Restoration of chronic- Δ⁹-THC-induced decline in sexuality in male rats by a novel benzoflavone moiety from Passiflora incarnata Linn

Abstract

1. The present study comprised treatment of healthy male rats with Δ⁹-tetrahydrocannabinol (THC, 10 mg kg⁻¹, p.o.), and combinations of THC with benzoflavone moiety (BZF, 10 and 20 mg kg⁻¹, p.o.) isolated from Passiflora incarnata Linneaus, over a period of 30 days.

2. Upon 30-days chronic administrations, the THC-treated male rats had a significant loss of libido (mounting behaviour with non-oestrous female rats), decrease in sperm count, and number of impregnated pro-estrous female rats.

3. Co-administration of BZF (10 and 20 mg kg⁻¹ p.o.) afforded significant protection against the chronic-THC-induced decrease in libido, mating performance and fertility during 30-day experimental regimen. The 20 mg kg⁻¹ dose of BZF exhibited better results.

4. Upon discontinuation of THC, treatment with BZF (10 and 20 mg kg⁻¹ p.o.) also facilitated the early restoration chronic-THC-induced decline in libido, sperm count and sexual fertility within 7 days.

Introduction

Since time immemorial, the plant Cannabis sativa (family Moraceae) has been used by human beings for altering mood, thoughts, for the enhancement of sensual pleasures, and during religious occasions (Chopra, 1971). The resinous exudates from the dried flowering and fruiting tops of Cannabis contain over 61 cannabinoids, amongst which Δ⁹-tetrahydrocannabinol (Δ⁹-THC) is the main psychoactive phyto-moiety of Cannabis which accounts for all the biological effects of Cannabis. As per the latest report by the WHO, the abuse of cannabinoids (slang names like marihuana, marijuana, hash, pot, grass and hemp) over the last decade has become a very serious socio-economic health-care issue all-over the world (UNDCP, 2001). Besides addiction, chronic intake of cannabinoids has been reported to cause severe impairment of sexual functions in humans and animals (Russo et al., 2002). The decrease in libido, sperm count and fertility, induced by chronic Cannabis consumption in male species are some of the important issues which must be taken care of before prescribing cannabinoids in AIDS and cancer chemotherapy.

While working on a traditional plant Passiflora incarnata Linneaus (Passifloraceae, synonyms – passionflower, maypops, marucuja, prempushpi), the methanol extract of the leaves of P. incarnata was confirmed to possess anxiolytic (Dhawan et al., 2001b, 2001c), effects at a dose of 100 mg kg⁻¹ per oral (p.o.) dose in mice. The bioactive methanol extract was further subjected to fractionations and chromatographic procedures to finally isolate a pure bioactive fraction (Dhawan, 2002). From this pure bioactive fraction, a tri-substituted benzoflavone moiety was isolated and its chemical identity was established using the standard spectroscopic techniques, i.e., UV, LC-MS, IR, ¹H-NMR and ¹³C-NMR (Dhawan et al., 2001d, 2001e; Dhawan, 2002). The isolation of a tri-substituted benzoflavone moiety (BZF) as the main bioactive phyto-constituent of P. incarnata has been an encouraging breakthrough in elucidating the mode of action of this plant, which finds mention in the ancient ayurvedic medical writings as a promising cure for male-impotence, post-menopausal decline in libido in females, menstrual irregularity, morphinism, alcoholism and tobacco addiction. Our subsequent pharmacological studies on the BZF moiety also confirmed that the BZF moiety isolated from P. incarnata was very effective in countering the menace of addiction-prone substances like morphine (Dhawan et al., 2002e), cannabinoids (Dhawan et al., 2002d), nicotine (Dhawan et al., 2002c) and ethanol (Dhawan et al., 2002f) in laboratory animals (Dhawan et al., 2002a). Treatments with 10 mg kg⁻¹ p.o. dose of BZF for 30 days resulted to significant increase in the libido (mounting behaviour with non-estrous female rats,), sperm count, fertilization potential (number of pro-estrous female rats impregnated) and greater litter size, in 2-year-old male rats, relative to control group of animals which were given vehicle only (Dhawan et al., 2002b).

The encouraging results of our two recent studies on BZF showed (a) that BZF (10 and 20 mg kg⁻¹, p.o.) upon co-administration with Δ⁹-THC (10 mg kg⁻¹, p.o.) twice daily, in a 6-day regimen, significantly attenuated the development of tolerance and dependence of cannabinoids in mice (Dhawan et al., 2002d), and (b) also increased the sperm count, libido, and sexual fertility during 30-day administration in 2-year-old male rats (Dhawan et al., 2002b), and prompted us to evaluate the BZF moiety for its probably usefulness in ameliorating or inhibiting the detrimental gonadal effects of chronic treatment with Δ⁹-THC.

Methods

Plant material and the bioactive BZF moiety

Axial parts of cultivated P. incarnata at Rati Ram Nursery in the village of Khurammpur, Kalsia in the district of Saharanpur (UP, India) were picked in January 1999 and the identity was confirmed at the Department of Systematic Botany, Forest Research Institute, Dehradun (UP). A voucher specimen (code no. 1325/2000) was deposited in the Herbarium-cum-Museum of the Forest Research Institute, Dehradun, India. Studies were performed with the BZF moiety recently reported by us to have been isolated from this plant (Dhawan et al., 2001a, 2001b, c, 2001d, 2001e; Dhawan, 2002).

Animals

Healthy adult male albino rats of Wistar strain (200–230 g) procured from the Disease Free Small Animals House, Haryana Agriculture University, Hisar (Haryana) were bred at the Central Animal House of the Panjab University, Chandigarh. The experimental protocols were approved by the Institutional Ethical Committee of the Panjab University, Chandigarh. The animals were divided as:

1. Control group – given vehicle (sesame oil containing 1% w w⁻¹ Polysorbate 80)

2. THC group – given 10 mg kg⁻¹ Δ⁹-THC in vehicle

3. THC+BZF-10 group – given THC (10 mg kg⁻¹) concurrently with BZF (10 mg kg⁻¹)

4. THC+BZF-20 group – given THC (10 mg kg⁻¹ concurrently with BZF (20 mg kg⁻¹)

5. BZF-10 group – given 10 mg kg⁻¹ BZF moiety

6. BZF-20 group – given 20 mg kg⁻¹ BZF moiety

 All the treatments were administered per oral (p.o.) once a day for 30 days. Each treated group comprised eight male rats (n=8; however in THC group n=12). At the end of treatments, the treated male rats were paired with non-oestrous female rats for 15 min and the number of mounts was recorded to assess the male rats libido. On the 31st day the male rats were mated with fertile pro-oestrous female rats (1 : 3) in separate cages for each different group. The presence of spermatozoa in the vaginal plug was taken as a parameter for mating. The number of female rats who became pregnant, was considered to be a parameter of fertility.

Mounting behaviour at the end of treatments on day 30

To observe the libido-oriented mounting behaviour, non-oestrous female rats were paired with treated male rats. The male rat assuming the copulatory position over the female but failing to achieve intromission was considered as a mount (Subramoniam et al., 1997). Male rats from each group were randomly chosen and suitably marked. The rats were placed in a clear aquarium and allowed to acclimatize for 15 min. After that, a non-oestrous female was introduced into the arena. The number of mounts was recorded for 15 min.

Sperm count and fertilization determined on day 31

The spermatozoa were counted as per the method of Zaneveld & Polakoski (1997). Sperm suspension was placed on both sides of Neubauer's hemocytometer and allowed to settle for 1 h. The number of spermatozoa in the squares of the hemocytometer was counted under the microscope at 100×magnification. The relative sperm count after different treatments, and the female rats impregnated by male rats (3 : 1) after receiving various treatments were counted.

Determination of sexual behaviour upon BZF administration to THC-treated rats

After evaluating their sexual behaviour and sexual parameters, the 12 male rats of THC groups were further sub-divided into control, BZF-10 and BZF-20 groups. The control group animals were given vehicle, whereas the other animals were given 10 and 20 mg kg⁻¹ BZF, p.o., once a day. The rationale for doing this study was to determine the beneficial effects of BZF-administration in restoring sexual parameters after cessation of chronic treatment with cannabinoids. On the 7th day after cessation of these treatments the sexual behaviour and parameters were again evaluated.

Results

As shown in the Table 1, rats subjected to 30-days chronic THC-treatment incurred loss of libido which was evident from their mounting behaviour pattern. Chronic administration of THC to male rats also rendered them sterile (sperm count=72.8×10⁶), and these rats failed to impregnate any female rat. However, the concurrent administrations of THC with BZF for 30 days, afforded a significant protection of the loss of libido and sexual parameters in rats receiving these co-treatments. Male rats receiving 10 mg kg⁻¹ p.o. dose of BZF+THC for 30 days exhibited elevated sperm count, i.e., 381.1×10⁶, and impregnated 62.5% of the female rats with reference to the THC group. The male rats receiving THC and 20 mg kg⁻¹ dose of BZF exhibited still better results (sperm count=418.0×10⁶; and female rats impregnated=87.5%). Administrations of BZF (10 and 20 mg kg⁻¹, p.o.) for 30 days in male rats increased libido, sperm count and number of female rats impregnated (Last two rows of Table 1).

Table 1.

Mounting behaviour, sperm count, and mating performance after treatments for 30 days

Sexual parameters, with and without the co-administration of BZF (10 and 20 mg kg⁻¹ BZF) in chronic cannabinoid-treated rats were evaluated on day 7 (Table 2). Upon measuring the various sexual parameters on day 7, the male rats receiving BZF-10 exhibited improvement in the sexual parameters. However mice which were given BZF-20 exhibited very significant recovery (almost 100%) in sexual performance, libido, and sperm count, relative to the rats of the control group (these are originally the male rats who received THC for 30 days, as indicated in Table 1, and were again treated with vehicle only for the next 7 days) and were not given BZF at all during the entire experimental paradigm.

Table 2.

Sexual behaviour and parameters recorded on day 7 after treatment of THC group with BZF

Discussion

It is evident from the findings reported in Table 1 and 2 that the co-treatment of BZF+THC, significantly prevents the deleterious sexual-effects of chronic THC treatments in male rats. In addition to this, BZF also facilitates the early restoration of sexual parameters after cessation of chronic treatment with THC. In both studies, the 20 mg kg⁻¹ dose of BZF affords better results than the BZF-10 dose. The rats, which received only BZF (10 and 20 mg kg⁻¹), exhibited increased libido, increased sperm count, and had increased virility.

Chronic intake of cannabinoids (Block et al., 1991; Rosenkrantz & Esber, 1980) leads to severe detrimental effects on the sex hormones production (by increasing oestrogen levels). The increase in free oestrogens in males species causes loss of libido, enlarged prostrate, fatigue and loss of muscle tone. Further, the increased oestrogen levels cause the disruption of normal testicular production of testosterone, and saturation of testosterone receptors in the hypothalamus in brain, thereby reducing the signal sent to the pituitary gland which in turn reduces the secretion of LH (Kaliszuk et al., 1989). This ultimately reduces testosterone production in gonads. Increased oestrogen due to excessive cannabinoid-intake also increases the production of sex-hormone binding globulin (SHBG) (Sharpe, 1998). The SHBG binds testosterone and reduces the free testosterone in the blood. The mode of action of the BZF moiety is an interesting and simple neuro-steroidal phenomenon. The BZF moiety, reported for the first time from a plant source, is a very potent inhibitor (Kao et al., 1998; Kellis & Vickery, 1984) of the liver microsomal enzyme aromatase, which is a member of cytochrome P-450 family (Lee et al., 1994b). The aromatase (P-450 3A4) enzyme is accountable for the metabolism of more than half of the drugs in use today (Guengerich, 1999), including the sex steroids, e.g., testosterone (Lee et al., 1994a). A specific functional aromatase gene ‘CYP 19' is necessary for the expression of aromatase enzyme in mice and human beings (Robertson et al., 1999). The enzyme aromatase is instrumental in the metabolism of testosterone to estradiol (Wilson, 1995). The BZF moiety, the strongest aromatase-inhibitor, prevents the metabolic de-activation of testosterone to estradiol (Wilson, 1995). The BZF moiety, the strongest aromatase-inhibitor, prevents the metabolic de-activation of testosterone to estradiol and, thus, increases free testosterone and decreases free oestrogen (Chen et al., 1997; Merken & Beecher, 2001). The increased plasma-testosterone levels directly influence the gonadotropins (luteinizing LH and follicle-stimulating-hormone FSH), which regulate spermatogenesis and maturation of sperms (Crémoux, 2000). High levels of testosterone in male species mean sexuality, increased libido, mental energy, stamina and virility.

The BZF moiety isolated from aerial parts of P. incarnata acts: (a) through its anti-aromatase function, and (b) by eliminating oestrogen's negative feedback loop (Chen et al., 1997). The concomitant administration of BZF with THC is postulated to maintain a high level of testosterone in the blood by stopping the aromatization of testosterone to oestrogen. Secondly, BZF may also facilitate the body to produce more testosterone by eliminating the so-called ‘negative feedback' loop, that otherwise reduces natural testosterone production. High testosterone accounts for an increase in sperm count, fertility, as well as sexual drive. Thus, from these studies the authors feel encouraged that the novel BZF moiety, being a highly potent anti-oxidant and the strongest reported aromatase inhibitor, affords significant prevention against the deleterious effects of substances like cannabinoids upon male sexuality, vigour and fertility.

Abbreviations

BZF

tri-substituted benzoflavone

THC

delta-9-tetrahydrocannabinol