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1.  Crystal Structure Elucidation and Anticancer Studies of (-)-Pseudosemiglabrin: A Flavanone Isolated from the Aerial Parts of Tephrosia apollinea 
PLoS ONE  2014;9(3):e90806.
Tephrosia apollinea is a perennial shrublet widely distributed in Africa and is known to have medicinal properties. The current study describes the bio-assay (cytotoxicity) guided isolation of (-)-pseudosemiglabrin from the aerial parts of T. apollinea. The structural and stereochemical features have been described using spectral and x-ray crystallographic techniques. The cytotoxicity of isolated compound was evaluated against nine cancer cell lines. In addition, human fibroblast was used as a model cell line for normal cells. The results showed that (-)-pseudosemiglabrin exhibited dose-dependent antiproliferative effect on most of the tested cancer cell lines. Selectively, the compound showed significant inhibitory effect on the proliferation of leukemia, prostate and breast cancer cell lines. Further studies revealed that, the compound exhibited proapoptotic phenomenon of cytotoxicity. Interestingly, the compound did not display toxicity against the normal human fibroblast. It can be concluded that (-)-pseudosemiglabrin is worthy for further investigation as a potential chemotherapeutic agent.
doi:10.1371/journal.pone.0090806
PMCID: PMC3946547  PMID: 24608571
2.  Syzygium campanulatum korth methanolic extract inhibits angiogenesis and tumor growth in nude mice 
Background
Syzygium campanulatum Korth (Myrtaceae) is an evergreen shrub rich in phenolics, flavonoid antioxidants, and betulinic acid. This study sought to investigate antiangiogenic and anti-colon cancer effects of S.C. standardized methanolic extract.
Methods
Betulinic acid was isolated from methanolic extract by crystallization and chromatography techniques. S.C. methanolic extract was analyzed by UV-Vis spectrophotometry, FTIR, LC-MS, and HPLC. Antiangiogenic effect was studied on rat aortic rings, matrigel tube formation, cell proliferation and migration, and expression of vascular endothelial growth factor (VEGF). Antitumor effect was studied using a subcutaneous tumor model of HCT 116 colorectal carcinoma cells established in nude mice.
Results
Analysis by HPLC, LC-MS and FTIR confirm presence of betulinic acid in S.C. methanolic extract. Quantitative analysis by HPLC indicates presence of betulinic acid in S.C. extract at 5.42 ± 0.09% (w/w). Antiangiogenesis study showed potent inhibition of microvessels outgrowth in rat aortic rings, and studies on normal and cancer cells did not show any significant cytotoxic effect. Antiangiogenic effect was further confirmed by inhibition of tube formation on matrigel matrix that involves human endothelial cells (IC50 = 17.6 ± 2.9 μg/ml). S.C. extract also inhibited migration of endothelial cells and suppressed expression of VEGF. In vivo antiangiogenic study showed inhibition of new blood vessels in chicken embryo chorioallantoic membrane (CAM), and in vivo antitumor study showed significant inhibition of tumor growth due to reduction of intratumor blood vessels and induction of cell death.
Conclusion
Collectively, our results indicate S. campanulatum as antiangiogenic and antitumor candidate, and a new source of betulinic acid.
doi:10.1186/1472-6882-13-168
PMCID: PMC3717079  PMID: 23842450
3.  Vascular Reactivity Concerning Orthosiphon stamineus Benth-Mediated Antihypertensive in Aortic Rings of Spontaneously Hypertensive Rats 
Orthosiphon stamineus Benth has been traditionally used to treat hypertension. The study aimed to investigate the vascular reactivity of water extract (WOS) and water : methanolic (1 : 1) extract (WMOS) of Orthosiphon stamineus Benth and AT1 receptors blocker in the mechanisms of antihypertensive mediated by α1-adrenergic receptor and EDNO and PGI2 releases in the SHR aortic rings. SHR (230–280 g) were divided into four groups: control, WOS, WMOS, and losartan. After being fed orally for 14 days, the aorta was harvested and subjected to PE (10−9 to 10−5 M) and ACh (10−9 to 10−5 M) with and without L-NAME (100 µM) and indomethacin (10 µM), respectively. WOS, WMOS, and losartan significantly reduced the contractile responses to PE intact suggesting the importance of endothelium in vasorelaxation. Losartan significantly enhanced the ACh-induced vasorelaxation. L-NAME significantly inhibited the ACh-induced relaxation in all groups. Indomethacin enhanced ACh-induced vasorelaxation in WMOS. Collectively, Orthosiphon stamineus leaves extract reduced vasoconstriction responses by the alteration of α1-adrenergic and AT1 receptors activities. The involvement of EDNO releases was clearly observed in this plant. In WOS, PGI2 releases might not participate in the ACh-induced vasorelaxation. However, in WMOS, enhancement of vasorelaxation possibly due to continuous release of PGI2.
doi:10.1155/2013/456852
PMCID: PMC3710645  PMID: 23878738
4.  Genotoxicity and acute and subchronic toxicity studies of a standardized methanolic extract of Ficus deltoidea leaves 
Clinics  2013;68(6):865-875.
OBJECTIVE:
Ficus deltoidea leaves have been used in traditional medicine in Southeast Asia to treat diabetes, inflammation, diarrhea, and infections. The present study was conducted to assess the genotoxicity and acute and subchronic toxicity of a standardized methanol extract of F. deltoidea leaves.
METHODS:
Sprague Dawley rats were orally treated with five different single doses of the extract and screened for signs of toxicity for two weeks after administration. In the subchronic study, three different doses of the extract were administered for 28 days. Mortality, clinical signs, body weight changes, hematological and biochemical parameters, gross findings, organ weights, and histological parameters were monitored during the study. Genotoxicity was assessed using the Ames test with the TA98 and TA100 Salmonella typhimurium strains. Phytochemical standardization was performed using a colorimeter and high-performance liquid chromatography. Heavy metal detection was performed using an atomic absorption spectrometer.
RESULTS:
The acute toxicity study showed that the LD50 of the extract was greater than 5000 mg/kg. In the subchronic toxicity study, there were no significant adverse effects on food consumption, body weight, organ weights, mortality, clinical chemistry, hematology, gross pathology, or histopathology. However, a dose-dependent increase in the serum urea level was observed. The Ames test revealed that the extract did not have any potential to induce gene mutations in S. typhimurium, either in the presence or absence of S9 activation. Phytochemical analysis of the extract revealed high contents of phenolics, flavonoids, and tannins. High-performance liquid chromatography analysis revealed high levels of vitexin and isovitexin in the extract, and the levels of heavy metals were below the toxic levels.
CONCLUSION:
The no-observed adverse effect level of F. deltoidea in rats was determined to be 2500 mg/kg.
doi:10.6061/clinics/2013(06)23
PMCID: PMC3674303  PMID: 23778480
Ficus deltoidea; Oral Toxicity; OECD; Genotoxicity; Isovitexin; Vitexin
5.  Antiangiogenesis and antioxidant activity of ethanol extracts of Pithecellobium jiringa 
Background
Angiogenesis plays a critical role in embryonic development and various physiological processes. However, excessive angiogenesis is associated with several pathological conditions including cancer. Pithecellobium jiringa (Jack) Prain is a traditional medicinal plant from the family Leguminosae. It is native to the Southeast Asia, where it has been used traditionally for treatment of various ailments such as hypertension and diabetes. The present work is aimed to study antioxidant and antiangiogenesis activities of P. jiringa ethanol extracts.
Methods
P. jiringa fruit rinds were extracted with ethanol and 50% ethanol. The antioxidant property was analysed using, 1,1-diphenyl-2-picryl-hydrazyl free radical scavenging assay. Phytochemical analysis was performed using thin layer chromatography and colorimetric methods. Then, cell growth inhibition was studied against a panel of human cell lines by MTT test. In vitro inhibition of angiogenesis was studied by the following assays: isolated rat aortic rings cell viability, colony formation, endothelial cell migration, endothelial tube formation on matrigel, and expression of vascular endothelial growth factor by endothelial cells. In vivo antiangiogenesis effect was studied by utilising fertilised chick embryos assay. The results were statistically analysed by analysis of variance.
Results
Ethanolic and 50% hydro-ethanolic extracts showed relatively high concentration of total phenolics associated with potent antioxidant activity. The rat aortic rings study conducted showed potent inhibition of the microvessels outgrowth with IC50s 5.27 ± 0.81 μg/ml (ethanolic) and 4.45 ± 0.63 μg/ml (50% hydro-ethanolic). Both extracts arrested the growth of human endothelial cells via down-regulation of VEGF expression, leading to inhibition of other angiogenesis cascades including migration of endothelial cells, and formation of capillary network on matrigel matrix. The extracts also inhibited the neovascularisation of chick embryo chorioallantoic membrane.
Conclusions
P. jiringa extracts inhibit angiogenesis by blocking the VEGF expression thus inhibiting endothelial cells proliferation, migration and differentiation most likely due to presence of the antioxidant phenolics.
doi:10.1186/1472-6882-12-210
PMCID: PMC3522529  PMID: 23126282
Pithecellobium jiringa; Antiangiogenesis; Antioxidant; Phytochemical analysis
6.  In vitro and in vivo anti-colon cancer effects of Garcinia mangostana xanthones extract 
Background
Xanthones are a group of oxygen-containing heterocyclic compounds with remarkable pharmacological effects such as anti-cancer, antioxidant, anti-inflammatory, and antimicrobial activities.
Methods
A xanthones extract (81% α-mangostin and 16% γ-mangostin), was prepared by crystallization of a toluene extract of G. mangostana fruit rinds and was analyzed by LC-MS. Anti-colon cancer effect was investigated on HCT 116 human colorectal carcinoma cells including cytotoxicity, apoptosis, anti-tumorigenicity, and effect on cell signalling pathways. The in vivo anti-colon cancer activity was also investigated on subcutaneous tumors established in nude mice.
Results
The extract showed potent cytotoxicity (median inhibitory concentration 6.5 ± 1.0 μg/ml), due to induction of the mitochondrial pathway of apoptosis. Three key steps in tumor metastasis including the cell migration, cell invasion and clonogenicity, were also inhibited. The extract and α-mangostin up-regulate the MAPK/ERK, c-Myc/Max, and p53 cell signalling pathways. The xanthones extract, when fed to nude mice, caused significant growth inhibition of the subcutaneous tumor of HCT 116 colorectal carcinoma cells.
Conclusions
Our data suggest new mechanisms of action of α-mangostin and the G. mangostana xanthones, and suggest the xanthones extract of as a potential anti-colon cancer candidate.
doi:10.1186/1472-6882-12-104
PMCID: PMC3457913  PMID: 22818000
7.  Bioactive Markers Based Pharmacokinetic Evaluation of Extracts of a Traditional Medicinal Plant, Piper sarmentosum 
In vitro assays are economical and easy to perform but to establish relevance of their results to real clinical outcome in animals or human, pharmacokinetics is prerequisite. Despite various in vitro pharmacological activities of extracts of Piper sarmentosum, there is no report of pharmacokinetics. Therefore, the present study aimed to evaluate ethanol extract of fruit of the plant in dose of 500 mg kg−1 orally for pharmacokinetics. Sprague-Dawley rats were randomly divided into groups 1, 2, and 3 (each n = 6) to study absorption, distribution and excretion, respectively. High performance liquid chromatography (HPLC) with ultraviolet detection was applied to quantify pellitorine, sarmentine and sarmentosine in plasma, tissues, feces and urine to calculate pharmacokinetic parameters. Pellitorine exhibited maximum plasma concentration (Cmax) 34.77 ng mL−1 ± 1.040, time to achieve Cmax (Tmax) 8 h, mean resident time (MRT) 26.00 ± 0.149 h and half life (t1/2) 18.64 ± 1.65 h. Sarmentine showed Cmax 191.50 ± 12.69 ng mL−1, Tmax 6 h, MRT 11.12 ± 0.44 h and t1/2 10.30 ± 1.98 h. Sarmentosine exhibited zero oral bioavailability because it was neither detected in plasma nor in tissues, and in urine. Pellitorine was found to be distributed in intestinal wall, liver, lungs, kidney, and heart, whereas sarmentine was found only in intestinal wall and heart. The cumulative excretion of pellitorine, sarmentine and sarmentosine in feces in 72 h was 0.0773, 0.976, and 0.438 μg, respectively. This study shows that pellitorine and sarmentine have good oral bioavailability while sarmentosine is not absorbed from the gastrointestinal tract.
doi:10.1093/ecam/nep143
PMCID: PMC3137875  PMID: 19770264
8.  Antiangiogenic properties of Koetjapic acid, a natural triterpene isolated from Sandoricum koetjaoe Merr 
Background
Angiogenesis, the formation of new blood vessels, has become an important target in cancer therapy. Angiogenesis plays an important role in tumor growth and metastasis. Koetjapic acid (KA) is a seco-A-ring oleanene triterpene isolated from S. koetjape. The solvent extract of this plant species was shown previously to have strong antiangiogenic activity; however the active ingredient(s) that conferred the biological activity and the mode of action was not established. Given the high concentration of KA in S. koetjape, an attempt has been made in this study to investigate the antiangiogenic properties of KA.
Results
Treatment with 10-50 μg/ml KA resulted in dose dependent inhibition of new blood vessels growth in ex vivo rat aortic ring assay. KA was found to be non-cytotoxic against HUVECs with IC50 40.97 ± 0.37 μg/ml. KA inhibited major angiogenesis process steps, endothelial cell migration and differentiation as well as VEGF expression.
Conclusions
The non-cytotoxic compound, KA, may be a potent antiangiogenic agent; its activity may be attributed to inhibition of endothelial cells migration and differentiation as well VEGF suppression.
doi:10.1186/1475-2867-11-12
PMCID: PMC3111336  PMID: 21524294
9.  Simultaneous determination of secondary metabolites from Vinca rosea plant extractives by reverse phase high performance liquid chromatography 
Pharmacognosy Magazine  2011;7(26):92-96.
Background:
Vinca rosea (Apocynaceae) is one of the most important and high value medicinal plants known for its anticancer alkaloids. It is the iota of the isolated secondary metabolites used in chemotherapy to treat diverse cancers. Several high performance liquid chromatography (HPLC) methods have been developed to quantify the active alkaloids in the plant. However, this method may serve the purpose in quantification of V. rosea plant extracts in totality.
Objective:
To develop and validate the reverse phase (RP)-HPLC method for simultaneous determination of secondary metabolites, namely alkaloids from V. rosea plant extracts.
Materials and Methods:
The quantitative determination was conducted by RP-HPLC equipped with ultraviolet detector. Optimal separation was achieved by isocratic elution with mobile phase consisting of methanol:acetonitrile:ammonium acetate buffer (25 mM) with 0.1% triethylamine (15:45:40 v/v) on a column (Zorbax Eclipse plus C18, 250 mm % 4.6 mm; 5 μm). The standard markers (vindoline, vincristine, catharanthine, and vinblastine) were identified by retention time and co-injected with reference standard and quantified by external standard method at 297 nm.
Results:
The precision of the method was confirmed by the relative standard deviation (R.S.D.), which was lower than 2.68%. The recoveries were in the range of 98.09%-108%. The limits of detection (LOD) for each marker alkaloids were lower than 0.20 μg. Different parts of the V. rosea extracts shows different concentrations of markers, flower samples were high in vinblastine content, while methanol extract from the leaves contains all the four alkaloids in good yield, and there is no significant presence of markers in water extracts.
Conclusion:
HPLC method established is appropriate for the standardization and quality assurance of V. rosea plant extracts.
doi:10.4103/0973-1296.80662
PMCID: PMC3113361  PMID: 21716929
Catharanthine; isocratic; quality assurance; Vinca rosea; vincristine; vindoline; vinblastine
10.  7-Hy­droxy-6-meth­oxy-2H-chromen-2-one 
The title compound, C10H8O4, is one of the coumarins existing in Morinda citrifolia L (Noni). The chromenone ring system is approximately planar with a maximum deviation of 0.0208 (14) Å. The meth­oxy group does not deviate from this plane [C—O—C—C torsion angle = −1.5 (3)°], indicating that the whole mol­ecule is almost planar. In the crystal packing, inter­molecular O—H⋯O hydrogen bonds link the mol­ecules into chains. These are further connected by C—H⋯O hydrogen bonds.
doi:10.1107/S1600536810029296
PMCID: PMC3007346  PMID: 21588426
11.  SDS-PAGE-Based Quantitative Assay for Screening of Kidney Stone Disease 
Biological Procedures Online  2009;11:145-160.
Kidney stone disease is a common health problem in industrialised nations. We developed a SDS-PAGE-based method to quantify Tamm Horsfall glycoprotein (THP) for screening of kidney stone disease. Urinary proteins were extracted by using ammonium sulphate precipitation at 0.27 g salt/mL urine. The resulted pellet was dissolved in TSE buffer. Ten microliters of the urinary proteins extract was loaded and separated on 10% SDS-PAGE under reducing condition. THP migrated as single band in SDS-PAGE. The assay reproducibility and repeatability were 4.8% CV and 2.6% CV, respectively. A total of 117 healthy subjects and 58 stone patients were tested using this assay, and a distinct cut-off (P < 0.05) at 5.6 μg/mL THP concentration was used to distinguish stone patients from healthy subjects. The sensitivity and specificity of the method were 92.3% and 83.3%, respectively.
doi:10.1007/s12575-009-9007-y
PMCID: PMC3055900  PMID: 19495911
Kidney stone disease; SDS-PAGE; Tamm Horsfall Protein and diagnostic device
13.  Qualification and application of an ELISA for the determination of Tamm Horsfall Protein (THP) in human urine and its use for screening of Kidney Stone Disease 
Kidney stone disease affects 1 - 20% of the general population. At present, the diagnosis of a stone is done using radiography method when noticeable symptoms appeared. We developed a non-invasive quantitative assay for urinary THP, namely ELISA; whereby our previous study and other reports had shown the usefulness of THP as biomarker for kidney stone disease. Since urine is biological fluid that is easily obtainable, this method could be used as a screening assay for kidney stone prior to confirmation with radiography. The ELISA gave assay linearity r2 > 0.999 within the range of 109 ng/mL to 945 ng/mL THP. Assay precisions were < 4% (C.V.) for repeatability and < 5% (C.V.) for reproducibility. Assay accuracy range from 97.7% to 101.2% at the various THP concentrations tested. Assay specificity and sensitivity were 80% and 86%, respectively. The cut-off points at P < 0.05 were 37.0 and 41.2 μg/mL for male and female, respectively. The assay is cost effective and rapid whereby the cost for assaying each urine sample in duplicate is approximately USD0.35 and within 5 hours, 37 samples can be assayed alongside full range of standards and 3 QC samples in each plate. Furthermore, sample preparation is relatively easy where urine sample was diluted 10 times in TEA buffer. The usability of the ELISA method for diagnosis of kidney stone disease is evaluated with 117 healthy subjects and 58 stone formers.
PMCID: PMC2500153  PMID: 18695745
Kidney stone disease; ELISA; Tamm-Horsfall protein; Screening assay
14.  Development of Multichannel Artificial Lipid-Polymer Membrane Sensor for Phytomedicine Application 
Sensors (Basel, Switzerland)  2006;6(10):1333-1344.
Quality control of herbal medicines remain a challenging issue towards integrating phytomedicine into the primary health care system. As medicinal plants is a complicated system of mixtures, a rapid and cost-effective evaluation method to characterize the chemical fingerprint of the plant without performing laborious sample preparation procedure is reported. A novel research methodology based on an in-house fabricated multichannel sensor incorporating an array of artificial lipid-polymer membrane as a fingerprinting device for quality evaluation of a highly sought after herbal medicine in the Asean Region namely Eurycoma longifolia (Tongkat Ali). The sensor array is based on the principle of the bioelectronic tongue that mimics the human gustatory system through the incorporation of artificial lipid material as sensing element. The eight non-specific sensors have partially overlapping selectivity and cross-sensitivity towards the targeted analyte. Hence, electrical potential response represented by radar plot is used to characterize extracts from different parts of plant, age, batch-to-batch variation and mode of extraction of E. longifolia through the obtained potentiometric fingerprint profile. Classification model was also developed classifying various E. longifolia extracts with the aid of chemometric pattern recognition tools namely hierarchical cluster analysis (HCA) and principal component analysis (PCA). The sensor seems to be a promising analytical device for quality control based on potentiometric fingerprint analysis of phytomedicine.
PMCID: PMC3909399
Lipid membrane sensor; Fingerprint profiling; Quality control; Phytomedicine; Eurycoma longifolia; Chemometric

Results 1-14 (14)