Introduction
Free radicals or highly reactive oxygen species formed in the human body by exogenous chemicals or endogenous metabolic processes. These are capable to oxidize bio-molecules viz nucleic acids, proteins, lipids and DNA. They can initiate different degenerative diseases like neurological disorders, diabetes, cancer, cirrhosis, atherosclerosis, arthritis etc.1, 2 Diabetes is a chronic disorder in the metabolism of proteins, fats, and carbohydrates.1, 2 It is characterized by chronic hyperglycemia due to defects in insulin secretion, insulin action or both.3 Free radicals inducing oxidative damage are the important factors to enhance risk of hyperglycemia in human being. Nowadays, different treatments, such as insulin therapy, pharmacotherapy and diet therapy are available to control diabetes. These treatments have some disadvantages such as drug resistance (reduction of efficiency), side effects, and even toxicity. Antioxidants are the compounds, which terminate the attack of free radicals and thus reduce the risk of diabetic disorder.3 Prior and Cao, (1999)4 reported that antioxidant supplements or dietary antioxidants protect against the damaging effects of free radicals. Presently, much attention has focused on the use of natural antioxidants to protect the human body from the oxidative damage caused by free radicals. In last decades, several medicinal plants have shown such effectiveness through the traditional methods.5 Therefore, it is better for diabetes patients to use the medicinal plant for controlling and treating the diabetes symptoms.
Cichorium (C.) intybus L., commonly known as chicory belonging to family Asteraceae is an erect fairly woody perennial herb, approx. 1m in height with a fleshy taproot of up to 75 cm in length and large basal leaves and it is widely distributed in Asia and Europe.6, 7 Chicory plant is considered important domestic plant usually grown for food, animal fodder, and to make traditional medicines. It contain various important constituents such fructo-oligosaccharides prebiotic, sesquiterpene lactones, caffeic acid derivatives (chicoric acid, chlorogenic acid, isochlorogenic acid, dicaffeoyl tartaric acid), inulin, proteins, hydroxycoumarins, flavonoids, alkaloids, steroids, terpenoids, oils, volatile compounds, vitamins.8, 9 They have possessed lot of benefits for the human health like hepato-protective, gastro-protective, cardioprotective, antioxidant, hypolipidemic, anticancer, reproductive, anti-inflammatory, analgesic, sedative, immunological, antimicrobial, anthelmintic, anti-protozoal, antidiabetic, wound healing and many other pharmacological effects. 6, 10 Presently, the chicory plants were cultivated at foothill area of Kumaun region of Uttarakhand for the evolution of their medicinal property for the development of herbal product. Hence, the current study was aimed to determine the phytochemical characterization, antioxidant activity, and effects on hyperglycemic rats of C. intybus. The antioxidants compounds such as total phenols, flavonoids, tannins and ascorbic acid were also determined to correlate with their antioxidant activity.
Material and Methods
Plant material and extract preparation
The leaves and roots of C. intybus were collected from the farm of DIBER, DRDO Haldwani, Nainital, Uttarakhand (India). The leaves and roots were shade dried and grinded into fine powder with the help of mechanical grinder. The freshly prepared dried powder of plant material was subjected to overnight in 20% ethanolic solution for hydroalcoholic extract (HAE) over a period of 48 h for cold extraction. The HAE was filtered and the solvent was removed by evaporation using rotary evaporator under reduced pressure at 40 ºC. The samples were stored in an airtight container at (-) 4 0C for further use.
Chemicals
Aluminium chloride, Potassium acetate, Methanol, Folin–Ciocalteau solution, Folin-Denis solutuion, 2, 6-dichlorophenolindophenolwere purchased from E. Merck. Catechol, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), Ascorbic acid, D-glucose, Bovine serum albumin, Quercetin, streptozotocin, Tannic acid and Oxalic acid were purchased from Sigma Chemical Co. Ltd, USA.
Phytochemical characterization of roots and leaves extract of C. intybus
The analysis of phytochemicals such as carbohydrates, proteins, total phenols, flavonoid, tannins, ascorbic acids in hydro-alcoholic extracts of leaves and roots of C. intybus were carried out.
Carbohydrates are the important components of storage and structural material in the plants. They exist as free sugars and polysaccharides. Carbohydrate contents can be measured by hydrolysis of polysaccharides into simpler sugars by acid hydrolysis and estimating the resultant monosaccharaides. Estimation of Total carbohydrate was done by Anthrone method.11 Total carbohydrate was calculated with the help of reference curve using D-glucose as standard.
Proteins are present in the living word, irrespective of size of the organism, since from the structural and functional basis of the cell. In some cases, identification of presence or absence of proteins is very necessary. Now a day different methods are available for the determination of total proteins. Estimation of total protein was done by Lowry’s method.12 In this method the blue colour developed by the reduction of the phosphomolybdic–phosphotungstic components in the Folin-Ciocalteau reagent with the amino acids present in the protein. The amount of protein present in the sample was calculated by using the standard graph. The bovine serum albumin (BSA) was used as standard.
Phenols, the another important aromatic compound are wide spread in plant kingdom having free radical scavenging ability due to presence of hydroxyl group.13 Natural polyphenolic compounds have inhibitory effects on mutagenesis and carcinogenesis in humans.14 Phenolic compounds include phenolic acids, flavonoids, tannins, stilbenes, curcuminoids, coumarins, lignans, quinones, etc are found in medicinal herbs and dietary plants. Phenols play important roles in resistance power of the plants against disease and pests. High amount of polyphenols to give resistant against bird attack. Total phenols were determined by Folin–Ciocalteau method. The standard curve was prepared by using different concentrations of catechol.15 The total amount of phenol present in the test samples was estimated by using the standard curved and expressed as mg phenols/100 g.
Flavonoids were determined by Aluminium chloride colorimetric method.16 1.0 mg/ml extract was mixed with 0.1 ml 10% aluminium chloride, 0.1 ml of 1.0 M potassium acetate and 2.8 ml distilled water. The mixture was vortexed for 30 seconds and then allowed to stand for 30 minutes at room temperature. The absorbance was recorded at 415 nm. Quercetin used as standard and standard curve was plotted. Flavonoid contents were represented as mg Quercetin equivalent/gm of dry weight
Tannins also possess very high antioxidant activity due to their tremendous free radical scavenging ability and thus they protect the body from harmful effect of free radicals. Tannin is an astringent, polyphenolic biomolecule that binds to and precipitates proteins and various other organic compounds including amino acids and alkaloids. The tannin compounds are widely distributed in many species of plants, where they play a role in protection from predation, and perhaps also as pesticides, and might help in regulating plant growth. Tannins were estimated by Folin-Denis method.17 Tannin like compounds reduces phosphotungstomolybdic acid in alkaline solution to produce blue colour, the intensity of which is proportional to the amount of tannins and measured in a spectrophotometer at 700 nm. The tannin content present in the test samples was calculated from the standard graph using tannic acid.
Ascorbic acid is a naturally occurring antioxidant compound found in medicinal plants, vegetables, fruits and whole grains. Total ascorbic acid content in plant extract was determined by 2, 6- dichlorophenolindophenol method.18 2 g dried powdered sample was extracted with 4% oxalic acid and the volume was made up to 100 ml. It was centrifuged at 10,000 rpm for 10 min. 5 ml supernatant liquid was transferred to a conical flask and 10 ml of 4% oxalic acid was added. It was titrated against standard dye solution (2, 6-dichlorophenolindophenol) to a pink end point. The procedure was repeated with a blank solution (without adding sample). 5 ml ascorbic acid of 100 ppm was used as standard. Ascorbic acid content was calculated using the formula:
Ascorbic acid (mg/100 g) = [0.5 mg x titer vol against
Screening of antioxidant activity as free radical scavenging activity (FRSA) by DPPH method in roots and leaves extract of C. intybus
The stable 1,1-diphenyl-2-picryl hydrazyl radical (DPPH) was used for determination of FRSA of the plant extract. Different aliquots of hydroalcoholic extract were taken in test tubes and volume was made upto 1 ml with methanol. Then added 2 ml methanolic solution of DPPH (0.1 Mm) in all test tubes. After 30 minute at room temperature, the absorbance was recorded at 517 nm. Ascorbic acid was used as standard.19, 20 Percentage inhibition was calculated as % free radical scavenging activity = [(Abs control — Abs sample)/Abs control x 100]
Where, Abs control is the absorbance of DPPH radical with methanol; Abs sample is the absorbance of DPPH radical with sample extract/standard. All test were performed in triplicates.
Effects of C. intybus on hyperglycemic rats
Animals used
Male Wistar rats (180-250 gm, age 10-14 w) were selected from the Experimental Animal House of the Institute for this study. They were kept in the polycarbonate plastic cages under standard management conditions (Temperature 25±3 °C, Relative Humidity - 50–70 %, and 12 h/12 h dark/light cycle). The animals had ad libitum access to food and drinking water. All procedures in the present study were carried out under strict compliance of approved directions of CPCSEA, Ministry of Environment and Forest, Government of India, New Dehli. The experiments were conducted according to protocols given below.
Acute toxicity study
The extract of Chicory leaf was administered orally to different groups of rats at different dosage viz 50, 500 and 2000 mg/kg body weight for acute toxicity. Acute toxicity study was carried out according to the OECD Guideline 423 21 and animals were observed for 14 days for any toxic symptoms and mortality.
Streptozotocin induced hyperglycemia in rats
Diabetes was induced in the rats (overnight fasted) by a single injection of streptozotocin (60 mg/kg b.w., i.p.). After 72 hrs of STZ injection, blood sample was collected from the retro-orbital of the rat eyes and plasma glucose level were determined. The confirmed hyperglycemic animals (plasma glucose levels ≥200 mg/dl) were separated and used for evaluate the effects of C. intybus on hyperglycemic rats22 in groups II-IV.
The selected rats were divided into following groups. Each group contains five rats.
Group I. Normal control group: Normal rats were treated with 0.5 mL saline orally for 21 days.
Group II. Hyperglycemic (diabetic) control group: Hyperglycemic rats were treated with 0.5 mL saline orally for 21 days.
Group III. Hyperglycemic test group: Hyperglycemic rats were treated with extract of C. intybus (50 mg/kg b.w.) orally for 21 days.
Group IV. Hyperglycemic test group: Hyperglycemic rats were treated with extract of C. intybus (200 mg/kg b.w.) orally for 21 days.
Group V. Hyperglycemic standard group: Hyperglycemic rats were treated with glibenclamide (5 mg/kg body weight) as standard drug orally for 21 days.
During the experimental period, body weight, food intake and water intake of all treated animals were monitored weekly. At the end of experimental period, the rats were fasted overnight, euthanized and the blood was withdrawn by retro-orbital puncture, collected and allowed to stand for 30 min at 37°C. Then the serum was separated from blood with the help of centrifugation at 3000rpm for 10 min, transferred to eppendorf tubes and stores at -20 0C for further estimation of serum biochemical parameters.
Blood serum biochemical parameters analysis
The serum biochemical parameters such as glucose, serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP), serum glutamic oxalacetic transaminase (SGOT), total bilirubin (TB,) direct bilirubin, total protein, albumin, cholesterol, triglycerides, creatinine, uric acid and BUN were measured of all treated animals by commercial kit with the help of biochemistry analyzer (MEDICA).
Statistical analysis
The results in the study were expressed as Mean ± S.D. Statistical analysis of the results were done by using one way ANOVA followed by post-hoc analysis by Dunnett’s ‘t’ test for all groups compared vs. control group and Student-Newman-Keuls Test for compared all pairs of groups.
Results and Discussion
Screening of phytochemicals
Phytochemicals such as total carbohydrate, total proteins, total phenolics, ascorbic acid, flavonoids and total tannins contents were quantitatively analysed in hydro-alcoholic extracts of leaves and roots of C. intybus. The results of analysis of above phytochemicals are shown in Table 1.
Table 1
Screening of phytochemicals in C. intybus
The total carbohydrate content in leaf and roots of C. intybus was found 6.314±0.38 g/100 g and 32.78±0.206 g/100 g respectively (Table 1). Similarly, in previous studies carbohydrate was reported 4.7 g/100g in leaves 23 and average 21% in roots.24 Hence, locally cultivated chicory plants shows good amount of carbohydrates in both leaf and root parts, but high content observed in leaf as compared to roots. The varied content of carbohydrates found in the chicory plant, which depend on their cultivation, climates, extraction method, and parts of the plant. Chicory plant is containing carbohydrates like saccharose, glucose and fructose, fructooligosaccharides, inulin etc. in leaves and roots parts. Inulin is a soluble polyfructan and belongs to a group of dietary fibre. Fructooligosaccharides is having prebiotic activity in humans.25 Use of chicory plant as food supplement will be beneficial for human being.
Along with carbohydrates, the Chicory plant is also good source of proteins and amino acids. Total protein content of C. intybus was recorded 1.89±0.01 g/100 g in leaf and in root 3.68±0.10 g/100 g showed inTable 1. Maximum content of protein was recorded in roots part than leaf part of C. intybus. In similar study, done by Saeed M et al., 2017 22 in C. intybus plant, the protein content was found to be 1.7 g/100 g in leaves.
Phenolics or polyphenols are secondary plant metabolites and ubiquitously present in plants and their products.26 In this study, total phenolics were found in leaves (55.4±2.2 mg Catechol equivalent (CE)/g) than roots (28.43±0.87mg CE /g) of C. intybus (Table 1). A study done by Shad et al. (2013)27 on C. intybus, total phenolic content was reported 25.2±2.6 mg GAE /g in methanolic extract (30%) of leaf and 4.7±0.7 mg GAE /g in methanolic extract (30%) of roots. Similarly Malik et al., 201728 reported total phenolic content 19.21±0.60 mg GAE /g in 80% methanolic leaf extract of cultivated C. intybus. Hence, results showed that the leaves are good source of phenolics than roots of C. intybus. Tanaka et al., (1998)14 suggested that the natural polyphenolic compounds are having inhibitory effects on mutagenesis and carcinogenesis in humans. Phenolics or polyphenols are having antioxidant property due to the presence of hydroxyl groups and it has protective role against the oxidative damage caused by endogenous free radicals.26, 29 They act through scavenging or chelating process.30, 31
In present study, maximum content of flavonoids was found in the leaf part (3.2±0.09 mg Quercetin equivalent (QE) /g) as compared to roots part (1.26± 0.103mg QE/g) of C. intybus shown in Table 1. Shad et al. (2013)27 has reported flavonoid 1.0±0.2 mg catechin equivalent (CE) /g in methanolic extract (30%) of leaf and 0.5±0.3 CE mg/g in methanolic extract (30%) of roots of C. intybus. Similarly, Malik et al., (2017)28 has reported total flavonoids 9.51±0.43 mg GAE /g in methanolic extract (80%) of leaves of cultivated C. intybus. Hence, results show that the both parts leaves and roots of C. intybus are good source of flavonoids, but leafs are having high amount than roots. Therefore, C. intybus has been found to have high medicinal value due to rich source of phenolics and flavonoid compounds.
Tannins are polyphenolic compounds with high molecular weight and having protective role against harmful effects of free radicals. They are also having protective role in plants against micro-organisms, unfavourable climatic conditions and damage by animals/birds' attack.32 In present study, total tannins content was recorded 0.73±0.1 mg Tannic acid equivalent (TAE) /g in leaf extract and 0.052±0.01 mg TAE/g in roots extract of C. intybus (Table 1). Abaas et al., (2015) 33 has reported total tannin content 0.59±0.04 mg/g in leaf extract of chicory plant. While, Shad et al. (2013)27 has reported total tannin content 6.6±0.2 mg/g in methanolic extract (30%) of leaf and 15.1±0.3 mg/g in methanolic extract (30%) of roots of C. intybus. The leaf of chicory plant is also a good source of total tannins.
The leaves and roots of chicory plant are a good source of ascorbic acid, which was recorded 89.9 ± 0.75 mg/g and 71.1±0.122 mg/g in the leaves and roots respectively (Table 1). Hence, leaves and roots of chicory plant are good source of antioxidant compound like phenolics, flavonoids, tannins, and ascorbic acid. Antioxidants are the compounds, which terminate the attack of free radicals and reduce the risk of diseases that are capable of inducing oxidative damage to human body.
Evaluation of antioxidant activity as FRSA
The DPPH method is most widely used method for in-vitro estimating Free Radical Scavenging Activity (FRSA) of the plant extracts. In this method DPPH are the free radicals, which neutralized by the antioxidant compound like ascorbic acid, total phenolics, tannins etc. In this study DPPH radical scavenging based antioxidant potential of the extracts was evaluated using the parameter IC50. Here, IC50 means the concentration of antioxidant required for 50% scavenging of DPPH radicals.
The HAE of leaf of C. intybus exhibited FRSA 67.61 %, whereas, root (HAE) exhibited FRSA 32.26 % at dose of 400 µg. The IC50 value was recorded in chicory leaf 295.78 µg/ml, while, in root 619.94 µg/ml. Lower IC50 value in leaf of chicory as compared to root indicates high antioxidant property inleaf. Milala et al., (2009)24 has reported high antioxidant activity in leaf (51.8 mg TAEC/g) than root (6.5 mg TAEC/g) of chicory plants. Whereas, Malik et al., (2017) 28 has reported IC50 value 46.81 µg/ml in 80% methanolic extract of leaves of cultivated C. intybus. The root and leaf extracts of plant were also showed dose-dependent FRSA increased proportionate to the increase in concentration of the extracts. The present results of the study indicates that plant cultivated at foothill area of Himalayas showed higher antioxidant activity as compared to earlier reports33 on antioxidant property of chicory leaf. Hence, we concluded that leaf and roots of chicory plant showed very good antioxidant activity may be due to presence of high amount of phenolics, flavonoids, tannins, and ascorbic acid in the leaf part of the plants. Shad et al. (2013)27 also showed the similar tendency in leaves showing good free radical scavenging capacity against DPPH radicals.
Table 2
Antioxidant activity or FRSA of C. intybus by DPPH method
Acute toxicity study
A preliminary toxicity study was designed to demonstrate the appropriate safe dose range that could be used for subsequent experiments rather than to provide complete toxicity data on the extract. In acute toxicity study, no significant changes in food intake, body weight and behaviour parameters such as alertness, motor activity, sluggishness, paralysis, breathing, restlessness, diarrhoea, convulsions, coma etc. All rats were found physically active and normal in health as well as neurological behaviors. There was no signs of toxicity and mortality found in oral dose of hydroalcoholic leaf extract of C. intybus up to 2 g/kg body weight during observation period of 14 days.
Table 3
Effect of C. intybus on body weight of hyperglycemic rats
Effects of C. intybus on hyperglycemic rats
The various plants traditionally have been used as anti-diabetes, and some have been proven to have hypoglycemic effects. These plants are having some important phytochemicals such as polysaccharides,34 inulin,35 terpenoids and tannins,36 and alkaloids,37 which are responsible for antidiabetic effect. Administration of STZ in rats caused rapid destruction of pancreatic β-cells, which led to impaired glucose stimulated insulin release and insulin resistance, both of which are marked feature of type II diabetes.38 Oral hypoglycemic agents and insulin are currently available for treating diabetes, but in present scenario of world population, there is a growing interest in herbal remedies due to side effects associated with the existing drugs.39 The present investigation indicates the hypoglycemic, anti-lipidemic and liver & kidney protective effects of HA leaf extract of C. intybus on STZ induced hyperglycemic rats. The C. intybus plant contains several medicinally important compounds such as polysaccharides, coumarins, alkaloids, steroids, terpenoids, inulin, proteins, caffeic acid derivatives, flavonoids, vitamins etc. They have used in traditional medicine as hepatoprotective, gastroprotective, liver tonic, cholagogue, antioxidant, hypolipidemic, diuretic and also is used as a tonic and anticancer agent.6, 8, 10, 40
Effect of C. intybus on food intake of hyperglycemic animals
To evaluation of effect of orally administration of C. intybus leaf extract (50 mg/kg and 200 mg/kg) and glibenclamide (5 mg/kg) for 21 days on food intake of STZ induced hyperglycemic animals was cited in Table 4. The Food intake of hyperglycemic (induced by STZ) rats was significantly and continuously increased till the end of the study as compared to normal control (non hyperglycemic) animals. While, food intake of C. intybus extract and glibenclamide (5 mg/kg) treated hyperglycemic animals was not significantly change during study period as compared to hyperglycemic animals. Food intake of hyperglycemic animals treated with C. intybus extract (50 mg and 200 mg/kg was not increased similar to glibenclamide treated hyperglycemic animals. Hence, increases in food intake of STZ treated hyperglycemic animals was declined by sub-chronic administration of leaf extract of C. intybus in hyperglycemic (STZ treated) animals. A researcher has suggested that C. intybus may be used for nutritional and pharmaceutical purposes in livestock.23 Therefore, we can concluded that hydroalcoholic extract C. intybus leaf may have important role in appetite of hyperglycemic rats.
Table 4
Effect of C. intybus on food intake of hyperglycemic rats
Effect of C. intybus on water intake of hyperglycemic animals
The effect of C. intybus leaf extract (50 mg/kg and 200 mg/kg, orally for 21 days) and glibenclamide (5 mg/kg, orally for 21 days) on water intake of hyperglycemic (STZ induced) animals is presented in Table 5. The water intake was found significantly high during the study in hyperglycemic (STZ induced) rats as compared to normal control (non STZ treated) rats. While, water intake of C. intybus extract (50 mg/kg and 200 mg/kg) treated hyperglycemic animals was not significantly change as compared to non-treated hyperglycemic animals and found similar to standard drug glibenclamide (5 mg/kg) treated animals during study period. Hence, water intake induced by STZ in hyperglycemic animals, was significantly declined by administration of leaf extract of C. intybus at both doses (50 and 200 mg). Azay-Milhau (2013)41 suggests that the whole-plant methanolic extract of chicory positively influences glucose transport, while not inducing adipogenesis at the same time. Therefore, it was concluded that leaf extract of C. intybus might have important role in utilization/transportation of glucose in diabetic rats.
Table 5
Effect of C. intybus on water intake of hyperglycemic rats
Effect of C. intybus on body wt of hyperglycemic animals
The effect of sub-chronic administration of C. intybus leaf extract and glibenclamide on body weight of STZ induced hyperglycemic animals is given in Table 3. The body weight of hyperglycemic (STZ induced) rats was significantly and continuously decreased till the end of the study as compared to normal control (non hyperglycemic) rats. Whereas, body weight of C. intybus leaf extract (50 mg/kg) and standard drug glibenclamide (5 mg/kg) treated hyperglycemic animals was also decreased up to two weeks and then constant up to third week as compared to hyperglycemic animals. In C. intybus extract (200 mg/kg) treated hyperglycemic animals, body weight was significantly decreased in first week, but it was recovered in third week. Hence, sub-chronic treatment of leaf extract of C. intybus in hyperglycemic animals, initially decreased body weight of hyperglycemic animals was recovered similar to previous study of Abdel-Rahim et al., (2016),42 who has suggested that the improvement in body weight of hyperglycemic animals treated with chicory leaf extract was observed as compared to the hyperglycemic non treated animals. Urias-Silvas et al. (2007)43 has reported that chicory inulin is useful for regulation of appetite. Therefore, we can conclude that hydro-alcoholic extract C. intybus leaf might affect digestion of carbohydrates, proteins and lipid and their utilization in hyperglycemic rats.
Effect of C. intybus on serum glucose of hyperglycemic animals
The effect of sub-chronic administration of C. intybus leaf extract (50 mg/kg and 200 mg/kg, orally) and glibenclamide (5 mg/kg, orally) on serum glucose level in blood of STZ induced hyperglycemic animals was evaluated (Table 6). The blood serum glucose level was found significantly high in hyperglycemic rats as compared to normal control animals (no hyperglycemic animals) during the whole study period. While, serum glucose level was significantly decreases in C. intybus extract (50 mg/kg and 200 mg/kg) and glibenclamide (5 mg/kg) treated hyperglycemic animals as compared to non treated hyperglycemic animals at the end of the study. In hyperglycemic control animals, level of glucose in blood of rats did not decreased on 21st day as compared to normal control (non hyperglycemic) animals. Hence, increased blood serum glucose in hyperglycemic animals by STZ treatment was significantly declined by sub-chronic administration of both doses (50 mg/kg and 200 mg/kg) of leaf extract of C. intybus similar to standard drug glibenclamide (5 mg/kg) treated hyperglycemic animals at the end of the study. Therefore, it was observed that the both doses (50 mg/kg and 200 mg/kg) of hydro-alcoholic leaf extract of chicory plant exhibited hypoglycemic effect in hyperglycemic rats similar to effect of glibenclamide (5 mg/kg) in hyperglycemic rats. This result was similar to previous result of studies of Abdel-Rahim et al., (2016)42 and Mubeen et al., (2013). 44 The hypoglycemic effects of C. intybus leaf extract in STZ hyperglycemic animals may be through potentiating pancreatic secretion of insulin or enhanced transport/utilization of blood glucose or inhibition of endogenous glucose production.35 A scientist Ghamarian et al. [2012]45 suggested that chicory has insulin-sensitizing activity.
Table 6
Effect on glucose and other serological parameters of hyperglycemic rats
Effect of C. intybus on lipid profile of hyperglycemic animals
The effect of sub-chronic administration of C. intybus leaf extract (50 mg/kg and 200 mg/kg, orally) and glibenclamide (5 mg/kg, orally) on lipid profile of hyperglycemic animals is presented in Table 6. The levels of blood serum cholesterol and triglycerides were found significantly high in hyperglycemic rats as compared to normal control animals (no hyperglycemic animals) on last (21st) day of the study. In C. intybus extract (50 mg/kg and 200 mg/kg) treated hyperglycemic animals, levels of blood serum cholesterol and triglycerides were significantly low as compared to hyperglycemic animals and the result was observed similar to glibenclamide (5 mg/kg) treated hyperglycemic animals. Therefore, it was indicates that the hydro-alcoholic extract of chicory leaf exhibited hypolipidemic effect in hyperglycemic similar to previous study of Abdel-Rahim et al., (2016)42 and Mubeen et al., (2013).44 Diabetes is associated with hyperlipidemia due to destruction of β-cells leads to depletion of plasma insulin. Insulin activates enzyme lipoprotein lipase, which hydrolyzes triglyceride under normal conditions. Diabetogenic agents significantly increase the cholesterol and TG levels.46 The significant control of plasma lipid levels suggests that the chicory leaf extract may produce its action by improving insulin secretion. A study on 150 type 2 diabetes mellitus patients confirmed that ingestion of C. intybus seed extract has significantly decreased inflammation, oxidative stress, and hypertriglyceridemia.47 Cholesterol synthesis may be decreased via the action of chicory inulin that inhibits hydroxymethyl glutaryl-CoA reductase.48 Hence, we can conclude that increases levels of blood serum cholesterol and triglycerides in STZ- hyperglycemic animals, were significantly decreased by treatment of leaf extract of C. intybus (50 mg/kg and 200 mg/kg).
Effect of C. intybus on liver functions of hyperglycemic animals
The serum levels of SGOT, SGPT, Total bilirubin and direct bilirubin were found significantly high in blood of hyperglycemic rats as compared to normal control animals (no hyperglycemic animals) (Table 6). While, blood serum levels of SGOT, SGPT, Total bilirubin and direct bilirubin were significantly decreased in hyperglycemic animal streated with C. intybus extract (50 mg/kg and 200 mg/kg) and glibenclamide (5 mg/kg) as compared to non treated hyperglycemic animals. Hence, increased levels of SGOT, SGPT, total bilirubin and direct bilirubin in blood serum of hyperglycemic animals were significantly reduced by sub-chronic administration of both doses (50 mg/kg and 200 mg/kg) of leaf extract of C. intybus similar to standard drug glibenclamide treated hyperglycemic animals. This study was support by Abdel-Rahim et al., (2016),42 who has reported that the activities of AST, ALT and LDH were observed significant low in the diabetic rats administrated balanced diet with 20% of either chicory leaves in comparison to diabetic rats which feed on either high fat diet or balanced diet.
No significant changes were observed in the blood serum levels of total proteins and albumins in any treated hyperglycemic animal with C. intybus and glibenclamide as compared to control (non hyperglycemic) animals. Therefore, it was concluded that the hydro-alcoholic extract of chicory leaf exhibited hepato-protective effects in STZ hyperglycemic animals.
Effect of C. intybus on kidney functions of hyperglycemic animals
The levels of uric acid, creatinine and BUN in blood serum were found significantly high in hyperglycemic rats as compared to normal control animals (no hyperglycemic animals) on 21st day of the study. Whereas, C. intybus extract (50 mg/kg and 200 mg/kg) and glibenclamide (5 mg/kg) treated hyperglycemic animals, levels of uric acid, creatinine and BUN in blood of animals were significantly low as compared to hyperglycemic animals (Table 6). Hence, increased levels of uric acid, creatinine and BUN in blood serum of STZ- hyperglycemic animals were significantly reversed by sub-chronic administration of both doses (50 mg/kg and 200 mg/kg) of leaf extract of C. intybus similar to glibenclamide (5 mg/kg, for 21 days) treated hyperglycemic animals. This result was found similar to result of the study of Abdel-Rahim et al., (2016), 42 who has reported that the serum levels of creatinine and urea were significant decrease in the hyperglycemic rats administrated balanced diet with 20% of chicory leaves. Therefore, it was indicated that the hydroalcoholic extract of chicory leaf enhanced kidney functions in hyperglycemic rats.
Conclusion
In view of above the result we conclude that the root and leaf parts of C. intybus locally cultivated in foothill area of Uttarakhand are found rich in total carbohydrate, proteins and antioxidant compounds like phenolics, tannins, ascorbic acid, and flavonoid and having antidiabetic, kidney and liver protective properties, but both parts are having significant difference bio-chemically and medicinally. The root part of the plant has high amount of carbohydrates and proteins than leaf part, while leaf part has high amount of antioxidant compounds such as phenolics, tannins, ascorbic acid, and flavonoid than root part. The C. intybus has also very good antioxidant properties that are high in leaf as compared to root. Hence, leaf part may be more beneficial in oxidative damaged related disorders. While, in metabolism related disorders, root part might be more effective than leaves.
Results of animal trials on hydro-alcoholic leaf extract of C. intybus have shown very good hypoglycemic properties that also improve the liver and kidney functions in hyperglycemic animals. The body weight, food and water intake of hyperglycemic animals were regulated at normal level by continued administration of leaf extract. It might regulate metabolism via enhance the sensitivity/production of insulin. It also suggest that further investigation to identify the mechanism action of active principle of the plant for hypoglycemic activity and improvement of liver and kidney functions to be required.