December 2010, Volume 1, No.

2
International Journal of Chemical and Environmental Engineering

Evaluation of the hepatoprotective activity of Citrus

microcarpa Bunge (Family Rutaceae) fruit peel
against acetaminophen-induced liver damage in
male BFAD- Sprague Dawley rats
Casimiro,Marifel Franchesca 1 Margarita Gutierrez 2 Danice Romagne Leano 1
Judilynn N. Solidum3
1
BS Industrial Pharmacy Graduate, College of Pharmacy, University of the Philippines, Manila
2
Faculty member, College of Pharmacy University of the Philippines, Manila
3
Associate Professor, College of Pharmacy University of the Philippines, Manila

Abstract
The Philippine Department of Health stated that liver cancer is the third common forms of cancer for both males and
females, hence the need for more hepatoprotective agents. Silymarin, from milk thistle is the most well known
hepatoprotective agent (Prahan and Girish, 2006) but due to availability and economic concerns with the use of milk
thistle other sources were explored. Fruit peels constitute a bulk in Philippine wastes. If such wastes can be used as
hepatoprotective agents, then wastes will be decreased and new sources of important products may be discovered.
This study aimed to evaluate the hepatoprotective activity of Citrus microcarpa Bunge fruit peel extract relative to the
commercially available Silymarin preparations. The chemical components of the fruit peels were analyzed to ascertain
pharmacologic value.
The study used an experimental research design using BFAD- Sprague Dawley rats as subjects. The hepatoprotective
activity was evaluated based on changes in the liver morphology- gross examination and differences in serum liver
enzyme levels- bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase
(AP) within and among the groups of rats. There was a significant decrease in ALT, AST and AP levels among rats
administered with the fruit peel extract. Silymarin significantly decreased bilirubin levels. These suggest a comparable
hepatoprotective activity between Silymarin and the fruit peel extract tested. Phytochemical analysis showed that the
fruit peel extract contained flavonoids, tannins, and glycosides.
Quantitative analysis on the chemical components of the fruit peel extract is suggested to facilitate the study of its exact
mechanism of action. Research on the protective ability of the fruit peel extract on other organ systems is recommended.
It is also suggested that other chemical liver toxicity inducers be used to observe the range of hepatoprotective activity of
the fruit peel extract studied.

Keywords: hepatoprotective, Citrus microcarpa Bunge (Family Rutaceae), ALT,AST,AP

1. Introduction Citrus microcarpa Bunge plant, locally known as

1.1. Background and significance of the study
Hepatotoxicity is a growing concern of today’s
modern society. The increasing incidence of alcoholism,
cigarette smoking, substance abuse and other unhealthy
lifestyle options, like eating fatty foods, have contributed
to the morbidity and mortality due to liver disease. Many
researchers have investigated plants for use in a wide
variety of liver disorders [1]. Recent studies have shown
that plants under Family Rutaceae like Murraya koenigii,
Citrus limon, Citrus aurantium and Tephrosia purpure
have been reported to possess hepatoprotective activity
[2, 3, 4, 5].
kalamansi, is a member of Family Rutaceae that is native
and widely cultivated in the Philippines. This species is a
native of the Philippines and does not occur naturally
outside of the archipelago. Kalamansi is a common fruit
vegetable often used by Filipinos in seasoning dishes or
as dipping sauce to other viands. It is a smooth and
slightly spiny plant, growing to a height of 3 to 5 meters.
Leaflets are elliptic to oblong-elliptic, 4 to 8 cm long.
Petioles are narrowly and scarcely winged, about a cm
long. Flowers are axillary, solitary, rarely in pairs, white,
and short-stalked. Fruit is yellow when ripe, nearly
spherical, 2 to 3.5 cm diameter, 6- to 7-celled, and thin-
 skinned. The skin or peel is green to yellowish green or
yellow, loosely adhering to the flesh. The flesh contains a
few seeds. The fruit contains volatile oil at 0.9 to 1.06%
and its rind contains aldehydes, sesquiterpenes, beta-
pinene, linalool, linalyl acetate, tannin, glucoside and
cyanogenetic substances [6].
The present study was undertaken to study the
possible hepatoprotective property of ethanolic extract of
rind or peel from kalamansi. It was also the intention of
the researchers to address the foul odor emited by
decomposing kalamansi rind in household garbage that
attracts flies.
At pharmacological doses, acetaminophen is
considered relatively safe as antipyretic and analgesic. At
high doses, acetaminophen induceds acute liver damage,
which is attributed largely to a by-product of its
metabolism, N-acetyl-p-benzoquinone imine (NAPQI).
NAPQI promotes depletion of glutathione sources,
thereby increasing the risk of liver damage by oxidizing
tissue macromolecules such as lipid or SH group of
protein and alter homeostasis of calcium [7, 8].
According to Barnes and Denz [9], rats have been widely
used for evaluating hepatic toxicity and the criteria of the
toxic action in rats are reduction in the rate of bodyweight
gain, detection of gross and histologic abnormalities in
the organs, increased mortality, and changes in organ
weights.
Should the fruit peels of kalamansi be proven to be
hepatoprotective, a new source for drug discovery will be
presented which will help make medicinal formulations
more cost effective. Further the innovation will help
alleviate problems of wastes in the environment.
1.2. Conceptual framework
INPUT
I.Collection of kalamansi fruit peels
II. Preparation of kalamansi peels, animal models,
drugs for lab analyses
THROUGHPUT
Laboratory tests for effects of kalamnsi fruit peels
on liver toxicity of BFAD Sprague Dawley rats
OUTPUT
Hepatoprotective activity of Kalamansi fruit peels
1.3. Problems
Generally, does the fruit rind extract of Citrus
microcarpa Bunge (Fam. Rutaceae), locally known as
kalamansi, exhibit hepatoprotective activity against
acetaminophen-induced liver damagein male BFAD:SD
rat models?
Specifically ,how does the hepato-protective activity
of the kalamansi peel extract compare with the
128
commercially-available preparation (eg, silymarin
capsule)? What are the chemical components in the rind
of kalamansi that may prove to be of value in the fields of
medicine, and pharmacy?
1.4. Objectives
This study generally aimed to determine the
presence of hepatoprotective activity of Citrus
microcarpa (kalamansi) fruit peel against
Acetaminophen-induced liver toxicity to Sprague Dawley
rats. Specifically it aimed to evaluate the hepatoprotective
activity of kalamansi fruit peel against acetaminophen-
induced liver damage in BFAD: SD rat models; compare
the hepatoprotective activity (i.e. efficacy) of the
kalamansi fruit peel extract from the commercially-
available preparation (silymarin capsules); to identify the
chemical components present in the kalamansi peel,
which may be of value in the fields of medicine and
pharmacy, through phytochemical screening.
1.5. Scope and limitations
This study involved the determination of the
presence of hepatoprotective activity of the kalamansi
peel extract against liver toxicity induced by
acetaminophen alone. Furthermore, the basic
phytochemical components of the fruit peel extract were
also determined.The actual compound essential for
hepatoprotective activity was not identified nor
quantified.
Commercially-available Silymarin was utilized
as positive control. The parameters used to measure the
hepatoprotective activity of the plant was limited only to
the monitoring of mortality, bodyweight, food
consumption, water intake, gross examination of the liver,
liver weight, and measurement of blood serum levels of
total bilirubin, direct and indirect bilirubin, alanine
aminotransferase, alkaline phosphatase, aspartate
aminotransferase.
2. Materials and Methods
2.1 Research design
The study involved the extraction of plant material
followed by an evaluation of its hepatoprotective activity.
The evaluation of the hepatoprotective activity of Citrus
microcarpa Bunge employed a pretest – post test only
experimental study design.
2.2. Plant Material
Kalamansi fruits were obtained from Mindoro
Province, Philippines through the Valenzuela Market,
Polo, Valenzuela City, Philippines. Samples were
identified by the Philippine National Museum.
Kalamansi rind were separated from the flesh of the
fruit, air dried and reduced to size. The material was
macerated in 95% ethanol for 48 hours and then filtered.
The filtrate was evaporated to dryness under steam bath.
The resulting residue was weighed and diluted with
 distilled water to obtain a final concentration of 1g/mL
(w/v)..
2.3. Drugs and Chemicals
The Silymarin capsule (LiveraideTM , bearing FDA
registration no. 41322, lot no. 83, manufactured on June
2009 with expiry date on June 2011, manufactured by
Capsugel, France, and imported and distributed in the
Philippines by Herbs & Nature Corporation, Bagbag,
Novaliches, Quezon City), was used in the study.
Silymarin is a hepatoprotective drug[10]. Each capsule
has a net weight of 636 mg and contains 125 mg of
sylimarin from the seeds of Silybum marianum of
Compositate Family, lecithin and beeswax.
Acetaminophen was obtained from the College of
Pharmacy, University of the Philippines, Manila.
2.4.Animals
Eight week old Sprague Dawley male rats were
obtained from the closed colony of the Philippine FDA.
All animals were given drinking water and standard
laboratory diet before and during the experiment. All test
animals were subjected to a one week acclimatization
period. The environmental conditions and husbandry
practices were kept constant throughout the experiment.
The study was approved by the UP Manila Institutional
Animal Ethics Committee.
2.5. Study Design
Four groups with 10 rats each were chosen. The
entire study took eight days to finish. Pre-test liver
function test were conducted to determine homogeneity of
the groups of rat. Group I served as the negative control.
Group II, III and IV were given acetaminophen (500
mg/kg) by oral gavage on the fourth day to induce liver
damage. From the first day to the seventh day Group III
was given silymarin (950 mg/kg bwt) as hepatoprotective
agent and Group IV was given kalamansi rind ethanol
extract (4,000 mg/kg bwt) as the test sample. On the eight
day, fourth day post-acetaminophen toxicity, all rats were
sacrificed. Blood serum were obtained, major tissues and
organs were observed and liver organs were weighed.
2.6. Animal Performance
Body weight gained, as well as 24-hour water and
feed intake, were measured 24 hours post-
acetaminophen toxicity.
2.7. Liver Function Test
Blood samples collected and submitted to a DOH-
accredited clinical diagnostic laboratory in Marikina City,
Philippines. Blood serum liver enzyme levels of
bilirubin, aspartate aminotransferase (AST), alanine
aminotransferase (ALT) and alkaline phosphatase (AP)
were analyzed.
2.8. Statistical Analysis
Body weight gain, feed and water intake, and liver
function test results, liver weight were analyzed using
Student t-test, NCSS statistical software. All data are
reported as mean and SD.
3.Results and Discussion
3.1. Pre-Test Baseline Data
The data shown in Table 1 shows that there were no
differences in bodyweight, total bilirubin, direct bilirubin,
indirect bilirubin, AST, ALT and alkaline phosphatase
among the groups of rats at the start of the treatment.

Table. 1 Bodyweight And Blood Serum Enzyme For Liver Function

Body
Total Direct Indirect Alkaline
AST ALT
Oral Treatment Weight Bilirubin Bilirubin Bilirubin Phosphatase

gm mg/dL mg/dL mg/dL IU/L IU/L IU/L

I 256.1 ± 0.15± 0.13 ± 0.04± 101.31± 49.67± 331.15 ±

Distilled Water
1.75 0.01 0.01 0.01 2.64 1.89 26.42

Distilled Water + 256.9 ± 0.15± 0.11± 0.04± 99.11± 47.37± 325.51±

II Acetaminophen 1.77 0.01 0.004 0.01 4.70 2.23 29.26

255.4 ± 0.17± 0.12± 0.05± 112.03± 51.15± 307.89±

Silymarin + Acetaminophen
III 1.86 0.01 0.01 0.01 2.90 2.14 26.16

IV Kalamansi Rind Extract + 256.6 0.16± 0.12± 0.05± 101.20± 49.60 ± 314.51±
Acetaminophen ±1.63 0.01 0.01 0.01 4.63 1.70 24.62

129
 3.2 Test Proper
Table 3 Twenty Four Hour Feed And Water Intake 24 Hours Post
3.2.1 Mortality
Acetaminophen Toxicity
On the sixth day, second day post-acetaminophen Feed Water
toxicity, one rat in Group II, rat no. 7, was found dead Group Oral Treatment Intake Intake
inside the cage. Rat no. 7 was found emaciated inside the grams mL
cage. Gross examination of the liver organ revealed 28.9 ±
I Distilled Water 29.1 ± 1.13
hemorrhagic lesions. 1.68
B.1.1 Body Weight Gained
Distilled Water + 14.1 ± 18.50 ±
Analysis of the bodyweight loss and gained 24 hours II
Acetaminophen 1.83 a 2.62 a
post-acetaminophen toxicity are summarized below in
Table 2. The data shows that rats in Groups II, III and IV
significantly decreased in bodyweight compared to Group 18.8 ± 18.7 +/-
III Silymarin + Acetaminophen
I. However, the table also shows that rats in Group II 3.11 a 3.57 a
and Group III has significantly lower body weight loss Kalamansi Rind Extract + 26.5 ± 25.80 ±
than Group II. Group IV still managed to gain some IV
Acetaminophen 2.65 2.64
weight. a p<0.05 compared with Group I

Table 2 Body Weight Gain 24 Hours After Acetaminophen Toxicity The results revealed that the mean feed and water
Body Weight intake of rats in Group IV was not significantly reduced
Group Oral Treatment Gained
compared to rats in Group I. It was seen that food and
Gm
water intake of groups II and III decreased significantly.
I Distilled Water 3.70 ± 0.62 The findings are biologically significant since the rats
II Distilled Water + Acetaminophen - 1.33 ± 1.16 a were not trained to refuse feeds and water that are made
available and accessible ad libitum. The group of rats
III Silymarin + Acetaminophen - 0.10 ± 1.14 a, b which were pre-treated with kalamansi rind extract
IV
Kalamansi Rind Extract +
1.20 ± 0.93 a, b
performed better than the group pre-treated with sylimarin
Acetaminophen 24-hours after giving large doses of acetaminophen to
a p<0.05 compared with Group I induce liver damage.
b p < 0.05 compared with Groups II and III

3.2.2. Feed and Water Intake

The 24-hour feed and water intake between the
groups were studied 24 hours after acetaminophen
toxicity. The results are shown in Table 3.

Table.4 Result Of Blood Serum Enzyme For Liver Function Test

Total Bilirubin                                                                      Indirect bilirubin 
a  p<0.05 compared with Groups I and III                          a p<0.05 compared  with Groups I and III                                                                                    
b p<0.01 compared with Groups I and III                           b p<0.01 compared with Groups I and  III 
AST a p<0.05 compared with Groups I, II and III ALT a p<0.05 compared with Groups I, II and III

BILIRUBIN
AST ALT Alkaline
Group Oral Treatment (SGOT) (SGPT) Phosphatase
Total Direct Indirect
Bilirubin Bilirubin Bilirubin

mg/dL mg/dL mg/dL IU/L IU/L IU/L

I Distilled Water 345.22±

0.09 ± 0.96 0.05± 0.004 0.04± 0.01 150.8 ± 9.31 51.79± 1.78
18.96

II Distilled Water + 327.67±

0.20 ± 0.06 a 0.05 ± 0.01 0.15 ± 0.05 a 143.16 ± 9.22 51.66± 2.67
Acetaminophen 21.24

III Silymarin + 264.41±

0.11± 0.02 0.06 ± 0.004 0.05± 0.01 144.72 ± 2.90 63.56± 5.11
Acetaminophen 13.96

IV Kalamansi Rind Extract 44.17± 2.04 252.7±

0.22± 0.04 b 0.06± 0.01 0.16± 0.01 b 108.20± 9.65 a
+ Acetaminophen a 24.03
3.3 Blood Chemistry Liver Profile

130
 3.3.1 Bilirubin
Group III (silymarin + acetaminophen) has normal
level of total bilirubin which is comparable to Group I
(negative control). Group IV (kalamansi rind extract +
acetaminophen), have significantly elevated total bilirubin
compared with Groups I and III which may indicate some
form of parenchymal injury. Values obtained are near the
upper limit of normal bilirubin value of 0.18mg/dL [11].
3.3.2 AST and ALT
In this study the animals were sacrificed after 80
hours before blood serum for hepatic enzyme function
tests were collected. Acetaminophen toxicity
significantly increases ALT and AST values immediately
upon administration as indication of hepatic injury. As
shown in the table, AST and ALT of Groups I, II and III
increased after 80 hours post-acetaminophen dosing
relative to group IV. In humans, serum ALT (SGPT)
biological half-life varies from 48 to 60 hours [12]
In Group IV (kalamansi rind extract), after an 80 hour
post acetaminophen toxicity, the serum AST (SGOT) and
ALT (SGPT) were significantly lower than all the groups,
including Groups II and III (sylimarin). The major organs
affected by acetaminophen in cases of overdose are liver
and kidneys. The protective property of kalamansi rind
extract may not be specific to acetaminophen induced
damage to liver alone, but to other tissues as well, like the
red blood cells, heart and skeletal muscles, pancreatic
tissues and kidney tissues, which also contain the AST
enzyme. The protective effect of kalamansi rind extract
may be the result of stabilization of plasma membrane,
thus preserving the structural integrity of cell as well as
the repair of tissue damage.
        The  24‐hour  feed  and  water  intake  (24  hour  post‐
acetaminophen  toxicity  )  shown  in  Table  III  of  Group  IV  hepatoprotective activity of kalamansi peel extract is
were not statistically different from Group I, the negative  silymarin preparations. Similarities in mechanism of
control, as kalamansi rind extract may also be protective  action between the two plant extracts may be expected but
to other organs and tissues.  It was observed that when  the presence of other medicinally beneficial
protected  by  kalamansi  rind  extract  ,  rats  are  least  phytochemicals in the kalamansi peel extract (i.e. tannins
affected  when  given  large  doses  of  acetaminophen  (  additional modes of action. As such, the kalamansi peel
Table IV).  extract has also been inferred to confer multi-system or
3.3.3 Liver Weight and Lesions
The following table shows the absolute and relative
mean weight of liver in all groups. Groups II, III and IV
relative liver weights were significantly heavier than
Group I after 80 hours post-acetaminophen toxicity.
131
Table 5 Absolute And Relative Weights Of The Liver At The
Termination Of The Study
Liver Weight
Absolute Relative
Group Oral Treatment
Grams/100
grams
gram bwt
10.34 ±
I Distilled Water 3.71 ±- 0.06
0.15
Distilled Water + 10.98 ±
II 4.15 ± 0.10 a
Acetaminophen 0.28
Silymarin + 10.60 ±
III 4.03 ± 0.17 a
Acetaminophen 0.41
Kalamansi Rind Extract + 10.88 ±
IV 4.07 ± 0.14 a
Acetaminophen 0.47
a p<0.05 compared with Group I
Literatures reported that when acetaminophen was
given to mice at 1,000 mg/kg, extensive necrosis was
associated with hemorrhage and seen in the liver. When
dose was reduced to 500 mg/kg punctuate hemorrhage
necrosis of liver tissue was observed [13]. This study
showed that after 80 hours post-acetaminophen toxicity,
although liver weights of Groups II,III and IV increased
relative to group I, no punctuate hemorrhage or other
appreciable liver lesions, like swelling (grossly by
observing and cutting the liver margins), were observed
particularly with groups III and IV. This finding indicates
possible recovery from the initial acetaminophen toxicity.
4. Conclusion
From the results obtained, kalamansi peel extract
showed hepatoprotective activity against Acetaminophen-
induced liver damage in male SD rats. The
hepatoprotection it conferred was reflected by the
significant decrease in serum enzyme levels and lack of
any gross morphological injury to the rat’s liver. It may
also be concluded from the results of the study that the
comparable to that of the commercially available
and glycosides), aside from flavonoids, may offer
multi-organ protection including that of the kidneys.
With the observations of hepatoprotective activity in
the kalamansi peel extract (against acetaminophen-
induced liver damage), evaluation of the same activity
against damage induced by other chemical toxicants and
subsequent identification of the possible component/s
responsible for the activity is recommended.
Determination of the most effective extract concentration
must also be employed. Quantitative evaluation of the
phytochemical components of the extract should be given
priority as well so as to provide means for
standardizations for future product formulation Finally,
similar with other ‘new’ drugs and substances, researches
geared toward toxicological profiling of kalamansi peel
extract is suggested.
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