| Semen Cuscutae(Tu-Si-Zi)is dry and ripe seeds of convolvulaceous plant cuscuta australis R.Br.or cuscuta chinensis Lam,and it is a well-known traditional chinese medicine first recorded in the famous Shen Nong's Herbal as an upper grade drug.Tropism of taste:Gan Wen.It has been used to nourish liver and kidney,prevent miscarriage and improve eyesight for thousands of years.It could be used for medicine,dietary supplement and cosmetic due to its biological and pharmacological activities.Processed Tu-Si-Zi has a long history.In ancient times,the most commonly used method was wine processing method,salt processing method,frying method,huangjing juice and migan juice processing method.The species recorded on the "Tu-Si-Zi" item of China pharmacopoeia(2015 and 2010)is salt processed Tu-Si-Zi.In Qing Dynasty,Yan-Xiting concluded that processed by stir-heating with salt to fill kidney.The combination of processing and clinical application,it pointed out that processed by stir-heating with salt can change the drug into the kidney.Kidney essence of Tu-Si-Zi is closely related to the enhancement of the immune system in the theory of traditional Chinese medicine.Recently,many studies have revealed that flavonoids and polysaccharides are the main constituents of Tu-Si-Zi.They could regulate improve immune system,enhance reproductive system and have potent antioxidant and anti-aging benefits.The study is a part of the national natural fund project—the material foundation and principle of the research which Cuscuta australis processed by salt enhance the efficacy of protecting kidney.This study aimed at comparing the difference of flavonoids and polysaccharides between the Tu-Si-Zi and salt-processed Tu-Si-Zi,and to study the similarities and differences of the regulating function on the immune system.So we can analyze the changes of Tu-Si-Zi of before and after salt processing on the basis of the correlation analysis of material and drug effects.Besides,salt and heating are the two main factors of salt processing method,and the correlation of immune regulation and the influence to flavones and polysaccharide components of salt and heating have been studied,so we can clarify the key processing factors of salt processing method.Objective:1.Determination of the content of total flavonoids,total polysaccharide and monomer flavonoids of crude Tu-Si-Zi and its processed products,and to analyze the changes of flavonoids and polysaccharides before and after processing.So we can clarify the change law of material foundation before and after salt processing.2.Comparison of the monosaccharide composition of polysaccharides between the crude Tu-Si-Zi and its processed products,and to reveal the changes of polysaccharide components before and after salt processing.3.Comparative study on the immune function of kidney Yang deficiency rats of total flavonoids and total polysaccharides between the crude Tu-Si-Zi and salt processed products,and to explore the mechanism of immune regulation and its material base.4.Study the influence to flavones and polysaccharides of salt and heating,and to study the correlation between the effect of salt and heat on the immune function of rats with kidney yang deficiency.So we can clarify the key processing factors of salt processing method.Methods:1.Collection of Tu-Si-Zi from hebei,Heilongjiang,Inner Mongolia and Sichuan,and salt processing products,water processing products,water processing plus salt products and salt water mixing products have been made according to the standards.Besides,the flavonoid extracts and polysaccharide extracts were prepared of crude Tu-Si-Zi and its processed products from Hebei origin.2.Determination of the content of total flavonoids through the method of sodium nitrite-aluminum nitrate-sodium hydroxide spectrophotometry,hyperoside as the standard,and to compare the difference of the content of total flavonoids between raw products and processed products.3.Determination of the content of total polysaccharides through the method of phenol-sulfuric acid spectrophotometry,D-Mannose as the standard,and to compare the difference of the content of total polysaccharides between raw products and processed products.4.Determination of the content of five monomer flavonoid(Hyperoside?Astragalin?Quercetin?Kaempferol?Isorhamnetin)of crude Tu-Si-Zi and its processed products.Chromatographic column:Agilent TC C18(4.6 mm*250 mm,5 microns);detection wavelength:360 nm;Column temperature:45?;Flow rate:1 mL/min;Sample size:15 uL;0.4%phosphoric acid as mobile phase B,methanol as mobile phase A,gradient elution.5.A precolumn derivatization HPLC method was used to detect the monosaccharide composition of crude polysaccharides and refined polysaccharides.Chromatographic column:Agilent TC C18(4.6 mm*250 mm,5 microns);detection wavelength:250 nm;Flow rate:1 mL/min;Sample size:20 uL;0.1 mol/L phosphate buffer as mobile phase A,acetonitrile as mobile phase B,gradient elution.6.Extracts of flavonoids and polysaccharides of crude Tu-Si-Zi and its salt processed products were filled into the rat's stomach of kidney-yang deficiency for thirty days.The thymus and spleen were rapidly removed and weighed.Besides,the ability of macrophage cell,the number of white blood cells,T lymphocytes,immunoglobulin G and immunoglobulin M were detected.7.Extracts of flavonoids and polysaccharides of raw products and processed products were filled into the rat's stomach of kidney-yang deficiency for thirty days.The thymus and spleen were rapidly removed and weighed.Besides,the T lymphocytes,immunoglobulin G and immunoglobulin M were detected.Results:1.The content of total flavonoids of crude Tu-Si-Zi of hebei,Heilongjiang,Inner Mongolia and Sichuan was 1.98%?1.65%?1.56%?0.87%,respectively.Among them,the content of total flavonoids is higher of crude Tu-Si-Zi of hebei.2.Determination results showed that the change trend of the content of total flavonoids of raw products and processed products of the four areas was consistent.Tu-Si-Zi of hebei province as an example,the raw product(1.98%),salt processing product(2.07%),water processing product(2.05%),water processing plus salt product(2.06%),salt water mix product(1.99%).3.The content of total polysaccharides of crude Tu-Si-Zi of hebei,Heilongjiang,Inner Mongolia and Sichuan was 6.32%?9.80%?8.72%?8.67%,respectively.Among them,the content of polysaccharides is higher of crude Tu-Si-Zi of heilongjiang.4.Determination results showed the change trend of the content of total polysaccharides of raw products and processed products of the four areas was consistent.Tu-Si-Zi of hebei province as an example,the raw product(6.32%),salt processing product(8.56%),water processing product(7.91%),water processing plus salt product(7.79%),salt water mix product(6.08%).5.Determination results showed the content of five kinds of monomer flavonoid of crude Tu-Si-Zi was different,and the content was lower of Sichuan province.Tu-Si-Zi of hebei province as an example,the content of hyperoside,astragalin,quercetin,kaempferol,isorhamnetin in order to 0.28%,0.16%,0.009%,0.092%,0.006%.6.The change trend of the content of five kinds of monomer flavonoid of raw products and processed products of the four areas was consistent.The content of quercetin of the salt processing products,water processing products and water processing plus salt products was increases,and the content of remaining monomer flavonoid was reduced,but there was no significant change between raw products and salt water mixing products.7.The monosaccharide composition of coarse and refined polysaccharides was no different through determination.The main monosaccharide was mannose,and the rest monosaccharides were glucose,rhamnose,galactose,galacturonic acid,xylose,arabinose,etc.Besides,crude polysaccharide of Tu-Si-Zi as an example,mannose:glucose:rhamnose:galactose:galacturonic acid:xylose:arabinose for 1:0.144:0.053:0.628:0.096:0.017:0.072.8.The results of thymus,spleen,macrophage cells,white blood cells,T lymphocytes,immunoglobulin G and M showed that flavonoids and polysaccharides of raw products and salt processing products all have good immune regulation.Among them,the salt processing products had better immune effect,and polysaccharides had better immune effect.9.The results of thymus,spleen,T lymphocytes,immunoglobulin G and M showed that extracts of raw products and processing products all have good immune regulation.Among them,the immunoregulatory effect was better of salt processing products,water processing products and water processing plus salt products.Conclusions:1.The content of total flavonoids and total polysaccharides is significantly increased after salt processing.The reason may be because the processing process is a process of heating and improve the friability of seed,so improve the dissolution rate of flavonoids and polysaccharides.So the content of flavonoids and polysaccharides is increased after salt processing.2.After salt processing,the content of quercetin is increased significantly,the content of astragalin and kaempferol is reduced,and the changement of content of hyperin and isorhamnetin is not obvious.The reason may be because other glycosides composition hydrolyzed into quercetin,and astragalin and kaempferol is hydrolyzed in the process of processing.3.The precolumn derivatization HPLC method can be used to detect the monosaccharide composition of polysaccharides and refined polysaccharides.It has no obvious difference of monosaccharide composition of coarse and refined polysaccharides,and the main monosaccharide is mannose.4.The raw products and salt processing products of Tu-Si-Zi all have better effect on immunoregulation.The salt processing product has better immune effect than raw product,and polysaccharides have slightly better immune effect than flavonoids.So the salt processing product can enhance the immunomodulatory effect.5.The content of total flavonoids,total polysaccharides and monomer flavonoid of Tu-Si-Zi is different after processing.The content of total flavonoids,total polysaccharides and quercetin are all higher of salt processing products,water processing products and water processing plus salt products,but the content of remaining monomer flavonoid is reduced.It shows that heating is the main factor to affect the content of flavonoids,polysaccharides and monomer flavonoid of Tu-Si-Zi.6.The extracts of raw products and processing products all have better effect on immunoregulation.The immunomodulatory effect of salt processing products and water processing products is better than water processing plus salt products and salt water mixing products,it shows heating is the main factor to affect the immunomodulatory effect of Tu-Si-Zi.Besides,results show the way of adding salt in salt processed has certain effect on its immunomodulatory effects. |
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