High-resolution bacterial growth inhibition profiling combined with HPLC–HRMS–SPE–NMR for identification of antibacterial constituents in Chinese plants used to treat snakebites

Ethnopharmacogical relevance

Bacterial infection is one of the main secondary infections caused by snakebite. The 88 plant species investigated in this study have been used as folk remedies for treatment of snakebite, and it is therefore the aim of this study to investigate whether the plants contain compounds with bacterial growth inhibition.

Materials and methods

The water and ethanol extracts of 88 plant species were screened at 200 μg/mL against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa for their antibacterial activity by micro-broth dilution assay. The most active extracts were fractionated into microplates using analytical-scale RP-HPLC, and subsequently growth inhibition was assessed for each well. The biochromatograms constructed from these assays were used to identify compounds responsible for antibacterial activity. The structures of five compounds were elucidated by HPLC–HRMS–SPE–NMR.

Results

Crude extracts of Boehmeria nivea, Colocasia esculenta, Fagopyrum cymosum, Glochidion puberum, Melastoma dodecandrum, Polygonum bistorta, Polygonum cuspidatum and Sanguisorba officinalis showed MIC values below 200 μg/mL against either Bacillus subtilis, Staphylococcus aureus, Escherichia coli or Pseudomonas aeruginosa. The biochromatograms demonstrated that tannins play a main role for the bacterial growth inhibition observed for all above-mentioned plants except for Polygonum cuspidatum. Furthermore, the high-resolution bacterial growth inhibition profiling combined with HPLC–HRMS–SPE–NMR allowed fast identification of three non-tannin active compounds, i.e., piceid, resveratrol and emodin from ethanol extract of Polygonum cuspidatum.

Conclusion

The high-resolution bacterial growth inhibition profiling allowed fast pinpointing of constituents responsible for the bioactivity, e.g., either showing tannins being the main bacterial growth inhibitors as observed for the majority of the active plants, or combined with HPLC–HRMS–SPE–NMR for fast structural identification of non-tannin constituents correlated with antibacterial activity.     

In vitro screening on β-amyloid peptide production of plants used in traditional medicine for cognitive disorders

Aim of the study

The aim of the study was to investigate the activity on β-amyloid peptide production of crude extracts of 9 plant species traditionally used in Benin or in Madagascar for the treatment of cognitive disorders, in order to select candidates for Alzheimer's disease treatment.

Materials and methods

For each species, hexane, dichloromethane, ethyl-acetate and water extracts were tested, at non-toxic concentrations, on CHO cells overexpressing the human neuronal β-amyloid peptide precursor (APP695) to measure variations of APP processing (by Western-blotting) and, for the most active, of Aβ-amyloid production (by ECLIA).

Results

We observed, at non-toxic concentrations, a significant increase in CTF/APP ratio with Oldenlandia affinis cyclotide-enriched fraction, Prosopis africana EtOAc extract, Pterocarpus erinaceus aqueous extract and Trichilia emetica hexane extract. We also showed that the Pterocarpus erinaceus extract significantly decreased Aβ production, displaying effects similar to those of DAPT (γ-secretase inhibitor) on APP processing, but may act on another inhibition site.

Conclusion

These active extracts are worth further studies to isolate the compounds responsible for the observed activities, to analyze their mode of action and determine their clinical potentials.     

Effect of acupuncture－like stimulation on cortical cerebral blood flow in anesthetized rats

The effect of acupuncture-like stimulation of the forepaw on cortical cerebral blood flow (CBF) was examined using a laser Doppler flowmeter in anesthetized rats. One or two acupuncture needles were inserted into the skin and underlying muscles at a depth of about 5 mm. One needle was manually twisted to the right and left once a second for 1 min and two needles were used to pass an electrical current. Stimulation of the forepaw produced increases in CBF and mean arterial pressure (MAP) in spinal cord-intact rats After spinal transection at the first to second thoracic level, the blood pressure response to manual or electrical stimulation of the forepaw was suppressed, whereas an increase in CBF still occurred. The increase in CBF induced by forepaw stimulation was abolished by severing the somatic nerves at the brachial plexus. Forepaw stimulation enhanced the activity of the radial, ulnar, and median nerves.An increase in CBF was elicited by excitation of group II (At3) and group 1V (C) somatic afferent fibers. The CBF responese was almost abolished by the i.v. administration of muscarinic and nicotinic cholinergic blocking agents (atropine 5 mg/kg and mecamylamine 20 mg/kg), and by bilateral lesions in the nucleus basalis of Meynert. Acupuncture-like stimulation of a forepaw increased acctylcholine release in the cerebral cortex. We concluded that the increase in CBF, independent of systemic blood pressure, elicited by acupuncture-like stimulation is a reflex response in which the afferent nerve pathway is composed of somatic groups Ⅱand Ⅳ afferent nerves,and the efferent nerve pathway includes intrinsic cholinergic vasodilators in the nucleus basalis of Meynert.     

Protective effect of Danhong injection on cerebral ischemia–reperfusion injury in rats

Ethnopharmacological relevance

Danhong injection (DH), a Chinese medical product, is used extensively for the treatment of cerebrovascular diseases such as acutely cerebral infarction in clinic.

Aim of the study

To explore the protective effect and the relevant mechanisms of DH on cerebral ischemia–reperfusion (I/R) injury.

Materials and methods

Cerebral I/R injury was induced through four-vessel occlusion (4-VO) or middle cerebral artery occlusion (MCAO). Adult male SD rats were randomly divided into six kinds of groups: normal control group, sham-operated group, I/R injury group, DH-treated groups at doses of 0.5 ml/kg, 1.0 ml/kg and 2.0 ml/kg. The effects of DH on murine neurological deficits and cerebral infarct volume, 6-keto-prostagladin F_1α (6-keto-PGF_1α) level, malondialdehyde (MDA) level and superoxide dismutase (SOD) activity in brain tissue, as well as the activities of plasma tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) after I/R were evaluated. Moreover, the expressions of Bcl-2 and Bax protein were detected by immunohistochemistry.

Results

There was no significant difference between the control group and the sham-operated group based on the measurement indicators. Compared with the vehicle-treated group, rats treated with DH showed dose dependent reductions in brain infarction size, and improvement of neurological outcome. The level of 6-keto-PGF_1α and the activities of SOD and plasma t-PA were enhanced significantly, whereas the level of MDA and the activity of plasma PAI were declined significantly. The immunohistochemical staining results also revealed that the expression of Bcl-2 protein was up-regulated and that of Bax protein was down-regulated when exposed to DH.

Conclusion

DH demonstrates a strong ameliorative effect on cerebral I/R damage in rats by its anticoagulant, antithrombotic, antifibrinolytic and antioxidant activities. Furthermore, suppressing apoptosis through regulating Bcl-2 and Bax protein expressions should be another potential mechanism by which DH exerts its neuroprotective function.     

Antibacterial screening of some Peruvian medicinal plants used in Callería District

Nine ethanol extracts of Brunfelsia grandiflora (Solanaceae), Caesalpinia spinosa (Caesalpiniaceae), Dracontium loretense (Araceae), Equisetum giganteum (Equisetaceae), Maytenus macrocarpa (Celastraceae), Phyllanthus amarus (Euphorbiaceae), Piper aduncum (Piperaceae), Terminalia catappa (Combretaceae), and Uncaria tomentosa (Rubiaceae), medicinal plants traditionally used in Calleria District for treating conditions likely to be associated with microorganisms, were screened for antimicrobial activity against nine bacterial strains using the broth microdilution method. Among the plants tested, Phyllanthus amarus and Terminalia catappa showed the most promising antibacterial properties, inhibiting all of the strains tested with minimum inhibitory concentrations (MICs) ranging from 0.25 to 16 mg/ml. The extract from aerial part of Piper aduncum was significantly more active against Gram-positive (MICs ranging from 1 to 2 mg/ml) than against Gram-negative bacteria (MICs > 16 mg/ml).     

Effect of electroacupuncture on the expression of PGC-1α and UCP-1in the brown adipose tissue of obese rats

Objective

To explore the effect of peroxisome proliferators-activated receptor γ coactivator-1α (PGC-1α) and uncoupling protein-1 (UCP-1) in the brown adipose tissue (BAT) of obese rats in the process of acupuncture treatment for obesity.

Effect of moxibustion on expressions of HO-1 and MCP-3 protein in colon of rats with Crohn’s disease

Objective

To observe the effect of moxibustion therapy on heme oxygenase-1 (HO-1) and monocyte chemoattractant protein-3 (MCP-3) protein expressions in the colonic mucosa of rats with Crohn’s disease (CD), and to explore the intestinal mucosal immune mechanism of moxibustion therapy in treating CD.

Methods

The CD rat model was established using the internationally accepted Morris method. The rats were randomly divided into a model group, a herbal cake-partitioned moxibustion group, a mild moxibustion group, a cigarette moxibustion group and a hot compress group, which were compared with the normal group. Except the normal group and the model group, rats in the other groups accepted different moxibustion therapies on bilateral Tianshu (ST 25). Hematoxylin-eosin (HE) staining was conducted and the pathological changes of the colon were observed under light microscope; the expressions of HO-1 and MCP-3 protein in rat’s colonic mucosa were determined by immunohisto-chemistry.

Results

Compared with the normal group, rats in the model group showed mucosal defect, villus destruction or loss, submucosal congestion and edema, glandular destruction or disappearance, reduced goblet cells, ulcer formation, significantly increased positive target area and positive target integral optical density of HO-1 and MCP-3 protein expression (all P<0.01). After treatment, compared with the model group, colonic mucosa was significantly improved in the herbal cake-partitioned moxibustion group and the mild moxibustion group, which mainly showed that the intestinal glands were arranged regularly, ulcer surfaces were covered by the neoformative epitheliums, or intestinal ulcers were replaced by the nascent granulation tissue, and submucosal edema was alleviated, with a small amount of inflammatory cell infiltration. The total areas and the integral optical densities of the positive targets for rat’s colonic mucosa HO-1 and MCP-3 protein expressions were decreased (all P<0.01). Compared with the cigarette moxibustion group and the hot compress group, the total areas and the integral optical densities of the positive targets for rat’s colonic mucosa HO-1 and MCP-3 protein expressions were significantly decreased (all P<0.01) in the herbal cake-partitioned moxibustion group and the mild moxibustion group.

Conclusion

Herbal cake-partitioned moxibustion and mild moxibustion can significantly improve the inflammatory response of colonic mucosa in CD rats. It can down-regulate the expressions of HO-1 and MCP-3 proteins in the colonic mucosa of CD rats, which may be one of the mechanism in intestinal mucosal immunity caused by moxibustion therapy.

     

Antimicrobial activity of plants used as medicinals on an indigenous reserve in Rio das Cobras, Paraná, Brazil

Ethnopharmacological relevance

A considerable percentage of global biodiversity is located in Brazil, a country that also has rich cultural and ethnic diversity. In the community of Rio das Cobras, Paraná, plants are still widely used in the health care not only by indigenous people but also by the non-indigenous population that inhabits the region. The investigation of the efficacy and safety of these plants in the treatment of infectious diseases provides insights for future studies of these species allowing the appropriated use by the indigenous people, since few or none study has been conducted so far.

Aim of the study

Evaluate the antimicrobial activity and cytotoxicity of some plants used as medicinal on an indigenous reserve in Rio das Cobras, Paraná, Brazil.

Materials and methods

The aqueous extracts were obtained by decoction and the 50% and 70% hydroalcoholic extracts by turbo extraction. The extracts were tested against strains of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, Candida parapsilosis, Candida tropicalis, Leishmania amazonensis, Poliovirus and HSV-1. Cytotoxicity assay using VERO cells were also performed.

Results

None of the extracts had a selectivity index (SI)>1 for any of the tested bacteria. Only Campomanesia eugenioides and Schinus terebinthifolius had an SI>1.0 for all of the tested Candida species. The best anti-Leishmania activity was obtained with Zanthoxylum rhoifolium and Schinus terebinthifolius. Extracts of Cordia americana were the most effective against herpes simplex virus type 1. Zanthoxylum rhoifolium was the most effective against Poliovirus, and Ocimum gratissimum was effective against both Poliovirus and Herpes Simplex virus. Among the plants investigated in the present study, Zanthoxylum rhoifolium had the fewest cytotoxic effect.

Conclusions

The plants investigated in the present study exhibited potential for future pharmacological uses, but additional studies, especially with regard to in vivo toxicity, must be conducted. The results of this preliminary survey are important for the Rio das Cobras Reserve community for the safe and effective use of plants in the treatment of some infectious diseases.     

Observations on Therapeutic Effect of Back-Shu Acupoint in Treatment of Asthma

目的：观察背俞穴治疗哮喘的疗效。方法：将61例患者分为背俞穴组、针灸组、针刺组，三组均每星期治疗2次，10次为1疗程，治疗3疗程后观察疗效。结果与结论：背俞穴组和针灸组控显率均高于常规针刺（P〈0．05），针灸组控显率略高于背俞穴组，但没有统计学意义。结果表明背俞穴治疗哮喘有较好的疗效。     

Effect of moxibustion at Shénquè ( CV 8) on anti-exercise-induced fatigue in exhaustive rats in varying degrees

Objective

To explore the effect of moxibustion at Shénquè (

CV 8) on the anti-exercise-induced fatigue in rats after exhaustive exercise in varying degrees.

Effect of electroacupuncture on urodynamics,PACAP-38 and PAC1R of spinal cord in rats with neurogenic bladder✰

Objective

To investigate the effect and mechanism of electroacupuncture (EA) in the intervention of neurogenic bladder (NB) caused by suprasacral spinal cord injury (SCI).

Effect on blood pressure of a dietary supplement containing traditional medicinal plants of Côte d'Ivoire

Ethnopharmacological relevance

A medicinal composition containing salt (sodium chloride) is given as a traditional dietary supplement to hypertensive patients (TDSHP) in Côte d’Ivoire. It consists of whole plant of Bidens pilosa (Asteraceae) and fresh leaves of Moringa oleifera (Moringaceae).

Aim of the study

The aim of this study was to establish the scientific basis for the use of this traditional recipe rich in sodium chloride in hypertension settings.

Materials and methods

We used a total aqueous extract of this traditional dietary supplement containing medicinal plants (Bidens pilosa, Moringa oleifera) and salt (sodium chloride). Experiment was carried out to evaluate its effect on arterial blood pressure of rabbits. The experimental device used for recording blood pressure in rabbits is based on the principle of Ludwig mercury manometer.

Results

TDSHP between 5 × 10⁻⁸ and 5 × 10⁻² mg/kg caused a dose-dependent hypotension. TDSHP elicited drops in blood pressure ranging between 7.14 ± 4 and 100 ± 7.5%, compared to normal blood pressure of rabbits. Fifty percent effective dose of TDSHP was 3.95 × 10⁻⁴ mg/kg. Similarly as the hypotension induced by acetylcholine, the one caused by TDSHP at dose of 3.95 × 10⁻⁴ mg/kg in rabbit was progressively inhibited by atropine, dosed between 5 × 10⁻⁴ to 5 × 10⁻² mg/kg. The percentage drop of recorded blood pressure ranged from 50.3 ± 1.87 to 3.71 ± 1.09% compared to the normal value of blood pressure. In the presence of atropine, TDSHP effect was partially inhibited. The same increasing doses of TDSHP reduced significantly the increase of blood pressure induced by adrenaline dosed at 4.76 × 10⁻⁴ mg/kg from 89.3 ± 2.19 to 1.19 ± 0.59%.

Conclusion

The consumption of this traditional dietary supplement is justified in hypertensive patients according to its composition and its ability to reduce blood pressure has been demonstrated experimentally. TDSHP should not be considered as an antihypertensive drug, it remains to us a salt substitute to be taken with moderation with strict adherence to the traditional dose.     

Protective effect of Pycnogenol® on ovariectomy-induced bone loss in rats

Pycnogenol® (PYC) is a natural plant extract from the bark of Pinus pinaster and has potent antioxidant activities. The protective effect of PYC on bone loss was studied in multiparous ovariectomized (OVX) female rats. Pycnogenol® (30 or 15 mg/kg body weight/day) was administered orally to 8‐month‐old OVX rats for 3 months. At the end of the experiment, bone strength was measured by a three‐point bending test and bone mineral density was estimated by peripheral quantitative computed tomography. Ovariectomy significantly decreased femur bone strength and bone density. Supplementation with PYC suppressed the bone loss induced by OVX. The OVX treatment significantly increased serum osteocalcin (OC) and C‐terminal telopeptide of type I collagen (CTx). Supplementation with PYC reduced the serum OC and CTx in OVX rats to a level similar to that of the sham‐operated group. The results indicated that orally administered PYC can decrease the bone turnover rate in OVX rats, resulting in positive effects on the biomechanical strength of bone and bone mineral density. Copyright © 2011 John Wiley & Sons, Ltd.     

Effect of Zhuyeshigao granule on tumor necrosis factor-α and interleukins serum protein levels in rats with radiation esophagitis

Objective

To investigate the effects of Zhuyeshigao granule (ZSG) on tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-2, IL-6, and IL-8 in rats with radiation esophagitis.

Methods

Fifty Wistar rats were randomly divided into five groups (10 rats in each group): control (without radiation), saline-treated, and low, medium, and high-dose ISG-treated groups. Rats were given normal saline (10 mL/kg) or 1.15, 2.3, or 4.6 g/kg ZSG by intragastric administration once a day for 7 days. A rat model of radiation esophagitis was established by local irradiation of Co60 (490.25 cGy/min, totaling 30 Gy). The administration of ZSG was continued for another 7 days and on the 7th day post-irradiation, inferior vena cava blood was collected. The serum was separated, and TNF-α, IL-1, IL-2, IL-6, and IL-8 protein levels were determined.

Results

Inflammatory response factors were found in the serum of each group. However, levels in ZSG-treated groups were significantly lower than in the saline-treated group (P<0.05).

Conclusion

ZSG may prevent the development of radiation esophagitis, perhaps by inhibiting the generation and release of the inflammatory response factors TNF-α, IL-1, IL-2, IL-6, and IL-8.     

Effect of Acupuncture on Serum SOD and MDA of Rats in Menopause

目的：观察针刺对围绝经期大鼠血清超氧化物歧化酶（SOD）、丙二醛（MDA）的影响。方法：对确立为围绝经期的雌性大鼠随机分组，分为针剌组、药物组、空白对照组，分别进行针剌治疗、更年安药物治疗和空白对照，同时另设青年组作对照。经17d治疗后，采用生化法检测血清SOD、MDA。结果：与青年组大鼠比较，围绝经期大鼠血清SOD含量降低（P〈0．01），MDA含量升高（P〈0．01）。经针刺治疗后大鼠血清SOD含量升高（P〈0．05），MDA含量降低（P〈0．05）。结论：针刺能够提高围绝经期大鼠血清SOD含量，降低MDA含量。     

Antimalarial activity of medicinal plants used in traditional medicine in S. Tomé and Príncipe islands

The present study investigates the antimalarial activity of 13 medicinal plants used in traditional medicine in S. Tomé and Príncipe (STP) islands in the Gulf of Guinea, aiming at identifying the most effective plants for further research. Fieldwork was carried out with the collaboration of 37 traditional healers from both islands, during an ethnobotanical study, which was conducted from 1993 to 1999. Our results indicate that the traditional healers in STP use several medicinal plants against fever and/or 'malaria' which reveal strong antiparasitic activity in vitro: four of the plant extracts have evident antiplasmodial activity against chloroquine resistant Plasmodium falciparum, with IC(50) values <10 microg/ml, and also revealed hepatic schizontocidal activity (<5-35 microg/ml). In vivo, the extracts caused partial reduction of Plasmodium berghei parasitaemia in mice.     

An ethnobotanical study of medicinal plants used by the local people of Alaşehir (Manisa) in Turkey

Ethnopharmacological relevance

This paper represents the first large-scale ethnobotanical study in the Ala?ehir and its surrounding (Manisa/Turkey). There are scarcely any studies for using plants. There is urgency in recording such data. This is the first ethnobotanical study in which statistical calculations about plants are done by ICF (Informant Consensus Factor) method. This study aimed to identify plants collected for medicinal purposes by the local people of Ala?ehir, located in the Aegean Region of Turkey, and to document the traditional names, preparation and uses of these plants.

Materials and methods

Field study was carried out over a period of approximately 2 years (2010–2012) in Ala?ehir. During this period, 137 vascular plant specimens were collected. Demographic characteristics of participants, local plant names, utilized parts and preparation methods of the plants were investigated and recorded. In the scope of the study, medicinal plant species and related information were collected; herbarium materials were prepared; and the specimens were entitled. Field research was conducted by collecting ethnobotanical information during structured and semi-structured interviews with native knowledgeable people in territory. In addition, the relative importance value of species was determined and ICF was calculated for the medicinal plants included in the study.

Results

A total of 137 medicinal plants belonging to 58 families were identified in the region. Among them, 105 species are wild and 32 species are cultivated plant. The most dominant medicinal plant families were Asteraceae (>13%), Lamiaceae (>11%), Rosaceae (>7%), and Fabaceae (>4%), again; the most common preparations were infusion and decoction. It was found that Origanum onites L., Urtica urens, Thymus zygioides Griseb., Matricaria chamomilla L., Salvia tomentosa Mill., Cerasus avium (L.), Tilia argentea Desf. ex DC., Hyoscyamus niger L., Urtica pilulifera L., Anethum graveolens L., Euphorbia rigida Bieb., Hypericum perforatum L., Paliurus spina-christi Mill., Rosa canina L., Viscum album L. subsp. austriacum (Wiesb.) Vollman, Silybum marianum (L.) Gaertn., Elymus repens (L.) Gould and Juglans regia L. were the most commonly used species. The traditional medicinal plants have been mostly used for the treatment respiratory tract diseases (14.1%), gastro-intestinal diseases (10%), kidney problems (7.3%), diabetes (7.1%), cholesterol (5%), rheumatism (4%), cancer various (4%), cardiovascular problems (3.1%) and burn (3%).

Conclusion

Gathering, processing and consuming wild edible plants are still practiced in all the studied Ala?ehir areas. These plants, used in the treatment of many different diseases, are freely harvested in this region at abundant amounts. Due to the increasing health service facilities in the area, herbal medicine, seemed to be more related to health care and disease prevention than cure.     

Effect of acupuncture plus thunder-fire moxibustion on MMP-3, TIMP-1 and TGF-β1 in rats with knee osteoarthritis

Objective

To observe the effect of acupuncture plus thunder-fire moxibustion on the expressions of matrix metalloproteinase-3 (MMP-3), tissue inhibitor of metalloproteinase-1 (TIMP-1) and transforming growth factor-β1 (TGF-β1) in cartilage of knee osteoarthritis (KOA) rats, and to explore the mechanism of acupuncture plus thunder-fire moxibustion in the treatment of KOA.

Methods

Thirty Sprague-Dawley (SD) rats were randomly divided into a blank control group, a model group and an acupuncture-moxibustion group by random digits table, 10 rats in each group. Rats in the model group and the acupuncture-moxibustion group were injected with papain in the right posterior knee joint to prepare the models. The levels of MMP-3 and TIMP-1 in rat synovium of each group were measured by enzyme-linked immunosorbent assay (ELISA) after 2 weeks of treatment. The level of TGF-β1 was determined by Motic B5 Micro-camera system.

Results

The levels of MMP-3 and TIMP-1 in the cartilage of the model group were significantly higher than those in the blank control group (all P<0.01); the levels of MMP-3 and TIMP-1 in the acupuncture-moxibustion group were lower than those in the model group, and the between-group differences were statistically significant (all P<0.05). The levels of MMP-3 and TIMP-1 in the acupuncture-moxibustion group were higher than those in the blank control group, and the differences were statistically significant (all P<0.05). The level of TGF-β1 in cartilage tissues of the model group was significantly lower than that in the blank control group (P<0.01); the level of TGF-β1 in the acupuncture-moxibustion group was higher than that in the model group (P<0.05), but it was lower than that in the blank control group, and the between-group difference was statistically significant (P<0.05).

Conclusion

Acupuncture plus thunder-fire moxibustion can effectively recover the abnormal expressions of MMP-3 and TIMP-1 in KOA model rats and somewhat up-regulate TGF-β1, which may be one of its mechanisms of acupuncture plus thunder-fire for KOA.