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PHYTOCHEMICAL SCREENING, ANTIOXIDANT AND ANTIDIABETIC

EVALUATION OF LEAF EXTRACTS FROM DIOSPYROS BLANCOI A. DC

Article · August 2019

DOI: 10.13040/IJPSR.0975-8232.10(8).3951-56

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Demetillo et al., IJPSR, 2019; Vol. 10(8): 3951-3956. E-ISSN: 0975-8232; P-ISSN: 2320-5148

IJPSR (2019), Volume 10, Issue 8 (Research Article)

Received on 22 November 2018; received in revised form, 26 February 2019; accepted, 08 March 2019; published 01 August 2019

PHYTOCHEMICAL SCREENING, ANTIOXIDANT AND ANTIDIABETIC EVALUATION OF

LEAF EXTRACTS FROM DIOSPYROS BLANCOI A. DC.
*1
M. T. Demetillo , O. M. Nuñeza 2, M. M. Uy 3 and W. T. P. S. K. Senarath 4
Department of Biology 1, Caraga State University, Butuan City, Philippines.
Department of Biological Sciences 2, Department of Chemistry 3, Mindanao State University-Iligan
Institute of Technology, Iligan City, Philippines.
Department of Botany 4, University of Sri Jayewardenepura, Gangodawila, Nugegoda, Sri Lanka.
Keywords: ABSTRACT: The objective of the study was to determine the
Alloxan, DPPH, phytochemicals present in the leaf extracts of Diospyros blancoi A. DC
Diabetes, Ethanolic extract, and to evaluate its antioxidant and antidiabetic activities. Qualitative
Phytochemical phytochemical tests were used to detect the presence of bioactive
Correspondence to Author: compounds present in leaf extracts of D. blancoi. Antioxidant activity
Meljan T. Demetillo was measured through diphenyl-1-picrylhydrazyl assay, and antidiabetic
Ph.D., potential was done using alloxan induced white mice. Based on the
Department of Biology, results, the following bioactive constituents are present in the leaves:
Caraga State University, Butuan alkaloids, flavonoids, tannins, terpenoids, saponins, anthraquinones,
City, Philippines. steroids, and glycosides in both ethanol and water extracts of D. blancoi.
E-mail: [email protected] Moreover, results showed that the ethanolic extract had better antioxidant
activity compared to water extract. Furthermore, results also showed that
both ethanolic and waterleaf extracts had significant antidiabetic effects
to alloxan-induced diabetic white mice as shown in their ability to reduce
blood glucose level. Results indicate that extracts of D. blancoi leaves are
potential sources of natural antioxidant and antidiabetic compounds and
could have potential use in the management of diabetes.
INTRODUCTION: Traditional and folklore Studies on plant remedies not only serve to make
medicine play an important role in health services the public more conscious regarding sources of
around the globe. The significance of plants in alternative medicines but also to open more
medicine remains even of greater importance with avenues for future drug discovery 2.
the current globalization to obtain drugs from plant In this growing interest, many of the phytochemical
sources due to high safety, efficacy, and economy bioactive compounds from medicinal plants have
1
. The search for new plant-derived drugs has been been studied intensively and have shown many
receiving renewed interest among researchers pharmacological activities 3. Screening of various
throughout the world given discovering new drugs. bioactive compounds from plants has led to the
QUICK RESPONSE CODE discovery of new therapeutic drugs which have
DOI: adequate protection and treatment roles against
10.13040/IJPSR.0975-8232.10(8).3951-56
multiple diseases. Micro-scale bioassay techniques
provide a frontline screen and are attractive
The article can be accessed online on because of simplicity, rapidity, cost-effectiveness
www.ijpsr.com and reasonable reliability, therefore are used
extensively for the screening of biological activity
DOI link: http://dx.doi.org/10.13040/IJPSR.0975-8232.10(8).3951-56 of plant materials 4.

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Tropical countries like the Philippines are abundant Qualitative Analysis for Phytochemical
in medicinal plants. Herbal knowledge from the Components: Phytochemical analysis of the leaf
local indigenous community has long been the extracts of D. blancoi was done using standard
basis for investigating the potential of plants as qualitative methods 8-9. The compounds analyzed
therapeutic agents 5. Diospyros blancoi is not for phytochemicals were alkaloids, flavonoids,
commonly known as alternative medicine in most glycosides, phenols, saponins, steroids, tannins, and
areas in the Philippines but is commonly used in anthraquinones.
Claver, Surigao Del Norte to cure some illnesses. A
decoction of leaves of D. blancoi can cure DPPH Radical Scavenging Activity: The
dysentery and fever, diarrhea, and has antibacterial antioxidant activity of the extracts was measured
properties. Moreover, D. blancoi is used to treat by the scavenging activity of the stable 2, 2-
chest colds, scars, stress, hypertension, heart diphenyl- 1-picrylhydrazyl assay (DPPH) 10. Free
ailments, and diabetes. radical scavenging capacity was evaluated on the
basis of the scavenging activity of DPPH by
The objective of this study was to determine the measuring the reduction of absorbance at 517 nm.
secondary metabolites using a standard protocol for The DPPH solution in methanol was mixed with
qualitative phytochemical screening and to 1ml of plant extract solution of varying
determine the antioxidant and antidiabetic activities concentrations (50, 100 & 500 μg/ml). A
of D. blancoi to validate the claims of local healers corresponding blank sample was prepared, and L-
from Claver, Surigao Del Norte Philippines. Ascorbic acid (1.0-100 μg/ml) was used as
reference standard. The mixture of 1ml methanol
MATERIALS AND METHODS: and 1 ml DPPH solution was used as a control. The
Identification and Collection of Plant Samples: reaction was carried out in triplicate, and the
Fresh leaves of D. blancoi were collected from decrease in absorbance was measured at 517nm
Claver, Surigao del Norte, Philippines (9’29”21 N after 30 minutes in the dark using UV-Vis
and 125’50”40 E). Field guide book and taxonomic spectrophotometer. The inhibition % was
keys following the list of medicinal plants of calculated using the following formula.
Philippines were used to identify the plants used in
the area 6-7. Moreover, complete plant materials of % Free Radical Inhibition = [(A control – A sample) / A
D. blancoi were photographed in their natural control] × 100
habitat for proper documentation. Where A control and A sample are the absorbance
Extract Preparation: Leaf samples (300 g) of D. values of the control and test sample, respectively.
balncoi were thoroughly washed under running tap The effective concentration of sample required to
water to remove unwanted material, rinsed with scavenge DPPH radical by 50% (EC50) was
distilled water, cut into smaller pieces, and boiled obtained by linear regression analysis of dose-
in sufficient amount of distilled water for 5 min in response curve plotting between % inhibition and
low heat. The resulting decoction mixture was concentration.
filtered, cooled, freeze-dried, and stored in air-tight Experimental Animals: A total of 24 healthy adult
containers. One kg of fresh plant materials was air- mice of both sexes (20-30 g) were obtained from
dried for 2-4 weeks, homogenized to a fine powder the rodent laboratory of Caraga State University,
using an electric blender, and was soaked in 1 lit of Ampayon, Butuan City and were acclimatized for
95% ethanol for 72 h, filtered, and concentrated in- five days to 25 ºC, and 12 h dark/light cycle with
vacuo to give the ethanolic extract. The hydro- free access to commercial rodent food (pellets) and
ethanolic extract was also prepared by mixing 100 water ad libitum. Ethics clearance approval number
ml distilled water and 100 ml pure 95% ethanol or is (National Ethics Committee) NEC Code: 2018-
1:1 ratio. The resulting mixture was filtered, 023-Demetillo.
concentrated in-vacuo using a rotary evaporator,
freeze-dried, and weighed to provide the hydro- Induction of Diabetes in Experimental Animal:
ethanolic extract. The prepared extracts were used Alloxan monohydrate (Sigma-Aldrich Chemical
for the phytochemical tests. Corp. Germany) was dissolved in sterile distilled

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water and was then administered by a single Collection of Blood Samples and Blood Glucose
intraperitoneal dose (120 mg/kg) body weight) into Determination: Blood samples were drawn from
12 h fasted mice. The blood samples were taken the tail tip of mice at weekly intervals until the end
after 2 days of alloxan injection. Mice screened for of the study. This was done by sterilizing the tail
diabetes having glycosuria and hypoglycemia with with 10% alcohol and then nipping the tail. Blood
a blood glucose level of above 240 mg/dL were was then drawn from the tip using blood glucose
taken for the study. All animals were allowed free self-test (Almedicus Co. Ltd., South Korea). Blood
access to water and pellet diet. Extracts were glucose determination was done on days 0, 7, 14,
administered by oral gavage daily for 21 days from and 21 of the study.
the day of induction. All procedures in the
experiments were guided by the observance of All procedures in the experiments were guided by
animal ethics guidelines from Bureau of Animal the observance of animal ethics guidelines from
Industry. Bureau of Animal Industry.

Experimental Design: All diabetic surviving mice RESULTS AND DISCUSSION: Preliminary
were taken and divided into groups of three mice phytochemical screening revealed the presence of
each: normal control mice (NCR); diabetic control alkaloids, flavonoids, saponins, tannins, and
mice (DCR); diabetic mice given ethanol, steroids in all extracts Table 1. All the
decoction and hydro-ethanol extracts of (100 mg/kg phytochemicals tested were present except
body weight); diabetic mice given ethanol, anthraquinones which were absent in the water
decoction and hydro-ethanol extracts of (50 mg/kg extract. In terms of solvent, ethanolic extracts have
body weight); and diabetic mice given standard abundant phytochemicals compared to water
drug glibenclamide (100 and 50 mg/kg body extracts.
weight).
TABLE 1: PHYTOCHEMICAL SCREENING OF THE DIFFERENT EXTRACTS OF D. BLANCOI LEAVES
Plant extracts Alkaloids Saponins Flavonoids Steroids Tannins Cyanogenic glycoside Anthraquinones
DbD + + ++ + + + -
DbHE + + + + + + -
DbE + ++ ++ ++ ++ ++ +
Key: (+)-present; (++)-abundant; (-)-absent; (DbD, DbHE, DbE)-Diospyros blancoi decoction, hydroethanolic and ethanolic extracts respectively.

Phytochemical screening of the leaf extracts of D. been shown to have enormous significance as anti
blancoi revealed the presence of alkaloids, hyper-cholesterol, hypotensive, and have cardiac
saponins, steroids, tannins, anthraquinones, and depressant properties 14. Furthermore, the existence
flavonoids which are compounds known to have of different secondary metabolites, especially
curative activity against several pathogens and saponins, tannins, and flavonoids in plants has also
therefore could support the traditional use for the been linked to the antimicrobial activities of the
treatment of various illnesses in the community. plants 15-16. The result of DPPH free radical
These phytochemicals also exhibited a wide range scavenging activity on the leaf extracts of D.
of activity such as anti-inflammatory, antiviral, blancoi is shown in Table 2. The highest radical
antibacterial, antiulcer, anti-allergic, and anti- scavenging activity was shown by ethanol extract
hepatic action 12. Alkaloids are basic nitrogenous (94.27%). The results obtained were comparable to
compounds which are pharmacologically-active the standard used (L-ascorbic acid) with a percent
and may exhibit tranquilizing and stimulating inhibition of 96.99. Results revealed that the
activities on the nervous system, hypertensive and decoction leaf extract of D blancoi obtained the
hypotensive action, vasoconstrictor and vasodilator highest EC50 value of 63.17 μg/ml radical
effects on the cardiac system 13. Steroids have a scavenging (antioxidant) activity, followed by
chemical structure similar to cholesterols which ethanolic extract with an EC50 value of 105.38
have been reported to decrease cholesterol μg/ml. These results suggest that the plant extract
absorption and plasma Low-Density Lipoprotein contains components with radical scavenging
(LDL) values. Saponins which are abundant have potential.

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Demetillo et al., IJPSR, 2019; Vol. 10(8): 3951-3956. E-ISSN: 0975-8232; P-ISSN: 2320-5148

TABLE 2: DPPH FREE RADICAL SCAVENGING ACTIVITY OF DIFFERENT EXTRACTS OF D. BLANCOI

Extracts 500 μg/mL 100 μg/mL 50 μg/mL EC50
Ave. ABS* % Antiradical Ave. ABS* % Antiradical Ave. ABS* % Antiradical μg/mL
activity activity activity
DbD 0.056 93.99 0.276 71.03 0.622 33.47 63.17
DbE 0.053 94.27 0.315 66.73 0.673 28.11 105.38
DbHE 0.28 69.95 0.571 38.84 0.724 22.74 214.14
AA 0.028 96.99 0.086 90.81 0.464 50.26 21.31
Key: (DbD, DbHE, DbE)-Diospyros blancoi decoction, hydroethanolic and ethanolic extracts respectively; AA- Ascorbic acid;
* mean of 3 determinations

TABLE 3: MEAN BLOOD GLUCOSE LEVEL OF EACH TREATMENT OF ALLOXAN- INDUCED WHITE MICE
Plant Blood Glucose Level (mg/dL)
Extracts/ Initial (0 day) 7th day 14th day 21th day
Control Dose Dose Dose Dose
100 mg/ml 50 mg/ml 100 mg/ml 50 mg/ml 100 mg/ml 50 mg/ml 100 mg/ml 50 mg/ml
DbD 252 252.4 199.6a 178.9b 119a 110.6b 91.5a 63.3b
DbE 252.5 248.9 146.8a 188.6b 99.8a 126.4b 90.5a 113b
DbHE 260.8 257.4 200.5a 216.2b 144.3a 169.4b 125.1a 145.7b
Glibenclamide 261.5 251.8 149.5a 123.9b 112.5a 98.6b 90.3a 91.4b
DCM 252 247.3ns 249.1ns 247.8ns
NCM 132.5 137.7ns 134.2ns 137.4ns
Key: (DCM)- Diabetic control mice; (NCM)- nondiabetic control mice; (a) P<0.05 significant decrease as compared to Zero h in a
row @ 100mg/ml dose; (b) P<0.05 significant decrease as compared to Zero hr in a row @50mg/ml dose; (ns)-not significant

A dose-dependent reduction in Blood Glucose antioxidant activities and are dominant chain-
Level (BGL) was observed in alloxan-induced breaking antioxidants of which most of these are
diabetic mice treated with ethanolic, present in the leaves. Moreover, anthraquinones
hydroethanolic, and decoction extracts of D. have also shown antioxidant properties. The
blancoi. Following the treatment, the extracts saponins from plant extracts have already been
produced a significant reduction (p<0.05) in blood reported as antibacterial and have potent
glucose levels of the treated diabetic mice. The antioxidant activity 18-19. Most of the polyphenols
effect was significantly compared to the standard contribute significantly to the antioxidant activity
drug, glibenclamide Table 3. A remarkable and act as highly effective free radical scavengers
decrease in blood glucose levels was observed with which are mainly due to their redox properties,
a dose of 100 mg/kg compared to 50 mg/kg in all which can play an important role in absorbing and
plant extracts. Among the extracts tested, it was neutralizing free radicals. Another bioactive
found that ethanolic showed a gradual decrease in compound that is abundantly found from the leaf
glucose level throughout 21 days, even though the extract of D. blancoi is the flavonoids that have
onset of antidiabetic effect with these extracts was been found to possess antioxidant properties 20.
seen within 7 days of treatment.
The hypoglycaemic activity observed with
Many studies have shown that many polyphenols ethanolic and water extracts of D. blancoi can be
contribute significantly to the antioxidant activity accounted to the presence of different
and act as highly effective free radical scavengers phytochemicals which could act synergistically or
which are mainly due to their redox properties, independently in lowering the blood sugar level.
which can play an important role in absorbing and Phytochemical studies have been associated with
neutralizing free radicals 16. Flavonoids and tannins the hypoglycaemic activity. Nauclea latifolia, a
are major groups of phenolic compounds that act as plant screened to contain alkaloids, showed a
primary antioxidants or free radical scavengers. significant decrease of blood glucose levels in
The medicinal effects of plants are often attributed alloxan-induced diabetic rats. Flavonoids are
to the antioxidant activity of phytochemical known to regenerate the damaged beta cells in the
constituents 17. Many studies confirmed that alloxan-induced diabetic rats 21-22. The reduction in
phenolic constituents, such as flavonoids, phenolic blood glucose of diabetic mice could occur partly
acids, and tannins are well known for their high by stimulating insulin production from the

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Demetillo et al., IJPSR, 2019; Vol. 10(8): 3951-3956. E-ISSN: 0975-8232; P-ISSN: 2320-5148

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glibenclamide due to the presence of certain of Pharmacology 2011; 5: 746-50.
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ethanol extract of D. blancoi leaf pointed out the Studies 2017; 5: 230-236.
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How to cite this article:

Demetillo MT, Nuñeza OM, Uy MM and Senarath WTPSK: Phytochemical screening, antioxidant and antidiabetic evaluation of leaf
extracts from Diospyros blancoi A. DC. Int J Pharm Sci & Res 2019; 10(8): 3951-56. doi: 10.13040/IJPSR.0975-8232.10(8).3951-56.
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