Investigation Of Molecular Mechanisms Underlying The Anti-Tumor And Anti-Angiogenic Activities Of Orthosiphon Stamineus Towards Colorectal Cancer

Teh Orthosiphon stamineus Benth. (Lamiaceae) digunakan secara meluas dalam perubatan tradisional. Kajian terbaru menunjukkan bahawa 50% ekstrak ethanolik daripada Orthosiphon stamineus (EOS) dan sebatian aktif, asid rosmarinik (RA), memaparkan kesan-kesan anti-angiogenik, anti-radang dan anti-tumor yang ketara dalam pelbagai model eksperimen. Walau bagaimanapun, mekanisme yang mendasari sifat-sifat ini tidak dinilai dengan sepenuhnya. Kajian yang dijalankan ini bertujuan untuk menilaikan lagi mekanisme molekul yang mendasari anti-tumor dan anti-angiogenik. Dalam model eksperimen penghijrahan, perkembangan dan pembentukan tiub, cerakin kedua-dua EOS dan RA aktif menyebabkan perencatan ketara terhadap fungsi sel endothelial manusia (HUVECs) yang penting bagi merangsang proses angiogenesis. Dalam kedua-dua kajian in vitro dan in vivo, penindasan besar neovaskularisasi dalam model aorta tikus, CAM dan plug matrigel juga diperhatikan. Kajian cerakin multipleks menunjukkan pengurangan faktor pertumbuhan utama bagi lata pro-angiogenik dan perkembangan tumor iaitu faktor pertumbuhan endothelial vaskular (VEGF), faktor pertumbuhan fibroblast asas (b-FGF), transformasi faktor pertumbuhan transformasi (TGF-α), faktor nekrosis tumor (TNF-β) dan interleukin-1, 2, 7. Orthosiphon stamineus Benth. (Lamiaceae) tea is widely consumed traditionally for its vast medicinal value. Recent studies revealed that 50% ethanolic extract of Orthosiphon stamineus (EOS) and its active compound, rosmarinic acid (RA), displayed significant anti-angiogenic, anti-inflammatory and anti-tumor effects in various experimental models. However, the mechanisms underlying these properties have not been fully evaluated. The present work aims to further evaluate the molecular mechanisms underlying its anti-tumour and anti-angiogenic properties. In migration, proliferation and tube formation assay, both EOS and its active RA caused significant inhibition of human endothelial cell (HUVECs) functions crucial for promotion of angiogenesis. Both in vitro and in vivo studies revealed significant suppression of neovascularisation in rat aortic ring, CAM and matrigel plug. Multiplex array studies showed reduction of key growth factors for pro-angiogenic cascade and tumor development i.e. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (b-FGF), transforming growth factor alpha (TGF-α), tumor necrosis factor (TNF-β) and interleukin-1, 2, 7

Evaluation of in vitro cytotoxicity effect of Clinacanthus nutans (Brum. f.) Lindau standardized leaf extracts

Purpose: To standardize Clinacanthus nutans (CN) leaf extracts, evaluate their contents of orientin, vitexin and isovitexin using a reversed-phase high-performance liquid chromatography (RP-HPLC) method, and also to investigate in vitro cytotoxicity of CN. Methods: CN leaf powder was macerated in distilled water, methanol, methanol (50 %), ethanol, and ethanol (50 %) over a hot water bath at 50 - 55 °C for 24 h. The extracts were standardized for total phenolic, flavonoid, proteins and polysaccharides content by ultra-violet (UV) spectrophotometry. Moreover, RP-HPLC was used to determine the contents of orientin, vitexin and isovitexin in the extracts. The anti-proliferative effect of the extracts against human colorectal carcinoma cell line (HCT116) and human colon normal cell line (CCD-18Co) was assessed using 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) assay. The most active extract was fractionated using silica gel flash column chromatography to produce 20 fractions. All the fractions were subjected to the MTT test. Results: The extracts showed modest cytotoxicity against HCT-116 and non-cytotoxicity against CCD18Co cell lines. Of all the extracts tested, the methanol extract (CN-M) showed the highest activity of all the extracts and had the highest content of flavonoid and phenolic compounds. Twenty fractions were obtained from this extract. Fraction nos. F3, F4, F14 and F16 showed significant (p < 0.05) cytotoxicity against HCT-116, with F14 having the highest activity. Conclusion: Fraction F14 has the potential to be developed to anti-colon cancer agent. However, further studies including chemical profiling, mechanism of action and safety profile of this fraction are required

Pharmacokinetics and antiangiogenic studies of potassium koetjapate in rats

© 2020 Purpose: Koetjapic acid is an active compound of a traditional medicinal plant, Sandoricum koetjape. Although koetjapic acid has a promising anticancer potential, yet it is highly insoluble in aqueous solutions. To increase aqueous solubility of koetjapic acid, we have previously reported a chemical modification of koetjapic acid to potassium koetjapate (KKA). However, pharmacokinetics of KKA has not been studied. In this study, pharmacokinetics and antiangiogenic efficacy of KKA are investigated. Methods: Pharmacokinetics of KKA was studied after intravenous and oral administration in SD rats using HPLC. Anti-angiogenic efficacy of KKA was investigated in rat aorta, human endothelial cells (EA.hy926) and nude mice implanted with matrigel. Results: Pharmacokinetic study revealed that KKA was readily absorbed into blood and stayed for a long time in the body with Tmax 2.89 ± 0.12 h, Cmax 7.24 ± 0.36 μg/mL and T1/2 1.46 ± 0.03 h. The pharmacological results showed that KKA significantly suppressed sprouting of microvessels in rat aorta with IC50 18.4 ± 4.2 μM and demonstrated remarkable inhibition of major endothelial functions such as migration, differentiation and VEGF expression in endothelial cells. Further, KKA significantly inhibited vascularization in matrigel plugs implanted in nude mice. Conclusions: The results indicate that bioabsorption of KKA from oral route was considerably efficient with longer retention in body than compared to that of the intravenous route. Further, improved antiangiogenic activity of KKA was recorded which could probably be due to its increased solubility and bioavailability. The results revealed that KKA inhibits angiogenesis by suppressing endothelial functions and expression of VEGF

Anti-uterine fibroid effect of standardized labisia pumila var. Alata extracts in vitro and in human uterine fibroid cancer xenograft model

Background: Uterine fibroids are a common type of solid tumor presenting in women of reproductive age. There are very few alternative treatment available from conventional treatment involving surgeries. Labisia pumila var. alata or locally known as ‘Kacip Fatimah’ was widely used as traditional medicine in Malaysia. This plant has been used to maintain a healthy female reproductive system. The present study aimed to evaluate anti fibroid potential of L. pumila extracts through in vitro apoptosis activity against uterine leiomyoma cells (SK-UT-1) and in uterine leiomyoma xenograft model. Evaluation of bioactive markers content were also carried out. Methods: Apoptotic induction of the extracts was determined by morphological examination of AO/PI dual staining assay by flourescent microscopy and flow cytometry analysis on Annexin V-FITC/PI stained cells. In vivo study was done in immune-compromised mouse xenograft model. HPLC analysis was employed to quantify marker compounds. Results: Morphological analysis showed L. pumila induced apoptosis in a dose dependent manner against SK-UT-1 cells. In vivo study indicated that L. pumila significantly suppressed the growth of uterine fibroid tumor. All tested extracts contain bioactive marker of gallic acid and cafeic acid. Conclusion: This work provide significant data of the potential of L. pumila in management of uterine fibroids

Ethyl-p-methoxycinnamate isolated from kaempferia galanga inhibits inflammation by suppressing interleukin-1, tumor necrosis factor-α, and angiogenesis by blocking endothelial functions

OBJECTIVE: The present study aimed to investigate the mechanisms underlying the anti-inflammatory and anti-angiogenic effects of ethyl-p-methoxycinnamate isolated from Kaempferia galanga. METHODS: The anti-inflammatory effects of ethyl-p-methoxycinnamate were assessed using the cotton pellet granuloma assay in rats, whereby the levels of interleukin-1 and tumor necrosis factor-α were measured in the animals' blood. In addition, the levels of interleukin, tumor necrosis factor, and nitric oxide were measured in vitro using the human macrophage cell line (U937). The analgesic effects of ethyl-p-methoxycinnamate were assessed by the tail flick assay in rats. The anti-angiogenic effects were evaluated first by the rat aortic ring assay and, subsequently, by assessing the inhibitory effects of ethyl-p-methoxycinnamate on vascular endothelial growth factor, proliferation, migration, and tube formation in human umbilical vein endothelial cells. RESULTS: Ethyl-p-methoxycinnamate strongly inhibited granuloma tissue formation in rats. It prolonged the tail flick time in rats by more than two-fold compared with the control animals. The inhibition of interleukin and tumor necrosis factor by ethyl-p-methoxycinnamate was significant in both in vivo and in vitro models; however, only a moderate inhibition of nitric oxide was observed in macrophages. Furthermore, ethyl-p-methoxycinnamate considerably inhibited microvessel sprouting from the rat aorta. These mechanistic studies showed that ethyl-p-methoxycinnamate strongly inhibited the differentiation and migration of endothelial cells, which was further confirmed by the reduced level of vascular endothelial growth factor. CONCLUSION: Ethyl-p-methoxycinnamate exhibits significant anti-inflammatory potential by inhibiting pro-inflammatory cytokines and angiogenesis, thus inhibiting the main functions of endothelial cells. Thus, ethyl-p-methoxycinnamate could be a promising therapeutic agent for the treatment of inflammatory and angiogenesis-related diseases

Neuroprotective and Anti-Inflammatory Effects of \u3ci\u3eRhus coriaria\u3c/i\u3e Extract in a Mouse Model of Ischemic Optic Neuropathy

Modulating oxidative stresses and inflammation can potentially prevent or alleviate the pathological conditions of diseases associated with the nervous system, including ischemic optic neuropathy. In this study we evaluated the anti-neuroinflammatory and neuroprotective activities of Rhus coriaria (R. coriaria) extract in vivo. The half maximal inhibitory concentration (IC50) for DPPH, ABTS and B-carotene were 6.79 ± 0.009 µg/mL, 10.94 ± 0.09 µg/mL, and 6.25 ± 0.06 µg/mL, respectively. Retinal ischemia was induced by optic nerve crush injury in albino Balb/c mice. The anti-inflammatory activity of ethanolic extract of R. coriaria (ERC) and linoleic acid (LA) on ocular ischemia was monitored using Fluorescence Molecular Tomography (FMT). Following optic nerve crush injury, the mice treated with 400 mg/kg of ERC and LA exhibited an 84.87% and 86.71% reduction of fluorescent signal (cathepsin activity) respectively. The results of this study provide strong scientific evidence for the neuroprotective activity of the ERC, identifying LA as one of the main components responsible for the effect. ERC may be useful and worthy of further development for its adjunctive utilization in the treatment of optic neuropathy

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

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

Anti-tumor Activity of NuvastaticTM (C5OSEW5050ESA) of Orthosiphon stamineus and Rosmarinic Acid in An Athymic Nude Mice Model of Breast Cancer

The current treatment strategies for metastatic breast cancer depend on the cancer subtype by palliating symptoms and prolonging life. However, triple-negative breast cancers have no targeted treatment available. Orthosiphon stamineus (O.s) is a traditional folk medicine plant used in South East Asia to treat many diseases. The aim of this study is to evaluate the anti-tumor activity of O.s extract formulation (ID: C5EOSEW5050ESA trademarked as NuvastaticTM) and its major component, rosmarinic acid in a breast in vivo tumor xenograft model. Human triple-negative breast cancer cells were transplanted into the mammary fat pad of 20 athymic nude mice. Fourteen days post-injection, mice were randomly assigned to four groups. C5EOSEW5050ESA (200 and 400 mg/kg/day) and rosmarinic acid (32 mg/kg/day) were administered orally. The body weight and tumor size were measured twice a week. Histopathological analyses of tumor tissues were conducted. Tumor necrosis and tumor growth were determined by hematoxylin and eosin staining. A significant reduction in tumor size and growth was found in all treatment groups. No significant difference between C5EOSEW5050ESA at 200 mg/kg and rosmarinic acid in the reduction of tumor size and necrosis. However, a more significant reduction was found in tumor growth and necrosis with 400 mg/kg of C5EOSEW5050ESA treatment as compared to other treatments. These results highlighted the anti-tumor activity of C5EOSEW5050ESA in reducing breast tumor growth in mice compared to the lower dose of C5EOSEW5050ESA and rosmarinic acid. This study provides valuable insights into using C5EOSEW5050ESA as a treatment to target triple-negative breast cancers in vivo

Neuroprotective and Anti-Inflammatory Effects of \u3ci\u3eRhus coriaria\u3c/i\u3e Extract in a Mouse Model of Ischemic Optic Neuropathy

Modulating oxidative stresses and inflammation can potentially prevent or alleviate the pathological conditions of diseases associated with the nervous system, including ischemic optic neuropathy. In this study we evaluated the anti-neuroinflammatory and neuroprotective activities of Rhus coriaria (R. coriaria) extract in vivo. The half maximal inhibitory concentration (IC50) for DPPH, ABTS and B-carotene were 6.79 ± 0.009 µg/mL, 10.94 ± 0.09 µg/mL, and 6.25 ± 0.06 µg/mL, respectively. Retinal ischemia was induced by optic nerve crush injury in albino Balb/c mice. The anti-inflammatory activity of ethanolic extract of R. coriaria (ERC) and linoleic acid (LA) on ocular ischemia was monitored using Fluorescence Molecular Tomography (FMT). Following optic nerve crush injury, the mice treated with 400 mg/kg of ERC and LA exhibited an 84.87% and 86.71% reduction of fluorescent signal (cathepsin activity) respectively. The results of this study provide strong scientific evidence for the neuroprotective activity of the ERC, identifying LA as one of the main components responsible for the effect. ERC may be useful and worthy of further development for its adjunctive utilization in the treatment of optic neuropathy

Toxicological studies of Orthosiphon stamineus (Misai Kucing) standardized ethanol extract in combination with gemcitabine in athymic nude mice model

Pancreatic cancer has the highest mortality rate among cancers due to its aggressive biology and lack of effective treatment. Gemcitabine, the first line anticancer drug has reduced efficacy due to acquired resistance. The current study evaluates the toxicological effects of Orthosiphon stamineus (O.s) and its marker compound (rosmarinic acid) in combination with gemcitabine. O.s (200 or 400 mg/kg/day) and rosmarinic acid (32 mg/kg/day) were administered orally and gemcitabine (10 mg/kg/3 days) intraperitoneally either alone or in combination treatment for fourteen days. Parameters including blood serum biochemistry, hematology, myeloid-erythroid ratio, incident of lethality, and histopathological analysis of liver, kidney, and spleen tissues were studied. Neither, individual drugs/extract nor chemo-herbal combinations at tested doses induced any toxicity and damage to organs in nude mice when compared to control group. Toxicological data obtained from this study will help to select the best doses of chemo-herbal combination for future pancreatic xenograft tumor studies. Keywords: Orthosiphon stamineus, Rosmarinic acid, Medicinal herb, Gemcitabine, Pancreatic cance