| Panax notoginseng?Burk.?F.H.Chen belongs to Panax genus,Acanthopanax family.Its roots and rhizomes are one kind of the most valuable traditional Chinese medicine?TCM?which possess multiple effects of dissipating stasis to stop blooding,dispersing swelling and pain.In recent years,total saponins of P.notoginseng have been made into a variety of pharmaceutical preparations for cardiovascular and cerebrovascular diseases clinical treatment,and with high market application values.However,after hundreds of years of cultivation,the adaptability of P.notoginseng to the environment has gradually declined,and the continuous cropping obstacle has become increasingly prominent.At the same time,because the growth of P.notoginseng is limited by the suitable growth environment,the herbal resources of it are insufficient.According to the references,like its roots,the leaves of P.notoginseng are also rich in triterpenoid saponins.Therefore,more and more attention has been paid to the development and application of P.notoginseng leaves.But whether P.notoginseng leaves can replace its roots and rhizomes is still a problem.Although experts and scholars have done a lot of work,due to the lack of systematic research,there are still no enough evidences to accurately answer the above questions.As is known to all,the efficacy of TCM depends on its chemical composition.Therefore,before answering the question of whether P.notoginseng leaves could replace its roots and rhizomes for medicinal use,we need to solve two main problems firstly.Firstly,whether the chemical components in P.notoginseng leaves are coincide with its roots and rhizomes basically.Secondly,whether the pharmacological effects of them are basically consistent?Traditional pharmacological effect of P.notoginseng is to dissipate stasis and stop blooding,disperse swelling and pain.It is mainly used for treating cardiovascular and cerebrovascular diseases,injuries,blood stasis and swelling pain closely related to inflammation.Nitric oxide?NO?is involved in the occurrence and development of inflammatory response.Therefore,regulating NO production may be an important way to treat inflammation.Then,this paper will study the chemical constituents of P.notoginseng leaves systematically firstly.Based on this,the in vitro anti-inflammatory active ingredients were screened with the inhibition of NO production as the evaluation index.In conclusion,this paper would initially provide theoretical basis for wether the P.notoginseng leaves could take palce to its roots.Various chromatographic methods including D101 macroporous resin,silica gel,ODS,Sephadex LH-20 column chromatorgraphies and preparative HPLC chromatorgraphy?PHPLC?,kinds of spectrophotometric methods including UV,IR,1H NMR,13C NMR,1H 1H COSY,HSQC,HMBC,NOESY,MS,and[?]D,along with chemical reaction methods were combined to used to carry out the systematic chemical constituents study on the 50%ethanol extract of P.notoginseng leaves.As a result,sixty compounds were obtained and identified.Among them,twenty-four ones were new dammarane triterpene saponins,named as notoginsenosides NL-A1-NL-A4?1-4?,NL-B1-NL-B3?5-7?,NL-C1-NL-C3?8-10?,NL-D?11?,NL-E1-NL-E4?12-15?,NL-F1?16?,NL-F2?17?,NL-G1?18?,NL-G2?19?,NL-H1-NL-H3?20-22?,NL-I?23?,NL-J?24?.Among them,notoginsenoside NL-J?24?was with new skeleton.Moreover,the thirty-six known isolates including thirty-three dammarane triterpene saponins,3?,12?,20S-trihydroxy-25-hydroperoxydammar-23-ene-3-O-[?-D-glucopyranosyl?1?2?-?-D-glucopyranosyl]-20-O-[?-D-xylopyranosyl?1?6?]-?-D-glucopyranoside?25?,quinquenoside L3?26?,floranotoginsenoside A?27?,gypenoside LXIX?28?,notoginsenoside Fh5?29?,ginsenoside F2?30?,notoginsenoside Fe?31?,gypenoside IX?32?,gypenoside XVII?33?,20?S?-protopanaxadiol-3-O-?-D-glucopyranoside-20-O-?-D-xylopyranosyl?1?4?-?-L-arabin opyranosyl?1?6?-?-D-glucopyranoside?34?,vina-ginsenoside-R18?35?,ginsenosides Rd,Rc,Rb2,Rb3 and Rb1?36-40?,vina-ginsenoside-R7?41?,notoginsenosides FP2,FZ,Fc,Fa,S,Fh1,TandD?42-49?,3-O-?-D-glucopyranosyl-?1?2?-?-D-glucopyranosyl-20-O-?-D-xylopyranosyl?1?6?-?-D-gl ucopyranosyl-darmmar-25-ene-3?,12?,20S,24S-tetraol?50?,3-O-?-D-glucopyranosyl-?1?2?-?-D-glucopyranosyl-20-O-?-D-xylopyranosyl?1?6?-?-D-gl ucopyranosyl-darmmar-25-ene-3?,12?,20S,24R-tetraol?51?,3-O-?-D-xylopyranosyl-?1?2?-?-D-glucopyranosyl-?1?2?-?-D-glucopyranosyl-3?,12?,20S,24S-tetrahydroxydammar-25-ene-20-O-?-D-glucopyranosyl-?1?6?-?-D-glucopyranoside?52?,notoginsenoside-Ng3?53?,chikusetsusaponin L5?54?,ginsenoside Rg1?55?,notoginsenoside R1?56?,ginsenoside Re?57?,together with three flavonoid glycosides,kaempferol-3-O-?-D-glucopyranosyl-?1?2?-?-D-galactopyranoside?58?,quercetin-3-O-?-D-xylopyranosyl-?1?2?-?-D-galactopyranoside?59?,and quercetin-3-O-?-D-glucopyranosyl-?1?2?-?-D-galactopyranoside?60?.Among the known ones,quinquenosideL3?26?,vina-ginsenoside-R18?35?,and3-O-?-D-glucopyranosyl-?1?2?-?-D-glucopyranosyl-20-O-?-D-xylopyranosyl?1?6?-?-D-gl ucopyranosyl-darmmar-25-ene-3?,12?,20S,24R-tetraol?51?were firstly isolated from this plant.And the NMR data of vina-ginsenoside-R18?35?was reported here firstly.Depending on whether there's oxygen at C-6 of dammarane triterpene saponins,the fifty-seven obtained saponins were divided into protopanaxadiol?PPD?-type?1-10,12-53?and protopanaxatriol?PPT?-type.According to the differences of side chain,PPD-type saponins have been devided into eight types.Among them,the 20?S?-PPD type saponins with aglycones,?20S,24??-3?,12?,20,24,25-pentahydroxydammaraneor?20S,23R?-3?,20-dihydroxy-12?,23-epoxy-dammar-24-ene were only found from the leaves of P.notoginseng.Besides,the known compounds 29,52 and 53 have not been reported from other parts of P.Notoginseng yet.The above characteristics could be used to distinguish the extracts of P.Notoginseng leaves from its other medicinal parts.At the same time,comparing the experimental results with literatures,it could be found that there are significant differences between the saponins in P.notoginseng leaves and its roots.PPD-type saponins were the main saponins isolated from P.notoginseng leaves,while its roots mainly contained PPT-type ones.The difference of component types would lead to different pharmacological activities.Therefore,from the point of chemical composition,it remains to be discussed whether the leaves of P.notoginseng could replace its roots and rhizomes as clinical drugs.Moreover,by comparing the NMR of?20S,24??-3?,12?,20,24,25-pentahydroxy dammarane type saponins and?20S,24??-3?,12?,20,24-tetrahydroxy-dammar-25-ene type ones(??H22b-H22a and??H23b-H23a for?20S,24??-3?,12?,20,24,25-pentahydroxy dammarane type;??H22b-H22a and?C-26 for?20S,24??-3?,12?,20,24-tetrahydroxy-dammar-25-ene type)as well as chromatographic retention behaviors of their C-24 epimer,the corresponding rules were summarized,which would provide a basis for the rapid determination of C-24configuration in these two kinds of saponins and similar ones.On the basis of the separation and identification of the chemical composition.RAW264.7 cells were used as the in vitro anti-inflammation screening model to evaluate the inhibitory effect of novel saponins 1-24 on LPS induced NO release,and the structure-activity relationships were summarized as well.The results showed that compounds 1,2,5,6,8-10,which had no absolute configuration substituent group at C-22–24,could inhibit the NO release from RAW 264.7 cells at the concentration of 50?m,and the inhibition effects was in dose-dependent at 10,25,and 50?m.While the compounds 13-18,21,23,24,which had absolute configuration substituent group at C-22–24,could exert NO inhibitory activity at 25?M,and their inhibitory activities were in dose-dependent at 1,10,and 25?m.Especially,compounds 17 and 24 still possessed strong biological activity at 1?M.The structure-activity relationships of compounds 1,2,5,6,8-10 were summarized.It was found that when the C-6 of glucosyl was substituted by xylosyl,it would exhibit stronger activity than which was replaced by furanarabinosyl?1 vs 2;8 vs 9?.When substituent glycosyl was same,different aglycone types would lead to different activity for glycosides,and the order of activity was displayed as following:25-OH substituent glycosides>25,26-en-24-one substituent glycosides>25-OOH substituent glycosides?5>8>1?.The activity comparison of triterpenoids 12-18,21,23,and 24 indicated that the inhibition of triterpenoids on NO production increased with the increase of the number of substituted sugars?14>13>12;17>16?;compraring the activities of 15 and 17,it was found that the configuration of 24-OH in the structure of 24,25-dihydroxy-substituted saponins had a strong influence on the activity?24S>24R?;the comparison of the activities of compounds 18,21,and 23 showed that when the substituent sugar groups were same,the aglycone,?20S,23R?-3?,20-dihydroxy-12?,23-epoxy-dammar-24-ene exhibited stronger inhibitory activity on NO production?18,21 vs 23?.In addition,the different activities of above two kinds of saponins suggested that the activities of triterpenoids whose C-22-24 without absolute configuration substituent group was stronger than whose C-22-24 with absolute configuration substituent group?5>12;6>14;9>14,concentration at 25?m?.At the same time,according to the references,it was found that the main components of P.notoginseng?some known compounds?also represented a strong inhibitory effect on LPS induced NO release in RAW 264.7 cells,which further suggested that P.notoginseng leaves could be a potential TCM for the development of anti-inflammatory drugs.Consequently,in this project,the chemical constituents of P.notoginseng leaves were systematically isolated and identified by a variety of spectral,chromatographic and chemical reaction methods,and a series of novel dammarane-type triterpene saponins were obtained,which would further improve the cognition of experts and scholars on the chemical constituents of P.notoginseng.Through summing up the structural characteristics of the triterpenoids,the distinguishment of P.notoginseng leaves from other parts of its extracts could be more accurate.Furthermore,the chemical compositions of P.notoginseng leaves were compared with its roots,and it was further confirmed that the main saponins of leaves and roots were PPD and PPT type saponins,respectively.Thus,whether the leaves of P.notoginseng could replace roots and rhizomes as clinical drugs?It remains to be discussed.However,it is worth pointing out that PPD type saponins are superior to PPT ones in anticoagulant,anti-inflammatory and anti-tumor aspects.At the same time,on the basis of chemical separation,RAW 264.7 cells induced by LPS were used as the screening model of the in vitro anti-inflammatory activity,and the potential anti-inflammatory activities of new compounds were evaluated;the structure-activity relationships of the tested compounds was summarized,which provided reference for structural modification and transformation.However,the mechanism of the anti-inflammatory effect of each active component remains to be determined by molecular biological or pharmacological experiments.This study laid a foundation for the research and development of P.notoginseng leaves as an anti-inflammatory drug,and provided a reference for the reuse of P.notoginseng resources. |
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