The Alpha-Amylase Inhibition Potential of Endophytic Fungi from

Indonesian Bay Leaves (Eugenia polyantha WIGHT.)

Wahyu Hidayati, Ade Nur Padillah, Maharadingga, Ni Putu Ermi Hikmawanti, Rini Prastiwi, Ani

Pratiwi, Lady Farahmayuni, Rezza Syahputra, Muhammad Fahrul

Faculty of Pharmacy and Sciences, Universitas Muhammadiyah Prof. DR. HAMKA, Jakarta Timur, Indonesia

Keywords: Eugenia polyantha, Endophytic fungi, Antidiabetic, Alpha-amylase, Inhibition, Diabetes

Abstract: Indonesian people use bay leaves as spices in local culinary and as traditional medicine, particularly to treat

diabetes. The problems with the mass production of antidiabetic drugs from bay leaves can be solved by

utilizing endophytic fungi as an alternative source for antidiabetic compounds. This study aimed to isolate

endophytic fungi from bay leaves and identify their antidiabetic activity through the in vitro inhibition of

alpha-amylase. The leaves were processed on potato dextrose agar media, and five isolates were grown in

an agar medium. The fermentation used a potato dextrose yeast medium that was left for five days on an

orbital shaker at room temperature. The crude was extracted using ethyl acetate solvent. In the in vitro

alpha-amylase inhibition test, the antidiabetic assay used the ethyl acetate extract of the endophytic fungi.

The inhibition percentage was calculated from the absorbance value read by a microplate reader. All isolates

inhibited alpha-amylase activity, but only three of them had high inhibition percentages (14.385%,

12.849%, and 39.246%). As a conclusion, the endophytic fungi isolated from bay leaves are potential as an

alternative source for the production of secondary metabolites to cure diabetes.

1 INTRODUCTION

Eugenia polyantha WIGHT. is one of the plants in

Indonesia that are commonly used as spices in many

local dishes. Empirically, this plant, especially the

leaf, has been widely used as medicine since the

ancient time. People believe that the leaves can cure

some diseases, including diabetes (Aminov, 2010;

Lelono & Tachibana, 2013; Elya et al., 2015;

Murugan et al., 2017).

Diabetes is a chronic disease caused by

insufficient production of insulin hormones by the

pancreas. It is characterized by the elevation of

blood sugar in the body. It can trigger some chronic

metabolite syndromes, such as cardiovascular

diseases and kidney disorders. According to the

statistical data reported by World Health

Organization (2016), in 2012, diabetes directly

caused 1.5 million deaths, and another 2.2 million

deaths were caused by diabetes-related diseases,

such as cardiovascular and kidney disorders.

Furthermore, in 2014, an estimated 422 million

adults over 18 years of age world wide had diabetes.

The Indonesian Ministry of Health reports that 12

million people in Indonesia have diabetes.

Moreover, WHO reveals a slight increase in the

number of diabetic people in Indonesia from 1996 to

2014 (WHO, 2016).

There are several preventive and curative ways

to reduce the number of diabetic people. For

prevention, WHO designs early detection methods,

such as blood glucose measurements, oral glucose

measurements, and HbA1c test. This organization

also introduces new preventive methods, namely

telemedicine and mobile phone-assisted intervention

to reach remote areas. In 2014, the government of

Senegal launched a diabetes monitor application

called mRamadan, which received support from

WHO. This approach improves the diabetes

management in remote areas. Besides prevention,

diabetes requires treatments that focus on the

reduction of blood glucose level. Some of the

commonly used medicines are insulin (by injection

or oral administration), metformin, glibenclamide,

and sulfonylurea (Serrano-Cinca et al., 2005; WHO,

2016).

Nowadays, people still believe that plants can be

used as alternative medicines. As the results, bay

leaves remain as an alternative source to cure

diabetes. The high price of antidiabetic medicines

Hidayati, W., Padillah, A., Maharadingga, ., Hikmawanti, N., Prastiwi, R., Pratiwi, A., Farahmayuni, L., Syahputra, R. and Fahrul, M.

The Alpha-Amylase Inhibition Potential of Endophytic Fungi from Indonesian Bay Leaves (Eugenia polyantha WIGHT.).

DOI: 10.5220/0008240201070111

In Proceedings of the 1st Muhammadiyah International Conference on Health and Pharmaceutical Development (MICH-PhD 2018), pages 107-111

ISBN: 978-989-758-349-0

107

contributes to this preference. Most of them are not

affordable for some people from low- to middle-

income families, limiting their options to herbal

medicines. The high expectation of the therapeutic

benefits of bay leaves triggers herbal medicine

companies to commercialize any drugs made of bay

leaves. However, they have to face a major obstacle,

that is finding the area to plant E. polyantha

WIGHT. The utilization of microbes in the

production of metabolites to cure diabetes

experiences a corresponding situation. The

potentially explorable microbes, in this case, are

endophytes. Endophytes are groups of microbes that

live inside plant tissue and have a symbiotic

relationship with the plants. They are also

responsible in the biosynthesis of secondary

metabolitesin the host plants (Strobel & Daisy 2003;

Staniek et al., 2008; Mishra et al., 2014; Pimentel et

al., 2011).

Based on the previous information, this study

aimed to investigate the potential of endophytic

fungi for producing secondary metabolites that could

inhibit alpha-amylase.

2 MATERIALS AND METHOD

2.1 Materials

This study used fresh bay leaves from Bekasi, West

Java, Indonesia. It also used some media and

reagents for the experiments. The endophytic fungi

isolation used 70% ethanol, 5.3% NaOAc,

Chloramphenicol (Generik, Indonesia), Potato

Dextrose Agar or PDA (Merck, USA), aquadest.

The Potato Dextrose Yeast or PDY (Merck, USA)

and ethyl acetate solvents were also used for the

fermentation and extraction process. As for the

alpha-amylase inhibition assay, the materials were

alpha-amylase enzyme from Bacillus sp. (Sigma-

Aldrich, USA), Phosphate Buffer Saline or PBS

(Sigma-Aldrich, USA), soluble starch (Sigma-

Aldrich, USA), acarbose (Generik, Indonesia), and

the Lugol’s reagent.

2.2 Methods

This study consist of several steps, namely

endophytic fungi isolation, fermentation and

secondary metabolites extraction, and alpha-amylase

inhibition assay as the last procedure.

2.2.1 Endophytic Fungi Isolation

This step began with leaf surface sterilization as

described in Widowati et al. (2016). The sterilized

leaves were then cut into several pieces (1 cm x 1

cm), put into a sterile PDA medium, and incubated

at room temperature for seven days.

2.2.2 The Fermentation and Extraction of

Secondary Metabolites

The fermentation was based on the protocol

introduced by Suciatmih et al. (2011) with some

modifications. Two pieces of endophyte isolates (1

cm x1 cm) were added to 20 ml of PDY medium and

incubated in a shaking incubator at room

temperature for seven days at 170 rpm. Afterward,

the metabolites were harvested by extracting the

fermented medium. The extraction involved the

addition of 1 volume of ethyl acetate into the

fermented medium that had already separated from

mycelium. The extract was then dissolved in PBS

for 500 ppm dosage.

2.2.3 The Inhibitory Activity of Alpha-

Amylase Assay

This process followed the inhibition assay for alpha-

amylase described in Kellogg et al. (2014). It used

the extract as the samples, acarbose as the positive

control, the uninhibited enzyme as the negative

control, and the extracts or acarbose without enzyme

as blanks. For the samples and the positive control,

this research used 96-well plate filled with 35 ul of

the extract or acarbose and 5 ul of the starch

substrate. The plates were then incubated at 37

C for

5 min. Afterward, 10 ul of the alpha-amylase

enzyme solution was added to each well and

incubated at 37

C for 10 min. The addition of 150 ul

of Lugol’s solution to each well would stop the

reaction. Then, the inhibition percentage was

calculated according to the absorbance value read by

a microplate reader at a wavelength of 595 nm. The

calculation of the inhibitory activity of each sample

used the formula (1)

% ℎ 





−(



−



)





100%

(1)

con

is the absorbance of the uninhibited enzyme,

ext

is the absorbance of the enzyme treated with the

extract, and A

blank

is the absorbance of the extract

with the substrate (no presence of enzyme).

MICH-PhD 2018 - 1st Muhammadiyah International Conference on Health and Pharmaceutical Development

108

2.2.4 Data Analysis

The analysis results were the average value (in

mean±SD) of the triplicate assay.

3 RESULTS AND DISCUSSION

The term endophyte defines the community of

microbes living inside plant tissue without damaging

their host. These organisms are reported to be

responsible for the secondary metabolic biosynthesis

of their host plants (Strobel & Daisy 2003; Strobel

2006; Staniek et al., 2008; Alvin et al., 2014; Zhang

et al., 2012). Therefore, the microbes produce some

compounds that can be found in the host plants (Qiu

et al., 2015; Zhang et al., 2012; Tan & Zou 2001).

The isolation of microbes from their host is the

most difficult procedure in endophyte research due

to the risk of contamination. As a preventive

measure, some antibiotics were introduced to the

isolation medium. This study used Chloramphenicol

to inhibit the presentation of bacteria. The isolation

commenced with placing the part of the plants’

organ on the agar medium. Afterward, this medium

was incubated for several days. This research

obtained five endophytic fungi after seven days of

incubation (Figure. 1).

The secondary metabolites were cultivated in a

liquid medium using the liquid fermentation method.

The harvested metabolites were collected through

ethyl acetate extraction. This study used ethyl

acetate instead of other solvents because the

targetted compounds were flavonoids. The other

reason for choosing the solvent was polarity. Ethyl

acetate is a semipolar solvent, meaning that it

attracts polar to semi-polar metabolites (Haque et

al., 2005). Flavonoid is polar and, therefore,

removable by ethyl acetate.

The antidiabetic activity of extracts from plants

or microbes can be measured in vitro and in vivo.

There are two types of assay commonly used in in

vitro experiments, namely β-glucosidase and α-

amylase inhibition. Analyzing the inhibition of these

enzymes is necessarybecause they induce the

absorption of glucose and the associated

postprandial hyperglycemic spike (Kellogg et al.,

2014; Sahani et al., 2017; Ruzieva et al., 2017).

Such inhibition is a strategy in diabetes management

as it can control the serum glucose level.

Figure 1. The isolates of endophytic fungi from Indonesian bay leaves: (A) KSP-O1, (B) KSP-02, (C) KSP-03, (D)

KSP-04, and (E) KSP-05

The Alpha-Amylase Inhibition Potential of Endophytic Fungi from Indonesian Bay Leaves (Eugenia polyantha WIGHT.)

109

This study only performed the inhibition assay

for alpha-amylase. Alpha-amylase is an enzyme that

hydrolyzes carbohydrate into glucose, and in

advance, it is easily absorbable (Kellogg et al.,

2014). Most of this enzyme is present in saliva, and,

as aconsequence, the serum glucose level becomes

higher easily because the hydrolyzation of

carbohydrate already begins in the mouth (Sahani et

al., 2017). The inhibitio n of alpha-amylase activity

decelerates the conversion of starch into glucose and

effectively reduces glucose absorption (Ruzieva et

al., 2017; Sahani et al., 2017). Based on the data

presented in Ta ble 1, each isolate of the endophytic

fungi inhibited the enzyme at different rates. Among

the five isolates, KSP-4 had the lowest activity. On

the contrary, KSP-5 had the highest percentage of

inhibitory activity, but it was lower than acarbose

(Figure.2).

4 CONCLUSIONS

Nowadays, endophytes become increasingly

attractive to observe particularly due to the difficulty

of finding a large area to grow medicinal host plants

for product commercialization. One of the chronic

diseases that causes high amount of mortality is

diabetes. Diabetes can be handled with prevention

and medication. Reducing blood glucose level is the

effective management in increasing the survival rate

of diabetic people. This study showed that the

endophytic fungi from one of the Indonesian

medicinal plants could inhibit the activity of alpha-

amylase. Further investigation is needed to optimize

the extraction to obtain the lowest concentration

with the greatest inhibitory activity and to identify

the in vitro inhibitory ability of endophytic fungi

against the alpha-glucosidase enzyme.

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