REPORT

Study Title

EVALUATION OF EGMAX FOR FERTILITY ENHANCING ACTIVITY IN FEMALE

ALBINO WISTAR RATS

Sponsor

Natural Remedies Pvt. Ltd.

Bangalore, Karnataka
INDIA

Test Facility

Department of Pharmacology and Toxicology

Natural Remedies R&D Centre
Bangalore, Karnataka
INDIA.

Project Report No.: E08.16

 Project Report No.: E08.16R: Evaluation of Egmax for fertility enhancing activity in female
albino Wistar rats

General Statements

Study Title : Evaluation of Egmax for fertility enhancing activity in

female albino Wistar rats.

Sponsor : Natural Remedies Pvt Ltd.

Bangalore, Karnataka
INDIA

Test Facility : Department of Pharmacology and Toxicology

Natural Remedies R&D Centre
Bangalore, Karnataka
INDIA

Responsibilities

Study Director : Dr. Chandrasekaran. CV

Test Facility Management : Dr. Chandrasekaran. CV

Study Personnel : Mr. Sasi Kumar. M

Mr. Pasanna Raja C

Page 2 of 11 pages
 Project Report No.: E08.16R: Evaluation of Egmax for fertility enhancing activity in female
albino Wistar rats

Contents Page

Study Title 1
General Statements 2
1 Study Objective 4
2 Test Item and Vehicle 4
2.1 Test Item 4
2.2 Vehicle 4
2.3 Materials (Drugs and chemicals) 4
2.4 Preparation of Test Item Formulation 4
3 Reasons for Choice of Species and Route of Administration 4
4 Animals and Husbandry 5
4.1 Animal Specification 5
4.2 Randomization 5
4.3 Animal Identification 5
4.4 Husbandry 5
4.5 Food and Feeding 5
5 Experimental Design and Procedures 6
5.1 Administration of the Test Item and Vehicle 6
5.2 General Procedure 6
6 Observations 6
6.1 Body Weight 6
6.2 Serum FSH and LH 7
6.3 Histopathological Analysis 7
7 Statistical Analysis 7
8 Results 7
9 Conclusion 10
10 Guidelines and References 10
11 Signature sheet 11

Page 3 of 11 pages
 Project Report No.: E08.16R: Evaluation of Egmax for fertility enhancing activity in female
albino Wistar rats

1 Study Objective

To evaluate the fertility enhancing effect of Egmax in female albino Wistar rats.

2 Test Item and Vehicle

2.1 Test Item:

Product code : NR-FE-05 (Batch. No: Z005)

Product name : Egmax powder

Manufactured By : Natural Remedies Pvt Ltd.

Manufactured date : November 2016

Storage conditions : Room Temperature (Desiccator)

2.2 Vehicle

0.5 % CMC was used as vehicle to dissolve test substances.

2.3 Materials (Drugs and chemicals)

Clomiphene citrate was used as reference substance at the dose level of 50 mg/kg rat body
weight suspended in vehicle.

Serum Follicle Stimulating Hormone (FSH) and Luteinizing hormone (LH) assay kits were
procured from Cloud clone corporation, Houston, USA.

2.4 Preparation of Test/Reference Item

Egmax and clomiphene citrate were weighed accurately to obtain 100 mg and 50 mg
respectively using analytical balance. The weighed amounts of each were suspended in 10 ml
(Dose volume considered for one kg rat body weight) of 0.5 % CMC to obtain a concentration of
100 mg/kg and 50 mg/kg rat body weight. The dosing solutions were prepared freshly, prior to
dose administration.

3 Reasons for Choice of Species and Route of Administration

The rat was selected because it has been historically proved to be a suitable test system for
similar studies. The results of the study may be useful to detect the fertility enhancing activity of
the test item in rats. Oral route for the test substance has been selected, as it is the proposed route
of exposure in target species.

Page 4 of 11 pages
 Project Report No.: E08.16R: Evaluation of Egmax for fertility enhancing activity in female
albino Wistar rats

4 Animals and Husbandry

4.1 Animal Specification

Species : Rattus norvegicus (Rat)

Strain : Wistar
Sex : Male, healthy adult animals, individual body weights were within
20% of the mean body weight (at the time of dosing).

Number of animals : 18 animals

Supplier : In- house bred at Department of Pharmacology and Toxicology

Age : 8-10 weeks of age at the time of dosing

Acclimatization : Animals were acclimatized to the test conditions for at least

5 Days prior to treatment

4.2 Randomization

Randomization was done based on body weight. Only animals with individual body weights
within 20 % of the group mean were considered.

4.3 Animal Identification

Animals were identified by diluted picric acid solution and cage cards.

4.4 Husbandry

Housing

Rats were housed in group of 3 during the experiment.

Environmental conditions

The room temperature was maintained at 22 3C and the relative humidity was kept between
30 and 70 %. Artificial light was set to provide 12 hours light and 12 hours dark.

4.5 Food and Feeding

UV treated water and a conventional laboratory rodent diet supplied by approved vendor was
offered ad libitum.

Page 5 of 11 pages
 Project Report No.: E08.16R: Evaluation of Egmax for fertility enhancing activity in female
albino Wistar rats

5 Experimental Design and Procedures

Group Treatment Dose

G1 Vehicle control (0.5% CMC) 10 ml/kg

G2 Egmax 100 mg/kg

G3 Clomiphene citrate 50 mg/kg

5.1 Administration of the Test Item, Reference item and Vehicle

The animals were dosed orally by gavage with the help of graduated syringes. The maximum
volume of liquid did not exceed 10 ml/kg body weight rounded up to single decimal point as per
recently recorded body weight for individual animals.

5.2 General Procedure

Female albino Wistar rats were divided into 3 groups with 6 animals in each group, randomized
based on body weight. Animals were treated with respective treatment regimen as mentioned in
the study design after estrous synchronization via pheromone effect. G1 was administered CMC,
G2 was administered Egmax and G3 administered with clomiphene citrate for two weeks.
Animals were bleeded during the fourth proestrous phase (analyzed via vaginal smear) from the
retro orbital plexus and serum was analyzed for FSH and LH levels. The animals were sacrificed
during the fourth estrous phase (analyzed via vaginal smear) and ovaries were collected for
histopathological analysis.

6 Observations

6.1 Body Weight

Body weights were recorded for all animals at the start of dosing (data not provided) and at the
end of two weeks of treatment.

Page 6 of 11 pages
 Project Report No.: E08.16R: Evaluation of Egmax for fertility enhancing activity in female
albino Wistar rats

6.2 Serum FSH and LH

Serum FSH and LH levels were determined at the end of two weeks of treatment.

6.3 Histopathological analysis

Ovary sections were made and stained with H&E. The number of follicles and corpus luteum
were counted.

7 Statistical Analysis

Raw data was processed using statistical software SPSS and Graphpad prism. Dunnett test was
used as post hoc test after homogeneous analysis. If the test for homogeneity is not met, then
Dunnett T3 test was applied as post hoc test. The mean and standard error of mean are
summarized in tabular form. All data were subjected to statistical analysis.

8 Results

8.1 Serum FSH and LH

Treatment with Egmax has significantly increased the serum FSH and LH levels compared
to control (Table 1), whereas treatment with clomiphene citrate significantly increased the LH
level compared to control.
8.2 Histopathological analysis
Histopathological analysis revealed that, treatment with Egmax and clomiphene citrate
has shown non-significant increase in ovarian cell count as compared to control (Table 2).

Table 1: Effect of different test substances on FSH and LH

Group FSH (ng/ml) LH (pg/ml)

Normal control (0.5% CMC; 10 ml/kg) 10.5 1.92 376 27.7

Egmax (100 mg/kg b.w) 18.8 1.31* 670 88.7*

Clomiphene citrate (50 mg/kg b.w) 18.5 1.66* 615 78.2

Values are expressed as Mean SEM; n = 6

*p < 0.05, Treatment group Vs Control group

Page 7 of 11 pages
 Project Report No.: E08.16R: Evaluation of Egmax for fertility enhancing activity in female
albino Wistar rats

Table2: Effect of different test substances on ovarian cell count

Group Follicle Count Corpus Luteum

Normal control (0.5% CMC; 10

2.50 1.02 11.33 1.82
ml/kg)

Egmax (100 mg/kg b.w) 3.33 0.67 20.00 1.84

Clomiphene citrate (50 mg/kg b.w) 1.83 0.40 16.50 3.89

Values are expressed as Mean SEM; n = 3 (left & right ovary)

*p < 0.05, Treatment group Vs Control group

Histopathological images

Control:

Control: Ovary - various stages of corpus luteum (CL) were visible, (H&E stain-4X)

Page 8 of 11 pages
 Project Report No.: E08.16R: Evaluation of Egmax for fertility enhancing activity in female
albino Wistar rats

EGMAX

Egmax Ovary: NAD various stages of different follicles (GF, AF, CL) were evident, medulla
vascularised (MV) with dilated blood vessels (BV) were visible, (H&E stain-4X).

Clomiphene citrate

Clomiphene citrate (+ve Control): Ovary- NAD - Graafian follicle (GF) and CL were visible,
medulla vascularised (MV) with dilated blood vessels (BV) were evident, (H&E stain-4X)

Page 9 of 11 pages
 Project Report No.: E08.16R: Evaluation of Egmax for fertility enhancing activity in female
albino Wistar rats

9 Conclusion

The present study demonstrates that, treatment with Egmax enhances the serum FSH and LH
levels in female albino Wistar rats during pro-oestrus phase which supports the fertility
enhancing activity of Egmax.

10 Guidelines and References

CPCSEA guidelines

Guide for the care and use of laboratory animals, 8th edition, 2011

Uchiyama F, Jikyo T, Takeda R, Ogata M. Lepidium meyenii (Maca) enhances the serum
levels of luteinising hormone in female rats. J Ethnopharmacol. 2014 Feb 3;151(2):897-
902.

Page 10 of 11 pages
 Project Report No.: E08.16R: Evaluation of Egmax for fertility enhancing activity in female
albino Wistar rats

STUDY PERSONNEL

Mr. Sasi Kumar. M : 27.03.2017

Study Personnel Date

Dr. Prasanna Raja : 27.03.2017

Study Personnel Date

Dr. Chandrasekaran CV : 27.03.2017

Study Director Date

Dr. Chandrasekaran CV : 27.03.2017

Test Facility Management Date

Page 11 of 11 pages