Pharmacological and analytical aspects of alkannin/shikonin and their derivatives: An update from 2008 to 2022

Alkannin/shikonin (A/S) and their derivatives are naturally occurring naphthoquinones majorly found in Boraginaceae family plants. They are integral constituents of traditional Chinese medicine Zicao (roots of Lithospermum erythrorhizon). In last two decades significant increase in pharmacological investigations on alkannin/shikonin and their derivatives has been reported that resulted in discovery of their novel mechanisms in various diseases and disorders. This review throws light on recently conducted pharmacological investigations on alkannin/shikonin and their derivatives and their outputs. Various analytical aspects are also discussed and brief summary of patent applications on inventions containing alkannin/shikonin and its derivatives is also provided.     

Naphthaquinones of Alkanna Orientalis (L.) BOISS

The roots of Alkanna orientalis (L.) Boiss yielded α- methyl-n-butyl alkannin (compound 1) and alkannin acetate (compound 2). The compounds were identified by UV, MS, ¹H NMR and ¹³C NMR. Quantitative determination of α- methyl-n-butyl alkannin and alkannin acetate in Alkanna orientalis (L) Boiss roots was established by TLC densitometry.     

阿卡宁的氧葡萄糖醛酸化代谢通路研究

目的对紫草素的立体异构单体阿卡宁的氧葡萄糖醛酸代谢通路进行研究和表征。方法采用液相色谱和质谱联用方法检测阿卡宁和其葡萄糖醛酸化代谢产物;将阿卡宁在人肝微粒体(HLM)、人肾微粒体(HKM)以及重组人类葡萄糖醛酸转移酶(UGT)中孵育,观察代谢轮廓、筛选参与催化的重组单酶、考察酶动力学;通过相关性分析以及化学抑制实验阐明UGT单酶对阿卡宁的选择性。结果阿卡宁在含尿苷5’-二磷酸葡萄糖醛酸(UDPGA)的HLM孵育中,可以检测到1个UGT代谢产物。UGT单酶筛选发现UGT1A9高选择性催化阿卡宁。酶动力学研究显示在HLM、HKM和UGT1A9中阿卡宁的UGT代谢都呈底物抑制模式,并且表观亲和常数(Km)在3.75~4.50μmol/L。阿卡宁和已知UGT1A9探针底物异丙酚在12例个体人肝中的UGT代谢具有很好的相关性,R2为0.88。化学抑制实验显示在HLM中,厚朴酚和尼氟灭酸对阿卡宁的UGT代谢具有明显的抑制作用;睾酮、雷公藤红素和尼罗替尼对阿卡宁的UGT代谢均无明显抑制作用。结论 UGT代谢是阿卡宁(紫草素)在人体的重要代谢途径之一,阿卡宁是人类UGT1A9的一个高选择性探针底物。     

SYUNZ‐16, a newly synthesized alkannin derivative,induces tumor cells apoptosis and suppresses tumor growth through inhibition of PKB/AKT kinase activity and blockade of AKT/FOXO signal pathway

Alkannin is the major bioactive compound of Arnebia euchroma roots, which is used in many therapeutic remedies in Chinese traditional medicine. SYUNZ‐16 is a new derivative of alkannin. In this study, anticancer effects of SYUNZ‐16 on human lung adenocarcinoma cell line GLC‐82 and human hepatocarcinoma cell line Hep3B were tested in vitro. The results showed SYUNZ‐16 could obviously inhibit the proliferation of these cancer cell lines via induction of apoptosis, with the evidence of increasing AnnexinV‐positive cells and cleaved caspase‐3 and PARP fragments. More importantly, we found that SYUNZ‐16 could inhibit AKT activity in cell‐free system. Treatment of cancer cells with SYUNZ‐16 decreased the phosphorylation of AKT. Additionally, SYUNZ‐16 partially attenuated the phosphorylation levels of FKHR and FKHRL1 in a dose‐dependent and time‐dependent fashion, and led to an increase in the nuclear accumulation of exogenous FKHR, and upregulated the mRNA expression of Bim and TRADD in cancer cells. Further study showed that constitutively activated AKT1 transfection could reduce apoptosis induction mediated by SYUNZ‐16. The in vivo experiments showed that SYUNZ‐16 had inhibitory effects on S‐180 sarcoma implanted to mice. And in GLC‐82 xenograft models, SYUNZ‐16 at 20 mg/kg/qod remarkably inhibited the tumor growth with the T/C value of 45.3%. Taken together, SYUNZ‐16 might be a potent inhibitor of AKT signaling pathway in tumor cells. These data provide evidence for the development of SYUNZ‐16 as a potential antitumor drug candidate for further research and development.     

Naphthoquinone Components from Alkanna tinctoria (L.) Tausch Show Significant Antiproliferative Effects on Human Colorectal Cancer Cells

Our research to seek active compounds against human colorectal cancer from the root of Alkanna tinctoria (L.) Tausch led to the isolation of two naphthoquinones, alkannin (1) and angelylalkannin (2). The antiproliferative effects of the two compounds on human colon cancer cells HCT‐116 and SW‐480 were determined by the 3,4‐(5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium salt (MTS) method. Cell cycle profile and cell apoptosis were determined using flow cytometry. Both of the two compounds showed significant inhibitory effects on the cancer cells. For alkannin (1) and angelylalkannin (2), the median inhibitory concentration (IC₅₀) values were 2.38 and 4.76 µ m for HCT‐116 cells, while for SW‐480 cells they were 4.53 and 7.03 µ m , respectively. The potential antiproliferative mechanisms were also explored. At concentrations between 1–10 µ m , both compounds arrested the cell cycle at the G1 phase and induced cell apoptosis. Copyright © 2012 John Wiley & Sons, Ltd.     

紫草萘醌(阿卡宁)衍生物SYUNZ-4的抗瘤作用研究

 目的探讨半合成的一种新的阿卡宁衍生物-3,11-双(2-羟乙巯基)-6-异己萘茜(代号为SYUNZ-4)体外对各种人癌细胞的细胞毒作用和体内的抗瘤作用。方法体外细胞毒作用是应用四氮唑蓝(MTT)法测定,以半数抑制浓度(IC₅₀)进行评价。体内抗瘤作用是应用小鼠移植瘤模型和人癌细胞裸鼠移植瘤模型。结果体外细胞毒试验表明,SYUNZ-4对7种人癌细胞有强的细胞毒作用,它们的IC50为0.32～7.75mg·L^-1,对耐药细胞株MCF-7/Adr和KBV200的IC50分别为1.96和7.87mg·L^-1。体内抗瘤试验,SYUNZ-4在2.0,6.0和10.0mg·kg^-1,ip,q2d×10d,对小鼠肉瘤S-180和小鼠肝癌HepS的抑瘤率分别为31.5%～69.7%和45.9%～67.5%(P<0.05～0.01)。SYUNZ-4在2.0,6.0,10.0和14.0mg·kg^-1,对小鼠肿瘤艾氏腹水癌实体型ESC的抑瘤率为30.2%～58.1%(P<0.05～0.01)。在6.0和10.0mg·kg^-1,ip,q3d×18d条件下,SYUNZ-4对人肺腺癌(GLC-82)裸鼠移植瘤的抑瘤率为48.4%和61.7%(P<0.001);在2,6和10mg·kg^-1剂量下对人鼻咽癌细胞(CNE2)裸鼠移植瘤的抑瘤率分别为35.5%、41.9%和48.3%,P<0.01～0.001。结论新合成的紫草醌衍生物SYUNZ-4具有强的细胞毒作用和抗小鼠肿瘤作用。     

Radical scavenging activity of Alkanna tinctoria root extracts and their main constituents, hydroxynaphthoquinones

Alkannin and shikonin (A[sol ]S) are pharmaceutical substances with a wide spectrum of biological properties. Radical scavenging activity is involved in aging processes, antiinssammatory, anticancer and wound healing activities. Hence, in the present study the DPPH radical scavenging activity of alkannin and shikonin, both monomeric and oligomeric, and extracts of Alkanna tinctoria roots were studied and a structure-activity relationship was approximated.It was shown that both monomeric and oligomeric alkannin and shikonin and also A[sol ]S esters exhibited extremely high radical scavenging activity. The presence of the naphthoquinone moiety seems to be essential for that activity, while the side chain of A[sol ]S possibly plays a minor role. Esterification of A[sol ]S on the side chain hydroxyl group does not affect radical scavenging activity. Organic solvents and olive oil (extracted at room temperature) extracts of Alkanna tinctoria roots, which contain as active ingredients A[sol ]S esters, exhibited very good antiradical activity.Alkannin and shikonin and their esters and also extracts of Alkanna tinctoria roots could be used promisingly in pharmaceutical and cosmetic preparations for their radical scavenging activity and probably for their antiaging activity.     

新疆紫草化学成分研究

目的 研究新疆紫草Arnebia euchroma的化学成分。方法 采用硅胶、ODS、凝胶柱色谱等多种柱色谱分离技术进行分离纯化,通过质谱、核磁共振波谱、圆二色谱等技术并结合文献报道的数据,对化合物进行结构鉴定。采用CCK8法,研究化合物对体外培养的人乳腺癌MCF-7细胞增殖的影响。结果 从新疆紫草石油醚部位中共分离得到24个化合物,分别鉴定为(S)-1-(6-异丙基-2,3-二氢-1H-茚-4-基)乙-1-酮（1）、(Z)-2-(2-(2,5-二羟基苯基)-2-氧亚乙基)-6-甲基庚-5-烯酸乙酯（2）、2-(2Z)-(3-羟基-3,7-二甲基-2,6-辛二烯基)-1,4-苯二醇（3）、新藏紫草酚（4）、(+)-(R)-脱氧碘化叶黄素（5）、3-(4-甲基-3-戊烯-1-基)-6-羟基-9-甲氧基-2H-1-苯并氧杂环庚烯-5-酮（6）、对羟基苯甲醛（7）、香草醛（8）、乙酰香草酮（9）、2-羟基-4-甲氧基肉桂醛（10）、guttaquinol B(11)、岩大戟内酯E(12)、3-乙酰氧基齐墩果酸（13）、羟基何帕酮（14）、β-谷甾醇（15）、豆甾-4-烯-3,6-二酮（16）...     

阿卡宁下调Trx/Akt通路诱导急性髓系白血病细胞凋亡的作用研究

目的 探讨新疆紫草Lithospermum erythrorhizon代表成分阿卡宁对急性髓系白血病细胞HL-60的抑制作用及机制。方法 取对数生长期HL-60细胞，采用台盼蓝法检测阿卡宁、紫草素处理后的细胞增殖抑制率；Hoechst 33258染色观察细胞形态；流式细胞仪检测细胞凋亡率；通过系统药理学方法筛选出阿卡宁以及急性髓系白血病（acute myeloid leukemia,AML）的共同靶点并分析得到关键靶点。通过邻苯二甲醛（o-phthalaldehyde,OPA）荧光探针法、差示扫描荧光分析实验在体外分子水平验证化合物与靶点的结合能力。通过Western blotting检测凋亡通路标志分子半胱氨酸天冬氨酸蛋白酶-3(cysteinasparate protease-3,Caspase-3)、cleaved Caspase-3、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白（Bcl-2associated X protein,Bax）蛋白表达。结果 阿卡宁呈剂量相关性抑制HL-60细胞增殖。Hoechst染色后荧光显微镜下可见阿卡...