| Berberis plants are rich in alkaloids,and alkaloids are mostly physiologically active components of many traditional Chinese medicines.Studies on alkaloids of Berberis plants are mainly restricted to their roots and stems,but previous studies have already found that their fruits also contain alkaloids.Therefore,in order to promote the exploitation of the fruit resources of Berberis plants,the alkaloid content in its fruit was determined.In addition,the observation of the microscopic characteristics of pericarp powders also can provide basic data for the identification of fruit species in this genus.With the microscope,we have conducted comparative observations of the microscopic characteristics of pericarp powders of 13 wild species of Berberis(Berberidaceae),which were collected in Sichuan,Yunnan,Tibet and Shandong provinces.Single factor combined with orthogonal design method was used to optimize the extraction process of total alkaloids in the pericarps,and then the total alkaloid contents of the pericarps of 13 species of Berberis were determined by using acid dye colorimetric.The contens of five monomeric alkaloids(magnoflorine,berberamine,jatrorrhizine,berberine and palmatine)were determined in the pericarp of 13 species of Berberis by ultra performance liquid chromatography(UPLC),and their interspecies differences were analyzed.The main results are as follows:1.Among the colours of pericarp powders of 13 species of Berberis,the pericarp powders of B.pruinosa,B.verruculosa and B.wangii were brown-yellow or yellow,the pericarp powders of B.hemsleyana,B.amurensis and B.liophylla were red-brown,and the pericarp powders of the other 7 species were all purple brown.The cells of parenchymal tissues of the pericarps were basically outlined in quadrangle to hexagon,and were15~60 μm in length,10~50 μm in width,and 1~5 μm in thickness.The pericarp powders of 11 Berberis species all contained stone cells,except B.phanera and B.amurensis,and there were obvious interspecies differences in their shapes and sizes.The pericarp powders of 13 species of Berberis all contained spiral vessels and scalariform vessels,5~19 μm in diameters.2.The pericarps of 13 species of Berberis all contained calcium oxalate cluster crystals and square crystals,and there existed certain interspecific differences in both shape and size of the calcium oxalate cluster crystals,but variation range of the sizes of calcium square crystals was relatively big within and between species.The diameter of calcium oxalate cluster crystals was 6~35 μm,the larger cluster crystals were found in B.liophylla(14~25μm),B.pruinosa(12~25 μm),B.wangii(11~35 μm)and B.davidii(11~26 μm),while the smaller cluster crystals were found in B.taliensis(8~11 μm),B.lijiangensis(8~13 μm),B.phanera(8~12 μm),etc.The calcium oxalate square crystals were 5~36 μm in length,and3~25 μm in width,the larger calcium oxalate square crystals were found in B.pruinosa(8~36×5~19 μm),B.liophylla(6~36×5~19 μm),B.wangii(7~65×5~22 μm),while the smaller ones were observed in B.taliensis(9~13×3~8 μm),B.sanguinea(10~17×7~10μm),B.amurensis(8~15×8~11 μm),ect.3.Taking B.pruinosa as an example,the extraction process of total alkaloids in the pericarps had been optimized.According to orthogonal tests,the best extraction conditions were compared and summarized as follows: p H of extraction liquid 3.5,the extraction temperature 60 °C,the extraction temperature 110 minutes,the additive amount of cellulase and pectinase 2 mg,ratio of liquid to solid 250:1 m L·g-1,and the extraction power 72 W.UPLC conditions for the extraction of five monomeric alkaloids were as follows: Agilent ZORBAX Eclipse Plus C18 column(2.1×50mm,1.8 μm).0.2% phosphoric acid solution(A)and methanol(B)were selected as an mobile phase,the gradient elution mode being: 17% B at 0~5 min,17~30% B at 5~8 min and 30~36% B at 8~14 min,flow rate being 0.3 m L·min-1,detection wavelength 230 nm,column temperature 40°C,and the injection volume 2 μL.4.The total alkaloid contents in the pericarps of 13 species of Berberis were4.132~51.192 mg·g-1,the higher alkaloids content of tatal alkaloids were found in B.pruinosa(51.192±0.786 mg·g-1),B.sanguinea(33.351±0.565 mg·g-1),B.davidii(15.402±0.386 mg·g-1),B.wangii(12.942±0.324 mg·g-1),while,relatively lower content of total alkaloids were found in B.hemsleyana(4.132±0.102 mg·g-1),B.replicata(4.251±0.105mg·g-1)and B.liophylla(4.608±0.114 mg·g-1).5.Five monomeric alkaloids were detected in the pericarps of nine Berberis species,except B.davidii,B.verruculosa,B.taliensis and B.replicata,from which berbamine was not detected.The content of magnoflorine varied from 0.147 to 13.827 mg·g-1,the content of jatrorrhizine varied from 0.047 to 13.620 mg·g-1,the content of berberine varied from 0.081 to 7.695 mg·g-1,and the content of palmatine varied from 0.523 to 12.375 mg·g-1.In addition,the content of magnoflorine in B.sanguinea(13.827±0.035 mg·g-1)and B.davidii(12.077±0.033 mg·g-1)were highest,the content of jatrorrhizine,berberine and palmatine in B.pruinosa was 13.620±0.140 mg·g-1,7.695±0.145 mg·g-1 and 12.375±0.035 mg·g-1,respectively.Berbamine was detected in the pericarps of 9 species,the conent of berbamine varied from 0.020 to 0.434mg·g-1,but their contents were all lower than the other four monomeric alkaloids,with the highest content found in B.sanguinea(0.434±0.044 mg·g-1).According to the results of various analysis methods with the contents of five alkaloids as the evaluation index,the higher quality species were B.pruinosa,B.sanguinea,B.sanguinea and B.davidii. |
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