5. RESULTS AND DISCUSSIONshodhganga.inflibnet.ac.in/bitstream/10603/22924/14/14... ·...

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5. RESULTS AND DISCUSSION

5.1. Pharmacognostic studies

Pharmacognostical studies of leaves of Salvadora persica have brought to

light microscopic features as well as preliminary phytochemical data of diagnostic

values.

5.1.1. Anatomical studies

5.1.1.1. Microscopic features of leaf

The sections were prepared as described in materials and methods.The sections of

leaf, lamina, petiole and stem showed the following characteristic features.

Fig. 5.1 Microscopic features of leaf of Salvadora persica

Ads-Adaxial side La- Lamina X-Xylem Ph-Phloem MR-Mesophyll region

The leaf has fairly prominent midrib and thick, smooth and even

lamina.Midrib is slightly raised on the adaxial side and projects into a shallow hump

on the abaxial side.The midrib region is 300 µm thick.It has a single prominent

vascular bundle surrounded by parenchymatous ground tissue.The vascular bundle

has six radial parallel files of xylem elements and a broad pad of phloem elements.

(Fig. 5.1)

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5.1.1.2. Microscopic features of lamina

Fig. 5.2 Microscopic features of lamina of Salvadora persica

LV – Lateral vein AdE-Adaxial epidermis Scl-Scleroid MT-Mesophyll tissue

The lamina is 160 µm thick.It has thin epidermal layers of spindle shaped

cells with thick cuticle.The epidermal cells are less than 10 µm thick.The mesophyll is

not well differentiated into palisade and spongy parenchyma.It consists of several

layers of vertically oblong,compact cells. A few adaxial layers of cells have dense

chloroplast and resemble the palisade cells. Along upper part of the mesophyll tissue,

there are highly dilated wide oblong cells.They do not contain any specific cell

inclusions.There are massive circular clusters of foliar scleroids distributed in the

median part of the mesophyll zone.The vascular bundles of the lateral veins are also

placed in the median zone of the lamina.The vascular bundles are collateral and did

not possess the bundle sheath cells. (Fig. 5.2)

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5.1.1.3. Venation

Fig. 5.3 Venations of Salvadora persica

VT – Vein termination Tsc – terminal scleroid

The lateral veins and vein-branches are fairly thick and straight.They form

wide and distinct vein-islets of polygonal outline.The vein-terminations are long and

slender.The striking character of the veins is the presence of clusters of

brachyscleroids at the tips of the vein-terminations.The terminal scleroids are also

elongated and lobed. (Fig. 5.3)

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5.1.1.4. Microscopic features of petiole

Fig. 5.4 T.S.of petiole of Salvadora persica

Ep – Epidermis Ph – Phloem X – Xylem Sc – Sclerenchyma GT – Ground tissue

The petiole is circular in cross sectional outline and measures 450 µm thick at

the base and 750 µm at the apex.It has thin epidermal layer of small thickwalled and

cuticularised epidermal cells.The ground tissue has 8-10 layers of circular compact

parenchyma cells.The vascular strand has deep are of xylem and phloem with a

narrow adaxial gap or almost closed excentric ring. Xylem consists of circular thick

walled, radial chains of elements. Phloem occurs in wide sheaths around the

xylem.The vascular bundle has discrete nests of sclerenchyma cells around its

periphery. (Fig. 5.4)

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5.1.1.5. Microscopic features of stem

Fig. 5.5 Microscopic features of stem of Salvadora persica

Co - Cortex Iph- Included phloem Pi - Pith Mph – Medullary phloem

Scl - Sclerenchyma SX -Secondary xylem

Fairly old stem of 1.75 mm thick exhibits thick secondary xylem cylinder

with an anomalous feature which is known as included phloem or interxylany

phloem.The stem has narrow,less prominent epidermal layer with thick

cuticle.Cortical zone is wide having several layers of tangentially elongated

parenchyma cells and thick,irregular,scattered masses of sclerenchyma cells.Phloem

zone is narrow and uniformly encircles the xylem cylinder.Secondary xylem is thick

and continuous cylinder of 350 µm width.It consists of circular,thin walled,radial

multiples of two or three vessels,thick walled fibres,wide,straight xylem rays and

tangentially stretched or circular masses of included or interxylany phloem.Primary

xylem is found around the inner boundary of the secondary xylem.Primary xylem

consists of numerous radial files of metaxylem elements and obliterated protoxylem

elements.Adjoining the primary xylem, there are wide nests of pith-phloem or

medullary phloem.The pith is wide and homogeneous with uniform type of circular,

compact parenchyma cells. (Fig. 5.5).

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5.1.2. Powder microscopic observation of Salvadora persica

The leaf powder was prepared as described in materials and methods.The leaf

powder of S.persica exhibits the following characters.

5.1.2.1. Leaf powder

In the leaf powder, small pieces of abaxial and adaxial epidermis of the leaf were

observed.

Fig. 5.6 Abaxial epidermis with densely stomatiferious cyclocytic type of stomata

SC –Subsidiary Cells St – Stomata EC – Epidermal Cells

Abaxial epidermis was found to be densely stomatiferous.The stomata were

cyclocytic type. A stoma was surrounded by two polar and two lateral subsidiary cells

or five subsidiary cells surrounding all around.The epidermal cells are small,

polygonal in outline, thick walled and walls being straight and smooth. (Fig. 5.6)

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Fig. 5.7 Adaxial epidermis with sparse stomata

SC –Subsidiary cells St-Stomata EC – Epidermal cells

The adaxial epidermis has sparse stomata which are also cyclocytic type.The

epidermal cells are polygonal, random in orientation and have thick straight anticlinal

walls. (Fig. 5.7)

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Fig. 5.8 Broken veins – branched Each branch – spherical cluster of

brachyscleroids or stone cells TSc – Terminal Scleroids

The leaf powder also showed broken pieces of veins which were branched.At

tip of each branch there was a spherical cluster of brachyscleroids or stone cells.The

scleroids were also found to be scattered and detached from the veins.The scleroid

clusters are 100×60 µm in size.The individual scleroids are 20×30 µm. (Fig. 5.8)

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Fig. 5.9 Spherical, subsessile glandular trichomes circular, consists of a triangular,

densely staining cells GTr- Glandular Trichomes

Spherical,subsessile glandular trichomes were other type of inclusion in the

powder.The trichome as seen in surface view are circular,consists of a few

triangular,densely staining cells.The gland is 60-70 µm wide. (Fig. 5.9)

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Fig. 5.10 Foliar scleroids

Presence of foliar scleroids is another characteristic feature of the powder.Short

thick and lobed scleroids or long thin unbranched filiform scleroids were observed in

the powder. (Fig. 5.10)

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5.1.2.2. Stem powder

The stem maceration showed calcium oxalate crystals (Fig. 5.11) and different

types of xylem elements.These elements include xylem fibres, xylem parenchyma and

vessel elements.

Fig. 5.11 Prismatic crystals of calcium oxalate under polarized light microscope

Cr- Crystals

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Fig. 5.12 Xylem fibres, xylem parenchyma vessel elements

VE – Vessel Fi – Fibres

Xylem fibres are fairly abundant in the powder.They are long,narrow,thick

walled and narrow lumened.They have arrow of slit-like pits.Their walls are

lignified,They are 70-90 µm long and 10 µm wide.

Xylem parenchyma cells which are rectangular to squarish with thick walls are

also seen in the powder.They are either solitary or in groups.They have simple pits.

There are cylindrical,wide and thick walled vessel elements.They have

simple,wide,circular horizontal perforation plate or oblique perforation plate.The

lateral pits are circular and dense.The vessel elements are 100-160 µm long and 50

µm wide.(Fig. 5.12).

5.1.3. Leaf constants of Salvadora persica

The leaf constants vein islet number, vein terminal number,stomatal

number,stomatal index and palisade ratio were determined as described in

methods.Leaf constants of Salvadora persica are specified in Table 6.1.

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5.1.4. Physical parameters

Physical parameters such as ash values, crude fibre content, extractive values

and loss on drying are also studied as per the procedure described in methods.The

physical parameters of Salvadora persica are presented in Table 6.2.

Table 6.1 Leaf constants of Salvadora persica

Parameters Average values

Vein islet number 13.6

Vein terminal number 16.8

Stomatal number of upper epidermis 41.9

Stomatal number of lower epidermis 52

Stomatal index of upper epidermis 17.4

Stomatal index of lower epidermis 22.3

Palisade ratio 4.03

Table 6.2 Physical parameters of Salvadora persica

Parameters Percentage

Total ash 8.2

Acid insoluble ash 5.2

Water soluble ash 6.3

Sulphated ash 6.9

Crude fibre content 8.2

Alcohol soluble extractive 6.7

Water soluble extractive 7.3

Loss on drying 0.29

The aforementioned anatomical characters are diagnosed for first time in the

leaf of Salvadora persica.These characters can be used to fix the pharmacognostic

standards for Salvadora persica in future. Leaf constants and other physicochemical

parameters are corraborative evidences in standardization of leaf of Salvadora

persica.

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5.1.5. Heavy metal analysis

The leaf powder was digested as described in methods and heavy metal

contents of leaf powder of Salvadora persica was analysed.The permissible limits of

heavy metals in Ayurvedic drugs as per WHO and FDA and heavy metal contents

present in the leaf powder of Salvadora persica are presented in the Table 6.3.

Table 6.3 Heavy metal contents of Salvadora persica

S.No

Name of the

heavy metal

Maximum

permissible

limit

Amount of heavy metal present in the leaf

powder of Salvadora persica(μg/g)

1. Copper Not specified 1.29

2. Cadmium 0.3 μg/g 0.29

3. Lead 10 μg/g 0.12

4. Silver Not specified 0.06

5. Mercury 1 µg/g 0.003

6. Arsenic 103 ng/g NIL

These heavy metals are essential for performing several biological functions in

human body. If these metals are present in high concentrations in blood stream, then

they can accumulate in vital organs and can cause various toxic effects.105, 106 Heavy

metal analysis of Salvadora persica indicates that the heavy metals copper, cadmium,

lead, silver, mercury and arsenic are present within the maximum permissible limit.

Hence, the leaf powder of Salvadora persica will not produce toxic effects while it is

used for pharmacological actions.

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5.2. Pharmacological studies

5.2.1. Extraction of plant material

The leaf powder of Salvadora persica was extracted as described in methods

and the extractive values obtained by successive solvent extraction are given below.

(Table 6.4)

Table 6.4 Extractive values obtained by successive solvent extraction

Solvents Percentage of extractive values

Petroleum ether 1.6

Hexane 2.5

Chloroform 3.2

Ethanol 95% 4.7

Water 5.7

The extractive values indicated that more phytoconstituents are present in

aqueous and alcoholic extracts (95%) than other solvents.

5.2.2. Acute toxicity studies

Acute toxicity studies were carried out as described in materials and methods.

From the acute toxicity studies the LD50 of alcoholic and aqueous extract of leaves of

Salvadora persica were found to be 2000 mg/kg bodyweight (Table 6.5 and

6.6).Accordingly, 200 mg/kg body weight was used as effective dose for both the

extracts. Results of gross behavioral studies in mice on administration of alcoholic

extract of Salvadora persica (2000 mg/kg bodyweight) and aqueous extract of

Salvadora persica (2000 mg/kg bodyweight) are presented in Tables 6.5 and 6.6.

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Table 6.5

Results of gross behavioral studies in mice on administration of alcoholic extract of

Salvadora persica (2000 mg/kg bodyweight)

Observation Effects

Gross activity Up to

3 h

3½ h 4 h 4½ h 5 h 5½ h 6 h 12

h

24

h

Respiration + + + + + + + + +

Writhing - - - - - - - - -

Tremor - - - - - - - - -

Convulsion - - - - - - - - -

Hind limb

paralysis - - - - - - - - -

Sense of touch and

sound + + + + + + + + +

Salivation + + + + + + + + +

Urination + + + + + + + + +

Diarrhoea - - - - - - - - -

Mortality - - - - - - - - -

+ indicates normal

- indicates no effect

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Table 6.6

Results of gross behavioral studies in mice on administration of aqueous extract of

Salvadora persica (2000 mg/kg bodyweight)

Observation Effects

Gross activity Up to

3 h

3½ h 4h 4½ h 5 h 5 ½ h 6 h 12 h 24h

Respiration + + + + + + + + +

Writhing - - - - - - - - -

Tremor - - - - - - - - -

Convulsion - - - - - - - - -

Hind limb

paralysis - - - - - - - - -

Sense of touch and

sound + + + + + + + + +

Salivation + + + + + + + + +

Urination + + + + + + + + +

Diarrhoea - - - - - - - - -

Mortality - - - - - - - - -

+ indicates normal

- indicates no effect

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5.2.3. Assessment of antilithiatic activity

In the present study, male rats weighing between 150 and 200 g were selected

to induce lithiasis because the urinary system of male rats resembles that of humans130

and also earlier studies have shown that the amount of stone deposition in female rats

was significantly less.131

a. Analysis of urine

Urine was collected as described in methods and analysed for potassium,

oxalate, phosphate, calcium and magnesium.Effect of alcoholic and aqueous extracts

of Salvadora persica (200 mg/kg bodyweight) on urinary parameters in control and

experimental animals are presented in Table 6.7.

Urinary supersaturation with respect to stone-forming constituents is

generally considered to be one of the causative factors in calculogenesis. Evidence in

previous studies indicated that in response to 14 day period of ethylene glycol

(0.75%v/v) administration, young male albino rats form renal calculi composed

mainly of calcium oxalate.The biochemical mechanisms for this process are related to

an increase in the urinary concentration of oxalate. Stone formation in ethylene glycol

fed rats is caused by hyperoxaluria which causes increased renal retention and

excretion of oxalate.132, 133 (Table 6.7)

In the present study, oxalate and calcium excretion are progressively increased

in calculi-induced animals (Group II).However, aqueous and alcoholic extract of

leaves of Salvadora persica lower the levels of oxalate as well as calcium excretion.

Since it is accepted that hyperoxaluria is a far more significant risk factor in the

pathogenesis of renal stones than hypercalciuria,the changes in urinary oxalate levels

are relatively much more important than those of calcium.134,135 Increased urinary

calcium is a factor favouring the nucleation and precipitation of calcium oxalate or

apatite(calcium phosphate) from urine and subsequent crystal growth.136Alcoholic and

aqueous extract of Salvadora persica decreased the oxalate and calcium levels and

prevent the nucleation and precipitation of calcium oxalate in curative and preventive

regimens(Group III,IV,V andVI).(Table 6.7)

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An increase in urinary phosphate is observed in calculi-induced rats(Group

II).Increased urinary phosphate excretion along with oxalate stress seems to provide

an environment appropriate for stone formation by forming calcium phosphate

crystals, which epitaxially induces calcium oxalate deposition.137 Treatment of

aqueous and alcoholic extracts of Salvadora persica restores phosphate level to

normal revealing its inhibitory potential over calcium phosphate stone formation in

addition to calcium oxalate stone formation.(Table 6.7)

Magnesium level diminished in group II animals.The alcoholic and aqueous

extract of Salvadora persica increased the magnesium level in curative and preventive

regimens.(Group III,IV,V andVI) Supersaturation, a step in the pathogenesis of

lithiasis, occurs when urinary concentration of chemicals that inhibit stone formation

decreases. Inhibitors of crystallization include magnesium, potassium and

nephrocalcin etc.Low urinary magnesium content is also a common feature during

stone formation138.A similar condition was observed in group II animals. Aqueous

and alcoholic extracts of Salvadora persica elevated urinary magnesium level and

thus, reduced the propensity to crystallize, thereby creating an ambience unfavourable

for precipitation. (Table 6.7)

Table 6.7

Effect of alcoholic and aqueous extracts of Salvadora persica (200 mg/kg bodyweight) on urinary parameters in control and experimental

animals.

All values are expressed as mean ± S.E.M for six animals in each group. (One way ANOVA Tukey’s multiple comparison test.)

+statistically significant at p<0.001 *statistically significant at p<0.05

a Comparisons are made with group I d Comparisons are made with group IV

b Comparisons are made with group II e Comparisons are made with group V

c Comparisons are made with group III f Comparisons are made with groupVI

Parameter

Group I Control

Group II

Calculi induced

Curative regimen Preventive regimen

Group III Group IV Group V Group VI

Alc. ext. (200 mg/kg body wt)

Aq. ext (200 mg/kg body wt)

Alc. ext (200 mg/kg body wt)

Aq. ext. (200 mg/kg body wt)

Potassium (mg/dl) 33.79±0.71 28.60±0.42+a 34.60±0.99*b*d 33.09±0.27*b 33.42±0.5*b 29.53±0.93*b

Oxalate (mg/24 h urine) 0.37±0.01 3.40±0.10+a 0.44±0.01*b*d 1.87±0.02*b 0.54±0.01*b*f 1.77±0.06*b

Phosphate (mg/24 h urine)

4.5±0.36 9.1±0.62+a 3.4±0.58*b 3.3±0.25*b 2.8±0.05*b*f 3.4±0.67*b

Calcium (mg/24 h urine)

0.64±0.04 1.43±0.06+a 0.60±0.03*b 0.75±0.03*b 0.62±0.01*b 0.79±0.02*b

Magnesium (mg/24 h urine)

1.98±0.06 0.71±0.02+a 2.09±0.09*b*d 1.48±0.36*b 1.81±.0.20*b 1.75±0.05*b

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b. Serum analysis

Serum was collected from retro-orbital vein as described in methods and

analysed for urea, creatinine and uricacid. Serum urea, creatinine and uricacid in

control and experimental animals are given in Table 6.8.

In calculi-induced rats (group II), marked renal damage was seen as indicated

by the elevated serum levels of urea, creatinine and uricacid. In urolithiasis, the

glomerular filtration rate (GFR) decreases due to the obstruction to the outflow of

urine by stones in urinary system. Due to this, the waste products, particularly

nitrogenous substances such as urea, creatinine and uricacid get accumulated in

blood.139 However, the curative and preventive treatment with aqueous and alcoholic

extracts of Salvadora persica significantly lowered the serum levels of accumulated

waste products which is attributed to the enhanced GFR. (Table 6.8)

Table 6.8 Serum urea, creatinine and uricacid in control and experimental animals

All values are expressed as mean ± S.E.M for six animals in each group. (One way ANOVA followed by Tukey’s s multiple comparison test.)




c Comparisons are made with group III f Comparisons are made with groupVI

Parameter (Unit)

Group I

Control

Group II

Calculi

induced



Alc. ext.

(200 mg/kg body wt)

Aq. ext.

(200 mg/kg body wt)

Alc. ext.

(200 mg/kg body wt)

Aq. ext.

(200 mg/kg body wt)

Urea(mg/dl) 15.62±0.44 43.15±0.65+a 24.25±0.28*b 31.25±1.70*b 27.7±1.31*b 29.3±0.05*b

Creatinine (mg/dl) 0.78±0.02 3.73±0.10+a 1.3±0.14*b 1.21±0.12*b 1.57±0.05*b 1.7±0.06*b

Uric acid(mg/dl) 5.27±0.12 8.67±0.08+a 3.58±0.08*b 3.47±0.12*b 4.65±0.15*b 4.66±0.04*b

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c. Kidney and liver homogenate analysis

Liver and kidney homogenates were prepared as described in methods and the

enzymic and non-enzymic antioxidants levels were estimated.

In the present study, significantly increased activities of GAO in liver and

LDH in liver and kidney of ethylene glycol induced urolithic rats were

observed(Group II).Liver and kidney act as the main sites of endogenous oxalate

synthesis. Ethylene glycol disturbs oxalate metabolism by way of increase the

substrate availability that increases the activity of oxalate synthesizing enzymes in the

rats. GAO and LDH catalyses the coupling of oxidation and reduction of glyoxalate

results in the formation of glycolate and oxalate.13This is due to substrate mediated

induction of the enzymes. A similar increase was also observed in glyoxalate,

pyridoxine deficient diet and glycolate administered rats.140-142 Administration of

aqueous and alcoholic extracts of Salvadora persica brought about a significant

reduction in GAO activity in liver and LDH activity in liver and kidney of extracts

treated rats both in curative and preventive regimen(Group III,IV,V andVI).(Tables

6.9 and 6.10)

Increased activity of GAO and LDH confirmed their direct link to

endogenous oxalate deposition in ethylene glycol induced urolithiasis. Administration

of aqueous and alcoholic extracts of Salvadora persica reduced the oxalate level in

liver and kidney of urolithic rats.

The biochemical mechanism by which various factors lead to initiation of

calcium oxalate stone formation is still not known. However, free radical production

has been known to be intricately involved in the process of crystal deposition in the

tissues. Oxalate, the major stone forming constituent, has also been reported to induce

lipid peroxidation and to cause tissue damage by reacting with polyunsaturated fatty

acids in cell membranes.143Similarly, the kidney and liver of the lithogenic rats(group

II,Tables 6.9 and 6.10) exhibited an increased level of lipid peroxides measured as

TBA reactive substances. Upon treatment with aqueous and alcoholic extract of

Salvadora persica (groups III, IV, V and VI), the level of lipid peroxides were found

to be restricted. This is attributed to the ability of the extracts to reduce the level of

oxalate supersaturation in the tissues. Mitigation of LPO in turn rectifies the stress on

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the antioxidant potency, which leads to normalization of non-enzymic antioxidant

content and the activities of different antioxidant enzymes in kidney and liver.

(Tables 6.9 and 6.10)

Though there is induction of lipid peroxidation by the prooxidants under

different pathological conditions, the level of LPO in cells is controlled by various

cellular defence mechanisms consisting of enzymatic and non-enzymatic

scavengers.144The physiological defense strategy appears to be a complex process

involving a large number of components. These cellular antioxidants are armored with

the capacity to deal with the reactive radical species produced by normal metabolic

process.145 (Tables 6.9 and 6.10)

In the present study, the level of glutathione (GSH), an important reducing

power capable of reducing oxidized tissue components was found to be diminished in

calculogenic rats.(Group II,Table 6.9 and 6.10). A marked reduction in the level of

GSH in the kidney and liver of group II animals might be responsible for the abysmal

state of the other antioxidants like vitamin E and vitamin C.This is attributed to the

increased utilization of GSH during oxalate-induced oxidative stress. Although a

relationship is not established there is an inverse correalation between the lower

content of GSH and higher level of peroxides.146Accordingly, in this study both

alcoholic and aqueous extract diminished lipid peroxidation in the tissues and

increased GSH levels significantly.(Tables 6.9 and 6.10)

As seen from Tables 6.9 and 6.10 the levels of vitamin C and vitamin E were

also found to be reduced drastically in the tissues of calculogenic rats.(Group

II)Vitamin C and vitamin E act synergistically through the interactions between water

and lipid-soluble substances by both non-enzymatic and enzymatic mechanisms to

confer protection in tissues and membranes against oxidative damage.147 The loss of

these vitamins virtually depict the extent of potential peroxidative assault caused by

increased concentration of oxalate in the system. The tissues of animals treated with

the alcoholic and aqueuos extract of leaves of Salvadora persica showed significantly

higher level of these vitamins along with GSH, which exhibit the protective effect of

the extracts against calcium- oxalate induced tissue damage. (Tables 6.9 and 6.10)

87

SOD (superoxide dismutase) and glutathione (GSH) decreased in group II

animals. (Table 9 and 10).The alcoholic and aqueous extracts of Salvadora persica

increased the levels of SOD and GSH in curative and preventive regimens (Group III,

IV, and VandVI). Physiologically,the superoxide dismutase(SOD) and the glutathione

system are the primary contributors of the cellular antioxidant capacity.148SOD which

is responsible for dismutation of reactive radicals was found to be significantly

lowered in the tissues of calculogenic rats.(Tables 6.9 and 6.10).Such a change may

be attributed to the oxidative assault rendered by increased oxalate content in the

tissues.149 In the group of animals supplemented with the extracts, its activity was

found to be markedly increased when compared with kidney and liver of lithogenic

animals.(group II,Table 6.9 and 6.10)

The activity of glutathione peroxidase (GPx), the first line of defence against

membrane damaging peroxidative assault was found to be inhibited in both kidneys

and livers of calculogenic rats. Alcoholic and aqueous extract of Salvadora persica

elevated GSH and GPx. Kidneys, being most vulnerable tissue to damage by lipid

peroxides, has been shown to exhibit drastic alteration in the antioxidant capacity

during peroxidative changes.Inhibition of GPx, which disposes of cellular H2O2 by

utilising GSH as co-factor, might be due to depletion of GSH along with the high

degree of peroxides being formed.150The animals treated with alcoholic and aqueous

extract of Salvadora persica increased GPx and GSH and disposed cellular hydrogen

peroxide. (Table 6.9 and 6.10)

Table 6.9 Effect of alcoholic and aqueous extracts of Salvadora pesica in kidney enzymes

Parameter

(Unit)

Group I

Control

Group II

Calculi induced


Group III Group IV Group V Group VI Alc.ext.

(200 mg/kg body wt)

Aq. ext

(200 mg/kg body wt)

Alc. ext

(200 mg/kg body wt)

Aq. ext.

(200 mg/kg body wt)

LDH(units/mg protein) 2.0±0.23 3.51±0.28+a 2.23±0.20*b*d 2.63±0.19*b 2.24±0.2*b*f 2.50±0.2*b

GPx(µg/mg protein) 8.6±0.32 5.3±0.06+a 8.55±0.49*b,d 6.1±0.25*b 7.45±0.4*b*f 6.12±0.3*b

Oxalate(mg/g tissue) 1.3±0.04 2.8±0.38+a 1.43±0.27*b 1.73±0.35*b 1.29±0.2*b 1.64±0.0*b

LPO(n mols) 1.9±0.06 5.4±0.21+a 2.2±0.18*b*d 3.7±0.17*b 3.14±0.0*b 3.52±0.14*b

Vit C((µg/mg protein) 2.05±0.24 1.2±0.06+a 1.94±0.09*b 1.7±0.01*b 2.01±0.0*b 1.81±0.1*b

Vit E(µg/mg protein) 1.09±0.04 1.03±0.05+a 2.01±0.04*b 1.09±0.13*b 1.08±0.0*b 1.06±0.0*b

SOD(unit/mg protein) 5.9±0.20 3.2±0.31+a 4.4±0.25*b 4.3±0.09*b 4.5±0.12*b 4.2±0.17*b

GSH(µg/mg protein) 4.7±0.58 3.5±0.21+a 5.8±0.12*b 4.2±0.14*b 4.01±0.12*b 4.03±0.2*b

All values are expressed as mean ± S.E.M for six animals in each group. (One way ANOVA followed by Tukey’s multiple comparison test.)




c Comparisons are made with group III f Comparisons are made with group VI

88

Table 6.10

Effect of alcoholic and aqueous extract of Salvadora persica on liver enzymes of control and experimental animals






Parameter

(Unit)

Group I Control

Group II Calculi induced

Curative regimen Preventive regimen Group III Group IV Group V Group VI Alc. ext.

(200 mg/kg body wt)Aq. Ext

(200 mg/ kg body wt) Alc. ext

(200 mg/kg body wt)Aq. ext.

(200 mg/kg body wt) SOD(unit/mg protein) 7.82±.0.63 5.32±0.41+a 6.5±0.65*b*d 5.9±0.5*b 6.1±0.34*b 5.90±0.61*b

GSH(µg/mg protein) 12.85±0.95 11.35±0.8+a 12.01±.014*b 11.92±0.82*b 12.75±0.14*b*f 12.91±0.15*b

GPx(µmols) 10.11±0.81 7.91±0.75+a 9.55±0.21*b*d 8.75±0.07*b 9.01±0.17*b*f 8.73±0.64*b

LDH(units/mg protein) 2.04±0.23 4.02±0.18+a 2.02±0.67*b 3.05±0.16*b 2.05±0.62*b*f 3.02±0.92*b

Oxalate(mg/g tissue) 1.63±0.24 2.49±0.10+a 1.89±0.05*b 1.78±0.29*b*c 1.57±0.51*b*f 1.89±0.77*b

GAO(nmols) 2.9±0.21 3.25±0.39+a 1.75±0.06*b 2.15±0.35*b 2.12±0.47*b 2.10±0.9*b 7

LPO(nmols) 3.11±0.11 5.21±0.53+a 1.78±0.17*b*c 2.84±0.08*b 3.36±0.11*b 2.82±0.08*b

Vit C(µg/mg protein) 3.21±0.27 0.74±0.15+a 1.21±0.06*b 1.17±0.08*b 1.32±0.02*b 1.24±0.06*b

Vit E(µg/mg protein) 1.92±0.20 1.56±0.06+a 1.65±0.01*b 1.67±0.07*b 1.68±0.07*b 1.62±0.10*b

89

90

Since oxalate itself has been reported to incite tissue damage, the antioxidant

protective mechanisms of cells and tissues are disturbed when oxalate induced

oxidative stress evade or overwhelm the cellular balance of pro and antioxidants.

Hence, under such conditions control over the urinary concentration of oxalate and

further crystallization process seems to be only way out. The alcoholic and aqueous

extractof leaves of Salvadora persica are capable of achieving this object.

It is evident from biochemical parameters, that alcoholic extract offer

remarkable protection against lithiasis than aqueous extract. Hence, the compounds

present in the alcoholic extract of leaves of Salvadora persica were isolated.

d. Histopathological findings

The sections of kidney and liver were prepared as described in methods and

observed under microscope.

91

Histopathology of kidney and liver

Fig. 5.13 Section of kidney of control rats showing normal cellular structure

Fig. 5.14 Liver of control rats showing normal cellular structure

Fig. 5.15 Kidney of ethylene glycol treated rats

showing dilated tubules and degeneration of epithelial lining.

Fig. 5.16 Liver of ethylene glycol treated rats showing degeneration of epithelial

lining.

92

Fig. 5.17 Kidney of group III rats showing reduction in the features mentioned in figure III.

(Curative regimen)

Fig. 5.18 Liver of group III rats showing reduction in the features mentioned in figure III.

(Curative regimen)

Fig. 5.19 Kidney of group IV rats showing near normal renal structure.(Curative

regimen)

Fig. 5.20 Liver of group IV rats showing reduction in the features mentioned in fig

IV (Curative regimen)

93

Fig. 5.21 Kidney of groupV rats showing normal tubules and epithelial linings(Preventive regimen)

Fig. 5.22 Liver of groupV rats showing reduction in features mentioned in figure

V (Preventive regimen)

Fig. 5.23 Kidney of groupVI rats showing near normal renal

structure.(Preventive regimen)

Fig. 5.24 Liver of group VI rats showing normal liver structure.(Preventive regimen)

94

The histopatological studies showed the presence of microcrystals in the

kidney and liver samples from lithiatic control group II,confirming stone formation.In

these sections, epithelial cells were damaged and tubules were dilated.Inflammatory

cells were present in intersitial spaces, as shown in Figures 5.15 and 5.16.All these

features were reduced after treatment with the alcoholic and aqueous extract of leaves

of Salvadora persica both in curative and preventive regimen as evident in Figures

5.17-5.24.The kidney and liver section from normal control rats are shown in Figures

5.13 and 5.14.No dilatation of tubules was observed,and the epithelial lining was

intact in these figures.

5.2.4. Mechanism of action of alcoholic extract of Salvadora persica in the

treatment of lithiasis.151, 152,153

Mechanism of stone induction by ethylene glycol (0.75% w/v)

Alcohol

Ethylene glycol Glycoaldelyde

Dehydrogenase

Oxidation

Glyoxal Oxalic acid Glyoxalic acid

Ethylene glycol is converted to glycoaldehede by the enzyme alcohol

dehydrogenase.Glyoxalicacid is formed by oxidation of glycoaldehyde. Oxalicacid is

formed on further oxidation of glyoxalicacid. Hence, stone formation in ethylene

glycol fed rats is induced by hyperoxaluria which increases renal retention and

excretion of oxalate. The alcoholic extract of leaves of Salvadora persica significantly

reduced the renal content of stone forming constituents (oxalate, calcium and

phosphate) in both preventive and curative regimens and increased the magnesium

level which is inhibitor of crystallization. Magnesium prevents sticking of crystals

into the kidney tubules.

95

Ethylene glycol disturbs oxalate metabolism by way of increase the substrate (oxalate) availability that increases the activity of oxalate synthesizing enzymes (GAO and LDH) in the rats. GAO and LDH catalyses the coupling of oxidation and reduction of glyoxalate results in the formation of glycolate and oxalate. Administration of alcoholic extract of Salvadora persica brought about a significant reduction in GAO activity in liver and LDH activity in liver and kidney of urolithic rats.

In urolithiasis the glomerular filteration rate (GFR) decreases due to the obstruction to the outflow of urine by stones in urinary system. Due to this the waste products, particularly nitrogenous substances such as urea, creatinine and uricacid get accumulated in blood. The curative and preventive treatment with alcoholic extract of Salvadora persica lowered the serum levels of accumulated waste products (urea, creatinine and uricacid) which is attributed to the enhanced GFR of extract.

Oxalate has been reported to induce lipid peroxidation and to cause renal tissue damage by reacting with polyunsaturated fattyacids in cell membrane.Since oxalate itself has been reported to incite tissue damage, the antioxidant protective mechanisms of cells and tissues are disturbed when oxalate induced oxidation stress evade or overwhelm the cellular balance of pro and antioxidants. Kidneys being most vulnerable tissue to damage by lipid peroxides have been shown to exhibit drastic alteration in the antioxidant capacity during peroxidative changes.

Physiologically the superoxide dismutase (SOD) and glutathione system are the primary contributors of cellular antioxidant capacity.GSH is an important reducing power capable of reducing oxidized tissue components. There is an inverse correlation between the lower content of GSH and higher level of peroxides. Also vitamin C and E act synergistically through the interactions between water and lipid soluble substances by both non enzymatic and enzymatic mechanisms to confer protection in tissues and membranes against oxidative damage.

The levels of these enzymes (SOD, GSH) were found to be reduced drastically in the tissues of calculongenic rats. The tissues of animals treated with the extract showed significantly higher levels of these enzymic antioxidants along with vitamin C and E, which exhibit the protective effect of alcoholic extract of leaves of Salvadora persica against calcium oxalate induced tissue damage.

96

In conclusion, the mechanism underlying the antilithiatic activity of alcoholic

extract of leaves of Salvadora persica involve,

1. The alcoholic extract of leaves of Salvadora persica increases the magnesium

level, which is inhibitor of crystallization and decreases the stone forming

constituents. (calcium,oxalate and phosphate)

2. GFR rate is increased by the extract.

3. The extract decreases the levels of GAO and LDH which are oxalate synthesizing

enzymes.

The extract also inhibits the oxalate induced toxic manifestations and free radical

production.

5.3. Phytochemical studies

5.3.1. Preliminary phytochemical studies

Preliminary phytochemical studies were done as described in methods.The

leaf powder has no characteristic odour and bitter taste.

5.3.2. Qualitative chemical examination of leaf powder

Qualitative chemical examination of leaves of Salvadora persica revealed that

sterols, terpenoids, flavonoids, alkaloids, saponins and volatile oil are present in leaf

powder of Salvadora persica.

5.3.3. Column chromatography

Column chromatography was carried out as described in materials and

methods using alcoholic extract of Salvadora persica.Fraction 5 and 9 gave single

spot in TLC.

97

5.3.4. HPTLC of fractions 5 and 9

Furthur fraction 5 and 9 were subjected to HPTLC as explained in materials

and methods.In HPTLC both fractions recorded single peaks. (Figures 5.25 and 5.26)

Fig. 5.25 HPTLC of 5th fraction of alcoholic extract of leaves of Salvadora persica

Fig. 5.26 HPTLC of 9th fraction of alcoholic extract of leaves of Salvadora persica

98

5.3.5. Characterisation of compounds obtained from fraction 5 and fraction 9

The compounds obtained from fraction 5 and 9 were characterized by UV,

IR, NMR and Mass spectroscopical studies.

a. Charaterisation of compound obtained from Fraction 5

Colourless needles from methanol

Yield: 0.0018%

M.P:251-252οC

Fig. 5.27 UV spectrum of pure compound obtained from 5th fraction of alcoholic

extract of Salvadora persica

λmax

λ max

99

Fig. 5.28 IR spectrum of pure compound obtained 5th fraction of alcoholic extract of

Salvadora persica

100

Fig. 5.29 1H NMR of pure compound obtained from 5th fraction of alcoholic extract

of Salvadora persica.

101

Fig. 5.30 13C NMR of pure compound obtained from 5th fraction of alcoholic


102

Fig. 5.31 Mass spectrum of pure compound obtained from 5th fraction of

alcoholic extract of leaves of Salvadora persica

UV גmax (M

IR (KBr):

3373cm-1,31H NMR (C

δ 4.81(m,H13C-NMR (

δ 39.3(C-1)

EIMSm/z:

442[M+](70

191(5).

From

standard co

The structu

Fig. 5.32 S

b. Charate

Colou

Yield:

M.P:1

MeoH):240 n

3006cm-1,29

CDCl3, 300

H2-29),1.75(b

(CDCl3, 300

),37.4(C-10

0),424[M-H

the spectral

ompound th

ure of betuli

Structure of

erisation of

urless needle

0.040%

97-198 οC

nm and 400

961cm-1,285

MHz):

br.s,H3-30),

0 MHz):

0),27.15(C-1

H2O]+(100),4

l data (Figu

he compoun

n is given in

f betulin

f compound

es.

nm.

54cm-1,1712

,0.92(s,H3-2

15),29.1(C-

409[M-33]+

ures 5.27-5.

nd present i

n Fig. 5.32

d obtained

2cm-1,1610c

24),0.87(s,H

16),29.6(C-

+(20),234(13

31) and com

in fraction

from fracti

cm-1,1460cm

H3-23)

-21),19.7(C-

3),220(41),2

mparison w

5 was iden

ion 9

m-1,973cm-1

-29).

207(10)

with spectral

ntified as be

103

1

and

l data of

etulin154.

104

Fig. 5.33 UV spectrum of pure compound obtained from 9th fraction of alcoholic

extract of leaves of Salvadora persica

λ max

105

Fig. 5.34 IR spectrum of pure compound obtained from 9th fraction of alcoholic


Fig. 5.35 1

13C NMR off pure comp

of leav

pound obtai

ves of Salv

ined from 9

vadora pers

9th fraction o

ica

of alcoholic

106

c extract

Fig. 5.36

1H NMR off pure comp

of lea

pound obtai

aves of Salva

ined from 9t

adora persi

th fraction o

ica

of alcoholic

107

extract

Fig. 5.37 M

extract of le

Mass spect

eaves of Sa

trum of pur

lvadora per

re compoun

rsica

nd obtained from 9th frraction of a

108

alcoholic

109

UV ג max (MeoH): 240 nm

IR Vmax (KBr):

3302 cm-1, 2954 cm-1,2854 cm-1,1733 cm-1,1606 cm-1 1H NMR (CDCl3, 300 MHz):

δ 5.11(m,H-12),1.02(s,H3-26), 0.99(s,H3-25), 0.91(s,H3-28),0.89(s,H3-29 and H3-30). 13C NMR (CDCl3, 300 MHz) :

δ 77.3(d,C-3),29.2(t,C-15),31.8(t,C-21),22.6(q,C-29).

EIMS m/z:

426[M]+(15),411[M-Me]+(18),408[M-H2O]+(16),393[M-Me-H2O]+32,257[M-

C11H210]+(20).218[M- C14H280]+(100),207[M-C16H27O]+(10),203[M-C15H27O]+(40)

and 89[M-C16H29O]+(55).

From the spectral studies (Figures 5.33-5.37) and comparison with spectral data

of standard compound the compound present in fraction 9 was identified as β-

amyrin154. The structure of β-amyrin is given in Fig. 5.38.

Fig. 5.38 Structure of β-amyrin.

5.4. Antilithiatic activity of β-amyrin

By the column chromatography, it is known that less amount of betulin

(0.0018%) is present in the leaf powder.of Salvadora persica. Thus, β-amyrin was

evaluated for antilithiatic activity.

110

5.4.1. Acute toxicity studies of β-amyrin

The acute toxicity studies as per OECD 423 revealed that the LD50 of β-amyrin

was found to be 200 mg/kg body weight.Hence the effective dose of 20 mg/kg body

weight was used. (Table 6.11)

Table 6.11

Results of gross behavioral studies in mice on administration of β-amyrin (200 mg/kg

body weight)

Observations Effects

Gross activity Up to 3 h 3 ½ h 4 h 4½

h

5 h 5½

h

6 h 12 h 24 h

Respiration + + + + + + + + +

Writhing - - - - - - - - -

Tremor - - - - - - - - -

Convulsions - - - - - - - - -

Hind limb paralysis - - - - - - - - -

Sense oof touch and sound + + + + + + + + +

Salivation + + + + + + + + +

Urination + + + + + + + + +

Diarrhoea - - - - - - - - -

Mortality - - - - - - - - -

+ indicates normal.

- indicates no effect.

111

5.4.2. Assessment of antilithiatic activity

a. Urine analysis

Chronic administration of 0.75%v/v ethylene glycol to male Wistar rats

resulted in hyperoxaluria. Oxalate, calcium and phosphate excretion progressively

increased in group II animals.Magnesium and potassium excretion gradually

decreased in calculi induced rats. The changes in the concentration of aforementioned

ions are incriminated in stone formation in group II animals. β-amyrin enhanced

magnesium excretion, which is inhibitor of crystallisation and decreased the excretion

of calcium,oxalate and phosphate.(Table 6.12)

Table 6.12 Effect of β-amyrin on urinary parameters



c Comparisons are made with group III f Comparisons are made with group V

All values are expressed as mean ± S.E.M for six animals.One way ANOVA followed by Tukey’s multiple comparison test.


Parameters Group I Normal control

Group II Lithiatic control



β-amyrin 20 mg/kg bodyweight




Calcium (mg/24 h urine) 1.27±0.07

1.51±0.01+a

1.28±0.06*b

1.18±0.12*b

1.36±0.07*b

1.17±0.08*b

Oxalate (mg/24 h urine)

0.37±0.03

3.64±0.11+a

0.95±0.04*b

0.85±0.06*b

1.10±0.08*b

0. 83±0.03*b

Phosphate (mg/24 h urine)

6.87±0.11

11.77±0.04+a

8.30±0.09*b

9.10±0.08*b

8.50±0.06*b

8.01±0.06*b*e

Magnesium (mg/24 h urine)

0.92±0.01

0.50±0.01+a

0.63±0.01*b

0.84±0.01*b*c

0.61±0.08*b

0.79±0.02*b*e

Potassium (ppm) 3370±0.71

2810±0.2+a

3300±0.21*b

3420±0.99*b*c

3750±0.9*b

4340±0.5*b*d*e

112

113

b. Serum analysis

The stone formation damaged the kidney sufficiently leading to an elevated

serum level of urea, creatinine and uricacid in group II animals. However, the curative

and preventive treatment with β-amyrin diminished the level of urea, creatinine and

uricacid in serum by enhancing the glomeruler filtration rate. (Table 6.13)

Table 6.13 Effect of β-amyrin on serum parameters of control and experimental

animals

Table 6.13 effect of β-amyrin on serum parameters of control and experimental animals


+Statistically significant at p<0.001 *Statistically significant at p<0.05



c Comparisons are made with group III f Comparisons are made with group V

Parameters

Group I

Control

Group II

Calculi

induced



β-amyrin 20 mg/kg

body weight

β-amyrin

40 mg/kg

bodyweight

β-amyrin

20 mg/kg

bodyweight

β-amyrin

40 mg/kg

bodyweight

Urea(mg/dl)

45±1.8

55±0.51+a

27±3.8*b

16.01±2.01*b*c

15±1.02*b

14.1±1.04*b*e

Creatinie(mg/dl)

1.49±0.07

3.1±0.11+a

2.10±0.06*b

0.81±0.04*b*c

2.06±0.05*b

1.94±0.04*b*e

Uric acid(mg/dl)

0.75±0.01

3.6±0.03+a

2.72±0.01*b

1.11±0.04*b

0.91±0.02*b

0.85±0.0*b *e

114

115

c. Kidney and liver homogenate analysis

In the present study, there is an upsurge in LPO in kidney and liver of rats

during hyperoxaluric condition. The kidney being most vascular in nature, is more

susceptible to toxic effect of lipid peroxides secondary to erythrocyte

membrane155.LPO, a degenerative pathway of the membrane components mediated

through the free radicals produced in the cell, is a hallmark feature of oxidative stress.

Decrease in antioxidant enzymes (GSH, GPx and SOD) might also be patrly

attributed to the elevation in LPO. Abnormal rise in LPO was reverted to near

normalcy by β-amyrin. (Tables 6.14 and 6.15)

The enzymic antioxidants GPx, GSH, SOD and nonenzymic antioxidants

vitamin C and vitamin E decreased in group II animals. β-amyrin augmented the

enzymic and non enzymic antioxidant levels both in curative and preventive

regimens. Reduction in the activity of GPx is due to the decreased availability of its

substrate GSH and partly due to its inhibition by superoxide radicals accumulated due

to the decreased activity of SOD.GSH reduction can additionally explain a decrease

concentration of non-enzymic antioxidant vitamin C which enters the cell mainly in

the oxidized form, where it is reduced by GSH.The diminution of this vitamin is

detrimental, because additionally to its antioxidant function, vitamin C plays a role in

sparing other antioxidants like vitamin E.The decrease in the levels of these vitamins

increases the risk of LPO.This correlates with previous observations where induction

of hyperoxaluria causes a significant decrease in non-enzymic antioxidants.156. β-

Amyrin refurbish the levels of enzymic and nonenzymic antioxidants.(Table 6.14 and

6.15)

Oxalate synthesizing enzymes (GAO and LDH) is amplified in the present

study, which is due to increased availability of their substrate. GAO, catalyses the two

step oxidation of glycolate to oxalate, with glyoxalate an intermediate.This enzyme is

localized in the liver and its activity is found to be increased during hyperoxaluria157.

LDH, a cytosolic enzyme, catalyses the coupling of oxidation and reduction of

glyoxalate,present in kidney and liver was also increased during hyperoxaluria158.The

increase in oxalate metabolizing enzymes was circumvented with administration of

β-amyrin.All these observations indicates that β-amyrin is endowed with

antiurolithiatic activity.(Table 6.14 and 6.15)

Table 6.14 Effect of β-amyrin on kidney parameters of rats intoxicated with ethylene glycol

Parameters

Group I Control

Group II

Calculi induced

Curative regimen Preventive regimen Group VI Group III Group IV Group V


β-amyrin 40 mg/ kg bodyweight



GSH(µg/mg protein) 4.84±0.07 2.45±0.21+a 3.87±0.21*b 4.24±0.14*b*c 3.91±0.12*b 4.45±0.23*b*e

GPx((µg of GSH utilized/min/mg protein)

8.90±0.25 5.90±0.06+a 7.20±0.25*b 8.25±0.49*b*c 7.57±0.36*b 8.45±0.07*b*e

LDH(units/mg protein) 2.09±0.20 5.15±0.23+a 3.74±0.22*b 2.93±0.24*b 3.42±0.19*b 2.85±0.24*b*e Vitamin C (µg/mg protein)

1.56±0.01 0.62±0.06+a 1.21±0.01*b 1.25±0.08*b 1.25±0.03*b 1.37±0.14*b*e

Vitamin E(µg/mg protein)

1.76±0.08 1.02±0.05+a 1.52±0.03*b 1.90±0.12*b*c 1.40±0.01*b 1.41±0.03*b

LPO(nmol of MDA formed/mg protein)

2.01±0.05 4.41±0.21+a 3.21±0.16*b 2.81±0.18*b*c 3.52±0.13*b 2.92±0.07*b*e

SOD(units/mg protein) 5.60±0.23 3.52±0.17+a 4.42±0.25*b 5.22±0.09*b*c 4.78±0.78*b 5.01±0.11*b

All values are expressed as mean ± S.E.M for six animals in each group. (One way ANOVA followed by Tukey’s multiple comparison test.) +Statistically significant at p<0.001 *Statistically significant at p<0.05 a Comparisons are made with group I d Comparisons are made with group IV b Comparisons are made with group II e Comparisons are made with group V c Comparisons are made with group III f Comparisons are made with groupVI

116

Table 6.15 Effect of β-amyrin on liver enzymes of control and experimental animals

All values are expressed as mean ± S.E.M for six animals in each group. (One way ANOVA followed by Tukey‘s multiple comparison test.)

+Statistically significant at p<0.001 *Statistically significant at p<0.05




Parameters

Group I Control

Group II Calculi induced

Curative regimen Preventive regimem Group VI Group III Group IV Group V

β-amyrin 20 mg/kg

bodyweight




LPO(nmol of MDA formed/mg protein)

3.10±0.11 8.4±0.53+a 2.94±0.08*b 2.12±0.17*b 3.27±0.07*b 2.30±0.11*b*e

GSH((µg/mg protein) 13.01±0.92 11.09±0.18+a 12.07±0.13*b 12.94±0.82*b*c 13.04±0.15*b 13.45±0.40*b*e

GPx(µg of GSH utilized/min/mg protein)

10.12±0.83 7.75±0.05+a 8.25±0.21*b 9.56±0.07*b*c 9.34±0.64*b 10.56±0.17*b*e

GAO(nmol of glyoxalte formed/mg protein)

2.86±0.24 4.95±0.10+a 3.84±0.05*b 3.64±0.29*b 2.98±0.77*b 2.76±0.10*b

LDH(units/mg protein) 2.34±0.20 5.44±0.23+a 4.74±0.22*b 4.53±0.24*b 3.01±0.19*b 2.28±0.24*b*c*d VitaminC((µg/mg protein) 3.21±0.01 2.84±0.06+a 3.22±0.01*b 3.24±0.08*b 3.32±0.03*b 3.34±0.01*b Vitamin E(µg/mg protein) 2.11±0.08 1.02±0.05+a 1.52±0.03*b 1.90±0.12*b*c 1.40±0.01*b 1.41±0.03*b

SOD(units/mg protein) 8.12±0.05 6.02±0.21+a 7.61±0.16*b 8.32±0.18*b*c 7.85±0.13*b 8.9±0.07*b*e

117

118

Comparison of biochemical parameters of alcoholic extract of leaves of

Salvadora persica and β-amyrin depicted that alcoholic extract is more active than β-

amyrin.This is attributed to the presence of β-amyrin along with other compounds in

the alcoholic extract. (Figures 4.1 to 4.13)

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