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Efficacy and toxicity studies of the combined extracts

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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


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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.

6.2.2.2 Acute Oral Toxicity – Up-and-Down Procedure, (Adoption 425 OECD)

6.2.2.2.1 Limit test at 2000mg/kg

An overnight fasted rat was weighed and the dose was calculated as per the weight. Freshly

prepared aqueous suspension of the mixture in 0.5% CMC was administered orally and

observed carefully for 48 h. After 48 h additional 4 animals were dosed in same way and

observed continuously for 48 h and 14 days thereafter for mortality.

6.2.2.2.2 Limit test at 5000mg/kg

Limit test at 5000mg/kg was also performed in same manner as at 2000 mg/kg and

observations were made as stated above.


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6.2.2.3 Dose for therapeutic efficacy

The 1/10thof the safe dose (500 mg/kg) was selected as the first dose and 2/3rd of the initial

dose was taken as 2nddose (333 ~350 mg/kg).

6.2.2.4 Composition of normal and protein deficient diet(Table 6-1)

Normal food pellets obtained from Hindustan Lever Limited, Mumbai, India, with a

composition as given below was termed as standard diet. The deficient diet was prepared

using white flour (maida obtained from local market) containing only starch (carbohydrate).

Casein, corn oil and cellulose were added as a source of protein, fat and fiber respectively

while jiggery served a dual purpose i.e. as a sweetening agent and a source of minerals. The

deficient diet carried almost the same energy as that of the normal, but was deficient in

proteins and minerals especially calcium.

Table 6-1: Composition of the animal diet for the study.

Sr. no. Nutrients Standard diet (g/g %) Deficient diet (g/g %) 1 Proteins 21 5 2 Carbohydrates 53 87 3 Lipids (fat) 5 3 4 Fibers 7 8 5 Minerals (ash) 8 2 Total energy 341 Kcal 335.9 kcal

6.2.2.4 Experimental design

The antiosteoporotic activity of the combination was carried out using ovariectomized rat model with

the two different diets.

6.2.2.4.1Experimental protocol for in vivo Antiosteoporotic study in group fed with

Standard diet

Thirty healthy female albino rats of equal size with a maximum of 10% variation were divided into 5

groups of equal size (n=6). The experimental design and protocol was the same as described in

chapter 5. All animals were ovariectomized except the first group SHAM which served as basal

control. The 2nd group served as ovariectomized control (OVX) and was fed with equi-volume of

saline all through the study. The 3rd group was fed with raloxifene, a SERM and served as standard

control. Groups 4 and 5 (F1 and F2 respectively) were treated orally with the combination of extracts

at two different dose levels 350 mg/kg and 500 mg/kg respectively. All the animals were fed with the

normal diet.


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Table 6-2: Experimental protocol for in vivo Antiosteoporotic study (Standard diet).

Groups Description Abbren Animals(n)

1 Sham operated (Basal control) SHAM 6

2 Ovariectomy (OVX) (Control) OVX 6

3 OVX + Raloxifene (5.4 mg/kg) RALOX 6

4 OVX + Combined extracts 350 mg/kg F1 6


6.2.2.4.2 Experimental protocol for in vivo Antiosteoporotic study in group fed with

Deficient diet

Twenty four healthy female albino rats of Wistar strain of equal size and age (maximum allowed

variation in animal weight was 10%) were divided into 5 groups of equal size (n=6). The experimental

design and grouping was same as described previously with the exception of the lower dose group (4)

which was absent. The experimental animals in all the groups were fed with protein and mineral

deficient diet prepared in the laboratory.

Table 6-3: Experimental protocol for in vivo antiosteoporotic study (deficient diet).

Groups Description Abbren Animals(n)

1 Sham operated (Basal control) SHAM 6

2 Ovariectomy (OVX) (Control) OVX 6

3 OVX + Raloxifene (5.4 mg/kg) RALOX 6


6.2.2.4.3Assessment

Assessment was carried out as described in chapter 5 i.e. on the 91st day, animals were bled

(retro-orbital vein) under ether anaesthesia and serum was separated using cold centrifuge at

4 °C. The serum samples were subjected for biochemical estimations namely, serum calcium

(Ca), serum phosphate (P), alkaline phosphatase (ALP), cholesterol (CHOL) triglycerides

(TG) and Tartarate resistant acid phosphastase (TRAP). The animals of the respective groups

were then systematically necropsied and both femur bones along with right tibia and vertebra

were isolated and freed of tissues. Right tibia, right femur and vertebra were subjected to

biomechanical testing. The right femur from each group was subjected to histopathological

examination. The methodology has been described in chapter (5). A few additional

parameters that are included for the present investigation are described below.


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6.2.2.4.3.1 Weight variation

The weight of the animals was recorded once every week and at the end total weight gain or loss was

calculated.

6.2.2.4.3.2 Triglycerides and serum phosphate

The biochemical markers serum triglycerides and phosphate were estimated by using an auto

analyser (Cobas c111, Roche Labs) in FIST-DST Lab, Dept. of Pharmacognosy, Manipal.

The kits (manufactured by Roche Diagnostics GmbH, Mannheim, Germany) used were

procured from Hitech Biomedicals, Mumbai.

6.2.2.5 Statistical analysis

The data was analyzed using one-way ANOVA with Tukey’s post-test and was performed

using Graph Pad Prism version 5.00 for Windows, Graph Pad Software, San Diego California

USA, www.graphpad.com. P<0.05 was considered significant.

6.3 Results and discussion

6.3.1 Preparation of combination of extracts In the present study three active extracts at their effective therapeutic dose were combined

together for the evaluation of its antiosteoporotic activity. In our studies on the individual

extracts, the ethanol extracts of C. quadrangularis and M. citrifolia were both found to

exhibit promising activity at 750 mg/kg b.w. while C. mukul was found effective at 500

mg/kg. Based on the results of the efficacy studies a mixture of all three extracts in 1:1:1 ratio

as per their respective effective doses i.e. C. quadrangularis: M.citrifolia: C. mukul in the

ratio 1.5:1.5:1.0 was prepared.

6.3.2 Acute Oral Toxicity – Up-and-Down Procedure, (Adoption 425 OECD)

6.3.2.1 Limit test at 2000 and 5000 mg/kg

0 – Survival, X – Death

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.

S. No. Mixture Dose (mg/kg) Outcome LD50 Range

(mg/kg) 1 Suspension (combination of extracts) 2000 0 0 0 0 0 >2000 2 Suspension (combination of extracts) 5000 0 0 0 0 0 >5000

http://www.graphpad.com/


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Table 6-6: Toxicity profile of the mixture.

S. No Mixture Dose (mg/kg)

Behavioral Toxicity

Neurological toxicity

Additional Toxicity

Mortality

1 Suspension (0.5 CMC) 2000 N N N N

2 Suspension (0.5 CMC) 5000 N N N N

The oral toxicity of the individual ethanol extracts of C. quadrangularis, M. citrifolia and C.

mukul has already been performed at both 2000 mg/kg and 5000 mg/kg levels (chapter 5) and

found safe. In the present study a combination of all the three extracts in the ratio 37.5:

37.5:25 was tested for the possible toxicity. It is believed that extracts with similar effects

when combined together give synergistic effect [3]. In the light of this theory, the

combination of two or more extracts may precipitate toxic effects also; it is therefore essential

to carry out safety studies of the combined extracts. As the safety of the individual extracts

was already established, the limit tests in the adoption 425 of the OECD guidelines for acute

oral toxicity were employed in the current study. Administration of the mixture to an

overnight fasted animal at a ceiling dose of 2000 mg/kg did not cause any sign of abnormality

or mortality in 48 h which encouraged us to dose an additional 4 animals. Neither mortality

nor any sign of toxicity could be observed in any of the animals even up to 14 days. The LD50

was therefore considered to be above 2000 mg/kg.

The combination showed no sign of toxicity at 2000 mg/kg limit test suggesting a high safety

margin. As the drug under study was targeted towards a chronic illness which requires long

term therapy, it was important to establish the maximum possible safety limits, which

prompted us to carry out another limit test at 5000 mg/kg. No toxic changes were observed in

any of the animals after 14 days thus establishing the LD50 of the combination to be above

5000 mg/kg.

6.3.4 Pharmacological activity

6.3.4.1 Body weight: It is generally observed that, postmenopausal woman gain excessive

weight along with accumulation of cholesterol. Excessive weight is often a risk factor for

osteoarthritis and occasionally for fractures while low body weight is a potential risk factor

for osteoporosis or osteoporotic fractures [11]. To avoid fractures due to postmenopausal

bone loss, an optimal musculature is essential. Bone weight contributes significantly to body

weight [12] which is hence used as a marker for bone mass. An appropriate body weight also

represents the general health of the animal. Our test combination included guggul which is

known to lower cholesterol levels [13] and is often used in traditional medicine in weight


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reduction regimens. C. quadrangularis has also been used in the weight reduction programs

[14] in the west.

Standard diet

In our study following ovariectomy, the animals in the untreated group (OVX-N) showed an

abnormal gain in weight as against a constant weight gain in normal animals (group 1),

mimicking the human tendency to gain weight after menopause. All the treated groups were

observed to control the weight gain. The test mixture at the higher dose (group 5) reduced the

weight to below normal levels, while the lower dose (group 4) best managed the weight. The

presence of C. mukul and C. quadrangularis in the formulation could have been responsible

for weight reduction at higher dose levels.

Deficient diet

The weight variation observed in the deficient diet also showed a similar trend. The OVX-D

group (2) gained weight inspite of the deficient diet while the normal animals in group 1

showed a lower but constant weight gain. The mixture treated group exhibited good

management of weight. The treatment has significantly prevented weight gain in the

ovariectomized rats but increased body weight as compared to sham-D. The results suggested

that the test mixture can be safely used for management of abnormal weight in

postmenopausal women.

Table 6-7: Average weight gain per week in standard diet fed animals.

Weeks 2 3 4 5 6 7 8 9 10

SHAM-N 5.5 5.3 4.5 5.8 5.9 5 4.8 5.2 4.9

OVX-N 5.8 6.5 7.6 7.6 7.4 8.5 8.8 9.4 12.4

Ralox N 4.2 4.2 3.9 4.5 4.8 4.5 4.8 4.8 4.3

F1-N 3.9 3.9 4.1 3.8 3.3 3.6 3.8 4.4 4.4

F2-N 3.8 3.2 3.2 3.2 2.8 2.8 2.8 2.8 2.9

Table 6-8: Average weight gain per week in standard diet fed animals.

Weeks 2 3 4 5 6 7 8 9 10

SHAM-D 3.8 3.7 3.8 3.4 3.2 2.8 3.4 2.6 2.4

OVX-D 4.4 4.4 5.1 5.8 5.3 6.1 6.4 6.3 6.8

Ralox D 4.1 4.7 3.8 3.4 3.6 2.7 3.7 3.2 3.9

F2-D 4.8 4.8 4.3 4.3 4.2 3.8 4.2 4.2 4.5


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6.3.4.2 Biochemical markers

6.3.4.2.1 Standard diet

A marked increase in both ALP and TRAP levels in the 2nd

group was observed when

compared to the normal control (group 1 p<0.0001). As discussed previously, ALP is a

marker for osteoblastic function while TRAP represents osteoclastic activity. The rise in the

ALP and TRAP levels in the estrogen depleted state is an indication of accelerated bone loss,

because the resorption and reversal phases of bone remodelling are short and the period

required for osteoblastic replacement of the bone is long [15]. Thus any increase in the rate of

bone remodelling results in a loss of bone mass resulting in numerous harvesian canals and

howship lacunae [16] which renders bone its porosity. While serum calcium levels remained

unchanged statistically, suggesting proper functioning of the thyroid hormones [17], serum

phosphate levels showed a significant reduction in their levels in the ovariectomized control

(group 2) when compared to the first group (p<0.0001).

Postmenopausal women are at a higher risk for cardiovascular diseases due to higher

cholesterol levels [18]. Although debated for long [19,20,21], menopause is thought to be a

determinant of the high cholesterol levels [22, 23, 24]. The accumulation of cholesterol in

postmenopausal women is attributed, to the deprivation of estrogen as well as to the lipid

profile changes during perimenopause [25]. Oxidative stress has been implicated as one of

the leading causes for higher cholesterol and cardiovascular risk. Lean et al.,[26]

demonstrated a significant loss in thiol antioxidant enzymes leading to compromised defence

5.5 5.3 4.5 5.8 5.9 5 4.8 5.2 4.9 5.8 6.5

7.6 7.6 7.4 8.5 8.8 9.4

12.4

4.2 4.2 3.9 4.5 4.8 4.5 4.8 4.8 4.3 3.9 3.9 4.1 3.8 3.3 3.6 3.8 4.4 4.4 3.8 3.2 3.2 3.2 2.8 2.8 2.8 2.8 2.9

1 2 3 4 5 6 7 8 9

Fig. 6-1 Average weight gain per week with standard diet

SHAM-N OVX-N Ralox N F1-N F2-N

3.8 3.7 3.8 3.4 3.2 2.8 3.4 2.6 2.4

4.4 4.4 5.1 5.8 5.3

6.1 6.4 6.3 6.8

4.1 4.7 3.8 3.4 3.6

2.7 3.7 3.2 3.9

4.8 4.8 4.3 4.3 4.2 3.8 4.2 4.2 4.5

1 2 3 4 5 6 7 8 9

Fig. 6-2 Average weight gain per week with deficient diet SHAM-D OVX-D Ralox D F2-D


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the leading causes for higher cholesterol and cardiovascular risk. Lean et al.,[26]

demonstrated a significant loss in thiol antioxidant enzymes leading to compromised defence

against oxidative stress. It can be therefore, hypothesized that estrogen depletion following

ovariectomy increases serum cholesterol. In the present study a significant elevation was

observed in the serum cholesterol levels and the triglycerides (P< 0.0001) of the

ovariectomized group 2 as compared to the sham (group 1) which corroborates the

hypothesis.

The animals in group 3 treated with raloxifene showed significant reduction in ALP

(p<0.0001) and TRAP (p<0.0001) levels, marginal increase in phosphate levels and a sharp

decrease in both cholesterol and triglyceride levels (p<0.0001) as compared to group 2. In the

test combination treated groups, as exhibited previously by the individual extracts, a

significant reduction was observed in the TRAP at both the dose levels (p< 0.0001 Vs. OVX-

N) indicating a marked reduction in the osteoclastic activity and thereby suggesting a

reduction in the bone resorption. The ALP levels on the other hand showed an optimal

increase which was found to be significantly higher than the Sham (p<0.001) and markedly

less than OVX (p< 0.001). The effect of treatment on biochemical markers is suggestive of

increased osteoblastic function with remarkable reduction in osteoclastic activity and

therefore lower bone turnover. Since the mixture contains extracts that are good antioxidants

with some of them having a proven record of inhibiting cytokines [27] it may be postulated

that osteoclast inductive cytokine RANK Ligand could have been suppressed resulting in

reduced osteoclastic activity. Treatment with the hexane and ethyl acetate fractions of Cissus

quadrangularis have resulted in a significant increase in bone formation indicated by an

increase in ALP levels in our earlier study (chapter 5). Our data is inadequate to propose any

mechanism of action, however, there are only two possible pathway by which the osteoblastic

function increases, the first being production or supplementation of growth factors which

stimulates the formation of pre osteoblasts and help them mature; and the second being early

apoptosis of the osteoclast that signals for the maturity of osteoblast. It must be noted that the

life span of the osteoclast is controlled by estrogen. Thus it can be postulated that the

estrogenic nature of our test mixture due to the presence of estrogenic constituents might

have reduced the life span of the osteoclast thereby stimulating ostblastic activity. The

postulate supports our biochemical findings, as the early induction of apoptosis in osteoclasts

minimizes the resorption which has been indicated in our results by significant lowering of

TRAP levels and apoptosis triggers the osteoblastic function which is indicated by an


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increase in the ALP levels. Consequently, shortened osteoclastic life span or number along

with continuous supply of newer executive cells (osteoblast) at a basic multicellur unit

(BMU) results in a competent bone, with minimum or no lacunae or canals [15, Monalogas].

6.3.4.2.2 Deficient diet

The same experiment was carried out in animals fed with the deficient diet as was done for

the normal diet. The deficient diet composed of only 5% protein and traces of minerals

including calcium with almost same energy as standard diet. This compromised food model

was designed so as to simulate malnourished conditions which common postmenopausal

women face in India. It is a well-known fact that the dietary habits in India are poor and

unbalanced. Shatrugna [28] demonstrated low bone mass among women, from low income

groups, who consumed low calorie food, inadequate calcium, protein and micronutrients.

Prevention and management of osteoporosis, requires healthy nutrition, adequate, calcium

and vitamin D along with good exercise.

Our treatment combination was a balanced composition of anabolic steroids from Cissus

quadrangularis [29,30,31] antihyperlipidemic constituents from Commiphora mukul [13,32]

and vitamin and mineral supplements from Morinda citrifolia [33,34]. The study was

therefore designed to investigate the effect of the combined extracts of these drugs on

ovariectomy induced osteoporosis in food insufficiency induced by deficient diet.

In our study cholesterol and triglyceride levels were significantly lower in the normal group

(1) when compared to their counterparts in the standard diet fed groups. The depreciation

may be attributed to the lack of a proper diet. It is interesting and intriguing to note that, even

in compromised food conditions, ovariectomization caused significant elevation in both

cholesterol and triglyceride levels; a plausible explanation for this could only be, the

oxidative stress caused by the estrogen depletion leading to the development of

hypercholestremia. The bone turnover marker levels like ALP and TRAP were also found

elevated when compared to Sham-D. Serum calcium level remained unchanged, while a

small decline was observed in serum phosphate levels.

In the treatment groups, both raloxifene and the test mixture decreased the elevated levels of

TRAP, Cholesterol and triglycerides, while increasing the ALP levels. The extract

combination showed marginally higher reduction in osteoclastognesis along with a better

elevation in the osteoblastic activity as compared to the Raloxifene treated group. The bone


130

Fig 6-3 d:Serum phosphate levles

Sham

-N

Sham

-D

OVX-N

OVX-D

RALOX-N

RALOX-D

F1-N

F2-N

F2-D

0

2

4

6

8

a c

Groups

U/L

turnover marker results were similar to our previous findings in the normal diet groups. The

results however could be more meaningfully inferred with the supporting data of

biomechanical and histopathological evaluation.

Table 6-9: Effect of the mixture on biochemical markers in ovariectomized rats fed with standard diet.

Groups ALP U/L TRAP CA P CHOL TG

SHAM-N 121.4 ± 2.31 3.74 ± 0.16 10.19 ± 0.41 7.09 ± 0.44 87.72 ± 1.34 86.45 ± 3.01

OVX-N 165.2 ± 2.97a 6.2 ± 0.15a 10.87 ± 0.31ns 5.67 ± 0.19a 109.4 ± 2.92a 117.2 ± 1.92a

RALOX-N 114.4 ± 1.77d 3.8 ± 0.20d 9.96 ± 0.26ns 6.56 ± 0.29ns 81.17 ± 2.09d 75.9 ± 1.3d

F1-N 144.2 ± 6.0b,e 2.65 ± 0.24c,d 11.18 ± 0.2ns 5.98 ± 0.17c 80.89 ± 2.109d 97.43 ± 3.65e

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.

Groups ALP U/L TRAP CA P CHOL TG

SHAM-D 108.6 ± 0.97 4.79 ± 0.31 9.562 ± 0.63 5.535 ± 0.30 73.05 ± 4.17 88.18 ± 2.45

OVX-D 166.3 ± 6.58a 6.11 ± 0.2b 10.48 ± 0.47ns 5.15 ± 0.69ns 86.27 ± 3.17c 130.4 ± 7.24a

RALOX-D 100.2 ±2.69a,d 4.29 ± 0.17d 10.17 ± 0.3ns 5.558 ± 0.2ns 71.23 ± 1.48e 82.9 ± 3.2d

F2-D 194.4 ± 4.23a,d 3.33 ± 0.20a,d 10.44 ± 0.19ns 5.67 ± 0.14ns 72.19 ± 2.1f 81.38 ± 1.53d

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


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


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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


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


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.


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


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)

Fig 6-6j: Intestine; Treated grp; no. changes (10x)

Fig 6-6k: Adrenal gland; ctrl; normal histopath. (10x)

Fig 6-6l: Adrenal gland; Treated grp; no. changes (10x)


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.


138

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