李月婷等
[摘要] 研究牡荊Vitex negundo var. cannabifolia果實(shí)的化學(xué)成分及其生物活性。利用硅膠、Sephadex LH20柱色譜和半制備HPLC等色譜方法分離純化,結(jié)合其理化性質(zhì)和MS、NMR等波譜學(xué)數(shù)據(jù)鑒定化合物的結(jié)構(gòu)。從牡荊子70%丙酮提取物的二氯甲烷萃取部位分離鑒定了19個(gè)化合物,包括13個(gè)木脂素和6個(gè)酚類化合物,分別鑒定為6羥基4(4羥基3甲氧基苯基)3羥甲基7甲氧基3,4二氫2萘醛(1),6羥基4(4羥基3甲氧基苯基)3羥甲基5甲氧基3,4二氫2萘醛(2),vitexdoin F(3),去四氫鐵杉脂素(4),vitexdoin E(5),4氧代芝麻素(6),L芝麻素(7),(+)beechenol(8),ligballinol(9),2(4hydroxyphenyl)6(3methoxy4hydroxyphenyl)3,7dioxabicyclo[3.3.0]octane(10),松脂素(11),balanophonin(12),theroguaiacylglycerolβconiferyl aldehyde ether(13),對(duì)羥基肉桂醛(14),松柏醛(15),5,7二羥基色原酮(16),反3,5二甲氧基4羥基肉桂醛(17),覆盆子酮(18)和alternariol 4methyl ether(19)?;衔?~10,14,18,19為首次從馬鞭草科植物中分離得到,化合物13為首次從牡荊屬植物中分離得到,化合物6,7,12,15~17為首次從該植物中分離得到。對(duì)分離得到的化合物進(jìn)行了體外抗炎和細(xì)胞毒活性評(píng)價(jià),8個(gè)化合物(3,5,7,10,11,14,15,17)能夠明顯抑制LPS誘導(dǎo)的RAW 264.7細(xì)胞釋放一氧化氮(NO),其IC50在7.8~81.1 μmol·L-1;4個(gè)化合物(1~4)對(duì)HepG2細(xì)胞具有明顯的細(xì)胞毒作用,IC50在5.2~24.2 μmol·L-1。
[關(guān)鍵詞] 牡荊子; 木脂素; 酚類化合物; 抗炎; 細(xì)胞毒
Chemical constituents from the fruits of Vitex negundo var. cannabifolia and
their biological activities in vitro
LI Yueting1,2, PANG Daoran1,2, ZHU Zhixiang1, HUO Huixia1,2, REN Yi1,2,
PENG Zhitian1,2, ZHENG Jiao1, ZHAO Yunfang1*, TU Pengfei1, LI Jun1*
(1. Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China;
2. School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, China)
[Abstract] Chemical constituents from the fruits of Vitex negundo var. cannabifolia and their nitric oxide (NO) inhibitory and cytotoxic activities were investigated. The compounds were isolated and purified by various column chromatography, and their structures were identified by physiochemical properties and spectroscopic data. Thirteen lignans and six phenolic compounds were isolated from the CH2Cl2 extract of the fruits of V. negundo var. cannabifolia, respectively. Their structures were elucidated as 6hydroxy4(4hydroxy3methoxyphenyl)3hydroxymethyl7methoxy3,4dihydro2naphthaldehyde (1), vitedoin A (2), vitexdoin F (3), detetrahydroconidendrin (4), vitexdoin E (5), 4oxosesamin (6), Lsesamin (7), (+)beechenol (8), ligballinol (9), 2(4hydroxyphenyl)6(3methoxy4hydroxyphenyl)3,7dioxabicyclo[3.3.0]octane (10), (–)pinoresinol (11), balanophonin (12), theroguaiacylglycerolβconiferyl aldehyde ether (13), transpcoumaryl aldehyde (14), coniferyl aldehyde (15), 5,7dihydroxychromone (16), trans3,5dimethoxy4hydroxycinnamic aldehyde (17), frambinone (18), and alternariol 4methyl ether (19). Compounds 810,14,18,19 were firstly isolated from Verbenaceae family, compound 13 was obtained from Vitex species, and 6,7,12,1517 from V. negundo var. cannabifolia for the first time, respectively. The isolated compounds were evaluated for their antiinflammatory and cytotoxic effects in vitro. Eight compounds (3,5,7,10,11,14,15,17) showed inhibition against NO production in LPSstimulated RAW 267.4 cells (IC50 in the range of 7.881.1 μmol·L-1) and four compounds (14) showed cytotoxicity on HepG2 cells (IC50 in the range of 5.224.2 μmol·L-1).
[Key words] Vitex negundo var. cannabifolia; lignan; phenolic constituent; antiinflammatory activity; cytotoxicity
doi:10.4268/cjcmm20162219
牡荊子為馬鞭草科牡荊屬植物牡荊Vitex negundo var. cannabifolia的干燥成熟果實(shí),具有除濕祛痰,止咳平喘,理氣止痛的功效,主要用于咳嗽氣喘,胃痛,泄瀉,痢疾,疝氣痛,腳氣腫脹,白帶,白濁等[1]?;瘜W(xué)研究表明牡荊子主要含黃酮[24]、木脂素[3,5]、三萜[4]、環(huán)烯醚萜[3]等化合物?,F(xiàn)代藥理研究表明其具有平喘[6]、鎮(zhèn)痛[7]、抗菌[1]、抗炎[8]、抗氧化[5]和抗腫瘤[9]等作用。為進(jìn)一步闡明牡荊子的有效成分,本課題組對(duì)牡荊子70%丙酮提取物的二氯甲烷萃取部位進(jìn)行了系統(tǒng)的化學(xué)成分研究,共分離鑒定了19個(gè)化合物,包括13個(gè)木脂素(1~13)和6個(gè)酚類化合物(14~19)。其中化合物8~10,14,18,19為首次從馬鞭草科植物中分離得到,化合物13為首次從牡荊屬植物中分離得到,化合物6,7,12,15~17為首次從牡荊中分離得到。
1 材料
高效液相離子阱飛行時(shí)間質(zhì)譜儀(日本島津公司);Varian 500 核磁共振儀(美國(guó)Varian公司);半制備型高效液相色譜儀(日本島津公司);半制備HPLC色譜柱(YMCPack ODSA, 10 mm×250 mm, 5 μm)。Sephadex LH20 填料(Amersham Biosciences,瑞典);ODS(40~63 μm,德國(guó)Merck);柱色譜用硅膠(200~300目)及薄層色譜用GF254硅膠預(yù)制板均為青島海洋化工廠生產(chǎn)。丙酮、二氯甲烷、甲醇、乙酸乙酯、正己烷等均為分析純。
牡荊子于2012年9月采自河南省信陽市,由北京大學(xué)藥學(xué)院天然藥物學(xué)系屠鵬飛教授鑒定為V. negundo var. cannabifolia的干燥成熟果實(shí)。標(biāo)本存放于北京中醫(yī)藥大學(xué)中藥現(xiàn)代研究中心(JLIVNC201209)。
2 提取與分離
干燥并粉碎的牡荊子藥材(6.5 kg)在室溫下分別用70%丙酮浸泡提取3次(每次溶劑40 L,浸泡24 h),過濾,合并提取液,濃縮得浸膏(450 g)。取浸膏400 g,用90%甲醇溶解,依次用正己烷、二氯甲烷萃取,得到正己烷部位(180 g)、二氯甲烷部位(90 g)、水部位(110 g)3個(gè)萃取部位。
二氯甲烷萃取物(80 g)經(jīng)硅膠(200~300目)柱色譜分離,正己烷乙酸乙酯(3∶1~0∶1)、二氯甲烷甲醇(25∶1~0∶1)梯度洗脫得到11個(gè)流分(Fr.1~11),F(xiàn)r.11經(jīng)重結(jié)晶,得到1(8.0 g),F(xiàn)r.11的結(jié)晶母液經(jīng)半制備液相色譜純化得3(2.0 mg)。Fr.5經(jīng)Sephadex LH20柱色譜,二氯甲烷甲醇(1∶1)洗脫得到7個(gè)流分(Fr.5a~5g)。Fr.5e,5f合并后經(jīng)硅膠柱色譜和半制備液相色譜純化得7(2.0 mg)。Fr.6經(jīng)Sephadex LH20柱色譜,二氯甲烷甲醇(1∶1)洗脫得到4個(gè)流分(Fr.6a~6d)。Fr.6b經(jīng)硅膠柱色譜和半制備液相色譜純化得6(4.1 mg)和18(4.0 mg)。Fr.7經(jīng)Sephadex LH20柱色譜,二氯甲烷甲醇(1∶1)洗脫得到8個(gè)流分(Fr.7a~7h)。Fr.7e經(jīng)硅膠柱色譜和半制備液相色譜純化得14(2.0 mg),15(2.5 mg)和19(2.3 mg)。Fr.8經(jīng)Sephadex LH20柱色譜,二氯甲烷甲醇(1∶1)洗脫得到15個(gè)流分(Fr.8a~8o)。Fr.8i經(jīng)硅膠柱色譜和半制備液相色譜純化得5(4.5 mg)和8(3.6 mg)。Fr.8l經(jīng)硅膠柱色譜和半制備液相色譜純化得16(4.5 mg)。Fr.9經(jīng)Sephadex LH20柱色譜,以甲醇洗脫得到8個(gè)流分(Fr.9a~9h)。Fr.9d經(jīng)硅膠柱色譜和半制備液相色譜分離純化得17(2.5 mg)。Fr.9e經(jīng)硅膠柱色譜、ODS柱色譜和半制備液相色譜分離純化得9(3.4 mg),10(2.1 mg)和11(2.4 mg)。Fr.10經(jīng)硅膠柱色譜分離,正己烷乙酸乙酯(2∶1~0∶1)洗脫得到5個(gè)流分(Fr.10a~10e)。Fr.10c經(jīng)Sephadex LH20柱色譜得4(10 mg)。Fr.10d經(jīng)反復(fù)硅膠柱色譜、Sephadex LH20柱色譜、ODS柱色譜和半制備液相色譜純化得到2(5 g),12(2.3 mg)和13(2.6 mg)。
3 結(jié)構(gòu)鑒定
4 藥理活性篩選
4.1 體外抗炎活性測(cè)定[21] 取對(duì)數(shù)期生長(zhǎng)的RAW 264.7細(xì)胞制備細(xì)胞數(shù)量為2×105個(gè)/mL細(xì)胞懸浮液,加入96孔板,每孔100 μL,于37 ℃在5% CO2的培養(yǎng)箱中培養(yǎng)24 h,將待測(cè)化合物用完全培養(yǎng)基稀釋成不同濃度的工作液,每個(gè)濃度設(shè)置3個(gè)復(fù)孔。作用于RAW 264.7細(xì)胞1 h后,加入終濃度0.5 mg·L-1的LPS刺激RAW 264.7細(xì)胞,37 ℃孵育24 h后,利用Griess法測(cè)定細(xì)胞培養(yǎng)上清中NO濃度,利用MTT法測(cè)定96孔板中的細(xì)胞活力,并計(jì)算各濃度天然化合物對(duì)RAW 264.7細(xì)胞分泌NO的抑制率和對(duì)RAW 264.7細(xì)胞生長(zhǎng)的抑制率。結(jié)果顯示,化合物3,5,7,10,11,14,15,17能不同程度抑制LPS誘導(dǎo)的RAW 264.7細(xì)胞釋放NO,其IC50分別為(7.8±1.5),(51.0±2.2),(81.1±5.7),(27.9±17.5),(10.5±0.6),(9.4±0.2),(10.5±0.6),(21.4±1.0)μmol·L-1,陽性藥吲哚美辛的IC50為(44.1±5.8)μmol·L-1;其他化合物在100 μmol·L-1(最高實(shí)驗(yàn)濃度)時(shí)的NO抑制率小于50%。所有化合物在100 μmol·L-1時(shí)對(duì)RAW 264.7細(xì)胞生長(zhǎng)沒有明顯抑制作用。
4.2 MTT細(xì)胞增殖毒性測(cè)定 取對(duì)數(shù)期生長(zhǎng)的HepG2人肝癌細(xì)胞制備細(xì)胞數(shù)量為3.5×104個(gè)/mL細(xì)胞懸浮液,加入96孔板,每孔100 μL,于37 ℃在5% CO2的培養(yǎng)箱中培養(yǎng)24 h使其完全貼壁,將待測(cè)化合物用完全培養(yǎng)基稀釋成不同濃度的工作液,每個(gè)濃度設(shè)置5個(gè)復(fù)孔。于加藥后48 h,每孔加入10 μL MTT試劑,在37 ℃作用2 h。用酶標(biāo)儀測(cè)定在570 nm處的吸光度,計(jì)算細(xì)胞存活率。結(jié)果顯示化合物1~4能不同程度抑制HepG2人肝癌細(xì)胞的生長(zhǎng),其IC50分別為(5.2±0.13),(19.7±1.44),(8.3±0.44), (24.2±1.27) μmol·L-1。
5 討論
本研究從牡荊子70%丙酮提取物的二氯甲烷萃取部位分離鑒定了19個(gè)化合物,主要以木脂素類化合物為主,其中13個(gè)化合物為首次從牡荊子中分離得到。同時(shí),對(duì)分離得到的化合物進(jìn)行了體外抗炎活性和細(xì)胞毒活性篩選。結(jié)果表明,8個(gè)化合物能夠不同程度地抑制LPS誘導(dǎo)的RAW 264.7細(xì)胞釋放NO,具有一定的抗炎活性;4個(gè)化合物能顯著抑制HepG2人肝癌細(xì)胞的增殖,具有較強(qiáng)的細(xì)胞毒活性,這些活性成分主要以木脂素類化合物為主。實(shí)驗(yàn)過程中發(fā)現(xiàn),牡荊子中苯代萘型木脂素6羥基4(4羥基3甲氧基苯基)3羥甲基7甲氧基3,4二氫2萘醛(1)和6羥基4(4羥基3甲氧基苯基)3羥甲基5甲氧基3,4二氫2萘醛(2)含量很高,文獻(xiàn)報(bào)道此類木脂素具有多種藥理作用,如抗炎[8]、抗腫瘤[9]、神經(jīng)保護(hù)[22]等,根據(jù)本研究結(jié)果并結(jié)合文獻(xiàn)報(bào)道,推測(cè)牡荊子發(fā)揮藥效的物質(zhì)基礎(chǔ)可能是其中含量較大的苯代萘型木脂素類成分,其作用機(jī)制仍需進(jìn)一步探討與研究。
[參考文獻(xiàn)]
[1] 國(guó)家中醫(yī)藥管理局中華本草編委會(huì). 中華本草. 第6卷 [M]. 上海: 科學(xué)技術(shù)出版社, 1999: 599.
[2] 宋妍, 楊雪, 葛紅娟, 等. 牡荊子的化學(xué)成分 [J]. 中國(guó)實(shí)驗(yàn)方劑學(xué)雜志, 2014, 20 (19): 116.
[3] Yamasaki T, Kawabata T, Masuoka C, et al. Two new lignan glucosides from the fruit of Vitex cannabifolia [J]. J Nat Med, 2008, 62: 47.
[4] 顧湘, 楊雪, 葛紅娟, 等. 牡荊子的化學(xué)成分研究Ⅱ [J]. 西北藥學(xué)雜志, 2015, 30 (2): 114.
[5] Lou Z H, Li H M, Gao L H, et al. Antioxidant lignans from the seeds of Vitex negundo var. cannabifolia [J]. J Asian Nat Prod Res, 2014, 16(9): 963.
[6] 劉懋生, 劉昌林, 顧剛妹, 等. 牡荊子脂質(zhì)對(duì)實(shí)驗(yàn)動(dòng)物氣道平滑肌的影響 [J]. 中國(guó)藥理學(xué)通報(bào), 1993, 9 (4):307.
[7] 鐘世同, 邱光鐸, 劉元帛, 等. 單葉蔓荊子、蔓荊子、黃荊子和牡荊子的藥理活性比較 [J]. 中藥藥理與臨床, 1996 (1): 37.
[8] Zheng C J, Huang B K, Han T, et al. Nitric oxide scavenging lignans from Vitex negundo seeds [J]. J Nat Prod, 2009, 72: 1627.
[9] Zhou Y J, Liu Y E, Cao J G, et al. Vitexins, naturederived lignan compounds, induce apoptosis and suppress tumor growth [J]. Clin Cancer Res, 2009, 15: 5161.
[10] Ono M, Nishida Y, Masuoka C, et al. Lignan derivatives and a norditerpene from the seeds of Vitex negundo [J]. J Nat Prod, 2004, 67: 2073.
[11] Anjaneyulu A S R, Rao A M, Rao V K, et al. Novel hydroxyl lignans from the heartwood of Gmelina arborea [J]. Tetrahedron, 1977, 33: 133.
[12] 青山, 曾光堯, 譚健兵, 等. 黃荊子親脂性化學(xué)成分研究 [J]. 中南藥學(xué), 2011, 9(7): 492.
[13] Cheng M J, Wu C C, Tsai I L, et al. Chemical and antiplatelet constituents from the stem of Zanthoxylum beecheyanum [J]. J Chin Chem Soc, 2004, 51(5A):1065.
[14] Chau V M, Nguyen X N, Hoang T Y, et al. Chemical constituents of Trichosanthes kirilowii and their cytotoxic activities [J]. Arch Pharm Res, 2015, 38: 1443.
[15] Luo Y, Dai H F, Wang H, et al. Chemical constituents from dragon′s blood of Dracaena cambodiana [J]. Chin J Nat Med, 2011, 9(2): 112.
[16] 任鳳芝, 陳書紅, 李麗紅, 等. 橡膠籽種殼的化學(xué)成分研究 [J]. 中國(guó)新藥雜志, 2012, 21(19): 2311.
[17] Richard R, Stange J, James J S, et al. Isolation of a phytoalexin, transpcoumaryl aldehyde, from Cucurbita maxima, Cucurbitaceae [J]. Phytochemistry, 1999, 52: 41.
[18] 趙湘湘, 鄭承劍, 秦路平. 黃荊子的化學(xué)成分研究 [J]. 中草藥, 2012, 43(12): 2346.
[19] 呂靜, 賈凌云, 袁久志, 等. 紅松松針化學(xué)成分的分離與鑒定 [J]. 沈陽藥科大學(xué)學(xué)報(bào), 2011, 28(1): 21.
[20] Tan N, Tao Y W, Pan J H, et al. Isolation, structure elucidation, and mutagenicity of four alternariol derivatives produced by the mangerove endophytic fungus No.2240 [J]. Chem Nat Compd, 2008, 44(3): 296.
[21] Huang Z, Zhu Z X, Li Y T, et al. Antiinflammatory labdane diterpenoids from Leonurus macranthus [J]. J Nat Prod, 2015, 78:2276.
[22] Yang Z B, Tan B, Li T B, et al. Protective effect of vitexin compound B1 against hypoxia/reoxygenationinduced injury in differentiated PC 12 cells via NADPH oxidase inhibition [J]. NS Arch Pharmacol, 2014, doi: 10.1007/s0021001410060.
[責(zé)任編輯 丁廣治]