Antioxidant and Anticancer Constituents from the Leaves of Liriodendron tulipifera

Sixteen compounds were extracted and purified from the leaves of Liriodendron tulipifera. These compounds include aporphines, oxoaporphine, coumarin, sesquiterpene lactone, benzenoids, cyclitol and steroids. (+)-Norstephalagine (2) (an aporphine) and scopoletin (8) (a coumarin) were isolated from Liriodendron tulipifera leaves from the first time. The identified compounds were screened for their antiradical scavenging, metal chelating and ferric reducing power activities. The results have showed that these compounds have antioxidative activity. The study has also examined the chemopreventive property of the isolated compounds against human melanoma cells A375. The results shown that (−)-anonaine (1), (−)-liridinine (3), (+)-lirinidine (6), lysicamine (7) and epitulipinolide diepoxide (9) significantly inhibited the proliferation of melanoma cells. These results revealed that these compounds have antioxidative activity and chemopreventive activity in skin melanoma cells.


Introduction
Free radicals and reactive oxygen species (ROS) are formed of hydrogen peroxide or superoxide anions [1]. Free radicals play an important role in different diseases [2]. The imbalance between the formation of ROS and the defenses provided by cell antioxidants will cause diseases, including cancer [3]. Skin cancer is the most prevalent cancer worldwide [4]. Skin cancer can be divided into melanoma and non-melanoma skin cancer depending on the cell type [4,5]. A lot of studies have demonstrated that diet and phytochemicals containing high level of antioxidants can decrease the incidence of cancers [6,7]. We have found a lot of phytochemicals from different plants with antioxidative activity and chemotherapeutic activity [8][9][10][11].
The genus Liriodendron (Magnoliaceae) contains two species, L. tulipifera and L. chinense. L. tulipifera is known as American tulip tree and is a hardwood native plant used for pulp and wood in furniture and paper-making in the United States. The bark of L. tulipifera was used by Native Americans as a febrifuge and for the treatment of fevers associated with malaria. Numerous phytochemical contituents including sesquiterpene lactones, alkaloids and sugar derivatives have been isolated from this species, however, few pharmacological studies of these phytochemicals are described. The antioxidant and anticancer activities of bark extracts of L. tulipifera were examined in previous studies [9,10].
(+)-Norstephalagine (2) (an aporphine) and scopoletin (8) (a coumarin) were first isolated from the leaves of this species. The study also evaluates the antioxidant activity and anticancer activity of this species. To the best of our knowledge, this is the first study on investigating the antioxidant capacity and anticancer activities of scopoletin and (+)-norstephalagine from L. tulipifera.

Antioxidant Activities of Compounds 1 to 16 from L. tulipifera
The scavenging of radicals was measured in this study, as antioxidants act to inhibit the oxidation. The scavenging activities of compounds 1-16 at dosage of 100 μM were determined by a 1,1diphenyl-2-picrylhydrazyl (DPPH) assay. As shown in Table 1, (+)-lirinidine (6) has minor radical scavenging activity (6.5%) compared with vitamin C (88.5%) at the same dose. The ferrous ion  Data were expressed as a mean value of at least three independent experiments; a Vitamin C was used as a positive control on DPPH assay at 100 μM; b EDTA was used as a positive control on metal chelating ability at 100 μM; c BHA was used as a positive control on reducing power at 100 μM.

Figure 2.
Anti-proliferative effects of L. tulipifera compounds on A375 cells. Cell growth was determined by MTT assay after incubation with 10, 50, 100 μM of compounds 1-14 respectively. Results are expressed as the percent of the cell proliferation of the vehicle control at 24 h.

Plant Material
The specimen of L. tulipifera was collected from Chiayi County, Taiwan in December, 2007. A voucher specimen was characterized by Dr. Jin-Cherng Huang of Department of Forest Products Science and Furniture Engineering, National Chiayi University, Chiayi, Taiwan and deposited in the School of Medical and Health Sciences, Fooyin University, Kaohsiung, Taiwan.

Determination of DPPH·Radical Scavenging Capacity
DPPH·is a stable free radial with a violet color (absorbance at 517 nm) that changes its color to light yellow when the free radicals are scavenged. Various concentrations of the four compounds were added to 0.1 mL of stable DPPH (60 μM) solution. When DPPH reacts with hydrogen-donating anti-oxidant, it is reduced, resulting in a decrease in absorbance at 517 nm. The analyzed time interval was 10 min per point, up to 30 min by using UV-vis spectrophotometer (Jasco, Tokyo, Japan). Vitamin C was acted as a positive control. The DPPH· radical scavenging activity (%) was determined as:

Metal Chelating Activity
The ferrous ion chelating potential of the four L. tulipifera compounds was investigated according to a previously described method [9]. Briefly, various test concentrations of samples dissolved in DMSO were added to a solution of 2 mM FeCl 2 ·4H 2 O (0.01 mL). The reaction was initiated by the addition of 5 mM ferrozine (0.02 mL), and the mixture was vigorously shaken and left standing at room temperature for 10 min. The absorbance of the mixture was then read at 562 nm against a blank. EDTA was used as a positive control. The metal chelating activity was determined as:

Reducing Power
The reducing powers of our natural pure compounds were determined according to the method of [9]. Briefly, various concentrations of test samples were mixed with 67 mM phosphate buffer (pH 6.8, 0.085 mL) and 20% potassium ferricyanide [K 3 Fe(CN) 6 , 2.5 μL) The mixture was incubated at 50 °C for 20 min, and trichloroacetic acid (10%, 0.16 mL) was then added to the mixture that was then centrifuged for 10 min at 3000 g. The upper layer of the solution (75 μL) was mixed with 2% FeCl 3 (25 μL), and the absorbance was measured with a 96-well plate spectrophotometer at 700 nm. A higher absorbance demonstrates a higher reductive capability.

Cell Culture
Human melanoma cell lines A375 were obtained from the American Type Cell Culture Collection (ATCC, Manassas, VA, USA). It was maintained in monolayer culture at 37 °C and 5% CO 2 in DMEM supplemented with 10% FBS, 10 µg/mL of penicillin, 10 µg/mL of streptomycin and 0.25 μg/mL of amphotericin B.

Cell Viability Assay-MTT Assay
The MTT assay was used to determine cell viability and proliferation. The cell lines were seeded in 96-well culture plates (1 × 10 4 cells/well). After seeding cells for 24 h, various compounds with concentration 100 μM were added. Within 24 h of compound treatments, images of human melanoma A375.S2 cells were taken at suitable time intervals. MTT solution (5 mg/mL and dissolved in phosphate buffered saline; PBS) was diluted 1:10 in culture medium and added to a culture dish followed by an incubation at 37 °C. After 2 h of MTT treatment, the media was removed and each precipitate in a specific dish was dissolved in 100 μL of DMSO to dissolve the purple formazan crystals. After the dishes were gently shaken for 20 min in the dark to ensure maximal dissolution of formazan crystals, the optical density (OD) values of the supernatant were measured at 595 nm. All experiments were repeated at least three times. In consideration of the possible anti-proliferative effects of DMSO, a maximal amount (0.5%) of DMSO was added to culture and used as positive controls. DMSO at this amount was found not to affect the growth of the human melanoma A375.S2 cells.

Statistical Analysis
All data are the means ± SD from at least triplicate experiments. The significance of the differences was analyzed by a one-way analysis of variance (ANOVA), with p < 0.05 or 0.01 as considered significant.

Conclusions
One study has shown that the bark and leaves extracts from L. tulipifera have antiplasmodial activity [21]. The leaf essential oil and methanolic extract of the leaves from L. tulipifera display anticancer activity [22,23]. From our current and previous study, we found that the leaf and bark extracts from L. tulipifera is abundant of antioxidants [9,10]. This study shows the antioxidant activity and anticancer activity of 16 constituents from the leaves of L. tulipifera. These constituents include aporphines, oxoaporphine, coumarin, sesquiterpene lactone, benznoids, cyclitol and steroids. (−)-Liriodendritol (14) is the most abundant component of the extract. (+)-Norstephalagine (2) (an aporphine) and scopoletin (8) (a coumarin) are isolated for the first time from L. tulipifera.
Among these compounds, (+)-lirinidine (6) displayed medium ferric reducing power activity and minor radical scavenging activity. A previous study has shown that lirinidine exhibited significant inhibition of collagen, arachidonic acid and platelet activating factor-induced platelet aggregation [24]. We suggest that (+)-lirinidine is a candidate for the cosmetic business and the food industry.
Although scopoletin did not show potential anticancer and antioxidant activity in this study, many other studies have shown that scopoletin has anti-inflammatory and antioxidation activity in vivo and in vitro [25,26]. One study has shown that (+)-norstephalagine has relaxation activity on rat uterine smooth muscle. It is interesting that sesquiterpene lactone epitulipinolide diepoxide strongly inhibited melanoma cells (A375) with minor ferric reducing power activity. Moreover, one study has demonstrated that epitulipinolide diepoxide has cytotoxic activity against KB cells [27]. In the future, the mechanism of action of epitulipinolide diepoxide can be further examined in different cancer cells.