Investigating the cellular antioxidant and anti-inflammatory effects of the novel peptides in lingzhi mushrooms.

The lingzhi mushroom (Ganoderma lucidum) is well known for its medicinal properties and has long played a role in traditional oriental medicine due to its health-giving benefits and potential to extend life expectancy. The mushroom contains a number of highly bioactive compounds and can also act as an excellent source of protein. This research investigated the peptides obtained from the protein hydrolysates of lingzhi mushrooms to assess their free radical scavenging abilities. These peptides were acquired via different proteases (Alcalase, Neutrase, papain, and pepsin-pancreatin) and were tested at a range of different concentrations (1.0%, 2.5%, and 5.0% w/v). The highest levels of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) radical scavenging activities were presented by lingzhi mushroom hydrolysate using 2.5% (w/v) pepsin-pancreatin after 6 h of digestion. The hydrolysate was then fractionated using 10, 5, 3, and 0.65 kDa molecular weight cut-off membranes. The results showed that the MW 0.65 kDa fraction had the highest level of free radical scavenging activity. Further analysis of this MW 0.65 kDa fraction began with another RP-HPLC fractionation technique to obtain three further sub-fractions. De novo peptide sequencing using electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS/MS) was chosen as the optimum method for studying the F3 sub-fraction. DRVSIYGWG and ALLSISSF were discovered as new peptides with different antioxidant properties. Adenocarcinoma colon (Caco-2) cells showed the antioxidant action of these synthesized peptides. This activity was linked to peptide concentration. The peptides and their pure synthetic counterparts were found to reduce NO generation by RAW 264.7 macrophages without causing cytotoxicity. The results of gene expression reveal that the DRVSIYGWG and ALLSISSF peptides were able to cut the expression of the proinflammatory cytokine genes iNOS, IL-6, TNF-α, and COX-2 in the context of RAW 264.7 macrophages.

Quaternized chitosan-coated nanoemulsions: A novel platform for improving the stability, anti-inflammatory, anti-cancer and transdermal properties of Plai extract.

A new positively charged nanoemulsion using quaternized chitosan (QCS) as a protective layer was developed to improve the stability and bioactivity of lipophilic active components. The anti-inflammatory Plai extract was chosen as both an active ingredient and an oil phase of the system. Compared with chitosan-coated nanoemulsion (NE2-CS) and uncoated nanoemulsion (NE1), the QCS coating could improve the stability of the Plai extract during 28 days. The particle size of NE1 increased from 141 nm to 202 nm after coating with QCS, whereas zeta potential changed from -22.03 mV for NE1 to 20.23 mV for NE2-QCS, confirming the presence of QCS. A clear improvement in anti-inflammatory, anti-cancer, and transdermal properties of Plai extract was verified for NE2-QCS, which could be due to the NEs' fineness and the permanent positive charge of the protective layer. Therefore, we suggested that QCS-coated NEs can be used as an effective transdermal delivery system for lipophilic active components.

pH-responsive selenium nanoparticles stabilized by folate-chitosan delivering doxorubicin for overcoming drug-resistant cancer cells.

Herein, we first report pH-responsive SeNPs stabilized with modified folic acid-N-trimethyl chitosan (TMC-FA) as nanocarriers for delivery of doxorubicin (DOX) to overcome drug-resistant cancer cells, which could enhance the activity of DOX by approximately 10-fold for a reduced IC50 value compared to free DOX. When nanoparticles were taken up by cells, the DOX-loaded SeNPs@TMC-FA demonstrated a faster release rate under acidic conditions. The cumulative release amount of DOX at pH 5.3 was 54.1% within 2h and 95.5% at 6h, whereas the release rate at pH 7.4 was 12.3% in 2h and 42.2% for 6h; release was not completed at the end of the study, 72h. Mechanistic studies suggested that DOX-SeNPs@TMC-FA induced cell death through the apoptosis pathway by involvement of caspase-3 and PARP proteins. The results demonstrated that pH-responsive SeNPs@TMC-FA, as targeted nanocarriers, promoted the efficacy of DOX and overcame drug resistance in NCI/ADR-RES cells.

Antioxidant and antiproliferative activities of protein hydrolysate from the rhizomes of Zingiberaceae plants.

Plant proteins have been investigated for their antioxidant activities, but there are still no reports detailing the antioxidant activity levels of plants in the Zingiberaceae family, which are popular food agents and used in folklore medicine. In this study, the crude rhizome protein extract and associated pepsin/pancreatin protein hydrolysate of 15 plants in the Zingiberaceae family were screened using the DPPH method for antioxidant activity. The protein hydrolysate of C. zedoaria possessed the highest antioxidant activity (IC50of 25.7±6.3µg/mL), which was close to that of the reference ascorbic acid (IC50of 22.3±1.8µg/mL). After enrichment by Q Sepharose ion exchange chromatography using a five step elution gradient of increasing NaCl concentration (0, 0.25, 0.5, 0.75 and 1M), the fraction eluting in the 0.5M NaCl (F50) showed the highest antioxidant activity (IC50 of 41.78±2.9µg/mL), and was found to have weak in vitro cytotoxicity against the HEP-G2 and SW620 cell lines (IC50 of 200.8±11.8 and 241.0±9.3µg/mL, respectively), but not the BT474, CHAGO and KATO-3 cell lines. F50 had an estimated molecular weight by MALDI-TOF mass spectrometry of 12,400-12,800 Da.

Antioxidant activity and ultrastructural changes in gastric cancer cell lines induced by Northeastern Thai edible folk plant extracts.

BACKGROUND: Phytochemical products have a critical role in the drug discovery process. This promising possibility, however, necessitates the need to confirm their scientific verification before use. Hence, this study aims to evaluate (1) the antioxidant activity, (2) cytotoxicity potential, and (3) the effect on ultrastructural alteration in gastric cancer cell lines through exposure to fractions of three local Northeastern Thai edible plants. METHODS: Plants, Syzygium gratum, Justicia gangetica and Limnocharis flava were extracted with ethyl acetate, and each crude extract analysed for their total phenolics content by Folin-Ciocalteu method. Their antioxidant activity was assessed using the ABTS system. The extracts were then assayed for cytotoxicity on two gastric cancer cell lines Kato-III and NUGC-4, and compared with Hs27 fibroblasts as a control using the MTT assay. The cell viability (%), IC50 values, as well as the ultrastructural alterations were evaluated after treatment with one way analysis of variance (ANOVA). RESULTS: The total phenolic values of the ethyl acetate extracts were well correlated with the antioxidant capacity, with extracted product of S. gratum displaying the highest level of antioxidant activity (a 10-fold greater response) over J. gangetica and L. flava respectively. Exposure of S. gratum and J. gangetica extracts to normal cell lines (Hs27) resulted in marginal cytotoxicity effects. However, through a dose-dependent assay S. gratum and J. gangetica extracts produced cytotoxicological effects in just over 75 percent of Kato-III and NUGC-4 cell lines. In addition, apoptotic characteristic was shown under TEM in both cancer cell lines with these two extracts, whereas characteristics of autophagy was found in cell lines after post exposure to extracts from L. flava. CONCLUSIONS: From these three plants, S. gratum had the highest contents of phenolic compounds and antioxidant capacity. All of them found to contain compound(s) with cytotoxicity in vitro on cancer cells but not on normal cell lines as resolved in tissue culture and ultrastructural analysis. This is the first report to show the effect on cellular alteration as apoptosis of an ethyl acetate extract of S. gratum and J. gangetica. Further studies are now focused on individual isolates and their function, prioritizing on S. gratum and J. gangetica for the development of novel therapeutics and combatants against cancer.

In vitro antiproliferative/cytotoxic activity on cancer cell lines of a cardanol and a cardol enriched from Thai Apis mellifera propolis.

BACKGROUND: Propolis is a complex resinous honeybee product. It is reported to display diverse bioactivities, such as antimicrobial, anti-inflammatory and anti-tumor properties, which are mainly due to phenolic compounds, and especially flavonoids. The diversity of bioactive compounds depends on the geography and climate, since these factors affect the floral diversity. Here, Apis mellifera propolis from Nan province, Thailand, was evaluated for potential anti-cancer activity. METHODS: Propolis was sequentially extracted with methanol, dichloromethane and hexane and the cytotoxic activity of each crude extract was assayed for antiproliferative/cytotoxic activity in vitro against five human cell lines derived from duet carcinoma (BT474), undifferentiated lung (Chaco), liver hepatoblastoma (Hep-G(2)), gastric carcinoma (KATO-III) and colon adenocarcinoma (SW620) cancers. The human foreskin fibroblast cell line (Hs27) was used as a non-transformed control. Those crude extracts that displayed antiproliferative/cytotoxic activity were then further fractionated by column chromatography using TLC-pattern and MTT-cytotoxicity bioassay guided selection of the fractions. The chemical structure of each enriched bioactive compound was analyzed by nuclear magnetic resonance and mass spectroscopy. RESULTS: The crude hexane and dichloromethane extracts of propolis displayed antiproliferative/cytotoxic activities with IC(50) values across the five cancer cell lines ranging from 41.3 to 52.4 µg/ml and from 43.8 to 53.5 µg/ml, respectively. Two main bioactive components were isolated, one cardanol and one cardol, with broadly similar in vitro antiproliferation/cytotoxicity IC(50) values across the five cancer cell lines and the control Hs27 cell line, ranging from 10.8 to 29.3 µg/ml for the cardanol and < 3.13 to 5.97 µg/ml (6.82 - 13.0 µM) for the cardol. Moreover, both compounds induced cytotoxicity and cell death without DNA fragmentation in the cancer cells, but only an antiproliferation response in the control Hs27 cells However, these two compounds did not account for the net antiproliferation/cytotoxic activity of the crude extracts suggesting the existence of other potent compounds or synergistic interactions in the propolis extracts. CONCLUSION: This is the first report that Thai A. mellifera propolis contains at least two potentially new compounds (a cardanol and a cardol) with potential anti-cancer bioactivity. Both could be alternative antiproliferative agents for future development as anti-cancer drugs.

In vitro antiproliferative activity of partially purified Trigona laeviceps propolis from Thailand on human cancer cell lines.

BACKGROUND: Cancers are some of the leading causes of human deaths worldwide and their relative importance continues to increase. Since an increasing proportion of cancer patients are acquiring resistance to traditional chemotherapeutic agents, it is necessary to search for new compounds that provide suitable specific antiproliferative affects that can be developed as anticancer agents. Propolis from the stingless bee, Trigona laeviceps, is one potential interesting source that is widely available and cultivatable (as bee hives) in Thailand. METHODS: Propolis (90 g) was initially extracted by 95% (v/v) ethanol and then solvent partitioned by sequential extractions of the crude ethanolic extract with 40% (v/v) MeOH, CH2Cl2 and hexane. After solvent removal by evaporation, each extract was solvated in DMSO and assayed for antiproliferative activity against five cancer (Chago, KATO-III, SW620, BT474 and Hep-G2) and two normal (HS27 fibroblast and CH-liver) cell lines using the MTT assay. The cell viability (%) and IC50 values were calculated. RESULTS: The hexane extract provided the highest in vitro antiproliferative activity against the five tested cancer cell lines and the lowest cytotoxicity against the two normal cell lines. Further fractionation of the hexane fraction by quick column chromatography using eight solvents of increasing polarity for elution revealed the two fractions eluted with 30% and 100% (v/v) CH2Cl2 in hexane (30DCM and 100DCM, respectively) had a higher anti-proliferative activity. Further fractionation by size exclusion chromatography lead to four fractions for each of 30DCM and 100DCM, with the highest antiproliferative activity on cancer but not normal cell lines being observed in fraction# 3 of 30DCM (IC50 value of 4.09 - 14.7 µg/ml). CONCLUSIONS: T. laeviceps propolis was found to contain compound(s) with antiproliferative activity in vitro on cancer but not normal cell lines in tissue culture. The more enriched propolis fractions typically revealed a higher antiproliferative activity (lower IC50 value). Overall, propolis from Thailand may have the potential to serve as a template for future anticancer-drug development.

Trigona laeviceps propolis from Thailand: antimicrobial, antiproliferative and cytotoxic activities.

Propolis is one of the natural bee products which has long been used as a crude preventative and prophylactic medicine, and has been reported to possess antibacterial, antiviral, anti-inflammatory, antioxidative and anticancer properties. Propolis of the stingless bee, Trigona laeviceps, was extracted by water or methanol at 35% (w/v) yielding a crude water or a methanolic extract at 60 and 80 mg/ml, respectively, which is 17.1 and 22.9% (w/w) of the total propolis, respectively. The antimicrobial activity of both crude extracts was assayed on four selected pathogenic microbes by using the agar well diffusion method. The results suggested that both water and methanolic crude extracts have some antimicrobial activities, water extract has greater antimicrobial activity than methanolic extract. The relative order of sensitivity of the four microbes were, however, the same between the two extracts from the most to least sensitive, S. aureus > E. coli >> C. albicans >>> A. niger, with indeed no observed growth inhibition of A. niger at all. Antiproliferative and cytotoxic affects were tested on the colon carcinoma cell line, SW620, using the three parameters: (1) MTT assay; (2) cell morphology; and (3) the fragmentation of genomic DNA. The water extract of propolis showed a higher antiproliferative activity than that of methanolic extract to SW620 cells, additionally both appeared to cause cell death by necrosis.

New halimane diterpenoids from Croton oblongifolius.

Three new halimane-type diterpenoids, crotohalimaneic acid ( 1), crotohalimoneic acid ( 2) and 12-benzoyloxycrotohalimaneic acid ( 3), were isolated from the stem bark of Croton oblongifolius Roxb. Their structures were established on the basis of spectroscopic and X-ray analysis. Compounds 1 and 2 showed non-specific strong cytotoxicity against a panel of human tumor cell lines; whereas 3 was inactive.

Diterpenoids from the stem barks of Croton robustus.

Three compounds were isolated from the stem barks of Croton robustus. Their structures were elucidated as trachyloban-19-oic acid, trachyloban-19-ol and poilaneic acid by spectroscopic analysis. Among them, trachyloban-19-ol and methyl trachyloban-19-oate exhibited weak cytotoxic activity against gastric carcinoma and colon carcinoma with ED50 of 9.2, 9.6 and 8.3, 9.1 microg/mL, respectively.

Croblongifolin, a new anticancer clerodane from Croton oblongifolius.

A new furoclerodane, croblongifolin, together with one known clerodane, crovatin and one known labdane, nidorellol, were isolated from the stem bark of Croton oblongifolius. Structures were established based on spectroscopic and X-ray analysis. Croblongifolin showed a significant cytotoxicity against various human tumor cell lines including HEP-G2, SW620, CHAGO, KATO3 and BT474.