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Methodology from Related Studies

Source: http://www.herdin.ph/index.php/component/herdin/?view=research&cid=333
Authors: Alvin Florentino, Andre Lee Garcia
Title: A comparative in vitro study of the antibacterial effect against Staphylococcus aureus of patola extract (luffa acutangula linn.) and
gumamela extract (Hibiscus rosasinensis) versus mupirocin.
Objective: To determine antibacterial effects of Luffa acutangula linn. (Patola) extract and Hibiscus rosasinensis linn. (Gumamela) extract
against Staphylococcus aureus versus Mupirocin; to compare t eh efficacy of different concentrations (25 percent, 50 percent, 75 percent and 100
percent) of dried Patola leaf extracts, obtained by solvent extraction using ethanol, against Staphylococcus aureus as compared to Mupirocin; to
compare the efficacy of different concentrations of the dried Gumamela flower extracts (25 percent, 50 percent, 75 percent and 100 percent)
against Staphylococcus aureus obtained by soxhlet's extraction using ethanol as solvent; to compare the antimicrobial efficacy against
Staphylococcus aureus of different concentrations (25 percent, 50 percent, 75 percent and 100 percent) of the dried Gumamela flower extracts as
compared to Mupirocin.

METHODOLOGY: The air dried Patola leaves were ground to a powdery consistency and was placed in a closed vessel containing ethanol. The
filtrate was made into different concentrations. The dried Gumamela flowers were ground and subjected to the Soxhlet's apparatus with ethanol.
The filtrate was made into different concentrations. The disc diffusion assay of antimicrobial sensitivity was employed in this study.
Materials and Methods: This study is a randomized controlled double blind experimental study.

Methodology from Related Studies

Source: http://scinet.dost.gov.ph/union/ShowSearchResult.php?f=&p=&page=&sid=1&id=The+antimicrobial+activity+of+
(Hibiscus+rosasinensis)+gumamela+flower+bud+extract+on+%3Cem%3EPseudomonas+aerruginosa%3C%2Fem%3E+ATCC%23+27853+and+
%3Cem%3EStaphylococcus+aureus%3C%2Fem%3E+ATCC%23+25923.
Authors: Ang, Mary June et al
Title: The antimicrobial activity of (Hibiscus rosasinensis) gumamela flower bud extract on Pseudomonas aerruginosa ATCC# 27853
and Staphylococcus aureus ATCC# 25923.
Abstract: This experimental study was conducted to test the antibacterial activity produced by the flower bud
extract of gumamela (Hibiscus rosasinensis) on Staphylococcus aureusATTC#25923 and Pseudomonas
aeruginosa ATCC# 27853 by the Kirby Bauer Disk Diffusion Method.
Gumamela flower buds were collected, washed, and air dried before they was placed in the soxhlet extraction apparatus. The solvent used for
the extraction was diethly ether. One hundred grams of gumamela flower buds produced 6 millimeter of extract:
The Pseudomonas aeruginosa ATCC# 27853 and Staphylococcus aureus ATTC#25923 were first incubated at 37 degrees Celsius and then
inoculated into nutrient broth until the turbidity matched that of 0.5 Mc Farland. The bacteria were swabbed on the Mueller Hinton Agar plates.
Sterile paper disks were impregnated with 100% gumamela flower bud extract to serve as the experimental substance. Other sterile paper disks
were impregnated with diethyl ether, for the negative control. Commercial preparations of antibiotic disks were also used for the positive
controls, Penicillin forStaphylococcus aureus ATTC#25923 and Ticarcillin for Pseudomonas aeruginosa ATCC# 27853. All disks were placed on
the plates. The plates were then incubated at 37 degrees Celsius for 18 hours. 20 trials were done for each organism, therefore, a total of 40
Mueller Hinton Agar plates were used.
Zones of inhibitions were measured. The 100% gumamela flower bud extract did not produced and kind of inhibitory effect
on Staphylococcus aureus ATTC#25923. Thus, there was no antimicrobial effect of 100% gumamela flower bud extract against Staphylococcus
aureus ATTC#25923. However, zones of inhibitions were seen forPseudomonas aeruginosa ATCC# 27853 that ranged from 6 mm to 12 mm in
diameter and with an average of 8.95 mm. Further testing with the use of the Minimum Inhibitory Concentration test showed that the gumamela
flower bud extract possessed an antimicrobial effect at both 100% and 50% concentrations. The more dilute solutions of the gumamela flower
bud extract failed to produce the same antimicrobial effect that was seen at the 100% and 50% concentration Level

Methodology from Related Studies

Source: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3609315/
Authors: P Ruban and K Gajalakshmi
Title: In vitro antibacterial activity of Hibiscus rosa-sinensis flower extract against human pathogens
Objective: To access the in vitro antibacterial activity of Hibiscus rosa-sinensis (H. rosa- sinensis) flower extract against human pathogens.

METHODS: Antibacterial activity was evaluated by using disc and agar diffusion methods. The protein was run through poly acrylmide gel
electrophoresis to view their protein profile.
2. MATERIALS AND METHODS:
2.1. Collection of samples
The flowers were initially separated from the main plants body and rinsed with distilled water and shade dried and then homogenized
into fine powder and stored in air tight bottles.
2.2. Extraction of aqueous component
2.2.1. Cold extraction
A total of 10 g of dried flower was soaked in 50 mL of cold water in a conical flask for 24 h and then filtered off using sterile
Whatman No. 1 filter paper into a sterile conical flask and evaporated by using solvent distillation apparatus. The extract was got with
the help of muslin cloth and centrifuged at 10000 rpm for 5 min. The supernatant was obtained and stored at 4 C for further use [6].
2.2.2. Hot extraction
A total of 10 g of dried flower was soaked in 50 mL of hot water which was then boiled for 30 min and kept for 24 h undisturbed and
then filtered through sterile filter paper, evaporated by using solvent distillation apparatus. The extract was got with the help of a
muslin cloth, centrifuged at 10000 rpm for 5 min and the supernatant was stored at 4 C for further use [7].
2.3. Methanol and ethanol extractions
A total of 10 g of flower air dried powder was weighed and was placed in 100 mL of organic solvents (methanol and ethanol) in a
conical flask and then kept in a rotary shaker at 190-220 rpm for 24 h. And then it was filtered with the help of muslin cloth and
centrifuged at 10000 rpm for 5 min. The supernatant was collected and the solvent was evaporated by solvent distillation apparatus to
make the final volume of one-fourth of the original volume, giving a concentration of 40 mg/mL. It was stored at 40 C in air tight
bottles for further studies[8],[9].
2.4. Extraction of protein
One gram of dry weight of the flower was dissolved into 2 mL of phosphate buffer and mixed nicely. Centrifuged at 10 000 rpm for 15
min at 4 C, supernatant was taken. Further, the equal volume of acetone was added and centrifuged at 10 000 rpm for 15 min at 4 C.
The pellet was taken and dissolved in phosphate buffer. This was taken as pure sample. The process was preceded in the same way as
above for the extraction of crude sample which was dividing of acetone[10].
2.5. Protein profile
The estimated protein was resolved in poly acrylmide gel electrophoresis (PAGE) in which the sample was diluted with sample buffer
in the ratio of 1:1 in 2 sample buffer at 100 volt.
2.6. Test microorganism for antibacterial assay
For the in vitro antibacterial assay the following human bacterial pathogens were studied such as S. aureus,Streptococcus, B.
subtillis, E. coli, P. aeruginosa and Salmonella sp.
2.7. Culture preparation for antibacterial assay
The cultures were grown on nutrient agar at 37 C for 18 h and the colonies were suspended in saline (0.85% Nacl) and its turbidity
was adjusted to 0.5 Mac Farland standards (108 CFU/mL). This saline culture preparation was used to inoculate the plates[11].
2.8. Anti bacterial assay

2.8.1. Disc diffusion

In the agar disc diffusion method the test compounds, i.e. the flower aqueous and organic extract were introduced into a disc 0.5 mm
(hi-media) and then allowed to dry. Thus the disc was completely saturated with the test compound at concentration of 40 mg/mL.
Then these discs were placed directly on the surface of Muller Hinton agar plates, swabbed with the test organism and the plates were
incubated at 37 C for 24 h.
2.8.2. Agar well diffusion method
Muller Hinton agar plates were prepared and wells of 5 mm were cut and swabbed with different cultures. The cut wells were then
filled with 50 L of both aqueous and solvent extracts of flowers and leaves separately and the plates were kept for incubation at 37
C for 24 h[12].
2.9. Statistical analysis
The results were analyzed by using standard deviation (SD) statistical method[8]

References
6. Farombi EO. African indigenous plant with chemotherapeutic potential and biotechnological approval to the production of bioactive
prophylactic agent. Afr Biotech. 2003;2:662667.
7. Ekpendu TO, Akshomejce AA, Okogun J. Anti inflammatory antimicrobial activity. Lett Appl microbial.1994;30:379384.
8. Anu Kiruthika K, Amutha Jaisheeba A, Sornaraj R. Evaluation of the antimicrobial property of selected flower extracts when exposed in a
hospital environment. Int J Pharm Tech Res. 2011;2:769774.
9. Ikram M, Inamul H. Screening of medicinal plants for antimicrobial activities. Fitoterapia. 1984;55:6264.
10. Sambrook J, Fritsch EF, Maniatis T. 2nd ed. New York: Cold Spring Harbor Laboratory, Cold Spring Harbor; 1989. Molecular cloning: A
laboratory manual.
11. Artizzu N, Bonaiganorel, Cottiglia F, Loy G. Studies of the diuretic and antimicrobial activity of cynodon dactylon essential
oil. Fitoterapia. 1995;66:174175.
12. Kumar S, Narain S. Herbal remedies of wetlands macrophytes in India. Int J Pharm BioSci. 2010;1(2):112.

Methodology from Related Studies

Source: http://www.academicjournals.org/article/article1380190830_Nascimento%20et%20al.pdf
Authors: Andresa Piacezzi Nascimento, Nathlia Ursoli Ferreira, Edna Aparecida Barizon, Bruno Alves Rocha, Mirela Mara de Oliveira Lima
Leite Vaz and Andresa Aparecida Berretta
Title: Methodologies for the evaluation of the antibacterial activity of propolis

MATERIALS AND METHODS

Microorganisms
The following microorganisms were used: Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853 and
Streptococcus pneumoniae ATCC 49619. The strains of bacteria were acquired from the American Type Culture Collection (ATCC).
Propolis extract
In this study, a green propolis standardized extract (EPP-AF, batch 010/08, Apis Flora, Ribeiro Preto-SP, Brazil) was used. EPP-AF
presents 11% (w/v) of propolis dry matter and it was chemically characterized in a previous study by our group (Berretta et al., 2012).
Validation of the disk diffusion method
The equipment used during the validation was calibrated within the period of validity of the calibration. The S. aureus and P.
aeruginosa were grown on Mueller Hinton agar (Difco, Detroid, MI, USA) and incubated at 35 2C aerobically for 24 h. The S. pneumoniae
was inoculated on Mueller Hinton agar with 5% sheep blood (Plast Labor, Rio de Janeiro-RJ, Brazil) and incubated at 35 2C in 5% CO2 for 24
h.
After the incubation period, an inoculum was prepared in a sterile, 0.85% physiological solution, with turbidity equivalent to a 0.5
McFarland standard (approximately 10 8 CFU/ml). The turbidity was measured by spectrophotometer (Biospectro SP-22, China) at a wavelength
of 625 nm. A sterile cotton swab was dipped into the suspension, spun several times and pressed firmly against the inner wall of the tube, above
the liquid level, in order to remove any excess inoculum from the swab.
The swab containing the suspension was spread over the surface of agar contained in a plate (90x15 mm). The procedure was repeated
twice to ensure even distribution of inoculum. Mueller Hinton agar was used in the test with S. aureus and P. aeruginosa. For S. pneumoniae,
Mueller Hinton agar with 5% sheep blood was used.
Paper disks (Laborclin, Pinhais-PR, Brazil) were placed in a sterile flask containing 5 ml of EPP-AF, for 5 min. Following this, the
soaked disks were removed using forceps, gently pressed on the wall of the flask in order to remove any excess solution and applied to the agar
surface. Each disk was pressed down, so as to ensure complete contact with the surface of the agar.
For the accuracy test, the following disks of antimicrobial agents were evaluated: disk containing 1 g of oxacillin (Cefar, So
PauloSP, Brazil) (for S. aureus and S. pneumoniae) and disk containing 10 g of gentamicin (Cefar, So Paulo-SP, Brazil) (for P. aeruginosa).
The plates of S. aureus and S. pneumoniae were incubated as described previously. The plates of P. aeruginosa were incubated at 35
2C aerobically for 18 h. After the incubation period, the diameters of the zones of inhibition were measured using a ruler.
The experiments were replicated five times for each of the three microorganisms. The accuracy of the method was calculated
according to the equation below:

To determine the precision of the method, the diameters of the zones of inhibition in tests with the EPP-AF (analyst 1) were measured
and the relative standard deviation (RSD) was calculated according to the equation below:

To determine the intermediate precision, a second analyst (analyst 2) performed the method. The difference between the mean of the
results of both analysts was calculated according to the equation below:

Where Ui is the mean of the results of analyst 1 and Xi is the mean of the results of analyst 2. The specificity was demonstrated by the
growth of the microorganisms in appropriate media, as described previously.

Validation of the broth macrodilution method

S. aureus, P. aeruginosa and S. pneumoniae were grown on culture media and incubated as described for the disk diffusion method.
After the incubation period, an inoculum was prepared in a sterile, 0.85% physiological solution, with turbidity equivalent to a 0.5 McFarland
standard (approximately 108 CFU/ml). The turbidity was measured by spectrophotometer at a wavelength of 625 nm. The suspension was tenfold diluted in the culture medium (dilutions ranging from 1:10 to 1:100) to give an inoculum of approximately 106 CFU/ml. Mueller Hinton
broth (Difco, Detroid, MI, USA) was used in the test with S. aureus and P. aeruginosa. For S. pneumoniae, Mueller Hinton broth supplemented
with 5% lysed horse blood (Ebefarma Biolgica e Agropecuria, Cachoeiras de Macacu-RJ, Brazil) was used.
Samples were serially diluted in test tubes (13x100 mm) with 1 ml of culture medium. After dilutions were made, 1 ml of microbial
suspension (106 CFU/ml) was added to each tube. The final inoculum concentration in each test tube was of approximately 5x105 CFU/ml. The
final dry propolis extract concentrations ranged from 0.85 to 55 mg/ml, for the tests with EPP-AF.
For the accuracy test, the following antimicrobial agents were evaluated: tetracycline (Inlab, Diadema-SP, Brazil) (for S. aureus and S.
pneumoniae) and gentamicin (Inlab, Diadema-SP, Brazil) (for P. aeruginosa). The final concentrations of tetracycline and gentamicin ranged from
0.06 to 32 g/ml.
Control tubes consisted of the culture medium (1 ml) plus microbial inoculum (1 ml) or culture medium alone (2 ml); these controls
were used as growth and sterility controls, respectively. Test and control tubes were incubated aerobically at 35 2C for 20 h (S. aureus and P.
aeruginosa) or 24 h (S. pneumoniae). After the incubation period, the MIC of tetracycline and gentamicin was determined.
Because of the turbidity that occurred in test broth when EPPAF was diluted in the culture medium, it was not possible to determine
the MIC. Therefore, the antimicrobial activity of the EPPAF was assessed by means of the minimum bactericidal concentration (MBC), which
was determined by subculturing 20 l aliquots from each tube in the broth dilution series onto agar plates. Mueller Hinton agar was used in the
test with S. aureus and P. aeruginosa. For S. pneumoniae, Mueller Hinton agar with 5% sheep blood was used. The plates of S. aureus and P.
aeruginosa were incubated at 35 2C aerobically for 24 h. The plates of S. pneumoniae were incubated at 35 2C in 5% CO2 for 24 h. After
the incubation period, the MBC was determined. It was defined as the lowest concentration of the sample required to kill the microorganism
being tested.
The experiments were replicated five times for each of the three microorganisms.The accuracy of the method was calculated according
to the equation below:

To determine the precision of the method, the MBC of EPP-AF (analyst 1) was determined and the RSD was calculated. To determine
the intermediate precision, a second analyst (analyst 2) performed the method. The difference between the mean of the results of both analysts
was calculated.
The specificity was demonstrated by the growth of the microorganisms in appropriate media, as described previously.
Statistical analysis
The data of the antibacterial activity were submitted to the nonparametric Kruskal-Wallis test. The established significance level was
1%.

Methodology from Related Studies

Source: https://www.academia.edu/4292957/Antimicrobial_flavonoid_fro_Hibiscus_rosa-sinensis
Authors: Consolacion Y. Ragasa and Leslie Ann A. Rufino
Title: Antimicrobial Flavonoid from Hibiscus rosa-sinensis Linn.

MATERIALS AND METHODS

2.1. General Experimental Procedures
NMR spectra were recorded on a Bruker Avance 400 in CDCl 3 at 400 MHz for 1H and100 MHz for 13C. Column chromatography was
performed with silica gel 60 (70-230mesh), while the TLC was performed withplastic backed plates coated with silica gel F 254. The plates were
visualized with vanillin-H2SO4 and warming.
2.2. Sample Collection
H. rosa-sinensis flowers (2 kg) were obtained from Fairview, Quezon City in September 2004. It was identified as Hibiscusrosasinensis at the Philippine National Museum.
2.3. Isolation
The air-dried flowers (582 g) of H. rosa-sinensis were ground in an osterizer, soaked in dichloromethane for three days, then filtered.
The filtrate was concentrated under vacuum to afford a crude extract (9 g) which was chromatographed in increasing proportions of acetone in
dichloromethane at 10% increment. The 60% to 70% acetone in dichloromethane fractions were combined and rechromatographed (3x) in
dichloromethane:diethyl ether:acetonitrile(9:0.5:0.5) to afford 1 (5 mg, yellowish solid).The dichloromethane fraction was rechromatographed in
petroleum ether to afford a mixture of hydrocarbons and squalene (10mg, colorless oil).
2.3.1. Antimicrobial Tests
The microorganisms used in these tests were obtained from the University of the Philippines Culture Collection (UPCC). These are
Aspergillus niger UPCC 4219, Candidaalbicans UPCC 2168, Bacillus subtilis UPCC1295, Pseudomonas aeruginosa UPCC 1244,
Escherichia coli UPCC 1195, Staphylococcusaureus UPCC 1143, and Trichophytonmentagrophyte UPCC 4193. The test compound as dissolved
in 95% ethanol. The antimicrobial assay procedure reported in theliterature (Guevara & Recio, 1985) was employed. The activity index was
computed by subtracting the diameter of the well from the diameter of the clearing zone divided by the diameter of the well.
2.3.2. Cytotoxicity Tests
Compound 1 was tested for cytotoxicactivity against a human lung non-small celladenocarcinoma (A549) cell line and a normal
Chinese hamster ovarian (AA8) cell line at theInstitute of Biology, University of thePhilippines, Diliman, Quezon City.Doxorubicin was used as
the positive control,while DMSO was used as the negative control. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT)cytotoxicity assay reported in the literature was employed (Freshney, 1994).

Methodology from Related Studies

Source: http://www.ijera.com/papers/Vol2_issue6/BV26490496.pdf
Authors: Vibha Porwal, Pallavi Singh, Devendra Gurjar
Title: A Comprehensive Study On Different Methods Of Extraction From Guajava Leaves For Curing Various Health Problem
Abstract: Psidium guajava leaf is an important part of guava tree which is useful in curing many health problems. Guava leaves having
properties like antibacterial, anti-oxidant, anti-cancer, anti ulcer etc. used in many diseases. In this article different extraction methods are
discussed and yield of these methods represented. Extraction processes with different solvent such as ethanol, methanol, ethyl acetate and water
are discussed in this article. The purpose of this article is to introduce the different extraction processes for different compounds for curing
different health problems. In this study, aqueous extract shows its widely use in medical as compare to other solvents extract. Aqueous extract of
guajava leaf have antiglycation activity and prevent neurodegenerative and cardiovascular disease and also shows antiprostate cancer activity
BASIC EXTRACTION METHODS
A. Super critical fluid extraction
Extraction process is basically for separation of one component from other component by using extracting solvent. In SFE, we use
supercritical fluid as extracting solvent with some co-solvent to increase its capacity to separate. For this type of extraction, we take sample of
guava leaves 1 mg in a cell column. In SFE CO2 is mostly taken as super critical fluid with ethanol and methanol as co-solvent.
Fig.1 showing different components: a pressure cell, pressure controller, collecting vessel, heating and cooling system and pump. First
liquid is converted into supercritical condition then pass it to the extraction vessel where it could easily diffuse into solid matrix of sample and
dissolve the material which we have to extract.
The dissolved material will swept away from cell column at lower pressure and extracted material settle out.CO2 can be recycled.
Temperature and pressure conditions should be 45-55 C and 200-300 bar.

B. Soxhlet extraction
Different components which used in Soxhlet extraction like thimble, water cooling system, and reservoir, by pass tube, siphon tube
and condenser can be seen in figure 2. We will take 10 mg of solid material of leaves keep in thimble which is loaded into soxhlet vessel having
flask containing extractor solvent. Solvent vapor moves up to the column and floods into the chamber housing the thimble of solid. Some part of
nonvolatile compounds dissolves in solvent. Process repeats many times until we get desired concentrated compounds in flask. Process has been
done at boiling temperature of solvent and extraction has been done in 100 ml ethanol for 3.5 hours.

C. Steam distillation

This process is basically for natural aromatic compounds used for temperature sensitive compounds which decompose at high
temperature i.e. 25 mg of dried leaf material distilled in 300 ml water. Process carried out for 3-4 hours at boiling temperature of solvent and after
distillation, volatile compounds and water separated by methyl chlorate.

In the above figure grey represents steam, which lowers the boiling point of the compounds in the mixture so that they can be distilled
before the heat destroy them.
D. Ultrasound extraction
We will take 1.5 mg of solid sample in ultrasound apparatus. Ultrasound apparatus consist of water tank, ultrasound generator, digital
timer, rotary gear and others (shown in fig.5).process temperature should be 40-50 C and process time should be 1-2 hours. After this process,
material was filtered using a vacuum evaporator.

Methodology from Related Studies

10

A study on the antimicrobial property of the flower extract from gumamela(Hibiscus rosasinensis) on Staphylococcus aureus
Agustin, F
Inventory of Health Researches : 151 (1994-1996)
Abstract:
Extracts from fresh flowers of the Hibiscus rosasinensis (gumamela) were diffused into paper discs then placed in agar plates with S. aureus
growths. Cloxacillin discs and discs soaked in 95% ethyl alcohol were used as positive and negative control respectively. The zones of inhibition
were then observed and measured. Hibiscus extracts had a 13.2 mm mean zone of inhibition compared to 29.6mm mean zone of inhibition for
Cloxacillin while 95% ethyl alcohol discs showed only a mean value of 9.4% zone of inhibition. Hibiscus extracts have some inhibitory effect on
S. aureus.
Availability :
Philippine Council for Health Research and Development; Department of Science and Technology
Email: pchrd@pchrd.dost.gov.ph