Genotoxicity, acute and subchronic toxicity studies of nano liposomes of Orthosiphon stamineus ethanolic extract in Sprague Dawley rats.

BACKGROUND: Orthosiphon stamineus (OS) Benth is a medicinal plant and native in Southeast Asia. Pharmacological effects of OS are attributed to the presence of lipophilic flavones. However; lipophilic compounds suffer from poor aqueous solubility which limits the OS oral bioavailability and therapeutic applications. Therefore, OS was prepared in nano formulation form using liposomes from soybean phospholipids. The aim of the present study is to evaluate the in vitro genotoxicity and in vivo oral toxicity of nano liposomes of OS ethanolic extract (OS-EL). METHODS: In the acute toxicity study Sprague Dawley female rats were given a single dose of the OS-EL at 5000 mg/kg/day orally and screened for two weeks after administration. In the subchronic study, three different doses of OS-EL 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. High-performance liquid chromatography was performed for identification and quantification of the major marker compounds in OS-EL. 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 repeated dose 28-day oral toxicity study, the administration of 250 mg/kg, 500 mg/kg, and 1000 mg/kg/day of OS-EL per body weight revealed no significant difference in food and water consumptions, bodyweight change, haematological and biochemical parameters, relative organ weights, gross findings or histopathology compared to the control group. The Ames test revealed that the OS-EL did not have any potential to induce gene mutations in S. Typhimurium. CONCLUSIONS: Analyses of these results with the information of signs, behaviour, and health monitoring could lead to the conclusion that the long-term oral administration of OS-EL for 28 days does not cause sub-chronic toxicity.

Isolation, Characterization, Crystal Structure Elucidation of Two Flavanones and Simultaneous RP-HPLC Determination of Five Major Compounds from Syzygium campanulatum Korth.

Two flavanones named (2S)-7-Hydroxy-5-methoxy-6,8-dimethyl flavanone (1), (S)-5,7-dihydroxy-6,8-dimethyl-flavanone (2), along with known chalcone, namely, (E)-2',4'- dihydroxy-6'-methoxy-3',5'-dimethylchalcone (3) and two triterpenoids, namely, betulinic and ursolic acids (4 and 5), were isolated from the leaves of Syzygium campanulatum Korth (Myrtaceae). The structures of compounds (1 and 2) were determined on the basis of UV-visible, FTIR, NMR spectroscopies and LC-EIMS analytical techniques. Furthermore, new, simple, precise, selective, accurate, highly sensitive, efficient and reproducible RP-HPLC method was developed and validated for the quantitative analysis of the compounds (1-5) from S. campanulatum plants of five different age. RP-HPLC method was validated in terms of specificity, linearity (r2 ≤ 0.999), precision (2.0% RSD), and recoveries (94.4%-105%). The LOD and LOQ of these compounds ranged from 0.13-0.38 and 0.10-2.23 µg·mL-1, OPEN ACCESS respectively. Anti-proliferative activity of isolated flavanones (1 and 2) and standardized extract of S. campanulatum was evaluated on human colon cancer (HCT 116) cell line. Compounds (1 and 2) and extract revealed potent and dose-dependent activity with IC50 67.6, 132.9 and 93.4 µg·mL-1, respectively. To the best of our knowledge, this is the first study on isolation, characterization, X-ray crystallographic analysis of compounds (1 and 2) and simultaneous RP-HPLC determination of five major compounds (1-5) from different age of S. campanulatum plants.

A novel reverse phase high-performance liquid chromatography method for standardization of Orthosiphon stamineus leaf extracts.

BACKGROUND: Orthosiphon stamineus Benth. (Lamiaceae) is a traditional medicinal plant which has been used in treating various ailments such as kidney diseases, bladder inflammation, arthritis and diabetes. The leaves contain high concentration of phenolic compounds, thus, rosmarinic acid (RA), 3'-hydroxy-5, 6, 7, 4'-tetramethoxyflavone (TMF), sinensetin (SIN) and eupatorin (EUP) were chosen as a marker compounds for standardization of various O. stamineus leaf extracts. OBJECTIVE: The aim was to develop and validate a new high-performance liquid chromatography (HPLC) method for quantification of 4 marker compounds (RA, TMF, SIN, EUP) in various O. stamineus leaf extracts. MATERIALS AND METHODS: The method was developed and validated using RP-HPLC-diode-array detection at 320 nm for accuracy, precision and limits of detection and was applied for quantification of it markers in five different extracts prepared in solvents with increasing polarity, using a gradient mobile phase 0.1% formic acid: Acetonitrile at a flow rate of 1 ml/min on reverse phase acclaim polar advantage II C18 column (3 µm, 3 × 150 mm) with 18 min separation time. RESULTS: The developed method provided satisfactory precision, and the accuracy of this method was in the range of 90.2% to 105.5%. All of 4 compounds showed good linearity at R2 > 0.999. CONCLUSION: The developed method is a simple, cost effective with shorter run time (18 min) in comparison to previous methods (30 min) and utilization of environmental-friendly solvents system. Therefore, this method has the potential to replace currently used methods in the routine standardization work of O. stamineus extracts, raw materials and its commercial products.

Analysis of L-citrulline and L-arginine in Ficus deltoidea leaf extracts by reverse phase high performance liquid chromatography.

BACKGROUND: Ficus deltoidea (FD) is one of the native plants widely distributed in several countries in Southeast Asia. Previous studies have shown that FD leaf possess antinociceptive, wound healing and antioxidant properties. These beneficial effects have been attributed to the presence of primary and secondary metabolites such as polyphenols, amino acids and flavonoids. OBJECTIVE: The aim was to develop a reverse phase high-performance liquid chromatography method with ultraviolet detection that involves precolumn derivatisation with O-phthaladehyde for simultaneous analysis of two amino acids L-citrulline and L-arginine in FD leaf extracts. MATERIALS AND METHODS: An isocratic elution program consisting of methanol: acetonitrile: Water at 45:45:10 v/v (solvent A) and 0.1 M phosphate buffer pH 7.5 (solvent B) at A: B v/v ratio of 80:20 on Zorbax Eclipse C18 SB-Aq column (250 × 4.6 mm, 5 µm) were used. The flow rate was set at 1 ml/min and detection was carried out at 338 nm with 30 min separation time. RESULTS: Good linearity for L-citrulline and L-arginine was obtained in the range 0.1-1000 µg/ml at R (2) ≥ 0.998. The limit of detection and limit of quantification values for both L-citrulline and L-arginine were 1 and 5 µg/ml, respectively. The average of recoveries was in the range 94.94-101.95%, with relative standard deviation (%RSD) less than 3%. Intra- and inter-day precision was in the range 96.36-102.43% with RSD less than 2%. CONCLUSION: All validation parameters of the developed method indicate the method is reliable and efficient for simultaneous determination of L-citrulline and L-arginine for routine analysis of FD.

Isolation, Characterization, Crystal Structure Elucidation, and Anticancer Study of Dimethyl Cardamonin, Isolated from Syzygium campanulatum Korth.

Syzygium campanulatum Korth is an equatorial, evergreen, aboriginal shrub of Malaysia. Conventionally it has been used as a stomachic. However, in the currently conducted study dimethyl cardamonin or 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC) was isolated from S. campanulatum Korth, leaf extract. The structural characterization of DMC was carried out by making use of various techniques including UV, IR, NMR spectral followed by LC-MS, and X-ray crystallographic techniques. For determining the purity of compound, highly effective techniques including TLC, HPLC, and melting point were used. The cytotoxicity of DMC and three different extracts of S. campanulatum was evaluated against human colon cancer cell line (HT-29) by three different assays. DMC and ethanolic extract revealed potent and dose-dependent cytotoxic activity on the cancer cell line with IC50 12.6 and 90.1 µg/mL, respectively. Quite astonishingly to our knowledge, this is the very first report on S. campanulatum as being a rich source (3.5%) of DMC, X-ray crystallography, and anticancer activity on human colon cancer cells.

Preparation and characterization of nano liposomes of Orthosiphon stamineus ethanolic extract in soybean phospholipids.

BACKGROUND: O. stamineus is a medicinal herb with remarkable pharmacological properties. However, poor solubility of the active principles limits its medicinal value. This study sought to prepare nano liposomes of OS ethanolic extract in unpurified soybean phospholipids in order to improve its solubility and permeability. OS liposomes were prepared by the conventional film method, and were characterized for solubility, entrapment efficiency, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), particle size and zeta potential, release, absorption in everted rat intestinal sacs, and DPPH scavenging effect. RESULTS: OS liposomes showed substantial enhancement of extract's solubility from 956 ± 34 to 3979 ± 139 µg/ml, with entrapment efficiency of 66.2 ± 0.9%. FTIR study indicates interaction between soybean phospholipids and OS extract. TEM and dynamic light scattering showed presence of round anionic nano liposomes with particle size and zeta potential of 152.5 ± 1.1 nm and -49.8 ± 1.0 mV, respectively. A study using the fluorescent probe pyrene showed the critical micellar concentration is 9.2 ± 2.9 µg/ml. Release studies showed 94 ± 0.1% release in non-formulated extract and 62.4 ± 0.1% in OS liposomes. Released extract from OS liposomes showed improvement in DPPH scavenging effect, IC50 = 23.5 ± 1.1 µg/ml compared to 32.4 ± 0.5 µg/ml in non-formulated extract. OS liposomes were stable at pH 5.5 and 7.4, but showed reversible agglomeration at pH 1.6. Absorption in everted rat intestinal sacs showed substantial improvement in permeability of 3'-hydroxy-5, 6, 7, 4″-tetramethoxyflavone, sinensetin, eupatorin, and 3 other unknown compounds. CONCLUSIONS: Enhanced solubility, absorption and antioxidant effect may improve the overall pharmacological effects and medicinal value of OS ethanolic extract.

Proapoptotic and antimetastatic properties of supercritical CO2 extract of Nigella sativa Linn. against breast cancer cells.

Nigella sativa, commonly referred as black cumin, is a popular spice that has been used since the ancient Egyptians. It has traditionally been used for treatment of various human ailments ranging from fever to intestinal disturbances to cancer. This study investigated the apoptotic, antimetastatic, and anticancer activities of supercritical carbon dioxide (SC-CO2) extracts of the seeds of N. sativa Linn. against estrogen-dependent human breast cancer cells (MCF-7). Twelve extracts were prepared from N. sativa seeds using the SC-CO2 extraction method by varying pressure and temperature. Extracts were analyzed using FTIR and UV-Vis spectrometry. Cytotoxicity of the extracts was evaluated on various human cancer and normal cell lines. Of the 12 extracts, 1 extract (A3) that was prepared at 60°C and 2500 psi (~17.24 MPa) showed selective antiproliferative activity against MCF-7 cells with an IC50 of 53.34±2.15 µg/mL. Induction of apoptosis was confirmed by evaluating caspases activities and observing the cells under a scanning electron microscope. In vitro antimetastatic properties of A3 were investigated by colony formation, cell migration, and cell invasion assays. The elevated levels of caspases in A3 treated MCF-7 cells suggest that A3 is proapoptotic. Further nuclear condensation and fragmentation studies confirmed that A3 induces cytotoxicity through the apoptosis pathway. A3 also demonstrated remarkable inhibition in migration and invasion assays of MCF-7 cells at subcytotoxic concentrations. Thus, this study highlights the therapeutic potentials of SC-CO2 extract of N. sativa in targeting breast cancer.

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.

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.

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.

In vitro antimetastatic activity of koetjapic acid against breast cancer cells.

Breast cancer is the most common cancer in women, and it can metastasize very rapidly. Tumor metastasis is the primary cause of cancer deaths. In the present study, we investigated the capability of koetjapic acid, a natural triterpene, in the induction of apoptosis and the inhibition of metastasis in the breast cancer cell line (MCF 7). The effects of koetjapic acid against 4 steps of metastasis have been assessed, including cell survival, clonogenicity, migration and invasion. Koetjapic acid exhibited cytotoxic activity against MCF 7 cells with an IC(50) of 68.88±6.075 µg/mL. The mechanism of cell death was confirmed due to the induction of apoptosis machineries; early and late apoptosis-related changes were detected, including the stimulation of caspase 3/7 activities, apoptosis-related morphological changes such as membrane blebbing, chromatin condensation and DNA fragmentation. A mitochondrial apoptosis pathway was found to be involved in koetjapic acid-induced cell death induction. Moreover, at a sub-toxic dose (15 µg/mL), Koetjapic acid inhibited cell migration and invasion significantly. Finally, koetjapic acid inhibited the colony formation properties of MCF 7 significantly. These results indicate that koetjapic acid possesses significant antitumor and antimetastatic effects, and warrants further investigation.

Syzygium aromaticum extracts as good source of betulinic acid and potential anti-breast cancer

Syzygium aromaticum (L.) Merr. & L.M. Perry, Myrtaceae, is an evergreen tree with anticarcinogenic, antimutagenic, aphrodisiac, antimicrobial, antioxidant and antiinflammatory properties. This study aims to investigate the anti-breast cancer effect of extracts from leaves, stem and bark of S. aromaticum and to develop a method for preparation of betulinic acid fraction from the leaves. Betulinic acid, ursolic acid and oleanolic acid contents of the extracts were determined by HPLC. A betulinic acid fraction was prepared by simple crystallization of leaves extract and was characterized by HPLC and mass analysis. Anti-breast cancer effects were studied on MCF-7 and MDA-MB-231 cells. The extracts were found to contain high levels of betulinic acid particularly the leaves extract which contained 17% wt/wt. The betulinic acid fraction contains 75% betulinic acid. Cytotoxicity testing reveals high and selective cytotoxic effect of the stem extract on MCF-7 cells with IC50 33±1.6 µg/mL. Cytotoxic effect of the stem extract was due to activation of apoptotic machinery of cell death. Combination studies of stem extract with tamoxifen reveals antagonistic effect at high concentration of tamoxifen and enhancement effect at low concentration. The selective cytotoxicity of the stem extract of S. aromaticum on MCF-7 is not due to betulinic acid but due to other constituents yet to be discovered.

alpha-Mangostin enhances betulinic acid cytotoxicity and inhibits cisplatin cytotoxicity on HCT 116 colorectal carcinoma cells.

Despite the progress in colon cancer treatment, relapse is still a major obstacle. Hence, new drugs or drug combinations are required in the battle against colon cancer. α-Mangostin and betulinic acid (BA) are cytotoxic compounds that work by inducing the mitochondrial apoptosis pathway, and cisplatin is one of the most potent broad spectrum anti-tumor agents. This study aims to investigate the enhancement of BA cytotoxicity by α-mangostin, and the cytoprotection effect of α-mangostin and BA on cisplatin-induced cytotoxicity on HCT 116 human colorectal carcinoma cells. Cytotoxicity was investigated by the XTT cell proliferation test, and the apoptotic effects were investigated on early and late markers including caspases-3/7, mitochondrial membrane potential, cytoplasmic shrinkage, and chromatin condensation. The effect of α-mangostin on four signalling pathways was also investigated by the luciferase assay. α-Mangostin and BA were more cytotoxic to the colon cancer cells than to the normal colonic cells, and both compounds showed a cytoprotective effect against cisplatin-induced cytotoxicity. On the other hand, α-mangostin enhanced the cytotoxic and apoptotic effects of BA. Combination therapy hits multiple targets, which may improve the overall response to the treatment, and may reduce the likelihood of developing drug resistance by the tumor cells. Therefore, α-mangostin and BA may provide a novel combination for the treatment of colorectal carcinoma. The cytoprotective effect of the compounds against cisplatin-induced cytotoxicity may find applications as chemopreventive agents against carcinogens, irradiation and oxidative stress, or to neutralize cisplatin side effects.

Solid dispersions of α-mangostin improve its aqueous solubility through self-assembly of nanomicelles.

α-Mangostin is an oxygenated heterocyclic xanthone with remarkable pharmacological properties, but poor aqueous solubility and low oral bioavailability hinder its therapeutic application. This study sought to improve the compound's solubility and study the mechanism underlying solubility enhancement. Solid dispersions of α-mangostin were prepared in polyvinylpyrrolidone (PVP) by solvent evaporation method and showed substantial enhancement of α-mangostin's solubility from 0.2 ± 0.2 µg/mL to 2743 ± 11 µg/mL. Fourier transform infrared spectroscopy and differential scanning calorimetry indicated interaction between α-mangostin and PVP. Transmission electron microscopy and dynamic light scattering showed self-assembly of round anionic nanomicelles with particle size in the range 99-127 nm. Powder X-ray diffraction indicated conversion of α-mangostin from crystalline into amorphous state, and scanning electron microscopy showed the presence of highly porous powder. Studies using the fluorescent probe pyrene showed that the critical micellar concentration is about 77.4 ± 4 µg/mL. Cellular uptake of nanomicelles was found to be mediated via endocytosis and indicated intracellular delivery of α-mangostin associated with potent cytotoxicity (median inhibitory concentration of 8.9 ± 0.2 µg/mL). Improved solubility, self-assembly of nanomicelles, and intracellular delivery through endocytosis may enhance the pharmacological properties of α-mangostin, particularly antitumor efficacy.

Evaluation of antiangiogenic and antoxidant properties of Parkia speciosa Hassk extracts.

Parkia speciosa Hassk is a traditional medicinal plant with strong antioxidant and hypoglycemic properties. This study aims to investigate the total phenolic content, antioxidant, cytotoxic and antiangiogenic effect of eight extracts from P. speciosa empty pods. The extracts were found to contain high levels of total phenols and demonstrated strong antioxidant effect in DPPH scavenging test. In rat aortic rings, P. speciosa extracts significantly inhibited the microvessel outgrowth from aortic tissue explants by more than 50%. The antiangiogenic activity was further confirmed by tube formation on matrigel matrix involving human endothelial cells. Cytotoxic effect was evaluated by XTT test on endothelial cells as a model of angiogenesis versus a panel of human cancer and normal cell lines. Basically the extracts did not show acute cytotoxicity. Morphology examination of endothelial cells indicated induction of autophagy characterized by formation of plenty of cytoplasmic vacuoles. The extracts were found to work by decreasing expression of vascular endothelial growth factor in endothelial cells.

Koetjapic acid, a natural triterpenoid, induces apoptosis in colon cancer cells.

Deregulated cell signaling pathways result in cancer development. More than one signal transduction pathway is involved in colorectal cancer pathogenesis and progression. Koetjapic acid (KA) is a naturally occurring seco-A-ring oleanene triterpene isolated from the Sandoricum koetjape stem bark. We report the cellular and molecular mechanisms of anticancer activity of KA towards human colorectal cancer. The results showed that KA induces apoptosis in HCT 116 colorectal carcinoma cells by inducing the activation of extrinsic and intrinsic caspases. We confirmed that KA-induced apoptosis was mediated by DNA fragmentation, nuclear condensation and disruption in the mitochondrial membrane potential. Further studies on the effect of KA on cancer pathways show that the compound causes down-regulation of Wnt, HIF-1α, MAP/ERK/JNK and Myc/Max signaling pathways and up-regulates the NF-κB signaling pathway. The result of this study highlights the anticancer potential of KA against colorectal cancer.

Cat's whiskers tea (Orthosiphon stamineus) extract inhibits growth of colon tumor in nude mice and angiogenesis in endothelial cells via suppressing VEGFR phosphorylation.

Cat's whiskers (Orthosiphon stamineus) is commonly used as Java tea to treat kidney stones including a variety of angiogenesis-dependent diseases such as tumorous edema, rheumatism, diabetic blindness, and obesity. In the present study, antitumor potential of standardized 50% ethanol extract of O. stamineus leaves (EOS) was evaluated against colorectal tumor in athymic mice and antiangiogenic efficacy of EOS was investigated in human umbilical vein endothelial cells (HUVEC). EOS at 100 mg/kg caused 47.62 ± 6.4% suppression in tumor growth, while at 200 mg/kg it caused 83.39 ± 4.1% tumor regression. Tumor histology revealed significant reduction in extent of vascularization. Enzyme-linked immunosorbent assay showed EOS (200 mg/kg) significantly reduced the vascular endothelial growth factor (VEGF) level in vitro (211 ± 0.26 pg/ml cell lysate) as well as in vivo (90.9 ± 2 pg/g tissue homogenate) when compared to the control (378 ± 5 and 135.5 ± 4 pg, respectively). However, EOS was found to be noncytotoxic to colon cancer and endothelial cells. In vitro, EOS significantly inhibited the migration and tube formation of human umbilical vein endothelial cells (HUVECs). EOS suppressed VEGF-induced phosphorylation of VEGF receptor-2 in HUVECs. High performance liquid chromatography (HPLC) analysis of EOS showed high rosmarinic acid contents, whereas phytochemical analysis revealed high protein and phenolic contents. These results demonstrated that the antitumor activity of EOS may be due to its VEGF-targeted antiangiogenicity.

Antitumorigenicity of xanthones-rich extract from Garcinia mangostana fruit rinds on HCT 116 human colorectal carcinoma cells

This study aimed to investigate the antitumorigenicity of xanthones-rich extract from Garcinia mangostana L., Clusiaceae, fruit rinds which was obtained by a simple recrystallization of 75 percent ethanolic extract. α-Mangostin content of the extract was determined qualitatively by TLC and quantitatively by HPLC, and total xanthones content was quantified by UV spectrophotometry. The extract was evaluated for cytotoxicity, apoptosis and antitumorigenicity on HCT 116 human colorectal carcinoma cells. α-Mangostin was found to be the main constituent of the extract which was 71.2±0.1 percent, and the total xanthones content was 95±4.8 percent (wt/wt). The extract showed potent dose dependent cytotoxicity with IC50 value 9.2 μg/mL. Apoptosis studies revealed activation of caspases 3 and 7, DNA fragmentation, chromatin condensation and loss of mitochondrial membrane potential. Studies on cell migration and colony formation indicate reduced cell migration ability and clonogenicity of treated HCT 116 cells at sub-inhibitory concentrations. Taken together, the cytotoxic effect of the xanthones extract is mediated through the mitochondrial pathway of apoptosis. The reduced cell migration and clonogenicity of HCT 116 cells might prevent both primary and metastatic tumor growth in vivo which will be the topic of our future work using the metastatic orthotopic colon cancer model.