EVALUATION OF THE EFFECT OF ONE NOVEL NUTRACEUTICAL FORMULATION ON THE SERUM LEVEL OF VITAMIN B12 IN RATS FED A VITAMIN B12-DEFICIENT DIET.

EVALUATION OF THE EFFECT OF ONE NOVEL NUTRACEUTICAL FORMULATION ON THE SERUM LEVEL OF VITAMIN B12 IN RATS FED A VITAMIN B12-DEFICIENT DIET.

1.0 TEST SYSTEM DETAILS:

Species : Rattus norvegicus (Rats)

Strain: Sprague Dawley or Wistar

Age : 3 weeks

Sex: Male and Female

No. of animals : 12/Group (6 Male + 6 Female)

Total animals : 84

2.0 TEST ARTICLES DETAILS

HF1: Vitamin B12 supplement derived from Zea mays.

3.0 VEHICLE DETAILS

The test articles will be formulated by utilizing 0.5% methylcellulose as the vehicle.

4.0 ALLOCATION OF GROUPS:

Group No.	Group Description	Disease Induction procedure	Treatment administered	Dose Volume and Route
G1	Normal Control	Normal Laboratory Diet × 10 weeks	0.5% MC, p.o., b.i.d.	5 ml/kg, p.o.
G2	Disease Control	All animals will be given a customized Vitamin B12-deficient diet from the third week of their age × 8 weeks	0.5% MC, p.o., b.i.d.
G3	Reference Control		Mecobalamin 32 mg/kg, p.o., b.i.d.
G4	Treated with a low dose of HF1		HF1: 3-10 mg/kg, b.i.d.
G5	Treated with Intermediate  dose-1 of HF1		HF1: 10-30 mg/kg, b.i.d.
G6	Treated with Intermediate  dose-2 of HF1		HF1: 30-100 mg/kg, b.i.d.
G7	Treated with a High dose of HF1		HF1: 100-300 mg/kg, b.i.d.

            Abbreviations: MC: Methylcellulose, p.o.-per os., bid: bis in die.

5.0 METHOD:

· Healthy animals will be selected for the study immediately after completion of the weaning period, randomized based on body weight, and will be assigned to 7 groups consisting of 12 (6 male + 6 female) animals each.

· Animals of Group G1 will be designated as normal-control and administered 0.5% MC, p.o., b.i.d.

· Disease control animals (assigned to group G2) will receive 0.5% MC, p.o., b.i.d.

· Animals of group G3 will be treated with reference drug Mecobalamin at the dose of 32 mg/kg, p.o., q.d.

· Animals of group G4-G7 will be treated with HF-1, at different incremental dose levels as outlined in Section 4.0 of Annexure-I, b.i.d.

· The normal control group (G1) will be fed a Normal Laboratory Diet, whereas animals allocated to groups G2 – G7 will be fed Vitamin B12 (AIN-93M) Deficient Customized diet for the next eight weeks.

· Immediately after, the completion of week eight animals of group G2-G7 will be randomized based on Vitamin B12 level.

· Compound, reference standard, and vehicle administration will be initiated from day 56 for the next two weeks (till day 70), after confirmation of Vitamin B12 Deficiency in groups G2-G7.

· On day 0, before the initiation of Vitamin B12-deficient diet, blood parameters will be determined for the baseline data. Vitamin B12 deficient diet will be given for 8 weeks and Vitamin B12 level will be evaluated every two weeks. Animals will be treated with formulations/Reference drugs/vehicles for two weeks after confirmation. Subsequently, after week eight on day 71, all animals will be sacrificed under an overdose of thiopentone anaesthesia. After suitable anaesthesia but before the animal dies, blood will be collected from the retro-orbital plexus for the estimation of Vitamin B12 parameters. Immediately after the animal dies, the liver, kidneys, and spleen will be weighed and will be stored at -80°C for any biochemical evaluations.

6.0 PARAMETERS TO BE EVALUATED:

· Body weight: Twice a week.

· CBC count and Vitamin B12 level in blood serum

· Serum methylmalonic acid, total homocysteine levels

· Determination of Cobalamine level in Liver tissue

7.0 REFERENCE(S):

1. Sianipar, I. R., Ujianti, I., Yolanda, S., Murthi, A. K., Amani, P., & Santoso, D. I. S. (2019). Developing vitamin B12 deficient rat model based on duration of restriction diet: Assessment of plasma vitamin B12, homocysteine (Hcy), and blood glucose levels. doi:10.1063/1.5096672

2. Ujianti I, Sianipar IR, Prijanti AR, Hasan I, Arozal W, Jusuf AA, Wibowo H, Prihartono J, Amani P, Santoso DI. Effect of Roselle Flower Extract (Hibiscus sabdariffa Linn.) on Reducing Steatosis and Steatohepatitis in Vitamin B12 Deficiency Rat Model. Medicina. 2023 May 28;59(6):1044.

                                                                 

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