Preclinical evaluation of lime juice as a topical microbicide candidate.

BACKGROUND: The continued growth of the global HIV epidemic highlights the urgent need to develop novel prevention strategies to reduce HIV transmission. The development of topical microbicides is likely to take a number of years before such a product would be widely available. This has resulted in a call for the rapid introduction of simpler vaginal intervention strategies in the interim period. One suggested practice would be vaginal douching with natural products including lime or lemon juice. Here we present a comprehensive preclinical evaluation of lime juice (LiJ) as a potential intervention strategy against HIV. RESULTS: Pre-treatment of HIV with LiJ demonstrated direct virucidal activity, with 10% juice inactivating the virus within 5 minutes. However, this activity was significantly reduced in the presence of seminal plasma, where inactivation required maintaining a 1:1 mixture of neat LiJ and seminal plasma for more than 5 minutes. Additionally, LiJ demonstrated both time and dose-dependent toxicity towards cervicovaginal epithelium, where exposure to 50% juice caused 75-90% toxicity within 5 minutes increasing to 95% by 30 minutes. Cervicovaginal epithelial cell monolayers were more susceptible to the effects of LiJ with 8.8% juice causing 50% toxicity after 5 minutes. Reconstructed stratified cervicovaginal epithelium appeared more resilient to LiJ toxicity with 30 minutes exposure to 50% LiJ having little effect on viability. However viability was reduced by 75% and 90% following 60 and 120 minutes exposure. Furthermore, repeat application (several times daily) of 25% LiJ caused 80-90% reduction in viability. CONCLUSION: These data demonstrate that the virucidal activity of LiJ is severely compromised in the presence of seminal plasma. Potentially, to be effective against HIV in vivo, women would need to apply a volume of neat LiJ equal to that of an ejaculate, and maintain this ratio vaginally for 5-30 minutes after ejaculation. Data presented here suggest that this would have significant adverse effects on the genital mucosa. These data raise serious questions about the plausibility and safety of such a prevention approach.

Disturbances in Calcium and Zinc Homeostasis During Testicular Damage Induced by Citrus aurantifolia Juice in Wistar Rats.

Infertility rate is high globally and in Nigeria. The reported spermicidal activity of Citrus aurantifolia juice (CAJ)and its popular consumption may be a contributing factor to the rise in male infertility. This study examined the effects ofCAJ on testis and evaluated the role of calcium and zinc in these effects. Twenty-eight male rats (200-220g) were groupedinto four (n=7). Group I (control) received 0.5ml normal saline, while groups II, III and IV received 600mg/kg, 900mg/kgand 1200mg/kg of CAJ, respectively, orally for 35 days. Sperm analysis, testicular histology, testicular zinc and calciumconcentrations were evaluated. The results showed a significant decrease (P < 0.001) in body weight and gonad-somaticindex (GSI) of the rats in group IV. No sperm cells were found in the sperm samples of all the treatment groups in contrastto control. There was a significant decrease (P < 0.001) in zinc concentration of group III and IV animals and a significantincrease (P < 0.001) in testicular calcium content of group III and IV animals. Derangement of testicular cyto-architecture,shrinkage or complete destruction of seminiferous tubules as well as absence of spermatogenic cells were observed in thetreatment groups. It was concluded that CAJ induced a destructive effect on testes of rats as evidenced by damaged testiculartissue, reduced gonado-somatic index, azospermia and disruption in testicular electrolyte homeostasis. It was concluded thatCAJ caused hypercalcaemia and hypozincaemia in the testicular tissue of the treated rats. Concurrently, CAJ also causeddamage to testicular histology, azospermia and decreased GSI. Citrus aurantifolia juice should be consumed with cautiondue to its potential to cause infertility in males.

The tumor promoting effect of lime-piper betel quid in JB6 cells.

Betel quid chewing is a general behavior in Taiwan, India, southeastern Asian and South Africa. In this study, microculture tetrazolium test (MTT) showed that the extract of lime-piper betel quid (LPB) (1.0-20 mg/ml) was toxic to JB6 cells. Cells exposed of LPB (0.1, 0.5, 1.0 mg/ml) for 7 days resulted in changes in cytomorphology with characteristics of carcinogenesis. With a long-term treatment (approximately 30 days) of low doses of LPB (1, 5, 10 microg/ml), the production of H2O2 and the activity of myeloperoxidase (MPO) and ornithine decarboxylase (ODC) were increased in JB6 cells. Cell cycle analysis showed a decrease in the G1 phase and an accumulation in the S phase 48 h after LPB treatment. When treating with 0.5 mg/ml LPB for 15 days as a promoter, type III foci were formed in the JB6 culture. These results demonstrated the tumor promotional effect of LPB in JB6 cells.

The tumorigenic characteristics of lime-piper betel quid-transformed JB6 cells.

Betel quid chewing is a general oral habit in Taiwan, India, southeastern Asian and South Africa with or without the additive of tobacco, alcohol or lime. In this study, the tumor-promoting neoplastic transformation effect of Lime-Piper betel quid (LPB) was examined on JB6 cells. The treatment of LPB at a high dose (1.0 mg/ml) for over 5 days or at lower doses (0.1, 0.5 mg/ml) for over 15 days induced the formation of transformed foci. The transformed cells showed the characteristics of colony formation in soft agar, higher growth rate and multilayer on culture dish. A two-fold induction of the protein levels of c-fos and c-jun proto-oncogenes was observed in the cells from the 50th passage (Cl1/p50, Cl2/p50 and Cl3/p50), suggesting that LPB-transformed cells were oncogenic. In addition, the LPB-transformed cells possessed an elevated level of c-Myc and an increased cell population distributed in the S phase of the cell cycle. These results demonstrated the promotion effect of LPB and indicate that it could be a tumor promoter.