| Delphinium trichophorum, a Chinese endemic species, which belongs to Delphinium genus (Ranunculaceae), distributes at an altitude of 2100-4600m in Northwest China, inclu-ding the Tibet Autonomous Region, Qinghai and Gansu Provinces. Biological activity studies performed on this species have shown it has analgesia, antipyretic, anti-inflammato-ry, insecticide activities and so on.As a widely used Tibetan Medicine, D.trichophorum, has been recorded in Jingzhu Materia Medica and Tibetan Annals. The quality of D.trichophorum is mostly controlled by original plant identification and medicinal material properties, and the study on chemical constituents and quality evaluation is bare. In addition, there are no reports on quality evaluation of Delphinium genus, most of them focus on chemical constituents. Therefore, systematic separation and identification were studied on this medicine. On the basis, we screened suitable index components to establish quality evaluation method using chromato graphic fingerprint and quantitation of multiple marker compounds in the material medicine, which may help to establish the quality standard of this medicine.Our studies mainly concentrated on chemical constituents and quality analysis e.g. isolation of chemical constituents, identification of main constituents by Thin layer Chromatography fingerprint (TLC) and determination of main chemical constituents. The results showed as follows:1.24 compounds were isolated from methanol extracts by using various chrom-atographic techniques and the structures of 21 compounds were elucidated on the ba sis of extensive spectroscopic analysis. Including 13 alkaloids:2-dehydro-11a-acetyl-1 3a-isobutyrylhet-isine(DTF-a),2α-phenacetyl-12β13α-dihydroxydelnudine(DTF-b),2a,1 1α,13β-triacetylh-etisine(DTF-c), isoboldine(DTF-d),2a-hydroxy-11β-acetyl-13β-isobuty rylhetisine(DTF-e), hetisinone(DTF-g), browniine(DTF-h), d-magnoflorine(DTF-p),2a, 13a-diacetyl-11α-hydroxyhetisine(DTF-q),2a-acetyl-11α,13β-dihydroxyhetisine(DTF-r), 2α,11β-dihydroxy-13α-isobutyrylhetisine(DTF-s),2a-hydroxy-11α-acetyl-13a-isovalerylh etisine(DTF-t),2α-hydroxy-11β,13β-diacetylhetisine(DTF-u); 1 nucleoside:adenosine(D TF-o); 7 flavones:kaempferol-3-O-(6"-acetyl)-β-D-glucopyranoside-7-O-a-L-arabinopyr anoside(DTF-f), quercetin-3-O-β-D-glucopyranoside-7-O-a-L-arabinopyranoside(DTF-i), quercetin(DTF-j), kaempferol-3-O-(6"-acetyl)-β-D-glucopyranoside(DTF-k), kaempferol-3-O-(3",6"-dia-cetyl)-β-D-glucopyranoside-7-O-a-L-arabinopyranoside(DTF-1), quercetin- 3-O-(6"-acetyl)-β-D-glucopyranoside(DTF-m), quercetin-3-O-β-D-glucopyranoside(DTF-n). There are six new compounds, including DTF-a, DTF-b, DTF-e, DTF-q, DTF-s and DTF-t.The ditepenoid alkaloids isolated from D.trichophorum are mainly hetisine-type which seems different from that from other plants of genus delphinium. And the st ructures of the flavones are consistent with which of genus delphinium,2. The major chemical constituents of D.trichophorum were confirmed to be alk aloids and flavones. In this case, the determination method of TLC fingerprint of t-he alkaloids and flavones of D.trichophorum has been established. The fingerprint c-haracteristics have been constructed by priority of batches drug materials, and the c-ommon patterns of TLC fingerprint have been obtained through "CHROMAP 1.51" solution software.Comparing with reference substances, seven fingerprint peaks were identified as 2α,11β-dihydroxy-13β-isobutyrylhetisine (1#); 2a-acetyl-11α,13β-dihydroxyhetisine (2 #); 2a-hydroxy-11β,13β-diacetylhetisine (3#); 2a-hydroxy-11β-acetyl-13β-isobutyrylh-etisine (4#); 2a-hydroxy-lla-acetyl-13β-isovalerylhetisine (5#); 2a,13a-diacetyl-11α-hydroxyhetisine (6#); 2α,11α,13β-triacetylhetisine (8#) in the TLC fingerprint of t-he alkaloids of D.trichophorum. By means of similarity analysis, it showed that the similarities of 10 batches samples, including nine batches of aerial parts and one b-atch of leaf, were from 0.8262 to 0.9806. The similarities of stem, flower and root is 0.6608,0.6461 and 0.1797. However, no fingerprint peaks could been found in TLC fingerprint of Delphinium caeruleum and Delphinium grandiflorum.And three fingerprint peaks were identified as quercetin-3-O-β-D-glucopyranoside-7-O-α-L-arabinopyranoside(1#); kaempferol-3-O-(6"-acetyl)-β-D-glucopyranoside-7-O-a-L-arabinopyranoside(4#); kaempferol-3-O-(3",6"-diacetyl)-β-D-glucopyranoside-7-O-a-L-arabinopyranoside(6#) in the TLC fingerprint of the flavones of D.trichophorum. Based on the similarity analysis, the similarities of 11 batches samples, including ni-ne batches of aerial parts, one batch of leaf and one batch of stem, were from 0.96 91 to 0.9964, and that of the flower and root were 0.3388 and 0.5637. But the si-milarity of Delphinium caeruleum was 0.2674, and there was no matched fingerprint peaks in Delphinium grandiflorum Linn.This method for determination the fingerprints of D.trichophorum is simple, sp-ecificity and beneficial for identification of different plant parts and quality control of the material medicine.3. A HPLC-ELSD method has been established for content determination of six alkaloids (2a-acetyl-11α,13β-dihydroxyhetisine(1),2a,13a-diacetyl-11α-hydroxyhetisine (2),2a,11 a-dihydroxy-13a-isobutyrylhetisine(3),2a,11 a,13β-triacetylhetisine(4),2a-hyd-roxy-11β-acetyl-13β-isobutyrylhetisine(5),2α-hydroxy-11α-acetyl-13β-isovalerylhetisine (6) in D.trichophorum by using a gradient elution of acetonitrile-0.1% triethylamine and 15 batches samples have been determined. The results showed thatthe contents of six alkaloids in 13 batches of D.trichophorum are in the range of 0～0.293mg·g-1, 0～2.866 mg·g-1,0.378～2.299 mg·g-1,0.429～3.609 mg·g-1,0.363～4.034 mg·g-1, 0.080～1.126 mg·g-1; but they couldn’t be detected in Delphinium caeruleum and D elphinium grandiflorum.The contents of six index compounds in D.trichophorum of different plant parts were discrepant. The total contents of six alkaloids in leaf, stem and flower were 11.390mg·g-1, 1.726 mg·g-1 and 10.711 mg·g-1. It showed that leaf and flower are similar, and the stem is obviously less than them in the contents of alkaloids. Whereas, there were only three compounds could be detected in the root of the pla-nt, and the total contents of six alkaloids were 0.946 mg·g-1, that is the least among four parts of plant.After further study, the contents of 2a-acetyl-lla,13β-dihydroxyhetisine(1),2a,13 a-diacetyl-lla-hydroxyhetisine(2),2a,lla-dihydroxy-13a-isobutyrylhetisine(3) in four batches of D.trichophorum (No.1-4) collected in 2008 were 0.225～0.293 mg·g-1,2.1 64～2.866 mg·g-1 and 1.847～2.299 mg·g-1, and in five batches of D.trichophorum (No.9-13) collected in 2009 were 0.077～0.099 mg·g-1,1.269～1.708 mg·g-1,1.009～ 1.545 mg·g-1. It seems that the contents of the three compounds in the samples col-1 ected in 2008 were higher than in 2009, the same as the total contents of six alkal oids.In short, chromatographic fingerprint and a qulity evaluation method using qua-ntitation of multiple marker compounds in the material medicine can be used to id-entify and evaluate the quality of D.trichophorum.In this paper, the constituents of D.trichophorum have been investigated intensevely and the major chemical constituents were confirmed to be alkaloids and flavones. Aporphinoid alkaloids and nucleoside have been isolated from Delphinium gunes for the first time, which can rich the kind of compounds in gunes Delphinium and afford the scientific basis of chemotaxonomy.Establishing chromatographic fingerprint and a qulity evaluation method of D. trichophorum by using quantitation of multiple marker compounds in the material medicine, which was useful for the quality control and the further study of quality standard of D.trichophorum. |
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