FIELD OF THE INVENTION
The present invention relates to a phosphodiesterase type five (PDE5) inhibitor for
the treatment of erectile dysfunction and more particularly to an herbal composition
acting as a PDE5 inhibitor for the treatment of male sexual disorder.
BACKGROUND OF THE INVENTION
Erectile dysfunction, also known as impotence, refers to the inability to obtain or
maintain an erection that is firm enough or lasts long enough to have satisfactory
sexual intercourse. The problem affects almost all men at one time or another in their
lives. A successful erection involves several parts of the body namely brain, blood
vessels, nerves and certain hormones working together. Sexual stimulation resulting
from various erogenous thoughts or stimuli or local genital contact triggers the
erectile process. The brain and nerves of the nervous system course in the pelvic
region to the genitalia and direct the chemical messages that result in penile erection.
The process basically involves blood flow to the penis. The erectile tissue and blood
vessels supplying the penis open up and allow blood entry to cause engorgement and
rigidity of the penis under the control of chemical signals. Depending upon the level
of stimulation, the chemical signals determine whether the penis is in its flaccid or
erect state.
An “aphrodisiac” is defined as any food or drug that arouses the sexual instinct,
induces venereal desire and increases pleasure and performance. This word is derived
from Aphrodite, the Greek Goddess of love. Aphrodisiacs are derived from plants,
animals or minerals and since time immemorial they have been the passion of man. A
lot of natural substances have historically been known as aphrodisiacs in Africa and
Europe. For instance, Yohimbine, the mandrake plant (Mandragora officinarum), the
ground rhinoceros horns in the Chinese culture and Spanish fly which is actually
toxic. Even in today's culture, there are certain foods that are used as aphrodisiacs,
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including strawberries and raw oysters. Chocolate, coffee and honey are also believed
to have aphrodisiac potential. Although these natural items are claimed as
aphrodisiacs, there is no or little scientific confirmation supporting those assertions.
Sexual dysfunction is a serious medical and social symptom that occurs in 10-52% of
men and 25-63% of women. Erectile dysfunction (ED), the main reason of male
impotence, is considered as one of the most important public health problems since it
affects a great percentage of men.
The biochemistry of male erectile dysfunction and its mechanisms are well
documented in the literature. During arousal nitric oxide (NO) synthase is activated
for the release of nitric oxide (NO) from the axons of parasympathetic nerve endings
in the walls of the arteries and sinusoids of the penile corpora cavernosa. Nitric oxide
stimulates soluble guanylase cyclase (GC) and the activated GC then catalyse the
conversion of guanosine triphosphate to cyclic Guanosine Mono Phosphate (cGMP),
which activates cGMP-dependent protein kinase (cGKI) and to a lesser extent protein
kinase A. Activated cGKI and protein kinase A further phosphorylate
Phospholamban, a protein that normally inhibits the Ca2+ pump within the membrane
of the sarcoplasmic reticulum. The Ca2+ pump is then activated and as a result the
level of free cytoplasmic Ca2+ is reduced, resulting in smooth muscle relaxation. In
the same way, the protein kinases activate the cell-membrane Ca2+ pump, leading to a
decreased sarcoplasmic Ca2+ concentration which induces a loss of contractile tone of
the penile smooth muscle and an increased blood flow in cavernous body resulting in
erection.
Another mechanism which causes penile erection is through cyclic adenosine
monophosphate pathway (cAMP). Corporal smooth muscle relaxation is mediated via
cAMP. The activated membrane-bound adenylyl cyclase, which generates cAMP
activates protein kinase A and to a lesser extent, protein kinase G. Prostaglandin E1
also increases the intracellular concentrations of cAMP in the corpus cavernosum
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smooth muscle cells. The generation of cAMP activates the Ca2+ pump and
consequently the level of free cytoplasmic Ca2+ is reduced resulting in smooth muscle
relaxation. Similarly, the protein kinase activates the cell-membrane Ca2+ pump,
leading to a decreased sarcoplasmic Ca2+ concentration which induces a loss of
contractile tone of the penile smooth muscle and increase of blood flow in the
cavernous body resulting in erection.
One of the mechanisms by which cyclic nucleotides induce the relaxation of smooth
muscle is through the opening of potassium (K+) channels, which leads to the efflux
of K+ from the smooth muscle cell, down their electrochemical gradient. This results
in hyperpolarization and an inhibitory effect on trans membrane Ca2+ flux and
eventually, smooth muscle relaxation.
After cessation of erotic stimuli, the nitric oxide release from the parasympathetic
nerves of the penis declines and the cGMP level in the smooth muscle cells falls
because of a decrease in synthesis coupled with the ongoing degradation of cGMP by
phosphodiesterase type 5 (PDE5). These muscle cells return to the more contracted
state and the penis becomes more flaccid because of the reduced amount of blood in
the corpora. Alteration in either psychological, hormonal, neurological, vascular, or
cavernosal factors can cause some degree of erectile dysfunction
The past 20 years of research on erectile physiology revealed the biochemical factors
and intracellular mechanisms responsible for corpus cavernosal smooth muscle
contraction and relaxation and revealed that erectile dysfunction is predominantly a
disease of vascular origin. Phosphodiesterases type-5 (PDE-5) inhibitors such as
Sildenafil, Tadalafil and Vardenafil are generally used for the management of erectile
dysfunction. PDE5 inhibitors are contraindicated in those taking nitrate medication.
They are also contraindicated in men for whom sexual intercourse is inadvisable due
to cardiovascular risk factors. The occurrence of adverse drug reactions (ADRs) with
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PDE5 inhibitors appears to be dose related. Headache is a very common ADR,
occurring in >10% of patients. Other common ADRs include: dizziness, flushing,
dyspepsia, nasal congestion or rhinitis. In 2007, the U.S. Food and Drug
Administration (FDA) announced that a warning about possible sudden hearing loss
would be added to drug labels of PDE5 inhibitors. Since 2007 there is evidence that
PDE5 inhibitors can cause an anterior optic neuropathy. PDE5 inhibitors are
primarily metabolized by the cytochrome P450 enzyme CYP3A4. There is an
increased risk for adverse drug interactions with other drugs which inhibit or induce
CYP3A4, including HIV protease inhibitors, ketoconazole, and itraconazole.
Combination with NO-releasing drugs such as glyceryl trinitrate (nitro-spray) is
contraindicated because potentially life-threatening hypotension (low blood pressure)
may occur.
On the other hand, Ayurveda is known for its contribution in the management of
impotency. “Vajikarana chikitsa” is a branch of Ayurveda, Indian system of alternate
medicine that describes different herbal formulations for management of male sexual
disorders including erectile dysfunction. “Vajikarana” herbs/aphrodisiacs are the
herbs that have been used in the Ayurvedic system of medicine to treat erectile
dysfunction. Most importantly, the relative safety which is a key with natural
ingredients is an added advantage with herbs. Thus, there is a need for a natural
PDE5 inhibitor composition which is relatively free from the unpleasant side effects
of the synthetic drugs.
SUMMARY OF THE INVENTION
The present invention relates to a natural PDE5 inhibitor composition and a process
for preparing the same.
In accordance with an embodiment of the invention, there is provided a natural PDE5
inhibitor composition, comprising: an extract of Akarkara (Anacyclus pyrethrum); an
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extract of Nutmeg (Myristica fragrans); and a plurality of excipients; wherein the
ratio of the extract of Akarkara to the extract of Nutmeg ranges from 6:1 to 1:1 and
preferably is 3:1.
In accordance with another embodiment of the invention, the plurality of excipients is
selected from the group consisting of a diluent, a disintegrant, an anti-microbial
preservative, a glidant, a lubricant, a film coating material and combinations thereof,
and the composition is in the form of a tablet.
In accordance with another embodiment of the invention, the diluent is present in an
amount in the range from 43.57-72.80%.
In accordance with another embodiment of the invention, the disintegrant is present
in an amount in the range from 3.86-6.76%.
In accordance with another embodiment of the invention, the anti-microbial
preservative is present in an amount in the range from 0.05-0.39%.
In accordance with another embodiment of the invention, the glidant is present in an
amount in the range from 0.48-0.97%.
In accordance with another embodiment of the invention, the lubricant is present in
an amount in the range from 0.48-0.97%.
In accordance with another embodiment of the invention, the film coating material is
present in an amount in the range from 2.90-3.86%.
In accordance with another embodiment of the invention, there is provided a process
for preparing a tablet of the natural PDE5 inhibitor composition, comprising the steps
of: (a) admixing the extract of Akarkara, the extract of Nutmeg, the diluent and the
disintegrant to form a first mixture; (b) admixing the anti-microbial preservative, the
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glidant, and the lubricant to form a second mixture; (c) admixing the first mixture and
the second mixture to form a third mixture; (d) compressing the third mixture into an
un-coated tablet by a suitable compressing machine; and (e) coating the un-coated
tablet with the film coating material to obtain the tablet of the natural PDE5 inhibitor
composition.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1. Ex-vivo percent PDE-5 enzyme activity in rats after administration of
vehicle, reference compound (Sildenafil citrate), Akarkara extract, Nutmeg extract
and a combination of the extracts of Akarkara and Nutmeg in the ratio of 3:1, referred
to as test drug.
Animals in Group G1 served as vehicle control and were orally administered 0.25%
CMC.
Animals in Groups G2 and G3 received Nutmeg extract at doses of 15 & 30 mg/kg
respectively.
Animals in Groups G4 and G5 received Akarkara extract at doses of 15 and 30 mg/kg
respectively.
Animals in Groups G6, G7 and G8 received the test drug at doses of 7.5, 15 and 30
mg/kg respectively.
Animals in Group G9 received reference compound Sildenafil citrate at doses of 10
mg/kg.
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DETAILED DESCRIPTION OF THE INVENTION
Discussed below are some representative embodiments of the present invention. The
invention in its broader aspects is not limited to the specific details and representative
methods. The illustrative examples are described in this section in connection with
the embodiments and methods provided. The invention according to its various
aspects is particularly pointed out and distinctly claimed in the attached claims read
in view of this specification.
It is to be noted that, as used in the specification and the appended claims, the
singular forms "a," "an," and "the" include plural referents unless the context clearly
dictates otherwise. Thus, for example, a reference to a composition containing “a
compound” includes a mixture of two or more compounds. It should also be noted
that the term “or” is generally employed in its sense including “and/or” unless the
content clearly dictates otherwise.
The expression of various quantities in terms of “%” or “% w/w” means the
percentage by weight of the total solution or composition unless otherwise specified.
All cited references are incorporated herein by reference in their entireties. Citation of
any reference is not an admission regarding any determination as to its availability as
prior art to the claimed invention.
The present invention, in its product and process aspects, is described in detail as
follows:
Akarakarabha or Akarkara (Anacyclus pyrethrum DC) is native of North Africa from
where it was introduced to Southern Europe. Roots of Akarakarabha are used in
treating rheumatoid arthritis and toothaches, in preparation of toothpaste, for gargling
in case of sore-throat. In Europe, its tincture is used as local irritant (counter-irritant).
It is also used in sexual disorder in males.
Anacyclin, eneteriyne alcohol etc. are the major chemical constituents of
Akarakarabha. This herb contains an essential volatile oil and an alkaloid, pellitorin
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or pyrethrin. It has alkamides, lignane (including sesamine), inulin (fructosan) and
tannins. Said alkamides include deca-2,4-dien acid-isobutylamide, anacycline and
dehydroanacycline. Akarakarabha has no toxic effect on body when taken in normal
quantity. The diluted essential oil in the root is used in mouthwashes and to treat
toothaches. This oil should not be used internally, except under professional
supervision. [Chevallier. A. The Encyclopaedia of Medicinal Plants Dorling
Kindersley. London 1996 ISBN 9-780751-303148]. It is one of the ingredients of a
drug which cures male sexual disorders without any toxic effect (The dried root,
U.S.P. 1820 – 1910)
The use of herbal remedies including the herb Jatiphala or Nutmeg (Myristica
fragrans Houtt) is popular as an alternative to standard western allopathic medicine
for a variety of problems including its aphrodisiac properties. Nutmeg primarily
constitutes of β-pinene, β-terpinene, safrole, methyleugenol, myristicin, elemicin,
trimyristin, dihydro-disoeugenol, myristic acid, epicatechin, cyanadin, nectandrin B,
verrucosin, lignans and neolignans etc.
Accordingly, the natural PDE5 inhibitor composition of the present invention, which
is free from the side effects associated with syntheric drugs, comprises an extract of
Akarkara (Anacyclus pyrethrum); an extract of Nutmeg (Myristica fragrans); and a
plurality of excipients; wherein the ratio of the extract of Akarkara to the extract of
Nutmeg ranges from 6:1 to 1:1 and preferably is 3:1.
The natural PDE5 inhibitor composition of the present invention is formulated in the
form of a tablet wherein the excipients include a diluent, a disintegrant, an antimicrobial
preservative, a glidant, a lubricant, and a film coating material.
Diluent, also called as filler, is often added to tablet formulations to provide better
tablet properties, such as improved cohesion, improved compression manufacturing
and adjusting weight of the tablet as per dye capacity. Non-limiting examples of the
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diluents that can be used in the present invention include micro-crystalline cellulose,
dibasic calcium phosphate, starch, magnesium carbonate, calcium carbonate, lactose
and so forth, each of which can be used individually or in combination, in an amount
in the range from 43.57-72.80%. Micro-crystalline cellulose (MCC) is purified
partially depolymerized cellulose, prepared by treating α-cellulose with mineral acids.
It is a white, crystalline powder composed of agglomerated porous microfibers. After
purification by filtration and spray-drying, porous microcrystals are obtained.
Disintegrant is always added to tablet to induce breakup of tablet when it comes in
contact with aqueous fluid and this process of desegregation of constituent particles
before the drug dissolution occurs, is known as disintegration process. Non-limiting
examples of the disintegrants that can be used in the present invention include,
croscarmellose sodium, crospovidone, sodium starch glycolate, polacrillin potassium,
and so forth, each of which can be used individually or in combination in an amount
in the range from 3.86-6.76%. Croscarmellose sodium is an internally crosslinked
sodium carboxymethylcellulose, wherein the cross-linking reduces
water solubility while still allowing the material to swell (like a sponge) and absorb
many times its weight in water, thereby accelerating the dissolution of the tablet.
Antimicrobial preservatives are substances used in tablets to increase the shelf life of
the pharmaceutical substance by preventing the growth of microorganisms. Nonlimiting
examples of the anti-microbial preservatives that can be used in the present
invention include, potassium sorbate, sodium benzoate, sodium methylparaben,
methylparaben, benzoic acid, sorbic acid and so forth, each of which can be used
individually or in combination. The tablet contains the anti-microbial preservative in
an amount in the range from 0.05-0.39%.
Glidant is added to the tablet formulation to improve the flow properties of the
material which is to be fed into the die cavity and aid in particle rearrangement within
the die during the early stages of compression. Non-limiting examples of glidants that
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can be used in the present invention include colloidal silicon dioxide, starch, talc, and
so forth, each of which can be used individually or in combination, in an amount in
the range from 0.48-0.97%.
Lubricants are the agents that act by reducing friction by interposing an intermediate
layer between the tablet constituents and the die wall during compression and
ejection. Non-limiting examples of the lubricants that can be used in the present
invention include, magnesium stearate, sodium stearyl fumarate, polyethylene glycol
6000 and so forth, each of which can be used individually or in combination, in an
amount in the range from 0.48-0.97%.
An un-coated tablet is coated with an appropriate film coating material for protecting
unstable compositions, protection in the stomach against enzymes and acids,
improving the appearance of the tablet, and masking objectionable odours and taste.
Non-limiting examples of the film coating material that can be used in the present
invention for coating un-coated tablets, include coating ready-mix, ethyl cellulose,
hydroxyl propyl methyl cellulose, shellac, diethyl phthalate, talc, colouring agents,
and so forth, each of which can be used individually or in combination, in an amount
in the range from 2.90-3.86%. The coating ready-mix comprises of hydroxyl propyl
methyl cellulose, diethyl phthalate, ethyl cellulose, talc, titanium dioxide, lake
ponceau 4R, and lake sunset yellow. Prior to coating the un-coated tablets, the
coating ready-mix is dispersed in isopropyl alcohol for approximately 5 minutes.
Thereafter, methylene chloride was added and stirred for approximately 45 minutes
and filtered through a nylon cloth to prepare the non-aqueous coating dispersion,
which was finally used to coat the un-coated tablets.
The present invention is more particularly described in the following non-limiting
examples that are intended as illustrations only since numerous modifications and
variations within the scope of the present invention will be apparent to a skilled
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artisan. Unless otherwise noted, all parts, percentages, and ratios reported in the
following examples are on a weight basis, and all reagents used in the examples were
obtained or made available from the chemical suppliers.
Example 1
Process for preparing Akarkara (Anacyclus pyrethrum) extract.
The dried herb was extracted with a mixture of alcohol and water (in a ratio of
10:90). The decoction so obtained was concentrated using Centritherm evaporator
(CT-1) (Flavourtech make) at around 45 °C under reduce pressure. The concentrated
mixture thus obtained was further dried with the help of an adsorbent and powdered.
Example 2
Process for preparing Nutmeg (Myristica fragrans) extract.
The dried herb was extracted with a mixture of alcohol and water (in a ratio of
10:90). The decoction so obtained was concentrated using Centritherm evaporator
(CT-1) (Flavourtech make) at around 45 °C under reduce pressure. The concentrated
mixture thus obtained was further dried with the help of an adsorbent and powdered.
Example 3-7
Table 1. illustrates the ingredients and their amounts present in the tablet of the
natural PDE5 composition. The unit of each value is percent by weight.
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Table 1. Natural PDE5 inhibitor tablet formulation
No Ingredients Example
3
Example
4
Example
5
Example
6
Example
7
1 Extract of Akarkara
(Anacyclus
pyrethrum)
19.32 27.05 21.74 14.49 28.99
2 Extract of Nutmeg
(Myristica fragrans)
4.83 13.53 7.25 14.49 4.83
3 Microcrystalline
cellulose
67.27 48.46 60.29 60.06 54.20
4 Croscarmellose
sodium
3.86 5.80 5.80 5.80 6.76
5 Sodium benzoate 0.06 0.08 0.10 0.08 0.10
6 Potassium sorbate 0.12 0.15 0.10 0.15 0.19
7 Colloidal silicon
dioxide
0.58 0.77 0.68 0.77 0.77
8 Magnesium stearate 0.58 0.77 0.68 0.77 0.77
9 Coating ready-mix 3.38 3.38 3.38 3.38 3.38
Example 8:
Process for preparing a natural PDE5 inhibitor composition in the form of a
tablet.
The amounts of ingredients used in preparing the natural PDE5 inhibitor composition
are presented in Table 1. Extract of Akarkara (Anacyclus pyrethrum), Extract of
Nutmeg (Myristica fragrans), micro-crystalline cellulose and croscarmellose were
sieved through a mesh of size 30, weighed and mixed in a mixing vessel to form a
first mixture. Sodium benzoate, Potassium sorbate, colloidal silicon dioxide and
magnesium stearate were sieved through a mesh of size 60, weighed and mixed in a
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mixing vessel to form a second mixture. The first mixture and the second mixture
were mixed to form a third mixture. The third mixture was compressed into un-coated
tablets using a suitable compressing machine, such as a tableting machine. The term
“tableting machine” used herein refers to a machine that compresses powders or
mixtures into tablets of uniform shape and size. The un-coated tablets were finally
coated with a non-aqueous coating dispersion, in a suitable coating pan, to obtain the
tablet of the natural PDE5 inhibitor composition.
Example 9
To evaluate the safety of a combination of the extracts of Akarkara and Nutmeg
in a ratio of 3:1, henceforth referred to as test drug, by conducting Acute & Sub
Acute (28 days) Toxicity Studies.
I. Acute Oral toxicity study in Rats:
The study was conducted to determine the acute oral toxicity of the test drug to
Female Albino Wistar Rats. The test drug was administered by oral route at doses of
500 mg, 1000 mg and 2000 mg/kg body weight to 3 albino female Wistar rats at each
dose level. One set of animals served as control. The rats were kept under 24 h
intense clinical observation followed by 14 days observation for clinical signs of
toxicity, if any. No mortality or signs of toxicity were observed during the 24 h
observation period in all the groups of animals. During the 14 day observation period
an animal that was administered the test drug at a dose of 2000 mg/kg died on the
sixth day. Hence, the experiment was repeated with another set of animals (3 female
animals), who were administered the test drug at doses of 2000 mg/kg. However no
mortality was observed in this experiment and necropsy report of the dead animal
revealed no gross abnormalities.
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On the basis of clinical signs and mortality observations, it was concluded that the
test drug is non-toxic up to a dose of 2000 mg/kg/po.
II. Sub-Acute Oral toxicity study in Rats:
This study was conducted to assess the 28 day repeated oral toxicity (Sub acute
toxicity) following oral administration of test drug in Wistar rats. 18 animals were
divided into three groups of 6 animals each. Group 1 received test drug at doses of
4.5 mg/kg/po daily for 28 days. Group 2 received the test drug at doses of 9 mg/kg/po
daily for 28 days. Group 3 served as control and were administered vehicle only.
The animals were observed for any abnormalities periodically for 28 days. The body
weight gain and 24 hour food intake were monitored at weekly intervals.
Haematological & Biochemical parameters were recorded at the beginning and at the
end of the study period. After 28 days, the animals were sacrificed in order to
perform gross necropsy.
Key results of the study were as follows:
1) No mortality or morbidity of the test animals were observed during the test period.
2) No statistically significant increase in body weight was observed as compared to
control animals.
3) No change in fur coating, eyes and mucous membrane, respiratory as well as
somatomotor activity was observed in any of the groups.
4) No significant increase in the food consumption was observed as compared to
vehicle control.
5) No significant change in Hematological parameters was observed when compared
to vehicle control.
6) No significant change in biochemical parameters were observed when compared to
vehicle control.
7) No signs of intoxication were observed.
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8) During visceral examination, no lesions or abnormalities like change in size, color,
congestion, hemorrhages, abscesses, inflammation, and necropsies were detected.
9) No abnormalities or variations of organ to body weight ratio were noted when
compared to the control animals.
Based on the above results it may be concluded that, the test drug was safe when
administered orally.
Example 10
Ex-vivo PDE-5 inhibitory activity of bioavailable Phytochemical principles of
Extract of Nutmeg, Extract of Akarkara and the test drug at various
concentrations.
The objective of the present study was to investigate ex-vivo PDE-5 inhibitory
activity of bioavailable Phytochemical principles of the extract of Nutmeg, the extract
of Akarkara and the test drug at various concentrations in male Wistar rats.
Overnight fasted animals were randomized based on body weight and divided into
different groups (G1-G9).
Group G1 served as vehicle control and was orally administered 0.25% CMC.
Groups G2 and G3 received Nutmeg extract at 15 & 30 mg/kg respectively.
Groups G4 and G5 received Akarkara extract at 15 and 30 mg/kg respectively.
Groups G6, G7 and G8 received the test drug at doses of 7.5, 15 and 30 mg/kg
respectively.
Group G9 received reference compound Sildenafil citrate at 10 mg/kg. All the
formulations and reference compound were administered orally at the dose volume of
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10 mL/kg to the respective groups. After 1 h of extract or reference drug
administration, ~200 μL of blood was withdrawn by retro orbital puncture from all
the animals. Plasma was separated by centrifugation and stored in deep freezer. PDE-
5 inhibitory activity of bioavailable components/principles was analyzed by cGMP
ELISA kit method, in plasma samples collected at l h time-point after ethanol
precipitation.
All the plasma samples demonstrated PDE-5 inhibitory activity (Figure 1). Both
Nutmeg and Akarkara demonstrated dose dependent PDE-5 inhibition with highest
inhibition occurring at a dose of 30 mg/kg at 1 h time point. PDE-5 inhibition was
observed due to the test drug, but it was not dose dependent.
Example 11
Erectile dysfunction ameliorating effect of test drug in rats.
Animals:
Male rats (wistar albino), 4 months old, weighing between 280-300 grams and
females, 3 months old and weighing 220 grams, were selected from the animal
facility of C. L. Baid Metha College of Pharmacy. All animals were kept in clean
polypropylene home cages. While male rats were housed in individual cages, 3-5
female rats were kept in a single cage. All the cages were placed in well-ventilated
animal room. Room temperature was maintained between 22-24 °C with relative
humidity of 60%. Animals were exposed to a reversed light-dark cycle (lights
switched on from 7 p.m. to 7 a.m. and lights switched off from 7 a.m to 7 pm).
Animals were acclimatized for about 2 weeks before the experiments. They were
allowed free access to food pellets and water. Experiments were done in the dark
period in a dim red florescent lamp. All animal experiments were carried out in
compliance with CPCSEA (Committee for the purpose of control and supervision of
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experiment in animals) guidelines and was approved by Institutional Animal Ethics
Committee (IAEC) of C. L. Baid Metha College of Phamacy, Chennai.
Induction of oestrous
72 female Wister rats weighing 200-220 g were used for mating stimulus. The female
rats were bilaterally ovariectomized via lumbar incisions under ketamine (100
μl/100g of rat) and xylazin (10 μl/100g of rat) as anesthesia 10 days prior to the
experiments. The females were rendered sexually receptive by single subcutaneous
injections of estradiol benzoate (10 μg) 52 h and progesterone (1 mg) 4 h, prior to
pairing. Oestrous females displayed a high degree of lordosis responding and
perceptivity.
Experimental
A combination of extracts of Akarkara and Nutmeg in a ratio of 3:1 was used as the
test drug. Forty male rats were randomly grouped into 4 groups (A–D) consisting of
10 animals each. Animals in group A, which served as the control, were administered
Sodium carboxy methyl cellulose (CMC) at 1 mL/100g body weight orally. Animals
in groups B and C were administered the test drug at doses of 4.5 and 9 mg/kg/po,
respectively, in a constant volume of 1 mL/100 g weight of the animal. Animals in
Group-D were administered the reference compound, Sildenafil citrate, at doses of
4.5mg/kg/po, and served as positive control. Drug and vehicle administrations were
done daily between 8:00 am to 9:00 am. The animals were allowed free access to rat
pellets and water.
Monitoring of Sexual behavior in male rats
A cage (0.3 m × 0.5 m × 0.3 m) made of perplex glass (observatory cage) with a
stainless steel mesh plate in the middle was put on a stainless steel metal box. The
sexual behavior of vehicle treated rats and those not treated with either the test drug
or the reference compound, Sildenafil citrate, with sexually receptive rats was
monitored to get the pre-treatment data of sexual behavior. One hour after the
respective treatment, one male rat was placed in one of the chambers of this cage.
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The rat was given 10 minutes to acclimatize and thereafter, 3 to 5 sexually receptive
female rats were introduced into the opposite chamber of the cage (Anticipatory
phase). After about 10 minutes the middle stainless steel mesh plate was removed and
the rats were allowed to mingle freely for 30 minutes (Consummatory phase).
Proceptive and precopulatory behaviors of a male with female rats were observed
from the cage side. Sexual behavior parameters were monitored for 30 minutes by
two skilled observers and the mean of the values were taken for the data preparation.
If male rat did not perform an intromission within the first 15 minutes, it was
removed and replaced with a new one. The sexual behavior of an individual rat was
observed for 30 minutes, one hour after the administration of the test drug and the
reference compound, Sildenafil citrate. The animals were then removed from the
observatory cage and placed into home cages. The observatory cage was cleaned with
wet cloth and dried in a ventilated room. The next experiment was performed after 4
h.
The following guidelines/ precautions were followed in the study:
a) Male rats were kept individually but females were kept in groups (3-5);
b) Training of each male for 15 minutes was performed until sexual behavior was
elicited and when the behavior was noticed, males were exposed to receptive females
(1 male with 3-5 females);
c) Repeated training was given to the rats to overcome the lack of sexual response in
the presence of observers;
d) The study was conducted in a silent room under dim red light;
e) Any jerking movement of the mating area was avoided to enable the rats to chase
each other;
g) Cleaning of the mating area was performed after each trial, since the urine trails
left by one rat might alter the sexual behavior of the next rat.
The following male sexual parameters were observed during the observatory
period:
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1) Mount Latency (ML): time from introduction of the female until the first mount.
2) Intromission Latency (IL): time from introduction of the female until the first
intromission.
3) Ejaculation Latency (EL): time from the first intromission until ejaculation.
4) Mount Frequency (MF): the number of mounts in a series.
5) Intromission Frequency (IF): the number of intromissions in a series (the number
of intromission preceding ejaculation and the resumption of sexual activity, as
indicated by next intromission)
6) Ejaculation frequency (EF): the number of times there was expulsion of semen
by males after vaginal penetration- characterized by rhythmic contraction of the
posterior abdomen.
7) Post-Ejaculatory Interval (PEI): the time from the occurrence of ejaculation
until the initiation of a new series, as indicated by the next intromission.
Statistical analysis
Results were expressed as mean±SEM. Statistical analysis was performed with one
way analysis of variance (ANOVA) followed by Tukey’s test using Prism 5
Graphpad Software.
The results of the study of the sexual behavior parameters of rats are presented in
Table 2.
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21
Table 2. Effect of test drug on sexual behaviors of rats to assess the aphrodisiac activity
Sexual
behavi
or
Vehicle control Test drug Dose-1
4.5mg/kg/po
Test drug Dose-2
9.0mg/kg/po
Sildenafil 4.5mg/kg/po
0 hr 1hr 4hr 0hr 1hr 4hr 0hr 1hr 4hr 0hr 1hr 4hr
ML
(second
s)
235.70
±1.48
237.13±0
.85
245.10
±0.47
239.0
4±1.1
0
131.23
±1.20*
*
128.31±
2.35**
258.12
±1.30
103.14±
0.45***
125.30±
2.34**
248.12±1.
13ns
98±12
0.98**
*
120.10±1
.02***
IL
(Second
s)
350.45
±2.01
348.67±2
.13
379.31
±0.97
353±.
20±1.
32
243.08
±3.18*
*
194.40±
2.54***
314.12
±1.45
203.30±2
.02***
232.32±
1.26***
352.83±2.
15
201.69
±1.05*
**
185.40±1
.86***
MF
(counts)
11.40±
0.76
12.10±0.
96
11.54±
0.62
9.45±
0.84
11.43±
0.87*
14.17±0.
45**
10.05±
0.95
13.01±0.
64*
14.04±0.
05**
10.45±1.0
1
17.62±
0.86**
*
19.20±0.
16***
IF
(Counts
)
10.50±
0.76
11.05±1.
01
10.85±
0.90
11.17
±0.95
13.44±
0.65*
20.03±1.
13**
11.02±
1.14
14.32±0.
25*
27.04±0.
05**
11.23±1.2
5
36.67±
0.56**
*
42.23±1.
02***
PEI
(second
s)
230.20
±7.01
239.32±7
.5
2.37.4
2±8.45
260.2
2±10.
2
241.10
±9.3*
210.20±
5.2**
258.04
±7.92
232.55±4
.6**
211.25±
10.4**
240.17±7.
48
201.45
±7.8**
173.1±12
.30***
EL
seconds
)
210.21
±11.22
245.18±1
3.28
231.21
±18.63
218.2
2±12.
42
306.10
±15.55
*
368.23±
13.50**
192.40
±9.45
295.21±2
0.10**
372.45±
16.34**
*
234.23±11
.20
367.24
±17.56
***
401.10±2
0.45***
EF
seconds
)
3.07±0.
86
3.76±0.4
7
3.12±0
.25
3.30±
0.78
5.20±0
.53**
6.42±1.0
1**
3.54±0.
87
8.20±0.6
4***
9.08±0.8
8***
3.10±1.06 10.25±
1.45**
*
13.26±0.
86***
Values are presented as mean±SD. All latency intervals are expressed in seconds. Significant from control *p,05, **p, 01,
***p,0.001,
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22
The test drug produced an increase in Mounting frequency (MF) and Intromission
frequency (IF). The MF and IF are considered as indices of both libido and potency.
The significant increase in the ejaculatory latency (EL) suggests that the test drug
prolonged the duration of coitus. The significant decrease in Post Ejaculatory Interval
(PEI) suggests that the test drug intensified the sexual activity in a sustained manner.
The test drug also produced a significant reduction in the mounting latency (ML) and
intromission latency (IL) as compared to control, confirming the aphrodisiac activity
of the test drug. The increase of penile erection induced by the test drug could be due
to its PDE5 inhibitory activity. Hence, there is a correlation between the PDE5
inhibitory activity of the test drug and the reference compound, Sildenafil citrate.
It can be logically concluded from the study that the natural PDE5 inhibitor
composition of the present invention may be useful in solving the sexual problems
such as premature-ejaculation and erectile dysfunctions in men. Hence, the test drug
should be further evaluated in human volunteers to establish its clinical efficacy to
ameliorate the symptoms of loss of libido, erectile disorders and lack of orgasm in
adult population.

We claim:
1. A natural PDE5 inhibitor composition, comprising:
(a) an extract of Akarkara (Anacyclus pyrethrum);
(b) an extract of Nutmeg (Myristica fragrans);
(c) a plurality of excipients;
wherein the ratio of the extract of Akarkara to the extract of Nutmeg ranges
from 6:1 to 1:1 and preferably is 3:1.
2. The natural PDE5 inhibitor composition, as claimed in claim 1, wherein the
plurality of excipients is selected from the group consisting of a diluent, a
disintegrant, an anti-microbial preservative, a glidant, a lubricant, a film
coating material and combinations thereof, and the composition is in the form
of a tablet.
3. The natural PDE5 inhibitor composition, as claimed in claim 2, wherein the
diluent is selected from the group consisting of microcrystalline cellulose,
dibasic calcium phosphate, starch, magnesium carbonate, calcium carbonate,
lactose and combinations thereof and present in an amount in the range from
43.57-72.80%.
4. The natural PDE5 inhibitor composition, as claimed in claim 2, wherein the
disintegrant is selected from the group consisting of croscarmellose sodium,
crospovidone, sodium starch glycolate, polacrillin potassium, and
combinations thereof and present in an amount in the range from 3.86-6.76%.
5. The natural PDE5 inhibitor composition, as claimed in claim 2, wherein the
anti-microbial preservative is selected from the group consisting of potassium
sorbate, sodium benzoate, sodium methylparaben, methylparaben, benzoic
acid, sorbic acid and combinations thereof, and present in an amount in the
range from 0.05-0.39%.
6. The natural PDE5 inhibitor composition, as claimed in claim 2, wherein the
glidant is selected from the group consisting of colloidal silicon dioxide,
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starch, talc and combinations thereof and present in an amount in the range
from 0.48-0.97%.
7. The natural PDE5 inhibitor composition, as claimed in claim 2, wherein the
lubricant is selected from the group consisting of magnesium stearate, sodium
stearyl fumarate, polyethylene glycol 6000 and combinations thereof and
present in an amount in the range from 0.48-0.97%.
8. The natural PDE5 inhibitor composition, as claimed in claim 2, wherein the
film coating material is selected from the group consisting of coating readymix,
ethyl cellulose, hydroxyl propyl methyl cellulose, shellac, diethyl
phthalate, talc, colouring agents, and combinations thereof and present in an
amount in the range from 2.90-3.86%.
9. A process for preparing a tablet of the natural PDE5 inhibitor composition, as
claimed in claims 2-8, comprising the steps of:
(a) admixing the extract of Akarkara, the extract of Nutmeg, the diluent and
the disintegrant to form a first mixture;
(b) admixing the anti-microbial preservative, glidant and lubricant to form a
second mixture;
(c) admixing the first mixture and the second mixture to form a third mixture;
(d) compressing the third mixture into an un-coated tablet by a suitable
compressing machine; and
(e) coating the un-coated tablet with the film coating material to obtain the
tablet of the natural PDE5 inhibitor composition.