There are approximately 6.1 million people living with the Human Immunodeficiency Virus and Acquired Immune Deficiency Syndrome in South Africa (Unaids.org, 2014). People with HIV/Aids have a higher risk of getting secondary infections and diseases such as Tuberculosis, which is the cause of many deaths in South Africa (Tbfacts.org, 2014). About 5.5 million people in South Africa are infected with Tuberculosis (Salim S. Abdool Karim, 2009). I have chosen to research and experiment on the Sutherlandia frutescens because it has anti-bacterial and anti-HIV properties (Katerere and Eloff, 2014). I also have a keen interest in alternative medicines so researching and testing a plant with many diverse properties such as S. frutescens will be an interesting and fruitful experience for me.
Compounds extracted from S. frutescens have antibacterial properties.
To test three extraction methods (water, ethanol and acetone) on S. frutescens, to see which method will have the most effective anti-bacterial properties on two different strains of bacteria (E. coli, S. epidermidis).
Research and Experimental Methodology:
For this project I will rely on secondary research. Which includes research articles and information from websites on the S. frutescens, extraction methods of antibacterial compounds and statistics regarding specific diseases affecting South Africa. I will also be doing primary research such as using different extraction methods to extract the antibacterial compounds from S. frutescens. I will testing the extracts on two different strains of bacteria.
The limitations that I would face in my research task would be the reliability of the research articles I used with regards to the S.frutescens. The strains of bacteria that I’m using are harmful to human beings.
Review of Literature
A review of the taxonomy, ethnobotany, chemistry and pharmacology of Sutherlandia frutescens (Fabaceae).
B-E. van Wyk, C. Albrecht
Year of publication:
The article is a review of many different articles on S. frutescens. The article focuses on the chemistry and ethnopharmacology of S. frutescens. It names the ailments that S. frutescens is used to treat ailments such as urinary tract infections and HIV. It’s also used as an antibacterial and anti-inflammatory. Its has been shown that S. frutescens has been widely used as a medication by various groups in South Africa particularly the in the Western Cape.
This article is review of many different articles and most of the information is derived from other articles concerning S. frutescens and its properties and uses.
This article is from the Journal of Ethnopharmacology, which is published on the journal publishing site elsevier.com. The journal was also reviewed by a board of editors from many different countries.
Evidence use to support conclusion:
The leaves of the Sutherlandia frutescens have antibacterial properties. Recent studies on this plant have mostly focused on the anti-cancer, anti-HIV, anti-diabetic, anti-inflammatory, anti-oxidant, analgesic and antibacterial activities.
The article describes the many uses and properties of S. frutescens such as it’s antibacterial properties, it’s anti-inflammatory and its anti-HIV properties. The article also goes over the many uses of S. frutescens ov
The article doesn’t elaborate on much on the antibacterial activities of S.frutescens The article doesn’t show methods of extracting S.frutescens.
B-E. van Wyk is a professor at the University of Johannesburg and teaches undergraduate plant taxonomy, postgraduate taxonomy, systematics, chemosystematics of African plant families, medical plant chemistry and ethnobotany.
Antibacterial and Antioxidant Activity of Sutherlandia frutescens (Fabaceae), A Reputed Anti-HIV/AIDS Phytomedicine
David R. Katerere† and Jacobus N. Eloff*
Year of publication:
The article describes the extraction methods that were used to extract S.frutescens The article also describes the how the different extracts where tested on different strains of bacteria. The article is about the antibacterial and antioxidant activity of S.frutescens. The second method of extraction produced a greater yield than the first method of extraction.
The first extraction method used five grams of a commercially available leaf sample of Sutherlandia frutescens (Sutherlandia/ Unwele®). The Sutherlandia frutescens (Sutherlandia/ Unwele®) sample was consecutively extracted three times using different substances, first with Hexane (coded SF-H), then by dichloromethane (DCM) (SF-D), then by acetone (SF-A) and finally by ethylacetate (SF-E). The second method consisted of splitting a leaf sample of a Sutherlandia frutescens into three portions weighing 5g each. The portions where portions where extracted separately twice with acetone, ethanol and water. Each extract was then dried using a rotary evaporator and weighed. The aqueous extract was then freeze dried.
S, frutescens was extracted using two different extraction methods.
Evidence use to support conclusion:
The total yield of all four solvents in the first method of extraction was 10.5%. In the second extraction method, acetone extracted 5.6%. Ethanol extracted 12.6% while eater extracted 17.2%.
It’s useful as it gives methods to extract the active ingredient from the plant.
The article doesn’t give a testing method that I can easily perform at school.
David R. Katerere†: Specialist Scientist at SA MRC, Visiting scientist at Scynexis, visiting scientist at UNINA, trainee Pharmacist at Drug Tech Pharmacy, Chief Bioanalyst at PAREXEL, Postdoc at University of Pretoria
Jacobus N. Eloff*: Gold Medal for Science for Society Academy for Science of South Africa (September 2012), Gold medal of the South African Academy for Science and Art is awarded for Scientific and Technological Achievement, Bronze medal from the International Horticultural Society (December 2008) in recognition of the organising the World Conference on Medical and Aromatic Plants.
Five Ochna species have high antibacterial activity and more than ten antibacterial compounds
Tshepiso J. Makhafola1
Jacobus N. Eloff1
Year of publication:
The article is about the antibacterial activities of five Ochna species. Leaf samples where extracted using different mediums from the leaf. The extracts were tested against various strains of bacteria.
The dried leaf powder was extracted with 20mL of acetone.
The solution was then shaken in 50 mL centrifuge tubes and centrifuged for 15 minutes at 4000 rpm. The extracts were decanted through into glass vials through filter papers and the solution was concentrated to dryness with a stream of cold air.
Only clean and dry leaves were selected, the selected leaves had no blemishes or dirt. The leaves were not washed with water as the water would possibly extract some water-soluble compounds, and to limit the posibilty of fungal growth on the leaves due to the moisture left on the surface due to the water. The leaves were dried at room temperature in the dark. The leaves were then made into a fine powder, with the particles being less than 1 mm in diameter. The leaves were then stored in sealed glass bottles in the dark to reduce chemical changes in the compounds present in the leaves.
There were no competing interests the article.
Evidence use to support conclusion:
The percentage yield in acetone between the five species was: O. gamostigmata (8%), followed by O. pulchdra, (7.5%), O. serullata (7%) O. pretorienses and O. natalitia ((2.5%)
This article shows different extraction methods and it also gives a suggestion to which extraction method and solvent worked the best to extract the particular compounds. It provides detailed images, tables and graphs which makes it easier to view the data that was collected.
Only gives information about on genus of plant (Ochna) and there is no information of S. frutescens.
Kobus (Jacobus N) Eloff: Gold Medal for Science for Society, Eskom award for capacity development, Gold medal of the South African Academy for Science and Art is awarded for Scientific and Technological Achievement, Gold Medal for Botany
Tshepiso Makhafola: Attended the University of Pretoria from 2008-2010. He has skills and expertise in research, molecular biology and biotechnology.
Influence of Sutherlandia frutescens extracts on cell numbers, morphology and gene expression in MCF-7 cells
B.A. Standera, S. Maraisa, T.J. Steynberga, D. Theronb, F. Joubertc, C. Albrechtd and A.M. Jouberta
Year of publication:
The article is about the influence of S.frutescens on cell numbers, morphology and gene expression in MCF-7 cells. An extraction was made our of small twogs and leaves, the solution was then filtered. It was demonstrated that ethanolic extracts of S. frutescens inhibited multiplying of MCF-7 mammary adenocarcinoma cells.
Dulbecco’s minimum essential medium eagle (DMEM) with Glutamax™ (Gibco BRL, USA)
• Crystal violet DNA stain was used to determine the number of cells. (Spectrophotometrically)
• Heat inactivated fetal calf serum (FCS) was used to culture the MCF – 7 human breast cell line.
• Penicillin was used to culture the MCF – 7 human breast cell line.
• Streptomycin was used to culture MCF – 7 human breast cell line.
• Sterile cell culture flasks
• 96-well plates where used to house the culturing cells.
• MCF-7 human breast a denoma carcinoma cell line were cultured in DMEM
• Cell Morphology: Two hundred and fifty thousand
MCF-7 cells were put onto heat-sterilized coverslips in well plates and they were exposed to 1.5 mg/ml of Sutherlandia Frutescence extract for periods of 24, 36, 48, and 72 hours at 37°C cells where counted using a microsceope.
Sterile culture flasks and well plates where used, the cultures where kept at a constant temperature of 37°C and in a humidified atmosphere with 5% CO2, the specimens of Sutherlandia frutescens were air dried in the shade in the area of Murraysburg in the Karoo, to reduce the chance degradation of the specimens. The specimens where identified as Sutherlandia frutescens by the botany and biotechnology department at the university of Johannesburg.
1 gram of Sutherlandia frutescens was mixed with 10ml of 70% ethanol to produce a stock solution. After the extraction of the Sutherlandia frutescens it was centrifuged to remove any debris and then it was filtered twice to obtain a purified 100mg/ml stock solution.
The cells where cultured for 24 hours. Vehicle controles where used prove the effectiveness of the Sutherladnia frutescens.
The results that were obtained were statistically analysed for significance using analysis of variance factor model. This was then proceeded by a two-tailed Student’s t-test.
Evidence use to support conclusion:
The ethanol extracts of the Sutherlandia frutescens inhibited the growth of the MCF-7 mammary adencarcenoma cells of the period of 72 hours. 1.5 mg/ml of the Sutherlandia frutescens ethanol extract was statistically found to reduce 50% of the growth of MCF-7 cell over 24 hours when compared to the vehicle-treated control.
It shows different methods of extracting the Sutherlandia frutescens and different substances used to extract the plant. It also gives results that have been statistically proven.
There aren’t any tests to prove its antibacterial effectiveness.
The article doesn’t mention the chemical compounds present in the plant that prove it’s effectiveness.
B.A. Stander: Department of Physiology, University of
Pretoria, P.O. Box 2034, Pretoria 0001, South
S. Marais: Department of Physiology, University of Pretoria,
P.O. Box 2034, Pretoria 0001, South
T.J. Steynberg: Department of Physiology, University of Pretoria, P.O. Box 2034, Pretoria 0001, South Africa
D. Theron: ACGT Microarray Facility, University of Pretoria, 0002 Pretoria, South Africa
F. Joubert: Bioinformatics and Computational Biology Unit, University of Pretoria, 0002 Pretoria, South Africa
C. Albrecht: Cancer Association of South Africa, P.O. Box 2121, Bedfordview 2008, South Africa
A.M. Joubert: Department of Physiology, University of Pretoria, P.O. Box 2034, Pretoria 0001, South Africa
Antibacterial Activity of Leaf Extracts from Combretum micranthum and Guiera senegalensis (Combretaceae)
Stefano Banfi, Enrico Caruso, Viviana Orlandi, Paola Barbieri, Serena Cavallari, Paolo Viganò, Pierangelo Clerici and Luca Chiodaroli
Year of publication:
Guiera senegalensis and Combretum micranthum lwaves were used and tested on for the presence of antibacterial compounds.
Five solvents were used to extract the plant material; the solvents were used in increasing polarity. Escherichia coli C1a and Staphylococcus aureus MSSA were used to test the antibacterial effectiveness of the plants. A bioautographic method was used to monitor the antibacterial activity of the plants extracts throughout the purification steps. The Minimum Inhibitory Concentration and Minimum Bacterial Concentration of the most purified and active plant extracts were evaluated at the end of the procedure.
Dry leaves extraction procedure: Whole leaves of C.
micranthum and G. senegalensis, were dried immediately after obtaining them from the plant in a local drying room at 40°C.
The dried leaves were then sent to Varses. Dried whole leaves weighing 100g were poured in a 2.5 L bottle and treated with 600ml of cyclohexane (least polar solvent). After a period of 24 hours the leaves were separated from the solvent by means of a Buckner funnel. This procedure was repeated using progressively more polar solvents: toluene, acetone, EtOH and water respectively.
Agar diffusion assay: Between 4-5 isolated colonies of each strain were collected and resuspended in 5ml of PB. It was then put onto its respective solid growth medium by means of a sterile cotton swab. The plates were incubated at 37°C for a set amount of time required for each microorganism. The antibacterial effect of the extract was measured by measuring the growth inhibition halo. Pictures if the inhibition halos were taken using a camera to document the findings.
Incubation temperature was kept constant at 37°C. Evidence of the inhibition rings were taken by means of a photo camera and those images were later analysed. A fair test was performed as four different methods of extraction where used, each with increasing polarity.
Evidence use to support conclusion:
Cm4-P showed good activity against S. aureus and S. xylosus.
Cm4-P showed some activity against Gram negative strains. Gs2-Paq was found to be more active against the Gram positive strians compared to Cm4-P.
Gives an example of how an extraction could be done by ordering the solvents according to polarity. It shows how the inhibition rings can be measured and analysed i.e. By means of taking photographs.
The article doesn’t show extraction methods and testing methods for S. frutescens
Stefano Banfi: Degree in organic chemistry in February1980 at the University of Milan, Assistant Professor in Organic Chemistry.
Enrico Caruso: Graduated with a degree in organic chemistry in October 1998 from the University of Milan, Assistant Professor in Organic Chemistry,
Viviana Orlandi: 1995: Degree in Biological Sciences, University of Milan discussing a thesis on “Expression of oppioid receptor in primary coltures of murine cortex neurons: trasduction signal pathway and interaction with glutamate receptors”. Member of the Italian Society for General Microbiology and Microbial Biotechnology (SIMGBM).
Paola Barbieri: 1980: Degree in Biological Science at the University of Milan, Institute of Genetics. Member of the American Society for Microbiology (ASM)Member of the Italian Society for General Microbiology and Microbial Biotechnology (SIMGBM).
Paolo Viganò: Degree in Biological Sciences; Postgraduate Diploma in Microbiology, Doctor of Biological Sciences; Specialist in Microbiology
Source 1 deals with the general usage of S.frutescens as a medicinal plant in South Africa. Source 2 deals with the antibacterial and antioxidant properties of S. frutescens. It also shows extraction methods and bacterial testing methods. Source 3 shows the antibacterial activities of the Ochna species of plants. This source gives an indication of what types of bacteria that need to be used for testing the antibacterial activities of the S. frutescens. Source 4 is about the influence of S. frutescens extract on MCF-7 cells. It has a good indication of an extraction method that can be used. Source 5 is about the antibacterial activity of leaf exracts from Combretum micranthum and Guiera senegalensis. It gives an example of an extraction method that can be used for S. frutescens. All the sources deal with extraction method that can be used for certain plants. Not all the articles deal with the extraction methods and testing of S. frutescens.
B-E. van Wyk and C. Albrecht, 2008. A review of the taxonomy, ethnobotany, chemistry and pharmacology of Sutherlandia frutescens (Fabaceae). Journal of Ethnopharmacology, [Online]. 119, 621-629. Available at: http://def-sa.com/def/wp-content/uploads/2011/10/A-review-of-the-taxonomy-ethnobotany-chemistry-and-pharmacology.pdf [Accessed 20 April 2014].
David R. Katerere† and Jacobus N. Eloff . 2005. Antibacterial and Antioxidant Activity of Sutherlandia frutescens (Fabaceae), A Reputed Anti-HIV/AIDS Phytomedicine. [ONLINE] Available at: http://def-sa.com/def/wp-content/uploads/2011/10/Antibacterial-and-Antioxidant-Activity-of1.pdf. [Accessed 06 April 14].
Tshepiso J. Makhafola and Jacobus N. Eloff. (2011). Five Ochna species have high antibacterial activity and more than ten antibacterial compounds. South African Journal of Science [online]. 108, 689.Available From:http://www.sajs.co.za/five-ochna-species-have-high-antibacterial-activity-and-more-ten-antibacterial-compounds/makhafola-tshepiso-eloff-jacobus.
St, er, B., Marais, S., Steynberg, T., Theron, D., Joubert, F., Albrecht, C. and Joubert, A. (2007). Influence of Sutherlandia frutescens extracts on cell numbers, morphology and gene expression in MCF-7 cells. Journal of ethnopharmacology, 112(2), pp.312–318.
Banfi, S., Caruso, E., Orlandi, V., Barbieri, P., Cavallari, S., Vigano, P., Clerici, P. and Chiodaroli, L. (2014). Antibacterial Activity of Leaf Extracts from Combretum micranthum and Guiera senegalensis (Combretaceae). Research Journal of Microbiology, [online] 9(2), pp.66-81.
Salim S. Abdool Karim, S. (2009). HIV infection and tuberculosis in South Africa: an urgent need to escalate the public health response. Lancet, [online] 374(9693), p.921. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2803032/ [Accessed 14 May. 2014].
Fritz Lherisson, F. (2014). South Africa. [online] Unaids.org. Available at: http://www.unaids.org/en/regionscountries/countries/southafrica/ [Accessed 16 May. 2014].
ALT Associated Mutations in Histone H3, ATRX and DAXX
Besides the contribution of DNA sequence and the state of shelterin binding in telomeric recombination and ALT activity, telomere architecture, and specially histone modifications, are responsible for some phenotypic characteristics of ALT. Recent studies of ALT tumors correlate telomere maintenance mechanism of ALT with activity of the complex ATRX/DAXX.
ATRX and DAXX form a chromatin remodeling complex that moderates chromatin changes. ATRX is responsible for encoding a member of the switch/sucrose non-fermentable (SWI/SNF) family ATP-dependent helicases that have chromatin-remodeling activity namely α-thalassemia mental retardation X-linked protein. DAXX encodes another component of ATRX that is a binding partner death-associated protein 6. ATRX/DAXX complex is needed for chromatin deposition of a histone associated with transcriptionally active open chromatin namely H3.3. It has been reported that the inhibition of this complex suppresses the recombinogenic nature of repetitive telomeric DNA, resulting in loss of hetrochromatic marks. Limitation of expression or loss of the complex ATRX/DAXX show to limit the H3.3 incorporation in telomeric chromatin with a positive effect in telomere recombination. The recruitment of such proteins in detriment of others may affect the construction of ALT telomeres. An example of that are the mutations in H3F3A, the encoding gene of H3.3 that has demonstrated alterations in histone incorporation in the genome with alterations at chromatin remodeling and gene expression.
Loss of ATRX/DAXX activity can originate altered gene expression that can promote ALT activity. This complex has been related with ALT, being associated or not with p53, and the majority of cancers with prevalence of ALT have ATRX mutations. The first correlation between ALT and epigenetic and chromatin dysfunction was found in PanNET and GBM tumors. In these tumors there were observed remains of ALT-associated PML bodies (APBs) and loss of the ATRX (and often) DAXX expression.
It has been described a linkage between ATRX and DNA methylation, chromatin conformation, replication and transcriptional repression, particularly in tandem repeated sections. Loss of ATRX affect telomeres very similarly to ALT, and possibly contributes to ALT activation. The majority of ALT cells exhibit deregulated ATRX expression, however, the sequence is present in the genome, suggesting a role of ATRX protein in ALT suppression, after specific changes in protein maturation.
In addition to ATRX, DAXX and p53 mutations, some tumors exhibit heterozygous mutations in the histone H3.3 gene, H3F3A. It is suggest that these alterations facilitate the activation of TMM by ALT mechanism through the epigenetic changes in the telomeric chromatin. However, some ALT tumors have functional histone H3, ATRX and DAXX expression, which indicates that other fails may contribute to the expression of some ALT hallmarks.
Cells with different TMM have significant differences in telomeric chromatin construction; in ALT cells it is possible to observe more compact telomeric chromatin, relatively with telomerase-dependent cells, that is consistent with most interactions between telomeres observed in these cells allowing HR-dependent telomere maintenance and promoting telomere transcription.
In the majority of tumors with ATRX, DAXX or H3F3A mutations it is also observed p53 mutation, which is indicative of a possible interaction between the mutations.
Non-determined telomere maintenance mechanism
Telomere maintenance mechanism in tumor cells are usually classified in two groups: telomerase-dependent TMM or ALT-dependent TMM. However, this classification is not consensual and there are some studies that identified some groups of tumors with characteristics of both mechanisms or neither.
There are some evidences that ALT pathway may be activated when there is a loss or inhibition of telomerase function. According to the results obtained by Chen et al. ALT telomere elongation is present following telomerase inhibition. In a similar manner, in a study with ALT active cells, the fusion with normal somatic cells or telomerase-positive cancer cells leaded to ALT activity suppression. Unlike to the expected, in a research in which there were used keratinocytes, normal human cells showed the presence of ALT pathway active, but repression of this mechanism in cells telomerase-immortalized. The cells were able to maintain telomeres by both ALT and telomerase and when there is dominance of one mechanism there is competition and further elongation of telomeres. In the same way it has been documented in a research of human sarcomas that the two mechanisms were not mutually exclusive in some tumors being observed in the same tumor some cells having ALT-associated PML bodies, characteristic of ALT, and other expressing telomerase.
The immortalization of cells is considered a general feature of cancer, however, in some conditions the acquirement of limitless growth through the activation of the telomere maintenance mechanisms is not required. Some tumors have neither evidences of the presence of telomerase activity nor ALT. There are some hypothesis to justify that specific condition, that are the fact of the tumor rise in cells that start out with long telomeres; the absence of adverse tumor factors, increasing the cell surviving and the presence of genetic mutations.
At this time, there are no therapies specifically targeting ALT. In some cancer types, particularly some with difficult prognostic, the frequency of ALT mechanisms has increased and the somatic mutations in the ATRX, DAXX and in the histone variant H3.3 found in some tumors can be used as ALT hallmarks. One possibility to target ALT positive cells is through the development of synthetic lethal approaches to kill ATL cells deficient in ATRX or DAXX producing stress for which the genes will act in a protective function.
There are evidences that ALT-dependent cancers use homologous recombination to maintain their telomere length, and this fault in specific cancers can be a promising target to therapy. An efficient therapeutic targeting of TMM in cancers can include the development of ALT inhibitors, since telomerase inhibitors are not effective for ALT tumors. The recent results obtained by Flynn et al. shows hypersensitivity of ATL positive cells to ATR (a critical regulator of recombination) inhibitors and their application in vitro disturb and selectively kill the ALT positive cells. Despite being necessary further research, the application of ATR inhibitors to ALT-positive cancer cells is a promising discovery to clinical studies.
As previously referred, some tumors exhibit telomerase and ALT activity in the same cell and both mechanisms can co-exist. It has been reported in mouse models experiments that in mosaic tumors their survival was promoted by ALT pathway when telomerase was inhibited. In order to obtain an efficient therapeutic; the tumor cells have to be evaluated in what concern the TMM present and in the case of mosaicism, the tumor may be targeted in each type of cell through combination therapies.
In tumors with a single mechanism of telomere maintenance, the treatment with the corresponding inhibitor may lead to the appearance of drug resistance or can treat just the relevant TMM, can be applied selection pressure and activation of the other TMM. Similarly to the treatment of mosaicism tumors, either use of both telomerase and ALT inhibitors is required.
Final remarks and future perspectives:
The unlimited proliferation capacity is one of the hallmarks of cancer and the activation of telomerase maintenance mechanisms (TMM) is essential in tumorigenesis to perform replicative immortality and replication-induced senescence resultant of telomere shortening. The most common mechanism to extend critically short telomeres is the activation of telomerase, but fewer yet significant numbers of tumors extend their telomeres through alternative recombination-based methods- ALT.
In order to develop anticancer therapies targeting ALT mechanisms of TMM, it is needed to intensify the study and the exploration of these alternative mechanisms. There are a number of issues that must be solved, focusing mostly in the epigenetic mechanisms present in ALT telomeres it is necessary to understand the epigenetic landscape and the correlation of the mutations in ALT tumors. For this purpose, the effect of re-introduction the expression of ATRX and the effect of chromatin disruption in ALT cells are experiments that can be performed. It can be further studied the repetitive structure of ALT telomeres and the behavior of each shelterin protein in these cells, specially the TRF2 that seems to have great influence. Furthermore, the specific characteristics of ALT cells like the function of PML bodies and the dynamics of APB formation can contribute to understand the induction of these alternative mechanisms. Complemented with in vivo trials and the research of pharmacological inhibition of ALT tumors it is possible the development of preclinical and clinical studies to treatments and consequently maximize cancer therapeutic efficacy.
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