Anti-oxidant and Anti-hepatotoxic Activities of Taraxacum officinale Weber (Dandelion)

Pradhan et al., 2006, illustrated that liver is master chemist (metabolic clearing house) and most regenerative part of human body that plays astonishing vital functions in detoxification and metabolism of endogenous / exogenous toxic chemicals. Chronic liver diseases cause morbidity and mortality (hepatic coma – central coma – death) due to hepatotoxins, environmental pollutants; hepatic cancer; alcoholic and intoxicants and other drug therapy. Radha et al., 2005, revealed that drug induced liver diseases (DILI) are caused by overdose / prolonged therapy of paracetamol and idiosyncratic liver injury triggered by other drugs like anti-tubercular drug rifampicin (R-cin), Amoxicillin + Clavulanate, Flucloxacillin, Erythromycin, Ciprofloxacin. Luper, 1998, had reported that no significant allopathic medicines are available for the treatment of DILI. Stickel and Schuppan, (2007) hepatotoxicity can cause mortality and morbidity (hepatic coma followed by central coma then death) and accounts for 3.5%-9.5% of all adverse drug reaction (ADRs) reports and up to 14.7% of fatal adverse reaction (FDRs). Drug induced liver injury (DILI) is caused by overdose / prolonged therapy of anti-tuberculor drugs, paracetamol, antibiotics etc. and it affect large population across the globe. (Dienstag et al., 2001). Sudipta et al., 2012 explained Drug-induced liver injury (DILI) / Drug-induced hepatotoxicity (DIH) as the most important cause of mortality and morbidity. The primary cause of liver intoxication includes alcohol (71%) psycho-pharmaceuticals & industrial exposure (11%). Onkar et al., 2016 compiled hepatotoxicity caused due to lipid- peroxidation, activation of pro-inflammatory mediators, induction of nitric acid (NO) synthase, mitochondrial dysfunction and Cytochrome P450 activation. Various risk factors for hepatotoxicity like race, age (geriatrics are at high risk due to reduced hepatic blood flow; decreased clearance; variation in drug binding; drug-to-drug interactions), multiple drug therapy, gender (more common in females than in males), alcohol (due to depletion of glutathione), genetic factors (idiosyncratic reactions), AIDS (low glutathione level), Drug formulations (long-acting drugs) and fibrosis. liver toxicity. Now a days, it has become one of the most popular "over-the-counter" (OTC) drug and occupied 50 -60% of the total OTC analgesic market. PCM induced acute hepatotoxicity at the higher then 15-20g. On over dosage PCM exhaust glutathione stores and leads to necrosis of hepatocytes. Acute over-dosage may cause fatal hepatic damage, and the number of self-poisonings and suicides with PCM - especially in the United States - has grown alarmingly in recent years. (Shah et al., 2011) Parabia et al., 2007, revealed that Drug-induced hepatotoxicity (DIH) / Drug-induced Liver injury (DILI) is the most common cause of acute hepatic failure and affect huge population across throughout the globe. Symptoms of liver damage include digestive disturbances such as constipation; jaundice (elevation of biochemical parameters like SGPT, SGOT, ALKP, TBIL and Total Albumin / Liver Function Tests / LFTs); necrosis and degenerative changes in hepatocytes, ballooning and fatty changes / steotosis, depletion of glutathione in the liver, rupture of hepatic membrane, loss of proteins in blood, inflammation of hepatocytes, cirrhosis of the liver, edema; pruritus (itching), nausea and vomiting.

Sampath et al., 2010, further, revealed that herbal drugs are more widely used than allopathic drugs as hepatoprotective because of them are inexpensive, better cultural acceptability, better compatibility, with the human body and minimal side effects. There are plants drugs such as "Milkthistle", "Bhringraja" and ―tree turmeric‖ which can give definite protection against experimentally induced hepatotoxicity. Sheetal et al., 2004, summarised Alternative System of Medicine (ASM) / Complimentary System of Medicine (CSM) which include Ayurveda (since last 5000 years) and Siddha (originated in India) Unani system (Persia; about 3,000 years), Chinese traditional System (5,000 years), Tibetan Systems (3,000 years old). (Balunas et al., 2005) Hikino et al., 1984 summarised various hepatoprotective medicinal plants like Silybum marianum, Andrographic paniculata, Wedelia calendulacea, Phyllanthus emblica, Picrorhiza kurroa, and Eclipta alba Linn. (Newman et al., 2002) Qiu (2007), articulated that there are reputed hepatoprotective polyherbal formulations include Amlycure DS, Liva-16, Livodin DS, Livosin, Livotone, Livotrit, Livergen, Acilvan Hepa-10, Livomap, Vimliv, Livomycin, Amylcure, Liv-52, Sanliv, etc. marianum, Boerrhaavia diffusa, Andrographis panuculata, Ocimum sanctum Tinospora cordifolia, Solanum nigrum, Astercantha longifolia, Plumbago zeylanica, Circhorium intybus, Terminalia chebula, Oldenlandia corymbasa, Phyllanthus niruri, Solarium nigrum, Phyllanthus amarus, Taraxacum officinale, Curcuma longa, Solanum nigrum etc. Schuppan et al., 1999 reported that plants are excellent sources of phenolic antioxidants. It has been assessed that these plants have been formulated together in different doses and combinations to achieve maximum synergistic antihepatotoxic / hepatoprotective activity. Taraxacum officinale (dandelion weed plant; Asteraceae) contains bioactive compounds like phenolic compounds, flavonoids, phenolics, coumarins, sterols, sequiterpenes, as main constituents and have shown various pharmacological activities. But, surprisingly, no significant phytochemical and pharmacological investigations are reported in literature so far. In-vitro anti-oxidant and in-vivo anti-hepatotoxic (against paracetamol) activities of leaves, aerial parts and roots of dandelion in albino rats are not reported in scientific literature published. So, it was an attempt to perform research on Taraxacum officinale (dandelion) with following objectives:  Phytochemical screening and qualitative analysis by using scientific techniques.  Estimation of TPC and TFC in dandelion extracts.  In-vitro anti-oxidant activity by DPPH, NO, SOD, CAT and Peroxidase.  In-vivo anti-hepatotoxic activity of dandelion extracts against PCM.  Safety and toxicity evaluation of dandelion extracts.  Lipid / biochemical parameters assessment with histopathological studies.

(i) Various research papers were scientifically reviewed for literature search, summarised, assessed, statistically analysed to establish research protocols. (ii) Phytochemical analysis techniques (chromatographic methods, IR, UV etc.) were used for qualitative/quantitative analysis. (iv) Biochemical parameters (like SGPT, SGOT, total protein, serum albumin, serum billirubin and alkaline phosphatase etc.), histopathological analysis of liver - body weight analysis.

Fresh entire plants of Taraxacum officinale Weber (dandelion) were harvested and collected from Medicinal Plant Garden of the IEC-GI campus in January 2020. Dandelion plants were washed to clean, dried under shade and finally coarsely pulverised in grinder. Dandelion parts were scientifically analysed by pharmacognostical methods for its authentication. Phytochemical screening was done to detect PPMs and SPMs and herbarium specimens were also deposited.

Various chemical tests were performed to detect reagent test), Saponins (Foam test), Carbohydrates (Molisch‘s; Barfoed‘s; Seliwanoffs tests), Tannins (Ferric chloride and Lead acetate tests), Amino acids (Millon‘s reagent tests) and Protiens (Biuret test), tests were carried out. PPMs and SPMs like alkaloids, glycosides, tannins, saponins, anthraquinones, phenolic compounds, flavanoids, steroids, reducing sugars, and amino acids were found present in the drug samples. The petroleum ether, chloroform, methanol and aqueous extracts Plant aerial parts, leaves and roots (dried under shade, coarsely pulverized separately) of dandelion were subjected for PPMs and SPMs using standard procedures described by Harborne (1973); Sofowora (1993); Khandelwal (2008).

Dandelion shade dried and coarsely pulverized aerial parts, leaves, and roots (1000 gm each) were Soxhlet extracted with crude ethanol (80%; Soxhletion; separately) for 16 hours, filtered, distilled, vacuum evaporated and finally lyophilized.

The total phenolics content (TPC, possess antioxidant activity) in EETOP, EETOL and EETOR extract of dandelion were undertaken by method of Jeong et al., 2010.

The total flavonoids content (antioxidant potential is correlated with TFC content) were determined in EETOP, EETOL and EETOR of dandelion as per method reported by Kamtekar et al. 2014.

In-vitro antioxidant activity of EETOL, EETOP and EETOR of dandelion by DPPH, NO, SOD, catalase, peroxidase radical scavenging method was performed.

(1996).

(i) Superoxide dismutase (SOD) activity (Habbu et al., 2008; Chidambara et al. 2002); (ii) Peroxidase activity (Nicholas, 1962); (iii) Catalase Activity (Aebi, 1984)

Hepatic tissues - excised – cleaned - homogenized (cold 1.15% KCl and10 mM phosphate buffer with EDTA (pH 7.4) - centrifuged (10,000 rpm for 10 min) - supernatant centrifuged (13,000 rpm; 60 min) - cytosolic extract – SOD / CAT / peroxidase estimated.

SOD (Units/ mg) was estimated using Kit (Sun Pharma, India) and based upon reduction of nitrobluetetrazolium (NBT) to water insoluble blue formazan. Standard operating procedures were undertaken (Habbu et al., 2008).

Toxicity studies of EETOL, EETOP, EETOR of dandelion was carried out as per OECD guidelines and IAEC Form B approval (IEC/IAEC/2022/05 dated 25-03-2022) Physiological and behavioural changes were observed then RBC count, MCV, MCH (Dacie and Lewis, 2001), Hb content (Pla and Fritz, 1971; Crosby et al., 1954), hematocrit (Ht) (Dacie and Lewis, 1991), PLT / WBC count (Wu and Hoak, 1974) were analysed and diagnostic kits were also used for biochemical analysis (Trinder, 1969; Spencer, 1986; Bretaudiere et al., 1976).

Kulkarni, 1999 reported that barbiturates are extensively metabolized in the liver and damaged liver suffers with delay in barbiturates clearance (thiopentone sodium 40 mg/kg i.p; cause longer duration of hypnotic effect; Gujrati et al., 2007) Grouping of Animals

Group I animals were given water ad libitum / vehicle control. Group II (toxic control) animals were given PCM (1gm/kg) for 03 weeks. Group III to VI animals of Group II to VI were served as models for PCM EETOP / EETOR (400 mg/kg) with thiopentone sodium (40 mg/kg i.p) (Group III, IV and V) and silymarin (standard drug, 140 mg/kg) with thiopentone sodium (40 mg/kg i.p) (Group VI).

The procured plant drug was authenticated as dandelion with herbarium specimen number IEC/Pharm/Herb/2020/102. Qualitative analysis of dandelion aerial parts, leaves and roots showed the presence of PPMs and SPMs like glycosides; alkaloids; amino acids; tannins; saponins; flavanoids; steroids; phenolics; reducing sugars; anthraquinones. Practical yield EETOL, EETOP and EETOR extracts were 1.86%, 1.54% and 1.12% respectively. Different concentrations of EETOL, EETOP and EETOR extracts (200 / 400 mg in 1 % carboxymethylcellulose) were used in pharmacological studies. High concentration of TPC were found in the EETOL (34.78±1.84 mg GAE/100 gDW) and EETOP (26.28±1.62 mg GAE/100 gDW), whereas TPC concentration was low in EETOR (14.56±1.34 mg GAE/100 gDW) of dandelion. Standard Plot was prepared and TFC in EETOP, EETOL and EETOR were analysed and calculated. It has been assessed that there is a close relationship between antioxidant activity and the amount of TFC (Negro et al., 2003; Li et al., 2009). Comparatively high amount of TFC was found in the EETOL (9.6 mg) and EETOP (8.4 mg) than EETOR (7.2 mg) extract. Free radical-scavenging activities of the EETOL, EETOP and EETOR extracts increased with increasing concentrations (regression equations significant at p < 0.05; Table 4 and Figure 3). Results were means ± standard errors of mean (SEM) and data analyzed by ANOVA to determine the level of significance (P < 0.05).

EETOL extracts produced highest NO scavenging activity with IC50 value 97.10 μg/ml (Table 5). Antioxidant activity by reducing power of EETOL, EETOP and EETOR assay was increased with increased dose.

Antioxidant activity of EETOL and EETOP extracts were significant (data were expressed as means ± S.D.) and analysis of variance was performed by the ANOVA procedures (Table 6; Figure 4-6). Dandelion extracts dose dependently elevated the reduced levels of SOD, catalase, and peroxidase activity (antioxidant activity by preventing cell membrane oxidation). Polyphenol, tannins, flavonoids and bitter principles in dandelion extracts induced antioxidant activity through significant superoxide radical

Note: means ± SEM (n = 6); ANOVA.

EETOL, EETOP and EETOR extracts produced potential antioxidant activity dose dependently by preventing cell membrane oxidation. Polyphenol, tannins, flavonoids and bitter principles in EETOL, EETOP and EETOR extracts induced antioxidant activity through significant superoxide radical scavenging activity, inhibition of lipid oxidation and by protecting glutathione in liver.

In safety and toxicity evaluation studies, mortality rates were zero percent (no mortality reported even at the dose of 2000 mg/kg. body weight). So, dandelion extracts (EETOL/EETOP/EETOR) possess wide safety margin and classified as Non-Toxic Constituent.

Note : Significance difference from PCM group (Group II). *P<0.05 Animals of Group I was given water ad libitum and on eighth blood samples were collected from retro-orbital sinus and biochemical parameters / LFTs of liver and histopathological slides were assessed (Table 10 ; Figure 9-13).

glutathione depletion, necrosis of hepatocytes, elevated levels of plasma amino-transferases, and increased concentration of billirubin. Biopsy of the showed centilobular necrosis (which include necrosis, degeneration, and infiltration) and rise in the level of LFTs were correlated with the hepatic lesions produced. (Table 10; Figure 10) Administration of EETOL extract (400 mg/kg) for 02 week (Group IV) produced significant alleviation of the serum enzyme activities. EETOL produced significant reversal effects which were clearly indicated by reddish coloration, normal anatomical architecture of the hepatocytes. EETOL showed a remarkable anti-hepatotoxic activity (Table 10; Figure 11) but comparatively lesser then standard reference drug i.e. Silymarin (Group IX; Table 10; Figure 12) and data represented in Table 10 was analyzed by ANOVA.

aP < 0.001 relative to control group; ***P < 0.001 relative to Toxicant group *P < 0.05 relative to Toxicant group

1. Aebi H. (1984). Catalase in vitro. Methods Enzymol. 105: 121-6. 2. Agarwal SS.(2001) Pharmacology and Therapeutics in the New Millennium, New Delhi, pp. 357-358. 3. Balunas M.J., and Kinghorn, A.D., (2005). Life Sci., 8(5), pp 431-41. 4. Benzie, I.F; Strain, J.J. (1996). Anal. Biochem. 239, pp. 70–76. 5. Beauchamp, Charles and Fridovich, Irwin. (1971). Analytical Biochemistry. 44 (1), pp. 276-287. 6. Blois, M.S. (1958). Nature. 181, pp. 1199–1200. 7. Bretaudiere, J.P., Baily, M., Phung, H.T. (1976). Clin. Chem. pp. 1614-1617. 8. Chidambara, M, Singh, R.P., Jayaprakasha, G. K., (2002). J. Agric. Food Chem. 14;50(17), pp. 4791-4795. 9. Crosby, W.H., Furth, F.W., Wunn, J.I., (1954). Armed Forces Med. J. 5, pp. 693-703. 10. Dacie, J.V, Lewis, S.M., (2001). Practical Haematology. Longman Group. pp 11-17. 11. Dacie, J.V., Lewis, S.M. (1991). Prac. Haem. Edinburgh:Churchill Livingstone. pp. 212-214. 13. Godfried, 1935. Biochem. J., Vol. 29, pp 1337. 14. Gornall, A.C., Bardawill, C.J., and David, M.M. (1949). Journal of Biological Chemistry; 177:751-767. 15. Gujrati, V., Patel, N., Rao, V.N., Nandakumar, K., Shalam, M.D. (2007). Indian J Pharmacol. 39, pp.43-47. 16. Habbu, P.V., Shastry, R.A., Joshi, H., (2008). Afr J Tradit Complement Altern Med. 5: pp. 158-64. 1. 17. Harborne, J. B. (1973). Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis. Springer Publications. pp. 1-288. 17. Hikino, H, Kiso, Y. (1984). Vol. II, Academic Press, London. pp. 39‐67. 18. Jayaprakasha, G.K, Singh, R.P., Sakariah, K.K. (2001). Food Chem. 73, pp. 285-290. 19. Jeong, C.H., Choi, G.N., Kim, J.H. (2010). Food Chem. 118, pp. 278–282. 20. Kamtekar et al. (2014). Jour. of Appl. P’ceutical Science. 4 (09); pp. 61-65. 21. Khandewal, K.R. (2008). Prac. Pharmacognosy. Nirali Prakashan, ed. 19., pp. 1-67. 22. Kulkarni, S.K. (1999). Hand book of experimental pharmacology. 3rd ed. Vallabh Prakashan: New Delhi. pp. 1-78. 23. Larson, RA. (1994). Phytochemistry. 27(4), pp.:969-978. 24. Li, H.Y. Hao, Z.B., Wang, X.L. (2009). Bioresour. Technol. 100, pp. 970–974. 25. Lowry, O., Bessel, Crawford, E. (1949). Jour. of biological Chemistry, 180, pp. 399. 26. Luper S. (1998). Altern Med Rev. 3, pp. 410–421. 27. Mukherjee, P.K., Narayanan, N.K., Sahoo, A.K. (2009). Expert Opinion on Drug Discovery, 4(5), pp 545-576. 28. Negro, C., Tommasi, L. Miceli, A. (2003). Bioresour.Technol. 87, pp. 41–44. 30. Nicholas, M.A.,. (1962). Anal Biochem 4, pp. 341-345. 31. Onkar, B., Krishna R.V., Bijjem, P.K., Vinod, G. (2016). Indian J Physiol Pharmacol. 60(1), pp. 6–21. 32. Parabia, M.H,, Adhvaryu, M.R., Reddy, N. (2007). World J Gastroenterol, 13, pp. 3199–205. 33. Pla, G.W., Fritz, J.C. (1971). J. Analyt. Chem., 54, pp 13-17. 34. Pradhan, S.C., Girish, C. (2006). Indian J Med Res. 124, pp. 491–504. 35. Qiu. (2007). Nature Drug Discov. 6: 506–550. 36. Radha, K.D, Yogesh, K.C., (2005) Dig. Dis. and Sci. 50(10), pp. 1807–1812. 37. Sampath, K.P., Swertia, C. (2010). J Chem Pharm Res. 2(1), pp. 262-266. 38. Schuppan, D., Jia JD, Hahn, E.G. (1999). Hepatology. 30(4), pp. 1099-1104. 39. Shah, V.N., Deval, K. (2011). Int J Pharm. 1(2), pp. 59-66. 40. Sheetal, V., and Singh, S.P., (2004). Veterinary World, 1(11), pp 347-350. 41. Sofowora, A. J. (1993). Altern. Complement. Med. 2 (3), pp. 365-372. 42. Spencer, K., (1986). Ann Clin Biochem. 23, pp 1-25. 43. Sudipta, D., Choudhury, M.D., Talukdar, A.D. (2012). Indian Journal of Fundamental and Applied Life Sciences, 2(1), pp.84-97. 44. Stickel F, Schuppan D., (2007). Digestive and Liver Disease. 39, 293–304. 45. Trinder, P. (1969). J. Clin. Pathol., 22(2), pp. 246-252. 46. Wu, K.K., Hoak, J.C. (1974). Lancet, 11, pp. 924- 926.