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KESEMBUHAN PATAH TULANG TERTUTUP FEMUR TIKUS PUTIH

YANG DIBERIKAN EKSTRAK BIJI KELABAT (TRIGONELLA


FOENUM-GRAECUM) LEBIH BAIK JIKA DIBANDINGKAN DENGAN
TANPA DIBERIKAN EKSTRAK BIJI KELABAT
NYOMAN GDE ADITYA GB1, SIKI KAWIYANA IK1, SUYASA IK1
BAGIAN ORTHOPAEDI DAN TRAUMATOLOGI FAKULTAS KEDOKTERAN
UNIVERSITAS UDAYANA, BALI, INDONESIA

ABSTRAK

Penyembuhan patah tulang dipengaruhi oleh faktor internal dan eksternal.


Faktor internal terdiri dari lokasi, konfigurasi, vaskularisasi, usia pasien, penyakit
yang ada, status hormonal. Faktor eksternal terdiri dari status gizi, obat-obatan
dan olahraga. Pilihan suplementasi herbal yang dapat membantu penyembuhan
patah tulang adalah biji kelabat yang memiliki tiga senyawa aktif, diosgenin,
trigonellin dan 4-hidroksi-L-isoleusin yang dapat menginduksi pembentukan
tulang.
Tujuan dari penelitian ini adalah untuk mengetahui pengaruh pemberian
ekstrak biji kelabat secara oral terhadap diameter kalus, ekspresi osteocalcin dan
ekspresi kolagen tipe I pada penyembuhan fraktur tulang femur tikus putih.
Penelitian ini menggunakan model Post Test-Only Control Group. Tulang femur
tikus putih difrakturisasi kemudian diukur diameter kalus, ekspresi osteocalcin,
dan ekspresi kolagen tipe I antara kelompok dengan dan tanpa pemberian ekstrak
biji kelabat.
Diameter kalus, ekspresi osteocalcin dan kolagen tipe I pada kelompok
perlakuan secara signifikan lebih besar daripada kelompok kontrol dengan nilai p
masing masing 0.001, 0.000 dan 0.000 (p<0,05). Diameter kalus pada kelompok
pemberian ekstrak kelabat 250 mg selama 14 hari (2307,453 482,287m) lebih
besar dari yang diberikan selama 28 hari (1870,347 402,799m), namun
diameter kalus secara signifikan lebih besar pada pemberian ekstrak dengan dosis
250 mg (2307,453 482,287m) dibandingkan dengan dosis 500 mg (1728,818
182,586m) dalam waktu 14 hari dengan p = 0,020 (p <0,05). Sedangkan
osteocalcin (23,00 1,224) dan ekspresi kolagen tipe I (ekspresi padat 16.17%)
pada kelompok perlakuan lebih tinggi dengan 500 mg dosis untuk pemberian
selama 28 hari dengan nilai p=0,000 (p <0,05).
Pemberian ekstrak biji kelabat meningkatkan diameter kalus tergantung dosis,
tapi tidak tergantung pada lama waktu pemberian. Ekspresi osteocalcin dan
kolagen tipe I juga lebih tinggi dengan pemberian ekstrak biji kelabat yang dapat
membantu proses penyembuhan patah tulang.
Kata kunci: fraktur, diameter kalus, osteocalcin, kolagen tipe I

FRACTURE HEALING OF CLOSED FRACTURE IN WHITE RAT


FEMUR TREATED WITH FENUGREEK SEED EXTRACT IS BETTER
THAN THOSE WITHOUT FENUGREEK EXTRACT
NYOMAN GDE ADITYA GB1, SIKI KAWIYANA IK1, SUYASA IK1
ORTHOPAEDIC AND TRAUMATOLOGY DEPARTMENT MEDICAL FACULTY
UDAYANA UNIVERSITY, BALI, INDONESIA

ABSTRACT
Fracture healing of the bone is affected by internal and external factors.
Internal factors consist of location, configuration, vascularization, patient age,
existing disease, hormonal status. External factors consist of nutritional status,
medication and exercise. Suplementation choices that can help to force the
fracture healing that categorized as herbal are introduced recently. Fenugreek seed
has three active compound diosgenin, trigonellin and 4-hydroxy-L-isoleucine that
can induce bone formation.
The goal of this research are to know the effects of giving fenugreek seed
extract orally to the callus diameter, osteocalcin expression and colagen type I
expression on the bone healling of the femur fracture of the white rat. This is
experimental research using Post Test-Only Control Group Design. White rats had
its femur fracturized and the callus diameter, osteocalcin expression, and kolagen
type I expression measured compared between the group with and without
Fenugreek seed administration.
Callus diameter, osteocalcin and collagen type I expression on treatment
group significantly greater than control group with p value 0.001, 0.000 and 0.000
respectively (p<0.05). The callus diameter on fenugreek extracts 250 mg for 14
days (2307.453 482.287m) was greater than for 28 days (1870.347
402.799m), however it shows significant greater diameter with doses 250 mg
(2307.453 482.287m) than 500 mg (1728.818 182.586m) in 14 days with
p=0.020 (p<0.05). Whereas the osteocalcin (23.00 1.224) and collagen type I
(severe expression 16.17%) expression in treatment group were higher with in 500
mg dose for 28 day administration with p value 0.000 (p<0.05).
Fenugreek seed administration increase the callus diameter dose dependently
but did not depend on time administration. Osteocalcin and collagen type I
expression also higher with fenugreek administration aiding the fracture healing
proccess.
Keywords: funegreek, fracture, callus, osteocalcin, type 1 collagen

INTRODUCTION
The process of bone healing in fractures is affected by various factors,
both internal and external. Internal include fracture location, fracture
configuration, the vascularization condition in the fractured area, age,
comorbidities, and the patients hormonal status. External factors include
nutrition, drugs, and exercise(1).
The process of bone healing requires much more nutrition compared to
that of a normal condition for the skeletal system. The required nutrition can be in
the form of antioxidant substances such as vitamin E, vitamin C, or alpha-lipoic
acid, alng with some important minerals in bone turn over such as calcium,
phosphor, or zinc. Protein in the form of aminoacid is also required in the
formation of type I collagen product as the material of bone matrix(2,3).
The fenugreek plant (Trigonella foenum-graceum) is one of the many
planted herbal plants in several places in Europe, North Africa, Asia, Argentina,
Canada, America and Australia. In the skeletal system, a fenugreek seed has three
main active substances such as diosgenin which is a sapogenin steroid in the form
of glycosides (saponin) as much as 0.57 0.67% of trigonellin which is a
phytoestrogenic alkaloid as much as 0.24 0.73% along with 4-hydroxi-Lisoleucine which is an amino acid as much as 0.68 1.26%(4).
This plant is also often used as medicine due to its therapeutic effects
towards some illnesses, such as diebetes mellitus and hypercholesterol. A
fenugreek plants pleitropic effect is caused by some of the substances contents in
its seeds and leaves(5,6). The administration of fenugreek seeds extract has been
said to increase the speed of closed fracture healing in the femur of white rats that
can be evaluated through the callus diameter, osteocalcin expression and type I
collagen expression.
MATERIALS AND METHOD
This research is an experimental study using the randomized post-test
only group design with 30 male rats as its samples. All of the rats right femur
were fracturized then fixated using a k-wire and divided into 6 groups. Control
group 0 without fenugreek seed administration underwent saccharification on the

second week. Control group 1 without fenugreek seed administration underwent


saccharification on the fourth week. Treatment group 2 was administered with 250
mg/day of fenugreek extract per oral, and underwent closed fracturization after
retrograde nailing of the rats femur, and saccharification on the second week.
Treatment group 3 was administered with 250 mg/day of fenugreek seed extract
per oral and underwent saccharification on the fourth week. Treatment group 4
was administered with 500 mg/day of

fenugreek seed extract per oral and

underwent saccharification on the second week. Treatment group 6 was


administered with 500 mg/day of fenugreek seed extract per oral and underwent
saccharification on the fourth week.

RESULT
Table 1. Mean of callus diameter and osteocalcin in control and treatment groups

Variable

Callus Diameter
(m)
Osteocalcin
(n)

Group
Treatment
Treatment
Group with Group with
250 mg of
500 mg of
Fenugreek Fenugreek
Extract for Extract for
28 Days
28 Days
(n=5)
Hari 28
(n=5)
(Mean
(Mean
SD)
SD)

Control
Group
without
Fenugreek
Extract for
14 Days
(n=5)
(Mean
SD)

Control
Group
without
Fenugreek
Extract for
28 Days
(n=5)
(Mean
SD)

Treatment
Group with
250 mg of
Fenugreek
Extract for
14 Days
(n=5)
(Mean
SD)

Treatment
Group with
500 mg of
Fenugreek
Extract for
14 Days
(n=5)
(Mean
SD)

2307.453
482.287

1728.818
182.586

1870.347
402.799

1381.358
249.021

1037.570
329.743

739.787
461.390

16.2
1.483

21.60
0.894

27.00
3.162

23.00
1.224

10.60
2.073

15.20
1.303

Calus diameter on all case group bigger than those on control group and the
differences of mean among case and control group is statistically significant with
the p = 0.001 (p< 0.05). While the expression of osteocalcin on case group higher
than those on control group, the differences of means among case and control
group is statistically significant with p = 0.000 (p< 0.05).

Table 2. Result of post-hoc LSD test between research variables for treatment and
control groups

Variable
Callus
Diameter

(I) Group

(J) Group

250 mg of
500 mg of
Fenugreek Extract Fenugreek Extract
for 28 Days
for 14 Days

95% Confidence
Interval
Lower
Upper
Bound
Bound

578.635

0.020

98.492

1058.777

437.105

0.072

-43.037

917.247

500 mg of
500 mg of
Fenugreek Extract Fenugreek
for 14 Days
Extract for 28
Days

347.459

0.148

-132.683

827.601

250 mg of
500 mg of
Fenugreek Extract Fenugreek Extract
for 28 Days
for 28 Days

488.989

0.046

8.846

969.131

250 mg of
500 mg of
Fenugreek Extract Fenugreek Extract
for 14 Days
for 14 Days

-5.400

0.000

-7.859

-2.940

250 mg of
Fenugreek Extract
for 28 Days

-10.800

0.000

-13.259

-8.340

500 mg of
500 mg of
Fenugreek Extract Fenugreek Extract
for 14 Days
for 28 Days

-5.400

0.000

-7.859

-2.987

-2.600

0.039

-5.059

-0.140

250 mg of
Fenugreek Extract
for 28 Days

Osteocalcin

Mean
Difference

250 mg of
500 mg of
Fenugreek Extract Fenugreek Extract
for 28 Days
for 28 Days

There are significant differences on calus diameter after fenugreek extract


treatment based on the amount of dose that were given. There are also significant
differences on osteocalcin expression after given fenugreek extract based on dose
and duration of treatment.

Tabel 3. Result of Kruskal-Wallis on case and control group

Variabel

Collagen
type I
Expression

Case
Group
with 250
mg
Day 14
Fenugreek
Extract
(n=5)

Case
Group
with 500
mg
Day 14
Fenugreek
Extract
(n=5)

16.0

16.0

Mean Rank
Case
Case
Group
Group
with 250
with 500
mg
mg
Day 28
Day 28
Fenugreek Fenugreek
Extract
Extract
(n=5)
(n=5)

27.0

20.40

Control
Group
Day 14
without
Fenugreek
Extract
(n=5)

Control
Group
Day 28
without
Fenugreek
Extract
(n=5)

4.50

9.10

P Value

0.000

Collagen type 1 expression on case group with fenugreek extract 250 mg and 500
mg for 14 days and 28 days shows the mean rank higher compare to the control
group without fenugreek extract, significant statistically with p = 0.000 (p< 0.05).
DISCUSSION
In this research, There is significant difference in callus diameter after
fenugreek seed extract administration compared to the control group. Significant
difference also present in dose difference. But there is no significant difference in
duration of fenugreek seed extract administration. It caused by remodeling
process that bone reformation from the fractured bone to the original state (7).
Fenugreek seed extract adminitration with 500mg dose almost reach its
teratogenik effect, that can produce pro-oxidant effect. Fenugreek seed has tissue
effect by increase antioxidant effect on low dose and pro-oxidant effect on high
dose(8).
This study corresponds with the study done by Alcantera et al., that bone
callus increased because increased of calsium deposition and collagen type 1
synthesis in intracellular matrix after diosgenin administration (9). Study done by
Yen, angiogenesis of VEGF-A on fracture healing is caused by diosgenin and
trigonellin(10). Trigonellin increase phytoestrogenic activity(4). Phytoestrogenic
activity increase vasodilatation efect produce nitric oxide that increase
vascularization (early phase of fracture healing) and followed by ingrowth of less
nitric oxide dependent angiogenic vessels (next phase of fracture healing)(11).

In this research, higher osteocalcin level on rat after administration of 250


mg and 500 mg fenugreek seed extract for 2 weeks and 4 weeks after fracture
compared to the control group. This study corresponds with the study done by AlSobayil, increased of biomarker metabolism activity of osteocalcin compared to
the control group(12). The effect of fenugreek seed extract administration on
fracture healing associated with high amino acid level in fenugreek seed. Amino
acid of 4-Hydroxy-isoleucine in fenugreek seed increase insulin growth factor-1
(IGF-1) level that stimulated proliferation, differentiation, chemotactic effect of
the osteoblast(13,14).
In this research, expression of type I colagen increased on the rat with 250
mg and 500 mg fenugreek seed extract administration for 2 weeks and 4 weeks
after fracture compared to the control group. This study corresponds with the
study done by Alcantera et al., diosgenin increased bone formation by stimulating
type I colagen synthesis, ALP, Runx2 and osteopontin expression in low dose
diosgenin(9). Fenugreek seed also had flavonoid antioxidant (apigenin, luteolin,
orientin, quercetin, vitexin, and isovitexin), Calcium, vitamin C that important for
bone colagen protein matrix synthesis, that is type I collagen(15,16,17).
CONCLUSION
Based on the data analysis that had been done in this study, there are some
conclusions that can be drawn as follows:
1. Callus diameter in fracture healing after administration of fenugreek seed
extract was thicker than without administration of fenugreek seed extract.
2. Osteocalcin expression in fracture healing after administration of
fenugreek seed

extract was higher than without administration of

fenugreek seed extract.


3. Type I colagen expression in fracture healing after administration of
fenugreek seed

extract was higher than without administration of

fenugreek seed extract.

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