Ulvan, a sulphated polysaccharide extracted from the green algae Ulva lactuca, has a structure composed of sulphated rhamnose and glucuronic acid, iduronic acid or xylose units. Ulvan is endowed with unique properties of non-toxicity, biodegradability and biocompatibility and could possess physicochemical and biological features appropriate for biomedical applications, including osteoarthritis (OA). Hyaluronic acid (HA) is used as therapy in individuals with OA with limited success. The present study aimed to confirm whether ulvan presents improved physicochemical and biological properties over HA, namely via reducing oxidative stress, an important aspect in OA pathogenesis.
1- Physicochemical characterization: Structure elucidation, enzymatic stability and rheological studies of ulvan.
2- Biological characterization: Antioxidant and anticoagulant properties of ulvan and comparison to HA.
Ulvan extraction was performed as referred elsewhere.
Physicochemical characterization: rheological measurements were carried out on a Paar Physica MCR300 modular compact rheometer. 1H-NMR spectra were recorded on a Varian Unity Plus spectrometer. HPLC analysis was performed using a Knauer apparatus with a Sugar-Pak 1 column. Several enzymes were screened to test ulvan stability.
Biological characterization: antioxidant activity of ulvan and HA was evaluated through measurements of reducing power and scavenging capacity against superoxide and hydroxyl radicals. Anticoagulant activity of ulvan was also determined with respect to the activated partial thromboplastin time (APTT) and prothrombin time (PT).
Results and Discussion
1H-NMR spectra showed that the main repeating unit of the extracted ulvan is A3S and allowed development of a novel method to determine sulphur content (4-6%). Ulvan was resistant to enzymatic degradation by hyaluronidase, β-glucuronidase, collagenase and specific MMP-1.
The reducing power activity of ulvan at 0.5% (w/V) was 53% compared to the standard ascorbic acid test [40 μg/mL] and was much higher than HA (3%). Notably however, ulvan (1%, w/V) presented pronounced capacity to scavenge superoxide radicals (62%), contrary to HA, which was devoid of superoxide radical scavenging capability.
Regarding anticoagulant properties, unlike HA, ulvan prolonged the clotting time (APTT) and reached 336 s at 2% (w/V), which was approximately 14-fold compared with the control (23.3 s).
Ulvan demonstrated higher antioxidant performance than HA against reactive oxygen species, highlighting superiority of ulvan in providing cell protection in environments of oxidative stress. This study demonstrates non-thrombogenicity of ulvan, which is greatly relevant taking into account its potential application as an injectable biomaterial. It is important to highlight that ulvan exhibits enhanced resistance towards enzymatic degradation, which should increase its performance.