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.
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.
Colorectal cancer (CRC) is one of the most common human malignant tumors worldwide. Arising from the transformation of epithelial cells in the colon and/or rectum into malignant cells, the foundation of CRC pathogenesis lies in the progressive accumulation of mutations in oncogenes and tumor-suppressor genes, such as APC and KRAS. Resistance to apoptosis is one of the key mechanisms in the development of CRC as it is for any other kind of cancer. Natural products have been shown to induce the expression of apoptosis regulators that are blocked in cancer cells. In the present study, a series of in vitro assays were employed to study the apoptosis inducing attributes of Isoledene rich sub-fraction (IR-SF) collected from the oleo-gum resin of M. ferrea. Data obtained, shows that IR-SF inhibited cell proliferation and induced typical apoptotic changes in the overall morphology of all the CRC cell lines tested. Fluorescent staining assays revealed characteristic nuclear condensation, and marked decrease in mitochondrial outer membrane potential in treated cells. In addition, an increment in the levels of ROS, caspase-8,-9 and -3 was observed. Proteomic analysis revealed that IR-SF up-regulated the expression of pro-apoptotic proteins, i.e., Bid, Bid and cytochrome c. Cytochrome c in turn activated caspases cascade resulting in the induction of apoptosis. Moreover, IR-SF significantly down-regulated Bcl-2, Bcl-w, survivin, xIAP and HSPs pro-proteins and induced DNA fragmentation and G0/G1-phase arrest in HCT 116 cells. Chemical characterization of IR-SF by GC-MS and HPLC methods identified Isoledene as one of the major compounds. Altogether, the results of the present study demonstrate that IR-SF may induce apoptosis in human colorectal carcinoma cells through activation of ROS-mediated apoptotic pathways.
The present study reports a bioassay-guided isolation of β-caryophyllene from the essential oil of Aquilaria crassna. The structure of β-caryophyllene was confirmed using FT-IR, NMR and MS. The antimicrobial effect of β-caryophyllene was examined using human pathogenic bacterial and fungal strains. Its anti-oxidant properties were evaluated by DPPH and FRAP scavenging assays. The cytotoxicity of β-caryophyllene was tested against seven human cancer cell lines. The corresponding selectivity index was determined by testing its cytotoxicity on normal cells. The effects of β-caryophyllene were studied on a series of in vitro antitumor-promoting assays using colon cancer cells. Results showed that β-caryophyllene demonstrated selective antibacterial activity against S. aureus (MIC 3 ± 1.0 µM) and more pronounced anti-fungal activity than kanamycin. β-Caryophyllene also displayed strong antioxidant effects. Additionally, β-caryophyllene exhibited selective anti-proliferative effects against colorectal cancer cells (IC50 19 µM). The results also showed that β-caryophyllene induces apoptosis via nuclear condensation and fragmentation pathways including disruption of mitochondrial membrane potential. Further, β-caryophyllene demonstrated potent inhibition against clonogenicity, migration, invasion and spheroid formation in colon cancer cells. These results prompt us to state that β-caryophyllene is the active principle responsible for the selective anticancer and antimicrobial activities of A. crassnia. β-Caryophyllene has great potential to be further developed as a promising chemotherapeutic agent against colorectal malignancies.
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.
Chronic inflammation intensifies the risk for malignant neoplasm, indicating that curbing inflammation could be a valid strategy to prevent or cure cancer. Cancer and inflammation are inter-related diseases and many anti-inflammatory agents are also used in chemotherapy. Earlier, we have reported a series of novel ligands and respective binuclear Ag(I)-NHC complexes (NHC=N-heterocyclic carbene) with potential anticancer activity. In the present study, a newly synthesized salt (II) and respective Ag(I)-NHC complex (III) of comparable molecular framework were prepared for a further detailed study. Preliminarily, II and III were screened against HCT-116 and PC-3 cells, wherein III showed better results than II. Both the compounds showed negligible toxicity against normal CCD-18Co cells. In FAM-FLICA caspase assay, III remarkably induced caspase-3/7 in HCT-116 cells most probably by tumor necrosis factor-alpha (TNF-α) independent intrinsic pathway and significantly inhibited in vitro synthesis of cytokines, interleukin-1 (IL-1) and TNF-α in human macrophages (U937 cells). In a cell-free system, both the compounds inhibited cyclooxygenase (COX) activities, with III being more selective towards COX-2. The results revealed that III has strong antiproliferative property selectively against colorectal tumor cells which could be attributed to its pro-apoptotic and anti-inflammatory abilities.
We recently reported the antineovascularization effect of scopoletin on rat aorta and identified its potential anti-angiogenic activity. Scopoletin could be useful as a systemic chemotherapeutic agent against angiogenesis-dependent malignancies if its antitumorigenic activity is investigated and scientifically proven using a suitable human tumor xenograft model. In the present study, bioassay-guided (anti-angiogenesis) phytochemical investigation was conducted on Nicotiana glauca extract which led to the isolation of scopoletin. Further, anti-angiogenic activity of scopoletin was characterized using ex vivo, in vivo and in silico angiogenesis models. Finally, the antitumorigenic efficacy of scopoletin was studied in human colorectal tumor xenograft model using athymic nude mice. For the first time, an in vivo anticancer activity of scopoletin was reported and characterized using xenograft models. Scopoletin caused significant suppression of sprouting of microvessels in rat aortic explants with IC50 (median inhibitory concentration) 0.06μM. Scopoletin (100 and 200mg/kg) strongly inhibited (59.72 and 89.4%, respectively) vascularization in matrigel plugs implanted in nude mice. In the tumor xenograft model, scopoletin showed remarkable inhibition on tumor growth (34.2 and 94.7% at 100 and 200mg/kg, respectively). Tumor histology revealed drastic reduction of the extent of vascularization. Further, immunostaining of CD31 and NG2 receptors in the histological sections confirmed the antivascular effect of scopoletin in tumor vasculature. In computer modeling, scopoletin showed strong ligand affinity and binding energies toward the following angiogenic factors: protein kinase (ERK1), vascular endothelial growth factor A (VEGF-A), and fibroblast growth factor 2 (FGF-2). These results suggest that the antitumor activity of scopoletin may be due to its strong anti-angiogenic effect, which may be mediated by its effective inhibition of ERK1, VEGF-A, and FGF-2.
Fermented Virgin Coconut Oil (FVCO) is widely used in the Southeast Asia as food and traditional medicine. The objective of the present study is the evaluation of chronic safety of the commercialized FVCO of Malaysia and other Southeast Asian countries. A single dose of 5000 mg/kg of FVCO was administered orally in rats (each group, n = 5) for the acute toxicity study and 175, 550 and 2000 mg/kg for sub-chronic and chronic studies (each group, n = 10), respectively. The behavior, mortality, and body weight of the rats were assessed to determine the toxic effects of FVCO. The haematology, biochemistry and histopathology of the treated rats were evaluated. The treated rats were safe with the dose of 5000 mg/kg in acute, sub-chronic and chronic indication. Abnormal clinical signs and morphology (gross necroscopy), changes of organ weight, anomalous haematology and biochemistry indexes were not found in comparison with the control (p > 0.05). In general, food and water intake were higher in the treated rats related to control. It was concluded that the presence of the antioxidant active compounds of FVCO might be the reason of safety. The structure activity relationship (SAR) provides a comprehensive mechanism to determine the safety that is the presence of the electron donating phenolic groups, carbonyl groups, and carboxylic acid in the ortho and meta position of the aromatic rings. The SAR showed the antioxidant properties of myristic acid and lauric acid determined by GC-MS analysis. This result suggests the safety of FVCO for chronic use, nutritional activity that FVCO formulation complies the requirements of regulatory agencies.
Syzygium campanulatum Korth is a plant, which is a rich source of secondary metabolites (especially flavanones, chalcone, and triterpenoids). In our present study, three conventional solvent extraction (CSE) techniques and supercritical fluid extraction (SFE) techniques were performed to achieve a maximum recovery of two flavanones, chalcone, and two triterpenoids from S. campanulatum leaves. Furthermore, a Box-Behnken design was constructed for the SFE technique using pressure, temperature, and particle size as independent variables, and yields of crude extract, individual and total secondary metabolites as the dependent variables. In the CSE procedure, twenty extracts were produced using ten different solvents and three techniques (maceration, soxhletion, and reflux). An enriched extract of five secondary metabolites was collected using n-hexane:methanol (1:1) soxhletion. Using food-grade ethanol as a modifier, the SFE methods produced a higher recovery (25.5%‒84.9%) of selected secondary metabolites as compared to the CSE techniques (0.92%‒66.00%).
Tephrosia apollinea (Delile) DC (Leguminosae) has been used in folk medicine in Arabian countries to treat inflammatory disorders. The plant has been described to treat swelling, bone fracture, bronchitis, cough, earache and wounds.
CONTEXT: Orthosiphon stamineus is a medicinal herb widely grown in Southeast Asia and tropical countries. It has been used traditionally as a diuretic, abdominal pain, kidney and bladder inflammation, gout, and hypertension.
AIMS: This study aims to develop and validate the high-performance thin layer chromatography (HPTLC) method for quantification of rosmarinic acid (RA), 3'-hydroxy-5,6,7,4'-tetramethoxyflavone (TMF), sinensitin (SIN) and eupatorin (EUP) found in ethanol, 50% ethanol and water extract of O. stamineus leaves.
MATERIALS AND METHODS: HPTLC method was conducted using an HPTLC system with a developed mobile phase system of toluene: ethyl acetate: formic acid (3:7:0.1) performed on precoated silica gel 60 F254 TLC plates. The method was validated based on linearity, accuracy, precision, limit of detection, limit of quantification (LOQ), and specificity, respectively. The detection of spots was observed at ultraviolet 254 nm and 366 nm.
RESULTS: The linearity of RA, TMF, SIN, and EUP were obtained between 10 and 100 ng/spot with high correlation coefficient value (R2) of more than 0.986. The limit of detection was found to be 122.47 ± 3.95 (RA), 43.38 ± 0.79 (SIN), 17.26 ± 1.16 (TMF), and 46.80 ± 1.33 ng/spot (EUP), respectively. Whereas the LOQ was found to be 376.44 ± 6.70 (RA), 131.45 ± 2.39 (SIN), 52.30 ± 2.01 (TMF), and 141.82 ± 1.58 ng/spot (EUP), respectively.
CONCLUSION: The proposed method showed good linearity, precision, accuracy, and high sensitivity. Hence, it may be applied in a routine quantification of RA, SIN, TMF, and EUP found in ethanol, 50% of ethanol and water extract of O. stamineus leaves.
SUMMARY: HPTLC method provides rapid estimation of the marker compound for routine quality control analysis.The established HPTLC method is rapid for qualitative and quantitative fingerprinting of Orthosiphon stamineus extract used for commercial product.Four identified markers (RA, SIN, EUP and TMF) found in three a different type of O. stamineus extracts specifically ethanol, 50% ethanol and water extract were successfully quantified using HPTLC method. Abbreviations Used: HPTLC: High-performance thin layer chromatography; RA: Rosmarinic acid; TMF: 3'-hydroxy-5,6,7,4'-tetramethoxyflavone; SIN: Sinensitin; EUP: Eupatorin; E: Ethanol; EW: 50% ethanol; W: Water; BK: Batu Kurau; KB: Kepala Batas; S: Sik; CJ: Changkat Jering; SB: Sungai Buloh.
KEYWORDS: rosmarinic acid; Eupatorin; Orthosiphon stamineus; high-performance thin-layer chromatography; sinensitin; validation
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.
OBJECTIVE: Toinvestigate the anti-angiogenic activity and antioxidant properties of Myristica fragrans (M. fragrans) (nutmeg) and Morinda citrifolia (M. citrifolia)(mengkudu) oils.
METHODS: The nutmeg and megkudu essential oils were obtained by steam distillation. The antioxidant activities of both essential oils were determined by beta-carotene/linoleic acid bleaching assay and reducing power while the anti-angiogenic activity was investigated using rat aortic ring assay using various concentrations.
RESULTS: The results showed that nutmeg oil has higher antioxidant activity than mengkudu oil. The nutmeg oil effectively inhibited the oxidation of linoleic acid with (88.68±0.1)% while the inhibition percentage of oxidation of linoleic acid of the mengkudu oil is (69.44±0.4)%. The nutmeg oil and mengkudu oil showed reducing power with an EC(50) value of 181.4 μg/mL and 3 043.0 μg/mL, respectively. The antiangiogenic activity of nutmeg oil showed significant antiangiogenic activity with IC(50) of 77.64 μg/mL comparing to mengkudu oil which exhibits IC(50) of 109.30 μg/mL.
CONCLUSIONS: Bioactive compound(s) will be isolated from the nutmeg essential oil to be developed as antiangiogenic drugs.
In this study the novel caryophyllene type sesquiterpene lactone (aspfalcolide) has been isolated from the leaves of Asparagus falcatus (Linn.) and characterized by IR, 1D NMR, 2D NMR, EI-MS, HR-ESI-MS and X-ray single crystal diffraction analysis. The aspfalcolide crystallizes in the orthorhombic space group P2(1)2(1)2(1) with a = 6.37360(10), b = 7.6890(2), c = 27.3281(6) Å, α = β = γ = 90(°) and Z = 4. One intermolecular O-H⋯O hydrogen bond enforces these natural molecules to form infinite chains through the crystal. Aspfalcolide was screened for its anti-angiogenic activity in human umbilical vein endothelial cells (HUVECs) and the result showed the remarkable inhibitory effect of aspfalcolide on the proliferation (IC(50) 1.82 μM), migration and tube formation of HUVECs.
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.
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.
Morinda citrifolia L. has been used for the treatment of a wide variety of diseases, including cancer. This study was undertaken to evaluate the anti-angiogenic effect of M. citrifolia fruits and leaves. Anti-angiogenic activity was evaluated in vivo using the chick chorioallantoic membrane assay. Bioactivity-guided fractionation and isolation were performed to identify the active constituent, and high-performance liquid chromatography analysis was then used to quantify the amount of this active constituent in the active extracts and fraction. The methanol extracts of fruits and leaves of M. citrifolia and the subsequent chloroform fraction of the fruit methanolic extract were found to have potential anti-angiogenic activity and were more potent compared to suramin. Scopoletin was identified as one of the chemical constituents that may be partly responsible for the anti-angiogenic activity of M. citrifolia fruits. The present findings further support the use of M. citrifolia in cancer or other pathological conditions related to angiogenesis.
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).
Studies on selected metabolites profiling of Orthosiphon stamineus extracts using chromatographic and spectroscopic techniques combined with chemometric tools have not been fully elucidated. Thus present study was performed to profile selected metabolites in O. stamineus leaves extracts using HPLC and FTIR combined with chemometric tools and correlated with biological activities.
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.