Study On The Chemical Composition Of Coffee-water Extract And Its Mechanism Of Action On Bone Cells And Bone Metabolism

Coffee belongs to the genus Coffea in the Rubiaceae family and is an important perennial evergreen cash crop.Yunnan Arabica coffee is an important specialty industry in Yunnan,accounting for more than 98%of the country’s planted area and production,and defining the position of Chinese coffee in the world.Coffee is considered as one of three major beverages in the world,along with tea and cocoa.However,studies of the composition of coffee beans,quality indicators,quality evaluation and the safety risks of long-term consumption are extremely few around the world.In particular,the impact of coffee on human bone metabolism health is still controversial.Long-term intake of coffee or coffee consumption and the relationship between bone metabolism is still lacking sufficient scientific experimental basis,and urgent in-depth research.To further reveal the relationship between coffee consumption and bone metabolism,MC3T3-E1 and RAW264.7 cells were used to establish osteoblast and osteoclast differentiation models,respectively.And to assess whether coffee aqueous extract and caffeine（comparison groups）had inhibitory or promotes the differentiation and activity of osteoblasts and osteoclasts.To clarified the mechanism of it.Then a mice model of de-ovalized osteoporosis was constructed,and the effects of coffee aqueous extract and caffeine on serum bone resorption indexes were determined by ELISA assay.μCT,HE staining,and TRAP staining were used to investigate the bone mass effect,anti-osteoporotic activity,and inhibition of osteoclastic differentiation in vivo of aqueous coffee extract and caffeine in a de-ovalized mouse model of osteoporosis.The main research results were obtained as follows:1.Differences in the quality characteristics of green coffee beans in the main production areas of China were clarified.A comparative analysis of 56 green coffee bean samples revealed that the caffeine and chlorogenic acid contents of Robusta medium-grain coffee green beans from Hainan province were significantly higher than those of Catimor coffee green beans from Yunnan province（P<0.05）.The total polyphenol content,flavonoid content and trigonelline content of the samples from the city of Baoshan were high and the overall scores of each index were good for the green coffee beans of different origins in Yunnan Province.2.The results of UHPLC-QE-MS metabolomics analysis revealed:（1）15 shared differential metabolites,including 3-hydroxycoumarin,quinic acid,4,5-dicaffeoylquinic acid,cryptochlorogenic acid,arachidonic acid,L-malic acid,alginate,and L-glutamic acid,were screened by comparing the metabolite indicators of three Arabica species of Catimor,Typica,and origin Arabica,indicating that Catimor,Typica,and Arabica are related.（2）Comparison of green coffee beans from five continents revealed that the relative contents of chlorogenic acid,isochlorogenic acid,3-hydroxycoumarin,linoleamide,palmitamide,L-glutamic acid,D-aspartic acid and L-phenylalanine were significantly higher（P<0.05）in green coffee beans from Asia than from other regions.The relative content of cryptochlorogenic acid,arachidonic acid in green coffee beans from Oceania were significantly higher than other regions（P<0.05）.The relative content of alanine was significantly higher in North American green coffee beans than in other regions（P<0.05）.The relative content of succinic acid,L-malic acid were higher in both North and South America.（4）Comparative analysis of metabolites of green coffee beans from different regions of Yunnan yielded 44 different metabolites,including 14 amino acids,11 lipids,10phenols,3 organic acids,and sugars,terpenes,alcohols,and other substances.The results showed that Yunnan Arabica coffee is richer in phenols,terpenoids,amino acids and alkaloids due to environmental,climatic and altitude factors.3.The optimal extraction process for cold nextracted coffee aqueous extract was 30 min of ultrasonic treatment time,1:12 g/m L material-to-liquid ratio and 2 times of extraction,and the extraction rate reached 20.78%under these conditions.The optimal extraction process for the heat extraction of coffee aqueous extract was 30 min of sonication time,1:12 g/m L material-to-liquid ratio and 80°C extraction temperature,under which the extraction rate reached 19.98%.4.The total sugar content of heat extract aqueous extract of dark roasted coffee beans was the highest;the reducing sugar content of light roasted heat extract and medium roasted heat extract aqueous extract were the high;the polyphenol content of light roasted heat extract aqueous extract was the highest;the flavonoid content of medium roasted heat extract aqueous extract was the highest;the effect of roasting degree and extraction method on caffeine content was not significant;the chlorogenic acid and trigonelline content decreased with the deepening of roasting degree;the effect of different extraction methods at the same roasting degree on trigonelline content was not significant;light roast heat extract coffee aqueous extract have the best DPPH radical scavenging ability and FRAP reduction ability;medium roast heat extract coffee aqueous extract have the best antioxidant ability（TEAC）.Correlation analysis revealed that DPPH radical scavenging ability was correlated with total phenol content;FRAP reducing power was correlated with total phenol,flavonoid and chlorogenic acid content.5.MC3T3-E1 osteoblast experiments revealed:（1）different roasting and extraction conditions of coffee aqueous extract and caffeine at concentrations≤50μg/m L had no effect on cell viability,but increased the ALP activity of osteoblasts and promoted osteoblast proliferation and differentiation.Mineralization experiments revealed a significant promotion of osteoblast calcium nodule production by light/medium roasted heat extract coffee（P<0.05）,when50μg/m L caffeine significantly reduced calcium nodule formation（P<0.05）,posing a health safety risk.（2）The results of cytokine changes during osteoblast growth and differentiation showed that coffee aqueous extracts with different roasting and extraction conditions had inhibitory effects on IL-6and IL-1βcytokines;light roasted heat extract/cold extract and medium roasted heat extract coffee aqueous extract had inhibitory effects on TNF-ɑcytokines and promoted the secretion of COLⅠcytokines;12.5μg/m L caffeine had inhibitory effects on IL-6 and IL-1βcytokines and promoted the release of COLⅠcytokines;12.5μg/m L m L caffeine inhibited IL-1βand TNF-ɑcytokines,but50μg/m L caffeine promoted the release of IL-6 and IL-1βcytokines and inhibited the secretion of COLⅠcytokines.（3）Osteogenesis-related marker gene expression revealed that coffee aqueous extracts with different roasting and extraction conditions and 12.5μg/m L caffeine promoted the expression of Runx2,Osterix,ALP,and COL-1 m RNA;50μg/m L caffeine inhibited the expression of Runx2,Osterix,ALP,and COL-1 m RNA.6.Exploration of the molecular mechanisms of the effects of coffee aqueous extract and caffeine on osteoblasts by Western blot revealed that coffee aqueous extract promoted the protein expression of COL1A1,Runx2,and Osterix via inhibiting the phosphorylation of AKT,IκBɑ,P65,and ERK;12.5μg/m L caffeine inhibited the protein expression of AKT,IκBɑ,P65,ERK JNK and P38 phosphorylation and promoted the protein expression of Runx2 and Osterix.However,50μg/m L caffeine promoted the phosphorylation of AKT,IκBɑ,P65,JNK,inhibited the protein expression of Runx2 and Osterix,and decreased the osteogenic.7.RANKL-induced RAW264.7 osteoclast experiments revealed:（1）different roasting and extraction conditions of coffee aqueous extract and caffeine at concentrations≤50μg/m L had no effect on cell survival,but reduced the TRAP viability of osteoclasts and inhibited osteoclast proliferation and differentiation.However,50μg/m L caffeine increased the number of multinucleated cells and promoted osteoclast proliferation.（2）The results of osteoclast growth and differentiation cytokines showed that coffee aqueous extracts with different roasting and extraction conditions inhibited IL-6,IL-1βand TNF-ɑ;12.5μg/m L caffeine inhibited IL-1βand TNF-ɑ;50μg/m L caffeine promoted IL-6 and TNF-ɑrelease.（3）Expression of osteoclast-related gene markers revealed that aqueous coffee extracts with different roasting and extraction conditions,12.5μg/m L caffeine inhibited the expression of RANKL,TRAP,CTSK,c-FOS,and NFATc1 m RNA;the opposite result was found with 50μg/m L caffeine.8.The molecular mechanisms of the effects of coffee aqueous extract and caffeine on osteoclasts were probed by Western blot,which revealed that coffee aqueous extract inhibited the protein expression of TRAP,CTR,CTSK,MMP9,RANKL,c-FOS,and NFATc1 by inhibiting the phosphorylation of AKT,JNK,ERK,P38,IκBɑ,and P65;12.5μg/m L caffeine inhibited the protein expression of TRAP,CTR,and CTSK by inhibiting the phosphorylation of AKT,ERK,JNK,IκBɑ,and P65.However,50μg/m L caffeine promoted the phosphorylation of P38,P65,and JNK,and the protein expression of CTR,CTSK,TRAP,and RANKL.9.Caffeine and coffee aqueous extract had no adverse effects on the growth,development and organ health of de-ovulated mice.55.44 mg/kg caffeine and 1.16 g/kg coffee aqueous extract were effective in improving the left femur/right femur index,E₂level,OCN content,the content of Ca,ACP/ALP value,the content of P,and had a mitigating effect on maintaining bone homeostasis.However,higher doses of caffeine（110.88 mg/kg）as well as coffee aqueous extracts（2.32 g/kg）resulted in a loss of calcium content.μCT and HE staining revealed that 55.44 mg/kg caffeine and1.16 g/kg coffee aqueous extract significantly protected against bone loss in mice model of de-ovulatory osteoporosis（e.g.,significant increase in Tb.area,BMD,BV/TV,BS/TV,and Tb.N）.While higher doses of caffeine and coffee aqueous extract stimulated bone loss in mice model of de-ovulatory osteoporosis.TRAP staining revealed that 55.44 mg/kg caffeine and 1.16 g/kg coffee aqueous extract inhibited osteoclast differentiation in de-ovulated mice,while higher doses of caffeine and coffee aqueous extract promoted osteoclast differentiation.10.Compared sham group with the model group,49 different metabolites of mice serum were screened,mainly fatty acids,amino acids and alkaloids;39 different metabolites of mice serum were screened in the positive control group compared with the model group,mainly fatty acids,amino acids and phenols;the main different metabolites of coffee aqueous extract on mice serum were lipids,amino acids,alkaloids,nucleotides and sugars;The main differential metabolites of caffeine on mice serum were lipids,amino acids,organic compounds,hormones,and alkaloids.The pathogenesis of osteoporosis in de-ovalized mice is closely related to disorders of phospholipid metabolism,amino acid metabolism and fatty acid metabolism in the organism.Among the main pathways affected are sphingolipid metabolism,pantothenic acid and Co A biosynthesis,niacin and nicotinamide metabolism,valine,leucine and isoleucine biosynthesis,lysine metabolism,β-alanine metabolism,arginine and proline metabolism,and cysteine and methionine metabolism.In this study,the quality characteristics of green coffee beans from the main production areas of China and their differential components were clarified,and the main dominant components of Yunnan Arabica coffee were phenols,terpenoids,amino acids,and alkaloids.Coffee aqueous extract and caffeine（no more than 4-5 cups of 200 m L coffee and no more than 400 mg caffeine intake per day）have inhibited osteoclastic differentiation and function in vivo and in vitro,and significantly alleviated the bone loss due to de-ovulation.The molecular mechanism achieved by interfering with the activation of key signaling pathways NF-κB,MAPK,AKT and blocking the entry of transcription factors NFATc1 and c-FOS in vivo,thus inhibiting the expression of signature genes of mature osteoclasts（MMP9,Cathepsin K,CTR,TRAP）.Similarly,coffee aqueous extracts and caffeine promoted the proliferation and differentiation of osteoblasts and calcium junction formation by inhibiting the activation of key signaling pathways NF-κB,MAPK,and AKT and promoting the expression of signature genes of mature osteoblasts(Runx2,COL1A1,Osterix.