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RESULTS AND

DISCUSSION

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Results and Discussion

4.1. GROWTH AND DEGRADATION

4.1.1 Screening of cyanobacteria

Among the thirteen cyanobacteria screened, three strains such as Oscillatoria

laetevirens BDU 20801, Phormidium valderianum BDU 20041, Lyngbya sp. BDU

141961 were showed a better colonization on P. juliflora. However, Oscillatoria

laetevirens colonized the substrates and started degradation more rapidly when

compared to other cyanobacterial species (Table-1). Considering the high growth rate

and colonization on the selected lignocellulosic material, Oscillatoria laetevirens

(Plate-1a) was selected for further studies.

The microscopic observation of the selected cyanobacterium showed the

following morphological features. The culture possessed trichomes single or forming

mat or spongy thallus without sheath, motile, showing typical oscillatory movements,

terminal portion of the trichome cells hooked or spindle shaped hormogones present

(Desikachary, 1951).

Order - Nostacales

Family - Oscillatoriaceae

Genera - Oscillatoria

Species - laetevirens

4.1.2 Optimization of growth conditions

Optimization of O. laetevirens growth with different sized particle (100 µm-

1 mm, 1-2 mm, 2-3 mm) (Plate-2a) of P. juliflora with varying dry weight ratios (0.05,

0.1, 0.2, 0.3 and 0.4) were tested. The results showed better colonization and

degradation of lignocellulosic material of a particle size between 1-2mm by the


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O. laetevirens. Also, O. laetevirens and P. juliflora in 0.1:0.3 (wet:dry weight) ratio

was found to be optimum for colonization of cyanobacterium as well as degradation of

lignocellulosic material. Wood particles of P. juliflora with the size of 1-2 mm and 0.3

dry weight ratio enhanced the cyanobacterial colonization and growth, additionally it

also facilitated the penetration of O. laetevirens and enzyme secretion for the

degradation.

4.1.2a Biochemical analysis

Chlorophyll a

Chlorophyll a estimation was carried out to determine the biomass of

O. laetevirens which was grown along with P. juliflora. Results indicated that

O. laetevirens grown with P. juliflora with different particle size and ratio (Fig. 8) on

15th day showed a high amount of chlorophyll content when compared to the control

(O. laetevirens alone). Furthermore, O. laetevirens grown with 1-2 mm particle size

and 1:3 ratio of P. juliflora showed higher amount of chlorophyll a content when

compared with other particle size (100 µm-1 mm and 2-3 mm) and ratio (0.05, 0.1, 0.2

and 0.4) and control O. laetevirens alone.

Chlorophyll a has been used to determine the biomass of cyanobacteria

(Manoharan and Subramanian, 1992; Shashirekha et al., 1997; Ramirez et al.,

2000 Vijayakumar et al., 2005; and Saswati et al., 2004). Chlorophyll a content in

Oscillatoria pseudogeminata var. unigranulata was studied by Manoharan and

Subramanian (1993) to monitor its growth response on exposure to ossein effluent.

Malliga (1993) studied the variation in chlorophyll a content to determine the growth

response of Azolla pinnata to various nitrogen sources like nitrogen free medium,

sodium nitrate, ammonium chloride and ammonium nitrate.

Anabaena azollae ML2 grown in the presence of coir pith showed an increase

in growth rate in terms of chlorophyll a was reported by Malliga et al. (1996).

Evaluation of degradative efficiency of petroleum hydrocarbons by Oscillatoria

agardhi and Anabaena sphaerica revealed increase in biomass when compared with a

control culture (Gamila et al., 2003). Parikh and Madamwar, (2005) measured the

growth of cyanobacteria during textile dye decolorization by measuring chlorophyll a.


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Thus it can be concluded that the increased chlorophyll a content in test samples

indicated that the presence of lignocellulosic waste P. juliflora did not inhibit the

growth of O. laetevirens but, surprisingly O. laetevirens utilized the degraded

lignocellulosic materials as a source of nutrients for its growth and other metabolic

activities.

Reducing Sugar

The presence of reducing sugars in the supernatant can provide supporting

evidence for the lignolytic activity of O. laetevirens. Concentration of reducing sugar

was considerably higher in O. laetevirens degraded lignocellulosic’s culture filtrate

(0.1:0.3 of O. laetevirens with P. juliflora respectively) when compared with control

cyanobacteria and control lignocellulosic alone (Fig. 9). This may be due to cleavage of

complex cellulosic material to simple sugars by the cyanobacterial enzymes. The

obtained results were similar with the previous studies of Shasirekha et al. (2001) who

reported the release of reducing sugars and amino acids into media during the growth of

Pleurotus florida on rice straw. Also the reducing sugar content was found to be higher

in culture filtrate of degraded P. juliflora by a fresh water cyanobacterium Phormidium

sp. (Prabha et al., 2005). Yu et al. (2009) reported that biological delignification

enhances the sugars released during the enzymatic hydrolysis. Roy et al. (2001) and

Sigoillot et al. (2002) confirmed the xylanase activity in Aeromonas caviae and

Pycnoporus cinnabarinus by measuring the reducing sugars released into the medium.

Similarly, Ojumu et al. (2003) mentioned that the presence of reducing sugar in the

supernatant could confirm the presence of cellulase activity in Aspergillus flavus Linn.

isolate NSPR 101.

Furthermore, the presence of reducing sugars and aromatic compounds in media

of pulp and paper production was used to confirm the production of peroxidase and

endoxylanase from Streptomyces (Tuncer et al., 2004). Kaya et al. (2000) confirmed

the influence of lignin and its degradation products by enzymatic hydrolysis of xylan

and by estimating the amount of reducing sugars in the media.

Phenol

The release of phenol from control O. laetevirens and P. juliflora was much

lower when compared to culture filtrate of degraded P. juliflora by O. laetevirens. A


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higher amount of phenol content was observed in degraded P. juliflora culture filtrate

of 1-2 mm particle size and in ratio 0.1:0.3 (O. laetevirens with P. juliflora) when

compared to other particle size (100 µm-1 mm and 2-3 mm) and ratio (0.05, 0.1, 0.2

and 0.4) on 15th day (Fig. 10). This result was in accordance with the biodegradation of

P. juliflora in which phenol was quantified more in P. juliflora exposed to Phormidium

sp. culture filtrate (Prabha et al., 2005).

Shashirekha et al. (1997) reported the ability of a marine cyanobacterium

Phormidium valderianum to degrade phenol up to a concentration of 100 mg/ml.

Wurster et al. (2003) reported the extracellular degradation of phenol by a

cyanobacterium Synechococcus PCC 7002. Their investigations led to the identification

of muconic acid as the major product of phenol transformation by Synechococcus PCC

7002 and demonstrated that the cleavage of the aromatic ring system by cyanobacteria

for the first time. This pathway of phenol degradation seems to be similar to the well

known ortho-fission of phenolic compounds by heterotrophic bacteria and yeasts,

which give rise in cis, cis-muconic acid.

All the evidences clearly suggest and justify that the presence of phenol in the

media supernatant can be due to the degradation of phenolic polymers from

lignocellulosic material by Oscillatoria laetevirens.

Lignin

Analysis of the lignin content in the control P. juliflora after 15 days of

incubation revealed that there was no reduction in the lignin content. However, 1-2 mm

particle size and 0.1:0.3 ratio of P. juliflora treated with O. laetevirens showed

maximum reduction of lignin (26.8%) content when compared to other treatments (Fig.

11). Malliga et al. (1996) have reported that Anabena azollae while being used as a

biofertilizer, exhibited lignolysis and released phenolic compounds which include

profuse sporulation of the organism. This report gives the usefulness of coir waste as a

carrier for cyanobacterial biofertilizers with supporting enzyme studies on lignin

degrading ability of cyanobacteria and use of lignocellulosic coir waste as an excellent

and inexpensive carrier for cyanobacterial biofertilizers.


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Crawford et al. (1983) studied the lignin degrading ability of Streptomyces

viridosporus and reported that it degraded 19.7% lignin in 8 weeks. Perestelo et al.

(1994) studied the Kraft lignin degradation by Serratia marcescens and found only

15% degradation. Akin et al. (1995) also reported the delignification of Bermuda grass

by white-rot fungi. The biodegradation of Bermuda grass stems was improved by 29-

32% using Ceriporiopsis subvermispora and 63-77% using Cyathus stercoreus after 6

weeks in the studies with Daedalea flavida and two of the Phlebia sp. Furthermore,

degradation of intermediates and products of synthetic lignin by Phlebia tremallosa

was observed by Gutierrez et al. (1996).

Kannan et al. (1989) studied the mechanism of degradation of paper mill

sludge containing lignin and cellulose by Pleurotus sajor-caju. It degraded 78% lignin

and 73% cellulose after 30 days of incubation. Investigations by Wurster et al. (2003)

of the unicellular marine cyanobacterium Synechococcus PCC 7002 revealed its ability

to metabolize phenol under non-photosynthetic conditions up to 100 mg/L.

An investigation made by Wu et al. (2005) to explore the lignin degrading

capacity of five white rot fungi Phanerochaete chrysosporium, Pleurotus ostreatus,

Lentinus edodes, Trametes versicolor and S22 reported that they were individually used

to treat black liquor from pulp and paper mill. The treatment removed 71% of lignin

and 48% of chemical oxygen demand (COD) from the waste water in 16 days.

Anbuselvi and Rebecca (2009) reported that the selected three different cyanobacterial

species such as Phormidium sp, Oscillatoria sp. and Anabaena azollae degrade high

lignin containing coir waste efficiently and estimated that 89% of lignin and 92% of

hemicelluloses were found to be reduced in 30 days.

4.1.3 Biodegradation of optimized P. juliflora

The P. juliflora particle size 1-2 mm and the 0.1:0.3 (O. laetevirens and

P. juliflora) were selected for further investigation

4.1.3a Microscopic view of degraded P. juliflora

The mineralization of Prosopis juliflora by Oscillatoria laetevirens during

different days of incubation was observed under microscope and it revealed that


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O. laetevirens colonize the Prosopis wood particles and effectively degrade the intact

cell wall by their lignolytic enzyme activity (Plate-3a-3d). The degradation of coir pith

by Oscillatoria annae was observed under microscope during different stages showed

lignin breakdown (Anandharaj, 2007).

Confocal laser scanning microscopy (CLSM) has been used to examine

bacterial and fungal decay in wood (Kim and Singh, 1999). Furthermore, electron

microscopy has been used to observe the biological deterioration of wood-based

composites exposed to various kinds of molds and fungi (Chung et al., 1999). Electron

microscopic and biochemical studies of lignocellulose degradation by wood-rotting

fungi have shown that enzymes such as lignin peroxidases, manganese-dependent

peroxidases, laccases and cellulases are too large to penetrate undegraded secondary

wood cell walls. Degradation occurs by surface interaction between cell wall and

enzymes, but initiation of decay at a distance from the fungal hyphae must involve

diffusible low-molecular mass agents (Evans et al., 1994).

Thus the present study revealed that microscopic observation at 10th, 20th 30th

and 40th days of degradation showed gradual degradation of lignocellulosic particles

and high extent of O. laetevirens colonization with P. juliflora and it was merely higher

in 30th and 40th day sample when compared with 10th and 20th day.

4.1.3b Growth parameters

Chlorophyll a

Chlorophyll a estimation was performed to find out the growth status of

Oscillatoria laetevirens along with the substrate P. juliflora. The results showed

significant increase of chlorophyll a in Oscillatoria laetevirens when grown with

Prosopis juliflora (47.7 mg/g) in 30th day when compared with control O. laetevirens

alone (24.4 mg/g) and other days samples (Fig. 12). Supporting evidence proved that

the growth of a cyanobacterium namely Anabaena azollae ML2 with coir pith showed

an increase in growth rate in terms of chlorophyll a (Malliga et al., 1996).

Another report showed that two isolated cyanobacterial strains, Oscillatoria

agardhi and Anabaena sphaerica, were evaluated for their degradative efficiency of


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petroleum hydrocarbons. Both strains revealed high algal biomass when compared with

control (Gamila et al., 2003). Increase of chlorophyll a in O. annae when grown with

coir pith was reported by Anandharaj (2007). Similarly, the fresh water

cyanobacterium Oscillatoria annae while grown with Lantana camara showed the

highest growth rate followed by P. juliflora and coir pith (Viswajith, 2008).

4.1.3c Enzyme assay

Lignolytic enzyme profile of O. laetevirens was studied for justifying its lignin

degrading ability on P. juliflora. Optimization of temperature and pH of each lignolytic

enzymes were performed colorimetrically. Laccase showed a maximum activity at

temperature 35 °C and pH 7.0 (Fig. 13 & 14) whereas, polyphenol oxidase showed

optimal activity at temperature 35 °C and pH 5.0 (Fig. 15 & 16). However, Manganese

independent peroxidase showed optimum activity at 25 °C and pH 4.0 (Fig. 17&18).

It is showed that polyphenol oxidase activity is highly induced in Oscillatoria

annae exposed to coir pith when compared to control (O. annae alone) (Anandharaj,

2007). Viswajith, (2008) revealed the presence of other manganese independent

peroxidase, laccase, polyphenol oxidase, cellulolytic enzymes like endogluconase and

xylanase in O. annae. Pointing (2001) reported that Lignin peroxidase (LiP), mangnese

peroxidase (MnP), laccase and versatile peroxidase (VPs) are the major lignin

modifying enzymes involved in lignin and xenobiotic degradation by wood rot fungi.

Both, lignin peroxidase and manganese-dependent peroxidase have been found

in the extra-cellular filtrates of many white-rot fungi which can degrade wood cell

walls (Kirk and Farrell, 1987; Waldner et al., 1988). Enzymes including polyphenol

oxidases, laccases, H2O2 producing enzymes and quinone-reducing enzymes can

degrade lignin (Blanchette, 1991). The white-rot fungus Phanerochaete chrysosporium

produces lignin-degrading enzymes, lignin peroxidases and manganese-dependent

peroxidases during secondary metabolism in response to carbon or nitrogen limitation

(Boominathan and Reddy, 1992). The advantages of biological pretreatment include

low energy requirement and mild environmental conditions.

Earlier studies showed that two freshwater cyanobacteria were able to

metabolize and degrade phenol (Gibson, 1982). Further, Shashirekha et al. (1997)


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reported that a marine cyanobacterium Phormidium valderianum BDU 30501 was

capable of degrading phenol through the activities of polyphenol oxidase and laccase

which were found to be intracellular. The absence of detectable activity of these

enzymes in control cultures establishes the inducible nature of these enzymes in this

cyanobacterium.

Enzyme profile of fresh water cyanobacterium Oscillatoria annae on exposure

to lignocellulosic material Lantana camara revealed the profund induction of both

major and accessory lignolytic enzymes like laccase, polyphenol oxidase, peroxidase

and esterase (Viswajith and Malliga, 2008).

Estimation of Hydrogen Peroxide

It is revealed that O. laetevirens exposed to P. juliflora released the maximum

amount (2.14 µM/g) of H2O2 when compared to control O. laetevirens (0.65 µM/g)

alone (Fig. 19). Similar result was reported in Oscillatoria annae exposed to coir pith

(Viswajith, 2008).

The possible involvement of hydrogen peroxide (H2O2) derived hydroxyl

radical (OH) in lignin degradation by Phanerochaete chrysosporium was investigated

by Forney et al. (1982). According to their study when P. chrysosporium was grown in

low nitrogen medium (2.4 mM N), increase in specific activity for H2O2 production in

cell extracts was observed to coincide with the appearance of ligninolytic activity and

both activities appeared after the culture entered stationary phase. Faison and Kirk

(1983) studied the relationship between the production of reduced oxygen species,

hydrogen peroxide (H2O2), superoxide (O2), and hydroxyl radical (OH) and the

oxidation of synthetic lignin to CO2 was studied in the cultures of white-rot fungus

Phanerochaete chrysosporium. Also in-vitro studies showed that milled wood lignin

was depolymerised by laccase in the presence of hydrogen peroxide (Evans, 1985).

The kinetics of the synthesis of H2O2 coincided with the appearance of the lignolytic

system. Also, H2O2 production was markedly enhanced by growth under 100% O2

mimicking the increase in ligninolytic activity characteristic of cultures grown under

elevated oxygen tension (Viswajith, 2008). These reports clearly indicate that

increased release of H2O2 from test samples was due to the lignolytic activity of

O. laetevirens.


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4.1.3d Biochemical estimations

Reducing sugar

The presence of reducing sugars in the supernatant acted as supporting evidence

for the lignolytic activity of Oscillatoria laetevirens. From the results it was observed

that the concentration of sugar was considerably higher in Prosopis juliflora degraded

culture filtrate in 30th day sample when compared with other days and with control

cyanobacteria and Prosopis juliflora (Fig. 20). This might be due to cleavage of

complex polymers to simple sugars by cyanobacterial enzymes. The obtained results

coincide with the previous studies made by Anandharaj (2007) who reported the

release of reducing sugars into the media during degradation of coir pith by

Oscillatoria annae. Also, the concentration of reducing sugar was considerably higher

in O. annae degraded Prosopis juliflora, Lantana camara and coir pith culture filtrates

when compared with control cyanobacterium and control lignocellulosics of

experiments carried out both in laboratory and field conditions (Viswajith, 2008).

Hence, it can be concluded that the presence of reducing sugars in the supernatant acted

as a confirmation of degradation of Prosopis juliflora by Oscillatoria laetevirens.

Phenol

Degradation of Prosopis juliflora resulted in release of some organic

compounds such as phenol which resulted in color changes of media from colorless to

brown. The phenol content was considerably high in Prosopis juliflora treated

cyanobacterial culture filtrate when compared to control Oscillatoria laetevirens and

Prosopis juliflora alone (Fig. 21). The presence of phenolic compounds in the culture

filtrate was further confirmed by UV spectrum analysis. The results obtained clearly

indicated the degradation of lignocellulosic material of Prosopis juliflora containing

lignin and holocellulose by Oscillatoria laetevirens. The degradation of lignocellulosic

material was so far demonstrated by other microorganisms such as bacteria (Perestelo

et al., 1994), actinomycetes (Ferraz and Duran, 1995) and fungi (Bhat and Narayan,

2003).

It was reported that the release of phenol content was considerably higher in

coir pith treated cyanobacterial culture filtrate when compared to control O. annae in

laboratory and field conditions. Also, O. annae grown with coir pith in urea medium


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released higher amounts of phenol when compared to BG11 and other minimal media

like NPK and Complex (Anandharaj, 2007).

UV-Vis Spectrum analysis

During the degradation of Prosopis juliflora by Oscillatoria laetevirens an

appreciable amount of phenolic compounds were released into the surrounding medium

that was studied by spectroscopic analysis (Fig. 22). In general the lignocellulose

consists of 45% cellulose, 30% hemicellulose and 25% lignin. Lignin shows a

maximum absorption at 270-310 nm wavelength. Thus, spectrophotometric detection of

the media supernatant in the UV range of 270-310 nm indicates the degradation of

lignin and the release of some interesting compounds into the medium. From the result

it was confirmed that the selected strain Oscillatoria laetevirens has the ability to

degrade the lignocellulosic waste of Prosopis juliflora and release compounds which

may be of bioactive in nature.

The present finding correlates with the spectral analysis of culture filtrate of

degraded coir pith by Oscillatoria annae which showed absorbance peak at 270 nm

(Anandharaj, 2007). Also, Vendramani and Trugo (2004) studied the phenolic

compounds in acerola fruit (Malpighia punicifolia L.) and reported that the absorbance

in UV region denoted that the compound released is of aromatic nature. Thus, this

report confirmed that the increase in the absorbance may be due to an increase in

concentration of phenolic compound in the supernatant released as a result of

degradation of Prosopis juliflora by Oscillatoria laetevirens.

Nitrate and Ammonia

Nitrate and ammonia are the sources of nitrogen that are essential for plant

growth. But, to some extent non-availability can be compensated by nitrogen fixing

cyanobacteria through atmospheric nitrogen fixation. Quantifying nitrate and ammonia

are valuable for assessing the potential of the cyanobacteria to fix nitrogen. In this

investigation estimations of nitrate and ammonia were carried out to analyze the

nitrogen fixing ability of the selected strain Oscillatoria laetevirens grown in ASN III

media along with Prosopis juliflora (Fig. 23 & 24). The nitrate and ammonia present in

the supernatants were analyzed and the obtained results showed that the amount of


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nitrate and ammonia were higher in Prosopis juliflora treated along with cyanobacteria

samples when compared to control (cyanobacteria alone) in 30th day sample. The

increased concentration of nitrate and ammonia depends on the availability of the

nitrogen source during the culture growth. The control cyanobacterial sample exhibited

a lesser amount of nitrate and ammonia when compared with test samples.

From the investigations, it is evident that cyanobacteria grown with Prosopis

juliflora exhibited a high quantity of nitrate and ammonia released into the medium.

This may be due to the fixation of atmospheric nitrogen by cyanobacteria under

microaerophilic and dark conditions which were created by addition of P. juliflora. Stal

and Krumbein (1985) reported about the fixation of nitrogen under aerobic conditions

by the non-heterocystous cyanobacterium Oscillatoria sp. According to them

Oscillatoria sp. while grown under alternating light-dark cycles, fixes nitrogen

preferably in the dark. Similarly, providing light-dark condition in lab and natural

light-dark condition in field during the degradation of coir pith by O. annae might have

been resulted in nitrogen fixation and accumulation of nitrate and ammonia in the

media (Anandharaj, 2007).

Protein

The obtained results showed that the concentration of protein in P. juliflora

treated O. laetevirens samples were higher when compared to control O. laetevirens

and P. juliflora alone (Fig. 25). The increase in protein content of test samples may be

due to the release of lignolytic enzymes into the medium which was required for the

degradation of lignin present in P. juliflora. Also the increase in protein can be

attributed to the de novo synthesis of phenol degrading enzymes and stress-related

proteins (Bhagwat and Apte, 1989). Shashirekha et al. (1997) reported the growth of

Phormidium valderianum 30501 during phenol degradation and observed that the test

samples showed higher protein content on exposure to phenol.

Early reports showed that the heterocystic, filamentous cyanobacterium

Calothrix sp. belonging to section IV exhibited variation in its protein content while

grown in BG11 with varying concentration of nitrogen (Ramirez et al., 2000). This

report clearly supported the difference in protein content exhibited by O. annae on


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exposure to various media like BG11, NPK, complex and urea. This could be due to the

variation in the availability of nitrogen present in the media. Maximum protein content

was observed in O. annae grown in urea supplemented media which was an easily

available nitrogen source in its simplest form (Anandharaj, 2007). Viswajith (2008)

showed that the protein was quantified higher in O. annae exposed to coir pith,

P. juliflora and L. camara when compared to control cyanobacteria and lignocellulosic

wastes alone.

Lignin

In P. juliflora a wide range of lignin content (11.5-31%) was reported by

Pasiecnik et al. (2001). While studying the effect of O. laetevirens on the degradation

of P. juliflora, it was observed that the reduction of lignin content in control P. juliflora

was negligible whereas, there was considerable decrease of lignin content in P. juliflora

exposed to O. laetevirens samples. The maximum percent of lignin reduction (34.3%)

was recorded in 30th day test sample when compared with other samples and control

(Fig. 26). Supporting evidences showed that 27% of lignin was found to be reduced in

coir pith treated with Oscillatoria annae when compared to control coir pith alone

during minimum incubation of 7 days period (Anandharaj, 2007). A fresh water

cyanobacterium Phormidium sp. was found to degrade 47% of lignin in P. juliflora and

22% lignin in coir pith within 30 days (Prabha et al., 2005; Malliga and Viswajith,

2007).

Previous studies with Daedalea flavida and two of the Phlebia sp. showed their

capability of degrading lignin selectively when inoculated with wheat straw. The

degradation of intermediates of synthetic lignin by Phlebis tremallosa was observed by

Gutierraz et al. (1996). Arora et al. (2002) showed that Daedalea flavida and two of

the Phlebia spp. (Phlebia radiata and Phlebia floridensis) have the capability of

degrading lignin selectively when inoculated with wheat straw.

In a biodegradation study, the selected lignocellulosics showed highest lignin

content in coir pith (37%) followed by Prosopis juliflora (23%) and Lantana camara

(22%). However, Oscillatoria annae treated lignocellulosics showed maximum

reduction of lignin content in L. camara (18.2%) followed by P. juliflora (17.4%) and


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coir pith (16.9%) after 30 days of incubation (Viswajith, 2008). Gupta et al. (2010)

reported that Pycnoporus cinnabarinus degrades about 7.69-10.08% of lignin in

P. juliflora and 6.89-7.31% in L. camara and eventually enhanced the holocellulose

content by 2.90-3.97% and 4.25-4.61%, respectively.

Holocellulose

Reports on the chemical wood composition of other Prosopis species says that

70% of the wood tissue of P. juliflora is composed of holocellulose (Pasiecnik et al.,

2001; Scholz et al., 2005). Holocellulose estimation revealed feasible reduction of

holocellulose content in control (P. juliflora alone) (0.8%) while O. laetevirens treated

P. juliflora showed 45.3% of reduction (Fig. 27). Supporting evidences showed that

maximum reduction of holocellulose was observed in O. annae treated L. camara

(8.7%) followed by P. juliflora (8.3%) and coir pith (2.1%) which revealed that

O. annae mediated lignin degradation is very much similar to the first group of wood

rot fungi (Viswajith, 2008).

Basidiomycetes are the most potent degraders of holo-cellulose because many

species grow on dead wood or litter, in environments rich in cellulose (Baldrian and

Valaskova, 2008). Report showed that the six fungal isolates degrade lignin and

holocellulose in mangium wood meal over 1 to 4 weeks. An increase in incubation time

tended to decrease the amounts of lignin and holocellulose. Isolate 371 was found to be

best at degrading lignin and holocellulose in mangium wood meal (Djarwanto and

Tachibana, 2009).

4.1.3e Phytochemical analysis

A phytochemical analysis revealed the presence of alkaloids, flavanoids,

terpenoids, saponin and steroids in the control (P. juliflora alone) and test (O.

laetevirens treated P. juliflora) samples (Table-2). The previous reports showed two

new flavonoid glycosides, kaempferol 4′-methyl ether 3-O-β-D-galactopyranoside and

retusin 7-O-neohesperidoside which is characterized from the stem bark of Prosopis

juliflora (Ranjana and Misra, 1981). Similarly, plant growth inhibitory alkaloids were

isolated from the extract of mesquite leaves by Nakano et al. (2004a).


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Studies on phytochemical composition in ethanol extract of root and stem of

Prosopis africana showed the presence of saponins, tannins, alkaloids, phenols and

steroids. The presence of secondary metabolites in a significant amount in the

investigated parts may have conferred antimicrobial activity. In this regard, the higher

concentration of these phytochemicals in the stem extract may have been responsible

for significantly higher inhibition exhibited by the stem extract on Candida albicans,

when compared to root extracts (Kolapo et al., 2009). Sathiya and Muthuchelian,

(2008); Sivakumar et al. (2009); Seetha Lakshmi et al. (2010) reported the presence

of alkaloids, terpenoids and steroids in methanolic extracts of Prosopis juliflora.

4.2. COMPOUND IDENTIFICATION

4.2.1 Spectrum analysis

The UV-visible absorption spectra and retension time of phenolic compound

enable its identification using chromatographic peaks. The phenolic molecules can

absorb light by their functional elements and the benzene ring system found in simple

phenols absorbs in 260-280nm region (Waterman and Mole, 1994). The spectrum of

ethanolic extract of different days of degraded P. juliflora extract confirmed absorption

bands at 270nm and the peak indicates that the compound present in the extract was of

aromatic in nature (Fig. 28). According to literature all the flavanols present are quite

similar in UV absorption pattern, but appear at distinct retention times (Sirmah, 2009).

Anandharaj (2007) reported that during degradation of coir pith by Oscillatoria annae

an appreciable amount of phenolic compounds were released into the surrounding

medium and it was determined by spectral analysis. From this it is evident that

O. laetevirens degrades P. juliflora and releases phenolic compounds and other

metabolites into the media.

4.2.2 Thin Layer Chromatography (TLC)

Thin Layer Chromatography analysis of the ethanolic extract of P. juliflora

alone and degraded P. juliflora samples showed the presence of major phenolic

compounds in plates eluted with Hexane:Ethyl acetate (6:4) (Fig. 29). The retention

factor (Rf) value of separated spots were measured and matched with standard phenols

(Table-3) which showed the presence of 4- ethoxy benzoic acid (0.24), 3,4- diethoxy


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benzoic acid (0.62) and catechin (0.65) in both samples. Also a few other compounds at

Rf value of 0.60, 0.66, 0.74 were observed.

Nadeem (1992) reported the presence of juliprosin and isojuliprosine in the

polar fraction of P. juliflora leaf extract. Later, catechin was isolated and identified as

the main compound in the methanol extract of Prosopis alba trunk (Astudillo et al.,

2000). Sivakumar et al. (2009) reported the presence of two alkaloids, which were

identified as 3-oxo-juliprosopine and secojuliprosopinol from the methanolic extract of

Prosopis juliflora bark.

4.2.3 High Performance Thin Layer Chromatography (HPTLC)

Madan et al. (2010) used HPTLC method for the separation and identification

of six different phenolic compounds of gallic acid, ferulic acid, syringic acid, catechin,

protocatechuic acid and vanillin in a single analysis which was found to enable rapid,

precise, accurate, simple and cost-effective analysis of these important phenolic

compounds from different plant materials and herbal products.

The amount of phenolic compound present in the ethanolic extract of

P. juliflora and degraded P. juliflora was shown in Fig. 30 and listed in Table-4. From

the obtained results, the absorbance in UV region indicates the presence of phenolic

compounds and this result coincides with TLC pattern which showed similar number of

bands and presence of flavonoid like compounds.

Supporting evidences showed that HPTLC separation of catechin and

epicatechin was achieved by multiple gradient development (MGD) with increasing

concentrations of acetonitrile (from 20 to 22%) in the water-formic acid mixture and

the UV detection at λ = 282 nm and λ = 500 nm were compared for estimation of

catechin content (Olech et al., 2007). Furthermore, Dhalwal et al. (2008) reported that

HPTLC quantification of Bergenia ciliata and Bergenia ligulata extract revealed the

presence of high concentration of bergenin, catechin and gallic acid.

4.2.4 High Performance Liquid Chromatography (HPLC)

The phenolic compounds present in each sample were identified by comparing

chromatographic peaks with the retention time (Rt) of individual standard (Fig. 31).


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The obtained results showed the presence of catechin (Rt = 9.8) like compound in

Prosopis juliflora (Rt = 9.9) and degraded Prosopis juliflora (Rt = 9.2). Similar results

were obtained in HPLC analysis of crude acetonic extract of P. juliflora heartwood

which indicated the presence of mesquitol as sole compound which corroborate with

the present results (Sirmah et al., 2009).

Supporting evidence showed that catechin was identified as the main free

radical scavenger from the exudate of Prosopis alba. HPLC analysis of the methanol

soluble part of the exudate showed a mean peak with Rt = 24.80min, corresponding to

catechin (Astudillo et al., 2000).

Carrillo et al. (2008) reported the presence of major compounds in acetone and

water extracts of Prosopis laevigata were identified as (-)-epicatechin, (+)-catechin and

taxifolin which are quantitatively determined by liquid chromatography (RP-HPLC-

UV).

4.2.5 Gas Chromatography Mass Spectrometry (GC-MS)

The mass spectrum showed the m/z ratio (mass to charge) at 292 for P. juliflora

and 291 for degraded P. juliflora which match with the molecular mass of mesquitol

which is 291. These results confirmed the presence of mesquitol like compounds in

both the control and test samples (Fig. 32). Further confirmation and identification of

the compound was done by FT-IR and NMR analysis.

GC-MS analysis of toluene or ethanol bark extractives of P. juliflora indicated

the presence of numerous compounds among which 4-O-methylgallocatechin as the

main component. Further, identifications of different compounds from bark extracts

using National Institute of Standards and Technology (NIST) library and literature

information indicate the presence of epicatechins, catechins, methylgallocatechins,

gallocatechins, fatty acids and sugars (Sirmah, 2009). Also Tapia et al. (2000)

reported catechins, as the main constituent from exudates of Prosopis flexuosa.

4.2.6 Fourier Transform Infrared Spectrometry (FTIR)

FTIR spectrum for P. juliflora showed characteristic hydroxyl group absorption

at 3405 cm-1. Fatty acids groups were identified by the presence of C-H vibrations


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between 2955 and 2838 and carbonyl bands at 1631 cm-1. Aromatic C=C skeletal

vibrations at 1603, 1460 and 1361 cm-1 are typical of aromatic structure in flavanols. A

strong absorption at 1019 cm-1 is characteristic of C-O vibrations in sugar units.

Similarly, hydroxyl group absorption at 3396 cm-1, C-H vibration between 2948 and

2835, aromatic C=C skeletal vibration at 1599, 1456, 1364 cm-1 and C-O sugar unit at

1024 cm-1 were observed for degraded P. juliflora (Fig. 33).

Similar results were observed in FTIR spectrum of acetone extractives of

P. juliflora heartwood spectrum, indicated hydroxyl group absorption at 3350 cm-1 and

aromatic C=C skeletal vibrations at 1613, 1514 and 1475 cm-1 which resembles

flavanol structure. Also FTIR spectrum of acetone extractives of P. juliflora leaves

showed absorption bands at 3400 cm-1 characteristic of OH group and bands at 1119

and 1071 cm-1, which could be ascribed to sugars units. A strong absorption band at

2917 and 2849 cm-1 characteristic of C-H vibrations and 1728 cm-1 characteristic of

C=O vibrations can be attributed to the presence of fatty acids in their free or esterified

form. Also, bands at 1510 and 1450 cm-1 are ascribable to aromatic structures (Sirmah,

2009).

4.2.7 Nuclear Magnetic Resonance Spectroscopy (NMR)

NMR spectrum showed a higher complexity indicating the presence of different

families of product such as fat, sugar along with typical flavonol signal in control

P. juliflora while spectrum of degraded P. juliflora extract showed the presence of

main component of flavanol signal (Fig. 34). From the spectrum data it is revealed that

mesquitol is the main component of ethanolic extract without any noticeable impurities

in degraded P. juliflora.

Supporting evidence showed that high amounts (8%) and purity of the rare

flavonoid (-)-mesquitol was identified as the major metabolite in heartwood extractives,

while (+)-epicatechin, (+)-catechin, gallocatechins, methylgallocatechins, fatty acids

and free sugar are present in the bark of P. juliflora (Sirmah et al., 2009). Pods contain

important quantity of galactomanans, mannose, saturated and unsaturated fatty acids

and free sugar. Leaves of P. juliflora contain alkaloids such as tryptamine, piperidine,

phenethylamine and juliprosopine (Tapia et al., 2000).


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Based on the results of TLC, HPTLC, HPLC, GC-MS, FTIR and NMR it is

confirmed and concluded that the presence of mesquitol as major compound in the

degraded P. juliflora as well as in extract of P. juliflora alone that which has been

previously reported by Sirmah, (2009). Thus, from the accumulated data the structure

of the compound identified was mesquitol (C15H15O6).

4.3. PHARMACOLOGICAL STUDIES

4.3.1 Experimental animal- Rattus norvegicus

It is evidence that various vertebrate species have similar biochemical and

physiological systems and are most closely related to humans genetically or

evolutionarily. In the present study Rattus norvegicus was used for its sensitivity,

acceptable results and to eliminate the chance of variation. Rats have been used in

many experimental studies to understand their genetics, diseases and the effects of

drugs (Krinke, 2000).

Laboratory rats are used more often in research every year than any other

animal species. Mice, rats and hamsters make up over 90% of the animals used in

biomedical research. In addition to having bodies that work similar to humans, these

animals are small in size, easy to handle, relatively inexpensive to buy and keep and

produce many offspring in a short period of time (Logan, 2001). Moreover, the rat has

been made to fit the emerging metaphors through selective breeding and

standardization laboratory rats are now potent symbols of scientific endeavor indeed

they stand alongside the ubiquitous double helix as icons of the laboratory in modern

western culture (Lynda, 2003).

4.3.1a Mode of drug administration

Oral drug delivery is the most desirable and preferred method of administering

therapeutic agents for their systemic effects. Recent research has opened many novel

avenues for the more effective, sustained and rate-controlled oral delivery of both

existing and new therapeutic agents, including peptide and protein drugs emerging from

the biotechnology arena. Furthermore, the oral route offers an attractive approach of

drug targeting at the specific sites within the gastrointestinal tract for the treatment of

certain pathological conditions such as gastroesophageal reflux disorder,


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gastroduodenal ulcers, inflammatory bowel disease and stomach and colon cancers

(Brahma and Kwon, 2006). Hence, oral drug delivery as a mode of administration was

chosen for this study.

4.3.2 Acute toxicity study

Acute toxicity study was crried out to evaluate the toxic characteristics of the

P. juliflora extract. Shetty et al. (2007) reported that acute oral toxicity is usually an

initial assessment of toxic manifestations and is one of the initial screening experiments

performed with all compounds. According to the Organization for Economic

Co-operation and Development (OECD) guidelines for acute oral toxicity, the given

LD50 dose of the ethanolic extract of P. juliflora (30th day sample) did not cause any

mortality up to 200 mg/kg of body weight and was considered as safe (Table-5).

Similar acute toxicity study revealed that the methanolic extract of P. juliflora bark was

safe up to a dose level of 400 mg/kg of body weight (Sivakumar et al., 2009).

Quintas-Junior et al. (2004) in their preliminary study on the total alkaloid

fraction of the pods of P. juliflora reported that they induced behavioral changes in

rodents, suggesting some effect on the central nervous system. They also showed an

acute toxicity (LD50) of 10.3 mg/kg intraperetonially and 637 mg/kg orally. Cytotoxic

effects of the extract containing alkaloids on GL-15 (Glial cells of lineage) cell line as

confirmed by the MTT (Methyl Thiazol Tetrazolium) test, LDH (Lactate

Dehydrogenase) ctivity and Trypan blue staining. The cytotoxic effects of alkaloid

extract from P. juliflora pods upon the viability were confirmed by the MTT test, LDH

activity and Trypan blue staining (Hughes et al., 2005).

Lydia et al. (2010) reported the oral median lethal dose of the methanolic

extract of Prosopis africana was found to be 3.808 g/kg in mice and > 5 g/kg in rats

and the study results supports the folkloric claim of the use of P. africana in analgesic

and anti-inflammatory activities.

4.3.3 Sub-acute toxicity study

4.3.3a Morphological observation- Body weight

Individual animal weights were recorded shortly before the first treatment (0th

day), and at study termination (30th day). The body weight of the control rats was found


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to be normal while ethanolic extract of O. laetevirens alone and P. juliflora alone

treated male and female rats showed slightly decreased body weight. The ethanolic

extract of degraded P. juliflora treated rats showed moderate increase in body weight

(Fig. 35). The present study showed that there was no significant weight gain in both

male and female rats at 200 mg/kg body weight.

Similar results were observed in animals administered with coir pith based

cyanobacterial culture filtrate in which the experimental rat did not show any increase

in body weight (Prabha et al., 2009). The aqueous leaf extract of Anogeissus

leiocarpus containing alkaloids, flavonoids, tannins and saponins when administered

orally (100 and 200 mg/kg) up to 28 days showed average body weight while a control

group showed consistent increase in weight. Those group treated with highest dose

(300 mg/kg) showed a decline in weight in 3rd and 4th week of the study (Agaie et al.,

2007).

The efficacy of ethanolic extract of Cassia kleinii on streptozotocin-induced

diabetic rats showed increased body weight in normal control while, diabetic rat treated

with 200 mg/kg extract showed a significant prevention in the reduction of body weight

(Babu et al., 2003).

The compound benzylcarbamothioethionate isolated from the ethanolic extract

of the root bark of Moringa oleifera L. when administered to experimental rats did not

show any significant changes in body weight of all the rats (Farjana et al., 2009).

4.3.4 Haematology

4.3.4a Haemoglobin (Hb)

Adequate haemoglobin percent is needed for normal physiology of animals,

which depends on the erythrocyte count. From the results, it was observed that

haemoglobin content was normal in control and the extract of degraded P. juliflora

treated male and female rats. The extract of P. juliflora alone and O. laetevirens alone

treated rats showed slightly decreased haemoglobin content (Fig. 36). The variation in

hemoglobin content may be correlated with the presence of phenolic compounds and it

is suggested that intake of phenolic compounds should be as moderate as possible.


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Ingestion of high dose of phenolic compounds has the tendency to cause anemia.

Similar results were obtained in rats treated with culture filtrate of degraded coir pith by

O. annae (Prabha et al., 2009).

Supporting evidence showed that prolonged administration of Artemisia judaica

(0.5 and 1 g/kg body weight) containing flavonoid as the major component in diabetics

induced rats did not show significant effect on hemoglobin content (Salwa et al., 2009).

Similar results were observed in rats administered with ethanolic extract of

Pothomorphe umbellata L. Miq. at 500 mg/kg body weight (Barros et al., 2005).

Ibrahim et al. (1997) reported that leaf extract of Anogeissus leiocarpus

containing flavonoids have been shown to increase vascular integrity and also act as

antihaemerrhagic (Dharmancida, 1991).

The effect of short term (15 days) alcoholic extract of Cassia kleinii (200 and

400 mg/kg) administration on the hematological and serum biochemical parameters of

male mice did not result in any change in hemoglobin content, total leucocyte counts,

serum urea, cholesterol, total lipid, glutamate pyruvate transaminase, glutamate oxalate

transaminase and alkaline phosphatase (Babu et al., 2003).

The aqueous leaves extract of Anogeissus leiocarpus administered in rats did

not show any significant changes on haemoglobin content in both control and extract

treated rats. This may be an indication of the relative safety of the extract at the doses

(100, 200 and 300 mg/kg) used in the study on haemopoetic system (Agaie et al.,

2007).

Salwa et al. (2009) revealed the effect of prolonged administration of Artemisia

judaica extracts on hematological parameters did not show any significant effect on

hemoglobin concentration, packed cell volume percentage, erythrocytic count, total

leucocytic count and differential leucocytic count in rats allover the period of the

experiment.


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4.3.4b Erythrocyte Sedimentation Rate (ESR)

Robbins, (1976) reported that flavonoids exert an apparent regulatory action on

erythrocyte aggregation and concentration which are two major factors affecting blood

viscosity and flow. The methoxylated compounds exhibit a highly significant

antiadhesive action on erythrocytes and inhibit erythrocyte sedimentation rate (ESR),

while hydroxylated glycosides accelerate aggregation and ESR. The aggregating effect

of flavonoids has been interpreted as a mechanism whereby erythrocyte concentration

is reduced since clumped cells are sequestered and removed from circulation. Such

action appears to be selective and similar to that of polylysine which preferentially

aggregates old erythrocytes with less charge than young erythrocytes which are having

higher negative charges (Obdulio, 1997).

The erythrocyte sedimentation rate was found to be normal in control and

experimental males treated with extract of O. laetevirens, P. juliflora and degraded

P. juliflora while it was slightly decreased in control and experiment female rats

administered with ethanolic extract of O. laetevirens, P. juliflora and degraded

P. juliflora (Fig. 37). Thus the alteration of erythrocyte sedimentation rate within male

and female was insignificant and remained within the normal range.

Ratheesh et al. (2009) reported that arthritic rats exhibited a reduced RBC

count, reduced haemoglobin level and an increased ESR. The treatment with

methanolic extract of Ruta graveolens (20 mg/kg body weight) improved the RBC

count, haemoglobin level and the ESR to a near normal level indicating the significant

recovery from the anaemic condition. This may be due to sparing effect of the

antioxidant defense system as the extract has quenched the increased generation of free

radical by the presence of antioxidants.

The anti-arthritic activity of ethanolic extract of seeds of Moringa oleifera in

adjuvant-induced arthritis rats showed normal level of rheumatoid factor value,

erythrocyte sedimentation rate and histopathology studies in the entire tested group

(Shailaja et al., 2007).


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4.3.4c Packed Cell Volume (PCV)

The hematocrit (Ht or HCT) or packed cell volume (PCV) or erythrocyte

volume fraction (EVF) is the proportion of blood volume that is occupied by red blood

cells. It is normally about 40-64% for male rat and 36-47% for female rat. From the

obtained results, it was clear that the tested extracts in all rats had no significant effect

on the packed cell volume in all experimental groups and this agreed with the finding

that ethanolic extract of degraded P. juliflora caused no significant changes at

concentration of 200 mg/kg body weight by oral administration for 30 days (Fig. 38).

The oral administration of ethanolic extract of Artemisia judaica containing

flavonoids as a major component showed no significant changes in hematological

parameters in rats after oral administration of the plant extract for 3 months (Salwa

et al., 2009).

The effect of oral administration of aqueous extract of Pelargonium reniforme

root containing flavonoid and tannins as the principal phenolic contents when

administered orally showed significant increase in the level of PCV at 100 mg/kg body

weight of rat while there was no significant changes observed at 200 and 400 mg/kg

body weight when compared with control (Adewusi and Afolayan, 2009).

The methanol extract of the stem bark of Prosopis africana (at doses of 62.5,

125 and 250 mg/kg) evaluated for analgesic and anti-inflammatory activities using

acetic acid-induced writhing assay and carrageenan-induced inflammation in rats

significantly attenuated the writhing with the highest activity at 250 mg/kg (76.89%)

comparable to that of piroxicam (83.16%) the standard agent used. Also the extract

showed significant anti-inflammatory activity from the third hour (Lydia et al., 2010).

Ekor et al. (2010) revealed that alloxan-induced diabetes was associated with

decreases in total WBC count and PCV. Both ethanolic and methanolic leaf extracts of

Sida acuta significantly prevented the decrease in total WBC and PCV induced by

alloxan both at 200 and 400 mg/kg.


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4.3.4d Red Blood Cell (RBC)

The red blood cell (RBC) count determines the total number of red cells

(erythrocytes) in a sample of blood. An elevated RBC count may be caused by

dehydration, hypoxia (decreased oxygen), or a disease called polycythemia. The RBC

count is also decreased due to cancer, kidney diseases and excessive intravenous fluids.

From the result, it was observed that RBC count was normal in control, extract of

degraded P. juliflora treated male and female rats, while extract of P. juliflora alone

and O. laetevirens alone showed reduction in RBC count (Fig. 39). The variation of

RBC count may be due to uptake of higher phenolic compound which interfere with the

haematological system of the experimental rats.

Supporting evidence showed that acute dosages of 0.5, 1.0, and 3 g/kg body

weight and chronic dosage of 100 mg/kg/day of ethanolic extracts of Curcuma longa

rhizomes caused poor weight gain, changes in the heart and lungs weights, and reduced

levels of white and red blood cells (Qureshi et al., 1992). Prabha et al. (2009)

reported that open access to 0.3% coir pith based phenolic culture filtrate to albino rat’s

showed normal red blood cells count when compared with controls.

The arthritic rats exhibited a reduced RBC count which indicated the anaemic

condition that is a common diagnostic feature with chronic arthritis. The treatment with

methanolic extract of Ruta graveolens (20 mg/kg) improved the RBC count to a near

normal level indicating the significant recovery from the anaemic condition (Ratheesh

et al., 2009).

The chloroform soluble fraction of ethanolic extract of root bark of Moringa

oleifera containing benzylcarbamothioethionate when administered to rat revealed that

hematological parameters such as the total RBC (red blood cell), total WBC (white

blood cell), differential count of WBC, platelet count, hemoglobin and ESR

(erythrocytes sedimentation rate) remained unchanged in both experimental and control

groups (Farjana et al., 2009).

4.3.4e White Blood Cell (WBC)

Leucocytes are known to increase sharply when infection occurs, as one of the

first line of defense of the body. The experimental study revealed that no significant

variations in WBC count was recorded in all male rats administered with ethanolic


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extract of O. laetevirens, P. juliflora and O. laetevirens treated P. juliflora at 200

mg/kg body weight (Fig. 40). The control and experimental groups of female rats

showed normal WBC count. A slight elevation in WBC count was recorded in

degraded P. juliflora extract treated female rat. This increase in count may be due to

antioxidant property of degraded P. juliflora which helps in getting rid of free radicals

that make the body more susceptible to infectious diseases.

Supporting evidence showed that there were no significant changes in various

haematological parameters such Hb, total count (TC) of RBC and WBC, differential

count (DC) of WBC and platelet count compared to the control group, which indicates

that test extracts of Cleome rutidosperma root, Neolamarckia cadamba and Spondias

pinnatabark did not show any toxic effect on circulating red cells (Sumanta et al.,

2009).

The extract of Pelargonium reniforme caused an increase in the level of the

total WBC count and its differentials (neutrophils, monocytes, lymphocytes,

eosinophils and basophils). This is an indication that the principal function of

phagocytes has been enhanced (Jimoh et al., 2008). The observed changes in the levels

of the WBC count and its differentials may provide a basis for the antibacterial,

antifungal and antitubercular properties of P. reniforme (Mativandlela et al., 2006).

The anti-inflammatory and anti-oxidant effects of methanolic extract of Ruta

graveolens L. in adjuvant induced arthritis rats revealed that there is a significant

restoration of WBC count in extract treated groups when compared to indomethacin

treated groups and normal control (Ratheesh et al., 2009).

The methanolic extract of Arthrospira platensis which is known to be rich in

antioxidants such as phenolic acids, α-tocopherol, and β-carotene was examined for

subchronic toxicities at doses of 6, 12 and 24 mg/kg body weight daily for 12 week

showed normal white blood cells count in all treated groups and in control. Also no

significant changes were observed in differential count (Hutadilok et al., 2010).


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4.3.4f Neutrophil

Neutrophils play a crucial role in the development and manifestation of

inflammation and they are the major source of free radicals at the site of inflammation.

Neutrophil derived free radical is known to cause inflammation and cytokines produced

by neutrophils are also responsible for inflammation (Young et al., 1989). From the

result obtain it is evident that neutrophil content was found to be normal in control and

experimental male rats and showed slightly decrease in neutrophil level in control

female rats when compared to experimental female rats (Fig. 41). This activity is

related to the phenolic compounds present in the extract.

Muruganandan et al. (2005) reported that mangiferin on cyclophosphamide

induced immunotoxicity and it is also evident that mangiferin exhibits an

immunoprotective role mediated through the inhibition of reactive intermediate-

induced oxidative stress in lymphocytes, neutrophils and macrophages.

Chunlaratthanaphorn et al. (2007) revealed that rats treated with water extract

from root of Imperata cylindrica (1,200 mg/kg/day) showed significant difference in

neutrophil, lymphocyte and eosinophil count from the control values, the alteration of

hematological and white blood cell count values were insignificant and remained

within the normal range.

Ethanolic leaf extracts of Sida acuta (400 mg/kg) against alloxan-induced

diabetes rats significantly reversed the decrease in neutrophil count associated with

alloxan toxicity. Effects produced on neutrophil count in the hyperglycaemic rats by

other treatments were not significant when compared with the saline-treated alloxan

diabetic rats (Ekor et al., 2010).

4.3.4g Lymphocyte

WBC and indices relating to it such as lymphocytes usually shows increase in

activity in response to toxic environment (Robins, 1974). In this study, WBC was not

significantly altered while lymphocytes, the main effectors cells of the immune system

(McKnight et al., 1999) showed slightly decrease in counts in P. juliflora and

O. laetevirens alone treated male and female rats when compared to the controls while


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there is a marginal increase in experimental rats administered with degraded P. juliflora

extract (200 mg/kg) in both male and female, thus suggesting that the extract only

exerted minimal challenge on the immune system of the animals (Fig. 42).

Similar results were observed in rats treated with ethanolic extract of

Sphenocentrum jollyanum at 50, 100 and 200 mg/kg body weight showing increased in

serum lymphocyte count when compared to control (Mbaka et al., 2010).

It is reported that Artemisia afra caused no significant changes in hematological

parameters in rats after oral administration of the aqueous plant extract for a period of 3

months (Mukinda and Syce, 2007). Similarly, methanolic extracts of Cleistocalyx

nervosum which contained high levels of phenols, flavonoids and tannin when

administered orally at 100 and 500 mg/kg for four weeks showed no significant

changes in hemoglobin, hematocrit, platelets and white blood cells in rat male and

female treatment groups (Sirinya et al., 2009).

Adewusi and Afolayan (2009) reported effects of graded doses of the aqueous

extract of Pelargonium reniforme on haematological parameters of wistar rats showing

a significant increase in lymphocyte counts in all the three doses 100, 200 and 400

mg/kg body weight, respectively. The observed changes in the levels of the WBC count

and its differentials may provide a basis for the antibacterial, antifungal and

antitubercular properties of P. reniforme (Mativandlela et al., 2006).

4.3.4h Eosinophils

The obtained results revealed that eosinophil count were found to be average in

control group and slightly decreased in rats treated with extracts of O. laetevirens and

with degraded P. juliflora. A slight elevation of eosinophil counts was observed in

extracts of P. juliflora alone treated rats which may be due to allergic reactions

(Fig. 43).

Supporting evidence showed that apple condensed tannins (ACT) supplement in

patients with atopic dermatitis (AD) at oral doses of 10 mg/kg per day for 8 weeks

reduced the inflammation, lichenification, cracking, itching, sleep disturbance and


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peripheral blood eosinophil counts when compared with the control group. This may be

due to anti-allergic effects described by previous reports (Takatsugu et al., 2000).

Besides, it is reported that when trans-resveratrol administered orally to male

rats for 28 days at a dose of 20 mg/kg/day did not alter the normal levels of

hematologic and biochemical tests (Emilia et al., 2002).

Kumarappan and Subhash (2007) revealed that the polyphenolic extract

(PPE) of leaves of Ichnocarpus frutescens evaluated for antitumor activity in vivo

against a Murine Ehrlich ascites carcinoma (EAC) model showed increase in eosinophil

counts with EAC control; while PPE (100 mg/kg) treated animals restored the

eosinophil level to its normal range compared to the control.

The anti-stress potential of ethanolic extracts of Lagenaria siceraria in rats

showed elevated blood cell counts of neutrophils, eosinophils and monocytes in stress

induced control rats while it has been significantly reduced by the L. siceraria ethanol

extract in a dose dependant manner of 100-400 mg/kg treated groups (Lakshmi and

Sudhakar, 2009).

4.3.5 Biochemical studies

4.3.5a Protein

Albumin and globulin are proteins found in highest concentrations in the

plasma. Albumin transports many small molecules in the blood and also prevents the

fluid in the blood from leaking out into the tissues (Duncan et al., 1994). Since

albumin is produced in the liver, increased serum albumin may indicate that the extract

promotes positive functioning of the liver. An increase in globulin levels indicates the

potential of immune response by increasing antibody production (Puri et al., 1993).

Experimental results revealed that serum protein was found to be normal in

control and in ethanolic extracts of degraded P. juliflora treated male and female rats

while ethanolic extracts of O. laetevirens alone and P. juliflora alone treated rats

showed slightly decreased serum protein levels (Fig. 44). Similar results were observed


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in rats treated with 0.3% coir pith based cyanobacterial culture filtrate containing

phenolic compounds (Prabha et al., 2009).

Barros et al. (2005) reported that the ethanolic root extract of Pothomorphe

umbellata L. contains 6.5% of 4-nerolidilcatecol, a phenolic compound with high

antioxidant activity when administered at 500mg/ kg body weight for a period of 40

days did not show any significant variations in total protein concentration in males and

female rats.

The treatment with ethanolic extracts of Indigofera trita at 200 and 400 mg/kg

body weight showed a significant and dose dependant increase in the levels of albumin

and total protein in CCl4 intoxicated rats when compared to control rats suggesting the

possibility of the extract to give protection against liver injury (Kumar et al., 2008).

Phenolic compounds extracted from the leaves of Myrtus communis when

administered to streptozotocin induced diabetic rats at 800 mg/ kg body weight

recorded a normal level of total proteins and showed a drastic decrease in albumin and

a corresponding increase in globulin when compared to normal rats that indicates

hepatic damage (Fahim et al., 2009).

4.3.5b Glucose

Glucose is the primary energy source for somatic cells, being carried through

the bloodstream and absorbed by the cells with the intervention of insulin. The results

obtained indicate that the control group showed normal glucose levels. Extract of

O. laetevirens alone, P. juliflora alone and degraded P. juliflora treated animals show a

slight decrease in glucose levels in both male and female rats of the respective groups

(Fig. 45). This may be due to hypoglycemic activity of the test compound. Supporting

evidences are provided by hypoglycemic activity of 0.3% of coir pith based

cyanobacterial culture filtrate in treated rats (Prabha et al., 2009).

Kateina et al. (2007) reported that the use of certain dietary polyphenolic

compounds may alter glucose metabolism, thus decreasing the risk for type 2 diabetes.

The effect of phenolic acids (caffeic, chlorogenic, rosmarinic, and ferulic) and extracts


100

from Smallanthus sonchifolius and Prunella vulgaris on glucose production in rat

hepatocytes showed that the phenolics at 500 µM and after 1 hr incubation lowered

glucose production via both gluconeogenesis and glycogenolysis compared to

metformin.

Similarly phenolic compounds extracted from Myrtus communis when

administered at 800 mg/kg body weight have made a remarkable antihyperglycemic

effect in experimental animal models (Fahim et al., 2009). Turan et al. (2010)

investigated the effects of oral administration of extract of green tea (Camellia sinensis)

and ginseng (American ginseng-Panax quinquefolium L.), given alone or together

decreases blood glucose levels and increases the levels of serum insulin in the diabetic

rats. And also reported that these effects have largely been attributed to the most

prevalent polyphenol contained in green tea, the catechin or flavanol

(-) epigallocatechin-3-gallate (Sato and Miyata, 2000; Dona et al., 2003).

Sokeng et al. (2005) reported the ethanol extract of Bridelia ndellensis had no

hypoglycemic effect in type 1 diabetic rats and postprandial glucose load conditions in

type 2 diabetic rats in fasting conditions. However, ethyl acetate and dichloromethane

fractions significantly lowered blood glucose levels in type 2 diabetic rats when fed

simultaneously with glucose. The active principles responsible for the

antihyperglycaemic effect are concentrated in the ethyl acetate and dichloromethane

fractions of the extract.

4.3.5c Albumin and Globulin

Albumin is synthesized by the liver using dietary protein. Its presence in the

plasma creates an osmotic force that maintains fluid volume within the vascular space.

Experimental results showed that albumin content in control and degraded P. juliflora

extract treated male and female rats were found to be normal. Subsequently the rats

treated with O. laetevirens and P. juliflora showed a decrease in albumin content in

both groups (Fig. 46). This may be due to chronic infections (parasites, some cases of

viral and bacterial infection), liver disease (biliary cirrhosis, obstructive jaundice) or

kidney dysfunction (nephrosis). Globulin content was found to be normal in males and

females of all experimental and control groups.


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Supporting evidences showed that the reduction in plasma total protein and

albumin levels were observed in diabetic rats and this is consistent with the results

obtained by Tuvemo et al. (1997). The decrease in protein and albumin may be due to

microproteinuria and albuminuria, which are important clinical markers of diabetic

nephropathy (Mauer et al., 1981) or may be due to increased protein catabolism

(Almdal and Vilstrup, 1988).

The levels of albumin and albumin/ globulin ratio in plasma were found to be

normal in control rats while it was decreased in diabetic animals. This may be due to

microproteinuria and albuminuria which are important clinical markers of diabetic

nephropathy and/or may be due to increased protein catabolism. These lowered levels

of plasma albumin and albumin/globulin ratio levels were significantly reverted in

Helicteres isora (100, 200 mg/kg/p.o) treated diabetic rats due to proper protein

metabolism (Kumar et al., 2007).

This observation was corroborated by the findings of Ismail et al. (1997) who

reported that enhanced serum albumin content was reduced to normal levels after the

administration of 1000 mg/kg root bark powder of Salacia oblongo and leaf powder of

Azima tetracantha in inflamed rats. Reports also showed that the extract of Myrtus

communis when administered to streptozotocin induced diabetic rats at 800 mg/kg

recorded a normal level of total proteins but showed a drastic decrease in albumin and a

corresponding increase in globulin indicating hepatic damage (Fahim et al., 2009).

4.3.6 Lipid Analysis

4.3.6a Cholesterol

Cholesterol is insoluble in blood, but transported in the circulatory system

bound to lipoproteins like low density lipoproteins (LDL) and high density lipoproteins

(HDL) which carry cholesterol to the liver. Alterations in the concentration of major

lipids like cholesterol, high-density lipoprotein cholesterol and triglycerides can give

useful information on the lipid metabolism as well as on the predisposition of the

animals to atheriosclerosis and its associated coronary heart diseases (Yakubu et al.,

2008).


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From the results obtained it was renowed that the total cholesterol level in all

the experimental male and female rats treated with extracts of O. laetevirens,

P. juliflora and degraded P. juliflora were found to be at lower levels when compared

with that of control rats (Fig. 47). This may be due to a high consumption of phenolic

compounds that reduced cholesterol levels.

This result concurred with an early study made by Fahim et al. (2009) reporting

phenolic compounds extracted from the leaves of Myrtus communis (800 mg/kg body

weight) inducing diabetic rats to show a progressive decline in cholesterol levels

towards normal while diabetic rats treated with 400 mg/kg showed only a moderate

decline.

Flavonoids are known for their diverse biological activities including

hypolipidemic activities. Ethanolic extracts of Clerodendron phlomoidis showed the

presence of flavonoids and related phenolic compounds. Oral administration of ethanol

extract of leaves of C. phlomoidis resulted in a significant reduction of serum lipid

levels in rats with hyperlipidemia viz. triglyceride and total cholesterol (Dhanabal et

al., 2008). Such dual property has also been reported from methanol extracts of Prunus

dadidiana (Rosaceae) and its flavonoid constituent prunin (Choi and Yokozawa,

1991).

The effect of ethanolic extract of Cassia kleinii on streptozotocin-induced

diabetic rats showed normal cholesterol level in control rats while diabetic control rats

showed increased cholesterol level. Subsequently, rats treated with glibenclamide (500

μg/kg) as well as Cassia kleinii extract (200 mg/kg) showed significant reduction in

cholesterol content to normal range (Babu et al., 2003).

The ethanolic extract of the roots of Pseudarthria viscida was evaluated for anti

diabetic activity against alloxan induced diabetes in albino rats which showed

significant activity as compare to standard glibenclamide. The result revealed that both

ethanolic extract (100 mg/kg) and (200 mg/kg) has significantly reversed the diabetes-

induced hyperlipidemia compared to standard drugs (Masirkar et al., 2008).


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4.3.6b Triglycerides (TGL)

Reports showed that phenolic compounds, propenylbenzenes and some

alkaloids decrease the cholesterol and triglycerides in rats (Argueta, 1994). This

hypocholesterolemic effect might be due to their abilities to lower serum triglyceride

and lower the magnitude of lipoprotein cholesterol levels as well as slowing the lipid

peroxidation process and enhancing antioxidant enzyme activity (Ines et al., 2007). In

the present study it was observed that there is no significant variation in triglycerides

level of the control, O. laetevirens extract, P. juliflora extract and degraded P. juliflora

extract treated rats (Fig. 48).

Antia, (2005) reported the effect of leaf extract of Catharanthus roseus Linn.

When administered orally at 0.1, 0.5 and 1.0 mg/kg/day for seven consecutive days,

they showed a significant decrease in total serum cholesterol, total triglycerides,

LDL-cholesterol and VLDL-cholesterol of rats. However, the level of HDL-cholesterol

was not altered by any of the doses given. The reduction in the level of LDL and VLDL

could have resulted from the antioxidant effect of the fresh leaf juice of C. roseus,

whose phytochemical component included flavonoids which are known for their

antioxidant effects.

Murthy et al. (2009) reported that phenolic compounds or phytochemicals

present in fruits and vegetables have possess anti-obesity and lipid-lowering effects. In

vitro and in vivo effects of phenolic compounds on the induction of pre-adipocytic and

adipocytic apoptosis and inhibition of adipocytic lipid accumulation were also reported

(Chin and Gow, 2008).

The synergistic effect of silymarin and ethanolic extracts of Phyllanthus amarus

against CCl4 induced hepatotoxicity rats showed decreased triglyceride levels in rats

treated with silymarin and aqueous extracts of P. amarus when compared to control,

silymarine alone ethanolic and aqueous plant extract alone treated rat (Yadav et al.,

2008).

The effects of the oral administration of aqueous extracts of Pelargonium

reniforme roots at 100, 200 and 400 mg/kg body weight for 21 days showed a decrease


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in the level of triglycerides in rats treated with 100 and 200 mg/kg of the extract and a

significant increase at 400 mg/kg (Adewusi and Afolayan, 2009). The increase in the

serum triglyceride levels might be due to accelerated lipolysis and this implies

depletion in the storage of fatty acids at this dose (Yakubu et al., 2008).

4.3.7 Renal function test

Kidney function tests are a collective term for a variety of individual tests and

procedures that can be done to evaluate how well kidneys are functioning.

4.3.7a Urea

Urea is a product of the protein metabolism that is excreted in urine and its

retention in the body may indicate renal damage (Rabo, 1998). From the result

obtained it is evident that control and degraded P. juliflora treated rats showed normal

levels of urea in the blood serum while O. laetevirens alone and P. juliflora alone

treated animal groups showed slightly elevated levels of urea (Fig. 49). This could be

due to either increased levels of protein being metabolized or due to a decrease in the

body's ability to filter it out. It was reported that phenolic compounds and flavonoids

have the ability to remove excess ammonia and urea and offer protection against

hyperammonemia (Essa et al., 2006) which corroborates the present findings.

Supporting evidence showed that flavonoids containing leaf extract of

A. leiocarpus caused a significant dose-dependent decrease in urea level in all treated

groups when compared to the control (Agaie et al., 2007). Kumar et al. (2007)

reported a significant increase in the level of plasma urea in diabetic rats when

compared with control rats, whereas after the treatment of diabetic rats with the bark

extract of Helicteres isora (100 and 200 mg/kg) the levels of urea significantly

decreased.

Vijayalakshmi et al. (2000) revealed that acute (72 hr) and subacute (30 days)

treatment of the extracts of Semecarpus anacardium nuts with different dosages

(50, 100, 250 and 500 mg/kg body weight) showed increased levels of blood glucose,

plasma urea, uric acid and creatinine in 500 mg/kg treated rats. Prabha et al. (2009)


105

reported that there was no significant change in serum urea in control and 0.3% coir

pith based cyanobacterial culture filtrate administered rats.

Essa and Subramanian (2006) reported that the level of blood ammonia, urea,

uric acid, non-protein nitrogen and creatinine were found to be higher in

hyperammonemia induced rats while these levels were significantly restored to near

normal upon the administration of alcoholic extract of Hibiscus sabdariffa (250 mg/kg

body weight) leaves containing glycosides, anthocyanins, polyphenols and flavones as

major components.

4.3.7b Uric acid

The effect of oral administration of ethanolic extract of O. laetevirens,

P. juliflora and degraded P. juliflora for 30 days on serum uric acid was recorded. The

results showed no significant variations in extracts of O. laetevirens alone and

P. juliflora alone treated groups when compared to control group (Fig. 50). On the

contrary uric acid was found to be slightly decreased in serum of rats treated with

extracts of degraded P. juliflora. From this it became evident that phenolic compounds

alone do not exhibit any toxic effects in the tested animals.

Alderman and Redfern (2004) reported that elevated serum uric acid

(hyperuricemia) can result from high intake of purine-rich foods, high fructose or

impaired excretion by the kidneys. A slightly higher level of uric acid did not cause

much harm, but extremely high levels of uric acid lead to the formation of crystals

which accumulate in the joints and may lead to gout.

Aliquots of the crude juice of olive leaves (Kronakii cultivar) which contained

215 ppm of polyphenolic compounds were administered to rats daily for 6 weeks by

stomach tube at 600, 1200 and 2400 ppm. The liver, kidney function tests and serum

contents were measured to assess the safety limits of polyphenolic compounds and the

data of the aforementioned measurements indicated that the administration did not

cause any changes in liver and kidney functions (Farag et al., 2006).


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Mageid et al. (2009) reported the effect of water algae extracts of red algae

(Asparagopsis taxiformis) and brown algae (Sargassum vulgare) on the concentration

of serum urea and uric acid in hypercholesterolemic rats showed an increase in the urea

and uric acid levels in the control group and rats administered with algae extracts

resulted in significant decreases in urea and uric acid levels. Also, the total lipids and

triglycerides level was reduced as a result of administrating the rats with algal water

extract as polyphenols.

4.3.7c Creatinine

Creatinine, a by-product of muscle energy metabolism that similar to urea is

filtered from the blood by the kidneys and excreted into the urine. With normal kidney

function, the amount of creatinine in the blood remains relatively constantly low but an

elevated blood creatinine level is a more sensitive indicator of impaired kidney function

(Fountain et al., 2007).

Serum levels of creatinine showed normal range in control male and female

rats, while the female rats treated with Prosopis juliflora and Oscillatoria laetevirens

showed slightly elevated levels of creatinine when compared with male rats. Rats

treated with degraded P. juliflora showed a decrease in levels of serum creatinine

(Fig. 51). Early reports showed that oral administration of phenolic compounds

containing coir pith based culture filtrate in experimental animals did not show any

significant variation in serum creatinine when compared with that of the control

(Prabha et al., 2009).

Oral administration of catechin at 40 mg/kg, twice daily for 4 days in ischemia/

reperfusion (I/R) induced rats which showed impaired renal function, produced a

significant reduction in the serum levels of creatinine and urea nitrogen. This may be

due to free radical scavenging activity and antioxidant capacity of the catechin

(Devinder et al., 2005).

Farag et al. (2006) reported that aliquots of concentrated olive leaf juice at 600,

1200 and 2400 ppm as polyphenols and butylated hydroxy toluene (BHT 200 ppm)

when administered to rats daily for 6 weeks indicated that the administration of olive


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leaf juice did not cause any changes in liver and kidney functions. On the contrary,

BHT at 200 ppm induced a significant increase in the enzyme activities and the serum

levels of uric acid, urea and creatinine.

Kumar et al. (2007) reported that diabetic hyperglycemia induces the elevation

of plasma levels of urea, uric acid and creatinine, which are considered as reliable

markers of renal dysfunction. Their experimental results showed a significant increase

in the level of plasma urea and creatinine in the diabetic rats when compared with

respective control rats, while after the treatment of streptozotocin (STZ) induced

diabetic rats with the bark extract of Helicteres isora (100 and 200 mg/kg), the levels of

urea, uric acid and creatinine were significantly decreased.

4.3.8 Liver function test

4.3.8a Serum Glutamic Pyruvic Transaminase (SGPT)

Liver damage is associated with cellular necrosis, increased tissue lipid

peroxidation and depletion in tissue GSH levels. In addition to that serum levels of

many biochemical markers like SGOT, SGPT, triglycerides, cholesterol, bilirubin,

alkaline phosphatase can be elevated (Mascolo et al., 1998). From the obtained results

it was found that SGPT level were normal in control and experimental rats, whereas

they were elevated in rats treated with extracts of Oscillatoria laetevirens and

P. juliflora alone which is showing liver toxicity (Fig. 52). This may be due to the

toxic effect of the administered compound.

Supporting evidence showed that aqeous extracts of A. leiocarpus caused a

significant dose-dependent increase in the serum level of aspartate aminotransferase,

alanine aminotransferase and alkaline phosphatase in all treatment groups compared to

the control. This indicates hepatocellular damage (Agaie et al., 2007). Woodman,

(1988) reported that an increase in the activity of these enzymes in the plasma is often

seen following liver damage and it is attributed to the loss of the enzyme from the

damaged hepatocytes rather than its increased production.

Maheswari et al. (2008) reported that there is alteration in the levels of

biochemical markers of hepatic damage like SGOT, SGPT, ALP and lipid peroxides in


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rats of paracetamol induced hepatotoxicity. Treatment with methanolic extract of

Orthosiphon stamineus leaves containing phenolic compound and flavanoids as major

components (200 mg/kg) decreased the levels of lipid peroxidation and the elevated

levels of above mentioned biochemical markers to near normal levels.

The level of ALP, SGOT, SGPT enzymes were found to be decreased in rats

treated with coir pith based cyanobacterial culture filtrate when compared to control

rats (Prabha et al., 2009). The protective effect of extracts on liver cells might exist

due to the presence of flavonoids and their antioxidant effects (Sallie et al., 1991).

4.3.8b Serum Glutamic Oxaloacetic Transaminase (SGOT)

The SGOT level was found to be normal in control and experimental male rats,

whereas it was high in female rats treated with Oscillatoria laetivirens alone. This may

be due to toxic effects of the administered compounds (Fig. 53).

Supporting evidences showed that hepatotoxicity induced rat by carbon

tertrachloride showed extensive liver damage confirmed by the elevation of SGOT and

SGPT. Pretreatment with the extract of Leucas aspera (400 mg/kg) significantly

reduced the elevation in liver enzymes, thereby showing that Leucas aspera has a

hepatoprotective nature (Mangathayaru et al., 2005).

Kumar et al. (2007) also reported an increase in the activities of aspartate

transaminase and alanine transaminase in the liver of diabetic animals. The rise in the

activity of alanine transaminase is due to hepatocellular damage and is usually

accompanied with aspartate transaminase. Treatment with Helicteres isora (100 and

200 mg/kg) or tolbutamide (250 mg/kg) normalized these enzymes activities.

Induced hepatotoxicity in rats by CCl4 showed liver injury as indicated by the

elevation of enzymes SGOT, SGPT and ALP. Treatment of animals with ethanolic and

aqueous extracts of Phyllanthus amarus showed partial hepatoprotection, where as

these extracts in combination with silymarin exhibited hepatoprotection as indicated by

significant changes in various liver parameters (Yadav et al., 2008).


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Mohammed (2010) reported hepatoprotective activity of the ethanolic extract

of the leaves of Spinacia oleracea L. (EESO) in hepatotoxicity induced rats by carbon

tetrachloride (CCl4). Thus pretreatment of rats with EESO, at 250 and 500 mg/kg body

weight for 21 consecutive days significantly prevented the CCl4 induced hepatic

damage which is indicated by serum marker enzymes (SGOT, SGPT, ALP and GGT)

and bilirubin levels.

4.3.8c Total and Indirect bilirubin

Assessment of liver function can be made by estimating the activities of serum

ALT, AST, ALP and bilirubin, which are originally present in higher concentrations in

the cytoplasm. When there is hepatopathy, these enzymes leak into the bloodstream in

conformity with the extent of liver damage (Nkosi et al., 2005). Bilirubin is one of the

most useful clinical clues to the severity of necrosis, and its accumulation is a measure

of binding, conjugation and excretory capacity of hepatocytes.

It is observed that total bilirubin and indirect bilirubin levels are normal in

control and degraded P. juliflora treated rats, while extract of O. laetevirens alone and

P. juliflora extract alone treated rats showed mildly elevated levels of bilirubin which

may be due to toxic metabolites of the administered extracts (Fig. 54). Normal levels

were observed in degraded P. juliflora treated rats, probably due to the antioxidant

properties of the flavonoids present in the extract.

Supporting evidence showed that there is a significant increase in the total

bilirubin contents, SGOT, SGPT and ALP activities in thioacetamide induced hepatic

damage groups. Treatment with polyphenolic extracts of Silybum marianum and

Chicorium intybus leads to a decrease in total bilirubin, SGOT, SGPT and ALP

activities as compared with thioacetamide treated groups. This confirms that

polyphenolic extracts have protective effects against hepatic cell injury induced by

thioacetamide (Madani et al., 2008).

Agaie et al. (2007) reported that the aqueous leave extracts of Anogeissus

leiocarpus administered to rats caused significantly lowered bilirubin levels in all

treatment groups compared to the control. This may be attributed to the depressant


110

effect of the extracts. Odutola, (1992) observed that some depressant compounds are

known to decrease bilirubin levels in the serum.

Bairwa et al. (2010) reported that administration of the ethyl acetate fraction of

stem bark of Ceiba pentandra (400 mg/kg) containing tannin, C-glycoside, phenolic

compounds, flavonoids, reducing sugar and triterpenes possesses a hepatoprotective

potential in hepatotoxicity induced rats by paracetamol (3 g/kg), which showed a

significant reduction in serum enzymes SGOT, ALP and the total bilirubin content.

4.3.8d Alkaline phosphatase (ALP)

Increased levels of circulatory Alkaline phosphatase (ALP) may be due to the

liver damage caused by the generation of free radicals. Reports showed that ammonium

chloride treated rats showed a significant increase in the level of circulatory ammonia,

urea, TBARS (thiobarbituric acid and reactive substances), HP (hydroperoxides) and

liver marker enzymes such as AST (aspartate transaminase), ALT (alanine

transaminase) and ALP (alkaline phosphatase). These changes were significantly

decreased in rats treated with Hibiscus sabdariffa leaf extract (HSEt) and ammonium

chloride which indicates that HSEt offers hepatoprotection by influencing the levels of

lipid peroxidation products and liver markers in experimental hyperammonemia and

this could be due to its free radical scavenging property and the presence of natural

antioxidants (Essa et al., 2006) which upholded the present findings.

The untreated control and degraded P. juliflora rats showed normal ALP while

extracts of O. laetevirens alone and P. juliflora alone treated rats showed slightly

elevated ALP (Fig. 55). This may be due to toxic effects of metabolites present in the

extract. A normal range of ALP in degraded P. juliflora treated rat indicates the

antioxidant effect and scavenging activity of flavonoid compounds present in the

extract. This coincides with the scavenging activity results.

Reports showed that the treatment with polyphenolic extracts of Silybum

marianum and Chicorium intybus decreased the total bilirubin, SGOT, SGPT and ALP

activities which were elevated in thioacetamide treated group. And this confirms that


111

polyphenolic extracts have protective effects against hepatic cell injury induced by

thioacetamide (Pyo et al., 2004).

A remarkable elevation was observed in serum and tissue ALP, GPT and GOT

activities in hepatotoxicity induced rats by CCl4 administration. Thus, oral

administration of the extract (250 and 500 mg/kg body weight), significantly reduced

CCl4 induced hepatotoxicity in rats, as judged from the serum and tissue activity of

marker enzymes (Glutamate oxaloacetate transaminase (GOT), Glutamate pyruvate

transaminase (GPT) and alkaline phosphatase (ALP)) (Sunilson et al., 2008). Similar

intoxication with CCl4 (500 μL/kg, i.p) to vehicle control rats showed a significant

increase in ALT, AST and ALP levels when compared to normal control rats.

Anogeissus latifolia administered at 300 mg/kg produced a significant reduction in the

above enzymes. Thus, the presence of rutin, quercetin and other antioxidants in

Anogeissus latifolia may be the contributing factor towards its hepatoprotective activity

and justifies the folkloric use of the plant in liver diseases (Pradeep et al., 2009).

4.3.9 Sperm count

It is observed that sperm count was normal in control rats. Rats treated with

O. laetevirens, P. juliflora extract showed decreased sperm counts, while animals

treated with degraded P. juliflora extracts showed normal sperm counts (Fig. 56).

Microscopic observations of sperms showed that higher deformed sperms were

observed in ethanolic extracts of O. laetevirens alone, P. juliflora extract treated

animals and it was found to be less in rats treated with ethanolic extracts of degraded

P. juliflora.

Orisakwe et al. (2003) reported the subchronic effect of Hibiscus sabdariffa

calyx aqueous extracts on the rat testes and showed that a significant decrease in

epididymal sperm counts in rats treated with 4.6 g/kg when compared to 1.15, 2.30 g/kg

treated and control. From this it is concluded that our results are consistent with the

above reports in regards to a decrease in sperm counts. Similarly, rats treated with 400

mg/kg of Morinda lucida leaf extract for 13 weeks showed a significant reduction in

sperm motility, viability and epididymal sperm counts when compared to control rats

(Shittu et al., 2008).


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4.3.10 Histopathology

4.3.10a Kidney

No pathological symptoms were observed upon treatment with ethanolic extract

of degraded P. juliflora by O. laetevirens and control rats. While rats treated with 200

mg/kg extract of O. laetevirens alone and P. juliflora alone showed sclerosis in

glomeruli (Plate-4). Early reports revealed that kidney of rats treated with phenolic

compounds alone do not exhibit any toxic effects as evidenced by the absence of

histological changes in the liver and kidney in non diabetic control rats. It is also

revealed that phenolic compounds exhibit marked antidiabetic effects when

administered at 800 mg/kg body weight (Fahim et al., 2009).

Microscopic examinations of kidney cells of rats administered with the phenolic

compounds of olive leaf juice showed histological characters of control rats, whilst, the

administration of BHT at 200 ppm severely damaged the rat kidney tissues (Farag

et al., 2006).

Kumar et al. (2008) reported the antioxidant and hepatoprotective properties of

Indigofera trita (EIT) on CCl4 treated rats showed edema of lining epithelium of renal

tubules and lumen of some of the tubules containing eosinophilic material. The

treatment with ethanolic extracts of Indigofera trita (EIT) at 400 mg/kg body weight

and vitamin E treatment showed normal renal tubules and minimal edema and focal

lymphocytic collection in the interstitial tissue.

Histopathological study of rat kidneys treated with methanol extracts of Cleome

rutidosperma root, Neolamarckia cadamba and Spondias pinnata bark at doses of 600

mg/kg upto 14 days showed normal size and shape of glomeruli, tubules, interstitium

and blood vessels. Also acute tubular necrosis or glomerular changes were absent

(Sumanta et al., 2009).

Hutadilok et al. (2010) reported that histopathological assessment of the

internal organs did not produce any significant changes in heart, spleen and kidney

tissues of all the wistar rats orally administered with methanol extract of Arthrospira

(Spirulina) platensis at doses of 6, 12 and 24 mg/kg body weight daily for 12 weeks.


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4.3.9b Liver

The histopathological pattern of untreated control rats showed normal

hepatocytic cells whereas rats treated with O. laetevirens and P. juliflora extracts at 200

mg/kg showed scattered individual cell necrosis due to hepatotoxicity of the

administered phenolic compounds present in the extract. Interestingly, normal

hepatocytes were observed in experimental rats (Plate-5). This revealed the

hepatoprotective activity of the ethanolic extract of degraded P. juliflora. Early reports

showed that high percentages of quercetin, rutin and gallic acid in the extract justifies

potent antioxidant activities (Boyle et al., 2000). Thus, the reactive species-mediated

hepatotoxicity can be effectively managed upon administration of agents possessing

antioxidants (Labib et al., 2003), free radical scavenger (Sohn et al., 2003) and anti-

lipid peroxidant (Gao and Zhou, 2005) properties.

Green tea has been found to provide protection to the liver against a variety of

toxic insults. Catechins have been discovered to be powerful antioxidants, which is

though to be at least in part responsible for green tea's hepatoprotective activity (Frank

and Painter, 1999). Kumar et al. (2008) reported the histopathological profile of liver

of CCl4 intoxicated rats showing early degenerative changes in hepatocyte cells. Liver

of rats treated with ethanol extract of Indigofera trita (EIT) at 200 mg/kg showed

changes of the hepatocytes in few areas along with focal lymphocytic collections and a

few degenerated cells. The treatment at 400 mg/kg body weight of the extract and

vitamin E treatment recovered the normal structure of liver cells.

Recent reports on the histopathology of liver and kidney revealed hydrophobic

changes till the end of the trial as recorded in diabetic rats treated with 400 mg of

phenolic compounds. Also reported, the phenolic compounds alone do not exhibit any

toxic effects as evidenced by the absence of histological changes in the liver and kidney

in non diabetic control rats (Fahim et al., 2009).

Multiple sections of the liver of rat treated with methanolic extracts of Cleome

rutidosperma root, Neolamarckia cadamba and Spondias pinnata bark at doses of 600

mg/kg showed normal lobular architecture. Hepatocytes appear normal and are

arranged in single cell cords radiating away from the central vein. No sign of non


114

specific lobular hepatitis was observed at the tested dose and there was no evidence of

bile stasis, granuloma, dysplasia or malignancy (Sumanta et al., 2009).

4.3.11 Antioxidant properties

Research in the recent past has accumulated substantial evidences that revealed

that enrichment of body systems with natural antioxidants may correct the vitiated

homeostasis and can prevent the onset as well as treat diseases caused and/or fostered

due to free-radical mediated oxidative stress. These developments accelerated the

search for antioxidant principles that lead to the identification of natural resources, the

isolation of active principles and further modification and refinement of active

antioxidant molecules (Halliwell, 1994; Tiwari, 1999; Pietta, 2000).

Antioxidants may exert their effects by different mechanisms such as

suppressing the formation of active species by reducing hydroperoxides and H2O2 and

also by sequestering metal ions, scavenging active free radicals and repairing damage.

Similarly, some antioxidants also induce the biosynthesis of other antioxidants or

defense enzymes. The bioactivity of an antioxidant is dependent on several factors like

their structural criteria, physico-chemical characteristics and in vivo radical generating

conditions (Tiwari, 2001).

Generally heartwood extractives showed higher antioxidant properties in

comparison with bark and sapwood extractives. Hydrophilic extractives from the more

polar solvents such as acetone and toluene/ethanol mixture gave higher antioxidant

properties than lipophilic extractives, suggesting that flavanols such as (-)- mesquitol

present in Prosopis juliflora extracts are responsible for the antioxidant activity (Wang

et al., 2004; Haupt et al., 2003).

The free radical scavenging activity of the extract was analyzed by the free

radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). The radical scavenging activity was

represented as percentage inhibition of DPPH radicals. From the obtained results the

free radical scavenging activity was found to be higher in the extract of degraded

Prosopis juliflora (77.4%) when compared with that of Prosopis juliflora (74.46%)

alone (Fig. 57).

results and discussion - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/5348/12/12_chapter...

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