Lycium barbarum polysaccharides (LBP) extraction technology and its antioxidation activity

This study was carried out to investigate the technology of extracting Lycium barbarum polysaccharide (LBP) and its antioxidation activity, using orthogonal experimental method to optimize its extraction conditions. Fenton reaction and pyrogallol experimental method were adopted to determine the antioxidation activity of LBP. The three factors investigated in the orthogonal experiment affected the results. Different concentrations of LBP effectively cleared hydroxyl radical (OH ) and superoxide anion (O2 + ). It was concluded that the extraction of LBP by cold maceration as well as the three factors, (solvent volume, extraction time and extraction times) affects its yield, and that it has antioxidation activity.


INTRODUCTION
Barbary wolfberry is a beneficial traditional Chinese medicinal food.Barbary wolfberry, belonging to Solanaceae family, Lycium, is a perennial deciduous shrub, has more than 80 species in the whole world and is widely distributed mainly in North and South America and Eurasia.In China, it is mainly distributed in Gansu, Ningxia, Tianjin and other places.Ningxia wolfberry is considered the best (He et al., 1997).Barbary wolfberry contains a variety of active ingredients, including carbohydrate, amino acids, trace elements, vitamins, alkaloids, fatty acids, etc; of it species, Lycium barbarum polysaccharide (LBP) has the highest carbohydrate content and its total carbohydrate content is up to 46.5% (Jiangsu, 1977).
In modern pharmacology, it is confirmed that polysaccharides plants clear oxygen radicals, increase antioxidase activity, resist lipid peroxidation, reduce the production of PGs, LTs, malonaldehyde (MDA) and other lipid peroxidation metabolites.They also do not allow cell metabolism and morphological changes caused by molecular aggregation and cross linking of membrane proteins and enzymes; and finally they clear free radicals and delay senility.Some studies (Dai et al., 2010) show that barbary wolfberry can reduce serum MDA content in elderly mouse and increase the biological activity of superoxide dismutase (SOD).This indicates that it can effectively increase antioxidase activity, clear oxygen radicals and reduce lipid peroxidation level.That shows it has good antioxidation activity.
This experiment extracts, separates and purifies LBP, to prove its antioxidation activity and explore its action mechanism.

MATERIALS AND METHODS
Kunming mice with body weight of 20 ± 2 g were bought from the Institute of Laboratory Animal Sciences of CAMS; the needed reagents were domestically obtained and were of analytical grade.

Pretreatment
Ningxia wolfberry was dried at 60°C to constant weight, crushed and screened through a 100 mm diameter-mesh.The filtrate was stored for later use.

Orthogonal experimental design for LBP water extraction technology
LBP is a water-soluble polysaccharide, and its content in Barbary wolfberry is above 35%.It os mainly composed of arabinose, galactose, glucose, rhamnose, mannose and xylose (Huang et al., 1998).In order to increase LBP extraction efficiency, this experiment investigates the three main factors affecting the results namely, solvent volume, extraction time and extraction times.The levels of the factors are shown in Table 1.To precisely weigh 9 shares of sample and number them, the experiment was carried out in a 3 × 3 orthogonal design and LBP content was calculated.

Hydroxyl radical (OH -) clearing effect (Jin et al., 1996)
H2O2/Fe 2+ system (Chun and David, 2001) is adopted to generate OH -by Fenton reaction.Precisely weighed 1.5 ml phenanthroline solution (5 mmol/L) was added to 1.0 ml phosphate buffer (pH = 7.4), and simultaneously 0.2 ml 7.5 mmol/L FeSO4 and 1.0 ml 0.1% H2O2 were added.Immediately, it was mixed.Water was added to total volume of 10 ml thermostatic water bath at 37°C for 1 h; then the absorbance was measured at 536 nm.In the experiment, LBP extracts were prepared at different concentrations (original herbs had 200 mg, 400 and 600 mg/L) with the aforementioned methods, and A536 (sample) was measured.A536 (undamaged) was measured without adding sample and H2O2.The clearance rate was then calculated.

Superoxide anion (O2 + ) clearing effect (Zhang et al., 2004)
To adopt pyrogallol autoxidation in order to initiate in vitro chemical reaction, 4.5 ml 1 mmol/L Tris-HCl buffer (pH 8.2) was put in a test tube and preheated at 25°C for 20 min; 0.2 ml 3 mmol/L pyrogallol and 2 ml LBP solution of different concentrations were added to water bath at 25°C for accurately 4 min.Then A325 value, denoted as A sample was measured.The reaction solution without extract was taken as the reference.A325 value, denoted as A damaged, was measured and the clearance rate was calculated as follows: Clearance rate = (A damaged -A sample) / A damaged × 100%

Orthogonal experiment results
The results show that the amount of water, extraction time and extraction times affect LBP extraction yield.The ranges of the factors are 2.97, 1.16 and 5.65, showing that the impacts on the experimental results are C>A>B.
The highest LBP content is 18.66% and the lowest is 12.56%, indicating that A 3 B 1 C 3 (that is, 80 ml/g water, once and 3 h extraction) is the best option (Table 2).

Experimental result for LBP clearing hydroxyl radical (OH -)
The results in Table 3 show that LBP has significant clearing effect on hydroxy radical (OH -).As the concentration of the test sample increases, the radical clearing ability of LBP increases gradually.

Result of clearing effect of LBP on superoxide anion (O 2 + )
The result shows that LBP of different concentrations has certain clearing effect on superoxide anion O 2 + , and the clearing effect presents dose-effect linear relationship as the dose increases (Table 4).

DISCUSSION
In this investigation of extraction technology, the extraction method involves cold maceration, without heating, and the main consideration is that polysaccharide is changed easily in high-temperature extraction process.This affects its yield and thereby affects the accuracy of  the results.In addition, some pigments in barbary wolfberry dissolve at high temperature and react easily with polysaccharides.This does not only affect the polysaccharide technology results, but can also bring hidden troubles to subsequent polysaccharide antioxidation experiments.Free radicals are groups or molecules with unpaired electrons and generally have short life, are unstable, active in nature etc..Under normal circumstances, the human body produces appropriate amount of free radicals for maintaining normal operation of the body.However, as age increases, free radical metabolism in tissues and body fluids slows down, excessive free radicals remain in the body, and the damage caused by free radical reactions leads to human aging and diseases.Currently, synthetic antioxidants can inhibit oxygen radical reactions, but excessive oxygen radicals in the body can cause bio-membrane damage, protein denaturation, enzyme inactivation and deoxyribonucleic acid (DNA) replication error, thus causing various diseases in the body (Valavanidis et al., 2004;Pincemail et al., 2002).Valavanidis et al. (2004); Olorunnisola et al. (2012) and Saalu et al. (2012) found that cancer (Srivastava and Mittal, 2005;Kannan, 2006), cardiovascular and cerebrovascular diseases (Sagar et al., 1992;Olfat et al., 2012), acquired immune deficiency syndrome (AIDS) and other diseases are related with oxygen radicals; therefore, the adverse effects caused by oxidation reactions coupled with oxygen radicals are of high concern and have gradually become research hotspot.OH -is active oxygen with the most active chemical properties, and its life is very short.
In the body, OH -can react with the surrounding molecules and generate level-2 free radicals, so as to destroy cell structure.Studies (Gus'kov et al., 2000;Jin Yue et al., 2007;Li, 2007a;b) show that some ingredients in some natural plants can effectively clear free radicals, protect body cells and tissues from destruction, effectively resist diseases and delay senility.This experiment explores the clearing effect of LBP on OH -through Fenton reaction, and the result shows that different concentrations of LBP can clear OH -radicals.The principle of pyrogallol autooxidation method is to use O 2 + scavenger to reduce the absorption peak area of pyrogallol autoxidation product at 325 nm and to use ultraviolet spectrophotometer to measure and indirectly calculate the O 2 + clearance rate for evaluating the antioxidation activity of the test sample.The experimental result shows that LBP has significant effects in clearing O 2 + radicals, which is consistent with the studies made by other scholars (Meng et al., 2009).This experiment extracts, separates and purifies LBP, to prove the antioxidation activity of LBP.

Table 1 .
Investigation of the levels of the factors for LBP extraction technology.

Table 2 .
Orthogonal experiment results for LBP extraction technology.

Table 4 .
Result of clearing effect of LBP on superoxide anion O2 + .