Studies On Chemical Constituents And Multi-components Content Determination Of Tripterygium Wilfordii

Tripterygium wilfordii Hook.f., also called"Hill arsenic", is a climbing shrub of perennial, and distributes widely in south of Chang Jiang river and southwest of China. It is extremely poisonous, the debaked roots is used traditionally for treating diseases, and now the root barks and leaves are usable as well. T. wilfordii has long been used as traditional Chinese medicine to treat a number of autoimmune disorders such as rheumatoid arthritis and chronic nephritis. Pharmacology studies have demonstrated that T. wilfordii can treat inflammation, tumors, HIV/AID and Alzheimer's disease. However, T. wilfordii has low therapeutic index, and the incidence of side effects is much higher than that of other plants. The side effects of T. wilfordii mainly occur in the digestive system, urinary system, genital system, cardiovascular system, bone marrow and hematological system.T. wilfordii is of complex chemical constituents, especially some trace constituents have powerful bioactivity. The main active compounds of T. wilfordii are triptolide and celastrol, which are active components, and also toxic components. So far, nearly 300 compounds have been isolated and identified from the T. species, including sesquiterpenoids(including sesquiterpene alkaloids), diterpenes and triterpenes. About 100 compounds among them have been confirmed to be biological active, and the order of their bioactivity from strong to weak is diterpenes, alkaloids, and triterpenoids. Moreover, more and more new compounds have being isolated from T. wilfordii.To further clarify what components are mainly responsible for pharmaceutical and toxicological effects, and to ensure safety and effectiveness clinically, we have started the work on ethyl acetate extracted part of the root bark and debaeked root, to isolate their chemical constituents by various chromatographic separation technologies. On that basis, we establish determination methods for detecting contents of multi-components.1. Studies on chemical constituents of T. wilfordiiThe root barks of T. wilfordii is extremely poisonous, so the debarked roots is used for treating diseases traditionally. As there is significant difference in the chemical components between root bark and debarked root, the root of T. wilfordii was divided into two parts, and studied separately. From ethyl acetate extracted part of the root bark and debarked root, 23 compounds was isolated by various chromatographic separation technologies, including silica gel column chromatography, gel-filtration chromatograph and liquid chromatography, etc. Each compound was identified by kinds of organic spectral analysis methods, including HR MS, 1H NMR, 13C NMR, COSY, HMBC, etc.Five out of the 23 compounds are confirmed new without report previously. The 20 compounds identified from the root bark: 12,14-dihydroxy-3-oxo-abieta- 8,11, 13-triene(7); 3,4,6-trihydroxy-2-oxo-23,24-nor-D:A-Friedooleana-1,3,5(10),7- tetraene- 29-oic acid(14); 9′-hydroxy-2-nicotinoylwilforine(21); 1-desacetylwilforgine(22); 1-desacetlwilforine(23); triptophenolide (1); triptonide(2); triptolide(3); triptonolide(4); salaspermic acid(8); orthosphenic acid(9); celastrol(10); 29-hydroxyfredelan-3-one(11); demethylzeylasteral(12); wilfortrine(15); wilfordine(16); wilforgine(17); wilforine(18) and wilforidine (20), the compound 7, 14, 21, 22 and 23 are new compounds.The 4 compounds identified from the root core: triptonoterpene(5),triptoquinone A (6),orthosphenic acid(9),3β-22βdihydroxy-olcan-12-en-29-oic acid (13).Because both root barks and debarked roots contain orthosphenic acid, totally 23 compounds were isolated from the root barks and the debarked roots of T. wilfordii, which belong to such classes as diterpenes, triterpenes and sesquiterpene alkaloids in this study. Five out of 23 are novel compounds. Among the above compounds, compounds 21, 22 and 23 have been proved to be novel alkaloids with sesquiterpene alkaloids skeleton, which are all wilfordate type. Compound 7 has been proved to be a new diterpene with triptonoterpene skeleton, and there are two hydroxyls substituted in the benzene ring at the same time, which enriches the substituent groups of this kind of diterpene. Compound 14 has been proved to be a novel triterpene with friedelane skeleton. All of the new findings increase the structures of alkaloids and terpenes in T. wilfordii, expand the diversity of the compounds skeleton, which lay the foundation for further research and exploitation to T. wilfordii.2. Studies on multi-components content determination of T. wilfordiiThe method of Rapid Resolution Liquid Chromatography has been established for the determination of eleven bioactive components, namely triptolide, wilforidine, wilfortrine, triptophenolide, wilfordine, wilforgine, triptonolide, wilforine, demethylzeylasteral, celastrol and wilforlide A. The eleven target components were separated by ZORBAX Eclipse Plus C18 column (100mm×2.1 mm, 1.8μm) with a gradient mobile phase consisting of acetonitrile and water at a flow rate of 0.5 ml.min-1, and simultaneously quantified by detecting method of dual– wavelength of 220nm and 210nm. The results showed that the eleven compounds had a good linearity in their respective range of concentration, and the average recovery was 95.6% -103.5%, and RSD was 1.0% - 4.8%.As the significant difference in chemical components among the root barks, debaked roots, stems and leaves, the samples of T. wilfordii were divided into four parts, namely root barks, debaked roots, stems and leaves, and separated and enriched by OASIS? HLB 3cc(60mg)solid-phase extraction tube. The results showed that the stems and leaves contained smaller amount of compounds than that the root bark and debaked roots did. The stems and leaves mainly contain some large pore compounds and some triterpenoids non-identified, and little alkaloids. Eleven samples, eight from GAP-based and three from uncultivated, were divided into root barks, debaked roots, stems and leaves, and were analyzed. The results showed that triptolide, celastrol and alkaloids were mainly resided in the root barks. Among the 8 GAP-based samples, the contents of celastrol ranged 6030μg-14463μg/g in the root barks, and 0-569μg/g in the debaked roots. There is significant difference between the two parts. The content of triptolide is also higher in the root barks than that in the debaked roots, which shows 29-98μg/g in the root barks, and 0-18μg/g in debaked roots. The root barks mainly contains alkaloids (mg/g), such as wilfortrine, wilforine, wilforgine. Compared the samples from uncultivated, the samples from GAP base contain more active components and higher content. In comparison of the samples collected in different seasons from the GAP base, the contents of diterpene, alkaloids and triterpenes were all higher in October than in June, proving that the reasonable harvest season of T. wilfordii is in autumn. Compared the root samples collected in the years of 2007 and 2009 with a diameter of about 4-6cm, we found that the thiner roots had the higher content of triptolide than the crude roots did. This finding is of coincidence with previous report, which hints that using the roots of younger plant can increase the economic returns. The results also showed that the leaves collected in autumn had much higher content of triptolide than the debaked roots did, and the highest content is 90μg/g, which provided a basis for the reasonable utilization of T. wilfordii.Wilforlide A is the content-control index in Lei Gong Teng Duodai tablet, which is a preparation made from the debaked roots of T. wilfordii. This study also used the diode array detector of HPLC and the identification of the mass spectrum to identify the target compounds of samples analyzed, and the results demonstrated that the retention times of chromatographic peak, between the analyzed sample from the debaked roots and the stems with wilforlide A, had a tiny difference (±0.5min), and spectrogram were also coincidence with wilforlide A. Through the mass spectrum analysis, wilforlide A molecular ion peak was not observed, judged that the samples contain no wilforlide A preliminary.In a word, with the studies on chemical components and the content determinations of 11 compounds such as diterpenes, alkaloids and triterpenes from the different parts, namely root barks, debarked roots, stems, and leaves of T. wilfordii, we have proved that there is significant difference among different parts of T. wilfordii. The major bioactive and toxic compounds are in root bark. The higher content of triptolide, a diterpenoid epoxide and the major active compound of T. wilfordii, is also found in autumn-collected leaves. Those findings provide with technique support and solid data for the futher reasonable utilization, and for safety, effectiveness and quality control of T. wilfordii in clinical application.