大叶冬青叶中2个新的三萜皂苷

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[摘要] 大叶冬青为苦丁茶的主要基源植物，三萜皂苷为其主要的活性和特征性成分。该研究从大叶冬青叶中分离得到了2个新的三萜皂苷类化合物，大叶冬青皂苷 R （1）和大叶冬青皂苷S （2），并根据NMR，MS等波谱方法和理化性质鉴定了其化学结构。其中大叶冬青皂苷R为第1个含有1928内酯环的三萜皂苷。

[关键词] 冬青科； 大叶冬青； 三萜皂苷

[Abstract] Two new triterpenoid glycosides， latifolosides R and S （1 and 2）， were isolated from the leaves of Ilex latifolia by various column chromatographic methods. Their structures were elucidated based on NMR spectroscopic data and chemical evidence.

[Key words] Aquifoliaceae； Ilex latifolia； triterpenoid glycoside

doi：10.4268/cjcmm20161616

The leaves of Ilex latifolia Thunb. （Aquifoliaceae）， a Chinese bitter tea called as "KuDingCha"， have been widely consumed as health beverage in China for thousands years. In traditional Chinese medicine， the leaves are also used for the treatment of dysentery， sore throats， obesity， hypertension as well as various inflammatory diseases[1]. Flavonoids， polyphenolics， triterpenes and triterpenoid glycosides have been isolated and identified from the leaves of I. latifolia[27]. Among them， triterpenes and triterpenoid glycosides were considered to be the major and characteristic constituents of this plant， some of which showed antioxidant[8]， antibacterial [9] and lipidlowering[1011] activities. Our previous papers[7，1112] reported the isolation and structural identification of several new triterpenes and triterpenoid glycosides from the title plant. As a part of our continuing investigation， two new triterpenoid glycosides， latifolosides R and S （1 and 2） were further isolated from the same plant （Fig. 1）. This paper describes the isolation and structural elucidation of two new compounds.

Fig.1 Chemical structures of 1 and 2

1 Material

1.1 General experimental procedures Optical rotations were measured with a JASCO P1020 polarimeter. UV data were obtained on a JASCO V550 UV/vis spectrophotometer. IR spectra （KBr） were recorded on a JASCO FT/IR480 Plus Fourier Transform spectropolarimeter. 1H and 13C NMR spectra were acquired with a Bruker Avance 400 spectrometer. HRESIMS data were obtained using an Agilent AccurateMassQTOF LCMS 6210 instrument. The analysis HPLC was performed on an Agilent 1260 instrument equipped with multiple wavelength diode array detector （DAD）， using Cosmosil 5 C18MSⅡ column （4.6 mm × 250 mm， 5.0 μm）. Preparative HPLC was carried out using Cosmosil 5C18MSⅡ column （10 mm × 250 mm， 5.0 μm） at a flow rate of 6 mL・min-1. Column chromatography was conducted on silica gel （200300 meshes， Qingdao Haiyang Chemical Co.， Ltd.， China）， Sephadex LH20 （GE Healthcare， USA） and ODS （YMC， Japan）.

1.2 Plant materials The leaves of I. latifolia were collected in Yunnan province of China in October 2008， and identified by Professor Chen Weiping at Xinglong Suboffice of Medicinal Plant Research Institute of Chinese Academy of Medical Science. A voucher specimen （No. 20081025） was deposited in Institute of Traditional Chinese Medicine and Natural Products， Jinan University， Guangzhou， China.

2 Methods

2.1 Extraction and isolation The airdried and powdered leaves of I. latifolia （8.0 kg） were extracted with H2O under reflux for three times. The solution was combined and concentrated under vacuum to yield the crude extract， which was redissolved in H2O. After filtration， the water soluble solution was subjected to macroporous resin D101 column eluted with H2OEtOH mixtures （100∶0； 55∶45； 25∶75； 5∶95）. The 75% EtOH eluate （400 g） was subjected to column chromatography （CC） over silica gel eluting with gradient mixtures of CHCl3MeOHH2O （90∶10∶1 60∶40∶1） to obtain nine fractions （Fr.1Fr.9）. Fr.7 was further purified by silica gel CC using a stepwise gradient elution of CHCl3MeOH to yield ten subfractions （Fr.7aFr.7j）. Fr.7d was subjected to Sephadex LH20 column eluted with MeOH to give two fractions （Fr.7d1 and Fr.7d2）. Fr.7d2 was further purified by preparative HPLC with MeOHH2O （60∶40） as the eluent to obtain 1 （10 mg）. Fr. 8 was purified on silica gel CC using a stepwise gradient elution of CHCl3MeOH to give nine subtractions （Fr. 8aFr. 8l）. Fr. 8d was further purified by preparative HPLC with MeOHH2O （50∶50） to yield 2 （20 mg）.

2.2 Acid hydrolysis of 1 and 2 and determination of absolute configuration of monosaccharides [13] Compounds 1 and 2 （each 2 mg） were heated in 2 mol・L-1 HCl （10 mL） at 80 ℃ for 4 h in water bath， respectively. The reaction mixtures were concentrated to dryness under reduced pressure. Each residue was dissolved in pyridine （1 mL）， and then Lcysteine methyl ester hydrochloride （5 mg） was added to the solution. The mixture was heated at 60 ℃ for 1 h， and isothiocyanate （5 mg） was subsequently added， followed by heating at 60 ℃ for another 1 h. Finally， the reaction mixtures were immediately subjected to HPLC analysis. The analysis was performed on C18 column， and the mobile phase was acetonitrilewater （25∶75， containing 0.05% formic acid） with a flow rate at 0.8 mL・min-1 and wavelength at 250 nm under room temperature. The absolute configuration of the sugar moieties were elucidated by comparison of the retention times of standard monosaccharides prepared in a same manner. The retention times of standard Larabinose， Lrhamnose and Dglucose were 23.78， 35.43 and 21.22 min， respectively.

Latifoloside R （1） Colorless needles （MeOH）， mp 284286 ℃， [α]25D=-64.8° （c 0.1， MeOH）； IR（KBr）υmax： 3 406， 2 942， 1 760， 1 644， 1 074 cm-1； HRESIMS m/z 949.476 3 [M + Na]+ （calcd for C47H74O18Na： 949.476 7）； 1HNMR （pyridined5，400 MHz） and 13CNMR （pyridined5，100 MHz） see Table 1.

Latifoloside S （2） Colorless needles （MeOH）， mp 248251 ℃， [α]25D=-16.8° （c 0.1， MeOH）； IR（KBr）υmax： 3 389， 2 923， 1 734， 1 635， 1 074 cm-1； HRESIMS m/z 1 365.665 9 [M-H]-（calcd for C65H105O30：1 365.669 6）； 1HNMR （pyridined5，400 MHz） and 13CNMR （pyridined5，100 MHz） see Table 1.

3 Results and Discussion

Compound 1 was isolated as colorless needles （MeOH）. The HRESIMS spectrum of 1 exhibited a quasimolecular ion at m/z 949.476 3 [M+Na]+ （calcd for C47H74O18Na： 949.476 7）， corresponding to the molecular formula of C47H74O18. The IR spectrum displayed characteristic absorptions for hydroxy （3 406 cm-1）， carboxyl （1 760 cm-1） and double bond （1 644 cm-1） groups. The 1HNMR spectrum of 1 exhibited signals for seven tertiary methyls （δH 0.85， 0.90， 1.16， 1.24， 1.42， 1.50 and 1.78， each 3H， s）， two oxymethines [δH 3.28 （1H， dd，J=11.2， 4.8 Hz） and 5.04 （1H， br s）]， and three anomeric protons [δH 4.88 （1H， d，J=5.6 Hz）， 5.12 （1H， d，J=7.6 Hz） and 6.15 （1H， br s）]. In the 13CNMR and DEPT spectra， fortyseven carbon signals were observed including four oxygenated carbons （δC 64.2， 73.9， 88.2 and 89.8）， two olefinic carbons （δC 139.6 and 142.1）， one carbonyl carbon （δC 177.7）， as well as three anomeric carbons （δC 102.0， 104.7 and 104.8）. All the 1H and 13CNMR spectroscopic data of 1 were assigned based on the 1H1HCOSY， HSQC， HMBC， ROESY and TOCSY experiments （Table 1）. The above NMR data suggested the presence of 12hydroxy13，18eneursane type triterpene skeleton and three sugar units in 1 （Fig. 2）.

The NMR data assigned to the aglycone part of 1 were in good agreement with those of （3β，12β，19α）3，12，19，20tetrahydroxyurs13（18）en28oic acid28，19lactone [11] except for the downfield shift of C3 （Δδ+10.3）， indicating that the aglycone of 1 was identical to the known compound and the 3OH was glycosylated. The HMBC correlations between H329 （δH 1.78） and C18 （δC 139.6）/C30 （δC 24.8）， between H330 （δH 1.42） and C20 （δC 73.9）/C21 （δC 35.5）， between H216 （δH 1.58 and 2.54） and C28 （δC 177.7）， as well as between H222 （δH 1.85 and 2.03） and C28 （δC 177.7） further confirmed the existence of a lactone ring in 1. In addition， the βconfiguration of 12OH and 20OH could be confirmed by the ROESY correlations between H12 （δH 5.04） and H9 （δH 2.03）/H329 （δH 1.78）， as well as between H329 （δH 1.78） and H330 （δH 1.42） （Fig. 2）. Thus， the aglycone of 1 was elucidated as 3β，12β，19β，20βtetrahydroxyurs13（18）en28oic19β，28lactone.

Acid hydrolysis of 1 afforded Dglucose， Larabinose and Lrhamnose according to the HPLC analysis of their derivatives. In combination of the large 3JH1H2 coupling constants （5.6 Hz for Larabinose and 7.6 Hz for Dglucose）， the relative configuration of the anomeric centers of Larabinose and Dglucose were deduced as α and β， respectively. The αconfiguration for the Lrhamnose could be established by comparison of the NMR data with those reported [14]. The linkage and sequence of the sugar units were determined by the HMBC correlations between H1 （δH 5.12） of glucose

and C3 （δC 82.5） of arabinose， between H1 （δH 6.15） of rhamnose and C2 （δC 74.7） of arabinose， as well as between H1 （δH 4.88） of arabinose and C3 （δC 88.2） of the aglycone （Fig. 2）. Therefore， the structure of 1 was determined to be 3OβDglucopyranosyl（13）[αLrhamnopyranosyl（12）]αLarabinopyranosyl （3β，12β，19β，20β）tetrahydroxyurs13（18）en28oic acid19β，28lactone， and named as latifoloside R. This is the first example of 19β28 lactonic triterpenoid glycoside existed in the genus Ilex.

Compound 2 was isolated as amorphous powder. Its molecular formula was deduced as C65H106O30 by its HRESIMS （m/z 1 365.665 9 [M-H]-， calcd for C65H105O30：1 365.669 6）. The IR spectrum showed absorption bands at 3 389， 1 734 and 1 635 cm-1， suggesting the presence of hydroxy， carboxyl and double bond. The 1HNMR spectrum displayed the signals for seven tertiary methyls （δH 0.79， 0.85， 0.88， 1.07， 1.11， 1.17 and 1.25， each 3H， s）， an oxymethine [δH 3.25（1H， dd，J=11.4， 3.6 Hz）]， and a trisubstituted olefinic unit [δH 5.94 （1H， br s）]. In the 13CNMR and DEPT spectra of 2， sixtyfive carbon signals were observed including seven methyls （δC 15.7， 17.2， 17.4， 23.7， 25.9， 28.2 and 33.1）， one oxygenated methine （δC 88.4）， two olefinic carbons （δC 122.6 and 144.2）， as well as a carbonyl （δC 176.4）. Based on the analysis of the 1D and 2D NMR spectra， all the 1H and 13CNMR signals of 2 were assigned as shown in Table 1. The aglycone of 2 was identified as oleanolic acid by comparison of the NMR data with those published in literature [15].

Furthermore， the 1H and 13CNMR spectra of 2 showed six anomeric proton signals [δH 4.77 （1H， d，J=6.8 Hz）， 5.15 （1H， d，J=7.6 Hz）， 5.28 （1H， d，J=7.6 Hz）， 6.19 （1H， d，J=8.0 Hz）， 6.34 （1H， br s） and 6.61 （1H， br s）]， corresponding to six anomeric carbon signals （δC 94.8， 101.0， 101.4， 103.4， 105.2 and 106.3）. After acid hydrolysis of 2， the monosaccharides were identified as Larabinose， Dglucose， and Lrhamnose by HPLC analysis of their derivatives， respectively. The relative anomeric configurations of the sugar moieties were deduced to be β for the Dglucose and α for the Larabinose by 3JH1H2 coupling constants of the anomeric protons， respectively. The αconfiguration for the Lrhamnose could also be determined by comparison of the NMR data with those reported in literature [14]. The HMBC correlations between H1 （δH 5.28） of terminal glucose and C2 （δC 84.6） of inner glucose， between H1 （δH 5.15） of inner glucose and C3 （δC 82.8） of arabinose， between H1 （δH 6.34） of terminal rhamnose and C2 （δC 74.5） of arabinose， between H1 （δH 4.77） of arabinose and C3 （δC 88.4） of aglycone， between H1 （δH 6.61） of terminal rhamnose and C2 （δC 76.1） of glucose， as well as between H1 （δH 6.19） of glucose and C28 （δC 176.4） of aglycone revealed the structure of the sugar chains as shown in Fig. 3. On the basis of all the above evidences， 2 was elucidated as 3OβDglucopyranosyl（12）βDglucopyranosyl（13）[αLrhamnopyranosyl（12）]αLarabinopyranosyl oleanolic acid 28OαLrhamnopyranosyl（12）βDglucopyranosyl ester， and named as latifoloside S.

Fig.3 Key HMBC correlations of 2

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