Phytochemical Screening and In Vitro Hepatoprotective Activity of Japanese Glorybower (Clerodendrum japonicum) Leaves

Introduction

Herbs are used traditionally worldwide for the deterrence and therapy of liver diseases.¹ The hepatoprotective potential of the herbs and their components is supported by several scientific studies.² Clerodendrum japonicum, commonly known as Japanese Glorybower, a member of family Verbaneaceae, is distributed in different parts of the world.³ The ethnomedicinal importance of this plant includes its use as a remedy for leucorrhoea, menstrual disorders, jaundice, wound healing, impetigo, arthralgia, 10 10 osteodynia, lumbago, hypertension, gonorrhoea, haematochezia, insomnia, and for its antibacterial and anti-inflammatory properties.^4-6 Few phytochemicals such martinoside, monoacetyl martinoside, acetoside and clerodenoside A have been identified in this plant.⁷

Materials and Methods

Collection

Clerodendrum japonicum leaves were procured from Nepal and authenticated by Dr. R. Nijagunaiah of Bangalore University. The Voucher specimen was preserved (PESCP/PCOG/VB2) in our laboratory.

Extraction

The dried leaf powder was successively extracted using non-polar to polar solvents. The extracts were dried and used.

Phytochemical investigation

Phytochemical analysis was done using the standard screening procedures.^8-10

Cytotoxicity study

The study was conducted using the HepG2 cell line with 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay.¹¹ Extracts were prepared in concentrations ranging between 10 µg/mL to 320 µg/ mL and cultured using Dulbecco’s Modified Eagle’s Medium (DMEM). HepG2 cell line was obtained from ATCC (American Type Culture Collection). The cell suspension was diluted to 50,000 cells/well and 100 µL of this suspension was placed in microtiter plate well, followed by 100 µL of various test concentrations of extracts after 24 hours. Doxorubicin (100 µM) was used as control. The plates were kept for incubation at 37°C for 24 hrs in 5% CO₂. Later the test contents were removed from the wells, 100 µL of Methylthiazolyl diphenyl tetrazolium bromide solution was added and again incubated for four hours in controlled conditions. The clear liquid was discarded and 100 µL of dimethyl sulphoxide (DMSO) was added. The plate was gently shaken. The resultant mixture was checked at 590 nm for absorbance and percentage inhibition was calculated as follows.

Percentage inhibition = ((A Control - A Sample) / A Control) X 100

The IC₅₀ values were generated from the dose-response curves.^12,13 The viability was observed based on morphology of HepG2 cells.

In vitro hepatoprotective activity

Hepatoprotective activity was assessed using HepG2 cell line culture.¹⁴ The samples with non-toxic concentrations were checked for hepatoprotective activity. Samples were prepared in concentrations of 0.312 µg/mL to 20 µg/mL using DMEM media for treatment. 100 µL of diluted cell suspension with 50000 cells/well was added to a 96 well microtiter plate and the incubation conditions of 37°C, 5% carbon dioxide were maintained for 24 hrs. Hepatotoxicity in HepG2 cells was induced by Paracetamol (100 μM) and was incubated for four hours. After four hours, 100 µL of samples were added to each well and again incubated at same conditions for 24 hrs. Then the contents in the wells were discarded, 100 µL of MTT was added to each well and again incubated for four hours. After the removal of supernatant liquid, the well was filled with 100 µL of DMSO. The contents in the plates were thoroughly mixed. The absorbance of the test and the standard was measured at 590 nm. The percentage growth inhibition and proliferation were calculated as follows:

% Inhibition = (OD of Control - OD of sample/OD of Control) x 100

OD - Optical Density

Results

Extraction

The percentage yield of the different extracts obtained by the sequential solvent extraction is shown in Table 1.

Phytochemical investigation

The leaves of Clerodendrum japonicum revealed the presence of phytosterols, terpenoids, phenolics, tannins, f lavonoids, carbohydrates and saponins (Table 2).

Cytotoxicity study

Optical density values of the extract and standard are shown as percentage of cell growth inhibition. At 320 µg/mL concentration, the ethanolic extract of C. japonicum showed 38.50% growth inhibition indicating lower cytotoxicity of alcoholic extract compared to the other extracts (Table 3). The morphological changes in the HepG2 cells for control (untreated), Doxirubin treated and test (treated with 10 µg/mL and 320 µg/mL concentrations of extracts) are shown in Figure 1. The ethanolic extract showed less morphological changes in the HepG2 cells (Figure 1g & 1h).

In-vitro hepatoprotective activity

Ethanolic extract of C. japonicum showed 35.99% of cell viability at 20 µg/mL in HepG2 cells against paracetamol induced toxicity which was better compared to the other extracts at 20 µg/mL. Standard Silimyrin showed hepatoprotective effect of 66.58% at 12.5 µg/ mL in HepG2 cells (Table 4).

Discussion

Preliminary phytochemical analysis of leaves of Clerodendrum japonicum indicated the contents of phytosterols, terpenoids, phenolics, tannins, flavonoids, carbohydrates and saponins. Phenolics and flavonoids being the large groups of phytochemicals possess wide range of therapeutic properties like antioxidant, antimutagenic, anticarcinogenic activities, protective action against cardiovascular diseases, and age-related degeneration of cell components.^15,16 HepG2 cell lines have been suggested as a substitute to human hepatocytes for in vitro models owing to their drug metabolizing enzyme activities.^17,18 HepG2 cell line has also been extensively utilized for assessing various properties such as cell protective, antioxidative, hypocholesterolemic, hepatoprotective, homeostatic and anti-insulin resistant.¹⁹ In the present study, the ethanolic extract of C. japonicum showed 35.99% of cell viability at 20 µg/mL in HepG2 cells against paracetamol-induced toxicity which was better than the other extracts at 20 µg/mL.

Conclusion

The present study findings in terms of in vitro hepatoprotective activity suggest that the ethanolic extract is effective in bringing about functional improvement in hepatocytes. The availability of the f lavonoids and phenolic compounds in the ethanolic extract of Clerodendrum japonicum leaves could be responsible for the hepatoprotective activity. A further detailed study regarding the phytochemistry is required to study the phytoconstituents responsible for this activity.

Conflict of interest

None

Acknowledgments

The authors of the article are thankful to the Principal and the Management of PES College of Pharmacy, Bengaluru, for providing the facility and the moral support.