This document describes a study that evaluated the anti-diabetic effects of an Ayurvedic herbo-mineral formulation called Trushanadi loha. The formulation contains 14 plant and mineral ingredients. An aqueous extract was tested in rats. Blood glucose levels were measured. The extract produced a 40% reduction in blood glucose levels in normal rats, demonstrating significant anti-diabetic activity. The formulation was also found to be non-toxic in acute oral toxicity tests in rats at doses up to 5,000 mg/kg. Overall, the study provides evidence that Trushanadi loha has anti-diabetic properties and a safe toxicity profile.

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* Corresponding author: K.V.Ramasubbarao
E-mail address: subbaraoketharaju@ymail.com
Available online at www.ijrpp.com
Print ISSN: 2278 – 2648
Online ISSN: 2278 - 2656 IJRPP | Volume 2 | Issue 1 | 2013 Research article
Phytochemical screening and in-vivo evalution of anti diabetic studies of an
ayurvedic herbo mineral formulation trushanaadiloha.
*1
K.V. RamSubbarao, 2
M.L. Naidu
1
Asst. Research Officer, Govt. Research Dept.(Ayurveda) Erragadda. Hyderabad, Andhra
Pradesh, India.
2
Ex Principal & Professor Department of Kayachikitsa, Dr. N.R.S.Govt. Ayurvedic
College, Vijayawada, Andra Pradesh, India
ABSTRACT
The Herbomineral formulation Trushanadi loha is a mixture of fourteen drugs. All drugs used in this formulation are
from different parts of plant sources. The mixture of different 14 drugs was extracted with aqueous and evaluated for
Anti diabetic activity. 40 % reduction in blood glucose levels was observed and found the herbomineral formulation
produced significant reduction in blood glucose of normal rats.
Key words: Anti Diabetic activity, Trushanadiloha, Aqueous extract, Herbo mineral formulation.
INTRODUCTION
The Herbomineral formulation Trushanadi loha is
mentioned by Yogaratnakara in the chapter of Medo
Roga chikitsa. It is Medohara, Mehara, Kustahara,
Sleshmavyadiara, Athi Stholya hara, Best Rasayana.
During the administration of medicine no restriction
in ahara and vihara is advised by him. To evaluate the
Mehahara (antidiabetic) activity In- Vivo study is
conducted.
MATERIALS AND METHODS
Borosil soxhlet extractor
Solvent evaporator
Digital balance
(ELB 300, Manufactured from – SHIMADZU)
All reagents and chemicals used were – AR Grade.
Preparation of Extracts
1kg of herbomineral powder Trushanadiloha sample
was crushed to coarse powder and passed through
sieve # 44. The sieved powder was stored in air tight,
high density polyethylene containers before
extraction. Extraction was performed by using sox let
apparatus (12 hours), carried out first with petroleum
ether (60-80 ºC) to de-fat the material. The de-fatted
material was then extracted with Benzene,
Chloroform, Acetone, ethanol and water to get
respective extract1
. The extracts was concentrated for
further studies at reduced pressure and temperature in
a rotary evaporator and tested for the presence of
secondary metabolites by different phyto chemical
tests. Different concentrations of extract were
prepared by dissolving the fine powder of extract in
10% aqueous dimethylsulfoxide (DMSO) for further
study.
International Journal of Research in
Pharmacology & Pharmacotherapeutics

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Preparation of Trushanadiloha
The drugs 1,3,4,5,6,7,8,9,10,12,13 mentioned in table
no - 1 are powdered and kept separately No.2 i.e.
Pippali are fried in cow’s ghee then powdered no.9
i.e. Bakuchi seeds purification was done in cow’s
urine for above 7 days. After 7 days it was dried and
powdered. No.14 i.e. loha (Iron) was taken in the
form of pure bhasma (ash/catalyst) and all 14
ingredients in equal quantity are mixed together
thoroughly and kept in a glass container.
Table.1. Composition of Trushanadiloha
Sl.No. Name of the Drug Latin Name Parts Used
1
Shunti Zingiber officinalis - Roxb Rizome
2 Pippali Piper langum linn Roots,Dried spices
3 Pippalimoola Piper langum root Root
4 Maricham Piper nigrum linn Fruits
5 Hareethaki Terminalia chebula ret Z Fruit coat
6 Amlaki Emblica officinalis gaertn Fruit coat
7 Vibhithaki Terminalia bela rica roeb Fruit coat
8 Chitramoola Plumbago zeylanica linn Root bark
9 Backuchi Psoralia carlifolia linn Seeds(Purified)
10 Saindava lavana Rock salt -
11 Souvarcha lavana - -
12 Bida lavana - -
13 Kacha lavana - -
14 Loha bhasma Purified iron catalyst(ash)
-
Preliminary Phytochemical Analysis
The Petroleum Benzene, Chloroform, Acetone,
ethanol and aqueous extract were screened for the
phytochemical constituents using the standard
method .The phytochemical components analyzed
were alkaloids, steroids, starch, proteins,
anthraquinone glycosides, saponins, flavonoids,
tannins, and cardiac glycosides.
Table.2. Phytochemical Screening of Herbomineral formulation with cow’s ghee and honey as
vehicle.
Test
Name of the extract
Petroleum
Ether
Chloroform Benzene Ethanol Acetone Aqueous
Alkaloids + + + + + -
Steroids + + + - + -
Carbohydrates - - - - - -
Starch - - - - - -
Proteins - - - + + +
Glycosides - - - - + +
Tannins &
Phenolics
+ - - + + +
Test for flavonoids - - - + + +
Note: ‘+’ Indicates Presence, ‘-’ Indicates Absence

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Acute oral toxicity studies
Animals
For the experiment Swiss albino mice of either sex, 3-4
weeks of age, weighing between 20-25 g, were
procured from Mahaveer Enterprises, Hyderabad, India
were used in the studies. Animals were maintained
under standard environmental conditions
(temperature: (24.0±1.0°), relative humidity: 55-65%
and 12 h light/12 h dark cycle) and had free access
to feed and water ad libitum. The animals were
acclimatized to laboratory condition for one week prior
to experiments. All protocols for animal experiment
were approved by the Institutional Animal Ethical
Committee (IAEC bearing No. 439/01/a/CPCSEA),
Shri Vishnu College of Pharmacy, Bhimavaram Andhra
pradesh.India.
Administration of doses
The Aqueous extract of Trushanaadiloha was
administered in a single dose by gavage using a
stomach tube or a suitable intubation canula.
Animals should be fasted prior to dosing (e.g. in mouse,
food but not water should be withheld for 3-4 hours).
Following the period of fasting, the animals should be
weighed and the test substance administered. After the
substance has been administered, food may be withheld
for 1-2 hours in mice. Where a dose is administered in
fractions over a period of time, it may be necessary to
provide the animals with food and water depending on
the length of the period. Groups of animals of a single
sex are dosed in a stepwise procedure using the fixed
doses of 5, 50, 300 and 2000 mg/kg (exceptionally an
additional fixed dose of 5000 mg/kg may be considered.
The initial dose level is selected on the basis of a
sighting study as the dose expected to produce some
signs of toxicity without causing severe toxic effects or
mortality. Clinical signs and conditions associated with
pain, suffering, and impending death, are described in
detail in a separate OECD Guidance Document .
Further groups of animals may be dosed at higher or
lower fixed doses, depending on the presence or
absence of signs of toxicity or mortality. This procedure
continues until the dose causing evident toxicity or no
more than one death is identified, or when no effects are
seen at the highest dose or when deaths occur at the
lowest dose. Exceptionally, and only when justified by
specific regulatory needs, the use of an additional upper
fixed dose level of 5000 mg/kg may be considered. For
reasons of animal welfare concern, testing of animals in
GHS Category 5 ranges (2000-5000mg/kg) is
discouraged and should only be considered when there
is a strong likelihood that results of such a test have a
direct relevance for protecting human or animal health
or the environment. Period of at least 24 hours will be
allowed between the dosing of each animal. All animals
should be observed for at least 14 days. The sample was
negative for carbohydrates and starch. Steroids,
proteins, glycosides, tannins and for flavonoids. The
presence of the bioactive compounds in the crude drugs
have been linked to their activities against disease
causing micro organisms and also offering the plants to
protect themselves against infections by pathogenic
micro organisms. The present study was conducted to
evaluate the acute oral toxicity of Herbomineral
formulation with cow’s ghee and honey as vehicle in
albino wistar rats. In the sighting study, the test
substances were administered in sequential manner to
one animal each at 2000 and 5000 mg / kg body weight
followed by four animals at 5000 mg/ kg-1 body weight
in the main study; whereas the test materials with well
documented traditional use were evaluated at 5000 mg /
kg-1 body weight. The treated animals were observed
for mortality, untoward clinical/toxic signs, and
alterations in body weight gain during the study. The
treated animals survived throughout the study period
and did not reveal any treatment related major abnormal
clinical signs at the tested dose levels for all the
products. In conclusion, acute oral toxicity testing of
screened Herbomineral formulation did not produce any
treatment-related adverse effects up to the dose level of
5000 mg/ kg-1 body weight.
Anti Diabetic activity
Animals
For the experiment Swiss albino mice of either sex, 3-4
weeks of age, weighing between 20-25 g, were
procured from Mahaveer Enterprises, Hyderabad, India
were used in the studies. Animals were maintained
under standard environmental conditions
(temperature: (24.0±1.0°), relative humidity: 55-65%
and 12 h light/12 h dark cycle) and had free access
to feed and water ad libitum. The animals were
acclimatized to laboratory condition for one week prior
to experiments. All protocols for animal experiment
were approved by the Institutional Animal Ethical
Committee (IAEC bearing No. 439/01/a/CPCSEA),

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Shri Vishnu College of Pharmacy, Bhimavaram Andhra
Pradesh.
Administration of doses
The Trushanaadiloha was administered in a single dose
by gavage using a stomach tube or a suitable intubation
canula.
Experiment design
Rats were fasted for 18 h before and during the
experiment they were withdrawn from food and water.
Rats were administered orally 18mg/200g of herbal
formula without addition of honey and ghee. Blood
samples were withdrawn by retro orbital puncture at 0,
1, 2, 3, 4, 6 and 8h and were analyzed for blood glucose
by glucose oxidase/peroxidase (GOD/POD) using
commercial glucose kits. 40 percent of serum glucose
reduction is observed on 3rd hour.
RESULT AND DISCUSSION
40 % reduction in blood glucose levels was observed
and found to the herbomineral formulation produced
significant reduction in blood glucose of normal rats.
The sample was negative for carbohydrates and starch.
It was positive to tests of alkaloids, steroids, proteins,
glycosides, tannins-phenolics and for flavonoids. The
presence of these bioactive compounds in the crude
drugs have been linked to their activities against disease
causing micro organisms and also offering the plants to
protect themselves against infections by pathogenic
micro organisms. The present study was conducted to
evaluate the anti diabetic activity of aqueous extract of
herbomineral formulation in albino Wister rats. In
conclusion anti diabetic testing of screened
herbomineral formulation produced significant
reduction in blood glucose of normal rats.
EFFECT OF HERBOMINERAL FORMULATION
ON NORMAL RAT BLOOD GLUCOSE
Dose given to each rat =18mg/200g of rat
Table. 3. After administration of herbaomineral formula rat Serum Glucose changes.
Time 0hr 1hr 2hr 3hr 4hr 6hr
Mg/dl 113 105 92 79 90 105
Mg/dl 120 89 76 65 102 82
Mg/dl 117 82 60 66 70 79
Table. 4. Percentage of glucose reduction
Time 1hr 2hr 3hr 4hr 6hr
% 7.00 19.00 30.00 20.00 7.00
% 26.00 37.00 46.00 32.00 15.00
% 30.00 48.00 44.00 40.00 32.00
mean 21.00 34.67 40.00 30.67 18.00

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Graphical representation of blood glucose reduction.
ACKNOWLEDGEMENT
The authors are thankful to the Principal, Shri Vishnu
College of Pharmacy, Bhimavaram Andhra
pradesh.India, for their cooperation and guidance to
complete this Research work.
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