Efficacy and toxicity studies of the combined extracts 120 6. Efficacy and toxicity studies of the combination of the extracts 6.1 Introduction Nature stands as a golden mark to exemplify the outstanding phenomena of symbiosis. Natural products from plants, animals and minerals have been the basis of the treatment for countless human diseases [1]. The various indigenous systems such as Siddha, Ayurveda, Unani [2] and homoeopathy use several plant species or their combinations to treat different ailments. Mostly these systems recommend using herbs in their crude state in the formulations in contrast to the modern medicine which believes in purification and isolation of bioactive principles from the crude botanicals. Modern medicine has successfully isolated a number of bioactive molecules from traditionally used herbs such as psoralen, piperidine, phyllanthin etc. [3]. Patwardhan and Mashalkar [3] suggest that that drug discovery need not be always confined to the discovery of a single molecule as the current ‘one drug fits all’ approach may be unsustainable in the future. They advocate that rationally designed polyherbal formulations could be explored as an option for multi-target therapeutic and prophylactic applications. Zimmermann et al. [4] showed a renewed interest in multi-ingredient synergistic formulations for the management of certain polygenic syndromes. As complex mixtures of diverse chemical species, herbal extracts may act as a synergistic multiple target therapeutic or prophylactic [5] agents to address polygenic syndromes like postmenopausal syndrome. The use of herbal medicine has been on increase in many developing and industrialized countries [6], mostly influenced by patients dissatisfaction with conventional allopathic medicines in terms of effectiveness and/or safety as against the satisfaction with therapeutic outcome [7,8] of the botanicals and the perception that herbal medicines are inherently safe. However, botanicals have also been reported for potential side effects and toxic reactions including teratogenicity [7,9,10]. As purified extracts of the herbal agents are more likely to trigger toxic reactions than the crude drug, it is therefore important to carry out toxicity studies on purified extracts, especially when such purified extract of one or more herbs are mixed together in a combination. A combination of different extracts with similar activities generally adds synergistic effect to the combination and is likely to potentiate side effects
20
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Efficacy and toxicity studies of the combined extracts
120
6. Efficacy and toxicity studies of the combination of the
extracts
6.1 Introduction
Nature stands as a golden mark to exemplify the outstanding phenomena of symbiosis.
Natural products from plants, animals and minerals have been the basis of the treatment for
countless human diseases [1]. The various indigenous systems such as Siddha, Ayurveda,
Unani [2] and homoeopathy use several plant species or their combinations to treat different
ailments. Mostly these systems recommend using herbs in their crude state in the
formulations in contrast to the modern medicine which believes in purification and isolation
of bioactive principles from the crude botanicals. Modern medicine has successfully isolated
a number of bioactive molecules from traditionally used herbs such as psoralen, piperidine,
phyllanthin etc. [3].
Patwardhan and Mashalkar [3] suggest that that drug discovery need not be always confined
to the discovery of a single molecule as the current ‘one drug fits all’ approach may be
unsustainable in the future. They advocate that rationally designed polyherbal formulations
could be explored as an option for multi-target therapeutic and prophylactic applications.
Zimmermann et al. [4] showed a renewed interest in multi-ingredient synergistic formulations
for the management of certain polygenic syndromes. As complex mixtures of diverse
chemical species, herbal extracts may act as a synergistic multiple target therapeutic or
prophylactic [5] agents to address polygenic syndromes like postmenopausal syndrome.
The use of herbal medicine has been on increase in many developing and industrialized
countries [6], mostly influenced by patients dissatisfaction with conventional allopathic
medicines in terms of effectiveness and/or safety as against the satisfaction with therapeutic
outcome [7,8] of the botanicals and the perception that herbal medicines are inherently safe.
However, botanicals have also been reported for potential side effects and toxic reactions
including teratogenicity [7,9,10]. As purified extracts of the herbal agents are more likely to
trigger toxic reactions than the crude drug, it is therefore important to carry out toxicity
studies on purified extracts, especially when such purified extract of one or more herbs are
mixed together in a combination. A combination of different extracts with similar activities
generally adds synergistic effect to the combination and is likely to potentiate side effects
Efficacy and toxicity studies of the combined extracts
121
besides the pharmacological activity [7]. Development of standardized, synergistic, safe and
effective herbal formulations with robust scientific evidence can also offer faster and more
economical alternatives [3].
A systematic preclinical testing of extract or combination of extracts under investigation is
highly essential to prove the safety and efficacy in the management of the disease for which it
is developed for. The present study was therefore, undertaken to study the extract
combination for its antiosteoporotic activity.
6.2 Experimental
6.2.1 Preparation of combination of extracts
Ethanol extracts of each of C. quadrangularis, C. mukul and M. citrifolia, at their effective
therapeutic dose, as already established previously(see chapter 5)were mixed together in a mortar
and pestle by trituration in the ratio, 37.5:25:37.5. The freshly prepared mixture in the above
mentioned ratio, was suspended in water using 0.5% CMC as a suspending agent for dosing.
6.2.2 Pharmacological and Toxicological studies of the combination of extracts
6.2.2.1 Animals
Female Wistar Albino rats (170-200 g) were obtained from Manipal central animal house and
were acclimatized to the experimental room having temperature 23 ± 2°C, controlled
humidity conditions and 12- h light - dark cycle. Animals were caged in polypropylene cages
with maximum of two animals per cage. The rats were fed with standard food pellets and
water ad libitum. Study was conducted after obtaining ethical committee clearance from the
Institutional Animal Ethics Committee of KMC, Manipal. No. IAEC/KMC/73/2009-2010.
Table 6-4: Weights of individual extract for preparation of mixture. Extract %ge Wt. for 2000 mg Wt. for 5000 mg C. quadrangularis 37.5 750 1875 M. citrifolia 37.5 750 1875 C. mukul 25 500 1250 Table 6-5: Oral toxicity study outcome and LD50.
F2-N 146.2 ± 3.43d 2.89 ± 0.22d 11.49 ± 0.18ns 6.98 ± 0.17ns 91.15 ± 4.03d 69.8 ± 1.77d Values are expressed as Mean ± SE Compared with one way ANOVA followed by Tukey’s post hoc using GraphPad Prism version 5.00.statistical software; Significance a=p<0.0001 Vs. Sham; d=p<0.0001vs. OVX; b=p<0.001 Vs. Sham; e=p<0.001 Vs. OVX; c=p<0.01 Vs. Sham; f=p<0.01 Vs. OVX
Table 6-10: Effect of the mixture on biochemical markers in ovariectomized rats fed with deficient diet.
Values are expressed as Mean ± SE Compared with one way ANOVA followed by Tukey’s post hoc using GraphPad Prism version 5.00.statistical software; Significance a=p<0.0001 Vs. Sham; d=p<0.0001vs. OVX; b=p<0.001 Vs. Sham; e=p<0.001 Vs. OVX; c=p<0.01 Vs. Sham; f=p<0.01 Vs. OVX
Fig 6-3 c:Serum Calcium levels
Sham
-N
Sham
-D
OVX-N
OVX-D
RALOX-N
RALOX-D
F1-N
F2-N
F2-D
0
5
10
15 No significance observed
Groups
mg/
dl
Fig 6-3 a: Alkaline phosphatase levels
Sham-N
Sham-D
OVX-N
OVX-D
RALOX-N
RALOX-DF1-N F2-N F2-D
0
50
100
150
200
250
a a
d a,d
b,ed
a,d
Fig 6-3 b:Tartarate resistent acid phosphatase
Sham-N
Sham-D
OVX-N
OVX-D
RALOX-N
RALOX-DF1-N
F2-N F2-D
0
2
4
6
8
a b
dd
dc,da,d
Groups
U/L
Fig 6-3: Bar graphs showing effect of extract combination on biochemical parameters
Efficacy and toxicity studies of the combined extracts
131
6.3.4.3 Biomechanical parameters
Osteoporosis is characterized by low bone mass and structural deterioration of bone tissue
leading to reduced bone strength and consequently high fracture risk [35]. Treatment for
osteoporosis focuses on slowing down the bone loss and strengthening the brittle bone by
facilitating mineralization which eventually increases bone mass and in turn BMD. A direct
measure of the bone strength can be attributed to the bone mass and thereby BMD. Ulku and
co-workers [36] demonstrated a definite association between mechanical strength of the bone
and BMD by comparing the mechanical strength of a bone with that of X-ray analysis and
computed tomography. The biomechanical strength testing models of Peng et al., [37] and
Ogeyet al., [38] have been commonly employed for antiosteoporotic evaluations. In our study
the three point bending and load testing of femoral neck was experimented as per the
methodology of Penget al., while compression test of IV lumbar vertebra was as perOgey et
al. The tests were performed using Electrolab’s Tablet Hardness Tester with digital output.
The stage of the machine was suitably modified to fit the rat bones. All three tests were
performed using the same machine.
6.3.4.3.1 Standard diet
The test results in our study revealed a clear distinction between the bone strength of sham
group (1) and that of ovariectomized (group2) control (p<0.0001) thereby indicating the
development of osteoporosis in the 2nd group. The femoral neck and vertebra offer a large
trabecular area which is more susceptible for resorption and weakening and which are
therefore, the commonest sites of fractures. Our study showed a lowest strength at the
Fig 6-3 e: Serum Cholesterol levels
Sham-N
Sham-D
OVX-N
OVX-D
RALOX-N
RALOX-D
F1-NF2-N
F2-D
0
50
100
150
a
c dc
dd
f
Groups
mg
/dl
Fig 6-3 f: Serum Triglycerides levels
Sham-N
Sham-D
OVX-N
OVX-D
RALO
X-N
RALO
X-DF1-
NF2-
NF2-
D
0
50
100
150
a
a
dd
dd
e
Groups
mg
/dl
Efficacy and toxicity studies of the combined extracts
132
femoral neck in the OVX group as compared to the sham (p<0.0001). The treated groups
showed a significant gain in the lost bone strength which was close to normal.
6.3.4.3.2 Deficient diet
Food plays a vital role in providing essential elements and vitamins to the body. The food
insufficiency was reflected in our findings as the normal group (Sham-D) showed
significantly lower biomechanical strength when compared the bone strength of normally fed
animals. The biomechanical strength was further deteriorated (p<0.0001Vs Sham D) in the
ovariectomized rats (group 2) indicating intense bone loss. A reversal was witnessed in all the
treated animals of the deficient food groups. It is of interest that the gain in bone strength in
these compromised food conditions was almost as close to the gain observed in the normally
fed animals, especially in the test mixture treated animals. The findings suggest an
uninterrupted mineralization even in the absence of minerals from the diet probably sourced
from the extract combination.
Table 6-11 Effect of the mixture on biomechanical parameter in ovariectomized rats fed with normal food.
Groups 3.pt.bend Comp Load on femur SHAM-N 111.6 ± 2.28 140.8 ± 4.417 38.6 ± 3.34 OVX-N 75.02 ± 2.72a 86.03 ± 3.18a 14.25 ± 1.65a RALOX-N 99.28 ± 2.13d 126.2 ± 1.92d 32.46 ± 2.43d F1-N 83.63 ± 1.92a 113.1 ± 2.23d 40.63 ± 3.28d F2-N 94.38 ± 2.78b,e 114.09 ± 3.0d 46.57 ± 1.21d Values are expressed as Mean ± SE Compared with one way ANOVA followed by Tukey’s post hoc using GraphPad Prism version 5.00.statistical software; Significance a=p<0.0001 Vs. Sham; d=p<0.0001vs. OVX; b=p<0.001 Vs. Sham; e=p<0.001 Vs. OVX; c=p<0.01 Vs. Sham; f=p<0.01 Vs. OVX Table 6-12 Effect of the mixture on biomechanical parameter in ovariectomized rats fed
with deficient food. Groups 3. pt. bend Compression Load on femur SHAM-D 97.97 ± 4.38 118 ± 3.57 33.68 ± 1.77 OVX-D 71.78 ± 1.834a 79.98 ± 4.51a 10.13 ± 0.41a RALOX-D 92.22 ± 2.45d 105.5 ± 2.86d 29.25 ± 2.15d F2-D 85.02 ± 2.87f 112.2 ± 4.38d 42.54 ± 2.10d Values are expressed as Mean ± SE Compared with one way ANOVA followed by Tukey’s post hoc using GraphPad Prism version 5.00.statistical software; Significance a=p<0.0001 Vs. Sham; d=p<0.0001vs. OVX; b=p<0.001 Vs. Sham; e=p<0.001 Vs. OVX; c=p<0.01 Vs. Sham; f=p<0.01 Vs. OVX
Efficacy and toxicity studies of the combined extracts
133
6.3.4.4 Histopathology
The histopathological observations endorsed the biochemical, biomechanical and histopathological
findings. The intensified bone turnover that predisposes to porosity of bone in the ovariectomized rats
and a significant reduction in the bone strength in the mechanical testing could be visualized in the
histopathological slides of the femurs of the ovariectomized rats. Almost complete disruption of
trabecular bone with thinning of cortical bones was very clear. Number of BMUs and relatively lower
deposition of the bone was very evident in the OVX as compared to the sham in the normally fed
groups which showed compact and competent trabeculae with no recognizable damage. However the
histomicrogaph of the sham in the deficient food groups revealed weakening of bone which is a clear
sign of nutritional deficiency, yet no lytic changes could be observed. Treatment with Raloxifene had
a reversal effect on the osteoporotic bone as seen in the histomicrographs of both standard and
deficient diet groups. The nutritional deficiency was obvious in all the slides of deficient food groups.
Treatment with the extract combination showed relatively less bone damage and was indicative of a
normalization process with almost complete ossification at both dose levels. At the higher dose the
combination showed almost the same microarchitecture as the Sham. The histomicrograph of mixture
treated animals in the deficient food group revealed good bone restoration with effective
mineralization, though the dietary insufficiency was evident.
Fig 6-4: Bar graphs showing effect of extract combination on biomechanical parameters.
Fig 6-4 a: 3 Point Bending of Tibia
Sham-N
Sham-D
OVX-N
OVX-D
RALOX-N
RALOX-DF1-N
F2-NF2-D
0
50
100
150
aa
dd
ef
Groups (n=6)
New
tons
Fig 6-4 b: Load testing of femoral neck
Sham-N
Sham-D
OVX-N
OVX-D
RALOX-N
RALOX-DF1-N
F2-NF2-D
0
20
40
60
a
a
dd
dd
d
Groups (n=6)
New
tons
Fig 6-4 c: Compression of IV lumbar vertebra
Sham-N
Sham-D
OVX-N
OVX-D
RALOX-N
RALOX-DF1-N
F2-NF2-D
0
50
100
150
200
a a
d
d d d d
Groups (n=6)
New
tons
Efficacy and toxicity studies of the combined extracts
134
Fig 6-5a: Sham-N showing
competent undamaged bone
(H& E stain 10X)
Fig 6-5b: Sham-N showing
BMUs and normal deposition
of bone (H& E stain 40X)
Fig 6-5c: Sham-D, bone thinning
increased howship & harvesian
canals (H& E stain 40X)
Fig 6-5d: Sham-D, increased
BMUs; incomplete deposition
of bone (H& E stain 40X)
Fig 6-5e:OVX-N, Disruptive
and lytic changes in the
trabeculae(H& E stain 40X)
Fig 6-5f:OVX-N, Disruptive and
lytic changes evident bone
resorption (H& E stain 40X)
Fig 6-5g:OVX-D, more
prominent lysis and bone loss
in the trabeculae (H& E stain
10X)
Fig 6-5h:OVX-D, Prominent
resorption and bone loss in the
trabeculae (H& E stain 10X)
Fig 6-5i:Ralox-N, Reversal
process bone formation is
distinct in trabeculae (H& E
stain 10X)
Fig 6-5j:Ralox-N, Reversal
process bone formation is
distinct in trabeculae (H& E
stain 40X)
Fig 6-5k:Ralox-D, Reversal
process thin bone deposition
nutritional deficiency (H& E
10X)
Fig 6-5l:Ralox-D, Reversal but
incomplete deposition nutritional
deficiency (H& E 10X)
Fig 6-5: Histomicrographs showing histopathological changes in rat bones.
Efficacy and toxicity studies of the combined extracts
135
Fig 6-5m:F1-N, Distinct
reversal process competent
bone deposition (H& E stain
40X)
Fig 6-5n:F1-N, Distinct but
incomplete reversal, bone
deposition process (H& E
40X)
Fig 6-5o:F2-N, Near
normalization with competent
bone deposition (H& E stain
10X)
Fig 6-5p:F2-N, Marked
slowdown in resorption;
distinct bone formation (H& E
stain 40X)
Fig 6-5q:F2-N, Distinct
reversal but thin deposition
due to nutritional deficiency
(H& E10X)
Fig 6-5r:F2-N, Thin deposition
is clear due to nutritional
deficiency (H& E10X)
Admin
Line
Admin
Line
Admin
Line
Admin
Line
Efficacy and toxicity studies of the combined extracts
136
Fig 6-6a: Untreated control ; normal
histopathology in kidney (H&E 10x)
6.3.4.5 Histopathology of the vital organs:
The histopathology of the vital organs was carried out at the end of our study to assess for any possible damage in the tissues after dosing for a period of 75 days. No obvious damage could be observed in any of the organs. The extract combination can thus be considered safe for long duration consumption even at a higher dose of 500mg/kg.
Lysis Cortical
bone
Fig 6-6: Histomicrographs showing histopathological observations in the vital organs of
the animals treated with mixture for 75 days and the normal group Under H& E stain.
Fig 6-6f:Treatedgroup; no
histopathology changes in Liver (H&E
Fig 6-6b: kidney; Treated grp; no histopath. changes (10x)
Fig 6-6a: kidney; Untreated ctrl; normal histopath. (10 x)
Fig 6-6c.Heart; Untreated ctrl; normal histopath. (10x)
Fig 6-6d: Heart; Treated grp; no histopath. changes (40x)
Fig 6-6e: Liver; Untreated ctrl; normal histopath.(10x)
Fig 6-6f: Liver; Treated grp; no histopath. changes (10x)
Fig 6-6g: Pancreas; untreated ctrl; normal histopath. (10x)
Fig 6-6h: Pancreas; treated grp; no changes (10x)
Fig 6-6i: Intestine; untreated ctrl; normal histopath. (10x)
Efficacy and toxicity studies of the combined extracts
137
6.3.5 Conclusion
A well planned and systematic study was carried out to determine the efficacy and toxicity of
the extract combination. Individual extracts were initially evaluated to establish the effective
dose of each extract. The extracts were combined in justified ratios corresponding to their
individual effective dose. Despite well-established safety profile of individual extracts, the
combined extract was tested for acute oral toxicity as per the standard recommendations and
found safe up to a dose of 5000 mg/kg. Antiosteoporotic efficacy of the mixture was tested at
two different dose levels of 350 mg and 500 mg/kg body weight wherein the combination
was observed to exhibit a dose dependent activity.
An important achievement is the inclusion of certain parameters performed for the first time
in our study. Serum cholesterol and triglycerides were tested for the first time in the
ovariectomized rat model. Post-menopausal women are highly prone to cardiovascular risks
triggered by gain in cholesterol and triglycerides. A drug or a medication that takes care of
associated signs and symptoms of a polygenic syndrome would be a panacea for a patient.
Herbal extracts being multi component mixtures may act on multi- targets i.e. a multipill
concept. Our test combination showed a comprehensive reduction in the elevated levels of
the cardiovascular determinants along with bone loss.
Another important inclusion in the model was a protein and mineral deficient diet. A well
referred literature survey for the ovariectomized rat model suggests that tests have been
carried out earlier using a calcium deficient diet for the evaluation of antiosteoporotic
activity. A protein and mineral deficient diet for the test has been used for the first time in our
study. A large number of women, who generally are prone to osteoporotic attack in the
subcontinent, are usually the victims of poor dietary conditions. Our model simulated the
conditions of the malnourished women. Administration of the extract combination however
did not fully supplement the malnourishment; but effectively corrected the bone loss and
improved the strength to normal bone strength, thereby suggesting its mineral
supplementation capability.
Our findings are very important step toward the development of a standardized, effective
herbal dietary supplement for the prevention and (or) management of postmenopausal
osteoporosis. The results of our study testify to the efficacy and safety of the test mixture
nonetheless clinical studies are required before they can be prescribed to humans.
Efficacy and toxicity studies of the combined extracts
138
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