lOMoAR cPSD| 3172 0 42

A PROJECT REPORT ON
PHYTOCHEMICAL SCREENING OF OF ANTI-DIABETIC PLANT

For partial fulfillment of B.pharma 8th semester

Session: 2023-24
Dr. A.P.J. Abdul Kalam Technical
University Lucknow

Institute of Pharmacy
HCPG College
Bawan Bigha , Varanasi, 221002

SUBMITTED BY :-

RITESH KUMAR SINGH Supervisor

B.pharma IVth year Dr. Brijyog
Roll No 2002050500068 (ASSISTANT PROFESSOR)
 CERTIFICATE
This certify that Ritesh Kumar Singh student of the Institute of Pharmacy Harish Chandra P.G. College,
Varanasi has undertaken this project on “ PHYTOCHEMICAL SCREENING OF OF ANTI-DIABETIC
PLANT”.

The project has been carried out by the student fulfillment of therequirement under our my supervision
& guidance.

DIRECTOR OF INSTITUTION: -

Director
Dr. Manish Kumar Gupta
Institute of Pharmacy, Harish Chandra P.G.
College, Varanasi-221002
 DECLARATION

I hereby declare that the project entitled “PHYTOCHEMICAL SCREENING OF OF ANTI-DIABETIC

PLANT” is as my own effort the guidance of Dr. Brijyog, ASSISTANT PROFESSOR. This project is
submitted to the Instituteof Pharmacy Harish Chandra P.G. College, Varanasi for the partial fulfilment
of the “BACHELOR OF PHARMACY”.
 ACKNOWLEGEMENT

I would like to express my regards and deep sense of gratitude towards our mentor Dr. Brijyog ASSISTANT
PROFESSOR who gave us the golden opportunity to work on the project entitled “PHYTOCHEMICAL
SCREENING OF OF ANTI-DIABETIC PLANT.” and provide continuous support and guidance that made this
project completed successfully.

Thanking you RITESH KUMARSINGH

B.pharma IVth year
Roll No 2002050500068
 Abstract
Diabetes mellitus is a major and current epidemic disease of the human race implicated with numerous
clinical manifestations. A number of protein-rich seeds such as that of Citrullus lanatus (watermelon) are
commonly used in traditional medicine with increasing acclaimed efficacy against diabetes mellitus. In this
study the effects of petroleum ether and ethanol extracts of the seeds of Citrullus lanatus on blood glucose
levels in alloxan-induced diabetes in mice have been investigated. Hyperglycemia was induced by the
injection of 150 mg/kg (i.p.) of alloxan monohydrate freshly dissolved in physiological saline. Doses (150,
200 and 250 mg/kg) per os, of the extracts were separately administered to a group of five diabetic mice in
the study. The activity was compared with reference standard glibenclamide (2 mg/kg, p.o.) and negative
control of physiological saline. Treatment ofalloxan-induced diabetic mice with the crude extracts of C.
lanatus seeds brought down the raised blood glucose levels significantly (P < 0.05) in a dose-dependent
manner. The ethanol extract was found to have moreantidiabetic effect than the petroleum ether extract.
Phytochemical screening of the seed extracts of Citrullus lanatus indicated the presence of steroids,
alkaloids, flavonoids, terpenoids, and saponins in both the ethanol and petroleum ether extracts. In
addition, anthraquinones, tannins and reducing sugar were detected in the ethanol extract.
 1. Introduction
The current epidemic of diabetes mellitus (DM) in Africa, coupled with impinging poverty, clearly indicates the
urgent need to develop new therapeutic drugs of cheaper and more available to face this growing health challenge. A number
of plants products among which the protein-rich seeds including Citrullus lanatus, Telfairia occidentalis, Lagenaria siceraria,
Cucumeropsis mannii and Cucurbita moschata are commonly used in traditional medicine with increasing acclaimed efficacy
against DM (Teugwa et al., 2013). Diabetes mellitus is a major illness of the human race implicated with numerous clinical
manifestations. It is a clinical syndrome characterized by chronic hyperglycemia (defects in insulin secretion, insulin action or
both) resulting in aberration in carbohydrate, fat and protein metabolism. It has been reported that the chronic hyperglycemia
of diabetes is associated with complications like renal failure, coronary artery disorder, neurological complications, cerebro-
vascular disease, blindness, and limb amputation, long term dysfunctions and failure of various organs and eventually premature
death (Lyra et al., 2006)). According to the World Health Organization (WHO) projections, the diabetes population is likely
to increase to 300 million or more by the year 2025 (Tielmans etal., 2007). The high cost of conventional drugs and their
unavailability in many rural areas, coupled with their high incidence of side effects, posed a dare need for a change of the
affair in case of modern medicine. Thus, the management of diabetes without any side effect is still a major challenge.
According to the WHO, approximately 80% of the world's population currently uses herbal medicines in healing different
ailments (Latha et al., 2010). However, among the estimated 400,000 plant species, only 6% have been studied for biological
activity, and about 15% have been investigated phytochemically (Cragg et al., 1997; Srinivasan, 2005). This shows a need for
planned activity guided phyto-pharmacological evaluation of herbal drugs. The aim of this study was to phytochemically
analyse and evaluate the antidiabetic activity of the seeds of Citrullus lanatus (Kankana in Hausa).
MATERIALS AND METHODS

Plant material
Seeds of Citrullus lanatus were collected from Unguwar Rimi market, Kaduna north, Kaduna. The seeds
were bench dried for eleven days and pulverized into coarse powder and kept in polythene bags at room
temperature, ready for extraction.

Extraction of plant material

The dried pulverized seed (300g) was macerated with 1000 mls of petroleum ether (60-80 °C) for three
days at room temperature and filtered and this was repeated twice.
The combined filtrate was distilled using a rotary evaporator and then air-dried to obtain the petroleum
ether extract. This procedure was repeated using ethanol on the residue of the seeds and after
filtration the combined filtrate was then distilled to afford the ethanol extracts.

Phytochemical screening
Phytochemical screening was carried out on the two extracts (petroleum ether and ethanol) of Citrullus
lanatus peel using standard procedures and tests (Trease and Evans, 1989; Sofowora, 1993) to
determine the presence of alkaloids, tannins, terpenoids, flavonoids, reducing sugar, anthraquinones, and
saponins.

Pharmacological study
Animals
White albino mice (22-32g) of both sexes obtained frombiochemistry department Kaduna state University,
Kaduna were used for the experiment. The animals were kept under standardenvironmental conditions of
temperature, relative humidity, andfed with standardized pellets and water ad libitum during theperiod in
aluminum cages. The mice were fasted for 12hrs beforeexperimentation but were allowed free access to
water.

Experimental design
Hyperglycemia was induced by intraperitoneal injection of 150mg/kg body weight alloxan monohydrate,
freshly dissolved in regular saline 0.9% physiological saline immediately before use, to overnight fasted
albino mice. After 7 days, animals with fasting blood glucose level ≥126 mg/dl (≥7.0 mmol/dl) (Vijan,
2010) or more were considered diabetic and employed in the study. The mice were then grouped into 5
groups of five mice each as follows:

Group 1: Served as positive control and received glibenclamide (2ml/kg body weight)
Group 2: Served as negative control receiving physiological saline(10 ml/kg)
Group 3: Received petroleum ether extract at 150 mg/kg bodyWeight
Group 4: Received petroleum ether extract at 200 mg/kg
Group 5: Received petroleum ether extract at 250 mg/kg
Group 6: Received ethanol extract at 150 mg/kg body weight
2.5 Preparation and extraction of plant materials
All plant samples were air-dried and pulverized into coarse powder by using a milling machine type Y
(Hangyu®, China) available at ITM, MUHAS. About 400 gm of each ground plant material was extracted
with 80% aqueous ethanol using percolation method at 25-33oC and after 24 h filtered through whatman
number 1 filter paper. The procedure was repeated two times to ensure exhaustive extraction of the
plant material. The extracts were pooled together. The filtrate was evaporated under reduced pressure
using rotary evaporator (Büchi Labortechnik, Flawil, Switzerland) at 40 to 50°C. The extracts were further
dried by freeze-drying using the Edwards freeze drier (Edwards High Vacuum International Crawley,
Sussex, England).

2.6 Phytochemical screening

Qualitative phytochemical analysis for secondary metabolites was carried out for the crude extracts as per
standard methods16,17.

2.6.1 Test for terpenoids

1 gm of each extract was dissolved in 2 ml of chloroform followed by adding carefully 3 ml of concentrated
sulphuric acid (H2SO4) to form a layer. A red brown colouration on the interface was indicative results for
presence of terpenoids.
2.6.2 Test for alkaloids
An amount of 0.5 gm of each extract was dissolved in 5 ml of 1% hydrochloric acid (1% HCl) and warmed
on water bath. Then, Dragendorff’s Test was done in which filtrates were treated with Dragendorff’s
reagent (solution of Potassium Bismuth Iodide). Formation of red precipitate was indicative results for
presence of alkaloids.
2.6.3 Test for phenolics
An amount of 0.1 gm of each extract was heated in 15ml of water (H2O) for 15min on a water bath and
filtering the solution. To 2 ml of filtrate, 2 ml of 5% aqueous ferric chloride was added; formation of blue
colour was indicative results for presence of phenols.
2.6.4 Test for flavonoids
Extracts were treated with few drops of sodium hydroxide solution (NaOH). Formation of intense yellow
colour, which becomes colourless on addition of dilute hydrochloric acid (HCl), was indicative results for
presence of flavonoids.
2.6.5 Test for saponins: An amount of 2 gm of each extract was boiled with 20 ml distilled water in
a water bath and filteredthen 10 ml filtrates was mixed with 5ml distilled water and shaken vigorously and
observe persistence of froth. The frothing was further mixed with 3 drops of olive oil then shaken
vigorously and observed for the formation of emulsion to indicate presence of saponins .
2.6.6 Test for glycosides
An amount of 0.5 gm of each extract was stirred with 10 ml of boiled distilled water. This mixture was
filtered and 2 ml of the filtrate hydrolyzed with few drops of concentrated hydrochloric acid (HCl) and the
solution rendered alkaline with a few drops of ammonia solution. Then, about 5 drops of this solution was
added to 2 ml of Benedict’s quantitative reagent and boiled. Appearance of reddish brown precipitate was
indicative results for presence of glycosides.
2.7 Determination of the anti-diabetic activity
Anti-diabetic potential of each extract was evaluated at a dose of 200 mg/kgbwt via in vivo test (using
albino mice) in oral glucose loading animal model by OGTT18,19.
2.7.1 Oral glucose administration
Test animals acclimatized for 5 days were fasted for 18 hours before the beginning of the experiment and
were then orally loaded by gavage with freshly prepared glucose (1 gm/kgbwt) 30 minutes after
solvent/extract/chlorpropamide administration.
2.7.2 Experimental design

At the beginning of the experiment body weight to the nearest gram and fasting blood glucose levels of the
animals were determined.
Seven groups of white albino mice, six albino mice (n = 6) in each received the following treatment
schedule:
Group I: Negative control (distilled water, 5 ml/kgbwt orally)
Group II: Positive control (chlorpropamide, 100 mg/kgbwt orally)
Group III: 80% aqueous ethanol Bridelia duvigneaudii roots extract (200 mg/kgbwt orally)
Group IV: 80% aqueous ethanol Dioscorea praehensilis tubers extract (200 mg/kgbwt orally)
Group V: 80% aqueous ethanol Ficus fischeri stem barks extract (200 mg/kgbwt orally)
Group VI: 80% aqueous ethanol Afzelia quanzensis roots extract (200 mg/kgbwt orally)
Group VII: 80% aqueous ethanol Cyphomandra crassifolia fruits extract (200 mg/kgbwt orally)

2.7.3 Blood glucose determination

Blood glucose level in blood collected from each mouse by partial tail amputation procedure from the tail
vein was measured after glucose loading at 0.5, 1, 2, 3 and 4 hours by commercially available glucose kit
based on a glucose oxidase enzymatic assay and determined by a glucose meter known as ACCU-
CHEK® Active (Roche Diabetes care GmbH, Mannheim –Germany)20,21.
2.8 Data and statistical analysis
The results of blood glucose levels were expressed as mean ± Standard Error of the Mean (SEM) with
sample size (n = 6). The statistical analysis of results was carried out using Student t-test followed by one-
way Analysis of variance (ANOVA) and Tukey’s multiple comparisons probability value (p ˂ 0.05).
2.9 Ethical approval
During the study the following issues were taken into consideration: Few mice were kept in each cage to
enable mice to express their normal behavior. Clean water and appropriate feed was given and mice were
kept at the temperature of 22oC (±3oC) and the relative humidity was 55 %. An ethical clearance of the
protocol for this study was given by the Director of Research and Publications of MUHAS.
3.0 RESULTS AND DISCUSSION

Phytochemical screening

Table 1.0 gave the results of phytochemical studies on both the petroleum ether and ethanol extracts of
the seeds of Citrullus lanatus.

Previous studies reported that medicinal plants contain phytochemical compounds responsible for anti-
diabetic activity which include terpenoids, alkaloids, phenolics, flavonoids, saponins, and glycosides13.
Phytochemical screening revealed the presence of terpenoids, phenolics, saponins and glycoside in B.
duvigneaudii. These constituents may in part be responsible for the observed significant activity of B.
duvigneaudii roots extract either singly or in synergy with one another.
3.2 Anti-diabetic potential
At a dose of 200 mg/kgbwt, B. duvigneaudii roots extract revealed its anti-diabetic potential by lowering
blood glucose on glucose loaded normal albino mice statistically different from that of untreated group (p ˂
0.05) at 0.5, 1, 2 and 3 hrs after administration (Table 2). The extracts of the other plants (D. praehensilis,
F. fischeri, A. quanzensis and C. crassifolia) did not show significant reduction in blood glucose levels
after administration (p ˂ 0.05) as shown in Table 2.
Evaluation of anti-diabetic potential of 80% aqueous ethanol extracts of the selected Tanzania medicinal
plants was carried out in oral glucose loaded white albino mice. Loading oral glucose in animals is
reported to cause physiological induced diabetes mellitus because the blood glucose level of the animal is
momentarily increased without damage to the pancreas 22. The 80% aqueous ethanol extract of B.
duvigneaudii roots was observed to demonstrate significant anti-hyperglycaemic activity in oral glucose
loaded white albino mice when compared to other medicinal plants selected for this study.
Chlorprpamide is a synthetic anti-diabetic drug belonging to the group of sulphonylureas anti-diabetic
drugs which cause hypoglycemia by stimulating insulin secretion from the pancreas20. The observed
reduction in blood glucose level of the oral glucose loaded mice by chlorprpamide in this study shows a
reverse state of diabetes. In this study, treatment with the 80 % aqueous ethanol roots extract of B.
duvigneaudii caused significant decrease in blood glucose level of treated oral glucose loaded mice
compared to untreated oral glucose loaded mice which is a reverse diabetic state characteristic.
Previous studies revealed that, medicinal plant have different mechanisms of anti-diabetic action
through which their effects are exhibited that might include promoting regeneration of β-cells of Islets of
Langerhans in the pancreas, enhancement of insulin release and activity on the cells, decrease glucose
uptake at the duodenal cellular level and other aspects of small intestine and the presence of high level
of fiber in plants which interferes with carbohydrate absorption23. The 80 % aqueous ethanol roots extract
of B. duvigneaudii may have acted through one of the above mechanisms. Phytochemical compounds
like terpenoids, phenolics, saponins and glycosides present in this extract have been reported to exert
anti-diabetic activity13 through one of the mechanisms mentioned above. However, some of the medicinal plants for
this study were observed to posses two (A. quanzensis and C. crassifolia), three (F. fischeri) or four (D. praehensilis)
of the phytochemicals but they did not exhibit anti-diabetic activity. It is reported that phytochemicals within the
complex mixture of phytochemicals tend to interact resulting into either potentiating the effect of bioactive
phytochemicals or interfering with their activity24. Therefore, the interaction which causes interference of bioactivity
of phytochemicals in their complex mixture may have occurred for the plants which did not exhibit anti-diabetic
activity for this study.
 CONCLUSION

The results of this study, using extracts of the seeds, for the first time have shown that also the seeds have
some potentiality as antidiabetic medication. Both the petroleum ether and ethanol extracts of the Citrullus
lanatus seeds exhibited significant (P<0.05) antidiabetic effect. The specific secondary metabolites
responsible for the antidiabetic activity are needed to be found and further investigation fractionation of the
extracts to to determine active compounds could be done in future. This might lead to the development of
new drugs. On the otherhand, this study supports the traditional use of the plant for the purposes already
mentioned.
This study indicates that the 80 % aqueous ethanol roots extract of B. duvigneaudii contains bioactive
compounds with anti-diabetic potential. Therefore, this study serves as a first step in revealing anti-
diabetic potential of B. duvigneaudii before isolation and identification of pure bioactive compound(s)
responsible for anti-hyperglycaemic activity for development and formulation of anti-diabetic agent(s).
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