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Asian Journal of Biological and Life Sciences
Original Research
Effect of short and long term intake of traditional aphrodisiac
Cantharis Q on liver of male albino rat
Roshan Clarence D'Souza*, Raghunandan P. Athalye1
* Department of Zoology, Sophia College for Women, Mumbai-400 026, India.
1 Department of Zoology, B. N. Bandodkar College of Science, Thane-400 601, India.
E-mail : [email protected]
Submitted : 12.05.2014
Contact No : +91 98674 08921
Accepted : 25.06.2014
Published : 30.08.2014
Cantharis Q has a long history in both folk and traditional medicine being used as an aphrodisiac. It is a crude alcoholic extract of blister beetle Lytta
vesicatoria commonly known as Spanish fly having cantharidin, a venomous substance present in it. The aim of the present study was to evaluate the
clinical application of Cantharis Q by assessing its effect on the vital organ, liver, for a short and long period in male albino rat. The recovery study was
also carried out in order to establish the reversibility of the treatment. Male albino rats of Wistar strain were given daily oral dosage of 500μg/Kg body
weight for 15 and 60 days. One group of 60 days treated rats was then kept for recovery without dosing for 30 days. The biochemical analysis of liver
showed that the activity of alkaline and acid phosphatase enzyme decreased in the treated rats for both the durations of treatment. Livers of 15 days
treated animals had RNA and total protein increased significantly. Sixty days treatment led to a significant increase in cholesterol and significant
decrease in glycogen, total protein and RNA levels in liver. Recovery animals had liver glycogen content significantly depleted whereas cholesterol,
total protein and RNA increased significantly. The histopathology showed loss of cytoarchitecture in various areas of liver, vacuolation and
inflammatory cellular infiltration in all the groups. Thus, it was observed that oral intake of Cantharis Q had a negative impact on the hepatic function
of male rats and the recovery was also not found to be significant.
Key words : Cantharis Q, aphrodisiac, clinical application, liver, biochemical analysis, histopathology
n many developing countries, traditional medicines are
widely utilized in the treatment of various ailments
including male reproductive function on an empirical basis. Fruits
of Piper guineense are used as aphrodisiac and have been shown
to stimulate sexual behaviour of mature male rats [1, 2]. Litsea
chinensis and Ochis macualta are used for their aphrodisiac
activity [3]. Leaves of Hibiscus macranthus and Basella alba have
androgenic property [4, 5]. A recent review states that randomized
clinical trials have shown that extracts of Lepidium meyenii,
known commonly as maca, has favorable effects on energy and
mood, may decrease anxiety and improve sexual desire [6]. Smallscale clinical trials performed in men have shown that extracts of
maca can heighten libido and improve semen quality [7, 8], improve
sperm production, sperm motility, and semen volume [6] and hence
is used as an aphrodisiac. Thus study of medicinal plants in order
to develop a new fertility enhancing drug is an attractive
preposition. Similarly in homeopathy a variety of plant, animal,
mineral and even toxic substances in very small doses are used
medicinally to stimulate the body's natural healing powers and to
bring the body back into balance. Sometimes crude extracts
(mother tinctures) are also used as medicine, which is termed as
non-homeopathic use of the substance as is the case with
Cantharis Q. It is a crude alcoholic extract of blister beetles called
Lytta vesicatoria, commonly called blister beetle and popularly
known as Spanish fly. Cantharis Q has been used nonhomeopathically from ancient times as an aphrodisiac [9, 10]. Lytta
vesicatoria of Fabricius is a beetle belonging to Phylum
Arthropoda, Class Insecta, Order Coleoptera, Family Meloidae.
These beetles carry a venomous substance diffused throughout
their body, especially in their blood called cantharidin, a potent
chemical that possesses caustic or blistering properties when the
insects are accidentally crushed or handled roughly [11]. Cantahris
Q is used in diluted potentized form for treatment of many
disorders such as baldness, rheumatism, burning and cystitis,
kidney and genital disorders [12]. At what period these beetles were
introduced into the practice of medicine is a matter of uncertainty
but find their most prominent sphere of action in exciting the
animal passions. It leads to irritation, i.e. pains with burning due
to vesication and produces unbounded sexual desire; also sexual
erethism and excitability. Genito urinary systems come under its
sphere of action [13]. The toxic principle, cantharidin is an
odourless, colourless compound that is soluble in various organic
solvents but only slightly soluble in water. It is a bicyclic
terpenoid having the molecular formula C10H12O4 [14]. After
ingestion, it is absorbed from the GI tract and is rapidly excreted
by the kidneys. During excretion it irritates the entire urinary
tract. The irritation of the urethra increases the blood flow to this
region and might result in priapism, a persistent abnormal
erection of the penis. It is likely that the priapism is the origin of
use of Spanish fly as an aphrodisiac [15]. The blister beetles have
been reported to be consumed by humans for aphrodisiac
purposes and there have been studies regarding the poisoning
effect of this extract [16, 17]. Lesions in the kidney and the efferent
urinary tract have been reported in some cases where it was used
as an aphrodisiac or to induce abortion. Though, Cantharis Q has a
long history in both folk and traditional medicine and has been
used since ages as a sexual stimulant and it has a wide spread
reputation of being an aphrodisiac [18], the effect on the vital organ
such as liver due to long term or short term use of this agent has
not been investigated in the past by a systematic study using a
model animal. Hence, the objective of this research study was to
carry out the primary assessment of the agent mainly through the
biochemical markers and histopathological study of liver which
could bring out the most relevant clinical features of the effect of
this animal drug on this vital organ of rat. Hence, through this
Asian J. Biol. Life Sci. | May-Aug 2014 | Vol-3 | Issue-2
research study there is an attempt to confirm the potential clinical
application of this agent and also the reversal of the effect, if any,
after withdrawing the drug treatment.
Drug / study agent
Crude alcoholic extract (mother tincture) of Lytta vesicatoria
manufactured by Sarada Homeo Laboratory (SHL), Kolkatta
700 015 is available in the market as Cantharis Q. Drug from one
batch was used throughout the experiment.
Experimental Animals
Thirty male adult albino rats (Rattus novergicus albinus)
Wistar strain of body weight of about 200-250 gms were kept
under standard laboratory conditions with a 12-hour light-dark
cycle. Regular rat food and water was provided ad libitum. All the
experimental animals were procured from the Haffkine
Biopharma. Corpn. Ltd., (A Govt. of Maharashtra undertaking),
Parel, Mumbai 400 012. The animals were kept under the
standard laboratory conditions as per the CPCSEA guidelines at
the Animal Testing Centre of Ruia College, Matunga, Mumbai,
Registration Number 315/CPCSEA.
Experimental Design
The animals were acclimated for a week. After acclimation,
the animals were divided into three groups of five animals each as
Group I, II and III as experimental animals for 15, 60 days of
treatment and recovery after 60 days treatment kept for 30 days
without dosing respectively. Corresponding groups of control
animals were kept for all these durations. Experimental animals
of group I, II and III were orally administered the drug at the
dosage of 500g / Kg body weight of the animals, diluted in 1 ml of
distilled water as a single dose per day using GI gavage for the
specified period as per the groups mentioned. Control animals
were orally administered 1 ml of distilled water, as a single dose
per day. Group III animals of 60 days duration treatment were
then kept for 30 days without any dosing in order to assess the
reversibility of the drug effect.
Biochemical analysis
Sets of control and experimental animals were sacrificed
using anaesthetic ether. Liver was excised from each of the
dissected animals. It was cleaned in cold normal saline, blotted
and immediately weighed and stored at -20oC for further
biochemical analysis. From each of the liver sample 40% tissue
homogenate was made in cold distilled water using Remi Tissue
Homogenizer. Different dilutions of this homogenate were used
as per the different standardized biochemical methods.
Biochemical assays of enzyme activities and that of metabolic
parameters were carried out using Diagnostic Reagent Kit from
Span Diagnostic. Extraction and of DNA and RNA was done as
per modified method of [19] using refrigerated centrifuge Rota 4,
Plastocraft Ltd. Chemicals of AR grade were purchased from
SISCO Research Laboratories Pvt. Ltd. and Qualigens Ltd. All
the colorimetric readings were taken on Systronics UV-Visible
Histopathological analysis
After sacrificing, one of the liver lobes of each animal was
fixed in alcoholic Bouin's fixative for at least 24 hours for
histological studies. They were washed with several changes of
70% alcohol, dehydrated in ethyl alcohol gradients, cleared in
chloroform and embedded in paraffin blocks. Sections of 7
thicknesses were taken on microtome (manufactured by Panchal
Scientific, India). Sections were stained with hematoxylin and
eosin[20]. Photographs were taken using Olympus CH20i
microscope with digital imaging facility.
Statistical analysis
All the data of biochemical analysis were expressed as Mean +
SD. Statistical analysis was done using two - way analysis of
variance ANOVA and Dunnett's test. Differences were considered
statistically significant when p-values were less than 0.05, P <
Effect on activity of liver biochemical parameters
As depicted in Table 1, in case of 15 days treated rats, the
activity of acid phosphatase decreased significantly with only
slight decrease in the activity of alkaline phosphatase and level of
cholesterol and glycogen increased insignificantly; as was the
case with DNA. However, total protein content and RNA of liver
increased significantly. The chronic treatment of 60 days resulted
in maximum alterations in various biochemical parameters. There
was a significant decrease in the activity of both the hepatic
marker enzymes for 60 days treatment. The cholesterol content
was significantly higher than the normal controls whereas the
glycogen content was significantly depleted. Even, the total
protein and RNA level of liver was diminished remarkably than
the control group. However, DNA content of liver of the treated
animals decreased only slightly i.e. insignificantly as that of their
controls. Chronically treated rats that were kept for the recovery
period of 30 days showed fluctuations in the liver biochemical
parameters like cholesterol, RNA and total protein which were
significantly increased whereas, the glycogen content
significantly decreased. Activities of both the hepatic marker
enzymes as well as the DNA content were at par with those of their
control counterparts. The changes in the biochemical parameters
did not indicate significant recovery.
Effect on liver histology
There was a significant decrease in the weight of liver for
treated animals of 15 and 30 days recovery. A significant increase
was observed for the 60 days duration of treatment [Table 1].
As depicted in Fig 1, treatment of 15 days resulted in damage
to the liver parenchyma of midzonal area. A ballooning
degeneration of hepatocytes was noticed in form of vacuolation
with fat droplets. A lot of congestion was observed in the portal
triad showing cellular inflammation which was also observed in
the periportal area.
Fig 2 shows that histopathology of chronically treated rat
livers depicted the picture like that of the acute hepatitis. There
was chronic inflammatory infiltration of portal triad with
lymphocytes and plasma cells. Injury was also seen in the
centrilobular area showing vacuolation and loss of hepatocytes.
The damage was also extended to the midzonal area with the loss
of hepatic cytoarchitecture. Histological studies also gave similar
observations in case of recovery rats where infiltration of
lymphocytes and plasma cells was seen as intense as in the
chronically treated group. However, midzonal area appeared to be
normal with slight damage still persistent in the centrilobular area
(Fig 2). Hence, the damage to the portal triad was not significantly
Asian J. Biol. Life Sci. | May-Aug 2014 | Vol-3 | Issue-2
Table 1. Effect on liver enzymes activity and liver metabolic parameters and organ weight of male albino rat
after15, 60 days treatment and 30 days recovery after 60 days treatment with Cantharis Q
The number of animals in each group, n = 5. Values are expressed as Mean + SD. Differences that are
significant between the respective control and experimental groups at P < 0.05 are marked as *
Biochemical and histopathological changes in the tissues,
affect the overall weight of that organ. Chronic treatment of 60
days with Cantharis Q resulted in a significant increase in the
weight of liver which corroborates with other reports based on
chronic toxicity studies [21, 22]. Acid phosphatase (E.C and
Alkaline phosphatase (E.C are said to be the marker
enzymes of liver indicating its various pathological conditions.
Decrease in liver acid phosphatase enzyme activity, as was
observed in the present study, has been reported in many
hepatotoxic studies which were accompanied with detrimental
histopathological changes [23-26]. Similar research studies have
reported lowered liver alkaline phosphatase activity in rats
associated with damage to liver by hepatotoxins [24, 27, 28] thus
confirming impairment in liver physiology. Thus it could be
inferred that short as well as long term treatment with Cantharis Q
affected the liver which is further supported by the histological
studies that revealed damage to the hepatocytes which probably
lowered the phosphatase activities in liver. Liver plays an
important role in the metabolism of carbohydrates, lipids and
proteins. Glycogen synthesis and degradation are coordinated by
several amplifying reaction cascades. Protein phosphatase (PP) 1
accelerates glycogen synthesis and hence decreases the rate of
glycogen breakdown [29]. Cantharidin, the main component of
Cantharis Q, is a strong inhibitor of the activity of serine /
threonine PP1 and PP2A [30-35]. This inhibitory effect of Cantharis
Q on PP1 has been reflected in the chronically treated rats for 60
days as well as the recovery group where a significant decrease in
glycogen content of liver was reported in the present study. PP2A
plays an important role in fatty acid metabolism [29]. Cantharidin,
therefore, most probably interferes with fatty acid metabolism
thus having its negative impact on liver lipid metabolism. It
decreases LH induced StAR protein levels [33, 36] due to which
transport of cholesterol to its site for steroid synthesis is in turn
Asian J. Biol. Life Sci. | May-Aug 2014 | Vol-3 | Issue-2
Fig 1. T. S. of liver of 15 days treated rats showing histopathological changes. Control rat showing (A) normal hepatic architecture of
the centrilobular area and the midzonal area and (B) normal structure of the portal triad area and the periportal area (400x). (C)
Experimental rat showing degenerative patches marked by the thick arrows (400x). (D) Experimental rat showing inflammation of the
portal triad as well as the periportal area as shown by the thick arrows (400x).
H- normal hepatocytes, S- sinusoids, CV-centrilobular vein, PTR- portal triad, BD- bile duct, A- branch of the hepatic artery, V- branch
of the portal vein.
Asian J. Biol. Life Sci. | May-Aug 2014 | Vol-3 | Issue-2
Fig 2. T. S. of liver of 60 days treated and recovery rats showing histopathological changes. Control rat showing (A) normal hepatic
structure of the midzonal area and (B) normal structure of the portal triad area with normal hepatocytes, the sinusoids and the
collagenous tissue. (400x). (C) Experimental rat of recovery group showing centrilobular area. Thick arrow 1- degenerated vacuolated
hepatocytes Thick arrow 2- loss of hepatocytic architecture, Thick arrow 3 dilated sinusoid, (D) 60 days experimental rat. Thick arrow
1- portal triad area with inflammatory cellular infiltrate. Thick arrow 2 - periportal area with degenerating hepatocytes lacking nuclei.
(E) Experimental rat of the recovery group showing normal hepatic architecture of the centrilobular and the midzonal area except for a
few patches having vacuolation marked by Thick arrow. (F) Experimental rat of the recovery group showing infiltration of the portal
triad area by lymphocytes and plasma cells marked by Thick arrows.
H- normal hepatocyte, S- sinusoid, BD- bile duct, CT- collagenous tissue, A- branch of the hepatic artery, V- branch of the portal vein,
CV- centrilobular vein
impaired. Thus, there could have been an increased accumulation
of cholesterol in hepatocytes there by increasing the liver
cholesterol content which was in the light microscopic histology
studies of the liver sections of the treated rats where ballooning of
hepatocytes due to fat accumulation was revealed. Further this
could also result in the impaired steroid synthesis that could in
turn affect the normal spermatogenesis process. Further, various
hepatotoxicity studies like effect of beryllium [37], ethanolic
extract of Crotolaria juncea (Leguminosae) seeds [38] and CCl4
treatment [39] have reported significantly decreased glycogen
content in liver of such treated albino rats. Histological damages
have also been reported in such treatments. Cholesterol is
reported to accumulate at higher levels than normal in liver during
hepatic malfunctioning [22, 28, 40]. Nature of fluctuations of protein
levels may vary as per the toxicant or drug under study and also
the dosage; though decrease rather than increase in liver protein
levels is more often seen in such hepatic damage studies. [38, 41-45].
The present study with Cantharis Q also reported alterations such
as significant increase for 15 days as well as recovery period and
decrease for 60 days, it being significant only for the latter. The
initial increase in protein could be due to decreased catabolism of
protein by hepatocytes due to their damage as reported in the
histological observations. However, as the treatment progressed,
probably there was an inhibition of protein synthesis. This is in
accordance the role of cantharidin as PP1 and PP2A inhibitor.
Histological analysis of the present study indicated damage to the
hepatic architecture which also explains the significant
alterations in hepatic protein metabolism. Recovery animals
showed the effect similar to that of 15 days treated which could be
due to withdrawal of long term treatment. However, significant
alterations in liver histology continued with respect to their
controls, which therefore did not indicate a good deal of recovery.
Protein synthesis is mediated by the coordinated interplay of
DNA and RNA, liver DNA and RNA assays are important.
Changes in RNA indicate toxicant induced changes in protein
synthesis [46]. Cell division, cell repair mechanisms could also be
influenced especially by DNA synthesis [29]. The trends observed
in fluctuations of DNA and RNA levels could be quite different in
different studies when related with liver protein content as
reported in various hepatotoxic studies [42, 47-50]. This could be due
to the much complicated mechanisms involved in nucleic acid
and protein metabolism. In the present study increased DNA as
well as RNA levels in liver of 15 days treated animals was
probably due to decreased catabolism of nucleic acids as in the
case with the associated protein metabolism where protein
degradation was affected. This could be corroborated with the
hepatotoxic study results of [43]. The recovery animals showed a
marginal increase in DNA levels and a significant increase in
RNA and protein levels. This result could be due to the stopping
of treatment of Cantharis Q since increase in DNA and RNA
levels also indicate tissue rejuvenation [45]. This may hold good as
Cantharis Q is known to be an anti-proliferative agent where it
affects the cell cycle and DNA synthesis by alternating the
dephosphorylation of extra cellular regulated kinases 1 and 2 [35].
The activity of these kinases is switched on by the serinethreonine specific phosphatase which then initiates mitosis [29].
Cantharis Q as mentioned earlier has been known to be the
inhibitor of such protein phosphatases thereby affecting cell
repair mechanism. Withdrawal of the treatment of the Cantharis
Q could slightly alleviate this effect. However, histological
analysis did not indicate a total recovery of normal hepatic
architecture. Hence damage caused by the chronic treatment with
cantharis Q might be an irreparable damage to certain hepatic
areas like periportal and portal triad as in case of chronic type of
hepatic damage there is a cellular inflammation of the portal tract
which may also extend into the parenchyma; the portal triads
show a conspicuous infiltration with the lymphocytes and plasma
cells and the lobular architecture is disturbed [51]. Various
toxicological studies have shown such histopathological changes
associated with the biochemical changes in the liver. [21, 22, 38, 40, 52-54].
Asian J. Biol. Life Sci. | May-Aug 2014 | Vol-3 | Issue-2
In the present study, there was an attempt to comprehensively
evaluate the function and physiology of the treated animals
through biochemical and histological investigations of liver
which is the largest gland that performs many important
functions. It could be concluded from the results and the
supporting references that hepatic damages caused due to
Cantharis Q treatment altered the biochemical and histological
status of the organ. The damage had been proportional to the
duration of the treatment. The recovery animals of the treated
group also did not show improvement of the lesions reported for
the chronic treatment. Hence it could be inferred that withdrawal
of the treatment for a period of 30 days did not lead to a significant
recovery. These findings could help in establishing the clinical
potential of this animal drug.
We would like to thank Dr. R. T. Sane, Former Principal of R.
Ruia College, Mumbai for granting the facility of the CPCSEA
registered Animal House of this institution. We are grateful to The
University Grants Commission for awarding the financial grant
through fellowship. We acknowledge Dr. C. V. Rao, Former Head
of Zoology Department, St. Xavier's College, Mumbai for his
valuable guidance in liver histopathological studies.
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