Stability and expression of K-ras mimotopes in freeze-dried recombinant Lactococcus lactis NZ3900-fermented milk powder during storage in vacuum packaging.

AIMS: This study aims to evaluate the storage stability of the freeze-dried recombinant L. lactis NZ3900-fermented milk powder expressing K-ras (Kristen rat sarcoma viral oncogene homolog) mimotopes targeting colorectal cancer in vacuum packaging. METHODS AND RESULTS: The freeze-dried L. lactis-fermented milk powder stored in 4-ply retortable polypropylene (RCPP)-polyamide (PA)-aluminium (AL)-polyethylene terephthalate (PET) and aluminium polyethylene (ALPE) were evaluated throughout 49 days of accelerated storage (38°C and 90% relative humidity). The fermented milk powder stored in 4-ply packaging remained above 6 log10 CFU g-1 viability, displayed lower moisture content (6.1%), higher flowability (43° angle of repose), water solubility (62%), and survivability of L. lactis after simulated gastric and intestinal digestion (> 82%) than ALPE packaging after 42 days of accelerated storage. K-ras mimotope expression was detected intracellularly and extracellularly in the freeze-dried L. lactis-fermented milk powder upon storage. CONCLUSIONS: This suggests that fermented milk powder is a suitable food carrier for this live oral vaccine.

Development of optimized functional clove fortified probiotic yoghurt.

We focused on assessing the antimicrobial effects of functional yoghurts supplemented with clove and probiotics. The formulation of aqueous clove extract (ACE) incorporated with probiotic yoghurt (Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactococcus lactis) was optimised in terms of aqueous clove extract concentrations (2.5-7.5% v/v), fermentation temperature (32-42 °C), and total culture concentration (1.5-4.5% v/v). pH, titratable acidity, syneresis, water holding capacity, viscosity, springiness, color difference, lactic acid bacteria viability, and the antibacterial property of 17 runs were determined as responses using Box-Behnken design. The results indicate that elevated ACE concentration leads to a significant (p < 0.05) increase in titratable acidity, antibacterial effectiveness (against K. pneumoniae and P. aeruginosa), springiness, and color profile. Conversely, an elevated fermentation temperature significantly (p < 0.05) reduces pH and L. bulgaricus viability (log CFU/mL). Additionally, there is a significant (p < 0.05) decline in S. thermophilus and L. lactis viability (log CFU/mL) as well as springiness with an increased culture concentration. The optimal conditions identified are 7.5% (v/v) ACE concentration, a fermentation temperature of 36.6 °C (37 °C), and a total culture concentration of 4.5% (v/v), resulting in a 79% desirability score. The spectra of components were mainly obtained at wavelength of 3258 cm-1, 1636 cm-1 and 1075 cm-1 in Fourier transform infrared (FTIR) spectroscopy of optimized functional yoghurt. Where, 3258 cm-1 corresponds to the stretching vibration of O-H (hydroxyl) groups, 1636 cm-1 corresponds to the C=O (carbonyl) stretching vibration, and 1075 cm-1 corresponds to the C-O (ether or alcohol) stretching vibration.

A comprehensive review of Bifidobacterium spp: as a probiotic, application in the food and therapeutic, and forthcoming trends.

Recently, more consumers are interested in purchasing probiotic food and beverage products that may improve their immune health. The market for functional foods and beverages that include Bifidobacterium is expanding because of their potential uses in both food and therapeutic applications. However, maintaining Bifidobacterium's viability during food processing and storage remains a challenge. Microencapsulation technique has been explored to improve the viability of Bifidobacterium. Despite the technical, microbiological, and economic challenges, the market potential for immune-supporting functional foods and beverages is significant. Additionally, there is a shift toward postbiotics as a solution for product innovation, a promising postbiotic product that can be incorporated into various food and beverage formats is also introduced in this review. As consumers become more health-conscious, future developments in the functional food and beverage market discussed in this review could serve as a reference for researchers and industrialist.

3D Printed gelatin film with Garcinia atroviridis extract.

Active packaging, such as edible film with antibacterial properties, can help extend the shelf life of food. The research aimed to develop a 3D printed gelatin edible film by using glycerol and Garcinia atroviridis extract (GAE). Mechanical properties of gelatin gel, physical, mechanical, and antimicrobial properties of edible film with glycerol and GAE were determined. Water solubility, total colour difference, and elongation of break of gelatin edible film increased as glycerol concentration increased (0-25% w/w), whereas tensile strength and Young's modulus value decreased from 26.5 to 4.64 MPa and 3.04 to 0.13 MPa, respectively. On the other hand, increasing GAE from 1 to 4% (w/w) increases elongation at break from 40.83 to 98.27%, while decreasing edible film tensile strength and gelatin gel hardness value from 8.94 to 6.21 MPa and 1848.67 to 999.67 g, respectively. Using 20% (w/w) glycerol and 4% (w/w) GAE, the best 3D printed film with low tensile strength (6.21 MPa), high elongation at break (98.27%), and antibacterial activity against S. aureus with 7.23 mm zone of inhibition was developed. It seems to have a great potentiality as an active packaging material for 3D printed gelatin edible film.

Synergistic enhancing effect of xanthan gum, carboxymethyl cellulose and citric acid on the stability of betacyanins in fermented red dragon fruit (Hylocereus polyrhizus) drink during storage.

Nowadays, the demand for using healthy natural pigments (betacyanins) in the food industry is increasing. The present study aimed to overcome the circumstances that render the betacyanins instability in the red dragon fruit drink using mild approaches. These included optimised fermentation, incorporation of anionic polysaccharide mixture solution [xanthan gum (XG, 0.30-0.40 %, w/v) and carboxymethyl cellulose (CMC, 0.50-0.90 %, w/v)] and also addition of citric acid (CA, 0.05-0.20 %, w/v). The results of this study showed that the hydrocolloid mixture solution of XG and CMC significantly increased the samples' viscosity, pH and °Brix but reduced the aw, while betacyanins concentration had no significant change. The incorporation of CA at increasing concentration only reduced the samples' pH significantly without affecting the viscosity, aw and °Brix. Among all fermented samples, Formulation 3E (0.40 % XG + 0.50 % CMC + 0.20 % CA) had achieved the desired commercial reference viscosity while also successfully minimised betacyanins degradation from 60.18 % to 14.72 %, had the best pH stability and no significant change in viscosity, aw and °Brix values after 4-week storage at 25 °C. The fermented red dragon fruit drink with betacyanins stabilised by Formulation 3E can be produced and served as an independent functional drink product and as a stable, functional ingredient (natural colourant) for the food industry.

Immunomodulatory action of Lactococcuslactis.

Fermented foods are gaining popularity due to health-promoting properties with high levels of nutrients, phytochemicals, bioactive compounds, and probiotic microorganisms. Due to its unique fermentation process, Lactococcus lactis plays a key role in the food business, notably in the manufacturing of dairy products. The superior biological activities of L. lactis in these functional foods include anti-inflammatory and immunomodulatory capabilities. L. lactis boosted growth performance, controlled amino acid profiles, intestinal immunology, and microbiota. Besides that, the administration of L. lactis increased the rate of infection clearance. Innate and acquired immune responses would be upregulated in both local and systemic compartments, resulting in these consequences. L. lactis is often employed in the food sector and is currently being exploited as a delivery vehicle for biological research. These bacteria are being eyed as potential candidates for biotechnological applications. With this in mind, we reviewed the immunomodulatory effects of different L. lactis strains.

The indigenous microbial diversity involved in the spontaneous fermentation of red dragon fruit (Hylocereus polyrhizus) identified by means of molecular tools.

Red dragon fruit (RDF) is well-known for its high nutritional content, especially the red pigment betacyanins that possess high antioxidant activity. Natural fermentation is an ancient yet outstanding technique that relies on the autochthonous microbiota from fruits and vegetables surfaces to preserve and improve the nutritional values and quality of the food product. The present study was to evaluate and identify the indigenous microbial community (bacteria and fungi) that are involved in the natural fermentation of RDF. Results revealed a total of twenty bacterial pure cultures and nine fungal pure cultures were successfully isolated from fermented red dragon fruit drink (FRDFD). For the first time, the PCR amplification of 16S rRNA and ITS regions and sequence analysis suggested nine genera of bacteria and three genera of fungi (Aureobasidium pullulans, Clavispora opuntiae, and Talaromyces aurantiacus) present in the FRDFD. Four dominant (≥10 % isolates) bacteria species identified from FRDFD were Klebsiella pneumonia, Brevibacillus parabrevis, Bacillus tequilensis and Bacillus subtilis. The carbohydrate fermentation test showed that all the indigenous microbes identified were able to serve as useful starter culture by fermenting sucrose and glucose, thereby producing acid to lower the pH of FRDFD to around pH 4 for better betacyanins stability. The present study provides a more comprehensive understanding of the indigenous microbial community that serves as the starter culture in the fermentation of RDF. Besides, this study provides a useful guide for future research to be conducted on studying the rare bacterial strains (such as B. tequilensis) identified from the FRDFD for their potential bioactivities and applications in medical treatment and functional foods industries.

Polysaccharide-Based Edible Films Incorporated with Essential Oil Nanoemulsions: Physico-Chemical, Mechanical Properties and Its Application in Food Preservation-A Review.

Edible films with essential oils (EOs) are becoming increasingly popular as an alternative to synthetic packaging due to their environmentally friendly properties and ability as carriers of active compounds. However, the required amounts of EOs to impart effective antimicrobial properties generally exceed the organoleptic acceptance levels. However, by nanoemulsifying EOs, it is possible to increase their antimicrobial activity while reducing the amount required. This review provides an overview of the physico-chemical and mechanical properties of polysaccharide-based edible films incorporated with EOs nanoemulsions and of their application to the preservation of different food types. By incorporating EOs nanoemulsions into the packaging matrix, these edible films can help to extend the shelf-life of food products while also improving the quality and safety of the food product during storage. It can be concluded that these edible films have the potential to be used in the food industry as a green, sustainable, and biodegradable method for perishable foods preservation.

Chitosan-Sodium Caseinate Composite Edible Film Incorporated with Probiotic Limosilactobacillus fermentum: Physical Properties, Viability, and Antibacterial Properties.

Single-use synthetic plastics that are used as food packaging is one of the major contributors to environmental pollution. Hence, this study aimed to develop a biodegradable edible film incorporated with Limosilactobacillus fermentum. Investigation of the physical and mechanical properties of chitosan (CS), sodium caseinate (NaCas), and chitosan/sodium caseinate (CS/NaCas) composite films allowed us to determine that CS/NaCas composite films displayed higher opacity (7.40 A/mm), lower water solubility (27.6%), and higher Young's modulus (0.27 MPa) compared with pure CS and NaCas films. Therefore, Lb. fermentum bacteria were only incorporated in CS/NaCas composite films. Comparison of the physical and mechanical properties of CS/NaCas composite films incorporated with bacteria with those of control CS/NaCas composite films allowed us to observe that they were not affected by the addition of probiotics, except for the flexibility of films, which was improved. The Lb. fermentum incorporated composite films had a 0.11 mm thickness, 17.9% moisture content, 30.8% water solubility, 8.69 A/mm opacity, 25 MPa tensile strength, and 88.80% elongation at break. The viability of Lb. fermentum after drying the films and the antibacterial properties of films against Escherichia coli O157:H7 and Staphylococcus aureus ATCC 29213 were also evaluated after the addition of Lb. fermentum in the composite films. Dried Lb. fermentum composite films with 6.65 log10 CFU/g showed an inhibitory effect against E. coli and S. aureus (0.67 mm and 0.80 mm inhibition zone diameters, respectively). This shows that the Lb.-fermentum-incorporated CS/NaCas composite film is a potential bioactive packaging material for perishable food product preservation.

Studies on the storage stability of betacyanins from fermented red dragon fruit (Hylocereus polyrhizus) drink imparted by xanthan gum and carboxymethyl cellulose.

Red dragon fruit is rich in health-benefited betacyanins that are susceptible to degradation. The present study was to improve the fermented red dragon fruit drink (FRDFD) betacyanins stability by incorporating hydrocolloids solution of xanthan gum (XG, 0.15-0.30%, w/v) and carboxymethyl cellulose (CMC, 0.3-0.5%, w/v) to produce Improved-FRDFD-dH2O. Results revealed the viscosities of all samples were significantly increased as the hydrocolloids concentration increased. All the samples' pH, aw, total soluble solids (TSS) and betacyanins content were not significantly affected by the hydrocolloids solution added. After four-week storage (25 °C), the formulation of 0.3% XG and 0.5% CMC had significantly reduced the betacyanins degradation from 60.55% to 30.66%. Meanwhile, all samples added with 0.3% XG and 0.3-0.5% CMC remained no significant change in viscosity, pH, aw and TSS after storage. These conclude the hydrocolloids solution of 0.3% XG and 0.5% CMC successfully stabilise the betacyanins in the FRDFD at 25 °C over four-week storage.

Spray Drying Encapsulation of Pediococcus acidilactici at Different Inlet Air Temperatures and Wall Material Ratios.

Pediococcus acidilactici has gained research and commercial interest due to its outstanding probiotic properties, yet its survival during storage and consumption requires improvement. This study aims to enhance P. acidilactici survival using spray drying encapsulation. Different inlet air temperatures (120 °C, 150 °C, and 170 °C) and whey protein isolate (WPI):gum arabic (GA) ratios (1:1, 3:1, 1:3) were tested. Cell viability was significantly (p < 0.05) affected by the inlet temperature but not the WPI:GA ratio. Increasing the inlet temperature to 170 °C significantly decreased P. acidilactici viability by 1.36 log cycles, from 8.61 log CFU/g to 7.25 log CFU/g. The inlet temperature of 150 °C resulted in a powder yield (63.12%) higher than at 120 °C (58.97%), as well as significantly (p < 0.05) lower moisture content (5.71%) and water activity (aw 0.21). Viable cell counts in all encapsulated P. acidilactici were maintained at 5.24−6.75 log CFU/g after gastrointestinal tract (GIT) simulation, with WPI:GA of 3:1 and inlet temperature 150 °C having the smallest log reduction (0.3 log cycles). All samples containing different WPI:GA ratios maintained sufficient viability (>7 log CFU/g) during the first three weeks of storage at 25 °C. These results could provide insights for further developing P. acidilactici as commercial probiotic products.

Development of chitosan edible film incorporated with Chrysanthemum morifolium essential oil.

BACKGROUND: Biodegradable food packaging has improved in quality with recent research incorporating natural extracts for functionality purposes. This research aims to develop chitosan film with Chrysanthemum morifolium essential oil to improve the shelf life of fresh raw chicken and beef. METHODS: 1.5% (w/v) chitosan films with Chrysanthemum morifolium essential oil (0% to 6% (v/v)) were produced through homogenization, the casting of a film solution in a petri dish and convection drying. The edible film was evaluated in terms of its physical (color, thickness, water vapor permeability), mechanical (puncture strength, tensile strength, elongation at break) and chemical properties (antioxidant assay, Fourier Transform Infrared Spectroscopy (FTIR)). RESULTS: With an increasing concentration of Chrysanthemum morifolium in the chitosan film, the test values of physical properties such as tensile strength, puncture force, and elongation at break declined significantly. However, the thickness, water permeability, and color profile (L*, a*, b*) values of the chitosan film increased. Similarly, the scavenging effect of antioxidant assay increased (from 4.97% to 18.63%) with a rise in Chrysanthemum morifolium concentration. 2%, 3%, and 4% of Chrysanthemum morifolium in the chitosan film showed a significant inhibition zone ranging from 2.67 mm to 3.82 mm against Staphylococcus aureus, a spoilage bacterium that is commonly found in chicken and beef products. The storage and pH tests showed that 4% of Chrysanthemum morifolium in the film maintained pH level (safe to consume), and the shelf life was extended from 3 days to 5 days of meat storage. CONCLUSIONS: This study demonstrated that the incorporation of 4% (v/v) Chrysanthemum morifolium extract into 1.5% (w/v) chitosan film extends the storage duration of raw meat products noticeably by reducing Staphylococcus aureus activity. Therefore, it increases the quality of the edible film as an environmentally friendly food packaging material so that it can act as a substitute for the use of plastic bags. Future studies will be conducted on improving the tensile strength of the edible film to increase the feasibility of using it in the food industry. In addition, the microstructure and surface morphology of the edible film can be further determined.

Edible Oils Adulteration: A Review on Regulatory Compliance and Its Detection Technologies.

Various events of edible oils adulteration with inferior ingredients were reported regularly in recent years. This review is aimed to provide an overview of edible oils adulteration practices, regulatory compliance and detection technologies. Many detection technologies for edible oils adulteration were developed in the past such as methods that are based on chromatography or spectroscopy. Electrochemical sensors like electric nose and tongue are also gaining popularity in the detection of adulterated virgin olive oil and virgin coconut oil. It can be concluded that these detection technologies are essential in the combat with food adulterers and can be improved.

Effects of inlet temperature and carrier concentration on spray-dried 'cempedak' (Artocarpus integer) fruit powder and its reconstitution properties.

BACKGROUND: 'Cempedak' (Artocarpus integer) is an aromatic fruit which is similar to jackfruit. Although it is rich in vitamin A and is consumed fresh, the fruit has a short shelf life. Hence, it can be converted through a spray-drying process, to form powder, which is more stable. Powder flow properties are important when considering storage, while its reconstitution characteristics are critical for the consumer to make juice from the product. METHODS: The parameters of spray-dried 'cempedak' fruit powder under study include inlet air temperature (140-180°C) and maltodextrin (DE 10) concentrations (5-15% w/w). Response surface methodology involving 14 runs was used to assess the effects of inlet temperature and maltodextrin on the powder flow properties and reconstitution properties of the spray-dried 'cempedak' powder. RESULTS: Out of the tested responses, only bulk density, change in cake height ratio, and water solubility index had a high coefficient of determination value. Inlet air temperature was found to be the main parameter to affect the bulk density, caking and water solubility index, when compared to maltodextrin concentration. By setting minimization of caking and maximization of water solubility index as the main determinants, the optimal parameters of 160°C inlet temperature and 15% (w/w) maltodextrin DE10 were generated, with a desirability of 0.697. CONCLUSIONS: The powder produced under optimal conditions (160°C and 15% w/w maltodextrin) had a low bulk density (480.01 kg/m3), low caking properties (0.17 change in cake height ratio), and a high solubility index (88.69). This indicates that the powder is stable to be stored (without caking) and will have good reconstitution when added to water.

Production of ‘kedondong’ (Spondias cytherea Sonnerat) powder as affected by different drying methods.

BACKGROUND: ‘Kedondong’ fruit is regarded as an exotic fruit that is gaining popularity due to its deliciousness and pleasant flavour. However, this fruit has a short shelf life, leading to problems with postharvest loss. In order to prevent losses, the fruit could be produced as a value-added product. In this study, the ‘kedondong’ fruit was preserved by drying into powder using different drying methods. METHODS: The kedondong powder was dried using five methods: convection oven drying, vacuum drying, spray drying, drum drying and freeze drying. The physical properties, flowability and DPPH radical scavenging ability of dried kedondong powder were examined. RESULTS: Spray-dried powder provided the significantly (p ≤ 0.05) highest process yield, which was 54.93%. All the powder produced had a low moisture content (3.03 to 5.66%) and water activity (0.19–0.37). Visually, whitish and fine powders were observed on spray-dried and freeze-dried samples, while convection oven-dried and vacuum-dried powder appeared yellowish and coarse. The pH of the reconstituted powders varied from 2.71 to 2.83, where drum-dried powder was the most acidic. Spray-dried powder showed the highest wettability and shortest dissolution time, which was 172.65 s and 10.55 s, respectively. With the exception of drum-dried powder, all the dried powders were classified as non-caking powders. The bulk and tapped density of the powders ranged from 0.32 to 0.70 g/mL and 0.38 to 0.86 g/mL, respectively. Vacuum-dried powder had very good flowability, convection oven-dried and drum-dried powder had good flowability, while spray-dried and drum-dried powder had fair flowability. Antioxidant assay showed that freeze-dried powder exhibited the highest free radical scavenging activity (IC50 = 701.29 μg/mL). CONCLUSIONS: This study indicates that spray-dried kedondong powder has great potential in the food industry due to its high process yield and better powder quality. Meanwhile, freeze drying best preserved the antioxidant properties of the powder, which could potentially be used as a functional ingredient as a result. This study is important for the fruit processing industry as it offers an alternative for the farmer to produce kedondong fruit powder because the fruit has a short shelf life. Converting the fruit into powder can diversify the resulting produce into different applications, such as fruit juice, beverages, jam and other food products.

Red Palm Oil: A Review on Processing, Health Benefits and Its Application in Food.

This review is aimed to provide a comprehensive overview of the physicochemical properties and extraction processes of red palm oil, its nutritional properties and applications in food. Crude palm oil is firstly extracted from the fruit mesocarp and processed into red palm oil using pre-treatment of crude palm oil, with deacidification steps, and deodorization via short-path distillation. These processes help to retain ß-carotene and vitamin E in red palm oil. Palmitic, stearic and myristic acids are the saturated fatty acids in red palm oil, while the unsaturated fatty acids are oleic, linoleic and linolenic acids. It is reported to overcome vitamin A deficiency, promote heart health and have anti-cancer properties.

Optimization of mango-pineapple jelly sphere production by frozen reverse spherification using a full factorial design.

BACKGROUND: The bite-sized jelly sphere with a gelatinous exterior and fruit puree interior is a type of innovative fruit-based dessert. This study aimed to produce jelly spheres with a gelatinous exterior and mangopineapple puree interior by using frozen reverse spherification. METHODS: A full factorial design (23) was applied to study the effects of mango-pineapple ratio (x1), immersion time in sugar solution (x2), and concentration of sugar solution (x3) in the production  of mango-pineapple jelly spheres using frozen reverse spherification. The responses studied were the physicochemical properties (color, total soluble solids, and texture) and sensory evaluation of mango-pineapple jelly spheres. RESULTS: Mango-pineapple ratio had a positive effect on a* and b* while having a negative effect L* value on the jelly sphere. Total soluble solids of jelly spheres were influenced by both immersion time in sugar solution and concentration of sugar solution. Immersion time in sugar solution had a positive effect on the peak force of the compression cycle and deformation at peak load while having a negative effect on the total soluble solid of jelly spheres. On the other hand, the concentration of sugar solution had a positive effect on the sensory evaluation in terms of flavor, texture, and overall acceptability. The desirability function approach was used to optimize the factors, and an overall desirability of 0.89 for all responses was achieved with 1.28:1 mango-pineapple ratio, 30 mins immersion time in sugar solution, and 22°Brix sugar solution. A proximate analysis of the optimized mango-pineapple jelly spheres had an energy content of 73.18 kcal/100 g and showed nutrient values of 81.11% moisture, 0.10% ash, 0.46% protein, 0% fat, 0.97% total dietary fiber, and 17.35% digestible carbohydrate. CONCLUSIONS: The development of the optimal mango-pineapple jelly sphere allows food producers to produce a dessert that is low in calories, with a good appearance and consumer acceptability.

Characterization of crude 5'-phosphodiesterase from germinated adzuki (Vigna angularis L.) beans.

BACKGROUND: 5'-Phosphodiesterase (5'-PDE) is an enzyme that hydrolyzes RNA to form 5'-inosine monophosphate (5'-IMP) and 5'-guanosine monophosphate (5'-GMP). These 5'-nucleotides can function as flavor enhancers. Adzuki beans (Vigna angularis L.) are found to be high in 5'-PDE. METHODS: 5'-phosphodiesterase (5'-PDE) enzyme was characterized from adzuki beans, in which the optimum pH and temperature were determined. In addition, the stability of 5'-PDE was assessed at different pH and temperature. The effects of cations and EDTA were evaluated to characterize the 5'-PDE enzymes further. RESULTS: The alkaline 5'-phosphodiesterase has an optimum pH of 8.5. This enzyme is also thermostable, with an optimum temperature of 80°C. The stability in terms of temperature and pH was also determined, and was found to be stable in the pH range of 7.0-8.5. This enzyme was found to retain more than 80% of its activity for 4 days at 60 and 65°C. In addition, the effects of 14 different metal ions, 4 types of detergents and ethylenediaminetetraacetic acid (EDTA) on 5'-PDE were studied. Ca2+, K+, Mg2+ and Li+ activated 5'-PDE while Na+, Zn2+, Ni+, Hg+, Cu2+, Pb2+, Fe2+, Al3+, Ba2+ and Co2+ were inhibitory. EDTA, Triton X-100 and sodium dodecyl sulfate (SDS) were strong inhibitors of 5'-PDE, while Tween 80 and Tween 20 were slightly inhibitory. The effects of cations and EDTA suggest that 5'-PDE from adzuki beans is a metalloenzyme. CONCLUSIONS: Although 5'-PDE from adzuki beans has a high temperature optimum of 80°C, the enzyme is more stable at 60°C, and different cations affected the activity of the enzyme differently.

Clinacanthus Nutans Hexane Extracts Induce Apoptosis Through a Caspase-Dependent Pathway in Human Cancer Cell Lines

Background: Clinacanthus nutans (C.nutans) is a plant consumed as a cancer treatment in tropical Asia. Despite the availability of numerous anecdotal reports, evaluation of active anticancer effects has remained elusive. Therefore we here examined antiproliferative, reactive oxygen species (ROS)-inducing and apoptosis mechanisms of whole plant extracts in different cancer cell lines. Methods: Antiproliferative actions of five solvent extracts (hexane, chloroform, ethyl acetate, methanol and water) of C.nutans were tested on non-small cell lung cancer (A549), nasopharygeal cancer (CNE1) and liver cancer (HepG2) cells using MTT assay. The most potent anticancer extract was then assessed by flow cytometry to study cell cycle changes . Intracellular levels of ROS were quantified by DCFH-DA assay. Involvement of the caspase pathway in induction of apoptosis was assessed using caspase assay kits. GC-MS analysis was performed to identify phytoconstituents in the extracts. Results: Hexane and chloroform extracts were antiproliferative against all three cell lines, while the ethyl acetate extract, at 300 µg/mL, was antiproliferative in the CNE1 but not A549 and HepG2 cases. Methanol and water extracts did not inhibit cancer cell proliferation. The most potent anticancer hexane extract was selected for further testing. It induced apoptosis in all three cell lines as shown by an increase in the percentage of cell in sub-G1 phase. Dose-dependent increase in ROS levels in all three cell lines indicated apoptosis to be possibly modulated by oxidative stress. At high concentrations (>100 µg/mL), hexane extracts upregulated caspases 8, 9 and 3/7 across all three cell lines. GC-MS analysis of the hexane extract revealed abundance of 31 compounds. Conclusion : Among the five extracts of C.nutans, that with hexane extract demonstrated the highest antiproliferative activity against all three cancer cell lines tested. Action appeared to be via ion of intracellular ROS, and induction of apoptosis via intrinsic and extrinsic caspase pathways.