}((NASFIT --For Medical Research--)){

Carried out at Amman University Jordan

Acute and Subchrinic toxicity of the Plant Extract NASFIT

Objectives: Evaluation of the possible toxic effects of the mentioned compound on acute and subchronic toxicity using different dose concentration according to the body weight for different time intervals.
This study will be carried over using the Amman University experience and facilities.

Experimental procedure:
   1) The solution will be filtered of particulate material to get free clear solution.
   2) Spargue Dowely Rats will be exposed to the plant extract through the drinking
   3) Animal Test.

Acute Toxicity
A group of 7 male Rats (100-150gm wt) will be given the extract diluted in drinking water in addition to a control group as shown in table 1.

                                   Table 1 . A protocol of the acute toxicity of the plant extract NASFIT

Number of animals

7
7

Treatment

Drinking water

Plant Extract (1:50)

The animals will be monitored for 14 days during administration for the followings:
    • Physical activity and general appearance: Physical parameters including behavior, activity and tendency to
       sleep as well as the appearance of treated rats.
    • Eating and drinking: Food consumption and weight loss/gain as well as water consumption.
    • Mortality

Subchronic Toxicity
Five groups each of 7 male Rats will be treated as shown in table 2

Table 2. Experiment protocol showing the treatment dilutions and the number of experimental animals for the subacute toxicity.

Number of animals

7*
7 7 7 7*

Treatment

Drinking water
1:5 1:10 1:25 1:50

                                      *Same animals in table 1 will be further investigated in the subchronic phase

1. The animals will be monitored for 8 weeks as in signs as mentioned previously.
2. Blood samples will be collected from retro-orbital plexus in 4 time intervals, before treatment, 4 week, 6 weeks and 8 weeks after exposure.
3. Analysis of blood samples for blood sugar, hematology, liver function and kidney function parameters. See table 3.
4. Postmortem analysis: Macroscopic examinations of any pathological changes of body organs will be done after the killing of Rats (experiment termination).
5. Statistical analysis. Data will be analyzed using t test
6. Report preparation. A signed, confidential report will be prepared by investigator.

Supervisor :                                          Co-advisor:                                                 Research assistant:
Dr.Mohammad Salahat                          Dr.Awni Shanableh                                     Mrs.Makbula Al-Haron
PhD.physiology                                      M.D.                                                           Clinical Biochemist.

Acute and Sub-chronic toxicity of the Plant Extract NASFIT
The plant extract NASFIT solution was supplied by Dr.Mohammed Nasser El-Fituri through International Labs for Medical Technology and conducted by Amman University .
The tested formula is of herbal origin, the liquid complex extract composed of: Amino Acids, Sugar (Fructose, Glucose, Sucrose), Fatty Acids and Sterols, very long chain Fatty Acids, and Organic Acids.
Moreover it contains a number of minerals, vitamins, albumin and other trace elements.
The drug has an effect on a wide spectrum of diseases, including some group of retrovirus, cancer of virus etiology, some autoimmune disorders and diseases associated with immune system deficiency, also qualified to rebuild of the immune system structures.

The main objective of this toxicological study is:
The Evaluation of the possible toxic effects of the mentioned extract on acute and sub-chronic toxicity using different dose concentration according to the body weight for different time intervals.

Acute and Sub-chronic Toxicity of Plant Extract NASFIT:

Objectives :
The Evaluation of the possible toxic effects of the mentioned extract on acute and sub-chronic toxicity using different dose concentration according to the body weight of the rats for different time intervals .

Experimental procedure:
1) Aqueous plant extract will be prepared . The solution will be filtered of particulate material to get free clear solution.
2) Sprague Dowely Rats will be exposed to the plant extract through the drinking 0.1 ml/kg of the extract three times daily . See table 1.

Acute Toxicity
Two group each of 7 male Rats (100-150gm wt) will be fed as shown below in table 1.

Number of animals

7*
7

Treatment

Drinking water
Plant extract
1:10

The animals will be monitored for 14 days during administration for the followings:
    • Physical activity and general appearance: Physical parameters including behavior, activity and tendency to
       sleep as well as the appearance of treated rats.
    • Eating and drinking: Food consumption and weight loss/gain as well as water consumption.
    • Mortality

Sub-chronic Toxicity
six groups each of 7 male Rats will be treated with 0.1 ml/kg three time daily from the extract, as shown in table 2

Table 2. Experiment protocol showing the treatment dilutions and the number of experimental animals for the sub-chronic toxicity.

Number of animals	7*	7*	7	7	7	7
Treatment	Drinking water	1:10	1:20	1:30	1:40	1:50

*Same animals in table 1 will be further investigated in the sub-chronic phase

1. The animals will be monitored for 8 weeks for physical and behavior change as mentioned previously.
2. Blood samples will be collected from retro-orbital plexus in 3 time intervals: 4 week, 6 weeks and 8 weeks after exposure, parallel with a control group of 7 rats.
3. Analysis of blood samples for: blood sugar, hematology, liver function and kidney function parameters. See table 3.
4. Postmortem analysis: Macroscopic examinations of any pathological changes of body organs will be done after the killing of Rats (experiment termination).
5. Statistical analysis. Data will be analyzed using t-test

Number

Test

1

Red Blood Cell count (RBC)

2

White Blood Cell count (WBC)

3

Hemoglobin

4

Platelets count

5

Blood Glucose Level

6

Total protein

7

Amylase

8

Uric Acid

9

Glutamate Pyruvate Transaminase (GPT)

10

Glutamate Oxaloacetate Transaminase (GOT)

11

Triglyceride

12

Cholesterol

13

Alkaline Phosphates (ALP)

14

Gamma GT

- Acute Toxicity Results :
The extract in oral dose of 0.1 ml/kg three times daily did not cause any mortalities or any sign of toxicity or change in behavior over 14 days following its administration.

- Sub-chronic Toxicity:

1- The extract did not induce any decrease in body weight, on the contrary, an apparent dose dependent increase in the body weight was observed in extract treated groups .
2-extract treated rats did not show any significant change in blood hematological parameters 4 week, 6 weeks and 8 weeks after extract exposure .
3-extract treatments did not induce any liver or kidney pathological, functional or metabolic, changes as demonstrated by serum biochemical analysis 4 week, 6 weeks and 8 weeks after extract exposure .
4- During 60 days observations period, no gross abnormalities attributable to drug toxicity were observed in the extract treated groups.
5- Postmortem macroscopic examination reveals no changes in body organs (kidney, spleen, liver & heart).

Table 3*: Blood hematological and Biochemical parameters of rats receiving orally extract with different concentrations daily after 8 weeks.

Test	Units	Control	1:10	1:20	1:30	1:40	1:50
WBC**	Cumm	15200	16300	15100	13400	12600	13900
RBC**	10¹²/l	8.75	8.25	7.55	8.95	8.86	8.46
Hemoglobin	g/dl	17.1	16.6	16.75	16.2	15.75	16.7
Platelets	Cumm	520000	410000	390000	415000	510000	495000
Blood Glucose	mg/dl	129	137	125	121	115	112
Total protein	g/dl	9.2	8.1	7.89	8.17	8.96	9.35
Amylase	IU/l	1197	1233	1267	1210	1121	1243
Uric Acid	mg/dl	2055	2.13	1.85	2.45	2.12	3.35
SGPT**	IU/l	58	62	67	78	76	83
SGOT**	IU/l	94	86	83	78	89	73
Triglyceride	mg/dl	83	92	84	98	102	97
Cholesterol	mg/dl	136	128	136	143	127	153
ALP**	IU/l	227	215	233	218	239	235
Gamma GT	IU/l	3.50	3.89	3.35	2.56	3.11	3.15

* Mean values for 7 rats
** WBC: White Blood Cells, RWC: Red Blood Cell , SGPT: Serum Glutamate Pyruvate Transaminase,
SGOT: Serum Glutamate Oxaloacetate Transaminase, ALP: Alkaline Phosphates.

The study confirmed that the tested preparation designated, has no detectable toxic effects even on repeated high dosage administration in rats.

Faculty Of Pharmacy:

Dr.Mohammad Salahat                          Dr.Awni Shanableh                          Dr.Hatem Amin Hejaz
Ph.D physiology                                      M.D.                                                     Ph.D Drug Design &
                                                                                                                          Medical chemistry