Protective Role of Magic Fruit and Honey Bee against Human Hepatocarcinogenesis

Background: Hepatitis C is an infectious disease that affects the liver. Its complication is a major high-risk group that may be lead to hepatocellular carcinoma HCC, so natural prevention is required. The combination of magic fruit and honey bee was used in the treatment of HCV patients were treated with 4 g from Magic fruit and 1 g from Honey bee 3 times daily for three months. Methods and findings: Our group study was conducted on 50 patients with chronic hepatitis C (the male number was 35 while the female was 15; the median age was 45 years) was taken from the outpatient clinics of NCI, Egypt. Results: The mean values of virus C level, which was determined by real time PCR and FoxP3 protein which was measured by ELISA in sera of patients with chronic hepatitis infection (CHI), showed highly significant decrease after treatment. Hence the results have shown improvements in liver function, kidney function, and CBC tests for the HCV patients post-treatment. Moreover, the results revealed also that, highly significant decreased of CD4 + CD25 expression by flow cytometric analysis was observed in the mononuclear leukocytes isolated from the patients after treatment. Conclusion: A combination of magic fruit and honey bee could be useful protective natural agents against human hepatocarcinogenesis induced by HCV infection. Further study is strongly recommended for large populations and prolonged treatment for 9-12 months.


Introduction
Hepatitis C virus (HCV) attacks the liver and leads an inflammation [1]. 3% of the world's population liver injure is infected with HCV as mentioned in recent WHO report; which was estimated that more than 170 million chronic carriers are developing liver cirrhosis and/or liver cancer [2].
HCV symptoms rarely occur in infected population; as dark urine, yellow tinged skin, fever and abdominal pain are occurring. 75% to 85% of chronic infection patients has no symptoms, hence leads to liver disease and cirrhosis [3,4] while cirrhotic patients will be developing other complications as liver failure, liver cancer [5].
Magic fruit (Ziziphus jujube) strengthens the liver and is used by mixing with their salivary and enzymatic secretion [9]. Honey is well known as an antifungal, antibacterial and antioxidant behavior because of phenolic compound [10]. The main constituents of honey are 50% fructose and 44% glucose [11] which gives the sweety flavor, while the remaining constituents of honey, chrysin, pinobanksin, catalase, pincocembrin and vitamin C which acting as an antioxidant [12].

Patients
The present study has been conducted on a total of 50 patients, all with chronic hepatitis C (The male number was 35 while the female was 15; The median age was 45 years) taken from the outpatient clinics of the NCI-Cairo University in Egypt. Blood samples were obtained during the time from July 2014 to January 2015. Treatment the patients who have (virus C) by taken them 4 g from Magic fruit and 1 g from Honey bee 3 times daily for three months. Blood samples were collected from patients before and after treatment. Serum samples were permitted to clot for 30 min at 37°C [13,14]. Then, they were centrifuged at 3000 RPM for 10 min. The sera were collected and divided to several aliquots and were stored at -20°C until starting our tests. According to Giardina's recommendation, the AFU activity levels were done within 30 days after collection [15].
HCV-test, HCV PCR analysis, FoxP3, and T-regulatory cell (CD4, CD25) in HCV patients have been performed before and after treatment.
HCV-PCR levels were quantitatively determined by instrument of Applied Biosystems (AB) step one real-time PCR system (Singapore), while CD4, CD25 were analyzed by instrument of Flow Cytometery NAVIOS Flow Cytometery. Beckman Coulter (USA). The biochemical parameters as CBC, Liver function tests, kidney function tests were done by integrated automation in clinical laboratories in NCI.

Statistical Analysis
Data were expressed as Mean ± SD. The SPSS program version 15 was used in analysis. One-way analysis of variance (ANOVA) followed by Duncan post hoc test and/or t-test were used in analysis. Chi-square test was used for categorical data. Pearson correlation coefficient was used to study correlations. The percent change was calculated as (values after treatment -values before treatment)/values before treatment × 100, and tabulated as absolute values. P-values less than 0.05 were significant.

Study population
Our study was done on 50 HCV patients from the outpatient clinics at the NCI, Egypt in the period between 2014-2015. They included 35 males and 15 females (66% / 34%). The age range was between 23 and 65 years with a median of 45 years.

Effect of treatment on HCV RNA/PCR (quantitative)
The median PCR was 0.53 × 10 6 before treatment, a value decreased to 0.12 × 10 6 after treatment with a percent reduction of 63.94 ± 27.51% ( Table 1).

Effect of treatment on human Fox 3p expression
The mean human Fox 3p after treatment, was 244.43 ± 107.6, that highly significantly decreased after treatment, 33.80 ± 31.14 (p<0.001) with a percent decrease of 85.67 ± 13.08% (Table 2)

Effect of treatment on human CD4CD25 expression
The same was observed for CD4CD25 where the mean expression was 21.43 ± 8.63 before treatment, a value significantly decreased to 9.13 ± 3.84 (P<0.001) after treatment with a percent reduction of 54.55 ± 18.57% (Table 3).

Effect of treatment on liver function tests
After treatment with the magic fruit and honey bee, highly significant decreases were detected for bilirubin, ALP, ALT, AST and AFU (p<0.001).

Effect of treatment on kidney function tests
After treatment with magic fruit and honey bee, highly significant decreases were detected for creatinine and urea (p<0.001 for each) whereas no significant effect was detected for uric acid level (p=0.308). The percent of reduction of creatinine and urea were 25.19 ± 13.8 and 22.48 ± 12.3, respectively ( Table 5).

Effect of treatment on blood picture
After treatment with magic fruit and honey bee, highly significant increasing was detected for RBCs and platelets count as well as   Table 6 Non-parametric correlations between PCR, FoxP3 and CD4CD25 in patient before and after treatment.

Archives in Cancer Research ISSN 2254-6081
We observed that; there are correlations between PCR, Fox P3 and CD4 CD25 before and after treatment. There is positive correlation for PCR and Fox P3 before treatment (R=0.377, P<0.01), while a negative correlation was observed between PCR before treatment and CD4 CD25 after treatment (R=-0.359, P, 0.01).

Discussion
The researchers were emphasized a strong association of cirrhosis and HCC due to chronic viral hepatitis. Although the use of surgery, chemotherapy and radiotherapy have led to increasing the survival rates for patients with hepatocellular carcinoma, there are still problems of the marked side effects of the previously mentioned treatments in addition to cancer metastasis that sometimes occurs among those patients. Most cases of HCV infection are asymptomatic and clinical presentation is mild. Resolution of the infection occurs 3-4 months after, 70% of cases do not resolve but progress to chronic hepatitis [16].
Saeed in 1991 was the first to report in Saudi Arabia the very high incidence of HCV-Ab among Egyptian blood donors, which was 19.5%. Darwish in 18 reported that the incidence was 15% among healthy blood donors. Yet, Kamel in 1992 reported that the incidence is 6% among health care workers. The prevalence of HCV in Egyptian blood donors was reported to be the highest among the world [17][18][19][20].
Magic Fruit (Ziziphus jujube): In the last few decades there has been an exponential growth in the field of Herbal medicine [21]. Our result elucidates the protective ability of Z. jujube against hepatic injury and chronic inflammation that was confirmed in previous study [22].
Z. jujube showed significant protection against CCl4 intoxicated HepG2 cell lines by means of increased cell viability and decreased LDH levels compared to control group. It was reported for anticancer, anxiolytic, anti-complementary and hypoglycemic activities [23][24][25][26]. Jujuba methanol extraction with different concentrations inhibited the lipid peroxidation and significantly restored the liver function markers (Ast, Alt, Alp, LDH) IN Acute liver damaged) [27].
Previous study demonstrated the role of honey supplementation that is improving the immunological behavior which gives a useful result that prevents hepatic damage which caused by obstruction of the common bile duct.
The present work represents the first trial carried out in Egypt to show the effects of the mixture of magic fruit and honey bee on the hepatocarcinogenesis as well as the level of PCR, T-regulatory cell (CD4, CD25), and Fox P3 gene expression. The results showed the highly significant decrease in (PCR, T-regulatory cell (CD4, CD25), and Fox P3 gene expression) level after treatment by mixture of magic fruit and honey bee. The results also showed that the activation of virus-C has been significantly decreased after treatment by the mixture than before treatment indicating the possibility of using the mixture as an inhibitor the activation of virus-C in patients to be active.

Conclusion
In conclusion, the present work casted some light on the probability of using the mixture of magic fruit and honey bee in the natural therapy of virus C through the activation of immune system and decreasing the level of (PCR, T-regulatory cell (CD4, CD25), and Fox P3 gene expression). Future studies are recommended to examine the possibility of using the mixture in the treatment of other types of cancer.