Evaluation of Anti-Diabetic Activity of Novel Bioactive Compounds of Carthamus Tinctorius Linn

INTRODUCTION

Diabetes is a complex metabolic disorder that is increasingly affecting the world’s population. Its health and economic consequences are considerable. The WHO Global Strategy for the Prevention and Control of non-communicable diseases recognizes diabetes as one of its priority conditions. Thepotential of plant as a source for new drugs is yet to be unexplored systematically. Among the estimated 250,000-400,000 plant species,only 6% have been studied for biological activity and about 15% have been investigated phytochemically.^1,2 The long-term, relatively specific complications of diabetes mellitus are predominantly vascular and include the development of retinopathy, nephropathy and neuropathy.People with diabetes also have a significantly increased risk of cardiac, peripheral arterial, and cerebrovascular diseases. It is estimated that 415 million people have diabetes in 20153 and about 70% of them live in developing countries.The burden of diabetes is well illustrated by the fact that 3%of a total population is treated for diabetes and its complications, the disease consumes around 10–15% of the entire health care budget.The diagnosis of diabetes is confirmed by demonstrating elevated blood plasma glucose levels either in fasting or post-prandial states. It is reported that the medicinal value is due to the presence of some chemical substance that produces a physiological action on the human body and therefore researchers always try to isolate these chemical substances from plants. In the present investigation, the anti-diabetic activity was assayed

The Carthamus tinctorius 4-7 is an erect, branched, smooth herb of 30-90 cm height. Leaves are stalkless, half-clasping, lanceolate, 5-10 cm long, 1-2.5 cm wide, but smaller towards the top, with margins minutely spiny toothed. Flowering heads are large, surrounded by a cluster of leafy bracts which gradually become the bracts of the involucre, 2.5-4 cm across. Flowers are orange-red. The achenes, often deformed, are obovoid, usually 4-ribbed and truncate at the top. Pappus is absent or scale like. Used in the conditions of rheumatic and painful joints and skin eruption. It also possesse wound healing, antioxidant, antiinflammatory and anti-carcinogenic activities.

MATERIALS AND METHODS

Collection of plant material

Fresh dried seeds were procured during early winter season from young matured plants from Aalagilawada, Davangere District, Karnataka State. Garbled seeds were procured, passed through sieve no.40 to get coarse powder and was used for studies.

Extraction

Supercritical extraction⁸ : The dried seeds were powdered using a mixer to obtain powder of the raw seeds of Carthamus tinctorius. Carbon dioxide (purity 99.99%) as solvent contained in a dip tube cylinder is installed. Supercritical CO2 was obtained by using SFT-10 Supercritical Fluid Pump. The SFT-10 is a high precision carbon dioxide pump designed to deliver liquid carbon dioxide at pressures up to 10,000 psi (68.9 MPa) (Fig.1).

Experimental procedure

The power of the SFT-110 SFE unit was turned on. Peltier cooler was turned on at least thirty minutes prior to the start of the experiment. Fifty grams of dried Carthamus tinctorius seeds was powdered. The powdered of Carthamus tinctorius seeds was placed into the SFT-110 Unit’s processing vessel and sealed. Both the static/dynamic valve and restrictor valve are closed.

The carbon dioxide tank valve was opened to allow the carbon dioxide to come into the unit (~750 psi). The oven temperature was set to 40o C and the restrictor block to 40^o C. The pressure on the pump was set to 2000 psi. The pump was begined to pressurize the sample vessel. It took 12-15 min. Once the pressure was up to 2000 psi, the sample was allowed to “soak” at that pressure for 15 min for 40^o C.

The static/dynamic valve was opened to allow free flow of carbon dioxide through the restrictor valve. The restrictor valve was adjusted to achieve about 24 mL/min of liquid carbon dioxide flow dynamically for 15 min. The pump was actuated and continued to maintain sample vessel pressure. The static/dynamic valve was closed and allowed to “soak” for an additional 15 min before repeating the dynamic flow step above. The static soak and dynamic flow step was repeated five more times.

The vessel temperature was set to ambient and the pressure control was set down to ambient. The unit was allowed to vent. When the vessel has reached ambient pressure, the inlet and outlet fittings were disconnected, the vessel was opened and the Carthamus tinctorius powder was removed. Similar procedure was carried out by varying the parameters such as temperature (45, 50, and 55o C), pressure (2000, 3000, and 4000 psi) and time (20, 25, and 40 min). Phytochemical investigations of all extracts were carried out in order to detect the presence of the following class of compounds.

Isolation

Isolation of pure components involved the following steps: 1. Chromatographic separation using silica gel (100-200 mesh) 2. Chromatographic separation using silica gel (60-120 mesh).

The SFE extract (10 g) was chromatographed over silica gel (100-200 mesh) on column 55 cm length and 6 cm diameter. Elution was carried out with solvent mixtures of increasing polarities. Fractions were collected in 100 mL portions and monitored by TLC (silica gel ‘G’ as adsorbent) and the fractions showing similar spots were pooled together. Elution with ethyl acetate: ethanol (EA: ET-OH) (40: 60) gave brown crystalline solid (450 mg) and named as CT1. Similarly, elution with EA: ET-OH (50:50) yielded shiny brown coloured crystalline solid (10mg) and was designated as CT-2.

Phytochemical screening⁹

Phytochemical investigation on seeds extracts of Carthamus tinctorius was carried out for the presence of steroids, alkaloids, saponin glycosides, flovones, coumarins, tannins, phenolic compounds, anthraquinone, chalcones, aurones, irridoids, and lignans.

Experimental animals

Adult Wistar albino rats either sex weighing between 150-250 g were used.

Chemical substances

Alloxon (SD-fine chemicals, Mumbai), EDTA (Loba chemicals, Mumbai), and Insulin (Appolo Pharmacy, Davangere) were used as received.

Equipment

The apparatus used for extraction is SFT-110 (Super critical fluid technologies, Inc, Newark, DE 19711.302-738-3420, U.S.A)

Acute toxicity studies¹⁰

Acute toxicity was evaluated on albino mice weighing between 30-35g. The fixed dose method was adopted as per OECD (Organization for Economic Co-operation and Development) Guideline No.423 of CPCSEA. The therapeutic dose fixed was 200mg/kg body weight i.e., 10%of the lethal dose.

Anti-diabetic activity¹¹

Anti-diabetic activity was performed byGOD/ POD-method, Beacon-Diagnostic Pvt. Ltd., India. Wistar rats were made diabetic by injecting alloxan monohydrate with a dose of 60 mg ⁄kg (i.p.) and the body was chilled (citrate buffer, pH 4.5). After 8 h, the rats showing blood glucose levels more than 250-350 mg/dl were considered as diabetic and were employed in the study. The study was carried out following the guidelines of principles of laboratory animal care.

Group I: Served as solvent control; Group II: Served as diabetic control (alloxaninduced); Group III: Received insulin (0.6 U/ Kg, sc.); Group IV: Received SCFE (100 mg/kg, p.o); Group V: Received SCFE (50 mg/Kg, p.o); Group VI: Received fraction-1(CT1) (50 mg/kg, p.o).

All the test samples were administered daily and the treatment period for all groups was two weeks.The treated animals fasted for 18 h and blood samples were drawn fromretro-orbital sinus puncture under mild anesthesia. The blood samples were collected in Eppendorf’s tube that contained 50µl of EDTA (anti-coagulant). The determination of serum glucose was performed by enzymatic method.

Statistical analysis

Data were statistically analyzed as mean±SEM and expressed as non-significant P<0.001 using One way ANOVA followed by Dunnett’s test.

RESULTS

Phytochemical screening

Phytochemical active constituents are reported in Table 1.

Anti-diabetic activity

The various extracts and fractions of seeds of Carthamus tinctorius Linn. showed excellent anti-diabetic activity (Fig.2) and reports are tabulated in Table 2.

DISCUSSION

The present study demonstrates the efficacy of super critical fluid extraction of Carthamus tinctorius (CT) and fraction CT1 . Several investigation reports have positive witness that they decrease the glucose level concentration in diabetic induced animal model. SCF

Soxhlet extraction with simple organic solvents were practiced usually, where as in this study new technique of extraction SCFE was adopted and obtained embody quality of extract. This study has substantiated due to SCFE; isolation of active moieties, which are responsible for anti-diabetic activity which has been mentioned in the results.

The alternative therapy has been gaining a momentum in recent years. Natural product chemistry provides scientific answer to many ailments. The bio- isolates of CT illustrate in curing Diabetes type-I and type-II providing a permanent and safe remedy. It was planned to screen indigenous plants like CT with view to discover a new bio-active compound with characterization of lead compound.

CONCLUSION

The data supported the traditional belief that Carthamus tinctorius (CT) has excellent antidiabetic proporties in alloxan induced diabetic rats, which significantly reduces the plasma glucose level promptly at 3rd week, it could improve diabetic conditons by virtue of its action on the endocrine pancreas. This activity was due to stimulation of insulin release via modulation of β-cell with Ca²⁺ handling. Based on these evidences, CT-extract could provide health benefits concerned to regulating the glucose concentration in blood plasma. Further characterization of active compounds and to establish its molecular level of mechanism of actions will be required for justification of active moieties of its clinical value.

CONFLICTS OF INTEREST

The authors declare no conflicts of interests.