5. PHARMACOLOGICAL SCREENING OF GISEKIA...

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5. PHARMACOLOGICAL SCREENING OF

GISEKIA PHARNACEOIDES

5.1 ACUTE ORAL TOXICITY STUDY

5.1.1 Introduction

Medicinal plants play a key role in the human health care because of

their efficacy, safety and lesser adverse effects. When herbal drugs are used

as a therapeutic substance for treating various ailments, it becomes an

essential requirement to fulfill the guidelines formulated by World Health

Organization (WHO). WHO guidelines have given one of the important

criteria to establish the safety profile of the herbal preparation (WHO report,

1991),

Numerous plants have been screened for their various biological

activities in an attempt to replace the standard drugs. However, the herbal

preparations have shown to control different diseases (Argueta et al., 1994)

such as diabetes (Virdis et al., 2003), cancer (Hecker, 1968), syphilis, asthma,

chest infections, rheumatism (Correia, 1994), leprosy (Bunkill 1985);

hypertention (Tchikaya et al., 2003), mouth ulcer and throat problems,

gastrointestinal disorders (kudi et al., 1999; Akinpelu 2001 Goncalves 2005;

Taylor 2005) etc. Most of these plant species have not been subjected to

chemical, toxicological, pharmacological or clinical investigation and have

been ignored by national health authorities for many decades. The lack of

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standardization of ingredients and their preparation usually are limited by the

toxicity or relative lack of efficacy compared with standard medications.

Hence in the present study the petroleum ether, chloroform and

methanolic extracts of Gisekia pharnaceoides whole plant obtained in

succession were subjected to acute oral toxicity evaluation in order to assess,

whether the extracts have any adverse effects or toxic manifestations for long

term use. Toxicological study results play an important safety assessment for

pharmaceuticals, pesticides and other chemicals. This study therefore was

designed to evaluate their toxicity in Wister albino rats as toxicity may result

from the overdose of these extracts.

5.1.2 Materials and methods

Preparation of the plant extracts for experimental studies

The crude extracts of Gisekia pharnaceoides in petroleum ether,

chloroform and methanol are designated as GPP, GPC and GPM respectively

as mentioned in the scheme I in Chapter 3. These extracts were used in the

form of suspension in water with 1% sodium carboxy methyl cellulose

(SCMC) as the suspending agent, for oral administration in rats.

Animals

Wister albino mice of either sex (25 – 30 g) and Wister albino rat of

either sex (180 – 200 g) were obtained from the inbred colony of Department

of pharmacology, C.L.B.M College of pharmacy, Thorapakkam, Chennai-96.

The animals were kept in polypropylene cages at 25±2°C with relative

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humidity of 45 – 55% under 12 h light and 12 h dark cycles. They were fed

with standard laboratory animal fed obtained from (Poultry research station,

Tamil Nadu Veterinary and Animal Science University, Chennai, India) and

tap water ad libitum.

All the pharmacological and toxicological experimental protocols were

approved by the Institutional Animal Ethics Committee (IACE) on

Committee for the Purpose of Control and Supervision on Experimentation on

Animals (CPCSEA), vide sanction letter No IAEC 21 / XV / CLBMCP.

The procedure was followed by using OECD (Organization of

Economic Cooperation and Development) guidelines 423 (Acute toxic class

method). Preliminary acute toxicity tests did not show any toxic signs or

symptoms in rats administered up to 2000 mg Gisekia pharnaceoides extract

per kg body weight. In this assay, 4 groups consisting of control, methanolic

extract, petroleum ether extract, chloroform extract-treated with 6 animals in

each group each containing an equal number both male and female were

formed. A dose limit at 2000 mg/kg body weight of each extract dissolved in

sodium carboxy methyl cellulose (SCMC) was administered orally to the

animals of test group. The animals of control group received SCMC. In each

case the volume administered was 10 ml / kg body weight. Following the

administration, the animals were closely observed during the first 3 h, and

48 h, there after, 14 days, for toxic sign and symptoms such as convulsions,

salivation, diarrhea, lethargy, sleep and coma and death. The weight of the

animals was monitored during the experimental period.

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5.1.3 Results

No abnormality in the general behaviour of the test animals either in

the short term or long term, was observed. Treated animals exhibited normal

behaviour as that of control group. No death was observed in any of the group

and all the animals lived upto 14 days (Table. 8). There was no body weight

loss during the observation period. But all the animals exhibited a gain in

body weight (Table. 9). The daily consumption of food and water in each sex

and group was observed to exhibit the same pattern.

5.1.4 Discussion

In general, folk medicine uses plant extracts without taking into

account their toxicity aspects. Though the Gisekia pharnaceoides is reported

to be used as an anthelmintic agent in folklore medicine (Wealth of India

2002), the toxicity of this plant have not been studied so far. In the present

study, the observations of acute toxicity test revealed that all the three extracts

of this investigation upto 2000mg/kg body weight showed no toxicity.

According to Clarke and Clarke (1977), substances with LD50 of 1000 mg/kg

body weight per oral route are registered as being safe or low toxicity.

Therefore, the Gisekia pharnaceoides which did not register LD50 even upto

2000m/kg body weight in the present investigation may be regarded as non

toxic either in folk medicine or in the diet. The result of Tables 8 and 9 also

suggest that the solvent extracts of Gisekia pharnaceoides did not affect the

increase in body weight as well as water and food intake, indicative of non-

toxic nature of the plant.

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5.2 ANTI-INFLAMMATORY ACTIVITY

5.2.1 Introduction

Inflammation or phlogosis is a pathological response of mammalian

tissues to a variety of hostile agents including infectious organisms, toxic

chemical substances, physical injury or tumor growth leading to local

accumulation of plasmic fluid and blood cells (Sobota et al., 2000). Although

inflammation is a defense mechanism, the complex events and mediators

involved in the inflammatory reaction can induce maintain and aggravate

many disorders. Hence, the employment of anti-inflammatory agents may be

helpful in the therapeutic treatment of those pathologies associated with

inflammatory reactions (Sosa et al., 2002). The clinical treatment of

inflammatory diseases is dependent on drugs which belong either to the non-

steroidal or steroidal chemical therapeutics. The non-steroidal anti-

inflammatory drugs (NSAIDs) such as aspirin, indomethacin and ibuprofen

inhibit early steps in the biosynthesis pathway of prostaglandins by inhibition

of COX enzymes and are the main drugs used to reduce the untoward

consequences of inflammation (Albert et al., 2002). However, the side effects

of the currently available anti-inflammatory drugs pose a major problem in

their clinical use. For instance, NSAIDs cause several serious adverse effects

like gastric injury and ulceration, renal damage and bronchospasm due to

their non-selective inhibition of both isoforms of the COX enzyme (Tapiero

et al., 2002). The use of steroidal drugs as anti-inflammatory agents is also

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becoming highly controversial due to their multiple side effects (Van den

Worm et al., 2001). Therefore, a need arises for the development of newer

anti inflammatory agents from natural sources with more powerful activity

and with lesser side effects as substitutes for chemical therapeutics.

Data concerning the inflammatory disease in the rural area is

sometimes, scarce. It is therefore not surprising that inflammatory diseases

are the common health problems treated with traditional remedies which

mainly comprise medicinal plants (Degif and Hahn 2001). For instance

several plants species of seseli growing in china are used in folklore for

treating the inflammatory diseases like swelling rheumatism, pain and

common cold (Hu et al., 1990). Sphenocentrum jollyanum, herbal species of

menispermeaceae family has been reported to be of use for the treatment of

rheumatic pains in Nigeria. (Moody et al., 2006). Cinnamomum camphora

sieb, commonly known as camphor as long been prescribed in traditional

medicine for the treatment of inflammation related diseases such as

rheumatism, sprains, bronchitis, asthuma and muscle pains. The materia

medica and wealth of India indicates the names of several plants having anti-

inflammatory property. In this direction the present study contributes more on

therapeutic use of plants for treating inflammatory disorders. The plant

identified was Gisekia pharnaceoides belong to the family of molluginaceae.

The aim of this part of the work is to test the various extracts of Gisekia

pharnaceoides, viz. GPP, GPC and GPM extract for the anti-inflammatory

activities in vivo.

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Principle

The inflammatory reaction is readily produced in rats in the form of

paw edema with the help of irritants or inflammagens. Substances such as

carrageenan, formalin, bradykinin, histamine, 5-hydroxy tryptamine, mustard

and egg white, when injected in the dorsom of the foot in rats produce acute

paw oedema within a few minutes of the injection.

5.2.2 Material and Methods

GPP, GPC and GPM were separately made into suspension SCMC.

A 1% suspension of diclofenac sodium in SCMC was prepared, as reference

drug.

Carrageenan induced hind paw edema model

Dose fixation

A pilot study was performed in which anti inflammatory activity by

carrageenan induced paw oedema in rat model. Wistar albino rats of either

sex were divided into six groups (n=6). The extracts were administered orally

in geometric progression starting from 100 mg/kg, and 250 mg/kg 500 mg/kg,

750 mg/kg and 1000 mg/kg respectively. The volumes of edema of the

injected and contra lateral paws were measured at +1, 2, 3 and 4 h after

induction of inflammation using a plethysmometer and percentage of

anti-inflammatory activity was calculated.

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Anti-inflammatory activity of test was performed at two dose levels. In

case of the petroleum ether, chloroform and methanolic extracts of Gisekia

pharnaceoides, the dose selected was 250 mg/kg, and 500 mg/kg based upon

the dose fixation studies. The reference drug diclofenac sodium, the dose is

fixed as 5 mg/kg.

Treatment protocol

The Wistar albino rats of either sex were divided into eight groups

containing six animals in each group and were fed with SCMC (Control) or

the crude extracts in SCMC (Test) or the diclofenac sodium in SCMC

(Reference) orally, as mentioned below. The test group of rats received either

250 mg or 500 mg of crude extract per kg body weight.

Group I - 1% SCMC 10 ml/kg/ body weight - Control

Group II – GPP 250 mg/kg body weight

Group III -GPP 500 mg/kg/ body weight

Group IV - GPC 250 mg/kg/ body weight - Test

Group V - GPC 500 mg/kg/ body weight

Group VI - GPM 250 mg/kg/ body weight

Group VII - GPM 500 mg/kg/ body weight

Group VIII- Diclofenac sodium 5 mg/kg body weight - Reference

The carrageen an assay procedure was carried out according to the

method of Winter et al., 1962. Oedema was induced by injecting 0.1 ml of a

1% solution of carrageenan in saline into the plantar aponeurosis of the left

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hind paw of the rats. The extracts or reference drug or the control vehicle was

administered 60 min prior to the injection of the carrageenan. The volumes of

edema of the injected and contra lateral paws were measured at +1, 2, 3 and 4

hrs after induction of inflammation using a plethysmometer. Results were

expressed as percentage inhibition oedema, calculated according to the

following formula: (Garcia et al., 1995)

Control-Test Percentage inhibition = ___________ x 100 Control

5.2.3 Results

In the carrageenan induced rat hind paw oedema test, the average left

back paw volume and percentage of inhibition by different extracts and the

reference drug are given in Table.10. For the Control (untreated) group

the injection of carrageenan caused localized oedema well in advance

compare to other groups. In this untreated group, the swelling increased

progressively to a maximum volume at 3rd h after the carrageenan injection.

The rats pre-treated with the crude extracts of Gisekia pharnaceoides had

significant reduction in the oedema volume on 3 rd h post dosing, at all dose

levels used. The percentage inhibition was as high as 73% for 500 mg/kg body

weight GPM of Gisekia pharnaceoides at 3 h. The effect of GPM

administration in controlling the paw oedema was almost comparable to the

reference drug (Table 10 Fig 25). An increase in the volume of paw oedema

was observed upto 3 h in all the groups, and there after it decreased. GPM at a

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dose level of 500 mg/kg body weight showed highest percentage inhibition of

(73.68%) oedema followed by the GPP that exhibited 63.15%. The GPC

seemed to be less effective than the other two extracts. The highest percentage

inhibition for 250, 500 mg/kg body weight dose was observed to be 56.5 and

73.68 % respectively.

5.2.4 Discussion

The anti inflammatory effect of the different solvent extracts of Gisekia

pharnaceoides (GPP, GPC and GPM) were investigated in the present study.

The carrageenan test was selected because of its sensitivity in detecting orally

active anti-inflammatory agents particularly in the acute phase of

inflammation (DiRosa et al., 1971; DiRosa, 1972). The intraplantar injection

of carrageenan in rats leads to paw oedema reportedly in two different phases:

the initial phase which occurs between 0-2 h after injection of carrageenan

has been attributed to the release of histamine, serotonin and bradykinin on

vascular permeability (Vinegar 1987). Where as the late phase has been

reported to be complement dependent reaction due to the over production of

prostaglandin in tissues (DiRosa et al., 1971). Also increased production of

nitric oxide (NO) and prostaglandins E2 (PGE2) has been noted in carrageenan

challenged animals (Selvemini et al., 1996). Though the Gisekia pharnaceoides

is known as a dietary supplement among the rural folk, its pharmacological

effects have not been explored yet. The data presented in this report indicate

that Gisekia pharnaceoides can exert a significant immuno modulatory effect

on various inflammatory responses, though the exact mechanism is yet to be

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understood. According to the results, the different extracts of Gisekia

pharnaceoides were shown to posses a different degree of anti-inflammatory

activity which is again dose dependent.

The variation of anti-inflammatory activity among the crude extracts of

Gisekia pharnaceoides may be due to the compositional variation of the

individual extract. For instance, the anti-inflammatory activity of coumarins

in TPA-induced ear oedema model in mice depends on their individual

substitution on the aromatic ring (Garcia-Argaez et al., 2000). The observed

anti-inflammatory property of the extracts of Gisekia pharnaceoides might

possibly due to their ability to inhibit the reactive oxygen species. As the

kaempferol (a member of flavonoid family), free radical scavenger

(Panichayupakaranant and Kaewsuwan 2004), this property would in turn

result in the inhibition of lipid peroxidation and subsequent inflammation, that

is noticed in the form of swelling.

Accumulating evidence indicates that excessive production of NO

plays a pathogenic role in both acute and chronic inflammation (Clancy

1998). NO is responsible for the vasodilatation, increase in vascular

permeability oedema formation and including synthesis of prostaglandins at

the site of inflammation (Moncada1991; Grisham 1999). The role of NO in

relation to carrageenan induced paw oedema was also reported (Selvemini

1996). Manipulation of NO free radical can be a potential and promising

therapeutic area in treating inflammations. Approaches being currently used

for inflammatory disorders include NO scavenger as well as NO inhibition

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(Mittal 2003). Since all the test drugs (GPP, GPC and GPM) exhibited

significant in-vitro NO free radical scavenging activity as discussed in the

section 5.4.3. The same may be, in part, attributed to the observed anti-

inflammatory effect of these test drugs. The increased rate of inhibition of

oedema Fig. 26 displayed GPM its constituent is actively involved in the

reactions of first phase as well as the second phase of the inflammatory

response. The inhibition in the first 2 h (ie first phase) was observed to be

53% and from 2-3 h (second phase) the inhibition increased further to 73%

showing the active participation of GPM in the anti-inflammatory reactions.

Where as the response recorded by the extracts of GPP and GPC are much

slower compared to that of GPM (Fig.25) and more particularly in the second

phase (after 2h). However, the pretreatment by GPP and GPC seemed to have

strengthened the defence mechanism and there by the paw oedema was

reduced to as high as 58% in the initial stage itself, and there after dose not

seem to have participated in the anti-inflammatory response.

5.3 ANALGESIC ACTIVITY

5.3.1 Introduction

The analgesics are one of the highest therapeutic categories on which

research efforts are concentrated (Elisabetsky and Castilhos 1990) in recent

years. Analgesic compounds available in the market, still present in wide

range of undesired effects (Katzung, 2001) leaving an open door for new and

better compounds. Natural products are belived to be an important source of

new chemical substance with potential therapeutic applicability. A large

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number of Indian medicinal plants are attributed with various

pharmacological activities due to their diversified phytochemical content.

Most of these medicinal plants have been identified and described by different

authors (Chopra et al., 1956). In this direction, attempt has been made in this

study to identify an undisclosed plant with analgesic activity. The efficacy of

the crude extract of the plant of this study is reported here.

Principle

Exposing heat to mice tail is one way of stimulating pain in cutaneous

receptors, which in turn excites the thermo selective and nociceptive fibers

(Luttinger 1985). Immersion of rat tail in hot water provokes an abrupt

withdrawal of the tail from the heat source. The time difference in the

withdrawal of tail of opioid and other rest drug treated animal was monitored.

Tail immersion method is most widely and reliably used for revealing the

potency of opioid analgesics (Jansen 1963).

Animals

Swiss albino mice of either sex were used for the experiments study.

5.3.2 Materials and Methods

GPP, GPC and GPM were separately made into suspension of SCMC.

Morphine prepared in the form suspension in SCMC of 1% (w/v) was

administered orally to animals.

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Dose fixation

In order to fix the dose for the extracts of Gisekia pharnaceoides, a

pilot study was performed in which analgesic activity by tail immersion

method was chosen as the model. Albino mice of either sex were divided into

six groups (n=6). The extracts were administered orally in geometric

progression at a dose of starting from 50 mg/kg, 100 mg/kg, and 250 mg/kg

500 mg/kg, 750 mg/kg and 1000 mg/kg body weight respectively. The basal

reaction time was recorded and the % production of analgesia was calculated.

Analgesic activity of test was performed at two dose levels. In case of

GPP, GPC and GPM extracts of Gisekia pharnaceoides, the dose selected was

250 mg/kg, and 500 mg/kg based upon the dose fixation studies. The

reference drug morphine, the dose is fixed as 5 mg/kg.

Tail immersion method

Tail immersion test was conducted as described by Aydin et al (1999).

Eight groups of Swiss albino mice of either sex weighing between 20-25 gm

were used for the study. This involved immersing the extreme 3cm of the

mice tail in water bath containing water at 55 ± 0.50C followed by the

observation for tail flicking of mice. The basal reaction time was monitored

by observing tail flicking. The interval of time between the dipping and

flicking of tail is noted as basal reaction time. The test groups were given

GPP, GPC or GPM SCMC 250, 500 mg/kg p.o in SCMC or SCMC alone

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(Control). The reference group animals received morphine (5 mg) in SCMC.

In a different expression, the animals were divided and treated as follows.

Group I - 1% SCMC 10 ml/kg/ body weight - Control

Group II – GPP 250 mg/kg body weight

Group III – GPP 500 mg/kg/ body weight

Group IV – GPC 250 mg/kg/ body weight - Test

Group V – GPC 500 mg/kg/ body weight

Group V I –GPM 250 mg/kg/ body weight

Group VII – GPM 500 mg/kg/ body weight

Group VIII - Morphine 5mg/kg body weight - Reference

After administration of the drugs, the tail flick response was tested at

an interval of 30, 60, 90,120,150 and 180 minutes. A cut off period of 10 sec

is considered as a maximum analgesic and the tail is removed from source of

hot water to avoid the damage.

5.3.3 Results

A significant reduction of the painful sensation due to tail immersion

in warm water was observed following oral administration of the different

solvent extracts at a dose of 250 and 500 mg/kg. The effect was seemed to be

noticeable after a period of 30 min (Fig. 27) and it was dose dependent

(Table. 11). The heat tolerance of the animal was observed to be low in all the

three solvent extract (GPP, GPC and GPM), with respect to 250 mg/kg dose

compare to that of 500 mg/kg dose (Fig. 28). However, the basal reaction

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time recorded by these low dose fed animals is higher than that of the control

(SCMC) group. (Fig. 28 and Table.11). The inhibitory effect of the extracts

became more pronounced between 60-120 min post dosing. Of all the

experimental samples, the methanol extract have shown highest analgesic

activity compared to other extracts for a particular dosage, in the tail

immersion test.

5.3.4 Discussion

Compare to the crude extracts of Gisekia pharnaceoides, morphine a

centrally active analgesic drug (Heidary and Darban 1999; Hajhashemi et al.,

2002), produced increased analgesic effect in the tail immersion method.

Therefore, the potency of the crude extracts (250 or 500 mg) was lower than

morphine (5 mg). However, this composition cannot be made as the matched

quantity of the active principle (Kaemphferol) of Gisekia pharnaceoides was

not administered to the mice in this study. The calculations, based on the

yield, say that each gram of crude extract contains 2 mg kaemphferol the

biologically active substance. In other words, 1 mg of biologically active

substance is present in 500 mg crude extract. Therefore it is clear that test

animals were administered with 0.5 mg or 1 mg of active substance as against

5 mg morphine in the control group was able to pronounce a basal reaction

time of 5.83 sec for 500 mg extract (Table. 11) as against 6.83 sec for morphine

(Fig. 28). The phytochemical studies (Chapter 3) Gisekia pharnaceoides

revealed the presence of many chemicals such as alkaloids, phenolic

compounds, flavanoids and tannins in the crude extracts. Determination of the

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role of each compound in the analgesic effect of this plant is a wide field for

more investigation. But the presences of flavanoids in Echium amoenum have

been reported to possess analgesic activity in vivo (Delorme et al., 1977;

Milis and Bone 2000). The presence of flavanoids in an extractable amount in

the Gisekia pharnaceoides was detected and therefore the analgesic effect

observed in this study may be attributed to the flavanoids, particularly the

kaemphferol. This finding is in good agreement with that of the earlier

investigators who have worked on other plant species. (Pathak et al., 1991;

Meyre- Silva et al., 1999; Bittar et al., 2000). These authors claim that the

flavanoids may have action on the peripheral anti-nociceptive effect. The

Kaempferol being the major component of the crude extract of Gisekia

pharnaceoides, this flavanoid could be responsible for the peripheral anti-

nociceptive effect. However, the exact mechanism of action of flavanoids

remains to be explored.

5.4 IN-VITRO ANTIOXIDANT ACTIVITY

5.4.1 Introduction

Gisekia pharnaceoides, the wild plant is already a part of the diet of

the rural population in certain parts of India. However, the pharmacological

activity of this plant is yet to be reported. Many plants often contain

substantial amounts of antioxidants including vitamins C and E, carotenoids,

flavonoids and tannins etc. and thus can be utilized to scavenge the excess

free radicals from human body (Pratt 1992; Shahidi et al., 1992; Rice –Evans

et al., 1996). Many investigations indicate that these compounds are of great

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value in preventing the onset and/or progression of many human diseases

(Hallilwell and Gutteridge 1989; Halliwell et al., 1992; Willet 1994; Tsao and

Akthar 2005). The oxidative stress in a biological system is exerted by the

free radicals like superoxide, hydroxyl, nitric oxide etc., which are known a

Reactive Oxygen Species (ROS). Most living species have a number of

defence mechanism against oxidative stress and toxic effects of normal

oxygen metabolism. In the case of plants, when used for edible purpose, it is

necessary to determine the efficacy of natural antioxidants to combat the free

radicals that are involved in the development of degenerative diseases

(Campbell and Abdulla 1995). Thus total antioxidant capacity of an edible

vegetable could be a parameter to evaluate the quality of vegetable foods

(Arnao et al., 1998; Cano et al., 1998), apart from its nutritional value. Hence,

this study was designed for the evaluation of possible beneficial antioxidant

potency of Gisekia pharnaceoides extracts by employing different methods

and techniques. The free radical system employed here are DPPH, Nitric

oxide, hydroxyl, superoxide and ABTS.


The crude extracts of Gisekia pharnaceoides in petroleum ether,

chloroform and methalnol are designated as GPP, GPC and GPM respectively

as mentioned in the scheme I in Chapter 3.

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5.4.2.1 Assay of diphenyl picryl hydrate radical scavenging Principle

1,1, Diphenyl 1,2-picryl hydrazyl (DPPH) is a stable free radical

which shows deep violet colour in ethanol solution, characterized by an

absorption maximum, at 517 nm. When a solution of DPPH is mixed with

that of a substance that can donate a proton, the free radical DPPH is reduced

to hydrazine.

Basically this technique is a decolourization assay which evaluates the

absorbance decrease at 517 nm, produced by the addition of the antioxidant to

a DPPH solution in ethanol. DPPH assay is considered as a valid and easy

assay to evaluate scavenging activity of antioxidants, since the radical

compound is stable and does not have to be generated as in other type of

radical systems.

Method

This assay is based on the measurement of the scavenging ability of

antioxidant substances towards the stable radical. In this experiment the free

NO2

N O2

O2

N N N

NO 2

NO 2

2N N H

NO

H+

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radical scavenging activity of the crude extracts was examined in-vitro by

using DPPH radical (Yokazawa et al., 1998). Extracts were prepared with

different concentration from a maximum of 800 to minimum of 25 μg/ml.

Briefly, the reaction mixture consisted of 1 ml of 0.1 mM DPPH in ethanol,

0.95 ml of 0.05 M Tris-HCl buffer (pH 7.4), 1 ml of ethanol and 0.05 ml of

the extract. The absorbance of the mixture was measured at 517 nm exactly

30 sec after adding the crude extracts. The experiment was performed, in

triplicate, and % scavenging was calculated using the formula:

Control-Test Percentage inhibition = x 100 Control

Parallely, a blank was made with all the reagents except the extracts. The

activity was compared with Vitamin E, which was used as a reference

antioxidant.

5.4.2.2 Assay of nitric oxide scavenging activity

Nitric oxide (NO) is a hydrophobic molecule and highly diffusible

free radical, generated through the oxidation of L-argenine to L-citrulline by

nitric oxide synthase (Alderton et al., 2001). Low concentration of nitric

oxide acts as a signaling molecule with dichotomous regulatory roles in many

physiological process, whereas high level of nitric oxide produced by

inflammatory cells can damage DNA, RNA, lipids and proteins, leading to

increased mutations and altered enzyme and protein function important to

multi stage carcinogenesis process (Patel et al., 1999, Li and Wogan, 2005).

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Effects of nitric oxide on cell is ultimately dependent on a complex of

factors including existing biological milieu; rate of nitric oxide production

and its rate of diffusion; interaction with other free radicals, metal ions and

proteins; levels of protective enzyme such as catalase and superoxide

dismutase; levels of antioxidants such as glutathione. However, the fate of

nitric oxide in biological systems is broadly governed by three main reaction

processes: diffusion and intracellular consumption, auto oxidation to form

nitrous anhydride (N2O3) and the reaction with superoxide to form peroxy

nitrite (ONOO-) (Chen et al., 2001).

Principle

The assay is based on the principle that, sodium nitro prusside in

aqueous solution at physiological pH spontaneously generates nitric oxide,

which interacts with oxygen to produce nitrite ions and the same can be

estimated using Griess llosviy reaction (Garrat et al., 1964).In the present

investigation, Griess llosviy reagent is modified by using 0.1% (w/v) napthyl

ethylene diamine (NED) dichloride, instead of 5 % (w/v) 1-napthylamine

scavengers of nitric oxide compete with oxygen leading to reduced

production of nitrite ions.(Marcocci et al., 1994).

Method

Sodium nitroprusside (5μM) in standard phosphate buffer solution was

incubated with different concentration (25-800 μg/ml) of the extracts

dissolved in standard phosphate buffer (0.025 M, pH 7.4) and the tubes were

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incubated at 25°C for 5 h (Sreejayan and Rao, 1997). After 5 h, 0.5 ml of

incubated solution was removed and diluted with 0.5 ml Griess reagent

(prepared by mixing equal volume of 1% sulphanilamide in 2 % phosphoric

acid and 0.1% naphthyl ethylene diamine dihydrochloride in water). The

absorbance of chromophore formed during diazotization of nitrite with

sulphanilamide and its subsequent coupling with napthyl ethylene diamine

was read at 546 nm. The control experiment was also carried out in similar

manner, using distilled water in the place of extracts. The experiment was

performed in triplicate and % scavenging was calculated using the formula:




antioxidant.

5.4.2.3 Measurement of Hydroxyl radical scavenging activity

Principle

Hydroxyl radical (OH•) scavenging activity can often be calculated

using the “deoxyribose assay”: a mixture of ferric chloride (FeCl3) and

ethylenediamine tetra acetic acid (EDTA) in the presence of ascorbate reacts

to form iron (II)-EDTA and oxidized ascorbate, H2O2 then reacts with iron

(II)-EDTA to generate iron (III)-EDTA plus HO• in the so-called Fenton

reaction.

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Fe2+ +H2O2 Fe3+ + OH- +HO•

That radical not scavenged by other components of the reaction

mixture attack the sugar deoxyribose and degrades it into a series of

fragments, some or all of which react on heating with thiobarbituric acid at

low pH to give a pink chromogen. Thus the scavenging activity towards OH•

of a substance added to the reaction mixture is measured on the basis of the

inhibition of the degradation of deoxyribose (Halliwell 1990 ; Aruoma et al.,

1993).

Method

The hydroxyl radical scavenging activity was measured by studying

the competition between deoxyribose and the extracts for hydroxyl radicals

generated in the Fe3+ / ascorbate / EDTA / H2O2 system. The hydroxyl radicals

attack deoxyribose, which eventually results in TBARS formation (Elizabeth

et al., 1990). The reaction mixture contained deoxyribose (2.8 mM), Fecl3

(0.1 mM), EDTA (0.1 mM), H2O2 (1 mM), ascorbate (0.1 mM), KH2PO4 -

KOH buffer (20 mM, pH 7.4) and various concentration (25-800 μg/ml) of

extracts and reference drug (Vitamin E) in a final volume of 1 ml. The

reaction mixture was incubated for 1 h at 37°C. Deoxyribose degradation was

measured as TBARS (Thiobarbituric acid reactive substance) at 532 nm

(Ohkawa 1979) and percentage scavenging was calculated using the formula:

Control-Test Percentage inhibition = x 100

Control

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antioxidant.

5.4.2.4 2, 2-Azinobis-(3-ethylbenzothiazoline - 6 - sulphonic acid) (ABTS)

radical cation decolorization assay

Principle

The ABTS assay is based on the inhibition by antioxidants of the

absorbance of the radical cation ABTS•+. The ABTS•+ radical cation is

generated directly by chemical reduction by manganese dioxide in the

absence of hemeprotein and H2O2 (Rice-Evans and Miller 1997a). A

modification of this method has been introduced by a decolorization

technique in which the radical is generated directly in a stable form using

potassium / ammonium persulphate (Re et al., 1999). Afterwards the formed

radical is mixed with antioxidant in the reaction medium and the percentage

inhibition at 745 nm is calculated.

Method

The anti oxidant capacity of each extract was evaluated by studying its

ability to bleach the radical (ABTS•+). ABTS radical cation was produced by

reaction of ABTS solution (7 mM) with 2.45 mM, ammonium persulfate and

the mixture was allowed to stand in a dark room temperature for 12 -16 h

before use. In this study, different concentrations (25-800 µg/ml) of the crude

extract (0.5 ml) were added to 0.3ml of ABTS solution and the final volume

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was made up with ethanol to make 1 ml. The absorbance was read at 745nm

(Auddy et al., 2003; Miller 1997) and the percentage scavenging calculated

by using the formula as given below:


Parallely, a blank was made with all the agents except the extracts. The


antioxidant.

5.4.2.5 Assay of superoxide radical scavenging activity

Principle

The scavenging activity towards superoxide radical of a wide range of

antioxidants is measured in terms of inhibition of generation of O2•- with the

hypoxanthine-xanthane oxidase superoxide generating system (Robak and

Gryglewski, 1988 Halliwell, 1990; Mitsuya et al., 1990). In this method O2•-

is generated using a non-enzymatic reaction of potassium superoxide in the

presence of alkaline DMSO (Nishikimi et al., 1972; Robak and Gryglewski,

1988). This O2•- reduces nitro-blue tertazolium (NBT) into formazan at pH 7.4

and room temperature, and the formazan generation is followed by

spectrophotometry at 560 nm. Any added molecule capable of reacting with

O2•-, inhibits the production of formazan.

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Method

The method was performed by using alkaline Dimethyl sulphoxide

(Henry et al., 1976). Potassium superoxide and dry DMSO were allowed to

stand in contact for 24 h and the solution was filtered immediately before use.

A 200 µl of the filtrate was added to 2.8 ml 10 mM potassium phosphate

buffer containing 56 µM nitroblue tetrazolium (NBT) and10µM EDTA. To

this, crude extract of Gisekia pharnaceoides (1 ml) of various concentrations

(25 – 800 µg/ml) was added and the absorbance was recorded at 560 nm,

against a control in which pure DMSO was added instead of alkaline DMSO

and the percentage scavenging calculated by using the formula as given

below:


Parallely, a blank was made with all the agents except the extracts. The


antioxidant.

5.4.3 Results

Several concentrations, ranging from 25 – 800 µg/ml, of the

methanolic, chloroform and petroleum ether extracts of Gisekia

pharnaceoides were tested for their antioxidant activity in different in vitro

models. It was observed that free radicals were scavenged by the test

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compounds in a concentration dependent manner upto 800 µg/ml

concentration in all the models (Table 12 – 16 and Fig 29 - 33). In GPM the

maximum percentage inhibition in all the models viz, ABTS, DPPH, hydroxyl

radicals, nitric oxide and super oxide radical was found to be 87.79, 77.78,

65.45, 63.62 and 51.78% respectively at 800 µg/ ml. In GPC the percentage

inhibition in all these models was found to be 60.83, 58.24, 52.13, 49.14 and

45.49 % respectively at 800 µg/ ml. In GPP percentage inhibition in all the

models was found to be 55.66, 50.54, 44.86, 42.08 and 40.42% respectively at

800µg/ml. On comparative basis the methanolic extract showed better activity

in quenching ABTS radicals with an IC50 value of 150 µg/ml and DPPH

radicals with an IC50 155 µg/ml. However, the extracts show encouraging

response in quenching hydroxyl radicals with an IC50 value of 150, 180 and

250 µg/ml respectively for GPM, GPC and GPP. The quenching activity was

moderate against the remaining oxidant models investigated in this study.

5.4.4 Discussion

Oxidative stress has been implicated in the pathology of many diseases

and conditions including diabetes, cardiovascular disease, inflammatory

conditions, cancer and aging (Marx et al., 1987). Antioxidants may offer

resistance against oxidative stress by scavenging the free radicals, inhibiting

the lipid peroxidation and by many other mechanisms and thus prevent

diseases (Youdim and Joseph 2003).

DPPH is a relatively stable free radical. The assay is based on the

measurement of the scavenging ability of antioxidants towards the stable

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radical DPPH. From the present results it may be postulated that Gisekia

pharnaceoides reduces the radical to the corresponding hydrazine when it

reacts with the hydrogen donors in the antioxidant principles. Such

observations have also been made by Sanchez-Moreno (2002) in his studies

on the free radical scavenging activity in foods and biological systems. From

methodological point of view, the DPPH method is recommended as easy and

accurate with regard to measuring the antioxidant activity of fruit and

vegetable juices or extracts. The results are highly reproducible (Gil 2000;

Cano 1998). The results presented in Table 12 and Fig 29. suggest that the

solvent extracts of Gisekia pharnaceoides must be a source of reducing

equivalents, and therefore donates an electron to reduce the DPPH radicals to

its corresponding hydrazine (Bolis et al., 1958).

Nitric oxide is a free radical produced in mammalian cells, involved in

the regulation of various physiological processes (Lalenti 1993 & Ross 1993).

However, excess production of NO is associated with several diseases such as

cancer (Nguyen 1992), diabetes (Corbett 1992), trauma (Szaboc 1994), sepsis,

(Thiemermann 1997) atherosclerosis, multiple sclerosis (Parkinson 1997) and

arthritis and in inflammations (Ohshima 2003). Herbal drugs having nitric

oxide radical scavenging property are gaining importance (Sarkar 2005; Rao

2006) because they possess various biological activities related to antioxidant

mechanisms (Shirwaikar et al, 2004). In the present study the nitrite produced

by the incubation of sodium nitro pruside in standard phosphate buffer at 25°c

was reduced by the extracts of Gisekia pharnaceoides (Table 13 and Fig 30).

This may be due to the antioxidant principles in the extracts which compete

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with oxygen to react with nitric oxide (Marcocci et al., 1994) thereby

inhibiting the generation of more deleterious products such as nitrous

anhydride (N2O3) and perhydroxy nitrite (ONOO-) (Chen et al, 2001).

Ferrous salts can react with hydrogen peroxide and form hydroxyl

radical via Fenton’s reaction. The iron required for this reaction is obtained

either from the pool of iron or the heme-containing-proteins (Cotran 1999)).

The hydroxyl radical (OH•) thus produced may attack the sugar of DNA

deoxy causing ribose fragmentation; base loss and DNA strand breakage

(Kaneko 1999). The generation of OH• in the Fenton reaction is due to the

presence of iron ions. When the Fe2+ / Fe3+ redox couple is bound by certain

chelators, the OH• formation is prevented. Whereas the increased colour

formation in the absence of crude extracts were observed in the deoxyribose

assay, as has been observed by (Sanchez-Morero 2002). Hence it is presented

that the crude extracts of this investigation acts as the chelators of iron ions,

binding to them, and preventing the formation of radical, though the extracts

not directly involved in the OH• scavenging. The results (Table 14 and

Fig 31) indicate that the extracts of Gisekia pharnaceoides play a major role

in the inhibition of ribose fragmentation and hence the decreased colour

formation in the deoxyribose assay.

The ABTS assay is applicable for both lipophilic and hydrophilic

antioxidants. The assay is based on the inhibition of the absorbance of the

radical cation, ABTS•+, which has a characteristic long wavelength absorption

spectrum (Sanchez-Morero 2002 ; Miller and Rice- Evans 1997). The ABTS

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chemistry involves direct generation of ABTS radical mono cation with no

involvement of any intermediary radical. It is a decolorization assay, thus the

radical cation is performed prior to addition of antioxidant test system, rather

than the generation of the radical taking place continually in the presence of

antioxidants. The results (Table 15 and Fig 32) obtained implied the activity

of the extract either by inhibiting the formation of or scavenging the ABTS•+

radicals since both the inhibiting and scavenging properties of antioxidants

towards ABTS•+ radicals, have been reported (Re 1999; Rice- Evans1997)

earlier. The results of this study were compared with that of the vitamin E

which is used as a reference scavenger.

Super oxides are produced from molecular oxygen by the oxidative

enzymes (Sainani et al., 1997) of body as well as via non enzymatic reaction

such as auto oxidation by catecholamines (Hammani et al., 1998). Superoxide

dismutase catalyses the dismutation of the highly reactive superoxide anion to

oxygen and hydrogen peroxide (Kamalakkannan et al., 2003). Superoxide

anion is the first reduction product of oxygen (Ray et al., 2002) which is

measured in terms of inhibition of generation of O2-. The various phyto

compounds of Gisekia pharnaceoides, that are observed in the preliminary

phytochemical screening, including tannins (Lin et al., 1974; Yoshida et al.,

1989), polyphenols (Brand Williams et al., 1995; Bondet, et al., 1997;

Yokozawa et al., 1998) and flavanoids (Okawa et al., 2001; Cao et al., 1997a)

are reported to have antioxidant properties and hence, these compounds may

be responsible for the scavenging of superoxide, generated potassium

superoxide-alkaline DMSO system, in addition to other reactive species

117

studied in this investigation. The overall superoxide radical scavenging

activity of the various extracts viz GPM, GPC and GPP suggest (Table 16 and

Fig 33) that the GPP exhibit increased activity compared to other two extracts

probably due to its higher tannin content, as tannins are easily extractable in

low polar solvents, such as petroleum ether, compare to high polar solvents

like chloroform and methanol.

The free radical scavenging property of the crude extracts of Gisekia

pharnaceoides against DPPH, NO, OH•, O2•- and ABTS•+ radicals is clearly

understood from the results of this chapter. The phytochemical screening of

the extract (Chapter 3) revealed the presence of flavonoids as well as tannins

and other compounds. Further purification of the crude extract ended up with

one compound namely kaempferol, confirmed the presence of flavonoids in

Gisekia pharnaceoides. Therefore the antioxidant property exhibited by the

crude extract against the radicals studied might be due to the tannins and

flavonoids. Such observation has been made by earlier investigators for

methanolic extract of other plants (Ho et al., 1992; Wang et al., 1996). Since

the free radical scavenging properties of the extracts have been confirmed in

vitro, the Gisekia pharnaceoides as such, by all means should also be

potential antioxidant in vivo. In human body superoxide, nitric oxide and

hydroxyl free radicals are produced endogenously. These radicals of normal

metabolism cause extensive damage to DNA, proteins and lipids and

constituting a major contribution to aging and also to degenerative diseases of

aging such as cancer, cardiovascular disease, brain dysfunction, and cataracts

(Ames et al., 1993). This oxidation process could be prevented or delayed, if

the antioxidants or the anti oxidant rich food is added to the diet. Gisekia

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pharnaceoides, in this study, is found to be an effective free radical scavenger

and hence can serve as a potential anti oxidant diet for those in the

advancement of age or aging related disorders and diseases.

Flavanoids are natural products, which has been shown to posses

various biological properties related to anti oxidant mechanisms (Perrisoud

1982; Glyglewski 1987; Corvazier 1985; Middleton 1984; Robak 1988 and

Ratty 1998). Thus, the antioxidant potential of Gisekia pharnaceoides

observed is due to the presence of flavonoids therein.

5.5 WOUND HEALING ACTIVITY

5.5.1 Introduction

Wound: Wound is defined as a loss or breaking of cellular and

anatomic or functional continuity of living tissues (Ayello, 2005).

Experimental wounds can be categorized into two types namely, full and

partial thickness. While the partial thickness wounds heal by mere

epithelialization, the healing of full thickness wound which extends through

the entire dermis involves more complex well regulated events (Treget et al.,

1997; Taun et al., 1998). Skin injury is highly complex owing to its structural

complexity. The skin is the most affected organ following an injury. Skin

consists of usually a cellular epidermis and acellular connective tissue dermis

and these two layers are interconnected through the basement membrane or

basal lamina. Skin, may therefore, be considered as an interesting structure in

which wound healing involves both regeneration and repair, and the most

common site of injury (Melissa Calvin 1998).

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Wound healing: Cutaneous wound healing is a self motivated

physiological process of cell regeneration which occurs without any external

stimuli. The process of wound healing occurs in different phases such as

hemostasis, inflammation, proliferation and tissue remodeling (Fig 34). Each

phase overlaps with the next (Manley and Bellmann 2000).

1. Homeostasis

Tissue injury initiates a response that first clears the wound of

devitalized tissue and foreign material, setting the stage for subsequent tissue

healing and regeneration. The initial vascular response involved a brief and

transient period of vasoconstriction is followed by active vasodilation

accompanied by an increase in capillary permeability. Platelets aggregated

with in a fibrin clot secrete a variety of growth factors and cytokines that set

the stage for an orderly series of events leading to tissue repair.

2. Inflammation

The Inflammation phase, present itself as erythema, swelling and

warmth and is often associated with pain. The inflammatory response

increases vascular permeability, resulting in migration of neutrophils and

monocytes into the surrounding tissue. The neutrophils engulf debris and

microorganisms, providing the first line of defense against infection.

Neutrophils ceases after the first few days of post injury if the wound is not

contaminated. If this acute inflammatory phase persists, due to wound

hypoxia, infection, nutritional deficiencies, medication use, or other factors

120

related to the patients immune response, it could interfere with the late

inflammatory phase (Stadelmann et al., 1998). In the late inflammatory phase,

monocytes are converted in the tissue to macrophages, which digest and kill

bacterial pathogens, scavenge tissue debris and destroy remaining neutrophils.

Macrophages, begin the transition from wound inflammation to wound repair

by secreting a variety of chemotactic and growth factors that stimulate cell

migration, proliferation and formation of the tissue matrix.

3. Proliferation

Proliferative phase is dominated by the formation of granulation tissue

and epithelialization. Its duration is dependent on the size of the wound.

Chemotactic and growth factors released from platelets and macrophages

stimulate the migration and activation of wound fibroblasts that produce a

variety of substances that is essential for wound repair, including

glycosaminoglycans (mainly hyaluronic acid, chondroitin-4 sulphate,

dermatan sulphate, heparin sulphate) and collagen (Stadelmann et al.,1998).

This forms an amorphous, gel like connective tissue matrix necessary for cell

migration. New capillary growth must accompany the advancing fibroblasts

into the wound to provide metabolic needs. Collagen synthesis and cross–

linkage is responsible for vascular integrity and strength of new capillary

beds. Improper cross-linkage of collagen fibers has been responsible for non-

specific post operative bleeding in the patients with normal coagulation

parameters. Early in proliferation phase, fibroblast activity is limited to

cellular replication and migration. Around the third day after wounding, the

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growing masses of fibroblast cells begin to synthesize and secrete measurable

amount of collagen. Collagen levels rise continually for approximately three

weeks. The amount of collagen secreted during this period determines the

tensile strength of wound.

4. Wound tissue remodeling

The final phase of wound healing is wound remodeling; the events in

remodeling are responsible for the increase in tensile strength, decrease in

erythema and scar tissue bulk, reorganization of new collagen fibres and the

final appearance of the healed scar. This process continues upto two years,

achieving 40 – 70 percent of the strength of undamaged tissue at four weeks

(Stadelmann et al.,1998). Type I collagen becomes the major collagen present

in the mature scar or normal skin, reversing the earlier type III collagen

predominance.

Factors involved in wound healing

In most of the cases, the complication in wound healing is due to

inflammation. Inflammation results in a continuous generation of reactive

species, such as the super oxide radical or the non radical hydrogen peroxide

(Pawlak et al., 1998). An imbalance between oxygen species and the

antioxidant defence mechanisms of a cell, leading to an excessive production

of oxygen metabolites leads to condition of oxidative stress. Oxidative stress

results in lipid peroxidation, DNA breakage and enzyme inactivation

including free radical scavenging enzyme (Wisemann et al., 1996). Evidence

122

for the potential role of antioxidants in the pathogenesis of many diseases

suggests that antioxidants may be of therapeutic use in these conditions

(Skaper et al., 1997). Flavanoids, a group of naturally occurring benzo-γ-

pyrone derivatives, have been shown to posses several biological properties,

including hepatoprotective, antithrombotic, anti inflammatic and antiviral

activities, many of which may be related partially atleast to their antioxidants

and free radical scavenging ability (Chen et al., 1990).

Fig. 34. The phases of cutaneous wound healing

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(i) The Gisekia pharnaceoides extracts of methanol (GPM),

chloroform (GPC) and petroleum ether (GPP) were prepared as

mentioned in Chapter 2.

(ii) Soframycin ointment was purchased locally.

(iii) Simple ointment containing as placebo wool fat (5%) hot

paraffin (5%), cetosteryl alcohol (5%) and white soft paraffin

(85%) was prepared (Anonymous, 1953) and used as placebo

control.

(iv) Preparation of test ointment:- About 50 gm of semisolid plant

extract was incorporated into the 1000 g of simple ointment

base.

5.5.2.1 Wound creation and treatment

Albino rats weighing 150-180 g were used for the study. Rats were

maintained in individual metabolic cages throughout the experiment, in

hygienic conditions and they were fed with commercial balanced diet and

water ad libitum. The fur on the backside of the rats was shaved under mild

ether anesthesia. Subsequently, open excision wounds of standard size of

2 x 2 cm were made by using a template. The ointment was applied daily

twice a day till the wound completely healed (Chatterjee and Chakravorty,

1993). The progress of wound healing was evaluated periodically by

monitoring the histological changes of the skin tissue obtained at 5th, 10th and

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15th days after wound creation. The anti-oxidant level after complete healing

of the wound was also assayed.

The animals were divided into five groups containing six animals each

and were treated as given below.

S.No. Groups Treatment

1 Group I – Control - simple ointment 2 Group II – GPM extract ointment 3 Group III – GPC extract ointment 4 Group IV – GPP extract ointment

5 Group V – Soframycin ointment

5.5.2.2 Wound contraction assessment by planimetry

The contour of the individual wounds of both control and experimental

animals was periodically measured using transparent graph sheet and the rate

of healing was calculated and expressed as percentage contraction (Morgen

1994). The observation on percent wound contraction and epithelialization

period were measured from initial day (Rashed et al., 2003). The following

formula was used to calculate the percentage of wound contraction.

Wound area on day 1 – wound area on day ‘n’ Wound contraction % = x100 Wound area on day ‘n’

125

5.5.2.3 Histological studies

Skin tissue from the wounded site of individual rat was removed after

sacrifice. They were then fixed in 10% buffered formalin, dehydrated through

graded alcohol series (30 - 100%), cleared in xylene and embedded in paraffin

wax (m.p. 56°C). Serial sections of 5 µm thickness were cut using microtome,

and stained with hematoxylin – eosin and were examined under light

microscope for epithelialization, fibrosis and angiogenesis (Yeo et al., 2000).

5.5.2.4 Preparation of skin homogenate

A 10% homogenate of skin tissue was prepared in 0.02M Tris HCl

buffer pH 7.0 using a Teflon homogenizer in ice-cold condition. The

homogenate was centrifuged at 5000 rpm for 10 min. The supernatants

collected were used for the determination of non-enzymatic antioxidants such

as ascorbic acid (AA) and enzymatic antioxidants such as catalase (CAT) and

superoxide dismutase (SOD) and glutathione peroxidase (GPx). Apart from

this, malondialdehyde (MDA), the product of lipid peroxidation was also

determined using the supernatant.

5.5.2.5 Protein determination

Protein content of the skin homogenate was determined according to

the method of Lowry et al, (1951). The aromatic amino acids tyrosine and

tryptophan present in proteins react with Folin –Ciocalteau reagent, which

contains phosphomolybtic acid and phosphotungstate to produce a blue colour

compound, which shows absorption maximum at 660 nm.

126

Reagents

Lowry’s reagent

Solution A : 2% sodium carbonate in 0.1N NaOH

Solution B : 0.5% copper sulphate in 1% sodium

Potassium tartarate

Solution C : 50 ml of solution A was mixed with 1.0 ml

of solution B, just before use

Folin –Ciocalteau reagent: 1:2 dilutions with water just before use

Standard solution : Standard bovine serum albumin (BAS)

containing 20 mg /100 ml were prepared.

Procedure

A 0.1 ml of the sample was made upto 1.0 ml with water, and a 4.5 ml

of lowry’s reagent (Solution C) was added, mixed well and allowed stand for

10 min. To this, 0.5 ml of Folin –ciocalteau reagent was added, mixed well

and kept at room temperature for 20 min. A standard solution containing BSA

at a concentration of 20 – 100 μg and reagents blank were treated in a similar

manner. The colour developed was read at 660 nm.

The total protein was expressed as mg/100mg of tissue.

127

5. 5.2.6 Measurement of antioxidant level

5. 5.2.6.1 Estimation of activity of Glutathione peroxidase

The activity of Glutothione peroxidase was estimated according to the

method of Rotruck et al., (1973). Reduced glutathione (GSH) is converted

into oxidized glutathione (GSSG) in the presence of the enzyme Glutothione

peroxidase (GPx) and its reaction is as follows

GPx 2GSH + H2O2 GSSG + 2H2O

Reagents

1. Phosphate buffer 0.1M, pH 7.0: 420 ml of 0.1M disodium

hydrogen phosphate was mixed with 0.1M sodium dihydrogen

orthophosphate until the pH adjusted to 7.0.

2. Ethylene diamine tetra acetic acid (EDTA), 1mM: 29.2 mg of

EDTA was dissolved and made upto 100 ml using distilled water.

3. Sodium azide 10 mM: 65 mg of sodium azide was dissolved in

distilled water and made upto 100 ml.

4. Glutathione (GSH), 4 mM: 125 mg of GSH was dissolved in 100

ml of distilled water.

5. Hydrogen peroxide, 2.5 mM: 0.03 ml of hydrogen peroxide

solution (30%) was made up to 100 ml with water.

128

6. Trichloro acetic acid (TCA), 10%: 10 gm TCA was dissolved and

made upto 100 ml with distilled water.

7. Disodium hydrogen phosphate (0.3 M): 4.25 gm of disodium

hydrogen phosphate was dissolved and made upto 100 ml with

distilled water.

8. 5, 5 – Dithio bis (2-nitrobenzoic acid) substrate (DTNB), 0.6mM:

2.37 mg of DTNB was dissolved in 100 ml of 1% tri sodium

citrate.

9. Glutathione standard (200 µg/ml): 20mg of reduced glutathione

was dissolved in 100ml of distilled water.

10. Trisodium citrate (1%w/v): 1gm of trisodium citrate was dissolved

and made upto 100 ml with distilled water.

Procedure

Triplicate samples of, 0.1 ml of skin homogenate taken in separate

tubes was mixed with 0.4 ml of phosphate buffer and added 0.1ml each of

sodium azide, EDTA and H2O2. Then, 0.2ml of GSH was added to all the

tubes and the reaction was arrested by the addition of 10 % TCA after 3 min

incubation at 37°C intervals. The tubes were then centrifuged and 1.0 ml of

the supernatant was transferred to fresh tubes. The blank contained 1ml of

distilled water. The standard glutathione was prepared in separate tubes at a

concentration range of 5 to 20 μg in a final volume of 1 ml. To all the above

tubes 4 ml of disodium hydrogen phosphate solution was added followed by

129

0.5 ml of DTNB and the colour developed was read at 412 nm in a

spectrophotometer against the blank.

The activity of Glutathione peroxidase is expressed as Units/mg

protein. One unit of enzyme activity is defined as the amount of the enzyme

that converts 1 µmole of GSH to GSSG per minute in the presence of H2O2.

5. 5.2.6.2 Ascorbic acid estimation

Ascorbic acid was estimated according to the method of Omeye et al.,

(1979). Ascorbic acid is oxidized by copper to form dehydro ascorbic acid and

diketogluconic acid. These products are then treated with dinitrophenylhydrazine

(DNPH) to form a derivative i.e., bis-2-4-dinitrophenylhydrazone. This

compound, in strong sulphuric acid undergoes a rearrangement to form a

yellowish orange product, which was measured spectrophotometrically.

Reagents

1. Trichloro acetic acid (TCA-10%): 10 g TCA was dissolved and

made upto 100 ml with distilled water.

2. Dinitrophenylhydrazine / Thiourea / Copper sulphate (DTC)

reagent: 0.4 g of thiourea, 0.05 gm of copper sulphate and 3.0

gm of DNPH was mixed and made upto 100 ml with 9N H2SO4.

3. Sulphuric acid H2SO4 (65%): 65ml of Conc.H2SO4 was made

upto 100 ml with distilled water.

130

4. Sulphuric acid H2SO4 (9N): 25 ml of Conc.H2SO4 was made

upto 100 ml with distilled water.

5. Ascorbic acid standard: A standard solution of ascorbic acid

was made by dissolving 100 mg in 100 ml of distilled water.

Procedure

1.0 ml of skin homogenate was made upto 3.0 ml by addition of 10%

TCA and the precipitated protein was cleared off by centrifugation. To 1.0 ml

of the supernatant was added 0.5 ml of DTC reagent and was incubated at

37°C for 3 h. After incubation, 2.5 ml of ice cold 65% H2SO4 was added,

mixed well and allowed to stand at room temperature for an additional period

of 30min. The yellowish orange colour formed was measured at 520 nm. The

standard tubes containing ascorbic acid were also treated in the same manner.

Ascorbic acid values were expressed as mg/gm protein.

5. 5.2.6.3 Superoxide dismutase

The activity of superoxide dismutase was estimated according to the

method of Marklund and Marklund (1974). Pyrogallol, auto oxidizes rapidly

in aqueous solution at a faster rate at a higher pH (8.0) to produce several

intermediate products. The inhibition of pyrogallol auto oxidation by the

enzyme present in the sample is employed in the quantification of activity of

superoxide dismutase. The inhibition of auto oxidation brought about by the

addition of enzyme is evaluated at the early stage as an increase in absorbance

at 420 nm.

131

Reagents

1. Tris - HCl buffer (0.1M), pH 8.2: 1.576 g of Tris-HCl was

dissolved in distilled water and the pH was adjusted to 8.2

using1N NaOH and the volume was made up to 100 ml with

distilled water.

2. Pyrogallol solution (0.2 mM): 126 mg of pyrogallol was

dissolved in 100 ml of Tris- HCl buffer and stored in an

aluminum foil wrapped stopperd test tube.

3. Diethylenetriamine penta acetate (DTPA-1mM): 19.67 mg of

DTPA was dissolved in 100 ml of distilled water.

4. Ethylene diamine tetra acetic acid (EDTA-1mM): 29.22 mg of

EDTA was dissolved in 100 ml of distilled water.

Procedure

A mixture containing 2.6 ml of Tris-HCl buffer, 0.1 ml of EDTA and

0.5 ml of DTPA was prepared. To this mixture, 0.5 ml of pyrogallol was

added and the increase in absorbance was read at 420nm against the blank for

3 min to determine the rate of auto oxidation of pyrogallol. The test sample,

0.1 ml of skin homogenate taken in separate tube was mixed with 2.5 ml of

Tris-HCl buffer, 0.1 ml of EDTA and 0.5 ml of DTPA. To this mixture,

0.5 ml of pyrogallol was added and the increase in absorbance was read at

420 nm using a spectrophotometer against the blank for a period of 3 min.

132

This measurement constituted the rate of inhibition of auto oxidation of

pyrogallol brought about by the enzyme present in the tissue homogenate.

The reagent blank contained a mixture of 3.1 ml of Tris-HCl buffer, 0.1ml of

EDTA and 0.5 ml of DTPA and this was used to set 100% absorbance.

The enzyme activity of superoxide dismutase was expressed as

units/mg of protein and it is defined as the 50% inhibition of auto oxidation of

pyrogallol per min by the enzyme.

5. 5.2.6.4 Estimation of activity of Catalase

The activity of catalase was estimated by the method of Sinha (1972).

The method is based on the fact that dichromate in acetic acid is reduced to

chromic acetate when heated in the presence of hydrogen peroxide (H2O2),

resulting in the formation of perchromic acid as an unstable intermediate. The

chromic acetate, thus produced was measured spectrophotometrically.

Reagents

1. Dichromate-acetic acid reagent (1:3 v/v): one volume of 5%

potassium dichromate in water was mixed with 3 volume of acetic

acid. The solution was further diluted to 1:5 with distilled water.

2. Phosphate buffer (0.01M, pH 7.0): 84 ml of 0.01M disodium

hydrogen phosphate was mixed with 0.01M potassium dihydrogen

orthophosphate until the pH adjusted to 7.0.

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3. Hydrogen peroxide (0.2M): 0.02 ml of H202 (S.G-1.01) was taken

and immediately mixed in 1 litre of distilled water. This solution

was used for initiation of reaction with tissue homogenate.

4. Potassium dichromate, 5% (K2Cr2O7. 7H2O): 5 gm of K2Cr2O7 was

dissolved in distilled water and made upto 100 ml.

Procedure

Triplicate sample of 0.1 ml of skin homogenate was taken, to which

1.0 ml of phosphate buffer and 0.5 ml of hydrogen peroxide were added. The

reaction was arrested immediately by the addition of 2.0 ml dichromate-acetic

acid reagent at 0, 30, and 60 second intervals. The reagent blank was prepared

by the addition of 1.6 ml of buffer and 2.0 ml of dichromate acetic acid

reagent taken in separate tubes. The test and blank tubes were heated in

boiling water bath for 10min for the green colour develop. The intensity of

green colour developed was measured at 570 nm using spectrophotometer

against blank, after the tubes were cooled at room temperature.

The activity of Catalase in the tissue homogenates is expressed as

µmoles of H2O2 consumed / min/mg protein at 37°C.

5. 5.2.7 Estimation of Tissue lipid peroxidation

Lipid peroxidation products (as Malondialdehyde) were determined

by the thiobarbituric acid reaction as described by Droper and Hadley (1990).

Malondialdehyde, a secondary product of lipid peroxidation reacts with

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thiobarbituric acid to form a pink chromogen (Thiobarbituric acid – 2

Malondialdehyde adduct), which was measured spectrophotometrically.

Reagents

1. 20% TCA solution : 20 gm of TCA was dissolved in

100ml of double distilled water.

2. 0.12M thiobarbituric acid: This was prepared freshly before

use.

3. Standard solution : 1,1,3,3-tetramethoxy propane

100 nmoles/ml

Procedure

To 0.5 ml skin homogenate, 2.0 ml of 20% TCA was added. The

contents were mixed well and centrifuged at 4000 rpm for 20 minutes. 2.0 ml

of the supernatant was mixed with 2.0 ml of TBA reagent. Reagent blank and

standards (5 – 20 nmoles) were also treated similarly. The contents were

heated for 20 minutes in a boiling water bath the tubes were cooled to room

temperature and the absorbance was read at 532 nm in a shimadzu UV-visible

double beam spectrometer. The lipid peroxide content was expressed as

nmoles MDA / mg protein.

135

5.5.3 Results

Rate of wound contraction

The percent wound contraction was calculated from the wound size,

measured periodically on day 5, 10, 15. It was observed to be 91, 96 and

100% on day 15 for GPP, GPC and GPM treated groups respectively. The

planimetry of the wound of the animals at different period of intervals was

depicted in Table 17 & Fig 35. The results of this study show a remarkable

difference between the control and GPM treated group. The other two extract

treated groups viz., GPP, GPC also seem to have positive influence on the

wound contraction but, are less effective compare to the GPM. The

differences in the healing efficacy among the extracts are clearly seen in the

first 10 days, after that the rate of healing is more or less same in all the three

groups. The healing pattern of soframycin (Reference) treated group follows

the results of GPM treatment. However, the real advantage of using an herbal

drug can be assessed by the histological examination of the re-epithelialised

skin specimens.

Period of epithelialization

The progression of wound contraction was evaluated by macroscopic

observations and parallely, the contour of wound size was monitored by

planimetry at regular intervals of time. The rate of wound healing in terms of

wound closure was observed to be in the following order

Group II > Group V> Group III> Group IV> Group I

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The macroscopic analysis of wound revealed that the methanolic

extract (GPM) treated groups required a total period of 14 days for complete

eplithelilaization, whereas the petroleum ether extract (GPP) treated group

and chloroform extracted (GPC) treated groups required 18 and 16 days

respectively for complete epithelialization.

Histology

Histological studies provided a good evidence of suitability of

progression of wound healing. The status of epithelialization in the tissue was

studied by examining hematoxylin-eosin stained specimen. The specimens

were observed for re-epithelialization, migration of cells, distribution of

collagen fibers, formation of blood vessels etc and a comparison was made

among the treated and control groups, and are presented as below.

Group I: Figure 36 shows the histology of control specimen on 5th, 10th

and 15th days. The untreated wound specimen of this group shows that the

process of healing is still in the early stage of inflammation (Fig 36.a) with

few numbers of fibroblasts and absence of keratinization. Appearance of

loosely packed fiber, incomplete epithelialization and presence of active

fibroblasts are seen on 10th day specimen of control. On 15th day the specimen

showed few numbers of blood vessels as a mark of angiogenesis in the dermis

region.

Group II: Figure 37 show the histology of GPM treated specimen on

5th, 10th and 15th days. The post wounding tissue on 5th day (Fig 37.a) showed

137

undiffentiated keratinization with active fibroblast. The 10th day specimen

showed (Fig 37.b) almost complete epithelialization with fairly good amount

of collagen and neo-vascularization. Where as on 15th day there was an (Fig

37.c) uniform accumulation of collagen with prominent thick bundles

consisting of moderate number of fibroblast. The epidermal layer appeared

continuous, consisting of well defined epithelium and no inflammatory cells

were observed in the wound site. In this group, the healing process was

completed within 15 days.

Group III: Figure shows the histology of the tissue specimen of GPC

treated group on 5th 10th and 15th days respectively. The specimens of day 5

(Fig 38.a) post wounding showed the onset of undifferentiated keratinization

and few number of fibroblast. On day10 was nearing to completion and few

numbers of blood vessels are seen in the midst of and loosely packed collagen

fibers in dermis region (Fig 38.b). Almost complete epithelialization, clearly

differentiated epidermis, and closely packed but more irregular arrangement

of collagen fibers are seen on 15th day showed (Fig 38.c).

Group IV: shows the histology of GPP treated wound tissue specimen

on 5th 10th and 15th days respectively. The histological result of this group on

day 5 (Fig 39.a) resembles the same as that of the Group III (Fig 38.a),

revealing that it is in the process of inflammation. On day 10, the specimen

showed the progression of the process of epithelialization, angiogenesis and

collagen deposition (Fig 39.b). On 15th day (Fig 39.c) epithelialization was

almost complete but with irregular and loose distribution of collagen in the

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dermis region.

Group V: The histology of reference group (soframycin treated)

specimens is shown in figures 40 a, 40.b and 40.c 50, 51 and 52 for 5th 10th

and 15th days respectively. The onset of keratinocyte differentiation and

accumulated active fibroblasts are seen on 5th day (Fig 40.a). Specimen of 10th

day (Fig 40.b) showed the progression of epithelialization towards completion

with irregular deposition of collagenous substance and under-developed blood

vessels. Complete epithelialization and uniform distribution with more active

fibroblasts were seen in dermis region on day 15. The collagen deposition was

still in the amorphous state (Fig 40.c) unlike that is seen in Group II

(Fig 37.c).

Protein content

Table 18 shows the water soluble protein levels in the re-epithelialised

tissue of treated and control animals. As seen from the results (Table 18), the

Group II animals show higher level of protein compare to other groups. More

particularly, it (Group II) shows significant increase against the protein value

of control group (p<0.001).

Antioxidant level

Glutothione peroxidase (GPx)

Table 19 shows the glutathione peroxidase level in the skin tissue of

different experimental groups. As shown in the results (Table 19), the GPM

treated wound tissue seems to produce or display more glutathione peroxidase

139

activity then any another group of animals. This particular group exhibits as

high as 2 fold of GPx level compare to the tissue of control animals.

Ascorbic acid

Though there was not much variation observed in the tissue ascorbate

level among the treated groups, an obvious increase in the ascorbate level was

seen in Group II, compare to its control, which is statistically significant

(p<0.001).

Superoxide dismutase

The SOD level in the GPM treated wound tissue was found to show an

increase to an extent of 40% to that of the control. Even amongst the other

treated groups, the Group II animal specimens showed higher level of SOD

activity (Table 19).

Catalase

The level of catalase, another antioxidant enzyme, in the wounded skin

specimens of various treatment groups also records a relatively an increased

activity in the GPM treated skin specimen compared with the other treatments

as well as the control.

140

Lipid peroxidation

Oxidative damage to tissue or cell depends upon the defence system

provided by the antioxidants. The resultant product of oxidative damage is

identified by measuring the level of malondialdehyde. The results of Table 19

shows that the GPM treated wound tissue exhibited least MDA value, of all

the other treatments, where it was observed to be about 2.6 fold less lipid

peroxidation compare to the control.

5.5.4 Discussion

Cutaneous wound healing involves repair and regeneration of tissue.

The healing process begins with the clotting of blood and is completed with

remodeling of the cellular layers of the skin. Healing process, a natural body

reaction to injury, initiates coagulation which controls excessive blood loss

from the damaged vessels. The next stage of the healing process is

inflammation and debridment of the wound followed by re-epithelialization,

which includes proliferation, migration and differentiation of squamous

epithelial cells of the epidermis. In the final stage of the healing process

collagen deposition and remodeling occurs within the dermis (Chettibi and

Ferguson 1997; Hj Baie and Sheikh, 2000). The involvement of each phase

varies over a spectrum dependent largely on the type, location, milieu factors

influencing the wound. Epithelialization is the process where keratinocytes

migrate from the lower skin layer and divide. Contraction is the process

where the wound contracts, narrowing or closing the wound. Matrix

formation is facilitated by the movement of fibroblast in the wound area and

141

collagen is laid down over and throughout the amorphous material. (Rudolph

1979; Sunilkumar et al.,1998). Recent studies in the process of wound healing

reveal that secretion of biologically active substances like growth factors,

integrins, fibronectin and decon is inevitable for the initiation of healing

process (Knighton et al., 1982; Dorminguez et al., 2002). Formation of

granulation tissue, the primary step during the healing process is a complex

event involving inflammatory cells, histocytes, plasma cells, mast cells and in

particular in fibroblast that promote the growth of tissue by collagen

production. Development and differentiation of granulation tissue are often

influenced by endogenous and exogenous factors, which can promote the

healing process (Tsuboi and Rifkin 1990).

In the present study, three different solvent extracts of Gisekia

pharnaceoides designated as GPM, GPC, GPP applied topically on the wound

and observed for its efficacy to promote the healing. Since the rate of wound

healing is higher in GPM treated group followed by that of GPC and then the

GPP, it is presumed that the crude methanolic extract of Gisekia

pharnaceoides that was applied exogenously might have enhanced a

centripetal migration of fibroblasts that advanced the wound contraction by

7 days compared to the control group. Many plant extracts and medicinal

herbs have shown potential flavanoids, as main components of many extracts,

act as powerful free radical scavengers (Tran et al., 1997; Berkercioglu et al,

1998). Role of antioxidants from plant extracts in wound healing has been

reported earlier by Tran et al., (1996). In the present investigation,

Kaempherol, a member of flavanoid family has been isolated in pure form

142

from methanolic extract and characterized (Chapter 3). This information

provides authenticity that the enhanced wound healing in GPM treated group

is due to Kaempherol. This apart, the preliminary phytochemical studies

revealed the presence of tannins too, that are known to promote wound

healing process mainly due to their astringent and antimicrobial property (Ya

et al., 1988). Therefore the combined effect of flavanoid and tannins in the

GPM would have triggered the wound contraction in 14 days. An observed

2 days delay of healing in GPC treated followed by 4 days delay in GPP

treated groups suggests the absence or the presence of negligible quantity of

flavanoids in GPC and GPP as they are the product of low polar solvents and

therefore might not have extracted flavanoids into it.

The wound healing property of the plant extracts was evidenced by

histological examination that revealed an increase in the level of collagen

deposition and in the number of fibroblast cells compared to control. A more

pronounced deposition of collagen fibers, well defined keratinocyte

differentiation, presence of relatively moderate number of fibroblasts and

absence of inflammatory cells observed in the GPM treated group support the

claim that the combined effect of flavanoid and tannins accelerate the normal

healing process and hence the increased rate of epithelialization compared to

all other treatments. (Fig. 35)

Reactive oxygen species (ROS) such as superoxide radical (O2•-),

hydroxyl radicals (•OH) or reactive nitrogen species (RNS) and nitric oxide

(NO•) arise from inflammatory cells, which are strongly implicated in the

143

pathogenesis of several disease including chronic ulcer (Rojkind et al. 2002;

Moseley et al., 2004 and Abd-El-Aleem et al., 2000). While ROS / RNS play

an important role in the normal wound healing process by killing the invading

microorganisms, the excessive overproduction of these species causes

indiscriminate cellular damage, thus resulting in delayed healing. Flavanoids

have been shown to possess free radical scavenging ability (Chen et al., 1990),

therefore prevent the cell death (Jovanoic et al., 1996) by protecting them

against lipid peroxidation (Dechameux et al., 1992). Studies on topical

application of compounds with free radical scavenging properties on patients

have been shown to significantly improve wound healing and protect tissue

from oxidative damage (Martin 1996). The in vitro free radical scavenging

ability of the different solvent extracts Gisekia pharnaceoides have been

demonstrated in the early part of this chapter. These extracts are found to

scavenge variety of ROS and RNS apart from DPPH• and ABTS+• radicals. In

particular the NO radical is easily scavenged by the extracts and particularly

the GPM was observed to be more active against all the radicals studied. This

observation is fully reflected in the study of wound healing as well. This

confirms the protective role of GPM, against the free radicals that is injurious

to cell, even in the in vivo model.

To support this claim, the antioxidant status of the re-epithelialised

tissue of different treatment of this study was assessed. The results of the

emzymic and non enzymic antioxidant levels suggests that there is a

significant increase in the antioxidant defence mechanism of treated animals

compare to that of the control, and therefore showed better healing activity.

144

The phytochemical work of this investigation concluded that the

extracts of Gisekia pharnaceoides contain certain bioactive compounds such

as flavanoids and tannins that are found to be advantageous for various

biological applications or therapies as discussed in earlier part of this report.

So scavenging effect might be one of the most important components

of wound healing. Since the GPM treated wound tissue showed higher

antioxidant levels, than that of other groups, a noticeable increase in the rate

of healing was observed in this particular group (Group II). Therefore the

enhanced rate of wound healing in the GPM treated group is due to the free

radical scavenging action of flavanoid – tannin combine in addition to

strengthening the invivo defence mechanism at the wound site. Many plant

extracts and medicinal herbs have shown potent antioxidant activity.

Flavanoids, the main components of many plants extracts act as powerful free

radical scavengers (Tran et al., 1997 and 1996, Berkercioglu et al., 1998).

The protective role of GPM against the ROS is generally ascertained

by monitoring the malandialdehyde (MDA), the lipid peroxidation product. A

higher level of MDA in the control group specimen may be correlated with

high level of unscavenged free radicals as against low level of MDA in the

GPM treated group, attributing to the high order of free radical scavenging

ability.

145

5.6 IN VITRO ANTHELMINTIC PROPERTY OF GISEKIA PHARNACEOIDES

5.6.1 Introduction

The prevalence of intestinal helmenthiasis is apparently very high, and

on a global scale these infections cause severe health problems in man and

domestic animals. More than one billion people are infected with Ascaris

lumbricoides, and hundreds of millions are infected with hookworms and

Trichuris (Guyatt and Evans, 1992). These infections cause intestinal

disorders, discomfort and loss of productivity through direct or indirect

interference with host nutrition and metabolism.

In livestock, gastrointestinal parasitic infections constitute a major

obstacle to animal production all over the world, particularly in tropical and

subtropical areas. The infections cause high economic losses in the form of

impaired performance and reproduction, and sometimes significant weight

losses and mortality rates (Fabiyi, 1986). A number of control measures to

combat these infections are available, and several classes of modem synthetic

anthelmintics have been shown to be very effective when used strategically in

the right epidemiological context. However, increasing problems of

development of resistance in helminths (Geerts and Dorny, 1995; Coles, 1997)

against anthelmintics have led to the proposal of screening medicinal plants

for their anthelmintic activities. These plants are known to provide a rich

source of botanical anthelmintics (Satyavati et al., 1976; Lewis and Elvin

Lewis, 1977). A number of medicinal plants have been used to treat parasitic

infections in man and animals (Nadkarni, 1954; Chopra et al., 1956; Said,

146

1969; Akhtar et al., 2000; Iqbal et al., 2004). Latex collected from young

papaya fruits were shown to possess anthelmintic activity against Ascaridia

galli infections in chickens (Mursof and He, 1991) and Heligmosomoides

polygyrus in mice (Satrija et al., 1995). The use of Calotropis procera flowers

as an anthelmintic in sheep have also been reported (Iqbal et al., 2005). Thus

plants with anthelmintic properties offer an alternative to manufactured

anthelmintics that is both sustainable and environmentally acceptable. Such

plants could have a more important role in the future control of helminth

infections in the tropics. With this view, the Gisekia pharnaceoides which

also has the potential to serve as nutritional supplement (Chapter 4) is studied

for its anthelmintic property against Annelida.

5.6.2 Material and Methods

(i) Pheretima pothuma (earth worm) nearly equal size (8±1cm)

was collected from the local Horticulture Department.

(ii) Solution of GPP, GPC or GPM was prepared by dissolving each

of extract substance in propylene glycol. Different

concentration (25, 50, 100 and 200mg) of each of extract

solution was prepared by diluting the stock solution, in

propylene glycol, using normal saline. This serves as test drug.

(iii) The reference drugs- Piperazine citrate or albendazole was

prepared by dissolving them in normal saline at a concentration

of 15mg/ml.

147

(iv) A 10% propylene glycol in normal saline was used as

experimental control – treatment.

(v) Saline was prepared and used to treat the normal control

group.

Experiment

Pheretima pothuma was placed in petridish containing four different

concentrations (25, 50, 100 and 200mg) each of GPP, GPC and GPM

solutions. Each petridish was placed with 6 worms and observed for paralysis

or death (Nargund 1999; Vigar 1984). The mean time for paralysis was noted

when no movement of any sort could be observed, except when the worm was

shaken vigorously; time of death of worm (min) was recorded after

ascertaining that worms neither moved when shaken nor when given external

stimuli. In the same manner piperazine citrate or albendazole was included as

reference compound. These observations were compared with those of

controls.

5.6.3 Results

The crude extracts samples which were used to evaluate anthelmintic

activity, showed variable times at different concentrations and the mean time

values were calculated for each parameter (Kulkarni 1999) and presented in

Fig 41 and Table 20. The crude extract of GPP showed the significant

anthelmintic effect causing death of the worm at all the concentrations but the

time of death was different in each case. However, when we observed the

148

response of worms in case of paralysis, there was significant variation among

the results produced by the different extracts at different concentration like

25, 50, 100 and 200 mg/ml. The GPP showed more significant effect on

paralyzing the worms, in terms of paralysis time, at every concentration

compared to that of GPC and GPM. Similar observations were made in the

anthelmintic activity as well.

5.6.4 Discussion

The effect of extracts on the paralysis or helminthiasis of the worm,

according to the results (Table 20) may be indicated as GPP > GPC > GPM.

In particular, the GPP (Petroleum ether extract) exhibited an increased

paralytic as well as helminthiatic effect over albendazole at the given

experimental concentrateons (Table. 20). This may be due to the increased

level of extraction of tannins in the GPP followed by GPC and then GPM, as

the GPP is the low polar solvent and hence tend to extract more amount of

tannin. The data presented in the Table. 20 and the observations made there

of, lead to the conclusion that the different degree of helminthiasis of the

different extracts are due to the level of tannins present in the compounds.

Tannins, the secondary metabolite, occur in several plants have been

reported to show anthelmintic property by several investigators. (Yesilada et

al., 1993; Sezik et al., 1997; Niezen et al., 1995; Khan and Diaz-Hernandez,

1999; Athnasiadou et al., 2001; Waller et al., 2001 ; Niezen et al., 1998).

Tannins, the polyphenolic compounds, are shown to interfere with energy

generation in helminth parasites by uncoupling oxidative phosphorylation

149

(Martin, 1997) or, bind to the glycoprotein on the cuticle of the parasite

(Thompson and Geary, 1995), and cause death. Coming to the chemistry of

nematode surface, it is a collagen rich extracellular matrix (ECM) providing

protective cuticle that forms exoskeleton, and is critical for viability. The

collagen is a class of proteins that are modified by a range co- and post-

translational modification prior to assembly into higher order complexes or

ECMs (Page and Winter 2003). The mammalian skin also consists largely of

collagen in the form of fibrous bundles. In leather making industry, vegetable

tannins are commonly used in the tanning operation of leather processing that

imparts stability to collagen of skin matrix through its reactivity and hence

make the collagen molecule aggregate into fibres. This results in the loss of

flexibility in the collagen matrix and gain of mechanical property with

improved resistance to thermal or microbial / enzymatic attack. Similar kind

of reaction is expected to take place between the nematode cuticle (the

earthworm) and the tannin of Gisekia pharnaceoides, possibly by linking

through hydrogen bonding, as proposed in this study. This form of reactivity

brings toughness in the skin and hence the worms become immobile and non

functional leading to paralysis followed by death.

STATISTICAL ANALYSES

The data were subjected to analysis of variance and the significant

differences among the mean compared with students `t' test using SPSS PC+

software.

150

REFERENCE

Abd-El-Aleem SA, Ferguson MW, Appleton I, Kairsingh S, Jude EB, Jones K, McCollum CN, Ireland GW. 2000; Expression of nitric oxide synthase isoforms and arginase in normal human skin and chronic venous leg ulcers. J Pathol ,191:434–42.

Akhtar, M.S., Iqbal, Z., Khan, M.N., Lateef, M., 2000. Anthelmintic activity of medicinal plants with particular reference to their use in animals in the Indo-Pakistan subcontinent. Small Ruminant Research 38, 99–107.

Akinpelu,D.A., 2001. Antimicrobiol activity of Anacardium occidentale bark. Fitoterapia 72, 286-287.

Albert, D., Zundorf, I., Dingermann, T., Muller, W.E., Steinhilber, D.,Werz, O., 2002. Hyperforin is a dual inhibitor of cyclooxygenase-1 and 5-lipoxygenase. Biochemical Pharmacology 64, 1767-1775.

Alderton WK, Cooper CE, Knowels RG. 2001. Nitric oxide synthes, structure, function and inhibition. Biochemistry Journal. 357:593 -615.

Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1993. Oxidants, antioxidants, and the degenerative diseases of aging. Proceedings of the National Academy of Science of the United States of America 90, 7915-7922.

Anonymous, 1953. British Pharmacopoeia. General Medical Council, the Pharmaceutical Press. 17, Bloomsbury square, London, W CI.P.

Argueta, A., Cano,A., Rodarate,M.E., 1994. Atlas de las plantas Medicinales de la Medicina Tradicional Mexicana. Instituto Natcional Indigenista, Mexico, DF, pp 1786.

Arnao M.B., Cano A. and Acosta M. 1998. Total antioxidant activity in plant material and its interest in food technology. Recent Research and Development in Agriculture and food chemistry 2: 893-905.

151

Aruoma O.I., Murcia A., Butler J. and Halliwell B. 1993. Evaluation of the antioxidant and prooxidant actions of gallic acid and its derivatives. Journal of Agricultural and Food Chemistry 41: 1880 – 1885.

Athnasiadou, S., I. Kyriazakis,F. Jackson, R. L. Coop. 2001. Direct anthelmintic effects of condensed tannins towards different gastrointestinal nematodes of sheep: In vitro and in vivo studies. Vet. Parasitol. 99, 205-219.

Auddy B, Ferreira M, Blasina F, Lafon L, Arredondo F, Dajas F, Tripathi P C, Seal T & Mukherjee B, 2003. Screening of antioxidant activity of three Indian medicinal plants, traditionally used for the management of neurodegenerative disease, J Ethanopharmacol, 84,132.

Aydin,S., Demir, T., Ozturk,Y., Baser,K.H.C., 1999. Analgesic activity from roots of Anagelica pubescens. Planta Medica 61, 2-8.

Ayello, E.A., 2005. What does the wound say? Why determining etiology is essential for appropriate wound care. Advances in Skin Wound Care 18, 98-109.

Bekerecioglu M, Tercan M, Ozyazan I 1998. The effect of Ginkgo biloba (Egb 761) as a free radical scavenger on the survival of skin flaps in rats. Scand J Plast Reconstr Hand Surg 32: 135–139.

Bittar, M., de Sousa, M.M., Yunes, R., Lento, R.A., Delle-Monache, F., Cechinel-Filho, V., 2000. Antinociceptive activity of I3, II8-Binaringenin, a bioflavanoid present in plants of the Guttiferae. Planta Medica.

Blois M.S., 1958. Antioxidant determinations by the use of stable free radical, Nature, 26, 1199.

Bondet.V., Brand –Williams.W and Berset C.1997. Kinetics and mechanisms of antioxidant activity using the DPPH free radical method. Lebensmitted Wissenschaft and Technologie 30: 609-615.

152

Brand –Williams W., Cuvelier M.E., and Berset C. 1995. Use of a free radical method to evaluate antioxidant activity. Lebensmitted Wissenschaft and Technologie 28: 25-30.

Bunkill., H.M., 1985. In: Farinhes, A.-D. (Ed), The Useful Plants of West Tropical Africa, vol. 1, 2nd ed. Royal Botanical Gardens, Kew, pp 346-349.

Campbell I C & Abdulla E.M., 1995. Strategic approaches to in vitro neurotoxicology, in Approaches and methods: Neurotoxicology (Academic Press London) 495.

Cano A., Hernandez-Ruiz.J., Garcia-Canovas F., Acosta M. And Arnao M.B. (1998). An end-point method for estimation of the total antioxidant activity in plant material. Phytochemical analysis 9: 196-202.

Cao G., Prior R.L., Cutler R.G. and Yu B.P. 1997a. Effect of dietary restriction on serum antioxidant capacity in rats. Archives of Gerontology and Geriatrics 25: 245-253.

Chatterjee, T.K., Chakravorty,A., 1993. Wound healing properties of the new antibiotics (MT81) in mice. Indian Drugs 30, 450 – 452.

Chen B, Deen W M. 2001. Analysis of the effects of cell spacing and liquid depth on nitric oxide and its oxidation products in cell culture. Chemical Research and toxicology 14: 135 – 147.

Chen Y, Zhen R, Jia Z, Ju Y. 1990. Flavinoids as super oxide scavengers and antioxidants. Free Radic Biol Med; 9: 19-21.

Chettibi S., Ferguson, M.W.J., 1997. Wound repair – an overview In: Gallin, J.I., Snyderman, R (Eds), Inflammation – Basic Princilpes and Clinical Correltes, Lippincot, Williams and Wilkins, Philadelphia, pp 865- 881.

Chopra, R.N., Nayar, S.L., Chopra,I.C., 1956. Glossary of Indian Medicinal Plants. National Institute of Science Communication, New Delhi, India.

153

Clancy RM, Amim AR , Abramson SB. 1998. The role of nitric oxide in inflammation and immunity. Arthritis and Rheumatism, 41: 1141- 1151.

Clarke, E.G.C., Clarke.M.L., 1977. Veterinary Toxicology Cassel and Collier Macmillan Publisher, London, pp 268-277.

Coles, G.C., 1997. Nematode control practices and anthelmintic resistance on British sheep Farms. Veterinary Record 141, 91–93.

Corbett JA, MC Daniel ML. 1992. Does NO mediate autoimmune destruction of betacells? Diabetes 41: 897-890.

Correia, M.P., 1994. Dicionario de plantas Uteis do Brasil e das Exoticas Cultivadas, vol.63. Instituto Brasileiro de Desenvolvimento Florestal, Rio de Janeiro.1.

Corvazier E & Maclouf J, 1985.Interference of some flavanoids and non-steroidal anti-inflammatory drugs with oxidative metabolism of arachidonic acid by human platelets and neutrophils, Biochim Biophys Acta 835, 315.

Cotran R S, Kumar V & Collins T, 1999. In Robbins pathological basis of diseases, 6th ed (Thomson Press (I) Ltd, Noida India) Association of clinical Biochemists of Indian publications, pp 577-580.

Dechameux T, Dubois F, Beauloye C, Wattiaux-de Conink S, Waniaux R. 1992. Effect of various flavanoids on lysosomes subjected to an oxidative stress. Biochem Pharmacol; 44: 1243 -1248.

Degif, T., Hahn,H.Z., 2001. Traditional treatment of skin disorders in Buta-jira, South Central Ethiopian Pharmaceutical Journal 19, 48-56.

Delorme, P., Jay. M., Ferry, S., 1977. I nventaire phytochimique des boraginaceae indigenes. Planta Medica 11, 5-11.

DiRosa,M., 1972. Biological properties of carrageenan. Journal of pharmacy and pharmacology. 66, 84-86.

154

DiRosa,M., Giround,J.P., Willoughby,D.A.,1971. Studies of the acute inflammatory response induced in rats in different sites by carrageenan and terpentine. Journal of pathology 104, 15-29.

Dorminguez E.T, Castro – Munozledo.F, Kuri-Harcuch, 2002. Growth factors and extracellular matrix proteins during wound healing promoted with frozen cultured sheets of human epidermal karatinocytes, Cell Tissue Res. 307 (1), 79 -89.

Draper H.H, and Hadley, M. 1990. Malondialdehyde determination as index of lipid peroxidation, Methods Enzymol. 186, 421 – 431.

Elisabetsky, E., Castilhos, Z.C., 1990. Plants used as analgesics by Amazonian caboclos as a basis for selecting plants foe investigation. International Journal of Crude Drug Research 28, 309-320.

Elizabeth K &Rao M N A, (1990). Oxygen radical scavenging activity of curcumin, Int J Pharm, 58 ,237.

Fabiyi, J.P. (1986) Production losses and control of helminths in ruminants of tropical regions. In: Parasitology – Quo radii? Proceedings of the Sixth International Congress of Parasitology, Australian Academy of Science, Canberra, 435-442.

Garcia, M.D., Fernandez,M.A., Saenz, M.T., Ahumada, M.C., 1995. Anti-inflammatory effects of different extracts and harpagoside isolated from Scrophularia Frutescens. II Farmaco 51, 443-446.

Garcia-Argaez, A.N., Ramirez, Apan T.O., Delgado, H.P., Velazquez, G., Martinez-Vazquez, M., 2000. Anti- inflammatory activity of coumrins from Decatropis bicolor on TPA ear mice model. Planta Medica 66, 279-281.

Garret D.C. 1964. The quantitative analysis of drugs. 3rd edition. Vol(3). Chapman and Hall, Japan 456 – 458.

155

Geerts, S., Dorny, P., 1995. Anthelmintic resistance in helminths of animals and man in the tropics. Bull. Seance. Acad. r. Sci.Outre-Mer Meded. Zitt. K. Acad. overzeese Wet. 41, 401– 424.

Gil M.I., Tomas – Barberan F.A., Hess – Pierce B., Holcroft D.M., and Kader A.A., (2000). Antioxidant activity of pomegranate juice and its relationship with Phenolic composition and processing. Journal of Agricultural and Food Chemistry. 49: 2480-2485.

Glyglewski R., Korbut R, Robak J & Swis J, 1987. On the mechanisms of antithrombotic action of flavanoids, Biochem Pharmacol, 36, 317.

Goncalves, J.L.S., Lopes, R.C., Oliveira, D.B., Costa, S.S., Miranda, M.M.F.S., Romanos, M.T.V., Santos, N.S.O., Wigg, M.D., 2005. In-vitro anti rotavirus activity of some medicinal plants used in Brazil against diarrhea. Journal of Ethnopharmacology. 99, 403-407.

Grisham MB, Heuil JD, Wink DA. 1999. Nitric oxide I. Physiological chemistry of nitric oxide and its metabolites: Implication in inflammation. American Journal of physiology; 276: 315-321.

Gutteridge J.M.C., 1985. Age pigments and free radicals: fluorescent lipid complexes formed by iron and copper containing proteins, Biochim Biophys Acta, 834, 144.

Gyuatt, H.L. and Evans, D. 1992. Economic considerations for helminth control. Parasitology Today 8, 397-402.

Hajhashemi, V., G hanadi, A., Pezeshkian, S.K., 2002. Antinoceptive and anti-inflammatoru effects Satreja hortensis L. extracts and essential oil. Journal of Ethanopharmacology 82, 83-87.

Halliwell B. and Gutteridge J M C.1990. Role of free radicals and catalytic metal ions in human diseases. Methods in Enzymology 186: 1-85.

Halliwell, B., Gutteridge J.M.C., Cross, C.E., 1992. free radicals, antioxidants and human disease: where are we now? Journal of Laboratory and Clinical Medicine 119, 598-619.

156

Hecker, E., 1968. Co-carcinogenic principles from oil of Croton tiglium and other Euphorbiaceae. Cancer Research 28. 2338-2349.

Heidari, M.R., Darban. M., 1999. Evaluation of the analgesic effect of Mellissa officinalis extracts by tail-flick test in mice. Physiology and Pharmacology 3, 81-87.

Hemmani T & Parihar M.S., 1998. Reactive oxygen species and oxidative DNA damage, Indian J Physiol Pharmacol, 42, 440.

Henry L E A, Halliwell B &Hall D O ,1976. The superoxide dismutase activity of various photosynthetic organisms measured by a new and a rapid assay technique, FEBS Lett, 66,303.

Hj Baie , Sheikh, K.A., 2000. The wound healing properties of Channa striatus – cetrimide cream – wound contraction and glycosaminoglycan measurement. Journal of Ethanopharmacology 73, 15 -30.

Ho, C. T.; Chen, Q.; Shi, H.; Zhang, K. Q.; Rosen, R. T. 1992.Antioxidative effect of polyphenol extract prepared from various Chinese teas. Prev. Med. 21:520–525.

Hu. C.Q., Chang, J.J., Lee, K.H., 1990. Antitumor agents, 115. Seselidiol. A new cytotoxic polyacetylene from seseli mairei. Journal of Natural Products 53, 932-935.

Ialenti A., Moncada S & Di Rosa M., 1993. Modulation of adjuvant arthritis by endogenous nitric oxide, Brit J Pharmacol, 110, 701.

Iqbal Z, Muhammad Lateef, Abdul Jabbar, Ghulam Muhammadb, Muhammad Nisar Khan. 2005, Anthelmintic activity of Calotropis procera (Ait.) Ait. F. flowers in sheep. Journal of Ethnopharmacology 102 ,256–261

Iqbal, Z., Lateef, M., Ashraf, M., Jabbar, A., 2004. Anthelmintic activity of Artemisia brevifolia in sheep. Journal of Ethnopharmacology 93, 265–268.

157

Jannsen PAJ. Niemegeers CJE, Dony JGH. 1963; The inhibitory effect of fentanyl and other morphine like analgesics on the warm water induced tail with drawl reflex in rats. Arzneimittel-Fersch .13:502-507.

Jovanovic SV, Steenken S, Tosic M, Majanovic A, Simic MG, 1994 Flavanoids as antioxidants. J.Am Chem Soc; 116: 4846 -4851.

Kamalakkannan N& Stanley Mainzen Prince P. 2003. Effect of Aegle marmelos fruit extract on tissue antioxidants in streptozotocin diabetic rats, Indian Journal of Exp Biol, 41 :1288.

Kaneko T, Tahara S& Matsu M, 1996. Retarding effect of dietary restriction on the accumulation of 8-hydroxy-2-deoxyguanosine in organs of Fischer 344 rats during aging, journal of Free Radical Biology and Medicine 23, 76.

Katzung, B.G., 2001. Basic and Clinical Pharmacology. 8th edition. Appleton and Lange, USA, pp. 523-525, 602-605.

Khan, L., P.A. Diaz-Hernandez 1999. Tannins with anthelmintic properties international workshop on Tannins in livestock and Human Nutrition. Waite campus, University of Adelaide.ACIAR proceeding, 92, pp. 130-139.

Knighton .D.R., Hunt T.K, Thakral, W.H., Goodson, 1982. Role of platelets and fibrin in the healing sequence: an in vitro studt of angiogenesis and collagen synthesis, Ann. Surg. 196 ,379 -388.

Kudi, A.C., Umoh, J.U., Eduvie, L.O., Gefu, J., 1999. Screening of some Nigerian medicinal plants for antibacterial activity. Journal of Ethnopharmacology 67, 225-228.

Kulkarni S.K., In; 1999, Handbook of experimental pharmacology, 3rd Edn., Vallabh Prakasham, Delhi, 179.

Lewis, W.H., Elvin Lewis, M.P.H., 1977. Medicinal Botany Plants Affecting Man’s Health. John Wiley & Sons, New York.

158

Li CQ, Wogan GN, 2005 Nitric oxide as a modulator of apoptosis. Cancer Letters 226:1-15.

Lin J.S., Smith V. and Olcott H.S. 1974. Loss of free radical signal during induction period of unsaturated lipids containing nitroxide antioxidants. Journal of Agricultural and Food Chemistry 22: 682-684.

Lowery, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193, 265 -275.

Luttingar D. 1985. Determination of antinociceptive activity of drugs in mice using different water temperatures in a tail-immersion test. Journal of pharmacological methods 13:351-357.

Manley, K. and Bellman, L. (Eds), 2000. Surgical Nursing : Advancing practice. Churchill living stone, London. 67.

Marcocci L, Packer L, Droy – Lefaiz M T, Sekaki A& Gardes – Albert M, 1994. Antioxidant action of Ginko biloba extract Egb 761, Methods of Enzymol, 234 ,462.

Marklund SL and Marklund G . 1974. Invervement of superoxide anion radical in the auto-oxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J. Biochem. 47: 469-474.

Martin A. 1996. The use of antioxidants in healing. Dermatol Surg; 22: 156 -160.

Martin, R. J. 1997: Mode of action of Anthelmintic drugs. Vet. J. 154, 11-34.

Marx J.L. Oxygen free radicals linked too many diseases. Science, 235, 529.

Melissa Calvin. 1998. cutaneous wound repair. In: Wounds. A compendium of clinical research and practice. 10, 12-32.

159

Meyre-Silva, C., Yunes, R., Santos, A.R.S., Magro, J.D., Monache, F.D., Cechinel-Filho, V., 1999. Isolation of a C-Glycoside Flavonoid with antinociceptive action from Aleurites moluccana Leaves. Planta Medica 65, 263-294.

Middleton E, 1984.The flavanoids, Trends Pharmacol Sci, 5, 335

Miller, N. J.; Rice Evans, C. A. 1997. Factors influencing the antioxidant activity determined by the ABTS•+ radical cation assay. FreeRadic. Res. 26:195–199.

Mills, S., Bone,K., 2000. Principles and Practice of Phytotherapy. Churchill Livingstone, Edinburgh, pp. 23-24. 31-34, 229-231.

Mitsuya k., Mizuta T., Kohno M., Hiramattsu M. and Nori A (1990). The application of ESR Spin-Trapping technique to the evaluation of SOD- like activity of biological substances. Bulletin of the Chemical Society of Japan 6: 187 – 191.

Mittal CK, Mittal R, Galiotos JK. 2003. Nitric oxide: Regulations and clinical Revalance. In : Trends in clinical Biochemistry and Laboratory medicine, 1st , 83-87.

Moncada S, Palmer RMJ, Higgs EA. 1991, Nitric oxide physiology, pathology and pharmacology. Pharmacological Review; 43: 109-142.

Moody.J.O., Robert.v.a., Connolly.J.D., Houghton.J.P., (2006) Anti-inflammatory activities of the methanolic extracts and an isolated furanoditerpene constituent of Sphenocentrum jollyanum Pierre (Menispermaceae). Journal of Ethanopharmacology 104, 87-91.

Morgen P.W, A.G.Binnington, C.W.Miller, D.A.Smith, A.Valliant, J.F.Prescott,1994. The effect of occlusive and semi occlusive dressings on the healing of acute full thickness skin wounds on the forelimbs of dogs, Vet. Surg. 23. 494 – 502.

160

Moseley R, Hilton JR, Waddington RJ, Harding KG, Stephens P, Thomas DW. 2004. Comparison of oxidative stress biomarker profiles between acute and chronic wound environments. Wound Repair Regen;12: 419–29.

Mursof, E.P. and He, S.1991. A potential role of papaya latex as an anthelmintic against patent Ascaridia galli infection in chicken. Hemera Zoa 74, 11-20.

Nadkarni, A.K., 1954. Indian Materia Medica. Popular Prakashan, Bombay, India.

Nargund L.V.G., 1999. Anthelmintic activity of 8-Fluoro-9-substituted (1, 3)–1,3,4-triazoles on pheretima pothuma , Indian Drugs, 36(2), 137-139.

Nguyen T, Brunson D, Crepsi CL, Penman BW, Wishnok JS, Tannenbaum SR. 1992. DNA damage and mutation in human cells exposed to nitric oxide in-vitro proceeding of national Academy of science USA. 89: 3030-3034.

Niezen, J. H., G. C. Waghorn, W. A. G. Charleston, 1998: Establishment and fecundity of Ostertagia circumcincta and Trichostrongylus colubriformis in lambs fed lotus (Lotus pedenculatus) or perennial rye grass (Lolium perenne). Vet. Parasitol. 78, 13-21.

Niezen, J. H., T. S. Waghorn, W. A. G. Charleston, G. C. Waghorn 1995: Growth and gastrointestinal nematode parasitism in lambs grazing either Lucerne (Medicago sativa) or sulla (Hedysarum coronarium), which contains condensed tannins. J. Agric. Sci. 125, 281-289.

Nishikimi M., Rao N.A and Yagi K. 1972. The occurrence of superoxide anions in the reaction of reduced phenazine methosulphate and molecular oxygen. Biochemical and Biophysical Researcation Communication 46: 894 – 854.

Ohkawa H, Ohishi N and Yagi K. 1979. Assay for lipid peroxides in animal tissue by thiobarbituric acid reaction. Anal Biochem; 95: 351-358.

161

Ohshima H, Tatemichi M, Sawa T. 2003.Chemical basis of inflammation induced caranogenesis. Archieves of Biochemistry and Biophysics. 417: 3-11.

Okawa M., Kinjo J., Nohar T., and Ono M., 2001. DPPH radical Scavenging activity flavanoids from some medicinal plants Biological and Pharmaceutical Bulletin 24: 1202-1205.

Omeye S.T, Turnball .J.D and Sanbrlich H.E., 1979, Selected methods for the determination of ascorbic acid in animal cells, tissues and fluids, Meth Enzymol. 62: 1-11.

Page A.P., Winter.A.D 2003. Enzymes involved in the biogenesis of the nematode cuticle. 53: 85-148.

Panichayupakaranant.P., Kaewsuwan .S., 2004. Bioassay – guided isolation of the antioxidant constituent from Cassia alata L.leaves. Songklanakarin J.Sci.Technol. 26, 103-107.

Parkinson JF, Mitrovic B, Merrill JE, 1997. The role of nitric oxide in multiple sclerosis. Journal of molecular medicine. 75: 174-186.

Patel RP. MC Andrew J, Sellak H, White CR, JOH, Freeman BA, 1999. Biological aspects of reactive nitrogen species. Biochemica et Biophysica Acta, 1411:385-400.

Pathak,D., Pathak, K., Sigla, A.K., 1991. Flavonoids as medicinal agents: recent advances. Fitoterapia 62, 371-388.

Pawlak W, Kedziora J, Zolynski K, Kedziora-Koranatowska K, Blazezyk J, Witkowski P. 1998. Free radicals generation by granulocytes from men during bed rest. J Gravit Physiol; 5 (1): 131-132.

Perrisoud D & Testa B, 1982. Hepatic Pharmacology: Mechanisms of action and classification of antinecrotic hepatoproductive agents, Trends Pharmacol Sci, 3, 365.

162

Pratt D.E, 1992. Natural antioxidants from plant material, in Phenolic compounds in foof and their effects on health II: Antioxidants and cancer prevention (Acs Symposium Series 507) edited by M.Hang, C.Ho& C.Lee (American chemical society; Washington DC) 54.

Rao YK, Fang SH, Tzeng YM. 2006. Anti-inflammatory activities of constituents isolated from phylanthus polyphyllus. Journal of Ehtanopharmacology. 103: 181-186.

Rashed, A.N., Afifi, A.M., 2003. Simple evaluation of healing activity of a crude extract of Portuloca oleracea linn. (growing in Jordan). In Mus musculus JVI-1. Journal of Ethanopharmacology 88, 131-136.

Ratty A.K., Sunamoto J & Das N.P., 1998. Interaction of flavanoids with 1,1,-diphenyl-2-picryl hydrazyl free radical, liposomal membranes and soybean lipoxygenase-1, Biochem Pharmacol, 37, 989.

Ray Gibanananda &Husain Syed Akhtar, 2002. Oxidants, antioxidants and carcinogenesis, Indian J Exp Biol, 40, 1214.

Re R, Pellegrini .N, Proteggente A, Pannala A, Yang M & Rice- Evens C, (1999) Antioxidant activity applying an improved ABTS radical cation assay, Free Rad Biol Med, 26 ,1231.

Rice –Evans C., Miller N.J., 1997. Factors influencing the antioxidant activity determined by the ABTS•+ radical cation assay. Free Radical Reasearch 26:195-199.

Rice-Evens, C.A., Miller, N.M., Paganda, G., 1996. Structure – Antioxidant activity relationships of flavanoids and phenolic acids. Free Radical Biology and Medicine 20, 933—956.

Robak J and Gryglewski R.J. (1988). Flavonoids are scavengers of superoxide anions. Biochemical Pharmacology 37: 837-841.

Rojkind M, Dominguez-Rosales JA, Nieto N, Greenwel P. 2002. Role of hydrogen peroxide and oxidative stress in healing responses.2, 86-88.

163

Ross R,1993.The pathogenesis of atherosclerosis: a perspective for the 1990’s, Nature 362, 801.

Rotruck JT, Pope AL and Ganther HE Swanson AB, Hafeman DG and Hoekstra WG.Selenium: 1973 .Biochemical role as a component of glutathione peroxidase. Science; 179: 588-590.

Rudolph. R , (1979). Location of the force of wound contraction, Surg. Gynecol. Obstet. 4, 547 – 551.

Saboc Thiernermannc. 1994. Invited orion: Role of nitric oxide in hemorrhagic, traumatic and anaphylactic shock and thermal injury shock.2: 145-155.

Said, M., 1969. Hamdard Pharmacopoeia of Eastern Medicine. Hamdard National Foundation, Karachi, Pakistan.

Sainani G S, Manika J S & Sainani R G.1997. Oxidative stress: a key factor in pathogenesis of chronic disease, Med Update, 1, 1.

Sanchez- Moreno C, 2002. Methods used to evaluate the free radical scavenging activity in foods and biological systems, Food Sci Tech Int, 8 ,121-137.

Sarkar D, Dutta A, Das M, Sarkar K, Mandlac chatterjee M. 2005. Effect of aloe vera on nitric oxide production by macrophages during inflammation Indian Journal of pharmacology . 37(6).371-375.

Satrija F, P. Nansen , S. Murtini , S. He. (1995) Anthelmintic activity of papaya latex against patent Heligmosomoides polygyrus infections in mice Journal of Ethanopharmacology 48 ,161-164.

Satyavati, G.V., Raina, M.K., Sharma, M., 1976. Medicinal Plants of India, vol. I. Indian Council of Medical Research, New Dehli, pp: 167–170, 201–206.

164

Selvemini D, Wang ZQ, Wyatt PS, Bourden DM, Morino MH, Manning PT, Currie MG, 1996. Nitric oxide: A key mediator in the early and late phase of carrageenan induced rat paw inflammation. British Journal of pharmacology; 118(4): 829-838.

Sezik, E.,Yesilada, E., Honda, G., Takaishi, Y., Takeda, Y., Tanaka, T., Traditional medicine in Turkey. X . Folk medicine central Anatolia. Journal of Ethanopharmacology 75, 95-115.

Shahidi, F., Janitha, P.K., Wanasundara, P.D., 1992. Phenolic antioxidant Critical Review. Food Sciences and nutrition 32, 67-103.

Shirwaikar Annie, K. Rajendran and Dinesh. 2004. Invitro antioxidant studies of Annona squamosa, Indian J. Exp. Biol. 142. 803

Sinha AK 1972. Colorimetric assay of catalase . Anal Biochem., 47; 389-394.

Skaper SD, Fabris M, Ferrari V, Carbonare MD, Leon A. 1997. Quercetin protects cutaneous tissue associated cell types including sensory neurons from oxidative stress induces by glutathione depletion: cooperative effects of ascorbic acid. Free Radic Biol Med; 22 (4): 669-678.

Sobota,R., Szwed, M., Kasza, A., Bugno, M., Kordula, T., 2000. Parthenolide inhibits activation of signal transducers and activators of transcription (STATs) induced by cytokines of the IL-6 family. Biochemical and Biophysical Research Communications 267, 329-333.

Sosa, S., Balick, M.J., Arvigo, R., Esposito, R.G.,Pizza, C., Altinier, G., Tubaro, A., 2002. Screening of the topical anti-inflammatory activity of some Central American plants. Journal of Ethanopharmacology 81, 211-215.

Sreejayan N and Rao MNA 1997. Nitric oxide scavenging by curcuminoids, J.Pharm. Pharmacol., 49: 105-107.

Stadelmann, W.K., Digenis, V.D., and Tobin, G.R. 1998. Impediments to wound healing . Am. J. Surg., 176, S 39-47.

165

Sunilkumar.S.P., Sivakumar . H.G. 1998. Evaluation of topical formulations of aqueous extract of Centella asiatica on open wounds in rats, Indian J. Exp. Biol. 36 ,569 -572.

Tapiero, H., Ba, G.N., Couvreur, P., Tew, K.D., 2002. Polyunsaturated fatty acids (PUFA) and eicosanoids in human health and pathologies. Biomedicine and Pharmacotherapy 56, 215-222.

Taun, T.L. and Nichter,L.S. 1998. The molecular basis of keloid and hypertropic scar formation. Mol. Med. Today., 4, 19.

Taylor, L.N.D., 2005. The healing power of Rainforest Herbs: A Guide to Understanding and Using Herbal Medicinals. Square One Publisher, Garden City Park. P.535.

Tchikaya, F.O., Datte,V.J., Bantsiele, G.B., Offoumou, A.M., 2003. Effects pharmacologiques de l’extrait aqueux de Anacardium occidentale (Anacardiaceae) sur la pression sanguine arterielle de Lapin et sur I’ artere aorte de cobaye. Revue de Medicine et Pharmacopea Afriaine 17, 41- 46.

Thiemermann C. 1997. Nitric oxide and septic shock, General pharmacology. 29: 159-166.

Thompson, D. P., T. G. Geary 1995: The structure and function of helminth surfaces. In: Biochemistry and Molecular Biology of Parasites (J. J. Marr, Ed.), 1st ed. Academic Press, New York, pp. 203-232.

Tran VH (1997) Antioxidant and free radical scavenging effects of some polyphenolic extracts from medicinal plants on wound healing. Abstract, PAN/Asian European Tissue Repair Society Post-satellite Meeting, November 2–3, Hanoi, Vietnam.

Tran VH, Hughes MA, Cherry GW .1996. The effects of polyphenolic extract from Cudrania cochinchinenesis on cell response to oxidative damage caused by H2O2 and xanthine oxidase. Abstract 27, First Joint Meeting of Chinese and Europian Tissue Repair Society, September 22–27, Xi’an, China.

166

Treget, E.E., Nedelec.B Scott.P.G. and Granary. A. 1997. Hypertrophic scars keloides and contractures. The cellular and molecular basis for therapy. Surg. Clin. North Am., 27, 701-730.

Tsao,R., Akhtar, M.H., 2005. Nutraceutical and functional foods. I.Curre trend in phytochemical antioxidant research. Journal of Food Agriculture and Environment 3, 10-17.

Tsuboi R, Rifkin.D.B, 1990. Recombinant basic fibroblast growth factor stimulates wound heaaling – impaired db/db mice, J. Exp. Med. 172 ,S37-S37.

Van den Worm, E., B eukelman, C.J., Van den Berg, A.J.J., Kores, B.H., Labadie R.P., Van Dijk.H., 2001. Effects of methoxylation of apocynin and analogs on the inhibition of reactive oxygen species production by stimulated human neutrophils. European Journal of Pharmacology 433, 225-230.

Vigar Z., 1984. Atlas of medical Parasitology, 2nd edn., Publishing house, Singapore, 216-218.

Viniger R, Schriber Wand Hugo R. 1969. Biphasic deneopment of carrageenan oedema in rats. Journal of pharmacology and Experimental Therapeutics; 166:96-103.

Virdi,J., Sivakami, S., Shahani,S., A.C., Banavalikar,M.M.,Biyani,M.K., 2003. Antihyperglycemic effects of three extracts from Momordica charantia. Journal of Ethanopharmacology 88, 107-111.

Waller, P. J., G. Bernes, S. M. Thomsborg, A. Sukumar, S. H. Richter, K. Ingebrigtsen, J. Hoglund 2001: Plants as de-worming agents of livestock in Nordic countries: historical perspective, popular beliefs and prospects for the future. Acta Vet. Scand. 42, 31-44.

Wang, H.; Cao, G.; Prior, R. L. 1996. Total antioxidant capacity of fruits. J. Agric. Food Chem. 44:701–705.

WHO. 1991. Progress report by the director general, document No A44/20, 22nd march, World Health Organization, Geneva.

167

Willet, W.C., 1994. Diet and health-what should we eat? Science 26, 532-538.

Winter CA, Risley EA and Nuss GW 1962. Carrageenan induced in hind paw of the rats as an assay for anti-inflammatory drug. Proc.Soc. Exp.Biol. Med., 11: 544-547.

Wisemann H, Halliwell B. 1996. Damage to DNA by reactive oxygen species and nitrogen species: role in inflammatory disease and progression to cancer. Biochem J; 313: 17-19.

Ya C, Gaffney SH, Lilly TH, Haslam E. 1988. Carbohydrate – polyphenol complexation. In: Hemingway RW, Karchesy JJ, eds. Chemistry and significance of condensed tannins. New York: Plenum Press;

Yeo J.H., Lee, K.W., Kim, H.C., Lyun, Y., Kim, S.Y., 2000. The effect of PVA /chitosan/fibran blended spongy sheets on wound healing in rats. Biology and Pharmaceutical Bulletin 23, 1220 -1223.

Yesilada, E., Sezik, E., Honda, G., Takaishi, Y., Takeda, Y., Tanaka, T., 1999. Traditional medicine in Turkey. IX . Folk medicine in north – west Anatolia. Journal of Ethnopharmacology 64, 195-210.

Yokozawa T., Chen C.P., Dong E., Tanaka T., Nonaka G.I., and Nishioka I. 1998. Study on the inhibitory effect of tannins and flavanoids against the 1,1-diphenyl -2-picrylhydrazyl radial. Biochemical Pharmacology. 56: 213-222.

Yoshida T., Mori K., Hatano.T., Okumura T., Uehara I., Komagoe K., Fujita Y. and Okuda.T., 1989. Studies on inhibition mechanism of autoxidation by tannins and flavanoids. V.Radical – Scavenging effects of tannins aand related polyphenols on 1,1-diphenyl -2-picrylhydrazyl radial. Chemical Pharmaceutical Bulletin 37: 1919-1921.

Youdim K.A & Joseph J A, 2003. A possible emerging role of phytochemicals in age – related neurological dysfunctions: a multiplicity of effects, Free Rad Biol Med, 30, 583.

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