Pharmacognostical Studies and Pharmacological Investigations for Anti-diabetic Activities of Taraxacum officinale Weber (Dandelion)

Mukherjee et al., 2009, illustrated that various medicinal plants phyoto-formulations are clinically proven as anti-diabetic and their use is strengthened due to their promptly accessibility, complete cure and minimal side effects. There are more than 500 anti-diabetic medicinal plants which include Gymnema sylvestre, Momordica charantia, Aegle marmelos, Trigonella foenum, Syzygium cumini, Combretum micranthum, Tinospora cordifolia, Berberis aristata, Curcuma longa, Elephantopus scaber, Aegle marmelos, Pterocarpus marsupium, Glycyrrhiza glabra, Liriope spicata, Azadirachta indica, Commiphora wighti, Syzygium cumini, Phyllanthus emblica, Ficus glomerata, Terminalia chebula, Asparagus racemosus, Tribulus terrestris, Piper nigrum, Plumbago zeylanica, Picrorrhiza kurroa, etc. Grover et al., 2002 revealed that in India unconventional medicines / natural medicines are used to cure diabetes. World wellbeing association has likewise prescribed the assessment of conventional plant separates treatment for diabetes. Vašut et al., 2015 reported that the genus Taraxacum (30–57 varieties ; many microspecies; divided into 9 sections; dandelion : plant's jagged-edged leaves / toothed leaves; 3–35cm height; single yellow flowers; florets). Koo et al., 2004 summarised that TO has terpenoid; sterol (taraxacin; taraxacerin); terpene/sterol (taraxasterol); free sterols (sitosterin, stigmasterin, and phytosterin). Further, Ali et al., 1989 reported that dandelion contains vitamins A, B, C and D; iron. It contains flavonoids, phenolics, coumarins, sterols, sequiterpenes, high beta-carotene, vitamin C and iron (more iron and calcium than spinach) as main constituents and shown various pharmacological activities. T. officinale is commercially cultivated and used as a traditional medicine and food (soup/salad) in Europe, North America, and China.

 Phytochemical screening and physio-chemical evaluation of ethanolic extract of T. officinale leaves (EETOL), ethanolic extract of T. officinale aerial parts (EETOP) and ethanolic extract of T. officinale roots (EETOR),  HPLC chromatography of various extracts of dandelion,  Anti-diabetic activity of EETOP, EETOL and EETOR against STZ.  Comparison of anti-hyperglycemic effects of dandelion extracts with glipizide.

(i) Various research papers were scientifically reviewed for literature search, summarised, (ii) Phytochemical analysis techniques (chromatographic methods, IR, UV etc.) were used for qualitative/quantitative analysis. (iii) Blood glucose level analysis were performed for anti-diabetic activity.

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.

Extracts of dandelion were subjected to preliminary phytochemical screening using standard procedures described by Sofowora (1993); Khandelwal (2008); (Table 1):

In the experiment freshly procured, washed, air-dried and pulverised / powdered aerial parts, leaves and roots of dandelion were subjected to physico-chemical analysis for parameter like foreign matter, extractive values (alcohol / water), swelling index, ash value (acid insoluble / water soluble), fluorescence analysis in various solvents (normal light / UV light), and loss on drying (LOD) etc.

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.

System Used: HPLC – diode array detector (DAD) - electrospray ionization (ESI) - tandem mass spectrometry (6410B HPLC-DAD-ESI-MS/MS; Agilent Technologies). Mobile Phase: Acetonitrile (eluent A) with formic acid (1.0% v/v, eluent B). Gradient Elution Flow Rate: 1 ml/min. Injection volume: 10 μl. Detection wavelength: 280 nm & 320 nm. ESI source was kept at 110˚C with desolvation temperature at 400˚C. Nebulizer pressure, fragmentor voltage, and capillary voltage were maintained at 35 psi, 135 V, and 3,000 V, respectively and spectra were recorded in the negative ionization mode over the m/z range of 100-1,000 Da.

Animals were treated with EETOL, EETOP and EETOR and the normal group received water. Animals were anaesthetised with Ketamine+Xylazine ((80 mg/kg+10 mg/kg i.p.) and blood was withdrawn from retro orbital plexus and blood glucose levels were estimated with Glucometer or by GOD/POD method at 1, 2, and 3 h.

Oral glucose tolerance test (OGTT) was estimation was carried out in overnight fasted normal rats. Animals were administered EETOL, EETOP and EETOR (400 mg/kg). Glucose (2 g/kg b.w.) was given orally (60 min after dosing) and glucose levels were estimated.

STZ in 0.1M citrate buffer (pH 4.5) was given to animals and glucose levels were analyzed after 07 days. Animals were treated with EETOL, EETOP and EETOR (Group III-VIII) and Glipizide (Standard, Group IX) as prescribed and after 21 days blood glucose levels were analysed. Determination of Effect of EETOL, EETOP and EETOR on Lipid Profile in diabetic rats Total cholesterol : Cholesterol estimated with Agappe Liqui CHEK kit (CHOD –PAP method.

Absorbance of standard Where 200 is the standard concentration

Enzymatic determination of triglycerides based on the following reactions:

Morphology of dandelion leaf showed spatula-like leaf (lion's-tooth), oblanceolate / oblong / obovate, length: 2.0–17.7 in; width 0.39–3.94 inch with shallow, lobed margin. Besides, morphology of flower of dandelion florets (rosette; 40 to over 100 per head), ligulate and bisexual, 12-18 segments. 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.

HPLC profile of EETOL indicated ferulic acid, caffeic acid and quercetin (detection was improved in chromatograms with absorbance at 320 nm compared that detected at 280 nm), high chlorogenic acid and chicoric acid contents (chicoric acid and chlorogenic acid in leaves were as high as 24.03 mg/g DW and 2.82 mg/g DW). Concentration of caffeic acid in EETOL was 0.237 ± 0.025 mg/g DW. Rutin and quercetin were also present. EETOL also contains flavonoids like luteolin-7-O-glucoside, luteolin-7-O-rutinoside and quercetin-7-O-glucoside. HPLC profile of EETOP confirmed flavonoids, rutin, quercetin (flower at 5.43 mg/g DW). Chlorogenic, syringic, caffeic and vanillic acid were found in EETOR fraction. Besides, flavonoids like isorhamnetin, luteolin, apigenin, and quercetin derivatives were found in EETOR. Dandelion EETOL has induced glucose lowering effect after 2 hr. Besides, sugar levels were restored after 3 hr in all treatment groups (Table 6). EETOL had alleviated blood glucose (maximum reduction

Note: Mean ± SEM (triplicate measurements)

Note: Mean ± SEM.

hand blood glucose in animals treated with EETOR was 204.2 (Day-1), 182.4 (day-7), 162.6 (day -14) and 154.8 on day-21. Glipizide (standard drug) significantly lowered glucose level from 206.4 (day 1)-162.2 (day -7)-124.6 (day-14) to 98.4 mg/dl after 21 days.

Observations recorded in Table 9 (Figure 9) indicated the effect of EETOL, EETOP and EETOR on cholesterol and triglycerides level in STZ induced diabetic rats. STZ toxicity induced increase in serum cholesterol (after 7 days 266.4; after 21 days 221.6), triglycerides [200.2 (7 days); 174.8 (21 days)] (P < 0.0001). EETOL treatment alleviated the level serum cholesterol [142.4 (7days); 120.0 (21 days)], triglycerides [132.4 (7 days); 84.4 (21 days)]. Treatment with EETOP alleviated cholesterol [154.6 (7days); 134.6 (21 days)], triglycerides [148.6 (7 days); 92.4 (21 days)]. Standard reference drug glipizide (5mg/kg) decreased cholesterol the [112.6 (7 days); 98.4 (21 days)], triglycerides [98.6 (7 days); 88.6 (21 days)]. EETOL has prominent hypolipidemic / hypocholesterolemic activity but lesser than Standard drug (P < 0.0001).

In normal animals HDL (good cholesterol) level were found to be 60.4 (7 days) and 40.5 (after 21 days). In diabetic control / toxic control group HDL level were 21.2 (7 days) and 27.2 (21 days). HDL was significantly lowered in diabetic rats when compared to normal control (P < 0.0001). EETOL restored HDL level 34.4 (7 days) and 42.6 (21 days) and EETOP treatment the produced HDL level 32.4 (7 days) and 43.2 (21 days). In glipizide (5mg/kg) treated group serum HDL was 38.6 (7 days) and 46.8 (21 days). Beneficial effect of EETOL and EETOP on HDL level in treated animals was therapeutic. (Table 10; Figure 10). In healthy animals (normal group) LDL (bad cholesterol) level were found to be 32.4 (7 days) and 36.0 (after 21 days). In toxic control group LDL level were 192 (7days) and 134.4 (21 days). LDL was significantly increased in STZ toxic group animals (P < 0.0001). EETOL alleviated LDL level 80.06 (7 days) and 58.2 (21 days) and EETOP treatment the produced LDL level 91.2 (7 days) and 62.4 (21 days). In glipizide treated group serum LDL was 72.8 (7days) and 48.6 (21 days). Beneficial effect (LDL alleviating effect) of EETOL, EETOP and Standard on LDL level in treated groups was good. In normal group VLDL level were found to be 20.6 (7 days) and 14.4 (21 days). In diabetic toxic control group VLDL were 41.2 (7days) and 40.2 (21 days). EETOL alleviated VLDL level 26.4 (7 days) and 15.6 (21 days) and EETOP treatment the produced VLDL EETOP on LDL level group III-IV were therapeutically positive. (Table 10; Figure 10). EETOL has prominent hypolipidemic / hypocholesterolemic activity but lesser than Standard drug (P < 0.001). Possible mechanisms of action (MoA) for anti-diabetic activity include restoration / repairing beta cells (protective effect) in diabetic animals; revitalization of damaged beta cells; flavonoids rejuvenate damaged beta cells (insulin secretagogues effect). In diabetic control group, HDL was significantly lowered in diabetic rats when compared to normal control. EETOL, EETOP and glipizide treatment restored / elevated HDL level. In STZ toxic control group LDL level was significantly increased. EETOL and EETOP extracts treatment alleviated LDL level. Standard glipizide alleviated LDL level. LDL alleviating effect of standard, dandelion EETOL and EETOP was good.

Both EETOL and EETOP produced significant anti-diabetic activity by repairing and revitalization of damaged beta cells and insulin secretagogues effect (lowered blood glucose level significantly). EETOL, EETOP and glipizide treatment induced prominent hypolipidemic activity with restored HDL level and alleviated LDL and VLDL level. In future, it is proposed that research work is to be conducted to isolate and characterize the pharmacologically active principles of EETOL and EETOP extracts T. officinale Weber.

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IEC Department of Pharmacy, IEC Group of Institutions, Greater Noida, Uttar Pradesh, India