INTRODUCTION:

Excess reactive oxygen species (ROS) causing increased oxidative stress induced tissue damage¹ and the ensuing anomalous inflammatory response causing hepatocellular and pancreatic injury pave way for the assumption that antioxidants may ameliorate the damages^2,3. Several plants used in the treatment of diabetes mellitus (DM) are endowed with various free radical scavenging molecules and thus exhibit good antioxidant activity⁴.

Salacia oblonga (S.oblonga) belonging to family Celastraceae is widely distributed in India and other Asian countries⁵. Due to the antioxidant properties of phytochemicals like quercetin and gallic acid⁶ and potent α-glucosidase inhibitory activity of the phytochemical ingredients salacinol and kotalanol along with several phenolic compounds^7,8, S.oblonga is known to reduce postprandial hyperglycemia, and insulinemia ^9,10. Knowledge about the pancreatic and hepatic structure and function in protracted DM on treatment with S.oblonga is incomplete.

The present study was aimed at studying the effect of a hydroalcoholic root extract of S.oblonga (SOE) on liver antioxidants, oxidants, serum enzymes and histology of liver and pancreas of streptozotocin (STZ) induced diabetic rats for a period of 16 weeks. Glibenclamide (Glb), the standard sulphonylurea drug, was taken as a reference.

MATERIALS AND METHODS:

Plant extract:

Required quantity of standardized SOE was obtained from Natural Remedies Private Limited, Bangalore, India, as a single lot. As per the product insert, the brown coloured powdered extract had α-glucosidase inhibition with IC₅₀<75.0µg/ml using [NR/BSY/AGIA/01] test protocol, α-amylase inhibition with IC₅₀<35.0µg/ml using [NR/BSY/SOP/004/02] test protocol and aldolase activity with IC₅₀<100.0µg/ml using [NR/BSY/ALDIA/01] test protocol¹¹. LD₅₀ for SOE was assessed to be 2g/kg body weight (the data is not produced in the present study). The safe dose in acute toxicity study was 1g/kg body weight, one tenth of this, 100mg/kg body weight/day, was used as the optimum dosage. Half of the first dose, 50mg/kg body weight/day was selected as the second dose. 0.5% carboxy methyl cellulose was used as solvent to dissolve the extract.

Animals used:

Domestically bred wistar strain albino rats of both sexes weighing 100±10g were selected for the study. They were placed in a non-infective area with an approximate humidity of 53±10%, temperature of 23±2^oC and controlled illumination. Polypropylene cages were used with paddy husk as bedding. The rats were fed normal laboratory pellet diet and water ad libitum and were kept for one week for acclimatization to the laboratory conditions prior to initiating the experiment. The study was carried out after obtaining the approval from the Institutional Animal Ethics Committee (213/PO/Re/S/2000/CPCSEA). All the experimental procedures were carried out as per the guidelines issued by CPCSEA and the Government of India guidelines for the use of laboratory animals.

A single dose of intraperitoneal injection of STZ (Sigma –Aldrich Corporation, 3050 Spruce, St. Louis, Missouri 63103. United States), 50mg/kg body weight in cold citrate buffer (0.1M) of pH 4.0, was given to the rats after fasting for 18-20 hours for induction of DM. The rats were kept under observation for 72 hours to check their survivability. On day -1 of the experiment, Fasting glucose (FBG) was determined using ACCU CHECK Active glucometer. The rats were assigned to the following treatment groups of eight rats each and fed using intragastric tube:

1. Group I – Normal control (NC) fed with vehicle.

2. Group2 – Diabetic control (DC) fed with vehicle.

3. Group3- Diabetic rats with SOE at the dose of 100mg/kg body weight/day (D+S-100)

4. Group4- Diabetic rats with SOE at a dose of 50mg/kg body weight/day (D+S-50)

5. Group5- Diabetic rats with Glb at the dose of 500µg/kg body weight/day (D+Glb).

Study protocol:

The duration of the study was 16 weeks, at the end of which, the animals were sacrificed with a high dose of ether. Blood was collected immediately by cardiac puncture in a plain vacutainer. Obtained serum was used for the estimation of enzymes Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT) by standard procedures suited for autoanalyser using kits from Roche Diagnostics (Mannheim, Germany).

The liver and pancreas were separated, washed thoroughly using normal saline and then weighed. A weighed amount of the liver and pancreas were gently homogenized separately using 0.9% saline in a final volume of 10 times the weight of the tissue. The homogenates were centrifuged at a speed of 3000 rpm for 10 minutes and supernatant was collected for estimation of vitamin C and carbonyl protein (CP) by the reaction with 2,4-dinitrophenyl hydrazine^12,13, reduced glutathione (GSH)and protein thiols (PT) levels by their reaction with 5,5’-dithio nitrobenzoic acid^14,15, malondialdehyde (MDA) by measuring the concentration of TBARS¹⁶.

The rest of the liver and pancreatic tissues were fixed with 10% buffered formalin for 24 hours and embedded in paraffin wax blocks. Uniform sections of 5µm were cut and stained with hematoxylin and eosin. These were examined under microscope for histopathological findings and photomicrographs were obtained.

Statistical analysis:

The grouped data of different parameters were analysed using the statistical software, SPSS-20 (SPSS, Chicago, IL, USA). The results were expressed as mean±SD for each group. Various parameters were compared between the groups by applying Kruskal-Wallis test. Correlation was checked by applying Pearson’s correlation test. P-value of <0.05 was considered to be statistically significant.

RESULTS:

Effect of SOE on biochemical parameters:

Table – 1: Levels of hepatic weight, antioxidants and oxidants in the experimental groups at 16^th week.

	NC	DC	D+ S-100	D+ S-50	D+ Glb
Vitamin C (µg/g tissue)	165.63±39.10	86.75±13.77^a	100.88±13.77^a	123.75±17.33^a,b	101.75±14.65^a
GSH(µg/g tissue)	431.25±70.93	307.38 ± 43.15^a	627.0±179.77^{a, b,c}	532.13±39.26^b,c	347.00±43.98
PT(µmol/mg protein)	66.50±6.52	61.46±18.71^a	76.69±10.79	68.36±15.67	68.08±12.78
MDA(µmol/g tissue)	16.91±4.89	18.28 ± 1.64^a	3.03 ± 0.31^{a, b,c,d}	13.43±1.91^b	14.44±0.77^b
CP(mU/mg protein)	2.56±0.78	3.91±1.80^a	1.96±0.19^b,d	2.50 ± 0.62^b	2.14±0.52^b
Wt of liver (g/100g body wt)	3.19±0.42	4.79±0.65^a	4.53±1.09^a	4.16±0.87^a	4.87±1.20^a

NC- Normal control, DC – Diabetic control, D+ S-100 – diabetic rats treated with 100mg/kg/day SOE, D+ S-50 – diabetic rats treated with 50mg/kg/day SOE, D+ Glb- diabetic rats treated with 500µg/kg/day Glb. p ≤ 0.05 - ^a-compared to NC, ^b- compared to DC, ^c- compared to D+ S-50, ^d-p compared to D+ Glb

Table – 2: Levels of pancreatic weight, pancreatic antioxidants and oxidants in the experimental groups at 16^th week.

	NC	DC	D+ S-100	D+ S-50	D+ Glb
Vitamin C(µg/g tissue)	73.63±17.61	29.13±5.25^a	51.25±10.99^{b, c}	71.63±3.58^b,d	43.13±15.41^b
GSH(µg/g tissue)	591.88±144.6	318.5±57.72^a	340.88±74.06^d	302.25±59.52^d	212.75±63.27^b
PT(µmol/mg protein)	35.63±9.74	12.21±1.87^a	28.39±9.32^{b, c}	15.06±2.97^b,d	32.31±5.49^b
MDA(µmol/g tissue)	2.23±0.27	7.9±0.94^a	2.60±0.60^{b, c,d}	3.33±0.66^b,d	5.54±1.17^b
CP(mU/mg protein)	0.39±0.04	1.18±0.33^a	0.90±0.15	0.96±0.44	0.78±0.20^b
Wt of pancreas (g/100g body wt)	0.65±0.04	0.47±0.11^a	0.64±0.15^b	0.61±0.11^b	0.55±0.12

Table – 3: Levels of serum hepatic enzymes in the experimental groups at 16^th week.

	NC	DC	D+ S-100	D+ S-50	D+ Glb
AST (U/L)	225.38±25.43	478.63±40.45^a	415.00±120.57^d	328.00±135.49^d	588.50±49.55^b
ALT (U/L)	74.13±5.22	220.38±39.05^a	215.00±44.85	216.38±57.95^d	170.88±12.93^b
ALP (U/L)	144.25±33.06	662.38±132.05^a	1001.13±227.29^b	824.02±144.92^b	874.13±144.96^b
GGT (U/L)	7.05±2.45	44.50±5.32^a	61.13±11.61^{b, c, d}	49.38±4.37^d	32.25±7.80^b

A comparison of hepatic antioxidants vitamin C, GSH and PT and that of oxidants MDA and CP and the weight of the liver among the various test groups are displayed in Table-1. A lower trend of antioxidants and raised oxidant status was observed in the DC group as compared to NC with statistical significance. Treatment with SOE brought up the levels of antioxidants with the D+S – 100 group showing a definite increase in GSH and PT. A consequent dose dependent decrease in the levels of MDA and CP were observed. Increase in GSH and decrease in MDA in the SOE treated groups statistically scored better over the Glb reference group. Weight of the liver was found to be significantly more in all diabetic groups compared to NC. SOE at higher dose showed elevated weight compared to the lower dose.

Pancreatic antioxidants vitamin C, GSH and PT and that of oxidants along with pancreatic weight are depicted in Table-2. There was decreased antioxidant levels with increased oxidants in DC group as compared to NC. SOE caused elevation of the levels of antioxidants and lowered those of oxidants and was statistically appreciable. The decrease in MDA and CP in SOE treated groups were also significant and dose dependent. The increase in GSH and decrease in MDA in D+S-100 group was significant over Glb treated group. Pancreatic weight in DC group was low compared to NC group. Treatment with SOE caused increased pancreatic weight which was dose dependent.

Table-3 gives the levels of serum hepatic enzymes in different study groups. SOE treatment led to insignificant changes in the level of AST and ALT in comparison to the DC group. Contrarily, a significant increase in ALP and GGT were observed with both doses. D+Glb group showed elevated levels of AST and ALP with insignificant changes in ALT and GGT compared to DC group. However, ALT and GGT of D+Glb group were significantly lower compared to D+S-100 group, whereas AST was high.

Effect of SOE on histology of liver and pancreas:

Fig- 1. Histopathology of liver of NC, DC, D+S-100, D+S-50 and D+ Glb (A,B,C,D,E) respectively.

A – NC (Normal control) X100, B- DC(Diabetic control) X400, C- D+ S-100(diabetic rats treated with 100mg/kg/day SOE) X100, D- D+ S-50(diabetic rats treated with 50mg/kg/day SOE) X400, E- D+ Glb(diabetic rats treated with 500µg/kg/day Glb) X100.

Fig-1 shows the histopathological findings of rat liver of different study groups. Normal hepatocyte architecture was maintained in NC. DC showed intracellular hydropic changes, focal fatty changes and sinusoidal congestion. The group receiving SOE at lower dose produced mild portal triaditis, and mild hyaline changes of blood vessels. The group receiving higher dose of SOE showed venous congestion and necrotic changes. Sinusoidal congestion and dilatation of the portal vein with portal triaditis were also seen in the Glb treated group.

Fig- 2. Histopathology of pancreas of NC, DC, D+S-100, D+S-50 and D+ Glb (A,B,C,D,E) respectively.

A – NC (Normal control) X100, B- DC(Diabetic control) X100, C- D+ S- D+ S-100(diabetic rats treated with 100mg/kg/day SOE) X100, D- 50(diabetic rats treated with 50mg/kg/day SOE) X100, E- D+ Glb(diabetic rats treated with 500µg/kg/day Glb) X100.

Histopathology of pancreas of different study groups is shown in Fig-2. NC group showed the normal pancreatic histology. Enzymatic necrosis of exocrine, lymphocyte infiltration and depleted islets of Langerhans were observed in DC. D+S-50 showed acute pancreatitis with necrosis of both exocrine and endocrine parts, where as in D+S-100 there was islet regeneration. Other findings in this group were congestion and oedema of the ducts. Glb treated group also showed depleted islets.

DISCUSSION:

In this study where the effects of prolonged treatment of hydroalcoholic extract of S. oblonga was tested to understand its influence on the antioxidants, oxidants and structure of liver and pancreas over 16 weeks, we found a significant increase in the levels of hepatic and pancreatic antioxidants and decrease in that of oxidants indicating the antioxidant potential of SOE. In a study on the effect of a hydroalcoholic root bark extract of S.oblonga on serum anti-oxidants, sperm morphology and count in Mitomycin-C induced testicular damaged rats¹⁷, the antioxidant activity was observed in 7 days of study period. In our study, we have demonstrated that the anti-oxidant effect of SOE is sustainable over the duration of 16 weeks and the effect of SOE was better than that of Glb. The phenolic compound, epicatechin has a protective effect on thiol groups¹⁸, and by this protective mechanism, the formation of advanced glycation end products may be controlled. At the cellular environment, both GSH and PT act as good antioxidants and the sparing action of these two may help in preservation of vitamin C making it available for its potent antioxidant action.

Elevated ALP and GGT in diabetic SOE treated rats indicates hepatic biliary obstruction. Since the values were higher than that of DC group, it may be safe to assume that the adverse effect seen in the treated groups is due to SOE. Same can also be appreciated in Glb treated group. The ongoing oxidative stress and altered inflammatory response have been proven to be the basis for liver and pancreatic injury manifesting as raised hepatic enzymes¹. S.oblonga and other plants have shown protective effect on rat kidney^19,20, liver²¹ and pancreas^22,23. In a review on anti-diabetic and anti-hyperlipidemic effects and safety of Salacia species, animal trials of maximum period of 90 days and human trial for a period of 3 months did not show any toxic effects. In contrast to these, hepatic hypertrophy was observed in 28 days of treatment with S.oblonga²⁴in one of the studies. Various phytochemicals like pyrrolizidine alkaloids, monoterpenes and sesquiterpenes induce hepatotoxicity²⁵. Sesquiterpenes and triterpens along with salacinol and kotalanol have been isolated from S. oblonga⁸. We have previously shown that oral administration of Phyllanthus niruri Linn. to CCl4 induced hepatotoxic rats for 7 days protected the liver, on the contrary, resulted in renal and testicular damage²⁶. The risk and beneficial effects need detailed evaluation before adopting the plant extracts for human consumption. In the present study, the hepatotoxic effect can be envisaged in SOE intake at higher doses over a protracted period, supported by increased liver weight as an indicator of hepatomegaly and histopathological finding of necrosis.

The sustained antihyperglycemic activity observed over a period of 16 weeks with a substantial decrease in the FBG and HbA1c and improved insulin levels shown earlier proves the superior efficacy of SOE over Glb²⁷. The mechanism of antihyperglycemic activity of the plant extract is due to its β-cell regeneration and insulinogenic properties as evidenced by the improvement in the insulin levels and the pancreatic β-cell mass. In the present study, however, we also observed mild necrosis of pancreas.

Assuming the safety of a treatment option just by the evidence of its effect in regularizing certain parameters without a study on its safety on the structure and function of vital organs may prove perilous. Though scientific verification and validation of several alternative medicines is done, there are reports of toxic hepatitis, renal damage and possible gonadal involvement following consumption of folklore medicines consistently in literature^25,26. In this regard, an attempt to know the safety of SOE on the liver and pancreas when used over a long period was addressed.

CONCLUSION:

SOE has antioxidant role and pancreatic regenerative capacity. On the contrary, the cytotoxic effect on hepatocytes and the indiscernible histological changes in the pancreas on intake at higher dose for a prolonged time recommends more controlled studies for validation of the dosage and its safety profile.

CONFLICTS OF INTEREST:

The authors have no financial or proprietary interests in any material discussed in this article.

ACKNOWLEDGMENTS:

The authors are thankful to the Indian Council of Medical Research (ICMR) for their monetary support for carrying out this project (Ad hoc research project No. 59/18/2005/BMS/TRM).

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Received on 25.09.2021 Modified on 22.02.2022

Research J. Pharm. and Tech 2023; 16(2):879-884.

DOI: 10.52711/0974-360X.2023.00149