Microfluidic Diffusion Sizing Applied to the Study of Natural Products and Extracts That Modulate the SARS-CoV-2 Spike RBD/ACE2 Interaction.

The interaction between SARS-CoV-2 spike RBD and ACE2 proteins is a crucial step for host cell infection by the virus. Without it, the entire virion entrance mechanism is compromised. The aim of this study was to evaluate the capacity of various natural product classes, including flavonoids, anthraquinones, saponins, ivermectin, chloroquine, and erythromycin, to modulate this interaction. To accomplish this, we applied a recently developed a microfluidic diffusional sizing (MDS) technique that allows us to probe protein-protein interactions via measurements of the hydrodynamic radius (Rh) and dissociation constant (KD); the evolution of Rh is monitored in the presence of increasing concentrations of the partner protein (ACE2); and the KD is determined through a binding curve experimental design. In a second time, with the protein partners present in equimolar amounts, the Rh of the protein complex was measured in the presence of different natural products. Five of the nine natural products/extracts tested were found to modulate the formation of the protein complex. A methanol extract of Chenopodium quinoa Willd bitter seed husks (50 µg/mL; bisdesmoside saponins) and the flavonoid naringenin (1 µM) were particularly effective. This rapid selection of effective modulators will allow us to better understand agents that may prevent SARS-CoV-2 infection.

Targets and mechanisms of dietary anthocyanins to combat hyperglycemia and hyperuricemia: a comprehensive review.

Hyperglycemia and hyperuricemia are both metabolic disorders related to excessive amount of metabolites in blood, which are considered as high risk factors for the development of many chronic diseases. Enzymes, cells, tissues and organs, which are relevant to metabolism and excretion of glucose and UA, are usually regarded to be the targets in treatment of hyperglycemia and hyperuricemia. Several drugs have been commonly applied to combat hyperglycemia and hyperuricemia through various targets but with unignorable side effects. Anthocyanins have become promising alternatives against hyperglycemia and hyperuricemia because of their bio-activities with little side effects. Structurally different anthocyanins from berry fruits, cherries and purple sweet potato lead to the diverse functional activity and property. This review is aimed to illustrate the specific targets that are available for anthocyanins from berry fruits, cherries and purple sweet potato in hyperglycemia and hyperuricemia management, as well as discuss the structure-activity relationship, and the underlying mechanisms associated with intracellular signaling pathway, anti-oxidative stress and anti-inflammation. In addition, the relationship of hyperglycemia and hyperuricemia, and the possibly regulative role of anthocyanins against them, along with the effects of anthocyanins in clinical trial are mentioned.

Effect of ultrasonic and ball-milling treatment on cell wall, nutrients, and antioxidant capacity of rose (Rosa rugosa) bee pollen, and identification of bioactive components.

BACKGROUND: Bee pollen has been regarded as a complete nutritional human dietary supplement but its nutrient absorption and biological effects may be restricted by the complex pollen wall. The aim of this study was to explore the effects of ultrasonic and ball-milling treatment on the release of nutritional components and on in vitro and in vivo antioxidant effects of rose (Rosa rugosa) bee pollen. RESULTS: Bee pollen walls were broken to varying degrees, nutrients were released, and in vitro and in vivo antioxidant effects of bee pollen were improved. The scavenging effects of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzothiazolone-6-sulfonic acid) (ABTS) radicals, and oxygen radical absorbance capacity (ORAC) were improved. In aging mice, wall-breaking treatment led to better organ recovery, enhanced superoxide dismutase (SOD) and catalase (CAT) effects, and malondialdehyde (MDA) reduction. Eight compounds of rose bee pollen ethanol extract, including isorhamnetin 3-O-diglucoside and N', N″, N‴-dicaffeoyl p-coumaroyl spermidine were identified by ultra-performance liquid chromatography electrospray ionization quadrupole time of flight mass spectrometry (UPLC-ESI-QTOF-MS/MS) assay. CONCLUSION: This study showed that ultrasonic treatment had greater wall-disruption effects of bee pollen on nutrient release and antioxidant effect promotion. In conclusion, rose bee pollen, with wall-breaking treatments, may have potential value as an ingredient in functional food processing. © 2019 Society of Chemical Industry.

Antioxidant activity against H2O2-induced cytotoxicity of the ethanol extract and compounds from Pyrola decorate leaves.

CONTEXT: The leaves of Pyrola decorate H. Andr (Pyrolaceae), known as Luxiancao, have long been used for treating kidney deficiency, gastric haemorrhage and rheumatic arthritic diseases in traditional Chinese medicine. OBJECTIVE: The phytochemicals and antioxidant capacities in vitro of P. decorate leaves were investigated. MATERIALS AND METHODS: Ethanol, petroleum ether, acetidin, n-butyl alcohol and aqueous extracts of Pyrola decorate leaves were prepared by solvent sequential process, and then isolated and purified to obtain phytochemicals. Cell viability was measured by MTT assay. PC12 cells were pretreated for 24 h with different extractions of P. decorate leaves at concentrations of 0.1, 0.5, 1, 5 and 10 mg/mL, then H2O2 of 0.4 mM was added in all samples for an additional 2 h. The antioxidant capacities of betulin, ursolic acid and monotropein were determined in PC12 cells against H2O2 induced cytotoxicity in vitro as well. RESULTS: Nine compounds (1-9) were isolated and structurally determined by spectroscopic methods, especially 2D NMR analyses. Ethanol extract treated groups showed inhibitory activity with IC50 value of 10.83 mg/mL. Betulin, ursolic acid and monotropein were isolated from P. decorate, and demonstrated with IC50 values of 6.88, 6.15 and 6.13 µg/mL, respectively. DISCUSSION AND CONCLUSIONS: In conclusion, Pyrola decorate is a potential antioxidative natural plant and worth testing for further pharmacological investigation in the treatment of oxidative stress related neurological disease.

Mechanisms for the biological activity of Gastrodia elata Blume and its constituents: A comprehensive review on sedative-hypnotic, and antidepressant properties.

BACKGROUND: Insomnia and depressive disorder are two common symptoms with a reciprocal causal relationship in clinical practice, which are usually manifested in comorbid form. Several medications have been widely used in the treatment of insomnia and depression, but most of these drugs show non-negligible side effects. Currently, many treatments are indicated for insomnia and depressive symptom, including Chinese herbal medicine such as Gastrodia elata Blume (G. elata), which has excellent sedative-hypnotic and antidepressant effects in clinical and animal studies. PURPOSE: To summarize the mechanisms of insomnia and depression and the structure-activity mechanism for G. elata to alleviate these symptoms, particularly by hypothalamic-pituitary-adrenal (HPA) axis and intestinal flora, aiming to discover new approaches for the treatment of insomnia and depression. METHODS: The following electronic databases were searched from the beginning to November 2023: PubMed, Web of Science, Google Scholar, Wanfang Database, and CNKI. The following keywords of G. elata were used truncated with other relevant topic terms, such as depression, insomnia, antidepressant, sedative-hypnotic, neuroprotection, application, safety, and toxicity. RESULTS: Natural compounds derived from G. elata could alleviate insomnia and depressive disorder, which is involved in monoamine neurotransmitters, inflammatory response, oxidative stress, and gut microbes, etc. Several clinical trials showed that G. elata-derived natural compounds that treat depression and insomnia have significant and safe therapeutic effects, but further well-designed clinical and toxicological studies are needed. CONCLUSION: G. elata exerts a critical role in treating depression and insomnia due to its multi-targeting properties and fewer side effects. However, more clinical and toxicological studies should be performed to further explore the sedative-hypnotic and antidepressant mechanisms of G. elata and provide more evidence and recommendations for its clinical application. Our review provides an overview of G. elata treating insomnia with depression for future research direction.

Diacylated anthocyanins from purple sweet potato (Ipomoeabatatas L.) attenuate hyperglycemia and hyperuricemia in mice induced by a high-fructose/high-fat diet. / ç´«è–¯åŒé °åŸºèŠ±è‰²è‹·å¯¹é«˜æžœç³–/é«˜è„‚è‚ªé¥®é£Ÿè¯±å¯¼çš„å°é¼ é«˜è¡€ç³–å’Œé«˜å°¿é ¸è¡€ç—‡çš„è°ƒèŠ‚ä½œç”¨.

Studies have shown that targeting xanthine oxidase (XO) can be a feasible treatment for fructose-induced hyperuricemia and hyperglycemia. This study aimed to evaluate the dual regulatory effects and molecular mechanisms of diacylated anthocyanins from purple sweet potato (diacylated AF-PSPs) on hyperglycemia and hyperuricemia induced by a high-fructose/high-fat diet. The body weight, organ index, serum biochemical indexes, and liver antioxidant indexes of mice were measured, and the kidneys were observed in pathological sections. The relative expression levels of messenger RNAs (mRNAs) of fructose metabolism pathway enzymes in kidney were detected by fluorescent real-time quantitative polymerase chain (qPCR) reaction technique, and the expression of renal transporter protein and inflammatory factor pathway protein was determined by immunohistochemistry (IHC) technique. Results showed that diacylated AF-PSPs alleviated hyperuricemia in mice, and that this effect might be related to the regulation of liver XO activity, lipid accumulation, and relevant renal transporters. Diacylated AF-PSPs reduced body weight and relieved lipid metabolism disorder, liver lipid accumulation, and liver oxidative stress, thereby enhancing insulin utilization and sensitivity, lowering blood sugar, and reducing hyperglycemia in mice. Also, diacylated AF-PSPs restored mRNA levels related to renal fructose metabolism, and reduced kidney injury and inflammation. This study provided experimental evidence for the mechanisms of dual regulation of blood glucose and uric acid (UA) by diacylated AF-PSPs and their utilization as functional foods in the management of metabolic syndrome.

Effective Therapeutic Verification of Crocin I, Geniposide, and Gardenia (Gardenia jasminoides Ellis) on Type 2 Diabetes Mellitus In Vivo and In Vitro.

For many centuries, Gardenia (Gardenia jasminoides Ellis) was highly valued as a food homologous Chinese herbal medicine with various bioactive compounds, including crocin I and geniposide. However, the functional mechanism underlying the hypoglycemic effect of gardenia is absent in the literature. To evaluate the effect of gardenia and its different extracts on type 2 diabetes mellitus (T2DM) in in vivo and in vitro experiments, the dried gardenia powder was extracted using 60% ethanol and eluted at different ethanol concentrations to obtain the corresponding purified fragments. After that, the active chemical compositions of the different purified gardenia fragments were analyzed using HPLC. Then, the hypoglycemic effects of the different purified gardenia fragments were compared using in vitro and in vivo experiments. Finally, the different extracts were characterized using UPLC-ESI-QTOF-MS/MS and the mass spectrometric fragmentation pathway of the two main compounds, geniposide and crocin I, were identified. The experimental results indicated that the inhibitory effect of the 40% EGJ (crocin I) on the α-glucosidase was better than the 20% EGJ (geniposide) in vitro. However, the inhibitory effect of geniposide on T2DM was better than crocin I in the animal experiments. The different results in vivo and in vitro presumed potentially different mechanisms between crocin I and geniposide on T2DM. This research demonstrated that the mechanism of hypoglycemia in vivo from geniposide is not only one target of the α-glucosidase but provides the experimental background for crocin I and the geniposide deep processing and utilization.

Male-Biased Gut Microbiome and Metabolites Aggravate Colorectal Cancer Development.

Men demonstrate higher incidence and mortality rates of colorectal cancer (CRC) than women. This study aims to explain the potential causes of such sexual dimorphism in CRC from the perspective of sex-biased gut microbiota and metabolites. The results show that sexual dimorphism in colorectal tumorigenesis is observed in both ApcMin/ + mice and azoxymethane (AOM)/dextran sulfate sodium (DSS)-treated mice with male mice have significantly larger and more tumors, accompanied by more impaired gut barrier function. Moreover, pseudo-germ mice receiving fecal samples from male mice or patients show more severe intestinal barrier damage and higher level of inflammation. A significant change in gut microbiota composition is found with increased pathogenic bacteria Akkermansia muciniphila and deplets probiotic Parabacteroides goldsteinii in both male mice and pseudo-germ mice receiving fecal sample from male mice. Sex-biased gut metabolites in pseudo-germ mice receiving fecal sample from CRC patients or CRC mice contribute to sex dimorphism in CRC tumorigenesis through glycerophospholipids metabolism pathway. Sexual dimorphism in tumorigenesis of CRC mouse models. In conclusion, the sex-biased gut microbiome and metabolites contribute to sexual dimorphism in CRC. Modulating sex-biased gut microbiota and metabolites could be a potential sex-targeting therapeutic strategy of CRC.

The Effects of Velvet Antler of Deer on Cardiac Functions of Rats with Heart Failure following Myocardial Infarction.

Velvet antler of deer (VAD) is a commonly-used kidney-Yang supplementing traditional Chinese medication. According to the heart-kidney-related theory, heart Yang originates in kidney Yang and heart failure due to heart Yang deficiency can be treated by tonifying kidney Yang. In this study, we investigated therapeutic effects of VAD on cardiac functions in rats with heart failure following myocardial infarction. Forty-eight male Wistar rats were subjected either to left coronary artery ligation (N = 36) or to sham operation (N = 12). One week after the surgery, rats with heart failure received daily treatment of double-distilled water, captopril or VAD by gavage for consecutively four weeks, while sham-operated animals were given double-distilled water. Ultrasonic echocardiography was adopted to examine cardiac structural and functional parameters and serum brain natriuretic peptide (BNP) concentration was measured using radioimmunoassay. We found that VAD partially reversed changes in cardiac functional parameters and serum BNP levels in rats with heart failure. These results provide further evidence for the heart-kidney-related theory and suggest that VAD might be a potentially alternative and complementary medicine for the treatment of heart failure.

[Analyzing the Chinese medicine pathogenesis of stroke].

Both ischemic and hemorrhage stroke pertain to the category of wind stroke in Chinese medicine (CM). Up to date, it is deemed that the etiology and pathogenesis of wind stroke are wind, fire, sputum, qi, stasis, and deficiency. Among them, it is regarded that wind and fire are the key factors triggering wind stroke. By analyzing the time order and causality, it is found that wind stroke is prior to the onset of wind and fire, wind and fire are the secondary outcomes of wind stroke. By parallel comparing stroke with thromboembolism and hemorrhagic diseases in other Zang-organs, it can be comprehended that the reason why wind symptoms appear in stroke is due to its physiological feature of brain itself. Based on Neijing, the pathogenesis of wind stroke is proposed as follows. Tunnels of viscera (vessels) get lesions. The old pathogenic factors of sputum and stasis or the stasis formed by bleeding inside viscera consume qi, and blood of viscera and damage the spirits hidden in them. The damage of Gan-spirit causes symptoms of stroke, such as hemiplegia, deviation of eyes and mouth, and so on. Wind and fire symptoms are caused by the injury of Gan blood and yin, and/or the stagnation of fire in pericardium (the pathway organ) due to obstruction by old pathogenic factors and stasis (formed by bleeding).

Triterpenoid acids from medicinal mushroom Inonotus obliquus (Chaga) alleviate hyperuricemia and inflammation in hyperuricemic mice: Possible inhibitory effects on xanthine oxidase activity.

The purpose of this study was to explore the hypouricemic effect in hyperuricemia mice of triterpenoid acids from Inonotus obliquus (TAIO), and decipher of the underlying xanthine oxidase inhibitory mechanism. Measurement of xanthine oxidase (XO) inhibitory activity was assayed. Organ indexes and serum biochemical indicators were measured in potassium oxonate-induced hyperuricemia mice. Studies showed that TAIO had the strong inhibitory effect on XO activity, and its inhibition type was mixed and reversible. In vivo, TAIO decreased efficiently uric acid level, hepatic XO, serum blood urea nitrogen activities in hyperuricemia mice. Indicating that TAIO may ameliorate kidney damage and relieve inflammation in hyperuricemic mice, and had the inhibitory effect on XO activity. Furthermore, eight triterpenoids were identified by Ultra performance liquid chromatography electrospray quadrupole time of flight mass spectrometry. These findings proved that triterpenoids from Inonotus obliquus would have potential biological characteristics and effect on controlling hyperuricemia and gout as an active supplement. PRACTICAL APPLICATIONS: There are a large amount of evidence indicating that hyperuricemia and gout are related to the hypertension and obesity. And gout and hyperuricemia are also possible connection with cardiovascular disease and metabolic syndrome. Currently, xanthine oxidase is the target of many kinds of chemical drugs at present, but the therapeutic drugs used in clinical medicine will produce more or less side effects. Therefore, the aim of this study was to explore the material basis of effective substances for reducing uric acid in Inonotus obliquus and to evaluate its effect. This study can provide a promising application of Inonotus obliquus in the fields of functional foods or medicines for gout and hyperuricemia.

An Optimized Two-Herb Chinese Food as Medicine Formula Reduces Cisplatin-Induced Nephrotoxicity in the Treatment of Lung Cancer in Mice.

Chemotherapy is considered a most effective way to treat cancer. However, it is very common that chemotherapy causes unbearable mental and physical side effects to cancer patients, which ultimately reduces the patients' confidence of overcoming diseases and compromises the treatment of chemotherapy. Cisplatin (DDP), a widely used anticancer agent for various types of cancers, also damages nontumor cells and tissues, which are mostly related to the activation of the inflammation pathway. Previously, we have discovered a few rational formulas of food as medicine materials that reduced systemic inflammation in in vitro and in vivo models. Hence, this study reports the ability of an optimized traditional Chinese anti-inflammatory formulation capable of synergizing the antitumor effect of DDP in vitro and in vivo. More significantly, by formulation of two anti-inflammatory herbal medicine, the Chrysanthemum × morifolium (Ramat.) Hemsl [Asteraceae] and Lonicera japonica Thunb [Caprifoliaceae] with a mediator Glycyrrhiza uralensis Fisch. ex DC [Fabaceae], a best formula relieved the kidney damage imposed by DDP. Treatments of various combinations of major chemical components of the three herbs also exhibited a similar trend for lowering the DDP-induced nephrotoxicity; however, contrary to that of the formula of herbal extracts, all chemical formulas could not recover the body weight and food intake of the tumor-bearing mice treated by DDP. Our findings suggested that the therapeutic index of DDP-based chemotherapy was able to be improved by minimizing toxicities from the two-herb formula to inhibit the inflammation in mouse tumor models and DDP-induced acute kidney injury mouse models.

Malignant potential of H22 hepatocarcinoma cells increases after recovery from IFN-γ-mediated inhibition.

IFN-γ (interferon γ) can effectively suppress tumours, but it has also been found to promote tumour progression. However, the underlying mechanisms by which it enhances malignancy have not been fully elucidated. By using a mouse model that expresses IFN-γ locally in muscle, we found that the growth potential of tumours was increased after a quick decrease of IFN-γ. Furthermore, the up-regulation of IRF-2 (IFN regulatory factor 2) and down-regulation of IRF-1 were also found in the tumour cells. Along these lines, IFN-γ led to down-regulated expression of cyclin-D1, Bcl-2 and Bcl-xL and up-regulated expression of p21WAF1 and Bax in tumour cells. Yet, the expression of these genes, as well as activation of ERK (extracellular signal-regulated kinase) and NF-κB (nuclear factor-κB), was also reversed shortly after a decrease in IFN-γ, all of which resulted in increase tumour cell proliferation and apoptosis resistance. These findings indicate that the malignant potential of tumour cells may be suppressed by interfering with IRF-2 signalling pathways during and after decreased IFN-γ in tumour microenvironments.

Inhibition mechanism of diacylated anthocyanins from purple sweet potato (Ipomoea batatas L.) against α-amylase and α-glucosidase.

This study aimed to evaluate inhibitory activity of anthocyanins from purple sweet potato and blueberries against α-amylase and α-glucosidase, as well as investigate the inhibition mechanism of diacylated anthocyanins (Diacylated AF-PSP). Diacylated AF-PSP better inhibited α-amylase (IC50 = 0.078 mg mL-1) and α-glucosidase (IC50 = 1.56 mg mL-1) than other anthocyanin fractions, which was a mixed-type inhibitor. Fluorescence analysis indicated that Diacylated AF-PSP bound to the enzymes mainly through hydrogen bonds and influenced the microenvironments of proteins. Additionally, surface hydrophobicity and circular dichroism spectra results confirmed conformational changes in the enzymes induced by Diacylated AF-PSP. Molecular docking further demonstrated the interaction of Diacylated AF-PSP with enzyme active site, which might be stabilized by its acyl groups. Finally, 160 mg kg-1 Diacylated AF-PSP significantly decreased (p < 0.01) blood glucose level peak by 20.52% after starch administration in SD rats. This study provided theoretical evidences for utilization of diacylated anthocyanins in hyperglycemia-management functional foods.

Enzymolysis peptides from Mauremys mutica plastron improve the disorder of neurotransmitter system and facilitate sleep-promoting in the PCPA-induced insomnia mice.

ETHNOPHARMACOLOGY RELEVANCY: For many centuries, Mauremys mutica is highly valued as a food homologous Chinese herbal medicine. It has been considered useful to sedate, nourish brain and promote sleep. However, the animal experimental evidence of its sleep-promoting activity is missing in literature. AIM OF THE STUDY: In this study, PCPA-induced insomnia model was used to explore the sleep-promoting mechanism of enzymolysis peptides from PMM, and its main composition and chemical structure were analyzed. MATERIALS AND METHODS: Experiments were performed using PCPA-induced insomnia model, all animals were intraperitoneally injected with PCPA (350 mg/kg·d) for two days. The sleep-promoting effect evaluated using measuring content of 5-HT, GABA, DA, IL-1, BDNF and expression of 5-HT1A receptor and GABAA receptor α1-subunit in mice brain. Primary structure of peptides was identified by HPLC-ESI-QqTOF-MS/MS. RESULTS: Compared with the model group, the content of 5-HT, GABA, IL-1, BDNF in mice brain of PMM peptide groups was increased to varying degrees, the content of DA was decreased, and the gene transcription and protein expression of 5-HT1A receptor and GABAA receptor α1-subunit were almost all returned to normal levels. In addition, the primary structures of most abundant nine typical peptides in PMM peptides were identified. CONCLUSIONS: The results showed that PMM peptides could improve the disorder of neurotransmitter system, restore compensatory over-expression 5-HT1A receptor and GABAA receptor α1-subunit, and have a good sleep-promoting effect. The specific amino acid composition, sequence and glycosylation modification of PMM peptides may be the key reason for their activity, which lays a foundation for the subsequent development of sleep-promoting peptide products.

Addition of Vegetable Oil to Improve Triterpenoids Production in Liquid Fermentation of Medicinal Fungus Antrodia cinnamomea.

The liquid fermentation of Antrodia cinnamomea is a promising alternative source for fungus production compared to the wildly grown fruiting body. Elicitation is a strong tool to enhance the productivity in microbial cells to obtain more compounds of interest. In this study, in order to improve the fungus growth and its terpenoids production, various vegetable oils were added in the fermentation broth of A. cinnamomea. It was found that corn oil from a group of vegetable oils exhibited the best effect on the biomass and triterpenoid content. After optimization, the initial addition of 1% (v/v) corn oil plus the inoculation of 10% (v/v) mycelia led to a maximum triterpenoid yield (532.3 mg L-1), which was increased as much as fourfold compared to the blank control. Differential transcriptome analysis demonstrated that corn oil significantly enriched several metabolic pathways including glycolysis/gluconeogenesis, propanoate metabolism and transmembrane hydrophobins. The enriched pathways interacted with deferentially expressed genes (DEGs) induced by corn oil treatment. Our research provides a potential strategy for the large production of triterpenoids by the improved fermentation of A. cinnamomea.

Deciphering the Formulation Secret Underlying Chinese Huo-Clearing Herbal Drink.

Herbal teas or herbal drinks are traditional beverages that are prevalent in many cultures around the world. In Traditional Chinese Medicine, an herbal drink infused with different types of medicinal plants is believed to reduce the 'Shang Huo', or excessive body heat, a status of sub-optimal health. Although it is widely accepted and has a very large market, the underlying science for herbal drinks remains elusive. By studying a group of herbs for drinks, including 'Gan' (Glycyrrhiza uralensis Fisch. Ex DC.), 'Ju' (Dendranthema morifolium (Ramat.) Tzvelev), 'Bu' (Microcos paniculata L.), 'Jin' (Lonicera japonica Thunb.), 'Xia' (Prunella vulgaris L.), and 'Ji' (Plumeria rubra L.), the long-term jargon is connected with the inflammation of modern immunology through a few pro-inflammatory markers. In vitro studies have indicated that cellular inflammation is lowered by Ju and Jin either individually or synergistically with Gan. Among all herbs, only Gan detoxicated cellular toxicity of Bu in a dose dependent manner. The synergistic formulation of Ju and Gan, or Jin and Gan, in a reduction of Shang Huo, was tested in vivo. Both combinations exhibited a lower percentage of neutrophils, monocytes, and CD4+/CD8+ ratio in the blood, as well as inflammatory cytokines. Furthermore, body weight in the combinatory groups was more stable than treatments using single herbs. The combination of old traditional oriental methods with Western science logistics, has resulted in the formulation of different herbs into one concoction for the use of detoxification and synergism.

Structural analysis and antitussive evaluation of five novel esters of verticinone and bile acids.

Shedan-Chuanbei powder, a complex of traditional Chinese medicine preparation, which consists of Snake Bile (Chinese name "Shedan") and Fritillariae Cirrhosae (Chinese name "Chuanbei"), is the most popular antitussive and expectorant formulation in Chinese communities. However, the clinical application of Shedan-Chuanbei powder is now stringently limited because of the shortage of the two crude medicinal materials, especially for the sake of animal protection. In addition, the inherent defects of the most of the complex of traditional Chinese medicine such as the indistinct basal pharmacodynamic materials and the difficulties in quality control had blocked them heading into the international medicinal market. So we attempted to seek new substitute for Shedan-Chuanbei powder for antitussive drugs. In order to gain some new compounds with better bioactivity and attenuated toxicity, we tried to combine two kinds of drugs through ester bond. Enlightened with "combination principle" in drug discovery, we synthesized five novel esters of verticinone and bile acids, both of which are the major bioactive components in Shedan-Chuanbei powder. We then evaluated the antitussive activity and the acute toxicity of the five ester-linked compounds. The five ester-linked compounds had much more potent antitussive activity and expectorant activity than single bile acids at the same doses, and had equivalent antitussive activity and expectorant activity in comparison with about double moles dose of the monomer verticinone. Especially, cholic acid-verticinone ester had much more potent antitussive effects than the monomer verticinone or cholic acid at the same dose. A further acute toxicity study showed that the LD(50) values of the five ester-linked compounds exceeded 3.5g/kg by intraperitoneal injection in mice. Based on the studies of pharmacology and acute toxicity, the five ester-linked compounds have synergic pharmacodynamic action and attenuated toxicity compared with single verticinone and single bile acids.

Addictive evaluation of cholic acid-verticinone ester, a potential cough therapeutic agent with agonist action of opioid receptor.

AIM: The purpose of this work was to search for potential drugs with potent antitussive and expectorant activities as well as a low toxicity, but without addictive properties. Cholic acid-verticinone ester (CA-Ver) was synthesized based on the clearly elucidated antitussive and expectorant activities of verticinone in bulbs of Fritillaria and different bile acids in Snake Bile. In our previous study, CA-Ver showed a much more potent activity than codeine phosphate. This study was carried out to investigate the central antitussive mechanism and the addictive evaluation of CA-Ver. METHODS: Testing on a capsaicin-induced cough model of mice pretreated with naloxone, a non-selective opioid receptor antagonist, was performed for the observation of CA-Ver's central antitussive mechanism. We then took naloxone-induced withdrawal tests of mice for the judgment of CA-Ver's addiction. Lastly, we determined the opioid dependence of CA-Ver in the guinea pig ileum. RESULTS: The test on the capsaicin-induced cough model showed that naloxone could block the antitussive effect of CA-Ver, suggesting the antitussive mechanism of CA-Ver was related to the central opioid receptors. The naloxone-urged withdrawal tests of the mice showed that CA-Ver was not addictive, and the test of the opioid dependence in the guinea pig ileum showed that CA-Ver had no withdrawal response. CONCLUSION: These findings suggested that CA-Ver deserved attention for its potent antitussive effects related to the central opioid receptors, but without addiction, and had a good development perspective.

Intervention on immunodeficiency mice and structural identification of enzymatic peptides from Mauremys mutica and Cuora trifasciata.

ETHNOPHARMACOLOGY RELEVANCY: Mauremys mutica (Asian yellow pond turtle, YPT) and Cuora trifasciata (Chinese three-striped box turtle, TBT) are traditional Chinese medicine. They possess many biological characteristics, such as immune-enhancement, anti-inflammatory, anti-cancer effects. They have been used as folk anti-cancer drugs in central and southern China for a long time. However, there was no reports of comparing the immune-enhancement effect of YPT and TBT, nor of identifying the structures of YPT peptides and TBT peptides. AIMS OF THE STUDY: The aim of this study was to evaluate the protective efficacy of YPT and TBT on immunodeficient mice and to compare the primary structures of YPT peptides and TBT peptides. MATERIALS AND METHODS: The protein extracts were extracted using 100 °C water, and peptides were obtained by hydrolyzing protein extracts using alkaline protease. Cyclophosphamide (CTX) was used to induce immunodeficiency in mice. The immune enhancement effect was evaluated by measuring body weight gain curve, thymus index, spleen index, serum SOD activity and GSH-Px activity. Primary structure of peptides was identified by HPLC-ESI-MS/MS. RESULTS: The protein extracts and peptides of the YPT and TBT had certain recovery effects on immunodeficient mice. YPT peptide has the best effect on the recovery of damaged immune organs and the improvement of SOD and GSH-Px activities in mice. In the identification of the primary structure of the polypeptide, we find that YPT and TBT contain some similar peptides as well as different peptides, and the concentration of the peptide segments in HPLC data is very different. The difference of biological activity may be determined by both the difference of specific peptide structure and concentration. CONCLUSIONS: Two kinds of healthy turtle protein extracts and peptides could have immune-enhancement function, and peptides obtained by enzymatic hydrolysis of YPT protein extracts have the best immune-enhancement effect. The identification of the primary structure of the peptide segment preliminarily showed that its biological activity was affected by the amino acid sequence and the concentration of part of the peptide segment. It laid a foundation for the follow-up search of immune-enhancement peptides and the development of high-value YPT products.