Study On Pharmacognosy And Product Development Of Fomitopsis Pinicola

Fomitopsis pinicola has a long history as a folk medicinal fungus in northeastern China.The fungus tastes slightly bitter,flat,and has been used for the treatment of cold leg disease.It has a variety of pharmacological effects such as anti-tumor,hypoglycemic,hypolipidemic,anti-oxidant and anti-inflammatory.The main anti-tumor active ingredient is3-acetoxy-8,24-wooldiene-21-acid?tsugaric acid A?.In order to better develop and utilize the resources of F.pinicola,this paper carried out pharmacological research,including authenticity evaluation,effectiveness evaluation and safety evaluation,and developed new products.In the evaluation of the authenticity,this paper completed the resource distribution and source,trait,microscopic,thin layer chromatography analysis,molecular identification,and content determination of anti-tumor active ingredient tsugaric acid A of F.pinicola by field sampling and observation of a large number of samples.The authenticity of the four goods called F.pinicola sold in the online market was verified.In terms of traits,the fruiting body of F.pinicola is semicircular or horseshoe-shaped,woody,and has an obvious mushroom aroma.The edge of the cap is from milky white to pale yellow,and has many ring edges.The color of the cap is distinct,the edges of each layer have obvious groove marks,and it is divided into the four layers of milky white,red,reddish brown and dark brown from the outside to the inside.The color of each ring growth cycle is from milky white,red,reddish brown,and finally changes to dark brown.Each ring represents one year,and most of the F.pinicola have a red band.the longer the growth period,the narrower the band.But a few red bands of F.pinicola will be dark brown due to picking in autumn and winter or after sun exposure.In terms of microscopic features,The mycelium of F.pinicola is colorless,no branches;spore colorless,smooth,elliptical,5.3-7.2×3.6-4.5?m.In the analysis of thin layer chromatography,the specific shift value was spotted at 0.3,which contained the chemical composition of tsugaric acid A.After optimizing the process of determination of active components,80%ethanol reflux extraction method was used for three times,the extraction time was 3 hours,2 hours and 1.5 hours,respectively,the amount of alcohol was 6 times,5 times and 5 times.The content of tsugaric acid A in F.pinicola was at least 0.123%under the optimized conditions.There are similar in the hyphae and spores of four kinds of F.pinicola sold in the online markets.The microscopic identification of the goods A reveals that the red band of the cap is covered with sterile silk,the edges are obvious,there are a lot of oily bubbles and red clumps,which is slightly discolored in water.After the identification of the traits,it was found that the two ends of the red band on the cover of the goods A were dark brown,and there is a trench mark in the middle of the strip,which did not conform to the growth law of the F.pinicola.It is indicating that the red band on the cover of the goods A is artificially coated with a certain paint,so the goods A is fake.After molecular identification,the B,C and D sequences of the goods are highly consistent with the gene sequences in the GenBank-registered Fomitopsis pinicola?accession number:DQ491408,HM052826?.From this,it is judged that the goods B,C,and D are genuine,and it is presumed that the goods B is 9-10 years old by the trait identification,the goods C is 3 years old,and the goods D is 6-7 years old.In the evaluation of the effectiveness,In order to verify the traditional efficacy of treating the old cold legs,this paper studied the anti-inflammatory,analgesic and antipyretic pharmacological activities of F.pinicola,and whether tsugaric acid A has been evaluated for this effect component or not.The anti-inflammatory activity of mouse ear swelling caused by xylene and acetic acid-induced peritoneal capillary permeability was investigated,and serum superoxide dismutase?SOD?and malondialdehyde?MDA?.Tissue fluid SOD,MDA,prostaglandin E₂?PGE₂?and tumor necrosis factor-??TNF-??levels were also measured in mice;using acetic acid-induced mouse writhing and hot plate induced mouse pain method to study its analgesic effect;using dry yeast to cause rat fever and endotoxin-induced rat fever model explored its antipyretic activity.The results showed that compared with the model group,the water extract and n-butanol extract with high polarity of F.pinicola could significantly reduce abdominal capillary permeability,mouse acetic acid writhing and hot plate pain response,and reduce the body temperature of fever induced by yeast and endotoxin in rats?P<0.01 or P<0.05?,followed by methanol extract?P<0.05?.Methylene chloride extract,petroleum ether extract and tsugaric acid A significantly reduced xylene-induced ear swelling in mice?P<0.01?,and significantly increased SOD levels in mouse serum and right ear tissue fluid?P<0.01 or P<0.05?,and significantly decreased MDA in mouse serum and MDA and PGE2 levels in mouse right ear tissue fluid?P<0.01 or P<0.05?.It is concluded that tsugaric acid A is one of its anti-inflammatory ingredients.The extract of F.pinicola has significant anti-inflammatory,analgesic and antipyretic effects,mainly concentrated in water and n-butanol extract with high polarity,followed by methanol extract.But the anti-inflammatory activities of different polar solvent extracts are different.In the evaluation of safety,In this paper,the mice were given six different dosages of F.pinicola fruiting body powder of 500,1000,1500,2000,4000 and 8000 mg/kg by pretest and formal test,and observed for 14 days.The results showed that the mice after administration had no abnormalities and all survived.The food intake and body weight of the mice in the administration group were higher than those in the blank group,and no lesions occurred in each organ of the mice,and the LD₅₀0 value could not be measured.The virulence of F.pinicola was extremely low,and the powder of F.pinicola at a dose of 8000 mg/kg was within the safe drug range of mice.In product development,this paper developed two products.First,the Fomitopsis pinicola beverage,under the guidance of the theory of traditional Chinese medicine,selected raw materials such as red bean,coix seed,hazelnut and honey,and developed a kind of beverage with prevention and cure rheumatism and joint pain,drinking for a long time has nutrition and health care function.Using color,odor and mouthfeel as the evaluation indicators,the optimum formula of Fomitopsis pinicola beverage was determined by orthogonal test.The optimum formula of Fomitopsis pinicola beverage was determined as 10 g F.pinicola,20 g red bean,15 g coix seed,20 g lycium barbarum fruit and 3 g honey.A more consistent quality standard has been established.The second is the Fomitopsis pinicola tsugaric acid tablets.Fomitopsis pinicola tsugaric acid tablets were prepared with tsugaric acid A as anti-tumor and anti-inflammatory.The best formula was selected by a single factor and orthogonal test methods.It was determined by experiments that 85%microcrystalline cellulose,9%sodium carboxymethyl starch,3%PVPk30 and 1.5%magnesium stearate were the optimum formulations.Three batches of Fomitopsis pinicola tsugaric acid tablets were prepared according to the optimal formula,and the quality detection of content,weight difference,disintegration time,friability,microbial limit,heavy metals and arsenic salts were carried out.The test results showed that each tablet of Fomitopsis pinicola tsugaric acid tablets contains about 0.5 mg of active ingredient,and other test indicators are in line with the 2015 edition of the Chinese Pharmacopoeia.