Staminane- and isopimarane-type diterpenes from Orthosiphon stamineus of Taiwan and their nitric oxide inhibitory activity

From the MeOH extract of Taiwanese Orthosiphon stamineus, two new staminane-type diterpenes, staminols C (1) and D (2), and three new isopimarane-type diterpenes, orthosiphonone C (3) and D (4) and 14-deoxo-14-O-acetylorthosiphol Y (5), have been isolated together with 16 known diterpenes, orthosiphols A, B, D, K, M, N, O, X, and Y, nororthosiphonolide A, neoorthosiphol B, orthosiphonone A, secoorthosiphols B and C, 3-O-deacetylorthosiphol I, and 2-O-deacetylorthosiphol J. Their structures were determined on the basis of the spectroscopic data. All the newly isolated diterpenes exhibited dose-dependent inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-activated macrophage-like J774.1 cells, and 2-O-deacetylorthosiphonone A showed the most potent activity, with an IC(50) value of 35.0 microM, comparable to that of the positive control N(G)-monomethyl-L-arginine (L-NMMA; IC(50), 35.7 microM).     

Five novel highly oxygenated diterpenes of Orthosiphon stamineus from Myanmar

Five novel highly oxygenated diterpenes, orthosiphols K (1), L (2), M (3), and N (4) and norstaminone A (5), were isolated from the aerial part of Orthosiphon stamineus, together with three known diterpenes, orthosiphols A (6) and B (7) and neoorthosiphol A (8). Orthosiphol L (2) is an isopimarane-type diterpene with a hydroxyl group at C-12, which supports the biogenesis of staminane-type diterpenes, i.e., migration of a vinylic group from C-13 of isopimarane to C-12. Norstaminone A (5) has a staminane carbon framework and supports the biosynthetic pathway from staminols to norstaminols via staminolactones. All the isolated compounds showed mild to weak antiproliferative activities toward highly liver metastatic colon 26-L5 carcinoma and human HT-1080 fibrosarcoma cell lines.     

不同产地肾茶HPLC指纹图谱研究△

目的:建立肾茶药材的HPLC指纹图谱分析方法。方法:采用Waters ODS柱(150mm×4.6mm,5μm),流动相为乙腈-水(含0.1%的磷酸),梯度洗脱,检测波长为205nm,流速为1mL·min-1。采用《中药色谱指纹图谱相似度评价系统(2004A)版》,计算不同产地肾茶样品的HPLC指纹图谱的整体相似度。结果:共有15个共有峰,13批被测样品的色谱指纹图谱与对照指纹图谱的整体相似度在0.908～0.985。结论:该指纹图谱检测方法简便、重现性好,可作为肾茶药材质量控制的重要方法。     

Bioactive diterpenes from Orthosiphon labiatus and Salvia africana-lutea

The known (+)-trans-ozic acid (1) and two new labdane diterpenoids (2 and 3) have been isolated from an ethanol extract of Orthosiphon labiatus. The structures of 2 and 3 were established mainly by 1D and 2D NMR spectroscopic means. The ethanolic extract of Salvia africana-lutea afforded the known abietane diterpenoids carnosol (4), rosmadial (5), and carnosic acid (characterized as its derivative 6). Compounds 3 and 6 exhibited MICs of 157 and 28 microM, respectively, against Mycobacterium tuberculosis, while 2 and 6 showed cytotoxic activity with IC50 82 and 69 microM, respectively, against a breast (MCF-7) human cancer cell line.     

Evaluation of the genotoxicity of Orthosiphon stamineus aqueous extract

Aim of the study

Orthosiphon stamineus, Benth, also known as Misai Kucing in Malaysia and Java tea in Indonesia, is traditionally used in Southeastern Asia to treat kidney dysfunctions, diabetes, gout and several other illnesses. Recent studies of Orthosiphon stamineus pharmacological profile have revealed antioxidant properties and other potentially useful biological activities thereby lending some scientific support to its use in folk medicine. So far the genotoxicity of Orthosiphon stamineus extracts has not been evaluated. In this study the genotoxic potential of Orthosiphon stamineus aqueous extract was investigated by the Salmonella/microsome mutation assay and the mouse bone marrow micronucleus test.

Materials and methods

Chemical composition of Orthosiphon stamineus aqueous extract was analyzed by High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD). The Salmonella/microsome assay (TA97a, TA98, TA100 and TA1535; plate incorporation method) was performed in the presence or in the absence of extrinsic metabolic activation (S9 mixture). In the mouse micronucleus assay, Orthosiphon stamineus extract was administered by gavage (0, 500, 2000 and 4000 mg/kg body weight/day for 3 days) to male and female Swiss Webster mice (N = 6 per dose per sex) and bone marrow cells were harvested 24 h after the last dose. Ethoxy-resorufin-O-dealkylase (EROD) and benzyloxy-resorufin-O-dealkylase (BROD) activities were determined in mouse liver microsomes.

Results

The chemical analysis revealed that the Orthosiphon stamineus extract contained flavonoids (sinensetin, eupatorin), caffeic acid, and rosmarinic acid (44.00 ± 1.879 μg/mg), the latter seemed to be one of its major constituent. Tested at doses up to 5000 μg/plate, the Orthosiphon stamineus extract was not toxic to Salmonella tester strains and did not increase the number of revertant colonies over the background incidence. In the mouse bone marrow assay, the extract did not alter the polychromatic:normochromatic erythrocytes (PCE:NCE) ratio, nor did it increase the incidence of micronucleated polychromatic erythrocytes (MNPEs). No overt toxicity and no change of CYP1A (EROD) and 2B9/10 (BROD) activities were noted.

Conclusions

Based on the aforementioned findings, it is concluded that the use of Orthosiphon stamineus in the traditional medicine poses no genotoxic risk.     

Antioxidant and hepatoprotective effects of Orthosiphon stamineus Benth. standardized extract

Orthosiphon stamineus (OS), Benth. (Lamiaceae) is widely used in Malaysia for treatments of various kidney and liver ailments. In the experiment, DPPH* radicals scavenging, Fe(3+)-induced lipid peroxidation inhibiting activities and trolox equivalent antioxidant capacity (TEAC) of methanol/water extract of Orthosiphon stamineus (SEOS) were determined. The results indicated that SEOS exhibited antioxidant, lipid peroxidation inhibition and free radical scavenging activities. The hepatoprotective activity of the SEOS was studied using CCl(4)-induced liver toxicity in rats. The activity was assessed by monitoring liver function tests through the measurement of alanine transaminase (ALT) and aspartate transaminase (AST). Furthermore, hepatic tissues were also subjected to histopathological studies. Pretreatment of SEOS (125, 250, 500 and 1000 mg/kg p.o.) dose-dependently reduced the necrotic changes in rat liver and inhibited the increase of serum ALT and AST activities. The results of the present study indicated that the hepatoprotective effect of Orthosiphon stamineus might be ascribable to its antioxidant and free radical scavenging property.     

Nitric oxide inhibitory substances from the rhizomes of Dioscorea membranacea

Thai medicinal plants locally known as Hua-Khao-Yen were examined for their inhibitory activities against lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW 264.7 cell lines. Among the plant species studied, an ethanolic extract of Dioscorea membranacea exhibited the most potent inhibitory activity, with an IC(50) value of 23.6 microg/ml. From this extract, eight compounds [two naphthofuranoxepins (1, 2), one phenanthraquinone (3), three steroids (4-6) and two steroidal saponins (7, 8)] were isolated and further investigated for their inhibitory properties of NO production. It was found that diosgenin-3-O-alpha-L-rhamnosyl (1-->2)-beta-D-glucopyranoside (7) possessed the highest activity (IC(50)=3.5 microM), followed by dioscoreanone (3, IC(50)=9.8 microM) and dioscorealide B (2, IC(50)=24.9 microM). Regarding structural requirements of diosgenin derivatives for NO production inhibitory activity, compound 7 which has a rhamnoglucosyl moiety at C-3 exhibited much higher activity than compounds that have either a diglucosyl substitution (8) or its aglycone (9); whereas, hydroxyl substitution at position 8 of naphthofuranoxepin derivatives conferred higher activity than the methoxyl group. It is concluded that diosgenin-3-O-alpha-L-rhamnosyl (1-->2)-beta-D-glucopyranoside (7), dioscoreanone (3) and dioscorealide B (2) are active principles for NO inhibitory activity of Dioscorea membranacea. Compounds 1-3 were also tested for the inhibitory effect on LPS-induced TNF-alpha release in RAW 264.7 cells. The result revealed that 3 possessed potent activity against TNF-alpha release with an IC(50) value of 17.6 microM, whereas, 1 and 2 exhibited mild activity. The present study may support the use of Dioscorea membranacea by Thai traditional doctors for treatment of the inflammatory diseases.     

Toxicity evaluation of a standardised 50% ethanol extract of Orthosiphon stamineus

Aim of the study

The present investigation was carried out to evaluate the safety of standardised 50% ethanol extract of Orthosiphon stamineus plant by determining its potential toxicity after acute and subchronic administration in rats.

Materials and methods

For acute toxicity study, up and down method (limit dose) was adapted. A single dose of 5000 mg/kg of the standardised 50% ethanol extract of O. stamineus was given orally to 5 healthy Sprague-Dawley (SD) female adult rats. The rats were observed for mortality and clinical signs for 3 h and then periodically for 14 days. While in the subchronic toxicity study, the extract was administered orally at doses of 1250, 2500 and 5000 mg/kg per day for 28 days to female and male SD rats, respectively. The animals were sacrificed, followed by examination of their organs and blood serum.

Results

In the acute toxicity study, standardised 50% ethanol extract of O. stamineus at a dose of 5000 mg/kg caused neither visible signs of toxicity nor mortality. All five rats survived until the end of observation period. While in subchronic toxicity, administration of the standardised 50% ethanol extract of O. stamineus at 1250, 2500, and 5000 mg/kg for 28 days did not produce any mortality and there were no significant differences in the general condition, growth, organ weights, heamatological parameters, clinical chemistry values, or gross and microscopic appearance of the organs from the treatment groups as compared to the control group.

Conclusions

Standardised 50% ethanol extract of O. stamineus did not cause any death nor did it cause abnormalities in necropsy and histopathology findings. There were no acute or subchronic toxicity observed and this extract could be devoid of any toxic risk. The NOAEL for the standardised 50% ethanol extract of O. stamineus is 5000 mg/kg per day for 28 days.     

Studies on diuretic and hypouricemic effects of Orthosiphon stamineus methanol extracts in rats

AIM OF THE STUDY: Orthosiphon stamineus (Labiatae) is a traditional folk medicine widely used in Southeast Asia for the treatment of several kidney disorders, gout and as a diuretic. This study was conducted to examine the diuretic and hypouricemic effects of Orthosiphon stamineus leaf extracts. MATERIALS AND METHODS: The diuretic effect of different methanol extracts was examined by treating different groups of Sprague-Dawley rats with single (2g/kg) oral doses of methanol and methanol:water (1:1) extracts. Hydrochlorothiazide (10mg/kg) was used as positive control in acute study. Methanol and methanol water (1:1) extracts at 0.5 g/kg were administered for a period of 7 consecutive days. Cumulative urine volume and electrolytes (Na+ and K+) concentrations at different time intervals were measured. On the other hand, hypouricemic activity of methanol:water extract (1:1) was experimented using different oral single doses (0.25, 0.5, 1 and 2g/kg). Allopurinol was used as positive control. Uric acid concentration in serum was analyzed by using RP-HPLC at 280 nm. RESULTS: Sodium and potassium excretion increased significantly (p<0.05 and <0.01) in the first 8h of treatment with a single dose (2g/kg) of the extracts in a pattern comparable to that of the known diuretic hydrochlorothiazide. Meanwhile, repeated administration of 0.5 g/kg methanol:water (1:1) extract showed a significant increase in urine volume (from day 3 to day 7) (p<0.01) and electrolytes excretion (Na+ and K+) from day 2 to day 7 (p<0.05 and <0.01). On the other hand, 0.5, 1 and 2g/kg of methanol:water (1:1) extract and the standard allopurinol reduced the serum urate level in hyperuricemic rats at hour 6. CONCLUSION: These results provided an evidence of the high tendency of methanol:water (1:1) extract of Orthosiphon stamineus towards diuretic and hypouricemic effects in rats.     

Antidiabetic Properties and Mechanism of Action of Orthosiphon stamineus Benth Bioactive Sub-fraction in Streptozotocin-induced Diabetic Rats

Orthosiphon stamineus is a popular folk medicine widely used to treat many diseases including diabetes. Previous studies have shown that the sub-fraction of chloroform extract was able to inhibit the rise of blood glucose levels in a glucose tolerance test. This study was carried out to evaluate the chronic effect and possible mechanism of action of the bioactive chloroform sub-fraction of O. stamineus using streptozotocin-induced diabetic rats and in vitro methods. Administration of the chloroform extract sub-fraction 2 (C?2-b) at a dose of 1 g/kg twice daily on diabetic rats for 14 days showed a significant lowering (p < 0.05) of the final blood glucose level compared to the pretreatment level. However, there were no significant differences in the plasma insulin levels post-treatment compared to the pretreatment levels for all doses of C?2-b. Conversely, C?2-b at a concentration of 2 mg/mL significantly increased (p < 0.001) the glucose uptake by the rat diaphragm muscle. The increase in glucose uptake was also shown when the muscle was incubated in a solution containing 1 IU/mL of insulin or 1 mg/mL of metformin. Furthermore, the effect of this sub-fraction on glucose absorption in the everted rat jejunum showed that C?2-b at concentrations of 0.5 mg/mL, 1 mg/mL and, 2 mg/mL significantly reduced the glucose absorption of the jejunum (p < 0.05–0.001). Similarly, the absorption of glucose was also inhibited by 1 mg/mL and 2 mg/mL of metformin (p < 0.001). These results suggest that the effect of C?2-b may be due to extra-pancreatic mechanisms. There was no evidence that the plant extract stimulated the release of insulin in order to lower the blood glucose level.     

Effects of Orthosiphon stamineus aqueous extract on plasma glucose concentration and lipid profile in normal and streptozotocin-induced diabetic rats

The objective of this study was to investigate the effects of Orthosiphon stamineus Benth. aqueous extract on plasma glucose concentration and lipid profile in normal and streptozotocin-induced diabetic rats. The chemical screening of the extract showed phenolic compound and flavonoid content were 13.24+/-0.33 mg/g and 1.73+/-0.14 microg/g, respectively. In oral glucose tolerance test, the extract (0.2-1.0 g/kg) significantly decreased plasma glucose concentration in a dose-dependent manner in both normal and diabetic rats. The extract at 1.0 g/kg was most effective in decreasing plasma glucose concentrations and the response was closed to the result of glibenclamide (5 mg/kg). After repeated daily oral administrations of the extract (0.5 g/kg) for 14 days, the extract significantly reduced plasma glucose concentration in diabetic rats at days 7 and 14. By the end of the study, plasma triglyceride concentration was lower in the extract-treated diabetic rats than untreated ones. Furthermore, plasma HDL-cholesterol concentration was significantly increased in diabetic rats treated with the extract. In perfused rat pancreas, the extract did not increase insulin secretion in the presence of 5.5 mM glucose, but 100 microg/ml extract potentiated glucose-induced insulin secretion. Our findings suggested that Orthosiphon stamineus aqueous extract is effective for alleviating hyperglycemia and improving lipid profile in diabetic rats.     

猫须草水提物对痛风性肾病大鼠肾脏URAT1、OAT1及病理的影响

目的探讨猫须草水提物对痛风性肾病大鼠肾脏尿酸盐转运体1(URAT1),有机阴离子转运蛋白1(OAT1)及病理的影响。方法采用酵母粉+腺嘌呤法建立大鼠痛风性肾病模型,测定其血尿酸、血肌酐、血尿素氮、尿尿酸、尿肌酐、24 h尿液排泄量及尿酸分级排泄率,光镜下观察肾组织病理变化,免疫组化法检测肾组织URAT1、OAT1蛋白表达。结果与模型组比较,猫须草水提物高、中剂量组大鼠血尿酸、血肌酐、血尿素氮水平及URAT1蛋白表达降低,而尿尿酸、尿肌酐水平,尿酸分级排泄率,OAT1蛋白表达升高;高、中、低剂量组大鼠24 h尿液排泄量升高程度无明显变化。结论猫须草水提物可能通过上调OAT1蛋白表达促进尿酸排泄、下调URAT1蛋白表达抑制尿酸重吸收的双重调节功能来促进尿酸在肾脏中的排泄,并减轻尿酸对肾脏的病理损伤。     

Nitric oxide production inhibitory activity of flavonoids contained in trunk exudates of Dalbergia sissoo

Methanolic extracts of trunk exudates of Dalbergia sissoo yielded two new open-chain neoflavonoids (1, 2), a new flavonoid (3), a new flavanone (4), and 26 known compounds. Their structures were elucidated by detailed spectroscopic analyses. The ability of the isolated compounds to prevent nitric oxide (NO) production by LPS-stimulated J774.1 cells was also studied. All of the isolated compounds except 4, formononetin, and zenognosin B exhibited significant activity in a concentration-dependent manner. Compounds 2 and 3 were among the most potent NO production inhibitors, with IC50 values of 3.19 and 6.22 microM, respectively, and compound 1 had an IC50 of 31.6 microM.     

A Simple Isocratic HPLC Method for the Simultaneous Determination of Sinensetin, Eupatorin, and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone in Orthosiphon stamineus Extracts

Orthosiphon stamineus extracts contain three flavonoids (3′-hydroxy-5,6,7,4′-tetramethoxyflavone, sinensetin, and eupatorin) as bioactive substances. Previous reported high performance liquid chromatography- ultraviolet (HPLC-UV) methods for the determination of these flavonoids have several disadvantages, including unsatisfactory separation times and not being well validated according to International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) standard guidelines. A rapid, specific reversed-phase HPLC method with isocratic elution of acetonitrile: isopropyl alcohol: 20 mM phosphate buffer (NaH₂PO₄) (30:15:55, v/v) (pH 3.5) at a flow-rate of 1 ml/minute, a column temperature of 25°C, and ultraviolet (UV) detection at 340 nm was developed. The method was validated and applied for quantification of different types of O stamineus extracts and fractions. The method allowed simultaneous determination of 3′-hydroxy-5,6,7,4′-tetramethoxyflavone, sinensetin, and eupatorin in the concentration range of 0.03052–250 μg/ml. The limits of detection and quantification, respectively, were 0.0076 and 0.061 μg/ml for 3′-hydroxy-5,6,7,4′-tetramethoxyflavone, 0.0153 and 0.122 μg/ml for sinensetin and 0.0305 and 0.122 μg/ml for eupatorin. The percent relative standard deviation (% RSD) values for intraday were 0.048–0.368, 0.025–0.135, and 0.05–0.476 for 3′-hydroxy-5,6,7,4′-tetramethoxyflavone, sinensetin, and eupatorin, respectively, and those for intraday precision were 0.333–1.688, 0.722–1.055, and 0.548–1.819, respectively. The accuracy for intraday were 91.25%–103.38%, 94.32%–109.56%, and 92.85%–109.70% for 3′-hydroxy-5,6,7,4′-tetramethoxyflavone, sinensetin, and eupatorin, respectively, and those for interday accuracy were 97.49%–103.92%, 103.58%–104.57%, and 103.9%–105.33%, respectively. The method was found to be simple, accurate and precise and is recommended for routine quality control analysis of O stamineus extract containing the three flavonoids as the principle components in the extract.     

Cyclooxygenase (COX)-1 and -2 inhibitory labdane diterpenes from Crassocephalum mannii

Two new labdane diterpenes, 8alpha,19-dihydroxylabd-13 E-en-15-oic acid (1) and 13,14,15,16-tetranorlabdane-8alpha,12,14-triol (2), as well as an acetylated derivative, 8alpha-O-beta-D-glucopyranosyllabd-13 E-ene-15,19-diol-8alpha-2',3',4',6'-hexaacetate (3a), were isolated from the aerial parts of Crassocephalum mannii. The structures of 1, 2, and 3a were elucidated by spectroscopic data analysis. Selective inhibitory activity for 1 and 2 and their acetate derivatives, 1a and 2a, against cyclooxygenases (COX-1 and COX-2) was detected.     

一氧化氮与骨质疏松

 目的揭示一氧化氮(nitric oxide,NO)与骨质疏松的关系,探讨NO供体型药物在骨质疏松症治疗中的可能应用前景。,方法综述国内外近期的相关研究成果,并进行归纳、总结。结果与结论NO是骨代谢的重要调节因子,适宜浓度的NO是维持破骨细胞和成骨细胞活性所必需的,相对高浓度的NO则主要表现为抑制破骨细胞活性,诱导破骨细胞凋亡。因此,向骨组织中引入一定量的外源性NO使之选择性地抑制破骨细胞的骨吸收,可以达到治疗骨质疏松的目的。     

Cassane- and norcassane-type diterpenes from Caesalpinia crista of Indonesia and their antimalarial activity against the growth of Plasmodium falciparum

The CH2Cl2 extract of the seed kernels of Caesalpinia crista, which exhibited promising antimalarial activity against Plasmodium berghei-infected mice in vivo, was examined and resulted in the isolation of seven new furanocassane-type diterpenes [caesalpinins C-G (1-5) and norcaesalpinins D and E (6, 7)] together with norcaesalpinins A-C (8-10) and 11 known compounds (norcaesalpinins A-C, 2-acetoxy-3-deacetoxycaesaldekarin e, caesalmin B, caesaldekarin e, caesalpin F, 14(17)-dehydrocaesalpin F, 2-acetoxycaesaldekarin e, 7-acetoxybonducellpin C, and caesalmin G). Their structures were determined on the basis of spectroscopic analysis. The isolated diterpenes showed significant dose-dependent inhibitory effects on Plasmodium falciparum FCR-3/A2 growth in vitro. Their IC50 values ranged from 90 nM to 6.5 microM, and norcaesalpinin E (7) showed the most potent inhibitory activity (IC50, 90 nM).     

Tigliane diterpenes from the latex of Euphorbia obtusifolia with inhibitory activity on the mammalian mitochondrial respiratory chain

Six diterpenes isolated from the latex of Euphorbia obtusifolia Poir. (Euphorbiaceae) were evaluated for their inhibition of the NADH oxidase activity in submitochondrial particles from beef heart. 4,20-Dideoxyphorbol-12,13-bis(isobutyrate) was the most potent inhibitor and showed an inhibitory concentration with IC(50) value of 2.6+/-0.3mM. In the present study, some structure-activity trends are suggested for the inhibitory activity of the mammalian mitochondrial respiratory chain of these natural product derivatives of 4-deoxyphorbol esters.     

Neo-clerodane diterpenes from Salvia herbacea

Chemical investigation of the aerial parts of Salvia herbacea led to the isolation of eight new neo-clerodane diterpenes (1-8), named tehuanins A-H, and three known compounds. The structures of these compounds were determined by analysis of their spectroscopic data. Three of the new diterpenes possess a 1,8-epoxy group (1-3). This unusual structural feature was confirmed by X-ray diffraction of 1. The structure of the previously isolated 1α,10α-epoxysalviarin was revised. The absolute configuration of 6 was established by X-ray diffraction analysis of its bromo derivative 6a. Cytotoxic and anti-inflammatory activities of these diterpenes were examined. None of the compounds were considered to be cytotoxic; however, compound 7 exhibited anti-inflammatory activity comparable to that of indomethacin.     

Furanolabdane diterpenes from Hypoestes purpurea

Four new furanolabdane diterpenes, hypopurin A (1), hypopurin B (2), hypopurin C (3), and hypopurin D (4), together with eight lignans, alpha-O-methylcubebin, beta-O-methylcubebin, hinoquinin, helioxanthin, 7-hydroxyhinokinin, dehydroxycubebin, justicidine E, and (-)-hibalactone, as well as two triterpenes, lupeol and betulin, were isolated from the dried aerial part of Hypoestes purpurea. The structures of 1-4 were elucidated mainly on the basis of NMR and MS. Compound 1 was found to be moderately cytotoxic toward the KB cell line with an IC(50) value of 9.4 microM.