Research Journal of Pharmaceutical, Biological and Chemical

Sciences

(ISSN: 0975-8585)

RESEARCH ARTICLE

Investigation of Phytochemical, Pharmacological Activities of Daucus

Carota Subs P. Sativus Extract on Streptozotocin Induced Diabetic Rats.
T Venkatachalam1*, N Senthil Kumar1, K Mounika2, D Saravanan3, and
Sattanathan Kumar4.
1Department of Pharmaceutical Chemistry, JKKMMRF- Annai JKK Sampoorani Ammal College of Pharmacy B.
Komarapalaym, Namakkal -638 183, Tamil Nadu, India.
2Department of Pharmaceutical chemistry, Shree Venkateshwara college of paramedical sciences, Othakudirai,

Erode, Tamil Nadu, India.

3Department of Pharmaceutical chemistry, Jaya college of Pharmacy, Thiruninravur Chennai- 602 204, Tamil

Nadu, India.
4Department of Pharmaceutical chemistry, Paavai college of Pharmacy and Research, R. Puliyampatti, Namakkal-

637018, Tamil Nadu, India.

ABSTRACT

The present study is to explore the methanolic extracts of leaves of Daucus carota subsp sativus
and fractions were studied streptozotocin induced diabetic rats. The dried leaves were powder and
extracted with methanol solvent by using hot continuous percolation (soxhelt) method. The alkaloids,
carbohydrate, flavonoids, phenolic compounds, and tannins contents of methanolic extract and its
fractions were also determined and correlated with its antidiabetic activity. The methanol extract and
its fractions curtained for streptozotocin induced diabetic rats in this study gilbenclamide as cast-off
for standard. Methanolic extract of Daucus carota subsp sativus revealed that LD50>2000mg/kg, the
biological dose was fixed at methanolic extract Daucus carota subsp sativus 200mg and 400mg of body
weight for the extract. The methanolic extract of Daucus carota subsp sativus leaves result that revealed
of alkaloids, carbohydrate, flavonoids, phenolic compounds, and tannins. The pharmacological activity
of methanolic extract of Daucus carota subsp sativus leaves all values are expressed as statistically
significant at a*= (p<0.001) for glucose tolerance and change in blood glucose level.
Keywords: Daucus carota subsp sativus, Solvent extraction, Streptozotocin and Glibenclamide, Oral
glucose tolerance and In-vivo Anti-diabetic studies.

https://doi.org/10.33887/rjpbcs/2021.12.4.12

*Corresponding author

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 INTRODUCTION

Diabetes mellitus is a cause for growing public health concern in both developed and
developing countries. In many countries, it is now a leading cause of death, disability, and high health
care cost. The WHO estimates that diabetes affects millions of people worldwide1. Developing nations,
comprising most of the world’s population may find responding to this resolution particularly
challenging since many are now facing the double burden of both infectious and chronic, non-
communicable disease [1].

Diabetes mellitus (DM) is a group of metabolic disorders characterized by a chronic

hyperglycaemic condition resulting from defects in insulin secretion, insulin action or both. Permanent
neonatal diabetes is caused by glucokinase deficiency and is an inborn error of the glucose-insulin
signalling pathway [2].

Type 1 diabetes and type 2 diabetes are heterogeneous diseases in which clinical presentation
and disease progression may vary considerably3. The major clinical classes of glucose intolerance
include insulin- dependent diabetes mellitus (IDDM or Type1) non-insulin- dependent diabetes
mellitus (NIDDM or Type 2), malnutrition- related diabetes mellitus, Impaired glucose tolerance (IGT)
and gestational diabetes (GDM). Type 1 diabetes is thanks to autoimmune β-cell destruction, usually
resulting in absolute insulin deficiency. Type 2 Diabetes is due to a progressive loss of adequate b-cell
insulin secretion frequently on the background of insulin resistance. Gestational diabetes occur
diabetes diagnosed in the second or third trimester of pregnancy that was not clearly overt diabetes
prior to gestation [3].

Diabetes has both acute and chronic complications. They are of variable speed of onset and
severity; often adversely affect the individual’s quality of life and they result in considerable premature
disability and death. Acute metabolic complications include diabetic ketoacidosis, hypoglycaemia, and
hyperosmolar coma. Chronic complications are nephropathy, retinopathy, neuropathy, and
cardiovascular, cerebra-vascular, and peripheral vascular diseases [4].

Currently available therapy for diabetes includes insulin and various oral hypoglycemic agents
such as sulfonylureas, metformin, glucosidase inhibitors, troglitazone, etc. But these are reported to
produce serious adverse side effects such as liver problems, lactic acidosis and diarrhea [5]. Biological
actions of the plants are related to chemical composition that are rich in phenolics, alkaloids,
flavonoids, terpenoids, coumarins, and glycosides usually show positive effects. On the other hand,
many conventional drugs for treatment of diabetes, such as metformin are secretagogues which have a
plant origin [6]. The conventional drugs are used to treat diabetes by improving insulin sensitivity,
increasing insulin production, and decreasing the amount of glucose in blood [7]. The adverse effect of
drug treatment is not always satisfactory in maintaining normal levels of blood glucose and this view
many medicinal plants have been provided a potential source of antidiabetic principle which are
widely used for the treatment of diabetes mellitus in various traditional system of medicine worldwide
and many of them are known to be effective against diabetes [8]. The hypoglycaemic effect of
pharmacologically active component of plant decreases the effect on α-amylase and various direct and
indirect effects of different blood parameters responsible for development of diabetes [9].

Diabetes and its consequences are the world's most serious disease. Various mechanisms, such
as DPP-4 inhibitors, α-glucosidase inhibitors, α-amylase inhibitors, and insulin refunctioning, are
involved in diabetes patients, the therapeutic agents, such as insulin and oral hypoglycemic
medications. The current study's goal is to compare the pharmacological activity of herbal extracts to
that of commercially available diabetic drugs. In this study, a healthy rat was given streptomycin to
induce diabetes, and the pharmacological effect of Daucus Carota Subs P.Sativus extracts was compared
to that of a commercially available medication.

MATERIALS AND METHODS

Collection and Authentication of plant material

The leaves of Daucus carota subs p sativus were carefully collected from the Ooty hills,
Udhagamandalam and it were authenticated by Dr. C. Murugan (Scientist & Head Office) Botanical

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Survey of India, Coimbatore.

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Drugs and chemicals

Streptozotocin (STZ) (LOBA Chemie, Mumbai, India) was purchased, preserved at 25ºC and
used for this study. Glibenclamide is an oral antidiabetic preparation with an efficient hypoglycemic
action. Diaonil (Glibenclamide) [10] manufactured by Aventis Pharma Ltd. Goa, India, was collected
from market and preserved at room temperature. All other chemicals and reagents used in the study
were of analytical grade.

Preparation of Plant Extract

The fresh leaves of Daucus carota subs p sativus were washed thoroughly with tap water and
then in distilled water. The washed leaves were a shade drying to treat fungus until complete dryness
of leaves. Then the leaves dried at room temperature and powdered by the electronic grinder until to
get coarse powder. About 200g of dry powder was extracted in the various solvent by continuous hot
percolation using Soxhlet apparatus. In extraction time keeps it temperature at 30 -400C.The
extraction was continued for 7 days. After completion of solvent extract, it is distillation and
concentrated to a dry mass by using rotary evaporator [11].

Animals

Swiss albino rats of Sprague–Dawley strain (200–250 g) of either sex obtained from animal
house of our Institute were used. The animals were fed a standard pellet diet and water ad libitum.
They were maintained in a controlled environment and temperature (22±5 ˚C with 12-h of light/dark
cycle). All experimental protocols were approved by the Institutional Animal Ethical Committee
(12/2009/CPCSEA) [12].

Acute toxicity study

Acute toxicity study of various solvent extract of Daucus carota subsp sativus was carried out in
Swiss Albino rats of either sex (190-250g) according to OECD (Organization for Economic Cooperation
and Development) guidelines No. 423. Extract at different doses up to 2000mg/kg p.o. was
administered and the animals were observed for behavioural changes, toxicity, and mortality up to 48
hours [13-16].

Experimental oral glucose tolerance

The overnight fasted (18hr) normal rats were taken and divided into four groups consists of six
animals. They were provided with drinking water only. Normal saline solution was administered to
group I animals. Group II animals were received glibenclamide (3mg/kg,b.w) as a standard. Daucus
carota subsp sativus methanol extract (200 and 400 mg/kg) was administered by oral route to group III
and IV Glucose (2mg/kg) load was fed 30 minutes after the administration of extracts. Blood was
withdrawn from tail vein under mild ether anaesthesia initial, 30,60 and 90minutes after glucose
administration and glucose level were estimated using glucose strips and a glucometer (standard
diagnostics Ltd). Blood glucose levels were noted and reported.

Experimental oral glucose tolerance

Female albino-Wistar rats weighing 150-250g were used in the present study. All rats were
kept at room temperature of 22-25ºc in the animal house. All the animals were followed the
internationally accepted ethical guidelines for the care of laboratory animals. Prior to the experiments,
rats were fed with standard food for one week in order to adapt to the laboratory conditions in
accordance with the recommendations for the proper care and use of laboratory animals. Fasting
blood glucose (FBG) of all rats was determined before the start of the experiment. Blood sample was
collected at weekly intervals from tail vein puncture till the end of study. In the continuous 21 days of
drug treatment, a blood glucose level of all animals was determined at the 0, 7, 14, 21 day by using one
touch glucometer (SD Check) method. On day 21, overnight fasted animals were under mild ether
anaesthesia, the blood was collected by direct cardiac puncture. Blood was collected in tubes
containing EDTA as anticoagulant for estimation of fasting plasma glucose and HbA1c.

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Histological study

After blood sampling for the biochemical analysis, the animals were sacrificed, quickly
dissected and small slices of pancreas were taken and fixed in 10% formalin. The specimens were
dehydrated in ascending grades of ethanol, cleared in xylene and embedded in paraffin wax. Sections
of 6µm in thickness were prepared and stained with Haematoxylin and Eosin then examined under
microscopy [17].

Statistical study

All the values of body weight, fasting blood glucose level, and biochemical parameter
estimations were expressed as mean ± standard error of mean (S.E.M) and was analyzed for
significance by ANOVA and groups were compared by Tukey-Kramer multiple comparison test, using
InStat v.2.02 software (Graph Pad Software Inc.). Differences between groups (p Value) were
considered significant at P<0.05 level. All data were graphically represented by using Prism Software V
2.02.

RESULT AND DISCUSSION

The preliminary phytochemical studies were done in the methanolic extract of Daucus carota
subsp sativus leaves result that revealed of alkaloids, carbohydrate, flavonoids, phenolic compounds
and tannins (Table 1).

Table 1: Phytochemical analysis of Dacus carota subsp sativa leaves extract

Test For Phytoconstituents C.E E.A.E M.E

Saponins + - +
Alkaloids + + +
Glycosides - - -
Tannins and phenolic compounds + + +
Carbohydrates + + +
Fixed oils - - -
Flavanoids + + +
Steroids - - -
M.E-Methanolic extract, E.A.E-Ethyl acetate extract, C.E-Chloroform extract
+ Present, – Absent
Acute toxicity study

The acute oral toxicity of the methanolic extract of Daucus carota subsp sativus was carried out
as per OECD 423-guidelines (Acute toxic class method). Acute toxicity studies revealed that
LD50>2000mg/kg for the extract. Hence, the biological dose was fixed at MEDC 200mg and 400mg
of body weight for the extract.

Effect On Glucose Tolerance

The doses of MEDC 200 mg/kg and 400 mg/kg increased the tolerance for glucose suggesting
increased peripheral utilization of glucose. The reduction in blood glucose level was dose dependent
(Table 2 and Figure 1).

Table 2: Effect of Glucose tolerance of methanol extract on streptozotocin induced diabetic rats.

Groups Change in blood glucose levels (mg/dL)

Treatment
Fasting After 30 After 60 After 90
Minutes minutes minutes
Group 1: Normal Control 62.92±2.10 152.10±2.76 160.80±2.90 156.90±3.10
(Vehicle only)
Group 2: MEDC 200mg/kg 68.76±1.5 130.08±3.87b 146.52±3.26c 141.76±3.18c
Group 3: MEDC 400mg/kg 66.42±2.12a 107.88±4.90a 123.26±2.23a 122.88±2.91a
Group 4: Glibenclamide 3mg/kg 70.69±2.08 101.17±2.38a 119.60±3.20a 116.18±3.10a
All values are expressed as mean ± SEM for six animals each statistically significant
data*=p<0.001b=p<0.01;c=p<0.05.

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 MEDC treated groups (II,III) and standard group(IV) compared with control(I) group.

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 Figure 1: Effect of Glucose tolerance of methanol extract on streptozotocin induced diabetic rats.

Changes In Blood Glucose

A significant increase in the level of blood glucose was observed in diabetic control rats when
compared to control rats. Administration of MEDC and glibenclamide to diabetic rats significantly
decreased the elevated level of blood glucose, near to control level (Table 3 and Figure 2).

Table 3: Changes in Blood Glucose in methanol extract on streptozotocin induced diabetic rats.

Treatment Blood glucose level (mg/dL)

Day 0 Day 7 Day 14 Day 21
Normal control rats (vehicles only) 78.60±3.14 79.10±3.12 82.96±2.56 76.84±2.91
Diabetic control rats 350.71±7.92a 366.18±12.96a 391.96±12.36a 397.10±11.64a
Diabetic group + Glibenclamide 84.91±3.86a 81.76±4.36b 78.16±3.21a 66.67±1.22a
3mg/kg
Diabetic group + MEDC (200mg/kg) 364.91±9.18a 324.21±5.90a 289.69±6.10a 260.71±9.90a
Diabetic group + MEDC (400mg/kg) 303.10±12.10b 266.90±0.20a 253.12±9.23a 220.86±11.92a
All values are expressed as mean ± SEM for six animals each statistically
significantata*=p<0.001b=p<0.01;c=p<0.05.MEDC treated groups (II ,III) and standard group(IV) compared
with control(I) group.

Figure 2: Changes in Blood Glucose in methanol extract on streptozotocin induced diabetic rats.

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Histopathology Observation

3a 3b

3c 3d

3f
Figure 3: Histopathological slides of pancreas of different animal groups, Normal control (A);
Diabetic control (B); Diabetic group with glibenclamide (C); Diabetic group with 200 mg MEDC (D);
Diabetic group with 400 mg MEDC.

The anti-diabetic activity of methanolic extract was further confirmed by a histopathological

study of the pancreas (Figure 3A-3D). Histology of the pancreas sections of the control rats showed the
normal pancreatic β-cell. The pancreas sections of carbon streptozotocin treated rats showed the
complete destruction of pancreatic β-cell due to the induction of streptozotocin when compared to
normal control rats. The pancreatic sections of methanolic extract treated rats showed an increase in
pancreatic β-cell count and remodelling of the structure of the pancreas when compared to the
glibenclamide treated and control group’s rats.

CONCLUSION

In the present study on the methanolic extract of Daucus carota subsp sativus leaves having
antidiabetic activity more over nearest activity of glibenclamide. This study shows that flavonoids
present in this extract may be possibly responsible for the antidiabetic activities. Histopathological
studies on isolated pancreas revealed that methanolic extract of Daucus carota subs psativus reversed
the changes which produced due to diabetes caused by streptozotocin. The normal pattern of histology
of pancreas was observed and further pharmacological and biochemical investigation is to be done to
find out the active constituent responsible for the antidiabetic activity.

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 ACKNOWLEDGEMENT

Authors are thankful to, JKKMMRF’S – Annai JKK Sampoorani Ammal College of Pharmacy
B. Komarapalayam–638 183 Tamil Nadu, India for providing all the facilities and support to carry out
the work.

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