CHAPTER 4 RESULTS AND DISCUSSIONS - …shodhganga.inflibnet.ac.in/bitstream/10603/15501/9/09_chapter 4.pdf · 59 Purkayastha et al (2008) and Hemalatha and Vadivel (2010). In contradiction

58

CHAPTER 4

RESULTS AND DISCUSSIONS

4.1 MICROPROPAGATION

4.1.1 Explant Selection

Selection of suitable explants is an important aspect for establishing

a successful regeneration system (Lorraine 1990). Therefore, shoot tips and

nodal explants were selected from eight months old A.lineata ex vitro raised

plant because of its promising agronomic characteristic, high proliferation

efficiency and maintenance of clonal fidelity (Vijaya and Padmaja 1999).

Plant regeneration from shoot tips and nodal explants has yielded encouraging

results in medicinal plants like Catharanthus roseus, Cinchona ledgeriana

and Digitalis spp, Rehmannia glutinosa, Rauvolfia serpentine, Isoplexis

canariensis (Roy et al 1994; Paek et al 1995 and Perez-Bermudez et al 2002).

4.1.2 Effect of Cytokinins in Shoot Induction

Cytokinins in general favor in vitro shoot proliferation (Thrope

1993). Cytokinins naturally fall into the category of N6-isopentyl adenine

derivatives. Unlike the purine derivatives, there exist other synthetic diphenyl

ureas that are even more active than their counterparts especially purines

(Thomas and Katterman 1986). Successful regeneration protocol for

Andrographis paniculata and Andrographis echoides in MS medium

containing only cytokinins for shoot induction have already been reported by

59

Purkayastha et al (2008) and Hemalatha and Vadivel (2010). In contradiction

to the present study, MS medium containing BAP, KN, TDZ and 2-iP at 0.5-

2.5 mg/l were fortified to study the efficiency of shoot induction which

showed slow emergence and insignificant proliferation of shoots in both the

explants (Data not shown).

4.1.3 Effect of AdS in Multiple Shoot Induction

AdS provides an available source of nitrogen to cell and can be

taken up more rapidly than inorganic nitrogen (Thom et al 1981). The benefits

of adenine are often noticed only when it is associated with ammonium nitrate

or cytokinins such as BAP or KN (Van et al 2008). Hence, the shoot

proliferation efficiency was improved by supplementation of AdS in

combination with cytokinins for promotion of adventious shoot formation in

the study. There was quick emergence of shoot bud from the shoot tip

explants after the 5th

day of inoculation, whereas, the nodal segments

responded only on the 8th

day. Frequency of adventious shoot formation was

high (25.7 ± 0.11 per explant) (Figure.4.1 a and b) within four weeks in the

shoot tip explants at an optimized concentration of 1.5 mg/l BAP along with

30 mg/l AdS compared to the nodal explants (15.0 ± 0.19 shoots per explant)

(Figure 4.1 c and d) (Table 4.1). Thus, BAP when added along with AdS

exhibited synergetic effect and improved the cell proliferation efficiency.

Similar strategy in using AdS as an adjuvant has also been adopted effectively

for many other plant species such as Holarrhena antidysenterica (Raha and

Roy 2001), Curcuma angustifolia (Shukla et al 2007) and Bacopa monnieri

(Ramesh et al 2006).

60

Table 4.1 Effect of various concentrations of cytokinins supplemented

with 30.0 mg/l AdS for multiple shoot induction in shoot tip and

nodal explants of A. lineata

PGR

(mg/l)

Explant source

Shoot tip Nodal segment

Response

(%)

Average

No. of

shoot per

explants

Mean

shoot

length

(cm)

Response

(%)

Average No.

of shoot per

explants

Mean

shoot

length

(cm)

BAP

0.5 85cd

8.5±0.13ef

2.4±0.02a

60ij

6.3±0.63d

2.0±0.03a

1.0 88bc

11.3±0.22c

2.2±0.01bc

72de

8.1±0.58b

1.9±0.07b

1.5 95a

25.7 ±0.11a

2.1±0.01cd

85a

15.0 ±0.19a

1.7±0.04bc

2.0 82ef

18.5±0.26b

2.0±0.06de

80bc

4.2±0.25g

1.4±0.06de

2.5 80fg

7.8±0.21h

1.9±0.03f

71ef

3.0±0.21lm

0.9±0.05gh

KN

0.5 75hi

4.8±0.23l

2.3±0.05ab

52no

2.01±0.54n

1.7±0.04bc

1.0 82ef

8.1±0.17fg

2.0±0.08de

59k

3.12±0.26kl

1.5±0.03cd

1.5 83de

8.5±0.30ef

1.3±0.03hi

60ij

3.0±0.17lm

0.8±0.07hi

2.0 89b

11.8±0.26c

1.6±0.04gh

68g

5.6±0.14f

1.2±0.06ef

2.5 74ij

5.2±0.34j

1.0±0.02ij

55lm

3.0±0.32lm

0.7±0.07ij

TDZ

0.5 55q

3.4±0.33mn

1.8±0.04fg

54mn

2.0±0.34n

1.7±0.04bc

1.0 60no

5.2±0.23j

1.0±0.07ij

62hi

4.0±0.12gh

0.8±0.09hi

1.5 57p

3.6±0.45m

1.3±0.09hi

57kl

3.5±0.45ij

1.2±0.03ef

2.0 39q

3.0±0.34no

0.8±0.03kl

48p

3.3±0.23jk

0.7±0.02ij

2.5 28r

3.0±0.45no

0.7±0.01lm

40pq

3.0±0.34lm

0.7±0.01ij

2-iP

0.5 72jk

4.7±0.44 2.0±0.02e

68g

3.6±0.23i

1.9±0.04b

1.0 70kl

8.7±0.23e

1.9±0.06f

77d

4.2±0.34g

1.5±0.06cd

1.5 78h

9.2±0.26d

1.3±0.05hi

82ab

7.6±0.21c

1.0±0.04fg

2.0 63m

7.4±0.27hi

0.9±0.07k

71ef

6.0±0.22de

0.8±0.03hi

2.5 62mn

5.0±0.34jk

0.7±0.08lm

65gh

3.1±0.22kl

0.7±0.02ij

Each experiment was repeated thrice with 50 explants.Values with the same letter within the same

column are not significantly different according to Duncan Multiple Range Test (DMRT) at 5%

interval.

61

4.1.4 Shoot Elongation

Table 4.2 Effect of GA3 on elongation of in vitro regenerated shoots in

A. lineata

Concentration of

GA3 (mg/l)

Response

(%)

Elongation of

shoots (cm)

Mean No. of

nodes

0.1 55e

3.3±0.2e

2.5±1.1de

0.2 72b

4.8±0.4d

3.0±0.3cd

0.3 87a

7.0±0.6a

4.0±0.1a

0.5 68c

6.5±0.8b

3.8±0.2b

1.0 60cd

5.1±0.2c

3.5±0.4bc

Each experiment was repeated thrice with 50 explants.Values with the same letter within the same


interval.

The stimulative effect of GA3 on elongation of shoots is well

known as it has been found to promote cell division and elongation in the

apical zone of shoots (George et al 1993). GA3 at 0.3 mg/l induced maximum

shoot elongation within two weeks and produced 4.0 nodes/shoot (Table 4.2;

Figure 4.1e). Similar effect was observed in Andrographis paniculata

(Purkayastha et al 2008), Graptophyllum pictum (Justin and Wilson 2010) and

Andrographis echoides (Hemalatha and Vadivel 2010).

4.1.5 Rooting

Although the promotive effect of auxins was achieved in eliciting

rooting response (D’Silva and D’Souza 1992) their type and level in the

nutrient medium were found to vary from tissue to tissue and species to

species (Rao and Padmaja 1996). In our study, elongated shoots regenerated

from shoot tip and nodal explants failed to produce roots when cultured on

media containing half- or full-strength MS medium without any growth

regulator even after 35 days of culture (Data not shown).

62

This was in accordance with the reports of Purkayastha et al (2008)

and Hemalatha and Vadivel (2010). The elongated shoots were inoculated in

MS medium containing IBA, NAA and IAA at 0.5, 1.0 and 2.0 mg/l. Among

the various concentrations of auxins tested IBA at 1.0 mg/l produced

maximum number of roots /explant (11.94±0.45) within a week compared to

IAA or NAA (Table 4.3; Figure 4.1 f).

Table 4.3 Effect of different auxins on rooting of the in vitro raised shoots

of A. lineata

PGR

(mg/l)

Root induction

(%)

Mean No. of

shoots per

explant

Mean root

length (cm)

Emergence

of roots

(Days)

IBA

0.5 68c

8.22 ±0.12b

2.81±0.81b

15-18

1.0 85a

11.94±0.45a

3.02±0.56a

07-10

2.0 75b

6.11±0.45c

2.62±0.88d

13-17

IAA

0.5 65cd

3.61±0.67d

2.70±0.45c

14-08

1.0 51ef

2.41±0.44f

1.73±0.78i

14-20

2.0 57e

3.29±0.32de

2.11±0.79f

15-21

NAA

0.5 45gh

2.11±0.14hi

2.22±0.23e

17-25

1.0 51ef

2.34±0.56fg

2.10±0.88g

18-23

2.0 47g

2.17±0.99gh

2.05±0.22h

18-25Each experiment was repeated thrice with 50 explants.Values with the same letter within the same


interval.

The roots were induced directly from the shoot base without an

intervening callus phase on media supplemented with IBA. However, rooting

was observed with an intervening callus in IAA and NAA. Similarly, IBA

was found to be effective in inducing roots in Beloperone plumbaginifolia

(Shameer et al 2009), Adhatoda vasica (Khalekuzzaman et al 2008) and

Andrographis paniculata (Purkayastha et al 2008).

63

Figure 4.1 Direct organogenesis of A.lineata

a)Shoot tip explant supplemented in MS+30.0 mg/l AdS + 1.5 mg/l BAP;

b)Multiple shoot induction in shoot tip explant; c) Nodal explant

supplemented in MS+30.0 mg/l + 1.5 mg/l BAP; d)Multiple shoot induction

in nodal explant; e)Elongation of regenerated shoots in GA3 0.3 mg/l;

f)Root induction of elongated shoots in MS +1.0 mg/l IBA; g)Hardening;

h)and i)Acclimatized plant in green house.

c

64

4.1.6 Acclimatization

For acclimatization, well rooted plants were transferred to pots

containing a mixture of sterilized sand, soil and vermiculate (2:1:1, v/v/v),

covered with a clear plastic bag and grown at 25±2ºC with 85% relative

humidity. When signs of new shoot growth were evident (3-4 weeks), the

plants were acclimatized to ambient temperature (Figure 4.1 g) for two weeks.

Plants were misted manually with sterilized water once a day during this

period to avoid desiccation. At the end of 3rd

week, the acclimatized plantlets

were successfully established in greenhouse with 70% survival rate (Figure

4.1 h and i).

4.2 RANDOM AMPLIFIED POLYMORPHISM DNA ANALYSIS

4.2.1 RAPD Analysis of the ex vitro /in vitro Raised A.lineata

The genetic diversity of the plants are analysed by using

morphological as well as genetic based tools, DNA techniques (Bennici et al

2003) and advanced molecular methods (Barazani et al 2002 and Shiran et al

2007). The PCR based method for DNA profiling and random amplified

polymorphic DNA (RAPD) techniques (Mir and Nabulsi 2003 and Fracaro et

al 2005) have been extensively applied in assessment of genetic diversity of

various plant species and is also quite helpful in detecting genetic variability

within short time (Khan et al 2005). RAPD markers have been successfully

applied to detect the genetic similarities or dissimilarities in various plants

(Sikdar et al 2010). Although, micropropagated plants derived from shoot tip

and nodal explants have been previously reported to maintain clonal stability,

there is still a chance of obtaining somoclonal variation through adopting

tissue culture approach (Ostray et al 1994 and Rani and Raina 2000).

65

Figure 4.2 PCR amplification of RAPD primers

Lane 1: OPA-20; Lane 2: OPA-7; Lane 3: OPA-9; Lane 4: OPA-10; Lane

5: OPA-11; Lane 6: OPA-13; Lane 7: OPA-18 and Lane 8: OPA-19.

Table 4.4 Description of amplified RAPD primers

Lane Primer SequenceAmplified

fragments

1 OPA-20 5’GTTGCGATCC3’ 2

2 OPA-7 5’GAAACGGGTG 3’ 7

3 OPA-9 5’GGGTAACGCC 3’ 2

4 OPA-10 5’GTGATCGCAG 3’ 7

5 OPA-11 5’CAATCGCCGT 3’ 2

6 OPA13 5’CAGCACCCAC3’ 3

7 OPA-18 5’AGGTGACCGT3’ 2

8 OPA-19 5’CAAACGTCGG3’ 2

66

Figure 4.3 RAPD analyses of in vitro/ex vitro grown plants (A.lineata)

using OPA-7 primer

Lane1:1kb DNA Ladder; Lane 2: Field grown plant; Lane 3-7: In vitro

regenerants.

Table 4.5 Gel scoring data analysis for OPA-7 primer

Band size

(bp)

Field

grown

plant

In vitro regenerants

1 2 3 4 5

400 1 1 1 1 1 1

450 1 1 1 1 1 1

500 1 1 1 1 1 1

1000 1 1 1 1 1 1

1300 1 1 1 1 1 1

1500 1 1 1 1 1 1

Total number of bands obtained for OPA-7 primer = 6

Number of monomorphic bands for OPA-7 primer = 6

Number of polymorphic bands for OPA-7 primer = 6

Percentage of polymorphism for OPA-7 primer = Nil

10000 bp

500 bp

1300 bp

1000 bp

1750 bp1500 bp1300 bp1000 bp500 bp450 bp400 bp

67

Figure 4.4 RAPD analysis of in vitro/ex vitro grown plants (A.lineata)

using OPA-10 primer

Lane1:1kb DNA Ladder; Lane 2: Field grown plant; Lane 3-7: In vitro

regenerants.

Table 4.6 Gel scoring data analysis for OPA-10 primer

Band size

(bp)

Field

grown

plants

In vitro regenerants

1 2 3 4 5

350 1 1 1 1 1 1

600 1 1 1 1 1 1

750 1 1 1 1 1 1

900 1 1 1 1 1 1

1100 1 1 1 1 1 1

Total number of bands obtained for OPA-10 primer = 5

Number of monomorphic bands for OPA-10 primer = 5

Number of polymorphic bands for OPA-10 primer = 5

Percentage of polymorphism for OPA-10 primer = Nil

Based on the above views, RAPD analysis of field grown and in

vitro regenerants of A.lineata plants were performed in this study to confirm

10000 bp

625 bp

1000 bp

1100 bp

900 bp750 bp

600 bp350 bp

68

the clonal stability. Eight primers (OPA-7, OPA-9, OPA-10, OPA-11, OPA-

13, OPA-18, OPA-19, OPA-20) (Figure 4.2; Table 4.4) were chosen for PCR

amplification. Among them, seven fragments were amplified with two

primers OPA-7 (Figure 4.3; Table 4.5) and OPA-10 (Figure 4.4; Table 4.6).

The size of the band produced by OPA-7 and OPA-10 primers ranged from

400 to 1600 bp and 350 to 1100 bp respectively. Similar pattern was reported

in Lavandula angustifolia (Echeverrigaray and Agostini 2000) and Ocimum

gratissimum (Vieira et al 2001) with OPA-7 and OPA-10 primers. No

polymorphism was detected in the ex vitro and in vitro grown plants of

A.lineata , which indicates that they maintain their genetic stability during in

vitro culture. This confirms the usefulness of tissue culture for the production

of certified plant material to obtain herbal medicines. This is in agreement

with Samataray and Maiti (2010) who reported that the micropropagated

plants derived from shoot tip and nodal explants of Chlorophytum

Borivillanum did not show any genetic variation following RAPD analysis.

4.3 PHYTOCHEMICAL ANALAYSIS

4.3.1 Qualitative and Quantitative Analysis of Phytochemicals

Different phytochemicals posses various protective and therapeutic

effects which are essential to prevent diseases and maintain a state of well

being. The medicinal value of these plants depends on the chemical

substances that have a definite physiological action on the human body. The

most important of these bioactive constituents of plants are alkaloids, tannins,

saponins, terpenoids, steroids, glycosides, flavanoids and phenolic

compounds (Hill 1952).

69

Table 4.7 Qualitative analysis of phytochemicals from A.lineata leaf

extract

PhytoconstituentsPetroleum

etherChloroform Ethanolic Aqueous

Alkaloid - - ++ +

Saponin - + + -

Terpenoid - - +++ -

Flavanoid ++ ++ +++ +

Phenol - - +++ ++

Tannin - - +++ +

Glycosides - - ++ _

Steroids ++ - + _

Gums and mucilage + - + +

Each value represents mean value ± SD of three experiments carried out each in triplicate.

+++ = High; ++ = Medium; + = Low; - = Absent

Hence, in the present investigation, qualitative analysis of four

different extracts (petroleum ether, chloroform, ethanol and aqueous extract)

of A.lineata leaves was analysed for its phytoconstituents (Table 4.7). Among

the various solvents tested, maximum separation of phytochemicals was

observed with ethanolic extract. There are reports available indicating the

maximum extraction of phytochemicals in the ethanolic extract (Ahamad et al

1998 and Panda et al 2009).

Table 4.8 Quantitative analysis of phenols and flavanoids in A. lineata

leaf extract

SolventsPhenolic content

(mg /g)

Flavanoid content

(mg /g)

Petroleum ether 3.21±0.36 3.71±0.22

Chloroform 4.37±0.76 12.08±0.53

Ethanolic 22.31±0.02 95.36±0.41

Aqueous 10.44±0.65 6.25±0.29


70

Plants are conceived as sources of antioxidants due to the presence

of polyphenols and flavonoids which possess wide biological properties

(Durga et al 2006). In the present investigation, the quantification of phenolic

and flavanoid content of ALL was carried out in various extracts. The

phenolic and flavanoid extraction was efficient with ethanol when compared

to solvents tested (Table 4.8). Luo et al (2002) related the separation of the

polyphenols (phenol and flavanoid) to antioxidant and antidiabetic activity in

many plants. This shows that ethanolic leaf extract of A.lineata (EtALL) may

possess both antioxidant and antidiabetic activity (Chiasson et al 1994 and

Djilani et al 2011). Hence, further studies were performed with the EtALL

extract.

4.4 IN VITRO ANTIOXIDANT ACTIVITY

4.4.1 DPPH Scavenging Assay

DPPH assay is one of the most widely used methods for screening

antioxidant activity of plant extracts (Nanjo et al 1996). DPPH is a stable,

nitrogen-centered free radical which produces violet color in ethanol solution.

It was reduced to a yellow colored product, diphenyl picryl hydrazine with the

addition of the extract in a concentration-dependent manner. The reduction in

the number of DPPH molecules can be correlated with the number of

available hydroxyl groups.

71


Figure 4.5 Effect of DPPH scavenging activity of EtALL extract

In this study, DPPH scavenging activity was investigated in EtALL

extract. The IC50 value of EtALL extract was closely comparable to vitamin

C. This observation suggested that EtALL extract (Figure 4.5) may contain

compounds such as polyphenolics that can easily donate electron/hydrogen

easily (Nanjo et al 1996). Similar findings were reported in the same family in

Coccinia grandis (Umamaheswari and Chaterjee 2008), Andrographis

paniculata (Lin et al 2009) and Justica wyanaadensis (Sudha et al 2011).

4.4.2 Lipid Peroxidation Activity

Lipid peroxidation is initiated by radicals attacking unsaturated

fatty acids and propagated by a chain reaction cycle (Shimazki 1994). Since

unsaturated fatty acids are most important components of biological

membranes and impart desirable properties upon the fluidity of cellular

membrane structure, the peroxidation of unsaturated fatty acids in biological

membranes leads to disruption of membrane structure and function (Machlin

and Bendich 1987).

72


Figure 4.6 Effect of lipid peroxidation activity of EtALL extract

In particular ·O2 and ·OH induces various injuries to the

surrounding organs and play a vital role in some clinical disorders. Therefore,

removal of ·O2 and ·OH is the most effective defense of the body against

diseases (Lin et al 1995). In the present study, there was a linear increase in

the inhibiton of EtALL extract in dose dependent manner along with vitamin

E where lipid peroxidation activity was induced by iron/ADP/ascorbate

complex in the rat liver homogenate (Figure 4.6). This is in agreement with

the earlier findings of Andrographis paniculata (Lin et al 2009) and Vitex

trifoliate (Sreedhar et al 2010).

4.4.3 Superoxide Scavenging Activity

Super oxide is biologically important since it can be decomposed to

form stronger oxidative species such as singlet oxygen and hydroxyl radicals,

which is harmful to cellular components in the biological system

(Oyaizu 1986). Therefore, the superoxide anion radical scavenging activity of

EtALL extract was assayed.

73


Figure 4.7 Effect of superoxide scavenging activity of EtALL extract

The EtALL extract was found to scavenge superoxide radicals

generated in riboflavin –NBT–light in vitro. There was a corresponding

increase in the superoxide scavenging activity with the increase in the

concentration of EtALL extract (Figure 4.7) which was comparable to the

standard (Vitamin C). Similar inhibition activity was reported in

Andrographis paniculata (Sheela et al 2009), Asystasia nemorum (Panarat

et al 2010), Hygrophila schulli (Vijaya et al 2010) and Hygrophila difformis

(Nripendra and Priyanka 2011).

4.4.4 Nitric oxide Scavenging Activity

Nitric oxide is a potent pleiotropic mediator of physiological

process such as smooth muscle relaxation, neuronal signaling, inhibition of

platelet aggregation and regulation of cell mediated toxicity. It is a diffusible

free radical which plays vital roles as an effector molecule in diverse

biological systems including neuronal messenger, vasodilatations and

antimicrobial and antitumour activities (Hu and Kitts 2000).

74


Figure 4.8 Effect of nitric oxide scavenging activity of EtALL extract

In the present investigation, the EtALL extract moderately inhibited

nitric oxide radicals present in the extract (Figure 4.8) in a dose dependent

manner lesser (43.6% inhibition) than vitamin C. Lower nitric oxide

scavenging activity was also reported in Spondias pinnata (Bibhabasu et al

2008), Ecbolium viride (Ashoka et al 2011), Barleria prionitis (Chavan et al

2011) and Acanthus ilicifolius (Tirunavukkarasu et al 2011).

4.4.5 Reducing Power Ability

The reducing capacity of a compound may serve as a significant

indicator for its potential antioxidant activity. Earlier authors (Tanaka et al

1988) have observed a direct correlation between antioxidant activity and

reducing power of certain plant extracts. The reducing properties are generally

associated with the presence of reductones (Duh et al 1999), which have been

shown to exert antioxidant action by breaking the free radical chain by

donating a hydrogen atom (Gordon 1990).

75


Figure 4.9 Effect of reducing power ability of EtALL extract

In the present study, the reductive capability of the EtALL extract

was found to be remarkable as the concentration of the extract increased,

closely to Vitamin C (Figure 4.9). The reducing power of EtALL extract may

be due to the presence of hydrophilic and polyphenolic compounds (Chandler

et al 1993). Significant reducing power was also reported in ethanolic whole

plant extract of Andrographis paniculata (Rakshamani and Kamath 2007),

Coccinia grandis (Umamaheswari and Chaterjee 2008), Phaulopsis

fasicepala (Adesegun et al 2009) and Asystasia gangetica (Suvarchala et al

2010).

4.5 IN VITRO ANTIDIABETIC ACTIVITY

4.5.1 -glucosidase Inhibition Effect of EtALL Extract

One of the therapeutic approaches for treating diabetes is to

decrease the post prandial hyperglycemia. This is performed by retarding the

absorption of glucose through the inhibition of the carbohydrate hydrolyzing

enzyme ( -glucosidase) in the digestive tract delaying carbohydrate digestion

time, (Chiasson et al 1994). Many -glucosidase inhibitors such as flavonoids,

76

alkaloids, terpenoids, anthocyanins, glycosides and phenolic compounds have

been isolated from plants.

Table 4.9 -glucosidase inhibitory activity in EtALL extract

SampleConcentration

(µg/ml)

Inhibition

(%)IC50 (µg/ml)

EtALL

10 46.34±0.23

10.1950 68.02±0.18

100 73.14±10.19

Acarbose

0.1 31.53±0.14

0.3120.5 72.49±0.09

1.0 82.92±0.12


In this context, the -glucosidase inhibitory effect of EtALL

extract was investigated at various concentrations (10, 50 and 100 µg/ml)

which was compared with the standard drug (Acarbose). There was a

progressive increase in percentage of inhibition with increase in

concentration. However, there was a increase in percentage of inhibitory

activity at lower concentration of Acarbose (Table 4.9). This may be one of

the possible reasons for the antidiabetic activity of this medicinal plant in in

vitro model. Substantiating our results, there were several reports available in

many plants such as Cuscuta reflexa (Anis et al 2002), Hypoestes serpens

(Lalao et al 2003), Alstonia scholaris (Anurakkun et al 2007), Adhatoda

vasica (Gao et al 2008), Andrographis paniculata (Edwin et al 2008),

Cecropia obtusifolia (Cetto et al 2008) and Asystasia dalzelliana

(Satish et al 2011).

77

4.5.2 MTT Assay, Insulin Mimicking and Sensitization Activity of

EtALL Extract in 3T3-L1 Cell Line

The MTT assay is used to assess the viability and the proliferation

of cells (Freshney 2000). It is also used to determine the cytotoxicity of the

crude extracts obtained from medicinally important plants.The assay works

with the principle of reduction of yellow color (MTT) to purple formazan by

the enzyme reductase which is present in living cells.


Figure 4.10 Cyctotoxic effect of EtALL extract

Control- Untreated 3T3-L1cells without extract

The cytotoxicity of EtALL extract on 3T3-L1 cell line at various

concentrations was evaluated in the presnt study. The control (untreated cells)

showed 100% cell viability. The cells treated with EtALL extract showed no

toxicity between 6.5 µg/ml to 50 µg/ml concentration. However, beyond 100

and 200 µg/ml it was found to be toxic (Figure 4.10). The cytotoxic nature of

EtALL extract is well supported by the previous work done in various plants

such as Vismia schultesii (Ivana et al 2006), Annona squasoma (Beena and

78

Remani 2008), Embelia ribes (Beena and Laskhmi 2010) and Terminalia

arjuna (Alam et al 2011).

Table 4.10 Insulin mimicking activity of EtALL extract in 3T3-L1 cell

lines

EtALL extract concentration

(µg/ml)

Insulin mimicking activity (%)

(Glucose uptake)

1.0 nmol/l Insulin (Standard) 100.21±2.34

12.5 42.68±3.57

25 63.8±3.56

50 52.6±5.21

75 41.7±3.64


Table 4.11 Insulin sensitizing activity of EtALL extract in 3T3-L1 cell

lines

EtALL extract concentration +

1.0 nmol/l insulin (µg/ml)

Insulin sensitization activity

(Glucose uptake) (%)

1.0 nmol/l Insulin (Standard) 195.03±9.25

12.5 220.32±3.47

25 290.44±5.07

50 246.34±3.56

75 230.62±2.03


Diabetes is characterized by increased blood glucose levels and

disturbances in the carbohydrate, fat and protein metabolism (Apparao et al

2003). The insulin stimulated glucose uptake in adipose tissue is a critical

factor for reducing post prandial blood glucose concentration. 3T3-L1 cells

are an excellent experimental model to quickly screen the effects of crude

drugs on glucose uptake (Liu et al 2001). Dysregulation of this process is one

of the important factors in diabetes. Many oral pharmacotherapies for the

79

management of diabetes have emerged out with this interest and are widely

used till today (Kameshwara et al 2001). This therapy may act by mimicking

insulin or either by stimulating insulin release or by potentiating insulin action

or by reducing hepatic glucose production (Yu-Chiao et al 2003). Therefore in

vitro antidiabetic activity was undertaken to evaluate the insulin

mimicking/sensitizing activity of the EtALL extract.

All observed values of glucose uptake activity are compared using

the control (untreated cells). These values are normalized with MTT cell

viability assay values (12.5 25, 50 and 75 µg/ml) for the EtALL extract. The

insulin mimicking activity was found to be the best at 25 µg/ml concentration

of EtALL extract. Further increase or decrease in the concentration does not

have any significant impact (Table 4.10).

The insulin sensitization activity was performed to determine the

synergestic effect of EtALL extract with insulin. A higher synergistic activity

was exhibited by 25 µg/ml concentration of EtALL extract. When compared

to the standard, the insulin sensitization activity was more than 90% (Table

4.11). The property of the bioactive compound plays a crucial role in

determining the mechanism of action for antidiabetic agents. For instance,

there were reports on medicinal plants exhibiting insulin mimicking activites

Agaricus campestris (Gray and Flatt 1998), Vernonia amygdalina (Atangho et

al 2010) and Curcuma longa (Mohankumar and McFarlane 2011). Some

medicinal plants were reported for insulin sensitization activity by Yu-Chiao

et al (2003) in Toona sinesis, Guy et al (2007) in Lagerstroemia speciosa,

Patrick et al (2008) in Psidium guajava and Morinda citrifolia, Padmanabha

and Kaiser (2011) in Eugenia jambolana and Aruh and Issac (2011) in

Dennettia tripetala.

Hence, we further subjected the EtALL extract for in vivo

evaluation of antidiabetic effect.

80

4.6 IN VIVO ANTIDIABETIC ACTIVITY

4.6.1 Antidiabetic Effect of EtALL Extract in Streptozotocin Induced

Model

It was intended to study the antidiabetic effect of EtALL extract in

STZ induced rat model. STZ is well known for its selective pancreatic -cell

cytotoxicity which has been extensively used to induce diabetes in animals. It

is less toxic than alloxan and allows a consistent maintenance of diabetes

(Raju and Balaraman 2008). A low dose of STZ (40 mg/kg b.w.) has been

used in this study to induce diabetes where half of the population of

pancreatic –cells are destroyed leaving behind residual -cells which secrete

insufficient insulin causing type 2 diabetes (Eliza et al 2009). Over-

production (excessive hepatic glycogenolysis and gluconeogenesis) and

decreased utilization of glucose by the tissues are the fundamental basis of

hyperglycemia in STZ induced diabetes (Patil et al 2011).

4.6.1.1 Acute toxicity

The dose range of the EtALL extract was fixed based on the acute

toxicity studies. In performing preliminary test for pharmacological activity in

rats, LD50 studies revealed the non toxic nature of EtALL extract upto 200

and 400 mg/kg b.w. Similar dosage level was also reported in Flacourtia

jangom (Ajay and Jyoti 2010), Cassia osscidentalis (Emmanuel et al 2010)

and Citrus limetta (Sripama et al 2011).

4.6.1.2 Effect of EtALL extract on oral glucose tolerence test

Oral glucose tolerance testing (OGTT) is a standard procedure that

is used to diagnose diabetes. Each year 1 – 5 % of people with impaired

glucose tolerance (IGT) develop diabetes (Rammohan et al 2008). Impaired

oral glucose tolerance (IGT) is an indicative of a predisposition of an animal

81

to diabetes condition. Agents that exhibit antihyperglycaemic effects are

capable of bringing blood glucose concentration to normal limits which helps

further to arrest the progression of impaired glucose tolerance (Raju and

Balaraman 2008).

Table 4.12 Effect of oral glucose tolerence test in EtALL extract

Groups 0 min 30 min 60 min 120 min

Normal 78.02±2.41 153.29±1.15 144.05±1.35 122.68±1.70

EtALL

(200 mg/ kg

b.w.)

82.07±1.15 132.25±0.60 *

126.47±0.71*

111.16±1.88*

EtALL

(400 mg/ kg

b.w.)

80.93±1.20 125.61±1.34**

116.38±1.15 **

96.15±1.12**

Glibenclamide

(600 µg /kg

b.w.)

75.94±0.92 116.42±0.66**

94.64±0.75 **

78.85±1.76**


*: p<0.05;

**: p<0.01.

The OGTT was performed using the normal, EtALL extract treated

(200 and 400 mg/kg b.w.) and glibenclamide (standard) treated diabetic rats.

Progressive decrease in the blood glucose level was observed with increasing

time. Although 200 and 400 mg/kg b.w. dosages of EtALL extract exhibited

glucose lowering ability, the higher dosage (400 mg/kg b.w.) showed better

activity when compared to the normal rats but slightly lower than

glibenclamide treated diabetic rats (Table 4.12). Many researchers reported

similar pattern of reduction in the blood glucose level in the OGTT analysis in

Andrographis paniculata (Rammohan et al 2008), Coccinia cordifolia and

Catharanthus roseus (Islam et al 2009), Telfaria occidentalis (Olorunfemi

et al 2010) and Calotropis gigantean (Nanu et al 2011).

82

4.6.1.3 Effect of EtALL extract on body weight, water and food intake

STZ-induced diabetes is characterized by a severe loss in body

weight (Al-Shamaony et al 1994) and increased food intake (Szkudelski and

Szkudeslka 2002). Loss in body weight might be the result of protein wasting

due to unavailability of carbohydrate as an energy source (Chen and Ianuzzo

1982).

Table 4.13 Effect of EtALL extract on body weight, water and food

intake in normal and streptozotocin induced diabetic rats

Groups

Changes in body weight

(g)

Water Intake

(ml/rat/day)

Food Intake

(g/rat/day)

Initial

0th

day

Final

28th

day

Initial

0th

day

Final

28th

day

Initial

0th

day

Final

28th

day

Normal 171.31±8.0 178.65±6.7 75.56±6.7 98.23±12.1 15.54±8.2 14.29±7.8

Diabeticcontrol

(STZ-40 mg /

kg b.w.)

174.62±15.5 136.32±8.9 155.0±8.9 165.6±6.8 40.5±5.6 56.53±8.4

Diabetes+EtALL

(200 mg/ kgb.w.)

174.67±15.7 202.66±2.4*

124.38±4.5 80.58±7.6*

20.75±5.8 27.69±6.5*

Diabetes+EtALL

(400 mg/ kg

b.w.)

173.83±15.3 201.01±4.5**

120.34±10.1 90.16±6.2**

25.44±8.9 30.34±6.2**

Diabetes +Glibenclamide

(600 µg/ kgb.w.)

170.16±10.1 203.08±3.6**

126.4±11.2 92.57±6.2**

25.13±9.0 32.5±12.3**


*: p<0.05;

**: p<0.01.

The normal, diabetic control, EtALL extract (200 and 400 mg / kg

b.w.) and glibenclamide treated diabetic rats were investigated for a period of

4 weeks for changes in body weight, food and water intake. The body weight,

83

food and water intake remained unchanged on the beginning of the 0th

day

(Table 4.13). By the end of the experiment (28th

day) the diabetic rats showed

a gradual decrease in body weight, whereas there was increase in food and

water intake compared to the normal rats. Oral administration of EtALL

extract (200 and 400 mg/kg b.w.) showed a significant gain in the body

weight, with reduced food and water intake compared to the diabetic control

rats. This may be due to improved insulin secretion and glycemic control in

extract treated rats. (Genet et al 1999). Glibenclamide (600µg/ kg b.w.) treated

diabetic rats also showed increase in body weight, decrease in food and water

intake compared to the diabetic control rats. Similarly, gain in body weight

and decrease of food and water intake were observed in diabetic induced rats

that were later treated with extracts of Trifolium sp. (Maisaa and Rawi 2007),

Costus speciosus (Eliza et al 2009) and Rhinacanthus nasutus

(Visweswara et al 2010).

4.6.1.4 Effect of EtALL extract on blood glucose and plasma insulin

level

There may be several causes for persistant hyperglycemia and the

most important among them is the failure of blood sugar regulation

(Bolkent et al 2000). In the present study, the blood glucose level was

evaluated on the 0th

day, 14th

day and 28th

day in the normal, diabetic control,

EtALL extract treated (200 and 400 mg/kg b.w.) and glibenclamide treated

diabetic rats. The diabetic control rats showed increase in blood glucose level

from the 0th

day to 28th

day when compared to the normal rats. There was a

gradual decrease in the blood glucose level in EtALL extract (200 and 400

mg/kg b.w.) treated diabetic rats. However, EtALL extract at 400 mg/kg b.w.

showed fivefold decrease in the blood glucose level when compared to

diabetic control rats. This hypoglycemic effect of EtALL extract may be due

to the presence of bioactive compounds which triggers the pancreatic

84

secretion of insulin from the existing -cells. The glibenclamide treated

diabetic rats also showed a similar glucose lowering ability compared to the

diabetic control rats (Table 4.14).

Table 4.14 Effect of EtALL extract on blood glucose and plasma insulin

level in normal and streptozotocin induced diabetic rats

Groups

Blood glucose level (mg/dl) Plasma

insulin (U/ml)

(28th

day)0

th day 14

th day 28

th day

Normal 77.5±4.39 91.5±1.27 89.5±4.21 130.08±1.66

Diabetic

control

(STZ-40 mg /

kg b.w.)

259.3±7.41 400.7±1.21 390.2±1.56 54.48±1.56

Diabetes+

EtALL

(200 mg/ kg

b.w.)

297.2±4.62 312.6±7.39*

200.6±2.45*

98.32±2.52*

Diabetes+

EtALL

(400 mg/ kg

b.w.)

290.6±3.62 283.6±2.11**

156.8±1.22**

110.21±0.67**

Diabetes +

Glibenclamide

(600 µg/ kg

b.w.)

294.5±3.51 308.2±2.42**

212.4±2.11**

116.23±0.54**


*: p<0.05;

**: p<0.01.

High plasma levels of insulin and glucose due to insulin resistance

are a major component of the metabolic syndrome. If insulin resistance exists,

more insulin needs to be secreted by the pancreas. If this compensatory

increase does not occur, blood glucose concentrations increases leading to

diabetes (Graham et al 2001). The plasma insulin level was observed in all the

groups at the end of 28th

day (Table 4.14). More than two fold decrease in

secretion of plasma insulin was observed on the 28th

day in the diabetic

control rats than the normal rats. Oral treatment of EtALL extract (400 mg /

85

kg b.w.) showed enhanced secretion of plasma insulin when compared to the

diabetic control rats. Glibenclamide treated diabetic rats also showed an

increase in plasma insulin level compared to the diabetic control rats

(Table 4.14). This hypoglycemic effect of EtALL extract may be due to the

presence of bioactive compounds which triggers the pancreatic secretion of

insulin from the existing -cells (Li et al 2004). The glucose lowering ability

and insulin seretory activity was well corelated by Eliza et al (2009) in Costus

specious, Kondeti et al (2010) in Petrocarpus santalinus and Arokiyaraj et al

(2011) Hypericum perforatum.

4.6.1.5 Effect of EtALL extract on Hb and HbA1C level

Increased non enzymatic glycosylation is one of the possible

mechanism linking vascular hyperglycemia and vascular complications of

diabetes. During diabetes, excess glucose present in the blood reacts with

hemoglobin to form HbA1C (Kondeti et al 2010). In uncontrolled or poorly

controlled diabetes, there is an increased glycosylation of a number of

proteins, including Hb (Alberti and Press 1982). HbA1C was found to

increase up to 16% in diabetic patients (Koeing et al 1976) and hence it is a

reliable index of glycemic control in diabetes (Gabbay 1976) which reflects

the average blood glucose concentration (Murray et al 2000).

Table 4.15 Effect of EtALL extract on Hb and HbA1C level in normal

and streptozotocin induced diabetic rats

Groups Hb (mg/dl) HbA1C (% total Hb)

Normal 15.6±1.4 0.45±3.7

Diabetic control

(STZ-40 mg/ kg b.w.)

6.5±1.8 0.79±1.0

Diabetes+ EtALL

(200 mg/kg b.w.)

9.6±1.9*

0.50±1.4*

Diabetes+ EtALL

(400 mg/ kg b.w.)

14.8±2.3**

0.46±1.6**

Diabetes + Glibenclamide

(600 µg/ kg b.w.)

13.7±2.7**

0.53±1.9**

Each value represents mean value ± SD of three experiments carried out each in triplicate.*: p<0.05;

**: p<0.01.

86

In this study, the Hb and HbA1C parameters were investigated in

the normal, diabetic control, EtALL extract treated and glibenclamide treated

diabetic rats. The increase in HbA1C in the diabetic control rats was in

correlation with decrease in Hb content when compared to normal rats. Oral

administration of EtALL extract at 400 mg/kg b.w. increased the Hb and

decreased the HbA1C level significantly. Glibenclamide treated rats also

showed increase in Hb and decrease in HbA1C compared to the diabetic

control rats (Table 4.15). The extracts of Gymnema sylvestre (Shanmugasudaram

et al 1990), Tinospora cordifolia (Rajalakshmi et al 2009), Costus specious

(Eliza et al 2009) and Zizyphus spins-christi (Michel et al 2011) were also

found to posses properties that control glycemic index.

4.6.1.6 Effect of EtALL extract on muscle and liver glycogen level

Glycogen is the primary intracellular storable form of glucose and

its level in various tissues is a direct reflection of insulin activity as insulin

promotes intracellular glycogen deposition by stimulating glycogen synthase

and inhibiting glycogen phosphorylase (Golden et al 1979).

Table 4.16 Effect of EtALL extract on muscle and liver glycogen level in

normal and streptozotocin induced diabetic rats

Groups

Muscle glycogen

(mg /100 mg wet

weight)

Liver glycogen

(mg /100 mg wet

weight)

Normal 9.8±1.2 46.3±2.2

Diabetic control

(STZ-40 mg / kg b.w.)

6.5±3.4 12.5±2.4

Diabetes+ EtALL

(200 mg/ kg b.w.)

8.0.±2.3*

40.5±3.6*

Diabetes+ EtALL

(400 mg /kg b.w.)

10.6±3.6**

44.3±2.8**

Diabetes + Glibenclamide (600 µg/

kg b.w.)

11.2±2.9**

42.8±2.9**


*: p<0.05;

**: p<0.01.

87

The glycogen content of skeletal muscle and liver markedly

decreased in diabetic rats (Welihinda and Karuanayake 1986) in proportion to

insulin deficiency (Stalmans et al 1997). In this study the level of muscle and

liver glycogen was analysed among the normal, diabetic control, EtALL

extract (200 and 400 mg/kg b.w.) and glibenclamide treated diabetic rats.

Decrease in muscle glycogen (79.5%) and liver glycogen (60.73%) content

was observed in the diabetic control rats when compared to the normal rats.

This may be due to the impairment of glucose synthesis in the liver and

skeletal muscles of rat during diabetes (Hwang et al 1997).

Hence, glycogen content of skeletal muscle and liver markedly

decreased in diabetic induced rats. When EtALL extract was administered at

400 mg/kg b.w. increased muscle and liver glycogen content was increased

(78.50% and 58.83%) when compared to the diabetic control rats (Table

4.16). This may be due to the stimulation of insulin release from -cells

(Lolitkar et al 1996). Glibenclamide treated rats also showed a significant rise

in the muscle and liver glycogen content compared to the diabetic control rats.

The extracts of Eugenia jambolana (Sharma et al 2003) and Costus specious

(Eliza et al 2009) showed increased level of muscle and liver glycogen

content in diabetic rats.

4.6.1.7 Effect of EtALL extract on carbohydrate metabolizing enzyme

in liver

Hexokinase, glucose -6-phosphatase and fructose -1, 6- bisphosphatase

are rate limiting glycolytic enzymes that are severely impaired during diabetes

condition. These enzymes play a very important role in the final step of

glucogenolysis and gluconeogensis (Hassan et al 2009). A decrease in the

activity of hexokinase, glucose-6-phosphatase and fructose -1, 6- bisphosphatase

has been shown to slow down the pentose phosphate pathway under diabetic

conditions (Abdel-Rahim et al 1992).

88

Table 4.17 Effect of EtALL extract on carbohydrate metabolizing

enzyme in liver of normal and streptozotocin induced

diabetic rats

GroupsHexokinase

(U/g protein)

Glucose-6-

phosphatase

(U/g protein)

Fructose -1, 6-

bisphosphatase

(U/g protein)

Normal 142.81±4.8 0.162±0.01 0.331±0.01

Diabetic control

(STZ-40 mg / kg

b.w.)

109.33±4.7 0.272±0.02 0.517±0.03

Diabetes+ EtALL

(200 mg/kg b.w.)127.21±4.3

*0.210±0.06

*0.495±0.04

*

Diabetes+ EtALL

(400 mg/kg b.w.)139.12±8.1

**0.171±0.02

**0.413±0.04

**

Diabetes +

Glibenclamide (600

µg/kg b.w.)

137.14±9.4**

0.182±0.02 **

0.435±0.04 **


*: p<0.05;

**: p<0.01.

In the present study, the normal, diabetic control, EtALL extract

treated and glibenclamide treated diabetic rats were subjected to glycolytic

enzyme analysis. There was a significant decrease in hexokinase activity and

an increase in glucose-6-phosphatase and fructose -1, 6-bisphosphatase

activity in the diabetic control rats compared to the normal rats. Oral

administration of EtALL extract at 400 mg/kg b.w. to diabetic rats

significantly increased liver hexokinase and decreased glucose-6-phosphatase

and fructose -1, 6- bisphosphatase activity when compared to the diabetic

control rats. Similar pattern of activity was observed with the glibenclamide

treated rats (400 mg/kg b.w.) (Table 4.17). These results are in agreement

with the previous reports of Gymnema montamum (Ananthan et al 2003),

Tinospora cordifolia (Rajalakshmi et al 2009) and Hypericium perforatum

(Arokiyaraj et al 2011).

89

4.6.1.8 Effect of EtALL extract on serum lipid profile

The most common lipid abnormalities in diabetes are

hypertriglyceridaemia and hypercholesterolaemia (Khan et al 1995 and Mitra

et al 1995). The development of hypertriglyceridemia in uncontrolled diabetes

is a consequence of a number of metabolic abnormalities that occur

sequentially (Lopez 2001). Insulin deficiency or insulin resistance may be

responsible for dyslipidemia, because insulin has an inhibitory action on

HMG-CoA reductase, a key rate limiting enzyme responsible for the

metabolism of cholesterol rich LDL-c particles (Havel et al 1970). VLDL,

which is a major carrier of plasma triglycerides in blood, becomes rich in

cholesterol and acts as a carrier of cholesterol (Lee et al 1994). Significant

lowering of total cholesterol and rise in HDL-c is a very desirable

biochemical state for prevention of atherosclerosis and ischemic conditions

(Lin et al 1995).

Table 4.18 Effect of EtALL extract on serum lipid profile in normal

and streptozotocin induced diabetic rats

Groups

Serum lipid profile

Total

Cholesterol

(mg/dl)

HDL-c

(mg/dl)

LDL-c

(mg/dl)

Triglycerides

(mg/dl)

Normal 95.85 ±3.7 56.43 ±4.8 80.42±0.9 16.16±1.4

Diabetic control

(STZ-40 mg / kg

b.w).

247.12 ±4.5 26.6 1±1.5 146.44±1.2 40.23±2.8

Diabetes+ EtALL

(200 mg/ kg b.w.)187.34±3.6* 35.81±1.3* 91.81±5.3* 30.64±2.5*

Diabetes+ EtALL

(400 mg/ kg b.w.)109.42±10.3 ** 50.22 ±3.2 ** 80.67±5.1** 13.52±0.7 **

Diabetes +

Glibenclamide

(600 µg/ kg b.w.)

124.34 ±5.1 ** 44.87±3.8 ** 90.68±1.1 ** 6.37±0.8 **


*: p<0.05;

**: p<0.01.

90

In this study, the serum lipid profiles of normal, diabetic control,

EtALL extract and glibenclamide treated diabetic rats was evaluated. There

was a significant decrease in HDL-c cholesterol and increase in total

cholesterol, triglycerides and LDL-c level in diabetic control rats when

compared to the normal rats. Oral administration of EtALL extract at 400

mg/kg b.w. showed significant increase in HDL-c cholesterol level and

decrease in level of total cholesterol, triglycerides and LDL-c cholesterol

compared to the diabetic control rats. The levels of HDL-c, total cholesterol,

triglycerides and LDL-c were close to the levels of normal rats in the 400

mg/kg b.w. extract treated diabetic rats. The glibenclamide treated diabetic

rats showed an increase in HDL-c level, triglycerides and LDL-c level when

compared to diabetic rats (Table 4.18). These findings are in agreement with

Ravi et al (2005) in Eugenia jambolina and Daisy et al (2009) reported in

Elephantopus scaber.

4.6.1.9 Effect of EtALL extract on serum urea, creatinine and uric acid

Renal diseases is one of the most common and severe

complications of diabetes (Rhodes et al 2006). The diabetic hyperglycemia

induces elevation of the serum levels of urea, creatinine and uric acid

which are considered as significant markers of renal dysfunction

(Almdal et al 1987). Degradation of protein and nucleic acid results in

the formation of non-protein nitrogenous compound such as urea and

creatinine. Uric acid clearance has been associated with insulin resistance

(Yassin et al 2004).

91

Table 4.19 Effect of EtALL extract on serum urea, creatinine and uric

acid in normal and streptozotocin induced diabetic rats

GroupsUrea

(mg/dl)Creatinine (mg/dl)

Uric acid

(mg/dl)

Normal 24.21 ± 3.79 3.61 ± 0.89 1.35± 0.10

Diabetic control

(STZ-40 mg / kg

b.w.)

41.11 ± 4.5 5.73 ±1.96 2.59 ±0.52

Diabetes+ EtALL

(200 mg/kg b.w).

38.56 ± 4.8*

4.72 ± 1.36*

2.13 ±0.21*

Diabetes+ EtALL

(400 mg/kg b.w.)

26.25± 10.3**

4.28 ±1.24**

1.95 ±0.14**

Diabetes +

Glibenclamide

(600 µg kg/b.w.)

33.33 ±5.1**

4.35 ±1.52**

2.04 ±0.34**


*: p<0.05;

**: p<0.01.

In the present study, the renal parameters such as urea, creatinine

and uric acid has been investigated among the normal, diabetic control,

EtALL extract and glibenclamide treated diabetic rats. There was a significant

increase in urea, creatinine and uric acid level in the diabetic control rats

when compared to the normal rats. However, administration of EtALL extract

at 400 mg/kg b.w. lowered urea, creatinine and uric acid level when compared

to the diabetic control rats. Glibenclamide treated diabetic rats also showed a

linear decrease of the urea, creatinine and uric acid level compared to the

diabetic control rats (Table 4.19). The extracts of Vinca rosea (Ghosh et al 2001),

Costus specious (Daisy et al 2008) and Elephantopus scaber (Daisy et al

2009) were also reported to control these renal dysfunction.

92

4.6.1.10 Effect of EtALL extract on albumin, globulin and total protein

Insulin deficiency leads to renal alterations which are demonstrable

in experimental diabetes, leading to a negative nitrogen balance, enhanced

proteolysis and lowered protein synthesis (Bhavapriya et al 2001).

Table 4.20 Effect of EtALL extract on albumin, globulin and total

protein in normal and streptozotocin induced diabetic rats

GroupsTotal Protein

(g/dl)

Albumin

(g/dl)A/G ratio

Normal 8.12±1.47 3.92±1.2 1.92± 1.6

Diabetic control

(STZ-40 mg / kg

b.w.)

3.63±2.8 1.21±1.7 0.51 ±1.3

Diabetes+ EtALL

(200 mg /kg b.w.)5.82±5.6

*2.01±2.3

*1.03 ±1.7

*

Diabetes+ EtALL

(400 mg/ kg b.w.)7.86±0.7

**3.22±2.6

**1.63 ±1.1

**

Diabetes +

Glibenclamide

(600 µg /kg b.w.)

7.32±0.8**

3.0 ±1.8**

1.52 ±1.2**


*: p<0.05;

**: p<0.01.

In this study, the total protein, albumin and A/G ratio of the normal

rats, diabetic control, EtALL extract and glibenclamide treated diabetic rats

were estimated. The diabetic control rats showed reduced level of total

protein, albumin and A/G ratio compared to the normal. This decline may be

due to the inhibited oxidative phosphorylation processes which lead to the

decrease of protein synthesis, increase in the catabolic process and reduction

of protein absorption (Trag and Reaven 1972 and Jefferson et al 1993).

93

Adminstration of EtALL extract at 400 mg/kg b.w. increased the levels of

total protein, albumin and A/G ratio compared to the diabetic control rats.

Moreover, the EtALL extracts at 400 mg/kg restored all the biochemical

parameters to near normal. Glibenclamide treated diabetic rats also showed

closer values to normal compared to diabetic control rats (Table 4.20). These

finding are in accordance with Kaleem et al (2006) and Jai and Loganathan

(2010).

4.6.1.11 Effect of EtALL extract on TBARS and hydrogen peroxide in

tissues and plasma

Numerous studies have demonstrated that oxidative stress is key

pathogenic factor in the development of diabetic complications. Oxidative

stress includes the production of highly reactive oxygen species that are toxic

to the cell, particularly the cell membrane in which these radicals interact with

the lipid bilayer and produce lipid peroxides. The increased level of lipid

peroxidation could be associated to increase in free radicals generation in

diabetes caused primarily due to high blood glucose levels, which upon

autoxidation generates free radicals and secondarily due to the effects like

STZ (Ivorra et al 1989). Previous studies have also reported increased lipid

peroxidation in the liver and kidney tissues of diabetic rats (El-Missiry and

El-Gindy 2000).

94

Table 4.21 Effect of EtALL extract on TBARS and hydrogen peroxide

in tissues and plasma of normal and streptozotocin induced

diabetic rats

Groups Normal

Diabetic

control

(STZ-40

mg /kg

b.w.)

Diabetes+

EtALL

(200 mg/

kg b.w.)

Diabetes+

EtALL

(400 mg/ kg

b.w.)

Diabetes +

Glibenclamide

(600 µg/ kg

b.w.)

TBARS

Liver

(mM/100 g

tissue)

0.76±0.6 1.72±0.3 1.56±0.5*

1.23±0.5 **

1.30±0.3**

Kidney

(mM/100 g

tissue)

1.80±1.2 3.02±0.5 2.49±0.6*

2.08±1.2 **

2.65±1.2**

Brain

(mM/100 g

tissue)

1.05±0.5 3.70±0.3 2.43±0.1*

1.23±0.4**

1.45±0.3 **

Plasma

(mM/dl)0.15±0.3 0.36±0.1 0.30±0.2

*0.20±0.1

**0.24±0.2

**

Hydrogen peroxide

Liver

(mM/100 g

tissue)

75.6±6.3 93.58±2.5 85.35±2.3*

78.71±4.4 **

81.53±3.8 **

Kidney

(mM/100 g

tissue)

50.71±6.5 79.23±0.7 70.97±4.5*

59.16±5.3 **

65.28±1.4 **

Brain

(mM/100 g

tissue)

117.0±0.3 131.40±0.1 130.56±2.6*

120.90±0.2 **

125.0±0.2 **

Plasma

(mM/dl)9.95±5.3 14.68±6.4 13.91±4.7

*10.75±4.3

** 12.96±5.7

**


*: p<0.05;

**: p<0.01.

95

In the present study, the lipid peroxide markers namely TBARS and

hydroperoxide were analysed in the tissues (liver, kidney and brain) and

plasma of normal, diabetic control, EtALL extract treated and glibenclamide

treated rats. The diabetic control rats showed a marked increase of TBARS

and hydroperoxide levels in the tissues and plasma compared to the normal

rats. Administration of (200 and 400 mg/kg b.w.) EtALL extract treated and

glibenclamide treated diabetic rats showed a significant decrease of TBARS

and hydroperoxides level in the tissue and plasma compared to the diabetic

control rats (Table 4.21). The treated lipid peroxide-mediated tissue damage

and plasma in diabetic rats were reported by Leelavinothan and Muniappan

(2004) in Scoparia dulcis, Daisy et al (2008) in Costus specious and Purnima

et al (2010) in Mimusops elengion.

4.6.1.12 Effect of EtALL extract on SOD and CAT in tissue and

haemolysate

Superoxide dismutase (SOD) has been postulated as one of the

most important enzyme in the enzymatic antioxidant defence system

which catalyses the dismutation of super oxide radicals to produce H2O2

(Baynes 1995). Catalase (CAT) is a haemoprotien which catalyses

the reduction of hydrogen peroxide and protects the tissues from highly

reactive hydroxyl radicals. The superoxide anion has been known to

inactivate CAT, which is involved in the detoxification of hydrogen

peroxide (Yan et al 1999).

96

Table 4.22 Effect of EtALL extract on SOD and CAT in tissue and

haemolysate of normal and streptozotocin induced diabetic

rats

Groups Normal

Diabetic

control

(STZ-40

mg/kg

b.w.)

Diabetes+

EtALL

(200

mg/kg

b.w.)

Diabetes+

EtALL

(400 mg/kg

b.w.)

Diabetes +

Glibenclamide

(600 µg/kg b.w.)

Superoxide dismutase (SOD)

Liver

(U/mg

protein)

6.10±0.6 3.62±0.3 4.56±0.2*

5.82±0.5 **

4.52±0.3 **

Kidney

(U/mg

protien)

14.90±1.2 10.34±0.5 11.52±0.4*

13.81±1.2 **

12.01±1.2 **

Brain

(U/mg

protein)

7.90±0.5 4.61±0.3 5.67±0.6*

7.01±0.4**

6.22±0.3 **

Haemolysate

(U/mg Hb)

2.18±0.3 1.52±0.1 1.61±0.2*

1.98±0.1**

1.94±0.2 **

Catalase (CAT)

Liver

(U/mg

protein)

73.54±6.3 46.32±2.5 50.86±0.5*

67.42±4.4**

58.72±3.8 **

Kidney

(U/mg

protein)

34.92±6.5 19.62±0.7 20.45±0.8*

28.41±5.3 **

25.02±1.4**

Brain

(U/mg

protein)

3.21±0.3 1.01±0.1 1.85±0.4*

3.01±0.2 **

2.33±0.2**

Haemolysate

( U/mg Hb)

60.45±5.4 45.72±6.4 50.36±0.5*

57.12±4.3**

58.92±5.7 **


*: p<0.05;

**: p<0.01.

97

In this study, the level of SOD and CAT in the tissues (liver,

kidney and brain) and hemolysate of normal, diabetic control, EtALL extract

and glibenclamide treated diabetic rats was determined. Diabetic control rats

showed a significant decrease in the levels of SOD and CAT in tissues and

hemolysate compared to the normal rats. On administration of 400 mg/kg b.w.

of EtALL extract the altered activities of SOD and CAT in tissue and

hemolysate reversed closely to the levels of normal when compared to the

diabetic control rats. There was linear increase of SOD and CAT levels

observed in glibenclamide treated diabetic rats compared to the diabetic

control rats (Table 4.22). Similar ameliorative effect of SOD and CAT was

reported in the extracts of Petrocarpus marsupium (Maruthupandian and

Mohan 2011) and Mimusopus elengion (Purnima et al 2010).

4.6.1.13 Effect of EtALL extract on the activities of GPx and GST in

tissues and haemolysate

GPx plays a primary role in minimizing oxidative damage. It has

been proposed that GPx is responsible for the detoxification of H2O2 in low

concentration, where as catalase comes into play when GPx pathway is

reaching saturation with the substrate. GSH metabolizing enzymes,

glutathione peroxidase and glutathione-S-transferase work in concert with

glutathione in the decomposition of H2O2 and other organic hydro peroxides

to non-toxic products, at the expense of reduced glutathione. Depletion of

these enzymes may result in deleterious oxidative changes due to the

accumulation of toxic products. As enzymatic antioxidants are saturated by

excessive levels of free radicals, the presence of non-enzymatic antioxidants

is presumably essential for the removal of these radicals (Allen 1991).

98

Table 4.23 Effect of EtALL extract on the activities of GPx and GST in

tissues and haemolysate of normal and streptozotocin

induced diabetic rats

Groups Normal

Diabetic

control

(STZ-40

mg/kg

b.w.)

Diabetes+

EtALL

(200 mg/kg

b.w.)

Diabetes+

EtALL

(400 mg/kg

b.w.)

Diabetes +

Glibenclamide

(600 µg/kg

b.w.)

Glutathione peroxidase (GPx)

Liver

(U/mg

protein)

6.21±0.4 3.21±0.3 4.25±0.5*

5.81±0.4**

4.98±0.4**

Kidney

(U/mg

protein

4.23±0.2 2.18±0.2 2.81±0.6*

3.89±0.2**

3.53±0.1 **

Brain (U/mg

protein)3.17±0.3 1.01±0.1 1.53±0.2

*2.73±0.1

**2.06±0.1

**

Haemolysate

(U/mg Hb)10.12±1.6 6.58±1.5 8.72±0.1

*9.28±1.3

**9.82±1.4

**

Glutathione-S-transferase (GST)

Liver (U/mg

protein)6.21±0.5 3.42±0.2 4.02±0.7

*5.82±0.3

**4.81±0.3

**

Kidney

(U/mg

protein)

5.03±0.4 2.15±0.1 3.53±0.8*

4.94±0.3**

4.52±0.3 **

Brain (U/mg

protein)5.58±0.4 1.43±0.1 3.64±0.2

*5.18±0.2

**4.35±0.2

**


*: p<0.05;

**: p<0.01.

99

Table 4.24 Effect of EtALL extract on level of glutathione in plasma

and tissue of normal and streptozotocin induced diabetic rats

GroupsReduced glutathione (mg/100 mg tissue)

Liver Kidney Brain PlasmaNormal 46.72±3.9 34.01±2.3 35.11±2.0 25.25±2.5Diabetic control(STZ-40 mg / kgb.w.)

23.23±1.8 19.06±1.2 20.04±1.2 12.03±0.4

Diabetes+ EtALL(200 mg/kg b.w.)

29.62±2.6*

21.85±1.7*

21.66±2.8*

15.81±0.5*

Diabetes+ EtALL(400 mg/kg b.w.)

40.01±2.8**

30.43±1.7 **

29.52±1.9 **

21.22±2.0 **

Diabetes +Glibenclamide(600 µg/kg b.w.)

34.72±2.9**

24.06±1.6 **

25.14±1.5**

18.07±1.2**


*: p<0.05;

**: p<0.01

In this study, the activities of GPx, GST and GSH in the tissues

(liver, kidney and brain) and plasma of normal, diabetic control, EtALL

extract and glibenclamide treated diabetic rats was evaluated. There was a

significant decrease of GPx, GST and GSH levels in the tissues and plasma of

diabetic control rats compared to the normal. Administration of EtALL

extract at 400 mg/kg b.w. treated diabetic rats showed a significant increase of

GPx, GST and GSH levels in tissue and plasma when compared to the

diabetic control rat. Glibenclamide treated diabetic rats also showed a linear

increase of these enzyme levels in tissue and plasma (Table 4.23 and Table

4.24). Our findings are very well correlated with the earlier reports of

Leelavinothan and Muniappan (2004) in Scoparia dulcis, Palani et al (2010)

in Chloroxylon swietenia and Kannampalli et al (2010) in Cassia fistula.

4.6.1.14 Effect of EtALL extract on liver enzyme markers in serum

Biochemical parameters are sensitive index to changes due to

xenobiotics and constitute important diagnostic tool in toxicological studies

100

(Dorman 2000). Phosphatases are important and critical enzymes in biological

processes, as they are responsible for detoxification, metabolism and

biosynthesis of energetic molecules for different essential functions

(Bengt and Kent 1975). Any interference in these enzymes leads to

biochemical impairment and lesions of the tissue and cellular function

(Khan et al 1995).

Table 4.25 Effect of EtALL extract on liver enzyme markers in serum

of normal and streptozotocin induced diabetic rats

GroupsAST

(U/dl)ALT (U/dl) LDH (U/dl) ALP (U/dl) ACP (U/dl)

Normal 34.22±2.5 55.17±4.7 1172.01±23.7 53.28±7.24 12.43±1.23

Diabetic

control (STZ-

40 mg/kg

b.w.)

64.17±3.9 88.23±3.0 1561.89±31.8 82.41±4.86 20.93±1.85

Diabetes+

EtALL (200

mg/kg b.w.)

50.45±3.0*

70.32±3.5*

1410.45±31.9*

77.80±4.67*

18.71±1.09*

Diabetes+

EtALL (400

mg/kg b.w.)

39.92±2.8*

*59.20±6.18

*

*1192.13±38.8

*

*64.73±5.27

*

*15.70±1.41

*

*

Diabetes +

Glibenclamid

e (600 µg/kg

b.w.)

47.54±3.0*

* 64.88±6.4** 1310.62±25.0

*

*67.63±4.46

*

*16.29±1.28

*

*


*: p<0.05;

**: p<0.01

In this study, it was attempted to determine the serum liver enzyme

markers in the normal, diabetic control, EtALL extract treated and

glibenclamide treated diabetic rats. The activities of biochemical markers

(AST, ALT, LDH, ALP and ACP) increased significantly in diabetic rats

compared to the normal which might be due to the necrotized state of the liver

101

in diabetic condition and leakage of these enzymes from the liver cytosol into

the blood stream (Ohaeri 2001). Oral administration of EtALL extract at 400

mg/kg b.w. has significantly reduced the level of biochemical markers

compared to the diabetic control rats. Glibenclamide treated diabetic rats also

showed a decrease in the level of biochemical markers compared to the

diabetic control rats (Table 4.25). Similar findings were reported by El-

Demerdash et al (2005) in Allium sativum, Daisy et al (2008) in Costus

specious and Muhammad et al (2011) in Digera muricata.

102

4.6.1.15 Histopathological observations in normal and experimental

diabetic rats (400X magnification)

Histopathological changes in liver, kidney and pancreas of normal,

diabetic control, EtALL extract (400 mg/kg b.w.) and glibenclamide (600

µg/kg b.w.) treated diabetic rats are given as follows:

Figure 4.11 Histopathological observations in tissue sections of normal

and diabetic rat liver

a) Normal rat: HEP-Normal radiating hepatocytes

b) Diabetic control: IF-Inflammated portal traid in sinusoidal space

c) Diabetic induced + EtALL treated: PT-Mild inflammated portal triad

d) Diabetic induced + Glibenclamide treated: HEP-Recovery of

hepatocytes;FC- fatty changes

103


and diabetic rat kidney

a) Normal rat: IT-Intact tubules; IG-Intact glomeruli (IG)

b) Diabetic control: DT-Degenerating tubules; FI-Fatty infiltration of

tubules

c) Diabetic induced + EtALL treated: FI-Mild fatty infiltration;

TU-Mild dilation of tubules

d) Diabetic induced + Glibenclamide treated: CG-Congested glomeruli;

NT- normal tubules

104


and diabetic rat pancreas

a) Normal rat: I-Normal architecture of pancreatic islets of langerhans;

AC-Acini

b) Diabetic control:SI-Shrunken islets of langerhans

c) Diabetic induced + EtALL treated: EI-Hyperplastic islets of

langerhans

d) Diabetic induced + Glibenclamide treated: NH-Normal hyperplastic

of islets of langerhans

105

Liver

The normal rats showed normal radiating hepatocytes

(Figure 4.11a) Pathological changes of liver of diabetic control rats include

hepatic nuclear condensation of portal triad with inflammation and sinusoidal

dilation (Figure 4.11 b). Piyachaturawat et al (1991) reported STZ exhibits

nephrotoxic and hepatotoxic activity. There was a remarkable reduction in

inflammation and sinusoidal dilation in the EtALL extract treated (400mg/kg

b.w.) diabetic rats (Figure 4.11 c) which were comparable with glibenclamide

diabetic rats (Figure 4.11 d). These results are similar to that observed in

A.paniculata in streptozotocin induced diabetic rats by Reyes et al (2006),

Costus specious by Eliza et al (2009) and Hypericum perforatum by

Arokiyaraj et al (2011).

Kidney

The normal rats showed intact tubules and glomeruli (Figure 4.12

a). Whereas, kidney of diabetic control rats showed glomeruli mesangial

capillary proliferation along with tubular epithelial damage (Figure 4.12 b).

De-Rubertis and Craven (1993) reported the progressive glomerulosclerosis

associated with decreased kidney function in streptozotocin diabetic rats.

There was a excellent recovery of renal function with EtALL extract treated

diabetic rats (Figure 4.12 c) explained by the regenerative capability of the

renal tubules which is comparable to glibenclamaide treated rats (Figure 4.12

d). The role of EtALL extract reversing the diabetic state at the cellular level

besides the metabolic normalization further proves its potential as an

antidiabetic assert. Similar results have been observed with the Annona

squamosa (Kaleem et al 2006), Tinospora cordifolia (Rajalakshmi et al 2009)

and Elephantopus scaber (Daisy et al 2009).

Pancreas

The normal rats showed unaltered exocrine acini and endocrine

islets (Figure 4.13 a). The ultra structure of streptozotocin induced pancreas

106

showed considerable shrinkage in the islet of langerhans (Figure 4.13 b).

These are in agreement with earlier reports (Sharma et al 2003 and Michel

et al 2011). The regenerative effect of the pancreatic cells in EtALL extract

treated diabetic rats (Figure 4.13 c) via exocrine cells of pancreas may

enlighten the positive effects of these agents on the production of insulin

better than glibenclamide treated diabetic rats (Figure 4.13 d). There are

previous reports on ameliorative effect of Annona squamosa (Kaleem et al

2006), Petrocarpus marsupium (Maruthupandian and Mohan 2011) and

Mimus elengi (Purnima et al 2010) on langerhan in the pancreas.

The above data showed that the EtALL extract (400 mg/kg b.w.)

was effective in improving the functions of liver, kidney and pancreas as well

as reducing the lesions associated with diabetic state in streptozotocin induced

diabetic rats.

4.7 ISOLATION AND CHARACTERIZATION OF BIOACTIVE

COMPOUNDS

4.7.1 Bioassay Activity Guided Fraction in 3T3-L1 Cell Line

Based on the promising outcome of the in vitro and in vivo

(antidiabetic and antioxidant) studies of EtALL extract an attempt was made

to isolate the bioactive fractions present in it using column chromatography

(Silica gel G (60-120)) and TLC technique.

Lipogenesis is the process by which simple sugars such as glucose

are converted into fatty acids, which are subsequently esterified with glycerol

to form the triacylglycerols that are packaged in VLDL and secreted from the

liver (Gregoire et al 1998). Adipocytes play a common link between diabetes and

obesity, which stores excess energy in the form of triglyceride, and releases

free fatty acids in response to energy requirements such as fasting. 3T3-L1

adipocytes cell line are used as in vitro models to evaluate the antidiabetic

action of the drugs since adipocytes mimic fat cells and induce insulin

resistance which is the major contributor of diabetes (Guilherme et al 2008).

107

Table 4.26 Bioassay guided fraction of EtALL extract Vs different

isolated fractions

Samples Concentration

(µg/ml)

Lipogenesis

(%)

EtALL extract

5

10

25

50

75

20.12±3.21

40.24±5.63

80.67±1.27*

60.83±7.04

46.65±4.24

Fraction I

(CHCl3= 100)

5

10

25

50

75

20.53±2.45

43.51±5.51

70.30±3.34*

51.53±1.56

45.44±1.58

Fraction II

(CHCl3:CH3OH=90:10)

5

10

25

50

75

19.56±7.34

38.87±3.38

72.45±5.26*

45.23±5.57

33.92±2.43

Fraction III

(CHCl3:CH3OH=80:20)

5

10

25

50

75

13.65±6.12

27.43±8.18

54.45±2.22*

53.23±2.25

45.30±3.29

Fraction IV

(CHCl3:CH3OH=70:30)

5

10

25

50

75

23.97±5.22

47.95±4.27

96.83±2.24**

68.22±1.21

51.02±4.22

Fraction V

(CH3OH=100)

5

10

25

50

75

17.33±6.52

35.62±3.59

60.39±3.44

66.28±2.49*

39.80±2.43

Control (3T3-L1untreated cells) 100.00

Rosiglitazone (1.0 µmol/l) 100.86±0.27**


*: p<0.05;

**: p<0.01.

108

EtALL extract was subjected to column separation which resulted

in 46 fractions with chloroform and ethyl acetate as mobile phases. Further,

these fractions were subjected to sub column separation with chloroform:

methanol combination (100, 90:10, 80:20, 70:30 and 100) as mobile phases.

In addition, fractions with similar refractive index were pooled together as

fraction I (fraction 1 to 5), fraction II (fraction 6 to 11), fraction III

(fraction 12 to 15), fraction IV (fraction 16 to 19) and fraction V (fraction 20

to 24). These fractions were collected, concentrated and bioactivity studies

were carried out using Oil-Red-O-staining in 3T3-L1cell line to evaluate the

percentage of lipogenic activity (Table 4.26).

EtALL extract, fractions I to V of concentrations 1, 10, 25, 50 and

75 µg/ml, rosiglitazone (1.0 µmol/l) (positive control) and untreated cells

(negative control) were used in the bioassay study. Adipocyte differentiation

of 3T3-L1 cells is a highly-controlled process that can be induced under a

hormonal cocktail of insulin, dexamethasone and IBMX (Abbasi et al 2004;

Lee et al 2009). Upon the completion of adipogenesis, preadipocyte

fibroblasts that were originally spindle-shaped transforms into round-shaped

cells. Simultaneously, accumulation of lipids and glucose uptake in response

to insulin are favoured. (Gregoire et al 1998 and Feve 2005). Therefore

intracellular lipid accumulation (lipogenesis) is commonly monitored in

Oil - Red - O - staining technique, as a general marker to indicate the extent

of adipogenesis in 3T3-L1 cells (He et al 2009; Shang et al 2007 and Hwang

et al 2009).

The lipogenic activity of the untreated cells was found to be 100%

in the Oil-Red staining (Figure 4.14 a). The EtALL extract at 25 µg/ml

showed 80.67% lipogenic activity (Figure 4.14 b). Among the various

fractions isolated fraction IV (25 µg/ml) (Figure 4.14 c) was found to inhibit a

109

maximum of 96.83% of lipogenic activity which may be due to the down

regulation of receptor (PPAR- ) which is expressed in 3T3-L1 cells.

However, rosiglitazone (PPAR- agonist) showed enhanced activity of

145.86% (Figure 4.14 d). The moderate lipogenic activity of fraction IV

(25 µg/ml) may be due to the PPAR antagonist effect and there was less

accumulation of intercellular lipids resulting in reduced side effects (Rayalam

et al 2008). Hence, 25 µg/ml could be the saturation limit of the isolated

bioactive fraction and EtALL extract. Therefore, an increase or decrease did

not exert any increment in lipogenic activity. The other fractions I, II, III and

V failed to exhibit significant lipogenesis activity. Similar findings were

reported in the isolated bioactive compounds and crude methanolic extracts of

Cinnamomum cassia (Baddireddi et al 2010) and Costus pictus (Shilpa

et al 2009).

Therefore, the active fraction IV was further purified using TLC

and characterized by NMR, IR and ESI-MS for structure elucidation of the

lead compound.

110

Figure 4.14 Oil-Red-O-Staining of EtALL extract and fraction IV

treated adipocytes of 3T3-L1 cell line (400X magnification)

a) Untreated 3T3-L1cells: FC-Pink colored fat cells

b) Adipocyte cells treated with EtALL extract: LA- Moderate lipid

accumulation

c) Adipocyte cells treated with fraction IV: LA-Higher lipid

accumulation

d) Adipocyte cells treated with rosiglitazone: FC-Increased lipid

accumulation

4.7.2 Characterization of Bioactive Principle

4.7.2.11H NMR analysis of the isolated bioactive compound

The isolated bioactive fraction IV (25 µg/ml) was characterized

using spectral analysis.

111

Figure 4.151H spectrum of the isolated bioactive compound

The light yellow gum like compound (8.0 mg) was isolated from

the EtALL extract. The1H NMR showed the following spectrum (Figure

4.15), the molecule was found to be a modified lignan with two

phenylpropenoid units coupled at the C-7 (C-7’) and C-8 (C-8’) positions,

from the observation of an AA’B’B system of methine signals at H 3.46 (2H,

dd like, H-7 and H-7’) and 3.30 ppm (2H, dd like, H-8 and H-8’). Other

resonance signals observed in the 1H NMR spectrum were assignable to two

aromatic rings at H 6.80 (2H, d, J = 2.1 Hz, H-2 and H-2’), 6.77 (2H, dd,

J = 2.1, 8.1 Hz, H-5 and H-5’), and 6.65 (2H, dd, J = 2.1, 8.1 Hz, H-6 and H-

6’), as well as carboxymethyl groups at H 3.68 (6H, s, H-10 and 10’). The

shape of the AA’B’B system of methine signals at H 3.46 and 3.30 ppm

implied a chemically equivalent but magnetically non-equivalent environment

for the cyclobutanoid proton set, and the relative configuration of the

112

cyclobutyl ring was determined to be the same as that of - truxinic acid,

based on a comparison of the 1H NMR chemical shifts of this compound with

reported data of various truxillic and truxinic acid derivatives (Ben-Efraim

and Green 1974). This conclusion was supported by a further comparison of

the NMR data of this compound with those of -truxinic acid derivatives

reported in recent years (Saito et al 2004).

4.7.2.213

C NMR analysis of the isolated bioactive compound

Figure 4.1613

C spectrum of the isolated bioactive compound

The13

C NMR showed the following spectrum (Figure 4.16),only

ten resonance signals, which suggested that the molecule is composed of two

identical units. These carbon resonance signals were assignable to two such

units, with each composed of an ester functional group [ C 173.6 (C-9 and 9’)

and 52.3 (C-10 and 10’)], an aromatic ring with substitution by two hydroxy

113

groups [ C 145.9 (C-3 and 3’), 145.0 (C-4 and 4’), 134.2 (C-4 and 4’), 119.1

(C-6 and 6’), 116.1 (C-5 and 5’), 114.8 (C-2 and 2’)], and two methine

carbons [ C 48.5 (C-7 and 7’) and 45.6 (C-8 and 8’)]. By deduction of the

unsaturation number of 10 due to the aromatic rings and ester functionalities,

it was evident that another ring is present in the molecule.

4.7.2.31H-

1H COSY spectrum of the isolated bioactive compound

Figure 4.171H-

1H COSY spectrum of the isolated bioactive compound

The assignments of the1H and

13C NMR signals of compound were

finalized with1H-

1H COSY, experiments (Figures 4.17). Therefore, the structure

of this compound was determined as dimethyl 3,3’,4,4’-tetrahydroxy - -

truxinate, which has not been reported so far in the literature of A. lineata.

114

4.7.2.4 FT-IR spectrum of isolated bioactive compound

Figure 4.18 FT-IR spectrum of the isolated bioactive compound

In the IR spectrum (Figure 4.18) IR (film) max 3350 (br), 3100,

2956, 1710, 1624, 1514, 1440, 1165, 825 cm-1 showed the corresponding

functional groups possibly present in the bioactive compound.

115

4.7.2.5 ESI-MS spectrum of isolated bioactive compound

Figure 4.19 ESI-MS spectrum of the isolated bioactive compound

The molecular weight was indicated by its molecular ion peak at

m/z 411.1061 [M + Na]+ (calculated for C20H20O8Na, 411.1057) in HRESIMS

(positive mode) m/z (Figure 4.19).

OH

OH

OH

OH

O

MeO

O

MeO

3

1678

7'8'

9

9'1'

6'

3'

Dimethyl 3, 3', 4,4'-tetrahydroxy- -truxinate

Figure 4.20 Structure of the isolated bioactive compound

116

Based on the spectral analysis, the isolated bioactive compound is

identified as Dimethyl 3, 3’, 4, 4’-tetrahydroxy- -truxinate (DT T) (Figure

4.20). They belong to the novel class of -truxinic acid derivative (Deng et al

2011). Further, the bioactive compound (DT T) was investigated for the rate

of glucose uptake, lipogenic activity and synergetic effect with insulin.

4.7.2.6 Insulin mimicking activity of Dimethyl 3, 3’, 4, 4’ - tetrahydroxy

- - truxinate in 3T3-L1 cell line

Glucose and fat are two major substrates for energy production in

animals. Coordination between their metabolisms in providing energy is

sophisticated and is regulated by many hormonal and metabolic factors

(Wei and Guangyuan 2007). Disturbance of the energy homeostasis may

cause serious clinical syndromes that are manifested by abnormal blood

glucose or fatty acid levels. Although adipose tissue accounts for only 5-20 %

of glucose disposal, much of the work on insulin stimulated glucose transport

has been performed in adipocytes, due to the fact that many mechanistic

studies with regard to insulin’s action have been easier to carry out in this

tissue (Rayalam et al 2008).


Control-3T3-L1 untreated cells

Figure 4.21 Effect of Dimethyl 3, 3’, 4, 4’-tetrahydroxy- -truxinate on

insulin mimicking activity in 3T3-L1 cell line

*: p<0.05;

**: p<0.01

117

The insulin mimicking activity was evaluated in the 3T3-L1 cell

line with untreated cells (control) compared with cells treated with various

concentrations of the isolated bioactive compound Dimethyl 3, 3’, 4, 4’-

tetrahydroxy- -truxinate (DT T) and insulin (1.0 nmol/l). Enhanced

(189.8%) glucose uptake activity was observed in insulin treated 3T3-L1 cell

line. Control (untreated cells) showed a sustained activity (100%). Among the

various concentrations of DT T tested, 25 µg/ml was found to exhibit

maximum rate of glucose uptake (131.2%) compared to the control cells

which might be due to the insulin mimicking activity or effective binding

exhibited by DT T to the receptors. In neither contrast, either a decrease nor

increase in concentration of DT T failed to show any significant rate of

glucose uptake. Hence, 25 µg/ml of DT T may be considered as the optimum

concentration in increasing the rate of glucose uptake (Figure 4.21). Similar

observations were also reported from the isolated natural compounds of

cinnamic acid in Cinnamomum cassia (Baddireddi et al 2010),

methyltetracosanote from Costus pictus (Shilpa et al 2009) and gallic acid

from Hippophae rhamnosides (Vishnu et al 2010).

4.7.2.7 Lipogenesis of Dimethyl 3, 3’, 4, 4’-tetrahydroxy- -truxinate

by Oil-Red –O-staining in 3T3-L1 cell

Antidiabetic drugs such as insulin and thiazolidinedione

(rosiglitazone) up-regulate both glucose transport and lipid biosynthesis in

adipocytes (Stevenson et al 1996 and Park et al 1997) which results in

obesity. Weight gain is a frequent side effect of insulin therapy in

combination with thiazolidinedione intake in diabetes patients (Andreelli et al

2000). The drugs with glucose-lowering activity and antilipogenic activity are

highly desirable. Hence, the effect of lipogenesis activity of DT T was

evaluated by Oil-Red-O-staining technique.

118


Control-3T3-L1 untreated cells

Figure 4.22 Effect of lipogenic activity of Dimethyl 3, 3’, 4, 4’-

tetrahydroxy- -truxinate using 3T3-L1 cell line

DT T (25 g/ml) showed maximum lipogenic (80.23%) activity.

Further increase or decrease in DT T concentration did not reveal significant

effect (Figure 4.22). Hence, DT T might have a combination of glucose

lowering ability and antiadipogenic activity. Compounds such as corosilic

acid from Eriobotria japonica (Wei and Guangyuan 2007) cinnamic acid

from Cinnamomum cassia (Baddireddi et al 2010), ellagic acid from

Lagerstroemia speciosa (Naisheng et al 2008), methyltetracosanote from

Costus pictus (Shilpa et al 2009) and gallic acid from Hippophae rhamnosides

(Vishnu et al 2010) were also reported to posses glucose lowering and

antiadipogenic activity.

4.7.2.8 Insulin sensitization activity of Dimethyl 3, 3’, 4, 4’-

tetrahydroxy- -truxinate in 3T3-L1 cell line

To study the synergetic effect of DT T (25 µg/ml) in combination

with insulin (0–20 nmol/l), the glucose uptake activity was performed.

Maximum glucose uptake activity of 810.02% was observed. Further increase

*

*: p<0.05;

**: p<0.01

*

119


Figure 4.23 Insulin sensitization activity of Dimethyl 3, 3’, 4, 4’-

tetrahydroxy- -truxinate at 25 µg/ml in 3T3-L1 cell line

or decrease in insulin did not have any impact on glucose uptake (Figure

4.23). Similar type of activity was reported by Wei and Guangyuan (2007)

with corosilic acid isolated from Eriobotra japonica, cirsimarin from

Microtea debilis (Zarrouki et al 2010) and anacardic acid from Annacardium

occidentale (Leonard et al 2010).

4.8 REVERSE TRANSCRIPTASE POLYMERIZED CHAIN

REACTION

4.8.1 Effect of Dimethyl 3, 3’, 4, 4’-tetrahydroxy- -truxinate on the

Expression of PPAR- and C/EBP- mRNA

Carbohydrate metabolism and differentiation of 3T3-L1 adipocytes

are associated with diabetes (Hoist and Grimaldi 2002). Peroxisome

proliferator-activated receptor (PPAR)- and the CAAT/enhancer binding

Protein C/EBP family (C/EBP- , , and ) are critical factors in 3T3-L1

preadipocyte differentiation (Mukherjee et al 2000). PPAR- is a member of

the nuclear receptor super family of transcription factors and is predominatly

expressed in adipose tissue (Tontonoz et al 1994). C/EBP families are basic

(DT T)

120

leucine zipper of transcription factors (Hattori et al 2003 and Wei et al 2005).

C/EBP- family and PPAR are sequentially expressed during 3T3-L1

preadipocyte differentiation (Wu et al 1999; Ron et al 1990; Lin et al 1992

and Freytag et al 1994).

Figure 4.24 Expression of PPAR- mRNA at various concentrations of

Dimethyl 3, 3’, 4, 4’-tetrahydroxy- -truxinate using 3T3-L1

cell line

Lane 1: control (3T3-L1untreated cells); Lane 2:10 µg/ml; Lane 3:25 g/ml;

Lane 4:50 g /ml


Figure 4.25 Relative mRNA expression of PPAR- at various

concentration of Dimethyl 3, 3’, 4, 4’-tetrahydroxy- -

truxinate using 3T3-L1 cell line

Therefore, PPAR- and C/EBP family are key transcription factors

for adipocytes differenation (Yamamoto et al 2002). Hence, in our present

study, the effect of DT T on the expression of PPAR- and C/EBP- in 3T3-

L1 adipocytes was analysed using RT-PCR. The expression level of PPAR-

*

*: p<0.05;

**: p<0.01

121

and C/EBP- mRNA was investigated at various concentrations of DT T.

Reduced expression level of PPAR- mRNA was observed with 25 µg/ml

DT T (Figure 4.24 and Figure 4.25).

Figure 4.26 Expression of C/EBP- mRNA in various concentration of

Dimethyl 3, 3’, 4, 4’-tetrahydroxy- -truxinate using 3T3-L1

cell line

Lane 1:control (3T3-L1untreated cell); Lane 2:10 g/ml; Lane 3:25 g/ml;

Lane 4:50 g/ml


Figure 4.27 Relative mRNA expression C/EBP- at various concentration

of Dimethyl 3, 3’, 4, 4’-tetrahydroxy- -truxinate using 3T3-

L1 cell line

Similarly, reduced level of C/EBP- mRNA was observed with

25 µg/ml DT T (Figure 4.26 and Figure 4.27). Further, increase or decrease

in concentration of DT T showed higher expression of PPAR- and C/EBP-

mRNA.

* *

*: p<0.05;

**: p<0.01

122

Thus, the effective concentration of DT T for reduced level of

PPAR- and C/EBP- expression was 25 µg/ml. This suggests that, unlike

most other antidiabetic drugs, DT T may reduce blood glucose without

increasing adiposity. The combination of these two activities of DT T makes

it ideally suited as a prototypic compound for further functional studies to

develop novel bioactive compound. Similar findings were reported in

corosilic acid isolated from Eriobotrta japonica (Wei and Guangyuan 2007).

This could be the first report in demonstrating the potentiality of

DT T in inhibiting adipocytes by down regulating the gene expression of

PPAR- and C/EBP- . Based on the promising results of gene expression

studies, the work was further extended to determine the binding affinity of

DT T to insulin receptor using molecular docking studies.

4.9 MOLECULAR DOCKING STUDIES

4.9.1 Structures of Dimethyl 3, 3’, 4, 4’-tetrahydroxy- -truxinate

and Rosiglitazone

The process of finding novel leads for a new target is the most

important and undoubtedly one of the most crucial steps in identifying a drug

and its development program (Holger et al 2000). The number of potential

therapeutic target proteins is proliferating rapidly, making it increasingly

important to develop techniques for rapidly discovering and optimizing novel

therapeutic agents for these new targets. Experimental combinatorial

chemistry has provided enormous libraries with millions of potential ligands

quickly accessible for experimental tests to find positive lead compounds

against specific target proteins (Wely et al 2004).

123

Molecular-docking methodologies ultimately seek to predict (or

often retrospectively reproduce) the best mode by which a given compound

will fit into a binding site of a macromolecular target (Hongming et al 2006).

There has been an extensive research focused on peroxisome proliferator

activated receptors (PPARs) belonging to nuclear receptor family and C/EBP-

CAAT enhancer binding proteins which are ligand-activated transcription

factors. PPAR- and C/EBP- is expressed most abundantly in adipose tissue

and mediates the antidiabetic activity of the insulin-sensitizing drugs

belonging to the thiazolidindione (Campbell 2005).

This key transcriptional factor plays a pivotal role in regulating

adipogenesis, insulin sensitivity and glucose homeostasis (Willson et al

2000). A drug molecule is triggered when the binding of small molecule to

the receptor protein is perfectly done. Such protein-ligand interaction is

comparable to the lock-and-key principle, in which the lock encodes the

protein and the key is ensembled by the ligand. The major driving force for

binding appears to be the hydrophobic interaction whose specificity is

however controlled by hydrogen bonding interactions (Hugo 1998).

124

Figure 4.28 Two dimensional structure of Dimethyl 3, 3’, 4,

4’tetrahydroxy- -truxinate

Figure 4.29 Two dimensional structure of rosiglitazone

In the present study, the molecular docking was carried out using

GOLD software to identify the best ligand receptor binding score among

DT T and rosiglitazone (ligand) Vs PPAR- and C/EBP- . It is estimated that

docking programs currently dock 70-80% of ligands efficiently (Priya et al

2011). The structure of the ligand DT T and rosiglitazone (Figure 4.28 and

Figure 4.29) was drawn using Chemsketch software.

125

4.9.2 Structures of PPAR- and C/EBP- Retrieved from Protein

Data Bank

Figure 4.30 3D Structure of PPAR- in a cartoon model visualized using

Rasmol

Pink color indicate helices, yellow color indicates strands, white color

indicates turns

Figure 4.31 3D Structure of C/EBP- in a cartoon model visualized using

Rasmol

Yellow color indicates strands, white color indicates turns

The structure of receptors PPAR and C/EBP- (Figure 4.30 and

Figure 4.31) were retrieved from the protein data bank.

126

4.9.3 Docked poses of Dimethyl 3, 3’, 4, 4’-tetrahydroxy- -truxinate

and rosiglitazone with PPAR-

Figure 4.32 Docked pose of PPAR- with Dimethyl 3, 3’, 4, 4’-

tetrahydroxy- -truxinate

The green dots represent the hydrogen bonding and amino acid bonding

with SER 382, LEU 419

Figure 4.33 Docked pose of PPAR- with rosiglitazone

The green dots represent the hydrogen bonding along with the amino acids

ASP381 and SER382

When DT T and rosiglitazone was docked with PPAR- the docking

score of DT T was lower than rosiglitazone (Figure 4.32 and Figure 4.33).

127

4.9.4 Docked poses of Dimethyl 3, 3’, 4, 4’-tetrahydroxy- -truxinate

and Rosiglitazone with C/EBP-

Figure 4.34 Docked pose of C/EBP- with Dimethyl 3, 3’, 4, 4’-

tetrahydroxy- -truxinate

The green dots represent the hydrogen bonding and amino acid bonding

THR 310, ASN 307 and ASN 307

Figure 4.35 Docked pose of C/EBP- with rosiglitazone

No hydrogen and aminoacid bonding

The docking score of DT T was lower with C/EBP- when

compared to rosiglitazone (Figure 4.34 and Figure 4.35).

128

Table 4.27 Docking score and docking modes of Dimethyl 3, 3’, 4, 4’-

tetrahydroxy- -truxinate and rosiglitazone

Protein Ligand nameAtom in

Protein

Atom in

ligand

H-bond

DistanceScore

PPAR-

Rosiglitazone ASP381:OD2

SER382:N

O18

O15

3.033

2.692

45.91

Dimethyl 3, 3’, 4,

4’-tetrahydroxy- -

truxinate

SER382:OG

LEU419: O

C19

O18

2.141

2.986

34.28

C/EBP-

Rosiglitazone No Hydrogen bonding 31.99

Dimethyl 3, 3’, 4,

4’-tetrahydroxy- -

truxinate

THR310:OG1

ASN307:OD1

ASN307:CB

O17

C13

C13

2.818

2.754

2.974

27.35

From Table 4.27 it can be inferred that rosiglitazone showed weak

interaction towards C/EBP- whereas DT T (bioactive principle) isolated

from A.lineata leaves showed strong interactions with PPAR- and C/EBP- .

Reports on molecular docking of PPAR- inhibitors have been postulated in

cis-parinaric acid (Yu et al 2004) heteroaryl propanoic acid derivatives of

PPAR agonists (Congreve 2005), chalcones as PPAR agonists (Manga et al

2010), vanillin semi carbazones (Venkatesan et al 2011) and -phenoxy

phenyl propionic acid derivatives are used as antidiabetic agents (Sivakumar

et al 2011). Since, no work has been published on molecular docking for

PPAR- and C/EBP- , this could be the first report of its kind.

Dimethyl 3, 3’, 4, 4’-tetrahydroxy- -truxinate has been

demonstrated as the best fit molecules in both the receptors (PPAR- and

C/EBP- ). Further, Dimethyl 3, 3’, 4, 4’-tetrahydroxy- -truxinate may be

structurally modified to enhance the antidiabetic potential and may serve as a

lead molecule in the discovery of novel antidiabetic drug.

Documents

CHAPTER 4 RESULTS AND DISCUSSIONS - …shodhganga.inflibnet.ac.in/bitstream/10603/15501/9/09_chapter 4.pdf · 59 Purkayastha et al (2008) and Hemalatha and Vadivel (2010). In contradiction