22

2. REVIEW OF LITERATURE

2.1. Literature review of Tephrosia calophylla Pers.

Botanical Name: Tephrosia calophylla Pers

Common Names: Telugu: Adavivempalli, Dumpavempalli, Gadda

vempalli, kommuvempalli.

Taxonomic classification:

Kingdom : Plantae

Division : Magnoliophyta

Class : Dicotyledons

Sub class : Polypetaceae

Series : Polypetaceae

Order : Rosales

Family : Fabaceae

Genus : Tephrosia

Species : Calophylla.

23

Figure: 2.1. Tephrosia calophylla Pers plant

24

Geographical distribution:

Tephrosia is a large tropical and subtropical genus belongs to

the family Fabaceae. Morphologically Tephrosia plants are perennial

woody shrubs, the genus Tephrosia possess more than 300 species, of

which 35 occur in India, and others are abundant in the equatorial

Africa, South Africa and South America regions39.

Tephrosia calophylla is a perennial under shrub found widely in

Andhra Pradesh, south India. It is mainly available in localities of hill

slopes, rare in shady locations. It is found widely in Talakona forest of

Andhra Pradesh40. Tephrosia calophylla is a perennial under shrub

which exhibits greater diversity. Leaves are simple, coriacius,

oblanceolate, entire, mucronate, petiolate, winged, anticulate at apex.

Flowers are Light pink, glabrous, composed mucronate, interminal

recemes plants start flowering in April to August every year. Roots are

Rhizomatous (or) tuberous.

Chemical constituents:

The genus Tephrosia contains a wide variety of flavanoids and

isoflavanoids. Earlier investigations on Tephrosia calophylla have

revealed the isolation of 23 different compounds of which 18 were

known and 5 are new.

A new coumestan derivative Tephcalostan from the whole plant

together with two known flavanoids, 7-Ο-methylglabranin and

kaempferol 3-o- -D-glucopyranoside were isolated and characterized

by Pannaka Hari kishore et al., 200341. The structure of Tephcalostan

25

was elucidated as 5'-(R)-8, 9-methylenedioxy-5-'-isopropenyl-4', 5-

dihydro forano-[2',3';2,3] Coumestan by extensive one-and two-

dimensional (1D-and 2D)-NMR techniques including ¹H-¹H correlation

spectroscopy (COSY), Hetero nuclear single quantum coherence

(HSQC), Hetero nuclear multiple bond connectivity (HMBC) and

Nuclear Overhauser enhancement spectroscopy (NOESY) experiments.

A benzil, Calophione A, 1-(6 -Hydroxy-1 ,3 -benzodioxol-5 -yl)-2-

(6 -hydroxy-2 -isopropenyl-2 ,3 -dihydro-benzofuran-5 -yl)-ethane-

1,2-dione and three coumestan derivatives, Tephcalostan B, C and D

were isolated from the roots of Tephrosia calophylla by Seru

Ganapathy et al., 200842. Their structures were deduced from

spectroscopic data, including 2D NMR ¹H-¹H COSY and 13C–1H COSY

experiments. Compounds were evaluated for cytotoxicity against RAW

(mouse macrophage cells) and HT-29 (colon cancer cells) cancer cell

lines and anti-protozoal activity against various parasitic protozoa.

Calophione A exhibited significant cytotoxicity with IC50 of 5.00 (RAW)

and 2.90 μM (HT-29), respectively

. Calophione A.

A chemical compound named Betulinic acid has been isolated

from the whole plant of Tephrosia calophylla by Rajagopal

Subramanyam et al., 200843. Betulinic acid has anti cancer and anti-

HIV activity and has been proved to be therapeutically effective

26

against cancerous and HIV-infected cells. Human serum albumin

(HSA) is the predominant protein in the blood. Most drugs that bind to

HSA will be transported to other parts of the body. Using micro TOF-Q

mass spectrometry, researchers have shown for the first time that,

Betulinic acid isolated from a plant (Tephrosia calophylla) binds to

HSA. The binding constant of Betulinic acid to HSA was calculated

from fluorescence data, indicating a strong binding affinity. The

secondary structure of the HSA-Betulinic acid complex was

determined by circular dichromism. The results indicates that the

HSA in this complex is partially unfolded Further, binding of Betulinic

acid at nanomolar concentrations to free HSA was detected using

micro TOF-Q mass spectrometry. The study revealed a mass increase

from 65199 Da (free HAS) to 65643 Da (HSA+drug), where the

additional mass of 444 Da was due to bound Betulinic acid. Based on

the results of this study, it is suggested that micro TOF-Q mass

spectrometry is useful technique for drug binding studies.

An another attempt has been made by the researchers to assess

the genotypic variability among twelve species of the genus Tephrosia

distributed in Andhra Pradesh, through DNA fingerprinting using

RAPD technique, P.Lakshmi et al.,200844. Twenty OPC (Operon

Biotechnologies GmbH, Germany) primers were used for this study.

The cluster analysis was made based on the similarity matrix, and

was performed using the unweighted pair Group Method with

Arithmetic Average (UPGMA) with the help of PHYLIP software ver.3.65

pooled from all the six primers. Present study has justified to a great

27

extent co-relating with the classification based on the morphological

traits. However a distinction between some members of the genus

Tephrosia is still a matter of debate. Hence further analysis are needed

to determined the correct intrageneric taxonomic treatment of

Tephrosia, since it represents one of the largest and most complex

groups in the core tribe Millettieae of the family Fabaceae. This study

represents the first approach in using nuclear DNA finger print

markers as a tool to study molecular systematic of the genus

Tephrosia.

Uses:

Many species in the Tephrosia genus have been used as

insecticidal, pesticidal agents and as poison particularly to fish in

connection with the high concentration of rotenone. The plants are

also used traditionally in folk medicine. According to Ayurveda, the

plant is useful as an anti-helmintic, anti-pyretic and as well as an

alexiteric drug. It is also active against leprosy, ulcers, and used as

alternative cures for diseases of the liver, spleen, heart and blood.

According to the Unani system of medicine, the root is diuretic, allays

thirst, enriches blood, cures diarrhea, it is also useful in bronchitis,

inflammations, boils and pimples. Leaves are tonic to intestines, and a

promising appetizer. The seeds can be used as substitute for coffee45.

28

The details of different compounds from Tephrosia calophylla

are enlisted below46.

Spinoflavanone B Caliphione A

Tephcalostan Tephcalostan A

Tephcalostan B Tephcalostan C

29

Glabranine Milletone

Betulinic acid Betulinic acid methyl ester

Stigmasterol -sitosterol

2-Methoxymaackiai Tephrosol(2-methoxymedicagol)

30

.

Didehydrovillosin(mixture of two Dehydro rotenone stereoisomers at c-6)

Substituted Dichromen-7-one Obovatin methyl ether

Obovatin 7-Methylglabranin

31

Candidone praecansone B

Ovalichalcone

32

2.2. Literature Review of Holostemma ada kodien Shcult:

Description of Holostemma ada kodien Shcult48, 49:

Botanical Name - Holostemma ada kodien Shcult.

Family - Asclepiadaceae.

Habit - Woody Climber.

Regional Names

Telugu - Palagurgu, Paalajilledu, Dudipala

tige, Bandi guruvindateega.

Hindi - Charivel, Ranimaoi, Rajain pardesi,

Kanju.

Marathi - Gaganthjuti, Haranadodi, Dudoli,

Dudurli.

Kannada - Arane bellu, Jeeva hale, Jeevanthi.

Malayalam - Atakodiyan, Atapatiyan.

Tamil - Palaikkirai.

33

Figure: 2.2 Holostemma ada kodien Shcult plant

Geographical distribution:

This plant is available in Adilabad, Kadapa, Tirupathi, East

Godavari, Kurnool, Visakhapatnam, Karimnagar, Chittoor,

Nizamabad, Nellore, Guntur, Srilanka, Myanmmar and western

China.

Description of plant:

An extensive hairless perennial climber.

Stems branched, puberulent to glabrous.

Leaves opposite, egg-shaped, base deeply heart-shaped, apex

bluntly acuminate, margin entire, hairless, papery. Lateral nerves

about 5 pairs and the lower 2 pairs arise from the base of the leaves.

Leaf stalks up to 3 cm long.

Inflorence extra-axillary, umbel-like or short raceme like,

occasionally branched, shorter than leaves, usually few flowered.

34

Flowers bisexual, 5-7 in axillary cymes, about 1.5 cm across,

pinkish purple, fleshy, distinctly stalked. Large Calyx without glands.

Corolla sub-rotate lobes overlapping to right.

Filaments connate. Anthers very large, decurrent to base of

column. Stigma head scarcely umbonate.

Follicles stout, cylindric-fusiform.

Seeds many, ovoid, about 1 cm long, falit winged along the

margin, with silky white hairs at apex.

Roots tuberous, about 3 cm across, whitish inside.

Special characters

The flowers are attractive, resembling Calotropis gigantean

(madar).

Fruits are thick, more or less boat-shaped in outline.

Sometimes all plant parts exhibit a pink tinge.

Roots taste sweet.

Chemical constituents

It also contains six amino acids like alanine, aspartic acid, valine,

glycine, serine and threonine50.

Medicinal uses47, 50

Root tubers of plant are used as stimulant, hepatoprotective,

aphrodisiac, expectorant and galactagouge.

Roots have cooling, alternative, tonic and laxative properties and

are also an astringent to the bowels and are sweet.

35

The root made into a paste is applied to eyes in ophthalmic and

also for scalding in gonorrhea.

In diabetes, the root rubbed into a paste is given in cold milk.

It is employed in decoction, as a remedy for cough and also for

orchitis.

It also cures ulcer, biliousness, disease of the blood worms,

itching and vesicular calculi.

2.3. Review on microbiological studies:

Introduction:

Micro organisms are the causative agents of a wide variety of

diseases in human beings as well as in animals and plants. To treat

the diseases, it is essential to know the details of concern causative

agents; the study of Microbiology enables this, and helps in the

treatment of diseases. Numerous numbers of herbal plants or their

agents are available to prevent or cure these infectious diseases. These

agents are called Antimicrobial agents.

An antimicrobial agent is a substance that kills or inhibits the

growth of microorganisms such as bacteria, fungi, or virus.

Antimicrobial drugs either kill the microbes (microbicidal) or prevent

the growth of microbes (microbistatic). The history of antimicrobial

agents begins with the observations of “Pasteur and Joubert” who

discovered that, one of type of bacteria could prevent the growth of

another.

36

Ideal features of an antimicrobial agent:

Quick acting

Few side effects

Quick “kill” of the pathogen

Broad spectrum in action

Water soluble

2.3.1. Classification of antimicrobial agents:

Antimicrobial agents can be classified by at least three different

schemes:

1. Effect on target cells:

Bactericidal

Ex: Streptomycin, Aminoglycosides, Penicillins

Bacteriostatic

Ex: Sulfonamides, Tetracyclins, Chloromphenicol.

2. Range of activity:

Narrow spectrum

Ex: Macrolides, Polypeptides

Moderate spectrum

Ex: Sulfonamides, Aminoglycosides

Narrow and moderate spectrum

Ex: Betalactins.

Broad spectrum

Ex: Chloromphenicol, Tetracycline

Anti-mycobacterial

Ex: Isoniazide, Ethambutol, Streptomycin, Rifampicin

37

3. Sites of activity within target cell:

Site of activity, destroyment of cell wall.

Ex: lyosozym Inhibition of cell wall synthesis

Ex: fasfomycin, bacitracin, betalactams, vancomycin

Inhibition of membrane integrity

Ex: surfactants, polyenesten

Inhibition of nucleic acid synthesis

Ex: 5-flurocytosine, acyclovin

Inhibition of protein integrity and protein synthesis

Ex: streptomycin, kanamycin, macrolides.

Antimicrobial activity of plants can be detected by observing the

growth response of various microorganisms to those plant tissues or

plant extracts, which are placed in contact with them. Many methods

are available to detect their antimicrobial activity. But all the methods

are not equally sensitive or even based on the same principle. But in

general the Biological evaluation can be carried out much more

efficiently on water soluble, nice crystalline substance than on

mixtures like plant extracts51, 52.

In order to detect the antimicrobial activity of plant extracts, the

following conditions must be full filled.

The plant extract must be brought into contact with the cell wall

of the micro organisms that have been selected for the test.

Conditions must be adjusted so that the microorganisms are

able to grow when no antimicrobial agents are present.

38

There must be some means of judging the growth, if any, made

by the test organism during the period of time chosen for the

test53.

2.3.2. General methods for antimicrobial screening:

Microbiological assays:

The inhibition of microbial growth under standardized conditions

may be utilized for demonstrating the efficacy of antimicrobial agents.

The microbial assay is based upon a comparison of growth of

microorganisms by measured concentrations of the antibiotics and

plant extract (to be examined) with that produced by known

activity.[known activity for anti biotic only].

Different types of methods are available to evaluate the antimicrobial

activity. They are,

1. Diffusion method

2. Dilution method

3. Bio-autographic methods

1. Diffusion method: (cup-plate method)

In the diffusion technique a reservoir containing the plant extract

to be tested is brought into contact with an inoculated medium (e.g.,

nutrient agar) and after incubation, the diameter of zone of inhibition

around the reservoir is measured. In order to lower the detection limit,

the inoculated system is kept at a low temperature for several hours

before the incubation, which favors diffusion over microbial growth

and thus increases the inhibition diameter. This method was

originally designed to monitor the amounts of antibiotic substances in

39

fermentation cultures and has also been used for obtaining bigrams

and for testing essential oils54. Different types of reservoirs have been

employed, including filter paper discs, porcelain or stainless steel

cylinders (borers) placed on the surface and cups (holes) are punched

in the medium. It is not necessary to sterilize the test samples since

any bacteria present will be confined to the reservoirs and will not

therefore be able to spread and ruin the place.

The cop-plate method, however, is only suitable diffusion

technique for testing aqueous suspensions of plant extracts. In this

method, the presence of suspended particulate matter in the sample

being tested is much less likely to interfere with the diffusion of the

antimicrobial substance into the agar than in the filter paper disc and

the cylinder plate methods. Precipitation of water-insoluble

substances in the cylinder or in the disc will indeed prevent any

diffusion of antimicrobial substances into the agar. Nevertheless, in

order to limit precipitation as much as possible in the cup-plate

methods, Pre-incubation should be carried out at room temperature

(25˚C) rather than 4˚C. Advantages of the diffusion method are, the

small size of the sample used in the screening and the possibility of

testing five or six compounds per plate against a single micro

organism.

2. Dilution method: (Tube dilution method)

In the dilution methods, samples being tested are mixed with a

suitable medium that has been previously inoculated with the test

40

organism. After incubation, growth of the microorganism may be

determined by direct visual or turbidimetric comparison of the test

culture with a control culture which did not receive the sample being

tested or by plating out both test and control cultures. Usually a

series of dilutions of the original sample in the culture medium is

made and then inoculated with the test organism. After incubation,

the end point of the test is taken as the highest dilution which will

just prevent perceptible growth of the test organism (MIC-Value).

These methods are the best for assaying water-soluble or lipophilic

pure compounds and to determine their MIC-Values, which can be

recorded using this method.

3. Bioautographic methods:

Bioautography, as a method to localize antibacterial activity on

a chromatogram, has found widespread application in the search for

new antibiotics from micro organisms. Most published procedures are

based on the agar diffusion technique; where by the antimicrobial

agent is transferred from the thin layer or paper chromatogram to an

inoculated agar plate through a diffusion process. Zone of inhibition

are then visualized by appropriate vital stains. The problems due to

the differential diffusion of compounds from the chromatogram to the

agar plate are simplified by direct bioautographic detection on the

chromatographic layer55. This method, however, requires more

complex microbiological equipment and is, in contrast to the contact

bioautographic methodology, easily affected by possible contamination

41

from air borne bacteria52. It should be noted that there are some

plants with antibacterial activity as shown in Table: 2.1.

Table: 2.1 Examples of some plant derived compounds with anti

microbial properties56.

Compound

Examples

Plant sources

Coumarins and

their derivatives

Asphodelin A 40-׳- -D-

glucoside

Asphodelus microcarpus

Simple phenols Epicatechin

Epigallocatechin

Epigallocatechin gallate

Calophyllum brasiliense

Camelliasinensis

. Camellia sinensis

Flavonoids Isocytisoside

Eucalyptin

Aquilegia vulgaris l.

Eucalyptus maculate

Flavones Luteolin

GB1hydroxybiflavanol

Senna petersiana

Garcinia kola

Tannins Ellagitannin Punica granatum

Alkaloids Berberine Mahonia aquifolium

Terpenes Ferruginol,(Diterpene)

Episiferol, (Diterpene)

Chamaecyparis

lawsoniana

Salvia viridis

42

2.4. Review on diabetes mellitus:

Diabetes mellitus is a heterogeneous metabolic disorder

characterized by altered carbohydrate, lipid and protein metabolism

caused by insulin deficiency, often combined with insulin resistance57.

It is considered as one of the five leading causes of death in the

world58. About 150 million people are suffering from diabetes

worldwide and it is almost five times more than the estimated ten

years ago and this may be doubled by the year 203059.

Hyperglycemia occurs because of uncontrolled hepatic glucose

output and reduced uptake of glucose by skeletal muscle with reduced

glycogen synthesis. When the renal threshold for glucose re

absorption is exceeded, glucose spills over into the urine (glycosuria)

and causes an osmotic diuresis (polyuria), which in turn, results in

dehydration, thirst and increased drinking (polydipsia). Insulin

deficiency causes wasting through increased breakdown and reduced

synthesis of proteins.

Various forms of diabetes mellitus60:

I. General-genetic and other factors not precisely defined

a) Type 1 diabetes mellitus (TIDM or IDDM).

Type 1A – Auto-immune type 1 diabetes mellitus.

Type 1B – Non-autoimmune or idiopathic type 1 diabetes

mellitus.

b) Type 2 diabetes mellitus (T2DM or NIDDM).

II. Specific defined gene mutations

a) Maturity onset diabetes of the young (MODY)

43

MODY 1 (Hepatic nuclear factor 4- gene mutations)

MODY 2 (Glucokinase gene mutations)

MODY 3 (Hepatic nuclear fact 1- gene mutations)

MODY 4 (Pancreatic determining fact x gene mutations)

MODY X (Unidentified gene mutation(s))

b) Maternally inherited diabetes and deafness (MIDD)

c) Mitochondrial leucine t–RNA gene mutations

d) Insulin gene mutations

e) Insulin receptor gene mutations

III. Diabetes secondary to pancreatic disease

Chronic pancreatitis

Surgery

Tropical diabetes (chronic pancreatitis associated with

nutritional and or toxic factors)

Gestations diabetes

IV. Diabetes secondary to other Endocrinopathies

Cushing’s disease

Glucocorticoid administration

Acromegaly

V. Diabetes secondary to immune suppression

VI. Diabetes associated with genetic syndromes

eg: Prader–willi syndrome

VII. Diabetes associated with drug therapy

a) Drugs with hypoglycemic effects

44

-adrenergic receptor antagonists

Salicylates

Indomethacin

Clofibrate

ACE inhibitor

b) Drugs with hyperglycemic effects

Epinephrine

Glucocorticoid

-adrenergic receptor agonist

Calcium channel blockers

Phenytoin

H2 receptor blocker

Virtually all forms of diabetes mellitus are caused by a

decrease in the circulating concentration of insulin (insulin deficiency)

and a decrease in the response of peripheral tissues to insulin (insulin

resistance). These abnormalities lead to alterations in the metabolism

of carbohydrates, lipids, ketones and amino acids. The central feature

of the syndrome is hyperglycemia characterized by a high blood

glucose concentration.

Hyperglycemia may become toxic and fatal as a result of

accumulation of non-enzymatically glycosylated products and

osmotically active sugar alcohols such as sorbitol in tissues, the

effects of glucose on cellular metabolism also may be responsible61.

Diabetic ketoacidosis is an acute emergency which develops on

45

prolonged hyperglycemia and develops in the absence of insulin

because of accelerated fat breakdown to acetyl CoA, which in the

absence of aerobic carbohydrate metabolism is converted to

acetoacetatae and -hydroxy-butyracetone, which causes acidosis62.

Hemoglobin undergoes glycosylation on its amino–terminal valine

residue to form the glucosyl valine adduct of hemoglobin, termed

hemoglobin A1c. The half-life on the modified hemoglobin is equal to

that of the erythrocytes (about 120 days). Since the amount of

glycosylated protein formed is proportional to the glucose

concentration and the time of exposure of the protein to glucose, the

concentration of hemoglobin, A1c in the circulation reflects the

severity of the glycemic state60.

Complications of the disorder63:

Most diabetic complications arise from prolonged exposure of

tissues to elevated glucose levels. These complications develop as a

consequence of the metabolic derangements in diabetes, often over

many years. Many of these result in diseases of blood vessels, either

large (macro vascular disease) or small (microangiopathy). Macro

vascular disease consists of accelerated atheroma, which is much

more common and severe in diabetic patients. Microangiopathy is a

distinctive feature of diabetes mellitus and particularly affects the

retina, kidney and peripheral nerves. Diabetes mellitus is the

commonest cause of chronic renal failure, which itself represents a

huge and rapidly increasing problem, the costs of which to society as

well as to individual patients are staggering. Coexistent hypertension

46

promotes progressive renal damage nephropathy and induces the risk

of myocardial infarction. Angiotensin–converting enzyme inhibitors or

antagonists of angiotensin AT1 receptors are more effective in

preventing diabetic nephropathy than other antihypertensive drugs,

perhaps because they prevent fibroproliferative actions of angiotensin-

II and aldosterone. Although the underlying mechanism of diabetic

complications is unclear much attention has been focused on the role

of oxidative stress, which contributes to the pathogenesis of different

diabetic complications.

Pancreatic islet hormones:

Maintenance of euglycemia, hyperglycemia or hypoglycemia

rests on the efficient functioning of the endocrine pancreas and the

hormones secreted by it. The islets of langerhans contain four main

cell types: cells, surrounded by a mantle of a cells interspersed

with cells. The cells also secrete islet amyloid polypeptide which

delays gastric emptying and opposes insulin by stimulating glycogen

breakdown. Glucagon also opposes insulin, increasing blood glucose

and stimulating protein breakdown.

Insulin therapy:

Insulin is the mainstay for treatment of virtually type-1 DM and

many type-2 diabetes mellitus patients. When necessary, insulin may

be administered intravenously or intramuscularly; however, long-term

treatment relies predominantly on subcutaneous injection of the

hormone. Subcutaneous administration of insulin differs from

physiological secretion of insulin in at least two major ways: The

47

kinetics do not produce the normal rapid rise and decline of insulin

secretion in response to ingestion of nutrients and the insulin diffuses

into the portal circulation; the direct action of secreted insulin on

hepatic metabolic processes is thus eliminated.

Screening methods of antidiabetic agents64, 92:

Before the advent of insulin and oral hypoglycemic drugs, the

major form of treatment involved the use of plants. More than 400

plants have been recommended and recent investigations have

affirmed the potential value of some of these treatments. The

hypoglycemic and/or anti-hyperglycemic effect of several plants used

as anti-diabetic remedies has been confirmed and the mechanisms of

their activity are being studied. Chemical studies directed at the

isolation, purification and identification of the substances responsible

for the antidiabetic activity are also being conducted. Various

diabetogens (Table: 2.4) have been discovered by the researchers to

assess the anti-diabetic potentials of the huge herbal resources.

Induction of experimental diabetes in the rat by particularly

destroying the pancreatic beta cells is the most convenient method

employed now days and the most commonly used diabetogenic agents

are alloxan and streptozotocin.

Herbal drugs used in the treatment of diabetes:

The antihyperglycemic effect of several plant extracts which

were used as antidiabetic remedies has been confirmed. The synthetic

hypoglycemic agents used in clinical practices have serious side

effects like hematological effects, coma, disturbance of the functions of

liver and kidney etc. In addition they are not suitable for use during

48

pregnancy. Compared with synthetic drugs, drugs derived from plants

are frequently considered to be less toxic with fewer side effects.

Therefore, the search for more effective and safer antidiabetic agents

has become an area of active research. Most commonly used plants in

herbal formulations in the treatment of diabetes are given in Table:

2.3. The list of poly herbal formulations used is shown Table: 2.4.

Table: 2.2 List of various diabetogens and doses used in diabetic

study.

S.No. Diabetogen Dose Animal

1 Alloxan 65-150 mg/kg Rat

2 Streptozotocin 40-60 mg/kg Rat

3 Cyclosporin A 40 mg/kg Rat

5 Dehydro ascorbic acid

650 mg/kg Rat

6 Methyl alloxan 53 mg/kg Rat

7 Ethyl alloxan 50-130 mg/kg Rat

8 Dexomethasone 2-5 mg/kg Rabbit

9 Sodium diethyl Ditiocarbonate

0.5 –1 g/kg Rabbit

10 Uric acid 1 g/kg Rabbit

49

Table: 2.3 Plants reported having anti-diabetic activity.65-86

Plants References

Aegle Marmelos

Aerva lanata

Alpinia galanga

Aporosa lindleyana

Averrhoa bilimbi

Azadirachta indica

Barleria lupulina

Bauhinia candicans

Berberis aristata

Boswellia glabra

Bougainvillea spectabillis

Caesalpinia bonducella

Curcuma longa

Gymnema sylvestre

Momordica charantia

Ocimum gratissimum

Syzygium cumini

Terminalia catappa

Terminalia chebula

Urtica dioica

Vinca rosea

Zizypus jujube

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

50

Table: 2.4 Some of the polyherbal formulations used in treatment

of diabetes mellitus.87-91

Formulation Reference

Ayushan- 82

Diabecon (D- 400)

Dianex

Diarun plus65

MA- 471

87

88

89

90

91

2.5. Review on isolation and characterization of phyto

constituents

Chromatography

The chromatography technique in 1901 during his research on

chlorophyll. He used a liquid-adsorption column containing calcium

carbonate to separate plant pigments. He first used the term

chromatography in print in 1906 in his two papers about chlorophyll

in the German botanical journal.

Principle

Chromatography is a powerful technique for separating

mixtures. It involves passing the sample, a mixture which contains

the analyte in the “mobile phase”, often in a stream of solvent,

through the “stationary phase”. The stationary phase retards the

passage of the components of the sample.

51

When components pass through the system at different rates

they become separated in time. Each component has a characteristic

time of passage through the system, called a “retention time”.

Chromatographic separation is achieved when the retention time of

the analyte differs from that of other components in the sample.

Various techniques for the separation of compounds rely on the

different affinities of substances for a gas or liquid mobile medium and

for a stationary absorbing medium through which they pass, such as

paper, gelatin, alumina or silica.

There are different types of chromatography such as paper, thin

layer or column chromatography each with its one advantages and

dis-advantages. Chromatography system has a stationary phase

(which can be solid or liquid) and a mobile phase (usually liquid or

gas). In column chromatography both phases are placed in a column

container.

Analytical chromatography is used to determine the identity and

concentration of molecules in a mixture.

Preparative chromatography is used to purify large quantities

molecular species.

Retention

The retention is a measure of the speed at which a substance

moves in a chromatography system. In continuous development

system like HPLC or GC, where the compounds are eluted with the

52

eluent, the retention is usually measured as the retention time Rt or

Tr, the time between injection and detection. In interrupted

development system like TLC the retention is measured as the

retention factor Rf, the distance moved by the compound divided by

the distance moved by the eluent front.

Rf = eluentby moved Distance

compoundby moved Distance

The retention of a compound often differs considerably between

experiment and laboratories due to the variations of the eluent, the

stationary phase temperature and the setup. It is therefore important

to compare the retention of the test compound to that of one or more

standard compounds under absolutely identical conditions.

Plate theory

The plate theory of chromatography was developed by Archer

John Porter Martin and Richard Laurence Millington Synge the plate

theory describes the chromatography system, the mobile and

stationary phase, as being in equilibrium. The partition coefficient is

based on this equilibrium and is defined by the following equation.

K = phase mobile in solute of ionConcentrat

phase stationary in solute of ionConcentrat

K is assumed to be independent of concentration and can

change if experimental conditions are changed, for example

temperature is increased or decreased. As K increases it takes longer

53

for solutes to separate for a column of fixed length and flow the

retention time (TR) and retention volume (Vr) can be measured and

used to calculate k.

Column chromatography

Column chromatography consists of a vertical glass column

filled with some form of solid support with the sample to be separated

placed on top of this support. The result of column is filled with a

solvent, which under the influences of gravity moves the sample

through the column. Similar to the other forms of chromatography,

difference in rate of movement through the solid medium translated to

different outlet times from the bottom of the column for various

compounds of the original sample.

In 1998 W.C. stills introduced a modified version of column

chromatography called flash column chromatography. The technique

is very similar to the traditional column chromatography, but the

solvent is driven through the column by applying pressure. When

applying pressure on the top of the column the separations were

performed within 20 minutes with improved separation.

The traditional thin layer chromatography or the high

performance thin layer chromatography (HPTLC) is widely used and

versatile separation method which shows a lot of advantages in

comparison to other separation techniques. In TLC a solution of the

sample is added to a layer of support material (i.e. grains of silica or

alumina) that has been spread out and dried on a sheet of material

such as glass. The support is known as the plate. The sample is added

54

as a spot at one end of the plate. The plate is put in a sealed chamber

that contains a shallow poll of chemicals (the solvent), which is just

enough to wet the bottom of the plate. As the solvent moves up

through the plate support layer by capillary action, the sample is

dragged along. The different chemical constituents of the sample do

not move at the same speed, however, and will become physically

separated from one another. The positions of the various sample

constituents and their chemical identities are determined by physical

methods (i.e. ultraviolet light) or by the addition of other chemical

sprays that react with the sample constituents 93.