Phytochemical and Pharmacological Evaluation of Ethanolic Extract of

Thorns of Mangrove Plant Species- Phoenix paludosa Roxb.

Presented By:
Examination Roll No.: 131027
Examination Session: 2013-2014
Phoenix paludosa Roxb
Department of Pharmacy
Jashore University of Science and Technology.
Jashore-7408, Bangladesh
 Plant Preview
Phoenix paludosa Roxb
Family: Arecaceae

Description:
Perennial palm growing up to a height of 6 m. Stem slender, cylindrical,
unbranched, distinct leaf scars.  Leaves pinnately compound, waxy,
glabrous, midrib strong, ending into strong sharp spine at apex, few
pairs of lower segments modified into sharp spines.

Distribution:
Andaman Is., Assam, Bangladesh, Cambodia, India, Malaya, Myanmar,
Nicobar Is., Sumatera, Thailand, Vietnam.

Traditional Uses
Anti-helminthic, Whole plant is used in construction of house, used as
medicine for insomnia.
 Literature Review of Phoenix paludosa Roxb
Findings Plant parts Reference

Lima, A., Parial, R., Das, M., & Das, A. K. (2010). Phytochemical and
Phytochemical and pharmacological studies of ethanolic extract from the leaf of mangrove
pharmacological studies Leaves plant Phoenix paludosa Roxb. Malaysian Journal of Pharmaceutical
Sciences, 8(2), 59-69.

Saha, S., Islam, M. K., Anisuzzman, M., Hasan, M. M., Hossain, F., &
antioxidant, analgesic and Leaves Talukder, C. (2012). Evaluation of antioxidant, analgesic and antidiarrheal
antidiarrheal activity activity of Phoenix paludosa roxb Leaves. International Journal of Basic
Medical Sciences and Pharmacy (IJBMSP), 2.

Samarakoon, S. R., Shanmuganathan, C., Ediriweera, M. K., Tennekoon, K.

H., Piyathilaka, P., Thabrew, I., & de Silva, E. D. (2016). In vitro cytotoxic
Cytotoxic and Antioxidant
Leaves and antioxidant activity of leaf extracts of mangrove plant, Phoenix
Activity paludosa Roxb. Tropical Journal of Pharmaceutical Research, 15(1), 127-
132.

Development and Akkak, A., Scariot, V., Marinoni, D. T., Boccacci, P., Beltramo, C., & Botta, R.
(2009). Development and evaluation of microsatellite markers in Phoenix
evaluation of microsatellite Leaves
markers dactylifera L. and their transferability to other Phoenix species. Biologia
Plantarum, 53(1), 164-166.
 Aims and Objectives

So the leaves of Phoenix paludosa 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 like Phytochemical
Screening, Antioxidant activity, Blood coagulation activity, Blood anti-coagulation activity, Anti-
hyperglycemic activity.

Phoenix paludosa Roxb

 Collection & Extraction
Leaves of P. paludosa 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
07 Test for Flavonoids
08 Test for Alkaloids Absent

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

60.00
50.00
40.00
30.00 Ascorbic acid 77.83
20.00
10.00
0.00
1 2 4 8 16 32 64 128 256 512 1024
P. paludosa extract 181.02
Concentration

Standard P. paludosa

Figure: Comparison of % inhibition by P. paludosa and

ascorbic acid to determine IC50.

Finding: P. paludosa activity of DPPH free radical scavenging is comparable with 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

80.00
Test Sample
% Scavenged

SC50 (µg/ml)
60.00

40.00
Ascorbic acid 89.63
20.00

0.00
6.25 12.5 25 50 100 200 400 800 P. paludosa extract 107.59
Concentration

Standard P. Paludosa

Figure: Comparison of % scavenged by P. paludosa and

ascorbic acid to determine SC50.

Finding: P. paludosa activity of H2O2 radical scavenging is comparable with 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 P. paludosa 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

P. paludosa extract.

Finding: P. paludosa had good amount 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
P. paludosa 89.05
0
0 0.25 0.5 0.75 1

Concentration (mg/ml)

Figure: Total flavonoid content determination of

P. paludosa extract.

Finding: P. paludosa 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)
Absorbance

0.6 0.56 Result:

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

P. paludosa 280.50
0
0 1 2 3 4 5 6 7

Concentration (mg/ml)

Figure: Total tannin content determination of

P. paludosa extract.

Finding: P. paludosa contains good amount of 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
P. paludosa 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 P. paludosa extract.

Finding: P. paludosa contains good amount of antioxidant capacity.

Umamaheswari, M., Chatterjee, T., 2008. African Journal of Traditional, Complementary and Alternative Medicines 5, 61-73.
 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

Fresh Blood
30 1.25 mg/ml 27.75

100 mg/ml
200 mg/ml
10 mg/ml

50 mg/ml
5 mg/ml

25 mg/ml
mg/ml

20
2.5

10 7.45 8.57
4.35 5.45
2.21
0
Phytomenadione vit k1 P. paludosa Negative Control

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

Result: P. paludosa 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 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

58.75

350 mg/ml
60

175 mg/ml
50
Coagulation Time (min)

87.5 mg/ml

0.9% saline
40
5 mg/ml

33.88

30
24.9

20
13.5
10 8.54

0
warfarin P. peludosa Control

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

Result: P. paludosa 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 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:
40

20
Test Sample IC50 (mg/ml)
0
0.1 0.2 0.3 0.4 0.5 1

-20
Acarbose 0.382
-40 P. paludosa extract 1.864
-60

-80

Figure: % inhibition by P. paludosa to determine IC50

Findings: P. paludosa 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.