Title:

Bioassay: The effectiveness of Morus nigra extract as an anti-bacterial property for vaginal
florae

Introduction:
In a few decades, many antibiotics were found to be resistance. This occurrence has led us to
search for a new, safe, clean, and effective antibacterial agents for natural plants. Morus Nigra or
Black Mulberry, a plant that grows in China, Korea and Japan, has been used in traditional
Chinese medicine, in particular as an herbal tea. Mulberry leaf tea's health benefits are attributed
to its naturally occurring compound, 1-deoxynojirimycin, or DNJ. DNJ is responsible for
mulberry's antidiabetic effects, which have been studied extensively. Mulberry leaf tea also has
powerful antioxidant properties and has been found to lower cholesterol and triglycerides and
reduce inflammation. (Karen Mccarthy, n.d.)

Bioassay
Bioassay is defined as estimation or determination of concentration or potency of physical,
chemical or biological agents by means of measuring and comparing the magnitude of the
response of the test with that of standard over a suitable biological system under standard set of
conditions. Bioassay is a successful tool in estimation and discovery of biologically active
substances and important application in sensitivity and specificity of pharmacological
applications. (Sunil J Panuganti, 2015).
Herbal Medicine
The use of herbal medicinal products and supplements has increased tremendously over the past
three decades with not less than 80% of people worldwide relying on them for some part of
primary healthcare. Although therapies involving these agents have shown promising potential
with the efficacy of a good number of herbal products clearly established, many of them remain
untested and their use are either poorly monitored or not even monitored at all. The consequence
of this is an inadequate knowledge of their mode of action, potential adverse reactions,
contraindications, and interactions with existing orthodox pharmaceuticals and functional foods
to promote both safe and rational use of these agents. (Martins Ekor, 2014)
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3887317/
According to an article published in the "International Journal of Food Sciences and Nutrition" in
2006, mulberry leaves contain calcium, iron and zinc. Mulberry also contains the antioxidants
ascorbic acid and beta carotene. Antioxidants inhibit cellular damage caused by free radicals,
which get created during food digestion and smoke and radiation exposure. Regularly consuming
foods and drinks rich in beta carotene may reduce your risk of cancer, according to PubMed
Health.
Vaginal Flora:
The vaginal flora are the bacteria that live inside the vagina. The normal vaginal flora are
dominated by various lactobacillus species. Lactobacilli help to keep the vagina healthy by
producing lactic acid, hydrogen peroxide, and other substances that inhibit the growth of yeast
and other unwanted organisms. They maintain the vagina at a healthy pH of around 4. This
mildly acidic environment helps protect against infection. So do the other substances they
produce. These bacteria are an important part of a healthy vaginal ecosystem. (Elizabeth Boskey,
2019)

Taman ra diri ang kopyaha sa

https://www.livestrong.com/article/265868-what-are-the-health-benefits-of-mulberry-leaf-tea/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3887317/

The use of herbal medicinal products and supplements has increased tremendously over the past
three decades with not less than 80% of people worldwide relying on them for some part of
primary healthcare. Although therapies involving these agents have shown promising potential
with the efficacy of a good number of herbal products clearly established, many of them remain
untested and their use are either poorly monitored or not even monitored at all. The consequence
of this is an inadequate knowledge of their mode of action, potential adverse reactions,
contraindications, and interactions with existing orthodox pharmaceuticals and functional foods
to promote both safe and rational use of these agents. Since safety continues to be a major issue
with the use of herbal remedies, it becomes imperative, therefore, that relevant regulatory
authorities put in place appropriate measures to protect public health by ensuring that all herbal
medicines are safe and of suitable quality. This review discusses toxicity-related issues and
major safety concerns arising from the use of herbal medicinal products and also highlights some
important challenges associated with effective monitoring of their safety. (2014)
Why is a Healthy Vaginal Flora Important?
A hallmark of bacterial vaginosis (BV) is disruption of this normal vaginal flora and a loss of
lactobacilli. This can not only be unpleasant in and of itself. It can also leave a woman more
susceptible to HIV and other sexually transmitted infections. Bacterial vaginosis is actually
caused by an overgrowth of bacteria that normally exist at low levels in the vagina. When the
lactobacillus population is disrupted, these bacteria take over. 
The bacteria associated with BV make a number of volatile amines. These chemicals are what
cause the distinctive odor associated with BV. This odor tends to be more present after sex,
particularly unprotected sex. This is because the amines become smellier at the higher pH
associated with semen. However, despite the association, BV is not caused by sperm. In fact, the
greatest evidence for sexual transmission of bacterial vaginosis is in lesbians. It is not clear
whether BV can be transmitted during vaginal intercourse. Bacterial vaginosis is usually
diagnosed by a wet mount.
How Can Healthy Vaginal Flora Be Restored?
One of the difficulties in treating BV and related conditions, such as yeast infections, is figuring
out how to restore the normal vaginal flora. Sometimes the bacterial populations return to normal
proportions after treatment. Other times they don't. In order to help restore a lactobacillus
dominated flora, a number of researchers are looking at probiotic pills and suppositories. These
treatments would contain lactobacillus species. The hope is that those bacteria would grow and
recolonize the vagina. To date, results have been somewhat positive, if preliminary. Still if they
are borne out, probiotics may be a new way to improve vaginal health and restore a healthy
vaginal flora.
https://academic.oup.com/cid/article/32/4/e69/467047

http://www.phcogj.com/sites/default/files/PharmacognJ-10-167.pdf

The leaf extract of Morus nigra were found to be ineffective against the vaginal flora. We
conclude that it is not recommended to use Morus nigra leaf as a way to aid vaginal flora.
However, further studies are needed.
The microbiological flora of the lower female genital tract provides a dynamic, complex example of
microbial colonization, the regulation of which is not fully understood. When an exogenous
bacterial species, with its array of virulence factors, is introduced into the host, disease does not
always occur. Conversely, under selected conditions, commensal endogenous bacteria—for
example, Gardnerella vaginalis and group B streptococci—can participate in disease processes.
Disease caused by both exogenous and endogenous bacteria correlates positively with a markedly
increased level of bacterial replication. The key question is what determines the quantity of a given
bacterium at any given time. For disease to occur, exogenous or endogenous bacteria that possess
pathogenic prerequisites must attain replicative dominance. Their ability to do so is potentially
governed by inhibitory or synergistic interrelationships with other microbes.

The microbiological flora of the lower female genital tract is a dynamic, complex example of
microbial colonization, the regulation of which is not fully understood. Much of what we know
about the bacterial composition of the female genital tract is derived from qualitative, descriptive
studies [1–10]. The fund of information that such studies have provided with regard to the
microbial flora of the lower female genital tract is weakened by the intrinsic technical limitations
that are inherent in the studies. Often, even the usefulness of qualitative data is negatively
affected by inappropriate or suboptimal methods of data collection, failure to use appropriate
transport systems or enriched media, or a lack of stringent anaerobic technique in the processing
and culture of specimens.

The importance of using specialized media is illustrated in a study of Clostridium difficile by

Bramley et al. [11]. These investigators evaluated cultures of vaginal specimens obtained from
522 women who made a total of 902 visits to a family planning clinic, and they found this
organism in only 1 patient. However, when a specialized medium that contained 0.2% para
cresol was used, a higher rate of isolation (11%) was obtained.

This science fair project involves the use of the bacteria E. coli. While E. coli is not considered a
biohazardous or dangerous bacteria, it is important to always properly clean and dispose of bacteria and
supplies that come in contact with it. See the Bacterial Safety guidelines below for more details on how to
handle bacterial cleanup and waste.

Preparing Plates for Disk Diffusion Test

For this experiment, it is important to inoculate the plate with a uniform distribution of bacterial colonies,
and to use the exact same procedure for each plate. Here are the steps for inoculating the control and
test plates.

1. Use a permanent marker to divide the bottom of 12 nutrient agar plates into four quadrants each.
In one quadrant of each plate write "C" for control. Write the name of a different disinfectant in
each remaining quadrant. In summary:
a. Every plate should have one control quadrant and 3 different disinfectant quadrants.
b. Each disinfectant should be represented on 6 different plates.
2. Follow the directions in the kit to reconstitute the dried E. coli. Let the reconstituted E. coli sit at
room temperature throughout the next step.
 3. Sterilize a cup of water by boiling it on the stove top for 5 minutes. Cover and wait for it to cool to
room temperature. Allowing the water to cool is critical; if it is too hot then it will kill the bacteria in
the next step.
4. Using proper sterile technique, inoculate each plate uniformly. While wearing gloves, gently
shake the container of reconstituted E. coli so that it is uniformly mixed. Put two drops of the E.
coli mixture on a plate. Dip a fresh (unused) sterile cotton swab into the sterile water. Use the
cotton swab to wipe the drops of bacteria around the entire surface of the plate. Cover the plate
and wait at least five minutes for the plate to dry. A few additional tips:
a. The sterile water on the cotton swab makes it easier to spread the bacteria uniformly.
b. Make sure to use a fresh swab for each plate. Do not dip a used swab back in to the
sterile water.
5. Hold a single sterile disk by the edge with sterile forceps and dip it into the disinfectant solution to
be tested. Touch the disk against the side of the container to drain off excess liquid.
6. Use sterile forceps to place a single disinfectant disk in the center of each of the quadrants on
your test plates. Use the forceps to gently press each disk against the agar surface to insure
good contact. Remember to use the exact same technique for each disk—consistency is very
important for this experiment. Make sure to carefully match the label on the plate with the right
disinfectant.
7. Place a plain (not dipped in any disinfectant) sterile disk in each control quadrant.
8. Incubate all of the plates, inverted (lid on the bottom and agar on top), overnight at 37°C. Use a
longer incubation time if necessary (for example, for incubation at lower temperature).

Measuring Zones of Inhibition

1. After overnight incubation, examine your plates (keep them covered at all times).
a. The control quadrants should show uniform colonies over the entire surface of the plate.
If the distribution is highly uneven, you will need to improve your inoculation technique
and repeat the experiment.
b. If your disinfectants are effective at the concentrations you tested, you should see zones
of inhibition around the disinfectant disks. The clear zones around each disk should have
a uniform width, since diffusion of the compounds through the agar should be uniform in
every direction. If this is not the case, suspect either your impregnation technique, or poor
contact of the filter paper with the agar.
2. Measure the diameter of the zone of inhibition for each disk. Keeping the lid of the plate in place,
use a ruler to measure the diameter of the disk plus the surrounding clear area in millimeters
(mm).
a. Include the diameter of the disk in your measurements. For example, if your disk has a
diameter of 6 mm and the clear area has a width of 3 mm beyond the disk, the diameter
of the zone of inhibition that you should measure and record would be 12 mm (6 mm +
3 mm + 3 mm). This is the standard way that zones of inhibition are measured.
b. You will get six separate measurements for each disinfectant, one from each of the three
test plates.
3. Are the diameters consistent across all three plates? Calculate the average and the standard
deviation of the diameter of the zone of inhibition for each disinfectant.
4. Use the values from Table 1 (below) to evaluate the bacterial response to each compound
(Johnson and Case, 1995).

Bacteria are all around us in our daily lives and the vast majority of them are not harmful. However, for
maximum safety, all bacterial cultures should always be treated as potential hazards. This means that
proper handling, cleanup, and disposal are necessary. Below are a few important safety reminders.

 Keep your nose and mouth away from tubes, pipettes, or other tools that come in contact with
bacterial cultures, in order to avoid ingesting or inhaling any bacteria.
 Make sure to wash your hands thoroughly after handling bacteria.
 Proper Disposal of Bacterial Cultures
 o Bacterial cultures, plates, and disposables that are used to manipulate the bacteria
should be soaked in a 10% bleach solution (1 part bleach to 9 parts water) for 1–2 hours.
o Use caution when handling the bleach, as it can ruin your clothes if spilled, and any
disinfectant can be harmful if splashed in your eyes.
o After bleach treatment is completed, these items can be placed in your normal household
garbage.
 Cleaning Your Work Area
o At the end of your experiment, use a disinfectant, such as 70% ethanol, a 10% bleach
solution, or a commercial antibacterial kitchen/bath cleaning solution, to thoroughly clean
any surfaces you have used.
o Be aware of the possible hazards of disinfectants and use them carefully.

Materials and Equipment 

 Neutralizing Bacteria Kit, available from our partner Home Science Tools. Needed from the kit:
o Nutrient agar plates (12)
o E. coli culture, freeze dried and ready to reconstitute
o Sterile disks (48)
o Sterile cotton swabs (6)
o Nitrile gloves
 You will also need to gather these items, not included in the kit:
o Permanent marker
o Pot
o Stove or other way of heating water to boiling
o Water, 1 cup
o Disinfectants (up to 6). Here are some ideas for different compounds to test:
 Solution of garlic powder
 Liquid bathroom cleaner
 Liquid floor cleaner (e.g., one containing pine oil)
 Mouthwash
 Contact lens cleaner
 Anti-acne product
 Household bleach (sodium hypochlorite)
o Optional: 37°C incubator for bacterial culture plates
o Ruler, metric
o Bleach
o Lab notebook
 Antimicrobial agents are chemicals that are used against bacteria. There are many such agents
available. Because there are many different situations where bacterial control is important, no
antimicrobial agent is effective in all situations. For example, you wouldn't use the same
compound to fight an ear infection as you would use to sterilize surfaces in an operating room.
The situations are completely different. In one case, you are trying to assist the body to fight off
an internal infection, and in the other case, you are trying to eliminate bacteria from inanimate
surfaces.
 There are many additional factors that you would have to consider in order to choose an
appropriate antimicrobial agent for a given situation. For example, are the chemical properties of
the agent (e.g., pH and solubility) appropriate for the situation? You would want to know whether
the compound is toxic—to humans, other animals, plants, or beneficial bacteria. Finally, you
would definitely want to know that the compound is effective against the organism(s) you are
trying to eliminate.
 This project shows you one method of measuring the effectiveness of an antimicrobial agent
against bacteria grown in culture. This is called the Kirby-Bauer disk-diffusion method, and here is
how it works. The bacteria of interest is swabbed uniformly across a culture plate. Then a filter-
paper disk, impregnated with the compound to be tested, is placed on the surface of the agar.
The compound diffuses out from the filter paper into the agar. The concentration of the compound
will be higher next to the disk, and will decrease gradually as distance from the disk increases. If
the compound is effective against bacteria at a certain concentration, no colonies will grow
wherever the concentration in the agar is greater than or equal to that effective concentration.
This region is called the "zone of inhibition." Thus, the size of the zone of inhibition is a measure
of the compound's effectiveness: the larger the clear area around the filter disk, the more effective
the compound. Figure 1, below, illustrates the idea.