Studies On Chemical Components Of Taxus Cuspidata Needles And Eupatorium Odoratum L, Microbial Transformation Research To The Active Components In Roots Of Inula Helenium L

Part one Chemical Study on the Components of Taxus cuspidata NeedlesTaxus cuspidata, also named yew and taxushouten, belongs to the genus of Taxus (Taxaceae). It mainly distributes in the mixed forest of needle broad leaf at isotherm of Chinese northeast, Japan, Korea and Russia area. The parts of Taxus cuspidata used for medical purpose are stems, branches, needles and roots, which commonly used to treat cancer, diabetes and heart disease.Constituents of Taxus cuspidata predominately are diterpenoids, as well as alkaloids, lignans and flavonoids. The clinical activity of Taxol? (paclitaxel) against breast, ovarian and other carcinomas, as well as the unique mechanism of anticancer and poor solubility in water, have spurred a worldwide search for better sources and improved analogues of this drug. Taxol? has already become the biggest sale anticancer drug in the world. But limited source, low content and slowly growth seriously restrain the development and utilization of this wonderful drug. In order to reduce the damage to wild Taxus spp. resources and relieve the situation of limited supply of Taxol?, we launched this program to investigate the constituents of Taxus cuspidata needles with the purpose to explore the new activity taxoids or taxol analoges as starting material for the semisynthesis of Taxol?.Objective: To study the components especially the taxanes in needles of Taxus cuspidata, and provides the foundation of searching for new anticancer leading compounds or Taxol? analoges. The methods such as silica gel column chromatography, preparative TLC, preparative HPLC, Sephadex LH-20 are used in the isolation and purification of the components. The structures of the pure compounds were elucidated by means of UV, MS, 1H-NMR, 13C-NMR, 1H-1H COSY, HMQC, HMBC and NOESY, etc. Methods: The air-dried Taxus cuspidata needles (3.2 Kg) were extracted three times with 95% alcohol at room temperature. The combined 95% alcohol extracts were concentrated to a residue under reduced pressure and partitioned between petroleum ether and water to de-fat and then extracted with EtOAc. The EtOAc-soluble portion was successively extracted with aqueous 5% HCl solution and aqueous 5% Na2CO3 solution. Finally we got the basic constituents (4.2 g), phenolic constituents (3.4 g) and neutral constituents (82 g) from the needles of Taxus cuspidata. The isolation and purification of the above three portions were carried out with the methods of silica gel column chromatography, preparative TLC, preparative HPLC and Sephadex LH-20 chromatography with different solvent system. The methods of TLC and HPLC were used to identify the purity of all the compounds isolated from this plant. The structures of the compounds were identified with the help of UV, MS, 1H-NMR, 13C-NMR, 1H-1H COSY, HMQC, HMBC and NOESY techiques.Results: Forty-one compounds were isolated from needles of Taxus cuspidata. Thirty-seven of them are taxanes, two of them are lignans, one sesquiterpenoid and one phenylpropanoid. The structures of them were identified as follows by spectroscopic methods:2'-Hydroxy taxine II (1), 2α, 9α, 10β, 13α-Tetraacetoxy-5α-(3'-methyl- amino-3'-phenyl)-propionyloxy-taxa-4(20), 11-diene (2), 9α-Hydroxy-2α, 10β, 13α-triacetoxy-5α-(3'-dimethylamino-3'-phenyl) butanoate-taxa-4 (20), 11-di- ene (3), 10β-Benzoxy-5α-(3'-dimethylamino-3'-phenyl) propanoxy-1β-hy- droxy-7β, 9α, l3α-triacetoxy-11(15→l)abeotaxa-4(20), 11-diene (4), Taxinine (5), 1β-Acetoxy isodrimeninol (6), 9α, 10β, 13α-Triacetoxy-5α-cinnamoyl- taxa-4(20), 11-diene (7), 2-Deacetoxyltaxinine J (8), Taxinine B (9), 9α, 10β-Diacetoxy-5α-cinnamoyltaxa-4 (20), 11-dien-13α-ol (10), Taxezopidine G (11), 7-Deacetoxytaxuspine J (12), 2-Deacetyltaxinine (13), 10-Deacetyl taxinine (14), 1-Hydroxy taxuspine C (15), Taxagifine (16), 2-Acetoxy-5- deacetyltaxusin (17), 2-Deacetoxy-5-decinnamoyltaxinine J (18), 2α, 9α, 10β- Triacetoxy-20-hydroxy-11,12-epoxytaxa-4-en-13-one (19), Taxinine A 11, 12- epoxide (20), Taxinine A (21), 5-Decinnamoyl taxinin J (22), Decinnamoyl taxagifine (23), 10-Deacetyl-13-oxobaccatin III (24), 9-Deacetyl-9-benzoyl- 10-debenzoyl brevifoliol (25), Baccatin III (26), 13-Oxobaccatin III (27), Yunnanxane (28), (3E, 7E)-10β,13α-Diacetoxy-2α, 5α, 20- trihydroxy-3, 8- secotaxa-3, 7, 11-triene-9-one (29), (3E, 7E)-2α, 10β, 13α-Tri acetoxy-5α, 20- dihydroxy-3,8-secotaxa-3, 7, 11-trien-9-one (30), Taxuspine G (31), Taxezopi- dine F (32), 9-Dihydro-13-acetyl-baccatin III (33), 10-Deacetyl baccatin III (10-DAB) (34), 7, 9-Deacetyl-5-decinnamoyl taxinine J (35), Taxol (36), 7-Epi-10-deacetyltaxol (37), 2α, 9α, 10β-Triacetoxy-13α-hydroxy-5α-O-(β- D-glucopyranosyl)taxa-4(20), 11-diene (38), Coniferyl aldehyde (39), (-)-Pinoresinol (40),α-Conidendrin ( 41).Conclusion: We investigated the constituents of Taxus cuspidata needles, and the results showed that all the methods used in this experiment were highly efficient and practical, especialy for the classification of basic consti- tuents, phenolic constituents and neutral constituents. Forty one compounds were isolated and identified, among them 2α,9α,10β-triacetoxy-13α-hydroxy- 5α-O-(β-D-glucopyranosyl)taxa-4(20), 11-diene (38) were reported for the first time in the nature. While 9α-Hydroxy-2α, 10β, 13α-triacetoxy-5α-(3'- dimethylamino-3'-phenyl)butanoate-taxa-4(20), 11-diene (3), 10β-Benzoxy- 5α-(3'-dimethylamino-3'-phenyl)-propanoxy-1β-hydroxy-7β, 9α, l3α-triace- toxy-11(15→l)-abeotaxa-4(20), 11-diene (4), 2α, 9α, 10β-triacetoxy-20- hydr- oxy-11, 12-epoxytaxa-4-en-13-one (19), 10-deacetyl-13-oxobaccatin III (24), 9-Deacetyl-9-benzoyl-10-debenzoylbrevifoliol (25), 13-Oxobaccatin III (27), (3E, 7E)-10β, 13α-diacetoxy-2α, 5α, 20-trihydroxy-3, 8-secotaxa-3, 7, 11- triene-9-one (29), (3E, 7E)-2α, 10β, 13α-triacetoxy-5α, 20-dihydroxy-3, 8- secotaxa-3, 7, 11-trien-9-one (30) were isolated for the first time from the plant of Taxus cuspidata.We also summarized the 1H-NMR characteristic of taxanes from the 1H-NMR data of all the taxanes isolated in our experiment and relevant literatures, and this will facilitate the elucidation of taxanes. Part two Chemical Study on the Components of Eupatorium odoratum LEupatorium odoratum L, also named Chromolaena odorata, is a perennial herb which belongs to the tribe of Eupatorium. It is one of the invasive species that do serious harm in our country at present. Eupatorium odoratum L is native to Central America, also distributed in South America, Asia, tropics of Africa. In China, this plant exists mainly in Guangdong, Hainan, Guangxi, Yunnan, and Guizhou provinces. As a traditional herb, Eupatorium odoratum L has the effects of eliminating stasis subdue swelling, detoxication, hemostasis, and insecticidal action, which mainly used to treat pain and swelling from injuries, sores swelling, dermatitis, and external bleeding. As to involve chemical constituents study about Eupatorium odoratum L, there are only a few records in the literatures and just flavonoids and essential oils were reported. Its biological activities still remained on the level of crude extracts. So comprehensive research on the chemical constituents and finding out active ingredients or leading compounds are very necessary for the exploitation and utilization of this plant in the fields of pharmacy and agriculture.Objective: To study the chemical constituents of Eupatorium odoratum L by methods of column chromatography, preparative thin layer chromatography and high-performance preparative liquid chromatography, and the structure elucidation of pure compounds recure to 1H-NMR, 13C-NMR, 1H-1H COSY, HMQC, HMBC, NOESY, UV, IR, MS. To find active constituents by means of the activity screen.Methods: Air-dried herbal of Eupatorium odoratum L (19 Kg) were extracted by reflux for three times with 95% alcohol. The residue was scattered in water, and then extracted with petroleum ether, CH2Cl2, EtOAc, respectively. Finally we got the petroleum ether for 29.5 g, CH2Cl2 portion for 101.2 g, and EtOAc portion for 40 g. Methods of silica gel column chromatography, preparative TLC, and Sephadex LH-20 were used in the isolation and purification of the above three portions. Structures of pure compounds were elucidated with the help of UV, IR, MS, 1H-NMR, 13C-NMR, 1H-1H COSY, HMQC, HMBC and NOESY. The biological activities of sixteen compounds were evaluated.Results: There were thirty-five compounds isolated from Eupatorium odoratum L, and among them, twenty-eight compounds were identified, including eight flavonoids, eight lignans, seven quinones, two sterols, one amide alcohol ester, one fatty acid, and one naphthoquinone derivative. The results of identification are as follows:Odoratin (1),β-Sitosterol (2), Aurantiamide acetate (3), 2, 6-Dimethoxy- 1, 4-benzoquinone (4), 1, 2-Methylenedioxy-6-methyl anthraquinone (6), 2-Methylxanthopurpurin 7-methyl ether (9), 6 or 7-Methylalizarin (10), EO-12 (12), 1, 2, 4-Trimethoxy-3-hydroxy-6-methyl-anthraquinone (13), 6-Methyl- anthragallol-1, 2-dimethyl ether (14), Ombuin (15), Rhamnocitrin (16), 4, 2'- Dihydroxy-4', 5', 6'-trimethoxychalcone (17), Persicogenin (20), Acacetin (21), Kaempferide (22), 3', 4'-Dimethoxy luteolin (23), Austrocortinin (24), (-)-Pinoresinol (25), Tianshic acid (27), 7-Mehtoxypinoresinol (28), 5, 7-Di- methoxypinoresinol (29), (-)-Olivil (30), Cleomiscosin C or D (31), (-)-Medioresinol (32), (-)-Syringaresinol (33), Cleomiscosin A or B (34),β-Daucostero (35).6-Methyl-anthragallol-1, 2-dimethyl ether (14) has anti-growth activity to PC-6 cell line (IC50, 15.56μg/mL). Odoratin (1) exhibited strongly stimulating activity to PPARγ(EC50, 1.10μg/mL).Conclusion: Twenty-eight compounds were isolated and identified from Eupatorium odoratum L, among them five compounds are new ones found in the nature and sixteen compounds are isolated for the first time from above plant.New compounds are as follows, 1, 2-Methylenedioxy-6-methylanthraqui- none (6), EO-12 (12), 1, 2, 4-Trimethoxy-3-hydroxy-6-methylanthraquinone (13), 6-Methylanthragallol-1, 2-dimethyl ether (14), and 5, 7-Dimehtoxy- pinoresinol (29). Compounds firstly isolated from Eupatorium odoratum L are compounds 3, 4, 9, 10, 16, 20, 23, 24, 25, 27, 28, 30, 31, 32, 33, and 34.Activity assay experiment shown that 6-methylanthragallol-1, 2-di- methyl ether (14) exhibited strong anti-growth activity to PC-6 cell line with IC50, 15.56μg/mL, and odoratin (1) showed stronger stimulating activity to PPARγ(EC50, 1.10μg/mL).Part three Microbial transformation research to the active components in roots of Inula helenium LMicrobial transformation, as known, is the process of metabolization and transformation occurred between exogenous chemical substance and internal enzyme of microorganisms. It can be applied in the field of natural medicinal chemistry. The structures of active components, with the aid of microbial transformation, can be modified to get a series of derivants among which to find more active or less side effects leading compound. Microbial transformation can also be used to study pharmacokinetics and metabolism of medicines.Isoalantolactone, a eudesmane sesquiterpene lactone, is the main component in roots of Inula helenium L. Results of content determination in more than ten batches of Inula helenium L collected from Hebei, Neimeng, Henan and Sichuan provinces indicated that the content of isoalantolactone reach up to 1.2~5.6% in the dried crud herbal material. Pharmacology study showed that isoalantolactone has the effects of desinsectization, antiprotozoon, and antibiosis.Objective: To get new or more active less toxicity derivants of isoalantolactone by method of macrobial transformation. To simulate in vivo metabolism process of isoalantolactone in mammals and help to find the substance which being to work in vivo.Methods: Air-dried roots of Inula helenium L (5 Kg) were extracted with 99% alcohol at room temperature for three times. The residue was scattered in water, and then extracted with petroleum ether and CH2Cl2, respectively. The method of AgNO3 silica gel column chromatography was used in the isolation and purification of alantolactone and isoalantolactone. Forty-seven strains of fungus were screened for their capabilities to transform isoalantolactone. Fermentation liquor was extracted with EtOAc and the biotransformation products were isolated by the method of silica gel column chromatography. The structures of pure compounds were elucidated from the spectral data of UV, IR, MS, 1H-NMR, 13C-NMR, 1H-1H COSY, HMQC, HMBC and NOESY.Results: Five compounds were isolated and identified from the roots of Inula helenium L, alantolactone (1a), isoalantolactone (1b),β-sitosterol (2), 5α-epoxyalantolactone (3), and caffic acid anhydride (4). Two strains of fungus M. spinosus AS 3.3450 and Aspergillus niger AS 3.1858 can be used in the biotransformation experiment, and we choosed M. spinosus AS 3.3450 for its productivity and biodiversity. Three biotranformation products were obtained and identified as 3α-hydroxy-isoalantolactone (Z-1a), 3α-hydroxy- 11, 13-dihydroisoalantolactone(Z-1b) and 2α-hydroxy-isoalantolactone (Z-2) on the basis of their spectral data.Conclusion: The five compounds isolated from the roots of Inula helenium L are all known compounds. The method of AgNO3 silica gel column chromatography can be used in the isolation of alantolactone and isoalantolactone for its good resolution. M. spinosus AS 3.3450 can be used in the biotransformation of isoalantolactone and three known biotranformation products were identified.Part four Studies on HPLC fingerprint and determination of alantolac- tone and isoalantolactone in Inula helenium L.Objective: To establish HPLC fingerprint of Inula helenium L. To establish a method for the determination of alantolactone and isoalantolactone simultaneously in Inula helenium L.Methods: The separation was carried out on a HICHROM C18 (250 mm×4.6 mm, 5.0μm) column with the mixture of acetonitrile and 0.1% formic acid as the mobile phase in a gradient elution model (flow rate: 1.0 mL/min, detection wave: 254 nm for fingerprint and 220 nm for determination ),and the column temperature was 25℃. Results: 1. The fingerprint of Inula helenium L. was established. Precision, the RSD values of relative retention time and peak areas were less than 0.2% and 1.7%. Peproducibility, the RSD values of relative retention time and peak areas were less than 0.2% and 2.8%. Stability, the RSD values of relative retention time and peak areas were less than 1.4% and 1.8%. The similarities of 15 batches of samples are above 0.848~0.989. 2. Determination. linear ranges of alantolactone and isoalantolactone are 6.66~48.00 mg/g and 12.64~55.67 mg/g, respectively. Precision, the RSD values of alantolactone and isoalantolactone are 0.46% and 1.13%, respectively. Peproducibility, the RSD values are 1.68% and 0.85%, respectively. The average recoveries are 98.5% and 99.4%, and the samples are stable in MeOH in 24 h. The results showed that contents of alantolactone and isoalantolactone in 15 batches of samples are between 6.66~48.00 mg/g and 12.64~55.67 mg/g, respectively.Conclusion: The method is sensitive,quick,and useful to actualize standardization planting. It can be used for the quality control for Inula helenium L . The HPLC fingerprint of Inula helenium L was established for the first time. The mutual pattern of HPLC fingerprint on Inula helenium L from Hebei province were established for the first time.