Lili Wng,Zhong Yun

a Medical and Nursing Branch,Panjin Vocational and Technical College,Panjin 124000,PR China;

b School of Traditional Chinese Materia Medica,Shenyang Pharmaceutical University,Shenyang 110016,PR China

Abstract More and more attention has been paid to the polysaccharide components of Glehnia littoralis.It has good immunoregulation,antioxidant,antitumor,hypoglycemic and other biological activities,as well as special health care functions.Therefore,it has a broad application prospect in medical,health care,food and other industries.In this paper,the extraction,purification,content determination and pharmacological activities of polysaccharides from Glehnia littoralis were reviewed,which provided a gauge for the further development and utilization of Glehnia littoralis.

Key words:Glehnia littoralis; polysaccharides; extraction process; pharmacological effect

1 Introduction

Glehnia littoralisfr.Schmidt exMiqis the dry root ofUmbelliferaeplant,which is mainly produced in Shandong,Hebei,Liaoning and other provinces.Shandong Laiyang is the real estate area,so it is also called LaiyangGlehnia.Glehnia littoralisis sweet,bitter and cold,it can return to lung and stomach channel.It has the effect of nourishing yin and clearing lung,benefiting stomach and promoting body fluid.It is used for hot dry cough of the lung,cough of phlegm and blood,hot disease,injury of saliva and thirst [1].There are many kinds of chemical components inGlehnia littoralis,such as polysaccharides,coumarins,polyacetylenes,lignins and so on [2].The content of polysaccharides is as high as 70% [3].Polysaccharide is made up of more than ten monosaccharides through glycosidic bond polymerization.In the past,it was abandoned as experimental waste for a long time.With the continuous development and utilization of polysaccharides activities,it has widely concerned by scholars and experts.The structural unit of polysaccharides is given in Fig.1.

Fig.1 Structural unit of polysaccharides

2 Research progress of extraction

The study on the activities of polysaccharides fromGlehnia littoraliswas carried out on the basis of extraction and separation,so the first task is to optimize its extraction process.At present,the main methods of polysaccharides extraction are water extraction and alcohol precipitation,acidalkali extraction,ultrasonic extraction,enzyme extraction,microwave extraction and so on.

2.1 Water extraction and alcohol precipitation

This method is to extract water-soluble polysaccharides.It is simple,and the factors that affect the extraction rate of polysaccharides need to be investigated.The factors usually include the ratio of material to liquid,extraction temperature,extraction time and so on.

Shen [4]et al.extracted the crude polysaccharides ofGlehnia littoralisby the method of water extraction and alcohol precipitation.Through the single factor test,orthogonal test was carried out on four factors:extraction times,extraction temperature,extraction time and material water ratio.Taking the extraction rate of the crude polysaccharide as the index,the best extraction process was obtained:extraction times were 3 times,extraction temperature was 90 °C,extraction time was 4 h,and the best material water ratio was 1:30.Under these conditions,the extraction rate of polysaccharides was 15.59%.Hou [5]et al.studied the four factors of ethanol concentration,extraction time,extraction times and extraction temperature.With the extraction rate of polysaccharides as the index,the best extraction process was determined by orthogonal test:ethanol concentration was 85%,extraction time was 2.0 h,times were 3,temperature was 80 °C.

Li [6]et al.extracted the polysaccharides ofGlehnia littoralisby water extraction and alcohol precipitation.The ratio of material to water,extraction temperature,extraction time,alcohol precipitation time and ethanol addition were investigated.The optimum extraction process was determined as follows:the ratio of material to water was 1:40,extraction temperature was 80 °C,and extraction time was 4 h.The yield of polysaccharides reached 34%.4 times volume of 95% ethanol,alcohol precipitation time was 8 h.Xiang [7]et al.extracted the polysaccharides ofGlehnia littoralisby hot water.The extraction temperature,time,ratio of liquid and material and times were investigated,with the extraction rate of polysaccharide as the index,the best technology was determined by star point design response surface analysis:25 times of distilled water was extracted twice at 94.9 °C for 120 min each time.

2.2 Acid alkali extraction

Acid water extraction method is under the condition of weak acid,plant cells swelling and breaking,releasing polysaccharides.Because of its strong acidity,it can break the glycoside bond and destroy the structure of polysaccharides,so it is often extracted with diluted acid.Alkaline water extraction is suitable for the extraction of acid polysaccharides,and the concentration of alkali should be diluted,otherwise the structure of polysaccharides will be destroyed,and the properties of polysaccharides will be affected,which is not conducive to the extraction of polysaccharides [8].This method is seldom used,because it can destroy the structure of polysaccharides.

Jing [9]et al.took the pretreatedGlehnia littoralisfor alkaline water extraction and acid water extraction respectively,he added the sample with 30 times volume of 0.3 mol/L sodium hydroxide solution at 25 °C for 2 h,extracted twice,and adjusted the pH of the extract to neutral with hydrochloric acid solution,thepolysaccharides alkali extraction was obtained,polysaccharides extraction rate was 24.80%.In addition,he added 30 times volume of 0.3 mol/L hydrochloric acid solution to the sample for 2 h at 50 °C,extracted twice,and adjusted the pH of the extract solution to neutral with sodium hydroxide solution,and the polysaccharides acid solution ofGlehnia littoraliswas obtained.The extraction rate of polysaccharides was 39.62%.

2.3 Ultrasonic extraction

This method can accelerate the release,diffusion and dissolution of effective substances in cells by using the cavitation,mechanical and thermal effects of ultrasound,it can significantly improve the extraction efficiency.This method does not need high temperature,the extraction time is short and the efficiency is higher than other methods.

Zhu [10]et al.used ultrasonic method to extract polysaccharides fromGlehnia littoralis.On the basis of single factor experiment,the ratio of water to material,ultrasonic power and extraction time were selected as the research objects,and the response surface method was used to optimize the extraction process.The optimal extraction process was determined as follows:the ratio of water to material was 19:1,the power of ultrasonic was 460 W,the extraction time was 23 min,and the extraction rate of polysaccharides was 60.15%.Compared with the traditional hot water extraction method,the extraction rate was increased by 60%.Jing [11]et al.obtained the sample ofGlehnia littoralisby pretreatment.On the basis of single factor investigation of ultrasonic extraction time,temperature and liquid-material ratio of polysaccharides extraction rate,the response surface analysis method of three factors and three levels was adopted.Taking the polysaccharides extraction rate as the index,the optimal extraction process ofGlehnia littoralispolysaccharides was:extraction temperature was 65 °C,extraction time was 22 min,liquid-material ratio was 22:1 (ML/g),under this condition,the extraction rate of polysaccharide was 12.13%.

2.4 Enzyme extraction method

This method is a new kind of biotechnology that can make the components of plant cell wall hydrolyze or degrade,it can make the effective components fully exposed and dissolve in the solvent.This method is mild and has high extraction rate.It is widely used in the extraction process of polysaccharides.The common enzymes are cellulase,protease,pectinase and so on.

Jing [12]et al.successively extracted the polysaccharides ofGlehnia littoralisby single enzyme and complex enzyme.Cellulase was selected by single enzyme method.The complex enzyme method selected cellulase and papain complex enzyme,and response surface analysis method was used to select the time,temperature,ratio of liquid to material,enzyme addition amount,ratio of cellulase and papain quality as the corresponding surface factors.The software design was used to optimize the extraction process,and the optimal extraction process of polysaccharides fromGlehnia littoralisby complex enzyme method was obtained as follows:enzymolysis temperature was 70 °C,enzymolysis time was 3 h,the ratio of liquid to material was 30:1 (mL/g),the amount of enzyme added was 3%,the mass ratio of cellulase to papain was 3:1,and the extraction rate of polysaccharides was 22.04% ±0.23%.Because of the specificity of enzyme,one enzyme can only hydrolyze one kind of substance,the effect of complex enzyme method is better than that of single enzyme method,besides,the papain can hydrolyze protein,which can improve the purity of polysaccharides and provide convenience for further separation and purification.

2.5 Microwave extraction

This method is to separate polysaccharides by using the effect of electromagnetic field and microwave heating.Compared with the traditional method,time and solvent are saved.

Zhou [13]et al.used microwave-assisted extraction to extract polysaccharides fromGlehnia littoralis,based on the single factor investigation of the influence of microwave power,microwave action time and ratio of material to liquid,he carried out orthogonal test and obtained the best extraction process:microwave power was 800 W,microwave radiation time was 100 s,ratio of material to liquid was 1:30 (mL/g).Under this process,the yield of crude polysaccharides was 39.3%,The content of polysaccharides was 65.4%.Wu [14]et al.extracted the water-soluble polysaccharides ofGlehnia littoralisby ultrasonic method,enzymolysis,microwave method and decocting method.The results showed that the extraction rate was 3.3% by decocting method ＞ 2.5% by enzymolysis ＞ 1.6%by microwave method ＞ 1.0% by ultrasonic method,the highest extraction rate was obtained by decocting method.

There are many ways to extract polysaccharides fromGlehnia littoralis,every method has its own advantages and disadvantages,it is very important to find efficient ways to maintain the structure and biological activity of polysaccharides.Table 1 is a comparison of the advantages and disadvantages of five extraction methods:

Table 1 Comparison of advantages and disadvantages of five extraction methods

3 Research progress of separation and purification

The polysaccharides extracted fromGlehnia littoralismust be separated and purified to get a pure polysaccharides with a single molecular weight.However,due to the complex structure and variety of polysaccharides,it is very difficult to separate and purify.At present,the separation and purification ofGlehnia littoralispolysaccharides mainly adopts column chromatography,which is relatively mild and has little impact on the structural damage and activity of polysaccharides.

Du [15]et al.extracted crude polysaccharides fromGlehnia littoralisby water extraction and alcohol precipitation,he removed protein by Sevage method,and depigmented activated carbon.After separation and purification by DEAE cellulose DE-52 column chromatography and Sephadex G-75 column chromatography,three polysaccharides components were obtained,further research showed that the components had better promotion on splenic lymphocyte proliferation in mice.

4 Research progress of content determination

As one of the main effective components,the content of total polysaccharides has been determined and some monosaccharides have been identified.

Ren [16]et al.determined the content of total polysaccharides ofGlehnia littoralisby phenol sulfuric acid method,the linear range was 1.96-6.86μg/mL (r=0.9990),and the average recovery was 97.6% (RSD=1.5%).The separation of six monosaccharides was realized by pre column Derivatization High Performance Liquid Chromatography and C18column ultraviolet detector.Xu [17]et al.qualitatively separated the polysaccharides ofGlehnia littoralisby TLC,and High Performance Liquid Chromatography was used to determine the content of polysaccharides ofGlehnia littoralisfrom different habitats and D-glucose after acid hydrolysis.The results showed that the content of polysaccharides ofGlehnia littoralisfrom different habitats were in the range of 46-54%,and the content of D-glucose was in the range of 71-75%.

5 Research progress of pharmacological activities

The polysaccharides ofGlehnia littoralishas a wide range of pharmacological effects,which can be used in the prevention and treatment of many diseases,such as tumor,low immune function,aging,hyperglycemia and so on.The details are as follows:

5.1 Immune function

The polysaccharides ofGlehnia littoralishas good immunoregulatory function,it is a natural immunoregulator.It can promote the growth of immune organs,activate immune cells,activate complement system and release cytokines.It is reported that the polysaccharides ofGlehnia littoraliscan not only inhibit the cellular immune function and the proliferation of T and B cells,but also inhibit the humoral immune function [18],which is the main active component of nourishing Yin and tonifying deficiency.

Liu [19]et al.extracted the crude polysaccharides ofGlehnia littoralis,he tested the immune indexes such as AFC,DTH,m Φ phagocytosis percentage,phagocytosis index,etc.Rong [20]et al.tested the killing activity of NK cells in spleen,the transformation function of T lymphocytes and the content of IgM of anti sheep red blood cell antibody in mice serum.Jie [21]et al.carried out the proliferation test of mouse spleen lymphocytein vitroon the polysaccharides ofGlehnia littoralis.The results showed that the sample concentration was 62.5μg/mL,the proliferation rate of mouse spleen lymphocytein vitrowas as high as 33.93%,with significant positive immune activity.

5.2 Free radical scavenging

The increase in free radicals can accelerate the aging process of human beings.The research shows that the polysaccharides ofGlehnia littoralishave a good free radical scavenging effect.

Li [22]et al.studied the free radical scavenging activity ofGlehnia littoralisand he determined the scavenging effect ofGlehnia littoralispolysaccharides on hydroxyl and superoxide radicals by colorimetry using the Fenton system and pyrogallol autoxidation reaction.The results showed that the semi inhibition rate ofGlehnia littoralispolysaccharides on hydroxyl radicals was 0.22 mg/mL,and the semi inhibition rate ofGlehnia littoralispolysaccharides on superoxide radicals was 0.25 mg/mL.Li [23]et al.prepared four polysaccharide components from Hebei genuine medicinal materials,includingGlehnia littoralis,he tested their scavenging effects on DPPH free radicals and hydroxyl free radicals.The results showed that the IC50values ofGlehnia littoralispolysaccharides to DPPH free radicals was 0.364 g/L,the IC50value of the scavenging rate of hydroxyl radicals was 4.229 g/L,both of which had a certain inhibitory effect.Zhang [24]et al.established a model of H2O2-induced neuronal oxidative damage of PC12 cells through the optimized polysaccharides extraction process ofGlehnia littoralis,she measured the survival rate of PC12 cells by MTT method,the protective effect of polysaccharides fromGlehnia littoralison H2O2induced PC12 cell damage was investigated.The results showed that polysaccharides had a certain scavenging effect on free radicals,the IC50value was 7.818 g/L (Vc was 0.072 g/L),it could reduce the degree of H2O2induced PC12 cell damage and had a scavenging effect on active free radicals.

5.3 Hypoglycemic eあect

The polysaccharides ofGlehnia littoraliscan promote insulin secretion,it can affect the activity of glucose metabolizing enzyme,and promote the effect of peripheral tissue on glucose and inhibit gluconeogenesis.

Jing [11]et al.determined the inhibitory effect of polysaccharides ofGlehnia littoralison α -glucosidase by ultraviolet spectrophotometry,he measured the absorbance value of the control and sample at 400 nm with acarbose as the control.The results showed that polysaccharides ofGlehnia littoralishad a certain inhibitory effect on α -glucosidase,when the concentration increased,the inhibitory effect increased.Its IC50value was 5.23 mg/mL.Jing [25]et al.also studied the activity of α -glucosidase inhibition of polysaccharides from ten different producing areas.The results showed that the IC50values measured were different,but the inhibition of α -glucosidase was positively correlated within a certain concentration range.Jing [26]et al.modified the polysaccharides with iron complex.The results showed that the polysaccharides and its iron complex both had a certain inhibition on α -glucosidase.When the concentration was 4 mg/mL,the inhibition rate of the polysaccharides was 23.6%,which was significantly lower than the inhibition rate of polysaccharide iron complex,the value of iron complex was 69.35%.The inhibitory effect of iron modified polysaccharides on α -glucosidase was significantly enhanced,but it still lower than that of acarbose.

5.4 Antitumor activity

Experiments showed that the compound preparations,water extractions and polysaccharides ofGlehnia littoralishad good antitumor activity and were used as adjuvant therapy for tumor.

Yuan [27]et al.treated 80 patients with advanced non-small cell lung cancer with sasanmaidong decoction.The results showed that sasanmaidong decoction had a certain effect on stabilizing tumor.Xiong [28]et al.treated patients with sasanmaidong decoction and sangjuyin for advanced non-small cell lung cancer,the effect was significantly better than that of patients with radiotherapy and chemotherapy alone.Zhang [29]et al.reviewed the literature on the treatment of nonsmall cell lung cancer with sasanmaidong decoction,he pointed out that sasanmaidong decoction could improve the immune function and life quality for patients with non-small cell lung cancer,it also had a certain impact on enhancing,reducing toxicity and stabilizing tumor.Liu [30]et al.showed that the three extracts of water-soluble substance,watersoluble substance and filter residue alcohol-soluble substance fromGlehnia littoralisall had anti lung cancer cell line (A549),anti gastric cancer cell line (SGC) and anti liver cancer cell line (HEP),especially had obvious inhibitory effects on lung cancer cell line (A549) and liver cancer cell line(HEP).Polysaccharides is the most abundant component inGlehnia littoralis,it is also the main component of water extraction.Therefore,it can be concluded that the compound preparation and water extract ofGlehnia littoralisare closely related to the function of polysaccharides in anti-tumor.Wu [31]et al.studied the anti-tumor effect with polysaccharides ofGlehnia littoralis,the result further proved that polysaccharides ofGlehnia littoraliscould effectively inhibit the growth of tumor cell lines,which was conducive to the treatment of tumor.

6 Conclusion

The polysaccharides ofGlehnia littoralisis rich in content and complex in structure.Its remarkable anti-oxidation and anti-tumor activities make it a good application in the treatment of anti-aging and anti-tumor.This paper reviews the extraction,separation and purification process,content determination and pharmacological action of polysaccharides fromGlehnia littoralis.In terms of extraction process,it is necessary to optimize the extraction process with multiple methods on the basis of ensuring the structure and extraction rate of polysaccharides.It is also necessary to elucidate the relationship between the structure and pharmacological activity of the polysaccharides,and provide the basis for the quality control of the polysaccharides fromGlehnia littoralisin the next step.