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Evaluation of In-Vitro Anti Diabetic Activity of a Siddha Herbo- Mineral Drug

Article · July 2015

DOI: 10.2174/2215083801666150701163117

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84 Current Traditional Medicine, 2015, 1, 84-90

Evaluation of in-vitro Anti Diabetic Activity of a Siddha Herbo-

Mineral Drug

S. Merish1*, M. Tamizhamuthu2, Thomas M. Walter3 and T. Sasi Priya4

1
Student Editor, [Link], Tirunelveli, Tamilnadu, India; 2Consultant,
Walter Siddha Research (Bethesda Siddha Research Centre), Tirunelveli, Tamilnadu,
India; 3Asst. Professor, Government Siddha Medical College, Palayamkottai,
Tirunelveli, India; 4Final BSMS, Government Siddha Medical College,
Palayamkottai, India
Abstract: The nature has provided abundant plant wealth for all the living creatures, which
possess medicinal virtues. Therefore, there is a necessity to explore their uses and to
ascertain their therapeutic properties. In that way, the Traditional Siddha Medicine plays a
vital role in preventing the progress of the disease. Saint Yugi (one of the Founders of Traditional Indian
Siddha system of Medicine) describes 21 types of Mega noigal, which includes Madhu megam (Madhu –
Sweet, Megam – Disease) [1, 2]. Diabetes mellitus is compared with Madhu megam in Siddha literature.
Diabetes mellitus is a complex and a diverse group of disorders that disturbs the metabolism of carbohydrate,
fat and protein. The number of diabetes mellitus cases has been increasing worldwide in recent years. Insulin
is a key player in the control of glucose homeostasis. Lack of insulin affects carbohydrate, fat and protein
metabolism. Management of diabetes without side effects is still a great challenge to the medical community
[3]. The aim of the present study is to investigate the in-vitro Anti-Diabetic activity of the selected Herbo-
Mineral drug, having its reference in age old Siddha classic text “Sarabhendhirar Vaithiya Ratnavali”. Thus,
validating the traditional claim of the Herbo-mineral drug exhibits a significant inhibitory effect on In-vitro
glucose diffusion and the result is being discussed.

Keywords: Anti-diabetic activity, Glucose uptake assay, Siddha medicine, Traditional diabetic drug.

1. INTRODUCTION along with the synthetic drugs, many agents of the

Diabetes mellitus (DM) is the commonest plant origin are also in use particularly for the
endocrine disorder that affects more 347 million treatment of Non-Insulin Dependent Diabetes
people throughout worldwide; More than 80% of Mellitus (NIDDM) [5].
people with diabetes live in low- and middle- Diabetes is fast gaining the status of a potential
income countries (WHO Diabetic Program report) epidemic in India with more than 62 million
[4]. The word “Diabetes” is adapted from the Diabetic individuals currently diagnosed with the
Greek word “a siphon” which means a “passer disease. In 2000, India (31.7 million) topped the
through”. It is caused by deficiency or ineffective world with the highest number of people with
production of insulin by pancreas which results in diabetes mellitus followed by China (20.8 million)
increase or decrease in concentrations of glucose with the United States (17.7 million) in second and
in the blood. It is found to damage many of the third place respectively. According to Wild et al.
body systems, particularly the blood vessels and the prevalence of diabetes is predicted to double
nerves (Nagappa et al., 2003). For its therapy, globally from 171 million in 2000 to 366 million
in 2030 with a maximum increase in India. It is
*Address correspondence to this author at the Student Editor, predicted that by 2030 diabetes mellitus may
[Link], Tirunelveli, Tamilnadu, India; afflict up to 79.4 million individuals in India,
Tel:/Fax: +919443056180; E-mail: drmerishjesus@[Link] while China (42.3 million) and the United States

2215-08/15 $58.00+.00 © 2015 Bentham Science Publishers

Evaluation of in-vitro Anti Diabetic Activity Current Traditional Medicine, 2015, Vol. 1, No. 2 85

(30.3 million) will also see significant increases in Valley (Northern India), 11.6 per cent in New
those affected by the disease [6]. Delhi (Northern India), and 9.3 per cent in West
India (Mumbai) compared with (13.5 per cent in
Hyperglycemia, or raised blood sugar, is a Chennai (South India), 16.6 per cent in Hyderabad
common effect of uncontrolled diabetes and over (South India), and 12.4 per cent Bangalore (South
time leads to serious damage to many of the body's India) [6, 8].
systems, especially the nerves and blood vessels
(WHO 1999). In the short term, hyperglycemia
Signaling Pathway
causes symptoms of increased thirst, increased
urination, increased hunger, and weight loss. When high levels of glucose are detected by the
However, in the long-term, it causes damage to beta cells in pancreas, insulin is released by the
eyes (leading to blindness), kidneys (leading to cells into the blood stream. This insulin circulates
renal failure), and nerves (leading to impotence in the blood and binds to the receptors embedded
and foot disorders/possibly amputation). As well, in the cell membrane of the muscle, liver, adipose
it increases the risk of heart disease, stroke, and and brain cells. Then the receptor became auto-
insufficiency in blood flow to legs. Several studies phosphorylated by adding phosphate group on its
have shown that good metabolic control prevents intracellular domain as well as tyrosine residues of
or delays these complications [7]. IRS1 (Insulin receptor substrate) protein. This
activation will lead to activation of a signaling
Current Scenario Statistics- India
cascade. The activated receptor protein then adds
Preliminary results from a large community the phosphate group to another protein called PI3
study conducted by the Indian Council of Medical kinase (phosphatidylinositol 3 kinase) that pass the
research (ICMR) revealed that a lower proportion signal to the next step in the signal pathway [9].
of the population is affected in states of Northern This PI3 kinase will phosphorylate the PIP2 and
India (Chandigarh 0.12 million, Jharkhand 0.96 that will activate other downstream kinases and
million) as compared to Maharashtra (9.2 million) finally the PKB became activated and this PKB
and Tamil Nadu (4.8 million). The National Urban will facilitate the translocation of Glut 4 (Glucose
Survey conducted across the metropolitan cities of transporter) to the plasma membrane. This will
India reported similar trend: 11.7 per cent in allow the transport of glucose into the cells.
Kolkata (Eastern India), 6.1 per cent in Kashmir

Fig. (1). Insulin signaling pathway.

86 Current Traditional Medicine, 2015, Vol. 1, No. 2 Merish et al.

Complications Associated with Diabetes glucose. Neuropathy can lead to sensory loss and
Mellitus damage to the limbs. It is also a major cause of
impotence in diabetic men. Diabetic foot disease,
Diabetic retinopathy is a leading cause of
due to changes in blood vessels and nerves, often
blindness and visual disability. Diabetes mellitus is
leads to ulceration and subsequent limb amputation.
associated with damage to the small blood vessels
It is one of the most costly complications of
in the retina, resulting in loss of vision. Loss of
diabetes, especially in communities with inadequate
vision due to certain types of glaucoma and cataract
footwear. It results from both vascular and
may also be more common in people with diabetes
neurological disease processes [10, 11].
than in those without the disease. Good metabolic
control can delay the onset and progression of
diabetic retinopathy. There is evidence that, even MATERIALS AND METHODS
in developed countries, a large proportion of those a) Ingredients of the Test Drug
in need are not receiving such care due to lack of
public and professional awareness, as well as an 1) Naval / Syzygium cumini
absence of treatment facilities. In developing 2) Padikaram / Alum
countries, in many of which diabetes is now
common, such care is inaccessible to the majority The reference for this preparation was taken
of the population [11]. from the Classic Siddha text, “Sarabhendhirar
Vaithiya Ratnavali”. The Trial drug was prepared
Diabetes is among the leading causes of kidney as per a Standard Operative Procedure (SOP).
failure, but its frequency varies between populations
and is also related to the severity and duration of
the disease. Several measures to slow down the b) Plant Material Authentication
progress of renal damage include control of high The fresh fruit seeds of Syzygium cumini were
blood glucose, control of high blood pressure, collected from Tirunelveli district and authenticated
intervention with medication in the early stage of by the Faculty of Department of Gunapadam (Herbal
kidney damage, and restriction of dietary protein. Pharmacology), Government Siddha Medical
Heart disease accounts for approximately 50% of all College, Palayamkottai, Tamilnadu, India.
deaths among people with diabetes in industrialized
countries. Risk factors for heart disease in people c) Purification of Drugs
with diabetes include smoking, high blood
pressure, high serum cholesterol and obesity. In Siddha system of medicine, purification of
raw drugs is an important procedure in making
Diabetic neuropathy is probably the most medicine in order to reduce its toxic effects and
common complication of diabetes. Studies suggest also to potentiate its therapeutic efficacy. The
that up to 50% of people with diabetes are affected dried Fruit seeds of Syzygium cumini were
to some degree. Major risk factors of this condition purified by removal of unknown particles and dust
are the level and duration of elevated blood from the sample and then sent to the powdering

Table 1. Information about the ingredients of the Medicine [15, 16].

Common name Botanical name/

S. No Phytochemistry Actions Uses in Siddha
Tamil/English Family

1 Naval/Jambul syzygium cumini/ β-sitosterol, Quercetin, Astringent Effective in treating Cough,

Kaempferol, Myricetin, Menorrhagia,
Myrtaceae Stomachic
Tannins, Gallic acid
Diuretic Skin disease & Blood stained
stools.
Tonic

2 Padikaram/Alum _ Alumen Astringent Cures Stomach ulcers

Styptic (APD), eye diseases, Arrests
bleeding.
Antiseptic
Anti-inflammatory
Evaluation of in-vitro Anti Diabetic Activity Current Traditional Medicine, 2015, Vol. 1, No. 2 87

Fig. (2). Purification process of Padikaram (Alum).

process [12]. Purification of Padikaram (Alum) is Principle

a Series of process (as shown in Fig 2) Glu cos e
‘Padikaram’ (Alum) was powdered and heated in D-Glucose + H2O2+ O2 ⎯Oxidase
⎯⎯ ⎯→
an earthenware. It was stirred until it changed into D-Gluconic Acid+H2O2
dry Crystalline form. After its crystalline water
evaporated completely and the end product was H2O2 + Reduced o-Dianisidine ⎯⎯ ⎯⎯→Peroxidase

collected [1, 13]. Oxidised o-Dianisidine

(Colourless) (Brown)
d) Preparation of Sample Oxidised o-Dianisidine ⎯⎯ ⎯→
H 2SO 4

The both powdered end product was filtered in Oxidised o-Dianisidine

pure white cloth (Vasthira kayam), weighed and (Brown) (Pink)
stored in an air tight container [14].
Glucose is oxidized to gluconic acid and
This End product was subjected to inhibitory hydrogen peroxide by glucose oxidase. Hydrogen
effect of glucose utilization using specific standard peroxide reacts with o-dianisidine in the presence
in vitro procedure. of peroxidase to form a colored product. Oxidized
o-dianisidine reacts with sulfuric acid to form a
e) Preparation of Extracts more stable colored product. The intensity of the
The shade dried powdered form of Fruit seeds pink color measured at 540 nm is proportional to
of Syzygium cumini and Purified powdered the original glucose concentration [17].
Sodium Bi-Borate were finely powdered and the Reagent Used
end Product were subjected to defatting using 80%
Petroleum ether and the filtrate was dried and Glucose Oxidase/Peroxidase Reagent
subjected to soxhilation. Briefly 50grams of plant
material was filled on extractor of a soxhlet o-Dianisidine Reagent
apparatus and subjected to soxhlation of 5-7 cycles Glucose Standard Solution
at 75oC. The extract was further condensed using
rotatory evaporator and used for further studies. Sulfuric Acid, ACS reagent
88 Current Traditional Medicine, 2015, Vol. 1, No. 2 Merish et al.

Fig. (3). Graphical representation of assay results.

Apparatus glucose consumption was measured in cell free

media using Glucose assay kit (Sigma Aldrich) as
1) Spectrophotometer or colorimeter suitable
per manufacturer’s.
for measuring absorbance at 540 nm.
Instructions. All experiments were repeated in
2) Cuvettes
triplicates and mean average was used for
3) Test tubes, 18 mm X 150 mm calculations [19, 20].
4) Pipettes capable of accurately dispensing OD of control − OD of test
volumes from 20 μl to 2.0 ml. % Glucose uptake = × 100
OD of control
5) Water bath capable of maintaining
temperature at 37 ± 1 °C. RESULTS AND DISCUSSIONS
Procedure Graphical Representation of Assay Results
Dilute sample with deionized water to It is concluded that the Siddha Herbo-mineral
approximately 20-80 μg glucose/ml. Filter or drug which is being used to control the diabetes,
deproteinize solution if necessary to clarify. exhibits 49.36% of inhibition at 100 µg/ml
Decolorize solutions that are strongly colored and concentration level when compared to the control
that have a low glucose concentration. Degas drug Pioglitazone which shows 73.14 % at 10
carbonated or fermented products [18]. µg/ml. This result clearly shows the Anti-diabetic
properties in higher the concentration. The present
60% confluent cells were starved overnight in study demonstrates the ability of the test drug to
PBS and individual cultures were exposed to inhibit glucose diffusion using an in-vitro model of
300mm glucose followed by Test drug plant glucose absorption. It can be concluded from the
extracts in increasing concentrations (10, 50, 100 data that the drug is beneficial in controlling the
µg/ml) and incubated for 2 hours. Pioglitazone blood glucose level, improves the lipid metabolism
was used as the standard drug and a positive and prevents diabetic complications.
control with glucose alone was maintained. The
Evaluation of in-vitro Anti Diabetic Activity Current Traditional Medicine, 2015, Vol. 1, No. 2 89

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Received: February 20, 2015 Revised: February 28, 2015 Accepted: March 06, 2015

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