Comparative in vitro Enzyme Inhibitory activities

of Different Plant Parts of Five Albizia Species

(Leguminosae)

Presented by

Abdullahi A. Adegoke, Queeneth A. Ogunniyi, Abraham

O. Nkumah, Omonike O. Ogbole

UCBR-8 2023 Global Trends In Translational Biomedical Research

September, 2023
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Outline
 Introduction
 Aim and Objectives
 Research design
 Methodology
 Results
 Conclusions
 Acknowledgement
 References
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DIABETES MELLITUS
 Diabetes is a chronic metabolic disorder
 associated with hyperglycemia

 Occurs when the pancreas is no longer able to

make insulin

 When the body cannot make good use of the

insulin it produces (IDF, 2019).

 Impaired metabolism of carbohydrates, proteins

and fats due to inadequate or inefficient activity
of insulin (Seung-Hyun et al., 2022).

Fig 1: Diabetes
www.worlddiabetesday.com
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Fig 2: Symptoms of Diabetes www.bigstock.com

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Fig 3: Types of Diabetes www.mycirclecare.com

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Fig 7: Management of Diabetes www.shutterstock.com

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MEDICINAL PLANTS
o Medicinal plants are regarded as rich resources of traditional medicines
from which many of the modern medicines are produced (Dar et al., 2017).

o Various parts of plants such as leaf, stem, bark and root are being used to
prevent, allay symptoms or revert abnormalities to normal.

o Traditional medicine is still recognized as the preferred primary health care

system in many communities.

o Treatment with medicinal plants is considered very safe as there is no or

minimal side effects.
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Justification of Study

o 60% of the world’s population

o About 80% in developing countries depend directly on medicinal plants for

medical issues.

o Reports on the α-amylase inhibitory activities of some species of the genus

Albizia (Reejo et al., 2014; Ambika and Jegadeesan, 2017)

o Hence, the need to compare the α-amylase and α-glucosidase inhibitory

activities of various plant parts from five species of the genus.

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Aim of Study

o To compare the in vitro α- amylase and glucosidase inhibitory activities

of different plant parts of five Albizia species.

Specific Objectives
o To select medicinal plants with α-amylase and α-glucosidase inhibitory
activity from previous studies.

o To extract and subject crude methanol extract of selected plants to in vitro

α-amylase and α-glucosidase inhibitory assays

o To subject active extracts to bioassay-guided fractionation.

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Significance of  -amylase inhibitors

  -amylase inhibitors play vital role in managing post-prandial

hyperglycemia because it lowers post-prandial blood sugar by

inhibiting α-amylase in the digestive organs.

 It retards the absorption of glucose in the digestive tract.

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Research Design

Plant Selection, Air drying and Concentration

Identification and Extraction in of crude extract
Authentication methanol

BIOASSAY BIOASSAY
 -amylase, - Fractionation  -amylase,  -
glucosidase of most active glucosidase and
extract Brine Shrimp
cytotoxicity
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Albizia lebbeck (L.)Benth. Albizia saman (Jacq.) F. Muell. Albizia ferruginea (Guill. & Perr.) Benth
Common names: Lebbek tree, Igbago Common names: Rain Tree, Local name: Ngu (Igbo), Ayinre-ogo (Yoruba)
(Yoruba) Monkey pod.

Albizia zygia (DC.) J.F.Macbr. Albizia glaberrima (Schum. & Thonn.) Benth
Local name: Nyie avu (Igbo), Ayinre were (Yoruba) . Local name: Ayunre funfun, silk tree
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Procedure for α-amylase Inhibitory Assay

• Samples were tested in different concentrations
• Absorbance, measured at 540 nm.
• Acarbose (standard drug)
• Percentage (%) inhibition and IC50 values were obtained.
(Bernfeld 1955; Ogbole et al., 2019).

• The inhibition of enzyme of -amylase is determined by:

Percentage inhibition = (Abs Control – Abs Sample) x 100

Absorbance control
Procedure for α-glucosidase Inhibitory
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Assay
• Samples were tested in different concentrations
• Absorbance, measured at 405 nm.
• Acarbose (standard drug)
• Percentage (%) inhibition and IC50 values were obtained.
(Kim, 2004; Ogbole et al., 2019).

• The inhibition of enzyme of α-glucosidase is determined

by:

Percentage inhibition = (Abs Control – Abs Sample) x100

Absorbance control
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RESULT
S
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Table 1: Phytochemical analysis of Crude Albizia species

S/N Test ALB ALL ALS ASB ASL ASS AFB AFS AFL AZB AZL AGB AGL

1 ALKALOIDS

Meyer’s reagent ++ ++ + ++ ++ + ++ - ++ ++ + + _

Dragendorff +++ ++ ++ ++ + - ++ - + ++ + + ++

Wagner reagent +++ + ++ ++ ++ - ++ + + ++ + ++ ++

2 SAPONIN

Demonstration of frothing +++ ++ + + ++ + + + ++ +++ + + +

3 ANTHRAQUINONE

Borntragers Test ++ ++ +++ - + + + - - ++ ++ + -

4 CARDIAC GLYCOSIDES

Keller- kellani’s ++ + + ++ + + + + - + - - -
Kedde’s +++ ++ ++ + + + + + - + + - -

5 GLYCOSIDES +++ ++ ++ + + + + + + ++ ++ - -

6 TANNIN +++ + ++ + + ++ + + + +++ + ++ +

7 FLAVONOIDS
Schinodas’s +++ ++ ++ + ++ + ++ + + ++ + + -

General +++ + ++ + - ++ + + + +++ + + -

8 STEROIDS
Liebarman burchard test + ++ ++ - + - - + + + - - ++

Present = +, Moderately present = ++, present in good quality=+++ and absent= -

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Table 2: Percentage (%) inhibition values and Acarbose values for

α- amylase inhibitory assay
Conc. Acarbose AFS ALL AGB ALS AGL AFL AZL AZB ALB ASL ASS ASB AFB

µg/mL

1000 86.44 65.23 62.60 71.37 57.82 45.09 56.89 70.53 73.58 75.42 73.68 89.91 88.86 87.74

500 67.71 54.00 57.48 61.55 56.02 41.70 51.39 51.33 69.62 73.64 58.86 51.41 57.78 51.61

250 42.05 35.33 52.04 59.02 53.41 36.89 50.61 50.99 69.00 70.33 57.51 49.99 33.72 32.48

125 28.25 33.03 44.23 53.96 52.81 36.84 40.44 38.62 67.50 67.35 49.99 26.83 23.47 21.15

61.5 9.69 31.81 43.40 48.33 51.14 33.88 30.90 35.98 65.32 53.35 48.53 15.01 19.36 12.58

31.25 1.90 24.83 33.60 30.85 22.78 32.16 28.25 23.033 62.61 13.34 40.93 13.25 5.70 0.13
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Table 3: Percentage (%) inhibition values and Acarbose values for

α- glucosidase inhibitory assay
Conc Acarbose AFS ALL AGB ALS AGL AFL AZL AZB ALB ASL ASS ASB AFB

µg/mL

1000 63.68 67.08 85.16 19.43 84.70 54.37 67.08 88.64 40.73 93.88 84.70 40.47 22.11 68.43

500 57.14 31.53 68.26 18.35 45.62 46.69 37.14 29.70 30.35 89.84 45.62 39.13 20.46 37.14

250 40.72 26.89 39.66 15.94 38.83 24.79 34.19 21.41 24.40 89.43 38.83 27.00 7.89 34.19

125 31.24 21.61 19.00 8.72 29.92 21.85 33.41 2.10 24.14 85.96 29.92 21.72 7.11 33.41

61.5 28.06 21.56 15.51 1.76 29.80 16.25 19.26 18.47 15.57 85.37 29.80 16.66 4.43 19.26

31.25 24.65 9.40 4.65 0 24.27 12.13 6.52 10.47 4.53 81.55 24.27 13.51 0 6.52
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Table 4: IC50 Values of α-amylase and α-glucosidase inhibition
assay for Crude Extracts of Albizia spp.
Plant Extracts IC50 values (µg/mL) for α-amylase IC50 values (µg/mL) for α-
glucosidase

Albizia lebbeck leaf (ALL) 237.40 265.50

Albizia lebbeck bark (ALB) 42.33 <31.20

Albizia lebbeck seed (ALS) 398.22 >1000

Albizia saman bark (ASB) 304.20 >1000

Albizia saman leaf (ASL) 158.62 517.20

Albizia saman seed (ASS) 263.00 1178.3

Albizia zygia leaf (AZL) 249.14 578.70

Albizia zygia bark(AZB) < 31.25 1158.2

Albizia ferruginea bark (AFB) 320.40 587.10

Albizia ferruginea leaf (AFL) 588.72 523.30

Albizia ferruginea seed (AFS) 495.66 508.40

Albizia glabberima leaf (AGL) 1316.12 739.30

Albizia glabberima bark (AGB) 119.88 >1000

Acarbose 121.50 121.50

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Table 5: Percentage (%) inhibition values for α- amylase inhibitory assay

for Albizia Lebbeck bark partitioned fractions
Conc Acarbose Hexane DCM EtoA Aqeous Butanol

µg/mL

1000 59.28±0.02 31.46±0.12 34.23±0.10 89.25±0.04 75.00±0.04 35.65±0.00

500 54.84±0.01 27.96±0.00 28.65±0.03 71.42±0.10 65.05±0.03 20.34±0.00

250 48.12±0.02 27.17±0.01 14.64±0.04 61.71±0.02 62.00±0.01 11.82±0.00

125 36.38±0.01 24.00±0.04 6.10±0.00 57.03±0.02 49.39±0.03 8.75±0.01

61.5 26.74±0.00 15.87±0.04 5.92±0.00 45.23±0.01 45.73±0.10 6.25±0.01

31.25 22.50±0.01 8.14±0.02 3.10±0.04 35.45±0.02 20.85±0.06 5.14±0.00

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Table 6: Percentage (%) inhibition values for Alpha glucosidase inhibitory

assay for ALB partitioned fractions
Conc Acarbose n-Hexane DCM EtoA Aqeous Butanol

µg/mL

1000 85.71±0.03 96.05±0.00 93.13±0.01 89.56±0.01 88.75±0.02 94.89±0.00

500 78.26±0.01 91.16±0.01 89.85±0.01 89.41±0.01 82.26±0.00 93.64±0.01

250 72.12±0.01 90.02±0.00 86.27±0.03 86.56±0.00 82.11±0.02 86.64±0.03

125 69.63±0.02 86.47±0.00 78.97±0.02 80.87±0.02 80.36±0.03 85.76±0.01

61.5 62.33±0.01 84.67±0.01 78.24±0.02 80.36±0.00 66.93±0.02 85.47±0.01

31.25 51.72±0.01 76.13±0.01 53.43±0.01 69.70±0.02 37.37±0.00 72.26±0.01

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Table 7: IC50 values of α- glucosidase assay for ALB partitioned fractions

60
IC50 values (µg/mL) for α-glucosidase
50

40
n- Hexane
Dichloromethane
30 Ethylacetate
Butanol
Aqeous
20 Acarbose

10

0
Series1
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Table 8: IC50 values α-amylase inhibition assay for ALB partitioned fractions
1200

1000
IC50 values (µg/mL) for α-amylase

800
n- Hexane
Dichloromrthane
600 Ethylacetate
Butanol
Aqeous
Acarbose
400

200

0
Series1
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Table 9: IC50 values of α- glucosidase and α-amylase inhibition assay for

ALB partitioned fractions
Samples IC50 values (µg/mL) for IC50 values (µg/mL) for α-amylase
α-glucosidase

n-Hexane 25.47 >1000

Dichloromethane 37.30 >1000

Ethylacetate 27.16 121.7

Aqeous 53.17 99.71

Butanol < 15.62 >1000

Acarbose 45.23 43.23

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Table 10: Pooled fractions from Vacuum Liquid Chromatograph (VLC)

S/N Fractions Solvent of elution

1 A (1-2) n-hexane/EtoAc

2 B (3-4) n-hexane/EtoAc

3 C (5) n-hexane/EtoAc

4 D (6-8) DCM/Methanol

5 E (9) DCM/Methanol

6 F (10-11) DCM/Methanol

7 G (12-14) DCM/Methanol
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Conclusion
• Plants still remain relevant to give novel remedies to mankind. For
their simple requirements, humans have depended on nature as the
major source of medicines, shelters, foods and other life inspiring
materials because the large proportion of medicinal plants in the
world's population continue to play a dominant role in the
healthcare system, and this is particularly used in developing
countries.
• From this study, it can be concluded that the inhibitory activity of
Albizia lebbeck bark could be due to the relative abundance of
secondary metabolites like alkaloids, saponin, flavonoids and
tannin.
• Albizia lebbeck bark could be a promising plant in the
management of Diabetes mellitus as it inhibited both α‐ amylase
and α‐ glucosidase enzymes used in this study.
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Recommendation
• This plant can be further be explored
pharmacologically by isolating bioactive
compounds for additional studies.

• I hereby recommend that Albizia lebbeck bark

should be formulated into capsule owing to it
hyperglycemia inhibitory potential.
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Selected References
Albizia lebbeck - A Promising Fodder Tree for Semi-Arid Regions, A quick guide to
useful nitrogen fixing trees from around the world, NFTA 88-03, August 1988.
Aridus, Unique Legumes on the University of Arizona Campus Part III Bulletin of the
Desert Legume Program of The Boyce Thompson Southwestern Arboretum
and The University of Arizona, 2006; Vol 18, Number 3.
Chaddha Varun., Nayak S., Solanki Sarita., 2011; Preliminary Phytochemical
Screening On Bark and Pods Of Albizzia Lebbeck Linn, International Journal Of
Comprehensive Pharmacy,11 (06).
Chinenye S, Uchenna DI, Unachukwu CN, et al. The pattern of diabetes mellitus in
Rivers State, Nigeria. Nig End Pract 2008; 2: 87–93.
Dar RA, Shahnawaz M, Qazi PH. Natural product medicines: A literature update. J
Phytopharmacol2017;6(6):349-351.
Joshi A, Sengar N, Prashad SK et.al. Wound-healing Potential of the Root Extract of
Albizia lebbeck. Journal of PlantaMedica 2013, 79(9):737-743.
Kokila K, Priyadharshini S. D, Sujatha V. Phytopharmacological properties of Albizia
species: a review. Int J Pharm Pharm Sci 2013;5:70-3.
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Thanks for the Audience