Chemical characterisation and hepatoprotective potential of Cosmos sulphureus Cav. and Cosmos bipinnatus Cav.

Abstract This study was conducted to validate the hepatoprotective activity of Cosmos sulphureus and Cosmos bipinnatus. Aqua-methanolic extracts of both plants were evaluated for the presence of various phyto-constituents through HPLC. Different doses of both plant extracts were administered to rats for nine days. Standard control was silymarin 100 mg/kg. Paracetamol 1 gm/kg was administered 3 h post treatment on 9th day for induction of hepatotoxicity. Blood was collected for the evaluation of liver biochemical markers and livers were removed for histopathological evaluation 24 h post-paracetamol treatment. HPLC analysis revealed the presence of quercetin, gallic acid, caffeic acid and chlorogenic acid in both plant extracts. The extracts of both plants decreased the level of alanine aminotransaminase and total bilirubin significantly (p < 0.05), dose dependently and protected hepatocytes from paracetamol-induced hepatotoxicity. It can be concluded that both plants may possess hepatoprotective activity possibly due to the presence of quercetin and phenolic compounds.


Introduction
The damage to liver is one of the major global health problems. The destruction of hepatocytes by drugs, chemicals or toxins is termed hepatotoxicity . A large population across the world relies on traditional remedies for the treatment and prevention of various diseases including non-viral hepatitis . It is due to low side effect profile, minimal cost and multiple beneficial effects of herbal medicines (Mehmood et al. 2016).
The plant species Cosmos sulphureus and Cosmos bipinnatus, belong to Asteraceae family. C. sulphureus is an annual plant, having yellow coloured leaves. It is native to Mexico, South and North America, and Thailand. It is effective as schistosomicidal agent (Aguiar et al. 2013). Nanoparticles of C. sulphureus enhance the membrane permeability of antibacterial drugs (Malaka et al. 2015). It may also interfere with metabolic enzymes of Plasmodium (Katsori and Hadjipavlou-Litina 2011). Flowers of C. sulphureus have also exhibited significant antioxidant activity, cytotoxic potential against gastric and colorectral adenocarcinoma cell lines and in vitro antidiabetic activity (Kaisoon et al. 2012). Traditionally, C. sulphureus is used as an anti-inflammatory, anti-fungal and hepatoprotective agent and has been used for treating splenomegaly, juandice, arthritis, gastric ulcer, flatulence and diabetes (Botsaris 2007). The other species C. bipinnatus is also an annual plant having violet coloured leaves. It is native to Mexico and South western America and is pharmacologically effective against leukaemia and bacterial infections (Olajuyigbe and Ashafa 2014). It also acts as an antioxidant and prevents oxidative DNA damage (Kaisoon et al. 2012). It is used traditionally as hepatoprotective agent and for the management of headache, juandice, spleenomegaly, stomach aches, flatulence and intermittent malarial fever (Jo et al. 2012).
Previous studies reported the total phenolic content and essential oil constituents in C. sulphureus and C. bipinnatus (Olajuyigbe and Ashafa 2014;Malaka et al. 2015). These species exhibit in vitro antioxidant potential (Kaisoon et al. 2012). Furthermore, the traditional hepatoprotective use of these species needs scientific validation (Jo et al. 2012). Therefore, the objective of present study was to determine the phytochemical constituents in C. sulphureus and C. bipinnatus for chemical characterisation and to evaluate their hepatoprotective potential for the validation of their folklore use.

Results and discussion
The percentage yields of aqua-methanolic extracts of C. sulphureus and C. bipinnatus were 18.18 and 27%, respectively. Preliminary phytochemical analysis of C. sulphureus and C. bipinnatus revealed that the fats and oils, carbohydrates, proteins, saponins, flavonoids, tannins, terpenoids, alkaloids, anthraquinones and steroids were present in both plant extract (Table S1).

Quantitative analysis
HPLC analysis revealed that the aqua-methanolic extract of C. sulphureus had chlorogenic acid, quercetin, gallic acid, caffeic acid, p-coumeric acid and vanillic acid, whereas C. bipinnatus contained quercetin, gallic acid, caffeic acid, chlorogenic acid and sinapinic acid. Summary of the phyto-constituents present in C. sulphureus and C. bipinnatus is presented in Table S2. All these phytochemicals are commonly present as secondary metabolites in edible plants which have their diverse role in human and herbivores ).

Liver function biomarkers
Hepatoprotective activity of aqua-methanolic extracts of C. sulphureus and C. bipinnatus was examined at 250, 500 and 750 mg/kg dose by assessing liver function biomarkers. Wistar rats treated with normal saline only showed the normal values of alanine aminotransaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin. Treatment with paracetamol raised the values of these parameters by more than 40%. The ameliorating effect of C. sulphureus extract was more pronounced on the raised level of ALT than that of C. bipinnatus at all the tested doses as the effect of latter was statistically significant only at 750 mg/kg dose (p < 0.05). The effect of C. sulphureus on ALT was statistically comparable to that of standard silymarin therapy. The effect of both plants on total bilirubin level was comparable to that of silymarin therapy and normal control (p < 0.0001). Both plants reduced the elevated levels of AST and ALP, however their ameliorating effect on ALP and AST were statistically insignificant when compared to disease control group. Increase in dose of the plant extracts improved the liver function tests. The variation in liver function biomarkers in treated and untreated rats is summarised in Table S3.
The hepatoprotective properties of C. sulphureus and C. bipinnatus may be due to the presence of hepatoprotective constituents such as quercetin, gallic acid, vanillic acid, caffeic acid, p-coumaric acid and chlorogenic acid. The presence of various flavonoids and phenolic compounds has been associated with the pharmacological and toxicological activities of medicinal plants . Antioxidant, free radical scavenging and antihepatotoxic activities of quercetin, gallic acid, p-coumaric acid, vanillic acid, caffeic acid and chlorogenic acid have been documented previously (Itoh et al. 2009;Kelly 2011). So the present study indicates that C. sulphureus and C. bipinnatus plant extracts may have distinctive hepatoprotective properties due to the presence of quercetin and phenolic compounds.

Histopathological evaluation
The gross anatomy of normal rats indicated a normal articulation. Hepatic cells and central vein were normally organised. Sinusoidal space was normal. Parenchyma covered the portal triads and liver tissue sections showed ordinary linings. Disease control group showed severe tissue necrosis and inflammation associated with paracetamol. Silymarin pretreated group showed that it defended against paracetamol induced liver damage and did not exhibit any inflammation. C. sulphureus and C. bipinnatus extract treated rats displayed minor enlargement in sinusoidal space. A slight inflammation was also seen in parenchymal cells. However, both plant extracts protected against paracetamol induced necrosis. Thus, the extracts of C. sulphureus and C. bipinnatus showed hepatoprotective activity against paracetamol-induced toxicity in the liver ( Figure S1).
The present experimental study indicates that C. sulphureus and C. bipinnatus plant extracts have distinctive hepatoprotective characteristics. The ameliorating effects of C. sulphureus against paracetamol-induced hepatotoxicity were more pronounced than the effects recorded with C. bipinnatus as indicated by liver biochemical parameters. The hepatoprotective activity of both plants may be attributed to the presence of quercetin, gallic acid, p-coumaric acid, vanillic acid, caffeic acid, chlorogenic acid and sinapinic acid (Itoh et al. 2009;Kelly 2011).

Disclosure statement
No potential conflict of interest was reported by the authors.