Effects of the Mixture of Melissa officinalis L., Cinnamomum zeylanicum and Urtica dioica on Hepatic Enzymes Activity in Patients with Nonalcoholic Fatty Liver Disease

MATERIALS AND METHODS

Plant material: The aerial parts of Melissa officinalis L. and Urtica dioica were collected in August 2011 from Botanical Garden of Arak University and identified as Melissa officinalis L. and Urtica dioica by Dr Salehi Arjmand from Department of Medicinal Plant, Faculty of Agriculture, Arak University. Cinnamon was supplied by Arak Medicinal Plants Company and identified as Cinnamon Zeylanicum. The leaves of Melissa officinalis L. and Urtica dioica were dried in shade at room temperature for 12 days.

Subjects: Thirty-five adults aged 26-71 years (15 men and 20 women) were randomly selected from patients who referred to the interdisciplinary ultrasound department of the Hospital of Shiraz University in Lar in the year 2011 for sonographic examination of the abdomen. After being recognized as belonging to the second stage of NAFLD, they were included in the study provided that they did not have a history of coronary artery disease, insulin-dependent diabetes, a bleeding diathesis, severe anemia, cancer within the past 5 years, any condition likely to lead to death within 5 years, use of anti-coagulants, cyclosporine and so none of the patients had suffered from viral hepatitis or autoimmune-related disorders or reported alcohol consumption. All participants were provided with specific written information about the aims of the study before written consents were obtained, in accordance with ethical rules of Pharmaceutical Sciences Research Center (PSRC) of Tehran University of Medical Science where the study protocol was approved. Prior to blood collection, each individual was extensively interviewed by a specialized physician who filled in a structured questionnaire about disease and habit diet. The included subjects were administered Lemon balm, Cinnamon and Urtica dioica mixture infusion (1.5, 0.5 and 0.25 g/100 mL) twice daily for 30 days at 7.5 am and 2 pm every day. Doses were obtained from our previous studies (Malekirad et al., 2011; Ranjbar et al., 2007; Mehri et al., 2011). A supervisor carefully checked to make sure that the volunteers were taking infusion properly. Blood samples were collected from all subjects before using mixture infusion and 12 h after the last dose of 30-day treatment with infusion.

Infusion preparation and protocol: Leaves of Lemon balm, Urtica dioica were dried and cleaned and then packed with Cinnamon in 1.5, 0.5 and 0.25 g bags. The subjects were instructed how to prepare the infusion by mixing a total of bags in 100 mL 98°C water for 30 min. A qualified expert supervised the whole procedure.

Biochemical analysis of serum parameters: All biochemical serum analyses were performed in the same laboratory including aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP).

Statistical analysis: Results are presented as Mean±SD. Statistical analyses were conducted using Stats Direct 2.7.8 software. The paired t-test and Wilcoxon Matched-Pairs Signed-Ranks Tests were applied. Relationships between parameters were determined by use of Pearson correlation analysis. The p-value of less than 0.05 was considered statistically significant.

RESULTS

The Mean±SD values for age and weight of subjects were 42.43±10.54 and 80.09±9.97, respectively. Of subjects, 15 (42.86%) were male and 20 (57.14%) were female.

After using the mixture infusion, ALT significantly (p = 0.001) reduced. The before and after Mean±SD were 133.49±76.65 and 114.08±66.98, respectively. A decrease in AST was observed after administration of infusion (119.43±154.18 before versus 87.8±68.46 after) but the decrease was not significant (p = 0.17). A decrease (p = 0.12) in ALP was observed by use of infusion.

Table 1:	The effect of infusion of Lemon balm, Cinnamon and Urtica dioica mixture on liver enzymes parameters

Table 2:	Correlation between demographic factors and liver enzymes parameters

The Mean±SD of before and after use of infusion were 113.03±103.71 and 91.29±41.63 (Table 1).

Analysis by Wilcoxon Matched-Pairs Signed-Ranks test showed a significant decrease in grade of sonographic examination (p = 0.003). A significant linear correlation was found between age and AST and ALT (r = 0.48, p = 0.003 and r = 0.4, p = 0.017, respectively) (Table 2). However, no linear correlation was found between gender and weight and liver enzymes.

DISCUSSION

In this study, the effect of Cinnamon, Melissa officinalis L. and Urtica dioica infusion on in NAFLD was tested and showed that the activity of ALT, AST and ALP decreased but only the decrease in ALT was statistically significant. Moreover, a significant linear correlation was found between age and AST and ALT. On the other hand, the ratio of AST/ALT after the treatment was decreased that is a good result.

Newly, the AST/ALT ratio greater than 1 is considered as a marker of disease severity (Clark, 2006). Moreover, NAFLD causes elevation of serum transaminase but ends up in fibrosis, cirrhosis and eventually hepatocellular carcinoma (Rodriguez et al., 2010). Of course, limited histological data support the association between improved aminotransferases and biopsy findings which require confirmation in a double-blind trial with appropriate statistical power based on liver histology (Bugianesi et al., 2005). The relationship between age and AST and ALT is reasonable since the rate of metabolic syndrome increases with age. Also, ALT is more specific to disorders of liver parenchyma cells.

Moreover, some studies reported that antioxidant therapy has beneficial effect on NAFLD or NASH. For example, one study indicated that Insulin sensitizing agents such as pioglitazone and anti-oxidant agents like vitamin E or IMOD^® help improving liver histology in patients with NASH (Smith and Adams, 2011; Rahimi et al., 2012). Other antioxidants such as N-acetylcysteine, betaine, etc. were found beneficial in the treatment of NASH (Mehta et al., 2002). Although, many pilot trials for antioxidants and lipid-lowering and hepatoprotective agents have yielded promising initial results in improving liver enzymes or features of liver histology (Park, 2008) but some studies do not confirm benefit of antioxidant on reduction of ALT in patients with pediatric NAFLD (Lavine et al., 2011).

However, the role of antioxidants in protection of liver seems important. Many experimental studies show that Urtica dioica has a protective effect on the liver but human studies are rare (Mehri et al., 2011). Kandis et al. (2010) reported that Urtica dioica has a protective effect on the liver in hepatic ischemia-reperfusion-injured rats and histopathological examination showed that liver tissue damage was significantly decreased in Urtica dioica group in comparison to control group (Kandis et al., 2010). Moreover, other results indicated that ethanolic and water extracts have more potent hepatoprotective action (Moselhy and Ali, 2009). Other study showed that administration of Urtica dioica extracts can cause a little modulating in the main morphometric indices of liver such as area of hepatocytes, nuclei and nucleolus in periportal and perivenous zones (Golalipour et al., 2009). Ozkol et al. (2011) reported that almost all doses of Urtica dioica prevents toxicity of cisplatin by decreasing AST, ALT, as well as increasing the reduced glutathione content, SOD, CAT, glutathione S-transferase and GPx.

On the other hand, it was shown that Cinnamon act as a hepatoprotective and reduces serum ALT, AST in rats (Amin and Abd El-Twab, 2009). Cinnamon extract was found as a potent hepatoprotective that elevates serum AST and ALT in rat CCl₄-induced liver damage model (Moselhy and Junbi, 2010).

The effects of Melissa officinalis L. extract on hyperlipidemic rats has been also studied. The Melissa officinalis L. extract reduced serum total cholesterol, total lipid, ALT, AST and ALP levels and RES in liver tissue. Moreover, it increased glutathione levels in the tissue (Bolkent et al., 2005).

Taken together, it seems that the present mixture is full of phenolic compounds with a lot of scavenging properties that result in improvement of NAFLD.

CONCLUSION

The consumption of the present mixture is beneficial for NAFLD patients. Conducting multi-center clinical trials with higher sample size is recommended.

ACKNOWLEDGMENT

The authors would like to thank all subjects and authorities of the Grash Hospital in Lar for their kind cooperation.