Phytochemical and Pharmacological Evaluation of Ethanolic

Extract of Barks of a Mangrove Plant Species –

Kandelia candel (L.)

Presented By:
Examination Roll No.: 141016
Examination Session: 2014-2015
Department of Pharmacy
Jashore University of Science and Technology.
Jashore-7408, Bangladesh

Kandelia candel(L)
 Plant Preview
Kandelia candel (L.)
Family: Rhizophoraceae

Description:
Leaves: opposite, blades shiny, mid-green, narrowly oblong, elliptic or
drop-shaped-oblong.
Flowers: whitish, with numerous protruding stamens.
Seed: viviparous, seedling hypocotyl narrowly cylindrical /club-shaped

Distribution:
Bangladesh, India, Myanmar, Thailand, Cambodia, Laos, Vietnam,
Malaysia, Indonesia

Traditional Uses
firewood, making enclosures and stakes and source of tannins.
 Literature Review of Kandelia candel
Findings Plant parts
Reproductive biology and
population genetic
structure
Effect of multiple heavy
metals on engymes
Condensed Tannins and
antioxidant activities
Proteomic Analysis of Salt-
Responsive Proteins
Reference
Sun, M., Wong, K., & Lee, J. S. (1998). Reproductive biology and
population genetic structure of Kandelia candel (Rhizophoraceae), a
Leaves
viviparous mangrove species. American Journal of Botany, 85(11), 1631-
1637.
Huang, G.-Y., Wang, Y.-S., Sun, C.-C., Dong, J.-D., & Sun, Z.-X. (2010). The
effect of multiple heavy metals on ascorbate, glutathione and related
Seedlings enzymes in two mangrove plant seedlings (Kandelia candel and Bruguiera
gymnorrhiza). Oceanological and Hydrobiological Studies, 39(1), 11-25.
doi:10.2478/v10009-010-0010-z
Zhang, L. L., Lin, Y. M., Zhou, H. C., Wei, S. D., & Chen, J. H. (2010).
Condensed tannins from mangrove species Kandelia candel and
Barks
Rhizophora mangle and their antioxidant activity. Molecules, 15(1), 420-
431. doi:10.3390/molecules15010420
Wang, L., Liu, X., Liang, M., Tan, F., Liang, W., Chen, Y., . . . Chen, W.
(2014). Proteomic analysis of salt-responsive proteins in the leaves of
Leaves mangrove Kandelia candel during short-term stress. PLoS One, 9(1),
e83141. doi:10.1371/journal.pone.0083141
 Aims and Objectives

So the leaves of Kandelia candel were selected for the present study that was arranged as follows.
A: Phytochemical tests: To identify the presence or absence of different phytochemical groups.
B: Pharmacological assessment: To evaluate different pharmacological activities.

Kandelia candel
 Collection & Extraction
Leaves of Kandelia candel was collected from Sundarbans, Khulna, Bangladesh in January, 2020 & Plants
were extracted by cold extraction method using 96% Ethanol.

Rotary
Collection Filtration
Evaporation

Vacuum
Chopping Maceration desiccation

Shade Drying Grinding Dried extract

 Phytochemical Screening
Test name Result
01 Tests for Reducing Sugar
02 Tests for Phenolic Compounds
03 Tests for Tannins
04 Tests for Terpenoids - Salkowski Test

05 Test for Saponins Present

06 Test for Steroids - Salkowski Test Absent
07 Test for Flavonoids
08 Test for Alkaloids
09 Test for Glycoside
10 Tests for Proteins & Amino acids - Xanthoprotein
Test
11 Tests for Acidic Compounds
 Evaluation of Antioxidant Activity
DPPH Free Radical Scavenging Assay
10 μl of each aliquots of the different concentrations (1-1024 μg/ml) of the extracts were added to 190 μl
of 0.007886% w/v methanolic solution of DPPH & absorbance at 517 nm was determined after 30 min
to obtain the IC50 (Inhibitory conc. 50%) (Sadhu et al., 2003).

% inhibition vs Concentration
100.00
Result:
90.00
80.00
70.00 Test Sample IC50 (µg/ml)
% inhibition

60.00
50.00
40.00
30.00 Ascorbic acid 46.25
20.00
10.00
0.00
1 2 4 8 16 32 64 128 256 512 1024
Kandelia candel extract 77.83
Concentration

Standard K. candel

Figure: Comparison of % inhibition by Kandelia candel and

ascorbic acid to determine IC50.

Finding: K. candel showed greater DPPH free radical scavenging activity than Ascorbic acid.
Sadhu, S.K., Okuyama, E., Fujimoto, H., Ishibashi, M., 2003. Chemical and Pharmaceutical Bulletin 51, 595-598.
 Evaluation of Antioxidant Activity (Cont.)
Hydrogen Peroxide Radical Scavenging Assay

Different concentrations (6.25 to 800 μg/ml) of extracts were added to a H 2O2 solution (40 mM) and at 230

nm absorbance was determined after ten minutes to obtain SC50 values (Keser et al., 2012).

% Scavenged vs Concentration
120.00 Result:
100.00
% Scavenged

80.00
Test Sample SC50 (µg/ml)
60.00
40.00
20.00 Ascorbic acid 89.63
0.00
6.25 12.5 25 50 100 200 400 800
Concentration
K. candel extract 107.59

Standard K. candel

Figure: Comparison of % scavenged by K. candel and

ascorbic acid to determine SC50.

Finding: K. candel showed higher H2O2 radical scavenging activity than Ascorbic acid.
Keser, S., Celik, S., Turkoglu, S., Yilmaz, O., Turkoglu, I., 2012. Chemistry Journal 2, 9-12.
 Evaluation of Antioxidant Activity (Cont.)
Determination of Total Phenolic Content (TPC)
Total phenolics of plant extracts were measured using Folin-Ciocalteu reagent and by evaluating the
regression equation of the calibration curve (y = mx + c), expressed in mg gallic acid equivalents (GAE) per
gram of dry powder (mg GAE/g) (Javanmardi et al., 2003).
Absorbance of Gallic Acid
Absorbance Linear (Absorbance)
Result:
Absorbance

1
0.77
0.8
f(x) = 5.55 x − 0.03 0.63 Total phenolic content
0.6
R² = 0.95 Sample
(mg GAE/g)
0.35
0.4 0.3
0.18
0.2 0.09 K. candel 451.63
0
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16

Concentration (mg/ml)

Figure: Total phenolic content determination of

K. candel extract.

Finding: K. candel had higher content of phenolic compounds.

Javanmardi, J., Stushnoff, C., Locke, E., Vivanco, J., 2003. Food Chemistry 83, 547-550.
 Evaluation of Antioxidant Activity (Cont.)
Determination of Total Flavonoid Content (TFC)
Total flavonoid content was determined by aluminum trichloride colorimetric method. The TFC was
calculated from the regression equation of the calibration curve (y = mx + c), expressed in mg quercetin
equivalents (QE) per gram of dry powder ( mg QE/g) (Marinova et al., 2005).
Absorbance of Quercetin
Absorbance Linear (Absorbance)

0.5
Result:
0.42
0.4 f(x) = 0.42 x + 0.03
R² = 0.99 0.34

0.27
Total flavonoid content
Absorbance

0.3 Sample
(mg QE/g)
0.2
0.13

0.1
0.01
K. candel 89.05
0
0 0.25 0.5 0.75 1

Concentration (mg/ml)

Figure: Total flavonoid content determination of

K. candel extract.

Finding: K. candel had good content of flavonoid compounds.

Marinova, D., Ribarova, F., Atanassova, M., 2005. Journal of the University of Chemical Technology and Metallurgy 40, 255-260.
 Evaluation of Antioxidant Activity (Cont.)
Determination of Total Tannin Content (TTC)
Total tannin content was measured by using the Folin-Ciocalteu reagent. TTC was calculated from the
regression equation of the calibration curve (y = mx+c), expressed in mg gallic acid equivalents (GAE) per
gram of dry powder ( mg GAE/g) (Tambe and Bhambar, 2014).
Absorbance of Gallic Acid
Absorbance Linear (Absorbance) Result:
Absorbance

0.6 0.56
0.48
f(x) = − 0.1 x + 0.66
R² = 0.99
0.4 0.35 Total tannin content
0.27
Sample
(mg GAE/g)
0.2 0.17

K. candel 51.2
0
0 1 2 3 4 5 6 7

Concentration (mg/ml)

Figure: Total tannin content determination of

K. candel extract.

Finding: K. candel had higher tannin content.

Tambe, V.D., Bhambar, R., 2014. Res Rev Journal of Pharmacology Phytochemistry 2, 41-47.
 Evaluation of Antioxidant Activity (Cont.)
Determination of Total Antioxidant Capacity (TAC)
The total antioxidant capacity was evaluated by the phosphomolybdenum method. TAC was calculated
from the regression equation of the calibration curve (y = mx+c), expressed in mg ascorbic acid equivalents
(AAE) per gram of dry powder ( mg AAE/g) (Umamaheswari and Chatterjee, 2008).
Absorbance of Ascorbic Acid

Absorbance Linear (Absorbance)

1.89
2 Result:
1.8
1.6 f(x) = 3.74 x − 0.09
R² = 0.98 1.37
1.4
Total antioxidant capacity
Absorbance

1.2
1
0.92 Sample
0.8 0.67
(mg AAE/g)
0.6
0.4
0.2 0
0.23
K. candel 249.59
0
0 0.1 0.2 0.3 0.4 0.5 0.6

Concentration (mg/ml)

Figure: Total antioxidant capacity determination

of K. Candel extract.

Finding: K. candel showed greater antioxidant capacity.

Umamaheswari, M., Chatterjee, T., 2008. African Journal of Traditional, Complementary and Alternative Medicines 5, 61-73.
 Evaluation of in vitro Blood Anticoagulation Activity
The anticoagulant activity was evaluated by prothrombin time test. Blood samples were obtained from
normal individuals and pure platelet plasma (PPP) was isolated by centrifugation. Plasma, plant extracts
and Cacl2 were added together and clotting time was recorded with stop watch (Mao et al., 2009).

Clotting Time (min) vs. Treatment Group

70

60 58.75

50
Coagulation Time (min)

40 37.7

30
21.95
20
13
10 8.59

0
warfarin K. candal Control

Figure: Effect of extract on clotting time (min) of blood in a blood anticoagulation activity test.

Result: K. candel extract exhibited considerable and significant blood anticoagulant activity.
Mao, W., Li, H., Li, Y., Zhang, H., Qi, X., Sun, H., Chen, Y., Guo, S., 2009. International Journal of Biological Macromolecules 44, 70-74.
 Evaluation of in vitro Blood Coagulation Activity
Coagulation was analyzed by measuring the prothrombin time (PT). Plant samples & standard solution were
added to freshly collected blood & time taken for the blood to clot (PT) was recorded (Ikese et al., 2015).

Clotting Time (min) vs. Treatment Group

60

50 48.48
43.92

40
Coagulation Time (min)

36.88

30 27.75

20

10 7.45 8.57
4.35 5.45
2.21
0
Phytomenadione vit k1 K. candel Negative Control

Figure: Effect of extract on clotting time (min) of blood in a blood coagulation activity test.

Result: K. candel extract, further evaluation was carried out to evaluate the in vitro blood anti-coagulation
activity.
Ikese, C., Okoye, Z., Kukwa, D., Adoga, S., Lenka, J., 2015. International Journal of Pharmaceutical Sciences and Research 6, 3391.
 Evaluation of Antihyperglycemic Activity (Cont.)
In vitro Evaluation of α-Glucosidase Enzyme Inhibitory Activity
Mixture of potassium phosphate buffer, enzyme solution and sample or standard solution was first incubated.
Then pNPG (substrate) and Na2CO3 solution was added to it and absorbance was measured at 405 nm to obtain

IC50 (Lawag et al., 2012; Qaisar et al., 2014).

% inhibition
Result:
60

40

20 Test Sample IC50 (mg/ml)

0
0.1 0.2 0.3 0.4 0.5 1
Acarbose 0.382
-20

-40
K. candel extract 1.149
-60

-80

Figure: % inhibition by K. candel to determine IC50

Findings: K. candel plant extracts exhibited antihyperglycemic activity by inhibition of α-glucosidase enzyme.

Lawag, I.L., Aguinaldo, A.M., Naheed, S., Mosihuzzaman, M., 2012. Journal of Ethnopharmacology 144, 217-219.
Qaisar, M.N., Chaudhary, B.A., Sajid, M.U., Hussain, N., 2014. Tropical Journal of Pharmaceutical Research 13, 1833-1836.
 Conclusion
According to the results of the present investigation, following results are

1. The plant extract revealed the presence of reducing sugar, phenolic

compound, tannins, terpenoids, saponins, steroid, flavonoids, alkaloids &
glycosides

2. The Plant extract exhibit good antioxidant activity like DPPH free radical
scavenging activity, H2O2 radical scavenging activity, TPC, TFC, TTC, TAC

3. Exhibited considerable and significant blood anticoagulant activity.

4. Plant extracts exhibited antihyperglycemic activity by inhibition of α-

glucosidase enzyme
 Acknowledgement

I wish to express my profound sense of gratitude to all the respectable teachers and other
members of Pharmacy Discipline for their support to carry out this thesis work.