The Antitumor Constituents from Hedyotis Diffusa Willd

As a TCM, Hedyotis diffusa Willd. has been using to treat malignant tumors, and many studies also showed that the extracts from Hedyotis diffusa Willd. possessed evident antitumor activities. Therefore, we carried out chemical study on Hedyotis diffusa Willd. and investigated the cytotoxicity of the obtained compounds on a panel of eight tumor cell lines. As a result, four new compounds were isolated from Hedyotis diffusa Willd., including three iridoid glycosides of Shecaoiridoidside A–C (1–3) and a cerebroside of shecaocerenoside A (4). Also, six known iridoid compounds (5–10) were also obtained. The cytotoxicity of all compounds against human tumor cell lines of HL-60, HeLa, HCT15, A459, HepG2, PC-3, CNE-2, and BCG-823 were also evaluated in vitro. New compound 3 exhibited evident cytotoxicity to all tumor cell lines except the Hela, and the IC50 values are from 9.6 µM to 62.2 µM, while new compound 4 showed moderate cytotoxicity to all the cell lines, and the IC50 values are from 33.6 µM to 89.3 µM. By contrast, new compound 1 and known compound 9 showed moderate cytotoxicity to HCT15, A459, and HepG2 selectively. Known compound 7 also exhibited moderate cytotoxicity to HCT15 and A459 selectively.


Introduction
As an annual herb, Genus of Hedyotis has been widely distributed in subtropical area of the world [1]. 62 species of Hedyotis are distributed in China, among which more than 20 species are used as medicines, ingcluding Hedyotis diffusa Willd. (H. diffusa Willd.). H. diffusa Willd. has been recorded in Chinese pharmacopoeia (2015 edt) and possesses the efficacies of diuresis to reduce edema, clearing away the heat evil and detoxifying, and promoting blood circulation to arrest pain [2]. Clinically, H. diffusa Willd. has been using to treat urinary tract infection, tonsillitis, appendicitis, pharyngitis, hepatitis, dysentery, diarrhea, and snake bites [1][2][3]. But more importantly, H. diffusa Willd. also showed significant effective on malignant tumors of breast, gastric, colon, rectal, and ovarian [2,4,5]. The components of iridoids, triterpenes, flavonoids, lignans, anthraquinones, alkaloids, cerebrosides, coumarins, and sterols were discovered during the chemical studies of H. diffusa Willd. [2,[6][7][8]. There are some differences of the chemical constituents if the H. diffusa Willd. grown in different parts of China. The contents of anthraquinones and iridoids in H. diffusa Willd. from Guangdong province were higher than the H. diffusa Willd. from the provinces of zhejiang, Jiangxi, Hubei, and Fujian. These include 2,7-dihydroxy-3-methyl anthraquinone, 2-hydroxy-3-methyl-1-methoxy anthraquinone, 2-hydroxy-3-methoxy-7-methoxy anthraquinone, 2-methyl-3-hydroxy anthraquinone, 2-methyl-3-hydroxy-4-methoxy anthraquinone, deacetyl asperulosidic acid, scandoside, E-6-O-p-coumaroyl scandoside methyl ester [9]. Various hepatoprotective, immunoloregulation, anti-tumor, anti-inflammatory, antibacterial, analgesia, sedative, and anti-oxidant activities can be found in pharmacological studies of H. diffusa Willd [3,6,[10][11][12][13], but more studies found that the extracts from H. diffusa Willd. possessed evident anticancer activities [1,2,[14][15][16][17][18][19][20]. H. diffusa Willd. has also been used to treat cancers adjuvantly for a long time in China. With increasing incidence and mortality in China, cancer has become the leading cause of death and caused serious public health problems. According to the latest report, in 2015, about 4,292,000 new cancer cases and 2,814,000 cancer deaths occurred in China, with lung cancer being the most common incident cancer and the leading cause of cancer death. Stomach, esophageal, and liver cancers were also commonly diagnosed and identified as leading causes of cancer death [21]. Therefore, screened active components from H. diffusa Willd. might be helpful. In this study, we carried out chemical study on H. diffusa Willd., and four new (1-4) along with six known (5-10) compounds were obtained. The structures of known compounds were determined by detailed NMR and ESI-MS spectra analyses, as well as comparing the data with the literature. In this paper, we describe the isolation of compounds 1-4 and elucidate their structures. The cytotoxic activity of all compounds against tumor cell lines of HL-60 (human leukemia cells), HeLa (human cervical cancer cells), HCT15 (human colon cancer cells), A459 (human lung cancer cells), HepG2 (human hepatoma cells), PC-3 (human prostate cancer cells), CNE-2 (human nasopharyngeal cancer cells), and BCG-823 (human gastric gland carcinoma cells) were also investigated in vitro. As a result, some active compounds will be screened, and the therapeutic basis of H. diffusa Willd. on tumors will also be revealed.

General
Column chromatographies such as Macroporous resin (AB-8 Crosslinked Polystyrene, Shanxi Lanshen Resin, Xi'an, China), silica gel (200-300 mesh, Hejie Technology Co. Ltd., Shanghai, China), and ODS-A (120A, 50 mm; DAISO, Kyoto, Japan) were used for isolations. Compounds were prepared on a preparative HPLC (Waters, Milford, MA, USA). Bruker AVANCE 400 MHz NMR instrument (Bruker SpectroSpin, Karlsruhe, Germany) was used to measure all the NMR spectra, including 1D-NMR and 2D-NMR spectra. Measured and analyzed the HRESIMS data was conducted on a Xero Q Tof MS spectrometer (Waters, Milford, MA, USA). IR Spectra data was recorded on FTIR-8400S (Shimadzu, Kyoto, Japan). The GC-MS (Angilent, Palo Alto, CA, USA) instrument was used to analysis the volatile derivatives from compounds. The growth of the tumor cell lines was monitored with a microplate reader (BMG FLUOStar OPTIMA, Ortenberg, Germany).

Plant Materials
The aerial part of H. diffusa Willd. was collected from Guangdong province of China and identified by Shuyuan Li of Guangdong Pharmaceutical University. The voucher specimen (No. 20160987) is deposited at the Herbarium of Guangdong Pharmaceutical University, Guangzhou, China.

Acid Hydrolysis of 1-3
Acid hydrolysis experiment was carried out as the method in reference [24]. Briefly, the sugar residues were obtained by hydrolyzing of compounds 1-3 (2.0 mg) with 2 mol/L H 2 SO 4 (2.0 mL), and then treated with trimethylchlorosilane, respectively. The sugar derivatives were further analyzed by GC-MS. As a result, the sugar derivatives from compounds 1 and 3 were determined to be D-galactose (t R = 19.46 min) and D-glucose (t R = 11.33 min), respectively. The sugar derivatives from compound 2 was determined to be D-glucose (t R = 11.33 min) and D-apiose (t R = 14.53 min).

Methanolysis of 4
Methanolysis of 4 was carried out according to the previous study [38]. In short, compound 4 (5.0 mg) was dissolved in in 82 % aqueous MeOH (20 mL) with 5% HCl and refluxed for 18 h. The FAME of 4 was obtained by extracting the reaction mixture with n-hexane. The FAME of 4 was a white amorphous powder,

Conclusions
We

Conflicts of Interest:
The authors declare no conflict of interest.