ACUTE TOXICITY STUDY OF ETHANOLIC EXTRACT OF FENUGREEK SEEDS (Trigonella foenum-graecum L.) ON WHITE RATS

  

ACUTE TOXICITY STUDY OF ETHANOLIC EXTRACT

OF FENUGREEK SEEDS (Trigonella foenum-graecum L.)

ON WHITE RATS

  

Kurnia Agustini, Sriningsih, Julham Effendi

  Center for Pharmaceutical and Medical Technology, Agency for the Asessment and Application of Technology, BPPT, Jakarta

  

correspondence auth

Abstract

  Fenugreek seed or bijiklabet (Trigonellafoenum-graecum L.) was known to have activity against degenerative diseases such as diabetes mellitus, hypercholesterolemia and postmenopausal symptoms. This study was conducted to investigate the safety of ethanolic extract of fenugreek on white rat, by determining Lethal Concentration 50 value (LC50). This in-vivo assay referred to the WHO protocols for the toxicity assay of natural medicines. We used Spraquedawley white rats, female and male, 6 weeks of age, which divided into a normal group and fivetreatment groups (1g/kgBW, 4g/kgBW, 8g/kgBW, 12g/kgBW, 16g/kgBW). Test sample was given once orally then the animal were monitored for two weeks. Observation of toxic effect includedphysical symptom of central nerve system, autonom nerve system and digestive system. Alldeathanimal were counted and LC50 were calculated. Result showed that there was no toxic effect and no death animal until 16g/kgBW dose of treatment. We concluded that ethanolic extract of Fenugreek is practically non toxic.

  Keywords : Fenugreek seeds, Trigonellafoenum-graecum L., acute toxicity

  INTRODUCTION

  Fenugreek seed or Foenigraeci semen is dried seed from Trigonellafoenum-

graecumL., Leguminosae, (WHO, 2007). In Indonesia, it was known asBijiKlabet.

Empirically, bijiklabetwas used to treathemorrhoids, asthma, ulcers, muscle pain and often used as a preventative hair loss and a skin softener.Many studies showed that it hasantidiabetic, anticancer and anti hypercholesterolemia (Mills, 2000). Fenugreek also has antiandrogenicactivity, due the presence of beta-sitosterol, palmitic-acid and stearic-acid.It also has the ability to reducetotal cholesterol, LDL, VLDLand triglycerides level significantly. The anti-hyperglycemic and anti-inflammatory properties investigated in fenugreek are additional benefit. Agustini (2007) showed that ethanolic extract of fenugreek has estrogenic effect on ovariectomized and immature female Wistar rats.

  Fenugreek contains sapogenin steroiddiosgenin, a precursor for sexual hormone (Evans, 2002), and its isomer (yamogenin, gitogenin, tigogenin, and trigoneoside)(Dewick, 1997; Wiryowidagdo, 2000). Fenugreek also contains fatty oil 20-30%, alkaloids (trigonelline, an alkaloid pyridine, gentianin and karpain), flavonoids (vitexinin glycoside or ester form, isovitexin, orientin, vicenin, quercetin and luteolin)

  9

  2002).

  This study was aimed to investigate the acute toxicity of ethanolic extract of Fenugreek in white rat.In a short term toxicity assay. Sample was given once in various doses and the observation was carried out for two weeks. All physical symptoms and death animal were analysed andthen compared to a normal group.The acute toxicity was expressed as LD50 which is defined as a dose that causes50% lethality of animal test. Sample is categorized as a safe material when the LD50 value is bigger than 15g/kgBW (Lu, 1995).

  METHOD

Sampel preparation. Fenugreek were obtained from Tawangmangu, Central Java,

  Indonesia. Seed were dried and grind, then were extracted with ethanol 96% food grade.Crude extracts were suspended inCarboxy Methyl Celulose (CMC) 0,5%.

  

Animal preparation. Experimental animals used in this study were 30 males and 30

  femalesSpraque Dawley (SD) white rats, 5-6 weeks of age, obtained from Indonesian Food and Drug Administration (FDA/BPOM). The animal were kept in the animal cage (25 + 2

  C) under 12 h light/dark cycle and fed with standard diet of pellet and free access to distilled water prior to the study, and theanimal were kept for acclimatization for one week. The experiment was done according to ethical clearance approved by Ethical Committee of BadanPenelitiandanPengembanganKesehatan/Balitbangkes.

  

Animal treatment. Animals weredivided into a normal group and five treatment

  groups (1g/kgBW, 4g/kgBW, 8g/kgBW, 12g/kgBW, 16g/kgBW). Test sample was given once orally then the animal were monitored for two weeks. Observation of toxic effect includedphysical symptom of central nerve system, autonom nerve system and digestive system. Alldeathanimal were counted and LC50 were calculated.

  Table 1.Group of Animal Treatment NO. GROUPS TREATMENT n

  1. N Normal Diet + CMC Na 0,5% suspense

  5 Male +5 Female

  2. D1 Normal Diet + Sample 1 g/kgBW

  5 Male + 5 Female

  3. D2 Normal Diet + Sample 4 g/kgBW

  5 Male + 5 Female

  4. D3 Normal Diet + Sample 8 g/kgBW

  5 Male + 5 Female

  5. D4 Normal Diet + Sample 12g/kgBW

  5 Male + 5 Female

  6. D5 Normal Diet + Sample 16 g/kgBW

  5 Male + 5 Female

RESULT AND DISCUSSION

  All animal treated with dose 1 (1g/kgBW) until dose 5 (16g/kgBW) showed no toxic effect significantly. Normally, after orally given sample treatment, all animal showed decrease of motoric activity for some minutes. Howefer after 30 minutes, this effectreverts to normal condition. This spontaneous effect is a normal response after orally gavage treatment. Theobservation of physical symptom of central nerve system, autonom nerve system and digestive system observation,were shown in table 2.

  10 NORMAL DOSIS1 DOSIS2 DOSIS3 DOSIS4 DOSIS5

• 2.
• 3.
• Stress hair - - - - - -
• : No Symptom + : There are Symptom +/- : Normal Both male and female rats in all groups demonstrrated a body weight gain almost similar to the normal control after 14 days.

  Constipation

  80 100 120 140 160 180

  60

  40

  20

  Figure 1. Body weight of male rats from all group for 14 days observation

  Note:

  Bloody Fesses

  Diarrhea

  11 OBSERVATION GROUPS N D1 D2 D3 D4 D5

  3. Urination - - - - - - Breath Rate +/- +/- +/- +/- +/- +/- Heart Rate +/- +/- +/- +/- +/- +/- Digestive System 1.

  2. Salivation - - - - - -

  1. Open eye +/- +/- +/- +/- +/- +/-

  4. Tremor - - - - - - Autonom Nerve System

  3. Convulsion - - - - - -

  2. Motoric Activity +/- +/- +/- +/- +/- +/-

  1. Sedasi - - - - - -

  Central Nerve System

  Day1 Day 6 Day 9 Day 12 Day 14 BW (gr ) Time Weighing- Males Body Weight

  12 Figure 2. Body weight of female rats from all group for 14 days observation

There was no lethal effect on animals in all group after 14 days observation.

Regulation from Indonesian FDA mentioned that if at dose 15g/kgBW no lethality

case occured, then it is not necessary to increase the dose. LD50 higher than

15g/kgBW is categorized “practically non toxic”.The lethality data waspresented in Table 3.

  

Table 3. Lethality case of animal from all groups

GROUPS MALE FEMALE TOTAL LETHAL % LETHALITY n LETHAL n LETHAL NORMAL

  5 5 0% D1 5 5 0% D2 5 5 0% D3 5 5 0% D4 5 5 0% D5 5 5 0%

  Table4. Catagorization of Toxic Effect(Lu,FC, 1996)

  Toxic Category LD

  50 VALUE Super toxic < 5 mg/kgBB Very hard toxic 5-50 mg/kgBB

  

Very toxic 50-500 mg/kgBB

Medium Toxic 0,5-5 g/kgBB

Mild Toxic 5-15 g/kgBB

Practically Non Toxic > 15 g/kgBB

  CONCLUSION No delayed toxic effect and lethality were observed after 14 days of observation in rats.

  We concluded that theethanolic extract of Fenugreek has a LD50 and categorized as “practically non toxic”.

  20

  40

  60

  80 100 120 140 160

  Day 1 Day 6 Day 9 Day 12 Day 14 BW (gr ) Time weighing

  

Females Body Weight

NORMAL DOSIS1 DOSIS2 DOSIS3 DOSIS4 DOSIS5