Title: Phytochemical Analysis and Anti-Microbial Activity against S. aureus, E.

coli and
C. albicans of Alupidan (Tetrastigma harmandii) Leaf and Stem Crude Extract
Brenda Balana, Mary Clarisse Beasares and Ann Ashley Galvez - Reserachers
Ime Glor D. Parcon and Ma. Aelyn Joy G. Jaleco - Advisers
Ramon Avanceña National High School - September 2017

INTRODUCTION

Medicinal plants have a long history of use and is widespread in both developing and
developed countries. According to reports of the World Health Organization, 80% of the
world’s population relies mainly on traditional therapies which involve the use of plant
extracts or their active substances (WHO, 1993).

Microorganisms have developed resistance against many antibiotics due to the

indiscriminate use of antimicrobial drugs (Ahmad et al., 1998). Furthermore, antibiotics
are sometimes associated with side effects (Cunha, 2001) whereas there are some
advantages of using antimicrobial compounds of medicinal plants, such as fewer side
effects, better patient tolerance, relatively less expensive, acceptance due to long history
of use and being renewable in nature (Vermani and Garg, 2002).

Tetrastigma harmandii, locally known as “Alupidan” in the Visayas, is a member of

the Vitacea genus which, comprised by approximately 95 species, distributed in tropical
and subtropical asia (Planchon 1887; Latiff 1983; Chen et al., 2011; Trias-Blasi et al.,
2012; Wen 2017)Most of the members of the Vitacea family show microbial activity (Lui
et al, 2003), aricidal activity (Adarsh et al, 2013), antiviral activity (Yang, 1989) have
phenolics, flavanoids contents and have antioxidant activity (Hossain et al, 2011).

Alupidan is woody vine; woody rough stems, one to two and a half centimeters in
diameter, having three to five elliptic-oblong leaflets, five to twelve centimeters long,
coarsely toothed, smooth and shinning. It has sour fruits and leaves that are usually used
in cooking to add flavor and is suitable for making preservatives. According to traditional
folklore, the decoction of the plant was used as a powerful diuretic and externally as
lotion to treat scabies. (Brown, 1957)

Specifically, this project aims to determine the following:

1. If there is a significant difference in the zone of inhibitions of the different
concentrations of Tetrastigma harmandii and commonly known as anti-microbial
agents against S. aureus, E. coli and C. albicans
2. The phytochemical properties of leaf and stem crude extract of Tetrastigma
harmandii (Alupidan)

Test Microorganisms
 Staphyloccocus aureus is a gram-positive, non-moving or non-motile cocci, round-
shaped bacterium that belongs to the family Staphylococcaceae, and is frequently
found in the nose, respiratory tract and on skin. (Wikipedia). It is one of the five most
common causes of infections after injury or surgery. It affects all known mammalian
species including humans. Due to its ability to affect a wide range of species, S.
aureus can be readily transmitted from one species to another. This includes
transmission between humans and animals. (news-medical.net) S. aureus was
discovered in Aberdeen, Scotland in 1880 by the surgeon Sir Alexander Ogston in
pus from surgical abscesses.
 Escherichia coli is a gram-negative, anaerobic, rod-shaped, coliform bacterium of the
genus Escherichia that is commonly found in the lower intestine of warm-blooded
organisms (endotherms). Most E. Coli strains are harmless but some can cause
serious food poisoning. https://en.wikipedia.org>Eschericheria
 Candida albicans is an opportunistic fungus (or form of yeast)that is the cause of
Candida Related Complex and many undesirable symptoms including fatigue, weight
gain, joint pain, and gas.A combination of factors can lead to Candida albicans
population getting out of control, establishing fast growing colonies and biofilms,
and start to dominate your gut. https://www.thecandidadiet.com
Positive Controls/Anti-biotics Used
*Erythromycin is used to treat certain infections caused by bacteria, such as infections of
the respiratory tract including bronchitis, pneumonia, Legionnaire’s disease (a type of
lung infection), and pertussis, diptheria, etc... It is in a class of medications called
macrolide antibiotics. It works by stopping the growth of bacteria.
https://medlineplus.gov>druginfo>meds

*Nystatin is an antifungal medication. It is used to treat Candida infections of the skin

including diaper rash, thrush, esophageal candidiasis and vaginal yeast infection.
https://enwikipedia.org>wiki>Nystatin
MATERIALS AND METHODS

This is a descriptive and experimental study to know what phytochemicals are

present in T. harmandii (Alupidan) and if there is a significant difference in the zone of
inhibitions of the different concentrations of T. harmandii against S. aureus, E. coli and C.
albicans.

Collection and Verification of Plant Specimen

Fresh Alupidan leaves and stems (Figure 1)

were collected at Brgy. Sto. Domingo,
Arevalo, Iloilo City. Identity of the plant
was verified online using monographs
from Flora of the Philippine Herbarium
by Robinson (1912) and Dr. Gerard P.
Penicilla, Chair, Biological Science
Department of West Visayas State
University.

Figure 1. Tetrastigma harmandii

Preparation of Materials for Sterilization

Materials used in the experiment were prepared and sterilized at West Visayas State
University Central Lab following the standard laboratory procedures.

Media Preparation

Muller Hilton Agar served as the medium for bacterial growth. It was prepared by
dissolving 35.2 grams powdered Muller Hilton agar in 1L distilled water. It was then
autoclaved at 95 psi, 121⁰C for 30 minutes. Other materials used in the study were put in
the Bio Safety cabinet.

Preparation of the culture broth

Nutrient broth was prepared and was inoculated with the test organism for a sub-
culture. A loop full of microorganism was taken and inoculated in the NB and was
incubated at 37⁰C for 24 hours to obtain a viscous growth.

Standardization of Test Organisms using McFarland Standard

Test organisms were then standardized by comparing it to the McFarland standard.

Sterile Broth were added to test tubes containing the test organisms to reach the same
turbidity.

Preparation of plant extract

Fresh Alupidan leaves and stems were collected and washed with tap water for three
times then with distilled water. Juice from leaves were extracted using a juicer. The
extract or juice were then put in the test tube. The same process was done with the stems.
The different concentrations were then prepared; 100%, 75%, and 50%. This was
done by adding distilled water to the extract.

Preparation of Positive and Negative Control

The positive control for the experiment is Erythromycin for S. aureus and E. coli and
Nystatin for C. albicans while the negative control is distilled water.

Preparation of Mueller Hilton agar plates

The prepared fresh and autoclave MHA media was poured in the Petri plates after
cooling it to 45⁰C and was kept to solidify. The prepared fresh and autoclave MHA media
was poured in the Petri plates after cooling it to 45⁰C and was kept to solidify.

Agar Well Diffusion Assay

1 ml of the different standardized test organisms were put in the MHA plates using a
pipettor and were swabbed on the solidified media surface using a spreader.
The using a cork borer with a diameter of 6mm, an agar well is created on the MHA
plates. Then, 0.1ml of different treatments and controls were then put in the agar wells.
The plates were placed in an incubator in upright position at 37oC.

Gathering of Results and Data Analysis

After 24 hours incubation, the zone of inhibition of the treatments were measured
using a Vernier caliper. Data gathered were then analysed using Oneway-ANOVA on
SPSS.
Phytochemical Analysis

1. Detection of Saponins
Foam Test: 0.5 gm/1ml of extract was shaken with 2ml of water. If foam produced
persists for ten minutes it indicates the presence of saponins.
2. Detection of Tannins
Crude extract was mixed with 2ml 2% Ferric Chloride, a blue green color or black
coloration indicate the presence of tannins and phenols.
3. Detection of Flavanoids
Alkaline Reagent Test: Extracts were treated with few drops of dilute sodium hydroxide
solution. Formation of intense yellow color, which becomes colorless on addition of
dilute acid, indicates the presence of flavanoids.
4. Detection of alkaloids: Extracts were dissolved individually in dilute hydrochloric
acid and filtered.
a) Mayer’s Test: Filtrates were treated with Mayer’s reagent (Potassium Mercuric Iodide).
Formation of a yellow colored precipitate indicates the presence of alkaloids.
b) Wagner’s Test: Filtrates were treated with Wagner’s reagent (Iodine in Potassium
Iodide). Formation of brown /reddish precipitate indicates the presence of alkaloids.
5. Detection of Carbohydrates: Extracts were dissolved individually in 5 ml distilled
water and filtered. The filtrates were used to test for the presence of carbohydrates.
a) Molisch’s Test: Filtrates were treated with 2 drops of alcoholic -naphthol solution in a
test tube and few drops of concentrated sulfuric was added. Formation of a violet ring at
the junction indicates the presence of carbohydrates.
Results
Antibacterial Activity of Tetrastigma harmandii (Alupidan)

Table 1 shows the bacterial count in the three treatments using the leaf and stem
crude extract of T. harmandii (Alupidan)

Table 1. Diameter of Zone of Inhibition (Agar Well Diffusion Assay)

Antimicrobial Activity against Staphyloccocus aureus
T. harmandii leaf extract T. harmandii stem extract
100% ml (6mm, 6mm, 6mm) (6mm, 6mm, 6mm)
75% ml (6mm, 6mm, 6 mm) (6mm, 6mm, 6mm)
50% ml (6mm, 6mm, 6mm) (6mm, 6mm, 6mm)
Antimicrobial Activity against Candida albicans
100% ml (6mm, 6mm, 6mm) (6mm, 6mm, 6mm)
75% ml (6mm, 6mm, 6 mm) (6mm, 6mm, 6mm)
50% ml (6mm, 6mm, 6mm) (6mm, 6mm, 6mm)
Antimicrobial Activity against Echerichia Coli
100% ml (7.8mm, 8.2mm, 9.8mm) (6mm, 6mm, 6mm)
75% ml (6.75mm, 7.8mm, 8.35 mm) (6mm, 6mm, 6mm)
50% ml (6.4mm, 6.25mm, 6mm) (6mm, 6mm, 6mm)

To find out if there is a significant difference in the zone of inhibition of bacteria in

the three treatments, Nonparametric Test was used.

Nonparametric Test
Table 2. Hypothesis Test Summary
Null Hypothesis Test Sig. Decision
1 The distribution of T1Saureus is the Independent-
same across categories of Samples Retain the
Concentrate Kruskal- 1.00 null
Wallis Test hypothesis
2 The distribution of T2Saureus is the Independent-
same across categories of Samples Retain the
Concentrate Kruskal- 1.00 null
Wallis Test hypothesis
3 The distribution of T3Saureus is the Independent-
same across categories of Samples Retain the
Concentrate Kruskal- 1.00 null
Wallis Test hypothesis
4 The distribution of T1Calbicans is the Independent-
same across categories of Samples Retain the
Concentrate Kruskal- 1.00 null
Wallis Test hypothesis
5 The distribution of T2Calbicans is the Independent-
same across categories of Samples Retain the
Concentrate Kruskal- 1.00 null
Wallis Test hypothesis
6 The distribution of T3Calbicans is the Independent-
same across categories of Samples Retain the
Concentrate Kruskal- 1.00 null
Wallis Test hypothesis
7 The distribution of T1Ecoli is the Independent-
same across categories of Samples Retain the
Concentrate Kruskal- .368 null
Wallis Test hypothesis
8 The distribution of T2Ecoli is the Independent-
same across categories of Samples Retain the
Concentrate Kruskal- .368 null
Wallis Test hypothesis
9 The distribution of T3Ecoli is the Independent-
same across categories of Samples Retain the
Concentrate Kruskal- .368 null
Wallis Test hypothesis
Asymptotic significances are displayed. The significant level is .05

Table 2 shows that there is no significant difference in the zone of inhibition of

microorganisms in the different concentrations of T. Harmandii

Phytochemical Analysis
Phytochemical Analysis was done to determine whether the selected plant has
varieties of phytochemicals (chemical compounds produced by plants) such as phenolics,
flavanoids, and tannins and possesses various activities such as radical scavenging,
antioxidant and reducing activities.

Table 3. Phytochemical Analysis

Result
Leaf (Tetrastigma Stem (Tetrastigma
TEST
harmandii) harmandii)
 1 2 1 2

SAPONINS
+++ +++ +++ +++
Foam Test
TANNINS
Ferric chloride test
- - - -
FLAVANOIDS
Alkaline reagent test
+ + + +
ALKALOIDS
Wagner’s test
+ + + +
PROTEIN
Ninhydrin +++ +++ +++ +++
CARBOHYDRATE
Molish Test - - - -

LEGEND:
Abundant: +++
Moderate: ++
Present: +
Absent: -

DISCUSSION
The study arrived at the following findings: (1) there is no significant difference in
the zone of inhibitions of the different concentrations of Tetrastigma harmandii when
used as anti-microbial agent against S. aureus, E. coli and C. Albicans; and (2)
Phytochemical properties of leaf and stem crude extract of Tetrastigma harmandii
(Alupidan) showed abundance of saponins and protein content and few content of
flavanoids and alkaloids. Furthermore, there were no tannins and carbohydrates found.

There is no significant difference in the zone of inhibition of the different

concentrations of T. harmandii when used as antimicrobial agent against S. aureus, E. coli
and C. Albicans....

RECOMMENDATION
 From the study, the researchers recommend the following:
1. Try the effect of Alupidan against other kinds of bacteria
2. Try a different type of extraction method to single out the anti-microbial property
like ethanolic or other types of extractions.
3. Since it is rich in saponins, it may be used as an organic content of dish washing
soap
4. Test the effect of Alupidan in curing diarrhea since it had shown minimal reaction
against
E. Coli

CONCLUSION

Maam.
Please add these
 APA format sang sources.
 Specify and discussion maam. ano ang saponins kg proteins?
And alkaloids kg flavonoids. Ky diri kamo maam maka bawi.
Essential gid nga mabutang nio kung ano ang meaning kung
may tannins, alkaloids kg saponins.
 Maam. dugangi ang recommendation tani.
 Maam nagdugang ko bibliography/sources.. need nio ni sia
 Ari ang anova maam. isli lang to .. pero same lng result

ANOVA

Sum of Squares df Mean Square F Sig.

Between Groups 327.121 7 46.732 160.970 .000

Zone of Inhibition in E. coli
Within Groups 4.645 16 .290
(mm)
Total 331.766 23
Between Groups 1491.073 7 213.010 898.857 .000
Zone of Inhibition on S.
Within Groups 3.792 16 .237
aureus (mm)
Total 1494.865 23
 Between Groups 1234.188 7 176.313 141.747 .000
Zone of Inhibition in C.
Within Groups 19.902 16 1.244
albicans (mm)
Total 1254.090 23
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