米碎花的酚苷類化合物及其鼻咽癌細(xì)胞毒活性

梁慧君, 李倩然, 李百琳, 范鈺汶, 敖卓?jī)x, 張偉杰, 吳杰偉, 袁捷

米碎花的酚苷類化合物及其鼻咽癌細(xì)胞毒活性

梁慧君, 李倩然, 李百琳, 范鈺汶, 敖卓?jī)x, 張偉杰, 吳杰偉*, 袁捷*

(廣州中醫(yī)藥大學(xué)中藥學(xué)院，廣州 510006)

為了解米碎花()的化學(xué)成分及其生物活性，運(yùn)用多種色譜技術(shù)從其乙醇提取物分離得到11個(gè)化合物,并對(duì)化合物進(jìn)行體外抗鼻咽癌細(xì)胞增殖活性評(píng)價(jià)。經(jīng)波譜數(shù)據(jù)分析，分別為異落新婦苷 (1)、3,5,7-三羥基色原酮-3---l-鼠李糖苷 (2)、1--反式-桂皮?；?-d-葡萄糖 (3)、1--(4-羥基苯乙基)-6--反式-桂皮?；?-d-葡萄糖(4)、eutigoside D (5)、1--(3,4-二羥基苯乙基)-6--反式-香豆酰基--d-葡萄糖(6)、eutigoside A (7)、1--(4-羥基苯乙基)-6--反式-咖啡酰基--d-葡萄糖 (8)、grayanoside A (9)、1--(4-羥基苯乙基)-6--(4-羥基苯甲?；?--d-葡萄糖(10)、3---d-葡萄糖基-4-羥基-芐基苯甲酸酯(11)。其中，化合物4為首次從天然來(lái)源獲得，化合物2～4和8～11均為首次從該屬植物中分離得到。MTT法表明，化合物10具有中等抑制5-8F細(xì)胞增殖活性。

柃木屬；米碎花；酚苷類；5-8F細(xì)胞；細(xì)胞毒性

柃木屬()是山茶科(Theaceae)的第二大屬，全球約有130種，廣泛分布于亞洲熱帶和亞熱帶地區(qū)。我國(guó)有81種，主要分布于云南、福建、江西、廣東各省區(qū)[1–2]。目前從柃木屬植物中分離得到的化學(xué)成分主要有萜類、黃酮及其苷類、木脂素及其苷類，簡(jiǎn)單苯丙素及其苷類等。同時(shí)，柃木屬植物提取物或化學(xué)成分在抗腫瘤、抗炎、抗氧化等方面表現(xiàn)出顯著的藥理活性[3–9]。米碎花()是山茶科柃木屬常綠灌木，廣泛分布于我國(guó)南方各省，根據(jù)《中華本草》記載[10]，米碎花多以根部或莖葉入藥，具有清熱解毒，除濕斂瘡的功效，多用于預(yù)防流感、治療鼻咽癌以及水火燙傷。研究表明，米碎花中的二萜、香豆素成分具有抗炎、抗腫瘤等活性[6–9]，但關(guān)于該植物系統(tǒng)的化學(xué)成分及其生物活性的報(bào)道較少。

鼻咽癌是我國(guó)南方地區(qū)高發(fā)惡性腫瘤，目前放射治療仍是其首選療法，但其易轉(zhuǎn)移、易復(fù)發(fā)的特點(diǎn)仍然威脅著人的生命健康，因此尋求有效防治鼻咽癌的方法具有十分重要的意義。近年來(lái)，為發(fā)揮我國(guó)中藥資源優(yōu)勢(shì)，從天然來(lái)源獲取抗腫瘤有效成分已日漸取得成效，紫杉醇、喜樹(shù)堿、長(zhǎng)春新堿等目前已成為某些腫瘤的首選藥。本課題對(duì)米碎花乙醇提取物的乙酸乙酯萃取部分進(jìn)行化學(xué)成分的分離鑒定，以及基于米碎花治療鼻咽癌的民間用藥經(jīng)驗(yàn)對(duì)其進(jìn)行體外抗人鼻咽癌細(xì)胞增殖活性評(píng)價(jià)，以尋求具有抗鼻咽癌活性的天然產(chǎn)物。

1 材料和方法

1.1 材料

試驗(yàn)植物于2019年1月采集于廣東省恩平市大松嶺公園，經(jīng)廣州中醫(yī)藥大學(xué)中藥學(xué)院吳杰偉副研究員鑒定為山茶科柃木屬米碎花()的枝葉，樣品存放于廣州中醫(yī)藥大學(xué)中藥學(xué)院新藥先導(dǎo)化合物實(shí)驗(yàn)室，標(biāo)本號(hào)No. 2020032701。

人鼻咽癌細(xì)胞株5-8F購(gòu)于中國(guó)科學(xué)院上海生命科學(xué)院細(xì)胞資源中心。

1.2 儀器和試劑

Bruker AV-400 MHz核磁共振波譜儀；Waters 2695 LC-Waters Acquity ELSD-Waters 3100 SQDMS液質(zhì)聯(lián)用儀；Waters Sunfire?RP C18分析型色譜柱(3.5m, 4.6 mm×100 mm)；MCI Gel CHP20P (75～ 150m, 日本三菱化學(xué))；柱層析硅膠(100～200、200～300目)薄層層析硅膠板(青島海洋化工廠); SephadexLH-20 凝膠(Pharmacia公司)；Agilent 1100 制備型高效液相；Waters Sunfire?RP C18制備型色譜柱(5m, 30 mm×150 mm)；FM-500顯微鏡(上海普丹光學(xué)儀器)；CO2細(xì)胞培養(yǎng)箱(上海力申科學(xué)儀器有限公司)；垂直超凈工作臺(tái)(上海智城分析儀器制造有限公司)；MK3型酶標(biāo)儀(Thermo公司)。

RPMI-1640培養(yǎng)基(Gibco公司)；胎牛血清(Gibco公司)；PBS緩沖液(Gibco公司)；青霉素-鏈霉素(Thermo公司)；細(xì)胞級(jí)DMSO (Sigma公司); MTT噻唑蘭(Sigma公司)；色譜純乙腈(Merck公司)；純水、超純水系統(tǒng)(Millipore公司)；其他有機(jī)溶劑均為分析純。

1.3 提取和分離

35 kg米碎花干燥枝葉用95%乙醇室溫浸泡提取3次，每次7 d，過(guò)濾并合并3次提取液，減壓濃縮得到總浸膏2.4 kg。將總浸膏用水懸浮，乙酸乙酯萃取3次，減壓濃縮合并萃取液，得到乙酸乙酯部位浸膏1.5 kg。乙酸乙酯部位經(jīng)MCI柱層析分離，以乙醇-水[20:80、50:50、80:20，(下同)]梯度洗脫，得到Fr. 2、Fr. 5、Fr. 8共3個(gè)組分。Fr. 5 (600 g)用硅膠拌樣，經(jīng)硅膠柱層析分離，以石油醚-乙酸乙酯(1:1、1:2、0:1)梯度洗脫，根據(jù)薄層色譜結(jié)果合并組分，得到組分Fr. 5A～Fr. 5Q。Fr. 5I～Fr. 5L經(jīng)Sephadex LH-20凝膠柱色譜(甲醇洗脫)分離, 分別得到組分Fr. 5I1～Fr. 5I4、Fr. 5J1～Fr. 5J4、Fr. 5K1～ Fr. 5K3和Fr. 5L1～Fr. 5L4。其中，F(xiàn)r. 5I3 (521.5 mg)、Fr. 5I4 (970.5 mg)、Fr. 5J3 (1.89 g)、Fr. 5K1 (387.8 mg)經(jīng)制備型高效液相(乙腈-水=5:95～35:65, 20 mL/min, 0～120 min)純化，分別得到化合物1 (58.2 mg)、2 (26.1 mg)、3 (28.1 mg)、4 (3.0 mg)。Fr. 5L4 (1.29 g)經(jīng)硅膠柱色譜分離，以二氯甲烷-甲醇(20:1、10:1、5:1)梯度洗脫，根據(jù)薄層色譜結(jié)果合并組分，得到組分Fr. 5L4A～ Fr. 5L4R，再經(jīng)制備型高效液相(乙腈-水=5:95～35:65，20 mL/min，0～120 min)純化, 分別得到化合物5 (6.0 mg)、6 (6.7 mg)、7 (32.1 mg)、8 (8.2 mg)、9 (14.5 mg)、10 (6.2 mg)和11 (8.5 mg)。

1.4 結(jié)構(gòu)鑒定

化合物1 白色粉末；ESI-MS: 451.53 [M + H]+; 分子式C21H22O11;1H NMR (400 MHz, DMSO-6):H11.69 (1H, s, 5-OH), 10.91 (1H, br s, 7-OH), 8.84 (2H, each s, 3′, 4′-OH), 6.78 (1H, d,= 1.6 Hz, H-2′), 6.68～6.63 (2H, m, H-5′, 6′), 5.89 (1H, d,= 2.0 Hz, H-8), 5.86 (1H, d,= 2.0 Hz, H-6), 5.49 (1H, d,= 2.6 Hz, H-2), 4.74 (1H, d,= 4.3 Hz, H-3), 4.70 (1H, d,= 1.5 Hz, H-1″), 3.39～2.36 (4H, m, H-2″～H- 5″), 0.78 (3H, d,= 6.1 Hz, H-6″);13C NMR (100 MHz, DMSO-6):C80.0 (C-2), 73.4 (C-3), 193.1 (C-4), 164.0 (C-5), 96.2 (C-6), 167.1 (C-7), 95.2 (C-8), 162.5 (C-9), 100.3 (C-10), 126.4 (C-1′), 115.1 (C-2′), 145.0 (C-3′), 145.2 (C-4′), 114.1 (C-5′), 117.6 (C-6′), 98.8 (C-1″), 70.2 (C-2″), 70.3 (C-3″), 71.2 (C-4″), 69.0 (C-5″), 17.6 (C-6″)。以上數(shù)據(jù)與文獻(xiàn)[11]報(bào)道一致, 故鑒定為異落新婦苷。

化合物2 白色粉末; ESI-MS: 341.62 [M + H]+; 分子式C15H16O9;1H NMR (400 MHz, CD3OD):H8.11 (1H, s, H-2), 6.34 (1H, d,= 2.0 Hz, H-8), 6.22 (1H, d,= 2.1 Hz, H-6), 5.33 (1H, d,= 1.7 Hz, H-1′), 4.14 (1H, dd,= 1.8, 3.4 Hz, H-2′), 3.82 (1H, dd,= 3.2, 9.1 Hz, H-3′), 3.76 (1H, dd,= 6.2, 9.4 Hz, H-5′), 3.47 (1H, t,= 9.5 Hz, H-4′), 1.28 (3H, d,= 6.2 Hz, H-6′);13C NMR (100 MHz, CD3OD):C147.9 (C-2), 140.3 (C-3), 178.7 (C-4), 163.4 (C-5), 100.0 (C-6), 166.2 (C-7), 94.9 (C-8), 159.3 (C-9), 106.4 (C-10), 102.1 (C-1′), 71.6 (C-2′), 73.5 (C-3′), 71.1 (C-4′), 71.9 (C-5′), 17.9 (C-6′)。以上數(shù)據(jù)與文獻(xiàn)[12]報(bào)道一致，故鑒定為3,5,7-三羥基色原酮-3---l-鼠李糖苷。

化合物3 黃色粉末; ESI-MS: 311.37 [M + H]+; 分子式C15H18O7;1H NMR (400 MHz, CD3OD):H7.81 (1H, d,= 16.0 Hz, H-3′), 7.62 (2H, m, H-5′, 9′), 7.42 (3H, m, H-6′, 7′, 8′), 6.59 (1H, d,= 16.0 Hz, H-2′), 5.63 (1H, d,= 7.8 Hz, H-1), 3.88 (1H, dd,= 1.8, 12.0 Hz, H-6a), 3.72 (1H, dd,= 4.5, 12.0 Hz, H-6b), 3.50～3.32 (4H, m, H-2, 3, 4, 5);13C NMR (100 MHz, CD3OD):C95.9 (C-1), 73.9 (C-2), 77.9 (C-3), 71.0 (C-4), 78.8 (C-5), 62.3 (C-6), 167.1 (C-1′), 118.2 (C-2′), 147.7 (C-3′), 135.5 (C-4′), 129.3 (C-5′, 9′), 130.0 (C-6′, 8′), 131.8 (C-7′)。以上數(shù)據(jù)與文獻(xiàn)[13]報(bào)道一致，故鑒定為1--反式-肉桂?；?-d-葡萄糖。

化合物4 黃色粉末; ESI-MS: 431.19 [M + H]+; 分子式C23H26O8;1H NMR (400 MHz, CD3OD):H7.70 (1H, d,= 16.0 Hz, H-3′), 7.54 (2H, m, H-5′, 9′), 7.40 (3H, m, H-6′, 7′, 8′), 7.04 (2H, d,= 8.4 Hz, H-4″, 8″), 6.64 (2H, d,= 8.5 Hz, H-5″, 7″), 6.55 (1H, d,= 16.0 Hz, H-2′), 4.53 (1H, dd,= 2.2, 11.9 Hz, H-6a), 4.37 (1H, d,= 6.3 Hz, H-6b), 4.34 (1H, d,= 7.9 Hz, H-1), 3.95 (1H, m, H-1″a), 3.72 (1H, m, H- 1″b), 3.54 (1H, m, H-5), 3.37 (2H, overlapped, H-2, 3), 3.22 (1H, t,= 7.8 Hz, H-4), 2.84 (2H, t,= 7.6 Hz, H-2″);13C NMR (100 MHz, CD3OD):C104.6 (C-1), 75.0 (C-2), 75.4 (C-3), 71.8 (C-4), 77.9 (C-5), 64.7 (C-6), 168.5 (C-1′), 118.6 (C-2′), 146.6 (C-3′), 135.7 (C-4′), 129.2 (C-5′, 9′), 130.0 (C-6′, 8′), 131.6 (C-7′), 72.5 (C-1″), 36.5 (C-2″), 130.5 (C-3″), 130.9 (C-4″, 8″), 116.1 (C-5″, 7″), 156.8 (C-6″)。以上數(shù)據(jù)與文獻(xiàn)[14]報(bào)道一致，故鑒定為1--(4-羥基苯乙基)-6--反式-桂皮?；?-d-葡萄糖。

化合物5 黃色粉末; ESI-MS: 447.37 [M + H]+; 分子式C23H26O9;1H NMR (400 MHz, CD3OD):H7.69 (1H, d,= 16.0 Hz, H-3′), 7.55 (2H, m, H-5′, 9′), 7.38 (3H, m, H-6′, 7′, 8′), 6.61 (1H, d,= 2.9 Hz, H-4″), 6.59 (1H, d,= 8.7 Hz, H-7″), 6.55 (1H, d,= 16.1 Hz, H-2′), 6.47 (1H, dd,= 2.9, 8.6 Hz, H-8″), 4.53 (1H, dd,= 2.2, 11.8 Hz, H-6a), 4.36 (1H, d,= 7.8 Hz, H-1), 4.34 (1H, dd.= 6.0, 11.9 Hz, H-6b), 3.99 (1H, m, H-1″a), 3.79 (1H, m, H-1″b), 3.55 (1H, m,H-5), 3.37 (2H, overlapped, H-3, 4), 3.23 (1H, m, H-2), 2.89 (2H, t,= 7.5 Hz, H-2″);13C NMR (100 MHz, CD3OD):C104.7 (C-1), 75.4 (C-2), 77.9 (C-3), 71.7 (C-4), 75.0 (C-5), 64.9 (C-6), 168.5 (C-1′), 118.6 (C-2′), 146.6 (C-3′), 135.7 (C-4′), 129.3 (C-5′, 9′), 130.0 (C-6′, 8′), 131.5 (C-7′), 71.1 (C-1″), 32.2 (C-2″), 127.0 (C-3″), 118.3 (C-4″), 151.1 (C-5″), 149.4 (C-6″), 114.8 (C-7″), 116.9 (C-8″)。以上數(shù)據(jù)與文獻(xiàn)[15]報(bào)道一致，故鑒定為eutigoside D。

化合物6 黃色粉末; ESI-MS: 463.23 [M + H]+; 分子式C23H26O10;1H NMR (400 MHz, CD3OD):H7.62 (2H, d,= 8.7 Hz, H-5′, 9′), 6.87 (2H, d,= 12.8 Hz, H-3′), 6.74 (2H, d,= 8.7 Hz, H-6′, 8′), 6.66 (2H, overlapped, C-4″, 7″), 6.52 (1H, dd,= 2.0, 8.0 Hz,H-8″), 5.80 (1H, d,= 12.7 Hz, C-2′), 4.47 (1H, dd,= 2.1, 11.8 Hz, H-6a), 4.28 (1H, d,= 7.8 Hz, H-1), 4.27 (1H, dd,= 6.1, 11.9 Hz, H-6b), 3.89 (1H, m, H-1″a), 3.66 (1H, m, H-1″b), 3.48 (1H, m, H-5), 3.35 (1H, m, H-4), 3.29 (1H, m, H-3), 3.19 (1H, t,= 7.7 Hz,H-2), 2.75 (2H, m, H-2″);13C NMR (100 MHz, CD3OD):C104.1 (C-1), 75.0 (C-2), 77.9 (C-3), 71.7 (C-4), 75.3 (C-5), 64.5 (C-6), 168.2 (C-1′), 116.3 (C-2′), 145.3 (C-3′), 127.6 (C-4′), 115.9 (C-5′, 9′), 133.7 (C-6′, 8′), 160.0 (C-7′), 72.3 (C-1″), 36.6 (C-2″), 131.4 (C- 3″), 117.1 (C-4″), 144.6 (C-5″), 146.1 (C-6″), 116.3 (C-7″), 121.2 (C-8″)。以上數(shù)據(jù)與文獻(xiàn)[16]報(bào)道基本一致，故鑒定為1--(3,4-二羥基苯乙基)-6--反式-香豆酰基--d-葡萄糖。

化合物7 白色粉末; ESI-MS: 469.68 [M + Na]+; 分子式C23H26O9;1H NMR (400 MHz, DMSO-6):H7.52 (1H, d,= 15.9 Hz, H-3′), 7.48 (2H, d,= 8.6 Hz, H-5′, 9′), 6.98 (2H, d,= 8.4 Hz, H-4″, 8″), 6.76 (2H, d,= 8.6 Hz, H-6′, 8′), 6.59 (2H, d,= 8.4 Hz, H-5″, 7″), 6.38 (1H, d,= 15.9 Hz, H-2′), 4.37 (1H, dd,= 2.0, 11.9 Hz, H-6a), 4.22 (1H, d,= 7.8 Hz, H-1), 4.15 (1H, dd,= 6.6, 11.8 Hz, H-6b), 3.78 (1H, m, H-1″a), 3.59 (1H, m, H-1″b), 3.38 (1H, m, H-5), 3.13 (2H, overlapped, H-2, 3), 2.98 (1H, m, H-4), 32.70 (2H, t,= 7.5 Hz, H-2″);13C NMR (100 MHz, DMSO-6):C103.0 (C-1), 73.3 (C-2), 76.5 (C-3), 70.1 (C-4), 73.7 (C-5), 63.6 (C-6), 166.0 (C-1′), 114.5 (C-2′), 145.3 (C-3′), 125.0 (C-4′), 115.7 (C-5′, 9′), 129.7 (C-6′, 8′), 159.8 (C-7′), 70.2 (C-1″), 35.4 (C-2″), 128.5 (C-3″), 130.4 (C-4″, 8″), 115.0 (C-5″, 7″), 155.6 (C-6″)。以上數(shù)據(jù)與文獻(xiàn)[3]報(bào)道一致，故鑒定為eutigoside A。

化合物8 黃色粉末; ESI-MS: 485.61 [M +Na]+; 分子式C23H26O10;1H NMR (400 MHz, CD3OD):H7.56 (1H, d,= 15.8 Hz, H-3′), 7.05 (1H, m, H-5′), 7.03 (2H, m, H-4″, 8″), 6.89 (1H, dd,= 2.0, 8.2 Hz, H-9′), 6.77 (1H, d,= 8.2 Hz, H-8′), 6.65 (2H, d,= 8.3 Hz, H-5″, 7″), 6.28 (1H, d,= 15.9 Hz, H-2′), 4.49 (1H, dd,= 2.2, 11.8 Hz, H-6a), 4.34 (1H, dd,= 6.0, 11.9 Hz, H-6b), 4.33 (1H, d,= 7.9 Hz, H-1), 3.94 (1H, m, H-1″a), 3.73 (1H, m, H-1″b), 3.52 (1H, m, H-5), 3.36 (2H, overlapped, H-3, 4), 3.21 (1H, m, H-2), 2.83 (2H, m, H-2″);13C NMR (100 MHz, CD3OD):C104.5 (C-1), 75.0 (C-2), 77.9 (C-3), 71.7 (C-4), 75.4 (C-5), 64.6 (C-6), 169.1 (C-1′), 114.8 (C-2′), 147.2 (C-3′), 127.7 (C-4′), 115.0 (C-5′), 149.6 (C-6′), 146.2 (C-7′), 116.5 (C-8′), 123.1 (C-9′), 72.4 (C-1″), 36.5 (C-2″), 130.6 (C-3″), 130.9 (C-4″, 8″), 116.1 (C-5″, 7″), 156.7 (C-6″)。以上數(shù)據(jù)與文獻(xiàn)[17]報(bào)道一致，故鑒定為1--(4-羥基苯乙基)-6--反式-咖啡?；?-d-葡萄糖。

化合物9 白色粉末; ESI-MS: 476.63 [M + Na]+; 分子式C24H28O10;1H NMR (400 MHz, CD3OD):H7.62 (1H, d,= 15.8 Hz, H-3′), 7.14 (1H, d,= 1.9 Hz, H-5′), 7.03 (2H, d,= 8.3 Hz, H-4″, 8″), 7.01 (1H, overlapped, H-9′), 6.80 (1H, d,= 8.1 Hz, H-8′), 6.64 (2H, d,= 8.4 Hz, H-5″, 7″), 6.38 (1H, dd,= 15.9 Hz, H-2′), 4.51 (1H, dd,= 2.2, 11.9 Hz, H-6a), 4.35 (1H, dd,= 6.2, 11.9 Hz, H-6b), 4.33 (1H, d,= 7.8 Hz, H-1), 3.94 (1H, m, H-1″a), 3.86 (3H, s, 6′-OCH3), 3.71 (1H, m, H-1″b), 3.53 (1H, m, H-5), 3.37 (2H, overlapped, H-3, 4), 3.22 (1H, m, H-2), 2.83 (2H, m, H-2″);13C NMR (100 MHz, CD3OD):C104.5 (C-1), 75.0 (C-2), 77.9 (C-3), 71.8 (C-4), 75.4 (C-5), 64.7 (C-6), 169.1 (C-1′), 115.2 (C-2′), 147.1 (C-3′), 127.6 (C-4′), 111.6 (C-5′), 150.6 (C-6′), 149.3 (C-7′), 116.5 (C-8′), 124.2 (C-9′), 72.5 (C-1″), 36.5 (C-2″), 130.6 (C-3″), 130.9 (C-4″, 8″), 116.1 (C-5″, 7″), 156.7 (C-6″), 56.4 (6′-OCH3)。以上數(shù)據(jù)與文獻(xiàn)[18]報(bào)道一致，故鑒定為grayanoside A。

化合物10 黃色粉末; ESI-MS: 419.50 [M - H]–；分子式C21H24O9；1H NMR (400 MHz, CD3OD):H7.89 (2H, d,= 8.8 Hz, H-3′, 7′), 6.98 (2H, d,= 8.5 Hz, H-4″, 8″), 6.80 (2H, d,= 8.8 Hz, H-4′, 6′), 6.66 (2H, d,= 8.4 Hz, H-5″, 7″), 4.59 (1H, dd,= 2.2, 11.8 Hz, H-6a), 4.41 (1H, dd,= 6.3, 11.8 Hz, H-6b), 4.34 (1H, d,= 7.8 Hz, H-1), 3.90 (1H, m, H-1″a), 3.72 (1H, m, H-1″b), 3.58 (1H, m, H-5), 3.39 (2H, overlapped, H-3, 4), 3.22 (1H, m, H-2), 2.81 (2H, m, H-2″);13C NMR (100 MHz, CD3OD):C104.5 (C-1), 75.1 (C-2), 78.0 (C-3), 71.9 (C-4), 75.5 (C-5), 64.8 (C-6), 168.0 (C-1′), 122.1 (C-2′), 132.9 (C-3′, 7′), 116.2 (C-4′, 6′), 163.7 (C-5′), 72.3 (C-1″), 36.5 (C-2″), 130.5 (C-3″), 130.9 (C-4″, 8″), 116.1 (C-5″, 7″), 156.7 (C-6″)。以上數(shù)據(jù)與文獻(xiàn)[19]報(bào)道一致，故鑒定為1--(4-羥基苯乙基)-6--(4-羥基苯甲?；?--d-葡萄糖。

化合物11 黃色粉末; ESI-MS: 429.23 [M + Na]+; 分子式C20H22O9;1H NMR (400 MHz, CD3OD):H7.62 (1H, d,= 3.0 Hz, H-2′), 7.48 (2H, dd,= 1.8, 8.4 Hz, H-2, 6), 7.41 (2H, m, H-3, 5), 7.36 (1H, m, H-4), 7.32 (1H, dd,= 3.0, 9.0 Hz, H-6′), 6.89 (1H, d,= 9.0 Hz, H-5′), 5.39 (2H, d,= 4.0 Hz, H-7), 4.75 (1H, d,= 6.3 Hz, H-1″), 3.80 (1H, dd,= 2.3, 12.1 Hz, H-6″a), 3.69 (1H, dd,= 5.0, 12.1 Hz, H-6″b), 3.42 (3H, m, H-3″, 4″, 5″), 3.36 (1H, m, H-2″);13C NMR (100 MHz, CD3OD):C137.0 (C-1), 129.4 (C-2, 6), 129.7 (C-3, 5), 129.5 (C-4), 68.2 (C-7), 113.4 (C-1′), 118.4 (C-2′), 151.5 (C-3′), 158.4 (C-4′), 119.1 (C-5′), 127.3 (C-6′), 170.7 (C-7′), 103.6 (C-1″), 74.8 (C-2″), 77.9 (C-3″), 71.1 (C-4″), 78.0 (C-5″), 62.3 (C- 6″)。以上數(shù)據(jù)與文獻(xiàn)[20]報(bào)道一致，故鑒定為3---d-葡萄糖基-4-羥基-芐基苯甲酸酯。

圖1 化合物1～11的化學(xué)結(jié)構(gòu)式

1.5 細(xì)胞毒活性評(píng)價(jià)

采用四氮唑鹽還原法(MTT法)[21]評(píng)價(jià)米碎花化學(xué)成分1～11體外抗人鼻咽癌細(xì)胞(5-8F)增殖活性。取化合物1～11，收集對(duì)數(shù)生長(zhǎng)期的5-8F細(xì)胞，以含10%胎牛血清及1%青霉素-鏈霉素的RPMI-1640培養(yǎng)基制成細(xì)胞懸液，以1×104/孔接種于96孔板后置于37 ℃，5% CO2培養(yǎng)箱培養(yǎng)。培養(yǎng)12 h后，設(shè)置調(diào)零孔(培養(yǎng)基)、對(duì)照孔(細(xì)胞、培養(yǎng)基)和給藥孔，給藥孔分別加入濃度為100、50、25、12.5、6.25、3.12mol/L的待試藥液，并設(shè)置3個(gè)復(fù)孔，37 ℃，5% CO2培養(yǎng)。24 h后每孔加入20L MTT溶液(5 mg/mL)，繼續(xù)培養(yǎng)4 h后終止培養(yǎng)，棄去孔內(nèi)培養(yǎng)液，每孔加入150L DMSO，置于搖床上震蕩10 min，在酶標(biāo)儀490 nm處檢測(cè)各孔吸光值，計(jì)算細(xì)胞抑制率以及半數(shù)抑制濃度(IC50)。

結(jié)果表明，化合物10具有中等抑制5-8F細(xì)胞增殖活性，其IC50值為(29.52±3.34)mol/L，其他化合物均顯示出微弱的抑制5-8F細(xì)胞增殖活性(IC50值>100mol/L)。

2 結(jié)果和討論

本研究通過(guò)多種色譜技術(shù)對(duì)米碎花植物化學(xué)成分進(jìn)行分離，經(jīng)波譜數(shù)據(jù)分析得到11個(gè)化合物，分別為異落新婦苷 (1)、3,5,7-三羥基色原酮-3--- l-鼠李糖苷 (2)、1--反式-桂皮?；?-d-葡萄糖 (3)、1--(4-羥基苯乙基)-6--反式-桂皮酰基--d-葡萄糖(4)、eutigoside D (5)、1--(3,4-二羥基苯乙基)-6--反式-香豆?；?-d-葡萄糖(6)、eutigoside A (7)、1--(4-羥基苯乙基)-6--反式-咖啡?；?-d-葡萄糖(8)、grayanoside A (9)、1--(4-羥基苯乙基)-6--(4-羥基苯甲?；?--d-葡萄糖(10)、3--- d-葡萄糖基-4-羥基-芐基苯甲酸酯(11)。其中，化合物4為首次從植物中分離得到，化合物2～4、8～11均為首次從該屬植物中分離獲得。同時(shí)，本研究評(píng)價(jià)化合物1～11對(duì)人鼻咽癌細(xì)胞5-8F增殖活性的影響，結(jié)果表明，化合物10具有中等抑制5-8F細(xì)胞增殖活性。

研究表明，酚苷類化合物在抗氧化、抗腫瘤、抗病毒、抗炎等方面表現(xiàn)出廣泛的藥理活性[22–24]。根據(jù)報(bào)道，本研究鑒定的化合物2具有較強(qiáng)的細(xì)胞毒活性，能夠抑制人結(jié)腸癌細(xì)胞、口腔表皮樣癌細(xì)胞以及乳腺癌細(xì)胞的增殖[25]；化合物6對(duì)DPPH自由基具有較高的清除能力，從而表現(xiàn)出明顯的抗氧化活性[26]；化合物7通過(guò)抑制過(guò)氧化氫誘導(dǎo)的氧化應(yīng)激人結(jié)腸癌細(xì)胞中IL-8的分泌，表現(xiàn)出一定的抗氧化、抗炎活性[27]。

本研究所得部分成分具有一定的抗鼻咽癌活性，為米碎花的抗鼻咽癌應(yīng)用提供了科學(xué)依據(jù)，也為米碎花的進(jìn)一步研究與開(kāi)發(fā)提供依據(jù)。

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Phenolic Glycosides fromand Their Cytotoxic Activities Against Nasopharyngeal Carcinoma Cells

LIANG Huijun, LI Qianran, LI Bailin, FAN Yuwen, AO Zhuoyi, ZHANG Weijie, WU Jiewei*, YUAN Jie*

(School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine,Guangzhou 510006, China)

To clarify the chemical constituents and their biological activities ofeleven compounds were isolated from its MeOH extract by various column chromatography methods, and the anti- proliferative activity of compounds against human nasopharyngeal carcinoma cells (5-8F) were evaluated. Basis on spectral data, their structures were identified as isoastinlbin (1), 3,5,7-trihydroxychromone-3---l-arabino- pyranoside (2), 1---cinnamoyl--d-glucopyranose (3), 2-(4-hydroxyphenyl)ethyl-6--()-cinnamoyl--d- glucopyranoside (4), eutigoside D (5), 6--coumaroyl-1--[2-(3,4-dihydroxyphenyl)ethyl]--d-glucopyranoside (6), eutigoside A (7), 2-(4-hydroxypheny)ethyl-[6--()-caffeoyl]--d-glucopyranoside (8), grayanoside A (9), 4-hydroxyphenylethyl-1---d-[6′--(4-hydroxybenzoyl)]-glucopyranoside (10), benzyl-4′-hydroxybenzoyl-3′---d-glucopyranoside (11). Compound 4 was isolated from natural resources for the first time, while the compounds 2-4, 8-11 were obtained from the genusfor the first time. The compound 10 showed moderate inhibitory activity on the proliferation of 5-8F cells by MTT method.

;; Phenolic glycoside; 5-8F cell; Cytotoxicity

10.11926/jtsb.4622

2022-02-10

2022-03-28

國(guó)家自然科學(xué)基金項(xiàng)目(81903509)資助

This work was supported by the National Natural Science Foundation of China (Grant No. 81903509).

梁慧君，女，碩士研究生，研究方向?yàn)橹兴幮滤幯邪l(fā)。E-mail: jun-7387289@163.com

. E-mail: wujiewei@gzucm.edu.cn; yuanjie@gzucm.edu.cn