TAYUG NATIONAL

HIGH SCHOOL
TAYUG PANGASINAN

Antimicrobial Activity of Serpentina

(Rauvolfia serpentina) Leaves as

Syrup Against Escherichia

Coli Causing Diarrhea

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 moisture content of the syrup using serpentina leaves?

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

a. Appearance; and

b. Odor?

5. What is the shelf life of the syrup using Serpentna leaves?

Hypothesis

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

a syrup for Diarrhea.

1. Syrup using Serpentina leaves has high alkaloids and a presence of tannins which can inhibit

the growth of e.coli.

2. The antimicrobial activity of serpentina at concentrations ranging from 50-1000 μg against

7.0x109 cells of S. aureus, Y. enterocolitica, and S. Typhi. S. Typhimurium, E. coli, and C.

Albicans.

3. The moisture content of the field-grown serpentina leaves is 72.83% (Moisture content of leaf

part of field-grown plants of Rauvolfia serpentina at 45°C)

a. Appearance; and

b. Odor.

5. Syrup using serpentina leaves has a shelf life of five (5) to six months(6) at room temperature

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

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:

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 that is also

environmentally friendly.

Scope and Delimitations

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.

Microbial count: microbial count is an accepted laboratory method used to estimate the microbial

population of a tested substance.

Moisture test: this test involves taping an 18" x 18" polyethylene sheet to a concrete surface and then

waiting at least 16 hours.

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.

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.
Retrieved from: https://www.webmd.com/vitamins/ai/ingredientmono-787/indian-snakeroot

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.

Retrieved from: https://www.cdc.gov/ecoli/general/index.html

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.

Retrieved from:

https://www.researchgate.net/post/What_are_the_objectives_of_chemical_investigation_of_a_medici

nal_plant, and https://www.wiley.com/en-us/Phytochemical+Analysis-p-j00002690

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.

Retrieved from: https://emedicine.medscape.com/article/2103786-overview

Microbial Count

Microbiological count methods are accepted laboratory methods for estimating a tested

substance's microbial population. One of the most important indicators in the field of hygiene

management is the total bacteria count. It indicates the number of microorganisms present in a

sample. The total bacteria count must be monitored because the number of microorganisms should not

exceed certain guide values. These guide values, expressed in CFU (colony-forming units) per gram

or milliliter.

Retrieved ftom: https://emedicine.medscape.com/article/2103786-overview

Moisture Content

Moisture content is simply the amount of water in a product. The moisture content of a product

influences its processibility, shelf life, usability, and quality. As a result, accurate moisture content

determination is critical in many industries, including food, pharmaceuticals, and chemicals.

Furthermore, legislation may govern the maximum allowable moisture content in certain products.

Retrieved from: https://www.mt.com/ph/en/home/applications/Laboratory_weighing/moisture-

content-determination.html

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.

Retrieved from: https://www.ipsos.com/en/ipsos-encyclopedia-hedonic-scale

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 (20℃ to 25℃).

Retrieved from: https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/shelf-life

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.
Retrieved from:

https://www.researchgate.net/publication/285569298_Extraction_and_evaluation_of_indole_alkaloid

s_from_Rauwolfia_serpentina_for_their_antimicrobial_and_antiproliferative_activities

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.

Retrieved from: https://www.mdpi.com/2223-7747/10/11/2278

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.

Retrieved from: https://www.researchgate.net/publication/308935117_In-

vitro_Antimicrobial_Activity_of_Roots_of_Rauwolfia_serpentina_L_Benth_Kurz

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.

Retrieved from: https://www.researchgate.net/publication/349594742_Evaluation_of_a-

amylase_expression_and_analysis_of_phytochemical_in_the_leaf_callus_tissue_of_Rauvolfia_serpe

ntina_Linn_Benth_Ex_Kurz_exposed_to_Cyclodextrin

According to a study conducted by Anwar Hussain 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 indicates the presence of alkaloids, saponins,

tannins, steroids, flavonoids, and phenols.

Retrieved from: https://www.researchgate.net/publication/320881291_Phytochemical_and_GC-

MS_Analysis_of_n-Hexane_Extract_of_Rauvolfia_serpentina_L_Benth_ex_Kurz
 METHODOLOGY

Research design

This study will use 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.

Procedures

I. Gathering and Preparation of Materials

The researcher will be gathering Two Hundred Fifty grams (250g/0.25 kg) of Serpentina

leaves at the researcher's residence at Barangay C.Lichauco Tayug Pangasinan. The honey will be

bought at the town market located at Tayug Pangasinan.

II. Plant Identification

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

Pangasinan, for Plant Identification.

III. Formulation of syrup

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

Hundred and Fifty milliliters (750 mL/0.75L) of water and wait until it boils. When the water with

serpentina leaves boils, the serpentina leaves will be removed from the beaker, and two spoons of

honey will be added. Filter the hot syrup through cotton wool and wait until it cools.

IV. Laboratory Analyses

i. Phytochemical Analysis- The syrup using Serpentina leaves will be

brought to A.G.S in San Carlos Pangasinan for the phytochemical

analysis.

ii. Antimicrobial Test- The syrup using Serpentina leaves will be brought

to A.G.S in San Carlos Pangasinan for the Antimicrobial Test.

iii. Microbial Count- The syrup using Serpentina leaves will be brought to

A.G.S in San Carlos Pangasinan union for the Microbial Count.

iv. Moisture Test- The syrup using Serpentina leaves will be brought to

A.G.S in San Carlos Pangasinan for the Moisture test.

V. Evaluation of Appearance and Odor by Human Panels

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

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

the appearance and odor. 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.
 Table2. 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

Odor X X X X X X X X X

X X

Average

Weighted

Mean

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

Table3. Legend

Hedonic Tone Verbal Description Sign

9 Like Extremely LE

8 Like Very Much LVM

7 Like Moderately LM

6 Like Slightly LS

5 Neutral N
 4 Dislike Slightly DS

3 Dislike Moderately DM

2 Dislike Very Much DVM

1 Dislike Extremely DE

VI. Shelf life Determination

The serpentina-based syrup will be kept at room temperature (25 ℃ to 32 ℃). The researcher

will check the shelf life determination once a week. The following are markers of color, texture, and

odor deterioration.

Table 4: Indicators for the deterioration of the herbal medicine (liquid) using serpentina

Characteristics Normal Sign of deterioration

Appearance X X

Odor X X

Texture X X

X is the indicator of deterioration.

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

the phytochemical analysis, antimicrobial analysis, microbial count, and moisture test, will be

gathered. The data collected will be analyzed, and conclusions in the study will be drawn.

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Wahab, S., Saeed, M., & Khan, S. (2021). A Comparative Antibacterial, Antioxidant, and

Antineoplastic Potential of Rauwolfia serpentina (L.) Leaf Extract with Its Biologically

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