Comparing the pharmacokinetics of 13α,21-dihydroeurycomanone and eurycomanone exclusively enriched in Eurycoma longifolia extracts and their spermatogenesis enhancement in andrographolide-induced oligospermia in rats

Abstract

Objectives

The quassinoids eurycomanone (EN) and 13α,21-dihydroeurycomanone (DHY) of Eurycoma longifolia Jack are reported to enhance spermatogenesis. This study aims to profile the pharmacokinetics of DHY, a minor and hitherto unstudied constituent, evaluate its spermatogenesis enhancement property and compare these attributes with that of the predominant EN.

Methods

Crude Eurycoma longifolia extract was chromatographed into a DHY-enriched extract (DHY-F) and an EN-enriched extract (EN-F). Male Sprague–Dawley rats were administered intravenously and orally with both extracts and their plasma levels of both quassinoids were determined. The extracts were then tested for their spermatogenesis augmentation ability in normal rats and an andrographolide-induced oligospermia model.

Key findings

Chromatographic enrichment resulted in a 28-fold increase of DHY in DHY-F and a 5-fold increase of EN in EN-F compared with non-chromatographed crude extracts. DHY showed better oral bioavailability (1.04 ± 0.58%) than EN (0.31 ± 0.19%). At 5 mg/kg, EN exhibited higher efficacy in spermatogenesis enhancement in normal rats and restoration of oligospermia to normal sperm profile versus DHY.

Conclusions

Despite the better pharmacokinetic profile of DHY, EN remains the main chemical contributor to plant bioactivity. DHY-F and EN-F represent improvements in developing Eurycoma longifolia as a potential phytomedicine for male infertility particularly oligospermia.

Eurycoma longifolia, quassinoids, 13α, 21-dihydroeurycomanone, spermatogenesis, pharmacokinetic, chromatographic enrichment

Introduction

Infertility is generally defined as the inability of a couple to conceive after trying unprotected intercourse for 1 year.^[1] In many cultures, infertility poses a life crisis that can disrupt the stability of the individual, the relationship of the couple, the family and the community. An estimated 48.5 million or 15% of couples suffer from this disorder worldwide.^[2,3] A recent study estimated that between 20 and 70% of infertility problems may have arisen from the male partner.^[4] Most of the male problems were due to oligozoospermia, a clinical condition of low sperm count (<15 million/ml) and quality. Current treatment modalities for male infertility are in-vitro fertilisation, intrafallopian transfer, intra-cytoplasmic sperm injection, surgery and hormone replacement therapy. Although these methods are efficacious to some degrees, they are costly and invasive, and thus may be beyond the socioeconomic means of societies in the developing countries and in resource-poor settings.^[5] A phytotherapeutic alternative developed along the lines of its reputation as a traditional medicine may hold a better appeal.

Eurycoma longifolia Jack (Simaroubaceae) known by local ethnic populations of South East Asia as ‘Tongkat Ali’ (Malaysia), ‘Pasak Bumi’ (Indonesia), ‘Tung Saw’ (Thailand) and ‘Cây Bá Bệnh’ (Vietnam) is traditionally used for its aphrodisiac property and general reproductive health benefits.^[6–8] The roots of the plant used for this purpose contain in substantial quantities (5–10% w/w) a group of chemical constituents called the quassinoids which are unique to plants of the Simaroubaceae family. Our previous studies have shown that quassinoids, particularly the major eurycomanone (EN), 13(21)-epoxyeurycomanone (EP) and 13α,21-dihydroeurycomanone (DHY) (Figure 1A), are the key bioactive attributors to the enhancement in spermatogenesis.^[9] A standardised extract chromatographically enriched in the three quassinoids, TAF2 significantly reversed the poor sperm count and quality in an andrographolide-induced oligospermic rat model.^[10,11] EN was shown to enhance sperm production in rats by increasing testosterone levels in the rat testis which were maintained in a self-regulated manner via the pituitary–hypothalamus–gonadal axis,^[12] thus giving it an advantage over the alternative testosterone replacement therapy which may pose risk for the systemic adverse effects of testosterone.^[13,14]

Figure 1

(A) Structures of key bioactive quassinoids in Eurycoma longifolia. HPLC chromatograms of (B) Crude ethanolic Eurycoma longifolia extract, (C) HP-20 fraction 7 (DHY-enriched fraction) and (D) HP-20 fraction 6 (EN-enriched fraction).

Despite these promising attributes, the quassinoids are inherently highly polar compounds that showed low oral bioavailability (<1%) due to their poor lipid membrane permeability.^[15,16] In our ongoing effort to improve the bioavailability of the present TAF2, we shifted our attention to the minor quassinoid DHY which is present at only 0.5% w/w in a crude ethanolic root extract of Eurycoma longifolia.^[17] Compared with the major quassinoids, little has been studied of DHY in terms of its pharmacokinetics and bioavailability. DHY presence in TAF2 in low concentrations has hitherto prevented its detection in rat plasma and hindered pharmacokinetic profiling. Furthermore, its contribution to TAF2 bioactivity as an individual compound is possibly obscured by the major quassinoids such as EN. We posited that DHY, differing structurally from EN by having a saturated methylene at the 13- and 21-positions of the scaffold may be less polar than the other quassinoids and may, therefore, have better bioavailability. This has motivated us to develop a chromatographic method that selectively enriched this minor constituent to produce an extract with high levels of DHY and relatively low levels or devoid of the other quassinoids. With this newly derived DHY-enriched extract, we were able to profile DHY pharmacokinetics for the first time and compare the bioavailability of DHY with that of the major EN. In addition, we demonstrated here the spermatogenesis enhancing activity of DHY in both normal and oligospermic rats in the form of an improved version of TAF2.

Methods

Chemicals

Reagents were of analytical grade or higher unless otherwise mentioned. HPLC grade acetonitrile and methanol, perchloric acid, formalin and thin layer chromatography silica gel 60 F₂₅₄ plates were from Merck KGaA. Diaion^TM HP-20 and Sepabead^TM SP207 resins were purchased from Mitsubishi Chemicals Corp. Carboxymethyl cellulose (CMC) was from Sigma-Aldrich GmbH. Sodium heparin was from Acros Organics.

Plant material

Eurycoma longifolia roots were purchased from Fidea Resources (Registration No.: 001 709800-D). The plant was identified by a curator (Mr. Saul Hamid bin Pakir Mohamad). A voucher specimen of the plant (No. JTN 1189) was deposited at the Penang Botanical Garden, Penang, Malaysia. The roots were chopped, air-dried, and ground to coarse particles of 0.5–1.0 cm using a laboratory grinder (APEX Instrumentation Limited).

Plant extraction

Dried powdered Eurycoma longifolia root (5 kg) was extracted with 95% ethanol at 55ºC for 8 h per day for 5 days (1 l × 5) consecutively. The combined extract was collected and evaporated in vacuo to give a dark brown residue of crude extract (150 g, 4% w/w yield).

Chromatographic enrichment of DHY and EN

Crude ethanolic extract (30 g each) was reconstituted with 95% methanol (100 ml) and chromatographed using two macroporous adsorbent resin columns HP-20 and SP207. Briefly, the resin was soaked in 50% v/v methanol overnight and subsequently packed into a glass column (internal diameter 100 × length 650 mm) and preconditioned with deionised water. The crude extract was eluted first with deionised water and followed by eluents with stepwise increments of 5% v/v of methanol over deionised water (5: 95 → 30: 70). Fractions collected were screened with thin layer chromatography (TLC) for the presence of desired quassinoids. Fractions containing quassinoids were pooled and dried in vacuo and subjected to HPLC analysis.

Animals

Sprague–Dawley (SD) male rats aged between 6 and 8 weeks and weighing 180 – 250 g from the same generation were used. At the commencement of the study, weight variation between animals were within ± 20 % of the mean weight. The animals were obtained from the Animal Research and Service Centre, Universiti Sains Malaysia and housed in an animal facility at an ambient temperature of 20–26°C under a daily 12 h light: 12 h dark cycle and allowed free access to food pellets (Gold Coin Feedmills Sdn. Bhd., Penang, Malaysia) and tap water. The animals were acclimatised for 5 days prior to onset of experiments. Both pharmacokinetic and spermatogenesis experiments in this study have been approved by the Animal Ethics Committee of Universiti Sains Malaysia with approval letters no. USM/Animal Ethics Approval/2016/(726) and USM/Animal Ethics Approval/2016/(103)(783), respectively.

Pharmacokinetic profiling of DHY- and EN-enriched extracts

Twelve animals were divided into two groups. The animals were fasted for 12 h prior to the study and food pellets were given at the 4th hour after the experiment began. Extracts were prepared as suspensions in distilled water for both oral and intravenous administrations. The first group (n = 6) was given 170 mg/kg of DHY-enriched extract (DHY-F) containing 22 mg/kg of DHY via oral gavage. Similarly, the second group (n = 6) was given an oral dose of 74 mg/kg of EN-enriched extract (EN-F) containing 22 mg/kg of EN. Blood samples (approximately 200 μl per animal) were collected using the tail nipping method at several time intervals for 24 h. After a 2-week ‘wash-out’ period, animals of the first and second groups were given intravenous via the tail vein 8 mg/kg DHY-F and 3.5 mg/kg EN-F, respectively. Both dosages contained 1.1 mg/kg DHY and EN, respectively. Blood was collected in the same manner as with the oral dosages. Blood samples were stored in a heparinised microcentrifuge tube and centrifuged (3000 × g) to remove blood cells. Each sample (50 μl) was then deproteinised with 70% perchloric acid (2.5 μl) and centrifuged (10 000 × g) to yield supernatant plasma for HPLC analysis. Pharmacokinetic parameters of EN and DHY were generated using the open source software PKSolver 2.0 by Zhang et al.^[18] based on a two-compartment model. Absolute bioavailabilities (F_abs) of the compounds were obtained using the following equation:

$${\displaystyle \begin{array}{l}{\displaystyle F_{abs}(\mathrm{\%})=\frac{\mathrm{O}\mathrm{r}\mathrm{a}\mathrm{l}\mathrm{A}\mathrm{U}\mathrm{C}/\mathrm{o}\mathrm{r}\mathrm{a}\mathrm{l}\mathrm{d}\mathrm{o}\mathrm{s}\mathrm{e}}{\mathrm{I}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{o}\mathrm{u}\mathrm{s}\mathrm{A}\mathrm{U}\mathrm{C}/\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{o}\mathrm{u}\mathrm{s}\mathrm{d}\mathrm{o}\mathrm{s}\mathrm{e}}\times 100}\end{array}}$$

HPLC analysis

A HPLC system comprising an Agilent 1120 Compact LC system equipped with a variable wavelength photometric detector and an Agilent EZ-Chrom Compact software (Agilent Technologies, Santa Clara, CA, USA) was used. Samples were eluted through a Purospher STAR RP-18 endcapped (4.6 mm i.d. × 150 mm, 5μm) column (Merck KGaA, Darmstadt, Germany) using an isocratic mobile phase consisting 94% deionised H₂O and 6% acetonitrile at 1.2 ml/min. UV detection wavelength was set at 238 nm. For quantification of the quassinoids in the extracts (crude and chromatographically enriched) and in the rat plasma, pure EN, DHY and 13α(21)-epoxyeurycomanone (EP) (purities > 95 %) isolated from Eurycoma longifolia as previously reported^[19] were used as standards. NMR spectra and HPLC purities of the quassinoids, as well as the HPLC method validation are given in the Supplementary Material.

Evaluation of DHY-F and EN-F spermatogenesis augmentation in normal rats

Eighteen male SD rats weighing between 180 and 250 g were divided into three groups. Group I (n = 6) served as controls and were given distilled water. Groups II and III (each n = 6) were treated with oral administrations of 38 mg/kg of DHY-F (containing 5 mg/kg DHY) and 12.5 mg/kg EN-F (containing 5 mg/kg EN), respectively. Treatments were given for 48 days consecutively. At the end of the treatment period, the animals were sacrificed by carbon dioxide asphyxiation and dissected for evaluation of sperm motility, morphology and count.

Evaluation of DHY-F and EN-F in the andrographolide-induced oligospermic rat model

Thirty-six male SD rats weighing between 180 and 250 g were divided into six groups (n = 6 in each group). Initially, to verify the model, two groups A1 and B1 were given by oral gavage, a vehicle (1% w/v CMC) and 25 mg/kg of andrographolide (Biopurify Phytochemicals, Chengdu, China) in 1% w/v CMC, respectively, for 48 days. The andrographolide dose given has been reported to induce abnormal, oligospermic spermatogenesis.^[11,20] A longer period (96 days) study was next conducted with group A2 serving as the vehicle group. Group B2 was given 25 mg/kg of andrographolide in 1% CMC for 48 days followed by another 48 days of ‘wash-out’ period without any treatment. Animals in groups C and D were given the same dose of andrographolide throughout 96 days. At the 48th day, both groups C and D were initiated on oral administrations of 38 mg/kg of DHY-F (containing 5 mg/kg DHY) and 12.5 mg/kg of EN-F (containing 5 mg/kg EN), respectively. At the end of study period (48th day for A1 and B1, 96th day for the remaining groups), the animals were sacrificed and dissected immediately for the evaluation of sperm motility, morphology and concentration.

Rat epididymal sperm evaluation

The cauda epididymis was removed following a method by Remie^[21] and the seminal fluid containing the rat spermatozoa was collected by a diffusion method.^[11] The collected seminal fluid was pipetted onto a microscope slide and observed under a light microscope at ×400 magnification using the hanging drop method.^[19] Motility and morphology of 200 spermatozoa were graded in each replicate according to the WHO Laboratory Manual for the Processing and Examination of Human Semen^[22] and expressed as percentages of motile spermatozoa and with normal morphology. The seminal fluid was diluted 5-fold with bicarbonate-formalin and pipetted (10 µl) into a Nebauer haemocytometer chamber (Hawksley, Sussex, UK) and sperm concentrations were determined as ×10⁶/ml/g of testis.

Statistical analysis

Data collected were presented as means ± standard deviations or standard errors of mean. Statistical analysis was done using SPSS 20.0 (IBM Corp., New York, USA). Significant differences of log transformed pharmacokinetic parameters between treatments (DHY-F and EN-F) were analysed by Student’s t-test. Significant differences of the sperm concentrations and percentages of motile and normal sperms between groups were analysed by one-way ANOVA followed by post-hoc multiple comparison Tukey’s HSD test. A confidence level of P < 0.05 was considered statistically significant.

Results and Discussion

Enrichment of the quassinoids EN and DHY

In a typical ethanolic crude extract of Eurycoma longifolia, the three quassinoids EN, EP and DHY were present (Figure 1B) with DHY being the minor constituent. The HPLC peak of DHY was also in close proximity to that of the major EN. We attempted to increase the content of DHY by chromatographic elution through two macroporous, non-polar, polystyrene-based absorbents: Diaion HP-20 and Sepabead SP207 (a more lipophilic counterpart of HP-20). The fact that HP-20 resins proved useful in significantly enriching the three quassinoids to produce TAF2 governed the choice of these absorbents. The results of the chromatography are shown in Figure 1C and D and Table 1.

Table 1

Quassinoid content (% w/w) in fractions produced by chromatographic elution of the crude Eurycoma longifolia extract through HP-20 and SP207. Fractions in which quassinoids were not detected (by TLC) are not shown

Fractions	Quassinoids			Total
	EN	EP	DHY
Crude extract	6.13 ± 0.14	1.68 ± 0.25	0.54 ± 0.05	8.35
HP-20
Fraction 5	22.40 ± 4.81	19.79 ± 2.87	–	42.19
Fraction 6	30.40 ± 2.50	–	1.70 ± 1.21	32.10
Fraction 7	1.91 ± 0.60	–	13.96 ± 1.10	15.87
SP207
Fraction 5	6.47 ± 4.24	2.05 ± 0.52	–	8.52
Fraction 6	24.86 ± 4.53	–	1.06 ± 0.02	25.92
Fraction 7	4.0 ± 2.19	–	10.32 ± 0.35	14.32

EN = eurycomanone; EP = 13α(21)-epoxyeurycomanone; DHY = 13α,21-dihydroeurycomanone.

The highest content of DHY was found in fraction 7 of the HP-20 chromatography (HP-20-F7). Notably, in HP-20-F7, DHY content (14% w/w) was increased by more than 20-fold versus DHY level in the crude extract (0.5% w/w). The other quassinoid EN although still present in HP-20-F7 (Figure 1C), was reduced to a low level (2% w/w). In addition, fraction 6 of HP-20 chromatography showed the highest increase in EN (30 % w/w) among all fractions (Figure 1D), a significant improvement in enriching this quassinoid over TAF2 previously reported to contain approximately 15% w/w of EN.^[12] Despite showing some degrees of selective enrichment of the three quassinoids, SP207 chromatography did not fare as well as HP-20. Hence, we have produced separately, an extract enriched in DHY (henceforth named DHY-F) as well as an EN-enriched extract (EN-F).

Using the enriched extracts DHY-F and EN-F, the pharmacokinetics of the respective quassinoids were profiled in rats administered intravenously and orally with the extracts. As the molecular weights of DHY and EN do not differ significantly, the administered dosages of both extracts were set to deliver an equivalent amount of both compounds (1.1 mg/kg for intravenous doses and 22 mg/kg for oral doses) to ensure a valid comparison between the two quassinoids could be made.

Pharmacokinetics of EN and DHY

Semi-logarithmic plots of the plasma concentrations versus time for each rat appeared to be biphasic in nature for both EN and DHY (Figure 2A and B) indicating that the pharmacokinetics of the compounds were better fitted using a two-compartment model. Hence, using this model, the pharmacokinetic parameters of the quassinoids were calculated (Table 2). No statistical differences (P ≥ 0.05) were found when comparing the parameters of EN versus those of DHY except that the oral AUC_0-∞ for DHY was notably higher than that of EN (P ≤ 0.05). Indeed, oral administration of the DHY-F extract attained a higher C_max of DHY (0.58 ± 0.34 µg/ml) than the C_max of EN (0.19 ± 0.12 µg/ml) from an oral dose of EN-F extract. Absolute bioavailability of both quassinoids remained low (≤5%) but DHY showed a significantly better oral bioavailability (1.04 %) when compared with EN (0.3 %). This could possibly be attributed to the slightly less polar property of DHY (CLogP -2.17) compared with EN (CLogP -2.26).

Figure 2

Plasma concentration versus time curves of DHY and EN upon (A) intravenous administration and (B) oral administration. Each value is expressed as mean ± SD (n = 6 rats).

Table 2

Pharmacokinetic parameters of DHY and EN. Each value is expressed as mean ± SD (n = 6 rats)

Both EN and DHY had V_c ≈ 0.06 l/kg and V_ss ≈ 0.06 to 0.1 l/kg that were typical of drugs with low volumes of distribution and a tendency to remain in the circulating plasma rather than distributing into the peripheral tissues. Such distribution profile was consistent with the highly polar nature of the polyhydroxylated quassinoids EN and DHY. The rate of distribution from the peripheral compartment to the central compartment (K₂₁) for EN being significantly higher (P ≤ 0.05) compared with its rate of distribution from the central compartment to the peripheral compartment (K₁₂) further supported this notion. The relatively short elimination half-lives of the quassinoids (t_1/2β ≈ 7–11 h) may also suggest that these compounds tended to distribute into the plasma and highly perfused tissues such as the kidney resulting in fast clearance.

The previous TAF2 when given orally to rats in a dose of 100 mg/kg (delivering approximately 15 mg/kg EN) showed a similar availability profile of EN as in the present study but DHY failed to be detected in the blood plasma.^[16] Thus, by profiling two extracts, each enriched in one quassinoid exclusively, we were able in this study to obtain individually the pharmacokinetics of the two compounds and clearly discern their differences particularly their oral biovailabilities.

Spermatogenesis enhancement by EN-F and DHY-F in normal rats

In normal rats, the sperm count, motility and percentage of abnormal spermatozoa were found to be within the normal range (Figure 3A–C) and corresponded with those of previous studies.^[10,11] DHY-F and EN-F were given in doses set to deliver 5 mg/kg of DHY and EN, respectively. This dose was chosen based on the dose of TAF2 (25 mg/kg containing approximately 5 mg/kg EN) shown to be efficacious in previous studies.^[12,16] The minute presence of EN (~ 2%) in DHY-F ensured that the extract primarily contained DHY and a low to negligible level of EN (<1 mg/kg EN). Vice versa, EN-F contained primarily EN (5 mg/kg) and relatively low level of DHY (approximately 0.125 mg/kg DHY). As such, these DHY- and EN-enriched extracts made possible for the spermatogenesis augmenting effect of the two quassinoids to be evaluated on an individual basis.

Figure 3

(A) Sperm concentrations, (B) percentages of motile spermatozoa and (C) percentages of normal spermatozoa in normal rats treated with DHY-F and EN-F for 48 days. (D) Sperm concentrations, (E) percentages of motile spermatozoa and (F) percentages of normal spermatozoa of andrographolide-challenged rats and vehicle control. Statistical differences were discerned by one-way ANOVA, followed by post-hoc Tukey’s HSD test: *significantly different versus untreated control (P < 0.05); #significantly different versus DHY-F (P < 0.05). Statistical differences were identified by unpaired t-test: @significantly different versus vehicle at P < 0.05.

Rats orally fed with DHY-F and EN-F showed significant increases in sperm concentrations (P < 0.05 versus untreated control). Interestingly, rats given EN-F scored the highest sperm concentration (48.6 × 10⁶/ml/g) of the treatment groups and this was significantly higher (P < 0.05) than rats given DHY-F suggesting that EN was more potent than DHY in spermatogenesis enhancement. Indeed, when compared with spermatogenesis enhancement in previous studies using TAF2.^[12,16] EN-F gave a higher activity. Daily doses of 25 mg/kg TAF2 (delivering approximately 3.75 mg/kg EN) showed a sperm concentration of 36.5 × 10⁶/ml/g.^[16] Possibly, this is due to the higher EN content in the present extract EN-F. Sperm motility and morphology were not affected by all the extracts.

Spermatogenesis enhancement by EN-F and DHY-F in andrographolide-induced oligospermic rats

Having shown the ability of DHY-F and EN-F to enhance spermatogenesis in normal rats, we sought to evaluate the effects of the extracts in a rat model of oligospermia induced by andrographolide. Initially, for 48 days, group A1 was given andrographolide (25 mg/kg) and their sperm profiles (sperm count, motility and morphology) were shown to be deleteriously altered when compared with a vehicle control group (B1) (Figure 3D–F) thus simulating oligospermia.

Next, in a 96-day study, four groups (A2, B2, C and D) were given treatments following the treatment schedule shown in Figure 4A. Animals treated with andrographolide for 48 days and were treatment-free for the following 48 days (B2) still retained significant oligospermia that was not different (P > 0.05) from that of rats given andrographolide for 48 days (B1), indicating a prolonged suppressive effect by andrographolide. DHY-F and EN-F significantly attenuated oligospermia (P < 0.05 versus B2) in the andrographolide-challenged rats and reversed their sperm profiles (sperm concentration, percentages of motile sperm and abnormal spermatozoa) to those of the normal vehicle control (Figure 4B and C). Remarkably, EN-F treatment on the oligospermic rats led to the highest sperm concentration (38 × 10⁶/ml/g) among all groups, significantly exceeding (P < 0.05) that of the normal vehicle control. Sperm concentration of EN-F-treated group was also higher than that of DHY-F (P < 0.05). This again underscored the higher potency of EN over that of DHY.

Figure 4

(A) Treatment protocol for four treatment groups. (B) Sperm concentrations, (C) percentage of motile spermatozoa and (D) percentages of normal spermatozoa of the treatment groups. Statistical differences were identified by one-way ANOVA, followed by post-hoc Tukey’s HSD test: #significantly different (P < 0.05).

Despite DHY having a 3-fold higher oral bioavailability than EN, EN-enriched extract (EN-F) was more efficacious in enhancing sperm production than DHY-F. This suggested that the bioactivity of the quassinoids may be governed not so much by pharmacokinetic and physicochemical factors as by pharmacodynamic factors such as their intrinsic molecular interaction with the target site of action. A finding to support this notion is that repeated and prolonged quassinoid treatments were efficacious despite the relatively low bioavailability (<5%) and short half-lives of the compounds when compared with commercial oral drugs with acceptable (or ‘ideal’) pharmacokinetic profiles.^[23] Mouse Leydig cells treated with EN and TAF2 showed significant testosterone elevations only after a prolonged treatment of 48–96 h^[24] and the permeability rates of EN in parallel artificial membrane permeability assay (PAMPA) and across Caco-2 cells were relatively low^[25] give hints to the quassinoids displaying slow cell or membrane permeation leading to a delayed onset of action which necessitates repeated and sustained treatment with the compounds for efficacy.

A limitation to the interpretation of the present results needs to be mentioned here. The oral dose given for the quassinoid-enriched extracts for the pharmacokinetic study (22 mg/kg EN or DHY) was higher than the once daily oral doses (5 mg/kg EN or DHY) administered for the efficacy study. The once only 22 mg/kg dose of EN or DHY was given specifically for the purpose of profiling the pharmacokinetics of the quassinoids across a 24-h period while the repeated daily 5 mg/kg doses of quassinoids were selected based on known efficacious doses from previous study for the 48-day spermatogenesis evaluation. A pharmacokinetics to pharmacodynamics (PK/PD) relationship therefore could not be established in the present study.

Conclusions

The minor bioactive quassinoid DHY of Eurycoma longifolia was successfully enriched to a sufficient quantity to enable determination for the first time, of its bioavailability and pharmacokinetics. Compared with the major quassinoid EN, DHY showed a better (3-fold) oral bioavailability which supports the assumption of DHY being the more lipid membrane-permeable compound. Further enrichment of both DHY and EN has accentuated the spermatogenesis enhancing activity of the quassinoids, especially EN thus providing further evidence that the quassinoids particularly EN are the main chemical contributors to Eurycoma longifolia bioactivity. The quassinoid-enriched extracts produced in this work, DHY-F and EN-F, thus represent further improvements to the use of Eurycoma longifolia as a potential phytopharmaceutical for male infertility and reproductive health.

Funding

The authors gratefully acknowledged financial support by the National Key Economic Area (NKEA) EPP#1 Research Grant Scheme (NRGS) awarded by the Ministry of Agriculture and Agro-Based Industry of Malaysia (No. NH1014D043) and the Universiti Sains Malaysia Bridging Grant (No. 304/PFARMASI/6316274).

Conflict of interest

The authors declare no conflict of interest.