EVALUATION OF ACUTE AND SUB-ACUTE TOXICITY OF A STANDARDIZED POLYHERBAL FORMULATION (HC9): AN IN VIVO STUDY

Objective: In the present study, we have performed the acute and sub-acute toxicity of a standardized polyherbal formulation (HC9) in Swiss albino mice. Methods: In acute toxicity study, the mice were orally administered with different doses (1750 and 2000 mg/kg) of HC9 and monitored for 14 d. In the sub-acute toxicity study, animals received HC9 extract by oral gavage at the doses of 250, 500 and 1000 mg/kg/day (????=5/group/sex) for 28 d. At the end of the study, the animals were sacrificed and evaluated for effect of HC9 on biochemical, hematological and histopathological parameters. Results: HC9 did not produce any adverse effects in biochemical, hematological, urine and histopathological parameters in mice. HC9 did not induce any adverse effects in terms of mortality and clinical signs in the acute toxicity study. It was well-tolerated by mice up to 2000 mg/kg/body weight. In sub-acute toxicity study, no treatment-related adverse effects were found in the mice upto 1000 mg/kg/day dose. No significant changes were observed in biochemical and hematological parameters as well as histopathology of tissues (liver, kidney, spleen, heart, lung, thymus, adrenal gland, epididymis and testis/ovary) among mice of either sex. Conclusion: Our results showed that HC9 did not induce any acute and sub-acute toxicity in male and female mice, thereby, suggesting its safety for future clinical application

Aqueous Cinnamon Extract (ACE-c) from the bark of Cinnamomum cassia causes apoptosis in human cervical cancer cell line (SiHa) through loss of mitochondrial membrane potential

<p>Abstract</p> <p>Background</p> <p>Chemoprevention, which includes the use of synthetic or natural agents (alone or in combination) to block the development of cancer in human beings, is an extremely promising strategy for cancer prevention. Cinnamon is one of the most widely used herbal medicines with diverse biological activities including anti-tumor activity. In the present study, we have reported the anti-neoplastic activity of cinnamon in cervical cancer cell line, SiHa.</p> <p>Methods</p> <p>The aqueous cinnamon extract (ACE-<it>c</it>) was analyzed for its cinnamaldehyde content by HPTLC analysis. The polyphenol content of ACE-<it>c </it>was measured by Folin-Ciocalteau method. Cytotoxicity analysis was performed by MTT assay. We studied the effect of cinnamon on growth kinetics by performing growth curve, colony formation and soft agar assays. The cells treated with ACE-<it>c </it>were analyzed for wound healing assay as well as for matrix metalloproteinase-2 (MMP-2) expression at mRNA and protein level by RT-PCR and zymography, respectively. Her-2 protein expression was analyzed in the control and ACE-<it>c </it>treated samples by immunoblotting as well as confocal microscopy. Apoptosis studies and calcium signaling assays were analyzed by FACS. Loss of mitochondrial membrane potential (Δψ_m) in cinnamon treated cells was studied by JC-1 staining and analyzed by confocal microscopy as well as FACS.</p> <p>Results</p> <p>Cinnamon alters the growth kinetics of SiHa cells in a dose-dependent manner. Cells treated with ACE-<it>c </it>exhibited reduced number of colonies compared to the control cells. The treated cells exhibited reduced migration potential that could be explained due to downregulation of MMP-2 expression. Interestingly, the expression of Her-2 oncoprotein was significantly reduced in the presence of ACE-<it>c</it>. Cinnamon extract induced apoptosis in the cervical cancer cells through increase in intracellular calcium signaling as well as loss of mitochondrial membrane potential.</p> <p>Conclusion</p> <p>Cinnamon could be used as a potent chemopreventive drug in cervical cancer.</p

The Aqueous Extract of <i>Ficus religiosa</i> Induces Cell Cycle Arrest in Human Cervical Cancer Cell Lines SiHa (HPV-16 Positive) and Apoptosis in HeLa (HPV-18 Positive)

<div><p>Natural products are being extensively explored for their potential to prevent as well as treat cancer due to their ability to target multiple molecular pathways. <i>Ficus religiosa</i> has been shown to exert diverse biological activities including apoptosis in breast cancer cell lines. In the present study, we report the anti-neoplastic potential of aqueous extract of <i>F. religiosa</i> (FR_aq) bark in human cervical cancer cell lines, SiHa and HeLa. FR_aq altered the growth kinetics of SiHa (HPV-16 positive) and HeLa (HPV-18 positive) cells in a dose-dependent manner. It blocked the cell cycle progression at G₁/S phase in SiHa that was characterized by an increase in the expression of p53, p21 and pRb proteins with a simultaneous decrease in the expression of phospho Rb (ppRb) protein. On the other hand, in HeLa, FR_aq induced apoptosis through an increase in intracellular Ca²⁺ leading to loss of mitochondrial membrane potential, release of cytochrome-c and increase in the expression of caspase-3. Moreover, FR_aq reduced the migration as well as invasion capability of both the cervical cancer cell lines accompanied with downregulation of MMP-2 and Her-2 expression. Interestingly, FR_aq reduced the expression of viral oncoproteins E6 and E7 in both the cervical cancer cell lines. All these data suggest that <i>F. religiosa</i> could be explored for its chemopreventive potential in cervical cancer.</p></div

Ficus regulates the growth of cervical cancer cells.

<p>SiHa (<b>A</b>) and HeLa (<b>B</b>) were treated with FR<b>_aq</b>(0–80 µg/ml) for 24–72 h and the number of viable cells were counted using the trypan blue dye exclusion method. Data represent mean ± SD of three independent experiments. (<b>C</b>) The cervical cancer cell lines (SiHa and HeLa) were treated with FR<b>_aq</b>(0–80 µg/ml) for one week. The colonies were stained with crystal violet and photographed. The experiments were repeated three times. (<b>D</b>) Both SiHa and HeLa (5×10³) along with FR<b>_aq</b> (0–80 µg/ml) were grown in soft agar for two weeks. Colonies were counted from at least 10 different areas and the average of each has been plotted. The data represents mean ± SD of five independent experiments.</p

Ficus regulates invasion and migration of cervical cancer cells.

<p>Analysis of cell migration in SiHa (<b>A</b>) and HeLa (<b>B</b>) treated with FR_aq (0–80 µg/ml) was measured by wound-healing assay. The upper panel of the image shows the wound made at 0 h. The lower panel shows the migration of cells corresponding to the distance travelled at 16 h. (<b>C</b>) Graphical representation of wound closure in SiHa and HeLa cells at 16 h after FR_aq treatment has been shown. Values were represented as the percent wound closure and expressed as mean ± SD for three independent experiments. (<b>D</b>) Cell invasion assay showing the percentage of cells invaded per field in the presence or absence of FR<b>_aq</b>. The invaded cells were counted in ten random fields and the values have been expressed as mean ± SD for three independent experiments. (<b>E</b>) Gelatin zymography showing downregulation of MMP-2 expression in FR<b>_aq</b> (0–80 µg/ml) treated SiHa and HeLa. (<b>F</b>) Western blot analysis showing decrease in Her-2 expression in SiHa and HeLa treated with FR<b>_aq</b> (0–80 µg/ml). Tubulin was used as a loading control. (<b>G</b>) Densitometric analysis of the western blot showing fold change in HER-2 protein levels in SiHa and HeLa. The bands were quantified by densitometry using ImageJ 1.44p (Wayne Rasband, National Institutes of Health, USA, <a href="http://imagej.nih.gov/ij" target="_blank">http://imagej.nih.gov/ij</a>).</p

Ficus induces apoptosis in HeLa through mitochondrial dependent pathway.

<p>(<b>A</b>) Representative FACS pictograms of cells treated with FR<b>_aq</b>(0–80 µg/ml) are shown. Percent of annexin V-positive (early-apoptotic cells, lower right quadrant) and Annexin V/PI-double-positive cells (late-apoptotic cells, upper right quadrant) are indicated. (<b>B</b>) Flow cytometric analysis of the rapid calcium release in HeLa cells after treatment with FR<b>_aq</b>(0–80 µg/ml) has been shown. Ionomycin was used as a positive control. The data represents mean ± SD of three independent experiments. (<b>C</b>) FACS analysis following JC-1 staining of HeLa showed alteration of the mitochondrial membrane potential after FR<b>_aq</b>(0–80 µg/ml) treatment compared to untreated control cells. The data represents mean ± SD of three independent experiments. (<b>D</b>) Western blot shows the expression of cytochrome c from cytosolic fraction. Tubulin was used as a loading control. (<b>E</b>) Total protein was isolated and analysed for expression of p53 and caspase 3 by immunoblotting. Tubulin was used as a loading control. (<b>F and G</b>) Densitometric analysis of the western blot showing fold change in protein levels. The bands were quantified by using ImageJ 1.44p (Wayne Rasband, National Institutes of Health, USA, <a href="http://imagej.nih.gov/ij" target="_blank">http://imagej.nih.gov/ij</a>).</p

Ficus arrests the cell cycle in SiHa at G₁/S phase and modulates the expression of cell cycle regulatory proteins.

<p>SiHa cells were treated with different concentrations of FR_aq (0–80 µg/ml) for 24 h. (<b>A</b>) Enhanced accumulation of the cells in G₁ phase with a concomitant decrease in S-phase population was observed after treatment with Ficus (as indicated by histograms). Western blot shows the expression levels of p53 and pRb (<b>B</b>) as well as p21 and ppRb (<b>C</b>). Tubulin was used as a loading control. (<b>D, E</b>) Densitometric analysis of the western blot showing fold change in protein levels upon FR_aqtreatment. The bands were quantified by densitometry scanning using ImageJ 1.44p (Wayne Rasband, National Institutes of Health, USA, <a href="http://imagej.nih.gov/ij" target="_blank">http://imagej.nih.gov/ij</a>). The data represents mean ± SD of three independent experiments.</p