ANTI-HYPERGLYCEMIC ACTIVITY OF TALAHIB (Saccharum spontaneum Linn.

)
ETHANOLIC STALK EXTRACT IN ALLOXAN-INDUCED DIABETIC MICE

Kenneth John P. Chavez1*, Janesa A. Rinton1, Ledielyn S. Geronimo1, and Darwin A. Garbeles1
1
Partido State University, Goa Camarines Sur, 4424 Philippines
*Corresponding author, email: kjpchavez.pbox@parsu.edu.ph

Abstract. Diabetes is a chronic metabolic disorder that is considered the sixth leading cause of
death worldwide. Ethnobotanical studies show that Talahib (Saccharum spontaneum Linn.) is a
prospective candidate for antidiabetic treatment, however, further studies are required to prove this
claim. Therefore, this study’s main goal is to evaluate the anti-hyperglycemic activity of Talahib
by monitoring the effects of the extract in terms of its acute oral toxicity, blood glucose level, and
body weight on 18 ICR Mice induced with alloxan monohydrate. Three doses’ ranges were used
viz: 250 mg/kg bwt, 500 mg/kg bwt, and 750 mg/kg bwt. The extract showed no toxicity upon oral
administration. It also revealed that the Talahib stalk extract exhibits a potential dose-dependent
anti-hyperglycemic effect, where higher dosages of TSE are associated with a more efficient
reduction in blood glucose levels. A significant difference in body weight after treatment
application was noted and has been identified that a dose of 500 mg/kg bw.t is the optimum dosage
for weight loss for diabetic mice. The results provide support to the traditional claim on the
antidiabetic effect of Saccharum spontaneum Linn.

Keywords: Alloxan monohydrate, Diabetes, Talahib, Saccharum spontaneum Linn.

INTRODUCTION

Diabetes is a metabolic disorder in prevalence of type 2 diabetes to the report of

which carbohydrate and lipid metabolisms
are improperly mediated by insulin because
of the body’s incapability to produce enough
insulin hormone or when the produced
insulin is not effectively utilized. In a
protracted period, this inadequate control of
insulin secretion, action, or both, primarily
leads to high blood glucose or
hyperglycemia, a clinical indicator of
diabetes, which in due course, causes
diabetes mellitus (DM) (ADA, 2013; IDF,
2021).
The International Diabetes Federation
(IDF) reported that in 2021 around 536.6
million people worldwide have diabetes, with
the majority living in middle-income
countries (414 million) and the least number
from low-income countries (18.7 million),
with 6.7 million deaths. The increasing global
the International Diabetes Federation (IDF)
in 2021, is driven by several factors that
encompass population aging, economic
development, and increasing urbanization.
The occurrence of diabetes in the
Philippines is increasing due to rapid
urbanization with cumulative dependence on
electronic gadgets and a sedentary lifestyle
contributing significantly to this epidemic. In
2021, IDF reported that 4,303,900 Filipino
adults have diabetes, and based on the data of
the Philippine Statistics Authority, diabetes
mellitus caused an estimated death of 37.8
thousand Filipinos in the year 2020, ranking
fourth among the leading causes of death,
with a prevalence rate of 6.8%. The
Philippines is one of the lower-middle-
income countries with insufficient insulin
and routine diabetes care, thus Filipinos face
 Figure 1. Graphical Abstract
substantial barriers to health care as well as
inadequate health financing leading to
precluding diabetes care and insulin access
and high out-of-pocket expenditure,
respectively (Eala et al., 2022).
Despite the mass prevalence and
usage of commercial drugs and laboratory
treatments for T2DM, these medications still
accompany various side effects which
include hypoglycemia, weight gain,
cardiovascular risk, and cancer risk, to name
a few (Osadebe et al, 2014). These
constraints and adversities necessitate further
research on antidiabetic drugs from plant
sources with a better safety and efficacy
profile. Herbal preparations and plant
extracts remain conventional as treatments
for diabetes, especially in countries with low-
and middle-income economies (Salehi et al.,
2019). The adverse effect of conventional
drugs in diabetes treatment has resulted in the
shift of utilizing herbal medicine due to
indigenous availability of certain herbs with
hypoglycemic effects and the advantages
plant-based medicines provide, such as
marked efficacy, less toxicity and side
effects, low cost, and availability (Rahman et
al. 2021).
Talahib (Saccharum spontaneum
Linn.), with its limited commercial use, is a
potentially invasive species that pose a threat
to cultivated lands by reducing crop
production. Studies regarding the medicinal
uses of S. spontaneum are also limited,
however, in some parts of India it has been
used traditionally in treating diabetes mellitus
claiming to have antidiabetic efficacy but
lacking sufficient clinical evidence of
effectiveness (Hui Tag et al., 2012).
Phytochemical screenings from the
methanolic extracts of the whole plant
revealed the presence of phenolic
compounds, alkaloids, flavonoids, reducing
sugar, tannins, and saponins (Landingin et al.,
2018; Meera et al., 2018; Komathi & Durai,
2015).
Keeping the above in view, the
present investigation suggests conducting a
study on the potential anti-hyperglycemic
effect of the ethanolic stalk extract of S.
spontaneum on blood glucose levels in
alloxan-induced diabetic ICR mice (Mus
musculus L.).

METHODOLOGY
Procurement and Verification of Plant
Sample
Talahib were collected beside the
Rangas River of Brgy. Tambangan, San Jose,
Camarines Sur, where it is found to be
abundant. Voucher specimens were
deposited at the UP Baguio Department of
Biology, and was then verified that the plant
is identified as Saccharum spontaneum Linn.
Preparation of Plant Extract
One (1) kilogram of fresh Talahib
stalks was collected. The samples were
cleaned with tap water and air-dried a room
temperature for a whole day to ensure that the
samples were free from moisture and other
unnecessary contaminants. These stalk
samples were then cut into smaller pieces and
macerated using a blender until pulverized.
The powder was then sealed inside a glass jar
while soaked in 95% ethanol for 48 hours.
Whatman filter paper no. 1 was used to filter
the extract. It was then concentrated in a
rotary evaporator at 45ºC for 2 hours. The
concentration of filtrate yielded a total of
75mL of purified Talahib extract. It was then
sealed in an airtight container, sterilized
under a UV light for 15 minutes, and stored
at room temperature for further use.
Experimental Animals
The test subjects for this study were
eighteen (18) male mice from the
International Cancer Research (ICR) strain,
that were around 9-10 weeks old and weighed
from 25-35 grams. To maintain ethical
conduct, necessary protocols for handling
animal models in the laboratory were
observed as well as guidelines prescribed by
the Institutional Animal Care and Use
Committee (IACUC COA-22-05) and the
Philippine Association for Laboratory
Animal Science (PALAS). ICR mice, under
standard laboratory conditions (25+3˚C, 12-
hour light and 12-hour dark cycle, 30-80%
humidity) prescribed by the Philippine
Association of Laboratory Animal Science
(PALAS), were fed with a rodent diet (crude
protein 17% min, crude fiber 8% max, and
crude fat 3% min), ad libitum and had free
access to water. ICR mice were acclimatized
for 7 days under strict hygienic conditions
before experimenting.
Experimental Set-ups
This study utilized a total of 18 male
ICR mice which were divided into 6
subgroups with varying treatments as
follows:
NC. Normal Control: nondiabetic mice given
distilled water only
DCW. Diabetic Control: diabetic mice
treated with distilled water only;
DCM. Metformin Control: diabetic mice
treated with Metformin (500 mg/kg b.wt);
TSE250. Diabetic mice treated with Talahib
stalk extract (250 mg/kg b.wt);
TSE 500. Diabetic mice treated with Talahib
stalk extract (500 mg/kg b.wt)
TSE 750. Diabetic mice treated with Talahib
stalk extract (700 mg/kg b.wt)
Blood Glucose Determination
Blood collection was performed
through the tail venipuncture method, and a
drop of blood was placed onto a glucometer
strip. Cautiousness was practiced to avoid
coprophagy. The blood glucose of
experimental mice will be estimated using the
glucose estimation kit.
Induction of Alloxan Monohydrate
Fasting blood glucose was
determined after depriving food for 6 hours
with free access to drinking water. The
normal blood glucose level for mice is
100mg/dL and not greater than 200mg/dL
(Bhatia & Khera, 2013), hence mice with
effective and permanent elevated plasma
glucose levels of (>200 mg/dL) were
subjected to the experiment. Experimental
diabetes in mice was induced by a single
intraperitoneal administration injection of
alloxan monohydrate in normal saline (0.9%)
with a dosage of 150 mg/kg body weight.
Blood glucose levels were also observed
using a Glucometer 48 hours after the
induction of alloxan monohydrate.
Application of Treatments
All the treatments were suspended in
a 0.1mL sterile aqueous solution and were
administered orally by oral gastric intubation
once daily for 21 days.
Waste Disposal
Experimental animals were subjected
to euthanasia through cervical dislocation
and the carcasses were deposited in a
designated plot by the IACUC. Biological
and chemical wastes were disposed of
properly in compliance with the standard
chemical safety guidelines.
Data Collection
Acute Oral Toxicity
Acute oral toxicity tests were
administered in 9 ICR mice of the treatment
group, following the procedures outlined by
the Organization for Economic Co-operation
and Development (OECD) 423. Following
the overnight fasting period, the animal
subjects were weighed and single doses of
Talahib stalk extracts ranging from
250mg/kg, 500mg/kg, and 750mg/kg, as well
as a vehicle regulation, were given orally and
monitored. After testing the substance, food
was not given for 2 hours (OECD 423).
Observations for clinical signs in the AOT
test were recorded based on the modified
SHIRPA method of Brayton et al. (2019),
which provided basic evaluation for
abnormal behaviors, and absence or
reduction in normal behaviors or reflex
responses. The number of survivors was
noted within 2 days. Mortality was observed
and recorded from the time of administration.
The treatment groups that were subjected to
the AOT test underwent a week of
withdrawal after the test was conducted
before proceeding to further experiments. For
clinical signs and body weight variables, a
binary scoring system was employed. The
presence of abnormal clinical signs was
scored with 1, absence, or normal with 0.
Fasting Blood Glucose
Upon confirmation of the
hyperglycemic conditions induced in the
experimental groups, the Talahib stalk
extract was administered orally according to
the dosage assigned to each (Bhatia & Khera,
2013). The time of administration was noted
as the reference point for the time interval
allotted before each blood glucose test or
trial. Fasting blood glucose was determined
after depriving food for 6 hours with free
access to drinking water. The fasting blood
glucose was estimated after 18 hours of
injection through the tail prick method using
a glucometer. The mice with effective and
permanent elevated plasma glucose levels of
(>200 mg/dL b.wt), were selected for the
present study. The blood glucose of
experimental mice was estimated using the
glucose estimation kit of Aviva® Plus.
Body Weight Determination
Body weight is a subtle indicator that
reflects the state of health of experimental
animals and a decrease in body weight
correlates with defects in body metabolism
that is due to toxicity. An increase in body
weight implies that anabolic effects have
overridden the catabolic ones. No variation
means protection against weight loss. A
decrease in body weight would mean that
catabolism has persisted. In this study, the
body weight of animal models was monitored
daily by utilizing a digital weighing scale
while being confined inside a beaker. And to
further evaluate the significant change in 500 mg/kg b.wt (TSE 500), and 750 mg/kg
body weight of diabetic mice from the b.wt (TSE 750). Notably, observations
experimental groups throughout the indicated in the post-test of the mice's
treatment application period, values for physical behaviors revealed no toxicological
percent weight change were gathered and symptoms, indicating that TSE does not pose
calculated using the formula below. any direct risk to ICR mice, and provided that
no mortality was recorded. These findings
𝐹𝑖𝑛𝑎𝑙 𝑊𝑒𝑖𝑔ℎ𝑡 (𝑔) − 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑊𝑒𝑖𝑔ℎ𝑡 (𝑔)
𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 𝑐ℎ𝑎𝑛𝑔𝑒 =
𝐹𝑖𝑛𝑎𝑙 𝑊𝑒𝑖𝑔ℎ𝑡 (𝑔)
× 100 highlight the favorable safety profile of TSE
in the context of acute oral toxicity
Data Analysis assessment in ICR Mice. The study also
Statistical analysis was performed revealed that the TSE were safe up to a dose
using the software IBM SPSS Statistics level of 750 mg/kg body weight. Although no
version 20. This study utilized the one-way lethality was observed at higher doses, the
ANOVA in establishing the significant experimental mice treated with higher doses
differences among the groups in terms of still showed lethargic behavior. These
blood glucose levels of diabetic mice under findings suggest that TSE is not toxic and can
the different treatment conditions. Tukey’s be considered safe for use in ICR mice. These
multiple range test was used for post hoc results are consistent with previous studies
analysis. All the results were expressed as conducted by Amutha et al. (2015) and
Mean ± S.E.M. A value of P<0.05 was Sathya, & Kokilavani (2012) on the acute
considered statistically significant, whereas oral toxicity of Saccharum spontaneum roots.
P>0.05 was considered nonsignificant. Moreover, the findings of Saleem et al.
(2017), Severin et al. (2023), and Dasofunjo
RESULTS AND DISCUSSION & Abong (2013) examined the toxicity of
other species of Saccharum spp. such as S.
Acute Oral Toxicity munja Roxb., S. officinarum, and S. Barberi,
The present study investigated the acute oral respectively, further support the outcomes of
toxicity of Talahib (Saccharum spontaneum the current study.
Linn.) ethanolic stalk extract (TSE) using a
modified screening protocol based on the Fasting Blood Glucose
established SHIRPA Mouse Physical This study assessed changes in blood
Examination by Brayton et al. (2019), as glucose levels after a 6-hour fasting period
detailed in Table 2. ICR mice were using a fasting blood glucose test. The
administered a single dose of TSE at varying experimental animals, except the normal
concentrations of 250 mg/kg b.wt (TSE 250), control group, exhibited high levels of blood
glucose before the administration of the

Table 1. Acute Oral Toxicity Test Result

TSE 250 TSE 500 TSE 750

Behavioral
Parameters
Pre-Test Post-Test Pre-Test Post-Test Pre-Test Post-Test
SHIRPA Physical
Normal Normal Normal Normal Normal Normal
Screening
Weight Normal Normal Normal Normal Normal Normal

Mortality 0 0 0 0 0 0
 600
500
400
BGL (mg/dL)
300
200
100
0
0 1
EXPERIMENTAL DAYS
Note: NC (Normal Control), DCW (Diabetic Control + Distilled Water), DWM (Diabetic Control + Metformin), TSE 250
(Talahib Stalk Extract at 250 mg/b.wt), TSE 500 (Talahib Stalk Extract at 500 mg/kg b.wt), TSE 750 (Talahib Stalk Extract
at 750 mg/kg b.wt).
Figure 2. Result of Fasting Blood Glucose Level
Talahib stalk extract, indicating the
occurrence of diabetic conditions. The study
found that the TSE 750 group showed the
most significant reduction in blood glucose
levels of 190.00 ± 7.00 mg/dL, followed by
the TSE 500 and TSE 250 groups. In
comparison, the negative control group
showed an increase in blood glucose levels,
while the positive control group exhibited a
decrease in blood glucose levels after
treatment application. Statistical analysis
indicated that TSE 750 and TSE 500
exhibited highly significant values,
highlighting the potent anti-hyperglycemic
activity of TSE at these dosages in diabetic
mice. The study also found that all
experimental treatments (TSE) were not
significantly different compared to the
positive control group, suggesting that TSE
exhibited a comparable effect to metformin in
reducing blood glucose levels. These findings
provide compelling evidence of the
significant anti-hyperglycemic effect of
Talahib ethanolic stalk extract at specific
dosages. The study's results are consistent
NC
DCW
DCM
TSE 250
TSE 500
TSE 750
7 14 21
with previous research, supporting the
potential antidiabetic effects of Talahib
extract and phenolic compounds in
Saccharum spp. on liver and kidney function,
as well as lipid metabolism in diabetes.
Body Weight
This study investigated the effect of
Talahib (Saccharum spontaneum Linn.) stalk
extract on body weight in diabetic mice, as
illustrated in Figure 3. The analysis of weight
differences between pre-and post-tests in
diabetic mice treated with Talahib extract
revealed that TSE 250 exhibited the highest
weight loss of 16.07%, followed by TSE 500
with a weight loss of 3.18%. In contrast, TSE
750 showed a weight gain of 10.32%. When
compared to the control groups, the negative
control group (DCW) showed an exponential
weight loss of 33.96%, while the positive
control group (DCM) showed a weight gain
of 2.31%. Similarly, the normal control group
(NC) exhibited a weight gain of 6.11%. The
analysis of weight differences between pre-
and post-tests in diabetic mice treated with
 15.000
10.000
Body Weight Change (%)
5.000
0.000
-5.000
-10.000
-15.000
0 1
Time (day)
Note: NC (Normal Control), DCW (Diabetic Control + Distilled Water), DWM (Diabetic Control + Metformin), TSE 250
(Talahib Stalk Extract at 250 mg/b.wt), TSE 500 (Talahib Stalk Extract at 500 mg/kg b.wt), TSE 750 (Talahib Stalk Extract at
750 mg/kg b.wt).
Figure 3. Percent Change in Body Weight throughout the Treatment Period.
different doses of Talahib extract showed that
TSE has a significant impact on the weight
difference of diabetic mice, with higher doses
showing a more significant effect. The study
also revealed that TSE has a dose-dependent
effect in preventing weight loss in diabetic
mice, with higher doses showing a more
significant effect. However, weight gain in
the TSE 750 group may have negative
implications, as it can exacerbate the
symptoms of diabetes and contribute to the
development of other health issues such as
obesity and metabolic syndrome.
The findings of this study are
consistent with previous studies that have
shown that excessive weight loss in diabetic
mice may lead to increased euthanization
rates, indicating the severity of the condition.
On the other hand, weight gain in diabetic
mice may also have negative implications, as
it can exacerbate the symptoms of diabetes
and contribute to the development of other
health issues such as obesity and metabolic
syndrome. The study suggests that TSE has
NC
DCW
DCM
TSE 250
TSE 500
TSE 750
7 14 21
the potential to be used as a natural remedy
for managing weight loss in diabetes, which
could be particularly beneficial in the context
of diabetes management. However, further
studies are warranted to determine the
optimal dosage of TSE and to elucidate the
underlying mechanisms of action of TSE.
The study highlights the importance
of carefully determining the optimal dosage
of TSE for managing body weight in diabetes
and suggests that TSE could be considered an
alternative or complementary treatment
option to conventional medications, such as
metformin, for managing body weight in
diabetic individuals. However, further
research is needed to determine the long-term
safety and efficacy of TSE in human subjects.
CONCLUSIONS
The findings of the toxicity
assessment of Talahib stalk extract (TSE)
indicate that TSE does not exhibit any
apparent toxicity, as the behavioral patterns
of the ICR mice remained within the normal
range. This suggests that TSE is a safe
alternative for the treatment of severe
infections. Furthermore, the findings from
the fasting blood glucose (FBG) test reveal
that TSE exhibits a potential dose-dependent
anti-hyperglycemic effect, where higher
dosages of TSE are associated with a more
efficient reduction in blood glucose levels.
Additionally, TSE poses a dose-specific
effect on weight loss, having TSE 500
identified as the optimum dosage for
mitigating weight loss for diabetic mice.
Thus, it can be concluded that the
administration of 500 mg/kg b.wt Talahib
ethanolic stalk extract is the optimal dosage
for ameliorating diabetes, as it is associated
with reduced fasting glucose levels and
regulating body weight loss similar to the
effect of the positive commercial drug
metformin.
RECOMMENDATIONS
Future investigations could expand
on this study's findings by conducting in vitro
studies to identify the mechanisms
underlying Talahib ethanolic stalk extract's
anti-hyperglycemic and weight loss effects.
Histopathological evaluation is also
recommended to understand the extract's
cellular effects. Additional parameters for
study include insulin sensitivity, pancreatic
beta-cell function, lipid profile, oxidative
stress and inflammatory markers, and
markers of diabetic complications. Long-
term safety and tolerability of TSE, including
potential adverse effects and drug
interactions, should also be explored.
Addressing these research areas could
provide a comprehensive understanding of
Talahib's therapeutic potential in diabetes
and inform evidence-based clinical
applications.
ACKNOWLEDGMENT
The authors are grateful to the DOST-
SEI and DOST Regional Office for granting
the Thesis Fund of this study.
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