Screening And Determination Of Active Components And Analysis Of Volatile Compounds From Bamboo Leaves Of The Genus Bambusa

China is rich in bamboo resource. Bamboo leaves have been used in traditional Chinesemedicine and collected by Chinese Materia Medica. It was found that extract of bamboo leaveshas multiple biological activities, such as antioxidation, antifungi, and can be used as apharmaceutical intermediate and food additive. To develop new plant drug, dietary supplementand value-added products from bamboo resources. The leaves of B. multiplex cv. Alphonse-Karr, B. textilis McClure, B. multiplex cv. Silverstripe, B. ventricosa McClure, B. multiplex var.multiplex, B. multiplex cv. Fernleaf and B. multiplex var. riviereorum R.Maire were collectedfrom Nanjing forestry university and Jiangxi academy of forestry in China. In this study, high-performance thin-layer chromatography (HPTLC) and high-performance liquidchromatography (HPLC) methods were optimized and validated for the simultaneousdetermination of flavone C-glycosides. DPPH-TLC and DPPH spectrometry were used toscreen the compounds with antioxidant activity from bamboo leaves. Compounds showedbiological active were isolated by preparative chromatography and identified by liquidchromatography/quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) and nuclearmagnetic resonance (NMR). Volatile compounds were enriched through head space solid phasemicroextractions (HS-SPME) and hydrodistillation (HD) technologies, and analyzedqualitatively with gas chromatography-mass spectrometry (GC-MS).(1) A simple HPTLC method was developed for the simultaneous determination ofisoorientin, isovitexin, orientin and vitexin in the commercial samples of bamboo-leaf-flavonoids. The flavone C-glycosides were extracted from bamboo-leaf-flavonoids withmethanol and chromatographed on silica gel60plates in automatic developing chamber(ADC2) with tetrahydrofuran-toluene-formic acid–water (16+8+2+1,v/v/v/v) as mobile phase.Quantitation was obtained with UV detection at350nm. Polynomial calibration plots wereconstructed in the concentration range200-1200ng/zone for isoorientin and100-600ng/zonefor isovitexin and160-960ng/zone for orientin and30-360ng/zone for vitexin with good correlation coefficients (r≥0.9995). The average recovery was found to be93.95%forisoorientin,95.35%for isovitexin,99.79%for orientin and100.46%for vitexin. The proposedHPTLC method was found to be simple, precise, specific and accurate and can be used formanufacturing quality control of bamboo-leaf-flavonoids.(2) An HPLC method was optimized and validated for the simultaneous determination offlavone C-glycosides, including isoorientin, isovitexin, orientin and vitexin in the leaves ofdifferent species of bamboo. Extraction was carried out with60%methanol. The separationwas performed using a C₁₈ column (4.6×250mm,5μm) at30℃. The solvent system consistedof a mixture of water with0.5%(v/v) glacial acetic acid and acetonitrile (85/15, v/v) at a flowrate of1.0mL/min. The detection wavelength was set at345nm. Satisfactory separation ofthese marker compounds was obtained in less than30min. The optimized HPLC methodproved to be linear in the concentration range tested (0.2-100μg/mL, r2≥0.9997), accurate(88-106%). The proposed method was validated to be simple and reliable and can be a tool forquality control of bamboo leaf extract.(3) The DPPH radical scavenging activity of the leaf extract from seven different speciesof bamboo were investigated by UV-Vis spectrometry. The radical scavenging rates of sevensamples ranged from30.52%to51.48%. The same results achieved by DPPH-TLC methodrevealed that the extract of B. textilis McClure showed high antioxidant activity with an IC50of25.26mg/L. A MTT method has been used for screening the anti-tumor activity of the extractof B. textilis McClure. The results showed no cytotoxic activity against the tumor cells testedK562, BGC823andA549).(4) A qualitative survey using a simple derivatization steps with the DPPH and NP reagentwas carried out. The flavone C-glycosides and phenolic acids found in bamboo leavesexamined could be the main antioxidant compounds. To characterize the main antioxidantcompounds, the leaf of B. textilis McClure was extracted with60%methanol by ultrasonicwave technology. The leaf extract of B. textilis McClure was further fractionated usingpreparative chromatography. Three antioxidant fractions were isolated by DPPH-TLCbioactive-guided fractionation. According to the data of UV, MS, and NMR spectra, three main antioxidant compounds were identified as5,7,3',4'-tetrahydroxy-6-C-β-D-glu-xylflavonoside(1),5,7,4'-trihydroxy-6-C-β-D-glu-rha flavonoside (2) and isoorientin(3).Compound (1) and (2) were first isolated from bambusa.(5) LC-Q-TOF-MS method for analyzing the constituents in the water extract of theleaves of B. textilis McClure has been established. Of the20detected compounds includingflavonoids, phenolic acids, coumarins,10were identified from their MS, UV, NMR spectra, orby comparing the retention time and mass spectrometry data with that of reference compoundsand reference literatures. The identified compounds were as follows:5,7,3',4'-tetrahydroxy-6-C-β-D-glu-xyl flavonoside(1),5,7,4'-trihydroxy-6-C-β-D-glu-rha flavonoside(2), isoorientin(3),orientin(4), trans-p-coumaric acid(5), cis-p-coumaric acid(6), coumarin(7), vitexin(8),5,7,4'-trihydroxy-8-C-β-D-glucose-α-L-rhamnosyl flavonoside(9), isovitexin(10).(6) A valid chromatographic fingerprint method using LC-Q-TOF-MS is proposed forstudying the constituents in rabbit plasma after oral administration of the water extract of theleaves of B. textilis McClure. Three compounds, including cis-p-coumaric acid, trans-p-coumaric acid and5,7,4'-trihydroxy-8-C-β-D-glucose-α-L-rhamnosyl flavonoside, weredetected in both the water extract of the leaves of B. textilis McClure and rabbit plasma.The results prove that three compounds could be the bioactive compounds of bambooleaves.(7) To conduct comparative study on the volatile compounds from bamboo leaves.Volatile compounds were enriched through head space solid phase microextractions (HS-SPME) method, and analyzed qualitatively with gas chromatography-mass spectrometry (GC-MS), which were comparative by analyzed with the method of direct injection GC-MS basedon hydrodistillation extraction (HD). Area normalization method was employed for thesubsequent quantification. The results showed that a total of79volatile compounds wereseparated and checked out with the two methods, including acids, phenols, ketones, esters,aldehydes, etc., in which76compounds were checked out with HD-GC/MS, while18compounds with HS-SPME-GC/MS. Comparison analysis showed that there were significantsimilarity in type of volatile compounds from different bamboo leaves between the two methods. The main volatile compounds from seven different bamboo leaves by the twomethods included3-buten-2-one,4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-,(E)-,5,9-undecadien-2-one,6,10-dimethyl-,(E)-,3-buten-2-one,4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-,(E)-,3-buten-2-one,4-(2,2,6-trimethyl-7-oxabicyclo[4.1.0] hept-1-yl)-,264butylated hydroxytoluene,2(4H)-benzofuranone,5,6,7,7a-tetrahydro-4,4,7a-trimethyl-,(R)-,2-pentadecanone,6,10,14-trimethyl-,1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester. These compounds havebeen applied in the field of flavor and fragrance, food preservative, and daily-use chemical.In conclusion, validated HPLC and HPTLC methods for determination of flavone C-glycosides in bamboo leaves were developed. The bamboo leaves of B. textilis McClureshowed high antioxidant activity and three antioxidant compounds were identified. Threebioactive compounds from the water extract of the leaves of B. textilis McClure were identified.Flavor components were found in the bamboo leaves of the Genus Bambusa. These resultsshould very useful in promoting the fine production of bamboo leaves as natural antioxidantand natural medicines.