TAYUG NATIONAL

HIGH SCHOOL

TAYUG PANGASINAN

Antimicrobial activity of Serpentina

(Rauvolfia serpentina) leaves as

syrup against Escherichia

Coli Causing Diarrhea

Background of the study

Many people are suffering because of Diarrhea. According to the DEPARTMENT OF

HEALTH, this disease kills 1.8 million people yearly (Worldwide). In 2018, 137,468 cases of acute

watery Diarrhea around the country (Philippines) were recorded, and 20,290 cases of acute watery

Diarrhea in the Ilocos region(2018). It has been proven that there is an antimicrobial activity from

serpentina against E.Coli "In-vitro Antimicrobial Activity of Roots of Rauwolfia serpentina L. Benth

Kurz" (2016). It is also proven that R. Serpentina leaves a significant activity and supports their

traditional uses in medicine "Antidiarrhoeal activity of leaf methanolic extract of Rauwolfia

Serpentina" (2012). However, there are existing solutions against Diarrhea that you can try at home,

according to an article written by Aaron Kandola (2019). Here are five examples. Rehydrating. It is

critical to stay hydrated when it comes to healing from Diarrhea. Diarrhea produces a fluid deficiency,

which includes dehydration. The body loses electrolytes like salt and chloride as a result of this. It is

critical to replenish fluids to aid healing. A person may become dehydrated if this is not done. Eating

a recovery diet. When recovering from Diarrhea, a diet of small, frequent meals may be preferable to

three larger meals per day. Foods high in pectin, like fruit, may be included in a good diarrhea diet.

Potassium-rich foods include potatoes and sweet potatoes. Electrolyte-rich foods, such as miso soup

and sports drinks. Cooked, soft vegetables with sufficient protein. Avoiding certain foods. It can help

to avoid foods that irritate or put pressure on the gastrointestinal tract, such as high-fat foods, greasy

foods, spicy foods, artificial sweeteners, and foods high in fructose. Taking probiotics. Probiotics are

beneficial bacteria that can help the digestive system. They can assist the gut's functions and aid in the

fight against infection. Some yogurts and fermented foods contain probiotics, live bacteria, and yeasts.

Probiotic supplements are also available in health food stores and online. Trying medicine. Diarrhea

can be treated with a variety of over-the-counter drugs. Antimotility medication can assist in

alleviating symptoms and hasten recovery. Loperamide is a frequent example of this type of

medication (Imodium).
 But why am I still conducting research about a serpentina-based syrup where there are

remedies such as this five? Well, according also to this same article that medications are not

appropriate sometimes. Well, this serpentina-based syrup is also more affordable than other remedies.

It is much easier to get than medicine, fixes hormones and metabolism, has natural healing, stronger

immunity, fewer side effects, and is cost-effective.

Statement of the problem

This study aims to develop a syrup from serpentina leaves.

1. What is the phytochemical component of Serpentina leaves that can inhibit the growth of

E.coli?

2. What is the antimicrobial activity of Rauvolfia Serpentina against E.coli?

3. What is the result of the 9-point hedonic scale for human panels in terms of:

a. Appearance; and

b. Odor?

4. What is the shelf life of the syrup using Serpentina leaves?

Hypotheses

Hypotheses are formulated based on the problems of this study. Serpentina leaves as a

syrup for Diarrhea.

1. Alkaloids and Tannins are Present on Serpentina Leaves.

2. The antimicrobial activity of serpentina showed 65% effectiveness on inhibition with 25%

concentration (making it intermediate), 65% effectiveness on inhibition with 50%

concentration (making it intermediate), 70% effectiveness on inhibition with 75%

concentration (making it intermediate), and 80% effectiveness on inhibition with 100%

concentration (making t susceptible).

3. The result of the 9-point hedonic scale is from like slightly in terms of:
 a. Appearance; and

b. Odor

4. Syrup using serpentina leaves has a shelf life of 1 week and 4 days on room temperature (The

average room temperature in the Philippines is 25℃ to 32℃) and 1 month and 16 days on

refrigerator temperature.

Significance of the study

Community. It will help the less fortunate to use antidiarrheal medications.

Entrepreneur. New products can be made and released to the market

Environment. Serpentina that are widespread can be used, and it will give some places more space

and flora to form.

Farmer. This could be a substitute job for them while the soil rests after the harvest. (Harvesting

Serpentina that are widespread)

General public. This research can raise awareness and inform the general public on how Escherichia

coli can affect anybody, the implications of the disease, and how it can be treated with syrup using

serpentina leaves.

National. It will help the nation to reduce the number of deaths of children as it informs them that

serpentina has a content that can inhibit the growth of Escherichia Coli (Bacteria that Cause Diarrhea).

People. It will help people unable to swallow a whole tablet/capsule.

Researchers. It will help them to improve their studies.

Society. Society will benefit from this research by purchasing an effective treatment for Escherichia

Coli that is also environmentally friendly.

Scope and Delimitations

This study aims to produce a syrup using serpentina leaves and see if it is effective or not. The

tests that will determine if the herbal medicine using serpentina is effective or not will occur in San

Carlos City, Pangasinan, at the A.G.S Diagnostic and Drug Testing Laboratory. Human panels will use

the 9-point hedonic scale to evaluate the syrup using serpentina leaves' acceptance level in terms of

appearance and odor in Barangay C.Lichauco. The shelf life will be determined at Brgy. C.Lichauco,

mold formation, a foul odor, and discolorations are all signs of spoliation.

Definition of terms

Antimicrobial test: used to determine which specific antibiotics a particular bacteria or fungus is

sensitive to.

E.coli: bacteria that cause Diarrhea

Hedonic Scale: The Hedonic Scale is a scale that indicates the extent to which respondents like or

dislike something.

Medicine: medicine is a remedy for a lot of diseases.

Probiotics: These are live microorganisms that are intended to have health benefits when consumed

or applied to the body.

Phytochemical analysis: is to publish original research on the creation, advancement, validation,

and/or expansion of analytical methodology used in plant sciences.

Serpentina: serpentina is a species of flower in the milkweed family Apocynaceae.

Shelf life: Shelf life is the length of time that a commodity may be stored without becoming unfit for

use, consumption, or sale.

 CHAPTER II

REVIEW OF RELATED LITERATURE AND STUDIES

Related literature:

Figure 1. Image of Serpentina (Rauvolfia Serpentina)

R. Serpentina

Serpentina (Rauvolfia Serpentina) Is a flower species in the milkweed family Apocynaceae.

Serpentina has been used in traditional Ayurvedic medicine in India, mainly for high blood pressure

and mental illnesses like schizophrenia and anxiety. Also, one of the chemicals in Indian snakeroot is

the same as a prescription drug called reserpine. Reserpine has been used to treat high blood pressure,

schizophrenia, and some symptoms of poor circulation, and according to a study conducted by II

Ezeigbo,* MI Ezeja, KG Madubuike, DC Ifenkwe, IA Ukweni, NE Udeh, and SC Akoma that the

extract of R. serpentina leaves has a significant antidiarrhoeal activity and supports its traditional uses

in herbal medicine.

Escherichia Coli

Escherichia coli (E. coli) bacteria typically live in the intestines of people and animals. Most

E. coli are harmless and vital to a healthy human intestinal tract. However, some E. coli are
pathogenic, meaning they can cause illness, either Diarrhea or disease outside of the intestinal tract.

The types of E. coli that can cause Diarrhea can be transmitted through contaminated water or food or

contact with animals or persons. E. coli consists of a diverse group of bacteria. Pathogenic E. coli

strains are categorized into pathotypes. Six pathotypes are associated with Diarrhea and collectively

are referred to as diarrheagenic E. coli.

Phytochemical Analysis

Phytochemical analysis is used to screen, identify, extract, and isolate Phyto-constituents in

order to evaluate the therapeutic potential of the plant and to develop phytochemical standards for

medicinal plant materials for quality control. Phytochemical Analysis publishes original articles on

the development, improvement, validation, and/or extension of analytical methodology in plant

sciences.

Antimicrobial Test

Antimicrobial susceptibility tests are used to determine which antibiotics a specific bacteria or fungus

is sensitive to. This testing is frequently used in conjunction with a Gram stain and culture, the results

of which are obtained much faster. Antimicrobial susceptibility tests can help physicians choose the

best drug and dosage for difficult-to-treat infections. The minimal inhibitory concentration (MIC),

which is the lowest concentration of drug that inhibits organism growth, is commonly reported as the

result. Reports usually include a quantitative result in g/mL as well as a qualitative interpretation. It is

used because bacteria may develop resistance to certain antibiotics.

Hedonic Scale

The Hedonic Scale is a scale that indicates the extent to which respondents like or dislike

something, such as a product they tried or a concept they saw. the application of a rating scale to the

evaluation of liking or approval for or pleasure obtained from an object (e.g., brand, product,

concept). As an example, an overall liking rating, which is frequently used as the primary criterion of
overall product performance. The most common hedonic scale is the nine-point hedonic scale (which

this study will use for the evaluation of appearance and odor by human panels), with one (1)

indicating extreme dislike and nine (9) indicating extreme liking.

Shelf Life

The shelf-life of a product is the amount of time it could be consumed. The product will

remain safe and have the desired sensory, chemical, physical, and microbiological properties

throughout its shelf life. Understanding shelf life is critical for assuring product safety and quality.

The shelf life of a product is determined by its degradation mechanism. Most can be influenced by a

variety of factors, including exposure to light, heat, and moisture; gas transmission; mechanical

stresses; and contamination by microorganisms. The shelf of the product of this study will be

determined by room temperature (The average room temperature in the Philippines is 25℃ to 32℃).

REVIEW OF RELATED STUDIES

According to a study conducted by S.R. Deshmukh, D.S. Ashrit, B.A. Patil, entitled"

Extraction and Evaluation of Indole Alkaloids from Rauwolfia Serpentina for their Antimicrobial and

Antiproliferative Activities." (2012), that phytochemical analysis of rauwolfia serpentina and their

different biological activities are conducted, and it is also said that to evaluate the indole alkaloids

from the serpentina and the method of TLC and HPLC are used. In addition, antimicrobial activity

was assessed using a well diffusion assay, MIC, and MBC. This study found that root extract was

effective against S. Typhi, and it was found to be the superior alternative for drug development in the

future.

According to a study conducted by Mohammad Y. Alshahrani 1,†ORCID, Zeeshan Rafi

2,†ORCID, Nadiyah M. Alabdallah 3, Ambreen Shoaib 4ORCID, Irfan Ahmad 1ORCID, Mohammed

Asiri 1ORCID, Gaffar Sarwar Zaman 1ORCID, Shadma Wahab 5ORCID, Mohd Saeed 6,*ORCID

and Salman Khan 2, entitled "A Comparative Antibacterial, Antioxidant, and Antineoplastic Potential

of Rauwolfia serpentina (L.) Leaf Extract with Its Biologically Synthesized Gold Nanoparticles
(R-AuNPs)" (2021) This study has done a comparative assessment of the antibacterial, antioxidant,

and anti-cancer activity of R. serpentina aqueous leaf extract (RSALE) with encapsulated gold

nanoparticles (R-AuNPs). This study Their characterization was done by numerous parameters,

including UV-visible spectroscopy, transmission electron microscopy, dynamic light scattering, and

zeta-potential. A potent antibacterial activity was represented via RSALE and R-AuNPs when

examined by disc diffusion against various Gram-positive and Gram-negative bacterial strains.

Additionally, an enhanced antioxidant potential was detected in R-AuNPs compared to RSALE

evaluated by the 2,2-Diphenyl-1-Picryl Hydrazyl Radical Scavenging (DPPH) Ferric reducing

antioxidant power (FRAP) assay.

A study conducted by Aniel K Owk, Lagudu Mutyala Naidu entitled "In-vitro Antimicrobial

Activity of Roots of Rauwolfia serpentina L. Benth Kurz" (2016) This study was carried out to

investigate the antimicrobial activity of solvents, as well as aqueous extracts of Rauwolfia serpentina

roots and the sections were tested against Bacillus subtilis, Enterococcus faecalis, Escherichia coli,

Klebsiella pneumoniae, Micrococcus luteus, Pseudomonas aeruginosa, Proteus Vulgaris,

Staphylococcus aureus, Streptococcus pneumoniae, Aspergillus niger, Candida albicans and

Saccharomyces cerevisiae by agar well diffusion method. According to the findings, methanol

extracts of the roots of R. Serpentina could be utilized to cure disorders caused by human pathogenic

bacteria and fungus.

According to K.M Elizabeth (2001), the disk diffusion proved the antimicrobial activity of

Rauwolfia serpentina, concentrations ranging from 50-1000 μg against 7.0x109 cells of S. aureus, Y.

enterocolitica, S. typhi. S. typhimurium, E. coli and C. albicans. The study also said that the

Serpentina is more effective against S. aureus and Y. enterocolitica. But the zone of inhibition formed

at 1000 μg concentration suggests that this herbal extract was much more effective against Y.

enterocolitica. These results indicate that R. serpentine has a potent broad spectral antimicrobial activity.
 According to a study conducted by Ramkishore Prabakaran, Vikram v., R.Lakshmi Sundara,

Narayanan Selvapalam entitled "Evaluation of Antioxidant and Antibacterial Potential and α-Amylase

Expression in the Leaf Callus Tissue of Rauvolfia serpentina (Linn.) Benth. Ex Kurz" (2020) is to

develop an effective strategy for serpentina leaf explant regeneration, as well as to investigate amylase

expression and phytochemical profiling using the GC-MS (gas chromatography and mass

spectrometry) technique and silver, gold, and copper nanoparticles were used to investigate

antibacterial activities in more depth.

According to a study conducted by Hussain et at. entitled (Phytochemical and GC-MS

Analysis of n-Hexane Extract of Rauvolfia serpentina L. Benth. ex Kurz.)." (2015) That this study

showed the phytochemical and GC-MS analysis of an n-Hexane extract of Rauvolfia Serpentina and

Qualitative analysis of root extracts of R. serpentina. The study showed that the presence of alkaloids

and tannins are significantly high.

METHODOLOGY

Research design

This study used The posttest-only Control Group Design (Subjects are randomly

selected and assigned to two groups, the control group and the experimental, and only the

experimental group is treated) to determine the syrup's effectiveness using R. Serpentina.

Table 1. Research Design

Bacteria that cause Diarrhea Treatment Posttest

 Experimental X

Escherichia Coli

Control X

Legend: Where X is the results.

Materials

For the preparation of Syrup using Serpentina leaves

Fifty grams (50g/0.05 kg) of serpentina leaves, Seven Hundred and Fifty milliliters (750

mL/0.75L), beaker, spoons, honey, and cotton wool.

For Actual Experimentation

Proper laboratory attire, Product, Brain Heart Infusion Broth, Mueller Hinton Agar, Test

Tubes, Petri dishes, Inoculating Loops, Cotton Swab, Thumb Forcep, Alcohol lamp, Paper disk soak

with product, Caliper or Ruler, Incubator, Autoclave, Mac Farland standard.

Procedures

I. Gathering and Preparation of Materials

The researcher gathered Two Hundred Fifty grams (250g/0.25 kg) of Serpentina leaves at the

researcher's residence at Barangay C.Lichauco Tayug Pangasinan. The honey is bought at the town

market located at Tayug Pangasinan.

II. Plant Identification

 The plant sample is brought to the testing center for plant identification at the Pangasinan

State University, College of Agriculture, Sta. Maria campus located at Sta. Maria, Pangasinan, for

Plant Identification.

Fifty grams (50g/0.05 kg) of serpentina leaves was added to the beaker with Seven Hundred and Fifty

milliliters (750 mL/0.75L) of water and waited until it boiled. When the water with serpentina leaves

boils, the serpentina leaves are removed from the beaker, and two spoons of honey are added. The hot

syrup was filtered through cotton wool and waited until it cools. (The formulation of syrup was

primarily based on the methods released by CUTM Courseware entitled “To prepare and dispense

simple syrup I.P”).

III. Laboratory Analyses

Phytochemical Analysis (Screening for alkaloids and tannins)

Screening for alkaloids and tannins was conducted at A.G.S Diagnostic Laboratory

Aujan Medics with the help of a qualified scientist. The screening for alkaloids used the

Mayer’s Reagent, Wagner’s Reagent, Bouchardats’s Reagent, and Valser’s Reagent to know

the presences of alkaloids. The screening for Tannins used the Gelatin Test, Gelatin Block

Test, and the Ferric Chloride Test to know the presence of tannins (A.G.S. Diagnostic

Laboratory Aujan Medics).

Table 2. Phytochemical screening of alkaloids,

Screening for Alkaloids

Qualitative Test Positive Result Actual Results Remark

Mayer’s Reagent Production of ppt. X X

Wagner’s Reagent Production of ppt. X X

Bouchard’s Reagent Production of ppt. X X

Valser’s Reagent Production of ppt. X X

INTERPRETATION X
Legend: Where x is the actual results, Remark and Interpretation

Table 3. Phytochemical screening of Tannins

Screening for Tannins

Qualitative Test Positive Result Actual Results Remark

Gelatin Test Production of ppt. X X

Gelatin Block Test Production of ppt. X X

Ferric Chloride Test Greenish Blue/ X X

Greenish Black Blue

INTERPRETATION X

Legend: Where x is the actual results, Remark and Interpretation.

According to a study conducted by Hussain et at. entitled (Phytochemical and

GC-MS Analysis of n-Hexane Extract of Rauvolfia serpentina L. Benth. ex Kurz.)." (2015)

That this study showed the phytochemical and GC-MS analysis of an n-Hexane extract of

Rauvolfia Serpentina and Qualitative analysis of root extracts of R. serpentina. The study

showed that the presence of alkaloids and tannins are significantly high.

Formulation of syrup

Fifty grams (50g/0.05 kg) of serpentina leaves was added to the beaker with Seven

Hundred and Fifty milliliters (750 mL/0.75L) of water and waited until it boiled. When the

water with serpentina leaves boils, the serpentina leaves are removed from the beaker, and

two spoons of honey are added. The hot syrup was filtered through cotton wool and waited

until it cools. (The formulation of syrup was primarily based on the methods released by

CUTM Courseware entitled “To prepare and dispense simple syrup I.P”).
Antimicrobial Test ((With different concentration, 25%, 50%, 75%, and 100%).

According to K.M Elizabeth, the disk diffusion proved the antimicrobial activity of

Rauwolfia serpentina, concentrations ranging from 50-1000 μg against 7.0x109 cells of S.

aureus, Y. enterocolitica, S. typhi. S. typhimurium, E. coli and C. albicans. The study also said

that the Serpentina is more effective against S. aureus and Y. enterocolitica.

Preparation of different concentrations (25%, 50%, 75%, and 100%) of Serpentina Aqueous

Extract.

Place at least 25 grams (For 25%), 50 grams (For 50%), 75 grams (For 75%), and 100

grams (For 100%) of plant sample into 5oomL (25%, 50%, 75%, 100%) capacity

Erlenmeyer’s flask. Pour 75mL (For 25%), 50mL (For 50%), 25 mL (For 75%), and 100 mL

(For 100%) of distilled water measured using a graduated cylinder and seal the flask’s

opening with parafilm or cork. Set aside for 1 hour and then reflux for another 1 hour. Allow

it to cool and then filter the extract, using whatman filter paper and funnel, to another

Erlenmeyer’s Flask. The aqueous 25%, 50%, 75%, and 100% plant extract is now ready.

(A.G.S. Diagnostic Laboratory Aujan Medics).

In the preparation of inocolumns, pick around 3-5 well-isolated colonies of the

microorganism and use it to inoculate it to the Brain heart infusion broth. Then incubate at

37°C for 2-8 hours until light to moderate growth of turbidity appears. Then compare it to the

Mcfarland Standard. Inoculate the Mueller Hinton Agar using sterile cotton swab immersed

into the inoculums (Squeezing the swab against the tube wall to remove the excess) by

streaking. By using sterile forceps pick the paper disc soaked with the product and distribute it

to the inoculate agar pressing them into firm contact. Incubate the plate at 37°C for 24 hours.

After 24 hours of incubation observe the zone of inhibition measuring it by calliper or ruler.

And finally, record your result. (A.G.S. Diagnostic Laboratory Aujan Medics).

Table 4. Experimental setup for the antimicrobial activity test against Escherichia Coli
 Experimental Group

Trial 1 Trial 2 Trial 3 Trial 4

X X X X

Legend: Where X is the percentage result of the Laboratory tests.

The experimental set up was tested with 4 different concentrations (25%, 50%, 75%,

and 100%) for the antimicrobial test of the serpentina leaves. The antimicrobial test was

conducted at A.G.S. Diagnostic Laboratory Aujan Medics.

IV. Evaluation of Appearance and Odor by Human Panels

Twenty (20) people aged twelve (12) and above were observed for the evaluation of the

appearance and odor of the serpentina-based syrup. The 9-point hedonic scale were used to evaluate

the appearance and odor, according to Peryam, D. R., and Pilgrim, F. J. (1957). The midway is neutral,

whereas the additional points increase the degree of like or disliking. The 9-point hedonic scale is

shown in the table below.

Table 3. 9-point Hedonic Scale for Human Panel.

LE LVM LM LS N DS DM DVM DE

Score (9) (8) (7) (6) (5) (4) (3) (2) (1)

=X =X =X =X =X =X =X =X =X

Appearance

=X =X =X =X =X =X =X =X =X

Odor

=X =X

Average

Weighted X
 Mean

The average weighted mean will be computed using the following formula:

Table4. Legend

Hedonic Tone Statistical Limit Verbal Description Sign

9 9.01 - 10.00 Like Extremely LE

8 8.01 - 9.00 Like Very Much LVM

7 7.01 - 8.00 Like Moderately LM

6 6.01 - 7.00 Like Slightly LS

5 5.01 - 6.00 Neutral N

4 4.01 - 5.00 Dislike Slightly DS

3 3.01 - 4.00 Dislike Moderately DM

2 2.01 - 3.00 Dislike Very Much DVM

1 1.01 - 2.00 Dislike Extremely DE

V. Shelf life Determination

The shelf life can be influenced by a variety of factors, including exposure to light, heat, and

moisture; gas transmission; mechanical stresses; and contamination by microorganisms, according to

an article published by Science direct entitled “Shelf Life - an overview”, 2012. The serpentina-based

syrup was kept at room temperature (The average room temperature in the Philippines is 25℃ to

32℃) and Refrigerator Temperature.

Table 5: Indicators for the deterioration of the herbal medicine (liquid) using serpentina

Characteristics Normal Sign of deterioration

Appearance Mould-free Yellowish Discoloration and/or

Color Formation of molds

Odor Bittersweet Smell Foul smell and/ or loss of

Bittersweet Smell

VI. Disposal of product.

After treatment and laboratory analyses, the disc containing escherichia coli with syrup

(treated and untreated) are properly disposed through an autoclave method.

VII. Data gathering and analysis

Data were collected following the experiment. The result of the laboratory analyses, such as

the phytochemical analysis, antimicrobial analysis, microbial count, and moisture test, were gathered.

The data collected was analyzed, and conclusions in the study were drawn.

RESULT AND DISCUSSIONS

A. Plant Identification
 After submission of the plant sample at PSU Sta. Maria campus, the licensed plant botanist

identified and certified that the sample was Serpenyina leaves, scientifically known as Rauvolfia

serpentina.

B. Laboratory Analyses

i. Phytochemical Analysis

Screening for alkaloids and tannins was conducted at A.G.S Diagnostic Laboratory

Aujan Medics with the help of a qualified scientist. The screening for alkaloids used the

Mayer’s Reagent, Wagner’s Reagent, Bouchardats’s Reagent, and Valser’s Reagent to know

the presences of alkaloids. The screening for Tannins used the Gelatin Test, Gelatin Block

Test, and the Ferric Chloride Test to know the presence of tannins (A.G.S. Diagnostic

Laboratory Aujan Medics).

Table 1. Phytochemical screening of alkaloids

Screening for Alkaloids

Analyst
Qualitative Test Positive Result Actual Results Remark

Mayer’s Reagent Production of W/ ppt. + A.C Franza

ppt.

Wagner’s Production of W/ ppt. ++ A.C Franza

Reagent ppt.

Bouchard’s Production of W/ ppt. ++ A.C Franza

Reagent ppt.

Valser’s Reagent Production of W/ ppt. + A.C Franza

ppt.

INTERPRETAT Presence of Alkaloids A.C Franza

ION

Table 1 showed that serpentina leaves have a 1 remark on Mayer’s Reagent and

Valser’s Reagent, while the table also showed that Serpentina leaves had 2 remarks on

Wagner’s Radiant and Bouchard’s Reagent. Interpreting that alkaloids are present on

Serpentina leaves.
 Table 2. Phytochemical screening of tannins

Screening for Tannin and Phenolic Compounds

Analyst
Qualitative Test Positive Result Actual Results Remark

Gelatin Test Production of W/ ppt + A.C Franza

ppt.

Gelatin Block Production of W/ ppt + A.C Franza

Test ppt.

Ferric Chloride Greenish Blue/ Greenish Black + A.C Franza

Test Greenish Black sol’n
Blue

INTERPRETAT Presence of Tannin A.C Franza

ION

Table 2 showed that serpentina leaves have a 1 remark on all the tests to determine

the presence of tannins and phenolic compounds (The tests are the Gelatin Test, Gelatin Block

Test, and Ferric Chloride Test). Interpreting that tannins are present on Serpentina leaves.

According to a study conducted by Hussain et at. entitled (Phytochemical and

GC-MS Analysis of n-Hexane Extract of Rauvolfia serpentina L. Benth. ex Kurz.)." (2015)

That this study showed the phytochemical and GC-MS analysis of an n-Hexane extract of

Rauvolfia Serpentina and Qualitative analysis of root extracts of R. serpentina. The study

showed that the presence of alkaloids and tannins are significantly high.

ii. Formulation of syrup

Fifty grams (50g/0.05 kg) of serpentina leaves was added to the beaker with Seven

Hundred and Fifty milliliters (750 mL/0.75L) of water and waited until it boiled. When the

water with serpentina leaves boils, the serpentina leaves are removed from the beaker, and

two spoons of honey are added. The hot syrup was filtered through cotton wool and waited
until it cools. (The formulation of syrup was primarily based on the methods released by

CUTM Courseware entitled “To prepare and dispense simple syrup I.P”).

iii. Antimicrobial Test (With different concentration, 25%, 50%, 75%, and 100%).

Preparation of different concentrations (25%, 50%, 75%, and 100%) of Serpentina Aqueous

Extract.

Place at least 25 grams (For 25%), 50 grams (For 50%), 75 grams (For 75%), and 100

grams (For 100%) of plant sample into 5oomL (25%, 50%, 75%, 100%) capacity

Erlenmeyer’s flask. Pour 75mL (For 25%), 50mL (For 50%), 25 mL (For 75%), and 100 mL

(For 100%) of distilled water measured using a graduated cylinder and seal the flask’s

opening with parafilm or cork. Set aside for 1 hour and then reflux for another 1 hour. Allow

it to cool and then filter the extract, using whatman filter paper and funnel, to another

Erlenmeyer’s Flask. The aqueous 25%, 50%, 75%, and 100% plant extract is now ready.

(A.G.S. Diagnostic Laboratory Aujan Medics).

In the preparation of inocolumns, pick around 3-5 well-isolated colonies of the

microorganism and use it to inoculate it to the Brain heart infusion broth. Then incubate at

37°C for 2-8 hours until light to moderate growth of turbidity appears. Then compare it to the

Mcfarland Standard. Inoculate the Mueller Hinton Agar using sterile cotton swab immersed

into the inoculums (Squeezing the swab against the tube wall to remove the excess) by

streaking. By using sterile forceps pick the paper disc soaked with the product and distribute it

to the inoculate agar pressing them into firm contact. Incubate the plate at 37°C for 24 hours.

After 24 hours of incubation observe the zone of inhibition measuring it by calliper or ruler.

And finally, record your result. (A.G.S. Diagnostic Laboratory Aujan Medics).

Table 3. Result of the Antimicrobial Test (25% concentration)

25% Concentration of Serpentina (Rauvolfia serpentina) Extract Analyst

 Trial 1 Trial 2 Trial 3 Intermediate
65% Effective A.C Franza
11mm 13mm 13mm

Table 3 showed that the Serpentina leaves has a 65% effectiveness against

Escherichia coli at 25% concentration (11mm, 13mm, 13mm) making it intermediate,

50%-70% are considered intermediate (A.G.S. Diagnostic Laboratory Aujan Medics). The

zone inhibition was measured using a ruler or calliper by mm.

Table 4. Result of Antimicrobial Test (50% concentration)

50% Concentration of Serpentina (Rauvolfia serpentina) Extract Analyst

Trial 1 Trial 2 Trial 3 Intermediate

65% Effective A.C Franza
13mm 13mm 12mm

Table 4 showed that the Serpentina leaves has a 65% effectiveness against

Escherichia coli at 50% concentration (13mm, 13mm, 12mm) making it intermediate,

50%-70% are considered intermediate (A.G.S. Diagnostic Laboratory Aujan Medics). The

zone inhibition was measured using a ruler or calliper by mm.

Table 5. Result of Antimicrobial Test (75% concentration)

75% Concentration of Serpentina (Rauvolfia serpentina) Extract Analyst

Trial 1 Trial 2 Trial 3 Intermediate

70% Effective A.C Franza
15mm 15mm 15mm

Table 5 showed that the Serpentina leaves has a 70% effectiveness against

Escherichia coli at 75% concentration (15mm, 15mm, 15mm) making it intermediate,

50%-70% are considered intermediate (A.G.S. Diagnostic Laboratory Aujan Medics). The

zone inhibition was measured using a ruler or calliper by mm.

Table 6. Result of Antimicrobial Test (100% concentration)

 100% Concentration of Serpentina (Rauvolfia serpentina) Extract Analyst

Trial 1 Trial 2 Trial 3 Susceptible

80% Effective A.C Franza
18mm 20mm 17mm

Table 6 showed that the Serpentina leaves has a 80% effectiveness against

Escherichia coli at 100% concentration (18mm, 20mm, 17mm) making it susceptible, 75%

and above are considered susceptible (A.G.S. Diagnostic Laboratory Aujan Medics). The

zone inhibition was measured using a ruler or calliper by mm.

According to K.M Elizabeth, the disk diffusion proved the antimicrobial activity of

Rauwolfia serpentina, concentrations ranging from 50-1000 μg against 7.0x109 cells of S.

aureus, Y. enterocolitica, S. typhi. S. typhimurium, E. coli and C. albicans. The study also said

that the Serpentina is more effective against S. aureus and Y. enterocolitica.

C. Evaluation of Appearance and Odor by Human Panels

Ten (10) people aged twelve (12) and above were observed for the evaluation of the

appearance and odor of the serpentina-based syrup. The 9-point hedonic scale were used to evaluate

the appearance and odor, according to Peryam, D. R., and Pilgrim, F. J. (1957). The midway is neutral,

whereas the additional points increase the degree of like or disliking. The 9-point hedonic scale is

shown in the table below.

Table.9-point Hedonic Scale for Human Panel.

LE LV LM LS N DS DM DV DE TW/ Ran

Score (9) M (7) (6) (5) (4) (3) M (1) WM ked

(8) (2)

6 2 1 1 73 LM

Appearance =48 =14 =6 =5 7.3

 1 3 1 3 2 62 LS

Odor =9 =24 =6 =15 =8 6.2

6.75 LS

Average

Weighted Like Slightly

Mean

The average weighted mean will be computed using the following formula:

D. Shelf Life Determination

The shelf life can be influenced by a variety of factors, including exposure to light, heat, and

moisture; gas transmission; mechanical stresses; and contamination by microorganisms, according to

an article published by Science direct entitled “Shelf Life - an overview”, 2012. The serpentina-based

syrup was kept at room temperature (The average room temperature in the Philippines is 25℃ to

32℃) and Refrigerator Temperature.

Room temperature

Date of observation Appearance Odor

Day 1 (September 6, 2022) No change. No change.

(Mold-free and Yellowish (Bittersweet smell)
color)
 DISCUSSION

The plant sample was collected at Barangay Clichauco, Tayug Pangasinan, and brought to the

testing center for plant identification at the Pangasinan State University, College of Agriculture, Sta.

Maria campus located at Sta. Maria, Pangasinan, for Plant Identification and it was identified and

verified that it is Serpentina and is scientifically known as Rauvolfia serpentina.

According to a study conducted by Hussain et at. entitled (Phytochemical and GC-MS

Analysis of n-Hexane Extract of Rauvolfia serpentina L. Benth. ex Kurz.)." (2015) That this study

showed the phytochemical and GC-MS analysis of an n-Hexane extract of Rauvolfia Serpentina and

Qualitative analysis of root extracts of R. serpentina. The study showed that the presence of alkaloids

and tannins are significantly high. The serpentina leaves were determined at A.G.S. Diagnostic

Laboratory Aujan Medics. Based on the readings, the results showed the presence of Alkaloids and

Tannins.

The product was done at the Researcher’s Residence at Barangay C.Lichauco, Tayug

Pangasinan. The formulation of the syrup was primarily based on the methods released by CUTM

Courseware entitled “To prepare and dispense simple syrup I.P”.

According to K.M Elizabeth, the disk diffusion proved the antimicrobial activity of Rauwolfia

serpentina, concentrations ranging from 50-1000 μg against 7.0x109 cells of S. aureus, Y.

enterocolitica, S. typhi. S. typhimurium, E. coli and C. albicans. The study also said that Serpentina is

more effective against S. aureus and Y. enterocolitica. Based from the results of the laboratory testing

of antimicrobial (with different concentrations), serpentina is 65% effectiveness on growth inhibition

of e.coli with 25% concentration, 65% effective on growth inhibition of e.coli with 50%

concentration, 70% effective on growth inhibition of e.coli with 75% concentration, and 80%

effective on growth inhibition of e.coli with 100%. In conclusion, serpentina is effective against e.coli

with 4 different concentrations.

Using the 9-point Hedonic scale, results from the evaluation of Appearance and Odor by

human panels showed that the 10 human panels liked slightly the syrup in terms of appearance and

odor. In the shelf-life determination, the test shows that the syrup using serpentina leaves lasted for

one (1) week and six (6) days on room temperature and lasted for one (1) month and sixteen (16) at

refrigerator temperature.

CONCLUSION

Based from the results obtained, the conclusion of this study have been drawn:

1. Alkaloids and Tannins are Present on Serpentina Leaves.

2. The antimicrobial activity of serpentina showed 65% effectiveness on inhibition with

25% concentration (making it intermediate), 65% effectiveness on inhibition with

50% concentration (making it intermediate), 70% effectiveness on inhibition with

75% concentration (making it intermediate), and 80% effectiveness on inhibition with

100% concentration (making t susceptible).

3. The result of the evaluation of appearance and odor of syrup by human panels is from

like slightly in terms of appearance and odor.

4. Syrup using serpentina leaves has a shelf life of 1 week and 4 days on room

temperature and 1 month and 16 days on refrigerator temperature.

 RECOMMENDATIONS

For the improvement of this study, the following recommendations were made:

1. The researcher suggests that the presence of alkaloids and tannins should be

quantitatively analysed.

2. The researcher suggests that the microbial analysis of the product should be tested.

3. The researcher suggests that the moisture content of serpentina leaves should be

determined.

4. The researcher suggests that another bacteria should be added alongside Escherichia

coli.

5. The bacteria that the researcher recommends to be added alongside Escherichia coli

is S. aureus and/or Y. enterocolitica since serpentina is more effective against those

two recommended bacterias.

6. The researcher suggests lengthening the shelf-life of the product.

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