Plant-based pharmaceuticals have been employed since centuries in the management of various
diseases of man and animals (Temple et al., 1996). The primary benefit of using plant derived
medicines is that they are relatively safer than synthetic alternatives, offering profound therapeutic
benefits at more affordable rates (Aarts, 1998). Plants are recognized for their ability to produce a
wealth of secondary metabolites and mankind has used many species for centuries to treat a variety of
diseases (Cragg et al., 1999). WHO has pointed out that 70-80% of world's population use medicinal
plants (Stephen, 1999) and so far around 35,000 plants species have been used for medicinal purposes
(Narayan et al., 2003).
Recently focus on plant research has intensified all over the world. Large amount of evidence have
been collected to show immense potential of medicinal plants used in various traditional systems
(Dahanukar et al., 2000). A large number of medicinal plants have already been tested and found to
posses active principles useful for treating various diseases (Haider et al., 2001). Moreover, several
plants produce secondary metabolites, which possess biological and pharmaceuticals activities and
have played important role in development of new drugs (Verpoorte, 2000).
Gemmo-therapy is a lesser known but modern and exciting research division of phyto-therapy which
uses stem cells of buds and young shoots of trees and shrubs, gathered in the spring time when they are
at a key stage of their natural growth cycle. Young shoots and buds of medicinal plants are freshly
prepared in a process using water, glycerin and alcohol. Since these extracts are from fresh and
growing tissues, Gemmo-therapeutic remedies are unique in their intense combination of vitamins,
minerals and other powerful properties of whole plants, including flowers, leaves, fruits, sapwood and
Cardiovascular diseases are usually caused by a blockage in the circulatory system that prevents blood
from flowing to the heart or the brain. The most common cause is a build-up of fatty deposits on the
inner walls of the blood vessels that supply the heart or brain. The blood vessels become narrower and
less flexible, also known as atherosclerosis (Braunwald et al., 1997). Globally, myocardial infarction is
a major public health concern and leading cause of mortality (Tilak-Jain and Devasagayam, 2006). In
recent years, an increasing number of young people are succumbing to myocardial infarction due to
unusual risk factors characterized by high triglycerides, low high density lipoprotein cholesterol (HDLC),
glucose intolerance, insulin resistance abdominal obesity and increased lipoprotein levels (Farvin et
al., 2006; Packard et al., 2005). The major abnormalities noticed following myocardial infarction are
lipidemia, peroxidation and loss of plasma membrane integrity (Farvin et al., 2004). There is wellestablished
relation-ship between coronary artery diseases, serum lipid and lipoproteins levels (Haust
and More, 1972).
Withania somnifera (Family: Solanaceae) is cultivated in Pakistan and India. The main constituents of
this plant are alkaloids and steroidal lactones. It has been reported to possess anti-carcinogenic and
antioxidant properties and is being used as drug (Ichikawa, et al. 2006). In Ayurvedic medicine, it is
used as a home remedy for several diseases and is also mentioned as a herbal tonic and health food
(Sharma et al., 1985). It is known to contain alkaloids and a special group of compounds called
Withanolides, which are considered to be its active principles (Tripathi et al., 1996). Recently, it has
been shown to inhibit lipid peroxidation in stress induced animals (Dhuley, 1998). In this present study,
we have attempted to assess comparative preventive and curative antilipidemic effects of Gemmotherapeutically-
treated Withania somnifera in the experimental model of salbutamol-induced myonecrosis
and have determined their effects on total, HDL-C, LDL- Cholesterol and triglycerides.
MATERIAL AND METHODS
Sample Collection & Extract Preparation
Samples of Withania somnifera were collected from botanical gardens of University of Agriculture,
Faisalabad and were identified by the Plant taxonomist in Department of Botany, University of
Agriculture Faisalabad, Pakistan.
Withania somnifera leaves were washed with distilled water. After drying in a shed, about 500 g of
clean leaves were added to 250 ml of H2O, shacked and boiled for half an hour, taken into the beaker
and was left for one day. Next day the solution was filtered and filtrate was used for the dose
preparation of rabbits.
Fresh growing leaves were washed with distilled water and weighed. The samples were set aside so
that the water will become evaporated and the exact weight of plant material was determined. The fresh
plant material blended in a mixture of alcohol and glycerin having a ratio of 2:1, respectively. The
mixture was left to stand for one month in a cool, shaded environment, and shaken from time to time to
help the maceration process. It was then filtered under constant pressure. After standing for a further
forty eight hours, it was filtered ones again. The resulting liquid was known as the stock, it consists of
glycerin and alcohol in a ratio of 2:1 respectively. The stock solution was then evaporated in rotary so
that all the alcohol was removed. The solution was kept in an incubator at a temperature of 65° C, and
thus the remaining alcohol was be evaporated. This stock solution or extract (Gemmo-therapeutically
treated Withania somnifera) was used within five years from the date on which the plant material was
picked (Churchill, 2002).
Qualitative and quantitative analysis was done for identification of the major phyto-constituents of
Withania somnifera and was carried out as described by Brain and Turner (1975) and Siddique and Ali
Salbutamol plant extracts (Gemmo and native), syringes, cotton, centrifuge tubes, Eppendorf tubes, kits
of total LDL,HDL cholesterol and triglyceride were required.
Eighteen rabbits weighing about 1.25 kg were used in the study. Animals were kept in cages under
hygienic and standard environmental conditions. They were fed a regular rabbit chow and water ad
libitum. Animals were weekly weighed and were randomly allocated eight groups comprising three
animals each. Table I shows the treatment protocol followed in the study.
Biochemical & Pathological Tests
Blood samples were collected from jugular vein of rabbits (Behne, 1981) before starting experiment,
during and after 30 days of experiment. The blood samples were centrifuged and serum was separated
and stored in deep freezer for further biochemical measurements. Serum was used for the assay of
biochemical parameters including total cholesterol, low density lipoproteins (LDL) cholesterol, high
density lipoproteins (HDL) cholesterol and triglycerides (TAGS) using kits.
Table – I: Treatment protocols followed in the experiments
Table II shows results of qualitative phytochemical analysis of native and Gemmo modified extract of
the plant, which shows that common constituents present in large amounts were flavonoids, glycosides
and tannic acid, etc.
Table – II: Quantative analysis of phyto-constituents present in Withania somnifera
Oral administration of ssalbutamol (50mg/ kg) significantly increased (p<0.05) heart rate; in Gemmocurative
(115 beats/ 30 sec) and native-curative (112 beats/ 30 sec) as compared to normal (95 beats /30
sec). Heart beat of rabbits was checked regularly with an interval of 24 hours before and after
administration of salbutamol. Post-treatment of ischemic rabbits with Gemmo and native extracts of
Withania somnifera (50 mg/ kg) for five days significantly reduced (p<0.05) heart beat both in
Gemmo-curative (90 beats /30 sec) and Native-curative groups (98 beats /30 sec) as compared to
ischemic group (112 beats /30 sec). Gemmo-extract showed more significant results (90 beats /30 sec)
than native-extract (97 beats /30 sec).
Table – III: effects of Withania somnifera on different blood lipid levels (mg/dl)
Similarly in preventive therapy, pre-treatment of ischemic rabbits with Gemmo and native extracts of
Withania somnifera (50 mg/ kg) for 20 days significantly restored (p<0.05) the heart beat to normal,
both in Gemmo-preventive (94 beats /30 sec) and Native-preventive groups (95 beats /30 sec) as
compared to ischemic group (112 beats /30 sec). Both the Gemmo and native-extract showed similar
cardio-protective effects. The Table III shows the Tachycardia values (per 30 sec) of different groups.
Rabbits were pre-treated with native and Gemmo-extract of Withania somnifera for 20 days and then
treated with salbutamol (50mg/kg) once a day for two days to induce myocardial injury.
The effects native and Gemmo modified extracts of Withania somnifera on various fractious of blood
lipids are shown in Table III. Similarly in native-preventive group, pre-treatment with the native extract
of Withania somnifera significantly restored the serum enzyme levels to normal; cholesterol
(237.00+10.09) HDL cholesterol (76.32+17.58), LDL Cholesterol (128.67+14.19) and triglycerides
(210.0+9.58) as compared to ischemia group having serum enzyme levels of the cholesterol
(357.00+17.10) HDL cholesterol (32.33+1.45), LDL cholesterol (134.67+60.88) and triglycerides
Table – IV: Preventive effect of Withania somnifera on different enzyme levels (U/L)
Both the Gemmo- and native extracts of Withania somnifera have showed antilipidemic activity and
significantly restored lipid levels in Gemmo-preventive and native-preventive groups to normal levels.
Table – V: Curative effect of Withania somnifera on different lipid levels (mg/dl).
Ischemia was induced in rabbits by oral administration of Salbutamol (50mg/ kg) for two consecutive
days at an interval of 24 hours. Salbutamol significantly increased (p<0.05) the serum levels of Total,
LDL Cholesterol, Triglycerides and decreased the serum level of HDL Cholesterol in curative groups
as compared to normal. However, post-treatment of ischemic rabbits with Gemmo and native extracts
of Withania somnifera (50mg/ kg) significantly reduced (p<0.05) serum levels of Cholesterol, LDL
Cholesterol, Triglycerides and Increased the serum level of HDL Cholesterol in curative groups as
compared to ischemia group in a five-day trial. Table V shows the total, HDL, & LDL cholesterol and
triglycerides levels in mg/dl of different groups. Results showed that Gemmo extract was more
effective than the native extract.
Administration of salbutamol (50 mg/kg) for two consecutive days significantly (p<0.05) increased
serum level of total cholesterol in Gemmo-curative group (324.671+8.33) and native-curative group
(341.67+6.77) as compared to non-treated group (346.33+ 20.85) while in normal group
(225.67+7.80). Post-treatment of ischemic rabbits with Gemmo and native extracts of Withania
somnifera (50 mg/kg) for five days significantly (p<0.05) reduced serum levels of total Cholesterol
both in Gemmo-curative (231.00+8.33) and Native-curative groups (245.00+12.06) as compared to non
treated ischemic group (410.00+8.50) and in normal control group (230.67 + 3.67) mg/ul, respectively.
Gemmo-extract showed more significant value (231.00+8.33) than native-extract (245.00+12.06).
Subsequent to the administration of Salbutamol (50 mg/kg) serum level of HDL-C decreased
significantly (p<0.05) in Gemmo-treated group (44.67+.67.0) and native-curative group (61.33+5.78)
as compared to non treated group (55.33 + 1.45) and in normal group (64.33+3.38). However, posttreatment
of ischemic rabbits with Withania somnifera for five days (p<0.05) did not decrease rather
increased serum levels of HDL cholesterol both in Gemmo-curative (82.00+58.0) and Native-curative
groups (70.00+4.04) as compared to non treated ischemic group (34.67+20.3) and normal control group
(62.00 + 1.53) mg/ul while Gemmo-extract showed more significant results (82.00+58.0) than nativeextract
Oral administration of Salbutamol (50mg/kg) for two consecutive days significantly (p<0.05) increased
serum levels of LDL-C in Gemmo-curative (126.67+2.86) and native-curative groups (130.33+5.40) as
compared to untreated ischemic (134.67+0.88) and in normal group (114.33 ± 7.22). However, posttreatment
of ischemic rabbits with Withania somnifera for five days significantly reduced (p<0.05)
serum levels of LDL-C both in Gemmo-curative (113.33+4.70) and Native-curative groups
(123.67+1.45) as compared to non treated ischemic group (150.67+1.20) and normal control group
(113.67 +6.84) mg/ml, respectively. Gemmo-extract showed more significant result (113.33+4.70) than
native-extract (123.67+1.45 mg /dl) at the end.
Administration of Salbutamol (50mg/kg) for two consecutive days significantly increased serum level
of triglycerides in Gemmo-curative group were 239.67+17.33 and 241.67+18.67 in native-curative
group as compared to non treated ischemic group (246.33 + 2085) and non treated ischemic group
(246.33 + 20.85) normal group (167.33+5.49). Post-treatment of ischemic rabbits with Withania
somnifera for five days significantly reduced (p<0.05) serum levels of triglycerides both in Gemmocurative
(166.33+5.57) and Native-curative groups (203.00+10.58). The comparison showed. Gemmo
extract more effective than native-extract.
Gross pathology refers to macroscopic manifestations and immediately after sacrificing, the animals
gross pathology changes were studied by a veterinary doctor, various pathological effects produced are
shown in Table VII & VIII.
Table – VII: Gross pathological studies of different organs of rabbits
Table – VIII: Weights of different organs of rabbits of different groups
Results obtained showed dose-related lowering effects of Withania somnifera extract on serum Total,
LDL-cholesterol and triglycerides but dose-related elevation of serum HDL-C. Our plant showed
maximum flavonoids and tannins contents, which strongly suggest the antioxidant and
cardioprotective, potential of Withania somnifera. (Rastogi and Mehrotra, 1991; Ju, 2005; Lie and
As described above a decrease in the serum levels of Total LDL-Cholesterol, Triglycerides and
increase in serum level of HDL Cholesterol was observed in Salbutamol treated groups. Pre and posttreatment
with native and Gemmo-therapeutically treated Withania somnifera (50 mg/Kg) restored the
level of lipids to normal levels. The base-line contents were found to be normal, which reveals that W.
somnifera at a dose 50mg/ kg did not induce any cardio-toxic effects. This is according to the previous
report presented by Mohanty et al. (2004) that W. somnifera at 50 mg/kg dose produced maximum
antilipidemic effects. The decrease in serum total, LDL-C and triglycerides and increase in the levels of
HDL-C may be due to presence of high flavonoid content in Gemmo extract of Withania somnifera .
The presence of high levels of flavonoids may suggest a wide spectrum of biological actions including
hypoazotemic, hypotensive, hypo-glycemic, estrogenic, spasmolytic, cholagogic, anti-inflammatory,
antilipidemic and antioxidant activities (Oladele et al., 1995). The hypoglycemic and hypolipidemic
activities of flavonoids from different plant sources have also been reported. (Sudheesh et al., 2005).
The presence of flavonoids in high concentrations in the extract may account for its hypoglycemic and
antilipidemic effects. It was reported that flavonoids play a major role in reducing the risk of
cardiovascular diseases by decreasing the blood lipids. Our results showed that flavonoids mixture
significantly decreased the plasma triglycerides, total cholesterol, and free fatty acids with increasing
HDL cholesterol level (Narender et al., 2006).
Gemmo extract showed more antilipidemic potential than native extract in curative therapy with similar
results in preventive therapy. Antilipidemic effects of Gemmo-extract is significant than native-extract
and is probably due to embryonic part of the plant being particularly effective for drainage and
detoxifying actions on the human body. Synthetic pharmaceutical agents, most herbs, and homeopathic
remedies that are prepared from the whole plant (usually flowering) have less amount of many of the
key elements (growth factors, phyto-hormones and auxins) formed during growth stage of plants. This
is because the gemmae contain many active principles that start to disappear after a plant reaches a
certain stage of development (Daniel and Towle, 2002).
The gross pathological examination of different organs of animals suggests cardioprotective potential
of Withania somnifera. Gross pathological confirmation of cardio-toxic effect produced by salbutamol
(50 mg/kg), in the present investigation has established the suitability of this model for studying the
cardioprotective effect of Withania somnifera (Mohanty et al., 2004). Compared to curative therapy,
gross pathology findings showed relatively greater efficacy of preventive therapy. The
histopathological examination showed that Gemmo-extract showed results that are more significant in
curative therapy. Hemalatha et al (2006) have reported that histopathological examination of liver
tissues of treated hyperlipidemic rats showed lesser degenerative changes as compared with
The major active constituents of Withania somnifera exhibit medicinal actions of certain steroidal
alkaloids and lactones as a class and are called Withanolides. The root contains steroidal lactone
(Withaferin A) and related Withanolides, along with various alkaloids. It is reported that Sitoindosides
VII, VIII, IX and X are most probably adaptogenic active substances present in Withania somnifera.
The exact mechanism of such myocardial adaptation is not yet known. However, it has been proposed
to function through induction of certain antioxidant agents (Archana and Namasivayam, 1999).
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