Determination of dehydroevodiamine in Evodia rutaecarpa (Juss.) Benth by high performance liquid chromatography and classification of the samples by using hierarchical clustering analysis

A simple, sensitive and accurate liquid chromatographic method with photodiode-array detection was developed for determination of dehydroevodiamine with detection wavelength at 368 nm and column temperature at 30 °C. The separation was carried out on an Agilent Zorbax SB-C₁₈ column (250 mm × 4.6 mm, 5 µm) together with a C₁₈ guard column. The mobile phase was acetonitrile–water (containing 30 mM sodium acetate trihydrate and 0.15% acetic acid) in the ratio of 30:70 (v/v) delivered at a flow rate of 1 mL/min. Excellent linear behavior was observed over the concentration range investigated, with correlation coefficient (R²) = 0.9998. This validated method was applied to determine the contents of dehydroevodiamine in 36 samples from different regions of China, and hierarchical clustering analysis was firstly used to classify and differentiate Evodia rutaecarpa samples. The analysis is specific and can be successfully applied to analyze E. rutaecarpa which is helpful for quality control of the herb.     

Cytotoxic sesquiterpenoids from the fruits of Lindera communis

A new sesquiterpenoid, namely Linerenone (1), together with three known sesquiterpenoids (2–4), were isolated from the fruits of Lindera communis. Their structures were determined by extensive spectroscopic analysis including 1D, 2D-NMR and HR-MS spectra. Compound 1 showed significant cytotoxic activity against H460, ES2 and DU145 cancer cells with IC₅₀ of 2.1 μg/mL, 2.8 μg/mL and 3.0 μg/mL, respectively.     

Successive media to improve the in vitro rhizogenesis of Nothofagus nervosa (Phil.) Dim. et Mil.

In vitro rooting constitutes a difficult step during the micropropagation process of forest species. The successive media culture technique represents one way to overcome this barrier and includes modifying physical (e.g. photoperiod) and chemical (e.g. flavonoids) factors during the rooting phases. The aim of this study was to obtain a successive media protocol based on the incorporation of flavonoids during the in vitro rooting of Nothofagus nervosa. The factors evaluated were the type, concentration, and combination of flavonoids in relation to the rooting phases, the presence of IBA in the culture medium, the photoperiod, and the effect of flavonoids on total tissue peroxidase activity. The photoperiod used included a darkness period during the rooting induction stage and the presence of 0.61 µMIBA in the culture medium. The results showed that flavonoid incorporation at a concentration of 20 µM accelerated the appearance of roots and improved the quality of the already formed ones. Each type and concentration of flavonoid produced different responses, with (±)naringenin giving the best results. The latter caused a peak in the peroxidase activity that was absent in the control treatments. This work allowed identifying an optimized rooting protocol through a successive media culture technique that improved the speed of appearance, as well as the quantity and quality of roots for a single N. nervosa clone.     

In vitro metabolism in Sprague-Dawley rat liver microsomes of forsythoside A in different compositions of Shuang-Huang-Lian

Shuang–Huang–Lian (SHL), a traditional Chinese formula containing Lonicerae japonicae flos (LJF), Scutellariae radix (SR) and Forsythiae fructus (FF), is commonly used to treat acute upper respiratory tract infection, acute bronchitis and light pneumonia. Forsythoside A is one of the main active ingredients in Forsythiae fructus, a key herb in SHL. In the present study, effects of different compositions in SHL on the in vitro metabolism in Sprague–Dawley rat liver microsomes of forsythoside A were investigated. The observations from Sprague–Dawley rat liver microsomes in the presence of β-NADPH or UDPGA that forsythoside A may be the substrates of CYP3A4, CYP2C9, CYP1A2, UGT1A6, UGT1A3, UGT1A1 and UGT1A9; Chlorogenic acid may be the substrates of CYP3A4, CYP2C9, CYP1A2, CYP2C19, UGT1A6, UGT1A3 and UGT1A1; Baicalin may be the substrates of CYP3A4, CYP2C19, CYP1A2, UGT1A9, UGT1A1 and UGT1A3; Baicalein may be the substrates of CYP3A4, CYP2E1 and UGT1A6. It was also found that the residue of forsythoside A in SHL, FF + LJF and FF + SR was greatly increased compared with that in FF in Sprague–Dawley rat liver microsomes in the presence of β-NADPH or UDPGA, which indicated that the metabolism of forsythoside A in SHL may be influenced by chlorogenic acid in LJF acting on the CYP3A4, CYP2C9, CYP1A2, UGT1A6, UGT1A3 and UGT1A1; baicalin in SR acting on the CYP3A4, CYP1A2, UGT1A9, UGT1A1 and UGT1A3; baicalein acting on the CYP3A4 and UGT1A6 respectively.     

Iridoids from the roots of Valeriana jatamansi and their neuroprotective effects

Three new iridoids, valeriandoids A–C (1–3), together with three known analogues (4–6), were isolated from the roots of Valeriana jatamansi. Their structures and relative configurations were elucidated by spectroscopic methods (IR, ESIMS, HRESIMS, 1D and 2D NMR) and by comparison of their NMR data with those of related compounds. All the isolated compounds were evaluated for their neuroprotective effects and compounds 1, 3, 4 and 6 showed moderate neuroprotective effects.     

林分密度对蒙古栎林下草本植物多样性的影响研究

以内蒙古赤峰市阿鲁科尔沁旗沙日温都栎林自然保护区蒙古栎林为主要研究对象,研究不同林分密度对林下草本植物多样性的影响。结果表明,研究区内共有13科、14属、16种草本植物;Simpson指数、Shannon-Wiener指数、Pielou均匀度指数和物种丰富度指数均随着林分密度的增加呈先增大后减小的趋势,当林分密度为750株/hm²时,林下草本植物多样性指数达到最大值;林分密度与Shannon-Wiener多样性指数、Pielou均匀度指数、Simpson多样性指数、物种丰富度指数、树高、胸径均呈极显著负相关,与郁闭度呈极显著正相关,与枝下高不相关。综上所述,最合理的蒙古栎林密度为750株/hm²,该密度下最有利于蒙古栎林及林下草本植物生长发育。     

neo-Clerodane diterpenes from Ajuga ciliata Bunge and their neuroprotective activities

Three new (1–3) and three known (4–6) neo-clerodane diterpenes have been isolated from the whole plants of Ajuga ciliata Bunge. The structures of the new compounds were elucidated as (12S)-1β,6α,19-triacetoxy-18-chloro-4α,12-dihydroxy-neo-clerod-13-en-15,16-olide (1), (12S,2′S)-12,19-diacetoxy-18-chloro-4α,6α-dihydroxy-1β-(2-methylbutanoyloxy)-neo-clerod-13-en-15,16-olide (2), and (12S)-6α,18,19-triacetoxy-4α,12-dihydroxy-1β-tigloyloxy-neo-clerod-13-en-15,16-olide (3), on the basis of spectroscopic data analysis. All the diterpenes were evaluated for the neuroprotective effects against MPP⁺-induced neuronal cell death in dopaminergic neuroblastoma SH-SY5Y cells and compounds 2–5 exhibited moderate neuroprotective effects.     

Fire fighting truck-based emergency mosquito biolarviciding to prevent outbreaks of malaria and arboviral disease in Kabul, Afghanistan

Emergency control of disease vectors requires high efficacy, rapid reaction and safe use of biocides in order interrupt transmission cycles without harming humans, non-target animals and the environment. In countries with complex emergencies, air-borne large-scale vector control is often limited, or impossible, due to questionable security as well as military, safety, equipment, or logistical constraints. While facing a potential outbreak of malaria and dengue fever in the Kabul area, Afghanistan, combined with high abundance of anopheline and Culex mosquitoes, emergency mosquito larvae control with Bacillus thuringiensis var. israelensis (Bti) and methoprene were carried out. For the first time, larvicides were applied by using a modern fire fighting truck in combination with aerial imaging of potential breeding sites. Three treatments with 0.07 g a.i. sterilized, water-dispersible granular (WDG) Bti/m² in combination with 0.0014 g a.i. methoprene/m², or 0.07 g a.i. Bti/m² alone, were conducted on July 29, August 29, and September 28, 2004, and significantly reduced larval indexes of 96.5 (P < 0.0001), 96.7 (P < 0.0001), and 97.6% (P < 0.0001) against anopheline, and 98.2 (P < 0.0001), 95.6 (P < 0.0001), and 96.2% (P < 0.0001) against Culex larvae, respectively. Before the larviciding campaigns started, up to 222.4 ± 32.0 mosquitoes were captured per standardized light trap per night. During May until October 2004, the following anopheline and Culex species were abundant: A. superpictus (29.3%), A. fluviatilis (17.0%), C. pipiens (15.2%), and C. pseudovishnui (38.5%). After biolarviciding, abundance of adult mosquitoes, as measured by light trap catches 20 days post-treatment, decreased significantly in Anopheles at 81.4 (P < 0.0001), 87.1 (P < 0.0001), and 78.2% (P = 0.01), and in part significantly in Culex for 75.2 (P < 0.0001), 78.3 (P < 0.0001), and 30.8% (P = 0.463), respectively. After the treatment, mosquito-associated annoyance complaints and disease transmission decreased markedly, and not a single confirmed case of malaria or arbovirosis was reported. It is concluded that biolarviciding using a fire fighting truck as the application device is a highly successful, rapid, and cost-effective method to control vector mosquito larvae, and to reduce human exposure to mosquito-borne diseases during complex emergency situations.     

Seasonal change in the temperature coefficientQ 10 for respiration of field-grown hinoki cypress (Chamaecyparis obtusa) trees

The effect of temperature upon nighttime respiration was examined on four different sized sample trees in a 17-year-old hinoki cypress (Chamaecyparis obtusa (Sieb. et Zucc.) Endl.) stand over two years. Seasonal changes inQ ₁₀ values and their responses to mean temperature were investigated. On the basis of the monthly relationships between nighttime respiration (r) and temperature inside a chamber (θ),r=r ₀exp (kθ), theQ ₁₀ value (=exp(10k)) was calculated. TheQ ₁₀ values were high (Q ₁₀≥3.0) in winter when mean air temperature was low, and gradually decreased toward summer (Q ₁₀≤1.5) through spring with increasing temperature. TheQ ₁₀ values were negatively correlated with mean air temperature. The response ofQ ₁₀ values to mean air temperature was described by a single equation, regardless of tree size. This result, which might be characteristic of this species, shows that respiration ofC. obtusa trees is promoted by slight increases of air temperature in winter season. On the other hand, temperature sensitivity of total respiration reduced during growing season when ambient temperature was high. These chaning temperature sensitivity according to seasons may depend on the seasonal change of the ratio of growth respiration to total respiration. It is concluded that changes in temperature due to changing seasons not only change respiration rate, but also change the response of respiration rate to temperature by shiftingQ ₁₀ values.     

Screening antiacne potency of Indonesian medicinal plants: antibacterial,lipase inhibition,and antioxidant activities

Indonesian medicinal plants were screened as potential sources of antiacne agents. The screening methods were performed using antibacterial assay against Propionibacterium acnes, lipase inhibitor assay, and antioxidant assay. The results showed that from 40 plant materials extracted with methanol and 50% ethanol in water, Caesalpinia sappan was the best extract based on the combined activities: antibacterial (minimum inhibitory concentration 0.13 mg/ml; minimum bactericidal concentration 0.25 mg/ml), lipase inhibitory [50% inhibitory concentration (IC₅₀) 120.0 μg/ml], and antioxidative (IC₅₀ 6.47 μg/ml). Another prospective extract is Intsia palembanica based on its lipase inhibitory activity (IC₅₀ 4.1 μg/ml) and antioxidant activity (IC₅₀ 3.87 μg/ml). Part of this report was presented at the 58th Annual Meeting of the Japan Wood Research Society, Tsukuba, March 2008     

Ultra-performance liquid-chromatography with tandem mass spectrometry performing pharmacokinetic and biodistribution studies of croomine,neotuberostemonine and tuberostemonine alkaloids absorbed in the rat plasma after oral administration of Stemonae Radix

Stemonae Radix (Stemona tuberosa Lour, Bai Bu) is an important traditional Chinese medicinal (TCM) plant known for its antitussive activity. Croomine, neotuberostemonine and tuberostemonine alkaloids of Stemonae Radix are major components responsible for antitussive action. In this work, plasma pharmacokinetic and biodistribution characteristics of the three alkaloids after oral administration of Stemonae Radix are investigated using a rapid and sensitive UPLC-Q-TOF–HDMS method. Mass spectrometry (MS) was performed on a Waters Micromass high-definition technology with an electrospray ionization source in positive ion mode, with excellent MS mass accuracy and enhanced MS data acquisition. Separation of main alkaloids was achieved on a Waters BEH C₁₈ column by linear gradient elution. Data were analyzed and estimated by compartmental methods and pharmacokinetic parameters calculated using WinNonlin Professional version 5.1. It was found that croomine, neotuberostemonine and tuberostemonine had faster absorbed into the bloodstream, maintain the high plasma concentration, and pose a large AUC value. The biodistribution of neotuberostemonine and tuberostemonine showed that the higher levels were in liver, and lung. Croomine was discovered in brain and showed that it could cross the blood–brain barrier, indicating that croomine plays an antitussive effect as acting on the central nervous system. Neotuberostemonine and tuberostemonine were not discovered in brain, demonstrating that they play an antitussive effect as peripheral antitussive. This work suggests that the pharmacokinetics and biodistribution based-UPLC-Q-TOF–HDMS can provide a reliable tools for screening bioactive components contributing to pharmacological effects of medicinal herbs.     

Studies on antidepressant and antinociceptive effects of ethyl acetate extract from Piper laetispicum and structure-activity relationship of its amide alkaloids

Piper laetispicum C.DC. (Piperaceae), is an endemic climbing, glabrous plant distributed in the southern part of China. A novel alkaloid amide, Laetispicine, from it has been proven to possess antidepressant activity. In this present study, antidepressant and antinociceptive effects of the ethyl acetate extract (EAE) of P. laetispicum have been studied in forced swimming, open field, acetic acid writhing and formalin tests in KM mice. A significantly antidepressant-like effect was showing at doses of higher than 60 mg/kg, which was not due to an increase in locomotive activity. The EAE also presented an analgesic effect, in our studies. At lower doses (30 mg/kg) the antinociceptive effect was likely mediated via peripheral inflammation and changes in central processing, and at higher doses (120 mg/kg) that was due to both central and peripheral pathways. We also quantitatively analyzed the major components of EAE by HPLC and approached the structure–activity relationship between structure of amide alkaloids and its antidepressant activities. The antidepressant effective components of EAE might be Leatispiamide A and Laetispicine. In their molecular structures, the isolated double bond from benzene ring and conjugated double bond located at 2–3 and 4–5 were necessary for its antidepressant activity.     

Allelopathic behaviour of three medicinal plant species on traditional agriculture crops of Garhwal Himalaya,India

Allelopathic behaviour of three medicinal herbs viz. Rheum emodi, Saussaurea lappa and Potentilla fulgens, on some traditional food crops was examined. Germination of all the traditional food crops was reduced significantly under aqueous extracts of S. lappa and P. fulgens but radicle and plumule growth of Amaranthus caudatus and Eleusine coracana was reduced significantly under aqueous extracts of all three medicinal species. For marginal farmers sole medicinal plants cultivation is not possible, therefore, medicinal plants based agroforestry is being propagated.     

Screening of extracts of Japanese woods for melanin biosynthesis inhibition

The effects of 120 methanol extracts prepared from bark and heartwood of 69 types of Japanese wood on the melanin production of B16 melanoma cells were examined. The melanin content of B16 melanoma cells was determined spectrophotometrically at 405nm. The extracts were also examined for their effects on cell viability. We found that the methanol extracts of Fagus crenata (buna, wood, 100μg/ml), Sapium sebiferum (Nankinhaze, wood, bark, 10μg/ml), and Zelkova serrata (keyaki, wood, 10μg/ml) greatly inhibited the melanin production of B16 melanoma cells without significant cytotoxicity. However, these extracts did not inhibit tyrosinase activity at the concentration of 100μg/ml. These findings indicate that the depigmenting mechanism of these extracts involves the suppression of some pigmenting signals in stimulating melanogenesis rather than the inhibition of tyrosinase activity. Part of this study was presented at the 53rd Annual Meeting of the Japan Wood Research Society, Fukuoka, Japan, March 2003     

Mortality and F1 production of Rhyzopertha dominica (F.) on wheat treated with diatomaceous earth: impact of biological and environmental parameters on efficacy

A series of experiments were conducted in which label rate (0.3 g per kg of wheat) with diatomaceous earth (DE) formulation Protect-It. Exposure studies were carried out at two levels of relative humiditiy (40% and 55%), two levels of insect density (10 or 20 adults per vial), and three levels of exposure periods. Test insects were placed in vials containing 40 g of soft white winter wheat mixed with either 0 or 0.3 g Protect-It per kg of wheat. After relating exposure periods for both insect density and relative humidity levels, highest mortality was only 16%. Mortality, regardless of the dose rate applied (0 and 0.3 g) were not significantly increased as the exposure period increased. Regardless of relative humidity (r.h.) (40% and 55%r.h.), mortality in control were not significantly increased as the exposure period increased for the same insect density. However, significant differences among the three exposure periods in 10 adults’ density in 0.3 g DE application. Mortality was significantly increased in 10 adults’ density. In this case mortality of R. dominica adults increased with the increasing of the exposure intervals. As for 20 adults’ density, mortality in 0.3 g DE application was not significantly changed along with the exposure intervals. After insects were exposed for 1, 2, or 3 week, dead and live insects were removed, and the wheat in the vial were returned to relating humidity box and kept for 8 week until F₁ adults emerged. Then, the number of F₁ adult was counted. The mean numbers of F₁ adults on untreated wheat at all density and r.h. combinations, regardless of exposure periods were significantly higher than that of 0.3 g DE- treated wheat (Fig. 3). At each density and treatment, the mean number of F₁ adults at 55% r.h. were higher that that of 40%r.h. The comparison between adult densities at each r.h. and treatment showed that adult densities influenced the F₁ production of R. dominica and F₁ adults were always higher at 20 adults’ density. The mean number of F₁ adults at each density, regardless of treatment progressively increased as the exposure period increased. Fewer progeny were produced at 10 adults’ density compared to 20 adults’ density for same exposure periods. The highest reproduction occurred in 3 week of exposure period for both insect densities. Regardless of adult densities, the mean number of F₁ adults was significantly increased as the exposure period increased (Fig. 5) for both control and 0.3 g DE treatment. Reduction of F₁ adults at 0.3 g DE application compared to control were found to be 76.88, 74.78 and 67.63% for 1, 2 and 3 week of exposure period, respectively.     

The 1,4-naphthoquinone derivative from Pyrola rotundifolia activates AMPK phosphorylation in C2C12 myotubes

An aqueous ethanol extract of Pyrola rotundifolia L. induced AMP-activated protein kinase (AMPK) phosphorylation in C2C12 myotubes. The bioassay-guided fractionation of the extract led to the isolation a 2-methyl-7-hydroxymethyl-1,4-naphthoquinone, or a 7′-hydroxy-chimaphilin, which showed concentration-dependent AMPK phosphorylation activity at 2.5–20 μg/ml. At a concentration of 10 μg/ml (50 μM), an approximately four-fold increase in the AMPKα(Thr¹⁷²) phosphorylation level was observed. The stimulatory effect of naphthoquinone on AMPK activity was comparable to that of known compounds found in natural sources that activate the AMPK signaling pathway.     

Antimicrobial activity against <Emphasis Type="Italic">Streptococcus sobrinus</Emphasis> and glucosyltransferase inhibitory activity of taxifolin and some flavanonol rhamnosides from kempas (<Emphasis Type="Italic">Koompassia malaccensis</Emphasis>) extracts

Twenty plant materials collected from the islands of Java and Kalimantan in Indonesia were extracted with 50% aqueous ethanol (crude extract). The crude extracts were assayed for antimicrobial activities against Streptococcus sobrinus and for glucosyltransferase (GTase) inhibition. Fourteen extracts inhibited the growth of S. sobrinus by more than 50% and six extracts inhibited GTase activity by more than 50% at a concentration of 100 μg/ml. Koompassia malaccensis (kempas) extracts showed 90% depression of S. sobrinus growth and 80% inhibition of GTase activity at a concentration of 100 μg/ml. Kempas crude extracts were subjected to column chromatography using Sephadex LH-20 and then preparative high-performance liquid chromatography to isolate four compounds A, B, C, and D. These compounds were identified as taxifolin and the flavanonol rhamnoside isomers neoastilbin, astilbin, and isoastilbin, respectively, from ¹H and ¹³C nuclear magnetic resonance (NMR) spectra and other two-dimensional NMR techniques (COSY, HMBC, and HMQC). Each compound depressed the growth of S. sobrinus over a concentration range of 9.3242.7 μg/ml and showed GTase inhibitory activity with IC₅₀ values in the range 27.4–57.3 μg/ml. Taxifolin and flavanonol rhamnoside isomers isolated for the first time from kempas could be potent compounds for preventing dental caries. Part of this report was presented at the 57th Annual Meeting of the Japan Wood Research Society Conference, Hiroshima, 2007     

Influence of leaf portion and plant species on the egg-laying behaviour of the predatory ladybird Serangium parcesetosum Sicard (Col., Coccinellidae) in the presence of a natural enemy

The effectiveness of natural enemies against arthropod herbivores can depend on the characteristics of the plant on which they are found. The influence of the plant on the egg-laying behaviour of the promising whitefly predator, Serangium parcesetosum Sicard (Col., Coccinellidae) was examined in order to be able to use it effectively in biological control programs. The present work investigated the possible influence of the portion of the leaf on the number of eggs laid as well as the effect of plant species on the way in which eggs are deposited by S. parcesetosum. The experiments were conducted on cucumber and cotton leaves with Bemisia tabaci (Genn .) (Hom., Aleyrodidae) as prey in the absence and presence of a natural enemy, the lacewing, Chrysoperla carnea (Stephens) (Neur., Chrysopidae) at two different temperatures. The results showed that at 18°C, S. parcesetosum females significantly preferred to lay their eggs between the veins and close to the veins of cucumber leaves, mean of 10.1 and 7.5 eggs, in the absence of C. carnea, respectively, while in its presence significantly more eggs were deposited close to the veins and close to the petiole. On cotton leaves, close to the petiole, a mean of 8.4 eggs in the absence of the lacewing, as well as close to the veins, mean of 6.3 eggs in the presence of the lacewing, were found to be the most suitable leaf portions for egg-laying. At 30° C, the females laid their eggs preferentially close to the veins of cucumber leaves in the absence and presence of C. carnea. On cotton leaves, S. parcesetosum females significantly preferred to lay their eggs close to the petiole, mean of 7.6 and 6.1 eggs, as well as close to the veins, mean of 6.2 and 8.7 eggs, in the absence and presence of the lacewing, respectively. At both temperatures, the ladybird females laid their eggs singly on cucumber leaves in the absence and presence of C. carnea. While on cotton leaves, the females had a tendency to deposit their eggs together in the absence and presence of the lacewing, except at 30°C in its absence. Within the same plant species, significant differences were found in the total number of eggs laid by S. parcesetosum females on cotton leaves at 18°C as well as on cucumber leaves at 30°C in the absence and presence of the natural enemy. In addition to the effects of presence and absence of C. carnea, and where eggs were laid, some significant differences due to plant species was found at both temperatures.This article is dedicated to Prof. Dr Wolfgang Schwenke on his 84th birthday.     

Isolation and sequence analysis of a Dof protein gene (PtDof1) in Populus

Both cDNA and DNA clones of PtDof1 (GenBank Accession No. FJ402844 and FJ402845) were isolated from plants grown in tissue culture of Populus tomentosa. The DNA sequence is 1597 bp including two exons and one intron. The cDNA is 969 bp in length with a 765 bp open reading frame which is capable of encoding 255 amino acids. The deduced amino acids sequence of the PtDof1 protein shares 65%, 56% and 55% identity with Vitis vinifera (CAO48618), Nicotiana tabacum (CAA08755) and Glycine max (ABI16022) Dof protein by blast analysis in GenBank. Phylogenic analysis suggests PtDof1 gene could belong to the Dof gene family. PtDof1 protein contains an unusual conserved single zinc finger with the pattern of C-X2-C-X21-C-X2-C, which may play a functional role in tissue-specific expression and possibly the auxin response of endogenous plant genes.