Flower Pollen Extract and its Other Effects

A new herbal combination, Etana, for enhancing erectile function: an

efficacy and safety study in animals

N Qinna 1.3, H Taha2, KZ Matalka1 and AA Badwan3

1
Department of Pharmacology and Biomedical Sciences, Faculty of Pharmacy and Medical Sciences, Petra University, Amman,
Jordan; 2Delass Natural Products, Naor, Jordan and 3Central Research Lab, The Jordanian Pharmaceutical Manufacturing CO. PLC
(JPM), Naor, Jordan

We present herein a new herbal combination called Etana that is composed of five herbal extracts
including Panax quinquelotius (Ginseng), Eurycoma longifolia (Tongkat Ali), Epimedium grandiflorum
(Horny goat weed), Centella asiatica (Gotu Kola) and flower pollen extracts. Most of the above-mentioned
extracts have been a long historical and traditional use for erectile dysfunction (ED). On the basis of the
mechanism of action of each of the above, a combination is introduced to overcome several physiological
or induced factors of ED. This study was conducted to show an enhancement of erectile function in male
rats. The animals were observed for 3 h after each administration for penile erection, genital grooming
and copulation mounting, and the penile erection index (PEI) was calculated. The maximum response
was observed at the concentration of 7.5 mg kg-1 of Etana. At a 7.5 mg kg-1 single dose, the percentage
of responding rats was 53 ± 7 with a PEI of 337 ± 72 compared with 17 ± 6 with a PEI of 30 ± 10 for
control animals. This PEI was significantly (P<0.001) higher than each single component and than the
sum of any two herbal components of Etana. When compared with sildenafil citrate, Etana induced more
pronounced PEI than 0.36 mg kg-1, but similar to 0.71 mg kg-1 of sildenafil. Furthermore, full acute and
sub-acute toxicity studies showed no toxic effects of Etana. In conclusion, this study describes a new and
safe combination of herbal components that enhance erectile function in male rats. Clinical studies are
warranted for evaluating Etana’s significance in ED.

International Journal of Impotence Research (2009) 21, 315-320; doi: 10.1038/ijir.2009.18; published online 4 June 2009

Keywords: Ginseng; Centella; Epimedium; Eurycoma; pollen; erectile dysfunction; Etana; herbal

Introduction chronic prostatitis) and certain drugs.1, 2 A

number of pharmacological agents are
Erectile dysfunction (ED) affects 50% of men introduced to correct ED transiently, such as the
aged between 40 and 70 years and therefore is orally consumed phosphodiesterase type 5
considered to be an important health problem.1 inhibitors, testosterone therapy, or vasoactive
As men age, several physiological or induced agents inserted intraurethrally or injected
factors arise that contribute to ED, such as a intracavernosally.1-3
decline in the testicular production of
testosterone, vascular functionality, levels and Some natural products such as Panax
responsiveness to vasoactive amines and quinquelotius, Eurycoma longifolia and
neurotransmitters, diseases (for example, Epimedium grandiflorum have the ability to act
cardiovascular, hypertension, diabetes mellitus, as an aphrodisiac and to help restore ED. The

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A new herbal combination, Etana, for enhancing erectile function:

An efficacy and safety study in animals
medicinal activity of Panax quinquelotius extract (50 mg), Gotu Kola extract (40 mg), and
(Ginseng) has improved penile rigidity, libido flower pollen extract (135 mg). The preparation
and patient satisfaction in men with ED,4, 5 was dissolved in distilled water and given to rats
whereas using a Eurycoma longifolia extract by oral gavage.
(Tongkat Ali) and Epimedium grandiflorum
(Horny Goat Weed) in animals increased sexual The method of analysis of Etana components is
arousal, motivation and frequency of sexual based on the HPLC method to assay a marker
activity.6-10 Furthermore, there are some natural for each constituent (for example, icariin for
products that could play a role in improving Epimedium grandiflorum and malasiatic acid for
circulation to the prostate and penis such as Centella asiatica). The method is based on a
Centella asiatica (Gotu Kola) and flower solid stationary phase (C18 packed column),
pollen.11-13 Therefore, it was our hypothesis that mobile phase, and separation by partition
the development of a herbal combination of the adsorption or ion exchange process. The
above five plant extracts, called Etana,14 could gradient mixtures of acetonitrile: H3PO4 and the
work on several age-induced causes of ED. On detection wavelengths were different for each
the basis of the mechanism of action of each component.
component, this herbal combination could have
Animals
an additive or synergistic effect to restore
erectile function. Male and female Wister rats (220-300 g) were
obtained from the Yarmouk University animal
To introduce Etana as an enhancer of male
house unit (Irbid, Jordan). The animals were
erectile function, this study examines the
housed at the animal facility in Petra University
efficacy of Etana in relation to each of its
in a 12 h light or dark cycle at a constant
components, to its dose-response effect, in
temperature of 22° C. All animals were
comparison to sildenafil as a known drug to
acclimatized for 10 days before the experiments
restore erectile function and to different herbal
with free access to a standard diet and drinking
combinations. In addition, acute and subacute
water. All animal experiments were carried out in
toxicity studies of Etana were carried out to
compliance with relevant laws and institutional
establish the safety of this herbal combination.
guidelines.
Materials and Methods
Sexual behavior and penile erection index
Herbal Extracts
Each animal group consisted of 10 male rats
Panax quinquelotius, Eurycoma longifolia and weighing 200-300 g. Each test preparation was
Epimedium grandiflorum extracts were dissolved freshly in distilled water and doses
purchased from Hongjiu Ginseng, the Active were administered by oral gavage. Dosing of
Ingredients Group and from the Chengdu Etana was either as a single dose per day or as
Wagott Pharmaceutical Co., Chengdu, China, a triple dose per day, 3 h apart, to show any
respective. Centella asiatica (Gotu Kola) and changes in the efficacy obtained from multiple
flower pollen extracts were purchased from administrations per day. Control animals were
Graminex, USA and Ennagram, France, given the vehicle alone (distilled water). Rats
respectively. Sildenafil citrate was obtained from were placed in glass cages, allowed free access
JPM, Jordan. to food and tap water and were observed for 3 h
after each drug administration for penile
Etana preparation and method of analysis erection, genital grooming and possible
copulation mounting. The number of responding
Etana is a mixture of Ginseng extract (100 mg), rats was recorded along with the number of
Tongkat Ali extracts (200 mg), Epimedium sexual activity episodes (penile erection, genital

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A new herbal combination, Etana, for enhancing erectile function:
An efficacy and safety study in animals
grooming or copulation mounting). Penile analyzed using one-way ANOVA. In some
erection index (PEI) was calculated for each cases, Turkey’s post test was carried out after
group by multiplying the percentage of active ANOVA to show the differences between
rats (responding rats) by the total number of selected groups. For all of the statistical
activity episodes.15-17 comparisons, the level of significant difference
was defined as P<0.05.
Acute and subacute toxicity assessment

Acute toxicity for Etana was determined in rats

(250-300 g) consisting of 10 rats per group (five
males and five females). A single dose of 0, 7.5,
37.5, 75, 150, 225 and 300 mg kg-1 (that is, 1 x
to 40 x of the human recommended daily dose
based on 70 kg b.w.) was given by oral gavage
to each animal per group. The animals were
observed closely for any toxic or abnormal Figure 1. The PEI for different groups of rats administered
behavior in the first 2 h after dosing and were either a single component of Etana, Etana or distilled water.
kept under further observation for 2 weeks. The PEI was determined by multiplying the average scores
reported during a 6-day treatment by the average
percentage of responding rats. The rats were monitored for 3
A subacute toxicity study for 28 days was
h after a single administration. Each single group was
carried out according to ICH guidelines. A single administered the same amount (mg) as is present in Etana.
dose of 0, 7.5, 15 and 75 mg kg-1 (that is, 1 x, 2 Each bar point represents the mean of six experiments ± s.d.
x, 10 x of the human recommended daily dose
based on 70 kg b.w.) was given by oral gavage Results
to each animal per group. Each test group
Etana versus each single component on male
consisted of five males and five females, and
rat sexual behavior
different sex animals were kept in separate
cages to avoid pregnancy during the test period. The PEI after the administration of each single
Animals were monitored carefully and body component of Etana in comparison with Etana is
weights were measured daily. At the end of 28 presented in Figure 1. In all treated animals, PEI
days, all animals were killed. Just before being increased significantly (P<0.001) when
killed, blood samples were taken from the compared with control animals. In addition,
jugular vein for a full blood and chemistry Etana-treated rats showed significantly higher
analysis. All internal organs were carefully (P<0.001) PEI than did each single Etana
removed, weighed and then fixed with 10% component-treated rats (Figure 1). In addition,
buffered formalin for histological examination. the PEI of Etana is higher than the sum of any
two herbal components. Furthermore, the
Data analysis
number of responding rats after a single
All variables were analyzed using SPSS version administration of Etana was significantly higher
10 statistical package (SPSS Inc., Chicago, IL, (P<0.001) than each single Etana component-
USA) using different statistical tests. For sexual treated rat.
behavior and PEI analysis, Student’s t test was
Dose response of Etana on male rat sexual
carried out to compare the level of significance
behavior
between groups. As for the toxicity study,
statistics were generated for time interaction, The dose response of Etana showed a
gender effect and differences between each significantly higher (P<0.001) PEI at a dose of
treatment group and the control group. The 7.5 mg kg-1 of Etana when compared with 2.5,
overall differences between the groups were 15 mg kg-1 and controls (Figure 2). In addition,
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A new herbal combination, Etana, for enhancing erectile function:
An efficacy and safety study in animals
when Etana was administered thrice a day, 3 h multiplying the average scores reported during a 3-day
treatment by the average percentage of responding rats.
apart to the same rats, the PEI was significantly
Each bar point represents the mean of three experiments ±
higher (P<0.001) at 7.5 mg kg-1 dose when s.d.
compared with that in the other doses and
control, and the cumulative PEI did not change Etana versus a different mixture of herbal
after the second or third dose to the same rats components on male rat sexual behavior
(Figure 2).
The PEI after administration of different mixture
Efficacy and safety in animals components, group 1 (Centella asiatica,
N Qinna et al Eurycoma longifolia, Epimedium grandiflorum,
flower pollen extract and Gingko) and group 2
(Ginseng, Eurycoma longifolia and Epimedium
grandiflorum), is shown in Figure 4. The
cumulative PEI and the percentage of
responding rats after the administration of Etana
thrice a day were significantly higher (P>0.001)
than the PEI and percentage of responding rats
of group1 and 2 (Figure 4). In group 1, Gingko
was added instead of Ginseng because of its
Figure 2. The PEI for different groups of rats administered
different doses of Etana. Etana was administered thrice a
known function as an aphrodisiac. A single
day, and PEI was determined by multiplying the average administration of Gingko (0.86 mg kg-1) showed
scores reported during a 3-day treatment by the average PEI and percentage of responding rats at 80 and
percentage of responding rats. Each bar point represents the 22%, respectively.
mean of three experiments ± s.d.
Acute and subacute toxicity of Etana
Etana versus Sildenafil on rat sexual behavior
No deaths occurred after the administration of
In this set of experiments, the effect of Etana 7.5
any of the single doses tested (7.5-300 mg kg-1).
mg kg-1 was compared with the effect of two
After a 28-day administration of 7.5, 15 or 75 mg
therapeutic doses (0.36 and 0.71 mg kg-1, based
kg-1 (that is, 1 x, 2 x, 10 x of the effective dose),
on 70 Kg b.w.) of sildenafil citrate. The PEI after
no deaths occurred, and the body weight did not
Etana (7.5 mg kg-1) administration as a single or
show any significant changes in male or female
triple dose per day was similar to that of 0.71 mg
rats. In addition, the weights of the internal
kg-1 of sildenafil and was significantly higher
organs did not show any changes after a 28-day
(P<0.001) than that of 0.36 mg kg-1 of sildenafil
administration of any of the doses tested.
and the control group (Figure 3).
The chemistry results after a 28-day
administration of 1 x, 2 x, and 10 x dose of
Etana showed no significant differences in
triglycerides, ALT, AST, ALP, sodium,
creatinine, calcium and phosphorus. However, a
significant reduction in cholesterol, urea and
potassium levels (P<0.03-0.001) was observed
(Table 1). The reduction of cholesterol was dose
dependent (P<0.001) as the percent reduction
was 20, 26 and 34% for 7.5, 15 and 75 mg kg-1,
Figure 3. The PEI for different groups of rats administered respectively, whereas the reduction of urea was
two doses of sildenafil and Etana. Doses were administered dose dependent (15% for all doses; P<0.03) and
thrice a day, 3 h apart, and PEI was determined by the reduction of potassium was seen only at the

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A new herbal combination, Etana, for enhancing erectile function:
An efficacy and safety study in animals
75 mg kg-1 dose (10%; P<0.015). On the other enhances erectile function in male rats. Most of
hand, the glucose level increased significantly the single constituents of Etana have been
only at 75 mg kg-1 dose (68%, P<0.025) (Table widely used for enhancing erectile function, and
scientific evidence was reported to explain the
mechanism of each component. The idea was to
show the additive or synergistic effect of such
combination. The results indicate that Etana
showed a significantly higher percentage of
responding rats and PEI. Furthermore, the
Etana efficacy was dose dependent, showing
higher activity at either single dose or triple dose
of 7.5 mg kg-1 per day, and can be administered
for a long period of time without any toxic effect.

To confirm our hypothesis with regard to the

1).
efficacy of Etana combination versus other
Figure 4. The PEI for different groups of rats administered possibilities, it was compared with two other
different combinations: Centella asiatica, Eurycoma combinations. The choice of the two other
longifolia, Epimedium grandiflorum, flower pollen extract and combinations was based on the known
Gingko for group 1: and Ginseng, Eurycoma longifolia and
Epimedium grandiflorum for group 2 and Etana. Doses were
mechanism of each component. Group 1 was a
administered thrice a day, 3 h apart, and PEI was mixture of Centella asiatica, Eurycoma longifolia,
determined by multiplying the average scores reported Epimedium grandiflorum, pollen extract and
during a 3-day treatment by the average percentage of Gingko versus Ginseng, Eurycoma longifolia
responding rats. Each single group was administered the
same amount (mg) as is present in Etana. Each bar point
and Epimedium grandiflorum (group 2) and
represents the mean of three experiments ± s.d. Etana (Ginseng, Eurycoma longifolia,
Epimedium grandiflorum, and Centella asiatica
The hematological changes after a 28-day and flower pollen). Group 1 components are
administration of 7.5, 15 and 75 mg kg-1 dose of similar to Etana except that it contains Gingko
Etana showed a significant increase in the instead of Ginseng. Gingko has also been used
percentage of lymphocytes, and a significant for aphrodisiac effects but it has a different
decrease in the percentage of neutrophils in mechanism of action from Ginseng.18 Group 2
peripheral blood at the doses of 15 and 75 mg does not contain flower pollen and Centella
kg-1 (P<0.05-0.001) (Table 1). However, the asiatica. On the basis of the above, one of the
absolute number of the above cells in peripheral mechanisms of action of Etana as a herbal
blood did not change because there was an combination to enhance blood flow is consistent
apparent reduction in the total leukocytes count. with the synergistic effects observed by
combining the individual components.11-13
Discussion

This study describes a new and safe

combination of herbal components that

Table 1. The significant hematological and biochemical findings of rats treated with different doses of Etana for 28 days

Cholesterol Urea Potassium Glucose

Group Lymphocytes % Neutrophils %
(mg dl-1) (mg dl-1) (mmol-1) (mg dl-1)
Control 58 ± 2 37 ± 2 6.5 ± 0.2 55 ± 9 73 ± 3 13 ± 2
7.5 mg kg-1 46 ± 5* 31 ± 2* 6.7 ± 0.2 56 ± 5 70 ± 3 14 ± 2
15 mg kg-1 41 ± 3** 32 ± 2* 6.8 ± 0.3 75 ± 3 80 ± 2* 8 ± 1*
75 mg kg-1 38 ± 2** 33 ± 1* 5.8 ± 0.2* 92 ± 12* 87 ± 2* 5 ± 1*

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A new herbal combination, Etana, for enhancing erectile function:
An efficacy and safety study in animals
*P<0.05 when compared with the control group, **P<0.001 when compared with the control group.

On the basis of the published scientific evidence It has been shown that Panax ginseng lowers
of each Etana component, the mechanism of cholesterol and triglyceride levels by activating
action of Etana can be fourfold. First, it has been lipoprotein lipase in hyperlipidemic rats.22 In this
shown that ginsenosides, which are extracted study, however, the rats were normal and 28
from Panax ginseng, increased the plasma days of Etana administration did not cause any
levels of FSH, LH, testosterone (total and free significant change in the triglyceride levels.
forms) and spermatozoa concentration and
motility.4 This suggests that ginsenosides act on This paper describes a new combination of
the hypothalamus and or pituitary to increase herbal extracts that enhances erectile function
plasma FSH and LH, thus activating testes to and is safe after a long day of use. In addition,
increase testosterone levels and spermatozoa this herbal combination could also help in
formation. 4, 19 Second, it was found that reducing the serum cholesterol level and in
Eurycoma longifolia enhanced the testosterone managing chronic inflammation of the
prostate. 11-13 Clinical studies are warranted for
effect by increasing the sexual performance of
inexperienced castrated male rats. 6 Third, it has evaluating Etana’s significance in ED and in
been shown that ginsenosides and icariin, men with chronic prostatitis.
isolated from Epimedium grandiflorum,
Conflict of Interest
promoted the release of nitric oxide (NO)from
corpus cavernosum.10, 20 The release of NO The authors declare no conflict of interest.
induces the relaxation of the smooth muscle and
thus enhances erection. In addition, Acknowledgements
ginsenosides and icariin were found to increase
intracavernosal pressure.10, 20 Furthermore, This work was funded by The Jordanian
icariin was found to be a cGMP-specific Pharmaceutical Manufacturing Co. PLC (JPM),
phosphodiesterase 5 inhibitor in vitro,24 but not Naor, Jordan.
in vivo, after oral dosing for 4 weeks.10 In this
References
study, however, the dose response of Etana
showed a bell-shaped curve (Figure 2), 1. McVary KT. Erectile dysfunction. New Eng J Med. 2007;
suggesting a phosphodiesterase inhibition. 357: 2472-2481.
Fourth, the addition of flower pollen extract and 2. Rosen RC< Link CL, O’Leary MP, Giuliano F, Aiyer LP,
Centella asiatica improves blood circulation to Mollon P. Lower urinary tract symptoms and sexual
health: the role of gender, lifestyle and medical
the prostate and penis, thereby enhancing the
comorbidities. BJU Int 2009; 103: Suppl 3: 42-47.
level of the other components (or their effects) of 3. Feifer A, Carrier S. Pharmacotherapy for erectile
Etana to reach the genital tract.11-13 dysfunction. Expert Opin Investig Drugs 2008; 14: 679-
Furthermore, it is known that one of the major 690.
problems that could result in ED is chronic 4. Salvati G, Genovesi G, Marcellini L, Paolini P, De Nuccio
I, Pape M et al. Effects of Panax Ginseng C. A Meyer
prostatitis.2, 13 Both Centella asiatica (Gotu Kola)
saponins on male fertility. Panminerva Med 1996; 38:
and flower pollen have antioxidative activities 249-254.
that are important to reduce male infertility and 5. De Andrade E, de Mesquita AA, Claro Jde A, de
help in managing chronic prostatitis.11, 13 Andrade PM, Ortiz V, Paranhos M et al. Study of the
efficacy of Korean Red Ginseng in the treatment of
In addition to enhancing erectile function, Etana erectile dysfunction. Sian J Andol 2007; 9: 241-244.
has other benefits. It lowered the serum 6. Ang HH, Cheang HS, Yusof AP. Effects of Eurycoma
longifolia Jack (Tongkat Ali) on the initiation of sexual
cholesterol level after 28 days of oral dosing in a
performance in inexperienced castrated male rats. Exp
dose-dependent manner. This cholesterol-
Anim 2000; 49: 35-38.
lowering effect is mainly due to Panax ginseng
and flower pollen.21-23
6|Page
A new herbal combination, Etana, for enhancing erectile function:
An efficacy and safety study in animals
 7. Ang HH, Lee KL. Effect of Eurycoma longifolia Jack on norapomerphine in rats. Arch Int Pharmadyn 1979;
orientation activities in middle aged male rats. Fundam 242: 241-247.
Clin Pharmacol 2002; 16: 479-483. 16. El-Thaher TS, Matalka KZ, Taha HA, Badwan AA. Ferula
8. Ang HH, Lee KL, Kiyoshi M. Eurycoma longifolia Jack harmonis ‘zallouh’ and enhancing erectile function in
enhances sexual motivation in middle-aged male mice. rats: efficacy and toxicity study. Int J Impot Res 2001;
J Basic Clin Physol Pharmacol 2003: 14: 301-308. 13: 247-251.
9. Gu Y, Meng G. Preparation conditions for decoction of 17. El-Thaher TS, Khatib S, Saleem M, Shnoudeh A, Badwan
Epimedium grandiflorum Morr. Zhongguo Zhong Yao AA. A novel compound JPM8: in vivo penile activity
Za Zhi 1990; 15: 412-413, 446. promotion in rats, effect on the relaxation and
10. Liu WJ, Xin ZC, Xin H, Yaun YM, Tian L, Guo YL. Effects cGMP/cAMP accumulation in isolated rabbit corpora
of icariin on erectile dysfunction and expression of cavernosa. Int J Impot Res 2002; 14: 453-461.
nitric oxide synthasae isoforms in castrated rats. Asian 18. McKay D. Nutrients and botanicals for erectile
J Andol 2005; 7: 381-388. dysfunction: examining the evidence. Altern Med Rev
11. Brinkhaus B, Linder M, Schuppan D, Hahn EG. 2004; 9: 4-16.
Chemical, pharmacological and clinical profile of the 19. Chen X, Lee TJF. Ginsenosides-induced nitric oxide-
East Asian medical plant Centella asiatica. mediated relaxation of the rabbit corpus cavernosum.
Phytomedicine 2000; 7: 427-428. Br J Pharmacol 1995; 115: 15-18.
12. Pointel JP, Boccalon H, Cloarec M, Ledevehat C, 20. Choi YD, Rha KH, Choi HK. In vitro and in vivo
Joubert M. venous insufficiency of the lower limbs. experimental effect of Korean red ginseng on erection.
Angelology 1986; 37: 420-421. J Urol 1999; 162: 1508-1511.
13. Chen HJ, Wang ZP, Chen YR, Qin DS, Fu SJ, Ma BL. 21. Ji W, Gong BQ. Hypolipidemic effects and mechanisms
Effects of pollen extract EA-10, P5 on chronic prostatitis of Panax notoginseng on lipid profile in hyperlipidemic
or infertility with chronic prostatitis. Acta Pharmacol rats. J Ethnopharmacol 2007; 113: 318-324.
Sin 2002; 23: 1035-1039. 22. Wόjcicki Jm Samochowiec L., Bartlomowicz B, Kinek A,
14. Badwan A, Taha H, Qinna N. Multi-component herbal Jaworska M, Gawroñska-Szklarz B. Effect of pollen
composition for the treatment of male erectile extract on the development of experimental
dysfunction. Patent Pending Appl #. EP200700011673, atherosclerosis in rabbits. Atherosclerosis 1986; 62: 39-
Publ: 08/06/2008; International Classes A61K36/258; 45.
A61K36/185; A61K36/296; A61K36/25; A61P15/10. 23. Xin ZC, Kim EK, Lin WJ, Tian L, Yuan YM, Fu J. Effects of
15. Benassi-Benelli A, Ferrari F, Quarantotti BP. Penile icariin on cGMP-specific PDE5 and cAMP-specific PDE4
erection induced by apomorphine and N-nopropyl- activities. Asian Andol 2003; 5: 15-18.

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A new herbal combination, Etana, for enhancing erectile function:
An efficacy and safety study in animals