Study On The Chemical Constituents And Quality Evaluation Of Herba Nerviliae Fordii

Herba Nerviliae Fordii, the leaves or the whole plant of Nervilia fordii (Hance) Schltr. (Orchidaceae), possessing effectivenesses of clearing away heat and toxic material, arresting cough, removing blood statis and stopping pain, was mainly used for treatment of pulmonary diseases. This plant has obvious effects on treating asthma, acute bronchitis and chronic asthmatic bronchitis, and has been clinically used in tranditional Chinese medicine descriptions such as Tianlong Kechuanling Capsule, Tianlongcha, and so on. N. fordii is mainly distributed in in Southern China as Guangdong province, Hainan province, Sichuan province, Yunnan province, and Guangxi Zhuang Autonomous Region of China. It also distributes in Thailand.There are three commercial specifications of this drug as Big-leaf, Middle-leaf and Small-leaf according to the leaf size. Nervilia plicata (Andr.) Schltr. was used as the substituents of N. fordii in some places. Previous studies only considered water-soluble extractives and ethanol-soluble extractives as the evaluation indicators to control Herba Nerviliae Fordii’s quality, which, was unable to make a comprehensive assessment. In order to provide scientific basis for Herba Nerviliae Fordii’s rational development and quality control, this paper has made a systematic research about the chemical constituents of the whole plant of N. fordii. On the basis of systematic research about the chemical constituents, fingerprint analysis, and simultaneous determination of multiple flavonoids have been established. The results are as follows:1.14 compounds were isolated from methanol extracts by using various chromatographic techniques and their structures were elucidated on the basis of extensive spectroscopic analysis. They were dihydrocycloeucalenol (NF-PE-1), stigmaterol(NF-PE-2), rhamnetin(NF-PE-3), stigmasterol glucoside (NF-PE-4), cerevisterol(NF-PE-5), rhamnazin(NF-EtOAc-1),rhamnocitrin(NF-EtOAc-2), rhamnazin-3-O-β-D-glucopyranoside (NF-EtOAc-3),rhamnazin-3-O-β-D-xylopyranosyl-(1â†'4)-β-D-glucopyranoside (NF-EtOAc-4),rhamnocitrin-4’-O-β-D-glucopyranoside(NF-BuOH-1),rhamnazin-3-O-β-D-glucopyranosyl-(1â†'4)-β-D-glucopyranoside(NF-BuOH-2),rhamnocitrin-3-O-β-D-glucopyr anosyl-(1â†'4)-β-D-glucopyranoside(NF-BuOH-3),4’-O-β-D-glucopyranosylrhamnazin-3-O -β-D-xylopyranosyl-(1â†'4)-β-D-glucopyranoside(NF-BuOH-4),complanatusid(NF-BuOH-5), respectively. There are three novel compounds (NF-EtOAc-4,NF-BuOH-2, NF-BuOH-4), and the compound NF-PE-1 together with NF-PE-4 are obtained from this plant for the first time.The major chemical components were confirmed to be flavonoids.2. The TLC fingerpints of Herba Nerviliae Fordii and N. plicata have been studied. The fingerprints of Herba Nerviliae Fordii of Big-leaf and Middle-leaf together with N. plicata were eatablished. Of all the peaks, Five fingerprint peaks were identified as 4’-O-β-D-glucopyranosylrhamnazin-3-O-β-D-xylopyranosyl-(1â†'4)-β-D-glucopyranoside, complanatusid,rhamnazin-3-O-β-D-glucopyranosyl-(1â†'4)-β-D-glucopyranoside,rhamnazi n-3-O-β-D-xylopyranosyl-(1â†'4)-β-D-glucopyranoside and rhamnazin-3-O-β-D-glucopyr anoside, comparing with reference substances.The common patterns of TLC fingerprints have been obtained through’Chromafinger’ solution software. The fourth, fifth, sixth and seventh peaks could not be found in zoneâ…¡of N. plicata’s common pattern and all the N. plicata samples,while all or some of these peaks could be found in zoneâ…¡of all the Herba Nerviliae Fordii samples. Thus We could evaluate the quality of Herba Nerviliae Fordii and distinguish Herba Nerviliae Fordii from N. plicata by using the TLC fingerpints.3. Chromatographic fingerprints of 15 batches of Herba Nerviliae Fordii and 5 batches of N. plicata were analysed with HPLC using a gradient elution of acetonitrile-0.2% phosphoric acid and the HPLC fingerprints of Herba Nerviliae Fordii of Big-leaf and Middle leaf together with N. plicata were eastablished. Of all the peaks, nine fingerprint peaks were identified as complanatusid,4’-O-β-D-glucopyranosylrhamnazin-3-O-β-D-xylopyranosyl-(1â†'4)-β-D-glucopyranoside, rhamnocitrin-3-O-â†'-D-glucopyr anosyl-(1â†'4)-β-D-glucopyranoside,rhamnazin-3-O-β-D-glucopyranosyl-(1â†'4)-β-D-gluco pyranoside,rhamnazin-3-O-β-D-xylopyranosyl-(1â†'4)-β-D-glucopyranoside,rhamnazin-3-O-β-D-glucopyranoside, rhamnetin, rhamnocitrin and rhamnazin comparing with reference substances.The similarity of the characteristic HPLC fingerprint modes of Herba Nerviliae Fordii of Big-leaf and Middle-leaf together with N. plicata is 0.980,0.967 and 0.863, which was calculated by a soft ware named "Similarity evaluation system for chromatographic fingerprint of TCM". Thus these two species of the same genus could be effectively differentiated by comparing their chromatograms, several chromatographic peaks could not be found in N. plicata and its common peaks’ area was significantly lower than that of N. fordii.Both the HPTLC and HPLC fingerpints could be used to disdinguish Herba Nerviliae Fordii. Comparing to HPTLC fingerprint, HPLC fingerpint possesses higher separation efficiency and higher detection sensitivity, and has high degree automation. But a colorful and visualized chromatogram could be presented in HPTLC fingerprint, and several samples could be analyzed on one plate by using HPTLC method. Two methods both have their own advantages, we could choose one of them in practical applications according to experimental needs and actual situations.4. A method for content determination of seven flavonoids in Herba Nerviliae Fordii has been established. According to the test results, rhamnocitrin-3-O-β-D-glucopyranosyl-(1â†'4)-β-D-glucopyranoside, rhamnazin-3-O-β-D-glucopyranosyl-(1â†'4)-β-D-glucopyranoside,rhamnazin-3-O-β-D-xylopyranosyl-(1â†'4)-β-D-glucopyranoside, rhamnazin-3-O-β-D-glucopyranoside could be used as characteristic markers to disdinguish N. fordii from N. plicata. None of the above markers was detected in N. plicata while all the N. fordii samples contained all or some of the above markers. On the basis of test results, the contents of the above markers should not be less than 0.064 mg·g-1,0.033 mg·g-1,0.080 mg·g-1 and 0.030 mg·g-1.The contents of seven flavonoids in all the Herba Nerviliae Fordii samples have been determined. According to content determination of 15 batches of samples,The contents of rhamnocitrin-3-O-β-D-glucopyranosyl-(1â†'4)-β-D-glucopyranoside, rhamnazin-3-O-β-D-glucopyranosyl-(1â†'4)-β-D-glucopyranoside,rhamnazin-3-O-β-D-xylopyranosyl-(1â†'4)-β-D-glucopyranoside,rhamnazin-3-O-β-D-glucopyranoside, rhamnetin, rhamnocitrin and rhamnazin are in the range of 0.064～0.733 mg·g-1,0.033～1.165 mg·g-1,0.080～2.721 mg·g-1,0.030～1.390 mg·g-1,0.044～0.584 mg·g-1,0.098～3.698 mg·g-1 and 0.047～1.870 mg·g-1, respectively. RSD of the total content of seven flavonoids is less than RSD of the content of each flavonoid, besides, metabolic connections may be exsist between these flavonoids with similar structure.Thus the total content of seven flavonoids could be used as one of the markers to control the quality of Herba Nerviliae Fordii and the limit should not be less than 2.1 mg.A systematic research was carried out to enrich the study of chemical constituents by this paper. A kind of quality evaluation system containing fingerprints and simultaneous content determination of multiple marker components was used by this paper, which was useful for the quality control of Herba Nerviliae Fordii, and useful for the further study of quality standard of Herba Nerviliae Fordii as well.