1% phosphoric acid as mobile phase, and detected at 240 nm by conventional methodology validation. For fingerprint analysis, RSDs of relative retention times and relative peak areas of the characteristic peaks were within 0-1.10 and 0-4.08%, respectively. For determination of 2-methoxyanofinic acid, the calibration curve showed good linearity (R2 > 0.9999) within the test range. The RSDs of precision, repeatability and stability test did not exceed 2.46, 0.83 and 1.11%, respectively. The average recoveries were between 95.08 and 103.05% with RSDs ≤2.29%. The results showed that there was no significant difference among the four species of GMR, but there were significant differences among GMR, GRR and spurious breeds by principal component analysis and hierarchical cluster analysis. Anofinic acid and its derivatives, as the characteristic markers, could be used for the identification and quality control of GMR.

Extracts of compound Muniziqi granule suppressed uterus contraction and ameliorated oxytocin-induced primary dysmenorrhea.

ETHNOPHARMACOLOGICAL RELEVANCE: Compound Muniziqi granule (CMG) is usually used as a traditional Uighur medicine to treat acne, chloasma, skin inflammation, primary dysmenorrhea (PDM), and menopausal syndrome. However, there are no sufficient data to support the clinic uses of CMG in PDM. AIM OF THE STUDY: This work aims to examine the effect of CMG as a treatment for PDM and reveal its possible therapeutic mechanism. MATERIALS AND METHODS: In vivo and in vitro mouse PDM models were utilized in this study. The mouse uterine contraction was induced by oxytocin after progynova or estradiol benzoate pretreatment. CMG, alkaloid extracts from seeds of Peganum harmala (AEP), and 10% and 95% ethanol extracts from seeds of Nigella glandulifera (EEN10 and EEN95) were given to mice in three doses by gavage. The writhing times within 30 min after oxytocin treatment were recorded to evaluate the analgesic effect, and the glutathione peroxidase (GSH-Px), malondialdehyde (MDA), 6-keto-prostaglandin F1α (6-k-PGF1α), prostaglandin F2α (PGF2α), thromboxane B2 (TXB2), and nitric oxide (NO) levels in uterine tissues and PGF2α and MDA in serum were determined. The effects (contractile curve) of CMG, AEP, EEN10, and EEN95 on uterus contraction induced by oxytocin in isolated mouse uterus were recorded. RESULTS: In contrast to the control group, CMG, AEP, N10, and N95 could display analgesic activities dose dependently by reducing the writhing response of the PDM model mice. CMG, AEP, EEN10, and EEN95 could also remarkably decrease the level of PGF2α, 6-k-PGF1α, TXB2, NO and MDA in uterine tissues and PGF2α and MDA in serum, whereas the activity of GSH-Px in uterine tissues was increased. Furthermore, CMG, AEP, EEN10, and EEN95 could significantly inhibit the frequency and amplitude of isolated uterus induced by oxytocin in a concentration-dependent manner. CONCLUSIONS: CMG exhibited a significant protective effect on experimental PDM. The mechanisms are probably associated with abating lipid peroxidation and over-inflammatory reaction, and alleviating the contraction of isolated mouse uterus. The seeds of P. harmala and N. glandulifera in the CMG may play an important role in exerting protective effects on PDM. This study provides pre-clinic proof to the use of CMG in clinical practice of PDM.

In in vivo evaluation of the anti-inflammatory and analgesic activities of compound Muniziqi granule in experimental animal models.

BACKGROUND: Compound Muniziqi granule (MNZQ), a traditional Uighur medicinal preparation, comprises 13 species of medicinal plants. MNZQ is traditionally used for regulating body immunity, modulating inflammation and pain, detoxification, and inhibiting tumor growth. This study aims to scientifically evaluate the anti-inflammatory and analgesic activities of MNZQ, support its clinical use and further research with scientific evidence. METHODS: The analgesic activity of MNZQ was evaluated using hot plate test and acetic acid-induced abdominal writhing test. Acute inflammation was evaluated using xylene-induced ear edema and carrageenan-induced paw edema models, while chronic inflammation was evaluated using cotton pellet-induced granuloma model. RESULTS: MNZQ exerted analgesic activities with a significant dose-dependent increase in latency in the hot plate test. The percentage inhibition suggested that MNZQ exhibited analgesic activities in the central nervous system. Meanwhile, MNZQ at 0.8, 2.4, and 7.2 g/kg strongly inhibited the acetic acid-induced writhing response by 25.22% (p < 0.01), 44.60% (p < 0.001), and 49.41% (p < 0.001), respectively. MNZQ also exerted analgesic activities in the peripheral nervous system. Moreover, MNZQ was demonstrated a significant anti-inflammatory effect against xylene-induced edema in a dose-dependent manner. The percentage inhibition was 22.24% (p < 0.01) at the highest dosage of 7.2 g/kg. MNZQ at 1.62 and 4.86 g/kg significantly reduced carrageenan-induced rat hind paw edema by 82.43% and 84.32% (p < 0.001), respectively, 1 h after injecting carrageenan, and the inhibitory effect lasted for 5 h. MNZQ also exerted a significant anti-inflammatory effect against cotton pellet-induced granuloma formation. MNZQ at 1.62 and 4.86 g/kg could inhibit granuloma formation by 17.07% and 17.60%, respectively, whereas the percentage inhibition of diclofenac was 33.12%. CONCLUSIONS: The results obtained suggest that MNZQ possesses potential anti-inflammatory and analgesic activities. This study provides a scientific basis for the use of MNZQ in alleviating pain and treating inflammatory disorders.