22 2. review of literature - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/9561/8/08_chapter...
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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.
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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
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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.
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The details of different compounds from Tephrosia calophylla
are enlisted below46.
Spinoflavanone B Caliphione A
Tephcalostan Tephcalostan A
Tephcalostan B Tephcalostan C
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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.
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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
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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
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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.