ISBN: 978-602-96839-1-2
ISBN of Volume I: 978-602-96839-2-9
PROCEEDIN6 OF
INTERNATIONAL CONFERENCE. ON
MEDICINAL PLANTS
in occasion of
the 38
thMeeting of National Working Group on Indonesian Medicinal Plant
21-21 July 2010
Surabaya, Indonesia
Editors:
Elisabeth C. Widjajakusuma
Kuncoro Foe
Sendy Junedi
Bambang Soekardjo
Retno Andayani
Achmad Fudholi
Lucia Hendriati
Angelica Kresnamurti
Organizing Committee
FACULTY OF PHARMAcY'
WID}'1\.
MANDALA CATHOUC
UBJ\f£RSITY'
in collaboration with
ej 'rocccdill8 oji IItcm,/flOllaf( oll}'rcllcc 011 : IIcdi'cillafeJ'fa IItS -Su ra6a),(/' i1lifc.'IICSIII .} /-.} / jury .}O f()
PHALERIA MACROCARPA
FRUIT EXTRACT AS
INSULINOTROPIC AGENT IN STREPTOZOTOCIN-INDUCED
DIABETIC CYNOMOLGUS MONKEYS
(MACACA
FASCICULARIS)
Irma H.
Suparto
l,2,],:,
Erni SulistiawatiI, Bayu Febram Praseto-l, Wulan
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nwa
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ra an an, asmma aramas n
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t ·1I Primate Research Cel1ler or HogOJ' Agriculture Lni\'el'sity
cHiopharmaca ｒ･ｾ･｡ｲ｣ｨ＠ Ccnter or Hogor Agriculture Lni\'ersity
'Chemistl,)' Dcpartment oC J--'aculty oi' Mathematical and Science or Hogor Agriculture Lni\'l'rsity -lPharmacology Departillent oi' Veterinary Faculty or HogOJ' Agriculture Lniversity
'Corresponding author: . JL Lodaya 2 no 5 HogOJ' 16151
Abstract: Mahkll!a dnt'll (P/w/I'r;U 1I)(/(TIII'ur/)(/) rruit is widely used as an alternative medicine lill diahctic condition. Ilowe\'er. its mechanism or action has not heen Curther studied. The ohjective or this lesearch is to investigate the insulinotropic e1Tect or I/1l1hkIJ!a dl'lJ'i/ rruit extract in streptozotocin-induced diahetic cynomolgus monkeys (Mu('(/('u ji/,\I';nt/ur;.I). Diahetes was induced hy single intravenous injection or stl'l'ptozotocin 55 mg/kg hody weight. Animals were randomly divided into three groups and 5 animals in each group recei\'ing treatment orally once a day in the mOl'l1ing hefilre meal time lilr 7 days. The rirst tl'l'atment gl'Oup as cOl1lrol I'l'cei\'ed distilled water. the second and third gl'Oup given /IIuhk"!1I "I'II'U extract tahlet or 500 mg/kg and 100 mg/kg. respecti\e1y, Fasting hlood glucose concentration was monitored daily. Plasma insulin,concentration and total
I)
cells pancreas was analyzed at the end oC the study. Artel' one week or treatment. Casting hlood glucose concentration was not dillerent in all three groups (ANOV A p>O,05l. There was a trend or increased insulin concentration in animals receiving extracts compal'l'd to control animals. The totalI)
cells pancreas was highcst in gl'Oups receiving extl'act oC 100 mg/kg hody compared to the other two gl'Oups (,\NOV A P «U):'i) The conclusion oj' ｴｨｩｾ＠ study is /I1uhklllU "1'11'(/ rruit extlact improvedI)
cells ｰ｡ｮ｣ｲ･｡ｾ＠ hut not sullicient to reduced rasting hlood glucose conccntration, This can he dill' or the short ｴｬＧｬｾ｡ｴｭ･ｬｬｬ＠ time.KClwords: ;1l.1II1i1l1l!rllp;I', .Ilrep!II::II!IIC;Il·;lldl/l'('" ";U/N'!;t' (TIlIJ/IIII/gLI.I /IIOIl"-e.",\,
/i
l'ell/)(III1TI'U,\INTRODUCTION
World Health Organization (WHO 1994) reported that in 2025 there will be an estimation or ]00 million people in the world sufrers rrom diabetes. Indonesia which rank 4th in the world for
diabetes is predicted an increase to 12.4 million people in the year 2025 (Oepkes 20(5). This disease has been predicted by WHO as one of the leading causes or death and disability within the next 25 years.
Thererore. the need to develop an afTordable antidiabetic regimen is very cruciaL Many people are tLlrning to ahemative or complimentary medicine. Herbal medicine becomes a preference because considered cheaper and accessible Cor coml11on people.
One or the herbal medicines is tnahko/a deml (lJha/eria tnact'Oc{UI){l). A member of the Thymelaeaceae ramily. believed to have several benerits including the ability to lower blood pressure, cure hepatitis. ｩｮ｣ｲ･｡ｾ･＠ stamina, treat cancer and lower blood glucose (Harmanto 20m:
Winarto 20m).
In previous ill-I'ilro study, ethanol extract of I1whko/a dl'\\'{/ fruit showed increased activity in insulin secreting BRIN BD-I I cell (Suparto 2(07). Its hypoglycemic effect was confirmed in streptolOtocin-induced diabetic Sprague Dawley rats with dosage of toOO mg/kg body weight given orally ror 2 weeks (Supm10 2008). The reduction of blood glucose up to 40o/c compm'ed to controL However. this ill I'il'o efficacy should be further evaluate in animal model that is closer to
human in physiology and genetic. such as cynomolgus monkeys (Macaca jasciclI/aris). Therefore.
(Proceedil1g of /l1tcmationa(Coriference on ｾｬＱ｣､ｩｦｩＱＯ｡ＨＨｐｦ｡ｬｬｴｳ＠ -Sura6aya,/l1doncsia 21-21 Jury 2010
in this study, the goal is to evaluate the efficacy of mahkota deH'(l fruits extract as insulin secretagogue in streptozotocin-induced diabetic cynomolgus monkeys.
MATERIALS ANI) METHOI)S
Malerials
Fifteen adult male cynomolgus monkey:.. (Macac{/ fascicltlaris) with an age range of 5-7 years (determined by dentition) and body weight between 3-5 were obtained Ji'om the Primate Research Center of Bogor Agricultural Univer:..ity, West Java. Indone:..ia. All experimental procedures on these animals were conducted in compliance with the guidelines established by the Institutional Animal Care and Use Committee.
Prepam/ion
or
etllllllOl extrac/Ripe fruit (dark pink) of l1/ahko/a del)'{/ V'v!ocrocorpo pllolerial from the Faculty of Forestry Bogor Agricultural University Indonesia were sliced thinly. dried in oven (50"C) for 30 hours and made into powder. The powder was extracted with maceration techniques in ethanol 30S;( for three days then. dried using rotw'y evaporator. The extract then confirmed with phytochemical qualitative assay to determine its content need to be positive for alkaloid.
ｓｴＬＮ･ｻｊｉｏｾＨＩｬｯ｣ｩＱＷ＠ indllc/ion
Before receiving treatment, animals were injected with streplOzotocin (STZ Sigma SO 130) to
induced diabetic condition. After fasted overnight. animals were anesthetized with intramuscular injection of ketamine 10 mg/kg bw and single intravenous injection of STZ 55 mg/kg (Koulmandhaellil. 20(3). After ll1jection, animals were hydrated with normal saline 100-150 m!. Fa:..ting blood ｧｬｵ｣ｻｬｾ･＠ was measured daily. when blood glucose in the range of 200 mg/dl or ahove Cor 5 day:... animah started treatment 1'01' 7 days. If concentration above or equal to 350 mg/dl.
animals received short acting insulin suhcutaneous injection 2-4 Unit to control the blood glucose level.
Frc(//mell/ groups
All animals held in individual cages. after one month quarantine, they were randomly divided into three equal group:.. (11= 5 animals). The three groups were animals receiving treatment orally with
I) extraci of mollko/a ､･ｾｬＧｬｉ＠ fruit with do:..age 500 mg/kg. with dosage of 1000 mglkg. and the third groups as control receiving only distilled water.
Samples collectio/1
At the end or the :..tudy. all animals were euthmlized with sodium pentobarbital Iml/2kg. Blood samples were collected for blood glucose and insulin concentration. Pancreas was collected at necropsy. It was divided into head, body and tail. fixed in lOCI, neutral bulTered formalin then stained with Gomori chrom hematoxylin dye. This staining showed
B
cell as basophilic and a cell eosinophilic (Fisher & Haskell 1954, Datal' el (II. 20(6), Total number ofr)
cell pancreas was counted manually.Swtisli( al (l11(llnis
Analyses were done with Statistica (Statsoft version 6). Before analysis. all variables were evaluated for distrihution and equality of variances among groups, Analysis was carried out lIsing one-way analysis of variance ｾａｎｏｖａＩ＠ followed by Fisher post hoc test. if significant with P value <0.05. The results are expressed as means ± standard error of means (SEM).
RESULTS
ｓｴｲ･ｪｊｬＨｉｾｯｬｯ｣ｩｬｬＭｩＱＷ､ｌｬ｣･､＠ diabetic animals
(Proceedillg oj Internat/oua[COlljerellce 011 ｾｈ･､ｩ｣ｩｬＱ｡ｲＨｐｦ｡ｵｴｳ＠ 5ura6aya, lrlliollesia 2/-21 jufy 20!O
From 15 animals at the beginning of the study. 12 survived lIntil end procedure. Treatment was started after 5 days or hyperglycemic, extract was administered orally for 7 days.
l:.JlecI o( In(/Mota dell'a ji'/lit extracI Oil blood gll/cose
After end of treatment. average of morning fasting blood glucose. at noon and evening were analyzed and it was not significantly different (ANOYA p >OJl5) (Figme 1). There was a trend of lower blood glucose in animals receiving fruit extract with both dosages.
---+---II ｾ＠
Time
"-igure I Blood glucose concentration in the morning. noon time lllld evening, after 7 days treatment with control, treatment of l1luhkota dewa dosage 500 and 11100 mg/kg in induced diabetic monkeys
Plasma inslllin concentration
Plasma insulin concentration at end of treatment was not significantly different (ANOYA p >0.(5) in all groups. Howeyer there was a tendency of increased concentration of plasma insulin in animals treated with fruit extract dosage of SUO mglkg, also 1000 mg/kg (Figure 2).
2.50
E 2.00
c 1.50 .
'" 1.00
F
ｾ＠
";
0,50
Figure 2 Plasma insulin of streptozotocin.induced diabetes cynomolgus monkeys in groups of control.
Control 500 rcgdl 1000 mgfci animals with extract dosage 500 and IlIOO mg/kg.
000 .
rotal/i cell pancreas
Basophilic
B
cell pancreas with Gomori staining was counted rrom all sections. The totalB
cell pancreas was significantly different (ANOY A p <0.0(8) and post hoc Fisher showed that animals treated with extract of 1000 mg/kg had higher totalB
cell compared 10 animals treated with 500mg/kg of extract (p 0.0(1) or control group (p = (l.02).
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Ｎｾ＠ 8000
0:0
ｾ＠ 6000 .
ｾ＠
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:; 4000
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I I':: 2000 ""
-
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ｾ＠
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-Control 500 ｭｾｫｧ＠ 1000 mgkg
[image:6.611.181.382.93.226.2]Trc;uruclH group,
Figure 3 Plasma insulin of streptozotocin induced diabclcs in cynomolgus monkeys in groups of control, tnatmcnt with extract dosage SOO and l()OO mg/kg.
DISClJSSION
The primary objective or this study was to evaluate 1he hypoglycemic efrects or mahko/(l dn\'a fruit extract through insulin secretagogue in streptozotocin-induced diahetic cynomolgus monkeys. Administration of STZ has heen widely used to induce type I diahetes in various animal models including cynomolgus monkeys (Wagner cl al. 200 I. Koulmandha cl 01. 200]). This agent alTecting degeneration and necrosis or pancreatic ｾ＠ cells (S;:kudelski 2(01) causes railure to secret insul in.
The major finding of this study is ｉｾ＠ cell pancreas regeneration increased hy the administration of mahkota dewa rruit extract. III I'ilm study in insulin secreting cell BRIN BOll
support this result. Ethanol extract of mahkota dewa fruit with concentration 4.) mg/L triggers the secretion or insulin in the presence or glucose (Suparto 2(07).
Other plant medicine showed the same henefit such as AIlJlona II1l1ri("ol(/. Allillll1 .l"£Ilil'lIl11.
and Argymlohillm mscwn with various dosages in STZ induced diahetic rats (Eidi cl al. 2(0). Ahmed Cllli. ＲｉＩｏｾＮ＠ Adeyemi cilli. 2(10).
The etpanolic extract of the mahko/(l dn\'o fruit demonstrated a promising increasecl in total ｉｾ＠ cell pancreas and directly affect the inuease in plasma insulin concentration, especially for the higher dose. However. this increased in regeneration of ｾ＠ cell pancreas and plasma insulin was not adequate to decrease the hlood glucose level. Hahibuddin et al. ＨＲＰＰｾＩ＠ had reported hypoglycemic elTect of Clll"{lllllma sil7(linl in diahetic rahhits administered for three weeks. Also. Adeyemi cl 01. (20] 0) had reponed using diahetic rats given Annon(} mwicalo for two weeks resulted significant regeneration of ｉｾ＠ cell pancreas. Duration of treatment can influence the glucose lowering effect if the mechanism depends on the regeneration of pancreatic cells. Compared to the treatment ti me in the present study was only for 7 days. there is an increased of cell regeneration. however glucose level was still high. This finding needs to he fut1her studied hy giving longer time of treatment.
The present study is the first reports on the i nsulinotropic acti vity of lI1(lhko/(l ､ｃｾ｜ＬＨｬ＠ fruit extract in streptozotocin-induced diahetic cynomolgus monkeys.
CONCLlJSION
The conclusion of this study is 1I111!Jko/(l r/cml (Plwlcria marcocwjJ(l) fruit extract improved ｉｾ＠ cell pancreas but not surficient enoLlgh to show hypoglyceIl!ic effect. This can he due to the short treatment time.
ACKNOWLEDGEMENTS
(Proceedi1l9 ojll1iematiollafCOl1jereuce 011 :ltcdirirwf(Ffauts -511ra6aya.l11doucsia 21-21 Jufy 2010
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