Sirrapac powder is a milled root bark of Capparis erythrocapus (CE)
which belongs to the family Cappareceae, previously known as Cdapparidaceae.
The Capparis genus comprises some 650 sp., of small trees and shrubs
in 30 genera which are found principally in tropical and warm temperate regions.
However, few species may be cultivated (Hutchinson, 1967).
Members of this family contain thioglucosides also known as glucosinolates
which release isothiocynates (mustard oil) when the plant is damaged. The plants
yield methyl isothiocynate from methyl glucosinolate otherwise known as glucocapparin.
This mustard oil has skin irritant property and may also have contact allergenic
property (Mitchell and Jordan, 1974; Richter,
The seeds and the juice of pounded leaves of CE are used against child convulsive
fever and the pounded root is used in severe abscess whilst vapour from the
pounded root is used for the treatment of inflammation of the connective tissue
of the eye in Tanzania and India (Hedberg et al.,
1982). However, powdered root of CE has been processed and used for the
management of arthritis over 25 years at the Center for Scientific Research
in to Plant Medicine (CSRPM), Mampong, Akuapem, Ghana.
Despite its long period of usage in the management of arthritis, its safety
in chronic use has not been pre-clinically evaluated. The aim of this study
is to scientifically evaluate the safety of powdered CE in male Sprague-Dawley
rats on chronic administration to validate its clinical use.
MATERIALS AND METHODS
Reagents and chemicals: Test kits; aspartate aminotransferase (AST),
alanine aminotransferase (ALT), gamma-glutamyl-transferase (-GT),
bilirubin (Direct and Total), Albumin, Creatinine, Urea, Creatinine-kinase (CK-MB)
were purchased from Cypress Diagnostics (Belgium). Urine test strips (UroColorTM
10) were supplied by Standard Diagnostics Inc., Korea and pentobarbital was
obtained from Sigma Chemical Co. (St. Louis, Mo, USA). All other chemicals were
purchased in the purest form available from British Drug Houses (BDH) Ltd.,
Animals: Male Sprague-Dawley rats (150-200 g) were obtained from the
Animal Unit of Centre for Scientific Research into Plant Medicine, Mampong-Akuapem,
in the Eastern Region of Ghana. The animals were fed on powered feed obtained
from Ghana Agro Food Company (GAFCO) Tema, Ghana. The research was conducted
in accordance with the internationally accepted principles for laboratory animal
use and care as found in the US guidelines (NIH Publication No. 85-23, revised
Sirrapac powder preparation: Air dried root bark (20 kg) of CE was oven-dried
at 65°C for 10 h. This was milled into fine powder and re-heated at 37°C
for 3 h after which it was bagged at room temperature under aseptic conditions.
Treatment of animals: Three groups of six male rats were kept in 18
separate metabolic cages. Group 1 was kept as a control and received normal
chow ad libitum for 6 months. Groups 2 and 3 were treated with 18.0
mg kg-1 b.wt. day-1 (normal human dose) and 180.0 mg kg-1
b.wt. day-1 (10x the normal dose) of CE powder, respectively mixed
with their feed for 6 months. All animals received sterilized distilled water
ad libitum. The animals in the metabolic cages of each group were weighed
on day zero (baseline) and fortnightly thereafter.
Blood Sampling and blood clotting time: Blood samples of rats in each
treatment group were obtained by tail bleeding at baseline and at 3 and 6 months
into Eppendorf tubes and blood clotting time determined and then placed on ice.
These were centrifuged at 4000xg for 5 min (Denley BS 400, England) and the
supernatant (serum) obtained, stored at -40°C for biochemical analysis.
Another set of blood samples were collected into tubes already coated with tri-sodium
citrate (Westergreen ESR, UK) for haematological analysis within 24 h.
Serum biochemical and haematological analyses: Serum ALT, AST, GGT,
total bilirubin, direct bilirubin, albumin, CK-MB, creatinine and urea of control
and CE-treated rats were determined using protocols from cypress diagnostic
kits (Belgium) with a semi-autoanalyzer (Photometer 4040; Robert Riele G and
Cole-2000, Germany). In haematological analysis, Red Blood Cells (RBC), White
Blood Cells (WBC), haematocrit (HCT), haemoglobin (Hb) and platelet (PLT), Mean
Cell Volume (MCV), Mean Platelet Volume (MPV) and thrombocytes (PCT) of control
and CE-treated rats were determined with Haema-screen 10 (Hospitex Diagnostics,
Italy) in accordance with standard protocol. The system is computerized to automatically
determine and display the haematological data for each blood sample.
Urinalysis: Urine samples of control and CE-treated rats, produced as
a result of involuntary discharges, were collected on clean ceramic tiles at
baseline and after 3 and 6 months of treatment. Urine glucose, bilirubin, ketones,
specific gravity, pH, proteins urobilinogen, nitrate, blood and leukocytes were
determined using urine reagent strips (UroColorTM 10, Standard Diagnostic
Histology: At the end of the six month treatment period (termination),
two rats from the control and CE-treated groups were euthanized by cervical
dislocation and the heart, lungs, liver, kidney and spleen were excised and
weighed. These organs, with the exception of the spleen, were then fixed in
10% formaldehyde and dehydrated with increasing concentration of ethanol (85-100%).
The tissues were cleared with chloroform and impregnated with paraffin wax.
Sections were cut, stained with haematoxylin and eosin and mounted on slides
for light microscopic examinations. Tissue sections of the four organs of two
other rats at baseline were also prepared (Baker and Silverstone,
Pentobarbital-induced sleeping time: In vivo drug interaction
was assessed by the pentobarbital-induced sleeping time. Control and CE-treated
animals were each given intraperitoneal injections of pentobarbital. All animals
were placed on their backs after they had lost consciousness and the time between
the animals falling asleep and regaining consciousness was recorded (Nyarko
et al., 1999).
Statistical analysis: One-way analysis of variance (ANOVA) and independent
sample t-test was conducted between control and tests to determine statistical
significance. The 5% level of probability was used as criterion of significance
in all instances. All statistical tests were performed with spss statistical
software version 10.0.
Body and organ weights: The percentage change in mean body weight with
period of treatment and organ wet weights at termination of treatment in control
and CE-treated animals are shown in Fig. 1 and Table
|| Mean organs wet weight at termination of treatment with CE
|Results are Means±SEM for N = 6, aFor treatment
||Percentage changes in body weight of control and CE-treated
Sprague-Dawley rats with duration of treatment. Each point represents Mean±SEM
(N = 6). aValue significantly different from control, p<0.05.
For details of treatment regimen
The results showed a significant reduction (p<0.05) in the change in body
weight of CE-treated animals compared to control, although there was no significant
difference (p>0.05) in body weight changes between CE-treated animals with
time. The animals on CE appeared leaner than the controls although the length
of their hind legs and femur as well as food intake of all treatment groups
were similar (results not shown). There were insignificant changes (p>0.05)
in the organ weights, expressed as percentage of body weight, at termination
between control and CE-treated animals.
Serum biochemistry and urinalysis: The effects of chronic administration
of CE to rats on selected serum biochemical and urine parameters at termination
of treatment are shown in Table 2 and 3.
The serum biochemical indices indicate that CE caused significant dose-dependent
reductions (p<0.05) in the levels of ALT at the 18 mg kg-1 (21%)
and 180 mg kg-1 (35%) dose levels. However, there were insignificant
(p>0.05) changes in serum GGT, AST, total and direct bilirubin, albumin,
urea, creatinine and CK-MB in the CE-treated animals when compared to controls.
|| Serum biochemical analysis at termination of treatment with
|Results are Means±SEM for N = 6, *Values significantly
different from control, p<0.05, aFor treatment regimen
|| Effect on indices of urinalysis at termination of treatment
|Figures represent means of 6 determinations, -: Absent, N:
Normal, ±: Trace and +: Positive, aFor treatment regimen
|| Effect on hematological indices at termination of treatment
|Results are Means±SEM for N = 6, aFor treatment
Dipstick urinalysis data indicate that there were no significant differences
in the levels of urine haemoglobin, blood, bilirubin, urobilinogen, proteins,
ketones, glucose, nitrite, leucocytes, pH and specific gravity between control
and CE-treated animals. The results were similar to baseline values (results
Haematological studies: The effect of CE on certain haematological indices
at termination of treatment is shown (Table 4). The results
show that there were no significant differences (p>0.05) in all parameters
measured between control and CE-treated animals. These results were similar
to that at baseline (results not shown).
Histological studies: The effects of CE on the histopathology of the
liver, kidney, lung and heart tissues at termination of treatment are shown
in Fig. 2-5.
||Histological appearance of the liver of control (a) Animals
at termination showing (1) normal hepatocytes and (2) interstitial spaces
and animals treated with normal dose of CE-18 mg kg-1, (b) 10x
the normal CE dose-180 mg kg-1 and (c) At termination, showing
no differences in appearance of hepatocytes (1) and interstitial spaces
(2) compared to control. Morphology of control liver at termination was
not different from that at baseline. For details of treatment regimen, Magnification:
||Histological appearance of the kidney of control animals at
termination (a) Showing (1) normal tubular and (2) glomerular areas and
animals treated with normal dose of CE-18 mg kg-1, (b) 10x the
normal CE dose-180 mg kg-1 and (c) At termination, showing no
differences in tubular (1) and glomerular (2) areas compared to control.
Morphology of control kidney at termination was not different from that
at baseline. For details of treatment regimen, Magnification: x132
||Histological appearance of the lung of control animals at
termination (a) Showing (1) normal alveolar areas and (2) Clara cells lining
a normal bronchiolar epithelial wall and (3) animals treated with normal
dose of CE-18 mg kg-1, (b) 10x the normal CE dose-180 mg kg-1
and (c) At termination, showing slight inflammatory response at alveolar
areas (4) and slight Clara cell hyperplasia (2) without changes in the bronchiolar
epithelial lining (3). Morphology of control lung at termination was not
different from that at baseline. For details of treatment regimen, Magnification:
||Histological appearance of the heart of control animals (a)
Animals treated with normal dose of CE-18 mg kg-1, (b) 10x the
normal CE dose-180 mg kg-1 and (c) At termination, showing no
differences in morphology of cardiac tissue. Morphology of control heart
tissue at termination was not different from that at baseline. For details
of treatment regimen, Magnification: x132
||Effect of pre-treatment of rats with CE on pentobarbital-induced
sleeping time. Values are Means±SEM (N = 6), For details of treatment
||Effect on blood clotting time at termination of treatment
of rats with CE. Results are Means±SEM (N = 6). For details of treatment
Results showed that CE did not affect the morphology of the liver, kidney and
heart tissues (Fig. 2a-c, 3a-c,
5a-c). However, lungs of CE-treated animals
showed slight but insignificant inflammatory response in alveolar areas and
Clara cell hyperplasia without the thickening of alveolar septa and bronchiolar
epithelial wall (Fig. 4a-c).
Pentobarbital-induced sleeping and blood clotting times: The effects
of pre-treatment with CE on pentobarbital-induced sleeping time and blood clotting
time are shown in Fig. 6 and 7. Results
indicate that there were no significant differences (p>0.05) in pentobarbital-induced
sleeping time and blood clotting time between control and CE-treated animals.
DISCUSSION AND CONCLUSION
Arthritis is a major debilitating disease that afflicts many in developed and
developing countries. The crippling effects of the disease may lead to the loss
of many man h to a nation as well as huge costs in medical expenses in view
of the fact that some of the most effective anti-inflammatory allopathic drugs
are quite expensive. It is, therefore, of importance that new herbal alternatives
of equivalent safety and efficacy be developed to meet the needs of sufferers
of arthritis in developing countries who are among about 70% of the population
who depend on traditional medicine for their primary health care needs.
In this study, we have evaluated the safety of the root bark of C. erythrocarpus
(CE), used at the CSRPM with ethnomedical evidence for its use in the management
of arthritis, in Sprague-Dawley rats. To this end we evaluated the effects of
CE on the function and/or morphology of key organs that are essential for the
normal function of the body, namely the liver, kidney, lung and heart as well
as the bone marrow.
Results from the liver, kidney and cardiac function tests (Table
2) indicates that there was no effect of CE on any of the indices measured
when compared to controls with the exception of serum ALT levels which was significantly
reduced in a dose-dependent fashion, an indication that CE or its metabolite(s)
inhibits ALT activity. It is known that damage to these organs will lead to
the release of enzymes (e.g., AST, ALT and ALP) into the blood or the reduction
in synthesis (e.g., albumin) or clearance of certain substances (urea and creatinine)
from blood leading to the elevation or reduction of their levels in the blood
(Gaw et al., 1998). This, therefore, indicates
that these organs were not adversely affected by CE treatment. These findings
are supported by the histological data which shows that the morphology of these
organs was not adversely affected by CE (Fig. 2, 3
and 5). Furthermore, urinalysis data support absence of renal
dysfunction or damage.
The lung is the most sensitive organ that can easily be adversely affected
by toxic insult by xenobiotics. Damage to the lungs by pneumotoxicants may lead
to massive inflammatory response and consolidation in the alveolar areas, alveolar
septa thickening, severe Clara cell hyperplasia or hypertrophy and thickening
of the bronchiolar epithelial lining among others (Krijgsheld
et al., 1983; Okine et al., 1986, 2005).
This may lead to fibrosis as a result of collagen deposition and the lung becoming
hypertrophied or oedematous. CE treatment of the animals did not show such marked
changes in the alveolar and bronchiolar areas as seen with known pneumotoxicants,
suggesting that the lung was not adversely affected by CE.
The bone marrow is the source of the biosynthesis and release of blood components
such as RBCs, WBCs, platelets and others into the blood. Reduction in blood
cells may be due to damage or suppression of the bone marrow or direct damage
to blood cells. Some chemicals including drugs are known to suppress the bone
marrow leading to the impairment of erythropoeisis (Lewis
et al., 1997). That CE did not affect any of the haematological indices
suggests that it did not suppress or damage the bone marrow or directly affected
the blood cells. The lack of effect of CE on blood clotting time suggests that
it does not affect vitamin K levels or inhibit the synthesis of blood clotting
proteins. The latter is supported by the fact that serum albumin levels were
not affected by CE treatment, suggesting that hepatic protein biosynthetic activity
was not impaired.
One of the major problems in co-joint administration of drugs is the issue
of drug interactions. Herb-drug interactions are known to be caused by phytochemicals
which are capable of altering CYP activity (Venkataramanan
et al., 2006). For example, hyperforin a chemical constituent with
antidepressant properties from the plant Hypericum perforatum L., has
a strong affinity for Steroid Xenobiotic Receptor (Nathan,
1999). Its binding to the receptor promotes the expression of CYP3A4 gene,
thus inducing the enzyme in the liver and intestines resulting in enhanced reduction
in the levels of other compounds, whose clearance is mediated by CYP3A4 (Moore
et al., 2000; Barone et al., 2001).
Pre-treatment of animals with CE did not affect the metabolism and pharmacological
effect of pentobarbital expressed as pentobarbital-induced sleeping time. This
indicates that CE does not modulate CYP isozymes responsible for the metabolism
of pentobarbital. However, the ability of CE to modulate other CYP isozymes
responsible for the metabolism of other compounds cannot be overruled.
The most significant finding of this study is the fact that CE significantly
inhibited the weight gain of the CE-treated animals. This observation cannot
be easily explained since we did not observe any differences in the eating and
drinking patterns between CE-treated and control animals. Indeed, the food and
drink intakes were similar. However, we observed that the CE-treated animals
were leaner although the length of their hind legs and femur were similar to
the control (results not shown). It is possible that CE may affect the metabolism
and deposition of fat in the animals, thus making them leaner.
It may be concluded that CE does not cause any organ specific toxicity, but
its ability to reduce weight gain of animals makes it a candidate plant material
for weight loss in obese persons and warrants further studies.
The authors will like to express their sincerest gratitude to all the laboratory
staff of the Animal Unit of CSRPM, Mampong, Akwapim, Ghana and to Mr. Alex Dodoo,
a technical staff at the Electron Microscopy Unit, Noguchi Memorial Institute
for Medical Research, University of Ghana, Legon, Ghana.