Isolation and Screening Endophyte Bacteria From Sanrego (Lunasia amara)

1. AIP Conference Proceedings 2513, 020015 (2022); https://doi.org/10.1063/5.0100111 2513, 020015
© 2022 Author(s).
Isolation and screening endophytic bacteria
producing α-glucosidase inhibitor from
Sanrego plant (Lunasia amara Blanco)
Cite as: AIP Conference Proceedings 2513, 020015 (2022); https://doi.org/10.1063/5.0100111
Published Online: 16 November 2022
Sri Winarsih, Noorhamdani Noorhamdani, Tri Ardyati, et al.

2. Isolation and Screening Endophytic Bacteria Producing α-
Glucosidase Inhibitor from Sanrego Plant (Lunasia amara
Blanco)
Sri Winarsih1*
, Noorhamdani Noorhamdani2
, Tri Ardyati3
, Adriani Adriani4,5
1
Department of Pharmacy, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia
2
Department of Microbiology Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia
3
Department of Biology, Faculty of Mathematics and Science Universitas Brawijaya, Malang, Indonesia
4
Doctoral Program in Medical Science, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia
5
Department of Biology Education in STKIP Pembangunan Indonesia, Makassar, Indonesia
*
Corresponding author email: wien23.fk@ub.ac.id
Abstract. Endophytic bacteria can be a potential source of new bioactive molecules which has medicinal properties like
their host plant. One of the Indonesian herbs is Sanrego plant (Lunasia amara Blanco), which is empirically used as
antidiabetic by people in South Sulawesi. This study aimed to isolate the endophytic bacteria producing α-glucosidase
inhibitor from the Sanrego plant. The endophytic bacteria isolated from leaf and stem of Sanrego plant, and screening of
the target bacteria using thin layer chromatography (TLC) with scopoletin as the control due to scopoletin is the
secondary metabolite of the Sanrego plant which has antidiabetic activity by in-silico. Alpha-glucosidase inhibitory test
carried out by colorimetric method using pNPG substrate, at 405 nm wavelength compared to acarbose. This study found
14 isolates from Sanrego leaf and 25 isolates from Sanrego stem. The ten isolates of them showed α- glucosidase
inhibitory effect, which were three isolates from leaves (D1-D3) and seven isolates from stems (B1-B7). The appearance
of the endophytic bacteria was circular, convex, and yellow in color; while the cell morphology mostly were Gram-
positive bacteria. Statistical analysis indicated the D1 isolate had the highest inhibition value than the other isolates
(ANOVA, p = 0.002). Interestingly, the inhibition value of the D1 isolate (8.67% ± 2.56) was higher compared to
acarbose (3.73% ± 1.23). It can be concluded that the endophytic bacteria from the Sanrego plant produce α-glucosidase
inhibitor compound, and the D1 isolate can be further investigated its potency as a source of α-glucosidase inhibitor.
Keywords: endophytic, bacteria, Lunasia amara, α-glucosidase inhibitor, screening
INTRODUCTION
The number of diabetic patients worldwide increasing every year, and this is a serious problem in health.
Diabetes causes organ complications and even death if not treated appropriately. The number of diabetic patients
worldwide is estimated increase from 415 million to 642 million by 2040 [1]. Indonesia has ranks 7th with the
largest number of diabetes patients in the world in 2019 [2]. High blood glucose levels are the characteristic of
diabetes, thus controlling blood glucose levels is important for diabetes treatment [3, 4].
Alpha-glucosidase is a digestive enzyme involved in the hydrolysis of carbohydrates into glucose. Inhibition of
this enzyme activity will delay degradation of oligosaccharides into glucose and it can control postprandial
hyperglycemia [5]. Some of the α-glucosidase inhibitors currently in clinical use are acarbose, miglitol, and
voglibose. However, long-term use and high dose of these drugs cause side effects such as diarrhea, abdominal pain,
flatulence, and other digestive disorders [6]. Currently, herbal medicines with antihyperglycemic activity have been
The 3rd International Seminar on Smart Molecule of Natural Resources (ISSMART) - Asian Federation of Biotechnology (AFOB) 2021
AIP Conf. Proc. 2513, 020015-1–020015-7; https://doi.org/10.1063/5.0100111
Published by AIP Publishing. 978-0-7354-4219-1/$30.00
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3. used as alternative drugs for type 2 diabetes because of their fewer side effects and more affordable costs compared
to synthetic hypoglycemic drugs [7].
Sanrego (Lunasia amara) belongs to family Rutaceae has been used empirically as an antidiabetic by people in
South Sulawesi. This plant contains scopoletin which has an α-glucosidase inhibitor effect based on in-silico study
[8]. In vitro studies found that Sanrego extract lowers blood glucose levels [9, 10]. Every plant has normal flora such
as endophytic bacteria. They live in plant tissues and produce secondary metabolites similar to their host plants.
Therefore, the endophytic bacteria are useful and easier to obtain some active compounds compared to using the
plant extract. Endophytic bacteria activities include antimicrobial, antioxidant, anticancer, antimalarial and
antidiabetic [11-13). The purpose of this study was to isolate endophytic bacteria from the Sanrego plant and to test
its activity as an antidiabetic through the mechanism of α-glucosidase enzyme inhibition.
MATERIAL AND METHODS
Materials
Sanrego plant samples were taken from Batu Materia Medica (BMM) Batu, East Jawa. The process of
isolation and α-glucosidase inhibition testing of endophytic bacteria was carried out in Microbiology
Laboratory, Department of Microbiology, Faculty of Medicine, Universitas Brawijaya.
Methods
Preparation and Isolation Endophytic bacteria
Isolation methods refers to previous studies with slight modification [13]. Leaf and stem samples were sterilized
by soaking for 1 minute in 70% ethanol, followed by 5 minutes in 3% NaOCl, then 1 minute in 70% ethanol again,
and finally rinsing with sterile distilled water for three times. The dried samples were crushed in a sterile mortar and
diluted to 10-3 with NaCl. A 500 μL of diluted sample was inoculated on the surface of the Triptone Soya Agar
(TSA) medium (containing 25 uL/mL of fluconazole) and incubated at room temperature for 1-3 days. A part of the
last rinsed distilled aquadest of simplicia (leaf or stem) was inoculated on the surface of the TSA media as a control
for the successful of surface sterilization. After incubation, bacterial colonies were transferred to new agar plates and
purified until pure colonies were obtained. Observation of bacterial morphology was carried out by observing the
shape, elevation, edge, color, size, and surface of the colony, also microscopic characteristic with Gram stain.
Biochemical test for Endophytic bacteria
Pathogenicity test
Endophytic bacterial isolates were cultured on Blood Agar and incubated for 24 to 48 hours at 370
C. The clear
zone around the colony becomes a positive indicator that bacteria can lyse red blood cells that means the bacteria
may be a pathogen. Bacteria with positive results were not used in this study.
Catalase test
Endophytic bacteria on Brain Hearth of Infusion Broth (BHIB) for 24 hours culture was dripped on a glass slide.
The suspense of bacteria was added with 1-2 drops of 3% H2O2 solution and then homogenized. If there are bubbles,
it indicates positive results.
Citrate utilization test
Isolated bacteria were inoculated on Simmon Citrate Agar (SCA) medium and incubated at 30 °C for 24 hours.
The blue color of the medium was to indicate that bacteria can use citrate as an energy source.
Motility, H2S production and Indole test
The motility of each isolate was tested in Sulfide Indole Motility (SIM) medium. The isolate were inoculated into
test tubes containing SIM medium, and incubated at room temperature for 24 hours. Widespread bacterial growth
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4. around the inoculation area indicates their motility. The production of H2S by bacteria causes the medium to turn
black. The formation of a red ring on the medium after the addition of Kovac's reagent indicates the bacteria produce
indole.
Production and extraction of the endophytic bacteria metabolites
Bacterial cultures aged 24 hours were inoculated on YSP media (containing 0.1% yeast extract, 0.1% starch,
0.5% peptone, pH 7) at room temperature. After 48 hours the cultures were centrifuged at 4000 rpm for 20 minutes at
40
C. The supernatant was added with ethyl acetate (1:1 v/v) then homogenized for 60 minutes. The ethyl acetate
layer was separated from the water layer using a separating funnel and then evaporated at 400
C to obtain bacterial
metabolites.
Selection of α-glucosidase inhibitor-producer
Six microliters of each metabolite of endophytic bacteria were spotted on a thin layer chromatography (TLC)
plate (1 cm from one edge of the plate) and elution in the mobile phase using hexane: ethyl acetate (2:8) with
scopoletin as a control substance. The plates were dried and sprayed with 10% KOH before being observed under
UV light at a wavelength of 366 nm.
Assay of α-glucosidase inhibition
The α-glucosidase inhibition test was carried out with pNPG substrate. The reaction mixture consisted of 50 µL
of phosphate buffer (0.1 M, pH 6.8), 50 µL of pNPG substrate (4 mM), 10 µL of bacterial metabolites incubated for
5 minutes at 37 °C. After incubation, α-glucosidase enzyme (0.075 U/mL) 50 µL was added and incubated for 15
minutes at 37°C. The reaction was stopped by adding 100 µL of Na2CO3. The same reaction mixture without enzyme
as the control. All reactions were carried out in a 96-well microplate and were repeated six times. The absorbance of
the solution was measured using ELISA reader at 405 nm of wavelength. The acarbose standard was used as
comparison compound. The inhibition value was measured using the following formula:
α-glucosidase inhibition (%) = (Absorb. of control – Absorb. of sample) x 100
Absorb. of control
RESULTS AND DISCUSSION
Isolation of endophytic bacteria
Thirty-eight different bacterial colonies were obtained from the leaf and stem of Sanrego plant. Stem had more
endophytic bacteria (24 isolates) than leaf (14 isolates) but data are not shown. The high bacterial diversity in the
stem may be caused by the position of stem closed to the roots. Roots are the main entry point for rhizosphere
bacteria into plant tissues [14, 15]. Rhizosphere bacteria live around roots and consist of various bacterial species
from several phyla [16, 17, 18]. Rhizosphere bacteria can enter the root tissue and colonize as endophytes. Stems
have stomata and lenticels which are the entrance for bacteria from the environment. When it has entered the stem
tissue, the bacteria live in the xylem vessels and do not harm the host cell. Each plant tissue provides a different
substrate for the life of endophytic bacteria [17]. We estimate that endophytic bacteria in the Sanrego plant require
more nutrients for growth which is achieved in stem than in leaf, hence, bacterial diversity is greater in stem.
Screening of an α-glucosidase inhibitor-producer
Ethyl acetate extract of endophytic bacteria metabolite was used for bacterial screening of α-glucosidase
producer. Ethyl acetate was used because it was able to get α-glucosidase inhibitor compound based on previous
studies [10,19, 20]. The method for selecting bacteria producing α-glucosidase inhibitor compounds was TLC,
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5. because it is fast, easy, sensitive and requires a small sample [21]. The results showed 10 bacterial isolates suspected
to produce scopoletin based on the appearance of blue fluorescent under UV light (366 nm). This result of this study
is in accordance with previous studies which stated that scopoletin under UV light has blue fluorescence [22]. The
retention factor (Rf) of endophytic bacteria was 2.4 – 2.6 and it closed to the Rf value of the scopoletin standard
(2.9), but data not shown. The endophytic bacteria are similar to their host plants (Sanrego plant) in terms to produce
scopoletin because both have the same metabolite synthesis pathway, namely the shikimic pathway [23, 24]. This
pathway is used by bacteria and plants to produce coumarine and their derivates such as scopoletin. The results of
TLC plate observations showed in Figure 1.
Morphology and biochemical identification of Sanrego endophytic bacteria
Observation for morphology of colony and cell of the endophytic bacteria are shown in Table 1. Macroscopic
identification for colony showed that generally the isolates obtained were more circular, convex, and yellow colour.
Microscopic identification for bacterial cell showed that the isolates were mostly Gram-positive with bacilli and
coccus morphology.
TABLE 1. Morphological characteristic of endophytic bacteria from Sanrego
Part of
plant
Isolate Morphology of colony Morphology of cell
Shape Elevation Colour Shape Gram
Leaves D-1 Circular Convex Yellow Coccus Positive
D-2 Circular Convex Yellow Coccus Positive
D-3 Circular Convex Yellow Coccus Positive
Stem B-1 Circular Convex Yellow Bacil Positive
B-2 Circular Convex Yellow Bacil Negative
B-3 Circular Convex Yellow Bacil Positive
B-4 Circular Convex Yellow Coccus Positive
B-5 Circular Convex Yellow Bacil Positive
B-6 Circular Convex Yellow Bacil Positive
B-7 Circular Flat Yellow Bacil Positive
FIGURE 1. Visualization of TLC results from bacterial metabolites using UV light (366 nm). Bacterial
extract suspected to contain scopoletin (lane 1-8) and scopoletin standard (lane 9) in red box was shown
blue fluorescent.
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6. The results of biochemical analysis of endophytic bacteria isolates stated that all isolates were catalase positive,
non-pathogenic, indole negative, H2S negative and CO2 negative. Isolate B1-B3 was used citrate as an energy
source. Isolates D3 and B4 were motile. Data is shown in Table 2.
TABLE 2. The results of the biochemical test of Sanrego endophytic bacteria
Isolate Pathogenicity
test
Catalase test Citrate
test
Indole
test
Motility
test
CO2
test
H2S
test
D-1 - + - - - - -
D-2 - + - - - - -
D-3 - + - - + - -
B-1 - + + - - - -
B-2 - + + - - - -
B-3 - + + - - - -
B-4 - + - - + - -
B-5 - + - - - - -
B-6 - + - - - - -
B-7 - + - - - - -
Assay of α-glucosidase inhibition analysis
Assay of bacterial metabolites showed that all bacteria were able to inhibit α-glucosidase activity and soe isolates
have higher inhibition value than acarbose (ANOVA, p value- = 0.002). Acarbose is one of drugs using diabetes
treatment, it is a single compound and has been used as the standard of α-glucosidase inhibitor. Inhibition value of
Sanrego endophytic bacteria shown in Figure 2. The highest inhibition values were shown by isolates D-1 (8.67%),
was higher than acarbose value (3.73%). Bacterial supernatants containing various secondary metabolites that may
cause synergistic work to produce high inhibitory values. The interaction of several active compounds was given
better impact than a single compound [25]. Metabolites produced by endophytic bacteria similar their host plant
include alkaloids, steroids, terpenoids, peptides, polyketones, flavonoids, quinols, phenols, and coumarins [26-28].
These compounds are important for disease management because it used for treatment of various disease. Secondary
metabolites produced by endophytic have activities similar with their host plant. Most endophytic generally have
various activities such as antimicrobial, antioxidant, anticancer, anti-inflammatory properties, antimalarial and
antidiabetic [11-13, 27-28].
Previous similar study that used the α-glucosidase enzyme concentration of 1 U/mL and the substrate
concentration of 10 mM resulted the high inhibition value of 103.18% [29]. Another study using the same enzyme
concentration (1U/mL) with a lower substrate concentration (5 mM) had a lower inhibition value of 75.42% [30].
Other different result was shown by endophytic bacteria from Salak which had a low inhibition value (62.5%) with
an enzyme concentration of 0.25 U/mL and substrate concentration of 20 Mm [31]. As well as the result of this
study, the inhibition value was 8.67%, with enzyme concentration of 0.075 U/l and substrate concentration of 4 mM.
This indicates that the higher enzyme concentration used, results the inhibition value also increase. According to
[32], there are several factors that affect enzyme activity including type of substrate, concentration of enzyme and
substrate, pH, temperature, and concentration of inhibitor.
Our results showed that the endophytic inhibition value had the lowest standard deviation of 1.3 and the highest
was 3.2 (Figure 2). This value indicates a small variation/diversity in the endophytes of Sanrego plants. This
statement is in accordance with the opinion which states that the higher the standard deviation, the greater the
variation in the sample [33].
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7. CONCLUSION
The endophytic bacteria from the Sanrego plant (leaf and stem) produce α-glucosidase inhibitor compound. The
endophytic bacteria isolated from Sanrego leaf (D1 isolate) can be further investigated as a source for α-glucosidase
enzyme inhibitor.
ACKNOWLEDGMENT
The authors would like to thank the Faculty of Medicine Universitas Brawijaya for funding this research and
Laboratory of Microbiology Faculty of Medicine Universitas Brawijaya for facilitating the research process.
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