Fagonia Cretica Tea

FAGONIA CRETICA TEA: The only research based herbal remedy for Breast Cancer

The administration of an aqueous extract of Fagonia cretica is reported effective at reducing tumour size and improving the quality of life of breast cancer patients, is well tolerated and does not exhibit adverse effects like vomiting, diarrhoea or alopecia which are common side effects of standard cytotoxic therapy.
Fagonia cretica is a species of plant in the caltrop family (Zygophyllaceae). It is native to dry regions of the Mediterranean Basin in North Africa (in Cape Verde, Canary Islands, Morocco, Algeria, Tunisia, Libya and Egypt), Southern Europe (in the Balearic Islands, Portugal, Southeast Spain, Sicily, and Greece) and West Asia (in Saudi Arabia and the Sinai peninsula).
For the entire world it is presented by senior agriculture scientists of Pakistan as FAGONIA CRETICA TEA.

PRICE: USD 45 for 250 gram packet
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BREAST CANCER IS LIFE-THREATENING

Breast cancer is life-threatening, but early detection saves lives. Regular self-checks and timely care can protect your health and future.You may take herbal remedy Fagonia Cretica Tea to treat the cancerous growth.

FAGONIA CRETICA TEA IS THE ONLY HERB IN NATURE THAT KILLS BREAST CANCER CELLS SELECTIVELY. IT IS AVAILABLE WITH US.

NOW WE PRESENT FAGONIA CRETICA TEA AFTER TESTING BY THE MOST ACCREDITED SCIENCE LABORATORIES OF SOUTH ASIA: (PCSIR LABORATORIES) - PAKISTAN COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH LABORATORIES, PAKISTAN.

CYTOTOXIC ACTIVITY OF FAGONIA CRETICA AGAINST HUMAN BREAST CANCER CELLS
Doctoral Thesis › Doctor of Philosophy
Authors
Matthew Lam
Abstract
In many parts of the world, plants are directly utilised for their medicinal properties. Traditional medicine from Pakistan, India and the Far East is well documented and its history is embedded in folklore. It has been documented that an aqueous extract of the desert shrub, Fagonia cretica, is a popular treatment for breast cancer in Pakistan. The administration of an aqueous extract of Fagonia cretica is reported effective at reducing tumour size and improving the quality of life of breast cancer patients, is well tolerated and does not exhibit adverse effects like vomiting, diarrhoea or alopecia which are common side effects of standard cytotoxic therapy. In the past, many pharmacologically active and chemotherapeutic compounds have been isolated from plants which subsequently have proven to be successful in clinical trials and been used as primary compounds in therapeutic regimes. Fagonia cretica has historical use as a treatment for breast cancer, yet there is little scientific evidence which shows chemotherapeutic potential towards breast tumours. Preparation and analysis of an aqueous extract of Fagonia cretica may reveal novel chemotherapeutic agents that can be used to effectively target cancer cells. An understanding of the mechanism of any activity may improve our understanding of cancer cell biology and reveal novel therapeutic targets. This thesis describes for the first time that an aqueous extract of Fagonia cretica shows potent in vitro cytotoxic activity towards breast cancer epithelial cell lines which was not seen towards normal mammary epithelial cells. Elucidation and characterisation of the cytotoxic mechanism was undertaken by analysing DNA damage, cell cycle status, apoptosis, metabolic state and expression of transcription factors and their targets. Finally, methods for the isolation and identification of active compound(s) were developed using various chromatographic techniques. An aqueous extract of Fagonia cretica was able to reduce cell viability significantly in two phenotypically different breast cancer cell lines (MCF-7 and MDA-MB-231). This activity was markedly reduced in normal mammary epithelial cells (HMEpC).
Further investigation into the mode of action revealed that extract treatment induced cell cycle arrest and apoptosis in both MCF-7 and MDA-MB-231 cell lines. This coincided with the formation of DNA double stranded breaks and the DNA repair marker ?-H2AX. In MCF-7 cells, ATM/ATR activation resulted in increased p53 expression and of its transcriptional targets p21 and bax, suggesting a role for a p53-mediated response.

Furthermore, inhibition of extract-induced p53 expression with siRNA reduced the cytotoxic effect against MCF-7 cells. Extract treatment was also associated with increased FOXO3a expression in MCF-7 and MDA-MB-231 cells. In the absence of functional p53, siRNA knockdown of extract-induced FOXO3a expression was completely abrogated, suggesting that FOXO3a plays a vital role in extract-induced cytotoxicity. Isolation and characterisation of the active compound(s) within the extract was attempted using liquid chromatography and mass spectrometry in conjunction with a cell viability assay. Multiple fractionations generated an active fraction that contained four major compounds as detected by mass spectrometry.
However, none of these compounds were identified structurally or chemically due to constraints within the methodology.

CYTOTOXIC AND ANTITUMOR POTENTIAL OF FAGONIA CRETICA TEA

Research Cunducted by: Ahsan HUSSAIN, Muhammad ZIA, Bushra MIRZA

Department of Biochemistry, Faculty of Biological Science, Quaid-i-Azam University, Islamabad - PAKISTAN
Received: 03.10.2006

Abstract: According to traditional knowledge, Fagonia creticahas medicinal potential, especially against cancer and tumors. In the present study, this information was analyzed at laboratory level by performing cytotoxic, antitumor (potato disc) and DNA damage assay. Significant cytotoxic activity was found against brine shrimps at LD50 118.89 ppm, while antitumor assay showed that the extract inhibited tumor induction on potato discs. Significant antitumor activity was found against all the tumor-inducing Agrobacteriumstrains tested (At6, At10 and At77), with maximum tumor inhibition (77.04%) against At10. However, the extract did not show any lethal activity against Agrobacterium tumefaciens strains, and furthermore, no DNA damaging activity was observed. The overall results indicate a strong anti-cancerous potential of this plant.
Key Words: Fagonia creticaL, cytotoxic activity, potato disc assay, mutagenic activity

Introduction
Screening programs for biologically active natural products require the right bioassays. Detection of compounds with the desired activity in complex plant extracts depends on the reliability and sensitivity of the test systems used. Thus, bioassays are essential for monitoring the required effects throughout activity-guided fractionation and purification until the active mono-substances are obtained.
Fagonia cretica L., a member of the family Zygophyllaceae, is a small spiny undershrub (Figure 1), mostly found in dry calcareous rocks throughout Pakistan (1,2). It is reputed to be a medicinal plant in scientific and folkloric literature, and its medicinal values are well documented (3). Fagonia cretica is astringent, febrifuge and prophylactic against small-pox. The plant is bitter and used for the treatment of fever, thirst, vomiting, Figure 1. Pictorial representation of Fagonia cretica with its leaves, dysentery, asthma, urinary discharges, liver trouble, fruits and inflorescence. typhoid, toothache, stomach troubles and skin diseases (4). Boiled residue of the plant in water is used to induce abortion. It is externally applied as a paste on tumors and Considering the medicinal activity of Fagonia cretica other swellings of the neck. An aqueous decoction of the based on traditional information, the present study was plant is a popular remedy for cancer in the indigenous conducted to evaluate methanolic extracts of Fagonia system of medicine (3), but no scientific attempt has yet cretica for its anticancerous potential by utilizing been made to evaluate the effects of its extracts. cytotoxicity, antitumor and DNA damage assays.

Cytotoxic and Antitumor Potential of Fagonia creticaL.

Materials and Methods

Fagonia cretica was collected from Sarwar Shaheed Chowk, District Thur, Pakistan in July 2004. Fresh aerial parts of Fagonia creticawere rinsed with distilled water and kept under shade till drying.
Preparation of extract
Extraction was carried out by simple maceration process. Aerial parts (490 g) were ground and merged in
3.5 L methanol. Homogenate was kept for 4 weeks atroom temperature (25 ± 2 ºC) in extraction bottle. After 4 weeks, the mixture was filtered twice, first using ordinary filter paper and then Whatman-41 filter paper. Methanol was completely evaporated at room temperature. In total, 21 g dried methanolic extract of aerial parts was obtained.
Brine shrimp toxicity assay (cytotoxic activity)
For brine shrimp assay, the procedure described by Meyer et al. (5) was followed. In brief, a rectangular dish (22 x 32 cm) was compartmentalized into two unequal halves with plastic divider of 2 mm with several holes and filled with artificial seawater (28 g sea salt/L, Sigma). Approximately 25 mg eggs (Artemia salina Sera, Heidelberg, Germany) were sprinkled in the larger compartment, which was darkened, while the smaller compartment was illuminated. After 24 hours, phototropic nauplii (brine shrimp larvae) were collected by pipette from the lightened side.
0.5 ml of 100, 1000 and 10,000 ppm concentrationsof the extract prepared in methanol was poured in vials and kept at room temperature to evaporate methanol. Then about 2 ml of sea water was added and 10 shrimps were transferred to each vial. The vials were placed under the illumination at room temp (25–28 °C), and after 24 hours the number of survivors was counted. LD50 was calculated using probit analysis (6).
Potato disc assay (antitumor activity)
For antitumor activity, the procedure describe by Galsky et al. (7) was followed. In brief, Agrobacterium tumefaciens virulent strains (At6, At10 and At77) were grown for 48 hours in Lauria broth medium containing rifampicin (10 µg/ml). Red skinned potatoes were surface sterilized in 0.1% mercuric chloride solution for 10 minutes and thoroughly washed with autoclaved distilled water. Potato discs (5 mm x 8 mm) were made with cork
20 borer and placed on agar (2%) plates (10 discs per plate). Agrobacterium culture (2 ml) mixed with 50 µl each of 10, 100 and 1000 ppm of extract (prepared in DMSO) was applied on the surface of each disc of respective concentration. Petri plates were then placed at 28°C for 21 days.
After 21 days, the discs were stained with Lugol’s solution (10% KI and 5% I2) for 30 minutes and then observed under dissecting microscope. Numbers of tumors per disc were counted and percent inhibition for each concentration was determined by the formula given below.

Average number of tumors of sample Percent inhibition = 100 – x 100
Average number of tumors of control
ANTIBACTERIAL ASSAY
Antibacterial assay of different concentrations of the extract of Fagonia cretica was performed against three Agrobacterium strains, At6, At10 and At77, using agar well diffusion method (8).
DNA damage analysis
Single colony of E. coli possessing pBluescript was grown for 24 hours at 37 °C in Lauria broth containing
0.05 mg/ml ampicillin. pBluescript was extracted by usingstandard protocols. The plasmid DNA was treated with 5, 10, 20 and 30 mg/ml concentrations of the plant extract dissolved in DMSO and with EcoR1 as positive control. These reaction mixtures were kept at 37°C for 2 hours for the complete digestion of plasmid DNA (9). After incubation, plasmid DNA was observed on 1% agarose gel.

Results and Discussion
Bioassays offer special advantages for identification of medicinal botanical extracts. Most often, a desired biological response is not due to one component but rather to a mixture of bioactive plant components. Therefore, crude extracts must be screened for biological activity and then any “active” extract should be fractionated directed with bioassays to exploit the bioactive compounds (10). A. HUSSAIN, M. ZIA, B. MIRZA. In the present study, the extract of aerial parts was prepared from Fagonia cretica using methanol as a solvent. According to traditional knowledge, Fagoniahas significant anticancer potential (3). Based on this information, the extract was evaluated for antitumor activity. This study was divided into three parts, i.e. brine shrimp assay, potato disc assay and DNA damage assay. According to Geoffrey et al. (1994) (11), the search for anticancer agents can be simplified by coupling one or two cytotoxic assays associated with an in vivo murine lymphocytic model. Cytotoxicity screening models provide important preliminary data to help select plant extracts with potential antineoplastic properties for future work (12).
Considering these reports, cytotoxic assay using brine shrimps was carried out according to the method described by Meyer et al. (5). Brine shrimp bioassay results (Table 1) clearly indicated the toxic effects of the extract. Our results showed that the brine shrimp survival is inversely proportional to the concentration of the extract used with LD50 118.89 ppm. Toxic effects on brine shrimps by the plant extract indicated the anticancer potential of F. cretica as suggested by Atalay (13) and Anderson (14).
The extracts showing significant activities in the cytotoxic assay are usually subjected to potato disc assay to confirm the anticancer potential of medicinal plants (13). This assay can be routinely employed as a comparatively rapid, inexpensive, safe and statistically reliable prescreen for 3PS (in vivo murine leukemia) antitumor activity. Potato disc assay was carried out using Agrobacterium tumefaciens for tumor induction. Various Agrobacterium strains can be used for this purpose. In the present study, three A. tumefaciens strains (At6, At10 and At77) were used against three
At 6 At 10 At 77

Figure 2. Effect of the extract of aerial parts of Fagonia cretica on
three tumor-producing strains of Agrobacterium
tumefaciens.
different concentrations (10 ppm, 100 ppm and 1000 ppm) of the extract, and there was a significant difference in tumor induction by the three strains and also tumor inhibition by the three concentrations of extract (Table 2). These results are significant at P < 0.05 for bacterial strains as well as for concentrations used.
Our results showed that Agrobacterium tumefaciens 10 was more prominent for producing tumor (6.1 ± 0.690) than At77 (4.7 ± 0.0592) and At6 (4.6 ± 0.495), as shown in Table 3 and Figure 2. Maximum inhibition was observed in At10 (40.98-77.04%) and least inhibition in At77 (17.89–38.29%). Usually, ≥ 20% inhibition of tumor is considered as a significant value for plant extracts (15). However, our results showed that the extent of tumor inhibition by an extract depends on the strain being used for the assay.

Table 1. Effect of methanolic extract of aerial parts of Fagonia creticaon brine shrimps (Artemia salinia).
Concentration used
LD50 Value (ppm)
10ppm 100ppm 1000ppm
Number of Shrimps Used 30 30 30
Number of Shrimps Killed 3 9 28 118.8992

Cytotoxic and Antitumor Potential of Fagonia creticaL.

Table 2. Statistical analysis of tumor inhibition by the extract of and tumor induction by three strains of Agrobacterium tumefaciens.
Source Degrees of Sum of Mean F Value Prob Freedom Squares Square
Strains 2 18.31 9.15 4.66 0.0114
Concentration 3 163.36 54.45 27.71 0.0000
Interaction between 6 10.48 1.74 0.88
Strains and Concentration
Error 108 212.20 1.96
Total 119 404.36

Table 3. Effect of the extracts of aerial parts of Fagonia cretica on three tumor-inducing strains of Agrobacterium tumefaciens.
A. HUSSAIN, M. ZIA, B. MIRZA

Figure 3. Effect of the extract of aerial parts of Fagonia cretica on the viability of Agrobacterium tumefaciens(At10) strain. C = Cefixime-USP; R = Roxycithromycin.
1 2 3 4 5 On the basis of these results, antibacterial assay was performed against all three strains of A. tumefaciensto check whether extracts are lethal for bacteria or are inhibiting at any level that is necessary for the genetic transfer mechanism and finally induction of tumor. Antibacterial assay showed that extracts had no effect on the viability of any of the three strains of A. tumefaciens (Figure 3).
Finally, DNA damage assay was carried out by using pBluescript. Results showed that methanolic extract of F. cretica was unable to produce any damage in the phosphate-sugar backbone of DNA. Figure 4 shows that the extract at the concentration of 10 mg/ml, 20 mg/ml and 30 mg/ml did not break the DNA backbone, while the plasmid DNA treated with EcoRI produced a single band. Maria et al. (9) also did not find any mutagenic activity of pepper tree bark extract using the same procedure. Some plant derived alkaloids can damage or cause aberration in
performing different biological assays including cytotoxic, potato disc, plasmid DNA damage and antibacterial assays. Significant cytotoxic and antitumor activity was found, which varied from strain to strain, but all values fall in the acceptable range mentioned for specific assay by different authors. Further work is required to isolate and characterize the individual bioactive compound.

Corresponding author:
Bushra MIRZA
Department of Biochemistry,
Faculty of Biological Science,
Quaid-i-Azam University,
Islamabad - PAKISTAN
E-mail: [email protected]