Effect of Katakakhadiradi Kashayam on Lipid Profile and Pancreatic Damage in Type II Diabetes Mellitus

The lipid pattern and levels in diabetic patient are the same as those for subjects with cardiovascular disease. The tactic underlying the oral hypoglycemic agent is to adjust the lipid profile; which can be achieved by herbal therapy. The use of herbs and formulations for attenuation of hyperglycemia and to aid protection against the pancreatic damage is clinically very important. This study was intended to find the efficacy of Katakakhadiradi Kashayam(KKK)on lipid profile and pancreatic tissue damage in streptozotocin-nicotinamide (SN) induced diabetic rats. The diabetic rats were treated with Katakakhadiradi Kashyam orally at doses of 100, 200 and 300 mg/kg/bw. For Original Research Article Subbiah et al.; JPRI, 33(28B): 135-141, 2021; Article no.JPRI.68461 136 28 days and compared with the standard drug Glibenclamide. After the kashayam treatment triglyceride, HDL-cholesterol and total cholesterol level were assayed and pancreatic tissue damage caused by streptozotocin was analysed by histology study.Katakakhadiradi kashayam could restore the serum lipid profile by controlling the blood glucose level and reduce the pancreatic injury in diabetic rats. Supplementation of Katakakhadiradi kashayam showed a significant improvement in the serum lipid profile thus helping in retarding the secondary complications.


INTRODUCTION
Diabetes (T2DM) and associated cardiovascular complications are major public health challenges globally. Chronic inflammation of the pancreas in diabetic condition can damage the beta cells that secretes insulin. People with T2DM have two-to four-fold enhanced risk of coronary artery disease (CAD), the leading cause of death in people with T2DM [1]. Hypertension and dyslipidemia are major modifiable risk factors for T2DM and connected CAD, which account for more than 87% of disability in low-and middleincome nations [2].
High lipid levels are related with serious health risks and elevated mortality. Hypertension, insulin resistance, hyperlipidemia and glucose intolerance are recognized as cardiac risk aspects in diabetes patients. The persistence of hypercholesterolemic state causes increased oxidative stress, resulting in the progression of coronary artery disease and other complications of diabetes [3].
The cholesterol together with some other types of fats cannot be dissolved in the blood. Furthermore, in order to be shifted to and from cells, they have to be specially carried by specific molecules known as lipoproteins, which has an outer layer of protein with an inner core of cholesterol and triglycerides. Moreover, lipoproteins are found essential for cholesterol to travel around the body [4].
Lipid alterations in patients with diabetes, often known as "diabetic dyslipidemia", which are typically characterized by high triglycerides, high total cholesterol, low high density lipoprotein cholesterol and elevated levels of small dense LDL particles [5]. Low density lipoprotein cholesterol levels may be moderately enhanced or normal. Lipid abnormalities are common in people with T2DM and prediabetes but the pattern of the different lipids may alter between economic levels, ethnic groups, and access to health care [6].
Recently, there is growing interest in herbal medicines all over the world. Traditional herbs having antilipidemic effect and antidiabetic effect can prove to be useful source for the development of new oral hypolipidemic and hypoglycemic agents or simple dietary adjuvant to existing therapies [7]. Although Katakakhadiardi kashayam has not been studied for its biological activity, but the plant components were reported to contain flavanoids, lignins, alkaloids, triterpenoids and polyphenolic compounds [8]. Katakakhadiardi kashayam treatment was found to have beneficial effect on hyperglycemia, blood glucose level restored to a normal clinical range. The present work was designed to study the effect of Katakakhadiardi kashayam on lipid profile in streptozotocin induced diabetic rats. To evaluate the effect of Katakakhadiardi kashayam on the histological changes which occur in the pancreatic tissue of the diabetic rats and the possible mechanisms through which this formulation produces its antidiabetic effect.

Plant Materials and Formulation
All the plant materials were collected from the herbal garden and drug store of Ayurveda College, Coimbatore, Tamil Nadu, India. Katakakhadiradi Kashayam is a herbal decoction prepared from each 10 grams of the following ingredient plants. Strychnos

Animals
Adult male albino wistar rats (6 weeks), weighing 150 to 200 g were used for the present antidiabetic study. The rats were kept in clean polypropylene cages and maintained in a wellventilated temperature regulated animal cage with a continuous 12 h light/dark schedule. The rats were given standard rat pelleted diet and filtered drinking water was given ad libitum.

Experimental Induction of Diabetes
The rats were kept overnight fasting and checked the primary fasting blood glucose from the blood sample collected from the tip of rat tail vein. The streptozotocin (60 mg/kg) was dissolved in citrate buffer (pH 4.5) and intraperitoneally injected to the rats done overnight fasting, after 15 min and 120 mg/kg of nicotinamide was administered intraperitoneally to induce non-insulin dependent DM. Hyperglycemia was noted after 72 hrs by the rise in blood glucose levels. The rats with blood glucose level higher than 250 mg/dl were used for the present study [9].

Study Design
The rats were classified into 6 groups each having six animals.Group I rats received normal saline 1 ml/kg bw. orally; Group II, animals for diabetes induction experimentally injected with inj streptozotocin 60 mg/kg bw. along with nicotinamide 120 mg/kg as intraperitoneal injection; Group III diabetic rats treated with Glibenclamide 20 mg/kg orally for 28 days; Group IV diabetic rats treated with Katakakhadiradi kashayam (KKK) 100 mg/kg orally for 28 days; Group V diabetic rats treated with KKK 200 mg/kg orally for 28 days; Group 6 diabetic rats treated with KKK 300 mg/kg orally for 28 days.

Blood collection
About 3 ml of blood was collected in a heparinized syringe. The rest of the heparinized sample was transferred to microcentrifuge tube and run at 5000 rpm for 20 min at 4°C to obtain serum. The serum was transferred to a clean microcentrifuge tube and stored at −80°C (Kumar et al., 2017).

Estimation of total cholesterol
Cholesterol estimation was done conferring to the procedure of Parekh and Jung, (1970).
Ferric chloride-uranyl acetate reagent (2.9 ml) was added to sample (0.1 ml). After centrifugation, sulphuric acid-ferrous sulphate reagent (2.0 ml) was added gradually and mixed well. Blank containing ferric chloride-uranyl acetate reagent (3.0 ml) and sulphuric acidferrous sulphate reagent (2.0 ml). A calibration graph was made with standard cholesterol. In a Shimadzu UV spectrophotometer, the optical density was taken (530 nm after 20 minutes). The levels of cholesterol were measured in mg/dl [10].

Estimation of triglycerides
Triacylglycerol estimation was done according to the procedure of Rice, (1970). To 0.1 ml sample, activated alumina (50 mg) and isopropanol (3.9 ml) was added and mixed well. This was then kept for 15 minutes. Centrifugation was done and supernatant (2.0 ml) was taken for examination. Alkaline potassium hydroxide(0.6 ml) was added to every tube and were kept for 10 minutes at 60°C. The tubes were chilled and sodium metaperiodate (1.0 ml) reagent was added to the tubes followed by the adding acetyl acetone reagent (0.5 ml). The tubes were again cooled and the color formed was read in a Shimadzu UV spectrophotometer at 405 nm against blank. The levels of triacylglycerol were measured in mg/dl [11].

Estimation of HDL
The HDL-consisting supernatants and the wholeserum test samples were assayed in opposed to the otclipoprotein antiserum as 20and 40-fold dilutions, respectively, in aqueous sodium chloride (150 mmol/I). In assays against the lipoprotein antiserum, sera were diluted 20fold with the same solution but the supernatants were applied undiluted. All precipitated applicable material was made free from any occluded supernatant before application. Each precipitate was dissolved in a volume of aqueous sodium chloride (150 mmolfl) equal to that of the original serum aliquot and reprecipitated by addition of an equal volume of aqueous PEG solution, as described above. The secondary supernatant was thrown out and the process was redone once more. Lastly, the low-density lipoproteins were re-dissolved in the same volume of aqueous sodium chloride [12].

Masson trichrome Staining
For analysing histopathology of pancreas, the tissues were administered for paraffin sectioning. The sections were fixed in Bouin's solution at room temperature overnight to intensify the colors and increase the contrast between the tissue components. The slides were washed for 1-2 minutes under running tap water (18-26°C) to remove yellow color from sections and then rinse briefly in double distilled water. The sections were incubated for 5 minutes with Weigert's Iron Hematoxylin solution to stain the nuclei dark and then discarded the solution. The slides were placed in a glass chamber and washed under warm running tap water for10 minutes to remove excess of Hematoxylin and to intensify the black color. Then incubated the sections for 5 minutes with Biebrich Scarlet-Acid Fuchsin Solution to stain the fibers red. The images are viewed using a bright-light microscope (40x magnification)and photomicrographs were taken using a Sony digital camera.

Statistical Analysis
The mean value and standard error were measured for each parameter. The significant differences were observed, mean values were compared using one way ANOVA. A probability value of less than 0.05 was considered significant.

RESULTS
Figs. 1-3 depicts that there was a significant (p<0.001) increase in triglyceride, total cholesterol and (p<0.001) significant decrease in high density lipoprotein in diabetic rats. The present study showed that Glibenclamide (p<0.001) significant reduction in the serum lipid levels in diabetic rats. Similarly, katakakhadiradi kashayam intake from minimum to maximum doses (100, 200 and 300 mg/kg) significantly restored the serum lipid levels in near normalcy in diabetic rats (Figs. 1-3).
Histopathology results showed that normal control group has normal pancreatic acini with thin sheet of collagen around them. Diabetic rats showed shrunken islets with thick and dense collagen fiber layers seen around the pancreatic ductules (arrow mark) and acini indicative of fibrosis. Glibenclamide group showed normal islets and thin collagen fibres around the acinar cells and ductules. KKK at doses 100 and 200 mg of showed normal islets with mild thickening of collagen fibers around the ductules and in between the pancreatic acini. High dose 300mg KKKshowed normal islets with thin layer of collagen fibers around the ductules (Fig. 4).

DISCUSSION
Dyslipidemia (or diabetic dyslipidemia when diagnosed in T2DM patients) is an abnormality in lipid metabolism; i.e. quantitatively observed in abnormalities in blood lipids including increased triglycerides (TG) and low-density lipoprotein (LDL-C) and a decreased high-density lipoprotein cholesterol (HDL-C) concentration. These disturbances are established risk factors in the development of atherosclerosis. Dysregulation of intestinal lipoprotein metabolism is often seen in T2DM patients.   [13,14]. The antidiabetic components and antilipidemic components of this formulation are previously tested by GC-MS analysis and its invitro free radical scavenging activity were also proved [15,16]. this polyherbal formulation have more antioxidant constituents which prevents the pancreatic cell injury in diabetes condition. The findings are consistent with earlier report showed that administration of Glibenclamide at a dose of 0.6 mg/Kg bw to diabetic rats caused a significant decrease in total lipids [12].

Fig. 2. Effect of katakakhadiradi kashayam on triglyceride level in diabetic rats
The KKK reduces the lipids ad lipoprotein levels in streptozotocin and nicotinamide induced diabetic rats ad it is also confirmed in pancreatic tissues by Masson trichrome staining. The KKK at a dose of 300 mg reduces the blood glucose and insulin levels which is already published by the author. The ayurvedic herbs present in the formulation also plays a very important role to reduce the blood lipids and regulate the lipid metabolism in a well balanced manner.

CONCLUSION
This Katakakhadiradikashayam showed perceived effectiveness with fewer side effects and relatively low costs herbal drugs prescribed for diabetes and its associated disease. Therefore, Katakakhadiradi kashayamcan be used as current hypoglycemic formulation to improve management of diabetic patients associated with hyperlipidemia.

CONSENT
It is not applicable.

ETHICAL APPROVAL
All animal experiments were conducted after getting approval from the ethical committee and in accordance with the guidelines for the appropriate care and use of laboratory animals(IAEC No: KMCRET/Ph.D/16/2016-2017).