Perbandingan Pengaruh Pencernaan Secara In Vitro Terhadap Aktivitas Antioksidan Dan Antihyperglikemik Dari Ekstrak Kasar Andaliman (Zanthoxylum Acanthopodium Dc.) Dan Lada Jepang (Zanthoxylum Piperitum Dc.).

COMPARISON ON IN VITRO DIGESTION EFFECT OF ANTIOXIDANT
AND ANTIHYPERGLYCEMIC ACTIVITY FROM ANDALIMAN
(Zanthoxylum acanthopodium DC.) AND JAPANESE PEPPER
(Zanthoxylum piperitum DC.) CRUDE EXTRACT

VANESSA KARNADY

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015

STATEMENT OF THESIS AND
SOURCES OF INFORMATION AND DEVOLUTION OF
COPYRIGHT*
I hereby declare that my thesis entitled Comparison on In Vitro Digestion
Effect of Antioxidant and Antihyperglycemic Activity from Andaliman
(Zanthoxylum acanthopodium DC.) and Japanese Pepper (Zanthoxylum piperitum
DC.) Crude Extract, is an original piece of work, written and completed on my own,
based on sources as listed on the work cited section and not a duplication of other
writing that has been published in other Universities.

I hereby assign copyright of my paper to the Bogor Agricultural
University.
Bogor, August 2015
Vanessa Karnady
NIM F251120061

SUMMARY
VANESSA KARNADY. Comparison on In Vitro Digestion Effect Of Antioxidant
and Antihyperglycemic Activity From Andaliman (Zanthoxylum acanthopodium
DC.) and Japanese Pepper (Zanthoxylum piperitum DC.) Crude Extract. Supervised
under HANNY WIJAYA and ENDANG PRANGDIMURTI.
Hyperglycemia is one of important issue lately due to it will lead into other
complication such as micro and macro vascular disease. Hyperglycemia is caused
by the excess of glucose in the human body. This uptake of excess glucose causes
imbalance between oxidants and antioxidants in the human body.
Andaliman(Zanthoxylumacanthopodium DC.) is a traditional exotic spice
that grows in North Sumatra, Indonesia. This spice is one genus with Japanese
pepper (ZanthoxylumpiperitumDC.) that mainly grows in Japan. Both of spices
have been used as folk medicines since their phytochemical substances possessing
strong antioxidant activity and allegedly to possess α-glucosidase inhibitor activity.

Their activity, however, might be altered under gastrointestinal digestion due to the
structure alteration of the responsible compounds. Therefore, this study was aim to
study and compare the changes of α-glucosidase inhibitor and antioxidant activities
of andaliman and Japanese pepper crude extracts under in vitro gastrointestinal
digestion
The in vitro gastrointestinal digestion is mimicking the digestion condition
in the gastric and small intestine. While, the determination of activity were done in
vitro by using α-glucosidase enzyme inhibition assay and DPPH radical scavenging
activity methods. Acarbose, a commercial inhibitor was used as positive control for
α-glucosidase inhibition assay and ascorbic acid as positive control for DPPH
radical scavenging activity.
Before digestion simulation, the crude extract of Japanese pepper showed
better inhibition activity towards α-glucosidase enzyme (IC50 = 3930.21µg/mL)
compare to andaliman (IC50 = 20346.94µg/mL). The crude extract of Japanese
pepper also showed better radical scavenging activity towards DPPH (IC50
=104.03µg/mL) comparing to andaliman (IC50 = 20346.94µg/mL).
In vitro gastrointestinal digestion decreased the antioxidant activity and αglucosidase inhibition of both spices. Japanese pepper’s α-glucosidase inhibition
activity were lost 1.42 times while andaliman lost 1.77 times. However, in DPPH
radical scavenging activity, andaliman were lost only 2.77 times while Japanese
pepper lost 8.26 times. Antioxidant activity of andaliman was more stable than

Japanese pepper during the digestion simulation.
Extract of Japanese pepper had stronger antioxidant (IC50 =5580.66µg/mL)
and α-glucosidase inhibition activity (IC50 = 859.55µg/mL) compare to andaliman’s
antioxidant (IC50= 1224.10µg/mL and α-glucosidase inhibition (36089.58µg/mL)
after digestion simulation. Comparing to andaliman, Japanese pepper still showed
better antioxidant activitiy as well as inhibition activity towards α-glucosidase
enzyme and antioxidant.
Keywords: α-glucosidase inhibitor, antioxidant, in vitro
digestion,ZanthoxylumpiperitumDC.,ZanthoxylumacanthopodiumDC.

RINGKASAN

VANESSA KARNADY. Perbandingan Pengaruh Pencernaan Secara in vitro
Terhadap Aktivitas Antioksidan dan Antihyperglikemik dari Ekstrak Kasar
Andaliman (Zanthoxylum acanthopodium DC.) dan Lada Jepang (Zanthoxylum
piperitum DC.). Dibimbing oleh HANNY WIJAYA dan ENDANG
PRANGDIMURTI.
Kondisi hiperglikemia merupakan salah satu masalah yang penting akhirakhir ini mengingat hiperglikemia dapat menyebabkan komplikasi lain seperti
penyakit makro dan mikro vaskular. Hiperglikemia disebabkan oleh kelebihan
glukosa dalam tubuh seseorang. Kelebihan glukosa tersebut akan menyebabkan

ketidakseimbangan antara oksidan dan antioksidan dalam tubuh manusia.
Andaliman (Zanthoxylum acanthopodium DC.) adalah rempah-rempah
tradisional dan bahan aditif alami yang tumbuh di alam liar di Sumatera Utara,
Indonesia. Tanaman ini masih dalam satu genus dengan “shansho”, Lada Jepang
(Zanthoxylum piperitum DC.) yang tumbuh terutama di Jepang. Kedua tanaman ini
sudah banyak digunakan sebagai obat tradisional karena banyak mengandung zat
fitokimia yang memiliki aktivitas antioksidan yang kuat dan juga diduga memiliki
kemampuan menghambat α-glukosidase. Namun, aktivitasnya mungkin akan
berubah di bawah kondisi pencernaan disebabkan oleh berubahnya struktur kimia
senyawa-senyawa aktifnya. Oleh karena itu, penelitian ini bertujuan untuk
mempelajari aktivitas dan sekaligus perubahan aktivitas tersebut yang dimiliki oleh
ekstrak kasar lada Jepang dan andaliman saat pencernaan secara in vitro.
Simulasi pencernaan secara in vitro dilakukan dengan meniru kondisi
pencernaan dalam lambung dan usus kecil. Sementara, penentuan aktivitas
dilakukan secara in vitro dengan menggunakan pengujian penghambatan terhadap
enzim α-glucosidase dan penghambatan aktivitas radikal DPPH. Acarbose, yang
merupakan obat komersial digunakan sebagai kontrol positif dalam uji
penghambatan α-glucosidase dan asam askorbat sebagai kontrol positif untuk
penghambatan aktivitas radikal DPPH.
Sebelum simulasi pencernaan, ekstrak kasar lada Jepang memiliki aktivitas

penghambatan yang lebih baik terhadap enzim α-glucosidase (IC50=3930.21μg/mL)
dibandingkan dengan andaliman(IC50=20346.94μg/mL). Ekstrak kasar lada Jepang
juga menunjukkan aktivitas antiradikal yang lebih baik terhadap DPPH
(IC50=104.03μg/mL) dibandingkan dengan andaliman (IC50=20346.94μg/mL).
Simulasi pencernaan in vitro menyebabkan penurunan aktivitas antioksidan
dan penghambatan α-glukosidase dari kedua rempah. Aktivitas penghambatan
α-glucosidase dari lada Jepang hilang sebanyak 1,42 kali sedangkan penghambatan
α-glucosidase andaliman hilang sebanyak 1,77 kali. Namun, dalam aktivitas
penghambatan radikal DPPH, aktivitas antioksidan kehilangan andaliman lebih
rendah yaitu hanya sebesar 2,77 kali dibandingkan dengan lada Jepang yang
kehilangan aktivitas antioksidan sebesar 8,26 kali. Aktivitas antioksidan andaliman
lebih stabil dibandingkan dengan lada Jepang selama simulasi pencernaanin vitro.
Ekstrak lada Jepang memiliki aktivitas antioksidan (IC50 = 5580.66μg/ mL)
dan aktivitas inhibisi α-glucosidase (IC50 = 859.55μg/mL) yang lebih kuat

dibandingkan dengan kemampuan antioksidan (IC50 = 1224.10μg / mL dan inhibisi
α-glucosidase (36089.58μg / mL) dari andaliman setelah simulasi pencernaan. Lada
Jepang masih memiliki aktivitas penghambatan terhadap enzim α -glucosidase dan
aktivitas antioksidan yang lebih baik dibandingkan dengan andaliman.
Kata kunci: α-glucosidase inhibitor, antioksidan, in vitro pencernaan,

Zanthoxylum piperitumDC, Zanthoxylum acanthopodiumDC.

© All Rights Reserved IPB, 2015
Copyright Reserved

Quoting some or all of this paper without including or mentioning the source is
prohibited. Quoting is only for educational purposes, research, scientific thesis,
report writing, criticism, or review an issue; and citations are not detrimental to
the interests of IPB
Announcing and reproducing some part or wholes paper in any form without IPB
permission is strictly prohibited.

COMPARISON ON IN VITRO DIGESTION EFFECT OF ANTIOXIDANT
AND ANTIHYPERGLYCEMIC ACTIVITY FROM ANDALIMAN
(Zanthoxylum acanthopodium DC.) AND JAPANESE PEPPER
(Zanthoxylum piperitum DC.) CRUDE EXTRACT

VANESSA KARNADY

Thesis

partial fulfillment of the academic requirements to obtain
Magister Sains
on
Food Science Study Program

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2015

Examiners of Thesis Exam: Dr. Didah Nur Faridah, STP, M.Si.

ACKNOWLEDGEMENTS
Praise Lord for His endless blessings and continuous leading for the
writer throughout the research and completion of this thesis report titled
“Comparison on In Vitro Digestion Effect of Antioxidant and
Antihyperglycemic Activity from Andaliman (Zanthoxylum acanthopodium
DC.) and Japanese Pepper (Zanthoxylum piperitum DC.) Crude Extract”. The
writer clearly realizes that the research and this report would not possible to be
completed without the support from many people. Writer would like to express

gratitude to:
1. Prof.Dr.Ir. C. Hanny Wijaya, M.Agr and Dr.Endang Prangdimurti as thesis
supervisors for for the time and guidance during the research and
completion of the thesis report.
2. My beloved family, mama, papa and Manda for always be my rock
3. My 2012 IPN friends, Eron, Diana, Ka Tiwi, Rosana, Eren, Laras, Kamil,
Novan, Mas Syafii, Tuti, Mia, Trina, Ara, Faris, Wulan, Rina, Anis and
other friends, thank you for the precious friendship and the knowledge you
shared with me.
4. Irena a friend, my andaliman supplier, thank you for sharing me your
knowledge and sharing everything about andaliman, especially the pictures
and journal writing.
5. The Laboratory members, Bu Irdha and Mba Sherly. Thank you for your
guidance, knowledge that you shared with me and for always there when I
am in trouble.
6. All Laboratory Staff, Pa Taufik, Pa Yahya, Mba Ari, Pa Rojak, Mba Irin,
Teh Yayam and Pa Sob for your help and instruction when using the
instruments,
7. Biofarmaka Laboratory Staff, Mba Ella, Mba Wiwik, Bu Ninik and Mba
Ina, thank you for the help, guidance and knowledge sharing when I did the

experiment.
8. Hashidoko sensei, for your guidance in doing lab experiments, data
analyzing and presentation practice which is very precious.
9. Echochem Laboratory member, Reika, Yokota, Nishiyama, Sharon, Nie,
Bocky, Nizhisuka, Haba, Aki, Yoshida, Zetry, Wang Lei, Hiroyo Hanai,
thank you for the friendship, concern, guidance, help and knowledge shared
when I was alone in Hokudai Lab.
10. Dhina and Namfon thank you for being my friends so I can finish this thesis
master, and thank you for teaching me about chemistry and lab technique.
The author is aware that this report is far from perfect and it may contain
many mistakes. The author would like to deliver apology and welcome any
critics and/or suggestions given to this report with great pleasure. Finally, the
author hopes that this report would be useful for the readers.

Bogor, August 2015

Vanessa Karnady

TABLE OF CONTENT
LIST OF FIGURES

xi

LIST OF APPENDICES

xi

1 INTRODUCTION
Background
Research Problem
Research Objectives
Research Benefit

1
1
2
2
2

2 Literature Review

Zanthoxylum Genus
Zanthoxylum acanthopodium DC. (Andaliman)
Zanthoxylum piperitum DC. (Japanese pepper)
Hyperglycemia
Oxidative Stress in Hyperglycemia
Alpha glucosidase enzyme and its inhibitor

2
2
3
6
7
8
8

3 Methods
Materials
Research Methods
Crude Extract Preparation
In vitro Gastrointestinal Digestion
Antihyperglycemic Activity Assay
Antioxidant Activity Assay

9
9
9
10
11
11
12

4 Results and Discussion
Crude Extract Preparation
Antihyperglycemic Activity
Antioxidant Activity

13
13
14
18

CONCLUSION AND SUGGESTION
Conclusion
Suggestion

22
22
23

BIBLIOGRAPHY

23

APPENDICES

28

BIOGRAPHY

35

LIST OF FIGURES
1
2
3
4

5
6
7
8

9

10
11

Andaliman field in Goting Raya, Simalungun North Sumatra
4
Zanthoxylum acantophodium DC.
(A) Mature (red) andaliman
(B) Young (green) andaliman
(A) Young (green andaliman)
(B) Mature (red andaliman)
(C) Senescent (black andaliman, seeds exposed)
Zanthoxylum piperitum DC.
Research Flow Chart
(A) Andaliman
(B) Japanese pepper
(A) α-glucosidase inhibition activities of crude extracts
(B) α-glucosidase inhibition activities of in vitro post digestion
extracts
(A)IC50 value of α-glucosidase inhibition activity of crude extracts
versus in vitro post digestion extracts
(B) IC50 value of α-glucosidase inhibition activity of crude extracts
versus in vitro post digestion extracts
(A) DPPH scavenging activities of crude extracts
(B) DPPH scavenging activities of in vitro post digestion extracts
(A) IC50 value of DPPH radical scavenging activity of crude extracts
versus in vitro post digestion extracts
(B) IC50 value of DPPH radical scavenging activity of crude extracts
versus in vitro post digestion extracst

5
5
5
5
5
5
7
10
14
14
16
16
17
17
21
21
22
22

LIST OF APPENDICES
1
2
3

Moisture content and yield
Antioxidant activity
Antihyperglycemic activity

28
29
32

1 INTRODUCTION
Background
The type 2 diabetes represents by the asymptomatic hyperglycemia, which
is an elevation in blood glucose levels, result from the inadequate insulin secretion
or action. Moreover, chronic hyperglycemia will produce reactive oxygen species
(ROS) which is a main role in microvascular and macrovascular complication
(Singh & Poonam 2009). Thus, controlling hyperglycemia is believed to be
important on treating diabetes mellitus. According to Shibano et al. (2008)
combination of the antihyperglycemic inhibitor and antioxidant will be more
effective for the treatment and prophylaxis of hyperglycemia.
Alpha glucosidase is an enzyme located in the small intestine brush border,
which will catalyzes the final step of carbohydrate digestion to glucose, the
absorbable monosaccharide. Synthetic acarbose is used as well-known
antihyperglycemic inhibitor which will limit the availability of the glucose by
hampering the rate of final step hydrolysis of complex carbohydrate into glucose in
the intestine (Jaiswal et al. 2012). However the use of synthetic acarbose is not
suitable for all because of the contraindication in patient with inflammatory bowel
disease and renal impairment (Kim et al. 2005), beside that synthetic acarbose does
not possess natural antioxidant as in natural compound. Therefore, in the field of
food science much interest has been focused on the development of food functional,
including screening of natural bioactive compound which have less side effect and
considering the phytochemical from natural compound.
Zanthoxylum acanthopodium DC. (andaliman) is an endogenous plant from
North Sumatra, Indonesia. It has lemony aroma and unique taste which give tingling
sensation on the tongue. Batak tribes widely used it as spices and folk medicine
(treating stomachache). According to Wijaya (2000), the tingling sensation came
from the trigeminal compound that has similar structure with a compound that was
isolated from Zanthoxylum piperitum DC. (Japanese pepper) which is known as
sanshool. The similar uniqueness made them more interesting to be further
explored. Some studies already showed the bioactivity of Japanese pepper, it had
strong antioxidant activity (Yamazaki et al. 2007), antimicrobial activity (Lee et al.
2012), hepatoprotective effect (Lee & Kye 2008) and gave relaxation effect on
circular muscle of gastric (Hashimoto et al. 2001). Likewise, andaliman has also
shown some bioactive capability such as antioxidant (Tensiska et al. 2003),
antimicrobial (Parhusip et al. 2005), xanthin oxidase inhibitor (Kristanti et al. 2012)
and anti-inflammatory (Yanti et al. 2011) but the bioactivity of andaliman is still
less popular than Japanese pepper, therefore it will need a study which can compare
their bioactivity.
From most study that had been done the antihyperglycemic activity from
both of them never been studied before, where areas study about Zanthoxylum
rhesta (Yagnambhatla et al. 2014) and Zanthoxylum scinifolium (Oh et al. 2010)
have already showed their capability of diminishing the activity of the αglucosidase enzyme. On the other hand, antioxidant and antihyperglycemic activity
might be altered under gastrointestinal digestion. Since there are some conditions
in the gastrointestinal digestion (pH, temperature and enzymes) which might

influence the antioxidant and antihyperglycemic activity (Bermudez-Soto et al.
2007). However, antioxidant and antihyperglycemic activity of the extract obtained
from in vitro digestion has not been investigated yet.

Research Problem
Hyperglycemia has become common disease in both developed and
developing countries in connection with life style changes a dietary habits. The αglucosidase inhibitor generally used to medically treat hyperglycemia. Despite
powerful synthetic α-glucosidase inhibitor, such as acarbose, they usually induce
side effect such as hepatoxicity and disorder gastrointestinal symptoms (flatulence,
diarrhea and abdominal boating). Therefore, a natural α-glucosidase inhibitor from
food sources like andaliman or Japanese Pepper become an attractive therapeutic
approach on treating hyperglycemia. Moreover, the treatment of hyperglycemia
will be more effective if they have combination roles as antioxidant and αglucosidase inhibitors. Unfortunately, the alteration of activity under
gastrointestinal condition has not yet been studied before. Therefore a further study
about the changes of antioxidant and antihyperglycemic activities of both
andaliman and Japanese pepper during gastrointestinal simulation will give better
understanding of both spices’ efficacy as antihyperglycemic ingredients.
Objectives
The purpose of this study was to compare the activity of andaliman and
Japanese Pepper crude extracts as α-glucosidase inhibitor and antioxidant,
particularly the changes of the antihyperglycemic and antioxidant activity during
an in vitro gastrointestinal digestion simulation.

Research Benefit
The research provided information regarding the antihyperglycemic and
antioxidant efficacy from andaliman and Japanese pepper which could be
implemented for further development as a natural antidiabetic ingredient from
plant. The result also indicated the other possibility of andaliman utilization besides
it being use as regular spices.

2 LITERATURE REVIEW
Zanthoxylum Genus
Zanthoxylum is a genus belongs to Rutaceae family which is used as spices
and medicinal application. This genus sometimes called Xanthoxylum since the
word Zanthoxylum derives from Greek word “xanthon xylon”. There are about 549

species of Zanthoxylum distributed mainly in tropical area, most of the trees are
deciduous and shrubs. It also could be identified by its fruit which usually are
follicles or esquizocarp, contains from one to five carpels, commonly aromatic, and
they are ordinarily bivalve with a single red or black, shiny seeds. This genus are
popular because its phytochemistry activity (Patino et al. 2012).
In the food industry essential oil from Zanthoxylum species such as
Z. xanthoxyloides, Z. gillettii, Z. simulans are obtained from their leaves or fruits.
The bark from Z. Tessmannii are usually being utilized as emulsifying agent and
encapsulants (Adesina 2005). Many species has been traditionally used as a remedy
throughout world, such as in Taiwan they used it as remedy for snakebite and
aromatic tonic for fever. The bark of Z .liebmannianum, is used in Mexico for the
treatment of stomach pains, amebiasis and intestinal parasites and as a local
anesthetic agent (Ross et al. 2004). Z. monophyllum in Venezuela, it used as a
remedy for runny nose or nasal mucosal inflammation. In Batak tribes, the fruits of
Z. acanthopodium DC. has been used to heal stomachache and toothache (Suryanto
et al. 2004). In East Asia, all part of the plant from Z.piperitum commonly used to
heal vomiting, diarrhea and abdominal pain (Yamazaki et al. 2007).

Zanthoxylum acantophodium DC. (Andaliman)
Zanthoxylum acanthopodium DC. (Andaliman) is a traditional exotic spice
and natural food additive that grows in the wild in North Sumatra, Indonesia,
especially in the area of Lake Toba. It is widely available in the Dairi, North
Tapanuli, Tobasa (Malau et al. 2002), Humbang, Silindung, and Toba Holbung
areas (Napitupulu et al. 2004). Its habitat is sandy loam soil and grows at elevation
of 0.9 kilometers above sea level where there can be up to 2500 mm of annual
rainfall with rain on 170-180 days/year (Napitupulu et al. 2004). Nowadays,
andaliman is cultivated, for example, in Gotting Raya, Simalungun North Sumatera
(Fig 1).
Andaliman which belongs to Family Rutaceae, is often mistaken as a
member of Family Piperaceae and identified as Piper ribesiodes. According to
Hsuang (1978), andaliman is classified as follows:
Division
: Spermatophyta
Sub division : Angiospermae
Class
: Dicotyledoneae
Order
: Geraniales
Family
: Rutaceae
Genus
: Zanthoxylum
Species
: Zanthoxylum acanthopodium DC.
Andaliman (Fig 2) is called by several names in its native regions. These
include Sinyar-sinyar (Angkola), Intir-Intir (Simalungun), Tuba (Karo), and
Syarnyar (South Tapanuli) (Napitupulu et al. 2004). According to a description
given by Siregar (2003), andaliman is a perennial shrub or low branching small tree
that grows erect, up to 5 meters in height. There are thorns on the stems, branches,
and twigs of the plants. The leaves are scattered, stemmed, 5-20 cm long and 3-15
cm wide, and contain oil glands. The upper surfaces of the leaves are shiny green
and the lower surfaces are light green or pale. Young leaves have green upper

surfaces and reddish green bottoms. Flowers are in the auxiliary, small with flat or
conical shaped bases, 5-7 free petals, 1-2 cm long, pale yellow color, androgynous,
having 5-6 stamens in flowers sitting on the base, reddish anthers, 3-4 pistils,
boarded apocarp ovaries. The fruit is box or capsule shaped, or rounded, 2-3 mm
diameter, green for the young fruit and red in mature fruit (Fig 3). It has one seed
per fruit, hard skin, and a shiny black color. Andaliman is commonly used in the
green form. However, on some occasions the red is utilized. Senescent andaliman
turns black and black seeds come out (Fig 4). In this condition, the typical flavor of
andaliman no longer exists.
Andaliman has traditionally been used in Batak cuisine served at a variety
of cultural events. Recently, it has been used in daily food. Almost every dish at a
Batak birth and marriage ceremonies is cooked with andaliman. This spice is
believed to have medicinal properties that improve the appetite of sick persons
(Napitupulu 2004). Traditional foods are prepared using andaliman such as arsik
(yellow cooked gold fish), naniura (uncooked or fermented gold fish), tombur (a
condiment for roasted fish or meat), and is also used as sauce (with crushed green
chili).

Fig 1. Andaliman field in Goting Raya, Simalungun North Sumatra.
The andaliman fruit has been traditionally used to cure stomachache and
diarrhea which is caused by the release of toxin by pathogen leads to response by
immune system causing inflammation following the release of immune cells. The
scientific study was studied by Yanti et al. (2011) concluded that the fruit extract
from andaliman significantly inhibits selected inflammatory biomarkers (Tumor
necrosis factor, interleukin, inducible nitric oxide synthase, Cyclooxygenase and
Matrix Metalloproteinase) at the protein and gene expression in lipopolysaccharides
induced macrophages.

Fig 2. Zanthoxylum acantophodium DC.

A

B

Fig 3. (A) Mature (red) andaliman and (B) Young (green) andaliman
A

B

C

Fig 4. (A) Young (green andaliman), (B) mature (red andaliman),
and (C) senescent (black andaliman, seeds exposed).
Alongside its anti-inflammatory ability, andaliman also traditionally used
for preserving cooked meat for few days against rancidity and spoilage, the
scientific studied bear out that andaliman has antiradical activity on its ethanolic
extract, this extract at 1000 ppm has higher radical scavenging than 1000 ppm of αtocopherol (Suryanto et al. 2004). Food preservatives potency is also supported by

study which done by Parhusip et al.(2006), that etyl acetate extract of andaliman
has a high inhibition towards either Staphylococus aureus or Bacillus cereus cell
and cell protoplast. Both of S.aureus and B.cereus, they are pathogen bacteria which
often contaminating food.
Tensiska et al. (2003), the antioxidant extract from andaliman fruit has its
activity on aqueous system. However, on emulsion system antioxidant activity is
less intense than Butylated Hydroxy Toluene (BHT) which was used as the positive
control. As well as in oil system, the antioxidant activity also less intense than BHT
which was observe by its longer induction time than BHT has. As antioxidant the
fruit extract from andaliman is relatively heat stable on aqueous system on 175ºC
in 2 hour. The pH stability on emulsion system showed protection of antioxidant
increase consequently to increase from pH 3 to 7.
Antiradical activity which is isolated from n-butanol andaliman extract
compound were type of hydroxy ester sterol and cholesterol-3-o-β-glucosidase and
they have xanthine oxidase inhibition were IC50 0,34μg/mL and 0,06μg/mL
respectively, and their antioxidant activity were IC50 68,35μg/mL and 60,52μg/mL
respectively, which is higher than BHT or quercetin. It was shown that compound
hydroxy ester sterol has better activity in DPPH reduction, and cholesterol-3-o-βglucosidase was better as xanthine oxidase inhibitor (Kristanti et al. 2012).

Zanthoxylum piperitum DC. (Japanese Pepper)
Japanese pepper (Zanthoxylum piperitum DC) is a decidous and shruberry
tree distributed in Japan, China and Korea (Fig 5.). The plant can grow into 3 up to
6 meters high. Various parts of this plant such as leaves, flowers and fruits are used
in of Japanese cuisine for its unique flavor, such as for sprinkling on the broiled eel
dish. In Asia around 1980’s the pericarp of the fruits from Japanese pepper has been
used as an antihelmintic and also treatments of digestive organs. Whereas, the fruits
and leaves was founded to contain terpenoid, aliphatic acid amides, alkaloid,
flavonoids and other phenolics (Hur et al. 2003).
The methanol extract from the leaves was proven to prevent lipid
peroxidation which was induced by bromobenzene. The extract was capable of
reducing the activity of analine hydrolase, epoxide producing enzyme and by
enhancing activity of epoxide hydrolase which is an epoxide removing enzyme.
However, it did not give effect to aminopyrine, N-dimethylase and gluthahione Stransferase (Hur et al. 2003). Research which done by Jeong et al. (2011) stated
that Z.piperitum leaf also have a radical-scavenger and reducing agent. It was
presented by protective effects against H2O2 induced neurotoxicity in a dosedependent manner. This protective effect was lead to the fact that the extract has
strong antioxidative and neuronal protective effects that are correlated with its high
level of phenolics, particularly quercetin, afzelin, and hyperoside.
Further study that done by Hatano et al. (2004), reveal that the pericarp of
Japanese pepper contain novel amides. The amides which isolated from the pericarp
is responsible for the tingling sensation on the tounge, the amides then called
sanshool. According to Wijaya (2000) the compound which was isolated from
andaliman showed similar structure with sanshool. Then, recent report from
Yamazaki et al. (2007) showed the antioxidant activity of methanol extract from

Japanese pepper fruit was found to be equal to that α- tocopherol and stable under
heat treatment, and the compound were identified to be hyperoside and quercetin.
They were found to be a good scavenger for DPPH.

Figure 5. Zanthoxylum piperitum DC.
Source: Suehiro, 2014
Glycoprotein from Japanese pepper also has positive effect as
hepatoprotective agent as a natural antioxidant. The result showed that Japanese
pepper’s glycoprotein has an inhibitory effect on hypoxanthine/xanthine oxidaseor glucose/glucose oxidase-induced cytotoxicity in a dose-dependent manner. In
addition, administration of Japanese pepper glycoprotein (20 mg/kg) lowers the
levels of lactate dehydrogenase, alanine transaminase, and thiobarbituric acid
reactive substances, whereas increases that of nitric oxide, accompanying the
normalizing effects on the activity of hepatic anti-oxidant enzymes (superoxide
dismutase, catalase, and glutathione peroxidase) in mouse model of carbon
tetrachloride-stimulated acute liver injury (Lee et al. 2008).
From above information showed that Japanese pepper is one of important
aromatic and medicinal plant which used widely in East Asia. Hwang and Kim
(2012), assessed the potential health risk of Japanese pepper derived essential oil,
based on bone marrow micronucleus, bacterial reverse mutation, and chromosome
aberration tests. The Z.piperitum derived essential oil contains myrcene, octanal, dlimonene and linalool which used as antimicrobial agents against foodborne
pathogens, from the study it showed no indication of bone marrow micronucleus
abnormalities, mutagenicity, or chromosomal aberration.

Hyperglycemia
In order absorbed by the body cell, carbohydrate need to be degrade into its
simplest molecules. In small intestine, the carbohydrate breakdown into simple
sugar molecules by digestion enzyme which allow glucose to be passed into the
blood stream. When the blood stream reached pancreas, β cell will detect the rising
of the glucose level in the blood stream, in order to reduce the glucose level, the β
cell release insulin into the blood stream. Insulin is a main gluco regulatory
hormone which will inhibit hepatic glucose production and stimulates peripheral

glucose uptake. After insulin circulate with glucose in the blood stream it will exit
the tissue to reach body cell. Body cell is a place where insulin receptor will bind
to circulation of insulin. The receptor will act as a lock whereas the insulin will act
as the key, when the receptor act normal it will lead glucose enter the body cell in
normal level. Therefore, the body cell can produced energy (Giugliano et al. 2008).
Otherwise in people who ail with diabetes type 2, the problem will be either
the βcell in pancreas does not produce enough insulin or the cell resist the effect of
insulin, or the problem could be both of them. When the cell resist the effect of
insulin, insulin cannot unlock the cell to let glucose enter the body cell because of
the abnormality of the receptor. Which means the glucose will be locked up outside
the cell and the glucose level in the blood will be high, this is known as
hyperglycemia. To compensate the hyperglycemia, the βcell excrete more and more
insulin, this will lead to the overworked of βcell which will lose its ability to
produce enough insulin (McDonnell et al. 2012).

Oxidative Stress in Hyperglycemia
Oxidative stress is generated by an imbalance between the production of
reactive oxygen species (ROS) and the antioxidant defense system. The overworked
of β cell caused by continuous excretion of insulin will be toxic for β cell itself,
which called β cell dysfunction. According to Wu et al. (2004) the dysfunctional of
β cell accompanied by increasing activity of glycolysis with lower production of
ATP, increasing the accumulation of intracellular ROS, oxidative damage to
mitochondria and also increasing the apoptotic cell death. The increasing of
glycolysis rate is because when the blood glucose level is too high, glucose will be
deposit in the liver where the glycolysis took place.
Actualy in glucose metabolism which is one of the oxidative metabolism,
ROS is a normal product from glucose metabolism that formed from the reduction
of molecular oxygen or by oxidation of water to yield products such as superoxide
anion and hydrogen peroxide. When the metabolism rat, in this case glycolysis,
increase it means that will be an excessive ROS in pancreatic β-cell, this will make
cellular damage on pancreatic β cell. The damage of pancreatic β-cell will increase
the development and worsening hyperglycemia. Therefore, it will need a scavenger
for the ROS which will be expected to prevent the toxic effect of increased
glycolysis (Hartati et al. 2012, McDonnell et al. 2012, Wu et al. 2004).

Alpha glucosidase enzyme and its inhibitor
Alpha glucosidase enzyme (EC 3.2.1.20) is enzyme which catalyse the
degradation of α-1,6 glycoside. The function of this enzyme is to hydrolyze α-limit
dextrin into glucose. Alpha glucosidase in mammal digestion located on the surface
of the brush border membrane of the small intestine which catalyse the final process
of carbohydrate digestion in the digestion. (Jaiswal 2012, Berdanier et al. 2006).
Some study reported that α-glucosidase enzyme could be inhibited by plant
extracts: Alstonia scholaris, Eleutherine Americana, Chaenomeles sinensis (JongAnurakkun et al. 2007; Ieyama et al. 2011; Sancheti et al. 2009). It was suspected

that the bioactive component in Alstonia scholaris that could inhibited the activity
of α-glucosidase enzyme is quercetin 3-O-β-D-xylopyranosyl (1”-2”)-β-D—
galactopiranosid and (-)-lioniresinol 3-O-β-D-glucopiranosid (Jong-Anurakkun et
al. 2007). Another study showed that the whole flavonoid mixture from Crapesium
abrotanoides plant extract showed non-competitive inhibition against αglucosidase enzyme activity which originated from yeast (Mayur et al. 2010).
Chaenomeles sinensis was shown to be a potent α-glucosidase enzyme which
related to the high phenolic content that have high antioxidant activity (Sancheti et
al. 2009). According to Arsiningtyas et al. (2014) mentioned the caffeoylquinic
acid derivatives shown to be important for the inhibitory activity of α-glucosidase
enzyme.
Acarbose known as fermented product from some species of Actinoplanes.
Acarbose is effective in inhibiting some carbohydrate degradable enzim such as :
α-glucosidase, cyclomaltodextrin glucaniltransferase (CGTase), α-amylase and
dextran sucrase. Acarbosa is pseudo oligosaccharide which has pseudo
oligosaccharides ring [[4,5,6-trihydroksi-3-(hidroksimetil)-2-cyclohexene-1yl]amino]-alpha-D-glucopyranosil-1(1-4)-O-(alpha)-D-glucopyranosil-(1-4)-DGlucose. The acarbose inhibition mechanism against those enzymes above because
of cyclohexene ring and the nitrogen linkage that mimics the transition state for the
enzymatic cleavage of glycosidic linkages (Yoon & Robyt 2002).

3 METHODS
Materials
Fresh andaliman fruit was obtained from Simalungun farm, in North
Sumatra, Indonesia. Japanese pepper fruit was obtained from traditional market in
Kyoto, Japan. α- glucosidase enzyme (G5003), pancreatin (P1750), bile extract
(B8631), pepsin (P7000), substrate p-nitrophenyl-α-glucopyranoside (N-1377) and
1-1-diphenyl-2-picrylhydrazyl (D9132) were supplied by Sigma Aldrich (USA).
L-ascorbic acid (F951727) was supplied from Merck. Glucobay (100 miligram
acarbose) was supplied from Bayer (PT. Bayer Indonesia, Jakarta), ethanol and
ethyl acetate were supplied by JT Baker (USA), methanol and ethanol was supplied
from Merck (Darmstadt, Germany). All chemicals used were analytical grade.

Research Methods
The research consisted of crude extract preparation and in vitro
gastrointestinal digestion. Parameter that observed were DPPH radical scavenging
activity and α-glucosidase inhibition activity. The flow chart could be seen in Fig 6
below.

Crude Extract Preparation
The moisture content of samples were measured by AOAC direct
gravimetric method (AOAC 2000). For the extraction, 100 gram fresh andaliman
fruit was crushed in dry blender for 10 seconds and then extracted using maceration
method by Akyla et al. (2014) for 72 hours in room temperature, 100 gram of
andaliman was soaked in the 220 mL of ethanol: ethyl acetate (1:1). The crude
extract was obtained by filtration through filter paper (Whatman no.1) and then
evaporated under reduced pressure at 40⁰C (Rotary evaporator, Buchi, R-210). 45
minute was taken to evaporate 220 mL. The yield of crude extracts was weighed.
Fresh Japanese pepper fruit was extracted using the same procedure as andaliman.
1. Crude extract preparation

Fresh Andaliman
or
Fresh Japanese Pepper
2. In vitro gastrointestinal digestion*
Extracted by EtOH:EtOAc (1:1)

Andaliman and
Japanese Pepper
Crude Extract

Gastric digestion
 pH 2
 Pepsin enzyme
Small intestine digestion
 pH 6.8
 Pancreatin enzyme
 Bile extract

Andaliman and Japanese
Pepper Post in vitro
Digestion Extract

DPPH radical scavenging activity assay

α-glucosidase inhibitor assay

*Acarbose and Ascorbic acid as positive controls also went through in vitro
gastrointestinal digestion.
Fig 6. Research Flow Chart

In vitro Gastrointestinal Digestion
The crude extract was diluted in distilled water, then it was passed through
upper digestive tract (gastric and small intestine) simulation based on the following
in vitro method by Cilla et al. (2011). The crude extract pH value was adjusted (pH
Meter, Eutech Instruments pH 700) into pH 2 using HCl (6M) and allowed to stand
for 30 minutes, to make sure the adjusted pH was done. To start the gastric
simulation, freshly prepared pepsin sufficient to yield 0.02 g pepsin/g extract was
added to the crude extract, then it was incubated in the shaking water-bath
(Incubator Shaker, GFL 1083) at 37ºC for 2 hour. The enzyme was terminated in
the cold condition inside ice bath for 10 minutes. Then, the extract was adjusted to
pH 6.8 by NaHCO3 (1M) in order mimicking the condition in the small intestine
and an amount of pancreatin and bile extract solution sufficient to provide 0.005 g
pancreatin and 0.03 g bile extract/g extract was added, and incubation was
continued for another 2 h at 37ºC. The enzyme was terminated in the cold condition
inside the ice bath for 10 minutes. The extract pH was adjusted to pH 7.2 by
dropwise of 0.5 M NaOH. All post digestion extract was transferred to centrifuges
tubes and was centrifuged at 3500 g (Heraeus, Labofuge 400 R) for 1 hour, followed
by collection of the supernatants which continued to antihyperglycemic and
antioxidant assay.

Antihyperglycemic Activity Assay
Antihyperglycemic activity was determined using the α-glucosidase
inhibition method described by Sancheti et al. (2009), α-glucosidase stock (1
units/mL) was diluted with phosphate buffer (pH7, 0.1M) to a final concentration
of 0.04 units/mL. The inhibitory activity against the α-glucosidase was measured
using the following procedures. The working samples at difference concentration
(10µL) were added to the sample and control B, while distilled water (10µL) was
added to the control and blank, and substrate 4-nitrophenyl α-D-glucopyranoside
10 mM (25µL) was added to all. Then, the α-glucosidase solution (25µL) was added
to the sample and control A, then the phosphate buffer was added to the blank and
control B. Control A defines as the total glucose after hydrolysis of substrate and
enzyme, control B as the initial glucose in the sample and substrate, sample as the
total of hydrolysis between sample and substrate, the blank as the initial glucose in
the substrate. The reaction was carried out at 37⁰C for 30 minutes, and was
terminated by adding 100 µL Na2CO3 0.2M, the optical density of the wells was
measured at 410 nm using microplate reader (Epoch, Biotek). The experiments
were done in triplicate and acarbose (Glucobay) was used as positive control. The
concentration of crude extract of andaliman that were used were 209033.61,
104516.81, 52258.40, 26129.20 and 13064.60 µg/mL (dry weight of andaliman),
for post digestion extract were 418067.23, 209033.61, 104516.81, 52258.40, and
26129.20 µg/mL (dry weight of andaliman). The concentration of crude extract and
post digestion extract of Japanese Pepper were 47649.88, 23824.94, 11912.47,
4764,99 and 2382.49 µg/mL (dry weight of Japanese pepper). The acarbose
concentration before digestion were 0.3, 0.1, 0.05, 0.01 and 0.05µg/mL, while after

digestion were 0.5, 0.3, 0.1, 0.05 and 0.01µg/mL. The inhibitory activity was
calculated from the following equation.
% � ℎ�����

A1 = Abs Control A – Abs Blank
A2 = Abs Sample – Abs Control B

=

� −�
�

%

The result was presented as half the maximal inhibitory concentration value
(IC50 value). IC50 value defined as the concentration of the substance required to
inhibit 50% of α-glucosidase activity under the assay conditions. It was determined
by constructing a dose response curve between the logarithm of the concentration
of substances on the X-axis and the inhibitory activities on the Y-axis.

Antioxidant Activity Assay
Antioxidant activity was determined using DPPH radical scavenging assay
according to Awah et al. (2010). The procedure was started by adding 150 µL of
the working samples at different concentration and mixed with 75 µL of 0.2 mM
DPPH in ethanol solution inside the micro well. The mixture was allowed to stand
at room temperature in dark room for 25 minutes. Blank solution was consisted of
the sample solution (150 µL) and ethanol (75 µL), for the control was consisted of
DPPH solution (75 µL) and ethanol (150 µL). The experiments were done triplicate
and used ascorbic acid as the positive control. The absorbance was measured at 518
nm (Microplate reader, Epoch, Biotek). The concentration of crude extract of
andaliman that were used were 2090.34, 1045.17, 522.58, 261.29, and 130.65
µg/mL of dry weight of andaliman, for post digestion extract of andaliman were
5225.84, 2090.34, 1045.17, 522.58, and 261.29 µg/mL of dry weight of andaliman.
The concentration of crude extract of Japanese pepper that were used were 476.50,
238.25, 119.12, 59.56, and 29.78 µg/mL of dry weight Japanese pepper and for post
digestion extract were 2382.49, 953.00, 476.50, 238.25, and 119.12 µg/mL of dry
weight Japanese pepper. Ascorbic acid concentration before digestion were 20, 10,
5, 2.5 and 1.25 µg/mL, while post digestion concentration were 40, 20, 10, 5 and
2.5 µg/mL. The inhibition activity was calculated using the following equation:
DPPH Inhibition activity (%)=

− [{

A s s

�

p e−A s

�

} ×

]

The result was presented as half the maximal inhibitory concentration value
(IC50 value). The IC50 value defined as the concentration of the sample required to
reduce 50% of initial DPPH concentration.

4 RESULT AND DISCUSSION
Crude Extract Preparation
Extraction was purposed to separate compounds from a homogen mixture
using a solvent as the separator agent, in order to get the bioactive compound. In
this study the extraction from both andaliman and Japanese Pepper fruit were done
using organic solvent, which were ethanol and ethyl acetate (1:1) and the extraction
method was maceration for 72 hours in the room temperature (Akyla et al. 2014).
The extraction was using only the fruits of andaliman and Japanese pepper. Fruit
means the pericarp and seed of andaliman and Japanese Pepper (Fig 7A&B).
In this study the extraction was done based on the polarity since the active
compound which has the physiological active on the fruits have not been fully
discovered yet. Therefore the solvents were expected to extract most of the
compounds in andaliman and Japanese Pepper fruit including the flavour compound
in andaliman. The solvent were represent the polar (ethanol) and less polar (ethyl
acetate) solvent. The utilization of ethanol was expected to extract the phenolic
compounds which have antioxidant activity, this was proven according to Suryanto
et al. (2004) showed that ethanolic extract contained the highest phenolic
compounds which gave the highest radical scavenging activity. According to Akyla
et al. 2014 the crude extract from the mixture of ethyl acetate and ethanol was also
given a similar aroma to the fresh andaliman. However, in this study the mixture
was expected to yield an extract contain both polar and less polar compound since
it is still an early study.
The result from maceration showed that Japanese Pepper were shown to
have green colour and andaliman was shown to have brownish green extract. Both
of the samples were went through the same procedure and condition until became
a crude extract, the final step of crude extract preparation. Then, the step that was
not in control was the process after harvesting, especially for the Japanese pepper,
while the andaliman was available directly from the Simalungun farm. The colour
that formed were different since the polyphenol oxidase enzyme in the andaliman
fresh fruit while in the Japanese pepper the enzyme was might be already
inactivated by the blanching process after harvesting.
The blanching process was not only inactivate the enzyme but also preserve
the green colour of the Japanese pepper fruit. Polyphenol is the substrates for the
browning enzyme (polyphenol oxidase) and also it is the compound which are
responsible for the colour of plants. The browning enzyme will be activated when
the plants were exposed to the adverse condition, such as after the andaliman was
harvested and exposed to the oxygen from air, polyphenol oxidase will catalyze the
first steps in the biochemical conversion of polyphenol to produce quinones, which
will undergo further polymerization to yield dark colour, referred as melanins
(Holderbaum et al. 2010).

AA

B

Fig 7. (A) Andaliman (B) Japanese pepper
The differences also seen in the andaliman crude extract yield was 9.57%
(db), while Japanese Pepper crude extract yield was 20.98% (db). It was most likely
influenced by the compound that extracted with the solvent, Japanese Pepper
showed higher yield which represent the more compounds were extracted from
Japanese pepper than andaliman. This also indicated that the solvent ratio between
ethyl acetate and ethanol was suitable to give a high yield of extract, while the ratio
might be less appropriate to obtain higher yield. The other factor which might be
influenced was the differences of the moisture content of both of fresh fruit,
Japanese Pepper (75.13%) was shown to have higher moisture content than
andaliman (80.13%) this moisture content influenced the calculation of the yield.
The moisture content might be influenced by the higher hydration after the
blanching in the Japanese pepper fruit (Gowen et al. 2007).

Antihyperglycemic Activity
Hyperglycemia is a high blood glucose due to inadequate insulin in the
body. One of the way to treat hyperglycemia is by hampering the key enzyme in
the small intestine which catalysed carbohydrate breakdown into glucose. Alpha
glucosidase is the key enzyme which will catalyse the broken down of α-1,6
glycosidic bond in alpha limit dextrin to glucose. The inhibition of the reaction
could hamper the main metabolic pathway by preventing the production of
metabolite, which is glucose. The compound which has the inhibition activity might
be possible act as α-glucosidase inhibitor.
In this study, the crude extract of andaliman and Japanese pepper were
shown to have inhibition activity towards the α-glucosidase enzyme. The whole
series of concentration inhibition activity of andaliman and Japanese pepper were
shown in Fig 8 (A). The range of andaliman concentration were from
13064.60µg/mL of andaliman dry weight to 209033.61µg/mL of andaliman dry
weight, while Japanese pepper were from 2382.49µg/mL of Japanese pepper dry
weight to 47649.88µg/mL of Japanese pepper dry weight. The series of
concentration were determined based on the inhibition activity below and above
50% inhibition activity that were needed in the IC50 activity.
Andaliman’s crude extract were giving 92.28% inhibition in
209033.61µg/mL, while Japanese pepper were giving 93.505% inhibition in
47649.88µg/mL. The 40% inhibition were given by 13064.60µg/mL of andaliman
crude extract and 2382.49µg/mL of Japanese pepper crude extract. The
concentration indicated that the crude extract of andaliman showed lower activity

than Japanese Pepper’s crude extract, because andaliman need higher concentration
to inhibit α-glucosidase enzyme. This might be caused by different condition of the
bioactive compound between fresh andaliman and Japanese pepper, according to
some studies, the phenolic phytochemical has the natural α-glucosidase inhibitor
(Kim et al. 2005; McDougall et al. 2005, Wang et al. 2012). Therefore, the different
condition of the fresh samples affected most of the phenolic in andaliman which
might have oxidized by the polyphenol oxidase, while most of the phenolic
compounds in Japanese pepper still have not oxidized yet, because of the blanching
process which make the inhibition activity of Japanese pepper were stronger. The
other reason which made Japanese pepper were stronger Japanese Pepper fruit
contains quercetin which also found to be antioxidant, this might be one of the
responsible compound that have the α-glucosidase inhibition activity (Yamazaki et
al. 2007). However, andaliman could contain quercetin just like Japanese pepper
fruit, however its fresh fruit condition was differed because of the blanching after
the harvest. The uniformity of the fresh fruit condition possibly gave a great effect
on the inhibition activity as well.
Quercetin is a type of flavonoid, that have been known to inhibit αglcosidase enzyme such as quercetin 3-O-β-D-xylopyranosyl (1”-2”)-β-Dgalactopyranoside and (-)-lyoniresinol 3-O-β-D-glucopyranoside (Jong-Anurakkun
et al. (2007). According to Tadera et al. (2006), the glycosylation at the C-3
positions on the C rings of flavones enhances the inhibitory activity, C-3-OH are
favourable to the inhibitory activity. Glycosylation is the process by which a sugar
is covalently attached to a target protein which is the α-glucosidase. This
glycosylation at C-3 in the C-ring of quercetin plays an important part in αglucosidase inhibitory activities of flavonols (