In-vitro Anti-Cancer Activity of Extracts Dracaen Cinnabari Balf. F Resin from Socotra Island in Yemen Republic

Plants have a long history of use in treatment of cancer, Since many years, plants were known to possess anticancer activities against different cancer cell lines. In this paper, we report a study based on anticancer properties of Dragon cinnabari resin. The resin of plant material was collected, shade dried and extracted with different solvents using soxhlet extraction procedure. In vitro anticancer activity is assayed with standard MTT colorimetric procedure against MCF-7 cell line. From the analysis it was found that Ether and Ethyl acetate of dragon cinnabari Balf. f showed nearly 50% MCF7 cell line inhibition at 100 μg/ml tested dose, whereas other extracts did not display much anticancer activities against MCF-7 breast cancer cell line. Based on the cytotoxicity studies against MCF-7 cell lines the ether and ethyl acetate extracts could be used as potential source for anticancer drugs. In-vitro Anti-Cancer Activity of Extracts Dracaen Cinnabari Balf. F Resin from Socotra Island in Yemen Republic Yasser Hussein Eissa Mohammed* Department of Biological-Chemistry, Applied Science College, Hajja University, Yemen


Introduction
The plants can produce many metabolic compounds mainly during the secondary metabolites, plant extracts contain several compounds that have biological active which used as natural medicine [1]. Today herbal derivatives are considered as the basis for a large proportion of the medications in traditional and modern systems of medicine [2]. Bioactive compounds are normally accumulated as secondary metabolites in all plant cells but their concentration varies according to the plant parts. Resin is one of the highest accumulated plant part of such compounds and people are generally preferred it for therapeutic, purposes some of the active compounds inhibit the growth of disease causing microbes either singly or in combination [3]. Dragon's blood tree is a non-specific name for dark red resinous exudations from different plant species endemic to various regions around globe that belongs to four genera Dracaena spp. (Agavaceae), Croton spp. (Euphorbiaceae), Daemonorops spp. (Palmaceae) and Pterocarpus spp. (Fabaceae) have a long history of being used as a traditional medicine the world over. Medicinal use of dragon's blood dates back to the ancient Greeks, Romans, Chinese and Arabs [4]. However, Dracaena cinnabari Balf. f. (D. cinnabari) belongs to Agavaceae family, which is commonly known as Damm Al-akhwain in Yemen. It is endemic to the Socotra Island, Yemen. D. cinnabari resin has traditionally been used to treat diarrhea, wounds, fevers, ulcers, hemorrhage, control bleeding, fractures, and burns [5]. Plant has anti-microbial and cytotoxicity effect. Some constituents of Dracaena cinnabari have been identified: Dracophan, ametacyclophan, Cinnabaron, Abiflavonoids, Numerous phenolic compounds belong to the homoisoflavonoids and chalcons, Sterol, triterpenoids and a new biflavonoids were isolated from this plant. Despite its wide uses, little research has been done to know about its true source, quality control, bioactive compounds and clinical applications. Therefore, it is of great interest to carry out a screening of these plant parts in order to validate their use in folk medicine and to reveal the active principle by isolation and characterization of their constituents [5][6][7]. The systematic screening of them may result in the discovery of novel active compounds. Dracaena cinnabari Balf. F resin was collected from Socotra Island (Yemen) on May 2014. Thus, in the present study attempts were made to investigate its anticancer activity of the resin extract of dragon cinnabari on MCF-7 cell line by standard MTT colorimetric procedure.

Anticancer Activity
There are many different anticancer herbs that have been used by different cultures throughout time for medicinal purposes, anticancer herbs come in many forms one of which is a type of thistle plants [8][9][10][11][12][13][14][15][16][17][18][19] Cancer is considered one of the most common causes of mortality worldwide. Progress made in cancer therapy has not been sufficient to a significantly lower annual death rate from most tumor types, and there is an urgent need for new strategies in cancer control [11]. For centuries, people have been using plants for their therapeutic values. Today 85000 plants have been documented for therapeutic use globally [12]. The World Health Organization (WHO) estimates that almost 75% of World's population has therapeutic experience with herbal drugs. Cancer is one of the most dangerous diseases in humans and presently there is a considerable scientific discovery of new anticancer agents from natural products [13]. The potential of using the natural products as anticancer drugs was recognized in 1950's by U.S. Natural Cancer Institute (NCI) since 1950 major contributions have taken for the discovery of naturally occurring anticancer drugs [14]. Dracaen cinnabari Balf. F. It was traditionally used to treat diarrhoea, dysentery, leucorrhoea, hemorrhoids, wounds, and infection during confinement, toothache and also it is used by the people in Yemene to cure diarrhea [5,15]. Biological activities such as anti-inflammatory and hepatoprotective activities were reported [16][17][18][19]. However, no work has been reported on the anticancer property of this plant. Keeping in view, the present study has been undertaken to investigate anticancer activity of the different extract of Dracaen cinnabari Balf. F against MCF-7 cells lines.

Assay controls
(i) Medium control (medium without cells).
(ii) Negative control (medium with cells but without the experimental drug/compound).
(iii) Positive control (medium with cells treated with a known drug, Metformin; 5 mM).

Collection of plant material
Dragon's blood tree (D. cinnabari) resin was collected from Socotra Island (Yemen) On May 2014.

MTT assay
The amount of viable cells was determined by examining cell number with the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, 5 mg/ml) dye-reduction assay measuring mitochondrial respiratory function. The antiproliferation (cytotoxicity) of D. cinnabari extracts on MCF-7 was evaluated by the MTT assay. The monolayer cell culture was trypsinized and the cell count was adjusted, using DMEM containing 10% FBS, such that 200 μl of suspension contains approximately 25,000 cells. To each well of the 96 well microtitre plate, 200 μl of the diluted cell suspension (approximately 25,000 cells) was added. After 24 h, when a partial monolayer was formed, the supernatant was aspirated and 200 μl of different test concentrations of test drugs were added on to the partial monolayer in microtitre plate. The plate was then incubated at 37 o C for 24 h in 5% CO 2 atmosphere. After 24 h, the plate was removed from the incubator and MTT reagent was added to a final concentration of 10% of total volume. The plate wrapped with aluminum foil to avoid exposure to light and incubated for 3 h at 37 o C in 5% CO 2 atmosphere. The culture medium was aspirated without disturbing the monolayer. Then 100 μl of solubilisation solution (DMSO) was added and the plate was gently shaken in a gyratory shaker to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 570 nm. The percentage growth inhibition was calculated using the following formula and concentration of test drug needed to inhibit cell growth by 50% (IC 50 ) values is generated from the dose-response curves for each cell line. The MTT assay can be used reliably to measure metabolic activity of cell cultures in vitro for the assessment of growth characteristics IC 50 -values and cell survival [20].

4.
After the formazon crystals have formed remove media from all the wells and add 100 μl of DMSO to all the wells and shake the plate to dissolve the formazon crystals.

LDH assay
LDH assays can be achieved by evaluating LDH released into the media as a marker of dead cells or performing lysis LDH as an indication of remaining live cells. Apoptosis and necrosis are two most important forms of cell death observed in normal and disease pathologies. A key signature for necrotic cells is the permeabilization of plasma membrane. This plasma membrane leakage from necrotic cells causes the release of intracellular contents into extracellular environment. In this experiment we detect the release of the enzyme Lactate dehydrogenase (LDH). LDH is a soluble cytoplasmic enzyme that is present in almost all cells and is released into extracellular space when the plasma membrane is damaged. The cells are fixed and stained with fluorescence tagged antibodies specific to this particular enzyme which is then measured by flow cytometry. Therefore, necrotic cells show decreased fluorescence intensity when compared to nonnecrotic/healthy cells. 2. Pipettes. You will need one in the range of 2-10 μl, one in the range of 10-100 μl, and another ranging from 100-1000 μl.

Materials
3. Vortex mixer. You could mix by tapping or shaking the tubes, but a mixer will give much more reproducible results in most cases.

12 × 75 mm polystyrene tubes.
5. Ice bucket with cover. Generally, cells are more stable and tolerate insult better when they're cold. The cover keeps light out, which could bleach the fluorochromes.

Results
The results obtained from the MTT assay are as follows.

Discussion and Conclusion
Sample collected in this study was selected to include the plant resin that have suggested bioactivity on the basis of their non-reported traditional usage as medicines. The plant resin used on traditional treatments for various disease as fever, tonsillitis, cough, dysentery, diarrhea, skin disease. The major aim of this study was to identify potential anticancer extracts that were affective not by feature of high concentration alone, relatively by specific activity demonstrated even at low doses. In order to achieve this aim, the maximum concentration (µg/ml) used in the study was 100 µg/ml as above results as the criteria for identifying plant resin with potent activity within range. Plants with less than 50% inhibitory activity within the test range were excluded from father screening. The concentration that causes 50% inhibition of cancer cells by the crude extract of the dragon blood resin displayed. Screening of Ether and Ethyl acetate of dragon cinnabari Balf. F showed the cytotoxicity studies revealed that the half minimum inhibitory concentration (IC 50 ) for the Ether and Ethyl acetate extract are 48.117, 50.692 µg/ml respectively, which resulted in moderate anticancer activities against MCF-7 cell lines, while the benzene extract showed IC 50 115.218 µg/ml which is less than ether and ethyl acetate extract extract with IC 50 extract against MCF-7 cell lines, as well as the hexane extract revealed that the IC 50 is 93.1136 µg/ml which is the least extract. The inhibitory properties of these extracts are compared with standard Metformin for MCF-7 cell line. The Percentage cancer cell inhibition profiles were found to be concentration dependent. Based on the cytotoxicity studies against MCF-7 cell lines the Ether and Ethyl acetate extracts could be used as potential source for anticancer drugs. On the other hand, Benzene, Chloroform and Ether extract treated cells showed a significant decrease in LDH FITC fluorescence intensity due to LDH leakage which indicates a necrotic cell death mechanism. While, Ethyl acetate and Hexane extract treated cells showed high LDH FITC fluorescence intensity which indicates an intact cell membrane and a possible apoptotic cell death mechanism.