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A COMPARATIVE STUDY ON THE TOXICITY LEVEL OF THE LEAF AND

STEM OF PANYAWAN (Tinospora rumphii)

A Research Project Presented to the

Faculty of Senior High School Department
RC-Al Khwarizmi International College Foundation, Inc.
Basak Malutlut, Marawi City, Philippines

In Partial Fulfillment of the

Requirements for the Senior High School Curriculum
Science, Technology, Engineering, and Mathematics Strand

Norjannah M. Saanoding
Sittie Faisah A. Fahad
Khairanie S. Hadji Amen
Abdul Raffy L. Somirado
Nailah M. H. Mohammad Taib

January 2024
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Chapter 1

THE PROBLEM AND ITS SCOPE

Rationale

Plants have been used for medicinal and health care purposes since ancient times,

having a long history of aiding and giving treatments to various illnesses and ailments.

Further, Tinospora rumphii or commonly known as Panyawan, is a plant specie that has

been traditionally used in Southeast Asia for its purposed health benefits. Previous

studies have shows that this plant contains various bioactive compounds with potential

pharmacological properties. However, the toxicity of different parts of this plants has not

been fully elucidated. This study, aimed to investigate and compare the toxicity levels of

the leaf and stem of Panyawan (Tinospora rumphii). The findings of this study may

contribute for a better understanding of the safety profile use of this plant and its potential

use in pharmaceutical.

Saladino et al. (2022) stated that tinospora rumphii is one prospective plant for

utilization. It is a tropical climbing shrub endemic to lower elevations. The plant's stem is

green and succulent, covered in a thin brown bark, and encircled with warty lenticels. T

has yielded a wide set of pharmacologically active compounds. rumphii. Amino acids,

glycosides, flavonoids, phenols, saponins, steroids, and terpenoids are examples of

bioactive compounds.

(Tinospora rumphii), commonly known as Panyawan, is a medicinal plant found

in the Philippines. The plant has a wide variety of traditional uses in alternative medicine,

including its potential as a treatment for diabetes, liver disease, and fever. Although there

is limited literature focused on the comparative toxicity of the leaf and stem of Panyawan
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(Tinospora rumphii), several studies have been conducted to investigate the plant's

potential medicinal properties.

Brine shrimp, scientifically known as Artemia, is a tiny aquatic crustaceans that

inhabit saltwater environments. This fascinating organism has gained significant attention

in scientific research due to their unique biological characteristics, including their ability

to survive in extreme conditions and their role as a valuable live food aource in

aquaculture.

Gajardo and Beardmore (2012) stated that the brine shrimp Artemia is a

microcrustacean that has evolved to survive and reproduce under the hard conditions

imposed by severely hypersaline habitats. Adaptation to these conditions has occurred at

many functional levels or domains, ranging from the molecular-cellular-physiological to

the population level. Such conditions are encountered by relatively few comparable

macro-planktonic species; hence, Artemia can be regarded as a model animal

extremophile with a distinct set of adaptations that is the topic of this review. The most

noticeable is a very efficient osmoregulation system capable of withstanding up to ten

times the salinity of regular saltwater. Under extreme environmental conditions, such as

when seasonal lakes dry up, Artemia seeks refuge by producing a highly resistant

encysted gastrula embryo (cyst) capable of severe dehydration, allowing the population to

avoid extinction. Cysts can be thought of as gene banks that store genetic memories of

past population situations. Their occurrence is due to females' evolved ability to

"perceive" upcoming unstable environmental conditions, which is manifested by their

ability to switch reproductive mode, producing either cysts (oviparity) when

environmental conditions become deleterious or free-swimming nauplii (ovoviviparity)

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that can sustain the population under suitable conditions. Conspecific populations are

fragmenting into locally adapted populations at the population level, whilst species are

limited to salty lakes in specific places (regional endemism).

Brine shrimps are particularly intriguing for scientific investigation due to their

adaptability to varying salinity level, temperature ranges, and the capability to enter

cryptobiosis, a state of metabolic dormancy that allows them to endure harsh

environment conditions. These features make them an ideal subject for experimental

studies, offering insights into physiological adaptations and potential applications in

diverse fields such as ecology, biology, and environmental science. The rationale behind

exploring brine shrimp in this investigation project lies in the opportunity to delve into

their intriguing biological traits, which may have implications for broader scientific

understanding and practical applications. By investigating the response of brine shrimp to

specific environmental variables, we aim to contribute valuable data to the scientific

community, paving the way for a deeper comprehension of their resilience and potential

uses in various contexts.

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Statement of the Problem

The purpose of this study was to evaluate and determine the toxicity of panyawan

stem and leaf extracts to brine shrimps, which are extremophile organisms. This focused

on answering the following questions:

1. What is the mortality rate of brine shrimp in panyawan leaf extract through

decoction?

2. What is the mortality rate of brine shrimp in panyawan stem extract through

decoction?

3. Is there a significant difference in the mortality rate of brine shrimps between the

leaf and stem extract through decoction of panyawan?

Null Hypothesis

H0: There is no significant difference in the mortality rate of brine shrimps

between the leaf and stem extract through decoction of panyawan.

Conceptual Framework

The conceptual framework of this study illustrates the different concepts of the

variables involved and investigated.

The conceptual framework of this study shows the relationship of the independent

variables, which are the panyawan leaf extract and the panyawan stem extract, to the

dependent variable, the mortality rate of brine shrimp under decoction of panyawan. This

study, it aimed to investigate the toxicity levels of the leaf and stem of (Tinospora

rumphii) using a standardized cytotoxicity assay.

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Independent Variable

Dependent Variable
Panyawan Leaf Extract

Mortality Rate of
Brine Shrimp
Panyawan Stem Extract

Figure 1. Research Paradigm

Significance of the study

On this study of the toxicity of Panyawan (Tinospora rumphii) lies in its potential

to contribute to the development of safe and effective natural products for medicinal

purposes and improve public health outcomes by evaluating its safety, analyzing its

chemical composition, using a standardized cytotoxicity risks associated with its use.

Traditonal Medicine Pratitioners. They will benefit from knowing the extent of

the toxicity of the plant’s leaf and stem and may adjust their dosage or preparation

methods accordingly.

Consumers. They will benefit from knowing any potential health risks

associated with consuming the plant’s leaf and stem may make informed choices about

their consumption.

Agricultural and Enviroment. The study may provide insights into the toxicity

of panyawan, which could be useful in developing pest control strategies and promoting

sustainable farming practices.

Future Researchers. The study may identify potential medicinal properties of

panyawan, which could lead to the development of new drugs or therapies.

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Scope and Delimitation of the Study

This study involved the use of brine chrimp as toxicity level indicator of panyawan

leaf and stem. This study aimed to determine the toxicity level of panyawan leaf and stem

and the difference between their extracts. This study was only delimited to the usage of

panyawan leaf and stem extracts. This research only used decoction process in extraction

of leaf and stem of panyawan. This study excluded the varying of concentration uses on

extracts.

Defination of Terms

In order to clarify the meaning of some terms used in this study, certain

significant terms were conceptually and operationally defined, that is as they were used in

this study.

Brine Shrimp. It is any of genus (Artemia) of brandchiopod crustaceans that can

exist in strongly saline environments. It is known for their adaptability to extreme

conditions and their ability to enter cytobiosis. A state of metabolic dormancy. In this

study, it is the experimental subject which will be used to determine the toxicity level of

panyawan leaf and stem extracts.

Decoction.

Leaf. It is the outgrowth from a plant stem that is typically a flattened expanded

variably shaped grenish organ. In this study, this will be extracted through decoction and

which toxicity level will be tested.

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Mortality Rate. It is a measure of the number of deaths (in general, or due to a

specific cause) in a particular population, scaled to the size of that population, per unit of

time (Porta, M, ed. 2014). In this study the researcher

Panyawan. It is a native plant from tropical and subtropical rain forest in India,

Myanmar, Sri Lanka, and Philippines (Ashcroft, 2022). In this study, it is the plant’s

extracts that are being investigated using its toxicity level.

Stem. It is the main structural axis of a plant that supports leaves, flowers, and

fruits, and conducts water, nutrient, and photosynthetic products between roots and leaves

(Dasauni, 2020). In this study, the researchers are finding the toxicity level of the

panyawan stem.

Toxicity. It can be expressed as the amount of a substance that is required to

cause harm or as the severity of the harm caused at a given does (Colerangle, 2017). In

this study, it is what the researchers are looking for the panyawan stem.

Chapter 2
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REVIEW OF RELATED LITERATURE AND STUDIES

This chapter presents the various literature and studies which are related to this

comparative study on the toxicity level of the leaf and stem of panyawan (tinospora

rumphii).

Related Literature

Panyawan

Panyawan Leaf Extract

Panyawan Stem Extract

Brine Shrimp

Related Studies

Panyawan

A study of Tan and Bajo (2014), tinospora rumphii (T. rumphii) is a folklore

medicinal plant found in Asia and Africa. Locals have regularly utilized it to cure a

variety of ailments, including jaundice, a symptom of liver impairment. T's action was

investigated. rumphii crude extract in combination with zinc sulphate, a recognized tumor

regulator, on hepatic damage caused by intraperitoneal (i.p) quinoline injections in albino

mice. Bilirubin content in blood serum was used to assess the hepatotoxic effect, whereas

single-cell gel electrophoresis (SCGE) was used to measure the genotoxic effect.

Following quinoline exposure, mice given the crude extracts had lower serum bilirubin

levels and DNA damage. Mice given zinc sulphate, on the other hand, had significantly

less DNA damage on hepatocytes. T. rumphii has hepatoprotective properties, according

to our findings. T. Rumphii extract is dose-dependent, and its use as a medicinal cure

must be properly regulated, although zinc has been shown to reverse the genotoxic
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impact of quinoline. T's potential was revealed in this study. Rumphii extract and zinc as

key hepatoprotective agents for future treatment of hepatic damage caused by cancer

treatment chemotherapeutic drugs.

Panyawan Leaf Toxicity

In a study conducted by Pregoner et al. (2019), leaf extract of (Tinospora

rumphii) against American cockroach (Periplaneta americana). The study used a

quantitative experimental research design. Cockroach strains were gathered using traps

and hand catches. Panyawan leaf was harvested and extracted in seven different

concentrations. The usual contact glass jar bioassay was used to evaluate the

concentrations and controls against the strains. The positive control had the shortest

knock-down time at 1.08 minutes, while the extracts of Panyawan leaf had a knock-down

duration ranging from 1.42 to 20.25 minutes. According to Probit Regression, the fatal

concentration 50 of Panyawan leaf extract was 15.836%. ANOVA demonstrated a

significant difference in cockroach knock-down periods when subjected to varied

Panyawan leaf extract concentrations, negative and positive controls (p0.01).

Homogeneous subsets produced from the Post Hoc Test using Tukey's Honest Significant

Difference revealed no significant difference between the positive control and the Extract

100% (p>0.01).

Panyawan Stem Toxicity

A study of Esquilla, Sanchez, and Tenorio (2021), lymnaeid snail infestation and

snail-borne parasite diseases such as fasciolosis continue to be a problem in the

Philippines' livestock industry. Thus, effective molluscicides are required. However,

routine use of commercially available molluscicides may result in unintended

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environmental pollution. Natural alternatives to commercially available molluscicides are

needed, hence this study studied the molluscicidal activity and efficacy of ethanolic stem

extract of makabuhay (T. rumphii) against lymnaeid snails. A total of 150 mature snails

from Barangay Guiamalia, Esperanza, Sultan Kudarat, were immersed in T. Extracts of

Rumphii in ethanol. Treatment A or negative control (100% tap water), Treatment B (5

000 ppm), Treatment C (7 500 ppm), Treatment D (10 000 ppm), and Treatment E or

positive control (10 000 ppm Surekill® 70WP) were the five treatment groups. Snail

death was measured 24 hours, 48 hours, and 72 hours after exposure. The findings

revealed that all T had 100% treatment-specific mortality. rumphii extract concentrations,

as well as in the positive control after 24 hours. The efficacy of treatments B, C, D, and E

was 96.67%. These findings indicate that T. Rumphii ethanolic extracts at concentrations

of 5 000 ppm, 7 500 ppm, and 10 000 ppm are efficient natural molluscicides against

lymnaeid snails. The encouraging results indicate that T. Rumphii stem ethanolic extract

could be a good natural, low-cost alternative to commercially marketed molluscicides.

Brine Shrimp

The study of Gajardo and Beardmore (2012), the brine shrimp Artemia is a

microcrustacean that has evolved to survive and reproduce under the hard conditions

imposed by severely hypersaline habitats. Adaptation to these conditions has occurred at

many functional levels or domains, ranging from the molecular-cellular-physiological to

the population level. Such conditions are encountered by relatively few comparable

macro-planktonic species; hence, Artemia can be regarded as a model animal

extremophile with a distinct set of adaptations that is the topic of this review. The most

noticeable is a very efficient osmoregulation system capable of withstanding up to ten

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times the salinity of regular saltwater. Under extreme environmental conditions, such as

when seasonal lakes dry up, Artemia seeks refuge by producing a highly resistant

encysted gastrula embryo (cyst) capable of severe dehydration, allowing the population to

avoid extinction. Cysts can be thought of as gene banks that store genetic memories of

past population situations. Their occurrence is due to females' evolved ability to

"perceive" upcoming unstable environmental conditions, which is manifested by their

ability to switch reproductive mode, producing either cysts (oviparity) when

environmental conditions become deleterious or free-swimming nauplii (ovoviviparity)

that can sustain the population under suitable conditions. Conspecific populations are

fragmenting into locally adapted populations at the population level, whilst species are

limited to salty lakes in specific places (regional endemism). The Artemia model presents

adaptation as a complicated reaction to important life circumstances, integrating and

improving previous and present experiences at all organizational levels. Although we are

discussing an invertebrate that is restricted to a specific environment, the mechanisms

that will be presented are of universal biological relevance. Finally, we emphasize the

importance of understanding Artemia's stress response to human population well-being.

A study conducted by Meyer et al. (1982), as a simple bioassay for natural

product research, a technique based on brine shrimp (Artemia salina LEACH) is

proposed. The process determines the LC50 values of active compounds and extracts in

the brine medium in g/ml. The toxicity of a wide spectrum of known active chemicals is

expressed as shrimp toxicity. The cytotoxicities of 9KB and 9PS were compared to the

findings of screening with seed extracts of 41 Euphorbiaceae species. As an in-house

general bioassay tool, the approach is quick, dependable, affordable, and convenient.
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Chapter 3

RESEARCH METHODOLOGY
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This chapter includes the discussion of research design, locale of the study, data

gathering procedure, the research instrument and the statistical treatment of the data. The

methodology describing the process of using brine shrimp as a comparative method to

determine the toxicity level of the leaf and stem of panyawan (tinospora rumphii).

Research Design

This study used experemintal quantitative resaerch design to utilize the scientific

approach. The experiment done to explain or find out the effect of the given variable.

This study aims to identify the assessment of the toxicity of panyawan leaf and stem

extract. Specially, the researcher test a hypothesis and to study relationship systematically

and scientifically among variable.

Locale of the Study

The research experiment was conducted in the Chemistry Laboratory of RC-Al

Khwarizmi International College Foundation, Inc. Senior High School, a private and

prominent senior high school established in 2018, situated in Basak Malutlut, Marawi

City, Lanao del Sur, Philippines. RC-AKICFI SHS is an institution that provides and

advanced learning curriculum to its learners and committed in its efforts to provide

quality education to the Muslim populace. The researchers chose RC-AKICFI SHS as the

locale of the study because of the availability of the materials, equipment, and laboratory

apparatus that were required in the conduction of the experiment.

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Fig

ure 2. RC-Al Khwarizmi International College Foundation, Inc. Senior High School,

Basak Malutlut, Marawi City, Lanao del Sur, Philippines

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Research Instrument

The instruments used by the researchers in this self-conducted experiment. This

research was conducted to determine the toxicity level of the leaf and stem of panyawan

(Tinospora rumphii). The supplies and tools that will be utilized to conduct the

experiment are listed below.

Table 1

List of Materials and Equipments used in the Study.

MATERIAL DESCRIPTION MEASUREMENT

Brine Shrimp This will be used to test and extract the 180 pcs.
two variable in the study.
Panyawan Stem This will be used to test the brine shrimp 90 grams
and how it will be toxic.
Panyawan Leaves This will be used to test the brine shrimp 90 grams
in how it will be toxic.
Air Pump This will be used to hatching the brine 1 pc.
shrimp.
Basin This will be used to wash the Panyawan 1 pc.
leaves.
Salt This will be used to mixed it into the 1 tablespoon
water for hatching the brine shrimp.
Water The 1 liter will be used to boil the 2L
panyawan leaf and stem. The another 1
liter. Is used for hatching brine shrimp.
Stainer It will be used in separating the 1pc.
panyawan stem in the bioling water.
Hose This will be used for hatching the brine 1 pc.
shrimp.
Pot This will be used for boiling the water 2 pcs.
where the panyawan leaves and stem are
added.
Bottle Used for hatching the brine shrimp 1 pc.
including the water and the salt.
Petri Dish To use for the expirement for the brine 6 pcs.
shrimp nauplii each replicates consists of
6.
Syringe Used to piped out 30 brine shrimp in each 3 pcs.
replicates.
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Data Gathering Procedures

Gathering of the data procedures of comparing the toxicity level of the leaf and

stem of panyawan (Tinospora rumphii) is challenging the use of brine shrimp as an

indicator.

In this experimental research, the following procedure will be conducted.

Pre-conventional Phase

To conduct this experiment reasearch, the researcher prepared a bottle with water

to hatch the brine shrimp eggs, 1 tablespoon of salt wich will be mixed into the water

when hatching the brine shrimp eggs, an air pump to provide air to hatch the brine shrimp

eggs quickly, a basin to wash the panyawan leaves and stems, 90 grams of panyawan leaf

and stem to test the toxicity to the brine shrimp, 2 pots to boil the water with panyawan

leaf and stem, 450 milliliters of water to boil the panyawan leaf and stem, a electric stove

is used to boil the panyawan leaf and stem, a stainer to filtered the panyawan leaf and

stem extract, a beaker to measure the sample extract, a syringe to pie in and out the 30

hatched brine shrimp, and 6 petri dish to observe the toxicity of brine shrimp.

Inter-conventional Phase

When the researchers already prepared the materials needed, the brine shrimp will

be hatched by putting 500 milliliters of water in a bottle with 1 tablespoon of salt. The

researchers provided an airpump, attached the hose and put the end of the hose on the

bottle. The researcher washed 90 grams of panyawan leaf are washed in a basin, wich

will be boiled in a pot under an electric stove with 450 milliliters of water for 30 minutes.

After boiling and letting it cool, filtered using a stainer to get the panyawan leaf extact.
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With the same procedure, 90 grams of panyawan stem are washed in a basin which will

be put in a pot under an electric stove with 450 milliliters of water for 30 minutes. After

boiling and letting it cool, filtered using a strainer to get the panyawan stem extract. The

researcher will prepare the two extracts to test its toxicity. 6 petri dish will be prepared to

put 30 brine shrimps in each of it by using syringe. 10 milliliters of each of the two

extracts will be put in each of the said 6 petri dish.

Post-conventional Phase

The researcher set a 1 hour timer to observe the container periodically for specific

duration. The result were counted by the number of alive brine shrimp and dead brine

shrimp inside each container at earch obeservation interval.

Statistical Treatment Data

This study aimed to determine the comparative study on the toxicity level of

panyawan stem extract and panyawan leaf extract to brine shrimp. Since this research

required an experimental research design, descriptive Statistics and independent sample t-

test were used. Further description of the statistical treatments and tools are listed below.

Descriptive Statistics. This was used to provide another content, a richer pictures

on enhanced presentation, in which in determine the effectiveness of panyawan leaf and

panyawan stem as an extract.

Independent Sample T-Test. This was used to compare the two independent

sample means and determine if there is a significant difference between, before and after

process.
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Chapter 4

PRESENTATION, ANALYSIS, AND INTERPRETATION OF DATA

This chapter contain the summary of findings, conclusions drawn from the

analysis and interpretation of the data, and the recommendation and suggestion of the

research for further research.

To determine the toxicity level of the leaf and stem extracts of papaya, the

decoction method of extraction was used and brine shrimp was utilized as indicators of

toxicity with three replicates composed of 30 samples in each replicate. Table 2 and 3

presents the descriptive statistics of the findings of the experiment.

Mortality of the brine shrimp after exposure to Panyawan leaf extract

Table 2

Descriptive statistics of the mortality of the brine shrimp using leaf extract

Treatment Replicate 1 Replicate 2 Replicate 3 Proportion Std.

Deviation

Leaf Extract 5 3 7 0.17 0.04

Table 2 shows the mortality of the brine shrimp after exposure to panyawan leaf

extract. The proportion and standard deviation were used to describe the data. Based on

the table, the leaf extract has exhibited a relatively small amount of toxicity, (p = 0.17, sd

= 0.04).
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Mortality of the brine shrimp after exposure to Panyawan stem extract

Table 3

Descriptive statistics of the mortality of the brine shrimp using stem extract

Treatment Replicate 1 Replicate 2 Replicate 3 Proportion Std. Deviation

Stem Extract 0.00 0.00 0.00 0.00 0.00

Table 3 presents the mortality of the brine shrimp using the stem extract of the

panyawan. Data revealed that there was no sign of toxicity in the extract since all of the

sample in each replicate did not exhibit mortality (p = 0.00, sd = 0.00). Add supporting

studies

Significant difference in the mortality of brine shrimp using stem and leaf extract

Table 4

Independent t-test of the mortality of brine shrimp between stem and leaf extract

statistical test value df p-value remarks

independent t-test 4.33 4 .012 s

Note. s = Significant at .05 level

Table 4 shows the significant difference between the stem and leaf extracts of

Panyawan in terms of the mortality of the brine shrimp. Data analysis using independent

samples t-test revealed that there is a significant difference between the two extracts (t (4)

= 4.33, p = .012). This indicates that the leaf extract may have a higher level of toxicity

than the stem extract. Add supporting studies