Evaluation of anti-cancer potential of Excoecaria agallocha (L.) leaf extract on human cervical cancer (SiHa) cell line and assessing the underlying mechanism of action

Background The incidence of cervical cancer is increasing at an alarming rate in many countries and presently, it is the most common form of malignant cancer being reported among women in India. Development of novel approach for cervical cancer therapy, sparing healthy normal cells overcoming the limitations of prevailing therapies is of prime importance. Mangroves constitute a significant repository of medicinally important plants. Thus, in this study, we aimed to determine the anticancer activity of the mangrove Excoecaria agallocha L. leaf extracts on human cervical cancer (SiHa HPV 16+) cell line with subsequent characterization of the bioactive compounds conferring the anticancer activity and studying the probable underlying mechanism of action of the purified plant extract. Results The plant extract was subjected to silica gel column chromatography and the fractions obtained were analyzed for cytotoxic activity against SiHa cells by MTT assay. One out of the three eluted fractions exhibited selective toxicity against SiHa cells with an IC50 value of 15.538 ± 0.577 µg/mL, while it had no cytotoxic effect on normal healthy human peripheral blood mononuclear cells. High-resolution liquid chromatography mass spectroscopy, coupled to electron spray ionization and diode array detection analysis, led to the structure elucidation and identification of a few pharmacologically important compounds, with Bergenin being present in the highest abundance. Fluorescence microscopy results revealed that the plant extract fraction induced LC3 puncta formation, in EGFP- SiHa cells indicating the onset of autophagy, with simultaneous stimulation of mitophagy. The plant extract also inhibited proliferation of the SiHa-smac-mCherry cells by second mitochondria-derived activator of caspase (SMAC)—induced cytochrome c dependent apoptosis, that was further confirmed with Caspase-3 activation by colorimetric assay. The GFP-dgn in SiHa cells was remarkably protected from proteasomal degradation that might upregulate the survivability of the cells significantly. Flow cytometry followed by Western blot analysis further asserted the ability of the plant extract fraction to cause cell cycle arrest of SiHa cells in the G2/M phase by significantly reducing protein expression levels of cyclin B1 and D1, decreasing Cdc2 level and simultaneously increasing p21 and p53 levels. Conclusion It could be inferred that the aqueous extract of E. agallocha successfully decreased the proliferation of SiHa cervical cancer cells through induction of autophagy and apoptosis in a concerted manner, with simultaneous stimulation of mitophagy and G2/M phase cell cycle arrest, hinting at Bergenin being the major compound conferring the anti-cancer activity of the plant extract. Thus, isolation of the identified bioactive compounds from E. agallocha and their subsequent purification for drug development might serve as a novel medicinal approach for the treatment of cervical cancer in conjugation with existing therapeutic methods.

Cytotoxic Activity, and Molecular Docking of Indole Alkaloid Voacangine and Bisindole Alkaloid Vobtusine, Vobtusine Lactone from the Indonesian Plant: Voacanga foetida (Blume) Rolfe

ABSTRACT The purpose of this study was to isolate and test its cytotoxic activity starting from extract fraction and its isolate compound, then carried out molecular docking to confirm the potential biological activity of ligands (vocangine, vobtusine, and vobtusine lactone) against inhibition proteins (Bcl-2, Bcl-xL, and Mcl-1), activation protein Bax and activation of apoptotic execution protein Caspase-3. This research is an experimental quantitative study using column chromatography and HPLC methods in the isolation process, MTT assay in determining the cytotoxic activity, and molecular docking in determining the prediction of apoptotic mechanism. The cytotoxic activity of VFB-DA, VFB-DB, VFB-BuOH, VFB-DB4 fractions, voacangine compounds, vobtusine are very strong. while vobtusine lactone is moderate cytotoxic activity. The docking score for voacangine, vobtusine, and vobtusine lactone compounds against Bcl-2 is -9.93; -10.07; -9.03 kcal/mol, against Bcl-xl is -9.77; -11.69; -9.76 kcal/mol, against Mcl-1 is -10.70; -10.77; -9.53 kcal/mol, and for Bax is -8.99; -6.87; -6.99 kcal/mol, as well as against caspase3 is -12.05; -12.21; -12.02 kcal/mol. The cytotoxic activity of voacangine, vobtusine, and vobtusine lactone compounds is thought to cause cell death by suppressing Bcl-2 activity; Bcl-xl; and Mcl-1, increased Bax activity and increased caspase3 activity. Key words: Voacanga foetida, in vitro, in silico

An in vitro approach to combat multidrug resistance in Salmonella typhi and human colon cancer with Excoecaria agallocha L. extract

Background The incidence of antibacterial resistance and colon cancer is increasing in India. Antibacterial resistance and chemoresistance demand the need of developing herbal or natural chemotherapeutic agents. Our study thus, aims to determine the antibacterial and anticancer activities of the leaf extracts of the mangrove Excoecaria agallocha. Results Liquid chromatography–mass spectroscopy analysis of the ethanolic E. agallocha extracts revealed the presence of Bergenin. The plant extract fraction containing Bergenin had potent antibacterial action against a resistant strain of Salmonella typhi with an MIC value of 15.7 ± 0.04 µg/mL. Treatment of the bacteria with the plant extract made it moderately susceptible to the antibacterial drugs ampicillin, aztreonam, cefotaxime, chloramphenicol and imipenem. The plant extract caused membrane damage and disrupted the expression of a 33 kDa outer membrane protein (OmpA) in S. typhi. It was plausibly due to this mechanism of the plant extract that made the bacteria susceptible to the antibacterial drugs to a certain extent. Further, fluorescence microscopy analysis revealed the anticancer property of the extract against a human colon cancer (DLD-1) cell line by activation of Caspase-3followed by subsequent apoptosis and exhibited cytotoxicity against the cancerous cell line with an IC50 value of 17.99 ± 1.12 µg/mL. Caspase-3 activity was observed to increase in a dose-dependent manner as determined by spectrophotometric assays. Moreover, the expression of the metalloproteinase-7 (MMP-7) was significantly reduced in plant extract treated DLD-1 colon cancer cells. Conclusion The results indicate that E. agallocha is a novel source of Bergenin, and the plant extract fraction under study may be used in combination therapy along with antibacterial drugs to combat antibacterial resistance of S. typhi and also to alleviate the risks of colon cancers in human. However, further investigations may be undertaken for its therapeutic application and to explore its potential bioactivity against other bacterial strains and human cancer cell lines.

Extraction, Structural Characterisation, and Immunomodulatory Properties of Edible Amanita hemibapha subspecies javanica (Corner and Bas) Mucilage Polysaccharide as a Potential of Functional Food

This research aimed to extract mucilage polysaccharides (MP) from Amanita hemibapha subspecies javanica (Corner and Bas), and further fractionate them using anion-exchange chromatography, yielding two fractions (MPF1 and MPF2). The crude extract, and fractions mainly consisted of carbohydrates (83.5–93.2%) with minor amounts of proteins (5.40–7.20%), and sulphates (1.40–9.30%). Determination of the monosaccharide composition revealed that glucose was the major unit, followed by galactose, mannose, rhamnose, and arabinose. The average molecular weight (MW) of the crude extract and fractions was in the range 104.0–479.4 × 103 g/mol. Interestingly, the crude extract, and fractions did not cause any toxic effect in RAW264.7 cells. However, they stimulated the RAW264.7 cells to release nitric oxide and cytokines through the activation of nuclear factor-kappa B (NF-κB), and mitogen-activated protein kinase (MAPK) pathways via cell surface TLR4. Structural analysis of the most immunestimulating extract fraction, MPF2, revealed that the main backbone consisted of α-D-(1→6)-glucopyranoside. These results suggest that the MPs derived from A. hemibapha subspecies javanica (Corner and Bas) are potent in enhancing immunity; hence, they can be used as a functional ingredient in food products.

Isolation, Purification and Elucidation of Stigmasterol and β-Sitosterol Glucosides from the Stems of Solenostemma argel Grown in Sudan and their Application as Insecticides

Two new compounds were isolated from the chloroform extract fraction of stems of Solenostemma argel, purified by column chromatography and elucidated by phytochemical and spectroscopic methods as Stigmasterol and β-sitosterol glucoside. Chloroform had the highest insecticide activity against the growth of the third larval instar of Tribolium castaneum used as a test insect, when it was compared with n-hexane, ethyl acetate, n-butanol and water extracts.

Antibacterial activity test on ethanol extract fraction of Kirinyuh (Chromolaena odorata L.) leaves for multi-drug resistant organisms bacteria

The resistance of pathogenic bacteria to antibiotics is increasing due to antibiotics with incorrect doses, wrong diagnostics, and the wrong target. Bacteria that have been resistant to several antibiotics are called multi-drug resistant organisms (MDRO) bacteria. Bacterial resistance to some antibiotics requires alternative herbal treatments, one of which is the Chromolaena odorata L. Research must therefore be conducted on the antibacterial activity of the ethanol extract fraction of C. odorata L. leaves for MDRO bacteria, such as Staphylococcus lugdunensis methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa extended-spectrum beta-lactamase (ESBL), and Klebsiella pneumoniae ESBL. This study aims to determine the antibacterial activity of the ethanol extract of Kirinyuh leaves (C. odorata L.) and the antibacterial activity of the fractionation against MDRO bacteria. Test results of kirinyuh leaf ethanol extract for S. lugdunensis MRSA, P. aeruginosa ESBL, and K. pneumoniae ESBL bacteria each resulted in an inhibition zone with an average diameter of 11.6 mm (strong), 11.5 mm (strong), and 11.13 mm (strong), respectively. Testing the antibacterial activity of the ethanol fraction against MDRO bacteria can show antibacterial activity against all tested bacteria, namely Fraction 5. The results of the antibacterial activity of fraction 5 against K. pneumoniae ESBL, P. aeruginosa ESBL, and S. lugdunensis MRSA bacteria with the formation of inhibition zones formed of 10.2 mm (strong), 8.8 mm (moderate), and 7.9 mm (moderate), respectively. The results of thin-layer chromatography showed that the secondary metabolites contained in the fifth fraction were terpenoids, steroids, and flavonoids.

Polyphenolic-Rich Compounds From Dillenia pentagyna (Roxb.) Attenuates the Doxorubicin-Induced Cardiotoxicity: A High-Frequency Ultrasonography Assisted Approach

Cardiovascular complications are the foremost concern in patients undergoing anticancer therapy. There is an unmet need to address the problems arising from the drug-induced toxicity for the long-term benefit of the patients undergoing chemotherapy. Alternative medicines are gaining their prosperity in addressing the various drug-induced organ toxicity. Dillenia pentagyna Roxb (DP) is an ethnomedicinal plant rich in flavonoids and phenolic contents. In India & Nepal, DP is a common ingredient of traditional medicines used to treat multiple ailments like inflammation, cancer, and diabetes. However, its protective role against doxorubicin (Dox) induced cardiotoxicity remains unexplored. Herein, we investigated the potential effects of various extracts/fractions obtained from the DP’s bark against Dox-induced cardiotoxicity, both in-vitro and in-vivo. The anti-oxidant content of the extracts/fractions was evaluated by using DPPH, ABTS and FRAP chemical assays. The results indicated that the hydroalcoholic (HA) extract of DP has intense anti-oxidant potential. Further fractionation of DP revealed that the phenolic-rich fraction (F1) has a high anti-oxidant potential. The protective effect of extract/fraction was also investigated in the H9c2 cell line following the Dox-induced cardiotoxicity model. We observed that the pre-treatment of extract/fraction in cardiomyocytes had exhibited increased cell viability. Fluorescence-based chemical assays indicated a decreased ROS levels in the treated groups in comparison to the Dox control group. The effect of DP was evaluated further in balb/c mice by the Dox-induced cardiotoxicity model. Non-invasive techniques like high-frequency ultrasonography and electrocardiogram revealed that the mice pre-treated with DP had improved cardiac functionality (left ventricular ejection fraction and stroke volume) and normalized the electrocardiograms compared to the Dox control group. Further, biochemical analysis with the cardiac tissues revealed that the cytoprotective proteins like HO-1, SOD-2, and Nrf-2 were elevated in the DP treated groups compared to the Dox control group. Overall, our results suggested that the bioactive extract/fractions of DP helped alleviate the Dox-induced cardiotoxicity. LC-QTOF-ESI-MS analysis of DP and F1 indicated that polyphenolic anti-oxidant compounds like gallic acid, syringic acid, and sinapic acid could be responsible for the potent -cardioprotective effect. Future understanding of the pharmacokinetics and pharmacodynamic parameters can help translate from the bench to the bedside.

Comparative in vitro antioxidant and antibacterial activities of leaf extract fractions of Crimson bottlebrush, Callistemon citrinus (Curtis.) Skeels

ObjectivesThe present study aimed to analyze a comparative in vitro free radical scavenging and antibacterial potentials of leaf aqueous and the successive extract fractions of Callistemon citrinus.MethodsFor in vitro antioxidant activity assessments, 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity, Fe3+ ion reducing antioxidant power (FRAP) assay, and total antioxidant capacity of the extracts were tested. Antibacterial potentials were tested through Agar well diffusion method using both gram-positive and gram-negative bacterial strains.ResultsData indicate the polar successive aqueous fraction (AQF) possesses the highest free radicals scavenging capacity, with lowest EC50 (the required extract concentration to scavenge half of the free radicals) for DPPH and FRAP assays, and contains the highest total phenolics (308.2±5.9 µg tannic acid equivalent/mg dry extract, DE), flavonoids (516.7±3.5 µg quercetin equivalent/mg DE), total antioxidant capacity (441.48±12.8 µg ascorbic acid equivalent/mg DE). Phenolics and flavonoids contents were positively correlated with the in vitro antioxidant activities. The antibacterial study indicates the petroleum ether and chloroform are suitable solvents for extracting antibacterial phytochemicals from C. citrinus leaves that are effective against both gram-positive and negative bacterial strains.ConclusionThe most polar fraction i.e. the successive aqueous extract fraction of C. citrinus leaves exhibited the highest antioxidant activities while the most non-polar petroleum ether extract fraction showed the highest antibacterial potentials thus these extract fractions might have therapeutic importance.

Phytochemical Evaluation and Antioxidant Capacity of Ganoderma lucidum and Pleurotus pulmonarius in Ibadan, Nigeria

Aims: To evaluate the phytochemical constituents and antioxidant properties of two selected mushrooms in Ibadan, Nigeria. Study Design: An experimental and descriptive study was conducted using the fruit bodies of Ganoderma lucidum collected from the Botanical garden University of Ibadan and Pleurotus pulmonarius which was bought from a local store in Ibadan. Successive extraction was done on the mushrooms with four solvents of increasing polarity that is from the non-polar (N-hexane) to a more polar solvent(water).Phytochemical and antioxidant analysis were then carried out on the extract fractions. Place and Duration of Study: Department of Botany, University of Ibadan, Ibadan, Nigeria and Department of Microbiology, University of Ibadan, Ibadan, Nigeria between May 2018 and February 2020. Methodology: The N-Hexane, Ethyl acetate, Ethanol and Aqueous extract fraction of Ganoderma lucidum and Pleurotus pulmonarius were analyzed in different system, including DPPH, Ferric ion Reducing Antioxidant Power (FRAP) and Total Antioxidant Capacity (TAC). The various antioxidant activities were compared to the standard ascorbic acid. Phytochemical include: Phenol, Alkaloids, Flavonoids, Tannins, Steroids, Saponins, Terpenoids Anthraquinones, and Cardiac glycosides were also analysed. Results: The Phytochemical screening result shows the presence of Phenol, Alkaloids, Flavonoids, Tannins, Steroids, Saponins, Terpenoids Anthraquinones, and Cardiac glycosides. The quantitative phytochemical screening revealed that the Ethyl acetate fraction of Ganoderma lucidum recorded the highest percentage of Alkaloids (41.70±0.14%) while the Ethanolic fraction of Pleurotus pulmonarius has the lowest percentage of Tannins (0.10±0.24%). DPPH scavenging activity of the Ethanolic extract fraction of Ganoderma lucidum (at 200 µg/ml) was 94.96% higher than that of Pleurotus pulmonarius (22.39%). The extract fraction of all the mushrooms possesses significant TAC content with N-Hexane and Ethyl acetate fraction having the highest. The results of the DPPH, FRAP, and TAC assays indicate that both mushrooms examined showed significant antioxidant activities. Among these, Ganoderma lucidum extract seems to be more effective antioxidant. The antioxidant activity of theses mushrooms has significant importance as it greatly contribute to their nutraceutical properties thus enhancing their nutritive value. Cultivation and production of dietary supplements from Ganoderma lucidum is highly recommended. Conclusion: The Mushrooms species analyzed have demonstrated to be good sources of phytochemical and antioxidants hence it can be recommended to pharmaceutical industries for the manufacturing of drugs.