FAR EASTERN UNIVERSITY

HIGH SCHOOL

“The Antibacterial Activity of Tinospora cordifolia (Makabuhay Plant) against

Staphylococcus aureus”

A Research Proposal
Presented to the Far Eastern University High School

In Partial Fulfillment
of the Requirements for Senior High School – Grade 12

by

De Guzman, Alcyren Joy D.

Baricaua, Mayflor B.
Baydo, Nyzel Jheune Z.
De Guzman, Giselle Jane C.
Enciso, Slinnie C.
Landrito, Sofia Lubelle P.
Lorio, Alliah B.
Nituda, Jasmine Claire M.
Santos, Mernelle Mae O.

Grade 12 – STEM 2

SEPTEMBER 2018
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TABLE OF CONTENTS

Page
1.0 Introduction

1.1 Background of the Study 3

1.2 Conceptual Framework 4
1.3 Theoretical Framework 5
1.4 Statement of the Problem
1.5 Research Hypotheses
1.6 Significance of the Study
1.7 Scope and Delimitations
1.8 Definition of Terms

2.0 Literature Review

2.1 Biological and Phytochemical Analysis of Tinospora cordifolia 18

2.2 Title of RRL Section 2 19
2.3 Title of RRL Section 3 25

3.0 Methods

3.1 Design 30
3.2 Subjects and Study Site 31
3.3 Data Collecting Instruments 32
3.4 Data Gathering Procedure 33
3.5 Data Analysis 34
3.6 Ethical Considerations

References 36

Appendices
Appendix I: Permit to Conduct Research 40
Appendix II: Informed Consent Form 49
Appendix III: ______________ Questionnaire 50
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“The Antibacterial Activity of Tinospora cordifolia (Makabuhay Plant) against

Staphylococcus aureus”

Baricaua, F. B., Baydo, N. B., De Guzman, A. D., De Guzman, G. C., Enciso, S. C., Landrito, S.
P., Lorio, A. B.,Nituda, J. M., Santos, M. O.

1.0 Introduction

1.1 Background of the Study

Herbal medicines have been part of traditional health care in most parts of the world for

thousands of years. Back in the day when there are little or few resources and when some doesn’t

have the means to obtain the things they need, people learned to study their surroundings and

take full advantage of what is around them that can and will benefit them. One example is when

people back then needs to be treated because of some disease, illness or an injury they rely on the

available resources that they can get especially around the place they live in and that was when

they discovered the use of some plants as herbal medicines or as an alternative medicine.

One of the plants known for having many medicinal use in traditional system of medicine

is the Tinospora cordifolia (Wild) Hook. & Thomson (Menispermeaceae). It is a common

climbing shrub found in tropical deciduous forest of south Indian peninsular plains. The plant

has been reported to contain Tinosporin, Columbin and Tinosporin acid. It is well reputed in

traditional system of medicine to treat various ailments such as fevers, inflammations, skin

infections, urinary infections etc. (Jeyachandran, Xavier, & Anand, 2003). In the Philippines, it is
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commonly known as Makabuhay plant and can be seen in rainforests, nearby thickets and in

most or all islands of the country. There are tons of benefits of using the plant especially its stem

as a primary ingredient. The plant is also used in the Philippines for treating rheumatism and

arthritis, abdominal pains, scabies, skin ulcer and other diseases/ailments by mixing its extract

with coconut oil as traditionally done back then.

Staphylococcus aureus is a major bacterial human pathogen that causes a wide variety of

clinical manifestations. S. aureus is found in the environment and is also found in normal human

flora, located on the skin and mucous membranes (most often the nasal area) of most healthy

individuals. S. aureus does not normally cause infection on healthy skin; however, if it is allowed

to enter the bloodstream or internal tissues, these bacteria may cause a variety of potentially

serious infections. Transmission is typically from direct contact. However, some infections

involve other transmission methods (Taylor & Unakal, 2017). It is the leading cause of skin and

soft tissue infections such as abscesses (boils), furuncles, and cellulitis. S. aureus can cause

serious infections such as bloodstream infections, pneumonia, or bone and joint infections.

Considering the uses of Tinospora cordifolia in traditional system of medicine, the

researchers will study on the antibacterial activity of Tinospora cordifolia against

Staphylococcus aureus in order to know if the extract of the plant can inhibit the growth of this

bacteria.

1.2 Conceptual Framework

This experimental study focuses on the antibacterial activity of Tinospora cordifolia

against Staphylococcus aureus. This section explains the designed framework of the whole

dissertation and explain the relationship among different concepts in the study.
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Figure 1 shows the conceptual framework of the study. The input is the title of the study.

It involves the variables of the study.

Then the process which includes the materials for the disk-diffusion method. The

researchers will culture the Staphylococcus aureus bacteria using blood agar.

The output includes the experimentation itself. The researchers will used the Disk-

diffusion method and measure the zone of inhibition. The statistical analysis specifically,

Analysis of Variance (ANOVA), will be used to determine the differences of the samples.

Figure 1. Paradigm of the Study

1.3 Theoretical Framework

Germs are present and can be found everywhere, it’s in the air, water and even the human

body isn’t spared of having it. There are major types of germs such as bacteria and fungi.
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Germ Theory explains that some diseases are caused by the presence, actions and growth of

microorganisms within the body. It is considered as the cornerstone of modern medicine and

clinical microbiology. Germ theory led in to the development of antibiotics and hygienic

practices. A lot of scientist and researchers played a huge part and contributed to this theory.

Louis Pasteur is one of the most major contributor of this theory. Pasteur is one of the first to

link germs to diseases. He also developed ways of making bacteria and viruses less

dangerous.

It is relevant in this study to develop ways on how to treat, lessen and prevent the

growth of disease causing bacteria or organism in one’s body. With the help of Germ Theory,

we are expected to prove that by the use of plant extract of a Tinospora cordifolia, that some

plants have antibacterial properties that can prevent and stop the growth of Staphylococcus

aureus bacteria in one’s body.

1.4 Statement of the Problem

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This research attempts to understand the antibacterial activity of Tinospora cordifolia

against Staphylococcus aureus. More specifically it seeks to find the answers to the following

questions:

1. What are the antibacterial properties of Tinospora cordifolia?

2. Does the ethanolic extract of Tinospora cordifolia stem in 50%, 75%, and 100%

concentrations against Staphylococcus aureus capable of producing zone of

inhibition using Kirby-Bauer method?

3. Is there a significant difference between the 50%, 75%, and 100% concentrations

compared to Penicillin in inhibiting Staphylococcus aureus?

1.5 Research Hypotheses

1. Tinospora cordifolia contains antibacterial properties which are Tinosporin,

Columbin and Tinosporin acid.

2. The ethanolic extract of Tinospora cordifolia stem in 50%, 75%, and 100%

concentrations are capable of inhibiting the growth of Staphylococcus aureus.

3. There is a significant difference between the 50%, 75%, and 100%

concentrations in inhibiting Staphylococcus aureus.

1.6 Significance of the Study

The findings in which this study will reveal, may benefit certain group and the benefits
they may be able to gain are as follows:

 HOUSEHOLD

This study is beneficial to every household, especially to those who has Tinospora

cordifolia in their backyard and is currently using it in many ways and purposes. It is hoped that

this study will encourage families to use a natural antibacterial agent in killing or stopping the
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growth of harmful and dangerous micro-organisms, which can lead to serious major

complications in the near future.

 CONSUMERS

Staphylococcus aureus is present in contaminated objects and is often spread through skin-to-

skin contact. This study aims to give knowledge or information if the consumers can really use

Tinospora cordifolia in curing staph infections.

 ACADEME

This research aims to create a good image for the benefit of this institution. If the findings

and procedures stated were accurate and relevant, the school will gain credits.

 RESEARCHERS

This experimental research is a good opportunity to expand the knowledge of the

researchers that can help them in everyday living. The learnings obtain by the researchers can

also be used in conducting a new experimental research in the future and in using another plant

extract against a chosen bacteria.

 FUTURE RESEARCHERS

The result of this research will serves as a good source of accurate and useful

information. In conducting new research or in testing the validity of other findings, the ideas

presented can be used as a reference. This study will also give them an overview about the

antibacterial property of Tinospora cordofilia, Kirby-Bauer method and Staphylococcus aureus.

1.7 Scope and Delimitations

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This study will be conducted primarily to assess the antibacterial activity of Tinospora

cordifolia against Staphylococcus aureus. This will be tested using the Kirby–Bauer test where

according to Hudzicki (2009), the purpose of this is to measure the antibiotic sensitivity of

bacteria on an agar plate that is spread with bacteria and uses paper disks of antibiotics to test the

extent to which bacteria are affected by those antibiotics. Through experimental – comparative

method, the researchers can manipulate the amount of Tinospora cordifolia stem extract) and

ethanol (mixed to obtain the amount that has best to counter microorganisms. This study is only

limited to the antibacterial activity of ethanol extracts from the stems of Tinospora cordifolia to

the bacteria Staphylococcus aureus. The study shall be conducted inside the Far Eastern

University Senior High School Science Laboratory and shall be incubated at 25° for 24 hours to

compare the diameter of inhibition which forms around the paper discs where the best amount of

antiseptic will show the largest inhibition zone.

1.8 Definition of terms

In order to have a better understanding for the readers and for clarification, the

researchers defined the important terminologies that will be seen in this study.

Antibacterial- destroying or inhibiting the growth of bacteria and especially pathogenic

microorganisms

Ethanol- a colorless volatile flammable liquid that has bactericidal activity and is used often as a

topical disinfectant. It is widely used as a solvent and preservative in pharmaceutical

preparations as well as serving as the primary ingredient in alcoholic beverages

Infection- the act or result of affecting injuriously

Microorganism- an organism of microscopic or ultramicroscopic size

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Mueller Hinton Agar- Mueller and Hinton developed Mueller Hinton Agar (MHA) in 1941 for

the isolation of pathogenic Neisseria species. Nowadays, it is more commonly used for the

routine susceptibility testing of non-fastidious microorganism by the Kirby-Bauer disk diffusion

technique

Phytochemical Testing- refers to the extraction, screening and identification of the medicinally

active substances found in plants. Some of the bioactive substances that can be derived from

plants are flavonoids, alkaloids, carotenoids, tannin, antioxidants and phenolic compounds

Staphylococcus- any of a genus (Staphylococcus) of non-motile gram-positive spherical bacteria

that occur singly, in pairs or tetrads, or in irregular clusters and include causative agents of

various diseases (such as skin infections, food poisoning, and endocarditis)

Tinospora cordifolia or Makabuhay plant- A plant that has a lot of medicinal claims such as

being a disinfectant. This is the main ingredient used as antiseptic agent to kill and prevent

growth of bacteria.

2 Literature Review

2.1 Biological and Phytochemical Analysis of Tinospora cordifolia

Stuart Jr. (2018) stated that the Tinospora codifolia has a wide range of use in

terms of its ability as an alternative medicine in which the stems and leaves of the plant are the

main parts that are utilized. Its properties are considered febrifuge, vulnerary, tonic, antimalarial,

parasiticide, and insecticidal. Studies also suggests Tinospora cordifolia is effective as an

antioxidant, antidiarrheal, antimicrobial, anti-diabetic, anti-proliferative, cardiotonic,

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hypolipidemic, antimalarial, ant nociceptive. Even though it has many uses, there are cautions in

using the plant especially those who are pregnant or patients with cardiac disorders.

Patel & Ishnava (2014) cultured vegetative parts such as stem, leaf and nodal explants

from an elite in vivo grown mature plant of Tinospora cordifolia on MS medium supplemented

with different hormonal concentrations and reported callus induction and organogenesis. Callus

formation occurred from nodal segments, leaf and inter-node explants when planted on different

combinations of hormones. Tinospora cordifolia showed response for in vitro shoot growth from

the nodal segment. The use of Tinospora cordifolia for the treatment of general weakness and

fever, the immunomodulatory properties, hypoglycemic activities have been reported. Three

major groups of compounds— protoberberine alkaloids, terpenoids, and polysaccharides—are

considered as putative active constituents of Tinospora cordifolia. Protoberberine alkaloids such

as berberine and palmatine are reported to have anti-cancer, anti-infective, anti-diabetic and

immunomodulatory activities.

According to Sinha, Mishra, Singh and Khanuja (2004), Tinospora cordifolia is an

important drug in the Indian Medical System (IMS) and is used in medicines immemorial. The

roots, stem and leaves of the plant are also medicinal. In early times it was used as an Ayurvedic

medicine and is reported to be potent vegetable tonic. It was also used in folk and tribal

medicine. It has been established through modern testing and evaluation (pre-clinical and clinical

trials) in different disease conditions. These studies place this indigenous drug a novel candidate

for bio prospection and drug development for the treatment of such diseases as cancer, liver

disorders, ulcers, diabetes, heart diseases and postmenopausal syndrome where satisfactory cure

managements are still not available.

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According to Choudhary (2018), Tinospora cordifolia is a large, deciduous extensively

spreading climbing shrub with several elongated twining branches that is commonly found in

India. They grow in deciduous and dry forests. It has many medicinal properties like antibiotic,

immunosuppressant, anti-cancer, anti-spasmodic, anti-microbial, anti-osteoporotic, anti-

inflammatory, anti-arthritic, anti-allergic, anti-diabetic, anti-oxidant, anti-stress etc. A variety of

active components like alkaloids, steroids, diterpenoid lactones, aliphatics, and glycosides, have

been isolated from the different parts of the plant body, including the root, stem, and leaves of

the plant. Guduchi is a traditional Ayurvedic medicine, and used in the treatment of fever,

diabetes, diarrhea, cancer, pain, skin disease etc.

According to Hook and Thomas (2018), Tinospora cordifolia is a herbal medicine that

has a lot of use. It is a well-known medicinal plant and used to cure a number of disease. In

today’s time, it is also called as magical herb due to its property of curing a lot of diseases. It is

commonly used to cure migraine, asthma, fever, jaundice, diabetes, intestinal worms and piles.

According to Duraipandiyan, Ignacimuthu and Balakrishna (2012) Tinospora cordifolia

(Willd) Miers (Guduchi) belonging to Menispermaceae (Family), which is native to and widely

distributed in the plains of India. Its plant, including the powder of its leaves, stem bark and roots

are used to treat ailments such as fever, diarrhea, cough, skin disease and a lot more in folk and

tribal medicine. Other than that it is used traditionally for treatment of inflammations, wound

healing, antiseptics and fungal infections alike. With its diverse substance and natural properties,

T. cordifolia definitely has numerous benefits.

In a review on chemical and biological properties of T. cordifolia by Sankhala (2010) a

variety of chemical constituents isolated from the plant belong to different classes such as
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alkaloids, glycosides, diterpenoid, lactones, sesquiterpenoids and steroids that contains about

11.2% of protein and is rich in Ca and phosphorus. Along with that, it also contains some

biological properties that produces an antioxidant action mainly from its alcoholic root extract

where it is reported to have a defense mechanism in alloxan induced diabetic rats. It also

contains an Immuno-modulatory activity where both alcoholic and aqueous extract of

T.cordifolia have been tested successfully to improve the immune system and the body resistance

against infections. With the presence of ethanol, methanol and aqueous in the extract, it has a

wide range of antibacterial pathogens.

As stated by Saha & Ghosh S, (2012) Tinospora cordifolia is a versatile resource for all

forms of life. The traditional ayurdendic medicine used it for ages in treating fever, jaundice,

chronic diarrhea, cancer, dysentery, bone fracture, pain, asthma, skin disease, poisonous insect,

snake bite, eye disorders. The report shows the active components of the plant such as alkaloids,

glycosides, lactones and steroids. These compounds have different biological roles of different

types in targeting diseases. The major biological property of Tinospora cordifolia includes

Immunomodulatory property, anti-diabetes property, anti-toxic effects, Anti-arthritic, anti-

osteoporotic effects etc. It also indicates that the site of action can lead us into identification of

novel perspectives into our understanding of nature.

In people’s everyday task in life, hands can always be covered with a lot of germs. It may

look clean in the outside but in reality it can cause illness and it can also spread to other person

by simply holding their hands even though it is washed. Many prevalent species of bacteria can

be found in our hands at any given time. The study of Tinospora cordifolia: One Plant, many

roles by Saha and Ghosh (2012) showed the importance of the effectiveness of plant genetic
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resources that can help to prevent or to lessen the bacteria that can be the cause of illness in our

body.

According to a phytopharmacological review by Joshi and Kaur (2017) plants has been

one of the important sources of medicines since the beginning of human civilization. Tinospora

cordifolia is a glabrous, succulent, woody climbing shrub native to India. It has anti- cancer,

nerve cell protecting, it is used to improve the immune system, the body resistance against

infections and most importantly it has a potential in preventing different diseases. The stem of

the plants is useful for skin problems and it is also used for treating wounds, pneumonia, asthma

and coughs.

Tinospora cordifolia, Nutritional,Ethnomedical and Therapeutic Utility by Kavya (2015)

stated that Tinospora cordifolia belongs to a family Menispermaceae a well-known in Ayurveda

as Gudchi. It is a traditional plant medicine that contains an impressive level which in every

aspect it leads to a healthy and long life. There are many herbal plants in the world but Guduchi

has a higher medical value. Because of its stem and leaves it is used for treating various ailments.

For the study of Sharma and Pandey (2010) entitled “Beneficial Effects of Tinospora

cordifolia on Blood Profiles in Male Mice Exposed to Lead”, the stem and the leaves of

Tinospora cordifolia does not just apply or treats humans but also animals. But it is widely used

for humans because of the bacteria and the diseases that most people encounter.

According to Mishra, Kumar, and Pandey (2013), Tinospora cordifolia has its medicinal

efficacy which includes phytochemical screening, antimicrobial, antioxidant, and anticancer

activities of the plant. Secondary metabolites including anthraquinones, terpenoids, and saponins

were present in many extracts in addition to phenolics. Total phenol contents in various extracts
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were found in the range of 8.75–52.50 catechol equivalent per gram. In disc diffusion assays,

polar extracts exhibited considerable inhibition against Klebsiella pneumoniae. Several other

extracts also showed antibacterial activity against pathogenic strains of E.

coli, Pseudomonas spp., and Proteus spp. The study demonstrated the presence of various groups

of phytochemicals in Tinospora cordifolia extracts which are responsible for showing

considerable antibacterial, antioxidant, and anticancer activities.

2.2 Antibacterial Activity of Tinospora cordifolia

Mohammed, Manish, and Dinesh (2012) stated that Tinospora cordifolia occupies a very

important place in the field of medicinal plants and is widely used as a traditional medicine. It is

attributed to its ethno-medicinal uses since ancient times by traditional healers and physicians.

Local people use this plant for treating skin diseases, cleansing agent and many more. Since it

was also used as an antimicrobial plant, Mohammed, Manish, and Dinesh conducted a study on

the microbial property of Tinospora cordifolia (roots). Based on their study even though the

plant’s ethanol and chloroform extracts have provided the best results as an antimicrobial this

only showed the basic information on the antimicrobial potential of the plant so further studies

are required to substantiate new biologically potent active antimicrobial compounds.

Razo et al (2015) stated that Makabuhay (Tinospora cordifolia) plant has been identified

to be having antibacterial properties, giving rise to the formulated products such as ointments

and soaps were which are now commercially available. Since Staphylococcus aureus is the

causative agent of pimple and acne. The extract of Tinospora cordifolia was experimented in

different concentration against Staphylococcus aureus. The experiment confirms the antibacterial

property of Makabuhay (Tinospora cordifolia) plant extract as a formulated facial cleanser

against Staphylococcus aureus at different concentrations.

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Aminul, Ashraful, and Mohammad (2011) has stated in their study the antimicrobial,

cytotoxicity, and antioxidant activity of Tinospora cordifolia. The stem was extracted with

methanol then partitioned before undergoing different experimentation. The results then

indicated that the extracts that were collected possess antioxidant, cytotoxic, and few

antimicrobial substances which justify the traditional uses of this plant in the treatment of

different diseases. Furthermore more future researches are necessary for elucidating the active

principles.

According to the study of Singh, Panghal, Kadyan, Chaudhary, & Yadav (2014), that

Silver nanoparticles synthesized from Tinospora cordifolia possess very good antibacterial

activity which makes them a potent source of antibacterial agent. The antibacterial activity of

silver nanoparticles prepared from Tinospora cordifolia against multidrug resistant strains was

determined by agar well diffusion assay and Minimum Inhibitory Concentration (MIC) was

estimated by qualitative experimentation by resazurin based micro broth dilution method. The

silver nanoparticles of stem of Tinospora cordifolia showed the zone of inhibition ranges from

10 ± 0.58 to 21 ± 0.25mm. The MIC of AgNPs from stem extract was found to be 6.25 to 200

μg/ml against Pseudomonas aeruginosa (a courge of hospital burn units and its emergence as

multidrug-resistant strains is a major problem in the control of nosocomial infection) strains.

The antibacterial activity of the aqueous, ethanol and chloroform extracts from the stems

of Tinospora cordifolia was studied using disc diffusion method against Escherichia coli,

Proteus vulgaris, Enterobacter faecalis, Salmonella typhi (Gram-negative), Staphylococcus

aureus and Serratia marcesenses (Gram-positive). Previous study of Jeyachandran, Xavier, &

Anand (2003) suggest that the ethanolic extract has significant antibacterial activity against
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tested bacteria. The study justifies the claimed uses of Tinospora cordifolia in the traditional

system of medicine to treat various infectious diseases.

The medicinal plants are used in traditional cure of various diseases and an impressive

number of modern drugs have been isolated from these natural resources, many based on their

use in traditional medicine. Plants used in traditional medicines, contain a vast array of

substances that can be used to treat chronic and infectious diseases. The antibacterial activities

of extracts of stem and leaves of Tinospora cordifolia were investigated to evaluate its medicinal

potential. The extracts were screened against Escherichia coli, Bacillus subtilis, Pseudomonas

aeruginosa and Stapylococcus aureus by disc diffusion method. Based on the study conducted

by Verma & Rojan (2012) the methanol extracts of Tinospora cordifolia was active on all the

strains of pathogens. The result of the study validates the use of methanol extract of this species

in ethnomedicine and could provide a lead in the isolation of antibacterial agents from methanol

extract of Tinospora cordifolia.

According to Singh, Katiyar, Singh and Srivastava (2015), Tinospora cordifolia can be a

good and potential source for herbal drug preparations in treating UTI. The antimicrobial

activities of Tinospora cordifolia extracts against the pathogens causing urinary tract infections

were examined in the study. Tinospora cordifolia is one of the most versatile herbs. It is also

called as nectar of life, as it is extremely useful in curing and strengthening the immune system

of the body. The extract of the plant contains several bitter components such as glucosides,

alkaloids, a glycoside-giloin, a non-glucoside-gilenin, gilosterol, alkakoid tinosporin, tinosporic

acid, tinosporol, berberine, tinosporidine, sitosterol isolated, cordifol, heptacosanol, octacosonal

and a new furanoid diterpene-tinosporide. The stem of Tinospora cordifolia is used as an

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ingredient in Ayurvedic preparations used in general debility, dyspepsia, fevers and urinary

diseases. The use of plant extracts with known antimicrobial properties, can be of great

significance in therapeutic treatments.

A study conducted by Hipol, and Cariaga (2012) entitled “Anti-inflammatory Activities

of the Aqueous Extract of the Stem of Tinospora Crispa (Family Menispermaceae)” aims to

evaluate the anti-inflammatory properties of the extract of stem of Tinospora crispa. Forty five

albino rats (Rattus norvegicus) of either sex weighing 180-200g were used for the Carageenan-

induced paw edema assay. Tinospora crispa was found to cause stabilization of cellular

membranes at 5%w/v and 7.5% w/v concentrations and inhibition of protein denaturation. These

activities are therefore suggested as the possible mechanisms for the anti-inflammatory action of

Tinospora crispa. The experiment on the reduction of induced rat paw edema evidently

confirmed the anti-inflammatory activity of Tinospora crispa extracts.

Mutalik M. & Mutalik N. (2011) showed the links between what is believed and

what is known regarding the use of Tinospora cordifolia. Evidences from various

pharmacological studies suggest beneficial effects. Immunomodulation, protective actions, and

antioxidant properties helps in treating diabetes, obstructive jaundice, malaria, hepatic and

splenic injury, protection from allergens and toxins, infections, inflammation, rheumatoid

arthritis, gout, leprosy, and in preventing cancer and tuberculosis.

Castillo, et al. (2013) evaluated the clinical efficacy and safety of Tinospora cordifolia lotion

including its cure rate and clearance time compared with permethrin lotion. A clinical study was

performed in three government institutions to investigate clinical efficacy of Tinospora

cordifolia lotion in sixty-six scabies-infected patients. The patients were treated with Tinospora
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cordifolia or permethrin lotions for three consecutive days for two weeks and clinical assessment

of each patient was performed for five weeks. The researchers found out that Tinospora

cordifolia lotion exhibits anti-scabies activity comparable with permethrin.

Arcueno, Retumban, Echano & Guerrero (2015) aimed to compare the effect of

Tinospora crispa on healing of diabetic wound among albino mice when given and administered

along with an ointment topically applied to the wound. Results showed a significant reduction in

blood glucose level among mice given the plant extract.

Harmful microorganisms can be transferred to hands from contaminated surfaces individuals

come into contact in day by day life. These kind of surfaces contribute a lot to the bacterial load

of hands which in turn transmit illness to one self and in addition to others. A study by De Alwis

et al. (2012) showed that with 60 selected medical students taken randomly for bacterial count

from both hands before and after toilet use, bacterial load in the hands for both males and

females showed an increment after toilet use. The bacteria Staphylococcus aureus mainly

appeared to isolate the hands of selected students the most.

To conclude, Patil, Kulkarni and Pandey (2017) stated that the plants’ extract have been

reported to have potential against microbial infections. This was tested against gram positive and

gram negative organisms such as E. coli and S. Aureus with the use of disc diffusion method and

gained results that shows antifungal activity among the entire fungal organism.

Kumar et al. (2017) in their study revealed that Tinospora cordifolia leaves were tested

and observed on Escherichia coli on three different solvent. Ethanolic, methanolic and aqueous

extracts of the leaf were tested using slip disc method to amount the anti-microbial against E.
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coli. As the result, the medicinal plant proves that it can be an alternative form of medicine than

the consumers used with its fewer side effects and easy availability.

According to Nagaprashanthi et al. (2012), the present investigation was carried out to

evaluate the in vitro antifungal and antibacterial activity of hydro alcoholic extract of Tinospora

cordifolia creeped on Azadirachta indica Tree (TC1) in comparison with that of Tinospora

cordifolia (TC2) creeped on fencing. Hydroalcoholic extract of T.cordifolia stem was prepared

by maceration technique. The microorganisms used as antibacterial and antifungal were

Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Pseudomonas sp, Aspergillus

niger, Aspergillus fumigates, mucor sp and Pencillium. The extract of TC1 exhibit effective

antimicrobial activity against all the organisms, while the extract of TC2 exhibits inhibition zone

on limited species such like Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas sp,

Aspergillus niger, Aspergillus fumigates, and mucor sp. The researchers concluded from this

study that Tinospora cordifolia stem extract creping on Azadirachta indica has antimicrobial

activity similar that is similar to neem tree when compared to Tinospora cordifolia creping on

fencing. This can explain that the host plant will gain some of the activities when they survive on

medicinal plants.

According to Patil, Kulkarni and Pandey (2017), the present work was being carried out

to isolate and identify the active constituents of the plants responsible for antibacterial activity.

The aqueous, methanol, ethanol and acetone extract of Tinospora cordifolia, Azarchita indica

and Ocimum santum leaves extract were screened for the presence of phytochemical components

and tested for antibacterial activity against Escherichia coli, Staphylococcus aureus, Klebsiella

pneumonia, Enterobacter aerogenes, Pseudomonas aeruginosa, Salmonella typhimurium,

Salmonella typhi, Staphylococcus epidermidis and Proteus vulgaris. The results obtained in this
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study thus suggests that the identified phytochemicals may be the bioactive constituents

responsible for the efficacy of leaves extract of Tinospora cordifolia, Azarchita indica and

Ocimum santum against fever, syphilitic, ulcer, inflammatory disease wounds, conjunctivitis etc.

It suggested that the traditional medicinal use of Tinospora cordifolia, Azarchita indica and

Ocimum santum be continued and have a scientific evaluation of its active constituents given

serious attention.

This study aims to investigate the in vitro antibacterial activity of Tinospora cordifolia

stem extract against Escherichia coli O78. According to Mamta and Jakhar (2016), medicinal

plants have been clinically used in curing various human and animal disorders. Tinospora

cordifolia is one of the most commercially exploited plants in pharmaceuticals. The estimated

annual demand of this species used in the preparation of crude herbal drugs in the Indian system

of medicines (ISM) is 10,000 tonnes. The stem pieces were washed, shade dried and powdered in

a mixer. For preparation of extract, 15 g of powdered material was boiled in 100 ml of water and

filtered through Whatman`s No.1 filter paper (Singh et al., 2014). The extract was used for

assessment of in vitro antibacterial activity of Tinospora cordifolia stem extract against

Escherichia coli O78. The overall results of in vitro antibacterial activity of aqueous extracts of

Tinospora cordifolia against E. coli indicated that the extract has potential to be used

commercially for control of E. coli infection.

2.3 Staphylococcus Aureus

The Staphylococcus aureus bacterium, commonly known as staph, was discovered in the

1880s. In the 1940s, medical treatment for S. aureus infections became routine and successful

with the discovery and introduction of antibiotic medicine, such as penicillin. According to

Stoppler (2018), the name Staphylococcus comes from the Greek staphyle, meaning a bunch of
 22

grapes, and kokkos, meaning berry, and that is what staph bacteria look like under the

microscope, like a bunch of grapes or little round berries. It is considered as one of the major

human pathogen and the most common community-acquired etiological agents and nosocomial

bacterial infection. Other than that, it is an important opportunistic pathogen, where it is rarely

affecting those patients whose immune system is depressed, responsible for a variety of diseases

ranging from minor skin infections to life-threatening systemic infections. It is one of the

common bacterial infections in humans.

Staphylococcus is a group of bacteria that can cause a multitude of diseases and can

cause a number of infectious diseases in various tissues of the body. Staph infections are caused

by the bacteria staphylococcus that is most of the time, cause no problems or result to minor skin

contamination. But, these infections can in turn be deadly if the bacteria invade deeper into the

human body such as bloodstream, heart and lungs. According to Taylor and Unaka (2017) these

bacteria are present in up to 25% of healthy people and can be found in the normal human flora

associated with the skin and mucous membranes of every human being. Over 30 different types

of staphylococci can infect humans, but most infections are caused by Staphylococcus aureus.

Common symptoms such as Boils under the arms or around the groin or buttocks, a painful rash

caused by Impetigo, Cellulitis and the Staphylococcal scalded skin syndrome which affects

mostly babies and children that features fever, rash and blisters. Food poisoning may also be

caused by these bacteria where its symptoms come and disappear quickly, often lasting for half a

day. It also causes Septicemia and Bacteremia, known as blood poisoning where the bacteria

enter a person’s bloodstream. Therefore, Staph contaminations are infectious until the point

when the disease has settled. Direct contact with an infected sore, wound, or with personal care

items are common routes of transmission.

 23

In the study of Espedido, et al. (2012) Staphylococcus aureus bacteremia is an infection

with an incident rate ranging from 20% to 50% cases per year. Patients die from SAB between

10% and 30%. Pathogen-host interactions, especially the presence of shock and the source of

SAB, are strong predictors of outcomes. At this rate, this account for a greater number of deaths

than AIDS, and tuberculosis. Although antibiotic resistance was associated, pathogen-specific

factors and poor response to antibiotic therapy was observed. The attributable mortality of S.

aureus bacteremia (SAB) is as at yet higher and indicators for mortality and clinical results of

this condition are in need to be clarified. In patients with Staphylococcus aureus bacteremia,

infectious disease consultation remains a valuable device to enhance clinical result.

3 Method

3.1 Design

The research study is experimental in nature that aims to determine the antibacterial

activity of Tinospora cordifolia against Staphylococcus aureus. The specific type of bacteria to

use is the Staphylococcus aureus. A phytochemical testing was done to determine the active

components present in Tinospora cordifolia. The concentrations of Tinospora cordifolia extract

are 50%, 75%, and 100% that were produced from simple dilution. Kirby-Bauer method was

used in this study, to determine the resistance mechanism and susceptibility of Staphylococcus

aureus against the Tinospora cordifolia and Penicillin. Kirby-Bauer testing measures sensitivity

of bacteria to antibiotics by culturing bacteria on solid growth media surrounding sources of

drug. The zone of inhibition is an area of clear media where bacteria are not able to grow

surrounds the wafer. A lager zone of inhibition around an antibiotic-containing disk indicates

that the bacteria are more sensitive to the antibiotic in the disk.

3.2 Materials and Study Site

 24

The main materials that will be used are the ethanolic extract of Tinospora cordifolia in

50%, 75% and 100% concentrations, Staphylococcus aureus bacterium and one tablet of

Penicillin. The study will be held at Far Eastern University High School in the University Belt

area, West Sampaloc, City of Manila during the first semester of the school year 2018-2019. For

the extraction of Tinospora cordifolia plant, the plant will be brought to the Chemicals and

Energy Division of the Department of Science and Technology. For the phytochemical testing of

Tinospora cordifolia plant, the plant will be brought to the Standard and Testing Division of the

Department of Science and Technology.

3.3 Data Collecting Instrument

The following instruments that will be used in this study are specified in this section.

Alcohol lamp- Alcohol lamp will be used to sterilize the inoculating loop and specimen to avoid

contamination.

Beaker- Beaker will be used to measure the amount of distilled water needed for the preparation

of media.

Erlenmeyer flask- Erlenmeyer flask will contain the solution for the media preparation. It will

then be placed in a heat plate for the solution to be dissolved.

Heat plate- Heat plate will be used to dissolve the solution for media preparation. It is important

that the solution will be dissolved properly to obtain the standard of the media and its efficacy.

Incubator- Incubator will be used to assist the growth of the culture. The specimen is usually

incubated at 37°C for 24 hours or depending on the growth requirement of the organism.
 25

Inoculating loop- Inoculating loop will be used to inoculate the specimen into the media. It is

important to sterilize it first with alcohol lamp to avoid contamination.

Petri dish- Glass and disposable plastic petri dish of standard size will contain the media of

choice.

Puncher- 6 mm diameter puncher was used to produce blank disks.

Stirring rod- Stirring rod aids to the dissolving of the solution for media preparation.

Whatman no. 3- The 6mm disks for Kirby-Bauer method was prepared using Whatman filter

paper no. 3.

3.4 Data Gathering Procedure

 Tinospora cordifolia Extraction and Preparation

The Tinospora cordifolia will be collected at San Nicolas, Arayat, Pampanga,

Philippines. An adequate amount of plant will be brought to Department of Science and

Technology to undergo a process of extraction and phytochemical testing.

 Tinospora cordifolia Authentication

The Tinospora cordifolia will be submitted to the National Museum of the Philippines

located at Padre Burgos Avenue, Ermita, Metro Manila. This is to verify that the plant that the

researchers will be using is a Tinospora cordifolia (Makabuhay plant).

 Tinospora cordifolia Ethanol Extract

 26

The Tinospora cordifolia will be brought to the Chemicals and Energy Division of the

Department of Science and Technology where it will be with ethyl alcohol (ET) using the

Soxhlet apparatus.

 Phytochemical Components of Tinospora cordifolia

The Tinospora cordifolia will be brought to the Standard and Testing Division of the

Department of Science and Technology where it will be tested to determine the active

components present in the plant.

 Tinospora cordifolia Concentrations

The Tinospora cordifolia ethanolic extract will be further diluted with distilled water to

achieve the desired concentrations of 50%, 75%, and 100%. To be able to obtain those desired

concentrations, this study used simple dilution by volume/volume percent solution (vol/vol %)

(Physiology Web, 2013).

𝑣𝑜𝑙𝑢𝑚𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒

%= 𝑥 100
𝑣𝑜𝑙𝑢𝑚𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

 Staphylococcus aureus Preparation

Blood agar will be used for the sub-culture of Staphylococcus aureus, which is a selective

differential medium for isolation and identification of the bacteria.

 Preparation of Blood agar

In standard preparation of Blood agar, suspend nutrient agar powder in of distilled water.

Heat this mixture to fully dissolve all components then autoclave the dissolved mixture at 121

degrees Celsius for 15 minutes. After it is autoclaved, allow to cool to 45-50 ° C before adding
 27

the blood that has been warmed to room temperature and mix gently but well and avoid bubbles.

The blood agar was then dispensed into sterile plates while liquid.

The Staphylococcus aureus will be coming from a pure culture and then will be swabbed

into the Blood agar. Three (3) subcultures were enough for three trials. Incubation of the

specimen was done for 24 hours at 37 ° C or at room temperature.

 Preparation of Mueller-Hinton agar

Mueller-Hinton agar is commonly used in disk diffusion test or Kirby-Bauer test. Kirby-

Bauer method determines the resistance or sensitivity of facultative anaerobes which can be used

to determine the treatment for bacterial infections.

In standard preparation of Mueller-Hinton agar, 21.0 grams of the medium was added to

1 liter of distilled water. Then, it was boiled for 1 minute with frequent agitation to completely

dissolve the medium. Following that procedure, it was autoclaved for 15 minutes at 15 PSI

(121°C) and will then be cooled at room temperature (HiMedia Laboratories Pvt., Mueller-

Hinton Broth, 2017).

 Kirby-Bauer method/ Disk Diffusion Method

Kirby-Bauer method, also known as the Disk Diffusion method, is used to determine the

ability of a specific antibiotic to inhibit a particular bacterium. The result of this method is shown

by the different zones of inhibition produced around the disks which may be reported as

susceptible (able to inhibit the bacteria), intermediate, or resistant (unable to inhibit the bacteria).

The disks used in this study will be prepared from a Whatman filter paper no. 3 punched

into 6 mm disks. The disks will be autoclaved for sterilization. After it was incubated, the
 28

prepared extracts with predetermined concentrations will be incorporated into the disks. This

study used Penicillin as the positive control, and distilled water incorporated into blank disks as

the negative control.

3.5 Data Analysis

The researchers will used statistical analysis specifically T-test and ANOVA (Analysis of

Variance). ANOVA will be used to determine whether the concentrations are significantly

different with each other. The T-test will be used to assess the difference between the samples.

3.6 Ethical Considerations

There will be no humans or animals that could be subjected to harm in any ways. The

researchers acknowledges the works of other authors that were used in this research paper.

Staphylococcus aureus is the only test subject that the researchers will focus on.

3.7Methodology Flowchart
 29

References:

Aminul,H.,Ashraful,I.,& Mohammad,S. (2011). Antimicrobial, Cytotoxicity,and Antioxidant

activity of Tinospora cordifolia. Journal of Pharmaceutical and Biomedical Sciences.
Vol 13(12)

Arcueno, R., Retumban,J.,Echano,J., & Guerrero,J.(2015).Wound healing potential of Tinos

pora crispa (Wild.) Miers [Menispermaceae] stem on diabetic mice. Journal of Medi
cinal Plants Studies 2015; 3(2):106-109. Retrieved August 10, 2018 from http://www.
plantsjournal.com/archives/2015/vol3issue6/PartB/3-6-8.pdf

Bala,M., Pratap,K., Verma,P., Singh,B.,& Padwad,Y.(2015). Validation of ethnomedicinal

potential of Tinospora cordifolia for anticancer and immunomodulatory activities and
quantification of bioactive molecules by HPTLC. J Ethnopharmacol. Epub 2015 . Retrie
ved Aug 5, 2018 from PubMed PMID.

Castillo, A., Osi, M., Ramos, J., Francia, J., Dujunco, M., & Quilala, P. (2013). Efficacy and
safety of Tinospora cordifolia lotion in Sarcoptes scabiei var hominis-infected pediatric
patients: A single blind, randomized controlled trial. Retrieve August 8, 2018 from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3643341/

Choudhary, V. (2018). Description and medicinal uses of Guduchi – Tinospora cordifolia.

Retrieved August 12, 2018 from http://natureconservation.in/description-and-medicinal-
uses-of-guduchi tinospora-cordifolia

Dadar, M., Dhama,K.,Sachan S., Khandia, R., Munjal, A., Iqbal, H., Latheef, S., Karthik,K.,
Samad, H., & Tiwari, R. (2017).Medicinal and Beneficial Health Applications of Tinos
pora cordifolia (Guduchi): AMiraculous Herb Countering Various Diseases/Disorders
 30

and its Immunomodulatory Effects. Recent Pat Endocrine, Metabolic and Immune Drug
Discovery, 92-111.doi:10.2174/1872214811666170301105101

Duraipandiyan,V., Ignacimuthu,S., & Balakrishna,K. (2012). Antimicrobial Activity of

Tinospora cordifolia: An Ethnomedicinal Plant. Retrieved from: https://www.yumpu.

com/en/document/view/26663609/antimicrobial-activity-of-tinospora-cordifolia-an-

ethnomedicinal-plant

Espedido,B., Gosbell,I., Peterson,D., Jensen,S., Vaska,V., & van Hal,S.(2012). Predictors of Mort

ality in Staphylococcus aureus Bacteremia. American Society for Microbiology,Retrieved Augus

t 26, 2018 from https://cmr.asm.org/content/25/2/362

Hipol R. L., Cariaga M., Hipol R. M., (2012) . Anti-Inflammatory Activities of the aqueous
extract of the stem of Tinospora crispa (family menispermaceae). Retrieved August 10,
2018 from: http://www.journalofnaturestudies.org/files/88-95-Tinospora-full.pdf

Hook, F. & Thomas (2018) Uses of Tinospora cordifolia (Willd.) Retrieved August 8, 2018 from
http://nif.org.in/TINOSPORA-CORDIFOLIA

Joshi,G. & Kaur,R. (2017). Tinospora cordifolia : A Phytopharmacological Review. Interna

tional Journal of Pharmaceutical Sciences and Research. Retrieved August 5,2018 from
http://ijpsr.com/bft-article/tinospora-cordifolia-a-phytopharmacological review/?view
=fulltext

Kavya B, 2015. Tinospora cordifolia (WILLD.) MIERS.: Nutritional,Ethnomedical and Thera

peutic Utility. Retrieved August 12, 2018 from http://www.ijrap.net/admin/php/uploads
/1326_pdf.pdf

Kumar,D.V, Geethanjali,B., Avinash,K., Kumar,J.R, & Chandrashekrappa,G. (2017).Tinos

 31

pora cordifolia: The Antimicrobial Property of the Leaves of Amruthaballi. J Bacteriol

Mycol Open Access 5(5): 00147. DOI: 10.15406/jbmoa.2017.05.00147

Mamta & Jakhar,K. (2016) Veterinary Research International April-June, 2016 .4(2)
pp 74-77

Mishra,A., Kumar,S. & Pandey,A. (2013). Scientific Validation of the Medicinal Efficacy

of Tinospora cordifolia. The Scientific World Journal Volume 2013, Article ID

292934,p. 8

Mohammed,A.,Manish,G.,&Dinesh(2012).Antimicrobial Activity of Tinospora crispa Root

Extracts. International Journal of Research in Ayurveda and Pharmacy. Vol 3(3)

Mutalik,M.,& Mutalik N. (2011).Tinospora cordifolia and its Varied Activities: What is believed
and What is known?. International Journal of Current Research and Review Vol. 3
Retrieved August 11, 2018 from http://ijcrr.com/uploads/2025_pdf.pdf

Nagaprashanthi,C., Rafi khan.P, Gopi chand.K, Aleemuddin MA,and Rajiya Begum.G. (2012).
In vitro Antimicrobial Activity of Tinospora cordifolia and its Phytochemical screening.
International Journal of PharmTech Research. 4(3), pp 1004-1008

Patel, M., & Ishnava, K. (2014). In vitro Study of Tinospora cordifolia (Wild.) Miers(Menis
permacceae)- a Multipurpose Plant, by Using Different Plant Bark Extracts for Second
ary Metabolite Production. Journal of Herbs, Spices and Medicinal Plants, 341-349. doi\
:10.1080/10496475.2013.876482

Patil,P., Kulkarni,C., & Pandey,A. (2017). Antifungal and phytochemical properties of Tino

spora cordifolia, Azadirachta indica and Ocimum sanctum leaves extract. Journal of

Medicinal Plants Studies 2017 5(5): 23-26

 32

Patil,P., Kulkarni,C., & Pandey,A. (2017).Antibacterial and phytochemical analysis of Tinos

pora cordifolia, Azarchita indica and Ocimum santum leaves extract against common
human pathogens: An in vitro study Journal of Pharmacognosy and Phytochemmistry
Volume 6(5): pp 702-706

Razo,J., Abarientos,M., Corpuz,F., Menor,P., and Pelagio,C.(2015). Antibacterial potential of

Makabuhay (Tinospora cordifolia) Extract formulated as Facial Cleanser Retrieved
August 12, 2018 from http://itceprints.slu.edu.ph:8080/xmlui/handle/123456789/1271

Rekha, Tamilarasu,S., Viji.(2017).Tinospora cordifolia. Retrieved from http://vikaspedia.in/

agriculture/crop-production/package-of-practices/medicinal-and-aromatic-plants/tinos
pora-cordifolia

Saha S. & Ghosh S., (2012). Tinospora cordifolia: One plant, many roles. Retrieved August
8,2018 from: https://www.ncbi.nlm.nih.gov/pubmed/23661861

Sankhala, L.N., Saini,R.K., & Saini, B.S.(2010). A Review on Chemical and Biological Proper

ties of Tinospora cordifolia Vol. 2, No. 2, pp. 340-344., Int. J. Med. Arom. Plants,ISSN

2249 – 4340

Sharma,V. & Pandey,V .( 2010). Beneficial Effects of Tinospora cordifolia on Blood Profiles
in Male Mice Exposed to Lead. Retrived August 8,2018 from https://www.ncbi.nlm
.nih.gov/pmc/articles/PMC2964740/

Singh, K., Panghal, M., Kadyan, S., Chaudhary, U., & Yadav, J. (2014). Antibacterial Activity of
Synthesized Silver Nanoparticles from Tinospora cordifolia against Multi Drug Resistant
Strains of Pseudomonas aeruginosa Isolated from Burn Patients. Retrieved August 12,
2018 from https://www.omicsonline.org/open-access/antibacterial-activity-synthesized-
 33

silver-nanoparticles-tinospora-cordifolia-multi-drug-resistant-strains-pseudomonas-
aeruginosa- isolated-burn-patients-2157-7439-5-192.php?aid=25859

Singh, P., Katiyar, D., Singh, B. & Srivastava, A. (2015). Antimicrobial activity of Tinospora
cordifolia extracts against urinary tract infections causing bacteria. International Journal
of Pharma and Bio Sciences. 6. B571-B577

Stoppler,M.(2018). Staph Infection. MedicineNet. Retrieved August 26, 2018 from https://
www.medicinenet.com/staph_infection/article.htm#staph_infection_facts

Stuart Jr.,G.(2018).Tinospora crispa (L.) Hoook.f. & Thomson Heavenly Elixir. Philippine
Medicinal Plants. Retrieved August 10, 2018 from http://www.stuartxchange.org/
Makabuhay

Taylor, T. & Unakal, C.(2017). Staphylococcus Aureus. StatPearls. Retrieved August 26,2018
from https://www.ncbi.nlm.nih.gov/books/NBK441868/

Upadhyay A. K., Kumar K., Kumar A.,& Mishra H.S. (2010). Tinospora cordifolia (Willd.)
Hook. f. and Thoms. (Guduchi) – validation of the Ayurvedic pharmacology through
experimental and clinical studies. Retrived August 12, 2018 from: https://www.ncbi.nlm.
nih.gov/pmc/articles/PMC2924974/

Verma, K., & Rojan, B. (2012). In Vitro Antibacterial Activity of Leaf and Stem Extracts of
Tinospora cordifolia Miers. Vegetos - An International Journal of Plant Research, 25(1),
13-15. Retrieved fromhttp://vegetosindia.org/journal/Vegetos 25(1)2012/In-Vitro-
Antibacterial-Activity-of-Leaf-and-Stem-Extracts-of-Tinospora-cordifolia-Miers..html

Vermani,A., Navneet & Gautam,S. (2013). Screening of Antibacterial Activityof Tinospora

cordifolia Miers. Extracts Against Dental Pathogens. Journal of Pharmacology and
Toxicology, 8: 28-34. DOI 10.3923/jpt.2013.28.3410.3923/jpt.2013.28.34
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APPENDIX I

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APPENDIX II

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