Download College of Medicine, University of the Philippines

The Hepatoprotective effect in vivo and Antioxidant effects in vitro of Tinospora cripa stem
extract
Group 6 Biochemistry
Mark Manguerra, John Christian Aniban, Apple Balbiran, Roscel Cadeliña, Hazel Cappleman,
Lowe Chiong, Genevieve Cruz, Jeffrey de los Santos, Vienna Encila, Veronica Flores, Carmela
Gonzales, Darnel Hurtado, Joseph Anthony Lachica, Ronald Luna, Charles Mercado,
Christopher Ocampo, Pamela Patdu, Grace Quilloy, Carlo Rubio, Arunee Siripunvarapon, Diana
Tamondong, Roland Joseph Tan, Ma-am Joy Tumulak, and Diane Zaragoza
College of Medicine, University of the Philippines Manila
Adviser:
Dr. Mariluz Mojica-Henshaw
Department of Biochemistry, College of Medicine, University of the Philippines Manila
Rifampicin is used prevalently in the treatment of tuberculosis, but it usually results in
hepatotoxic effects such as jaundice. This study investigated the hepatoprotective effect of
Tinospora crispa root extract against rifampicin-induced liver toxicity in mice and its free radical
scavenging activity compared to ascorbic acid. Groups of 7 mice given a single 400 mg/kg dose
of rifampicin were treated with either the extract or silymarin, a common hepatoprotective drug.
Mortality of mice given rifampicin alone was similar to the treatment groups but the serum ALT
and bilirubin levels for both groups were decreased. Histopathological studies revealed that all
groups except for those given rifampicin alone did not have fatty metamorphosis of hepatocytes.
The free radical scavenging activity of the extract was comparable to silymarin, but both had
about 1/200 the antioxidant activity of ascorbic acid. Tinospora crispa may have a
hepatoprotective effect in mice but its mechanism is still unknown.
Keywords: Tinospora crispa, hepatoprotection, antioxidant
INTRODUCTION
Background of the Study
There is an estimated 1.9 billion people worldwide afflicted with tuberculosis. In 1999,
the total number of TB cases registered in the Philippines was 144,932. The World Health
Organization ranks the Philippines fourth among all countries in number of tuberculosis cases,
with up to 39% of children aged between 5 and 9 already infected (Easton 1998, Wallerstein
1999). Among the countries in the Western Pacific Region, the Philippines ranks third with a
high case notification rate of 198.1 per 100,000 population.
Rifampicin is included in the first-line combination therapy against tuberculosis along
with isoniazid (INH), pyrazinamide (PZA) and ethambutol (Chan and Iseman 2002). However,
jaundice and other manifestations of hepatotoxicity usually occur during treatment. The risk is
increased when rifampicin is given concomitantly with INH and PZA (Saraswathy et al. 1998).
Liver diseases may also occur due to excess consumption of alcohol, infections, and autoimmune
disorders (Visen et al. 1996, Zhang et al. 2002).
Despite advances in allopathic medicine, there is no available effective hepatoprotective
medicine, which has encouraged the development of herbal extracts that have shown a positive
hepatoprotective effect (Murthy and Srinivasan 1993). This study serves as a preliminary
screening of hepatoprotective compounds in Tinospora crispa which, in the future, may be
included in the TB regimen. Studies have validated Tinospora cordifolia extract as a possible
treatment for hepatotoxicity (Singh et al. 1984). Pyridoxine (Vitamin B6), for example, is given
to prevent peripheral neuropathy associated with INH. In this experiment, we will assess the
hepatoprotective activity of the fractions of ethanolic extract of Tinospora in rifampicin-treated
albino mice by measuring the levels of various biochemical parameters. We will also attempt to
propose a possible mechanism of activity by determining radical scavenging activity of the
fractions.
Objectives
The general objective of the study is to determine if crude extracts of Tinospora crispa
possess a hepatoprotective activity in rifampicin induced hepatotoxicity in male Swiss albino
mice.
The specific objectives are:
1) To determine serum ALT and bilirubin levels in mice treated with rifampicin alone,
Silymarin and the plant extract.
2) To determine if crude extracts of Tinospora crispa possess an antioxidant property
Hypotheses
Ho1: There is no difference in serum ALT and bilirubin levels between the mice treated with
Silymarin (the positive control), plant extract and rifampicin alone (the negative control).
Ha1: There is a difference in serum ALT and bilirubin levels between the mice treated with
Silymarin (the positive control), plant extract and rifampicin alone (the negative control).
Ho2: The plant extract possesses no free radical scavenging activity.
Ha2: The plant extract possesses a free radical scavenging activity
Significance of the Study
Due to the high prevalence of tuberculosis in the country, rifampicin has been used as a
common medication in treatment of the disease. This drug, however, has hepatotoxic effects.
Due to the high cost of living, impoverished Filipinos turn to herbal medicine as an alternative to
commercially produced drugs for many of their health complications. This study, therefore, will
not only make a valuable contribution to the growing number of studies devoted to
hepatotoxicity and hepatoprotection but will also add to the investigations currently being
undertaken to find medicinal plants with hepatoprotective functions. Results of the study may
confirm local information regarding the hepatoprotective potential of T. crispa. This may add to
the knowledge o the already established medicinal uses of T. crispa. Finally, the study could
pave the way to provide a much cheaper source of medicine of liver damage.
Scope and Limitations
This study assessed the hepatoprotective potentials of T. crispa on male Swiss Albino
mice. This was done by measuring the serum levels of ALT and bilirubin in the different
treatment groups. In addition, the free radical scavenging activity of the various treatment groups
was also compared. Histopathological analysis was also performed. The study utilized mice of
which died due to the toxic effects of rifampicin. This study is limited to the rifampicin induced
hepatotoxicity model. The extracts were derived from the stem and not from the other parts of
the plant.
REVIEW OF RELATED LITERATURE
Rifampicin-Induced Liver Damage
Rifampicin is a broad-spectrum antibiotic effective against both gram (+) and gram (-)
bacteria. It inhibits RNA synthesis by combining with RNA polymerase. It is active on tubercle
bacilli of lower metabolism and slower growth such as those persisting in the closed lesions of
lower O2 tension. Moreover, it is also useful in the treatment of leprosy.
Rifampicin (RIF) along with isoniazid (INH), pyrazinamide (PZA), and ethambutol is the
first-line combination therapy against tuberculosis. However, many studies have revealed the
hepatotoxic activity of rifampicin with significant increase in risk when given concomitantly
with isoniazid. In an experiment on male albino mice, combination of isoniazid with rifampicin
resulted in a higher activity of transaminase and alkaline phosphatases and a higher rate of
inhibition of biliary secretion and synthesis and excretion of bile acids, bilirubin, and cholesterol
with bile. Moreover, an increase was observed in the level of lipid peroxidation products of the
hepatocyte membranes in liver homogenates and blood. The increased hapatotoxicity of
isoniazid, was evident from a more pronounced decrease in the number of sulfhydryl groups
accompanied by an increase in the number of disulfide ones in the liver and blood. Potentiation
of isoniazid hepatotoxicity under the action of rifampicin was due to its inducing activity with
respect to the microsomal oxidation enzymes. According to Vavricka et. al. (2001), the
antibiotics rifamycin SV and rifampicin has been shown to interfere with hepatic organic anion
uptake systems in the liver with the inhibition of the organic anion transporting polypeptides or
OATPs. These polypeptides are responsible for the elimination of sulfobromophthalein (BSP)
and served as indicator of potent liver organic anion uptake system since it is a natural substrate
of the OATPs as well as bile salts, steroid conjugates, thyroid hormone and unconjugated
bilirubin among others.
According to one study, the antituberculosis therapy induces acute or chronic liver
damage in some individuals. Liver injury was characterized as being mild to moderate and the
type of injury associated was represented by pure cholestasis and hepatocanalicular lesions.
Other effects include rise in serum bilirubin and transaminase levels. Furthermore, rifampicin
may cause epigastric distress, CNS side effects, and immunological disturbances. It also imparts
a reddish-orange color to body fluids.
Some parallel studies have sought to test the protective effect of certain substances
against the hepatotoxicity of rifampicin and isoniazid as well. Oral treatment with the ethanol
extract of Hemidesmus indicus roots significantly prevented rifampicin and isoniazid-induced
hepatotoxicity in mice. Berberine compound from Berberis aristata was studied for its possible
antihepatotoxic action in rats. Pretreatment of animals with berberine prevented the
acetaminophen- or CCl4-induced rise in serum levels of alkaline phosphatase and transaminases,
suggestive of hepatoprotection. Post-treatment with three successive oral doses of berberine
reduced the hepatic damage induced by acetaminophen, while CCl4-induced hepatotoxicity was
not modified suggesting a selective curative effect against acetaminophen. Furthermore, the
hepatoprotective activity of N-acetylcysteine (NAC), a glutathione (GSH) precursor was
investigated in young Wistar rats. The oxidative hepatic injury in INH-RIF co-exposed animals
which showed histological lesions ranging from intralobular inflammation to patchy necrosis
was closely associated with significant decline of GSH and related thiols as well as with
compromised antioxidant enzyme system. The oxidative stress was furthered by increased lipid
peroxidation. The co-administration of NAC which supported the cellular antioxidant defense
mechanism, prevented the induction of oxidative stress.
The use of rifampicin in other diseases also manifested signs of hepatotoxicity. A case of
a patient with borderline tuberculoid Hansen’s disease (leprosy) who developed the
diaminodiphenylsulphone syndrome after multi-drug therapy comprising dapsone and rifampicin
demonstrated features consistent with drug-induced hepatitis, tubulo-interstitial nephritis and
myocarditis. In addition, rifampicin monotherapy is considered an effective second line therapy
for controlling pruritus in patients with chronic cholestatic liver disease. It is used as an antipruritic agent in the autoimmune cholestatic liver disease, primary biliary cirrhosis (PBC). The
patients experienced significant hepatitis or impairment of liver synthetic function.
Tinospora crispa
Tinospora crispa, known as Makabuhay plant in the Philippines is a climbing,
dioecious vine reaching a height of 4-10 meters. The stem is about 1 centimeter thick, somewhat
fleshy, with scattered protuberances. The leaves are thin, ovate, 6-12 centimeters long and 7-12
centimeters wide. The petiole is 3-6 centimeters long. Racemes are solitary or in pairs arising
from the axis of the leaves, pale green, and short-pedicelled. The fruit is 7-8 millimeters long
(Merrill 1974).
This plant is widely distributed in the Philippines usually flowering from March to
May. It also occurs in Malaya. (Merrill 1974).
Other local names of Tinospora crispa include paliaban, panauan, pangiauan,
pangiauban and taganagtagtua in the Visayas. It is also known as sangaunau in Baguio
(Quisumbing 1978).
The Filipinos and Malays in general regard this vine as a universal medicine. The plant
has long been used traditionally in the treatment of a number of varied diseases. It is commonly
prescribed as an aqueous extract in the treatment of stomach trouble, indigestion, and diarrhea. It
is also an effective remedy in the treatment of tropical ulcers. In powder form, it is prescribed in
fevers. A preparation with coconut oil is an effective cure for rheumatism and flatulence of
children. This preparation starts with chopping the stem into pieces of 1 or 2 inches long, and
placing them in a jar with coconut oil. The jar is put aside and not opened until a year has
elapsed. Prior to treatment, the preparation is first "cooked" under the sun. Other uses include the
decoction of the stem as an excellent vulnerary for itches, ordinary and cancerous wounds. It is
also used as a hot foot bath in the treatment of athlete's foot by boiling chopped 1 foot long
portion of the vine in 5 glasses of water for 15 minutes and a tub bath for scabies by boiling
chopped 1 meter long portion of the vine in 1 gallon of water for 15 minutes. Internally, it is used
as a tonic and antimalaria and externally, as a parasiticide (Quisumbing 1978).
More recent studies have verified the efficacy of Tinospora crispa for the treatment of
diabetes in animal models. Moreover, these studies have provided sufficient biochemical
evidence that support the traditional claims for the hypoglycemic effect of the plant. The
antihyperglycemic effect of T. crispa is not due to interference with intestinal glucose uptake but
rather is associated with increased insulin secretion (Noor et al. 1989, Noor and Ashcroft 1998).
In addition, current studies have also revealed the usefulness of T. crispa for the treatment of
malaria (Rahman et al. 1999) and various types of inflammation (Higashino et al. 1992).
Chemical studies on the plant report that the whole plant contains a bitter principle,
traces of an alkaloid, berberine and a glucoside. Further studies, however, have identified that the
bitter principle is glucosidal in nature. This bitter principle occurs as a white, crystalline powder
which is freely soluble in alcohol but very slightly soluble in other organic solvents, such as
ether and chloroform. It dissolves slowly in water. The bitter principle consists of 41.15%
carbon, 11.67% hydrogen and 47.18% oxygen (Marañon 1927).
More recently, two triterpenes have been isolated from the stems of T. crispa, namely,
cycloeucalenol and cycloucalenone. Both of these triterpenes possess cardiotonic activities
(Konghathip et al. 2002).
The hepatoprotective effect of T. cordifolia extract has been studied in carbon
tetrachloride-induced liver damage in rats. While acute damage was enhanced by prior exposure
to the drug, it proved effective in the prevention of fibrosis, and in stimulating regeneration in
hepatic tissue. Clinically, this drug has been tried as a therapeutic modality in rheumatoid
arthritis, jaundice and in diabetes (Singh et al. 1984).
Measurement of Serum Alanine Aminotransferase Activity
Transamination is the process in which an amino group is transferred from amino acid to
an -keto acid (Murray et al. 2000). The glutamic transaminase enzyme, serum alanine
aminotransferase (ALT), catalyzes the formation of glutamate and pyruvate from the reaction of
L-alanine and -ketoglutaric acid (Murray et al. 2000). Measuring blood levels of serum alanine
aminotransferase (ALT), is the most common way to test for liver disease. Blood enzyme levels
above a certain value indicate liver disease (ACP-ASIM 2002). Elevated serum ALT levels are
found in hepatitis cirrhosis and obstructive jaundice. Levels of ALT are only slightly elevated in
patients following a myocardial infarction (Biotron Diagnostics 2001).
Many methods and modifications have been proposed for the determination of serum
ALT.
The
various
methods
generally fall
into
two
categories:
colorimetric
and
spectrophotometric (Biotron Diagnostics 2001). The Reitman-Frankel colorimetric method
involves the addition of 2,4-dinitrophenyl hydrazine (DNPH) which reacts with pyruvate, the
product of transamination to form a hydrozone complex (Reitman and Frankel 1957).
Subsequent addition of sodium hydroxide breaks down the hydrozone complex and produces a
reddish coloration on the solution (Biotron Diagnostics 2001). The intensity of the color is
proportional to the enzymatic activity (Reitman and Frankel 1957).
With the advent of spectrophotometry, measurement of serum ALT has become much
more sensitive and accurate (Biotron Diagnostics 2001). The absorbance of the product from the
Reitman-Frankel colorimetric method is read at 550 nm. Increase in pyruvate production results
to a higher absorbance reading (Rafei 2001). However the presence of the co-substrate, -keto
glutarate in the assay contributes to the final absorbance reading such that the change in
absorbance is not linearly related to the theoretical value of pyruvate produced and hence the
enzyme activity (Rafei 2001).
Total Bilirubin Determination
Bilirubin is a breakdown product of hemoglobin. When hemoglobin is destroyed in the
body, the iron porphyrin or heme portion is catabolized by a complex enzyme system, heme
oxygenase. Hemin, which constitutes the ferric form of the oxidized iron, is reduced to heme
with NADPH, and with the aid of more NADPH, oxygen is added to the alpha-methenyl bridge
between pyrrole I and II of the porphyrin ring. Further addition of oxygen releases ferric iron and
produces carbon monoxide. Also splitting of the tetrapyrrole ring results in an equimolar quantity
of biliverdin IX-alpha. In mammals, a soluble enzyme called biliverdin reductase reduces the
methenyl bridge between pyrrole III and pyrrole IV to a methylene group to produce bilirubin
IX-a, a yellow pigment (Harper 2000).
Bilirubin metabolism occurs primarily in the liver. It is taken up by liver parenchymal
cells, conjugated in the smooth endoplasmic reticulum, and secreted into the bile (Harper 2000).
A low level of bilirubin (about 1 mg/dL) circulates throughout our bodies, too faint to be visible.
However, excessive levels of bilirubin cause the yellow pigment to be visible. This condition is
known as jaundice (Greene 1998). Jaundice, the discoloration of skin and sclera of the eye,
occurs when bilirubin accumulates in the blood at a level greater than approximately 2.5 mg/dL.
This is due to red blood cells being broken down too fast for the liver to process, liver diseases,
bile duct blockage, viral hepatitis, and liver scarring or cirrhosis (MedlinePlus Encyclopedia).
Drugs like allopurinol, anabolic steroids, diuretics, some antibiotics, antimalarials,
azathioprine, chlorpropamide, cholinergics, codeine, epinephrine, meperidine, methyldopa,
methotrexate, MAO inhibitors, morphine, nicotinic acid, oral contraceptives, phenothiazines,
quinidine, salicylates, steroids, sulfonamides, theophylline, and rifampicin cause a significant
rise on bilirubin levels (MedlinePlus Encyclopedia).
At very high levels of bilirubin in the bloodstream, permanent damage to certain areas of
the brain of newborn infants (kernicterus) occurs which causes a characteristic form of crippling
known as athetoid cerebral palsy (EncyMaster). There is also an increased risk for permanent
hearing loss, mental retardation, spastic quadriplegia, or even death (Greene 1998).
Conjugation is the process by which bilirubin is converted from a nonpolar form to a
polar one, which is readily excreted in the bile, by adding glucoronic acid molecules to it (Harper
2000). This conjugated bilirubin is called total bilirubin.
On the other hand, unconjugated bilirubin is called direct bilirubin. When this is elevated,
the cause is usually outside the liver, typically gallstones.
Total bilirubin testing measures the amount of bilirubin in the bloodstream. Normal total
bilirubin levels range from 0.20 mg/dL to 1.5 mg/dL.
Measurement of Antioxidant Activity
The ability of several plant extracts such as Solanum hainanense (Phuc 1998) and ‘Banzhi-lian’ (Lin 1997) to prevent drug-induced hepatotoxicity has been correlated with their free
radical scavenging property. Antioxidative activity (AOA) is conventionally used to indicate the
ability of an antioxidant to scavenge some radicals. Free radical colorimetry relies on the
reaction of an antioxidant with the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH,
C18H12N5O6, MW= 394.33 g/mol) dissolved in methanol (Buijnsterns 2001). DPPH is a
relatively stable paramagnetic free radical that accepts electrons or H+ radicals to become stable
diamagnetic molecule (Brand-Williams et al. 1995). The reduction of DPPH by an antioxidant
results in the formation of purple-blue colored solution. The activity is measured through
conventional spectrophotometry at 514 nm (Buijnsterns 2001).
MATERIALS AND METHODS
Preparation of extract. Tinospora crispa (Makabuhay) stems were procured from a
public market in Quiapo. These were homogenized in absolute ethanol and left to macerate for 3
days at 25ºC. The mixture was then filtered using filter paper and the resulting liquid was
concentrated under reduced pressure at 40ºC. The extract was dissolved using distilled water to
concentrations of 10 g/L and 20 g/L.
Test animals. A single strain of albino mice were maintained under standard husbandry
conditions and acclimatized for 10 days. The rats are given standard laboratory feed and water ad
libitum. Groups consisted of 7 rats each. For all tests, a double blind procedure was followed
wherein neither the data observer nor the data collector were aware of the particular treatment of
the mice.
Rifampicin-induced hepatotoxicity. The control group received distilled water 0.2 mL
four times at 12h intervals and 0.2 mL distilled water 30 mins after the first administration of
distilled water.
The rifampicin group received 0.2 mL distilled water four times at 12h intervals and a
single 0.2 mL dose of rifampicin suspension (400 mg/kg) 30 mins after the first administration of
distilled water.
The extract low group received crude extract solution 0.2 mL (100 mg/kg) four times at
12h intervals and a single 0.2 mL dose of rifampicin suspension (400 mg/kg) 30 mins after the
first dose of the crude extract solution.
The extract high group received crude extract solution (200 mg/kg) four times at 12h
intervals and a single 0.2 mL dose of rifampicin suspension (400 mg/kg) 30 mins after the first
dose of the crude extract solution.
The silymarin group received silymarin solution (100 mg/kg) four times at 12h intervals
and a single 0.2 mL dose of rifampicin suspension (400 mg/kg) 30 mins after the first dose of
silymarin solution.
Forty-eight hours after rifampicin administration, blood was collected using the cardiac
extraction method from all groups. The blood samples were placed in calcium-EDTA tubes at
room temperature. Plasma was separated by centrifugation at 2500 rpm at 37° for 15 mins and
analyzed for ALT (SGPT) and direct bilirubin.
Total Bilirubin (Jendrassik method, Randox kit). The following solutions were
pipetted into a cuvette (mL):
Blank
Sample
Sulfanilic acid
0.20
0.20
Sodium nitrite
-
0.05 ml
Caffeine
1.00
1.00
Sample
0.20
0.20
The contents were mixed and allowed to stand for 10 mins at 22ºC. To each cuvette, 1
mL of the tartrate solution was added, and the solutions were mixed and allowed to stand for 5
mins at 22ºC and absorbance of the sample was read against the blank at 578 nm. The total
bilirubin was calculated using the following equations:
Total Bilirubin (umol/L)= 185 x Abs or Total Bilirubin (mg/dL)= 10.8 x Abs
ALT/SGPT (Young-Karmen method, Randox kit). L-alanine was reconstituted with a
lactate dehydrogenase, NADH and -ketoglutarate solution. Into a cuvette, 1 mL of the
reconstituted solution and 0.1mL of the sample was pipetted and mixed. Initial absorbance at 340
nm was read and again after 1, 2 and 3 mins. The activity of alanine aminotransferase present in
the sample was calculated using the following equation:
U/L= 1746 x ΔA 340 nm/min
DPPH Scavenging activity. A 1 mg/mL DPPH solution, a 10 mg/mL T. crispa solution,
a 10 mg/mL silymarin solution, and a 1 µg/mL ascorbic acid solution were prepared using
methanol as a solvent. In a 10 mL volumetric flask, 0.5 mL of the DPPH solution and 1, 2, 3, 4,
5, and 6 mL of the test solutions (either silymarin and T. crispa extract) were mixed and diluted
to 10 mL using methanol. The procedure was repeated for ascorbic acid using 0.25, 0.5, 1, 2, 3,
and 4 mL volumes. Twenty minutes were allowed to elapse before the spectrophotometry
readings were taken. Initial absorbance (without sample) and final absorbance (with sample) was
read at 517 nm using methanol as blank. The EC50 or effective concentration which reduces the
absorbance by 50% using linear regression was determined using ascorbic acid as control.
Statistical analysis. The results of the serum alanine aminotransferase and bilirubin
assays were subjected to continuous summary descriptives and comparatives analyses since the
sample size was insufficient to analyze using analysis of variance.
RESULTS
The table reflects the number of deaths after 48 hours of rifampicin treatment. The
silymarin and extract treated groups showed higher mortality compared to rifampicin alone.
Table 1. Number of deaths observed per treatment group following administration of
rifampicin
Treatment
Doses
Deaths out of 7
Control
0
Rifampicin
400 mg/kg
5
Ethanolic Extract
100 mg/kg
7
Ethanolic Extract
200 mg/kg
5
Silymarin
100 mg/kg
5
As shown in the table and figures below, 48 hours after intoxication with a single dose of
rifampicin (400 mg/kg) an increase in serum ALT was observed relative to the control (without
rifampicin). The treatment with the crude ethanolic extract of T. crispa (200 mg/kg) induced a
decrease of serum ALT levels greater than that of silymarin (100 mg/kg). The difference was not
statistically significant (p=0.10).
The concentration of bilirubin in the serum was also markedly elevated when rifampicin
was administered alone, but with T. crispa the increase was suppressed but at a lesser extent
compared to silymarin. The results for this test were not statistically significant (p=0.10).
Table 2: Bilirubin serum concentrations
Trial
Bilirubin (mg/dL)
Control
Rifampicin
1
7.95
15.29
2
6.31
105.41
3
10.97
4
21.51
5
9.16
6
7.60
7
24.71
Average
12.60
60.35
Standard deviation 7.38
63.72
Silymarin
10.37
1.22
Extract
17.88
17.11
5.79
6.47
17.50
0.55
Table 3: Alanine Aminotransferase serum concentrations
Trial
Alanine Aminotransferase (U/I)
Control
Rifampicin Silymarin
1
146.66
290.53
2
205.33
108.95
10.13
3
22.35
4
0.00
5
11.17
6
5.59
7
12.57
Average
42.84
127.81
150.33
Standard Deviation 79.96
26.67
198.28
Extract
29.33
12.57
20.95
11.85
Figure 1: Comparison of ALT serum concentrations in the different treatment groups
400
350
300
Serum ALT, U/L
250
200
150
100
50
0
Control
Rifampicin
Silymarin
Extract
-50
-100
The table and figure show the scavenger activity and their correlation coefficients.
Ascorbic acid showed the maximum activity, followed by silymarin and the crude extract. The
scavenger activity shown by T. crispa is comparable to silymarin, however both possess only
about 1/200 the antioxidant power of ascorbic acid. All the samples tested were at a
concentration of 25 ug/mL of the reactive medium, DPPH.
Figure 2: Comparison of Bilirubin serum concentrations in the different treatment groups
140
120
total bilirubin, mg/dl
100
80
60
40
20
0
Control
Rifampicin
Silymarin
Extract
-20
Table 4: Percent Scavenging Activity of the Different Treatment Groups
Samples
Tinospora crispa extract
Silymarin
Acorbic acid
Effective Concentration
5.9 x 10-2
2.00 x 10-1
2.92 x 10-4
% Scavenging Activity
0.50
0.15
100
Fig. 3 Histopathological Findings of Mice Liver in Various Treatments
Rifampicin (400x)
Control (400x)
Extract, 200 mg/mL (400x)
Silymarin (100x)
DISCUSSION
In the present study, serum ALT and bilirubin levels were measured as indicators of
hepatotoxicity. Elevated levels of serum enzymes indicate cellular leakage and loss of functional
integrity of the liver cell membrane (Phuc et al. 1998).
Results show that mice treated with rifampicin alone showed highly significant increase
in serum ALT and bilirubin. This result is consistent with previous studies concerning the
harmful effects of this drug. Several mechanisms have been proposed regarding the role of
rifampicin in inducing liver damage. rifampicin is converted to its active metabolite 25desacetylrifampin, which in turn reduces drug-metabolizing enzymes and actively and
specifically binds to RNA polymerases thereby inhibiting nucleic acid and protein synthesis
(Rao, et al. 1998). Also the active metabolite can also induce liver injury when it is converted to
free radicals via liver enzymes such as cytochrome P450 which would cause lipid peroxidation,
disrupting the integrity of the hepatocellular membrane (Lin et al. 1997).
On the other hand, subjects treated with crude extracts of T. crispa (200 mg/kg) had
lower levels of serum ALT compared to those treated with rifampicin and Silymarin. Also, these
subjects exhibited reduced levels of bilirubin compared to those treated with rifampicin alone.
These results suggest that a hepatoprotective activity may be present in the T. crispa extracts.
Since rifampicin induced toxicity relies on cytochrome P450 to produce reactive free
radical metabolites, the hepatoprotective actions of T. crispa ethanolic extracts may be due to
inhibition of cytochrome P450, prevention of lipid peroxidation and stabilizing hepatocellular
membrane. Results of the experiment indicate that crude extract of T. crispa possesses only
about 1/200th the antioxidant activity of ascorbic acid but is three times more potent than
silymarin. The negligible scavenger activity T.crispa suggests that its hepatoprotective
mechanism may not be due to its ability to directly combine with free radicals but other
mechanisms such as inhibition of radical formation or membrane stabilization (Lin et. al. 1997).
Histopathologic examination of the liver of rifampicin-treated group revealed lipid vacuoles
within hepatocytes. The lipid accumulates when lipoprotein transport is disrupted and/or when
fatty acids accumulate. rifampicin is a hepatotoxin that interferes with mitochondrial and
microsomal function in hepatocytes, leading to an accumulation of lipid (Lin et al. 1997). This
was not evident in the control, silymarin and extract-treated groups.
These results, however, are not statistically significant due to the high mortality among
the test mice brought about by the toxic side effects of rifampicin. Symptoms, manifested twelve
hours after administration of rifampicin include red-orange discoloration in the urine and feces
and tremors in the subjects. Such side effects were also observed in previous studies (Skakun and
Shman’ko 1985).
In addition, the researchers were not able to determine if the crude extract or silymarin
alone was toxic. Further studies, however, may focus on this matter by determining the LD50 of
both the crude extract or silymarin.
RECOMMENDATIONS
The researchers recommend that further studies be made regarding the hepatoprotective
properties of Tinospora crispa using a larger sample size and various test dosages that are to be
administered to the test subjects. Different extraction procedures and solvents may also be
utilized to further track down the possible active component of Tinospora crispa involved in its
role in hepatoprotection. Other hepatotoxicity induced models may also be explored such as
CCl4, Trinitrotoluene, and Paracetamol in testing the plant extract. Enzymatic assays may also be
performed to further investigate the role of cytochrome P450 systems in free radical formation.
CONCLUSIONS
Tinospora crispa ethanolic extract at 200 mg/kg possesses hepatoprotective activity
against rifampicin-induced toxicity. Its activity is comparable to silymarin, a known
hepatoprotective agent.
Also, since the ethanolic extract posseses only about 1/200 the antioxidant activity of
ascorbic acid, its hepatoprotective activity cannot be correlated with its ability to scavenge free
radicals. Thus, further studies on its mechanism of action are warranted, as well as isolation of
compounds responsible for the activity.
LITERATURE CITED
American College of Physicians-Annals of Internal Medicine (ACP-ASIM). 2002. Should Blood
Enzyme Levels for Liver Disease Be Changed? http://www.acponline.org/college/
pressroom/tipsheets/02jul02.htm.
Attri S., S. V. Rana, et al. 2000. Isoniazid- and rifampicin-induced oxidative hepatic injury—
protection by N-acetylcysteine. Hum. Exp. Toxicol. 19: 517-522.
Biotron
Diagnostics
2001.
Alanine
Aminotransferase
(ALT)
Reagent
Set.
http://www.biotrondiagnostics.com/ALT_SGPT_REAGENT_COLORIMETRIC_ENDP
OINT_METHOD.htm.
Brand-Williams, W., M. E. Cuvelier, and C. Berset. 1995. Use of a free radical method to
evaluate antioxidant activity. Lebensm. Wiss. Technol. 28: 25-30.
Buijnsters, M., D. Bicanic, M. Chirtoc, M. C. Nicoli and Y. Min-Kuo. 2001. Evaluation of antioxidative activity of some antioxidants by means of a combined optothermal window and
a DPPH free-radical colorimetry. Anal. Sci. 17: s544-s546.
Chan, E. D. and M. D. Iseman. 2002. Current medical treatment for tuberculosis. BMJ 325:
1281-1286.
De Souza, A. F., A. de Oliveira e Silva, J. Baldi, T. N. de Souza, and P. M. Rizzo. 1996. Hepatic
functional changes induced by the combined use of isoniazid, pyrazinamide, and
rifampicin in the treatment of pulmonary tuberculosis. Arq. Gastroenterol. 33: 194-200.
Easton, A. 1998. Tuberculosis controls in Philippines have failed so far. BMJ 317: 557.
Guo J. T., H. L. Lee, S. S. Chiang, F. I. Lin, and C. Y. Chan. 2001. Antioxidant properties of the
extracts from different parts of Broccoli in Taiwan. J. Food Drug Anal. 9: 96-101.
Higashino, H., A. Suzuki, Y. Tanaka, and K. Pootakham. 1992. Inhibitory effects of Siamese
Tinospora crispa extracts on the carrageenin-induced foot pad edema in rats. Nippon
Yakurigaku Zasshi 100: 339-344.
Janbaz, K. H. and A. H. Gilani. 2000. Studies on preventive and curative effects of berberine on
chemical-induced hepatotoxicity in rodents. Fitoterapia 71: 25-33.
Kagaya, N., M. Kawase, H. Maeda, Y. Tagawa, H. Nagashima, H. Ohmori, and K. Yagi. 2002.
Enchancing effect of zinc on hepatoprotectivity of epigallocatechin gallate in isolated rat
hepatocytes. Biol. Pharm. Bull. 25: 1156-1160.
Kanai, K. 1991. Introduction to tuberculosis and mycobacteria. Tokyo, Japan: SEAMIC/IMF.
p.15.
Kongkathip, N., P. Dhumma-upakorn, B. Kongkathip, K. Chawananoraset, P. Sangchomkaeo,
and S. Hatthakitpanichakul. 2002. Study on cardiac contractility of cycloeucalenol and
cycloeucalenone isolated from Tinospora crispa. J. Ethnopharmacol. 83: 95-99.
Lau G. 1995. A fatal case of drug-induced multi-organ damage in a patient with Hansen’s
disease: dapsone syndrome or rifampicin toxicity? Forensic Sci. Int. 73: 109-115.
Lin, C., Shieh, D. and Yen, M. 1997. Hepatoprotective Effect of the Fractions of Ban-Zhi-Lian
on Experimental Liver Injuries in Rats. J. Ethnopharmacology. 56: 193-200.
Marañon, J. 1927. The bitter principle of makabuhay, Tinospora rumphii Boerlage. Philipp. J.
Sci. 33: 357-361.
Merrill, E. D. 1912. Flora of Manila. Manila: Bureau of Printing. p204.
Murray et al. 2000. Harper’s Biochemistry. Stanford. Appleton and Lange.
Murthy, M. S. R. and M. Srinivasan. 1993. Hepatoprotective effect of Tephrosia purpurea in
experimental animals. Indian J. Pharmacol. 25: 34-36.
Noor H. and S. J. Ashcroft. 1998. Pharmacological characterization of the antihyperglycaemic
properties of Tinospora crispa extract. J. Ethnopharmacol. 62: 7-13.
Noor, H., P. Hammonds, R. Sutton, and S. J. Ashcroft. 1989. The hypoglycaemic and
insulinotropic activity of Tinospora crispa: studies with human and rat islets and HITT15 B cells. Diabetologia 32: 354-359.
Prabakan, M., R. Anandan, and T. Devaki. 2000. Protective effect of Hemidesmus indicus
against rifampicin and isoniazid-induced hepatotoxicity in rats. Fitoterapia 71: 55-59.
Prince M. I., A. D. Burt, and D. E. Jones. 2002. Hepatitis and liver dysfunction with rifampicin
therapy for pruritus in primary biliary cirrhosis. Gut 50: 436-439.
Quisumbing, E. 1978. Medicinal Plants of the Philippines. Quezon City: Katha Publishing. p300301.
Rafei, U. M. 2001. Transaminases - Colorimetric end-point method. http://w3.whosea.org/micro/
10.htm.
Rahman, N., T. Furuta, S. Kojima, K. Takane, and M. A. Mohd. 1999. Antimalarial activity of
extracts of Malaysian medicinal plants. J. Ethnopharmacol. 64: 249-254.
Rao, K.S. and Mishra, S. H. 1998. Antihepatotoxic Activity of Monomethyl Fumarate Isolated
from Fumaria indica. J. Ethnopharmacology. 60: 207-213.
Reitman, S. and S. Frankel. 1957. A colorimetric method for the determination of glutamic
oxaloacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. 28: 56-63.
Saraswathy, S. D., V. Suja, P. Gurumurthy, and C. S. S. Devi. 1998. Effect of Liv.100 against
antitubercular drugs (isoniazid, rifampicin, and pyrazinamide) induced hepatotoxicity in
rats. Indian J. Pharmacol. 30: 233-238.
Singh, B., M. L. Sharma, D. K. Gupta, C. K. Atal, and R. K. Arya. 1984. Protective effect of
Tinospora cordifolia Miers on carbon tetrachloride induced hepatotoxicity. Indian J.
Pharmacol. 16: 139-142.
Skakun, N. P. and V. V. Shman’ko. 1985. Synergistic effect of rifampicin on hepatotoxicity of
isoniazid. Antibiot. Med. Biotekhnol. 30: 185-189.
Vavricka, S. R., J. V. Montfoort, H. R. Ha, P. J. Meier, and K. Fattinger. 2002. Interactions of
rifamycin SV and rifampicin with organic anion uptake systems of the human liver.
Hepatology 36: 164-172.
Visen, P. K. S., B. Saraswat, G. K. Patnaik, D. P. Agarwal, and B. N. Dhawan. 1996. Protective
activity of picroliv isolated from Picrorhiza kurrooa against ethanol toxicity in isolated
rat hepatocytes. Indian J. Pharmacol. 28: 98-101.
Wallerstein, C. 1999. Tuberculosis ravages Philippine slums. BMJ 319: 402.
Zhang, G. L., Y. H. Wang, W. Ni, H. L. Teng, and Z. B. Lin. 2002. Hepatoprotective role of
Ganoderma lucidum polysaccharide against BCG-induced immune liver injury in mice.
World J. Gastroenterol. 8: 728-733.