Active Cytotoxic Compounds and Essential Oil from Bougainvillea Alba Antiepileptic and Antipsychotic Effects of Ipomoea reniformis (Convolvulaceae) in Experimental Animals

Ipomoea reniformis Chaos is claimed in Indian traditional medical practice to be useful in the treatment of epilepsy and neurological disorders. In the present study, pretreatment effect of methanolic extract of Ipomoea reniformis on epilepsy and psychosis was evaluated in rodents using standard procedures. Besides evaluating epileptic and behavioral parameters, neurotransmitters such as Gamma-Amino Butyric Acid (GABA) in epilepsy and in psychosis dopamine, noradrenaline and serotonin contents in the rodent brain were estimated. The extract pretreatment reduced maximal electro shock; Isoniazid (INH) and Pentylenetetrazole (PTZ) induced seizures and also significantly inhibited the attenuation of brain GABA levels by INH and PTZ in mice. These results suggested that the observed beneficial effect in epilepsy may be by enhancing the GABAergic system. The test drug also inhibited the apomorphine induced climbing and stereotyped behavior and showed significantly reduced levels of brain dopamine, noradrenaline and serotonin which may be due to blocking of central dopaminergic, noradrenergic and serotonergic pathways or by enhancing the GABAergic system. The results obtained in present study suggest that the title plant possesses antiepileptic and antipsychotic activities in rodents. antimicrobial activity [10]. Further, the principle constituents of IR such as sinapic and ferulic acids have exhibited behavioural and pharmacological Abstract Chemical analysis of methanol extract of flowers and leaves of Bougainvillea alba led to isolation of four compounds identified as 3’, 4’, 6 - trimethoxy, 5 – hydroxyl 7-prenyl 3- O - (feruloyl (1→2) - rhamnpyranosyl) flavone[1], 3- O β -D-glucopyranosyl oleanolic acid 28- O - β -D-glucopyranosyl (1→3”) β -D-glucopyranosyl (1→3”’) α -L –xylopyrenoside [2], Lepidolide [3], E feruloyl 3- O - Coumaroyl α -L Rhamnopyranoside [4]. In addition, two oil fractions, the major identified components of oil are Elemene< β ->, Lanceol < cis ->, Nerolidol < cis ->, Methyl palmitate and Abietatriene. Cytotoxic activity of methanol extract of both leaves and flowers of Bougainvillea alba , isolated compounds and oil fractions were determined using the SRB Assay on human tumor cell line (Hepatocyte generation 2, HepG2). The isolated compounds showed cytotoxic activity at LC 50 15.2, 17.2, 15.6 and 2.82 µg/ml for compound 1,2, 3 and 4 respectively, while the two oil fraction showed activity at 2.67 µg/ml.


Introduction
Ipomoea reniformis (IR) also called as merremia emarginata (Burm. f.) is a procumbent herb belonging to the family convolvulaceae. In India, it is commonly known as Undirkana and Mushakparni. The plant is widely distributed in India, Sri Lanka, Philippines, Malaysia, Tropical Africa and mainly grows in rainy and winter season. In India, it is found in Southern part mainly counting Chennai, and some places of Andhra Pradesh [1]. Traditionally, IR has been used to treat diverse clinical conditions ranging from pain; fever to neurological disorders [2]. IR has been claimed to be useful for inflammation, headache, fever, cough, neuralgia, rheumatism and also in liver and kidney diseases [3]. The powder of leaves is used as a snuff during epileptic seizures. Juice acts as purgative and the root is having diuretic, laxative actions and applied in the disease of the eyes and gums [4].
The plant contains various neuroprotective chemical constituents such as caffeic, p-coumaric, ferulic and sinapic acid esters. Petroleum ether extract contains fats and fixed oil while aqueous extract contains amino acids, tannins (condensed and pseudo tannins) and starch [5]. IR has been reported to possess various pharmacological actions, mainly antidiabetic [6], antiinflammatory [7], nephroprotective [8], antibacterial [9], antioxidant and antimicrobial activity [10]. Further, the principle constituents of IR such as sinapic and ferulic acids have exhibited behavioural and pharmacological

Introduction
Cancer is considered one of the most common causes of mortality worldwide. The most common methods for treatment of cancer are chemotherapy, radiotherapy and surgery, most of the above methods exhibit severe toxicity and/or resulting undesirable side effects. There is a long history of medicinal use of plants; it is a most promising site for discovery of novel biologically-active substances [1].
Nyctaginaceae is a family of around 33 genera and 290 species of flowering plants, widely distributed in tropical and subtropical regions. Saponins [2], flavonoids [3,4] and phenolic acid [5] were isolated from the family.
Bougainvillea is a genus of flowering plants native to South America from Brazil to Peru and Argentina. It is a popular ornamental plants, rapid growing and flower all year in warm climate. Flavonoids were found as active principle isolated from the plant leaves where it showed a strong activity on xanthone oxidase inhibition [6].
Glycosides of oleanolic acid and Quercetin were isolated from the plant [7]. This work investigate the cytotoxic effect of methanol extracts of the leaves and flowers of Bougainvillea alba, four isolated compounds and two essential oil fractions using the SRB Assay on human tumor cell line HepG2.

Plant Material
Flowers and leaves of Bougainvillea glabra ' Alba' (Bunga Kertas) were collected from of Banha Agriculture university gardens, The plant was kindly identified by Mrs. Terasa Labib, General Manager and head of plant Taxonomy in El-Orman Botanical Garden, Giza, Egypt. Voucher specimens (BA-I) were deposited at laboratory of Medicinal Chemistry, Theodor Bilharz Research Institute. The flowers and the leaves of the plant were isolated, dried separately in shade, then finely powder with an electric mill. [Hz]) and 13 C-NMR spectra were recorded in CD 3 OD, operating at 300 MHz for proton and 75 MHz for carbon 13 spectrometer. Chemical shifts (δ) are reported in parts per million, using TMS as internal standard. Mass spectra were recorded on a finnigan TSQ 700 GC/MS equipped with a finnigan electrospray source (ESI-MS). Paper chromatography sheet [Whattman 1], using 15 % Acetic acid as solvent system, the chromatograms were visualized under UV light (at 254 and 366 nm). Column chromatography was performed using a glass column 120 x 7 cm and using polyamide as a stationary phase.

Equipment
GC/MS analysis of essential oil was performed with a finnigan mat 7000 gas chromatograph coupled to mass detector. The capillary column used was DB5, 30m x 0.25mm, 0.5 µm film thickness.
Operating conditions: Injector and ion source temperatures 220 o C/M interface temperature program 3 min. isothermal at 50 o C, then programmed from 59-250oC at 30oC/min Carrier gas: 0.88ml He/min. Ion source 70 e V.

Extraction and Isolation
The flower dry powder (1 Kg) were soaked in 85 % methanol (7 L x 3), reflux for 6 weeks to give 120 g crude methanol extract. The crude methanol extract was successively extracted with petroleum ether, chloroform, and ethyl acetate to give petroleum Ether extract (1.16 gm), chloroform extract (2.03 gm) and ethyl acetate extract (1.43 gm.).
The remaining methanol extract was desalted by dissolving the extract with water then precipitated with methanol. The filtrate evaporated till dry to give 90 gm which was subjected to polyamide column chromatography using elution system water, water: Methanol with different gradients, finally pure methanol [8].
Fractions (500 ml each) were collected; tested using paper chromatography (PC) developed with solvent system 15 % Acetic acid and/or TLC plates. Similar fractions were collected together to give 8 groups [A-H]. Group A was free from compound except traces of sugar. Group B subjected to more purification with organic solvent to give compound 1. Both group C and D separately were purified on silica gel sub column with elution system Chloroform: methanol: water 15:6:1 to give compound 2 and 3. Group E purified on SephadexLH20 sub column with elusion system Methanol: water, 8:2 to give compound 4. Group F give traces of unidentified compounds. Group G & H afforded oil collection which analyzed by GC/MS system.

Identification of Essential Oil
Qualitative identification was based upon matching the resulting mass spectra with those given by data system using the computer mass spectra identification program Amdis 2.0 (NIST, Gaithersburg, USA), retention time as well as comparison with published data R . The peak area measured for the total ion current was followed by quantitative determination of the different constituents (

Measurement of Potential Cytotoxicity (SRB Assay)
The sulforhodamine B (SRB) assay which was developed in 1990 remains one of the widely used methods for in vitro cytotoxicity screening [10]. The assay relies on the ability of SRB to bind to protein components of cells that have been fixed to tissue culture plates by trichloroacetic acid. SRB is a bright-pink amino oxanthene dye with two sulfonic groups that bind to basic amino acid residues under mild acidic conditions, and dissociate under basic conditions. The SRB method has proven to be sensitive, practical and suited to large scale screening applications as well as research.

Human Tumor Cell Lines
They were obtained frozen in liquid nitrogen (-180 0 C) from the American Type Culture Collection. The tumor cell lines were maintained in the National Cancer Institute, Cairo, Egypt, by serial sub-culturing.

Buffers
Tris base 10 mM (PH 10.5): It was used for SRB dye solubility. 121.1 gm of tris base was dissolved in 1000 ml of distilled water and PH was adjusted by HCl acid (2 M).

Sulphorhodamine-B (SRB) Assay of Cytotoxic Activity
This method was carried out according to that of Skehan et al. [11]. Cells were used when 90 % confluence was reached in T25 flasks. Adherent, cell lines were harvested with 0.025% trypsin. Viability was determined by trypan blue exclusion using the inverted microscope (Olympus 1x70, Tokyo, Japan The IC50 values (the concentrations of thymoquinone required to produce 50 % inhibition of cell growth). The experiment was repeated 3 times for each cell line.

Discussion
Methanol extract of both flowers and leaves of Bougainvillea alba was tested on PC chromatogram and TLC plates. They showed great similarity in number and kind of compounds but flowers showed more number of spots than that of leaves, the methanol extract of the flowers showed higher cytotoxic effect against human tumor cell line (Hepatocyte generation 2). The flowers methanol extract was subjected to chromatographic fractionation to yield four compounds and two oil fractions. The oil fraction showed significant cytotoxic effect, they were analyzed by GC/MS spectrophotometer.

Conclusion
Four compound and two essential oil fractions were isolated from Bougainvillea alba, they were tested for Cytotoxic activity using the SRB Assay on human tumor cell line. The oil fractions and compound 4 showed significant activity compared to original methanol extract and other three compounds.