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THE COMBINATION INFUSION OF Talinum paniculatum (Jacq.) Gaertn., Centella asiatica (L.) Urb. AND Curcuma xanthorrhiza Roxb. EFFECTS ON TESTOSTERONE LEVELS AND INTRODUCTION, CLIMBING AND COITUS FREQUENCIES IN MALE SPRAGUE-DAWLEY RATS

Authors:
  • Research Center for Pharmaceutical Ingredients and Traditional Medicine BRIN
  • Medicinal Plant and Traditional Medicin Research and Development Center

Abstract and Figures

Talinum paniculatum (som Java) reported to have many pharmacological activities such as increasing spermatozoa quality, cell regenerations, fertility, and antibacterial effect both empirically and scientifically. This study aimed to prove the combination infusion effects of som Java, gotu kola, and temulawak on introduction, climbing, and coitus levels as well as testosterone levels in male Sprague Dawley (SD) rats. SD rats were divided into 2 groups, control group received aquadest, treatment group received formula a mixture of som java Talinum paniculatum) root, gotu kola (Centella asiatica) herb, and Javanese turmeric (Curcuma xanthorrhiza) rhizome with the dose of 491,2 mg/200 g BW for 7 consecutive days. Artificial estrus of female SD rats were induced by administering estradiol valerat 2 mg/200 g BW 48 hours before observation. The determined parameters were levels of testosterone as well as introduction, climbing and coitus frequencies. Administration of estradiol valerat led to artificial estrus induction of female SD rats. The administration of the combination infusion dose 491.2 mg/200 g BW significantly increased testosterone level as well as introduction and climbing frequencies of male SD rats as of 27.77%; 86.39% and 69.17% respectively. ABSTRAK Som jawa (Talinum paniculatum) secara empiris dan ilmiah terbukti telah banyak dimanfaatkan oleh masyarakat untuk mengatasi berbagai masalah kesehatan diantaranya untuk meningkatkan kualitas dan kuantitas spermatozoa, regenerasi sel, fertilitas, dan sebagai antibakteri. Penelitian ini dilakukan untuk membuktikan efek peningkatan kadar hormon testosteron serta frekuensi introduction, climbing, dan coitus tikus jantan SD dari pengaruh infusa kombinasi som jawa, pegagan, dan temulawak. Hewan uji tikus galur SD dibagi menjadi 2 kelompok, yaitu kelompok kontrol diberikan akuades, sedangkan kelompok perlakuan diberikan infusa ramuan akar som jawa, herba pegagan, dan rimpang temulawak dosis 491,2 mg/200 g bb selama 7 hari berturut-turut. Induksi estrus artifisial tikus betina dengan pemberian estradiol valerat 2 mg/200 g bb dilakukan 48 jam sebelum pengamatan. Parameter yang diukur meliputi kadar testosterone, frekuensi introduction, climbing, dan coitus. Dibandingkan dengan kelompok kontrol, pemberian infusa ramuan tidak berefek pada frekunsi coitus, namun mampu meningkatkan kadar testosteron serta frekuensi introduction dan climbing tikus jantan sebesar 27,77; 86,39 dan 69,17%.
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Jurnal Tumbuhan Obat Indonesia ISSN: 1979-892X (print)
Vol. 12, No 1, Agustus 2019, hal 33-40 ISSN: 2354-8797 (online)
33
THE COMBINATION INFUSION OF Talinum paniculatum (Jacq.) Gaertn.,
Centella asiatica (L.) Urb. AND Curcuma xanthorrhiza Roxb. EFFECTS
ON TESTOSTERONE LEVELS AND INTRODUCTION, CLIMBING AND
COITUS FREQUENCIES IN MALE SPRAGUE-DAWLEY RATS
Efek Infusa Kombinasi Talinum paniculatum (Jacq.) Gaertn., Centella
asiatica(L.) Urb. dan Curcuma xanthorrhiza Roxb. terhadap Kadar
Testosteron dan Frekuensi Introduction, Climbing dan Coitus Tikus SD
Nuning Rahmawati 1*), Ika Yanti Marfuatush Sholikhah 2)
1 Medicinal Plant and Traditional Medicine Research and Development Centre
Jl Raya Lawu 11, Karanganyar, Central Java, Indonesia
*e-mail: nunrahmawati@gmail.com
ABSTRACT
Talinum paniculatum (som Java) reported to have many pharmacological activities such as increasing
spermatozoa quality, cell regenerations, fertility, and antibacterial effect both empirically and
scientifically. This study aimed to prove the combination infusion effects of som Java, gotu kola, and
temulawak on introduction, climbing, and coitus levels as well as testosterone levels in male Sprague
Dawley (SD) rats. SD rats were divided into 2 groups, control group received aquadest, treatment group
received formula a mixture of som java Talinum paniculatum) root, gotu kola (Centella asiatica) herb,
and Javanese turmeric (Curcuma xanthorrhiza) rhizome with the dose of 491,2 mg/200 g BW for 7
consecutive days. Artificial estrus of female SD rats were induced by administering estradiol valerat 2
mg/200 g BW 48 hours before observation. The determined parameters were levels of testosterone as
well as introduction, climbing and coitus frequencies. Administration of estradiol valerat led to artificial
estrus induction of female SD rats. The administration of the combination infusion dose 491.2 mg/200 g
BW significantly increased testosterone level as well as introduction and climbing frequencies of male
SD rats as of 27.77%; 86.39% and 69.17% respectively.
Keywords: Talinum paniculatum, testosteron, aphrodisiac, climbing, coitus
ABSTRAK
Som jawa (Talinum paniculatum) secara empiris dan ilmiah terbukti telah banyak dimanfaatkan oleh
masyarakat untuk mengatasi berbagai masalah kesehatan diantaranya untuk meningkatkan kualitas
dan kuantitas spermatozoa, regenerasi sel, fertilitas, dan sebagai antibakteri. Penelitian ini dilakukan
untuk membuktikan efek peningkatan kadar hormon testosteron serta frekuensi introduction,
climbing, dan coitus tikus jantan SD dari pengaruh infusa kombinasi som jawa, pegagan, dan
temulawak. Hewan uji tikus galur SD dibagi menjadi 2 kelompok, yaitu kelompok kontrol diberikan
akuades, sedangkan kelompok perlakuan diberikan infusa ramuan akar som jawa, herba pegagan,
dan rimpang temulawak dosis 491,2 mg/200 g bb selama 7 hari berturut-turut. Induksi estrus
artifisial tikus betina dengan pemberian estradiol valerat 2 mg/200 g bb dilakukan 48 jam sebelum
pengamatan. Parameter yang diukur meliputi kadar testosterone, frekuensi introduction, climbing,
dan coitus. Dibandingkan dengan kelompok kontrol, pemberian infusa ramuan tidak berefek pada
frekunsi coitus, namun mampu meningkatkan kadar testosteron serta frekuensi introduction dan
climbing tikus jantan sebesar 27,77; 86,39 dan 69,17%.
Kata kunci: Talinum paniculatum, testosteron, introduction, climbing, coitus
Jurnal Tumbuhan Obat Indonesia ISSN: 1979-892X (print)
Vol. 12, No 1, Agustus 2019, hal 33-40 ISSN: 2354-8797 (online)
34
INTRODUCTION
Aphrodisiac is an agent that contributes to an arouses sexual desire (Singh et al., 2012).
Nowadays, the prevalence of sexual dysfunctions was quite high and tended to increase with
age, especially in men (Nicolosi et al., 2004). Sexual dysfunction can be influenced by various
factors such as psychological disorders, neurological disorders, chronic disorders, systemic
diseases, and life styles (Singh et al., 2013). Tan et al. (2011) reported the correlation between
erectile dysfunctions and metabolic syndrome (Tan et al., 2011). Diabetes mellitus as one of
metabolic disorder also often associated with sexual dysfunction, fertility, and impotence
(Kamaruzaman & Noor, 2017). There are many kinds of sexual dysfunction. The most frequent
sexual dysfunctions for women are desire and arousal dysfunctions, while premature ejaculation
and erectile dysfunction are the most common sexual dysfunctions for men (Mccabe et al.,
2016). In human life, sexual relationships take a high priority in social and biological
relationship (Singh et al., 2013). It has been stated that aphrodisiac combined with healthy life
style will attain an improved sexual life (Erhabor & Idu, 2017).
Many natural substances have been empirically utilized for aphrodisiac. T. paniculatum
Gaertn is one of natural aphrodisiac widely used in Asia as reproductive tonic (Thanamool et al.,
2013). In Indonesia, T. paniculatum is well known as Som Java or Java Ginseng. Java Ginseng is
commonly used for many medicinal purposes. The daily oral administration of ethanol extract of
Java Ginseng roots in concentration of 1.4 mg/20 g bw for 9, 18, and 27 consecutive
experimental days could shorter mounting latency as well as increase mounting frequency
against Balb male mice induced by ethinylestradiol as of 0.56 ug/20 g bw/day (Winarni, 2007).
Previous research demonstrated that the root of T. paniculatum is the richest source of steroidal
saponins and potential to be used in many medicinal purposes. The multiple pharmacological
effects of T. paniculatum have been reported, include for vitality and maintain blood circulation,
testosterone levels as well as motility and number of sperm increasing whereas saponin was
determined as main chemical compound responsible to those effects (Solim & Manuhara, 2016).
Previous research revealed that the combination form of C. xanthorrhiza Roxb with Piper
retrofractum L showed aphrodisiac activity indicated by the increase of climbing frequency as
well as coitus frequency against male rats (Rahmawati & Bachri, 2012) . The safety data of those
formula compositions has proven in previous studies. Ethanol extract of C. xanthorrhiza Roxb
doses of 300, 2000 and 5000 mg/kg bw had no side effects as well as toxic effects and did not
cause the death of Swiss female mice (Devaraj et al., 2013). Standardized extract of C. asiatica
Urban leaves had been tested in Sprague-Dawley rats for acute oral toxicity, sub-chronic toxicity,
and mutagenic potential. The study found that the extract did not produce mortality or
significant changes in the clinical signs (Deshpande et al., 2015). Another acute toxicity studies
found that C. asiatica whole plant was safe to Swiss mice with a dose of 3, 5 and 7 g/kg body
weight orally (Pingale, 2008).
Jurnal Tumbuhan Obat Indonesia, 12(1): 33-40, Agustus 2019
Rahmawati, N, Sholikhah, I.Y.M: The Combination Infusion of............ 35
(a) (b)
Figure 1. Chemical structure of estradiol valerate (a) and testosterone hormone (b)
The combination formula of T. paniculatum, C. asiatica and C. xanthorrhiza were expected
to provide a better synergistic effect rather than in single formula administration. Many
researches related to T. paniculatum pharmacological activity in single composition has been
implemented, however the efficacy data in the combination form is not available yet. Therefore,
this research needs to be conducted to prove the effect of combination infusion of T. paniculatum
root, C. asiatica herb and C. xanthorrhiza rhizome against male Sprague Dawley (SD) rats’ libido.
METHODS
Preparation of Infusion of Simplicias
Simplicias of T. paniculatum, C. asiatica and C. xanthorrhiza were provided by Postharvest
Laboratory of Medicinal Plant and Traditional Medicine Research and Development Center,
Ministry of Health. Determination of main component dose of T. paniculatum root was based on
previous preclinical trials. The additional components doses, C. xanthorrhiza rhizome and C.
asiatica herb were based on empirical use, subsequently converted to rat dose (multiplied by an
extrapolation factor of 0.0182).
Table 1. Dosage of each formula composition
Composition
Dosage (mg/200 g bw)
Total dosage (mg)
Som Java
Gotu kola
Javanese turmeric
200
109,2
182
491,2
Based on table 1 and previous preclinical research, it is obtained a comparison of Som
Java, gotu kola and Javanese turmeric as of 10:5:9 with the total dose of 491.2 mg/200 g bb. The
ratio of the herbs weight and distilled water was 10% w/v with the calculation as of:
Table 2. Herbs weight for 50 ml infusion stock for SD rats
Som Java
Gotu kola
Javanese turmeric
200 mg x 50 ml =5000 mg
109,2 mg x 50 ml = 2730 mg
182 mg x 50 ml = 4550 mg
2 ml
2 ml
2 ml
Each ingredient was weighed according to calculations and homogenised with aquadest up to 50
ml, the mixture then be added aquadest with volume as of two times the herbs weight. An
infusion was made by heating the mixture for 15 minutes started after the temperature reached
90°C, the infusion was filtered into a glass beaker and given 2 ml/200 g bw orally to a rat.
Jurnal Tumbuhan Obat Indonesia, 12(1): 33-40, Agustus 2019
36 Rahmawati, N, Sholikhah, I.Y.M: The Combination Infusion of.........
Experimental Animals
As of twenty four Sprague Dawley 2-3 month-old, male and female rats were used in this
experimental study. Rats were obtained from the Experimental Animal Care Unit of Universitas
Gadjah Mada, Indonesia. Rats were divided into control and treatment groups, each group
consisted of 4 rats (1 male and 3 female) with 3 replication of each group (in total 6 groups). The
number of experimental rats was determined based on Ferderer Formula, (t-1) (n-1)>15, in
which “t” is groups number and “n” is the minimal number of animal used in each group (in this
study, n was 4 for six groups of it has fulfilled Ferderer Formula).
Animal Treatments
Before the study began, experimental rats were acclimatized for 7 days, then housed in the
experimental pharmacology laboratory of Medicinal Plant and Traditional Medicine Research
and Development Center (MPTMRDC). The rats were given ad libitum drink and fed with
standard pellets. Artificial estrus of female rats were induced by per oral administration of
estradiol valerate 2 mg/200 g bw 48 hours before conducting libido parameters observation.
The combination infusion of T. paniculatum root, C. asiatica herb, and C. xanthorrhiza rhizome
were given to male rats for 7 consecutive days. Rats body weight were recorded on day-0 and
day-7. Determination of testosterone levels was conducted before and after treatment using
testosterone ELISA kit whereas blood was taken via retro orbital plexus venous of rats. On the
7th day at 18.30 pm for the duration of 180 minutes, the experimental observation was
conducting on some parameters of aphrodisiac activities. The first parameter was the onset of
introduction phase, climbing as well as coitus phase. The 2nd parameter were the frequencies of
introduction, climbing and coitus of the control and treatment groups in separately individual
cages in which one male rat was caged with three female rats in estrus phase within lightless
condition. Male rats started introduction phase when they began to get closer around female
rats, kissing the female rats body, while climbing phase defined as when male rats climbed up
above the female rats body and coitus phase when sexual intercourse happened, the genitalia of
the male rat entered the female rats genitalia. The body weight of experimental rats was also
monitored in this current study before and after treatment.
Data Analysis
All data collected from control and treatment experimental groups were analyzed using
paired and unpaired t-test of SPSS.
Ethical Approval
All research involving experimental animal as subject need ethical approval. This study
had an ethical approval on 2011 from Ethics Committee of National Institute of Health Research
and Development.
RESULTS AND DISCUSSION
The mixture of T. paniculatum, C. asiatica and C. xanthorrhiza shown to have aphrodisiac
effect on Sprague-Dawley (SD) white rats (Rattus norvegicus) as observed animal in this
experimental pharmacological study. Induction of artificial estrus of female rats was performed
by administering estradiol valerate 2 mg/200 g bw 48 hours before observation. Estradiol
valerate known as natural estrogen going to replace the use of ethinyl estradiol in order to
minimize the risk for thromboembolism (Ahrendt et al., 2010). Within a few minutes, estradiol
valerate could be quickly absorbed, hydrolyzed and converted to estradiol, whereby exerting the
same estrogenic effects. One milligram of estradiol valerate is equivalent to 0.76 mg of ethinyl
estradiol. Two mg of estradiol valerate can have the same biological effect of 4 to 20 μg ethinyl
estradiol. A research reported the use of estradiol valerate might contribute in reducing the risk
Jurnal Tumbuhan Obat Indonesia, 12(1): 33-40, Agustus 2019
Rahmawati, N, Sholikhah, I.Y.M: The Combination Infusion of............ 37
for thrombosis. In different kind of concentrations, estradiol valerate showed inhibitory effect
on the follicle-stimulating hormone and the stimulation of the endometrium cell as well as
maturation of vaginal epithelial cells and protein synthesis in the liver. The research results
showed the administration of estradiol valerate 2 mg/200 g bw 48 hours before observation
induced artificial estrus on most of female rats (Ahrendt et al, 2010). Other research reported
that estrogen plays an important role in the female animal estrus phase in term of physiological,
behavioral, and ovulation manifestations as well as luteinizing hormone release and increase
blood circulation in the genital organs (Hafez & Hafez, 2000).
The administration of combination infusion of T. paniculatum, C. asiatica, and C.
xanthorrhiza at concentration of 491.2 mg/200 g bw for seven consecutive days led to increase
the percentage of introduction and climbing phase frequency of treatment male rats group
compared to the control group as of 86.39 and 69.17 % respectively. However there was a
difference starting time between introduction phase of treatment and control groups as of
30.30% as shown in table 2. The secondary metabolites contained in T. paniculatum determined
to be responsible for its estrogenic activity as reported in previous study (Thanamool et al.,
2013). Testosterone level of male rats was another parameter for determining aphrodisiac
activity of the formula. From previous studies, it is known that administration of medicinal plant
to experimental animals generally begins to show the aphrodisiac effect on the 7th day, so that in
this study the observations of the aphrodisiac parameters were made on the 7th day (Sugiarto &
Setiantiningrum, 2010). Traditional medicines have incomparable advantages and
disadvantages when used to develop new drugs, such as onset of any pharmacological effect,
abundant clinical experiences, and their unique diversity of chemical structures and biological
(Yuan et al., 2016).
Group
Replication
t intro
(minutes)
t coitus
(minutes)
Frequency
Introduction
Climbing
Coitus
Treatment
1
2
-
16
11
0
2
4
-
12
16
0
3
4
-
16
9
0
Average
3.3
14,7
12
0
Control
1
1
-
2
3
0
2
2
-
2
4
0
3
4
-
2
4
0
Average
2.3
-
2
3.7
0
Percentage (%)
30.30
86.39*
69.17*
0
This study revealed that the administration of T. paniculatum, C. asiatica and C.
xanthorrhiza effect on testosterone levels of male rats compared to control group. The provision
of the combination infusion for seven consecutive days shown positive effect on testosterone
hormone levels of male rats indicated by the presence of significantly increase of testosterone
levels before and after treatment compared to control group with p value < 0.05 as seen in table
4.
Table 4. Testosterone levels of male rats before and after treatment
Group
Replication
Testosterone level
P (95%)
Jurnal Tumbuhan Obat Indonesia, 12(1): 33-40, Agustus 2019
38 Rahmawati, N, Sholikhah, I.Y.M: The Combination Infusion of.........
Before treatment
After treatment
Treatment
1
1.046
1.415
0.012*
2
1.066
1.326
3
1.158
1.437
Control
1
0.896
0.945
0.460
2
0.516
0.486
3
0.605
0.661
*based on paired t-test with 95% of confident level (p value < 0.05, significantly different
Pair t-test statistical analysis showed a significant increase in testosterone levels in male-
treated rats before and after treatment (p<0.05), while there were no differences in testosterone
levels before and after treatment in male control rats (p>0.05). These results are in accordance
with the previous research in which single adduction of T. paniculatum could increase
testosterone level of male mice, shortening mounting latency and increase mounting frequency
(Winarni, 2007). Asiaticoside contained in C. asiatica as well as xanthorrhizol in C. xanthorrhiza
which contributed in rejuvenating body system expected to have synergistic effect in
combination with T. paniculatum on male rat libido (Bhavna & Jyoti, 2011). Stigmasterol and β
sitosterol contained in T. paniculatum considered to responsible on testosterone level increasing
by supplying cholesterol for pregnenolon forming of testosterone synthesis (Winarni, 2007).
Eseyin (2018) reported that stigmasterol’s chemical structure positively related to testosterone
synthesis and it was contributes to its aphrodisiac potentials. In male, testosterone levels do not
have a correlation in sexual activity but in female, testosterone levels are positively related to
sexual satisfaction and the frequency of sexual intercourse (Zitzmann & Nieschlag, 2001).
Table 4. Rats body weight before and after treatment
Groups
Rats body weight (g)
p
(95%)
Before treatment
After treatment
Treatment
187
191
0.321
190
210
162
162
Control
133
153
0.069
151
185
126
139
The rat body weights were measured on the day-0 and day-7. The results of the study
revealed that the administration of infusion combinations formula in experimental rats showed
no significant changes (p>0.05) in body weight between the treatment and control groups as
shown in table 4. This indicated that the combination of infusion for 7 consecutive days gave the
same effect on body weight changes both on treatment and control groups. Bias factor for the
use of laboratory animals became the limitation of this study in which introduction phase,
climbing and coitus frequencies are specified as parameters that can be influenced by several
factors, including stress and environmental factors.
Jurnal Tumbuhan Obat Indonesia, 12(1): 33-40, Agustus 2019
Rahmawati, N, Sholikhah, I.Y.M: The Combination Infusion of............ 39
CONCLUSION
In conclusion, the administration of combination infusion of T. paniculatum, C. asiatica and
C. xanthorrhiza at a dose of 491.2 mg/200 g bw for 7 days consecutively significantly increased
testosterone level as well as introduction and climbing frequencies of male SD rats as of 27.77%;
86.39% and 69.17% respectively.
ACKNOWLEDGEMENTS
Special gratitude for our research team for all contributions in aphrodisiac experimental
study. This research was funded by MPTMRDC, National Institute of Health Research and
Development, Ministry of Health, Republic of Indonesia.
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This paper presents a review of plants identified from various ethno botanical surveys and folklore medicinal survey with aphrodisiac activity. An aphrodisiac is defined as an agent that arouses sexual desire. Erectile dysfunction (ED) or male impotence is defined as the inability of a man to achieve and maintain an erection sufficient for mutually satisfactory intercourse with his partner. Sexual health and function are important determinants of quality of life. To overcome the problem of Male sexual (or) erectile dysfunction various natural aphrodisiac plants potentials are preferred. This review discuss about aphrodisiac potential of plants, its botanical name, Common name, family, part used and references, which are helpful for researcher to development new herbal aphrodisiac formulations.
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Aphrodisiac is the word derived from Aphrodite, the Greek goddess of sexual, love and beauty. An aphrodisiac is defined as an agent (food or drug) that arouses sexual desire. Current sexual dysfunction therapy lack satisfactory success due to adverse effect, hence patients are seeking complementary and alternative medicine to treat sexual dysfunction. Ayurveda and other Indian literature mention the use of plants in various human ailments. India has about more than 45 000 plant species and among them several thousand are claimed to possess medicinal properties. Researchers conducted in the last few decades on the plants mentioned in ancient literature or used traditionally for sexual dysfunction. This review reveals that some plants and their extract have aphrodisiac activity, which are helpful for researcher to develop new herbal aphrodisiac formulations. In the recent years, interest in drugs of plant origin has been progressively increased.
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Introduction The incidence and prevalence of various sexual dysfunctions in women and men are important to understand to designate priorities for epidemiologic and clinical research. Aim This manuscript was designed to conduct a review of the literature to determine the incidence and prevalence of sexual dysfunction in women and men. Methods Members of Committee 1 of the Fourth International Consultation on Sexual Medicine (2015) searched and reviewed epidemiologic literature on the incidence and prevalence of sexual dysfunctions. Key older studies and most studies published after 2009 were included in the text of this article. Main Outcome Measures The outcome measures were the reports in the various studies of the incidence and prevalence of sexual dysfunction among women and men. Results There are more studies on incidence and prevalence for men than for women and many more studies on prevalence than incidence for women and men. The data indicate that the most frequent sexual dysfunctions for women are desire and arousal dysfunctions. In addition, there is a large proportion of women who experience multiple sexual dysfunctions. For men, premature ejaculation and erectile dysfunction are the most common sexual dysfunctions, with less comorbidity across sexual dysfunctions for men compared with women. Conclusion These data need to be treated with caution, because there is a high level of variability across studies caused by methodologic differences in the instruments used to assess presence of sexual dysfunction, ages of samples, nature of samples, methodology used to gather the data, and cultural differences. Future research needs to use well-validated tools to gather data and ensure that the data collection strategy is clearly described.