Alpha amylase Inhibition and Brine Shrim
Alpha-amylase Inhibition and Brine Shrimp Lethality Activities of Nine Medicinal Plant
Extracts Selected from South-West Nigerian Ethnomedicine
Ogbole Omonike Oluyemisia, Aliu Latifat Olabimpea, Abiodun Oyindamola Olajumokeb,
Ajaiyeoba Edith Oriaburea*
a. Department of Pharmacognosy, University of Ibadan, Ibadan, Nigeria
b. Department of Pharmacology and therapeutics, University of Ibadan, Nigeria
* Author for correspondence
Email: edajaiye@gmail.com
Phone no: +2348023222796
Alpha-amylase inhibitors derived from plants posed a new dimension in drug design for the
treatment of postprandial hyperglycemia, a major concern for type-2 Diabetes. This has
necessitated the screening of a large number of ethno medicinally indicated plants from South–
West Nigeria for α-amylase inhibitory activity.
Nine medicinal plants were selected, extracted into methanol and subsequently screened for
alpha-amylase inhibition. Toxicity of plant extracts was determined by brine shrimp lethality
assay (BSLA). The most active plant, Nauclea diderrichii Merr., was fractionated into four
solvent systems (n-hexane, ethyl acetate, dichloromethane and n-butanol). Fractions obtained
were also tested.
Results indicated that all extracts showed varying degree of alpha-amylase inhibition, N.
diderrichii had IC50 = 248.30 ± 0.27 µg/mL, the highest inhibitory activity while its butanol
fraction with IC50 = 137.8 µg/mL had activity comparable to the standard drug, acarbose (IC 50 =
177.50 ± 0.42 µg/mL). The LC50 values for BSLA ranged from 11.35 to 1127.50 μg/mL.
Tetracera scandens L., was the most toxic with 11.35 µg/mL.
Most of the extracts showed potential alpha-amylase inhibitory activity and appeared safe in the
BSLA. Butanol fraction of N. diderrichii will be exploited for the presence of alpha amylase
inhibitory constituents.
KEYWORDS Alpha-amylase inhibitors, Postprandial Hyperglycemia, Brine Shrimp Lethality
Assay, Nauclea diderrichii.
INTRODUCTION
Health management through traditional herbal medicine is gaining importance as it is posing a
re-emergence for the treatment of various lifestyle disorders with minimum side-effects (2), Plant
based herbal medicine system continues to play a frontline role in health management with more
than 80% of the world’s population relying on traditional medicine for their primary health care
(9).
Diabetes mellitus is a group of metabolic disorders characterized by high blood glucose
(hyperglycemia) resulting from defects in insulin secretion, insulin action, or both. The chronic
hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of
different organs, especially the eyes, kidneys, nerves and heart (5). According to the World
Health Organization (WHO), the number of affected people in the world has doubled in the past
few years and it is expected to double again by the year 2025. Globally in 2012 and 2013,
diabetes resulted into 1.5 to 5.1 million deaths per year, making it the 8th leading cause of death
(10). On the African continent, Nigeria has the highest number of people with diabetes with over
1.2 million sufferers (11, 22).
One of the effective approaches to control diabetes is to inhibit the activity of alpha-amylase, an
enzyme responsible for the breakdown of starch, to simpler sugars like dextrin, maltotriose,
maltose and glucose (3). Alpha-amylase catalyses the hydrolysis of α-1,4-glucosidic and α-1,6glucosidic bonds of polysaccharides, starch and glycogen into oligosaccharides and thereby
liberating α-maltose, α-glucose, and α-limit dextrins. (12). Inhibition carried out by alphaamylase inhibitors delays carbohydrate hydrolysis rate and maintains the serum blood glucose in
hyperglycemic individuals (6).
The importance of these enzymes inhibitors has attracted interest because inhibition of the
catalytic activity of α-amylase leads to retardation of the absorption of glucose and the reduction
of postprandial blood glucose level.In addition, secondary metabolites that inhibit alpha-amylase
play a key role in plant defence against pest especially insect., Some inhibitors currently in
clinical use are acarbose and miglitol which inhibit glycosidases such as α-amylase and aglucosidase. However, many of these synthetic agents have their limitations, which include non-
specificity, failure to alleviate diabetic complications and numerous side effects such as bloating,
abdominal discomfort, diarrhea and flatulence (9).The fact that the inhibitors of this enzyme are
not readily available in the pharmaceutical industries underscores the continuous search for
inhibitors from medicinal plants (14).
Brine shrimp lethality (BSL) assay is a simple and inexpensive bioassay developed for screening
plant extracts to detect plant extracts or compounds that are relatively safe or non-toxic. The eggs
of Artemia salina (brine shrimp) have been used in the BSL assay, a simple bench top bioassay
which has yielded good results similar to the mammalian type (13, 20).
Medicinal plants are being used right from ancient times for they are an exemplary source of
drugs due to their high efficacy, reduced cost and minimal side effects and also their safety being
consider using brine shrimp lethality (BSL) assay as many of these plants are taken orally
without any consideration of the toxicity of their components (8).
MATERIALS AND METHODS
Chemicals and Reagents
Alpha (α)-amylase (Aspergillus oryzae), Sodium potassium tartrate, 3,5-Dinitrosalicylic acid,
Potatoes starch, Maltose, Sodium hydroxide pellets were purchased from Sigma Aldrich USA,
USA. Acarbose was purchased from (Serva Heidelberg, Germany). All other chemicals or
reagents used in this study were of analytical grade.
Plant collection and authentication
A total of nine plants collected and identified from an ethno botanical study in Oyo and Ondo
State in South-West Nigeria in November 2013 were used in this research. Plants were identified
and authenticated at Forest Herbarium Ibadan (FHI), Forestry Research Institute of Nigeria
(FRIN), Ibadan where voucher specimen numbers were assigned and plants, deposited.
Acanthospermum hispidum DC., Alchornea laxiflora Benth, Calyptrochilum christyanum
Summerh, Heliotropicum indicum L., Holarrhena floribunda G.Don., Ipomoea asarifolia Desr.,
Nauclea diderrichii Merr., Piper guineense Schumach, Tetracera scandens L. were the nine
plants that were used for the study. The plant materials were air-dried for three (3) weeks and
milled into coarse powder.
Plant extraction
A known quantity (150-400 g) of each plant material was macerated with distilled methanol at
room temperature (29°C) for 72 h. After removal of methanol invacuo using rotary evaporator
percentage yields were calculated and plant extracts were stored in the refrigerator (4 °C) until
used. Thereafter, the crude methanol extract of the most active plant was partitioned into hexane,
dichloromethane, ethyl acetate and butanol.
In vitro Alpha-amylase Inhibitory Assay
The assay was performed using a modified method described by (21). Plant extracts in
concentration range 31.25 to 1000 μg/mL range were prepared, of each extract (250 μL) was preincubated with 250 μL of α-amylase solution (50 µg/mL in 0.02 M sodium phosphate buffer, pH
6.9) at 25°C for 10 min. Thereafter 250 μL of 1% starch solution was added and then further
incubated at 25°C for 10 min. The reaction was terminated by adding 500 μL of dinitrosalicylic
acid (DNS) reagent (96 mM 3, 5-dinitrosalicylic acid and 5.31 M sodium potassium tartrate in 2
M sodium hydroxide). Tubes were then incubated in a water bath at 80°C for 15 min. The
reaction mixture was diluted with 5 mL distilled water and the absorbance was determined at 540
nm (SPECTRA max PLUX, Analytik and Biotechnologie, Germany). An individual blank
(containing 1 mL of buffer and 1 mL 3, 5 dinitrosalicylic acid solutions) was prepared for
correcting the background absorbance. Negative control experiments were representative of the
100% enzyme activity was prepared replacing the extract with distilled water. The α-amylase
inhibitory activity was calculated as percentage inhibition. Acarbose, a well-known α-amylase
inhibitor, was used as positive control. The same procedure was repeated for the standard
(maltose) at concentrations of 2.0 to 0.05 mg/mL and the calibration curve was constructed.
Then, amount of maltose generated from the tests was extrapolated from the equation from
maltose standard curve. All measurements were carried out in triplicates. The inhibition
percentage of α-amylase activity was evaluated by the following formulae;
% Inhibition = 100 – (Absorbance of test)/ (Absorbance of control) × 100
Brine shrimp lethality assay
The assay was carried out according to the method (13). Brine shrimp eggs (A. salina) were
obtained from Prof. Edith O. Ajaiyeoba of Department of Pharmacognosy,University of Ibadan,
Ibadan in Oyo State, Nigeria. They were hatched in natural seawater obtained from the Bar
Beach, Lagos, Nigeria. After 48 h incubation at room temperature and under illumination, the
resulting nauplii (larvae) were attracted to the other side of the tank with a light source and
collected with a Pasteur pipette. After hatching, ten brine shrimps (nauplii) were transferred into
each extract selected concentrations (five dilutions, 1.6 to5000 µg/mL) in 96 micros well plates.
Cyclophosphamide, (a reference cytotoxicity drug) was used as positive control while 3%
methanol was used as negative control. Measurements were carried out in triplicates.
Statistical analysis
All experiments were carried out in triplicates and were expressed as mean ± SEM.
The plant extract/drug concentrations were plotted against percentage inhibitory activity of plant
extract or standard drug. Fifty percent (50%) inhibitory concentration of plant extract/standard
drug (IC50) was determined using non-linear regression in a commercial available statistical
package Microcal Origin®. Similarly, The 50% lethal concentration (LC 50 value) at 95%
confidence interval was calculated for each plant extract in the BSLA.
RESULTS
The plants selected from the survey for this study, including their families, local names, parts
used and percentage yield of methanol extracts are displayed in Table 1. The in vitro alphaamylase inhibitory activity of all the extracts and acarbose, are presented in Table 2. Table 3
represents inhibitory activities of all the fractions with the standard drug acarbose, expressed as
IC50 values in μg/mL, while Figure 1 shows percentage (%) inhibition of the fractions and
acarbose.
All the extracts exhibited varying degree of activities ranging from IC50 values 248.30 ± 0.27 for
Nauclea diderrichii to >1000 μg/mL for Acanthospermum hispidum. Nauclea diderrichii
showing highest activity while A. hispidum had the lowest activity (Table 1). Butanol fraction of
N. diderrichii showed highest activity of all the fractions tested having IC50 value of 137.8 ± 0.37
μg/mL (Table 3).
Results of the Brine shrimp lethality (BSL assay) of all the extracts were displayed in Table
4.The results revealed that eight of the plant extracts appeared safe with LC 50> 1000 µg/mL Only
Tetracera scandens was toxic with LC50 of 11.35 µg/mL, which makes it more toxic than the
standard drug cyclophosphamide, which had LC50 of 16.3 µg/mL. Crude extracts resulting in
LC50 values of less than 100 μg/mL were considered significantly toxic (19).
Table 1.Plant selected for the study
Plant
FHI
Family
No
Acanthospermum 110050 Asteraceae
hispidum DC.
Local
name(Y)
Dagunro
gogoro
Part
used
Aerial
part
Amount
extracted
250g
Yield
(g)
15.88
%
Yield
6.35
Alchornea
laxiflora Benth.
110155
Euphorbiaceae
Pepe
Leaf
250g
33.59
13.44
Calyptrochilum
christyanum
Summerh
110054
Orchidaceae
Ela
Whole
plant
150g
3.25
2.17
Heliotropicum
indicum L.
110156
Boraginaceae
Apari
igun
Aerial
part
400g
22.62
5.66
Holarrhena
floribunda
G.Don.
Ipomoea
asarifolia Desr.
110053
Apocynaceae
Dagba
Leaf
400g
13.82
3.46
110052
Convolvulaceae
Gboro
ayaba
Leaf
400g
26.89
6.72
Piper guineense
Schumach
110051
Piperaceae
Iyere
Leaf
400g
25.71
6.43
Tetracera
scandens L.
Nauclea
diderrichii Merr.
---------
Dilleniaceae
Opon
400g
3.82
3.82
110049
Rubiaceae
Opepe
Aerial
part
Stem
bark
1400g
51
3.64
*Y=Yoruba name
Table 2. Alpha amylase inhibition and Brine Shrimp lethalityof the nine selected plant extracts
Plant Extract
BSLA
LC50 (μg/mL, n = 3)
Acanthospermumhispidum
Alchornealaxiflora
α-amylase inhibitory
activity. IC50 (μg/mL, n =
3)
>1000
295.60 ± 0.53
Calyptrochilumchristyanum
Heliotropicumindicum
467.20 ± 0.32
360.60 ± 0.30
294.80
391.30
Holarrhena floribunda
249.10 ± 0.24
595.10
Ipomoea asarifolia
420.70 ± 0.14
1484
Naucleadiderrichii
248.30 ± 0.27
1127.50
Piper guineense
580.00 ± 0.35
285.50
Tetracerascandens
273.20 ± 0.11
11.35
Acarbose
177.50 ± 0.42
-
Cyclophosphamide
-
16.3
183.70
142.40
n = no of replicates
Table 3. Alpha-amylase inhibitory activities of fractions of Nauclea diderrichii
S/N
N. diderrichii Fractions
IC50 (μg/mL)
1
Butanol
137.8 ± 0.37
2
Ethly acetate
212.8 ± 0.47
3
Dichloromethane
>500
4
Hexane
188.3 ± 0.34
5
Acarbose
177.5 ± 0.42
n=3
DISSCUSSION
Traditional plant remedies or herbal formulations have been in existence since times immemorial
and their use is on the increase despite huge controversies regarding their efficacy and safety
(18). Reports indicate that plants produce a large variety of amylase inhibitors and this provides
protection against insects, their larvae and a lot of microbial pathogens (7, 16).
All the plants used in this study showed varying degree of enzyme inhibition as shown in Table
2, with methanol extract of N. diderrichii exhibiting highest α- amylase inhibitory activity
(248.30 ± 0.27 μg/mL) while Piper guineense had the least inhibitory activity, the standard drug
acarbose had IC50 of177.50 ± 0.42 μg/mL, as presented in Table 2. Assaying various fractions
obtained from N. diderichii indicated that the n-butanol fraction exhibited the highest activity
among the fractions with an IC50 value of 137.8 ± 0.37 μg/mL and dichloromethane fraction had
the least activity with an IC50 value >500 μg/mL as seen in Table 3. This is an indication that the
compound primarily responsible for activity appears to reside in the polar component of the
extract. It is also noteworthy that the butanol fraction had better activity than Acarbose, the
standard drug.
Nauclea diderrichii is a subtropical or tropical moist lowland forests tree. Known as Opepe in
Yoruba language in Nigeria. It is dense and has been known to possess a great degree of
resistance to fungi and insects. Its resistance to termites makes it essentially valuable to the
furniture, art, building and construction industries in West Africa (1).This ability to resist insect
attack might be linked to the presence of enzyme inhibitors and vice versa. A large number of
proteinaceous and non-proteinaceous molecules in plants have been known to act as part of the
plant's natural defense against herbivores in order to prevent feeding (17,7). α-Amylase
inhibitors have been reported as compounds known to confer resistance against herbivore attack
(14,15).
CONCLUSION
Management of diabetes with a holistic approach, devoid of any side effects is now the major
challenge to the medicine and the pharmaceutical industries. One of the effective approaches in
doing this is to inhibit the activity of alpha-amylase enzyme which is responsible for the
breakdown of starch to simple sugars. Of the nine methanol extracts studied, the methanol
extract of stem bark of Nauclea diderrichii was observed to possess the highest alpha-amylase
inhibitory potential. The butanol fraction of N. diderrichii was more active than acarbose. The
study suggests that the inhibitory compound (s) might be in the polar fraction. Further work is
ongoing to isolate and purified inhibitory compound (s) from this plant by chromatographic
methods. In addition, this study confirmed the safety of the medicinal plant extracts being used
traditionally for the prevention and treatment of diabetes, almost all of them appeared safe.
Conflict of interest
The authors declare no conflict of Interest.
Ethical consideration
All applicable international, national, and/or institutional guidelines were followed.
REFERENCES
1. Adeoye, A. O., and R. D. Waigh. 1983. Secoiridoid and Triterpenic acids from the
stems of Nauclea diderrichii Phytochemistry, 22(4): 975–978.
2. Ali, H., P. J. Houghton, and A. Soumyanath. 2006. α- Amylase inhibitory activity of
some Malaysian plants used to treat diabetes; with particular reference to Phyllanthus
amarus. J Ethnopharmacol 107: 449-55.
3. Alexander, R., 1992. Maltodextrins: production, properties and applications. In:
Schenk F, Hebeda R (ed.) Starch hydrolysis products; worldwide technology:
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4. Balaraman, A. K., J. Singh, S. Dash, and T. K. Maity. 2010. Antihyperglycemic and
hypolipidemic effects of Melothriama deraspatana and Cocciniaindica in
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5. Davidson, S. 1995. Principles and practice of medicine. 17th edition; Churchill
Livingstone- Edinburgh.
6. Dineshkumar, B., M. Analava, and M. Manjunatha. 2010. A comparative study of
alpha amylase inhibitory activities of common antidiabetic plants of Kharagpur 1
block. Int J Green Pharmacy 4: 115-21.
7. Franco, J., O. L. Silva, M. Slivinski, C. T. Bloch, C. Rigden, and F. G. Sám. 2002.
Purification, biochemical characterisation and partial primary structure of a new αamylase inhibitor from Secalecereale (rye). Int J Biochemisty and Cell Biology
32:1195-1204.
8. Gauresh, S., C. Rahul, S. Jayant, and S. Sadhana. 2012. Inhibition of carbohydrate
hydrolysing enzymes by methanolic extract of Couroupita guianensis leaves. Int J
Pharm Bioscience 3: 511 – 20.
9. Hansawasdi, C., J. Kawabata, and T. Kasai. 2012. Alpha amylase inhibitors from
Roselle (Hibiscus sabdariffa Linn.) tea. Biotechology Biochemistry 64: 1041-3.
10. IDF DIABETES ATLAS. International Diabetes Federation. 2013. p. 7.ISBN
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11. IDF International Diabetes Federation: 2005. The International Consensus on the
Diabetic Foot and the Practical Guidelines on the management.
12. Lee, S. Y., S. Kim, R. M. Sweet, S.W. Suh. 1991. Crystallization and a preliminary Xray crystallographic study of α-amylase from Bacillus licheniformis. Arch Biochem
Biophy 291:255–257.
13. McLaughlin, J. L. 1991. Crown gall tumors on Potato disc and brine shrimp lethality.
Two simple bioassays for higher plant screening and fractionation. In: Hostettmann,
K. (Ed.), Assays for Bioactivity, vol. 1. Academic Press, London.
14. Mehrabadi, M., A. R. Bandani, and O. Kwon. 2011. Biochemical characterization of
digestive α-d-glucosidase and β-d-glucosidase from labial glands and midgut of
wheat bug Eurygastermaura. Entomological Res 41(3):81-87.
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15. Mehrabadi, M. and A.R. Bandani. 2011. Purification and characterization of midgut
α-Amylase of Eurygasterintegriceps. J Entomological Sciences 12:25-32.
16. Mehta, J. l., N. Rasouli, A. K. Sinha, and B. Molavi. 2006. Oxidative stress in
diabetes: A mechanistic over view of its effects on atherogenesis and myocardial
dysfunction. Int J Biochemistry Cell Biology 38: 794-803.
17. Mendiola-Olaya, E., A. Valencia-J-menez, S. Valdes-RodrA±guez, J. Delano-Frier,
and Blanco-Labra. 2000. Digestive amylase from the larger grain borer, prostephanus
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18. Ramkumar, K. M., B. Thayumanavan, T. Palvannan, and P. Rajaguru. 2010.
Inhibitory effect of Gymnemamon tanum leaves on α-glucosidase activity and αamylase activity and their relationship with polyphenolic content. Med Chem Res
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Annonanceousacetogenins. Phytochem Anal 4: 27-48.
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microwellcytotoxicitiy assay using Artemiasalina (brine shrimp). Planta Med
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21. Tundis, R., M. R. Loizzo, and F. Menichini. 2010. Natural products as alpha-amylase
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22. WHO. October 2013."Diabetes Fact sheet N°312". Retrieved 25 March 2014.
Extracts Selected from South-West Nigerian Ethnomedicine
Ogbole Omonike Oluyemisia, Aliu Latifat Olabimpea, Abiodun Oyindamola Olajumokeb,
Ajaiyeoba Edith Oriaburea*
a. Department of Pharmacognosy, University of Ibadan, Ibadan, Nigeria
b. Department of Pharmacology and therapeutics, University of Ibadan, Nigeria
* Author for correspondence
Email: edajaiye@gmail.com
Phone no: +2348023222796
Alpha-amylase inhibitors derived from plants posed a new dimension in drug design for the
treatment of postprandial hyperglycemia, a major concern for type-2 Diabetes. This has
necessitated the screening of a large number of ethno medicinally indicated plants from South–
West Nigeria for α-amylase inhibitory activity.
Nine medicinal plants were selected, extracted into methanol and subsequently screened for
alpha-amylase inhibition. Toxicity of plant extracts was determined by brine shrimp lethality
assay (BSLA). The most active plant, Nauclea diderrichii Merr., was fractionated into four
solvent systems (n-hexane, ethyl acetate, dichloromethane and n-butanol). Fractions obtained
were also tested.
Results indicated that all extracts showed varying degree of alpha-amylase inhibition, N.
diderrichii had IC50 = 248.30 ± 0.27 µg/mL, the highest inhibitory activity while its butanol
fraction with IC50 = 137.8 µg/mL had activity comparable to the standard drug, acarbose (IC 50 =
177.50 ± 0.42 µg/mL). The LC50 values for BSLA ranged from 11.35 to 1127.50 μg/mL.
Tetracera scandens L., was the most toxic with 11.35 µg/mL.
Most of the extracts showed potential alpha-amylase inhibitory activity and appeared safe in the
BSLA. Butanol fraction of N. diderrichii will be exploited for the presence of alpha amylase
inhibitory constituents.
KEYWORDS Alpha-amylase inhibitors, Postprandial Hyperglycemia, Brine Shrimp Lethality
Assay, Nauclea diderrichii.
INTRODUCTION
Health management through traditional herbal medicine is gaining importance as it is posing a
re-emergence for the treatment of various lifestyle disorders with minimum side-effects (2), Plant
based herbal medicine system continues to play a frontline role in health management with more
than 80% of the world’s population relying on traditional medicine for their primary health care
(9).
Diabetes mellitus is a group of metabolic disorders characterized by high blood glucose
(hyperglycemia) resulting from defects in insulin secretion, insulin action, or both. The chronic
hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of
different organs, especially the eyes, kidneys, nerves and heart (5). According to the World
Health Organization (WHO), the number of affected people in the world has doubled in the past
few years and it is expected to double again by the year 2025. Globally in 2012 and 2013,
diabetes resulted into 1.5 to 5.1 million deaths per year, making it the 8th leading cause of death
(10). On the African continent, Nigeria has the highest number of people with diabetes with over
1.2 million sufferers (11, 22).
One of the effective approaches to control diabetes is to inhibit the activity of alpha-amylase, an
enzyme responsible for the breakdown of starch, to simpler sugars like dextrin, maltotriose,
maltose and glucose (3). Alpha-amylase catalyses the hydrolysis of α-1,4-glucosidic and α-1,6glucosidic bonds of polysaccharides, starch and glycogen into oligosaccharides and thereby
liberating α-maltose, α-glucose, and α-limit dextrins. (12). Inhibition carried out by alphaamylase inhibitors delays carbohydrate hydrolysis rate and maintains the serum blood glucose in
hyperglycemic individuals (6).
The importance of these enzymes inhibitors has attracted interest because inhibition of the
catalytic activity of α-amylase leads to retardation of the absorption of glucose and the reduction
of postprandial blood glucose level.In addition, secondary metabolites that inhibit alpha-amylase
play a key role in plant defence against pest especially insect., Some inhibitors currently in
clinical use are acarbose and miglitol which inhibit glycosidases such as α-amylase and aglucosidase. However, many of these synthetic agents have their limitations, which include non-
specificity, failure to alleviate diabetic complications and numerous side effects such as bloating,
abdominal discomfort, diarrhea and flatulence (9).The fact that the inhibitors of this enzyme are
not readily available in the pharmaceutical industries underscores the continuous search for
inhibitors from medicinal plants (14).
Brine shrimp lethality (BSL) assay is a simple and inexpensive bioassay developed for screening
plant extracts to detect plant extracts or compounds that are relatively safe or non-toxic. The eggs
of Artemia salina (brine shrimp) have been used in the BSL assay, a simple bench top bioassay
which has yielded good results similar to the mammalian type (13, 20).
Medicinal plants are being used right from ancient times for they are an exemplary source of
drugs due to their high efficacy, reduced cost and minimal side effects and also their safety being
consider using brine shrimp lethality (BSL) assay as many of these plants are taken orally
without any consideration of the toxicity of their components (8).
MATERIALS AND METHODS
Chemicals and Reagents
Alpha (α)-amylase (Aspergillus oryzae), Sodium potassium tartrate, 3,5-Dinitrosalicylic acid,
Potatoes starch, Maltose, Sodium hydroxide pellets were purchased from Sigma Aldrich USA,
USA. Acarbose was purchased from (Serva Heidelberg, Germany). All other chemicals or
reagents used in this study were of analytical grade.
Plant collection and authentication
A total of nine plants collected and identified from an ethno botanical study in Oyo and Ondo
State in South-West Nigeria in November 2013 were used in this research. Plants were identified
and authenticated at Forest Herbarium Ibadan (FHI), Forestry Research Institute of Nigeria
(FRIN), Ibadan where voucher specimen numbers were assigned and plants, deposited.
Acanthospermum hispidum DC., Alchornea laxiflora Benth, Calyptrochilum christyanum
Summerh, Heliotropicum indicum L., Holarrhena floribunda G.Don., Ipomoea asarifolia Desr.,
Nauclea diderrichii Merr., Piper guineense Schumach, Tetracera scandens L. were the nine
plants that were used for the study. The plant materials were air-dried for three (3) weeks and
milled into coarse powder.
Plant extraction
A known quantity (150-400 g) of each plant material was macerated with distilled methanol at
room temperature (29°C) for 72 h. After removal of methanol invacuo using rotary evaporator
percentage yields were calculated and plant extracts were stored in the refrigerator (4 °C) until
used. Thereafter, the crude methanol extract of the most active plant was partitioned into hexane,
dichloromethane, ethyl acetate and butanol.
In vitro Alpha-amylase Inhibitory Assay
The assay was performed using a modified method described by (21). Plant extracts in
concentration range 31.25 to 1000 μg/mL range were prepared, of each extract (250 μL) was preincubated with 250 μL of α-amylase solution (50 µg/mL in 0.02 M sodium phosphate buffer, pH
6.9) at 25°C for 10 min. Thereafter 250 μL of 1% starch solution was added and then further
incubated at 25°C for 10 min. The reaction was terminated by adding 500 μL of dinitrosalicylic
acid (DNS) reagent (96 mM 3, 5-dinitrosalicylic acid and 5.31 M sodium potassium tartrate in 2
M sodium hydroxide). Tubes were then incubated in a water bath at 80°C for 15 min. The
reaction mixture was diluted with 5 mL distilled water and the absorbance was determined at 540
nm (SPECTRA max PLUX, Analytik and Biotechnologie, Germany). An individual blank
(containing 1 mL of buffer and 1 mL 3, 5 dinitrosalicylic acid solutions) was prepared for
correcting the background absorbance. Negative control experiments were representative of the
100% enzyme activity was prepared replacing the extract with distilled water. The α-amylase
inhibitory activity was calculated as percentage inhibition. Acarbose, a well-known α-amylase
inhibitor, was used as positive control. The same procedure was repeated for the standard
(maltose) at concentrations of 2.0 to 0.05 mg/mL and the calibration curve was constructed.
Then, amount of maltose generated from the tests was extrapolated from the equation from
maltose standard curve. All measurements were carried out in triplicates. The inhibition
percentage of α-amylase activity was evaluated by the following formulae;
% Inhibition = 100 – (Absorbance of test)/ (Absorbance of control) × 100
Brine shrimp lethality assay
The assay was carried out according to the method (13). Brine shrimp eggs (A. salina) were
obtained from Prof. Edith O. Ajaiyeoba of Department of Pharmacognosy,University of Ibadan,
Ibadan in Oyo State, Nigeria. They were hatched in natural seawater obtained from the Bar
Beach, Lagos, Nigeria. After 48 h incubation at room temperature and under illumination, the
resulting nauplii (larvae) were attracted to the other side of the tank with a light source and
collected with a Pasteur pipette. After hatching, ten brine shrimps (nauplii) were transferred into
each extract selected concentrations (five dilutions, 1.6 to5000 µg/mL) in 96 micros well plates.
Cyclophosphamide, (a reference cytotoxicity drug) was used as positive control while 3%
methanol was used as negative control. Measurements were carried out in triplicates.
Statistical analysis
All experiments were carried out in triplicates and were expressed as mean ± SEM.
The plant extract/drug concentrations were plotted against percentage inhibitory activity of plant
extract or standard drug. Fifty percent (50%) inhibitory concentration of plant extract/standard
drug (IC50) was determined using non-linear regression in a commercial available statistical
package Microcal Origin®. Similarly, The 50% lethal concentration (LC 50 value) at 95%
confidence interval was calculated for each plant extract in the BSLA.
RESULTS
The plants selected from the survey for this study, including their families, local names, parts
used and percentage yield of methanol extracts are displayed in Table 1. The in vitro alphaamylase inhibitory activity of all the extracts and acarbose, are presented in Table 2. Table 3
represents inhibitory activities of all the fractions with the standard drug acarbose, expressed as
IC50 values in μg/mL, while Figure 1 shows percentage (%) inhibition of the fractions and
acarbose.
All the extracts exhibited varying degree of activities ranging from IC50 values 248.30 ± 0.27 for
Nauclea diderrichii to >1000 μg/mL for Acanthospermum hispidum. Nauclea diderrichii
showing highest activity while A. hispidum had the lowest activity (Table 1). Butanol fraction of
N. diderrichii showed highest activity of all the fractions tested having IC50 value of 137.8 ± 0.37
μg/mL (Table 3).
Results of the Brine shrimp lethality (BSL assay) of all the extracts were displayed in Table
4.The results revealed that eight of the plant extracts appeared safe with LC 50> 1000 µg/mL Only
Tetracera scandens was toxic with LC50 of 11.35 µg/mL, which makes it more toxic than the
standard drug cyclophosphamide, which had LC50 of 16.3 µg/mL. Crude extracts resulting in
LC50 values of less than 100 μg/mL were considered significantly toxic (19).
Table 1.Plant selected for the study
Plant
FHI
Family
No
Acanthospermum 110050 Asteraceae
hispidum DC.
Local
name(Y)
Dagunro
gogoro
Part
used
Aerial
part
Amount
extracted
250g
Yield
(g)
15.88
%
Yield
6.35
Alchornea
laxiflora Benth.
110155
Euphorbiaceae
Pepe
Leaf
250g
33.59
13.44
Calyptrochilum
christyanum
Summerh
110054
Orchidaceae
Ela
Whole
plant
150g
3.25
2.17
Heliotropicum
indicum L.
110156
Boraginaceae
Apari
igun
Aerial
part
400g
22.62
5.66
Holarrhena
floribunda
G.Don.
Ipomoea
asarifolia Desr.
110053
Apocynaceae
Dagba
Leaf
400g
13.82
3.46
110052
Convolvulaceae
Gboro
ayaba
Leaf
400g
26.89
6.72
Piper guineense
Schumach
110051
Piperaceae
Iyere
Leaf
400g
25.71
6.43
Tetracera
scandens L.
Nauclea
diderrichii Merr.
---------
Dilleniaceae
Opon
400g
3.82
3.82
110049
Rubiaceae
Opepe
Aerial
part
Stem
bark
1400g
51
3.64
*Y=Yoruba name
Table 2. Alpha amylase inhibition and Brine Shrimp lethalityof the nine selected plant extracts
Plant Extract
BSLA
LC50 (μg/mL, n = 3)
Acanthospermumhispidum
Alchornealaxiflora
α-amylase inhibitory
activity. IC50 (μg/mL, n =
3)
>1000
295.60 ± 0.53
Calyptrochilumchristyanum
Heliotropicumindicum
467.20 ± 0.32
360.60 ± 0.30
294.80
391.30
Holarrhena floribunda
249.10 ± 0.24
595.10
Ipomoea asarifolia
420.70 ± 0.14
1484
Naucleadiderrichii
248.30 ± 0.27
1127.50
Piper guineense
580.00 ± 0.35
285.50
Tetracerascandens
273.20 ± 0.11
11.35
Acarbose
177.50 ± 0.42
-
Cyclophosphamide
-
16.3
183.70
142.40
n = no of replicates
Table 3. Alpha-amylase inhibitory activities of fractions of Nauclea diderrichii
S/N
N. diderrichii Fractions
IC50 (μg/mL)
1
Butanol
137.8 ± 0.37
2
Ethly acetate
212.8 ± 0.47
3
Dichloromethane
>500
4
Hexane
188.3 ± 0.34
5
Acarbose
177.5 ± 0.42
n=3
DISSCUSSION
Traditional plant remedies or herbal formulations have been in existence since times immemorial
and their use is on the increase despite huge controversies regarding their efficacy and safety
(18). Reports indicate that plants produce a large variety of amylase inhibitors and this provides
protection against insects, their larvae and a lot of microbial pathogens (7, 16).
All the plants used in this study showed varying degree of enzyme inhibition as shown in Table
2, with methanol extract of N. diderrichii exhibiting highest α- amylase inhibitory activity
(248.30 ± 0.27 μg/mL) while Piper guineense had the least inhibitory activity, the standard drug
acarbose had IC50 of177.50 ± 0.42 μg/mL, as presented in Table 2. Assaying various fractions
obtained from N. diderichii indicated that the n-butanol fraction exhibited the highest activity
among the fractions with an IC50 value of 137.8 ± 0.37 μg/mL and dichloromethane fraction had
the least activity with an IC50 value >500 μg/mL as seen in Table 3. This is an indication that the
compound primarily responsible for activity appears to reside in the polar component of the
extract. It is also noteworthy that the butanol fraction had better activity than Acarbose, the
standard drug.
Nauclea diderrichii is a subtropical or tropical moist lowland forests tree. Known as Opepe in
Yoruba language in Nigeria. It is dense and has been known to possess a great degree of
resistance to fungi and insects. Its resistance to termites makes it essentially valuable to the
furniture, art, building and construction industries in West Africa (1).This ability to resist insect
attack might be linked to the presence of enzyme inhibitors and vice versa. A large number of
proteinaceous and non-proteinaceous molecules in plants have been known to act as part of the
plant's natural defense against herbivores in order to prevent feeding (17,7). α-Amylase
inhibitors have been reported as compounds known to confer resistance against herbivore attack
(14,15).
CONCLUSION
Management of diabetes with a holistic approach, devoid of any side effects is now the major
challenge to the medicine and the pharmaceutical industries. One of the effective approaches in
doing this is to inhibit the activity of alpha-amylase enzyme which is responsible for the
breakdown of starch to simple sugars. Of the nine methanol extracts studied, the methanol
extract of stem bark of Nauclea diderrichii was observed to possess the highest alpha-amylase
inhibitory potential. The butanol fraction of N. diderrichii was more active than acarbose. The
study suggests that the inhibitory compound (s) might be in the polar fraction. Further work is
ongoing to isolate and purified inhibitory compound (s) from this plant by chromatographic
methods. In addition, this study confirmed the safety of the medicinal plant extracts being used
traditionally for the prevention and treatment of diabetes, almost all of them appeared safe.
Conflict of interest
The authors declare no conflict of Interest.
Ethical consideration
All applicable international, national, and/or institutional guidelines were followed.
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