| Anthocyanins are a group of flavonoid compounds and exhibit not only appealing color but also promising pharmacological activities.Anthocyanins have been used as natural pigments and functional ingredients in food,cosmetic and pharmaceutical industries.Nitraria tangutorun Bobr.and Lycium ruthenicum Murr.are widespread in Qinghai-Tibetan plateau,and their fruits have been used as traditional medicinal foods for thousands of years in China.The previous reports have shown that N.tangutorun and L.ruthenicum fruits are excellent sources of anthocyanins.Green chemistry aims to reduce or eliminate the utilization or generation of feedstocks,products,reagents,etc.,which are hazardous to human health or environment.Development and application of green extraction methods and green analytical methods are of significance in the extraction processes of natural bioactive compounds.The present study aimed to develop green approaches for extraction,separation,analysis and identification of anthocyanins from N.tangutorun and L.ruthenicum.The main researches are as follows:1.N.tangutorun fruit has been developed as many products,such as juice,red dry wine and compound seed oil soft capsules,as a result,N.tangutorun seed meal was substantially generated.Our preliminary experiments showed that N.tangutorun seed meal contained anthocyanins.Development of this by-product could solve the resource waste problem.Therefore,ultrasound-assisted extraction of anthocyanins from N.tangutorun seed meal was optimized using response surface methodology.Extraction at 70 ℃ for 32.73 min using 51.15%ethanol rendered an extract with 65.04 mg/100 g of total anthocyanins and 947.39 mg/100 g of total polyphenols.An in vitro antioxidant assay showed that the extract exhibited a potent DPPH radical-scavenging capacity.Eight anthocyanins in N.tangutorun seed meal were tentatively identified according to the UV-Vis spectrum and HPLC-MS data.2.Aqueous two phase extraction,one of liquid-liquid extraction techniques,has been considered as an environmentally benign technique.Salt-ethanol aqueous two phase extraction is promising to simultaneously extract and purify anthocyanins from plant materials,which combines the solid-liquid extraction and liquid-liquid extraction in a single step.For developing aqueous two phase extraction of anthocyanins,the partition behaviors of different polar anthocyanins in NaH2PO4/(NH4)2SO4-ethanol aqueous two-phase systems were investigated,and aqueous two-phase extraction of anthocyanins from N.tangutorun and L.ruthenicum were optimized.The partition coefficient of anthocyanins increased with the rise of their hydrophobicity,and the low-polar anthocyanins(e.g.acylated-anthocyanins)exhibited a higher preference for top-phase in NaH2PO4/(NH4)2SO4-ethanol aqueous two-phase systems.NaH2PO4-ethanol aqueous two-phase system gave higher total anthocyanin yield and better selectivity of total anthocyanins and total sugars than that of(NH4)2SO4-ethanol system.Extraction at 65 ℃ for 45 min and at 45.5 ℃ for 45 min using 28%NaH2PO4 and 26%ethanol(w/w),98.91 ± 0.03%of N.tangutorun total anthocyanins(3.62±0.05 mg/g)and 99.84±0.01%of L.ruthenicum total anthocyanins(13.16±0.29 mg/g)were extracted from raw materials,respectively;and more than 70%of total sugars were removed.3.Green analytical chemistry is one of the branches of green chemistry,and it emphasizes to minimize or eliminate the use of toxic substances and the generation of wastes.Acetonitrile and methanol are frequently used as mobile phases for anthocyanin HPLC analysis,and ethanol is a greener solvent and promising to replace them.Trifluoroacetic acid and formic acid solutions have been used to improve HPLC separation and resolution of anthcoyanins.Trifluoroacetic acid is highly eco-toxic and not easy to biodegrade.Formic acid is greener because of low toxicity and easy biodegradation to carbon dioxide and water.As a result,a HPLC-DAD method was developed to analyse N.tangutorun seed meal anthocyanins using a mixture of ethanol and a 5%(v/v)formic acid aqueous solution at a 20:80(v/v)ratio as the optimized mobile phase.However,formic acid is highly volatile and has a pungent odor,and direct contact with the liquid formic acid or inhaling formic acid vapor can damage health.Therefore,the safer and greener mobile additives should be developed for anthocyanin analysis.For this purpose,fruit acids(also known as alpha-hydroxy acids),including citric acid,malic acid,tartaric acid,lactic acid,glycolic acid and ascorbic acid,were used for anthocyanin HPLC analysis,and their effects on the chromatographic behaviors of N.tangutorun and L.ruthenicum anthocyanins were investigated.Tartaric acid solutions were promising for anthocyanin HPLC analysis.A mixture of ethanol and a 0.1 mol/L tartaric acid solution was used as the optimized mobile phase for analyzing N.tangutorun anthocyanins,and 15 anthocyanin peaks could be determined within 16 min.A mixture of ethanol and a 0.3 mol/L tartaric acid solution was used as the optimized mobile phase for analyzing L.ruthenicum anthocyanins,and 15 anthocyanin peaks could be determined within 17 min.4.Solvent is an important concern in green chemistry.As a kind of green solvent,the deep eutectic solvent has attracted the attention of scientists,and it is regarded as the solvent for the 21st century.In this study,deep eutectic solvents were used to extract anthocyanins from N.tangutorun and L.ruthenicum,,and the deep eutectic solvents based on choline chloride-1,2-propanediol were demonstrated promising for extraction of total anthocyanins from N.tangutorun and L.ruthenicum.For N.tangutorun total anthocyanins,an extraction yield of 1.413±0.054 mg/g could be achieved under the optimal conditions that extraction temperature of 68 ℃,mole ratio of 1:3.4,water content of 20%,liquid/solid ratio of 15:1 and extraction time of 45 min.And for L.ruthenicum total anthocyanins,an extraction yield of 4.45±0.07 mg/gcould be achieved under the optimal conditions that extraction temperature of 52 ℃,mole ratio of 1:3,water content of 10%,liquid/solid ratio of 20:1 and extraction time of 45 min.5.The lack of volatility of deep eutectic solvents makes it fairly challenging to isolate nonvolatile compounds from them.Among the separation and purification technologies,macroporous resin separation is particularly attractive.In this study,macroporous resin separation of anthocyanins from deep eutectic solvents was investigated.According to the static and dynamic adsorption/desorption tests,LX-32 macroporous resin was selected for preparative separation of N.tangutorun and L.ruthenicum total anthocyanins from deep eutectic solvents.More than 95%total anthocyanins could be recovered.6.A green UPLC-Q-TOF-MS method was used for anthocyanin identification using a mixture of ethanol and 0.1%formic acid solution as the mobile phase,and 13 and 16 anthocyanin-derivatives were tentatively identified from N.tangutorun and L.ruthenicum,respectively.The present study demonstrated that:N.tangutorun seed meal could be used for extracting anthocyanins;aqueous two-phase extraction and deep eutectic solvent-based extraction combined with macroporous resin separation could be used for preparative separation of anthocyanins;and a mixture of ethanol and fruit acid solution was promising for anthocyanin analysis.Our study could promote the applications of green extraction and green chromatography in research and development of natural bioactive compounds. |
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