2. Literature Review 2.1 Cassia absus - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/7205/5/05...f. Habit and general features Sida spinosa Linn. is a small erect, or suberect,

Chapter 2 Literature Review

Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, SVKM’s NMIMS,

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2. Literature Review

2.1 Cassia absus

2.1.1. Plant Profile 13, 14, 15:

Table 2.1: Botanical Classification of Cassia absus 1 Kingdom Plantae

2 Division Magnoliophyta

3 Class Magnoliopsida

4 Order Fabales

5 Subfamily Caesalpinioideae

6 Tribe Cassieae

7 Genus Cassia

8 Species Absus

Family

Botanical family: Fabaceae

Ayurvedic: Shimbi Kul

Other Names

Table 2.2: Synonyms of Cassia absus

Hindi Chaksu

Sanskrit Chakusya, Aranyakullithaka

Gujarati Chimed

Marathi Ivala, Rankulith, Ranhulge

Kannada Kann kutakin bij, Kadhulig

Bengali Bankulthi, Banku kirti kalay

Habitat

It is found all over the world in the tropical region. It is found everywhere in India.

From western Himalayas to Sri-Lanka it grows freely.

Parts used: Seeds and leaves

Geographical Sources

Africa, Australasia, Australia, Caribbean, Central America, Asia: Bangladesh,

Bhutan, East Timor, India, , Indonesia-ISO, Java, Myanmar, Nepal, Pakistan, Sri Lanka,

Sulawesi, Thailand, Vietnam.

India: Andhra Pradesh, Arunachal Pradesh, Assam, Bihar, Dadra-Nagar-Haveli,

Daman, Delhi, Diu, Goa, Gujarat, Haryana, Himachal Pradesh, Jammu-Kashmir,

Karnataka, Kerala, Madhaya Pradesh, Maharashtra, Manipur, Meghalaya, Nagaland,



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Orissa, Pondicherry, Punjab, Rajasthan, Sikkim, Tamil Nadu, Tripura, Uttar Pradesh,

West Bengal.

Morphology

Fig No. 2.1. Morphological features of Cassia absus.

An erect, sparingly branched annual 15-45 cm. high; stems and branches clothed with

spreading viscous glandular hairs. Leaves long- petioled; stipules 3mm. long, subacute.

Leaflets 2 pairs, very oblique, 1.6- 3.8 by 0.8-2.5 cm., elliptic- oblong or elliptic-

obovate, obtuse or subacute, minutely mucronate; glabrous or nearly so above, slightly

hairy but not glandular beneath; petioles’ 1.25mm long, densely hairy. Flowers in

terminal or leaf opposed erect narrow few flowered racemes; pedicle short, viscous

hairy; bracts beneath the pedicels ovate, acute; bracteole 1 about the middle of each

pedicel, small ovate. Calyx hairy 4mm long; segments oblong, obtuse, subequal. Petals

6mm long, obovate-cuneate, reddish yellow, tender, veined. Stamens 5 all perfect equal.

Ovary densely bristly with long hairs. Pods 2.5-4.5 cm. by 6-8mm. ligulate, nearly

straight, oblique, compressed, thin, and clothed with bristly hair. Seeds 4-6, trapezoid-

ovoid, 4.5 by 4mm. black, shining.



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2.1.2 Chemical Constituents 16

The seed pulp contains 1.5% Chaksine and Isochaksine. These are the major

constituents that are water soluble. Seeds also contain various oils. It has various

alkaloids and minerals like calcium, phosphorous, iron and zinc. Besides these it also

contains vitamins like thiamine and riboflavin.

Seeds reduced to fine powder loose 13.5 % at 100ºC; ash amounts to 3.7 % and contains

a trace of manganese. When extracted with water acidified with sulphuric acid indicated

the presence of an alkaloid principle along with a yellow resin insoluble in alkalies.

Petroleum ether extract contained a non- drying oil insoluble in alcohol and contained

trace of oily matter completely soluble in petroleum ether.

Structures of Chemical constituents:

Riboflavin

Chaksine

Quercetin



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Uses: 16

According to ayurveda it contains

• Gunna (properties) - ruksh (dry)

• Rasa (taste) - tickt (bitter), kashaya (astringent)

• Virya (potency) – sheet (cold)

• Prabhav (action) - chakshuy (eye tonic)

1. The seeds are bitter; astringent to the bowels, diuretic, attenuant, stimulant; cure

diseases of the eye; used in syphilitic ulcers, leucoderma.

2. Paste made of seeds- Its paste is used in local application for scrapping of dead

tissue on the skin. It also reduces inflammation on the wounds. It is very effective in

treating eye ailments like cataract, trachoma, ulcers, polyps etc. It also reduces

watering of eyes. It also works in eye infection. It is helpful in reducing the blood

letting though a wound. It also has a good role in skin aliments.

3. Powder- Powder made out of seeds is very helpful in relieving from diarrhea, and

accumulation of toxins in the body. It is also effective in renal stones, anurea and in

painful urination. It is also helpful in stopping of hemorrhages occurring in the

body.



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2.2 Pharmacognostical Review of plant (Sida spinosa Linn.) 17,18,19

a. Botanical Name Sida spinosa (L.), Malvaceae.

Fig 2.2.. Sida spinosa Linn. showing (a) whole plant and (b) flowers

b. Taxonomical Classification:

Kingdom: Plantae Plants

Subkingdom: Tracheobionta – Vascular plants

Division: Magnoliophyta – Flowering plants

Class: Magnoliopsida – Dicotyledons

Subclass: Dilleniidae

Order: Malvales

Family: Malvaceae

Genus: Sida L.

Species: Sida spinosa L.

c. Synonyms

i. Sida glandulosa Linn.

ii. Sida alba Linn.

iii. Sida angustifolia Linn

d. Vernaculars Names:

English: Prickly sida, spiny sida



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Sanskrit: Nagabala, kharyasta.

Malayalam: Kattu-venthiyam, Anakuruntotti

Tamil: Mayirmanikkam

Hindi: Bariyara, Janglimethi, Gulsakari.

Telgu: Chinamootam.

e. Geographical distribution

The plant is found throughout the hotter parts of India but is more common in the dry

districts. It has been reported occurring in Bengal, Bombay, Gujarat, Sind, the Circars,

Carnatic, and Madras. It is a weed of waste places roadsides and lands that have lately

been under cultivation, but is not very common in Kerala.

f. Habit and general features

Sida spinosa Linn. is a small erect, or suberect, grey pubescent suffruticose branched

herb or very rarely an under shrub with a slender erect stem winding from branch to

branch and many small branches, the young shoots covered with soft grey mealy stellate

down and bearing simple small ovate or rounded cordate, variable leaves usually with

two or occasionally three, small or minute, stiff some what spiny projections or

tubercles at the nodes adjacent to or just below the place of insertion of the leaves, and

small cream yellow solitary flowers on slender joined peduncles with the joint or

articulations near the flower. The plant is in flower usually during the rainy and cold

seasons mostly from Octobers to December, but may occasionally bear flowers all

through the year.

g. External Morphology

Habit: herb

Description: "Erect annual or perennial herbs 0.5-0.8 m tall, copiously but minutely

stellate puberulent, eventually glabrate. Leaf blades linear to narrowly oblong or ovate,

1-5 cm long, margins serrate to crenate, base obtuse to truncate, petioles usually " or

less the length of blades, stipules filiform, 2-5 mm long, each subtended by a short

tubercle. Flowers solitary or in clusters, often somewhat corymbose toward the ends of

the branches, pedicels slender, 0.2-2.5 cm long; calyx strongly 10-ribbed, 4-7 mm long;

petals pale yellow to yellowish orange, 5-6- (-7) mm long; staminal column included.



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Mericarps 5, 3-4 mm long, grading apically into 2 antrorsely pubescent awns 0.5-1.5

mm long, lower dorsal and lateral walls strongly reticulate, apical surfaces smooth,

puberulent. Seeds ca. 1.5 mm long, glabrous or with a few hairs around the hilum"

h. Cultivation: Typical growing conditions are full or partial sun and moist to mesic

soil that is loamy and fertile. Once the seeds germinate, this plant develops very

quickly.

i. Detection and Identification

Sida spinosa is 20-90 cm tall depending on growing conditions. The stem is tough,

upright, with many branches, covered with hairs. The main nodes carry 2-3 thorns.

Leaves are alternate, elongated, with toothed margins, 2-4 cm long, petiolate. Flowers

are axillary, situated at the end of short pedicels, sometimes in small clusters. The

corolla is composed of 5 light yellow petals. There is a single thin taproot. The fruit is a

ring of 5 one-seeded segments, each with 2 sharp spines at the tip. The ring breaks up

maturity, releasing the 5 seeds. The seeds are ovate, light brown to grey-brown, 2-3 mm

long.

Phytochemical Review of plant Sida spinosa Linn. 20,21

Chemical constituents reported from plant Sida spinosa and other sida species are as

follows:

1) A new Heptahydroxyergost-7-en-6-one from Sida spinosa plant

Synonyms: 2,3,14,20,22,25,28-Heptahydroxyergost-7-en-6-one

Biological Source: Constituent of Sida spinosa

1.1.1.1 Heptahydroxyergost-7-en-6-one

HO

HOH

OH

H

HOOH CH2OH

OH

O



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2) 2-(Methylamino)-1-phenyl-1-propanol

Synonym(s): -[1-(Methylamino) ethyl] benzenemethanol, 9Cl 2-(Methyl

amino)-1-phenyl-1-propanol

Biological Source: Aconitum napellus, Catha edulis, Taxus baccata, Sida

cordifolia, Roemeria refracta

OH

NHMe

(1R,2R) -form

2-(Methylamino)-1-phenyl-1-propanol

3) N-Methyltryptophan, 9Cl

Synonym(s): 3-(2-Indolyl)-2-methylaminopropanoic acid. Abrine

Biological Use/Importance: Antiinflammatory, antiophthalmic agent

Biological Source: Alkaloid from Aotus subglauca, Sida cordifolia and

Gastrolobium callistachys (Leguminosae, Malvaceae)

1.1.1.2 NMethyltryptophan,

COOH

MeNH C H

CH2

(S )-f ormNH



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4) Peganine

Synonym(s): 1,2,3,9-Tetrahydropyrrolo [2,1-b] quinazolin-3-ol, 9CI. 1,2,3,9-

Tetrahydro-3-hydroxypyrrolo [2,1-b] quinoline. Vasicine. Linarine

Biological Source: Alkaloid from the leaves of Adhatoda vasica, the roots of

Sida cordifolia, and from several other Sida spp. and Lunaria spp.

Biological Use/Importance: Bronchodilator and respiratory stimulant. Shows

antihypertensive props. Uterine stimulant and abortifacient. Expectorant

Peganine

5) Vasicinol

Synonym(s): 1,2,3,9-Tetrahydropyrrolo [2,1-b] quinazoline-3,7-diol, 9CI. 7-

Hydroxypeganine. 7-Hydroxyvasicine

Biological Source: Alkaloid from the roots, leaves and seeds of Adhatoda

vasica, the roots of Sida cordifolia and from other Sida spp.(Acanthaceae,

Malvaceae)

Biological Use/Importance: Transient hypotensive agent, cardiac depressant.

Histamine antagonist shows mild anticholinesterase activity

Vasicinol

6) Vasicinone

Synonym(s): 2,3-Dihydro-3-hydroxypyrrolo [2,1-b] quinazolin-9 (1H)-one, 9CI

N

N

OH

4 (R)-form

N

NHO

OH

(R)-form



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Biological Use/Importance: Shows bronchodilator and weak hypotensive

inotropic action

Biological Source: Alkaloid from Biebersteinia multifida, the seeds and above

ground parts of Peganum harmala, the foliage of Peganum nigellastrum, the

roots of Sida cordifolia

Vasicinone

Antimicrobial activity: 22

Antimicrobial activity of ethanolic extracts was carried out. Four bacterial and two

fungal were used in this study. Standard strain of S. aureus, B. subtilis, E.coli,

P.aeruginosa, C.albicans were used. All microbes were found to be sensitive to the

ethanolic extract of Sida spinosa and showed a potential activity agains growth of both

Gram positive and Gram negative bacteria and fungus. The activity was concentration

dependent.

Diuretic activity: 23

Aqueous and alcoholic extracts of Sida spinosa leaves were tested for diuretic activity

in rats. The parameters studied on individual rat were body weight before and after test

period, total urine volume, urine concentration of Na+, K- and Cl-. In the present study

alcoholic and aqueous extracts of Sida spinosa leaves (100mg/kg of body weight)

showed increase in urine volume, cation and anion excretion. Furosemide was used as

reference diuretic.

O

N

NOH



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2.3 Diabetes mellitus 24

Diabetes mellitus (DM) comprises a group of common metabolic disorders that share

the phenotype of hyperglycemia. Several distinct types of DM exist and are caused by a

complex interaction of genetics, environmental factors, and life-style choices.

Depending on the etiology of the DM, factors contributing to hyperglycemia may

include reduced insulin secretion, decreased glucose utilization, and increased glucose

production. The metabolic dysregulation associated with DM causes secondary

pathophysiologic changes in multiple organ systems that impose a tremendous burden

on the individual with diabetes.

2.3.1 Classification of diabetes mellitus 25,26

Types of Diabetes

On the basis of nature and causing factors, diabetes can be divided in to following

types:

Type-1 Diabetes

It usually occur in young generation, near about 5% of whole diabetic population have

type 1 diabetes. It is a slowly progressive autoimmune disease mediated through T-

cells.In this the immune system attacks the insulin producing beta-cells and destroys

them as a result hyperglycemia (high blood sugar level) occurs. The classical symptoms

of type 1diabetes appear when 70-80% of beta-cells have been destroyed. Therefore,

profound

insulin deficiency is requires insulin replacement therapy.

Type-2 Diabetes

This is most prevalent in aging and elderly population, representing more than 90% of

all

cases of the diabetes (Day, 1998). In type 2 diabetes body cells lose their ability to

properly respond to signals given by insulin. However,pancreatic beta-cells producing

the insulin 2-3 time the normal amount. Therefore, it is an ‘insulin resistance’ condition

and can usuallybe treated without insulin replacement therapy.



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Etiologic Classification of Diabetes Mellitus 27

I. Type 1 diabetes (β-cell destruction, usually leading to absolute Insulin deficiency)

A. Immune-mediated

B. Idiopathic

II. Type 2 diabetes (may range from predominantly insulin resistance with relative

insulin deficiency to a predominantly insulin secretory defect with insulin resistance)

III. Other specific types of diabetes

A. Genetic defects of β -cell function characterized by mutations in:

1. Hepatocyte nuclear transcription factor (HNF) 4 α (MODY 1)

2. Glucokinase (MODY 2)

3. HNF-1 α (MODY 3)

4. Insulin promoter factor (IPF) 1 (MODY 4)

5. HNF-1 β (MODY 5)

6. NeuroD1 (MODY 6)

7. Mitochondrial DNA

8. Proinsulin or insulin conversion

B. Genetic defects in insulin action

C. Diseases of the exocrine pancreas—pancreatitis, pancreatectomy, neoplasia,

cystic fibrosis, hemochromatosis, fibrocalculous pancreatopathy

D. Endocrinopathies—acromegaly, glucagonoma, pheochromocytoma,

hyperthyroidism, somatostatinoma, aldosteronoma

E. Drug- or chemical-induced—Vacor, pentamidine, nicotinic acid,

glucocorticoids, thyroid hormone, thiazides, phenytoin, protease inhibitors,

clozapine, beta blockers

F. Infections—congenital rubella, cytomegalovirus, coxsackie

G. Uncommon forms of immune-mediated diabetes—“stiff-man” syndrome,

anti-insulin receptor antibodies

H. Other genetic syndromes sometimes associated with diabetes—Down’s

syndrome, Klinefelter’s syndrome, Turner’s syndrome, Wolfram’s syndrome,

myotonic dystrophy, porphyria, Prader-Willi syndrome etc.



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IV. Gestational diabetes mellitus (GDM) MODY, maturity onset of diabetes of the

young.

Complication of Diabetes 25-28

Among acute complications hypoglycemia and ketoacidosis are the most important. The

patients using excessive insulin or oral drugs develop rapid and severe lowering of

blood sugar below certain critical limits (below 45-55mg/dl) resulting hypoglycemia

that may cause coma. When body can not use carbohydrate as fuel for energy, it utilizes

large amount of fats and proteins. This results in over production of metabolic product

ketones. The increase amount of ketones in blood stream cause ketoacidosis and

patients may enter into coma . Diabetic patients who have high blood sugar levels are at

increased risk of formation of blood clots. This is due to their stickier platelet cells

which cause several abnormalities . In patients with long standing diabetes impaired

vision, cataract,renal failure, sensory loss, gastrointestinal problems, foot ulcers,

hardening of blood vessels, stroke would be recognized as chroniccomplication.

2.3.2 Experimental models of diabetes mellitus 29,30

Animal models have been used extensively in diabetes research. Early studies used

pancreatectomised dogs to confirm the central role of the pancreas in glucose

homeostasis, culminating in the discovery and purification of insulin. Today, animal

experimentation is contentious and subject to legal and ethical restrictions that vary

throughout the world. Most experiments are carried out on rodents, although some

studies are still performed on larger animals. Several toxins, including streptozotocin

and alloxan, induce hyperglycaemia in rats and mice. Selective inbreeding has produced

several strains of animal that are considered reasonable models of type 1 diabetes, type

2 diabetes and related phenotypes such as obesity and insulin resistance. Apart from

their use in studying the pathogenesis of the disease and its complications, all new

treatments for diabetes, including islet cell transplantation and preventative strategies,

are initially investigated in animals. In recent years, molecular biological techniques

have produced a large number of new animal models for the study of diabetes, including

knock-in, generalized knock-out and tissue-specific knockout mice.



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2.3.2.1 Animal Models of Type 1 Diabetes Mellitus 30-32

Alloxan induced diabetes

Mechanism of Induction of Diabetes

The mechanism by which alloxan induces diabetes in susceptible species has not been

entirely clarified. It has been reported that alloxan has several effects on the β-cells of

the pancreas, and is likely that some combination of these effects results in destruction

of β -cells by alloxan. Reviews by Malaisse and Lenzen and Panten present two

different proposals to explain the mechanism. Alloxan is highly reactive molecule that

is readily reduced to dialuric acid, which is then auto-oxidized back to alloxan resulting

in the production of H2O2, O2¯ , and hydroxyl radicals. Alloxan has been shown to

reduce DNA strand breaks in isolated islets and in islets following in vivo

administration of alloxan. More recent works has shown that the DNA fragmentation is

mediated by H2O2. The induction of DNA strand breaks activates nuclear poly (ADP-

ribose) synthetase resulting in depletion of cellular NAD levels. Two factors appear to

make the islets especially sensitive to the effects of alloxan; the first factor is that

alloxan is rapidly taken up into islet cells, and the second factor is sensitivity of islets to

peroxides.

A second mechanism proposed for the diabetogenic effects of alloxan concerns its

ability to react with protein sulfydryl (SH) groups. The proposed mechanism involves

reaction of alloxan monohydrate with the SH groups on glucokinase, a signal

recognition enzyme in the pancreatic β -cells, which changes blood glucose

concentration with respect to the rate of insulin secretion. By this mechanism, inhibition

of glucokinase and other SH-containing membrane proteins on the β -cells would

eventually result in cell necrosis. One of the effects of alloxan on the β -cells is the

inhibition of glucose-stimulated insulin release, and this is likely related to the

inhibition of glucokinase. However, there is no convincing evidence that the reaction of

alloxan with protein SH groups would result in the cellular and nuclear necrosis that

occur within minutes when alloxan induces diabetes in rabbits and other animals. A

study of the effects of alloxan on glucose oxidation and viability of islets from humans,

rats, and mice showed that there were major species differences in response to alloxan.

The range of the diabetogenic dose of alloxan is quite narrow and even slight



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overdosing may generally be toxic and may cause loss of many animals. This loss is

likely to be due to kidney tubular cell necrotic toxicity, in particular when too higher

doses of alloxan are administered. The most frequently used intravenous dose of alloxan

in rats is 65 mg/kg, but when it is administered intraperitoneal (i.p.) or subcutaneously

(s.c), its effective dose must be higher. For instance, an intraperitoneal dose below 150

mg/kg may be insufficient for inducing diabetes in this animal species. In mice, doses

vary between 100 to 200 mg/kg by intravenous route .

Streptozotocin (STZ) induced diabetes 31

Streptozotocin (2-deoxy-2-(3-methyl-3-nitrosourea) 1-D-glucopyranose) is a broad

spectrum antibiotic which is produced from Streptomyces achromogenes. The

diabetogenic response to STZ was first detected by Upjohn Laboratories during testing

of potential antibiotics from this organism. However Rakieten et al 1963 were the first

to describe that β -cell necrosis and the ensuing diabetic state could be produced after a

single intravenous dose of STZ in rats and dogs.

Mechanism of the diabetogenic action of STZ 32

Chemical structure of STZ comprises a glucose molecule with a highly reactive

nitrosourea side chain that is thought to initiate its cytotoxic action. The glucose moiety

directs this agent to the pancreatic β -cell, where it binds to a membrane receptor to

generate structural damage. A decrease in diabetes induction efficacy after substitution

of glucose by other sugars supports the presence of stereospecific membrane receptor or

recognition site on the plasma membrane of the β -cell, identified as probably being the

glucose transporter GLUT2. However as no plasma membrane labeling was recorded.

Streptozotocin with radioactive 14C-STZ, another explanation for β -cell plasma

membrane damage is that it occurs secondary to other indirect actions of STZ. At the

intracellular level, three major phenomenons are currently held responsible for β -cell

death: [1] Process of methylation [2] free radical generation and [3] nitric oxide (NO)

production. Methylation: The deleterious effect of STZ results from the generation of

highly reactive carbonium ions (CH3 +), formed from decomposition of the nitroso

moiety. The CH3 + ions cause DNA breaks by alkylating DNA bases at various

positions, resulting in activation of the nuclear enzyme poly(ADP-ribose) synthetase as

part of the cell repair mechanism. As cellular pyridine nucleotide, particularly NAD+ is



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utilized as substrates for the nuclear enzyme, a profound decline in NAD+ occurs within

20 min. In effect, an abrupt and irreversible NAD+ exhaustion leads to cessation of

NAD+-dependant energy and protein metabolism, ultimately leading to cell death.

Inhibition of poly (ADPribose) synthetase by agents like 3-aminobenzamide and

nicotinamide are known to protect β –cells from NAD+ depletion and cell death after

STZ exposure .

2.4.Free radicals 33-36

Free radicals are highly reactive molecules or chemical species capable of independent

existence. Generation of highly reactive oxygen species (ROS) is an integral feature of

normal cellular function like mitochondrial respiratory chain, phagocytosis, arachidonic

acid metabolism, ovulation, and fertilization. Their production however, multiplies

several folds during pathological conditions. The release of oxygen free radicals has

also been reported during the recovery phases from many pathological noxious stimuli

to the cerebral tissues .

Oxygen, because of its bi-radical nature, readily accepts unpaired electrons to give rise

to a series of partially reduced species collectively known as (ROS) including,

superoxide (O2.-), hydrogen peroxide (H2O2), hydroxyl (HO), peroxyl (ROO), alkoxy

(RO), and nitric oxide (NO), until it is itself completely reduced to water. Most of the

superoxide radicals are formed in the mitochondrial and microsomal electron transport

chain. Except for cytochrome oxidase, which retains the partially reduced oxygen

intermediates bound to its active site, all other elements in the mitochondrial respiratory

chain, e.g., ubiquinone, etc.,transfer the electron directly to oxygen and do not retain the

partially reduced oxygen intermediates in their active sites . On the internal

mitochondrialmembrane, the superoxide anion may also be generated by auto-oxidation

of semiquinones. The majority of superoxide radicals generated by mitochondrial

electron transport chain are enzymatically dismutated to H2O2. The hydroxyl and

alkoxy free radicals are very reactive species and rapidly attack the macromolecules in

cells .

Damage due to free radicals caused by ROS leads to several damaging effects as they

can attack lipids, proteins/enzymes, carbohydrates, and DNA in cells and tissues. They

induce undesirable oxidation, causing membrane damage, protein modification, DNA



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damage, and cell death induced by DNA fragmentation and lipid peroxidation. This

oxidative damage/stress, associated with ROS is believed to be involved not only in the

toxicity of xenobiotics but also in the pathophysiological role in aging of skin and

several diseases like heart disease (atherosclerosis), cataract, cognitive dysfunction,

cancer (neoplasticdiseases), diabetic retinopathy, critical illness such as sepsis and

adult/acute respiratory distress syndrome, shock, chronic inflammatory diseases of the

gastrointestinal tract, organ dysfunction, disseminated intravascular coagulation,deep

injuries, respiratory burst inactivation of the phagocytic cells of immune system,

production of nitric oxide by the vascular endotheliums, vascular damage caused by

ischaemia reperfusion known as ischaemia/reperfusion injury and,release of iron and

copper ions from metalloprotein. Iron changes have been detected in multiple sclerosis,

spastic paraplegia, and amyotrophic lateral sclerosis, which reinforces the belief that

iron accumulation is a secondarychange associated with neuro degeneration in these

diseases, although it could also be related to gliosis (glia might produce free radicals) in

the diseased area,or the changes in the integrity of the blood brain barrier caused by

alteredvascularisation of tissue or by inflammatory events.

2.5. Overview of free radicals and diabetic complications 37,38

Superoxide anion radicals can also react with nitric oxide to form reactive peroxynitrite

radicals.39, 40 . Hyperglycemia is also found to promote lipid peroxidation of low density

lipoprotein (LDL) by a superoxide-dependent pathway resulting in thegeneration of free

radicals.40, 41. Another important source of free radicals in diabetes is the interaction of

glucose with proteins leading to the formation of an Amadori product and then

advanced glycation endproducts (AGEs). 42

These AGEs, via their receptors (RAGEs), inactivate enzymes and alter theirstructures

and functions, promote free radical formation and quench and block antiproliferative

effects of nitric oxide. By increasing intracellular oxidative stress, AGEs activate the

transcription factor NFβ, thus promoting up-regulation of various NF-_B controlled

target genes. NFβ enhances production of nitric oxide, which is believed to be a

mediator of islet beta cell damage. 43, 44, 45

Considerable evidence also implicates activation of the sorbitol pathway by glucose as a

component in the pathogenesis of diabetic complications,example, in lens cataract



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formation or peripheral neuropathy. Efforts to understand cataract formation have

provoked various hypotheses. In the aldose reductase osmotic hypothesis, accumulation

of polyols initiates lenticular osmotic changes.

In addition, oxidative stress is linked to decreased glutathione levels and depletion of

NADPH levels. Alternatively, increased sorbitol dehydrogenase activity is associated

with altered NAD+ levels, which results in protein modification by nonenzymatic

glycosylation of lens proteins.

Mechanisms linking the changes in diabetic neuropathy and induced sorbitol pathway

are not welldelineated. One possible mechanism, metabolic imbalances in the neural

tissues, has been implicated in impaired neurotrophism, neurotransmissionc

changes,Schwann cell injury and axonopathy. 46-54

2.5.1. Sources of oxidative stress in diabetes 55- 58

Direct evidence of oxidative stress in diabetes is based on studies that focused on the

measurement of oxidative stress markers such as plasma and urinary F2-isoprostane as

well as plasma and tissue levels of nitrotyrosine and •O2- . There are multiple sources of

oxidative stress in diabetes including nonenzymatic, enzymatic and mitochondrial

pathways.

Nonenzymatic sources of oxidative stress originate from the oxidative biochemistry of

glucose. Hyperglycemia can directly cause increased ROS generation. Glucose can

undergo autoxidation and generate •OH radicals. In addition, glucose reacts with

proteins in a nonenzymatic manner leading to the development of Amadori products

followed by formation of AGEs. ROS is generated at multiple steps during this process.

In hyperglycemia, there is enhanced metabolism of glucose through the polyol (sorbitol)

pathway, which also results in enhanced production of •O2-.

Enzymatic sources of augmented generation of reactive species in diabetes include

NOS, NAD(P)H oxidase and xanthine oxidase. All isoforms of NOS require five

cofactors/prosthetic groups such as flavin adenine dinucleotide (FAD), flavin

mononucleotide (FMN), heme, BH4 and Ca2+-calmodulin. If NOS lacks its substrate L-

arginine or one of its cofactors, NOS may produce •O2- instead of •NO and this is

referred to as the uncoupled state of NOS . NAD(P)H oxidase is a membrane associated

enzyme that consists of five subunits and is a major source of •O2- production



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The mitochondrial respiratory chain is another source of nonenzymatic generation of

reactive species. During the oxidative phosphorylation process, electrons are transferred

from electron carriers NADH and FADH2, through four complexes in the inner

mitochondrial membrane, to oxygen, generating ATP in the process . Under normal

conditions, •O2- is immediately eliminated by natural defense mechanisms. A recent

study demonstrated that hyperglycemia-induced generation of •O2- at the mitochondrial

level is the initial trigger of vicious cycle of oxidative stress in diabetes . When

endothelial cells are exposed to hyperglycemia at the levels relevant to clinical diabetes,

there is increased generation of ROS and especially •O2-, which precedes the activation

of four major pathways involved in the development of diabetic complications.

2.5.2. Natural defense against oxidative stress and antioxidants 59, 60

Reactive species can be eliminated by a number of enzymatic and nonenzymatic

antioxidant mechanisms. Another enzyme that is important is glutathione reductase,

which regenerates glutathione that is used as a hydrogen donor by glutathione

peroxidase during the elimination of H2O2. Maritim and colleagues recently (2003)

reviewed in detail that diabetes has multiple effects on the protein levels and activity of

these enzymes, which further augment oxidative stress by causing a suppressed defense

response. For example, in the heart, which is an important target in diabetes and prone

to diabetic cardiomyopathy leading to chronic heart failure, SOD and glutathione

peroxidase expression as well as activity are decreased whereas catalase is increased in

experimental models of diabetes.

Nonenzymatic antioxidants include vitamins A, C and E; glutathione; α-lipoic acid;

carotenoids; trace elements like copper, zinc and selenium; coenzyme Q10 (CoQ10); and

cofactors like folic acid, uric acid, albumin, and vitamins B1, B2, B6 and B12. Alterations

in the antioxidant defense system in diabetes have recently been reviewed . Glutathione

(GSH) acts as a direct scavenger as well as a co-substrate for GSH peroxidase. It is a

major intracellular redox tampon system. Vitamin E is a fat-soluble vitamin that

prevents lipid peroxidation. It exists in 8 different forms, of which α-tocopherol is the

most active form in humans. Hydroxyl radical reacts with tocopherol forming a

stabilized phenolic radical which is reduced back to the phenol by ascorbate and

NAD(P)H dependent reductase enzymes.



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2.6. Importance of Herbals 61

:

Over three-quarters of the world population relies mainly on plants and plant extracts

for health care. More than 30% of the entire plant species, at one time or other, were

used for medicinal purposes. It is estimated that world market for plant derived drugs

may account for about Rs.2,00,000 crores. Presently, Indian contribution is less than

Rs.2000 crores. Indian export of raw drugs has steadily grown at 26% to Rs.165 crores

in 1994-’95 from Rs.130crores in 1991-’92. The annual production of medicinal and

aromatic plant’s raw material isworth about Rs.200 crores. This is likely to touch US

$1150 by the year 2000 and US $5trillion by 2050. It has been estimated that in

developed countries such as United States, plant drugs constitute as much as 25% of the

total drugs, while in fast developing countries such as China and India, the contribution

is as much as 80%. Thus, the economic importance of medicinal plants is much more to

countries such as India than to rest of the world. These countries provide two third of

the plants used in modern system of medicine and the health care system of rural

population depend on indigenous systems of medicine.

Of the 2,50,000 higher plant species on earth, more than 80,000 are medicinal. India is

one of the world’s 12 biodiversity centres with the presence of over 45000 different

plant species. India’s diversity is unmatched due to the presence of 16 different agro-

climatic zones, 10 vegetation zones, 25 biotic provinces and 426 biomes (habitats of

specific species).Of these, about 15000-20000 plants have good medicinal value.

However, only 7000-7500 species are used for their medicinal values by traditional

communities. In India, drugs of herbal origin have been used in traditional systems of

medicines such as Unani and Ayurveda since ancient times. The Ayurveda system of

medicine uses about 700 species, Unani 700, Siddha 600, Amchi 600 and modern

medicine around 30 species. The drugs are derived either from the whole plant or from

different organs, like leaves, stem, bark, root, flower,seed, etc. Some drugs are prepared

from excretory plant product such as gum, resins and latex. Even the Allopathic system

of medicine has adopted a number of plant-derived drugs



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2.6.1.Indian Medicinal plants with hypoglycemic activity:62,63

The NAPRALERT database lists over 1200 species of plants representing 725

genera in 183 families extending from the marine algae and fungi with antidiabetic

activity. Over half of these have been used ethnopharmacologically in traditional

medicine as antidiabetics and 50% of these traditional remedies have been studied

experimentally. Antidiabetic plants have often used by practitioners in treating the

individuals with type 2 diabetes.

Some time immemorial various plants and plant derived compounds have been

used in the treatment of diabetes to control the blood sugar of the patients. The use of

herbs in the management of diabetes mellitus has been prevalent in Indian society from

a long time. Several medicinal plants have reported to possess potential hypoglycaemic

activity in Indian system of medicines. There have been several reviews on the

hypoglycaemic medicinal plants, more particularly use of Indian botanicals for

hypoglycaemic activity

2.6.2. Traditional plants vs other plants 63,64

The plants have been screened for the activity on the basis of

ethanopharmacology or on random basis. The Indian subcontinent has an extensive

indigenous collection of natural remedies such as Ayurveda, unani, Siddha and so on.

Based on such systems, we can find not only new remedies but also new lead molecules

may be obtained.

Plants Traditional Others

Total numbers tested 296 541

Total active 286(81%) 254(47%)

Most of the drugs from plant sources are secondary metabolites, which have no

role in plant metabolism, but are postulated to play a significant role in plant defense

mechanism.

There are about 200 pure compounds from plant sources reported to show blood

glucose lowering effect. The following table gives the summary of the chemical classes

of these compounds.



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2.6.3 Phytochemical principle in antidiabetics 63

Till now so many numbers of compounds, which are responsible for the activity,

have been isolated from the plants. Some of the antihyperglycaemic principle tested

clinically and it is also found to be more effective.

1. Charantin and P-Insulin

These active principles are isolated from the seeds of Momordica charantia

(Cucurbitaceae). Charantin is a mixture of steroidal glycosides, β - Sitosterol - D

Glucoside and 5,25 - Stigamasten - 3β-ol -D Glucoside.

A polypeptide compound, Polypeptide - P or P-Insulin isolated from the fruits,

seeds and tissue culture seedlings is composed of 17 aminoacids. On administration it

was shown to be isulininomimetic in rodents and primates.

2. Peptidoglycan

This active principle peptidoglycan composed of 7 aminoacids having total

molecular weight of 6K Da and an oligosaccharide of molecular weight 1.2 k Da is

isolated from seeds and the pulp of Eugenia jambolana (Myrtaceae). However the exact

structures of the two compounds still remains to be elucidated.

3. Gymnemic acid

Gymnemic acid is the active principle isolated from the leaves of Gymnema

sylvestre (Ascelpiadceae). It is reported to stimulate manufacture or rejuvenation of β

cells of islets in pancreas.

4.Trigonelline and Scopoletin

The constituent of Trigonella foenum graecum (Leguminosae), Trigonelline, an

alkaloid which is a N - methyl derivative of nicotinic acid is found to be more active.

Another reported antihyperglycaemic principle is Scopoletin.

5. Marsupsin and Epicatechin

The reported constituent from Pterocarpus marsupium (Leguminoosae)

responsible for hypoglycaemic activity is (-) epicatechin, which at the dose of 30mg/Kg

causes an ATP dependent enhancement of glucose stimulated insulin secretion from

islets. From the heartwood of this plant, three phenolics were isolated. These are



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marsupsin, Pterosupsin and pterstilbene. Of these, marsupsin and pteorstilbene were

found to be effective in STZ diabetic rats.

6. Saudin

Cluytia richardiana (Euphorbiaceae) contains a diterpenoid saudin, which

possess marked hypoglycemic effect. The use of whole plant for the treatment of

diabetes mellitus has been patented.

7. Stevioside

Stevia rebaudiana (Compositae) contains an active chemical constituent known

as stevioside, which exerts antihyperglycaemic and insulinotropic, effects in diabetic

rats. Steviosides significantly suppress the glucose response to glucose tolerance test.

8.Hypoglycin

The potent hypoglycaemic agent from Blighia sapida (Sapindaceae) is the cyclo

propanoid aminoacid known as hypoglycin A and hypoglycin B.

9. Allicin and Allin

Allicin and Allin are the two constituents of Allium cepa and Allium sativum.

The hypoglycemic action is due to allicin, a diallyl disulphide oxide and allin by virtue

of their thiol groups. These disulphides act as sparring agent for insulin by competing

with it for inactivating compounds.

2.6.4 Poly herbal formulation used in diabetes 65

The following are the few examples of poly herbal formulations used in the

treatment of diabetes mellitus.

1. Amrycard (Aimil pharmaceuticals)

2. Nimbola (Kee pharma)

3. Syndrex (Plethico labs)

4. Mersina (J & J De Chane)

5. Amree (Aimil Pharmaceuticals)

6. Amree plus Gran (Aimil pharmaceuticals)

7. Diabecon (Himalaya drug company)

8. Amrycard (Aimil pharmaceuticals)



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2.7. Antioxidant status in diabetes 66

2.7.1 Oxygen Toxicity

Oxygen is required for all living for their survival. But at the same time, Oxygen

is potentially toxic. Salvemini has described oxygen as a double-edged sword. It is vital

to life but leads to formation of by- products that are toxic such as formation of super

oxide anions.

Dissolved oxygen at high concentration is toxic to animals. Rats when subjected

to breathe pure oxygen at 2 atmospheric pressure undergo convulsions in 5 to 6 hours

and may die.

2.7.2. Molecular Oxygen

Molecular oxygen is an essential component for all living organisms, but the

formation of various reactive intermediates of molecular oxygen metabolizes the cell

aerobically, thus eventually leading to a process termed as "oxidation". Oxidation is one

of the destructive processes, wherein it breaks down and damages various molecules.53

On the one hand while oxidation In vitro involves the participation of oxygen, on the

other hand, most of the biological oxidation in vivo occurs in the absence of oxygen

resulting in biological metabolism.

2.7.3 Free Radicals

Free radicals are chemical species of atoms or molecules that possess an un-

paired electron on their outermost orbital. These free radicals are highly unstable and

can therefore react with other molecules by giving out or accepting a single electron.

2.7.4 Defense Systems 67

Free radicals and other oxygen-derived species are constantly generated in vivo,

both by "accidents of chemistry" and for specific metabolic purposes. The reactivity of

different free radicals varies, but some can cause severe damage to biological

molecules, especially to DNA, lipids and proteins. Antioxidant defense systems

scavenge and minimize the formation of oxygen derived species, but they are 100 %



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effective. Hence diet derived antioxidants may be particularly important in diminishing

cumulative oxidative damage and in helping us stay healthier.

Although the human body continuously produces free radicals, it possesses

several defense systems, which are constituted of enzymes and radical scavengers.

These are called "First line antioxidant defense systems", but are not completely

efficient because almost all components of living bodies, tissues, cells and genes

undergo free radical destruction's.

"The second line defense systems" are constituted of repair systems for

biomolecules, which are damaged by the attack of free radicals. Specific enzymes are

known to have been involved in this context and several of them have been identified in

prokaryotes and in eukaryotes. The function of these enzymes involved in repairing

directly damaged biomolecules such as lipids, polysaccharides, proteins, nucleic acids

etc, or in eliminating oxidized compounds.

2.7.5.Types of Free Radicals

� Super oxide anion

� Singlet oxygen

� Nitric oxide radical

� Hydrogen peroxide

� Hydroxyl radical

� Peroxy radical

� Hydroperoxides

� Transition metals.



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2.8.Antioxidants 67

Antioxidants are compounds which act as inhibitors of the oxidative process.

They are quite large in number and diverse in nature, and they oppose the process of

oxidation largely by neutralizing free radicals. Antioxidants at relatively small

concentrations have the potential to inhibit the oxidant chain reactions.

Classification of Antioxidants

Antioxidants are classified into two types.

(i) Enzymatic antioxidants

Super oxide dismutase

Catalase

Peroxidase

Glutathione peroxidase

Glutathione reductase

(ii) Non-enzymatic antioxidants

Vitamin C

Vitamin E

ß-carotene

Uric acid

Ubiquinone

Ceruloplasmin

2.8.1 Antioxidant Status in Diabetes Mellitus 68

Free radicals may play an important role in causation and complications of

diabetes. The increased oxidative stress and accompanying decrease in antioxidants may

be related to causation of diabetes mellitus. Diabetes complications have also been

suggested to involve free radical related processes such as disturbed antioxidant system

and oxidative damage of membrane. In diabetes, alterations in the endogenous free

radical scavenging defense mechanisms may lead to ineffective scavenging of reactive

oxygen species resulting in oxidative damage. The altered antioxidant status, during

diabetes causes increased production of free radicals leading to oxidative damage and



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tissue injury. During diabetes, liver shows a relatively more severe impairment in

antioxidant capacity than kidney.

Activities of antioxidant enzymes, glutathione reductase, glutathione peroxidase,

catalase and super oxide dismutase are decreased in liver of diabetic rats. The level of

natural antioxidants vitamin C and Vitamin E are lowered in plasma of diabetic rats.

Transition metals also play a role in glucose acceleration. Indeed, many of the

glucose induced oxidative modifications are likely to be mediated by Fenton reactions,

which are catalyzed by transition metals. It is now uncertain that whether free or

complex metals or both forms are involved. Under certain experimental conditions,

ceruloplasmin the major copper containing protein of plasma can induce oxidative

modification of lipo proteins, it is considered a model of oxidative damage by copper

bound to protein. Glucose has been reported to accelerate oxidative modification of

LDL induced by copper and iron, by a mechanism, which likely involves the Cu2+

reducing properties of this sugar.

STZ has been proposed to be acting as diabetogenic due to its ability to destruct

pancreatic ß-islet cells possibly by free radical mechanism. Significant increase in the

levels of lipid peroxides suggest that increased oxidative stress could be attributed to

number of factors,

� Generation of free radicals by hyperglycemia related to glucose auto oxidation.

� Glucose auto oxidation has been linked to non-enzymatic glycosylation and

glycosylated proteins have been shown a source of free radicals.

� The reduced regeneration of natural protective antioxidants such as Vit. E, Vit.

C, glutathione and others may be the reason for increased oxidative stress.

� Regeneration of antioxidants requires reduced form of glutathione as reduced

glutathione has thermodynamically and kinetically favorable reduction potential,

which reduces other low potential reducing substances such as Vit. E and Vit. C

� Reduction of oxidized form of glutathione requires NADPH, as cofactor and

enzyme glutathione reductase. The reduction and availability of NADPH which

could be either due to reduced synthesis or increased metabolisation of NADPH

through some other pathway could be responsible for low levels of reduced

glutathione in STZ treated rats.



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2.8.2.Glutathione 69

Glutathione is a tripeptide derived from Glycine, Glutamate and Cysteine. The

first step in its synthesis is a condensation of the γ - carboxyl group of glutamate with

the α - Amino group of cysteine. ATP to form an acyl phosphate intermediate, which is

then attacked by the cysteine amino group, first activates the carboxyl group. The

second step is similar, with the α - carboxyl group of cysteine activated to an acyl

phosphate to permit condensation with Glycine.

GSH is present in virtually all cells, often at high levels, and can be thought of

as a kind of redox buffer. It probably helps to maintain the sulfhydryl groups of

proteins in the reduced state and the Iron of heme in the ferrous (Fe2+) state and serves

as a reducing agent for glutaredoxin. Its redox function can also be used in removing

toxic peroxides that form in the course of growth and metabolism under aerobic

conditions.

Glutathione, Peroxidase, a Selenium (Se) – containing enzyme (MW– 85,000

Da) present in significant concentrations in the cytoplasm of cells, detoxifies H2O2 to

H20 through the oxidation of reduced glutathione (GSH). A seleno cysteine amino acid

has been identified as the active site of the enzyme. A second enzyme, glutathione

reductase may then reduce the oxidized form of glutathione, with NADPH as the

reducing agent.

In addition, glutathione peroxidase can metabolise lipid hydroperoxides to less

reactive hydroxy fatty acids. Therefore, the ability of glutathione peroxidase to reduce

H2O2 or other hydroperoxides is dependent on the activity of glutathione reductase as

well as the availability of NADPH.



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H 2O2 2 GSH (reduced) 2 NADP+

GSH Peroxidase GSH reductase HMP shunt

Path way

2H2O GSSG (oxidized) 2NADPH+H+

Figure 2.3 HMP shunt pathway

Mechanism of free radical scavenging action of cellular low molecular weight

antioxidants – α-tocopherol, ascorbate, and reduced GSH, through NADPH –

glutathione reductase (GR) system.

ROO. Tocopherol Ascorbate 2GSH NADP+

RO. radical

ROOH Tocopherol

ROH radical Ascorbate GSSG NADPH

+ H+

Figure 2.4: Free radical scavenging action of antioxidant

2.8.3. Lipid Peroxidation 70

In 1940's E.H.Farmer and his team at British Rubber Producers Association

Laboratories, USA established the mechanism by which unsaturated lipids undergo

"auto oxidation" or "peroxidation". This discovery put the free radicals on the map.

Since then the relevance of lipid peroxidation to biology and medicine has been

explored.



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Definition

Lipid peroxidation is a complex process known to occur in both plants and

animals which involves the formation and propagation of lipid radicals, the uptake of

oxygen, a rearrangement of the double bonds in unsaturated lipids and the eventual

destruction of membrane lipids producing a variety of break down products including

alcohols, ketones, aldehydes, and ethers. In simple terms, it can be defines as the

oxidative deterioration of lipids containing a number of carbon - carbon double bonds.

Mechanism of Lipid peroxidation

Lipid peroxidation usually begins with the,

� Abstraction of a hydrogen atom from an unsaturated fatty acid resulting in the

formation of a lipid radical.

� The rearrangement of the double bonds results in the formation of conjugated

dienes.

� Attack by molecular oxygen produces a lipid peroxy radical.

� Lipid peroxy radical can either abstruct a hydrogen atom from an adjacent lipid

to form a lipid hydroperoxide or form a lipid endoperoxide.

� The formation of lipid endoperoxides in unsaturated fatty acids containing

atleast three methylene interrupted double bonds can lead to teh formation of

malondialdehyde as a breakdown product.

Types of Lipid peroxidation

(a) Spontaneouis Lipid peroxidation.

(b) Stimulated Lipid peroxidation.

Reactive species which initiate Lipid peroxidation

Involvement of Reactive oxygen species

Although extensively studied, the reactive species which initiate Lipid

peroxidation has not been identified. Autooxidation of lipids in living organisms is a

slow circumscribed process because molecular oxygen is a weak oxidant. It is generally

assumed the superoxide radical plays a major role in their process.

O2 + e - ─────> O2- ·



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Hydroxyl radical

Since its discovery, the importance of Haber - Weiss reaction for the initiation

Lipid peroxidation has been emphasized.

O2- · +H2O2 + H+ —————> O2 + HO· +H2O

The hydroxyl radical (HO·) formed is highly reactive and has been shown to peroxidise

lipids in chemical systems.

L ― H + HO· ―――> L· H2O

However, chemically defined reaction was not observed.

Metal ions

Most biological studies of lipid peroxidation involve transition metal ions. When

Fe2+ ions, Cu+ ions or simple chelates of these ions are added to isolated biological

membrane peroxidation occurs. The oxidised form of these transition metal ions (Fe3+,

Cu2+ ) can also accelerate peroxidation if a reducing agent (e.g. ascorbate) is added.

Sometimes the membrane itself can provide the reducing power.

The iron ions are themselves free radicals and Fe2+ can take part in electron

transport reaction with molecular oxygen.

Fe2+ +O2 [Fe2+ ―O2 Fe3+ ― O2٠-] Fe3+ + O2٠

-

Perferryl ion

Inhibitory reactions

Primary defense systems prevailing lipid peroxidation interacts with the

initiation. Enzymes such as superoxide dismutase, catalase, glutathione peroxidase

dismutate the oxygen radicals formed and there by prevent it from participating in lipid

peroxidation. The primary defense systems are not specific for lipid peroxidation. They

defend against numerous biochemical lesions such as damage to proteins, nucleic acids,

sugars, metabolic intermediates etc.

In contrast, the secondary defense systems are highly specific for lipid

peroxidation reactions.



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2.8.4.Super Oxide Dismutase 71, 72

The discovery of the enzyme superoxide dismutase ushered a new era in

understanding of oxidative processes in biological systems. SOD was the first enzyme

found to use an oxygen free radical substrate. The removal of superoxide by SOD is the

vital link in the system of proteins, enzymes, water and lipid soluble substance involved

in antioxidant defense. A network of enzymatic and non-enzymatic reactions maintains

the delicate balance between oxidants and free radicals against "oxidative stress" and

susceptibility to oxidative damage.

The superoxide radical anion or its protonated form causes oxidative molecular

damage to lipids, proteins and other molecules. Within cells, organelles, and in extra

cellular fluids different forms of SOD help to maintain a lower steady state of

superooxide. The SOD action forms hydrogen peroxide, which is a strong oxidant,

scavenged by catalase and peroxidases, lowering its steady state levels. Superoxide

radicals are involved in diverse physiological and pathophysiological processes. Many

enzymes producing superoxide have been characterized.

The superoxide radical anion, which mediates oxidative damage in various

biologicals systems, is formed during normal metabolism as well as patho physiological

processes through the action of various drugs. Superoxide dismutases (SODs), which

are present in all aerobic organisms, provide a defense that is essential for their survival.

Such a defense is not complete and O2-/HO2· plays a role in oxidative stress. Therefore

the elevation of the level of SOD may have the therapeutic application. The

detoxification of O2- can be achieved catalytically or by reagents that stoichiometrically

remove it, and a distinction is generally sought between scavengers of O2- and catalysts.

Scavengers act in a stoichiometric fashion and, therefore, a high flux of O2- , or even a

low flux for an extended time, can deplete their level.



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2.8.5. Catalase 73, 74

Catalase is widely distributed in nature. It is found in all aerobic

microorganisms, in plant and animal cells. The catalase activity in mammalian tissues

varies greatly, it is highest in liver and kidney and low in connective tissues. In these

cells it is mainly particle-bound whereas in erythrocytes it exists in a soluble state.

Human erythrocytes are normally rich in catalase. Catalase exerts various physiological

functions.

On one hand the enzyme located in organelles acts as a regulator of H2O2 levels

and as a specific peroxidase, e.g. in the peroxisomes of the liver it is combined with a

number of H2O2 producing enzymes, such as D-amino acid oxidase and uricase. On the

other hand, in erythrocytes, catalase and glutathione peroxidase jointly exert a

protective function for haemoglobin and other SH proteins, the relative contributions of

each enzyme varying with species and experimental conditions. The lower the catalase

activity of red cells, the more effective the action of oxidizing agents.

Catalase is a tetrameric haemin - enzyme consisting of 4 identical tetrahedrally

arranged sub units of 60000 g/mol each. Therefore it contains 4 ferriprotoporphyrin

groups per molecule, its molecular mass being approx.240 000 Da.

2.8.6.Ceruloplasmin 75,76

Ceruloplasmin is a α2 – globulin that contains approximately 95% of the total

serum copper. Ceruloplasmin has a single polypeptide chain with 1046 amino acids and

3 glucosamine linked oligosaccharide side chains with a total carbohydrate content of 8

to 9.5%. The peptide chain and carbohydrate together result in mean molecular mass of

132KDa.

Primarily the hepatic parenchymal cells synthesize ceruloplasmin, with small

amounts apparently synthesized by macrophages and lymphocytes. The primary

physiologic role of ceruloplasmin involves plasma redox reactions. It can function as an

oxidant or antioxidant depending on other factors, such as the presence of free ferric

ions and ferritin binding sites. Acting as a ferroxidase, Cp is vitally important in

regulating the ionic state of iron in particular, oxidizing Fe2+ to Fe3+. Under physiologic



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conditions, Cp is also important in the control of membrane lipid oxidation, probably by

direct oxidation of cations, thus preventing their catalysis of lipid peroxidation.

Ceruloplasmin probably also transports copper to tissues, which have separate

membrane receptors for Cp and albumin bound copper. Albumin and trans cuprein are

also major copper transport proteins, especially after absorption from the intestinal tract.

2.8.7.Ascorbic Acid and Tocopherol 77, 78

Vit.C is one of a group of nutrients, which includes Vit. E and β-carotene that

are known as antioxidants. Supplementation of the diet with these compounds decreases

the incidence of chronic diseases. Although Vitamins C, E and β-carotene each show

different protein actions, the common chemical property thought to be central to their

biologic action is the ability to inactivate toxic oxygen free radicals.

Vitamin C is the enolic form of an α-ketolactone. The endiol groups at the

second and third carbon atoms are sensitive top oxidation and can easily convert into

diketo group, L-dehydroascorbic acid. This oxidised form is more effective. Vitamin E

activity in animal tissues is essentially due to α-tochopherol and more specifically d- α-

tochopherol in free form or as acetate ester.



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2.9 Analytical Method Development and Validation

Analytical Method Development

The primary goal of the pharmaceutical analysis is to assure drug quality. It is well

known that quality cannot be tested in to a product; however, well planned testing with

suitable methodology and instrumentation can help build quality in to a drug product.

Chromatographic methods are commonly used for quantitative and qualitative analysis

of pharmaceutical and herbal preparations. A qualitative method provides information

about the identity of sample, whereas, a quantitative method provides numerical

information as to the relative amount of one or more of these components. High

performance liquid chromatography (HPLC) and High performance thin layer

chromatography (HPTLC) analysis has become as the requirement in developing

pharmaceutical drug substances today because of its potential, speed and convenience

for use at routine work. HPLC is the most versatile instrument and user friendly

software available for the development of methods of analysis and their validations; it is

possible to maintain a high degree of assurance for the quality of drugs. 79,80

Analytical Procedure

The analytical procedure refers to the way of performing the analysis. It should describe

in detail the steps necessary to perform each analytical test. This includes: sample,

reference standard and reagent preparation, use of apparatus, generation of calibration

curve, use of formulae for the calculation etc.

Analytical Method Validation

Validation is a process of evaluating products or analytical methods to ensure

compliance as per the pre-determined criteria / specifications. It is important to check

that the requisites to fulfill these requirements are properly functioning, reliable and

documented. This includes the instruments (hardware and firmware), computer

(hardware and software) and the analytical methods. When equipment and a particular

method have been selected and found validated, the equipment and particular method

goes through a system suitability test before and between the sample analysis.



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Analytical Method Validation Guidelines

Method validation is a process of proving that an analytical method is acceptable for its

intended purpose for pharmaceutical methods. The guidelines from the United States

pharmacopoeia (USP), International Conference on Harmonization (ICH), and the Food

and Drug Administration (FDA) provide a framework for performing such validation.

In general, the methods for regulatory submission must include studies on specificity,

linearity, accuracy, precision, range, detection limit, quantification limit and robustness.

The process of validating a method cannot be separated from the actual development of

the method conditions, because the developer will not know whether the method

conditions are acceptable until validations studies are performed. The development and

validation of a new analytical method may therefore be as interactive process. 81

An analytical run includes analysis method, analytical system and sample analysis.

Analytical systems should be tested prior to and during routine use. All the analytical

methods must be well characterized, fully validated and documented, and should satisfy

the relevant requirements as to specificity, accuracy, selectivity and precision.

Knowledge of the stability of the test substance and / or biotransformation product in

the sample material is a prerequisite for obtaining reliable results. It should be noted

that,

• Validation comprises both, before study and within study phases.

• Validation must cover the intended use of assay.

• The calibration range must be appropriate to the study samples.

• If different studies are to be compared, the samples from these studies have been

assayed by different methods and these methods cover a similar concentration

range and the same matrix, should be cross-validated.

• Doing a thorough method validation may be tedious but the consequences of not

doing it right are wasted time, money and resources.

Validation Parameters

Various method Validation parameters are,

1) Specificity



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2) Accuracy

3) Precision

4) Limit of detection (LOD)

5) Limit of quantification (LOQ)

6) Linearity and Range

7) Robustness

8) System suitability

The parameters for method validation as defined by ICH (International

Conference on Harmonization) guidelines Q2 R1 are summarized below:

1. Specificity

Specificity is the ability to assess unequivocally the analyte in the presence of

components, which may be expected to be present. Typically these might include

impurities, degradant, matrix etc. Specificity is measured by resolution, plate count and

tailing factor. An investigation of specificity should be conducted during the validation

of identification tests, the determination of impurities and the assay. The procedures

used to demonstrate specificity will depend on the intended objective of the analytical

procedure. It is not always possible to demonstrate that an analytical procedure is

specific for a particular analyte (complete discrimination). In this case a combination of

two or more analytical procedures is recommended to achieve the necessary level of

discrimination. If pharmacopeial methods and impurity standards are not available,

specificity is measured by resolution, plate count and tailing factor.82

Identification 82

Suitable identification tests should be able to discriminate between compounds of

closely related structures, which are likely to be present. The discrimination of a

procedure may be confirmed by obtaining positive results (perhaps by comparison with

a known reference material) from samples containing the analyte, coupled with negative

results from samples which do not contain the analyte. In addition, the identification test

may be applied to materials structurally similar to or closely related to the analyte to

confirm that a positive response is not obtained. The choice of such potentially



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interfering materials should be based on sound scientific judgment with a consideration

of the interferences that could occur.

Assay and impurity test (s) 83

For chromatographic procedures, representative chromatograms should be used to

demonstrate specificity and individual components should be appropriately labeled.

Similar considerations should be given to other separation techniques. Critical

separations in chromatography should be investigated at an appropriate level. For

critical separations, specificity can be demonstrated by the resolution of the two

components, which elute closest to each other. In cases where a non-specific assay is

used, other supporting analytical procedures should be used to demonstrate overall

specificity.

2. Accuracy

The accuracy of an analytical procedure expresses the closeness of agreement between

the values, which is accepted either as a conventional true value or an accepted

reference value found. Accuracy should be established across the specified range of the

analytical procedure.

Assay (for drug substance)

� Application of an analytical procedure to an analyte of known purity (e.g.

reference material);

� Comparison of the results of the proposed analytical procedure with those of a

second well-characterized procedure, the accuracy of which is stated and/or

defined.

Assay (for drug Product)

� Application of the analytical procedure to synthetic mixtures of the drug

product components to which known quantities of the drug substance to be

analyzed have been added.

� In cases where it is impossible to obtain samples of all drug product

components, it may be acceptable either to add known quantities of the

analyte to the drug product or to compare the results obtained from a second,

well characterized procedure, the accuracy of which is stated and/or defined.



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Recommended data

Accuracy should be assessed using a minimum of 9 determinations over a minimum of

3 concentration levels covering the specified range (e.g. 3 concentrations /3 replicates

each of the total analytical procedure).

Accuracy should be reported as percent recovery by the assay of known added amount

of analyte in the sample or as the difference between the mean and the accepted true

value together with the confidence intervals.

3. Precision

The precision of an analytical procedure expresses the closeness of agreement (degree

of scatter) between a series of measurements obtained from multiple sampling of the

same homogeneous sample under the prescribed conditions. Precision may be

considered at three levels: repeatability, intermediate precision and reproducibility.

Precision should be investigated using homogeneous, authentic samples. However, if it

is not possible to obtain a homogeneous sample it may be investigated using artificially

prepared samples or a sample solution.

The precision of an analytical procedure is usually expressed as the variance, standard

deviation or coefficient of variation of a series of measurements.

Repeatability

Repeatability expresses the precision under the same operating conditions over a short

interval of time. Repeatability is also termed intra-assay precision.

Repeatability should be assessed using: A minimum of 9 determinations covering the

specified range for the procedure (e.g. 3 concentrations/ 3 replicates each) or a) a

minimum of 6 determinations at 100% of the test concentration.

Intermediate precision

Intermediate precision expresses within-laboratories variations: different days, different

analysts, different equipment, etc.

Reproducibility

Reproducibility expresses the precision between laboratories (collaborative studies,

usually applied to standardization of methodology).



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The standard deviation, relative standard deviation (coefficient of variation) and

confidence interval should be reported for each type of precision investigated

4. Limit of Detection

The detection limit of an individual analytical procedure is the lowest amount of analyte

in a sample which can be detected but not necessarily quantitated as an exact value. The

minimum concentration at which the analyte can reliably be detected is established.

LOQ, LOD and SNR

• Limit of Quantitation

• Limit of Detection

• Signal to Noise Ratio

noise

Peak A

LOD

Peak B

LOQ

Baseline

Figure 2.5 Graphical representation of signal to noise ratio, LOD, LOQ

Based on visual evaluatio

� Mostly for non-instrumental methods

Based on signal-to-noise

� Analytical procedures which exhibit baseline noise

� Compare measured signals from samples with known low concentration

of analyte with those of blank samples.

� A signal-to-noise ratio 3 or 2:1 is acceptable.

Based on the Standard Deviation of the Response and the Slope

� The Quantitation Limit (QL) may be expressed as:

� DL = 3.3(σ/S)

Where σ= the standard deviation of blank

S = the slope of the calibration curve

� The slope S may be estimated from the calibration curve of the standard.



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5. Limit of Quantitation

The quantitation limit of an individual analytical procedure is the lowest amount of

analyte in a sample which can be quantitatively determined with suitable precision and

accuracy. The quantitation limit is a parameter of quantitative assays for low levels of

compounds in sample matrices, and is used particularly for the determination of

impurities and/or degradation products.

Based on visual evaluation

� The quantitation limit is generally determined by the analysis of

samples with known concentrations of analyte and by establishing the

minimum level at which the analyte can be quantified with acceptable

accuracy and precision.

Based on signal-to-noise

� A typical signal-to-noise ratio is 10:1.

Based on the Standard Deviation of the Response and the Slope

� The Quantitation Limit (QL) may be expressed as:

� QL =10(σ/S)

Where σ= the standard deviation of blank

S = the slope of the calibration curve

� The slope S may be estimated from the calibration curve of the

standard

6. Linearity & Range

The linearity of an analytical procedure is its ability (within a given range) to obtain test

results which are directly proportional to the concentration (amount) of analyte in the

sample.

In these, samples of different concentrations are injected into the HPLC system and the

graph of sample area v/s concentration of sample is plotted.

Linearity is evaluated by:



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a) Coefficient of determination

b) Y-intercept,

c) Residual sum of squares

d) Slope of the regression line

e) Analysis of deviation of the actual data points from the regression line

and

f) Response factor

The range of an analytical procedure is the interval between the upper and lower

concentration (amounts) of analyte in the sample (including these concentrations) for

which it has been demonstrated that the analytical procedure has a suitable level of

precision, accuracy and linearity.

ICH recommends that, for the establishment of linearity, a minimum of five

concentrations normally be used. It is also recommended that the following minimum

specified ranges should be considered

Assay of a drug substance (or a finished product): from 80% to 120% of the test

concentration.

Determination of an impurity: from 50% to 120% of the specification.

For content uniformity: a minimum of 70% to 130% of the test concentration, unless a

wider or more appropriate range, based on the nature of the dosage form (e.g., metered-

dose inhalers) is justified.

7. Robustness

The robustness of an analytical procedure is a measure of its capacity to remain

unaffected by small, but deliberate variations in method parameters and provides an

indication of its reliability during normal usage.

Examples of typical variations are:

� Stability of analytical solutions

� Extraction time

In the case of liquid chromatography, examples of typical variations are

� Influence of variations of pH in a mobile phase

� Influence of variations in mobile phase composition

� Different columns (different lots and/or suppliers)



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� Temperature

� Flow rate

8. System Suitability Testing

System suitability testing is an integral part of many analytical procedures. The tests are

based on the concept that the equipment, electronics, analytical operations and samples

to be analyzed constitute an integral system that can be evaluated as such. System

suitability test parameters to be established for a particular procedure depend on the

type of procedure being validated. 84

Table 2.3 System Suitability testing factors to be considered

S.No Factors Limits

1 Reproducibility %RSD should be less than or equal to 2

2 Capacity factor (k’) should be greater than 2

3 Resolution (Rs) should be greater than 2

4 Tailing factor (T) should be less than or equal to 2

5 Theoretical plates (N) should be greater than 2000

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