MATERIALS AND METHODS

Preparation of plant extract: Rafflesia cantleyi buds were collected from the state of Perak, Malaysia during months of July-August 2013. The buds were botanically identified by Dr. Jumaat Adam. A voucher specimen (AZIE02, AZIE03) was deposited in laboratory G104, Biology Building, Faculty of Science and Technology, Universiti Kebangsaan Malaysia (UKM).

Preparation of extracts: In this study, we were interested to determine the effect of the water extract commonly used by traditional herbal practitioners. The bud was washed with tap water to remove dusts and then rinsed with distilled water and cut into small pieces (~2 cm) and air dried at room temperature (~25°C) for 5-7 days. The dried bud was ground to a fine powder using grinder (Panasonic Model, Malaysia) and stored at -20°C until used. The dried bud powder (372.08 g) was extracted by boiling in distilled water (1.5 L) for 30 min. Resulting water extract was filtered through a tea strainer. Boiling of the powder in 1.5 L distilled water was repeated and pooled. Filtrate was centrifuged in 50 mL batches at 2000 rpm for 10 min in a centrifuge (Eppendorf-5810 R). The resulting supernatant was filtered through filter paper Whatman (No. 1) placed on a funnel. Filtrate was dried in a freeze dryer and stored at 4°C in amber glass vials until use.

Animals: The 40 healthy adult male Sprague Dawley rats were assigned randomly into four main groups: A, B, C and D. Each comprised 10 adult male rats, weighing 250-300 g. They were obtained from the Animals House, UKM, Bangi and housed in plastic cages. The animals were acclimatized for a week to a 12 h light and dark cycle with free access to standard rodent pellet diet and water ad libitum before the experiment.

Experimental protocol: The experimental protocol and procedures used in this study has been approved by the UKM Animal Ethics committee (UKMAEC, No. 505). Group A rats received only distilled water and considered as the control group. The groups B, C and D were treated daily with 250, 500 and 1000 mg kg^–1 b.wt., of water extract of R. cantleyi bud, respectively. The doses were given orally using a gavage needle for 28 days. The animals were observed daily for clinical sings and mortality. Body weights and behavior were recorded every week during the study period and amounts of supplied food and water measured daily. On day 29, animals body weight were recorded before being anaesthetized. Blood samples were collected for serum testosterone analysis and the reproductive organs removed and weighed.

Mounting test: This test was performed weekly, 2 h prior to the experiment, the animals were weighed, transferred to a dark room, put in an individual cage and left for at least 1 h before the commencement of sexual behavior study. Mounting behavior of the male rats toward the receptive females were observed according to the method previously described by Meyerson et al. (1973) and Mahanem et al. (2004). Female rats used as mating stimuli were brought to heat with a single intramuscular injection of 0.125 mg kg^–1 estradiol benzoate and 1.25 mg kg^–1 of progesterone, 48 and 4 h before testing, respectively. Mounting behavioral test was conducted by placing a receptive female in the male cage. The male rat was expected to reach receptive female within 3 min. The time that the male started to mount the receptive female was considered as positive sexual behavior. Male which failed to respond within 3 min after being placed in the cage were considered as having negative sexual behavior. This test is useful to determine the willingness of male rats to sexual behavior towards the female.

Orientation activity: This activity was observed after the mounting test. Sexual behaviors of a male with female were observed from the cage side for prospective and precopulatory behaviors for 30 min. Orientation activity was analyzed in three segments as described by Islam et al. (1991), using the following scores:

The cumulative score for each orientation behavior noted in during the observation period was later calculated.

Mating test: The time taken for the male rat’s intromission activity was recorded. Male rats that couldn’t have an intromission within the first 15 min fail the mating test. The following male sexual parameters were recorded or calculated during the observation period:

Other computed male sexual behaviour parameters include:

Testosterone level: Serum testosterone was assayed using the enzyme immunoassay (EIA KIT; Cayman Chemical, USA, Catalog No. 582701). Samples were assayed in duplicate, according to the standard protocol provided in the assay kit.

Statistical analysis: Statistical analysis was performed with the Statistical Programs from the Social Science (SPSS) Version 17. Statistical significance of data was assessed by analysis of variance (ANOVA) and differences were considered significant at (p<0.05). Significant difference between control and experimental groups were assessed by Tukey test and Least Significant Difference (LSD). All data was expressed as Mean±Standard Deviations (SD).

RESULTS

Effect of water extract of R. cantleyi bud on body weight: During the 28 days of observation, there are significant change in the body weights of treated rats compared to control rats (p<0.05) as result shown in Table 1.

Relative organ weights: No significant change (p>0.05) in the epididymis weight and relative organ to body weight in animals treated with the water extract. An increase in testes and relative organ to body weight was significantly (p<0.05) in animals receiving 500 and 1000 mg kg^–1 doses as shown in Table 2.

Effect of R. cantleyi bud extract on testosterone serum levels: Figure 1 showed a dose dependent increase in the level of serum testosterone produced by male rats treated with water extract of R. cantleyi bud.

Table 1:	Weekly body weight (g) of rats treated with different doses of R. cantleyi bud water extract
Values are represented as Mean±SD of 10 replicates, *Significant difference in body weight compared to control at (p<0.05)

Table 2:	Relative organ weights of sprague dawley rats treated with water extract of R. cantleyi bud
Values are represented as Mean±SD, *Significant difference (p<0.05) compared to control

Fig. 1:	Levels of serum testosterone following oral administration of R. cantleyi water extract compared to control group. *significant difference of serum testosterone in treatment compared to control (p<0.05)

Table 3:	Mean of time for males (n = 10) to reach and subsequently mount the receptive female after being treated with R. cantleyi extract at different doses compared to control
NR: No. response even at 360 sec, *Significant difference (p<0.05) compared to control

The result revealed significant increase in serum testosterone level was observed in treated groups (500 and 1000 mg kg^–1) when compared to control group (p<0.05). There was no significant difference between control and 250 mg kg^–1 treated group (p>0.05).

Effect of R. cantleyi bud extract in mounting test: The administration of water extract of R. cantleyi for 28 days to male rats resulted in remarkable increase in mounting of the male rats after consuming of water extract of R. cantleyi bud (for treated group) compared (distilled water) control group as shown in Table 3. The extract at doses 500 and 1000 mg kg^–1 b.wt., treated male rats displayed higher sexual activity from the first week of treatment compared to treated at 250 mg kg^–1 and control group. The period of time to reach the female decrease significantly (p<0.05) with prolonged treatment with the extract. There was no significant difference between animals treated with 500 and 1000 mg kg^–1 (p>0.05) but significant difference was noted when compared to control (p<0.05).

Effect of R. cantleyi bud extract on orientation behavior: Water extract at the doses of 250, 500 and 1000 mg kg^–1 b.wt., markedly influenced the orientation behavior of the treated animals, which showed more attraction towards female rats. The result revealed significant increase in number of licking (p<0.05) and the sniffing (p<0.05) of treated males towards receptive female compared to control group. The behavioral assessment of rats towards environment (exploration and climbing) decreases in experimental animals (p>0.05), while rearing was significantly increase on treated animals at the dose level of 500 and 1000 mg kg^–1 b.wt. The result showed also significant increase (p<0.05) on the genital grooming of treated animals while decrease on nongenital grooming as compared to control group Table 4.

Effect of R. cantleyi bud extract on mating behavior: Water extract of R. cantleyi bud at doses 250, 500 and 1000 mg kg^–1 b.wt., showed significant increase (p<0.05) on Mount Frequency (MF), Intromission Frequency (IF) and Ejaculation Latency (EL) compare to distilled water control group. Moreover, it causes significant decrease (p<0.05) in the Mount Latency (ML) and Intromission Latency (IL) compared to control group as shown in Table 5.

The computed male sexual behaviour parameters which included percentage of mounted, intromitted, ejaculated, index of libido and copulatory efficiency were higher in the extract treated rats compared to the control group (Table 6). In contrast, the extract reduced the inter-copulatory interval of the treated animals in compared to control group.

DISCUSSION

Rafflesia has been used traditionally in medicinal practitioners as aphrodisiac. The preliminary phytochemical screening of aqueous R. cantleyi bud extract revealed the presence of alkaloids, flavonoids, tannin and steroids. The result indicates that the aqueous extract of R. cantleyi causes increase in animal body weight as well as in sexual organs such as testes and epididymis. Genesis of steroids is one of the causes of increased body and sexual organ weight and an increase in these parameters could be regarded as a biological indicator for effectiveness of the plant extract in improving the genesis of steroidal hormones. It has been reported that steroids constituents found in many plants possess fertility potentiating properties (Thakur and Dixit, 2007). Also, the study revealed the presence of flavonoids in R. cantleyi bud extract which has been implicated to have a role in altering androgen levels (Padashetty and Mishra, 2007). Since androgenic effect is attributable to testosterone levels in blood (Amini and Kamkar, 2005). The weight, size, secretary function of testes, epididymis and seminal vesicle are closely regulated by androgens. It is well established that androgens are the major regulators of growth, structure and functions of accessory sex organs (Agrawal et al., 1986). Testosterone is the most important androgen secreted from the interstitial leydig cells. It has many important roles in the male reproductive functions such as inhibition of Gonadotropin Releasing Hormone (GnRH) and Luteinizing Hormone (LH) secretion, induction and maintenance of the differentiation of male accessory reproductive organs (Dohle et al., 2003).

Sexual behavior in male is a complicated phenomenon under the control of endocrine, central and peripheral nervous systems.

Table 4:	Effect of water extract of R. cantleyi bud on orientation activity in male rats
Value are given as Mean±SD, N = 10, *Significant difference (p<0.05) compared to control

Table 5:	Effect of water extract of R. cantleyi bud on mating behaviour in male rats
Value are given as Mean±SD, N = 10, *Significant difference (p<0.05) compared to control

Table 6:	Effect of water extract of R. cantleyi bud on computed male rat sexual behaviour parameters
Value are given as Mean±SD, N = 10, *Significant difference (p<0.05) compared to control

Aphrodisiac activity in Eurycoma longifolia has been suggested to be associated with a 4.3 kDa bioactive peptide that was also detected in Rafflesia sp. (Asiah et al., 2007). This potential phytoandrogen has been reported to increase testosterone levels in rat leydig cell (Sambandan et al., 2004). The presence of similar peptide should therefore be confirmed if available in R. cantleyi.

Sexual behavior is enhanced by elevated testosterone levels. Another logical candidate that might attribute to the sexual behavior is the flavonoids constituent of the R. cantleyi bud extract (Bakoush et al., 2015). It has been reported that flavonoids alter the androgen levels (Gauthaman et al., 2002). The alteration in the androgen level was supported by the increase in testosterone content of the animals in the present study. In addition, the bud extract also has alkaloids that may have ergogenic properties by inducing vasodilation of blood vessels which consequently result in erection (Agmo, 1997). Alkaloids have also been reported to facilitate of sexual behavior (Adimoelja, 2000).

Sexual behavior in animal models is considered useful in predicting the potential similar effects of chemicals in human (Hull and Dominguez, 2007). The present investigation reveals that the aqueous extract of R. cantleyi can enhance male sexual behavior. In male rats, latency for mount and intromission are considered as indicators of the sexual motivation, whereas intromission and ejaculation frequencies are considered as behavioral indication of sexual performance and facilitation (Neill et al., 1990). The result showed significantly increase in Mount Frequency (MF) and Intromission Frequency (IF) as compared to control. The MF and IF are considered the indices of both libido and potency. The R. cantleyi bud extract significantly decreases the mount and intromission latencies indicating enhancement of sexual motivation. The significant increase in computed male sexual behavior parameters in this study are indications of sustained increase in sexual activity and aphrodisiac property inherent in the R. cantleyi bud extract. Nevertheless, caution must be taken on the dose consumed due to the effect on the organs especially liver and kidney as indicated by this study.

CONCLUSION

The results of this present study demonstrate that water extract of R. cantleyi bud enhance sexual behavior in male rats that may be attributed by the presence of alkaloids, steroid and flavonoids. This study supports the traditional medicinal use of R. cantleyi bud as an aphrodisiac from nature. Caution must be taken on the suitable dose. Another important point is that this plant is facing extinction. Results from this study should not be used as a reason for hunting the plant down. The unique property of the important constituent from R. cantleyi needs to be explored for synthetic production.

ACKNOWLEDGMENTS

The project was conducted from grants available from Universiti Kebangsaan Malaysia (grants numbers UKM DPP-2014-021, UKM-DPP-2014-084, DPP-2015-FST and Malaysian Ministry of Education (FRGS/1/2014/ST03/UKM /01/1). Doctoral scholarship from Ministry of Higher Education, Libya to the first author is duly acknowledged.