The protective effect of Scenedesmus dimorphus polysaccharide as an antioxidant and antiaging agent on aging rat model induced by D-galactose

Materials and chemical

Microalgae S. dimorphus collection of the Biochemical Laboratory, Department of Chemistry, Andalas University, D-gal (purity ≥ 99%) and vitamin E (purity 95%) were purchased from Sigma-Aldrich (St. Louis, MO). Vitamin E was dissolved in distilled water containing 1% Tween 80 solution. Lipid peroxidase (MDA) assay kit and superoxide dismutase assay kit were purchased from Abcam USA. CAT assay kit, Sirius Red, hematoxylin, xylene, and advanced glycation end products (AGEs) PromoKine Assay Kit were purchased from Elabscience.

Instrumentation

We used the following: ultrasonic bath (Krisbow DSA50-GL, 40 kHz), spectrophotometer UV/Vis (Spectronic Genesys 20 Visible), centrifuge (Nasco 2,000–12,000 rpm), freeze drying equipment (Biobase-BK FD12S), fluorescence spectrophotometer (JASCO FP-6500) with a Peltier thermostat (JASCO, Easton, MD), and ImageJ 1.49v software, National Institute of Health, Bethesda, MD.

Experimental design

The experiment was arranged in a randomized block design, comprising six groups with eight rats each: (1) normal control (NC), (2) polysaccharide control (PC), (3) aging control induced orally by D-gal (0.25 mg/g bw/days), (4) drug control (vitamin E), (5) aging rats treated with low-dose polysaccharides (D-gal + SDP 0.2 mg/g bw), and (6) high-dose polysaccharides (D-gal + SDP 0.8 mg/g bw) orally treated. Observations were made at intervals of 2, 4, and 8 weeks.

Extraction and purification of SDPs

The ultrasonic method was used to extract the polysaccharide S. dimorphus. The biomass extraction procedure was immersed in distilled water (130 w/v) in a flask that was placed into an ultrasonic cleaning bath (preheated at 60°C, 24 minutes with 300 W, and incubated at 90°C, 2 hours). The cell debris extract from the polysaccharides was removed by centrifuge. To concentrate the polysaccharide solution, a rotary evaporator was used and was precipitated with four volumes of 95% (v/v) ethanol for 24 hours at 4°C. The precipitate was separated by centrifugation (10,000 g/minutes, 15 minutes), and the associated protein was released by trichloroasetic acid and the SDP crude extract was freeze-dried (Wei et al., 2005).

Collecting blood

Blood specimens of Wistar rats were separated between serum and erythrocytes using centrifugation (5,000 g) for 30 minutes. Blood was collected from the neck veins under deep anesthesia and drawn using a blood tube, for analysis of SOD, CAT, MDA, and fluorescent-advanced glycation end products (F-AGEs) (Armaini et al., 2020).

In vivo enzymatic antioxidant activity test

Enzymatic antioxidant activity was determined by the colorimetric method; an SOD assay was carried out using an SOD Assay Kit (Abcam code ab.65354), a CAT (hydrogen peroxidase) assay was carried out using CAT Assay Kit (Elabscience, code E-BC-K102-S), and a lipid peroxidase assay was carried out using an MDA Assay Kit (Abcam code ab. 233471).

Measurement of F-AGEs

F-AGEs were measured with the spectrofluorometry method, using an AGE PromoKine Assay Kit. The wavelength of excitation is 360 nm and emission is 460 nm. The blanks used were phosphate buffer saline solutions. Fluorescence intensity is calculated in arbitrary units per milliliter (AU/ml) and total protein in AU/g, and the Biuret method was used for determining total protein.

Hydroxyl radical scavenging activity

A 1 ml SDP solution with various concentrations (0.4, 0.8, 1.2, 1.6, and 2.0 mg/ml) was prepared, and 1 ml of FeSO4 (9 mM), 1 ml of salicylic acid and ethanol (9 mM), and 1 ml H2O2 (9 mM) were added and mixed and incubated at 37°C for 30 minutes. Their absorbance was measured at 510 nm. Distilled water in the same volume replaced the sample used as the control group.

The hydroxyl radical scavenging activity was expressed as follows:

Scavenging rate of hydroxyl radical (OH^•) (%) = ([A₀ – A₁]/A₀) × 100,

where A₀ is the absorbance of the control group and A₁ is the absorbance of the sample.

Determination of superoxide radicals scavenging activity

The superoxide radical (O₂^•-) scavenging activity of SDP was determined using Halliwell’s method with some modifications. The sample was prepared with a volume of 4 ml and then 2.5 ml of Tris-HCl buffer (50 mM, pH 8.2) and 0.6 ml pyrogallol (25 mM) were added and mixed and incubated at 25°C for 5 minutes. HCl 1 ml was added to stop the reaction and the absorbance was measured at 560 nm.

Scavenging activity was calculated based on the following formula:

Scavenging rate of superoxide radical (O₂^•-) (%) = ([A₀ – A₁]/A₀) × 100,

where A₁ is the absorbance of the sample and A₀ is the standard absorbance (water not H₂O₂); ascorbic acid was used as a positive control.

Histopathological preparation procedures of rat skin tissue

The skin on the back of the rats was taken using an incisional biopsy technique, using a scalpel to make an incision. The rat was anesthetized using ether, an incision biopsy size of approximately 0.5 cm square was made, and the biopsy tissue was processed into histological preparation.

Staining procedure with Sirius Red

Rat skin tissue was fixed with 10% formalin and then processed into paraffin blocks. Paraffin blocks of 4 mm thickness were cut with a rotary microtome and placed on the slide. Blocks were deparaffinized with xylene (2 × 5 minutes) and rehydrated with multilevel alcohol, beginning with 100% ethanol, followed by 96% and then 70%, and distilled water, for 5 minutes each. The preparation was stained with hematoxylin for 8 minutes, rinsed with aquadest for 10 minutes, and then soaked in a Sirius Red solution in saturated picric acid for 1 hour. Subsequently, it was rinsed in acidified water (2 × 5 minutes) and dehydrated in graded alcohol, with 70%, 96%, and 100% ethanol, for 5 minutes each. Clearing in xylene 2 × 5 minutes, the glass deck was mounted with Entellan.

Assessment procedure

The fibrocollagen matrix was red due to the coloration with Sirius Red. Measurement of the fibrocollagen matrix area was carried out by taking photomicrographs at 100× magnification (objective 10×) in five different fields. The red-stained area was measured using the ImageJ program (ImageJ 1.49v software, National Institute of Health, Bethesda, MD), by isolating the red color in Sirius Red staining and calculating the proportion of pixels colored red compared to the total pixel unit area. The proportion of fibrocollagen matrix area was reported in percent.

Statistical analysis

The data expressed are mean ± standard deviation; sample (n = 8) at p < 0.05 was considered statistically significant. Statistical analysis was made using analysis of variance (ANOVA) (with two factors with replication) of Microsoft Excel 2016. Estimates of animal friction or mortality in this study expect a reduction of 10%.

Enzyme activity of SOD

Wistar rats induced by the D-gal group experienced an increase in ROS, which resulted in aging. Increased ROS resulted in a decrease in SOD activity of D-gal-induced rats compared to that of normal rats (NC). The activity of SOD enzymes in the body was higher in rats administered with SDP than in NC and drug control (vitamin E). The result of the SOD enzyme activity test in each treatment can be seen in Table 1. The administration of SDP for 2–8 weeks caused a significant increase in SOD activity compared with SOD activity in NC and rats administered with vitamin E. Statistical tests with ANOVA at p < 0.05 showed significant differences in each treatment group. Aging rats treated with vitamin E displayed a higher increase in SOD enzyme activity than that of NC at 8 weeks of observation time.

Table 1. SOD enzyme activity in various treatments with 2–8 weeks of observation time. [Click here to view]

Table 2. Activity of CAT enzymes in various treatments with 2–8 weeks observation time. [Click here to view]

Enzyme activity of CAT

Wistar rats induced by D-galactose caused ROS levels to high in the body so that it can reduce the activity of CAT enzymes and cause oxidative stress. Decreased CAT is triggered by an increase in ROS, which is observed 2–8 weeks after the D-gal treatment. In this study, the results of the CAT enzyme activity test in all treatment groups were observed for 2–8 weeks as shown in Table 2. The data obtained showed that rats administered with SDP for 2–8 weeks displayed a higher increase in the activity of CAT enzymes than the NC and rats administered with vitamin E as a drug control. An SDP dose of 0.8 mg/g bw for 8 weeks is an effective dose to increase CAT activity. Analysis of variances at p < 0.05 showed significant differences among treatment groups, NC and aging control (group treated with D-gal).

Enzyme activity of malondialdehyde (MDA)

Increased MDA activity was observed in aging control due to the induction by D-gal in Wistar rats for a duration of 2–8 weeks. D-gal causes oxidative stress due to increased ROS, causing lipid peroxidation. Table 3 shows MDA test results observed in 2–8 weeks in each treatment group. Increased MDA activity in aging control continues until week 8 as a result of increased lipid peroxidation. D-gal can induce lipid peroxidation in the epidermis as its structure comprises phospholipids. The administration of SDP can decrease the MDA activity more than the administration of vitamin E and no administration at all (NC), at each variation of the SDP dose at observations of 2–8 weeks. Based on the ANOVA test results, there were significant differences among treatment groups, the group administered with D-gal as aging control (^bp < 0.05), and the NC (^ap < 0.05).

In vitro test of antioxidant activity

An assay of in vitro antioxidant activity of SDP was done by measuring the scavenging activity against free radicals. Based on the results of testing, the SDP scavenging activity against hydroxyl radicals (OH^•) is quite high, including strong antioxidants with IC₅₀ = 0.1 mg/ml (100 ppm); superoxide radicals (O₂^•−) show the ability of low scavenging activity, including antioxidants with IC₅₀ = 0.174 mg/ml (174 ppm), as shown in Figure 1a and b. The SDP shows a high performance of scavenging activity on OH^• compared with O₂^•− scavenging activity. Measurement was conducted using vitamin E as a standard with IC₅₀ = 57.114 ppm. Based on IC₅₀ values, SDP is a strong antioxidant (IC₅₀ = 50–100 ppm) and an effective antiaging agent.

Formation of collagen in Wistar rat skin

Histopathological tests were carried out on the red-colored fibrocollagen matrix in Sirius Red staining. Measurement of the fibrocollagen matrix area was carried out by taking photomicrographs at 100× magnification (objective 10×) in five different fields. The ratio of collagen in the aging control group (induced with D-gal) was lower than in the normal group. After administration of SDP at doses of 0.2 and 0.8 mg/g bw, an increase of collagen ratio in the skin of aging Wistar rats was higher than in the skin of normal NC and group treated with vitamin E as a drug control. This shows that the administration of SDP is effective in repairing collagen damage in the aging rat model. The effective dose of SDP is 0.8 mg/g bw, which can increase the amount of collagen optimally (see Fig. 2).

F-AGEs formation

The formation of F-AGEs was increased in aging Wistar rats due to D-gal induction compared with NC. D-gal induction causes an increase in ROS that can trigger the formation of AGEs in Wistar rats. The administration of vitamin E as a drug control can reduce the formation of AGEs to achieve normal conditions (like that of NC). Treatment with SDP at low doses (0.2 mg/g bw) showed a decrease in the formation of AGE; at high doses (0.8 mg/g bw), after 8 weeks of observation, the decrease in the formation of AGEs was significantly higher in rats treated with SDP than those administered with vitamin E. The administration of SDP can inhibit the formation of AGEs. This could cause a decrease in ROS because SDP has antioxidant activity. Statistical analysis (ANOVA at p < 0.05) showed a significant difference in each treatment group for aging control (group treated with D-gal).

Table 3. Malondialdehid (MDA) activities on various treatments with 2–8 weeks observation. [Click here to view]

Figure 1. (a) Hydroxyl radical scavenging activity with linear regression y = 1.85 + 497.5 x and R = 0.985; IC50 = 0.1 mg/ml. (b) Superoxide radical scavenging activity with linear regression y = 13.35 + 273.54 x and R = 0.996; IC50 = 0.174 mg/ml. [Click here to view]

Figure 2. Collagen ratio at treatment, NC, PC, D-gal (D-gal induction for aging), Vit E (drug control), D-gal + SDP 0.2 mg/g BW (aging treated with low-dose S. dimorphus polysaccharide), and D-gal + SDP 0.8 mg/g BW (aging treated with high-dose S. dimorphus polysaccharide). [Click here to view]

Figure 3. F-AGEs expressed in AU mg/g protein (mean ± SD of eight rats) and formation of fluorescent-advanced glycation end products at treatment; NC, PC, D-gal (D-gal induction for aging), Vit E (Drug control), D-gal + SDP 0.2 mg/g BW (aging treated with low-dose polysaccharide S. dimorphus), and D-gal + SDP 0.8 mg/g BW (aging treated with high-dose SDPs). Statistical analysis (ANOVA) showed a significant difference in aging control (D-gal) at p < 0.05. [Click here to view]

Figure 4. Comparison of fibrocollagen matrix deposition in animal skins, control, and treatment. The fibrocolagen matrix is colored red in Sirius Red staining. (A) NC, (B) aging control (D-gal), (C) Vit E (drug control), (D) treatment with SDP 0.2 mg/g BW, and (E) treatment with SDP 0.8 mg/g BW. Caption index in the picture; epidermis (E), dermis (D), hair follicles (F), and collagen bundle (↓) with 40× and 100× magnification. [Click here to view]

Histopathology of Wistar rat skin tissue

Photomicrographs of the rat skin showed a difference in the deposition of the fibrocollagen matrix among treatment groups. The red fibrinogen matrix on Sirius Red staining on the skin of the aging Wistar rat due to D-gal induction (Fig. 3b) shows a thinner dermis with lower matrix deposition accompanied by lower collagen deposition compared to that in NC (Fig. 3A).

In the NC, fibrocollagen matrix in the dermis between the adnexa was clearly visible and colored red by Sirius Red. The provision of vitamin E as a drug control (Fig. 3c) in rats aging due to D-gal induction shows that the group treated with vitamin E could increase matrix deposition marked by a thicker collagen matrix than NC and aging control (group treated with D-gal). Treatment with SDP (0.2 mg/g bw) in aging rats increased matrix deposition, which was more evident in the treatment group with high doses of SDP (0.8 mg/g bw), with thicker fibrocollagen matrix, diffuse, and dense (see Fig. 3d and e). Thus, SDP is useful as an antiaging agent in rats aging due to the induction of D-gal, as it repairs damage to the fibrocollagen matrix and stimulates the increase of collagen synthesis.

Enzyme activity of SOD, CAT, and MDA

The induction of D-gal can cause overall aging in various organ systems. Xu (1985) reported that mouse induced by D-gal for 6 consecutive weeks can increase galactose in their bodies, which cause accelerated aging in mouse; he found remarkable signs of aging in ICR mouse after being injected with D-gal in the retrobulbar for 8 weeks. D-gal-induced mice have been used as animal models in aging research or antiaging pharmacology (Wei et al., 2005). The aldose reductase enzyme plays a role in turning galactose into galactitol, a compound that cannot be metabolized by the body and gets accumulated in cells, thereby causing osmotic pressure changes; this can cause cell swelling, cell dysfunction, and eventually causes aging (Ye et al., 2014).

The induction of D-gal causes aging in Wistar rats due to increased ROS leading to oxidative stress. When the level of ROS exceeds cell's defense mechanisms, the production of ROS during environmental stress can pose a threat to cells, resulting in lipid peroxidation, protein oxidation, nucleic acid damage, enzyme inhibition, and ultimately cell death (Sharma and Dubey, 2005; Srivastava and Dubey, 2011; Verma and Dubey, 2003). D-gal induction causes cell death in the skin of Wistar rats because an increase in ROS triggers aging faster than in normal conditions. Oxidative stress due to D-gal induction decreases endogenous antioxidant activity (SOD and CAT) in Wistar rats, as shown in Tables 1 and 2. SOD enzymes play a role in catalyzing the conversion of superoxide to oxygen and hydrogen peroxide; subsequently, CAT converts hydrogen peroxide into oxygen and water. Superoxide anions are products aimed specifically at enzyme signaling; they are by-products of several metabolic processes that are caused by SOD activity controlling the level of various ROS (Wang et al., 2018).

Chemically, the SOD activity accelerates the reaction of superoxide anions (O2⁻) by forming hydrogen peroxide (H₂O₂) and oxygen (Hayyan et al., 2016). SOD, CAT, and GSH-Px are the three main endogenous protein antioxidants with enzymatic activity, which are the first line of defense against oxidative stress on the body (Aguilar et al., 2016). The administration of SDPs can enhance the effectiveness of endogenous antioxidant SOD and CAT to near-normal values; it is better than giving vitamin E, since SDP as an antioxidant can scavenge superoxide and hydroxyl free radicals. SDP can inhibit oxidative stress and MMP expression, causing SOD and CAT to increase. Excessive oxidative stress or inflammatory response can activate MMP because oxidative stress increases MMP regulation, including MMP-1, MMP-3, MMP-9, and MMP-13, through binding of AP-1 to MMPs and proinflammatory cytokines; it also increases MMPs to regulate and decrease elastin collagen fibers (Pittayapruek et al., 2016).

The development of MMP inhibitors is a promising antiaging strategy. Wrinkles in the skin form due to the breakdown of extracellular matrix proteins through fragmentization of collagen and MMP secretion (Naylor et al., 2011). Natural polysaccharides from Psudoelteromonas sp., low-molecular-weight fucoidan, function as antiaging and anti-inflammatory agents, since they can reduce connective tissue damage by inhibiting MMP activity and increasing collagen synthesis (Kim et al., 2018). Native fucoidan modulates MMP and fibroblast expression in humans (Moon et al., 2009). Natural polysaccharides extracted from Porphyridium have strong antiaging characteristics that have shown significant inhibitory activity of both hyaluronidase and elastase enzymes, which play an important role in skin aging (Kenny et al., 2012).

In this study, owing to the induction of D-gal, a significant increase in MDA and accelerated aging in the skin of Wistar rats used as animal models were observed at periods of 2, 4, and 8 weeks compared with those of NC and drug control (vitamin E). D-gal induction can cause oxidative stress due to increased ROS, which can induce lipid peroxidation and epididymis as the structure consists of phospholipids. Lipid peroxidation due to D-gal induction triggers an increase in ROS so that MDA levels increase. Increased ROS and decreased endogenous antioxidant activity (SOD and CAT) can disrupt the balance of the reduction-oxidation reaction (redox), causing oxidative stress.

Hydroxyl radicals (OH^•), produced by H₂O₂ at high levels due to ROS, attack polyunsaturated fatty acids in membranes, forming reactive carbon radicals that react with oxygen to form peroxyl radicals. This product results in the self-amplifying generation of lipid peroxide, which causes extensive damage and, ultimately, the disintegration of the membrane. Decomposed lipid peroxides form various aldehydes, including MDA, which are used as analytic markers of lipid peroxidation (Packer et al., 2013). Prolonged oxidative stress results in ROS, which can damage the lipid membrane of the bilayer phospholipid component of the cell wall, causing cell death to continue damaging the tissue around the cell (Del Rio et al., 2005). D-gal reacts with macromolecules such as lipids, proteins, and DNA without enzymatic reactions. The end result of this reaction is the formation of AGEs, which can be significantly accelerated by oxidative stress and trigger the occurrence of lipid peroxidation reactions as well as an increase in MDA levels (Ahmed, 2005; Gkogkolou and Bohm, 2012).

F-AGEs formation

The administration of SDP to aging Wistar rats can reduce ROS, since SDP as an antioxidant can scavenge OH^• (IC₅₀ = 0.1 mg/ml) and O₂^•− (IC₅₀ = 0.174 mg/ml), thereby inhibiting the formation of AGE and lipid peroxidation; this reduces MDA levels. Cells have special receptors for AGE (RAGE), where the stimulation of these receptors leads to the activation of transcription factors to increase the transcription of proinflammatory genes (Bierhaus et al., 2005). RAGE is expressed in mRNA and also in terms of the level of protein in fibroblasts and keratinocytes, and the expression is increased in sun-exposed skin, causing skin aging (Lohwasser et al., 2006). Since AGE can react with a variety of biomolecules, its formation leads to collagen cross-linking, which decreases flexibility (Prasad et al., 2012; Yamagishi et al., 2012). AGE, when accumulated in cells, can accelerate the physiological aging process (Chen et al., 2018). D-gal induction in treated Wistar rats caused a decreased collagen ratio compared with that in normal and drug controls (group treated with vitamin E); this was owing to a degradation of the fibrocollagen matrix, resulting in the aging of Wistar rats.

The aging process was driven by the reduction of ROS due to the induction of D-gal, resulting in the activation of MMPs and degradation resulting from extracellular matrix components, especially collagen and elastic fibers. These changes in the extracellular matrix (ECM) cause the appearance of fine wrinkles; in the dermis extracellular matrix, disorders play an important role in intrinsic and extrinsic aging. Wrinkles are caused by a reduction in collagen, elastic fibers, and hyaluronic acid. Enzymes in the ECM are responsible for processing elastic fibers, collagen, and proteoglycans (Ritz-Timme et al., 2003; Shapiro et al., 1991). Elastic fibers are structures that are rich in fibrillin, microfibrils, glycoproteins, elastin, and proteins (Kielty et al., 2002). These glycoproteins are interconnected through hyaluronic acid and build up skin tissue (Naylor et al., 2011). AGE can react with a variety of biomolecules; for example, they can result in the formation of collagen cross-bonds, which causes a decrease in skin flexibility (Lal et al., 2002). Owing to the induction of fibroblast activation, collagen cross-linking and metalloproteinase (MMP 1, 2, and 9) production increase, and AGEs greatly affect the dermis (Pageon et al., 2007). The administration of SDP in aging Wistar rats increases the collagen ratio since SDP inhibits the activation of MMPs and enzymes in an ECM, namely elastase and collagenase, which causes damage to collagen and reduces skin elasticity; this is because SDP can reduce ROS so that the synthesis of collagen can be stimulated by the SDP.

Histopathology of Wistar rat skin tissue

There were differences in the deposition of the fibrocollagen matrix among treatment groups. In normal mice (NC), the fibrocollagen matrix was seen in the dermis between adnexa. Rat aging due to D-gal induction displayed visible lower collagen matrix deposition with a thinner dermis accompanied by lower collagen deposition than that of NC (Liao et al., 2016). The dermis provides structural support for skin blood vessels; its depletion causes structural changes in the skin.

In aging skin, fragmentation of the skin collagen matrix occurs, as a result of the action of specific enzymes (MMPs) and destroys the structural integrity of the dermis, triggered by the induction of D-gal. Fibroblasts functions to produce and regulate collagen matrices that cannot attach to fragmented collagen. Loss of attachment prevents fibroblasts from receiving mechanical information and leads to collapse (Fisher et al., 1996), resulting in lower collagen deposition. In old skin, collapsed fibroblasts cause decreased levels of collagen and increased levels of collagen-degrading enzymes. In aging rats given the drug (vitamin E), collagen deposition appears thicker than in normal and aging control. This is due to vitamin E, which acts as an antidote to free radicals (ROS) so as to prevent the occurrence of AGE and inhibit the activity of MMP-1; this causes collagenase to specifically decompose certain collagens or other proteins in the dermis ECM (Lapiere, 2005; Sanz, 2016). MMPs are responsible for the physiological degradation of various ECM proteins (Page-McCaw et al., 2007). Collagen fragmentation in skin aging is a consequence of the physical and functional interactions between fibroblasts and the skin matrix as well as the accumulation of fragmented collagen which causes changes in the appearance of the skin. Only interstitial collagenase (MMP-1) is involved in the replacement of normal skin collagen, of the four collagenases expressed in humans (Kenny et al., 2012). In healthy young skin, MMP-1 expression is very low.

SDP provides an illustration of increased collagen matrix deposition; at high doses, collagen deposition caused by it is more higher and slightly higher than that caused by vitamin E. Excessive D-gal administration disrupts the synthesis of the collagen matrix in the body including the dermis; if there is fat accumulation, the subcutis accompanying the collagen deposition of the skin can be lower, marked by thin skin and sagging/reduced skin elasticity. This is what happened in the aging control group in this study. With the administration of vitamin E as an antioxidant, an increase in ROS can be prevented. The ROS free radical antidote can prevent lipid peroxidase so that no decomposition of fat or ECM including skin collagen matrix occurs; this may be the basis for increased collagen matrix deposition and bundle in vitamin E administration (Dikalov and Harisson, 2014).

The administration of SDP contains beta-glucan monomers, which have antioxidant activities with a high ability to ward off free radicals (ROS); these monomers also have an effect like that of vitamin E in increasing matrix deposition and collagen bundle at higher polysaccharide doses, activity its antioxidants higher compared to vitamin E because polysaccharides have complex ingredients such as galacturonic acid, N-acetyl-d-glucosamine, and compound hyaluronic acid in addition to antioxidants. This apparently can encourage higher collagen matrix synthesis than that of vitamin E. Natural polysaccharides have shown significant inhibitory activity of the two enzymes that play an important role in the aging process, namely hyaluronidase and elastase enzymes (Kenny et al., 2012).