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Study of ameliorating properties of Tinospora cordifolia on Diabetes and

acute Pancreatitis in Alloxan treated Rats

Article  in  Der Pharmacia Lettre · November 2016

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Der Pharmacia Lettre, 2016, 8 (18):133-140

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ISSN 0975-5071
USA CODEN: DPLEB4

Study of ameliorating properties of Tinospora cordifolia on Diabetes and acute

Pancreatitis in Alloxan treated Rats
Kumud Ranjan Thakur*, S. R. Padmadeo, Bipin Bihari Mishra and Kumar Pranay

Department of Biochemistry, Patna University, Patna- 800005, Bihar, India

_____________________________________________________________________________________________

ABSTRACT

Diabetes and acute pancreatitis are major health concern globally. India has been growing as a diabetic capital of
the world. Diabetes and acute pancreatitis were induced by administration of Alloxan intraperitoneally
150mg/kg.b.wt in to Wistarnorvegicus. Diabetes induction was confirmed by blood glucose concentration above
400mg/dl. The desired plant Tinospora cordifolia (250mg/kg.b.wt) stem extract showed significant remedial action
by lowering blood glucose to normal (87mg/dl) and restoring Pancreatic enzymeslipase to (1.74IU/L) and amylase
to (583.1IU/L). These results were further authenticated by remodeling of pancreatic histoarchitecture which
showed increase in no. Islet of Langerhans, acinar cells, decreased vacuolar space, sinusoidal space and atrophy.

Key words: Alloxan, Acute pancreatitis, Tinospora cordifolia, Histoarchitecture

_____________________________________________________________________________________________

INTRODUCTION

Diabetes mellitus is characterized by chronic hyperglycemia with disturbance of carbohydrate, fat and protein
metabolism resulting from defect in insulin secretion, insulin action or both [1].

The effect of diabetes mellitus include long term damage, dysfunction and failure of various organs especially eye,
kidney, heart and vessels. Typical characteristics symptoms associated with diabetes are thirst, polyuria, blurring of
vision, weight loss, polyphagia and in its most severe condition with ketoacidosis or non ketotichyperosmolarity
which in the absence of effective treatment leads to stupor, coma and death. Diabetes causes heavy burden on the
income and leads to death. Annual reports of WHO 2016 has estimated that 422 million adult aged over 18 were
diabetic in 2014.WHO surveyed South East Asia and western pacific region which covers approximately half of the
world diabetic population. It was estimated that total death burden till 2012 to be 3.7 million in which 2.2 million
deaths from cardiovascular disease, chronic kidney disease and tuberculosis related to higher than optimal blood
glucose. It was also mentioned that 43% of all death due to high blood glucose occur before the age of age 70[2]. It
is estimated that 35 million in our country already have diabetes and it is expected to reach 70 to 80 million by
2030AD.

Acute pancreatitis is caused due to inflammation of pancreatic gland which may arise due to various reasons like
reactive oxidative stress, hypercalaemia, hyperlipidemia, viral infections, some drugs like sulfonamides, steroids etc.
However; the exact mechanism behind acute pancreatitis has not been understood yet and due to this there is
scarcity of proper treatment.

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The present research includes the condition called acute pancreatitis resulting from altered exocrine activity which is
the major concern now a day.

Experiments shows 50% prevalence of non pancreatic diabetes 35% in IDDM and NIDDM in case of exocrine
abnormality by using direct and indirect pancreatic function test [3]. Serum lipase is of pancreatic origin whose
concentration is found to be 5,000 times more than other tissues [4, 5].However lipase is also secreted by stomach,
duodenum, liver, heart and tongue [6, 7, 8].

More than threefold elevated level of serum amylase and lipase is found to be the characteristic of acute pancreatitis
and meant to be the clinical manifestation used for the diagnosis and its treatment [9, 10].

Intensive use of allopathic medicine causes various life threatening side effects to human body. However treatment
by means of natural products is relatively free of side effects and is also cost effective.

Tinospora cordifolia, the desired plant for experiment commonly called as “ Guduchi” in Sanskrit and ‘Giloy’ in
hindi belongs to Menispermaceae family[11]. It has a wide range of phytoconstituents which include cordifolide,
heptacosanol, clerodanefuranoditerpene, diterpenoidfuranolactone, tinosporine, tinosporide, berberine, magniflorine,
choline[12], N- trans- ferulolyl tyramine[13], tinocordiside B,C and D[14]. It has wide range of medicinal properties
so it is exploited as a most demanding Ayurvedic medicine for the treatment of allergy, arthiritis, inflammation,
lowering of fever, wound, pneumonia, asthma and cough[15,16].

Some of the active component isolated from stem of Tinospora cordifolia like magnoflorine, syringing, N-
formylannonain, cordifolioside, 11-hydroxymustakone and N- methyl -2- pyrolidone enhances immunomodulatory
properties, phagocytic activity of macrophages, ROS formation by neutrophil[17,18,19,20,21].It also shows most
potent activator of IL-6 cytokines which cures injuries, inflammation, activation of cytotoxic T cell and B cell
differentiation [22].In traditional folk medicine it is widely used as hypoglycemic agent which act by promoting
insulin secretion, inhibiting gluconeogenesis and by mitigating oxidative stress[23].

MATERIALS AND METHODS

For the present research work wistar rats (Rattusnorvegicus) were selected which were obtained from animal
market, Tripolia, Patna city, Bihar and breeded up to 3-4 generations in the Biochemistry lab of Patna University to
ensure the purity of strain.

Housing
Rats were kept separately in the ratio of two females per male in polypropylene cages of different size Small cage-
26 x 19x13 (h) cms for breeding and Large cage- 40 x 25 x 15 (h) cms for experimentation. Rats were kept in
environment that are compatible with life, health and comfort, in such a way that regular needs of the animals, like
feeding, cleaning, handling and the turnover of stock could be conveniently met. Water was provided ad libitum

Physical Environment
The temperature of the rat experimentation room was in the range of 24 o C–28o C. Twelve hours of light and twelve
hours of darkness were provided in the room for their optimal growth and reproduction. The light intensity and
humidity of the room were maintained at an optimal level.

Feeding
The laboratory rats were fed on laboratory prepared enriched bread constitutes wheat flour, jaggery, powdered milk
and gram flour. For providing vitamin supplement they were fed with carrot, sprouted gram and sprouted moong
bean.

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Details of rats grouping and treatment given to rats

Cage no Treatment Average weight No. of mice each cage Selected dose (mg/kg b.w)
1 Normal / Control 100-120 gm 5 Normal saline
2 Alloxan treated 100-120 gm 5 150 mg /kg b.wt
3 7daytreated Tinospora cordifolia 100-120 gm 5 250 mg/kg b.wt
4 14 days treated Tinospora cordifolia 100-120gm 5 250 mg/kg b.wt
5 21 days treated Tinospora cordifolia 100-120 gm 5 250 mg/kg b.wt

Preparation of ethanolic extracts of Tinospora cordifolia stem (TCS)

The collected leaves of Tinospora cordifolia were dried under shade and undergone crushing in electric blender to
form powdered and subjected to extraction by soxhlet's extractor using distilled ethanol as a solvent(90% ethanol)
in ratio of 1:5 (100 g powder with 500 ml solvent). The extraction was performed for 18 h. The extract was
concentrated by evaporating in vaccum using Rota vapour (Popular, India) at 60oC temperature to ensure minimum
denaturation of phytocemicals.

Administration of the extract

Suspension of ethanolic extract was prepared in normal saline. The extracts of Tinospora cordifolia(TC) were
administered in a dose of 250mg/kg. b.wt, which were selected as per our preliminary studies for its hypoglycemic
effect. And Control groups were given Alloxanin normal saline respectively.

Blood collection was done through tail clipping to obtainserum whereas tissue was collected from pancreas after
dissection of anaesthesized decapitated rats.

Biochemical assay: For Biochemical analysis was done in Semi-Automatic Chemistry Analyzer (BiosystemBTS-
350,Costra Brava 30, 08030, Barcelona).

All the biochemical assessments have been done for normal, control and Tinospora cordifolia treated rats. 3
observations have been taken in each group.

Glucose test was performed by using Dr. MorpenGluco(Model: BG-03) one blood glucose monitoring system .
Amylase was estimated through CNPG3 method obtained from Lifechem, Kamineni Life Sciences, India and lipase
by Kinetic colorimetric method obtained from EURO Diagonostic, Spain.

Histopathological assay:
After the rats were sacrificed, Pancreas was immediately separated devoid of fat bodies, chopped and fixed in 10%
neutral formaline. Thereafter the tissues were dehydrated in a graded series of alcohol and embedded in paraffin
wax. A 5 µm tissue section was cut on microtome, fixed on egg albumin, deparaffinized in xylene and then hydrated
descending series of alcohol up to water. Again dehydration was done up to 70% and then stained with Eosin and
Hematoxylene and mounted on DPX for microscopic study.

RESULTS

Pancreas plays a very important role as exocrine and endocrine organ. Endocrine role of pancreas is significant in
metabolizing glucose which is an important energy source, cells take up the glucose in the presence of insulin to
provide energy, in the absence of insulin cells starved and death .In the present experiment alloxan induced diabetic
rats has glucose concentration of 600.2±0.158mg/dl, which drastically decreased to 293.2±0.158mg/dl on treatment
with Tinospora cordifolia on 7thdays. On further treatment with Tinospora cordifolia on 14th days decreases to
149.2±0.158mg/dl and on 21st days glucose decreases to 87.4±0.158mg/dl showed hypoglycemic activity of
Tinospora cordifolia. In the present research work pancreatic function tests like serum amylase and lipase were
performed to study exocrine activity along with its histopathological correlation. Normal rats having 600±0.152
IU/L concentration of serum amylase when treated with alloxan has amylase level of 800±0.150IU/L. which on
treatment with phytochemical extract restoration was seen ie, 787±0.09IU/L, 725±0.014IU/L and 583.1±0.121IU/L
was seen in 7, 14 and 21 days respectively. Simultaneously in case of serum lipase, most potent pancreatic function
market similar trend was seen i.e., 1.09±0.08IU/L in case of normal, 6.26±0.120IU/L in case of diabetic but after
administration of extract to diabetic rats value significantly declined to 5.11±0.100IU/L, 4.05±0.06IU/L and
1.74±0.112IU/L in consecutive 7, 14 and 21 days.

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i
i

i i

v v

In this study it was found that both serum amylase and lipase were found to be elevated more than threefold the
upper limit shows the condition of acute pancreatitis on exposure to alloxanbut after treatment with Tinospora
cordifoliaphytoextract both serum lipase and amylase were found to be close to normal this result shows both the
property of Tinospora cordifolia, ie., damage caused in pancreatic acinar cells and regulating the digestive function
and mitigating intestinal disorder generally caused in the diabetic condition.

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Simultaneously looking towards the histopathological changes we got a positive result regarding repairing of cell
damage and its recovery to healthy condition.

Figure 1 shows the normal histoarchitecture of pancreas having normal acinar cells, islets of langerhans and other
cells. After treatment with Alloxan 150mg/kg.b.wt intraperitoneally (figure iii and iv) cellular damage had been seen
confirmed by tissue degeneration, atrophy, damaged islet of langerhans, widened sinusoids and degenerated acinar
cells. Figure v and vi shows phytoextract treated group of 7 and 14 days respectively which confirms regeneration of
Islets of Langerhans, Acinar cells, gradual increase in cellular density, decrease in sinusoidal space and atrophy was
seen. Figure vi shows pancreatic tissue of 21 days phytoextract treated group which shows therapeutic effect by the
recovery of damaged cellular part of pancreas like regeneration of islets of Langerhans, β cells, clear outline of
acinar cells with prominent granules, narrowness of tissues space, reduced atrophy and vacuole formation.

DISCUSSION

In the present study Rats demonstrated a significant change in biochemical parameters in terms of blood glucose
level, serum amylase, and lipase level as well as in histoarchitecture of pancreatic tissue when exposed to alloxan
(150mg/kg.b.wt) followed by treatment with ethanolic extract of Tinospora cordifolia (250mg/kg.b.wt) for 21 days
at an interval of 7 days respectively. As reported in previous works [24, 25, 26].

In the present investigation administration of alloxan significantly brought destruction of pancreatic tissue which is
evident from the increase in fasting glucose concentration compared with the control group. This result is in
agreement with [26, 27].

Level of serum amylase and lipase which are secreted from pancreas acinar cell [28] are the important diagonistic
marker of acute pancreatitis. In the present work treatment with alloxan caused increase in amylase and lipase
indicating pancreatitis effect in accordance with findings of [28, 29, 30]. Similarly Alloxan treatment leads to
significant change in histoarchitecture of pancreas as compared to normal which is evident by changes such as
vacuolization, necrosis, cellular atrophy and hyperchromic nuclei as reported by other workers [30].

However treatment of rats suffering from diabetes and pancreatitis due to alloxan exposure with ethanolic stem
extract of Tinospora cordifolia at a dose of 250mg/kg.b.wt significantly restored the blood glucose which is in
agreement with previous workers[31,32] indicating the anti hyperglycemic effect of the desired herbal extract [33].
In the present work treatment with Tinospora cordifolia extract leads to decrease in level of amylase and lipase level
to normal level on 21 days treatment, which may be due to presence of flavonoid[34]. Histopathological studies of
pancreatic section of rats treated with ethanolic stem extract of Tinospora cordifolia revealed significance
amelioration capability to this plane as compared to alloxan treated group which is evidenced by reduction in
atrophy, vacuole and necrosis followed by regeneration of acinar cell.

Figure shows (i) Normal rats histoarchitecture having normal cellular arrangement islet of Langerhans (IL) and
Acinar cell (Ac). (ii and iii) shows the Alloxan treated Diabetic rats atrophy, tissue degeneration , poor cellularity of
islets of langerhans (IL) and damaged islets cells along with hyperchromicity in diabetic control group. Widening of
sinusoids(s), necrosis(N) and degenerated Acinar cells (Ac). (iv and v)shows phytoextract treated of 7 and 14 days .
Increase in the density of cells, decrease in vacoulation, atrophy and sinusoidal space.

(vi) shows section of pancreas showing regenerating islet of langerhans (IL), β-cells (B) and clear outlines of Acinar
cell with prominent granules (Ac) in Tinospora cordifoliaethanolic extract treated pancreas of rat. Narrowing of
tissue space, reduced atrophy and vacuole formation.

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TEXT GRAPH-1

Effect of Tinospora cordifolia phytoextract on serum amylase in

alloxan induced diabetic rats
1000
Concentration of serum

800
600
Amylasein IU

400
200
0 Amylase(IU)
control Aloxan 5 Days 10 days 15 Days
treated Extract Extract Extract
Duration of dose in days
.

TEXT GRAPH-2

Effect of Tinospora cardifolia phytoextract on alloxan induced diabetic

rat
800
Concentration of blood

600
glucose in mg/dl

400
200 Random blood glucose in
0 mg/dl
Control Alloxan 5 Days 10 Days 15 Days
treated Extract Extract Extract
Duration of dose in days
.

TEXT GRAPH -3

Effect of Tinospora cordifolia phytoextract on serum lipasein alloxan

induced diabetic rats
Concentration of serum lipase

8
6
4
in IU

2
Lipase(IU)
0
Control alloxan 5 Days 10 Days 15 Days
induced Extract Extract Extract
Duration of dose in Days

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CONCLUSION

The present study showed after treatment with ethanolic extract of Tinospora cordifolia for three consecutive weeks
restored the damaged histoarchitecture to the normal level which can be justified by the normal blood glucose, lipase
and amylase level.

In this way extract pronounces its hypoglycemic effect as well as its remedial exocrine property by restoring lipase
and amylase concentration close to normal level. Although study was performed in a limited sample size and
parametes but the significant changes in the choosen parameters clearly indicates its prophylactic activity. However
further extensive research need to be done to elucidate its potential as therapeutic agent for pancreatitis and mode of
action.

Acknowledgements
For the present research authors are highly thankful to DST INSPIRE Govt. of India for financing and Head of the
Department of Biochemistry, Patna University for providing infrastructures and necessary equipments.

REFERENCES

[1] American Diabetes Association Diabetes Care, 2009 Jan, 32(Supplement 1), S13-S61.
[2] Global report on diabetes by WHO,2016, ISBN 9789241565257.
[3] P.D. Hardt,Pancreatology, 2003, 3, 395-402.
[4] J.A. Lott, C.J. Lu, Clinchem.,1991, 37 361-368.
[5] M. Pantcghini,Clinchem.,1992,38, 1712-1716.
[6] T. Terada, T. Keda, Y. Nakanuma,Hepatology1993, 18, 803-808 .
[7] A. Zambon, S. Bertocco, N. Vitturi, V. Polentarutti, D. Vianello,G. Crepaldi, BiochemSoc Trans2003, 31 1070-
10704.
[8] N.W. Tietz, D.F. Shney, Clin chem.,1993, 39, 746-756.
[9] P.B. Cotton, G. Lehman, J. Vennes, J.E. Geenen, R.C. Russell, W.C. Meyers,GastrointestEndosc,1991, 37,383-
93.
[10] D.C. Whitcomb, Gut, 2004, 53, 1710-7.
[11] V. Rana, K. Thakur, R. Sood, V. Sharma, T.R. Sharma, J Genet, 2012,91, 99–103.
[12] S.S. Singh, S.C. Pandey, S. Srivastava, V.S. Gupta, B. Patro, Indian Journal of Pharmacology2003, 35, 83.
[13] A.K. Upadhyay, K. Kumar, A. Kumar, H.S. Mishra, Int J Ayurveda Res, 2010, 1, 112–21.
[14] G.R. Rout G, Z Naturforsch C. 2006, 61,118–22.
[15] V. Joshi, R.P. Joshi,Journal of Pharmacognosy &Phytochemistry2013, 2, 269-75.
[16] M. Pandey, M.K. Vyas, R. Sharma, International Journal of Pharmaceutical & Biosciences, 2012, 3, 612-8.
[17] Y.B. Tripathi, M. Sharma, M. Manickam. Rubia, Indian J BiochemBiophys. 1997, 34, 302–6.
[18] B. Bishayi, S. Roychowdhury, S. Ghosh, M.H. Sengupta, J Toxicol Sci. 2002, 27,139–46.
[19] M. Subramanian, G.J. Chintalwar, S. Chattopadhyay, Redox Rep.2002, 7, 137–43.
[20] P. More, K. Pai, ImmunopharmacolImmunotoxicol. 2012, 34,368–72.
[21] R. Upadhyaya,V. Sharma, K.V. Anita, Res J ChemSci, 2011, 1, 71–9.
[22] D.S. Sudhakaran, P. Srirekha, L.D. Devasree, S. Premsingh, R.D. Michael, Indian J Exp Biol. 2006, 44, 726–
32.
[23] M.K.Sangeetha, H.R.RaghavendranBalaji, V.Gayathri, H.R.Vasanthi, J Nat Med, 2011, 65, 544–50.
[24] G.A.Jelodhar,M.Meliki,M.H.Motadayen,S.Sirus, Indian J. Med. Sci, 2005, 59(2), 64-69.
[25] K.Hamden, M.A.Baujbina, H.Masmoudi, FM.Ayadi, K.Jamoussi, A.Elfeki,BiomedPharmacother.2009, 63, 95-
99
[26] I.Dahecha, K.S.Belgitha, K.Hamdenb,A.Fekib, H.Belgithc, H.Mejdoub,Int. J. biol. Macromol, 2011, 49, 942-
947.
[27] M.Ramar, M.Beulaj, T.Raman, A.Priyadarshini, S.Polanysamy, M.Velayudam, A.Munusamy, N.M.Prabhu,
B.Vasheeharan, Eur. J. Pharmacol. 2012, 690, 226-235.
[28] S.Jasdanwala,M. Babyatsky, Integr. Mol. Med.2015, 2(3), 189-195.
[29] P.Suguna, A.Geetha, R.Arena, and G.V, Indian journal of experimental Biology, 2013, 51, 292-302.
[30] A.H.M.Vishwanathaswamy, B.C.Koti, A.Gore, A.H.M.Thippeswamy, R.V.Kulkarni, Indian Journal of
Pharmaceutical sciences 2011, 139-145.
[31] J.K.Grover, V.Vats, S.S. Rathi, Journal of Ethnopharmacology ,2000, 77, 461-470.

139
Scholar Research Library
 Kumud Ranjan Thakur et al Der Pharmacia Lettre, 2016, 8 (18):133-140
______________________________________________________________________________
[32] P.Stanley, P.Mainzen, and M.P.Venugopal,Phytother Res,2001, 15, 213-2178.
[33] H.A.Mansour, A.Newainy Al-Sayeda, M.I.Yusuf, S.A. Sheweita,Toxicology 2002, 170, 221-228.
[34] C.C.Velusami, A.Agarwal, V.Mookambeswaran, Evidanced based Complimentary and Alternative Medicine,
2013, 7

140
Scholar Research Library

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