Chemical constituents and In vitro anticancer activity of Tiliacora triandra leaves

Introduction: Tiliacora triandra (F. Menispermaceae) is edible and is also known for medicinal values. The leaves are a popular flavoring in Southeast Asia, especially in the northeast of Thailand. The objectives of this study were to determine the major constituents of the leaves of T. triandra, and assess their anticancer activities against human cancer cell lines. Methods: The leaves were extracted by a soxhlet apparatus with petroleum ether, dichloromethane, ethyl acetate and water. Major constituents were then purified and identified using chromatographic procedures and various spectroscopic techniques. In vitro anticancer activity tests of T. triandra extracts were performed by resazurinmicroplate assay (REMA), and tested with 3 cell lines: oral cavity cancer (KB), lung cancer (NCI-H187) and breast cancer (MCF-7) cell lines. Results: The result indicated that the main compound of T. triandra leaves was oxoanolobine. The methanol extract showed the highest cytotoxic activity against lung cancer (NCI-H187) cell line whereas the water extract exhibited the highest activity against oral cavity cancer (KB) cell line. The IC50 of oxoanolobine against the NCI-H187 cell line was 27.60 ± 4.30 μg/mL. Conclusion: T. triandra leaves contain oxoanolobine as the major constituent and have the potential of anticancer activity but are required to be investigated further.


INTRODUCTION
Tiliacora triandra Diels (Menispermaceae) is a species of flowering plant native to Southeast Asia.This plant is widespread in the northeast of Thailand and Lao PDR.It is a climbing plant with deep green leaves and yellowish flowers.Their leaves are used particularly in many cuisines of the northeast of Thailand and Lao PDR, especially in traditional bamboo shoot soup.The leaves are often used in traditional Thai medicine as antipyretic and anticancer agents. 1 Phytochemical study of its root revealed some bisbenzylisoquinoline alkaloids, including tiliacorine, tiliacorinine and nor-tiliacorinine A. 2 Previous study demonstrated that plants containing bisbenzylisoquinoline alkaloids have been used in traditional medicine for the treatment cancer. 3Chemical analysis revealed that T. triandra possesses high levels of beta-carotene and minerals such as calcium and iron. 4The root extract is used for treatment of fever and malaria. 5The water extract does not cause acute or subchronic toxicities in either male or female rats. 6ancer is the leading cause of death in developed countries and the second leading cause of death in developing countries.The most common cancers worldwide are lung cancer, breast cancer, large intestine cancer, stomach cancer and prostate cancer. 7Incidence and mortality rates for most cancers are increasing in several countries because of adoption of unhealthy lifestyles, such as smoking, physical inactivity and consumption of high calorie food. 8Nowadays, there has been huge attention towards natural products with their cancer prevention and therapeutic effectiveness.Evidence suggests that phytochemicals from fruits and vegetables may play an important role in reducing chronic disease risk including cancer. 9,10espite the traditional claims of anticancer properties of T. triandra leaves was reported and there has been minimal investigation of chemical constituents and anticancer activity in T. triandra leaves.Therefore, objectives of this study were to investigate the chemical com-position and to test anticancer activity of its extracts and the isolated compounds against human cancer cell lines.The results of the current study demonstrate the potential of this plant in cancer prevention and treatment.

Preparation of plant extracts
The plant sample (leaves) of T. triandra was collected from the Maha Sarakham province, Thailand and identified by the authors (Dr.Phadungkit M).The voucher specimens were deposited at Faculty of Pharmacy, Mahasarakham University, Thailand.The leaves were pulverized and sequentially extracted by a soxhlet apparatus using petroleum ether, dichloromethane, ethyl acetate and methanol.Some of the leaf powder was macerated in water for 7 days.The solvents were evaporated by a rotary evaporator, whereas the water extract was lyophilized by a freeze dryer.The crude extracts obtained were subjected to investigate the chemical constituent and to test anticancer activity.

In vitro anticancer assay
In vitro anticancer activity tests of T. triandra extracts were performed by resazurine microplate assay (REMA) and tested with 3 cell lines: oral cavity cancer (KB), lung cancer (NCI-H187) and breast cancer (MCF-7) cell lines. 11In brief, cells (5×10 3 cells/mL) were seeded on 96-well plates and cultured in a culture medium containing 10% fetal bovine serum at 37 o C for 24 h.The medium was then replaced with serum-free medium containing the herbal extracts at various concentrations (0, 0.5, 5 and 50/ mL).After incubation for 24 hrs at 37 o C under 5% CO 2 , the supernatant was removed and the MTT solution (0.5 mg/mL) was added to each well at 4 hrs prior to the end of the experiment.The formazan crystals that had formed in viable cells were measured using a wavelength of 540 nm in a spectrophotometer.All experiments were done in triplicate.The average data from the triplicates were expressed in terms of killing percentage relative to a negative control.The percentage of inhibition (%) of each of the test samples was calculated according to the following formula:

Percentage of inhibition (%) =[(A control -A sample )/A control ]x100
Cytotoxicity of each sample was calculated as IC 50 value.The IC 50 value is the concentration of the test compounds that cause 50% inhibition or cell death and was obtained by plotting the percentage inhibition versus concentration of the test compounds.

Isolation and structure elucidation of the isolate compound
The chemical component was isolated from the methanol extract of T. triandra leaves by chromatographic separation on a Silica gel column.It appeared as yellowish amorphous crystal.The IR spectrum revealed the vibration of the hydroxyl stretching (-OH) at 3367 cm -1 (broad), carbonyl stretching (C=O) at 1621 cm -1 .The electron impact mass spectrometry (EI-MS) showed molecular peak at m/z 291 which is in accordance with the structural formular of C 17 H 9 O 4 N.The 13 C-NMR spectrum exhibited 17 signals (Table 1), together with the information from a DEPT spectrum, corresponding to 1 methylene, 6 methines, and 10 quaternary carbons.In the 13 C-NMR spectrum data afforded 17 lines.The downfield region signal at d 182.16 ppm was in consistent with a carbonyl group while the resonance signals at d 164.58 and 164.29 ppm were assigned to the chemical shift of oxygen-bearing carbons of C-2 and C-6, respectively.The carbon signal at 161.47 was assigned to a nitrogen-bearing methylene (C-9) on the basis of a DEPT experiment and its chemical-shift value.The other carbon resonances were in accordance with those assignments reported with a known alkaloid, oxoanolobine. 12The 1 H-NMR (500 MHz, CD 3 OD) signals (Table 1) for the compound were assigned by comparing the NMR spectral data with those of oxoanolobine.The 1 H-NMR spectrum showed signals between 7.20-8.80ppm indicating the presence of aromatic protons.The singlet peak at 6.17 ppm was assigned to two methyl group protons of oxygen bearing carbons.From these data, the structure of the isolated compound was assigned as oxoanolobine (Figure 1).This is the first report to show that oxoanolobine is the major constituent in T. triandra leaves.

In vitro anticancer assay
The anticancer activity of the herbal extracts and the isolated compound is shown in Table 2.The methanol extract and the water extract showed potent activity against oral cavity cancer (KB) and lung cancer (NCI-H187), respectively, whereas the petroleum ether and the dichloromethane extracts appeared to be inactive in all human cancer (IC 50 >50 mg /mL).Oxonanolobine possessed moderate activity against NCI-H187 with IC 50 value of 27.6 ± 4.30 mg /ml when compared to the standard anticancer drugs, doxorubicin and ellipticine.Previous study showed that alkaloids exhibited the cytotoxic activity against human lung cell lines. 13In addition, the results confirm the potential use of this plant for the treatment of cancer in traditional medicine reported by Nanasombat, S and Teckchuen, N. 1

CONCLUSION
The present study showed that oxoanolobine was the main bioactive compound found in T. triandra leaves.The methanol extract showed the highest cytotoxic activity against lung cancer (NCI-H187) cell line whereas the water extract exhibited the highest activity against oral cavity cancer (KB) when compared to the other extracts.The study indicated that T. triandra leaves might be applicable in natural medicine for cancer prevention and or treatment.

Figure 1 :
Structure of the isolated compound, Oxoanolobine