Phytochemical Screening and Cytotoxicity of Crude Extracts of Vatica diospyroides Symington Type LS

Purpose: To investigate the phytochemistry and cytotoxic activity of the leaf, branch and fruit extracts of Vatica diospyroides Symington type LS, an endangered medicinal plant in the peninsula of Thailand. Methods: Extracts of the mature leaves, branches and fruit (both cotyledon and pericarp) of five-year old tree were obtained using various organic solvents. Thin–layer chromatography (TLC) and phytochemical screening were employed to identify the chemical constituents. Cytotoxicity was characterized by 50 % inhibition (IC 50 ) of human breast cancer cell lines (MCF-7 and MDA-MB-468) using 3-(4,5-dimethylthaizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: The extracts of the plant parts differed in chemical composition. Terpenoids and anthraquinones were present in all extracts, while cardiac glycosides and tannins were isolated from the branches and leaves, respectively. The acetone and methanol extracts of the fruit cotyledons were highly cytotoxic against MDA-MB-468 cell line (IC 50 = 3.1 µg/ml for both extracts), whereas acetone and dichloromethane extracts of the fruit pericarps were highly active against MCF-7 cell line (IC 50 = 18.2 and 21.0 µg/ml, respectively). The other extracts were less active. Conclusion: This work revealed the potentials of V. diospyroides fruit as a cytotoxic agent against human breast cancer cell lines. However, further studies, including identification and purification of the active compounds, will need to be pursued.


INTRODUCTION
The family Dipterocarpaceae has many medicinal plants containing valuable curative compounds [1], such as genera Dipterocarpus, Shorea and Vatica [2]. In Thai ethnobotanical medicine, flowers and stems of the genus Vatica have been used as functional ingredients in cardiac and blood tonic remedies. Modern drug discovery research has shown that their stems contain resveratrol derivatives, namely Vaticaphenol A and Vaticanol series [3][4][5]. These resveratrol derivatives have been found to play an important role in cardiovascular treatment [6], and to be cytotoxic against various human cancer cell lines [3,5]. Resveratrol derivatives for human cancer chemoprevention are being tested in clinical research [7]. Therefore, the stems of plants in the genus Vatica not only provide traditional medicines like Thai cardiovascular tonics, but are currently supplied worldwide as cardioprotective and anticancer agents.
The stem of V. diospyroides Symington, an endemic medicinal and fragrant dipterocarp of Thailand peninsula, has strong anticancer activity [3,5]. However, other plant parts such as leaves have only been documented as having secondary compounds unlike the stem [8]. However, extracting the valuable phytochemical constituents of V. diospyroides from leaves, branches and fruits has not been reported to date.
Cancer is a leading cause of death worldwide and accounts for 7.6 million deaths especially in low-and middle-income countries. Invasive cancers such as those of lung, stomach, liver, colon/rectum, and breast, cause about 50 % of all deaths [9]. Nowadays, plant secondary metabolites are accepted for use as anti-tumor agents, inhibiting the growth of human cancer cells. Most of the secondary compounds found in common medicinal plants are alkaloids, flavonoids and tannins [10]. The anti-cancer compounds, vinblastine and vincristine in common clinical use, are produced from genus Catharanthus, known in Thai as Phaeng phuai farang, and are sold in the U.S. market [1]. In a previous work [3], human oral epidermoid (KB), colon cancer (Col2), and breast cancer (BC1) cell lines were used in bioassay-guided fractionation of resveratrol tetramer, purified from the extract of V. diospyroides stem.
We had reported earlier that V. diospyroides can be classified into two distinct types of tree, namely the SS and LS forms [11]. It is reasonable to expect that these distinct types may have different activities against human cancer cells. The LS type is dominant in the population of Nong Thung Thong non-hunting area of Thailand, which may be the best genetic source of V. diospyroides. The present study aims to identify types of active constituents of various parts of the LS type of this plant and the cytotoxic activity of their extracts against human breast cancer cell lines (MCF-7 and MDA-MB-468). The plant samples were cut into small pieces and completely air-dried, and stored in glass containers until extraction. About 270 g of the dried plant material was extracted for five days with n-hexane (C 6 H 14 ), dichloromethane (CH 2 Cl 2 ), methanol (CH 3 OH), or acetone ((CH 3 ) 2 CO). The extracts were filtered through 2 layers of cotton fabric. The extract of each sample was evaporated at room temperature, under reduced pressure, to the dry residue and stored in sterile vial pending phytochemical and cytotoxic screening.

Thin layer chromatography (TLC)
Thin layer chromatography was applied to the extracts. The chromatograms were developed and dried on the silica gel TLC plates, and prior to spraying with vanillin-sulphuric acid, the spots visually observed under UV 254  MTT assay was employed to determine cell viability. After 72 h of incubation, 100 µl of 0.5 mg/ml MTT reagent was added into each well, and incubated for 30 min in a CO 2 incubator. MTT solution was then discarded and 100 µl dimethyl sulfoxide (DMSO) was added into each well to solubilize the cells and dissolve the color substance. For complete solubilization, the plates were vigorously agitated for 5 min at room temperature, and then read for optical density at 570 nm using a plate reader. A calibration curve based on optical density was constructed from which growth inhibition was determined. The 50 % growth inhibition concentrations (IC 50 ) of the extracts were calculated from fitted response curves. The cytotoxic activities of all the extracts against breast cancer cell lines were labeled according to the National Cancer Institute (NCI, USA) criteria (highly inhibiting activity means IC 50 ≤ 20 µg/ml) [12].

Phytochemical constituents
Phytochemical data (Table 1) show distinct patterns of chemical compositions in constituents of the extracts. The patterns of composition differed considerably in their quantitative values. The results of phytochemical evaluations are shown in Table 1. All the plant parts investigated were rich in terpenoids and anthraquinones whereas, alkaloids were absent. The n-hexane and methanol leaf extracts contained flavonoids, while only the n-hexane branch extract contained flavonoids was able to extract flavonoids from branches. Tannins and cardiac glycosides were present only in leaves and branches while terpenoids, anthraquinones and saponins were present in both pericarp and cotyledon extracts. Leaves and fruit contained saponins while branches did not. Table 2 presents the Rate of flow (R f ) values observed under UV 254 . The n-hexane leaf extract revealed 3 spots, and the branch extract 4 spots also, the dichloromethane branch extract presented 4 spots. Details of these and spots are contained in Table 2.

In vitro cytotoxicity
The criteria used to categorize the activity of V. diospyroides extracts against human breast cancer cell lines (MCF-7 and MDA-MB-468), based on IC 50 values, were modified from those of NCI and Geran et al [12] as follows: IC 50 ≤ 20 µg/ml = highly active, IC 50 21 -200 µg/ml = moderately active, IC 50 201 -500 µg/ml = weakly active and IC 50 > 501 µg/ml = inactive. The cytotoxicity data for the fruit extracts against human breast cancer cell lines are displayed in Table 3.

DISCUSSION
Various active phytochemical compounds were found in the fruit, branch and leaf extracts of V. diospyroides Symington type LS. Terpenoids and anthraquinones were the major constituents found in all the extracts. These compounds have previously been reported as principal components of resveratrol derivatives in various extracts of medicinal herbs [13]. In general, the resveratrol compounds found in the stems of dipterocarpaceae are semipolar and polar [2],     and extracting these from the leaves, branches and fruit of V. diospyroides require polar or highly polar solvents. Our results also indicate acetone and methanol are the best solvents for extraction, in terms of quantitative and cytotoxic properties of the extracts.
On the other hand, alkaloids were absent from all the extracts. The non-existence of alkaloids in V. diospyroides is a deviation from several other medicinal plants [10]. Alkaloids are commonly found in most medicinal plants, and they have various useful bioactivities which favours their use as active ingredients in anti-inflammatory medications [14]. In addition, they are widely used for treating various cancers [1] and malaria [15]. Previously, the cardioprotective effect of alkaloids from other plants in dipterocarpaceae family has been described [6]. Thus, if indeed V. diospyroides extracts has prophylactic cardiovascular effect, this is likely to be due to non-alkaloidal compounds.
Interestingly, cardiac glycosides were only present in the branch extract. Cardiac glycosides have been found in a number of plants used in blood tonics for treating heart failure via blood pressure regulation [16]. This may lend some support for the use of V. diospyroides stem extract in classic Thai blood tonic. Tannin, however, was present only in the leaf extract while saponin was absent only from only the branch extracts. Flavonoids, which were present both in leaf and branch extracts, are antioxidants with cardiovascular prevention and anticancer properties [1,2,17].
The anticancer activities of terpenoids, saponins, cardiac glycosides and anthraquinones have been reported for various plant extracts [10,18,19]. Terpenoids and saponins are biologically active against various human cancer cell lines [19]. Similarly, these compounds are potent against human breast cancer cell lines. However, these secondary metabolites exert their anticancer activity through different mechanisms. Carvalho et al [20] reported that plant extracts can decrease cell proteins, alter cell morphology, and fragment nucleosomal DNA of cancer cells, resulting in cell apoptosis. Therefore cardiac glycosides may induce immunogenic cancer cell death.
The human breast carcinoma cell lines used in this study -MCF-7 and MDA-MB-468 -are in common laboratory use. A high cytotoxic activity against MCF-7 was observed for acetone and dichloromethane extracts of pericarp, whereas the acetone and methanol extracts of cotyledon were highly active against MDA-MB-468. Thus each extract is a different cocktail of ingredients with potential activity that is specific to a particular cell line. This is not unexpected since MCF-7 is a luminal cancer cell line representing low tumor grade. In contrast, the basal-like carcinoma cell line, MDA-MB-468, represents a high tumor grade, and is triple negative for the immunophenotypic markers ER -/PGR -/HER2 -. MDA-MB-468 was expectedly more sensitive to the extracts and culture conditions than MCF-7. High cell proliferation of MDA-MB-468 corresponds to a poor prognosis.
Fruit extracts of other medicinal plants, e.g., some compounds produced from the seed kernels of the Thai medicinal plant, Entada rheedei Sprengel, have been evaluated against various human cancer cell lines [17]. However, the fruit extracts of V. diospyroides have not previously been described and reported in Thai ethnopharmacology, and the active components have not been identified. The current study is the first to demonstrate that the highly active constituents of the fruit extracts inhibit breast cancer cells.
No normal human cells were tested in this study, and cultured immortalized cell lines will not behave as they would in vivo. While in vitro toxicology may sound attractive, there is a view that it is currently unrealistic. Our findings have indicated that fruit extracts of V. diospyroides may be worth further investigation with a view to isolating the active compounds.

CONCLUSION
This work demonstrates that V. diospyroides fruit extracts have pharmacological potential. The pericarp and cotyledon extracts of this plant inhibit the growth of MCF-7 and MDA-MB-468, on a highly active level. Further studies are planned to identify and purify the in vitro active compounds for subsequent in vivo evaluation.