Atypical apoptosis in L929 cells induced by evodiamine isolated from Evodia rutaecarpa

Two alkaloids, evodiamine and rutaecarpine, isolated from the dried fruits of Evodia rutaecarpa Bentham were evaluated in vitro for antiproliferation activity on tumor cells versus human peripheral blood mononuclear cells (PBMC). Evodiamine had more potent cytotoxic effects on five tumor cell lines (human malignant melanoma A375-S2, human cervical cancer HeLa, human breast adenocarcinoma MCF7, human acute monocytic leukemia THP-1, murine fibrosarcoma L929) than rutaecarpine. Moreover, evodiamine did not affect PBMC viability for a 36?h culture period. Although apoptotic bodies were observed in evodiamine-treated L929 cells stained with Hoechst 33258, DNA fragmentation as a hallmark of apoptosis was not found. Caspases were involved in the protection of L929 cells against cell death. Evodiamine initiated atypical apoptosis in L929 cells by cycle arrest at the G0/G1 phase.     

34种吴茱萸中吴茱萸碱和吴茱萸次碱的含量测定

建立了吴茱萸中吴茱萸碱和吴茱萸次碱的反相高效液相色谱法 ,并用该法测定了 34种不同产地及不同炮制方法吴茱萸中吴茱萸碱和吴茱萸次碱的含量 .采用峰面积内标法定量 .吴茱萸碱和吴茱萸次碱在 0 5～ 2 0g/mL范围内均呈线性 ,相关系数分别为 0 9999、0 9998,平均回收率分别为 96 6 %、96 0 %     

A Comparison between Water and Ethanol Extracts of Rumex acetosa for Protective Effects on Gastric Ulcers in Mice

Rumex acetosa is a perennial herb that is widely distributed across eastern Asia. Although the hot water extract of R. acetosa has been used to treat gastritis or gastric ulcers as a folk medicine, no scientific report exists for the use of this plant to treat gastric ulcers. Hence, the present study was undertaken to assess the anti-ulcer activity of water and 70% ethanol extracts obtained from R. acetosa, using an HCl/ethanol-induced gastric ulcer model in mice. Anti-inflammatory and free radical-scavenging activities of these two extracts were also evaluated and compared. As a result, the administration of R. acetosa extracts significantly reduced the occurrence of gastric ulcers. However, significant differences in protective activity against gastric ulcers were observed between the two samples. In the case of the group pretreated with an ethanol extract dosage of 100 mg/kg, the protective effect (90.9%) was higher than that of water extract (41.2%). Under histological evaluation, pretreatment with R. acetosa extracts reversed negative effects, such as inflammation, edema, moderate hemorrhaging and loss of epithelial cells, presented by HCl/ ethanol-treated stomachs. Meanwhile, R. acetosa extracts showed potent DPPH radical-scavenging activity and decreased NO production in a murine macrophage cell line, RAW 264.7, in a dose-dependent manner without affecting cellular viability. The greater anti-ulcer and NO production inhibitory activities exhibited by ethanol extracts compared to water extracts could be ascribed to the higher emodin levels, a major anthraquinone component of this plant.     

吴茱萸不同炮制品中吴茱萸碱、吴茱萸次碱、柠檬苦素含量测定

目的:考察不同炮制方法及炮制前后吴茱萸中吴茱萸碱、吴茱萸次碱和柠檬苦素的含量变化情况。方法:采用高效液相色谱法,Hypersil ODS C₁₈（250 mm×4.6 mm,5μm）;流动相:乙腈-水-四氢呋喃-乙酸（41:59:1:0.2）;流速:1.0 ml·min^-1;检测波长:225 nm;柱温:25℃。结果:吴茱萸碱、吴茱萸次碱、柠檬苦素进样量分别在0.156～3.120μg（r=0.999 6,n=6）、0.116～2.320μg（r=1.000 0,n=6）、0.620～12.400μg（r=0.999 1,n=6）范围内与峰面积呈良好线性关系,平均回收率分别为97.2%（RSD=1.33%）、97.1%（RSD=1.56%）、97.3%（RSD=1.52%）。结论:不同炮制方法吴茱萸碱、吴茱萸次碱、柠檬苦素含量不同,砂烫法优于其他炮制方法。     

On the evaluation of the preparation of Chinese medicinal prescriptions. VI. The changes of the alkaloid contents by processing of Evodia fruit]

A crude drug, Evodia fruit (goshuyu) was processed to detoxicate and reduce the bitter taste. Following the procedure described in Shokanron, Evodia fruit was washed in hot water, and then dried. The alkaloid contents of processed Evodia fruit was analyzed by high performance liquid chromatography. The result shows that the content of hydroxyevodiamine decreased to 0.55 times, while the content of rutaecarpine and evodiamine hardly change in the final processed material. However, evocarpine content increased to 1.3 times comparing with the untreated Evodia fruit. The phenomena was ascribed to the flowing-out of the water-soluble portion, and also the weight of extract and the intense of bitterness in the processed fruit were reduced to about 1/3 times.     

HPLC法同时测定吴茱萸中吴茱萸碱、吴茱萸次碱与吴茱萸内酯的含量

目的:建立同时测定吴茱萸中吴茱萸碱、吴茱萸次碱与吴茱萸内酯含量的方法。方法:采用高效液相色谱法。色谱柱为迪马C1(8200mm×4.6mm,5μm),流动相为乙腈-0.04%庚烷磺酸钠溶液(48:52,V/V),流速为1.0mL·min-1,检测波长为225nm,柱温为35℃。结果:吴茱萸碱、吴茱萸次碱、吴茱萸内酯的进样浓度分别在5.38～53.80、5.02～50.20、10.30～103.00μg·mL-(1r均为0.9999)范围内与各自峰面积积分值呈良好线性关系;三者平均加样回收率分别为99.75%、97.66%、85.68%,RSD分别为0.67%、1.16%、1.54%(n=6)。结论:本方法简便、可行、重复性好,可用于吴茱萸中吴茱萸碱和吴茱萸次碱的含量测定;但吴茱萸内酯的回收率较低,需进一步改进其测定方法。     

Biochemical Evaluation of the Hypoglycemic Effects of Extract and Fraction of Cassia fistula Linn. in Alloxan-induced Diabetic Rats

Various extracts of flowers of Cassia fistula Linn (Leguminosae) such as petroleum ether (60-80°), chloroform, acetone, ethanol, aqueous, and crude aqueous extracts and two fractions of ethanol extract were tested for antihyperglycemic activity in glucose-overloaded hyperglycemic rats. The effective antihyperglycemic extracts and fraction were tested for their hypoglycemic activity at two dose levels, 200 and 400 mg/kg, respectively. To confirm their utility in higher models, the effective extracts and fraction of C. fistula were subjected to antidiabetic study in an alloxan-induced diabetic model at two dose levels, 200 and 400 mg/kg, respectively. Biochemical parameters like glucose, urea, creatinine, serum cholesterol, serum triglyceride, high-density lipoprotein, low-density lipoprotein, hemoglobin, and glycosylated hemoglobin were also assessed in experimental animals. The petroleum ether and ethanol extracts of C. fistula and the water-soluble fraction of ethanol extract were found to exhibit significant antihyperglycemic activity. The extracts, at the given doses, did not produce hypoglycemia in fasted normal rats, and the fraction exhibited weak hypoglycemic effect after 2 h of the treatment. Treatment of diabetic rats with ethanol extract and water-soluble fraction of this plant restored the elevated biochemical parameters significantly (P<0.05) to the normal level. No activity was found in the petroleum ether extract of the plant. Comparatively, the water-soluble fraction of ethanol extract was found to be more effective than the ethanol extract, and the activity was comparable with that of the standard, glibenclamide (5 mg/kg).     

Antioxidant and Anticholinesterase Activities in Various Parts of Sonneratia caseolaris (L.)

Cork tree, (Sonneratia caseolaris L.), family Sonneratiaceae, is a typical plant in mangroves. It is widespread in tropical and subtropical tideland throughout the World. It is reported to possess many medicinal properties. For searching new pharmacological activities of Cork tree, the total phenolic contents, antioxidant activities and the electric eel acetylcholinesterase inhibitions and the kinetics of extracts of various plant parts were determined. The graphs of trolox equivalent antioxidant capacity and ferric reducing antioxidant power of all extracts showed good linearity with P-value of slopes less than 0.05. The methanol extract of calyxs by maceration method and methanol extract of stamen by soxhlet method presented moderate trolox equivalent antioxidant capacity values. For ferric reducing antioxidant power assay, all extracts gave fair to low antioxidant activities. The tacrine, stamen extract and seed extract by maceration using methanol showed noncompetitive inhibition on acetylcholinesterase activity. While, luteolin, luteolin glycoside and calyx extract and seed extract by boiling using water presented partial noncompetitive inhibition on acetylcholinesterase activity.     

Quality evaluation of Evodia rutaecarpa (Juss.) Benth by high performance liquid chromatography with photodiode-array detection

A simple, sensitive and accurate HPLC-DAD method was developed for simultaneous determination of wuchuyuamide-I, quercetin, limonin, evodiamine and rutaecarpine in Evodia rutaecarpa that has been widely used as one of the traditional Chinese medicines (TCMs). Chromatographic separations were performed on a reverse-phase C(18) column with the gradient elution of acetonitrile-water and the simultaneous detection at five wavelengths. Good linear behaviors over the investigated concentration ranges were observed with the values of r higher than 0.999 for all the analytes. The recoveries measured at three levels varied from 98.77 to 102.36%. The validated method was successfully applied for the simultaneous determination of the five chemical constituents in 36 batches of samples collected from different regions or time that were investigated and authenticated as E. rutaecarpa (Juss.) Benth. Hierarchical clustering analysis (HCA) and principal components analysis (PCA) were performed to differentiate and classify the samples based on the contents of the five characteristic constituents. The total contents of evodiamine and rutaecarpine in different samples were calculated and the blending method proposed was demonstrated to be very useful in saving resources and in guiding rational herb use.     

The Effect of Polyphenols Isolated from Cynanchi wilfordii Radix with Anti-inflammatory,Antioxidant, and Anti-bacterial Activity

Recently, Cynanchi wilfordii Radix has gained wide use in Asian countries as a functional food effective for relieving fatigue, osteoporosis, and constipation, particularly in menopausal disorders. However, its anti-inflammatory and anti-microbial activities have not been explored in detail to date. The anti-inflammatory, antioxidant, and anti-bacterial properties of the Cynanchi wilfordii Radix extracts obtained with water, methanol, ethanol, and acetone were compared. All 4 polyphenol-containing extracts exhibited anti-inflammatory and antioxidant effects. The ethanol extract was found to elicit the most potent reduction of nitric oxide (NO), prostaglandin E₂ (PGE₂), and cytokine (IL-1β, IL-6, IL-10, and TNF-α) levels, as well as inhibit the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in a concentration-dependent manner. The evaluation of antioxidant activity also revealed the ethanol extract to have the highest free radical scavenging activity, measured as 85.3±0.4%, which is equivalent to 99.9% of the activity of α -tocopherol. In the assessment of anti-bacterial activity, only ethanol extract was found to inhibit the growth of the Bacillus species Bacillus cereus and Bacillus anthracis. These results show that polyphenols of Cynanchi wilfordii Radix have anti-inflammatory, antioxidant, and anti-bacterial properties that can be exploited and further improved for use as a supplementary functional food, in cosmetics, and for pharmaceutical purposes.     

Phase I and phase II metabolite identification of rutaecarpine in freshly isolated hepatocytes from male Sprague–Dawley rats

Rutaecarpine, an alkaloid originally isolated from Evodia rutaecarpa, has been used for the treatment of gastrointestinal disorders in Asia. In the present study, the phase I and phase II metabolites of rutaecarpine were investigated in freshly isolated hepatocytes from male Sprague–Dawley rats. The individual metabolites were characterized via liquid chromatography-tandem mass spectrometry. The incubation of rutaecarpine with freshly isolated hepatocytes for 2 h yielded five major phase I metabolites. In addition, three glucuronide conjugates and four sulfate conjugates were observed. Because the majority of metabolites observed in vivo were identified, freshly isolated hepatocytes might be useful for the identification of certain metabolites formed from drug candidates from a reduced number of experimental animals.     

Evaluation of Parmotrema reticulatum Taylor for Antibacterial and Antiinflammatory Activities

Lichens produce variety of secondary metabolites including depsides, depsidones and dibenzofurans having multifunctional activity in response to external environmental condition. Present study provides evidence for in vitro antibacterial and in vivo antiinflammatory activity of acetone and ethanol extracts of Parmotrema reticulatum. In vitro antibacterial activity was investigated against gram positive and gram negative bacteria. Cotton pellet-induced granuloma, xylene-induced ear swelling, carragennan-induced edema, histamine-induced and carboxymethylcellulose sodium-induced leukocyte emigration in mice models were used to quantify the antiinflammatory activity. Acetone and ethanol extracts were showed antibacterial activity against Bacillus subtilis (minimal inhibitory concentration: 100 and 150 μg/ml) and Staphylococcus aureus (minimal inhibitory concentration: 100 and 200 μg/ml), Escherichia coli (minimal inhibitory concentration: 200 and 250 μg/ml), and Pseudomonasa eruginosa (minimal inhibitory concentration: 200 and 300 μg/ml). Acetone extract was inhibited edema significantly at 200 mg/kg with xylene, cotton pellet, carragennan and histamine induced edema in vivo models. Ethanol extract was found effective at dose of 300 mg/kg with all in vivo antiinflammatory models. The results showed significant (P<0.01) antiinflammatory effects at 200 and 300 mg/kg dose of acetone and ethanol extracts, respectively, which can be concluded that significant activity may be due to presence of flavanoids in ethanol extract and usnic acid in acetone extract.     

Rutaecarpine attenuates osteoclastogenesis by impairing macrophage colony stimulating factor and receptor activator of nuclear factor κ‐B ligand‐stimulated signalling pathways

Rutaecarpine is a major alkaloid isolated from Evodia rutaecarpa. Here, we investigated the effects of rutaecarpine on osteoclast differentiation induced by macrophage colony stimulating factor (M‐CSF) and receptor activator of nuclear factor κ‐B ligand (RANKL) in bone marrow‐derived macrophages (BMMs). Treatment with rutaecarpine significantly inhibited osteoclastogenesis and prevented bone resorption of BMM‐derived osteoclasts. Mechanistically, rutaecarpine decreased the protein level of nuclear factor of activated T cells cytoplasmic‐1 (NFATc1) and the phosphorylation of other signalling pathways during the osteoclast differentiation. Thus, rutaecarpine may be useful as a therapeutic agent for the treatment of bone diseases.     

In vitro study on α-amylase inhibitory activity of an Indian medicinal plant, Phyllanthus amarus

Objective:

The objective of this study was to evaluate the α-amylase inhibitory activity of different extracts of Phyllanthus amarus against porcine pancreatic amylase in vitro.

Materials and Methods:

The plant extracts were prepared sequentially with ethanol, chloroform, and hexane. Each extract was evaporated using rotary evaporator, under reduced pressure. Different concentrations (10, 20, 40, 60, 80, and 100 μg/mL) of each extract were made by using dimethyl sulfoxide (DMSO) and subjected to α-amylase inhibitory assay using starch azure as a substrate. The absorbance was read at 595 nm using spectrophotometer. Using this method, the percentage of α-amylase inhibitory activity and IC₅₀values of each extract was calculated.

Results:

The chloroform extract failed to inhibit α-amylase activity. However, the ethanol and hexane extracts of P. amarus exhibited appreciable α-amylase inhibitory activity with an IC50 values 36.05 ± 4.01 μg/mL and 48.92 ± 3.43 μg/mL, respectively, when compared with acarbose (IC₅₀value 83.33 ± 0.34 μg/mL).

Conclusion:

This study supports the ayurvedic concept that ethanol and hexane extracts of P. amarus exhibit considerable α-amylase inhibitory activities. Further, this study supports its usage in ethnomedicines for management of diabetes.     

Evaluation of Antimicrobial and Wound Healing Potentials of Ethanol Extract of Wedelia biflora Linn D.C. Leaves

To rationalize scientifically the traditional claim on use of Wedelia biflora (Linn.) D. C. for the treatment of wounds and infections, the present study was designed to evaluate the antimicrobial and wound healing activity of ethanol extract of leaves of W. biflora. In in vitro assays the test extract was subjected to antimicrobial activity by agar well-diffusion method and minimum inhibitory concentration method in different microbial strains. Wound healing activity of the test extract was studied by excision wound model and incision wound model in Wistar albino rats. In excision wound model, 97.90% wound healing was recorded in 10% w/w extract treated group on 16^th days of postsurgery, whereas only 58.50% was observed in control group. In incision model, higher breaking strength, high hydroxyl proline content and histopathological study in extract treated groups revealed higher collagen redeposition than the control group. The agar well-diffusion evaluation and minimum inhibitory concentration established antimicrobial efficacy of ethanol extracts of W. biflora. These observations established the traditional claim and therapeutic activity of W. biflora and it could be a potent wound healing candidate for use in future.     

Studies on the alkaloid constituents of Evodia rutaecarpa (Juss) Benth var. bodinaieri (Dode) Huang and their acute toxicity in mice

Six alkaloids (1–6) have been isolated from the fruits of Evodia rutaecarpa (Juss) Benth var. bodinaieri (Dode) Huang, two of which are new compounds, identified as 2-undecyl-4(1H)-quinolone (4) and 1-methyl-2-undecanone-10′-4(1H)-quinolone (5); the known compounds were identified as rutaecarpine (1), evodiamine (2), 1-methyl-2-undecyl-4(1H)-quinoline (3) and 2-undecanone-10′-4(1H)-quinolone (6). Compounds 1–5 were evaluated for their acute toxicity.     

Antioxidant and antitumor activities of Artemisia campestris and Thymelaea hirsuta from southern Tunisia

The essential oil of Artemisia campestris and the ethanol-water, hexane and water extracts of A. campestris and Thymelaea hirsuta collected in southern of Tunisia were investigated for their antioxidant (DPPH, ABTS and beta-carotene methods) and antitumor growth inhibition of human colon cancer HT-29 cells using MTT test activities.All the A. campestris extracts tested at high concentrations (100 μg/ml) showed activity ranging from 19.5% for essential oil to 64.4% of negative control growth for infusion extract, except the hexane extract. With T. hirsuta, all the extracts tested (hexane and ethanol-water), except the infusion extract, also exhibited antitumor activity (58.2% and 65.5% of control growth respectively).The ethanol-water and infusion extracts of A. campestris showed higher antioxidant activity, polyphenol and flavonoid contents than those of T. hirsuta. These results show that there is a positive correlation between the antitumor activity and the antioxidant activity, and of these two activities and with the levels of polyphenols and flavonoids.The essential oil and the other extracts of A. campestris, which exhibited significant antitumor activity against the HT-29 cells deserve further research into the chemoprevention and treatment of colon cancer.     

Evaluation of Antioxidant Activity of Picrorhiza kurroa (Leaves) Extracts

Picrorhiza kurroa is a well-known herb in Ayurvedic medicine. Although it shows antioxidant, antiinflammatory and immunomodulatory activities, it is most valued for its hepatoprotective effect. The rhizomes are widely used against indigestion problems since ancient times due to improper digestive secretions. Aim of this study was to explore antioxidant study of P. kurroa leaves for a new source of naturally occurring antioxidants. Two pure compounds, luteolin-5-O-glucopyranoside (1) and picein (2) were isolated from butanol extract through column chromatography. Different extracts of P. kurroa leaves (ethanol, ethyl acetate, butanol) were quantified for isolated compound (2) by high-performance liquid chromatography. All the extracts and isolated compounds were evaluated for its antioxidant activity using two assays, 2,2-diphenyl-1-picrylhydrazyl radical and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) assay. The linear detection range was 1.56-200 μg/ml for picein. The limit of detection and limit of quantification for picein were 2.34 and 7.81 μg/ml, respectively. Butanol and ethyl acetate extract showed greater antioxidant activity as compare to ethanol extract. Compound 1 and ascorbic acid showed nearly similar antioxidant activity where as 2 showed no activity at standard concentration. The IC₅₀ values for 2,2-diphenyl-1-picrylhydrazyl radical and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) assay for ascorbic acid, compound 1, ethanol extract and its different fractions (ethyl acetate and butanol) were found to be 0.81, 1.04, 67.48, 39.58, 37.12 and 2.59, 4.02, 48.36, 33.24, 29.48 μg, respectively.     

Antidiabetic and antihyperlipidemic effects of Thespesia populnea fruit pulp extracts on alloxan-induced diabetic rats

Present study was carried to find out the antihyperglycemic and antihyperlipidemic activity of ethanol and aqueous extract of Thespesia populnea fruit pulp on alloxan-induced diabetic rats. Diabetes was induced in rats by administration of alloxan (150 mg/kg, i.p.). After the successful induction of experimental diabetes, the rats were divided into five groups each comprising a minimum of six rats. Phytochemical analysis and acute toxicity study of extracts was also done. The effects of extracts and metformin on fasting blood glucose and plasma lipid were examined for 28 days. Statistical analysis was carried out by using analysis of variance followed by Dunnet''s multiple comparison test and paired t-test were done as the test of significance using GraphPad Prism. P≤0.05 was considered as the minimal level of statistical significance. Therapeutic dose of extract was found to be 200 mg/kg on the basis of acute toxicity study. Aqueous and alcoholic extract showed a significant reduction in blood glucose levels as well as a lipid profile of diabetic rats at the end of 28^th day of treatment. However, in groups treated with plant extract the reduction in the blood glucose and improvement in lipid profile was slightly less than that achieved with the standard group (metformin). From this study, it can be concluded that ethanol and aqueous extract of Thespesia populnea exhibited significant antihyperglycemic and antihyperlipidemic effects on alloxan-induced diabetic rats.     

Evaluation of Ethanol and Aqueous extracts of Cinnamomum verum Leaf Galls for Potential Antioxidant and Analgesic activity

In the present study, ethanol and aqueous extracts of leaf galls of Cinnamomum verum were prepared to evaluate the antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay and superoxide radical scavenging assay with ascorbic acid as a standard, and analgesic activity by tail immersion test and acetic acid-induced writhing test methods using diclofenac sodium as the reference drug. Swiss albino mice maintained under standard laboratory conditions were used for analgesic tests. In the 2,2-diphenyl-1-picrylhydrazyl assay it was found that the aqueous and the ethanol extract possessed almost equal capacity to inhibit free radicals (IC₅₀=13.3 and 13.53 µg/ml) but found less than ascorbic acid (IC₅₀=9.96 µg/ml). And in superoxide assay the ethanol extract was found to be more potent in scavenging super oxide radicals when compared to ascorbic acid and the aqueous extract (IC₅₀=237.1 and 197.8 µg/ml) with the IC₅₀=119.7 µg/ml. For analgesic activity, ethanol extract showed the maximum time required for response against thermal stimuli (6.75±0.47 s) and maximum % of writhing inhibition (44.57%) when compared to aqueous extract (5.25±0.48 s and 32.61%), whereas diclofenac showed response in 7.25±0.25 s 67.39% inhibition in tail immersion and writhing tests, respectively. These results demonstrate that the ethanol extracts of leaf galls possessed high antioxidant and analgesic activity.