HEPATOPROTECTIVE EFFECT AND GC-MS ANALYSIS OF TRADITIONALLY USED BOSWELLIA SACRA OLEO GUM RESIN (FRANKINCENSE) EXTRACT IN RATS

Background: The water extract of oleo-gum-resin of Boswellia sacra Fleuckiger (Burseraceae) is used in the treatment of liver problems in the Middle East and Arab-African countries. The present study was carried out to evaluate its effect on liver injury induced by carbon tetrachloride in acute and chronic liver injury models. Materials and Methods: The Boswellia sacra extract was administered at doses of 2 ml/kg and 5 ml/kg. Silymarin, a known hepato-protective agent was used as standard. The extract was subjected to GC-MS analysis to determine the presence of various phyto-constituents. Results: The Boswellia sacra extract at tested doses of 2 ml/kg and 5 ml/kg significantly reduced the elevated serum levels of biomarkers in liver damage. The hepatoprotective effect was supported by changes in histopathology. The GC-MS analysis of the extract revealed the presence of several phyto-constituents that included menthol, 3-cyclohexen-1-ol and octanoic acid. Conclusion: The water extract oleo-gum-resin of Boswellia sacra possesses hepatoprotective activity, as claimed by traditional healers within the Middle East and Arab-African countries.


Introduction
The treatment of liver diseases with herbal drugs is a common practice across the globe, more so in developing countries.Several herbs, crude and standardized herbal extracts and isolated active components are used for the treatment of liver damage.It is estimated that around 700 herbal formulations from 100 plants are in clinical use (Roy et al., 2012).
Most of these herbal extracts/formulations have been tested scientifically to verify claimed benefits on liver function.We had earlier reported hepatoprotective effects of Psidium guajava (Roy et al., 2006), Boswellia serrata (Jyothi et al., 2006) and Tectona grandis (Majumdar et al., 2007) that are used widely for treatment of liver disorders.However, there are other herbal products that are marketed specifically for the treatment of 'liver problems', but their effectiveness has not ascertained through either experimental or clinical studies.One of such product is the water extract of Boswellia sacra Fleuckiger (Burseraceae) oleo gum resin (frankincense), known in Arabic as Luban.It is widely used in the Middle East and Arab-African countries for the treatment of cough, stomach ache and liver problems.Furthermore, the Boswellia oleo gum resin (frankincense) is also chewed as chewing gum for its essence.The Boswellia sacra oleo gum resin and its products are marketed by several companies throughout the world (www.alibaba.com,2014; frankincense, 2014; Boswellia sacra, 2014).
The present study was undertaken to determine the effect of administration of water extract of Boswellia sacra on carbon tetrachloride induced liver damage in rats.

Materials
The water extract of Boswellia sacra oleo gum resin was purchased from the market.The most widely used brand in Saudi Arabia (Luban-e-hakim) was used in the present study.The extract was a clear solution with a very strong essence similar to that of dried Boswellia sacra oleo gum resin.Preparation of water extract by traditional method in our laboratory, which involves boiling the oleo gum resin with water in a closed jar or soaking the Boswellia sacra oleo gum resin in water overnight did not produce an extract with such strong odorous extract.Hence, the marketed extract was used in the present study.All other chemicals used were of analytical grade from different companies.

Animals
Wistar albino rats of either sex weighing between 180-210 g were used.The animals were maintained under standard conditions of 12:12 hr light dark cycle at a temperature of 25±2 0 C. The experimental protocol was approved by the research committee of the institute for its ethical and scientific content.

Preliminary Phytochemical Analysis
The extract was subjected to preliminary phytochemical analysis to detect the presence of phyto-constituents such as carbohydrates, proteins, amino acids, saponins, tannins and phenolic compounds, flavonoids and glycosides.

Gas Chromatography-Mass Spectroscopy (GC-MS) Analysis of Extract
For performing the GC-MS analysis, the water extract of Boswellia sacra was extracted using dichloromethane and this extract was injected into GC-MS system (Shimadzu-QP2010S).For GC-MS detection, an electron impact ionization system was used.Helium gas (99.95%) was used as a carrier gas at a constant flow rate of 1 ml/min, and an injection volume of 1 μl was employed (a split ratio of 10:1).The injector temperature was maintained at 250 °C, the ion-source temperature was 200 °C, the oven temperature was programmed from 60 °C (isothermal for 2 min), with gradual increase to 325°C at 15 min.Mass spectra were taken at 1.4 kV.The relative percentage amount of each component was calculated by comparing its average peak area to the total areas.Interpretation of mass-spectrum GC-MS was conducted using NIST, WILEY and SZTRIP libraries.The spectrum of the unknown components was compared with the spectrum of known components.

Evaluation of Hepatoprotective Activity
Carbon Tetrachloride (CCl 4 ) Induced Acute Hepatic Damage (Roy et al., 2006) The animals were divided into five groups consisting of six animals and treated as follows; Group 1: Vehicle (1 ml/250 g, p.o.) Group 2: Distilled water for 9 days + CCl 4 (1 ml/kg, p.o.) on ninth day Group 3: Silymarin (100 mg/kg, p.o) for 9 days + CCl 4 (1 ml/kg, p.o.) on ninth day Group 4: Boswellia sacra extract (2 ml/kg/day, p.o.) for 9 days + CCl 4 (1 ml/kg, p.o.) on ninth day Group 5: Boswellia sacra extract (5 ml/kg/day, p.o.) for 9 days + CCl 4 (1 ml/kg p.o.) on ninth day The CCl 4 was administered after dilution with liquid paraffin the ratio of 1:1.Food was withdrawn 12 hr before carbon tetrachloride administration to enhance liver damage in animals of groups 2, 3, 4 and 5. Blood samples were collected 24 hr after the administration of CCl 4 .and serum was used for assay of marker enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and serum bilirubin.All the biomarkers were determined using commercially available kits.The animals were then sacrificed; liver was isolated, washed with normal saline, dried and weighed immediately.The liver was then subjected to histo-pathological examination.

Statistical Analysis
Values are expressed as mean ± standard error of mean (SEM).Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey's test for comparison of all parameters.The statistical analysis was done using computer software (Graphpad lnstat DATASET 1, ISD, software version 3.0 for Windows).Values of p<0.05 were considered to indicate statistical significance.

Results
The water extract of Boswellia sacra oleo gum resin did not shown presence of any of the tested phyto-constituents indicating that it contains only essential oils.

Evaluation of Hepatoprotective Activity Carbon Tetrachloride (ccl 4 ) Induced Acute Hepatic Damage
Administration of CCl 4 produced a significant increase in all the biomarkers of liver damage in serum when compared to vehicle control.Both doses of Boswellia sacra (2 ml/kg and 5 ml/kg) and silymarin (100 mg/kg p.o.) significantly reduced the levels of ALT, AST, ALP and bilirubin when compared to CCl 4 treated control (Table 3).Liver sections from CCl 4 treated animals showed severe liver damage indicated by hydropic degeneration, inflammation and steatosis in periportal region.In animals treated with extract or silymarin, inflammation, steatosis and congestion were reduced though it was difficult to differentiate the effect produced by silymarin with that produced by both doses of the extract.All values are mean ± SEM, n=6, *** P<0.001 compared to control, # P<0.05, ### P<0.001 compared to CCl 4 treated control.

Carbon Tetrachloride (CCl 4 ) Induced Chronic Hepatic Damage
A significant difference in biochemical markers, AST, ALT, ALP and bilirubin was observed between normal and CCl 4 treated animals (Table 4).Comparison of Boswellia sacra and silymarin treated groups with CCl 4 control revealed that both doses of Boswellia sacra extract (2 ml/kg and 5 ml/kg) and silymarin significantly reduced the hepatic damage as indicated by reduction in biochemical parameters and histological studies similar to that mentioned above.

Discussion
The water extract of Boswellia sacra oleo gum resin reduced the serum enzyme levels indicating good hepatoprotective activity at both tested doses.However, the effect was less compared to that of the standard drug; silymarin.As mentioned earlier, the Boswellia sacra oleo gum resin, commonly known as frankincense or incense is widely used in different parts of the world.In the Middle East and Arab-African countries, it is chewed as chewing gum because of its flavor, burnt and used as incense for its smell apart from its used for treatment of diseases.The water extract of Boswellia sacra oleo gum resin is a house hold medicine used for the treatment of cough, stomach problems and liver problems.The word 'liver problems' is confusing and discussion with traditional healers revealed that liver problems include; jaundice, liver infection and other liver diseases.
There are several methods for the preparation of the water extract of frankincense.The frankincense covered in a muslin cloth or a wire mesh is boiled with water in closed vessels for 15-30 min and the water extract is used after cooling.Another method used is soaking the frankincense in water overnight followed by filtering.Some people add few drops of frankincense oil to water and use it (Nancy Healing, 2014;

Figure 1 :
Figure 1: GC-MS chromatogram of water extract of Boswellia sacra

Table 1 :
List of major chemical constituents present in the water extract of Boswellia sacra

Table 2 :
Mass spectra of major constituents in Boswellia sacra water extract Compound name

Table 3 :
Effect on serum enzyme levels in CCl 4 induced acute liver damage