Influence of ascorbic acid and α-tocopherol on the autoxidation and in vitro antifungal activity of amphotericin B

Mohammed Habib Belhachemi, Zahia Boucherit-Otmani, Kebir Boucherit, Sara Belmir

Curr Med Mycol . 2021 Mar;7(1):12-18. doi: 10.18502/cmm.7.1.6178.

Abstract

Background and purpose: Amphotericin B (AmB) is the standard treatment for systemic fungal infections; however, the formation of reactive oxygen species reduces the efficacy and stability of this molecule. The present study aimed to evaluate the effect of the combination of AmB with ascorbic acid and α-tocopherol on its autoxidation and antifungal activity.

Materials and methods: The antifungal activity against Candida albicans was evaluated by the viable cell counting method and checking their morphological changes with a scanning electron microscope. Monomer state of AmB was assessed by scanning the UV absorbance in the range of 300-450 nm and the lipid peroxidation was measured using quantification of thiobarbituric acid reactive-substances (TBARS).

Results: Based on the findings, the addition of ascorbic acid (3×10² µg/mL) and α-tocopherol (16 µg/mL) to the reaction medium of AmB increased its antifungal activity while maintaining its molecular stability. Moreover, the level of TBARS formed in the reaction medium of AmB was significantly reduced after combination with ascorbic acid and α-tocopherol.

Conclusion: Given their availability, their anti-free radical activity, and their low toxicity, the incorporation of ascorbic acid and α-tocopherol into the reaction medium of AmB seems to be a promising approach to obtain an effective antifungal formulation.

M J Kuhn, L M Sordillo

J Dairy Sci . 2021 Mar 24;S0022-0302(21)00448-3. doi: 10.3168/jds.2020-19675. Online ahead of print.

Abstract

Diseases that occur during the transition period are exacerbated when cows are unable to cope with an increased pro-oxidant load that results in oxidative stress. Dairy cattle are routinely supplemented with the vitamin E analog α-tocopherol to mitigate the severity of oxidative stress. Nonetheless, oxidative stress remains a disease predisposing condition for many dairy cattle. A better method of optimizing the antioxidant functions of vitamin E is needed. α-Tocopherol is only 1 of 8 analogs of vitamin E, all of which have varying antioxidant properties in other mammals, albeit a shorter physiological half-life compared with α-tocopherol. A primary bovine mammary endothelial cell oxidant challenge model was used to determine functions of certain vitamin E analogs. The aim of this study was to determine if other analogs, namely γ-tocopherol or γ-tocotrienol, have antioxidative functions in bovine cells and if these functions may protect cellular viability and endothelial function from oxidant damage. Physiological (10 μM) and supraphysiological (50 μM) concentrations of γ-tocopherol and γ-tocotrienol had a greater capacity to reduce accumulated reactive oxygen species derived from a nitric oxide donating pro-oxidant antagonist, when compared with α-tocopherol, after 30 min to 6 h of treatment. Further, γ-tocotrienol (10 μM) decreased cell cytotoxicity to a greater amount than other analogs at like concentrations, whereas γ-tocopherol (10 μM) reduced lipid peroxidation and apoptosis more effectively than other analogs. Last, α-tocopherol (5 and 10 μM) and γ-tocopherol (5 and 10 μM) significantly slowed pro-oxidant induced loss of endothelial cell barrier integrity over a 48-h period using an electrical cell-substrate impedance sensing system. Concerningly, γ-tocotrienol drastically reduced the endothelial barrier integrity at only 5 μM despite no apparent effect on cellular viability at like concentrations. γ-Tocotrienol, however, was also the only analog to show significant cytotoxicity and reductions in viability at supraphysiological doses (25 and 50 μM). Our results suggest that γ-tocopherol has antioxidant activities that reduces cellular damage and loss of function due to oxidant challenge as effectively as α-tocopherol. These data set the foundation for further investigation into the antioxidant properties of vitamin E analogs in other bovine cells types or whole animal models.

Chao Yang, Yiying Zhao, Suji Im, Cindy Nakatsu, Yava Jones-Hall, Qing Jiang

J Nutr Biochem . 2021 Mar;89:108567. doi: 10.1016/j.jnutbio.2020.108567. Epub 2021 Jan 9.

Abstract

The gut microbiota play important roles in colon cancer. Vitamin E δ-tocotrienol (δTE) and its metabolite δTE-13′-carboxychromanol (δTE-13′) are known to have cancer-preventive effects, but their impact on gut flora during tumorigenesis and the role of the metabolite in δTE’s beneficial effects remain to be determined. In the murine colitis-associated colon cancer (CAC) induced by azoxymethane (AOM) and dextran sulfate sodium (DSS), we show that δTE and δTE-13′ inhibited the multiplicity of large adenomas (>2 mm²) by 34% (P<.05) and 55% (P<.01), respectively, compared to the control diet. δTE-13′ diminished AOM/DSS-increased GM-CSF and MCP-1, and δTE decreased IL-1β. Using 16S rRNA gene sequencing of fecal DNAs, we observe that δTE and δTE-13′ modulated the composition but not the richness of gut microbes compared to the control. Both δTE and δTE-13′ enhanced potentially beneficial Lactococcus and Bacteroides. The elevation of Lactococcus positively correlated with fecal concentrations of δTE-13′ and its hydrogenated metabolite, suggesting that the metabolite may contribute to δTE’s modulation of gut microbes. Furthermore, δTE-13′ counteracted AOM/DSS-induced depletion of Roseburia that is known to be decreased in patients with inflammatory bowel diseases. δTE uniquely elevated (Eubacterium) coprostanoloigenes. Our study demonstrates that δTE and δTE-13′ inhibited tumorigenesis, suppressed pro-inflammatory cytokines and modulated gut microbiota in a murine CAC model. These findings uncover new and distinct activities of δTE and δTE-13′ and support the notion that the metabolite may play a role in δTE’s anticancer and modulation of gut microbes.

Revathi Raviadaran, Mei Han Ng, Davannendran Chandran, Kah Kooi Ooi, Sivakumar Manickam

Mater Sci Eng C Mater Biol Appl . 2021 Feb;121:111808. doi: 10.1016/j.msec.2020.111808. Epub 2020 Dec 22.

Abstract

This work aimed to evaluate the effects of encapsulated tocotrienols (TRF) and caffeic acid (CA) in water-in-oil-in-water (W/O/W) multiple nanoemulsion with cisplatin towards cancer cells. This work is important considering the limited efficacy of cisplatin due to tumour resistance, as well as its severe side effects. A549 and HEP G2 cancer cell lines were utilised for evaluating the efficacy of the encapsulated W/O/W while HEK 293 normal cell line was used for evaluating the toxicity. TRF, CA and CIS synergistically improved apoptosis in the late apoptotic phase in A549 and HEP G2 by 23.1% and 24.9%, respectively. The generation of ROS was enhanced using TRF:CA:CIS by 16.9% and 30.2% for A549 and HEP G2, respectively. Cell cycle analysis showed an enhanced cell arrest in the G0/G1 phase for both A549 and HEP G2. TRF, CA and CIS led to cell death in A549 and HEP G2. For HEK 293, ~33% cell viability was found when only CIS was used while >95% cell viability was observed when TRF, CA and CIS were used. This study demonstrates that the encapsulated TRF and CA in W/O/W with CIS synergistically improved therapeutic efficacy towards cancer cells, as well as lowered the toxicity effects towards normal cells.

Nur-Vaizura Mohamad, Soelaiman Ima-Nirwana, Kok-Yong Chin

Biomed Pharmacother . 2021 Feb 11;137:111368. doi: 10.1016/j.biopha.2021.111368. Online ahead of print.

Abstract

Tocotrienol has been shown to prevent bone loss in animal models of postmenopausal osteoporosis, but the low oral bioavailability might limit its use. A self-emulsifying drug delivery system (SEDDS) could increase the bioavailability of tocotrienol. However, evidence of this system in improving the skeletal effects of tocotrienol is scanty. This study aims to evaluate the therapeutic efficacy of annatto tocotrienol with SEDDS in a rat model of postmenopausal bone loss. Ten-month-old female Sprague Dawley rats were randomized into six groups. The baseline group was euthanatized at the onset of the study. Four other groups underwent ovariectomy to induce estrogen deficiency. The sham underwent similar surgery procedure, but their ovaries were retained. Eight weeks after surgery, the ovariectomized rats received one of the four different regimens orally daily: (a) SEDDS, (b) annatto tocotrienol [60 mg/kg body weight (b.w.)] without SEDDS, (c) annatto-tocotrienol (60 mg/kg b.w.) with SEDDS, (d) raloxifene (1 mg/kg b.w.). After eight weeks of treatment, blood was collected for the measurement of delta-tocotrienol level and oxidative stress markers. The rats were euthanized and their bones were harvested for the evaluation of the bone microstructure, calcium content and strength. Circulating delta-tocotrienol level was significantly higher in rats receiving annatto tocotrienol with SEDDS compared to the group receiving unformulated annatto-tocotrienol (p < 0.05). Treatment with unformulated or SEDDS-formulated annatto tocotrienol improved cortical bone thickness, preserved bone calcium content, increased bone biomechanical strength and increased antioxidant enzyme activities compared with the ovariectomized group (p < 0.05). Only SEDDS-formulated annatto tocotrienol improved trabecular microstructure, bone stiffness and lowered malondialdehyde level (p < 0.05 vs the ovariectomized group). The improvement caused by annatto tocotrienol was comparable to raloxifene. In conclusion, SEDDS improves the bioavailability and skeletal therapeutic effects of annatto tocotrienol in a rat model of postmenopausal bone loss. This formulation should be tested in a human clinical trial to validate its efficacy.

Rajesh Sreedharan Nair, Nashiru Billa, Chee-Onn Leong, Andrew P Morris

Pharm Dev Technol . 2021 Feb;26(2):243-251. doi: 10.1080/10837450.2020.1860087. Epub 2020 Dec 15.

Abstract

Tocotrienol (TRF) ethosomes were developed and evaluated in vitro for potential transdermal delivery against melanoma. The optimised TRF ethosomal size ranged between 64.9 ± 2.2 nm to 79.6 ± 3.9 nm and zeta potential (ZP) between -53.3 mV to -62.0 ± 2.6 mV. Characterisation of the ethosomes by ATR-FTIR indicated the successful formation of TRF-ethosomes. Scanning electron microscopy (SEM) images demonstrated the spherical shape of ethosomes, and the entrapment efficiencies of all the formulations were above 66%. In vitro permeation studies using full-thickness human skin showed that the permeation of gamma-T3 from the TRF ethosomal formulations was significantly higher (p < 0.05) than from the control. The cumulative amount of gamma-T3 permeated from TRF ethosome after 48 hours was 1.03 ± 0.24 µg cm^-2 with a flux of 0.03 ± 0.01 µg cm^-2 h^-1. Furthermore, the flux of gamma-T3 across the Strat-M ^® and the epidermal membrane was significantly higher than that across full-thickness human skin (p < 0.05). In vitro cytotoxicity studies on HaCat cells showed significantly higher cell viability than the pure drug solution (p < 0.05). The enhanced skin permeation and high cell viability associated with this formulation suggest a promising carrier for transdermal delivery.

Phui Yee Tan, Beng Ti Tey, Eng Seng Chan, Oi Ming Lai, Hon Weng Chang, Tai Boon Tan, Yuanfa Liu, Yong Wang, Chin Ping Tan

Foods . 2021 Feb 7;10(2):358. doi: 10.3390/foods10020358.

Abstract

Calcium carbonate (CaCO₃) has been utilized as a pH-responsive component in various products. In this present work, palm tocotrienols-rich fraction (TRF) was successfully entrapped in a self-assembled oil-in-water (O/W) emulsion system by using CaCO₃ as the stabilizer. The emulsion droplet size, viscosity and tocotrienols entrapment efficiency (EE) were strongly affected by varying the processing (homogenization speed and time) and formulation (CaCO₃ and TRF concentrations) parameters. Our findings indicated that the combination of 5000 rpm homogenization speed, 15 min homogenization time, 0.75% CaCO₃ concentration and 2% TRF concentration resulted in a high EE of tocotrienols (92.59-99.16%) and small droplet size (18.83 ± 1.36 µm). The resulting emulsion system readily released the entrapped tocotrienols across the pH range tested (pH 1-9); with relatively the highest release observed at pH 3. The current study presents a potential pH-sensitive emulsion system for the entrapment and delivery of palm tocotrienols.

Geetha Maniam, Chun-Wai Mai, Mohd Zulkefeli, Ju-Yen Fu

Nanomedicine (Lond) . 2021 Feb;16(5):373-389. doi: 10.2217/nnm-2020-0374. Epub 2021 Feb 5.

Abstract

Aim: To synthesize niosomes co-encapsulating gemcitabine (GEM) and tocotrienols, and physicochemically characterize and evaluate the antipancreatic effects of the nanoformulation on Panc 10.05, SW 1990, AsPC-1 and BxPC-3 cells. Materials & methods: Niosomes-entrapping GEM and tocotrienols composed of Span 60, cholesterol and D-α-tocopheryl polyethylene glycol 1000 succinate were produced by Handjani-Vila and film hydration methods. Results: The film hydration produced vesicles measuring 161.9 ± 0.5 nm, approximately 50% smaller in size than Handjani-Vila method, with maximum entrapment efficiencies of 20.07 ± 0.22% for GEM and 34.52 ± 0.10% for tocotrienols. In Panc 10.05 cells, GEM’s antiproliferative effect was enhanced 2.78-fold in combination with tocotrienols. Niosomes produced a significant ninefold enhancement in cytotoxicity of the combination, supported by significantly higher cellular uptake of GEM in the cells. Conclusion: This study is a proof of concept on the synthesis of dual-drug niosomes and their efficacy on pancreatic cancer cells in vitro.

Yuedi Ding, Jun Fan, Zhenqiang Fan, Kai Zhang

J Steroid Biochem Mol Biol . 2021 May;209:105835. doi: 10.1016/j.jsbmb.2021.105835. Epub 2021 Feb 5.

Abstract

The problem of multidrug resistance (MDR) presents a major obstacle in the chemotherapy of cancer. The MDR phenotype is often linked to the overexpression of ATP-binding cassette (ABC) transporters, that pumps out and decreased intracellular drug accumulation. γ-Tocotrienol, an unsaturated tocopherol belonging to the vitamin E family, has been shown to reverse the MDR of MCF-7/Adr cell. To reveal the role of γ-tocotrienol-NF-κB-P-gp axis in the reversal process, the expression level of mdr1/P-gp was determined by real-time PCR and western blot, while NF-κB activity was detected by immunofluorescence and NF-κB transcriptional activity reporter assay. Besides, mdr1 promoter activity and P-gp transport capacity were measured with the effect of γ-tocotrienol and NF-κB agonist/antagonist. Results showed that γ-tocotrienol effectively inhibited the expression levels of mdr1 mRNA and P-gp protein. It is demonstrated that γ-tocotrienol also suppressed mdr1 promoter activity and the efflux activity of P-gp. In addition, the activation of NF-κB signaling pathway and the transcriptional activity of NF-κB were both reduced by γ-tocotrienol. Evidences also showed that the NF-κB pathway is really involved in the regulation of the expression and function of mdr1/P-gp. Taken together, we confirmed that γ-tocotrienol reversed the MDR of MCF-7/Adr through the signaling pathway of NF-κB and P-gp.

Mohd Saufi Harun, Tin Wui Wong, Chee Wai Fong

Int J Pharm . 2021 Jan 25;593:120099. doi: 10.1016/j.ijpharm.2020.120099. Epub 2020 Nov 28.

Abstract

This study investigated combination nanocarrier and microwave system for α-tocopherol and γ-tocotrienol delivery against dermatitis, without skin thinning effect of steroids. The vitamin E was formulated into water-rich/water-poor nanoemulsions, and had their droplet size, zeta potential, morphology, therapeutic content, encapsulation efficiency and release, in vitro skin therapeutics/nanoemulsion penetration, retention and permeation profiles, and in vivo pharmacodynamics characteristics examined, with skin pre-treated by precision microwave when applicable. The nanoemulsions had droplet sizes <150 nm and negative zeta potential values. The skin pre-treatment by microwave (1 mW/3985 MHz) promoted therapeutics accumulation in epidermis through enhancing nanoemulsion penetration into skin. The combination nano- and microwave technologies fluidized skin lipid and protein domains with epidermal microstructures being fluidized to a greater extent than dermis, allowing a relatively high epidermal-to-dermal nanoemulsion distribution. Microwave of lower or higher than 3985 MHz brought about lower skin therapeutics/nanoemulsion accumulation due to insufficient lipid/protein domain fluidization or microwave-skin interaction limiting at skin surfaces only. Using water-rich nanoemulsion with higher therapeutic release and skin pre-treatment with 3985 MHz microwave, dermatitis was alleviated in vivo without skin thinning of standard steroid. The use of combination microwave and nanotechnology promotes vitamin delivery and translates to positive dermatitis treatment outcome that warrants future investigation.