Protective effect of Emilia sonchifolia (L.) against high protein diet induced oxidative stress in pancreas of Wistar rats.

OBJECTIVES: The present study was undertaken to investigate the oxidative damage, the biochemical and histopathological changes in the pancreas of the Wistar rats which was fed high protein diet and the recovery after the oral administration of the n-hexane extract of the herb, Emilia sonchifolia.
MATERIALS AND METHODS: The rats fed with high protein diet for a period of 30 days and treated with n-hexane extract of Emilia sonchifolia (250 mg/kg body weight). Body weight, pancreatic weight, serum amylase, lipase, aspartate transaminase, alanine transaminase, urea, uric acid, creatinine, DNA and RNA content of the pancreas, pancreatic enzymatic and non-enzymatic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, reduced glutathione, and vitamin C were evaluated. RESULTS AND DISCUSSION: At the end of the study the rats gained less body weight and showed a significant (P < 0.05) increase in serum levels of amylase, lipase, aspartate transaminase, alanine transaminase, urea, uric acid, creatinine, tissue DNA, and RNA content and showed a significant (P < 0.05) decrease in the pancreatic antioxidants. Treatment with the n-hexane extract ameliorated the damage caused by high protein diet. This was also evidenced by histopathological studies.
CONCLUSION: From the results, it was suggested that the n-hexane extract of Emilia sonchifolia has an effective medicinal property and can act as a pancreato-protective herb.

Nutrition plays an important role in the management of health and prevention of diseases. Major shift in the proportion of one macronutrient results in compensatory changes in the other macronutrients. Protein is one of the macronutrients and is required for the body's structure and proper function. Proteins function as enzymes, hormones, and antibodies, as well as transport and structural components. Transamination and oxidation result in elimination of protein as water, carbon dioxide, and nitrogen.[1] In high-protein diets, weight loss is initially high due to fluid loss related to reduced carbohydrate intake, overall caloric restriction, and ketosis-induced appetite suppression. High-protein diets are not recommended because they restrict healthful foods that provide essential nutrients and do not provide the variety of foods needed to adequately meet nutritional needs. Individuals who follow these diets are therefore at risk for compromised vitamin and mineral intake, as well as potential cardiac, renal, bone, and liver abnormalities overall.[2]

The organ in greatest danger is pancreas and the normal operation of its function is essential for the digestion and absorption of nutrients. Nutritional imbalance can cause deleterious effects and may be one of the causative factors of oxidative stress. They cause redox imbalance and further lead to a number of diseases produced through accumulating reactive oxygen species (ROS) in vivo.[3] Earlier studies have confirmed that pathogeny of acute pancreatitis, which is one of the diseases with higher incidence, is attributed to the oxidative damage of free radicals on endocrine and exocrine function of pancreas.[45] Herbs play an important role in the absorption and digestion and also have been used as a food and for medicinal purposes for centuries.[6] Existing remedies from folk medicine should be rigorously examined for potential relevance in the treatment of disease, and it is an alternative solution to allopathic medicine embodied with severe side effects.[7] In plants, terpenoids occur in nearly all natural food and represent a chemical defense against environmental stress and provide a repair mechanism for wounds and injuries. Terpenoids were evaluated for their potential antineoplastic activity in various human cancer cell lines such as gastric, pancreatic, and colon carcinomas.[8] Effective ingredients in several plant derived medicinal extracts are terpenoid compounds of mono, di, sesqui, tri, and tetraterpenoid groups.[9] One such folk medicine is Emilia sonchifolia in which only the n-hexane extract of the plant showed the presence of terpenoids in high concentration. Emilia sonchifolia is an edible plant used in the ayurvedic system of medicine for the treatment of gastropathy, diarrhea, opthalmia, nyctolpia, cuts and wounds, intermittent fevers, pharyngodyma, and asthma.[10] The aqueous and the methanolic extracts of the leaves of Emilia sonchifolia progressively exhibit antitumour activities.[11] The n-hexane extract of Emilia sonchifolia (250 mg/kg) has anticancer effect and rich in terpenoids.[12] With these backgrounds, the present study was undertaken to its medicinal property against pancreatic damage in Wistar rats fed high protein diet.

Materials and Methods

Plant collection

Emilia sonchifolia was collected from Thrissur, Kerala, India. The plant was authenticated by Dr. G.V.S. Moorthy, Botanical Survey of India, TNAU Campus, Coimbatore. The voucher number is BSI/SRC/5/23/09-10/Tech/782. Fresh plant material was washed under running tap water, air-dried and powdered.

Preparation of extract

The powder soaked in n-hexane solvent was kept in the shaker for 48 h at room temperature. The extract was collected and concentrated at 40°C under reduced pressure using rotary evaporator. The dried extract was stored at 4°C until further use. The remaining residue was extracted again with the fresh solvent to ensure complete extraction.

Preparation of high protein diet

Soybeans purchased from the local store, Coimbatore was powdered by using electronic blenders and stored in an airtight container.

Animals

The male wistar rats weighing between 50 and 55 g were obtained from Animal house of Karpagam University, Coimbatore. The yet weaning animals were caged in well-ventilated hygienic conditions. At the beginning of the experiment they were given a normal food for a week. After this adaptation period, they were divided randomly into four groups each containing six. The study was approved by Institutional Animal Ethical Committee (IEAC) constituted for the purpose of CPCSEA, Government of India.

Experimental design

Group I: Control rats.

Group II: Rats fed high protein diet (100% raw soy flour) for 30 days.

Group III: Rats fed high protein diet (100% raw soy flour) were treated with n-hexane extract of Emila sonchifolia (250 mg/kg body weight for 30 days).

Group IV: n-hexane extract of Emilia sonchifolia alone (250 mg/kg body weight).

Biochemical assays

Determination of serum biochemical parameters

After the experimental period of 4 weeks, the animals were sacrificed under light chloroform anesthesia. The blood was drawn from the para-orbital venous complexes and serum separation tubes, allowed to clot for 30 min at room temperature and then centrifuged at 1000× g for 10 min and stored at 4°C. The biochemical parameters such as amylase,[13] lipase,[14] AST, ALT,[15] urea,[16] uric acid,[17] and creatinine[18] were estimated. The pancreas were excised immediately, cleaned free of extraneous material and perfused with ice cold saline (0.9%) and stored in 10% formalin, which are used for the antioxidant and histopathological studies, respectively.

Determination of pancreatic DNA and RNA content

The nucleic acid DNA and RNA were extracted from the tissues.[19] One hundred mg of tissues were homogenized in 5 ml of 10% TCA was added to allow complete precipitation of protein and nucleic acid for 30 min. The mixture was centrifuged for 10 min at 5000 rpm and the precipitate was washed thrice with 10% ice cold TCA to remove any soluble compounds.

The precipitate was later treated with 5 ml of 95% ethanol to remove the lipid content and centrifuged for 10 min. The lipid free tissue residue was suspended in 5 ml of 5% TCA and placed in a water bath maintained at 90°C for 15 min with occasional stirring. This facilitated the quantitative separation of nucleic acid from the precipitated protein. This was centrifuged for 10 min and the supernatant was used for the estimation of RNA by Orcinol method[20] and again the residue is added with 5 ml of 10% TCA centrifuged and the supernatant is used for estimation of by diphenyl amine method.[21]

Estimation of tissue lipid peroxidation

Lipid peroxidation (LPO)[22] was calculated on the basis of the molar extinction coefficient of malondialdehyde (MDA) and expressed in terms of nanomoles of MDA/mg protein.

Estimation of antioxidant assays

The enzymatic antioxidants such as superoxide dismutase (SOD),[23] catalase (CAT),[24] glutathione peroxidase (GPx)[25] and the non-enzymatic antioxidants such as reduced glutathione[26] and vitamin C[27] were evaluated in the pancreatic tissue homogenates.

Statistical analysis

The results obtained were expressed as mean ± SD. The Statistical comparison among the groups were performed with one way ANOVA and DMRT using a statistical package program (SPSS 10.0) at P<0.05.

Results and Discussion

The body weight gain of the rats fed with Raw soy flour (Group II) was significantly lower when compared to Group I normal rats. The gain in body weight and feed utilization efficiency are lower in rats fed with high protein diet which might be because of poor digestion, decreased food intake by rats and thus results in weight suppression. In contrast, the pancreatic enlargement was observed in Group II rats fed Raw soy flour and it was significantly greater than the Group I normal rats [Table 1]. It was reported that[28] raw soybeans had a growth depressing effect in rats and also caused pronounced growth of the pancreas and stimulated pancreatic enzyme secretion.[29] Administration of Emilia sonchifolila significantly (P < 0.05) brought back the body weight and the pancreatic enlargement to its normal levels.

Table 1

Body weight and the pancreatic tissue weight of the control and experimental rats

The markers most commonly used to diagnose pancreatic damage include serum amylase and serum lipase levels; a level two to three times the upper limit of normal indicates a pancreatic attack.[30] An elevated amylase and lipase is observed in Group II rats fed with high protein diet which is an evidence for pancreatic damage [Table 2].[31] Our findings also include increase in the serum aminotransferases (AST and ALT) in Group II rats above that of the Group I control rats [Table 3]. High-protein diets are designed to induce ketosis, a condition that occurs in uncontrolled diabetes mellitus and starvation. When there are not enough carbohydrates in the diet to provide glucose to the cells that rely on it as an energy source, ketone bodies are formed from fatty acids. An increase in circulating ketones can disturb the body's acid-base balance, causing metabolic acidosis, which can lead to hypophosphatemia, resorption of calcium from bone, osteoporosis, and kidney stones. High-protein diets are associated with reduced kidney function, which, over time, can lead to permanent loss of kidney function. These diets are associated with a significant decline in kidney function.[32] Excessive protein ingestion can increase amino acid oxidation and urea synthesis. High protein intakes provide a greater uric acid load to the kidney. Despite increases in urinary uric acid excretion, increases in serum uric acid are observed [Table 4].[33] Treatment with the n-hexane extract of Emilia sonchifolia significantly (P < 0.05) restored the serum enzyme levels, urea, uric acid, and creatinine to its normalcy.

Table 2

Effect of n-hexane extract of Emilia sonchifolia on the activities of serum amylase and lipase of experimental animals

Table 3

Serum AST and ALT of the control and the experimental rats

Table 4

The concentration of Urea, Uric acid and creatinine in serum of control and experimental groups

A high-protein diet could destroy ROS in the digestive gland. The pancreas is an important glandular organ in the digestive system and the normal operation of its function is essential for the digestion and absorption of nutrients. Moreover, many studies have confirmed that pathogeny of acute pancreatitis, which is one of the diseases with higher incidence, is attributed to the oxidative damage of free radicals on endocrine and exocrine function of pancreas.[34] The synthesis of DNA and RNA needs a large amount of ATP to synthesize purine and pyrimidine and activate amino acids, and the ATP-generating process is accompanied by free radical production, so that oxygen free radicals are excessively produced, which is the possible reason for oxidative stress induced by the excessive protein diet.[35] In this study, the results indicates that a high protein diet could stimulate cell differentiation and growth, and promote synthesis of DNA and RNA in pancreas of Group II rats fed high protein diet. The DNA and RNA content in pancreas of Group III rats was found to be significantly (P < 0.05) decreased when compared with Group II rats [Table 5].

Table 5

DNA, RNA content in the pancreatic tissue of the control and experimental rats

Short ingestion (2-3 weeks) of fairly high levels of Raw soy flour by rats leads to pancreatic hypertrophy or hyperplasia.[36] Chronic ingestion of raw soybean meal produces pancreatic hypertrophy in chicks[37] pigs[38] and rats[39] plus benign and malignant tumors[40] and an increased susceptibility to carcinogens[41] in the rats. Numerous studies also suggest that CCK promotes pancreatic carcinogenesis.[42] High amino acid content in the duodenum can stimulate cholecystokinin (CCK) release, which promotes the acini to secrete enzymes.[43] CCK binds to the specific receptors on pancreatic acinar cells and is known to stimulate the growth of normal pancreatic tissue. This growth effect is mediated by high affinity CCK receptor. Increase in levels of CCK caused hypersecretion of digestive enzymes and pancreatic growth in all the rats[44] and also increase number of pancreatic cells (hyperplasia) and their size (hypertrophy),[45] pancreatic neoplasia[46] and also increase in pancreatic DNA synthesis.[47] Thus Raw soy flour is an inducer of pancreatic acinar cell tumors through CCK mediated mitogenic stimulation.[48] Calam et al,[49] found that feeding of a meal containing Raw soy flour to human subjects led to an increase in the level of CCK in the blood. It has been found to stimulate the growth of human pancreatic cancer cell line both in vivo in nude mice and in vitro cell cultures.[50]

The high protein diet caused a remarkable change in antioxidant mechanism by increasing the process of lipid peroxidation and decreasing the activities of SOD, CAT, GPx, reduced glutathione and vitamin C in Group II rats where as treatment with Emilia sonchifolia significantly (P < 0.05) decreased the lipid peroxide content and enhanced the antioxidant activities in Group III rats. This shows that the n-hexane extract of Emilia sonchifolia protects the pancreas from the oxidative stress induced by high protein diet [Tables 6 and 7].

Table 6

Effect of n-hexane extract of Emilia sonchifolia on the activities of enzymatic antioxidants in pancreas of control and experimental animals

Table 7

Effect of n-hexane extract of Emilia sonchifolia on the activities of non-enzymatic antioxidants in pancreas of control and experimental animals

Administration of the n-hexane extract of inhibited the excessive secretion of the enzymes and enhanced the body weight, maintained normal absorption and digestion in the rat fed Raw soy flour (Group III) and this is evident from the histopathological study which showed the restoration of the damaged tissue [Figure 1]. It may be due to the fact that the active principle in the n-hexane extract of Emilia.sonchifolia possibly reached the pancreatic tissue via circulation to provide pancreatic protection. Similarly, n-hexane extract of Emilia.sonchifolia significantly ameliorated the hepatic oxidative stress induced by the high protein diet.[51]

Figure 1

(a): The normal section of the pancreas (Group I). (b): The pancreatic tissue with marked acinar atrophy and inter acinar spaces the acinar architecture was destroyed (Group II). (c): The pancreatic tissue with mild acinar damage. The pancreas appears almost normal (Group III). (d): The normal section of the pancreas (Group IV)

The present study is performed for the first time to elucidate that Emilia.sonchifolia can act as a natural antagonist and be called as a pancreato-protective herb. Further studies are required to determine its particular terpenoid group and also the active constituent which may lead to the development of natural plant based medicine.