Phytochemical and Pharmacological Evaluation of

Methanolic Extract of Allophylus cobbe (L.) Leaves

Presented By: Tanvir Ahamed

Examination Roll No.: 141004
Examination Session: 2014-2015
Department of Pharmacy
Jashore University of Science and Technology.
Jashore-7408, Bangladesh

Allophylus cobbe (L)

Allophylus cobbe (L.)
Plant Preview
Family: Sapindaceae

Morphology:
Leaves: 1-3(-4)- foliolate, often minutely glandular beneath,
green.
Flowers: small, slightly irregular, shortly pedicellate & stalked.
Fruits: Red, lobed & seeded, drupe like
Seeds: Straw-coloured, ellipsoid to globose, without an aril.

Distribution:
Bangladesh, India, Sri Lanka, South Africa, South America,
Southeast Asia, Papua New Guinea.

Traditional Uses
Bone fractures recovery, relief from joint pains, for rashes, for stomach
ache.
 Literature Review of Allophylus cobbe
Findings Plant parts Reference
Chavan, R., & Gaikwad, D. (2016). The Ethnobotany,
Phytochemistry and phytochemistry and biological properties of Allophylus species
Leaves & Barks
biological properties used in traditional medicine: a review. World Journal of Pharmacy
and Pharmaceutical Sciences, 5(11), 664-682.
Castillo, L., González-Coloma, A., González, A., Díaz, M., Santos,
E., Alonso-Paz, E., . . . Rossini, C. (2009). Screening of
Nematocidal activity Roots
Uruguayan plants for deterrent activity against insects. Industrial
Crops and Products, 29(1), 235-240
Marwah, R. G., Fatope, M. O., Al Mahrooqi, R., Varma, G. B., Al
Abadi, H., & Al-Burtamani, S. K. S. (2007). Antioxidant capacity
Antioxidant capacity Leaves
of some edible and wound healing plants in Oman. Food
chemistry, 101(2), 465-470.
 Aims and Objectives

So the leaves of Allophyllus cobbe 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 Antioxidant
activity, Blood coagulation activity, Blood anti-coagulation activity, Anti-hyperglycemic activity.
C: GC-MS analysis: For evaluation and identification of major components.

Allophylus cobbe leaves

 Collection & Extraction
Leaves of Allophylus cobbe was collected from Chittagong, Bangladesh in January, 2020 & Plants were
extracted by cold extraction method using 96% Methanol.

Rotary
Collection Filtration
Evaporation

Vacuum
Chopping Maceration %Yield
desiccation
~5.6%

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 +

06 Test for Steroids - Salkowski Test +
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 _

[(-) for absence; (+) for low; (++) for medium; (+++) for high concentration, based on color density]
 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).
Result:
% inhibition vs Concentration
100.00
90.00
80.00 Test Sample IC50 (µg/ml)
70.00
% inhibition

60.00
50.00 Ascorbic acid 46.25
40.00
30.00
20.00 A. cobbe extract 77.83
10.00
0.00
1 2 4 8 16 32 64 128 256 512 1024

Concentration
Finding: A. cobbe extract has substantial DPPH
Standard Allophylus cobbe
free radical scavenging activity, which is
Figure: Comparison of % inhibition by A. cobbe and comparable to the Ascorbic acid standard.
Ascorbic acid to determine IC50.

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
Result:
100.00
90.00
80.00
70.00 Test Sample SC50 (µg/ml)
% Scavenged

60.00
50.00
40.00
Ascorbic acid 64.35
30.00
20.00
10.00 A. Cobbe extract 103.37
0.00
6.25 12.5 25 50 100 200 400 800

Concentration
Finding: A. cobbe extract has substantial H2O2
Standard Allophylus cobbe radical scavenging activity, which is comparable to
Figure: Comparison of % scavenged by Allophylus cobbe that of ascorbic acid.
and ascorbic acid to determine SC50.

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 Result:

Absorbance Linear (Absorbance)
Absorbance

0.8
0.77 Total phenolic content
f(x) = 5.55 x − 0.03 0.63 Sample
0.6
R² = 0.95 (mg GAE/g)
0.35
0.4 0.3
A. cobbe 490.84
0.18
0.2 0.09

0
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16
Finding: A. cobbe contains high amount of phenolic
Concentration (mg/ml) compounds , that’s why it exhibits good
Figure: Total phenolic content determination of antioxidant activity.
A. cobbe extract.

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 Result:

Absorbance Linear (Absorbance)

0.5 0.42
0.4 f(x) = 0.42 x + 0.03 0.34 Total flavonoid content
R² = 0.99 Sample
Absorbance

0.27
0.3
(mg QE/g)
0.2 0.13
0.1
0.01
A. cobbe 145.46
0
0 0.25 0.5 0.75 1

Concentration (mg/ml)
Finding: A. cobbe had good content of flavonoidal
Figure: Total flavonoid content determination of compounds.
A. cobbe extract.

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 Result:
Absorbance

Absorbance Linear (Absorbance)

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

A. cobbe 51.6
0
0 1 2 3 4 5 6 7

Concentration (mg/ml)
Finding: A. cobbe had good proportion of
Figure: Total tannin content determination of tannin content.
A. cobbe extract.

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
Result:
Absorbance Linear (Absorbance)
2 1.89
1.8
1.6 f(x) = 3.74 x − 0.09
1.37
Total antioxidant capacity
1.4 R² = 0.98 Sample
(mg AAE/g)
Absorbance

1.2
0.92
1
0.8 0.67
0.6 A. cobbe 142.87
0.4 0.23
0.2 0
0
0 0.1 0.2 0.3 0.4 0.5 0.6
Finding: A. cobbe showed substantial
Concentration (mg/ml)
antioxidant capacity.
Figure: Total antioxidant capacity determination
of A. cobbe extract.

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 56.79

50
44.92
Coagulation Time (min)

40 36.62

30
25.76

oo
Fr
es

bl
h

d
51
g2

l5
20

g/
m

m
.
2

l
5

m
m

.
m

m
1
0

/
g
/

1m

m
2
0
0

0g
0/

ml

m
5
0
g

/m
9.37

2l

mg
g/

ml
10 7.49

l
4.46 5.46
2.36
0
Phytomenadione vit k1 Allophylus cobbe Negative Control

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

Result: A. cobbe extract possess no blood-coagulation property. 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).

70
Clotting Time (min) vs. Treatment Group

350 mg/ml
60 58.75

175 mg/ml
Coagulation Time (min)

50
5 mg/ml

41.36

87.5 mg/ml

0.9% saline
40

30 28.89

20 16.39

10 8.54

0
warfarin Allophylus cobbe Control

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

Result: A. cobbe extract exhibited considerable and significant blood anticoagulation 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 Antihyperglycemic Activity
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
inhibition
et al., 2012; Qaisar et al., 2014). Result:
60
50
40 Test Sample IC50 (mg/ml)
30
20
10
Voglibose 0.382
0
0.1 0.2 0.3 0.4 0.5 1
-10 A. cobbe extract 0.742
-20
-30
-40 Findings: A. cobbe plant extracts exhibited anti-
Figure: % inhibition by A. cobbe to determine IC50 hyperglycemic 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.
 GC-MS Analysis
One microliter of extract was injected in split less mode into the injection port of the GC system.
The inlet temperature was set at 250 °C, and the oven temperature was programmed as 60 °C for 0 min,
followed by ramping to 240 °C min−1 at 5 °C min−1 for 4 min. Total run time was 40 min. Helium gas
was used as the carrier gas at a constant flow rate of 1.0 ml/min. The interface transfer line temperature
was set at 280 °C.
MS detection was set in scan mode. Quadrupole analyzer temperature was 230 °C; ion source
temperature was 150 °C. Ions were obtained by electron ionization (EI) mode at 70 eV. The scan time
and mass
range were 1 s and 50–550 m/z, respectively

Béjaoui, A., Chaabane, H., Jemli, M., Boulila, A., & Boussaid, M. (2013). Essential oil composition and antibacterial activity of Origanum vulgare subsp.
glandulosum Desf. at different phenological stages. Journal of Medicinal Food, 16(12), 1115-1120
 GC-MS Analysis (cont.)
GC-MS analysis of the plant extract led to reliable identification of 20 different compounds. 20 characteristic peaks
were present. The major compounds detected in plant were bis(2-ethylhexyl) phthalate, n-benzylaniline, n-
trifluoroacetyl and phthalic acid, 4-methylhept-3-yl octadecyl ester, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate,
tetradecanoic acid, 10,13-dimethyl-, methyl ester, phytyl palmitate, coenzyme q12 (ubiquinone 60)

R.Time (min) Name MW Structure

29.254 BIS(2-ETHYLHEXYL) PHTHALATE 390

29.414 N-BENZYLANILINE, N- 279

TRIFLUOROACETYL-
 GC-MS Analysis (cont.)

R.Time (min) Name MW Structure

29.548 PHTHALIC ACID, 4-METHYLHEPT-3-YL 530

OCTADECYL ESTER

27.311 TETRADECANOIC ACID, 10,13-DIMETHYL-, 270

METHYL ESTER

38.498 COENZYME Q12 (UBIQUINONE 60) 998

 GC-MS Analysis (cont.)

Total ion chromatogram of methanolic extract of A. cobbe (1 µL of sample).

 GC-MS Analysis (cont.)
Chemical composition of A. cobbe
SL Retantion Time Chemical Name MW Area
(Min) %
1 18.315 ALPHA.-GUAIENE 204 0.77 %

2 18.388 BENZENE, 1-(1,5-DIMETHYL-4-HEXENYL)-4-METHYL- 202 1.11 %

3 18.402 BENZENE,1-(1,5-DIMETHYL-4-HEXENYL)-4-METHYL-(S,Z)-2-METHYL-6- 202 0.23 %

(PTOLYL)HEPT-2-EN-1-OL
4 19.319 BICYCLO[3.2.1]OCT-2-ENE, 3-METHYL-4-METHYLENE- 134 0.48 %

5 20.893 2,2,4-TRIMETHYL-1,3-PENTANEDIOL DIISOBUTYRATE 286 11.12 %

6 21.449 TRIDECANE, 3-METHYL-2,12-DIPHENYL-5,7,9-TRIS(1-PHENYLETHYL)- 662 1.46 %

7 24.799 MYO-INOSITOL, 4-C-METHYL- 194 3.19 %

8 25.388 CYCLOHEXANONE, 2-METHYLENE-5-(1-METHYLETHYL)- 152 0.83 %

9 25.569 2-PENTACOSANONE 366 0.51 %

10 25.763 1,5-DIPHENYL-2H-1,2,4-TRIAZOLINE-3-THIONE 253 0.72 %

 GC-MS Analysis (cont.)
Chemical composition of A. cobbe
SL Retantion Time Chemical Name MW Area
(Min) %
11 27.311 TETRADECANOIC ACID, 10,13-DIMETHYL-, METHYL ESTER 270 4.68 %

12 28.242 PENTADECANOIC ACID, 15-BROMO 320 1.38 %

13 29.254 BIS(2-ETHYLHEXYL) PHTHALATE 390 65.11 %

14 29.414 N-BENZYLANILINE, N-TRIFLUOROACETYL- 279 1.25 %

15 29.548 PHTHALIC ACID, 4-METHYLHEPT-3-YL OCTADECYL ESTER 530 2.26%

16 30.755 PHYTYL PALMITATE 534 1.95 %

17 30.895 13-OCTADECENOIC ACID, METHYL ESTER 296 1,95%

18 30.982 TETRADECANOIC ACID, 10,13-DIMETHYL-, METHYL ESTER 270 3.51 %

19 31.103 11,14-OCTADECADIENOIC ACID, METHYL ESTER 294 0.60%

20 38.498 COENZYME Q12 (UBIQUINONE 60) 998 0.40%

 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

5. GC-MS profiling indicated a total of 20 bioactive saturated and unsaturated fatty

acids and fatty acid methyl ester compounds which possess many desirable biological
activities
 Acknowledgement

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