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

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

The Chemotherapeutic Efficacy of Hyaluronic Acid Coated Polymeric Nanoparticles against Breast Cancer Metastasis in Female NCr-Nu/Nu Nude Mice

Polyethylene glycol (PEG) coated Poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) for cancer treatment are biocompatible, nonimmunogenic and accumulate in tumour sites due to the enhanced permeability and retention (EPR). Doxorubicin (DOX) is a potent but cardiotoxic anticancer agent. Hyaluronic acid (HA) occurs naturally in the extra-cellar matrix and binds to CD44 receptors which are overexpressed in cancer metastasis, proven to be characteristic of cancer stem cells and responsible for multidrug resistance. In this study, an athymic mice model of breast cancer metastasis was developed using red fluorescent protein (RFP)-labelled triple negative cancer cells. The animals were divided into four treatment groups (Control, HA-PEG-PLGA nanoparticles, PEG-PLGA nanoparticles, and Free DOX). The tumour size growth was assessed until day 25 when animals were sacrificed. Mice treated with HA-PEG-PLGA NPs inhibited tumour growth. The tumour growth at day 25 (118% ± 13.0) was significantly (p p < 0.05) less metastasis in liver, spleen, colon, and lungs as compared to control and to Free DOX groups. The efficacy of HA-PEG-PLGA NPs was proven in vivo. Further pharmacokinetic and toxicity studies are required for this formulation to be ready for clinical research

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 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

Increased aqueous solubility and proapoptotic activity of potassium koetjapate against human colorectal cancer cells

Objectives Recently, we have isolated koetjapic acid (KA) from Sandoricum koetjape and identified its selective anticancer potentiality against colorectal carcinoma. KA is quite likely to be useful as a systemic anticancer agent against colorectal malignancy. However, with extremely low solubility, KA has to be converted into a biocompatible solubilized form without compromising the bioefficacy. Objective of this study is to enhance solubility of KA and to evaluate anticancer efficacy of potassium koetjapate in human colorectal cancer cells. Methods (2-Hydroxypropyl)-β-cyclodextrin inclusion complex and solid dispersions (carboxymethyl cellulose, polyvinylpyrrolidone and sodium lauryl sulphate) of KA were prepared. In addition, a salt of KA, potassium koetjapate was synthesized. Key findings Potassium koetjapate demonstrated higher solubility than the other tested formulations with enhanced cytotoxicity against HCT 116 cells. The enhanced efficacy of potassium koetjapate is attributed to apoptotic induction of nuclear condensation and disruption of mitochondrial membrane potential in the cells. Interestingly, potassium koetjapate was found to be safe in rats after oral administration (LD > 2000 mg/kg). Conclusions The salt formulation of KA appears to modulate the capability of the parent compound by enhancing its solubility and improves its bioefficacy against colon cancer cells, suggesting attractive roles for its applications in medicine

Optimization of Cat’s Whiskers Tea (Orthosiphon stamineus) Using Supercritical Carbon Dioxide and Selective Chemotherapeutic Potential against Prostate Cancer Cells

Cat’s whiskers (Orthosiphon stamineus) leaves extracts were prepared using supercritical CO2 (SC-CO2) with full factorial design to determine the optimum extraction parameters. Nine extracts were obtained by varying pressure, temperature, and time. The extracts were analysed using FTIR, UV-Vis, and GC-MS. Cytotoxicity of the extracts was evaluated on human (colorectal, breast, and prostate) cancer and normal fibroblast cells. Moderate pressure (31.1 MPa) and temperature (60°C) were recorded as optimum extraction conditions with high yield (1.74%) of the extract (B2) at 60 min extraction time. The optimized extract (B2) displayed selective cytotoxicity against prostate cancer (PC3) cells (IC50 28 µg/mL) and significant antioxidant activity (IC50 42.8 µg/mL). Elevated levels of caspases 3/7 and 9 in B2-treated PC3 cells suggest the induction of apoptosis through nuclear and mitochondrial pathways. Hoechst and rhodamine assays confirmed the nuclear condensation and disruption of mitochondrial membrane potential in the cells. B2 also demonstrated inhibitory effects on motility and colonies of PC3 cells at its subcytotoxic concentrations. It is noteworthy that B2 displayed negligible toxicity against the normal cells. Chemometric analysis revealed high content of essential oils, hydrocarbon, fatty acids, esters, and aromatic sesquiterpenes in B2. This study highlights the therapeutic potentials of SC-CO2 extract of cat’s whiskers in targeting prostate carcinoma

Crystal Structure Elucidation and Anticancer Studies of (-)-Pseudosemiglabrin: A Flavanone Isolated from the Aerial Parts of <i>Tephrosia apollinea</i>

<div><p><i>Tephrosia apollinea</i> 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 <i>T. apollinea</i>. 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.</p></div

IC₅₀ Values of (-)-Pseudosemiglabrin (SSG) on Various Human Cancer Cell Lines^a.

a<p>The median inhibitory concentrations (IC₅₀) were determined by nonlinear regression analysis of log-concentration-response curves of 3 different tests (n = 3).</p

A) The photomicrographs depict the images of PC3 cells with Hoechst 33258 stain taken at 6 and 12 hours after treatment.

<p>The cells treated with SSG revealed clear signs of proapoptosis. The cells treated with 0.1% DMSO (Vehicle) showed prompt and evenly distributed nucleus with fully extended pseudopodial like projections of cell membrane. Whereas, the cells treated with SSG (10 µM) displayed blebbing of cellular membrane and the typical apoptotic changes in the chromatin structure. The arrows indicate the clear signs of nuclear condensation including the half moon (crescent) shaped apoptotic nuclei. The arrowheads indicate the chromatin dissolution, breakdown and fragmentation. The standard reference, betulinic acid also showed the similar induction of apoptosis in the cells. B) Graphical representation of percentage of apoptotic indices. The apoptotic index for each test group was expressed as a percentage of the ratio of apoptotic cells number to the total cell number in 10 different fields. Values are presented as mean ± SD (<i>n</i> = 10), * represents p<0.05 and ** represents p<0.01.</p