Antiosteoporotic effects and proteomic characterization of the target and mechanism of an Er-Xian Decoction on osteoblastic UMR-106 and osteoclasts induced from RAW264.7.

Er-Xian Decoction (EXD) has been used for the treatment of osteoporosis disorders, menopausal syndrome, and other aging diseases in the Chinese traditional healthcare system. However, the targets and mechanism of action have not been clarified. This study was designed to investigate the effects and possible target proteins of EXD on osteoblastic UMR-106 cells and osteoclasts induced from RAW264.7 cells using the proteomic analysis technique. We found that EXD at a concentration of 50-200 microg/mL significantly enhanced osteoblastic UMR-106 cell proliferation, alkaline phosphatase (ALP) activity and formation of bone nodules, and decreased tartrate-resistant acid phosphatase (TRAP) activity and the bone resorption action of osteoclasts induced from RAW 264.7 cells. In EXD-treated osteoblasts, there were increases in the expression of heat-shock protein 1, high mobility group protein (Hmgb1), acidic ribosomal phosphoprotein P0, histone 2, carbonyl reductase 1, ATP synthase, aldolase A, and Rho GDP dissociation inhibitor (GDI)-alpha; and reduction in the expression of carbonic anhydrase 3, prohibitin, hemiferrin, far upstream element (FUSE)-binding protein. In EXD-treated osteoclasts, there were increases in the expression of vimentin, protein disulfide isomerase associated 3 and alpha-fetoprotein; and reduction in the expression of calnexin. These results indicated that EXD modulates bone metabolism through regulation of osteoblastic proliferation, apoptosis, and cell activation, and osteoclastic protein folding and aggregation.

Antinociceptive activity of Paederosidic Acid Methyl Ester (PAME) from the n-butanol fraction of Paederia scandens in mice.

Antinociceptive activity of Paederosidic Acid Methyl Ester (PAME), a chemical compound isolated from the n-butanol fraction of Paederia scandens, was evaluated in mice using chemical and thermal models of nociception. PAME given by intraperitoneal injection at doses of 20, 40 and 60 mg/kg produced significant inhibitions on chemical nociception induced by intraperitoneal acetic acid, subplantar formalin or capsaicin injections and on thermal nociception in the tail-flick test and the hot plate test. In the pentobarbital sodium-induced sleep time test and the open-field test, PAME neither significantly enhanced the pentobarbital sodium-induced sleep time nor impaired the motor performance, indicating that the observed antinociceptive activity of PAME was unlikely due to sedation or motor abnormality. Core body temperature measurement showed that PAME did not affect temperature within a 2-h period. Moreover, PAME-induced antinociception in the hot plate test was insensitive to naloxone or nimodipine but significantly antagonized by L-NAME (N (G)-nitro-L-arginine methyl ester), methylene blue and glibenclamide. These results suggested that PAME-produced antinociception was possibly related to the pathway of NO-cGMP-ATP sensitive K(+) channels, which merited further studies regarding the precise site and mechanisms of action.

Authentication and quantitative analysis on the chemical profile of Xanthium fruit (Cang-Er-Zi) by high-performance liquid chromatography-diode-array detection tandem mass spectrometry method.

A high-performance liquid chromatographic method using diode-array detection and electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS/MS) was developed for the qualitative and quantitative analysis on Xanthium fruit, a commonly used traditional Chinese medicine. In this study, 7 characteristic components, 1-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, chlorogenic acid, 1,5-O-di-caffeoylquinic acid, 1,3-O-di-caffeoylquinic acid, 4,5-O-di-caffeoylquinic acid and 1,3,5-O-tri-caffeoylquinic acid were identified and quantified by a validated HPLC-DAD method, and a fingerprint comprised of 12 markers was established under the same operating conditions. Furthermore, HPLC-ESI-MS/MS method was successfully used to deduce the structure of three main constituents. On the basis of the established chromatographic profiles, 30 populations of cocklebur samples including 3 related species and 1 unknown species were divided into 3 chemotypes, indicated that place of origin significantly influences the kinds and content of components in cocklebur, and hence affects their quality. The simultaneous determination of 7 caffeoylquinic acids in the 30 samples showed a great variety in the amounts of caffeoylquinic acids present. The study indicated that some species such as Xanthium mongolicum of the genus Xanthium might be suitable for development as new alternative sources of caffeoylquinic acids to supplement the officially listed Xanthium species, and the abundant constituents such as chlorogenic acid perhaps should be recorded in some authorized publications and applied to the quality control or quality evaluation for Xanthium in China. The entire analytical procedure is reproducible and suitable for the authentication and quantification of Xanthium fruits.

Population genetic structure of the medicinal plant Vitex rotundifolia in China: implications for its use and conservation.

Vitex rotundifolia L. is an important plant species used in traditional Chinese medicine. For its efficient use and conservation, genetic diversity and clonal variation of V. rotundifolia populations in China were investigated using inter-simple sequence repeat markers. Fourteen natural populations were included to estimate genetic diversity, and a large population with 135 individuals was used to analyze clonal variation and fine-scale spatial genetic structure. The overall genetic diversity (GD) of V. rotundifolia populations in China was moderate (GD = 0.190), with about 40% within-population variation. Across all populations surveyed, the average within-population diversity was moderate (P = 22.6%; GD = 0.086). A relatively high genetic differentiation (G(st) = 0.587) among populations was detected based on the analysis of molecular variance data. Such characteristics of V. rotundifolia are likely attributed to its sexual/asexual reproduction and limited gene flow. The genotypic diversity (D = 0.992) was greater than the average values of a clonal plant, indicating its significant reproduction through seedlings. Spatial autocorrelation analysis showed a clear within-population structure with gene clusters of approximately 20 m. Genetic diversity patterns of V. rotundifolia in China provide a useful guide for its efficient use and conservation by selecting particular populations displaying greater variation that may contain required medicinal compounds, and by sampling individuals in a population at >20 m spatial intervals to avoid collecting individuals with identical or similar genotypes.

Anti-nociceptive activity of aqueous fraction from the MeOH extracts of Paederia scandens in mice.

AIM OF THE STUDY: We examined the effects of the aqueous fraction (AF) on nociception models mice induced by the chemical and the thermal stimuli so as to elucidate the analgesic activity and provide scientific basis for the clinical use of Paederia scandens. MATERIALS AND METHODS: The AF of MeOH extract from P. scandens was evaluated on anti-nociceptive activity in mice using chemical and thermal models of nociception. RESULTS: Given orally, the aqueous fraction at doses of 200, 400 and 800 mg/kg produced significant inhibitions on chemical nociception induced by intraperitoneal acetic acid and subplantar formalin injections and on thermal nociception in the tail-flick test and in the hot plate test. More significant inhibition of nociception was observed at dose of 800 mg/kg of this fraction. In the pentobarbital sodium -induced sleeping time test and the open-field test, the aqueous fraction neither significantly enhanced the pentobarbital sodium -induced sleeping time nor impaired the motor performance, indicating that the observed anti-nociception was unlikely due to sedation or motor abnormality. CONCLUSIONS: These results suggested that the aqueous fraction produced anti-nociception possibly related to the iridoid glycosides and polysaccharides in this fraction.

Regulative effects of essential oil from Atractylodes lancea on delayed gastric emptying in stress-induced rats.

Gastric motor dysfunction induced by psychological stress results in many symptoms of functional dyspepsia (FD). There are a number of herbal medicines that are reported to improve gastrointestinal motor. However, the mechanisms of considerable herbal medicines are not explicit. In the present study, the effects of an essential oil (EO) extracted from Atractylodes lancea on delayed gastric emptying, gastrointestinal hormone and hypothalamic corticotropin-releasing factor (CRF) abnormalities induced by restraint stress in rats were investigated and the mechanism of the EO is also explored. Oral administration of EO for 7 days did not affect normal gastric emptying, but accelerated delayed gastric emptying induced by restraint stress in rats. The EO significantly increased the levels of motilin (MTL) and gastrin (GAS) and decreased the levels of somatostatin (SS) and CRF. The EO did not modify gastric emptying in vagotomized rats that underwent restraint stress, but antagonized delayed gastric emptying induced by intracisternal injection of CRF. These results suggest that the regulative effects of the EO on delayed gastric emptying are preformed mainly via inhibition of the release of central CRF and activation of vagal pathway, which are also involved in the release of gastrointestinal hormones such as MTL, GAS and SS.

Correlation between the genetic diversity and variation of total phenolic acids contents in Fructus Xanthii from different populations in China.

Fructus Xanthii (Cang-Er-Zi) is a traditional Chinese medicine that is used in curing nasal diseases and headache according to the Chinese Pharmacopoeia. For the effective quality control of its medicinal values, reflected by chemical variation patterns, in addition to the relationship with genetic diversity, analyses based on UV spectrophotometry, HPLC fingerprinting and inter-simple sequence repeat (ISSR) molecular markers were carried out, involving 16 Xanthium populations from different locations in China. The HPLC data showed considerable variation of chemical constituents among the 16 Xanthium populations, and they were classified to three chemotypes by hierarchical clustering analysis. Abundant genetic diversity was detected among the Xanthium populations, which were also clustered into three groups based on their ISSR data and varied according to different species. Combining the genetic divergence and chemical differences showed an important result that, in the two chemotypes, the higher contents of total phenolic acids (TPA) in Fructus Xanthii showed greater genetic diversity (I). We suggest that genetic diversity affects the contents of TPA. Since variable phenolic acid contents may affect therapeutic efficacy, it is important to point out that combining the use of genetic base with chemotype will help control the favourable chemotypes and breed new cultivars with more desirable chemical constituents.

Antinociceptive activity of petroleum ether fraction from the MeOH extracts of Paederia scandens in mice.

The petroleum ether fraction of MeOH extract from Paederia scandens was evaluated on anti-nociceptive activity in mice using chemical and thermal models of nociception. Given orally, the petroleum ether fraction (PEF) at doses of 20, 40 and 80mg/kg produced significant inhibitions on chemical nociception induced by intraperitoneal acetic acid and subplantar formalin or capsaicin injections and on thermal nociception in the tail-flick test and in the hot plate test. More significant inhibition of nociception was observed at dose of 80mg/kg of the petroleum ether fraction. In the pentobarbital sodium-induced sleeping time test and the open-field test, the petroleum ether fraction neither significantly enhanced the pentobarbital sodium-induced sleeping time nor impaired the motor performance, indicating that the observed anti-nociception was unlikely due to sedation or motor abnormality. Moreover, the petroleum ether fraction-induced anti-nociception in both capsaicin and formalin tests was insensitive to naloxone, but was significantly antagonized by glibenclamide. These results suggested that the petroleum ether fraction produced anti-nociception possibly related to glibenclamide-sensitive K(+)-ATP channels, which merited further studies regarding the precise site and mechanism of action. The major constituents of the petroleum ether fraction (PEF) determined by GC/MS analysis, are linoleic acid, the sterols and vitamin E. Therefore it can be suggested that they exert synergetic effects and are together responsible for the antinociceptive activity of the PEF-fraction.

[Effect of Liriope platyphylla total saponin on learning, memory and metabolites in aging mice induced by D-galactose].

OBJECTIVE: To investigate the effects of Liriope platyphylla total saponin (LPTS) on learning, memory, neuromediators and metabolites in aging mice induced by D-galactose. METHODS: Ninety Kunming mice were randomly divided into nine groups: normal saline (NS)-treated group, untreated group, high- (100 mg/kg), medium- (50 mg/kg) and low-dose (10 mg/kg) LPTS-treated groups, Shuxuening-treated group, jiaogulanosidi-treated group, flunarizine-treated group and vitamin E-treated group. The Kunming mice in the NS-treated group were administered with NS by intraperitoneal injection, while the aging mice in the other eight groups were administered with D-galactose by intraperitoneal injection. At the same time, the aging mice in different groups were fed with corresponding drugs for 42 days, and the aging items of the mice in different groups were measured, respectively. RESULTS: LPTS could improve the memory of aging mice induced by D-galactose, promote its body weight, and increase the thymus and spleen indexes of the aging mice. LPTS could decrease the levels of MDA and lipofuscin, inhibit MAO activity and increase SOD activity and GSH-Px level. CONCLUSION: LPTS may improve the ability of learning and memory and delay aging.

Evaluation of the estrogenic activity of the constituents in the fruits of Vitex rotundifolia L. for the potential treatment of premenstrual syndrome.

The ethanol extract of the fruits of Vitex rotundifolia (VRE) and its four major compounds (casticin, luteolin, rotundifuran and agnuside) were tested for their estrogen-like activity by using the modified cell proliferation assay (E-SCREEN assessment system), as well as the estrogen receptor (ER(alpha)), estrogen receptor-regulated progesterone receptor and pS2 mRNA expression in MCF-7 cells. The results showed that only agnuside and rotundifuran could stimulate the proliferation of MCF-7 cells. These actions were dose dependent (range from 100 nM to 10 microM) and could be significantly inhibited by the specific estrogen receptor antagonist ICI 182,780. The estrogen receptor ER(alpha) and the estrogen receptor-regulated progesterone receptor and pS2 mRNA levels were increased by treatment with rotundifuran and agnuside within 24 h, and the effects could be reversed by ICI 182,780. The standardization of the extract and constituents were carried out by means of a high-performance liquid chromatography-fingerprint. It was concluded that VRE and its compounds showed estrogen-like activity and that the estrogenic effects of rotundifuran and agnuside were mediated by the estrogen inducible gene, which may be useful in regulating the hormone levels to treat related diseases. However, further studies are required to assess the physiological significance of VRE in animals and man.

Estrogen-like activity of volatile components from Vitex rotundifolia L.

BACKGROUND & OBJECTIVES: The fruits of Vitex rotundifolia L. are used as a traditional medicine in China. Dried fruits of V. rotundifolia (rich in essential oils) have been known to show strong estrogenic activity. Therefore, we investigated volatile components in the essential oil isolated from the fruits of V. rotundifolia and its estrogen-like biological activity in human breast adenocarcinoma cells. METHODS: Essential oil was extracted by SFE (supercritical fluid extraction) and its chemical composition was analysed by gas chromatography-mass spectrometry (GC-MS). The estrogenic activity of essential oil was evaluated in the MCF-7 cells by proliferation assay. RESULTS: The chemical composition of 40 compounds was determined and this represented 84.1 per cent of the total oil which was characterized by a high content of unsaturated fatty acids (54.84% of total oil). The oil contained mainly linoleic acid (47.46%), palmitic acid (5.18%), untriacontane (2.28%), bicetyl (2.61%) and stearic acid (2.2%). The essential oil strongly stimulated the proliferation of MCF-7 cells, and this effect was significantly inhibited by the specific estrogen receptor antagonist ICI 182,780. INTERPRETATION & CONCLUSION: Our results showed estrogen-like biological activity of the essential oil of the fruits from V. rotundifolia, and in future has a potential medical application.

[Studies on chemical constituents of Liriope platyphylla].

Seven chemical constituents were isolated from the CHCl3 fraction and n-BuOH fraction from EtOH ext. of Liriope platyphylla Wang et Tang. Their structures were elucidated as lupenone (I), lupeol (II), ursolic acid (III), beta-sitosterol (IV), diosgenin (V), LP-A (VI) and LP-B (VII) respectively. Compound I, II and V were isolated from this genus of Liriope for the first time. Compound III and IV were isolated from this plant for the first time.

Antinociceptive activity of n-butanol fraction from MeOH extracts of Paederia scandens in mice.

The n-butanol fraction of the MeOH extract from Paederia scandens was evaluated for antinociceptive activity in mice using chemical and thermal models of nociception. The n-butanol fraction, given orally at doses of 150, 300 and 600 mg/kg produced significant inhibition of chemical nociception induced by intraperitoneal acetic acid and subplantar formalin or capsaicin injections, and of thermal nociception in the tail-flick test and in the hot plate test. More significant inhibition of nociception was observed at a dose of 600 mg/kg. In the pentobarbital sodium-induced sleeping time test and the open-field test, the n-butanol fraction neither significantly enhanced pentobarbital sodium-induced sleeping time nor impaired motor performance, indicating that the observed anti-nociception was unlikely to be due to sedation or motor abnormality. Moreover, the n-butanol fraction-induced anti-nociception in both the capsaicin and formalin tests was insensitive to naloxone or glibenclamide but was significantly antagonized by nimodipine. These results suggest that the n-butanol fraction produced anti-nociception possibly related to nimodipine-sensitive L-type Ca2+ channels, which merits further studies regarding the precise site and mechanism of action. Furthermore, four iridoid glycosides isolated from the n-butanol fraction might be related to its antinociceptive action fraction. Therefore, the effect on analgesic activity of each chemical compound in large quantity might well be considered in a further study.

Prevention of bone loss by aqueous extract of Epimedii sagittatum in an ovariectomized rat model of osteoporosis.

OBJECTIVE: To investigate the prevention effect of aqueous extract of Epimedii sagittatum (ESE) on ovariectomy-induced (OVX) bone loss in rats. METHODS: Rats were divided into sham-operated and OVX groups. The OVX rats were divided into four groups treated with distilled water, 17beta-estradiol (1 mg/kg, ig) and ESE (0.5 and 1 g/kg, ig) for 11 weeks. Serum calcium, phosphorus, estradiol, bone gla protein concentrations and serum alkaline phosphatase activity were measured. Bone density was assayed by dual-energy X-ray absorptiometry. The undecalcified longitudinal proximal tibial metaphysical sections were cut and stained for the bone histomorphometric analysis. RESULTS: In OVX rats, alkaline phosphatase activity in serum was markedly increased by ESE treatment, which had no obvious influence on the body weight. Meanwhile, atrophy of uterus and descent of bone mineral density were suppressed by ESE treatment. In addition, ESE completely corrected the decreased concentrations of calcium and E2 in serum observed in OVX rats. Histological results also showed ESE prevented the increases in trabecular separation (Tb.Sp) in OVX rats whereas it did not alter trabecular thickness (Tb.Th) and trabecular number (Tb.N) in OVX rats. Moreover, ESE had remarkable effect on bone formation rate with bone volume as referent (BFR/BV) and bone formation rate with bone surface as referent (BFR/BS). CONCLUSION: The findings assessed on the basis of biochemical test, bone mineral density and histomorphometric parameters show that aqueous extract of Epimedii sagittatum has a definite antiosteoporotic effect and can prevent the OVX-induced bone loss in rats.

Antiosteoporotic activity of Er-Xian Decoction, a traditional Chinese herbal formula, in ovariectomized rats.

The antiosteoporotic effect of a herbal formula, Er-Xian Decoction (EXD), in ovariectomized (OVX) rats model of osteoporosis was investigated. The rats were divided into Sham and OVX groups. The OVX rats were further sub-divided into four groups administered orally with water, nylestriol (1 mg/kg, weekly) or EXD (300, 600 mg/kg, daily) for 12 weeks. In OVX rats, the increases of body weight, serum BGP and ALP were significantly decreased by EXD treatment. In OVX rats, atrophy of uterus and descent of BMD were suppressed by treatment with EXD and nylestriol. In addition, EXD completely corrected the decreased concentration of calcium, phosphorus, and estradiol in serum observed in OVX rats. EXD also significantly increased biomechanical strength comparable to the Sham group. This was also confirmed by histological results that showed its protective action. The findings assessed on the basis of biochemical, bone mineral density, biomechanical, and histopathological parameters strongly suggested that EXD had a definite antiosteoporotic effect, which is similar to estrogen.

Protective effect of steroidal saponins from rhizome of Anemarrhena asphodeloides on ovariectomy-induced bone loss in rats.

AIM: To investigate the protective effect of steroidal saponins from Anemarrhena asphodeloides (ATS) on ovariectomy (OVX)-induced bone loss. METHODS: Sprague-Dawley rats were divided into sham and OVX groups. The OVX rats were treated with vehicle, nylestriol or steroidal saponins extract for 12 weeks. Serum calcium, phosphorus, estradiol (E(2)), osteocalcin concentration and serum alkaline phosphatase activity were measured. Bone density was assayed by dual-energy X-ray absorptiometry. The undecalcified longitudinal proximal tibial metaphysical (PTM) sections were cut and stained for histomorphometric analysis of the bone. RESULTS: In OVX rats, alkaline phosphatase activities in serum were markedly increased and concentrations of osteocalcin were decreased by ATS treatment, which had no influence on the body weight. Meanwhile, atrophy of the uterus and descent of bone mineral density (BMD) was suppressed by treatment with ATS. In addition, ATS completely corrected the decreased the concentration of calcium and E(2) in serum observed in OVX rats. Histological results showed ATS prevented decreases in trabecular thickness and increases in trabecular separation of proximal tibia metaphysis (PTM) in OVX rats. However, it did not alter osteoclast number in OVX rats. Moreover, ATS (300 mg/kg) had a remarkable effect on promoting bone formation action in OVX rats. Nylestriol treatment decreased the bone formation rate and mineral apposition rate. CONCLUSION: An adequate supply of steroidal saponins of Anemarrhena asphodeloides prevented OVX-induced bone loss in rats through the promotion of bone formation but not the inhibition of bone resorption.

Phenolic acids in Fructus Xanthii and determination of contents of total phenolic acids in different species and populations of Xanthium in China.

OBJECTIVE: To study the chemical constituents of Fructus Xanthii and to determine the contents of total phenolic acids (TPA) in fruits of Xanthium from different populations for evaluating the quality of them. METHODS: Components in Fructus Xanthii were isolated and purified by various column chromatographies and the contents of TPA were determined by ultraviolet spectrophotometry with chlorogenic acid (CHA) as reference substance. RESULTS: Six caffeoylquinic acids along with caffeic acid and ferulic acid were isolated from Fructus Xanthii. The contents of TPA of the samples collected from 29 populations in China varied from 0.31% to 1.44%. Among the samples originated from two species and 1 variety of Xanthium, the contents of TPA in X. sibiricum var. subinerme samples with an average of 0.36% were relatively lower than those in other 2 species. While the content of TPA in Sample 3 collected from Shanghai was 1.44% and the highest among all the samples, and that in Sample 12 from Xinjian of Jiangxi Province was 0.38% and the lowest among the X. sibiricum samples. CONCLUSION: 5-O-caffeoylquinic acid, 1,4-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid were isolated from Xanthium plant for the first time. The difference of contents of TPA in samples from different species and different populations in China was relatively significant. Fructus Xanthii in Shanghai and Sanming of Fujian Province were considered high quality if contents of TPA were used as reference for quality evaluating.

[Effect of madecassoside on depression behavior of mice and activities of MAO in different brain regions of rats].

OBJECTIVE: To evaluate the effects of madecassoside (MC) on the depression behavior of mice and the activities of monoamine oxidase (MAO) in different rat brain regions. METHODS: Imipramine as the positive contrast medicine, effects of MC on the depression behavior of mice were observed by forced swimming test and reserpine antagonist test. Moclobemide and pargyline as the positive controlled medicines, the activities of monoamine oxidase-A (MAO-A) and monoamine oxidase-B (MAO-B) in different rat brain regions were determined after intragastric administration of MC in 3 different dosages for 3 days or 21 days. RESULTS: (1) The low, middle and high dosages of MC (i.g.) significantly reduced the immobility time of mice in forced swimming test (P<0.05). (2) MC in dosages of 10 mg/kg and 20 mg/kg prevented the lowering of temperature induced by reserpine (P<0.05), while 40 mg/kg had no significant effects on it (P>0.05). (3) With acute administration (3 days), the low, middle and high dosagey of MC (i.g.) significantly inhibited the activity of MAO-A in hippocampus (P<0.01), and the high dosage significantly inhibited the activity of MAO-A in hypothalamus (P<0.01), while the 3 dosages had no significant effects on the activity of MAO-A in cortex (P>0.05). With chronic administration (21 days), MC in 3 dosages had no significant effects on the activities of MAO-A in cortex and hypothalamus (P>0.05), and the high dosage (40 mg/kg) significantly enhanced the activity of MAO-A in hippocampus (P<0.01). (4) With acute administration, MC in dosages of 10 mg/kg and 20 mg/kg significantly inhibited the activity of MAO-B in cortex (P>0.05), and MC in dosage of 10 mg/kg significantly inhibited the activity of MAO-B in hypothalamus (P<0.05), and MC in dosage of 20 mg/kg significantly enhanced the activity of MAO-B in hippocampus (P<0.01). With chronic administration, MC of 3 dosages produced no significant effects on the activities of MAO-B in 3 different rat brain regions (P>0.05). CONCLUSION: These results support the idea that MC produces antidepressant effects through MAO inhibition in rat brain, which seems stronger with acute administration than chronic administration, while its mechanism remains to be further studied.

[Studies on identification of Gryllotalpa by near-infrared diffuse reflectance spectrometry].

OBJECTIVE: To identify and analyse the different species, same species in different regions and confusion species. METHOD: Near-infrared diffuse reflectance spectrometry was used. RESULT: Clustering analysis showed that clustering relations were far among different Gryllotalpa species and close among the same species from different regions, and there were close relations among the same species from near regions and between Teleogryllus emmus and G. orientalis. CONCLUSION: Near-infrared diffuse reflectance spectrometry method can be used in classification and identification of Gryllotalpa.

Total coumarins from fruits of Cnidium monnieri inhibit formation and differentiation of multinucleated osteoclasts of rats.

AIM: To determine the effects of TCFC (total comarins from the fruits of Cnidium monnieri) on the activity of osteoclasts in vitro. METHODS: Osteoclasts isolated from rat marrow cells were co-cultured with osteoblasts under the 1,25-dihydroxyvitamine D3. The tartrate-resistant acid phosphatase (TRAP) stain was used to identify osteoclast morphology. The activity of TRAP was measured by p-nitrophenyl sodium phosphate assay. The resorption pit area on the bone slices formed by osteoclasts was measured by computer image processing. Calcium concentration in the medium of co-culture of bone slices and osteoclasts was determined by atomic absorption spectra. RESULTS: TCFC 2.5-25 mg/L inhibited osteoclast formation and differentiation. TCFC 0.25-25 mg/L inhibited TRAP activity of osteoclasts and TCFC 25 mg/L decreased the TRAP activity by 26.3 % and 24.1 % after 48 h and 72 h, respectively. TCFC 25 mg/L decreased the osteoclastic bone resorption pit area by 25.05 % and Ca2+ release from bone slices by 41.73 %. CONCLUSION: TCFC reduced the bone lose by decreasing the osteoclast formation, its TRAP activity, and osteoclastic bone resorption.