An aqueous extract of Poncirus fructus activates the prokinetic activity of 5-HT receptor subtype 4 without hERG interaction

Aim of the study

Poncirus fructus (PF) - also known as the dried, immature fruit of Poncirus trifoliata (L.) Raf. (Rutaceae) - is a natural substance that has long been used for various gastrointestinal disorders in eastern Asia. An aqueous extract of PF (PF-W) has particularly potent gastroprokinetic effects, but its molecular mechanism was not well understood. Identification of the underlying prokinetic mechanism of PF-W was pursued in the present study.

Materials and methods

Changes in in vitro cAMP levels and in vivo intestinal transit rate (ITR) caused by PF-W were measured after pretreatment with GR125487, an antagonist for serotonin receptor subtype 4 (5-HT4R). An [³H] astemizole binding assay and electrophysiology experiments were performed to determine if PF-W has any interaction with the human ether-à-go-go related gene (hERG) potassium channel.

Results

PF-W induced an increase in intracellular cAMP in 5-HT4R-expressing HEK293T cells, indicating that PF-W does activate 5-HT4R. Moreover, pretreatment with GR125487 successfully blocked the increase, suggesting that the response was 5-HT4R-specific. More importantly, pretreatment of GR125487 in rats inhibited the elevation of ITR by PF-W, indicating that the prokinetic effect of PF-W was indeed exerted via 5-HT4R. On the other hand, both [³H]-astemizole binding assay and electrophysiological experiments revealed that PF-W did not interfere at all with the hERG channel.

Conclusion

It was found that PF-W exerts its prokinetic activity through a 5-HT4R-mediated pathway, with no interaction with hERG channels. Therefore, PF-W is a good candidate that might be developed as a prokinetic agent with fewer expected cardiac side effects.     

Long-term administration of an aqueous extract of dried,immature fruit of Poncirus trifoliata (L.) Raf. suppresses body weight gain in rats

Aim of study

The purpose of the present study was to examine the effects of daily administration of an aqueous extract of the dried, immature fruit of Poncirus trifoliata Raf. (Rutaceae) (PF-W) on body weight in rats.

Materials and methods

PF-W was used in following experiments: 10-week-long measurement of body weight and food intake, in vitro pancreatic lipase activity assay, in vivo triglyceride concentration study, and measurement of intestinal transit rate.

Results

A high dose of PF-W (200 mg/2 mL/animal/day, in aqueous solution) was administered intragastrically to rats for 10 weeks. PF-W suppressed body weight gain by 6%. However, administration of PF-W at a lower dose (20 mg/animal/day) did not reduce weight gain. Administration of low- or high-dose PF-W had no effect on food intake throughout the experimental period. Additional experiments revealed that the suppressive effect of PF-W on body weight gain was not related to pancreatic lipase activity. Moreover, co-administration of PF-W with a lipid emulsion did not reduce plasma triglyceride concentration. Of interest, the high dose of PF-W significantly increased the rate of intestinal transit, whereas oral administration of the lower dose did not. Control and PF-W-treated groups did not differ in hematological and serum biochemical parameters, or in relative organ weights after 10 weeks of high-dose PF-W administration.

Conclusion

PF-W does not suppress body weight gain by interfering with fat absorption in a pancreatic lipase-dependent manner. The suppressive effect of PF-W on weight gain is likely due to the increased rate of intestinal transit, and the consequent reduction in nutrient absorption. Moreover, it appears that PF-W is relatively safe for long-term use. Taken together, the results of the present study suggest that long-term, daily administration of PF-W successfully suppressed body weight gain—apparently due to accelerated intestinal transit and not to interference with pancreatic lipase activity. Due to its apparent long-term safety, PF-W is a potential therapeutic agent for reduction of body weight in humans.     

Poncirus trifoliate fruit modulates pacemaker activity in interstitial cells of Cajal from the murine small intestine

Ethnopharmacological relevance

Poncirus fructus (PF) has been widely used as a traditional medicine in Eastern Asia, especially to ameliorate the symptoms of gastrointestinal (GI) disorders related to abnormal GI motility.

Aim of the study

Poncirus fructus (PF), also known as Poncirus trifoliata (L.) Raf. (Rutaceae), is widely used as a traditional medicine in Eastern Asia mainly to ameliorate the symptoms of gastrointestinal (GI) disorders related to abnormal GI motility. In a previous study, a methanol extract of PF was found to have particularly potent gastroprokinetic effects. Interstitial cells of Cajal (ICCs) are pacemaker cells in the gastrointestinal tract, but the action mechanisms of PF extract in mouse small intestinal ICCs have not been investigated. Therefore, in the present study, we investigated the effects of a methanol extract of PF (MPF) in mouse small intestinal ICCs. In addition, we sought to identify the receptors involved.

Materials and Methods

Enzymatic digestions were used to dissociate ICCs from small intestines. The whole-cell patch-clamp configuration was used to record potentials (current clamp) from cultured ICCs. In addition, we analyzed intracellular Ca²⁺ concentrations ([Ca²⁺]_i).

Results

MPF decreased the amplitudes of pacemaker potentials in ICCs, and depolarized resting membrane potentials in a concentration dependent manner. Y25130 (a 5-HT₃ receptor antagonist) and RS39604 (a 5-HT₄ receptor antagonist) blocked MPF-induced membrane depolarizations, whereas SB269970 (a 5-HT₇ receptor antagonist) did not. Pretreatment with Na⁺ or Ca²⁺-free solution or thapsigargin (a Ca²⁺-ATPase inhibitor in endoplasmic reticulum) abolished the generation of pacemaker potentials and suppressed MPF-induced activity. [Ca²⁺]_i analysis showed that MPF increased [Ca²⁺]_i. Furthermore, treatments with PD 98059, SB203580, or JNK II inhibitor blocked MPF-induced membrane depolarizations in ICCs.

Conclusion

These results suggest that MPF modulates pacemaker potentials through 5-HT₃ and 5-HT₄ receptor-mediated pathways via external Na⁺ and Ca²⁺ influx, and via Ca²⁺ release from internal stores in a mitogen-activated protein kinase dependent manner. The study shows MPF is a good candidate for the development of a gastroprokinetic agent. In view of the effects of MPF on ICCs, further research is required, particularly to identify the active compound(s) involved and to determine their action mechanisms.     

Double directional adjusting estrogenic effect of naringin from Rhizoma drynariae (Gusuibu)

Ethnopharmacological relevance

Chinese traditional medicine Rhizoma drynariae (Gusuibu) is widely used for clinically treating osteoporosis and bone non-union. Naringin and its active metabolite naringenin are the main active ingredients of Rhizoma drynariae total flavonoids.

Aim of the study

The purpose of this paper is to confirm estrogenic and anti-estrogenic activity of naringin and naringenin, and provide the basic data to further study for the dose-effect relationship and the mechanism for Rhizoma drynariae in treatment of osteoporosis and other estrogen deficiency-related diseases.

Materials and methods

Naringin was extracted from Rhizoma drynariae. Naringin and its metabolin naringenin were tested estrogenic and anti-estrogenic activities through the experiment of cell proliferation and uterus weight gain in mice. Their estrogen-receptor binding abilities were tested by yeast two-hybrid experiment and nuclear receptor cofactor assays (RCAS) experiment, and their possible binding sites for ERβ were performed by computer aided molecular docking technology.

Results

Naringin and naringenin showed significant effects on the proliferation of estrogen-sensitive ER(+) MCF-7 cells in the absence of estrogen. Induction increased proliferation as the drug concentration, and the strongest proliferation appeared at a concentration of 8.6 × 10⁻⁵ M. When estradiol (10⁻¹⁰ M) and the different concentrations of naringin or naringenin were treated at the same time, naringin and naringenin could result in antagonistic effects on estradiol-induced MCF-7 cell proliferation, but they did not significantly affect proliferation of estrogen-insensitive ER(−) MDA-MB-231 cells. Naringin and naringenin exhibited higher binding capacity to estrogen receptor β (ERβ) than estrogen receptor α (ERα) in yeast two-hybrid experiments and nuclear receptor cofactor assays (RCAS) experiment. Docking simulation between naringin/naringenin and ERβ were performed, and the corresponding binding free energies of naringin-receptor and naringenin-receptor docked complexes were −7.95 and −10.45 kcal/mol. Hydrogen bonds were found between naringin and the amino acid residues Lys304 and His308. The oxygen atom (O11) of naringenin formed hydrogen bond to Arg346, and there may be hydrophobic space interactions between phenyl group (C13-C18) of naringenin and the amino acid residues Leu298, Met336, Met340, Phe356, Ile376 and Leu380.

Conclusions

Naringin and naringenin revealed a double directional adjusting function of estrogenic and anti-estrogenic activities. Both of them showed estrogenic agonist activity at low concentration or lack of endogenous estrogen. On the other hand, they also acted as estrogenic antagonists at high concentrations or too much endogenous estrogen. They produced estrogenic and anti-estrogenic effects primarily through selectively binding with ERβ, which could prevent and treat osteoporosis with the mechanism of estrogenic receptor agitation. This paper confirmed the estrogenic and anti-estrogenic activity of naringin and naringenin, and further studies were still essential to study their dose-effect relationship and the anti-osteoporosis mechanism for Rhizoma drynariae in the treatment of osteoporosis and other estrogen deficiency-related diseases.     

Fuzi-Lizhong pill compensates hypothyroid-hypothermia via ghrelin release

Ethnopharmacological relevance

Fuzi-Lizhong pill (FLZ) is a traditional Chinese medicine for treating patients with Spleen Yang deficient syndrome. Ghrelin, a peptide with 28 amino acid residues, plays multiple roles in thermogenesis. This study aims to explore FLZ regulating ghrelin to compensate hypothermia in rats with hypothyroid and indigestion.

Materials and methods

In litter-matched rats, hypothermia was developed with both thyroidectomy at d₁ and interscapular brown adipose (IBA) removal at d₄₂, indigestion was induced with both high fat diet and fasting-feeding cycle from d₅₆; the littermates with hypothermia and indigestion were administrated with FLZ from d₇₀. Adaptive thermogenesis, thyroid hormones, metabolites, ghrelin dynamics were measured at d₉₈.

Results

The results showed that plasma ghrelin levels were inversely correlated with the gastric ghrelin levels and adaptive thermogenesis in rats undergone both thyroidectomy and IBA removal. Fatty diet and FLZ enhanced the increase of plasma ghrelin of hypothyroid rats. These were supported by the changes of plasma thyroid related hormones, plasma metabolites, gastric ghrelin mRNA and protein, and the effects of fatty diet or FLZ.

Conclusions

Our results suggest that more ghrelin release compensate chronic hypothermia in rats with both hypothyroidism and indigestion. It could explain the mechanisms of FLZ in relieving chronic hypothermia.     

The influence of compatibility of traditional Chinese medicine on the pharmacokinetic of main components in Fructus Aurantii

Ethnopharmacological relevance

Fructus Aurantii is one of the commonly used traditional Chinese medicines and the compatibility of Fructus Aurantii, Radix et Rhizoma Rhei and Semen Crotonis Pulveratum were the main components in a Chinese traditional patent medicine Weichang'an pill. Naringin and neohesperidin are two main flavonoid glycosides in the extract of Fructus Aurantii. The pharmacokinetics of naringin and neohesperidin, as the main marker constituents were analysis by a simple and accurate method.

Materials and Methods

In the present study, pharmacokinetic differences of two flavonoid glycosides were detected after oral administration of the extract of single herb and different compatibility of Fructus Aurantii, Radix et Rhizoma Rhei and Semen Crotonis Pulveratum which at doses of approximately 700 mg/kg naringin and 300 mg/kg neohesperidin.

Results

It indicated that the pharmacokinetic process of naringin and neohesperidin in four groups were demonstrated bimodal phenomenon after oral administration. Comparing to pharmacokinetic parameters of the single herbs administration, there were no significant difference after the compatibility of Fructus Aurantii and Radix et Rhizoma Rhei. However, the absorption of two flavonoid glycosides was increased dramatically after the compatibility of Fructus Aurantii or Semen Crotonis Pulveratum. The same results were attained after co-administration of the extract of three herbs.

Conclusion

Some components in the extract of Semen Crotonis Pulveratum promoted the absorption of naringin and neohesperidin.     

Evaluation of Ginkgo biloba extract as an activator of human glucocorticoid receptor

Ethnopharmacological relevance

Ginkgo biloba, which is one of the most frequently used herbal medicines, is commonly used in the management of several conditions, including memory impairment. Previously, it was reported to decrease the expression of peripheral benzodiazepine receptor and the biosynthesis of glucocorticoids, thereby regulating glucocorticoid levels. However, it is not known whether Ginkgo biloba extract regulates the function of the glucocorticoid receptor.

Aim of the study

We determined whether Ginkgo biloba extract and several of its chemical constituents affect the activity of human glucocorticoid receptor (hGR).

Materials and methods

A hGR-dependent reporter gene assay was conducted in HepG2 human hepatocellular carcinoma cells and hGR target gene expression assays were performed in primary cultures of human hepatocytes.

Results

Multiple lots and concentrations of the extract and several of its chemical constituents (ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, and bilobalide) did not increase hGR activity, as assessed by a cell-based luciferase reporter gene assay. The extract did not influence the expression of hGR target genes, including tyrosine aminotransferase (hTAT), constitutive androstane receptor (hCAR), or pregnane X receptor (hPXR), in primary cultures of human hepatocytes. Moreover, hGR antagonism by mifepristone (also known as RU486) did not attenuate the extent of induction of hCAR- and hPXR-regulated target genes CYP2B6 and CYP3A4 by Ginkgo biloba extract.

Conclusion

Ginkgo biloba extract, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, and bilobalide are not activators of hGR. Furthermore, the extract does not influence the hGR-hCAR or the hGR-hPXR signaling pathway in primary cultures of human hepatocytes.     

Relaxant effect of flavonoid naringenin on contractile activity of rat colonic smooth muscle

Ethnopharmacological relevance

Disturbed gastrointestinal (GI) motility can be associated with smooth muscle abnormalities and dysfunction. Exploring innovative approaches that can modulate the disturbed colonic motility are of great importance for clinical therapeutics. Naringenin, a flavonoid presented in many traditional Chinese herbal medicines, has been shown to have a relaxant effect on different smooth muscles. The aim of the present study was to investigate the effect of naringenin on regulation of GI motility.

Material and methods

Mechanical recording was used to investigate the effect of naringenin on isolated rat colonic smooth muscle spontaneous contractions. Whole cell patch clamp, intracellular [Ca²⁺] concentration ([Ca²⁺]_i) and membrane potential measurements were examined on primary cultures of colonic smooth muscle cells (SMCs). A neostigmine-stimulated rat model was utilized to investigate the effect of naringenin in vivo.

Results

Naringenin induced a concentration-dependent inhibition (1–1000 μM) on rat colonic spontaneous contraction, which was reversible after wash out. The external Ca²⁺ influx induced contraction and [Ca²⁺]_i increase were inhibited by naringenin (100 μM). In rat colonic SMCs, naringenin-induced membrane potential hyperpolarization was sensitive to TEA and selective large-conductance calcium-activated K⁺ (BK_Ca) channel inhibitor iberiotoxin. Under whole cell patch-clamp condition, naringenin stimulated an iberiotoxin-sensitive BK_Ca current, which was insensitive to changes in the [Ca²⁺]_i concentration. Furthermore, naringenin significantly suppressed neostigmine-enhanced rat colon transit in vivo.

Conclusion

Our results for the first time demonstrated the relaxant effect of flavonoid naringenin on colon smooth muscle both in vitro and in vivo. The relaxant effect of naringenin was attributed to direct activation of BK_Ca channels, which subsequently hyperpolarized the colonic SMCs and decreased Ca²⁺ influx through VDCC. Naringenin might be of therapeutic value in the treatment of GI motility disorders.     

Meta-analysis of Traditional Chinese Medicine in treating functional dyspepsia of liver-stomach disharmony syndrome

Objective

To systematically assess the clinical effectiveness of traditional Chinese medicine (TCM) in the treatment of functional dyspepsia (FD) of liver-stomach disharmony syndrome by meta-analysis.

Methods

Random controlled trials (RCTs) were retrieved from databases, including Pubmed, China national knowledge infrastructure, Wanfang Data, VIP Information, and the Cochrane Library. Trials were selected according to inclusion criteria. The effects of traditional Chinese medicine (TCM) versus prokinetic agents in the treatment of functional dyspepsia (FD) of liver-stomach disharmony syndrome were compared by meta-analysis. RevMan 5.0.24 was used for data analysis. The effective rate was assessed by odds ratios (ORs), and 95% confidence intervals (Cls) were calculated. The cure rate was analyzed by the Peto OR. Simple statistical analysis was chosen to assess the frequency of prescribed Chinese herbs in treating this syndrome.

Results

Thirteen trials were included, involving 1153 patients, and these were of poor methodological quality. Twelve studies mentioned the effective rate and cure rate. TCM therapy showed a better clinical effect rate compared with that with prokinetic agents [OR: 3.2, 95% CI (2.27, 4.51)]. The TCM group also had a better cure rate than that in the group of prokinetic agents [Peto OR: 2.26, 95% CI (1.61, 3.18)]. With regard to the frequency of Chinese herbs used in these 13 trials, Baishao (Radix Paeoniae), Chaihu (Radix Bupleuri), and Gancao (Radix Glycyrrhizae) were mostly prescribed, followed by Xiangfu (Rhizoma Cyperi), Zhishi (Fructus Aurantii Immaturus), Zhiqiao (Fructus Aurantii), Foshou (Citrus medica var.sarcodactylis), and Chenpi (Pericarpium Citri Reticulatae). No serious adverse effects were reported.

Conclusion

TCM therapy shows a superior effective rate and cure rate compared with those in prokinetic agents in the treatment of FD of liver-stomach disharmony syndrome. However, further strictly designed RCTs are required because of the poor quality of included trials.     

Assessment of in vitro pharmacological effect of Neotropical Piperaceae in GABAergic bioassays in relation to plants traditionally used for folk illness by the Yanesha (Peru)

Ethnopharmacological relevance

A previous pilot ethnobotanical and ethnopharmacological study with the Q‘echi? Maya identified the family Piperaceae, as an important taxonomic group traditionally used for the treatment of epileptic and culture-bound anxiety disorders and possessing activity in the GABA system. Following that lead, a botanical survey was conducted in Peru, where 47 species of Piperaceae were collected including 21 plants traditionally used for folk illnesses by the Yanesha of Peru, an indigenous Amazonian group.

Materials and methods

Two high throughput bioassays were used to quantify the in vitro activity of botanical extracts on the GABA system.

Results

Plant extracts demonstrated moderate to high affinity to the γ-aminobutyric acid benzodiazepine (GABA-BZD) receptor. In addition, extracts demonstrated low to moderate activity in the inhibition of the GABA-transaminase, with select plants exhibiting significant activity. Plants indicated by the Yanesha showed comparable activity to the other Piperaceae plants collected. Piper cremii was the most active plant in the GABA-BZD receptor assay, and Drymaria cordata (Caryophyllaceae) in the GABA-T assay.

Conclusion

The study provides evidence that there is a pharmacological basis behind the use of plants in the treatment of susto and mal aire in both Central and South America, and we propose that the possible mechanism of action includes an interaction with the GABA-T enzyme and/or the GABA_A-BZD receptor.     

Effect of didrovaltrate on I-calcium current in rabbit ventricular myocytes

Objective

To investigate the effect of didrovaltrate on L-type calcium current (I_ca-L) in rabbit ventricular myocytes.

Methods

We used the whole cell patch clamp recording technique.

Results

Didrovaltrate at concentrations of 30 μg/L and 100 μg/L significantly decreased peak ICa-L (ICa-Lmax) from (6.01 ± 0.48) pA/pF to (3.45±0.27) pA/pF and (2.16 ± 0.19) pA/pF (42.6% and 64.1%, n=8, P<0.01), respectively. Didrovaltrate shifted upwards the current-voltage curves of ICa-L without changing their active, peak and reverse potentials. Didrovaltrate affected the steady-state inactivation of ICa-L. The half activation potential (V_1/2) was significantly shifted from (−26 ± 2) to (−36 ± 3) mV (n=6, P<0.05), with a significant change in the slope factor (k) (from 8.8 ± 0.8 to 11.1 ± 0.9, n=6, P<0.05). Didrovaltrate did not affect the activation curve.

Conclusion

Didrovaltrate blocks ICa-L in a concentration-dependent manner and probably inhibits ICa-L in its inactive state, which may contribute to its cardiovascular effect.     

Discovery of N-methyltetrahydroprotoberberines with κ-opioid receptor agonists-opioid receptor agonist activities from corydalis yanhusuo W. T. Wang by using two-dimensional liquid chromatography

Ethnopharmacological relevance

The need for an efficacious analgesic without unwanted side effects is urgent. κ-opioid receptor agonists are known to exhibit potent analgesic effects and elicited fewer side effects than other opioid agonists. Thus in this study we chose the κ-opioid receptor as the target to identify the active components from traditional Chinese medicines (TCMs).

Materials and methods

The κ-opioid receptor was expressed in human embryonic kidney-293 T cells (HEK293T). Fluorometric Imaging Plate Reader (FLIPR) assay was used for the determination of Ca²⁺ response when κ-opioid receptor was activated. A novel 2D separation system employing C18HCE as the first dimension and a strong cation exchange column (SCX) as the second dimension was conducted for the purification of the active principles.

Results

With the aid of HPLC-based activity profiling, activities could be linked to two peaks from Corydalis yanhusuo W. T. Wang (C. yanhusuo) extract. Two N-methyltetrahydroprotoberberines with κ-opioid receptor agonist activities were isolated for the first time from C. yanhusuo by using 2D-LC.

Conclusions

Our study suggests that N-methyltetrahydroprotoberberines may serve as a new scaffold for κ-opioid receptor ligands. The strategy that we adopted can be applied to other naturally-occurring active alkaloids acting at different receptors.     

Pharmacological basis for the medicinal use of muskmelon base (Pedicellus Melo.) for abdominal distention and constipation

Ethnopharmacological relevance

Muskmelon base (Pedicellus Melo.) has a long history (Ming Dynasty) as a Chinese traditional medicine. According to traditional use, it was prepared as rectal suppositories for treating abdominal distention and constipation. The present study was carried out on the pharmacological basis for the medicinal use of muskmelon base.

Aim of the study

The objective of this study was to determine the pharmacological basis for the medicinal use of the ethanol extract from muskmelon base (EMB) for abdominal distention and constipation.

Materials and methods

In this study, we report the gastrointestinal prokinetic action of EMB following single rectal or large intestinal administration. Laxative activity, gastric emptying and small intestinal transit tests were examined in ICR mice. SD rats were used to determine changes in large intestinal transit and contractile effects of the proximal colon in vivo. Guinea pigs were used to evaluate the contractile effects of the proximal colon and the possible mechanism or mechanisms on proximal colon activity ex vivo. Moreover, the acute toxicity of EMB was evaluated.

Results

In the in vivo experiments, the acute toxicity test showed that the maximum tolerated dose (MTD) of EMB was 400 mg/kg. A laxative effect was observed in mice at different dosages (6.5, 13 and 26 mg/kg). EMB showed a dose-dependent acceleration of gastric emptying (13 and 26 mg/kg). It also promoted both small intestinal (6.5, 13 and 26 mg/kg) and large intestinal (4, 8 and 16 mg/kg) transit activity. In the SD rat model, single rectal administration of EMB (8 and 16 mg/kg) showed a significant increase in both the frequency and amplitude of proximal colon smooth muscle contractility. These increases in amplitude and frequency peaked 30–60 min after EMB administration and corresponded with the results of the laxative activity test. The ex vivo experiments showed that varying doses of EMB (11.5, 23 and 46 mg/kg) had a direct prokinetic effect that was sensitive to atropine.

Conclusions

Our results show that EMB is a low dosage, fast acting drug with a large therapeutic window (4–400 mg/kg) and shows significant gastrointestinal prokinetic action after single rectal or large intestinal administration. This gastrointestinal prokinetic effect was stronger in the intestines than in the stomach. This effect was sensitive to atropine, suggesting that EMB acts mainly through cholinergic mechanisms.     

Melanin-concentrating hormone-1 receptor antagonism and anti-obesity effects of ethanolic extract from Morus alba leaves in diet-induced obese mice

Ethnopharmacological relevance

In Korea, Morus alba leaves have been traditionally administered as natural therapeutic agent for the alleviating dropsy and diabetes.

Aim of the study

The present study was performed to evaluate melanin-concentrating hormone receptor subtype 1 (MCH1) antagonism of the ethanol extract of Morus alba leaves (EMA) and its anti-obesity effect in diet-induced obese (DIO) mice.

Materials and methods

The binding affinity of EMA for the MCH1 receptor with europium-labeled MCH (Eu-MCH), the function of recombinant MCH1 receptors expressed in CHO cells, and the anti-obesity effects in DIO mice were evaluated.

Results

MCH1 receptor binding studies showed, EMA exhibited a potent inhibitory activity with IC₅₀ value of 2.3 ± 1.0 μg/ml. EMA (10–100 μg/ml) also inhibited the intracellular calcium mobilization with the recombinant MCH1 receptors expressed in CHO cells. In an anti-obesity study with DIO mice, long-term oral administrations of EMA for 32 consecutive days produced a dose-dependent decrease in body weight and hepatic lipid accumulation.

Conclusions

These results suggest that chronic treatment with EMA exerts an anti-obesity effect in DIO mice, and its direct MCH1 receptor antagonism may contribute to decrease body weight.     

Evidence of the mechanism of action of Erythrina velutina Willd (Fabaceae) leaves aqueous extract

Ethnopharmacological relevance

Erythrina velutina is traditionally used for sleepiness, convulsions and nervous system excitation in Brazil. Although central effects have been reported for Erythrina velutina, little is known about its mechanism of action.

Aim of the study

To investigate the pharmacological evidences of mechanism of action of Erythrina velutina leaves aqueous extract (AE).

Materials and methods

Terminal segments of the guinea-pig ileum (n = 5–8) were mounted in an organ bath and isotonic contractions were recorded. Phytochemical screening was carried out on AE.

Results

AE (0.025–2.50 mg/ml) produced contractile response in the guinea-pig ileum, yielding typical concentration–response curves (EC₅₀ = 0.63 mg/ml). Electrically evoked contractions were significantly increased in the presence of AE. AE-elicited contractions were significantly reduced by bicuculline, tetrodotoxin, atropine, verapamil or incubation in low calcium–high potassium solution. Atropine along with verapamil abolished AE contractile response. Alkaloids, catechins, steroids, flavonols, flavononols, flavonoids, phenols, saponins, tannins, triterpenoids, and xanthones were detected in AE.

Conclusions

AE contains important constituents for pharmacological activities. AE-induced contractions seem to involve GABA_A receptor activation, acetylcholine release, muscarinic receptor activation, augmentation of Ca²⁺ entry through L-type calcium channels, and calcium release from the intracellular stores. These findings provide further support for Erythrina velutina traditional uses.     

NMR metabolomics for identification of adenosine A1 receptor binding compounds from Boesenbergia rotunda rhizomes extract

Ethnopharmacological relevance

Boesenbergia rotunda Linn. (Zingiberaceae) is traditionally used in many Asian countries as medicine for stomach pain and discomfort, viral and bacterial infection, inflammation, and as diuretic agent.

Aim of the study

The study aimed to identify adenosine A1 receptor binding compounds from Boesenbergia rotunda rhizome extract by using comprehensive extraction coupled to the NMR metabolomics method.

Materials and methods

Dried and powdered Boesenbergia rotunda rhizomes were extracted with the comprehensive extraction method to obtain several fractions with different polarity. Each fraction was divided into two: for NMR analysis and for adenosine A1 receptor binding test. Orthogonal projection to the least square analysis (OPLS) was used to study the correlation between metabolites profile and adenosine A1 receptor binding activity of the plant extracts. Based on Y-related coefficient and variable of important (VIP) value, signals in active area of OPLS loading plot were studied and the respective compounds were then elucidated

Results and discussions

Based on OPLS Y-related coefficient plot and variable of importance value plot, several characteristic signals were found to positively correlate to the binding activity. By using 1D and 2D NMR spectra of one of the most active fraction, pinocembrine and hydroxy-panduratin were identified as the possible active compounds. Two signals from ring C of pinocembrine flavanone skeleton with negative coefficient correlations possibly overlapped with those of non-active methoxylated flavanones which were also presence in the extract. NMR based metabolomics applied in this study was able to quickly identify bioactive compounds from plant extract without necessity to purify them. Further confirmation by isolating pinocembrine and hydroxy-panduratin and testing their adenosine A1 receptor binding activity to chemically validate the method are required.

Conclusion

Two flavonoid derivatives, pinocembrine and hydroxy-panduratin, have been elucidated as possible active compounds bind to adenosine A1 receptor. Flavonoid was reported to be one of natural antagonist ligand for adenosine A1 receptor while antagonistic activity to the receptor is known to associate with diuretic activity. Thus, the result of this research supports the traditional use of Boesenbergia rotunda rhizome extract as diuretic agent.     

Ameliorative effects of Ocimum sanctum in sciatic nerve transection-induced neuropathy in rats

Objectives

The present study was aimed at investigating the ameliorative effect of Ocimum sanctum in sciatic nerve transection (axotomy)-induced peripheral neuropathy in rats.

Materials and methods

Sciatic nerve transection-induced axonal degeneration was assessed histopathologically. Paw pressure, Von Frey Hair, tail cold-hyperalgesia, motor in-coordination tests were performed to assess the extent of neuropathy. Biochemical estimations of thiobarbituric acid reactive species (TBARS), reduced glutathione (GSH), and total calcium levels were also performed. Methanolic extract of Ocimum sanctum at different doses (50, 100 and 200 mg/kg p.o.) was administered for 10 consecutive days starting from the day of surgery.

Results

Administration of Ocimum sanctum attenuated sciatic nerve transection-induced axonal degeneration, reduction of nociceptive threshold and motor in-coordination. Moreover, it also attenuated axotomy-induced rise in TBARS, total calcium and decrease in GSH levels in a dose-dependent manner.

Conclusion

Anti-oxidant and calcium attenuating actions may be responsible for observed ameliorative effects of Ocimum sanctum in axotomy-induced neuropathy.     

Ethnopharmacology of Q’eqchi’ Maya antiepileptic and anxiolytic plants: Effects on the GABAergic system

Ethnopharmacological relevance

The Q’eqchi’ Maya possess a large selection of plants to treat neurological disorders, including epilepsy and susto (fright), a culture-bound illness related to anxiety disorders.

Aim of the study

To investigate the activity of antiepileptic and anxiolytic plants in the GABAergic system, and determine if there is a pharmacological basis for plant selection.

Materials and methods

Ethanol extracts of 34 plants were tested in vitro for their ability to inhibit GABA-transaminase (GABA-T) or bind to the GABA_A-benzodiazepine (BZD) receptor, two principal drug targets in epilepsy and anxiety. Pharmacological activity was correlated with relative frequency of use, based on informant consensus.

Results

Ten plants showed greater than 50% GABA-T inhibition at 1 mg/ml, while 23 showed greater than 50% binding to the GABA_A-BZD receptor at 250 μg/ml. Piperaceae, Adiantaceae and Acanthaceae families were highly represented and active in both assays. There was a significant positive correlation between GABA-T inhibition and relative frequency of use for epilepsy, and an even stronger correlation between GABA_A binding and relative frequency of use for susto (fright).

Conclusions

Clearly, Q’eqchi’ traditional knowledge of antiepileptic and anxiolytic plants is associated with the use of pharmacologically active plants. Based on the evidence, it is suggested that the mechanism of action for some traditionally used plants may be mediated through the GABAergic system.