Antiproliferative Effect and the Isolated Compounds of Pouzolzia indica

Previous report showed the high potent antiproliferative effect of the methanolic part extracted from the aerial parts of Pouzolzia indica on NB4 and HT93A acute leukemic cell lines with the IC50 values of 28.5 and 49.8 μg/mL, respectively. The bioassay-guided fractionation of the methanolic part gave 5 fractions, that is, FFI–FFV. FFII, FFIII, and FFIV inhibited the above leukemic cell lines with the IC50 values of 15.1 (FFII), 14.4 (FFIII), 32.1 (FFIV), and 31.0 (FFII), 9.7 (FFIII), 10.5 (FFIV) μg/mL, respectively. The compounds in these fractions were isolated using chromatographic technique. FFII contained friedelin 1, 28-hydroxy-3-friedelanone 2, and 7-methoxy-coumarin 3. FFIII contained 6, 7-dimethoxy-coumarin 4, scopoletin 5, methyl caffeate 6. FFIV contained sitosteryl glucoside 7 and a supposed glycosphingolipid 8. The chemical structures were elucidated by spectroscopic methods.


Introduction
Pouzolzia indica Gaudich.var. angustifolia Wedd. (local name "Non tai baihong") is a Thai medicinal plant in the family Urticaceae [1,2]. It was used as remedy for the ailments in female infertility, cancer, and inflammation and as emmenagogue and insecticide [3]. The chemical constituents in P. indica were scarcely reported. Only lanceolone, an isoflavone compounds, was isolated [4]. Previously, the antiproliferative effect of the methanolic part of this plant was reported [5]. It could inhibit the growth of NB4 and HT93A cells with the IC 50 values of 28.5±0.1 and 49.8±0.7 g/mL [5], respectively. The apoptosis of NB4 cells treated with 75 g/mL of this fraction for 24 hours increased from 3.2% to 22.2%, whereas HT93A cells underwent apoptosis from 3.0% to 51.3% when treated with the methanolic part at 150 g/mL [5]. The previous results, therefore, showed high potent antiproliferative effect of this methanolic part on these acute leukemic cells. From this study, the active extract was further fractionated using column chromatography. The active fractions were determined by bioassay. The compounds in each fraction were isolated and structurally identified.

Results
The antiproliferative effect of FFI-FFV on human leukemic cell lines was investigated as shown in Figure 3. It was found that FFII, FFIII, and FFIV could inhibit growth of NB4 and HT93A (Figure 4). Therefore, FFII, FFIII, and FFIV were continued to evaluate the IC 50 values on these cell lines at varying concentrations ranging from 0 to 50 g/mL.
The results showed that FFII, FFIII, and FFIV had the IC 50 values on NB4 cell line at 15.1 ± 0.5 g/mL, 14.4 ± 0.6, and 32.1 ± 0.7 g/mL, respectively, whereas the IC 50 values of the HT93A cell line were 31.0 ± 0.1, 9.7 ± 1.3, and 10.5 ± 0.7 g/mL, respectively, as shown in Figure 4. Additionally, FFIII inhibited growth strongly on both NB4 and HT93A cell lines, while FFII inhibits growth strongly on NB4 more than HT93A. FFIV showed strong growth inhibition on HT93A more than NB4. The active fractions FFII, FFIII, and FFIV were further chromatographed on the silica gel columns repeatedly, and the isolated compounds were identified using spectroscopic 6 Evidence-Based Complementary and Alternative Medicine methods. FFII was composed of friedelin 1 and 28-hydroxy-3friedelanone 2 and 7-methoxy-coumarin or herniarin 3. FFIII was composed of 6,7-dimethoxy-coumarin or scoparone 4, scopoletin 5, and methyl caffeate 6. FFIV was composed of sitosteryl glucoside 7 and a supposed glycosphingolipid 8. Sitosteryl glucoside 7 (85 mg) was isolated which was the highest yield as shown in Figure 1.

Discussion
P. indica, which has been long used in Thai traditional medicine for treating various diseases including malignancies, was investigated in this study. Based on our previous report [5], the methanolic part of this plant showed high potent antiproliferative effect on NB4 and HT93A acute promyelocytic cell lines. Here, in this study, we demonstrated that eight compounds were isolated from this active methanolic part. It was chromatographed on CC repeatedly as shown in Figure 1. The bioassay determined the active fractions; they were FFII, FFIII, and FFIV. We found that FFII could inhibit growth on NB4 stronger than HT93A, while FFIII showed growth inhibition on both NB4 and HT93A. Interestingly, FFIV exhibited dominantly growth inhibition on HT93A. The differences in the antiproliferative effects of these fractions might arise from the different active compounds themselves and the interactions with the oncoproteins in these acute promyelocytic cell lines, that is, the long and short types of PML-RAR in NB4 and HT93A, respectively.
The antiproliferative effect of FFII might be caused by the presence of 2 triterpenes, that is, friedelin 1, and 28hydroxy-3-friedelanone 2, and one coumarin 3, namely, 7-methoxy-coumarin. Previously, the cytotoxicity of 28hydroxy-3-friedelanone against A549-human lung cancer cell line, LLC-mouse Lewis lung carcinoma, HL60-human promyelocytic cell line, and MCF7-human breast cancer cell line were demonstrated. Hence, some triterpenes could strongly induce apoptosis by attending the mitochondrial membrane potential and regulating the expression of Bcl-2 different compasses [14,20]. The IC 50 ( g/mL) of 7-methoxycoumarin 3 on HL60 and K562 human chronic leukemia cells was also demonstrated with the values of 28.9 and 19.3 g/mL, respectively [21,22].
For FFIII, the cytotoxic activity of this fraction might result from coumarins (4, and 5, namely, 6,7-dimethoxycoumarin, and scopoletin, resp.) including methyl caffeate 6. The previous reports demonstrated that coumarins could inhibit several human cancer cell lines such as QU-DB large cell lung cancer, and human leukemia HL60 cells [22,23]. The mechanism of action of coumarins was exerted from the inhibition of tubulin polymerization and the induction of cell cycle arrest at G2/M phase [23]. The involvement of cell cycle inhibition might be due to the inhibition of the release of cyclin D1, an essential enzyme in cell cycle progression [24]. Interestingly, high concentration of scopoletin can have antiproliferative effect on lymphoma cell line by inducing apoptosis [25]. In addition, methyl caffeate can inhibit growth of human cervical adenocarcinoma cell line (HeLa) [26]. Notably, methyl caffeate, which contains 2 hydroxyl groups  on aromatic ring, can induce cytotoxic activity via the strong antioxidant activity from these hydroxyl groups [27]. FFIV inhibited HT93A stronger than NB4 cells. It contained sitosteryl glucoside 7 and a supposed glycosphingolipid 8. The partial structures of 8 included two acyl chains, one of which was palmitic acid, -D-glucose, and an amino alcohol. 1 H-NMR spectrum of 8 showed that the typical resonances of amino alcohol part of glycosphingolipid were H-1a at 4.24 (1H, m), H-1b 4.66 (1H, m), and H-2 and H-3 4.75 (2H, m) ( Figure 5) [3,28]. One acyl chain was biosynthetically originated from palmitoyl-CoA which was shown by the long chain methylene protons of 8 appearing as multiplets at 1.1-1.3 [29]. The presence of sugar protons as complex multiplets at 3.90-4.50 ppm (7H, m, from H-1 to H-6 ) was substantiated by carbon signals at 105.6 (C1 ), 75.4 (C2 ), 78.6 (C3 ), 71.8 (C4 ), 78.4 (C5 ), and 63.0 (C6 ). The structure of 8 was thus supposed to be a glycosphingolipid. The sitosteryl glucoside 7 was previously reported to have the antiproliferative effect on human colon cancer cell by inducing the apoptotic pathway [27]. The glycosphingolipid 8, which contains sphingosine, can induce apoptosis involving with the ceramide and sphingosine-1phosphate-mediated pathway [30,31]. The result from our study pointed out that coumarins were promising anticancer agent [32]. The extract fraction containing mainly coumarins like FFIII could be developed as a drug material for anticancer phytopharmaceutical.

Conclusion
The methanolic part of P. indica extract inhibited the acute promyelocytic leukemia cell lines, NB4, and HT93A. The bioassay-guided fractionation of the active part got three different active fractions. They were FFII, FFIII, and FFIV. The FFII showed strong growth inhibition on NB4, whereas the FFIII exhibited strong growth inhibition on both NB4 and HT93A. The FFIV demonstrated strong growth inhibition on HT93A. The active compounds isolated from the FFII contained mainly triterpenoids (friedelin 1 and 28-hydroxy-3friedelanone 2) and some coumarins (7-methoxy-coumarin 3). The FFIII contained mainly phenolic compounds (scoparone 4, scopoletin 5, and methyl caffeate 6), and the FFIV contained mainly glycosides (sitosteryl glucoside 7 and glycosphingolipid 8). P. indica was the first report about antiproliferative effect on human leukemic cell lines, and the structures of compounds 1-8 were elucidated. The further investigation including drug development will be studied on these fractions especially the FFIII which demonstrated the best antiproliferative effect on both human leukemic cell lines (NB4 and HT93A).