The potential of sanrego (Lunasia amara) in

enhancing fertility and anti-hyperglycemic

effect in diabetic induced male rats
Cite as: AIP Conference Proceedings 1614, 579 (2014); https://doi.org/10.1063/1.4895266
Published Online: 17 February 2015

Nor Raidah R., Mahanem M. N. and Mohd Shazrul Fazry S.

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 The potential of Sanrego (Lunasia amara) in Enhancing
Fertility and Anti-hyperglycemic Effect in Diabetic Induced
Male Rats

Nor Raidah R., Mahanem M. N. and Mohd Shazrul Fazry S.

School of Biosciences and Biotechnology, Faculty of Science and Technology,

Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia.

Abstract. Study on the effects of Lunasia amara (LA) aqueous extract on male fertility and its anti-hyperglycemic
activity was carried out. Twelve adult male Sprague-Dawley rats were divided into two groups for fertility test; control
given orally distilled water (n=6) and treatment (n=6) given 60 mg/kg aqueous extract of LA for 42 days. On day 43, all
rats were sacrificed and cauda epididymis was isolated for sperm quality analysis that includes parameter of sperm count,
motility and viability. Anti-hyperglycemic study was done on five groups of male rats; I-normal control, II-Diabetic
control and three other groups induced diabetic given 500 mg/kg metformin, 60 mg/kg LA and 120 mg/kg LA
respectively. Diabetes was induced in the male rats by intravenous injection of 55 mg/kg streptozotocin. On day 7, the
fasting blood glucose level was measured from blood drawn by tail snip. Results showed that aqueous extract of LA
increased significantly (p < 0.05) sperm count (39.88 ± 2.33) x 106, viability 82.46 ± 1.91 % and progressive motility
76.00 ± 1.51and of sperm data in treated group compared to control group. LA aqueous extract at dose 120 mg/kg was
significantly reduced the fasting blood glucose in the diabetic rats by 49.53 %. In conclusion, the aqueous extract of LA
effective in increasing sperm quality of male rats and suggest that LA may possess anti-hyperglycemic property.
Keywords: sanrego, Lunasia amara, sperm quality, anti-hyperglycemic.
PACS: 87.19.X, 87.85.M

INTRODUCTION
Medicinal plants or herbs serve as therapeutic alternatives and safer choices of treatment available. A larger
number of these plants and their extract have shown beneficial therapeutic effects, including anti-oxidant, anti-
inflammatory, anti-cancer, anti-microbial, and immunomodulatory effects. Interestingly, some plants such as L.
amara may have beneficial effects on enhancing fertility [1] and anti-hyperglycemic [2].
Lunasia amara (LA) or commonly known as “sanrego”, belongs to the family of Rutaceae. Traditionally, LA is
used to treat sexual dysfunction and male fertility [3-4]. Its stem is used as traditional medicine for diabetic patients.
del Rosario [2] in her study on the anti-hyperglycemic activity of LA Blanco found that ethyl acetate extract of LA
has good potential in reducing the hyperglycaemia blood glucose compared to the hexane and chloroform extract. It
reduced about 38.7% of hyperglycaemic blood glucose level in non-diabetic rats.
About 90% of male diabetic patients experienced sexual dysfunction, impotence and infertility caused by
testicular failure associated with prolonged hyperglycemia [5]. Previous reports had demonstrated the effectiveness
of LA as a natural anti-hyperglycemic agent in non-diabetic rat but little is known of its potential on diabetic
subjects. The aim of this study is to discover the potential of LA in increasing male fertility and its effects on the
fasting blood glucose level of streptozotocin (STZ) induced male diabetic rats.

MATERIALS AND METHODS

Preparation of LA Stem Aqueous Extract

About 200 g of dried LA stem bark was ground to a fine powder and boiled in 1000 mL of distilled water for 60
minutes. The extract was filtered through Whatman paper no. 3 before freeze-dried.

The 2014 UKM FST Postgraduate Colloquium

AIP Conf. Proc. 1614, 579-582 (2014); doi: 10.1063/1.4895266
© 2014 AIP Publishing LLC 978-0-7354-1250-7/$30.00

579
 Fertility Test
A total of ten male Sprague-Dawley rats aged eight weeks are divided into two groups. Group 1 (n=5) is the
control group that was given 1 mL distilled water orally. Group 2 (n=5) are rats administered orally with 60
mg/kg/day of LA for 42 days. Rats were then sacrificed on day 43 and cauda epididymis was isolated for sperm
analysis including sperm count, motility and viability. The cauda epididymis were minced and suspended in 15 mL
of Biggers-Whitten-Whittingham (BWW) medium [6] prior to incubation in 5% CO2 incubator for 30 min at 37˚C to
allow sperm to swim up. Sperm count was assessed using ‘Improved Neubauer Haemocytometer’ while sperm
viability and motility was determined as per WHO laboratory manual [7].

Anti-hyperglycemic Study
Diabetes in rats was induced by a single dose of streptozotocin (STZ) (55 mg/kg/day, i.v.) dissolved in citrate
buffer (0.1 M, pH 4.5) after 16 h fasting. Diabetes was confirmed in 12 h-fasted rats by measuring blood glucose
level from tail snip after five days with glucometer AccuCheck® Performa (Roche Diagnostics GmbH, German).
Only those rats with blood glucose level above 13 mmol/L were included in the study. A total of 25 male rats are
divided into five groups (n=5). Group 1 is non-diabetic induced and four other groups of STZ-induced diabetic were
force fed with distilled water (control diabetic), metformin (500 mg/kg/day), 60 mg/kg/day LA and 120 mg/kg/day
LA respectively. The fasting blood glucose levels of the rats were measured at day 7 of treatment.

Statistical Analysis
Statistical Package for the Sosial Sciences (SPSS) version 18 was used for statistical analysis. The data are
presented as mean ± standard error of means (SEM) and statistical significance was tested by One-Sample t test. A
value of p < 0.05 was considered statistically significant.

RESULTS AND DISCUSSION

Fertility Assessment
The effect of LA aqueous extract on male fertility was demonstrated by sperm quality analysis. TABLE (1)
showed the effects of L. amara aqueous extract on sperm count, viability and motility. The sperm counts and sperm
viability of treated group LA aqueous extract showed significantly (p < 0.05) increased with (39.88 ± 2.33) ×106 and
82.46 ± 1.91 % respectively compared to control group. The percentage of sperm motility showed treated group has
significantly (p < 0.05) increase in the percentage of progressive sperm motility (76.00 ± 1.51) % compared to the
control group which is (69.15 ± 3.49) %. The administration of LA aqueous extract at a dose 60 mg/kg/day on male
rats significantly (p < 0.05) increase the sperm quality including sperm count, motility and viability compared to
control.
The result showed that LA aqueous extract has potential to increase the male rats’ fertility regarding to its sperm
quality. We believe that these increases of sperm quality were due to the present of steroid in LA. Pharmacognosy
and phytochemistry study revealed that LA contains steroids. Administration of steroid plant such as LA stimulates
the reproductive organs related to sexual behavior [8]. Studied done by Muhamad Jafar and Mahanem [1] also
showed that dose 60 mg/kg/day aqueous extract of LA were the most effective dose to increase male rat libido and
fertility.

TABLE (1). Effects of LA aqueous extract on the sperm count, viability and motility grade compared to
control (* significant difference p < 0.05).
Sperm quality parameters Group
Control L. amara
Sperm count (×106) 29.93 ± 1.62 39.88 ± 2.33*
Viability (%) 77.77 ± 0.72 82.46 ± 1.91*
Motility (%)

580
 - PR 69.15 ± 3.49 76.00 ± 1.51 *
- NP 2.85 ± 0.64 3.08 ± 0.43
- IM 29.60 ± 2.65 23.88 ± 1.80
Note for the grade of sperm motility according to WHO 2010:
PR – Progressive motility IM – Immotility
NP – Non progressive motility

Anti-hyperglycemic Effect
The effect of LA aqueous extract on STZ-induced diabetic male rat was demonstrated by fasting blood glucose
level after seven day of treatment. FIGURE 1 showed that LA aqueous extract at dose 120 mg/kg significantly (p <
0.05) reduce the fasting blood glucose level by 49.53 % while dose 60 mg/kg/kg of LA aqueous extract maintain the
fasting blood glucose on diabetic rats. Declining pattern at dose 120 mg/kg LA was similar to the metformin
treatment which is an established anti-diabetic drug that serves as a positive control in this study.
Dose 120 mg/kg LA aqueous extract has succeeded in reducing the fasting blood glucose level compared to
diabetic control group. Dose treatment at 120 mg/kg/day is suggested may have anti-hyperglycemic effect. Hormone
glucagon is released by body during fasting to release glucose into the blood stream. The rise of glucose levels in the
blood stream will stimulate insulin production to stimulate uptake the glucose by the cell. However in diabetes, this
process does not occur and caused glucose level normally remain high [9]. LA is suggested have involved in this
reducing blood glucose level mechanism.

FIGURE 1. Fasting blood glucose level of rats from a) normal, b) diabetes control, c) diabetes treated with 55 mg/kg
metformin, d) diabetes treated with 60 mg/kg LA and e) diabetes treated with 120 mg/kg LA. (* significant
difference p < 0.05 compared to diabetic control).

CONCLUSION
LA has demonstrated to enhance male fertility and showed anti-hyperglycemic potential in this study. LA is
suggested as natural product to treat diabetic patients who suffer from infertility. However, further study is required
for the effects of LA aqueous extract on fertility of male diabetic rats.

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 ACKNOWLEDGMENTS
The authors wish to thank Universiti Kebangsaan Malaysia for funding the project through Research Grant
University, No. UKM-GUP-2011-335. Facilities provided by the Animal House, Faculty of Science and
Technology, Universiti Kebangsaan Malaysia are gratefully acknowledged.

REFERENCES
1. L. Muhamad Jafar and M. N. Mahanem, Sains Malaysiana, 38 (5), 793-797 (2009).
2. M. F. del Rosario, “A Study on The Anti-diabetic Activity of Lunasia amara Blanco”, B.Sc. Thesis, University of Philippines
Diliman, 1999.
3. A. Muhtadi, Makalah Seminar Sehari Tumbuhan Afrodisiak Fakulti Farmasi UNTAG Jakarta (1999).
4. S. Nurbaeti, “Aktivitas Androgenic Fraksi Alkaloid Daun Maitan (Lunasia amara Blanco) pada anak ayam jantan”, B.Sc.
Thesis, University of Padjadjaran, 2000.
5. S. Amaral, P. J. Oliveira and J. R. Santos, Bentham Science Publishers Ltd, 4, 46-54 (2008).
6. J.D. Biggers, W.K. Whitten and D.G. Whittingham, in Methods in Mammalian Embryology, San Francisco: Jr. Freeman,
1971.
7. World Health Organisation, Laboratory Manual for the Examination and Processing of Human Semen,,Geneva: WHO Press,
2010.
8. Nurlaila, “Analisis Farmakognosi dan Fitokimia Simplisia Daun Maitan. (Lunasia amara Blanco)”, B.Sc. Thesis, University of
Padjadjaran, 2000.
9. G. Wilcox, Clin Biochem Rev. 26, 19-39 (2005).

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