Aloe barbadensis: how a miraculous plant becomes reality

Aloe barbadensis Miller is a plant that is native to North and East Africa and has accompanied man for over 5,000 years. The aloe vera plant has been endowed with digestive, dermatological, culinary and cosmetic virtues. On this basis, aloe provides a range of possibilities for fascinating studies from several points of view, including the analysis of chemical composition, the biochemistry involved in various activities and its application in pharmacology, as well as from horticultural and economic standpoints. The use of aloe vera as a medicinal plant is mentioned in numerous ancient texts such as the Bible. This multitude of medicinal uses has been described and discussed for centuries, thus transforming this miracle plant into reality. A summary of the historical uses, chemical composition and biological activities of this species is presented in this review. The latest clinical studies involved in vivo and in vitro assays conducted with aloe vera gel or its metabolites and the results of these studies are reviewed.     

Sporicidal efficacy of genipin: a potential theoretical alternative for biomaterial and tissue graft sterilization

Terminal sterilization of musculoskeletal allografts by gamma radiation minimizes the risk of disease transmission but impairs allograft mechanical properties. Commonly employed crosslinking agents can sterilize tissues without affecting mechanical properties adversely; however, these agents are toxic. Genipin is reported to be a benign crosslinking agent that strengthens mechanical properties of tissues; however, the antimicrobial capacity of genipin is largely unknown. The present study’s aims were: (1) to assess the sporicidal potential of genipin, (2) to improve antimicrobial capacity by changing chemical and physical treatment conditions. To establish genipin’s sterilization potential Bacillus subtilis var. niger spore strips were treated with 0–10 % genipin in PBS or in 1:1 DMSO:PBS up to 72 h at room temperature (RT). Sterilizing doses and concentrations of genipin were used to treat B. pumilus and Geobacillus stearothermophilus spores to assess broader spectrum sporicidal activity of genipin. Scanning electron microscopy (SEM) was performed to evaluate gross morphological changes after genipin treatment. Optimal sterilization conditions were determined by evaluating the effects of temperature (RT-50 °C), DMSO:PBS ratio (0:100–100:0), and treatment duration (24–72 h) on B. subtilis. Genipin penetration of full thickness bovine patellar tendon and cortical bone specimens was observed to assess the feasibility of the agent for treating grafts. Initial studies showed that after 72 h of treatment at RT with 0.63–10 % genipin/DMSO:PBS B. subtilis spore strips were sterilized; 0.63 % genipin/PBS did not sterilize spore strips at 72 h at RT. Genipin doses and concentrations that sterilized B. subtilis spore strips sterilized B. pumilus and G. stearothermophilus spore strips. SEM revealed no gross morphological differences between untreated and treated spores. Treatment optimization resulted in sterilization within 24 h with 100 % PBS, and DMSO facilitated sporicidal activity. Genipin penetrated full thickness patellar tendon specimens and 3.72 ± 0.58 mm in cortical bone specimens. Genipin sterilizes B. subtilis, B. pumilus, and G. stearothermophilus spore strips. It penetrates soft and hard tissues at doses previously shown to be non-toxic and to improve mechanical strength in collagen-rich soft tissues. Further studies are indicated to assess genipin’s effects on the mechanical properties of genipin-sterilized grafts, the ability of genipin to eradicate infectious species other than spores, and to assess whether sterilant activity persists after penetrating tissues and biomaterials.     

Comparative analysis of gelatin scaffolds crosslinked by genipin and silane coupling agent

Scaffolds based on gelatin (G) are considered promising for tissue engineering, able to mimic the natural extracellular matrix. G drawback is its poor structural consistency in wet conditions. Therefore, crosslinking is necessary to fabricate stable G scaffolds. In this work, a comparative study between the performance of two different crosslinkers, genipin (GP) and γ-glycidoxypropyltrimethoxysilane (GPTMS), is presented. Flat membranes by solvent casting and porous crosslinked scaffolds by freeze-drying were prepared. Infrared spectroscopy and thermal analysis were applied to confirm G chain crosslinking. Moreover, GP and GPTMS increased the stability of G in aqueous media and improved the mechanical properties. Crosslinking reduced the wettability, especially in the case of G_GPTMS samples, due to the introduction of hydrophobic siloxane chains. Both G_GP and G_GPTMS scaffolds supported MG-63 osteoblast-like cell adhesion and proliferation.     

Synthesis of a Semi-Interpenetrating Polymer Network as a Bioactive Curcumin Film

This study focused on the synthesis and characterization of a natural polymeric system employing the interpenetrating polymer network (IPN) comprising curcumin as a bioactive. Biopolymers and actives such as chitosan, hypromellose, citric acid, genipin, and curcumin were used to develop an effective, biodegradable, and biocompatible film employed therapeutically as a wound healing platform. The semi-IPN films were investigated for their physicochemical, physicomechanical, and biological properties by quantification by FTIR, DSC, and Young’s modulus. Following characterization, an optimum candidate formulation was produced whereby further in vitro and ex vivo studies were performed. Results revealed a burst release occurring at the first hour with 1.1 mg bioactive released when in contact with the dissolution medium and 2.23 mg due to bioactive permeation through the skin, thus suggesting that the lipophilic nature of skin greatly impacted the bioactive release rate. Furthermore, chemical and mechanical characterization and tensile strength analysis revealed that the degree of crosslinking and concentration of polymeric material used significantly influenced the properties of the film.KEY WORDS: biomaterials, crosslinker, curcumin, films, semi-interpenetrating polymer network, wound healing     

Effect of solvent-dependent viscoelastic properties of chitosan membranes on the permeation of 2-phenylethanol

The viscoelastic behaviour of chitosan was followed by dynamic mechanical analysis (DMA) while the sample was immersed in gradient compositions of water/ethanol mixtures. The swelling equilibrium of chitosan membranes, both crosslinked with genipin or not, increased linearly with the water content. Increasing the water content, it was simultaneously observed a peak in the loss factor (around 25 vol.%) and a reduction of the storage modulus, which was attributed to the α-relaxation of chitosan. This was the first time that the glass transition dynamics in a polymer was monitored in immersion conditions where the composition of the plasticizer in the bath is changed in a controlled way. The water content at which tan δ presented a maximum increased with both increasing frequency and increasing crosslinking density. The permeability decreased steadily with the ethanol content, reaching very low values around the glass transition. Therefore we hypothesize that conformational mobility of the polymeric chains may play an important role in the diffusion properties of molecules trough polymeric matrices.     

Effect of inorganic salts and glucose additives on dose–response,melting point and mass density of genipin gel dosimeters

Genipin gel dosimeters are hydrogels infused with a radiation-sensitive material which yield dosimetric information in three dimensions (3D). The effect of inorganic salts and glucose on the visible absorption dose–response, melting points and mass density of genipin gel dosimeters has been experimentally evaluated using 6-MV LINAC photons. As a result, the addition of glucose with optimum concentration of 10% (w/w) was found to improve the thermal stability of the genipin gel and increase its melting point (T_m) by 6 °C accompanied by a slight decrease of dose–response. Furthermore, glucose helps to adjust the gel mass density to obtain the desired tissue-equivalent properties. A drop of T_m was observed when salts were used as additives. As the salt concentration increased, gel T_m decreased. The mass density and melting point of the genipin gel could be adjusted using different amounts of glucose that improved the genipin gel suitability for 3D dose measurements without introducing additional toxicity to the final gel.     

Effects of fermented aloe vera mixed diet on larval growth of Protaetia brevitarsis seulensis (Kolbe) (coleopteran:Cetoniidae) and protective effects of its extract against CCl4‐induced hepatotoxicity in Sprague–Dawley rats

The present study was conducted to evaluate the efficacy of fermented aloe vera mixed diet on larval growth of Protaetia brevitarsis seulensis (Kolbe) (coleopteran: Cetoniidae) and protective effects of extract of larvae of P. brevitarsis seulensis fed fermented aloe vera mixed diet against Carbon tetrachloride (CCl₄)‐induced hepatotoxicity in Sprague–Dawley rats. To determine whether different diets can affect the larval growth of P. brevitarsis seulensis, six different diets were investigated in the experiment, and the results exhibited that 15% fermented aloe vera with 85% fermented oakwood sawdust was the best diet for larval growth of this insect, followed by 9% fermented aloe vera with 91% fermented oakwood sawdust. Adult male Sprague–Dawley rats (n= 30) were separated into five groups of six each, as follows: control group; CCl₄ group; CCl₄ plus larval extract group (without fermented aloe vera); CCl₄ plus 9% and 15% larval extract groups (with fermented aloe vera). All extract groups were fed with 30 mg/kg extracts of fermented oakwood sawdust only and fermented oakwood sawdust plus 9% and 15% fermented aloe vera, respectively, once every consecutive day, with administration of CCl₄ (1.5 mL/kg, 20% CCl₄ in olive oil) twice a week for 3 weeks. Administration of CCl₄ increased the serum alanine aminotransferase, aspartate aminotranseferase and thiobarbituric acid reactive substance levels in rats and reduced levels of glutathione in the liver. Treatment with extract of larval‐fed fermented aloe vera mixed diet significantly alleviated these changes to nearly normal levels. The histopathological changes induced by CCl₄ were also significantly attenuated by extract of larval‐fed fermented aloe vera mixed diet treatment. The results suggest that the beneficial effect of fermented aloe vera mixed diet on larval growth may be to stimulate the larval life cycle, and 15% larval‐fed fermented aloe vera mixed diet exhibits potent hepatoprotective effects on CCl₄‐induced liver injury in rats, likely due to increased glutathione contents and the inhibition of lipid peroxidation.     

Biologically active fibers based on chitosan-coated lyocell fibers

The possibilities of obtaining biologically active cellulose–chitosan fibers were examined. An effective two-stage method was developed. The first stage involves the formation of dialdehyde cellulose by the potassium periodate oxidation of lyocell fibers, which is able to form Schiff’s base with chitosan. In the second stage, chitosan-coated lyocell fibers were prepared by subsequent treatment of oxidized lyocell fibers with a solution of chitosan in aqueous acetic acid. The impact of this two-stage protocol on the chemical and physical properties of lyocell fibers was evaluated by determining carbonyl group content, fineness and tensile strength of fibers, as well as chitosan content in the composite cellulose–chitosan fibers. Antibacterial activity of the chitosan-coated lyocell fibers against different pathogenens: Staphylococcus aureus and Escherichia coli, was confirmed in vitro experiments.     

Biodegradablescaffolds based on chitosan: Preparation,properties, and use for the cultivation of animal cells

The influence of the conditions of the formation of chitosan hydrogels crosslinked with glutaraldehyde (GA) or genipin (the polysaccharide molecular weight, pH level, and concentration of the chitosan solution) on the gel time and the properties of biopolymer scaffolds for tissue engineering obtained by the freeze-drying of hydrogels was studied. The resulting scaffolds had different structures (morphology, degree of anisotropy, average pore size) and moisture-retaining capacities. The cytotoxicity of biodegradable scaffolds based on chitosan with a low content of genipin and GA was studied for the first time. Using the L929 mouse fibroblasts model line, we demonstrated that scaffolds based on chitosan with a molecular weight of 320 and 190 kDa crosslinked with genipin and GA (0.005 and 0.01 mol/mol of chitosan amino groups) are biocompatible. Using confocal laser microscopy, we demonstrated that the cells are uniformly distributed in all scaffold samples and they successfully grew and proliferated when cultured in vitro for 4 days.     

Novel bioactive maloyl glucans from aloe vera gel: isolation, structure elucidation and in vitro bioassays

In this study, three novel maloyl glucans were isolated at temperatures below 15 degrees C from aloe vera gel (Aloe barbadensis Miller). These compounds were characterized using NMR spectroscopy, ESIMS, MALDITOF-MS and capillary electrophoresis. The compounds were characterized as 6-O-(1-L-maloyl)-alpha-,beta-D-Glcp (veracylglucan A), alpha-D-Glcp-(1-->4)-6-O-(1-L-maloyl)-alpha,-beta,-D-Glcp (veracylglucan B) and alpha-D-Glcp-(1-->4)-tetra-[6-O-(1-L-maloyl)-alpha-D-Glcp-(1-->4)]-6-O-(1-L-maloyl)-alpha,-beta-D-Glcp (veracylglucan C). These unusual malic acid acylated carbohydrates were then tested in vitro for effects on cell proliferation and gene expression of proinflammatory cytokines, IL-6, IL-8 and ICAM-1, using RT-PCR. Veracylglucan B demonstrated potent anti-inflammatory and anti-proliferative effects, while Veracylglucan C, on the other hand, exhibited significant cell proliferative and anti-inflammatory activities. Veracylglucan A could only be isolated in smaller quantities, and it proved to be very unstable. Thus no biological effects could be observed in this respect. The in vitro bioassays also indicated that Veracylglucan B and C are antagonistic and competitive in their effects on cell proliferation. The results of this work represent a major step forward in the research on aloe vera gel. This is the first time that two fully chemically characterized compounds are shown to be responsible for known biological activities of aloe vera gel.     

Chitosan–caffeic acid–genipin films presenting enhanced antioxidant activity and stability in acidic media

The use of chitosan films has been limited due to their high degradability in aqueous acidic media. In order to produce chitosan films with high antioxidant activity and insoluble in acid solutions caffeic acid was grafted to chitosan by a radical mechanism using ammonium cerium (IV) nitrate (60 mM). Genipin was used as cross-linker. This methodology originated films with 80% higher antioxidant activity than the pristine film. Also, these films only lost 11% of their mass upon seven days immersion into an aqueous solution at pH 3.5 under stirring. The films surface wettability (contact angle 105°), mechanical properties (68 MPa of tensile strength and 4% of elongation at break), and thermal stability for temperatures lower than 300 °C were not significantly influenced by the covalent linkage of caffeic acid and genipin to chitosan. Due to their characteristics, mainly higher antioxidant activity and lower solubility, these are promising materials to be used as active films.     

Antibacterial Activity and Drug Release of Chitosan Sponge Containing Doxycycline Hyclate

The purpose of the present study was to develop and characterize the chitosan sponges loading with doxycycline hyclate and their antibacterial activities. The pore density of chitosan sponge prepared with freeze drying technique was increased as the higher concentrated chitosan solution was used. The sponge prepared from 10% w/w of the chitosan solution and crosslinking with glutaraldehyde solution was utilized for loading with doxycycline hyclate. The drug release and sustainable antibacterial activity of fabricated sponge were assessed using dissolution test and agar diffusion test, respectively. Drug release from non-crosslinked sponge into phosphate buffer pH7.4 was slower than that from crosslinked sponge since the former could absorb the medium and form gel to retard the initial drug diffusion. Sustainable antibacterial activity of developed sponge was evident against S. aureus and E. coli. In conclusion, the in vitro release profile and antibacterial efficiency indicated that doxycycline hyclate could be sustained using chitosan sponge.     

Staphylococcus aureus cell wall structure and dynamics during host-pathogen interaction

Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus missing penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which coincided with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease.     

Suppression of Splenic Lymphocyte Proliferation by Eucommia ulmoides and Genipin

We investigated the modulation of innate and adaptive immune cell activation by Eucommia ulmoides Oliver extract (EUE) and its ingredient genipin. As an innate immunity indicator, the phagocytic activity of macrophages was determined by measuring engulfed, fluorescently labeled Escherichia coli. As a surrogate marker for the respective activation of cellular and humoral adaptive immunity, concanavalin A (Con A) and lipopolysaccharide (LPS) induction of primary splenocyte proliferation was assayed in in vitro and ex vivo systems. EUE and genipin suppressed the proliferation of primary splenic lymphocytes induced by Con A or LPS, but not macrophage phagocytosis. Oral administration of EUE and genipin to mice decreased splenic lymphocyte proliferation induced by Con A or LPS. These results revealed that E. ulmoides and genipin suppressed cellular and humoral adaptive immunity, and they suggest that E. ulmoides and genipin are promising candidates for immunosuppressive drugs that target diseases that involve excessive activation of adaptive immunity.     

Evidence on antimicrobial properties and mode of action of a chitosan obtained from crustacean exoskeletons on Pseudomonas syringae pv. tomato DC3000

Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) causes bacterial speck of tomato, a widely spread disease that causes significant economical losses worldwide. It is representative of many bacterial plant diseases for which effective controls are still needed. Despite the antimicrobial properties of chitosan has been previously described in phytopathogenic fungi, its action on bacteria is still poorly explored. In this work, we report that the chitosan isolated from shrimp exoskeletons (70 kDa and 78 % deacetylation degree) exerts cell damage on Pto DC3000. Chitosan inhibited Pto DC3000 bacterial growth depending on its concentration, medium-pH, and presence of metal ion (Mg⁺²). Biochemical and cellular changes resulting in cell aggregation and impaired bacterial growth were also viewed. In vivo studies using fluorescent probes showed cell aggregation, increase in membrane permeability, and cell death, suggesting the chitosan antibacterial activity is due to its interaction as a polycation with Pto DC3000 membranes. Transmission electron microscopic analysis revealed that chitosan also caused morphological changes and damage in bacterial surfaces. Also, the disease incidence in tomato inoculated with Pto DC3000 was significantly reduced in chitosan pretreated seedlings, revealing a promising action of chitosan as nontoxic biopesticide in tomato plants. Indeed, a wider comprehensive knowledge of the mechanism of action of chitosan in phytopathogenic bacterial cells will increase the chances of its successful application to the control of spread disease in plants.     

Genipin cross-linked alginate-chitosan microcapsules: membrane characterization and optimization of cross-linking reaction

The genipin cross-linked alginate-chitosan (GCAC) microcapsule, composed of an alginate core and a genipin cross-linked chitosan membrane, was recently proposed for live cell encapsulation and other delivery applications. This article for the first time describes the details of the microcapsule membrane characterization using a noninvasive and in situ method without any physical or chemical modifications on the samples. Results showed that the cross-linking reaction generated the fluorescent chitosan-genipin conjugates. The cross-linked chitosan membrane was clearly visualized by confocal laser scanning microscopy (CLSM). A straightforward assessment on the membrane thickness and relative intensity was successfully achieved. CLSM studies showed that the shell-like cross-linked chitosan membranes of approximately 37 microm in thickness were formed surrounding the microcapsule. The reaction variables, including cross-linking temperature and time significantly affected the fluorescence intensity of the membranes. Elevating the cross-linking temperature from 4 to 37 degrees C drastically intensified the membrane fluorescence, suggesting the attainment of a high degree of cross-linking on the chitosan membrane. Extended cross-linking time altered the cross-linked membranes in modulation. Although genipin concentration and cross-linking time had little effects on the membrane thickness, cross-linking at higher temperatures tended to form relatively thinner membranes.     

Preparation of <Emphasis Type="Italic">in situ</Emphasis> Injectable Chitosan/Gelatin Hydrogel Using an Acid-tolerant Tyrosinase

An in situ injectable chitosan/gelatin hydrogel was formed under slightly acidic conditions (pH 4.0 ~ 4.5) using an acid-tolerant tyrosinase, tyrosinase-CNK. A homogeneous chitosan/tyrosinase-CNK solution was prepared in one part of a dual-barrel syringe, and highly soluble gelatin in distilled water was prepared in the other part of the syringe without any additional crosslinking materials. Chitosan/gelatin hydrogel was formed in situ by simple injection of the solutions at room temperature followed by curing at 37°C. However, conventional mushroom tyrosinase did not catalyze this permanent gel formation. Tyrosinase- CNK-catalyzed glycol chitosan/gelatin hydrogel was similarly formed by this in situ injection approach. The hydrogels exhibited a high swelling ratio of 20-fold their own weight, interconnected micropores with an average diameter of approximately 260 μm and in vitro biodegradability suitable for tissue engineering and drug delivery applications. These results showed that tyrosinase-CNK-mediated chitosan/gelatin hydrogel formation has remarkable potential for the development of novel formulations for in situ injectable gel-forming systems.     

Neurite growth in 3D collagen gels with gradients of mechanical properties

We have designed and developed a microfluidic system to study the response of cells to controlled gradients of mechanical stiffness in 3D collagen gels. An 'H'-shaped, source-sink network was filled with a type I collagen solution, which self-assembled into a fibrillar gel. A 1D gradient of genipin--a natural crosslinker that also causes collagen to fluoresce upon crosslinking--was generated in the cross-channel through the 3D collagen gel to create a gradient of crosslinks and stiffness. The gradient of stiffness was observed via fluorescence. A separate, underlying channel in the microfluidic construct allowed the introduction of cells into the gradient. Neurites from chick dorsal root ganglia explants grew significantly longer down the gradient of stiffness than up the gradient and than in control gels not treated with genipin. No changes in cell adhesion, collagen fiber size, or density were observed following crosslinking with genipin, indicating that the primary effect of genipin was on the mechanical properties of the gel. These results demonstrate that (1) the microfluidic system can be used to study durotactic behavior of cells and (2) neurite growth can be directed and enhanced by a gradient of mechanical properties, with the goal of incorporating mechanical gradients into nerve and spinal cord regenerative therapies.     

不同生境中国芦荟凝胶活性成分的分析

中国芦荟产于云南元江地区,是一种极具发展潜力的芦荟品种,在全国均有引种,特别在东北地区的温室中已开始大面积种植。本项研究对不同生境的中国芦荟凝胶中活性成分的含量进行了分析。生境不同的中国芦荟凝胶中活性成分含量有明显差异。从测定结果上看,云南元江产的中国芦荟凝胶中所含的活性成分要高于黑龙江温室内产的中国芦荟;积温高、土壤呈弱酸性的条件下,中国芦荟凝胶中的蛋白质、有机酸含量较高。     

Novel starch/chitosan blending membrane: Antibacterial,permeable and mechanical properties

Antibacterial membranes were prepared from a mixture of hydrolyzed starch and chitosan. Glycerin was incorporated in the membranes to as plasticizer agent. The effects of component ratio on the mechanical and permeable properties of the prepared membranes were investigated. The elongation-at-break and water vapor transmission rate of starch/chitosan blending membranes were largely improved compared with each single component due to the interaction formed between the hydroxyl groups of starch and the amino ones of chitosan, which was confirmed by FT-IR characterizations. With the help of optical microscope, the influence of component ratio on the morphologies of starch/chitosan membranes was systematically investigated. It comes to a conclusion that extreme low or high starch content will cause an asymmetric membrane surface. To prove the antibacterial activity of obtained membranes, Escherichia coli (E. coli) was chosen as the target bacteria via optical density method. The resulted starch/chitosan membranes exhibited an outstanding antibacterial activity against E. coli.