A NOVEL TANNIC ACID FROM GANODERMA LUCIDUM FRUITING BODIES EXTRACT AMELIORATES EARLY DIABETIC NEPHROPATHY IN STREPTOZOTOCIN INDUCED DIABETIC RATS

A NOVEL TANNIC ACID FROM GANODERMA LUCIDUM FRUITING BODIES EXTRACT AMELIORATES EARLY DIABETIC NEPHROPATHY IN STREPTOZOTOCIN INDUCED DIABETIC RATS

E.M Elhussainy¹, N.A Elzawawy ² and S.H. shorbagy ³

Physiology Department¹, Faculty of Medicine, Kafer el sheikh University, kafer el sheikh, Egypt.

Microbiology Department ², Faculty of Science, Tanta University, Tanta, Egypt.

Pathology Department ³, Faculty of Medicine, Tanta University, Tanta, Egypt

ABSTRACT: Aim: The present study evaluated the anti-diabetic, anti-hyperlipidimeic, antioxidant activities and renal protective effect of aqueous extract from Ganoderma. lucidum fruiting bodies in streptozotocin induced diabetic rats. Material and Results: Qualitative and quantitative phytochemical analysis for G. Lucidum aqueous extract showed the Presence of high concentration of tannins content. Thin layer chromatography (TLC) revealed the presence one tannin spot which high performance liquid chromatography (HPLC) analysis confirmed that the tannin compound is tannic acid. Extracted tannic acid showed highly antioxidant activity in vitro. Two doses of the extract were administered intraperitoneally. Tannic acid significantly decrease blood glucose levels and significant increase in serum insulin in addition, it improved serum lipid profile in diabetic rats compared to untreated diabetic group. Results: Showed that tannic acid also had potent antioxidant activity in vivo, as it significantly decreased MDA while GSH and GPx in erythrocytic and renal tissues were significantly increased. Histopathological examination of kidney tissue tannic acid treated rats showed mild glomerular atrophy and minimal tubular affection with noticeably dilated blood vessels compared to untreated diabetic rats. Conclusion: These results suggest that extracted tannic acid has antihyperglycemic, hypolipidemic and renal protective effect against STZ-induced diabetic nephropathy in rats.

INTRODUCTION: Diabetes mellitus (DM) is a major health problem all over the world, because its incidence and prevalence are elevated and increasing, reaching epidemic proportions ¹. Diabetes is likely to be the fifth leading cause of death worldwide ². The global percentage attributable to diabetes was estimated to be 260 million people ³. Much of the increase incidence of diabetes worldwide occurs in developing countries with such cases as ageing, unhealthy diets and obesity with malnutrition- related causes playing a pivotal role.

One of the most important complications of diabetes mellitus is diabetic nephropathy (DN) which considered a major long term complication occurring in 30-50% of diabetic patients ⁴. The early stages of diabetic nephropathy are characterized by an elevation of urinary albumin excretion, an increment of glomerular filtration rate and renal hypertrophy ⁵. Metabolic derangements, systemic and glomerular hypertension found to occur in the progression of diabetic nephropathy which is a leading cause of end-stage renal failure. Oxidative stress and lipid peroxidation is thought to play an important role in mediating the vascular complications of diabetes, including nephropathy ⁶. Therefore, a therapeutic approach targeting its causative mechanisms is urgently needed.

There are several classes of approved oral antidiabetic drugs, most of them exert undesirable side effects, drug interactions and are expensive since they comprise polypharmacy regimen. ⁷  Thus, natural products and their derivatives have been a successful source of bioactive molecules in medicine as they contain polyphenolic compounds which are found predominantly in several comestible fruits and medicinal fungi as a part of daily food consumption.

12. lucidumis a basidiomycetes fungi, belonging to the family of Polyporaceae. Its fruiting body has long been used in China, and Korea as a folk medicine for the treatment of wide varieties of diseases. Studies on this mushroom have demonstrated many interesting biological activities, including antitumour ⁸, antioxidant ⁹, cytoxicity ¹⁰, hepatoprotective ¹¹, and anti-inflammatory effects ¹².

Therefore, the aim of the present work was to study the effect of aqueous extract of G. Lucidum (tannic acid) on glucose homeostasis, lipid profiles and some kidney functions in diabetic nephropathy. In addition, to study the possible mechanism of action of tannic acid as a new antidiabetic drug andthis would help in testing the efficiency of G. lucidum extract as a natural source for medical treatment.

MATERIAL AND METHODS:

Macro-Fungi Organism:

Fruiting bodies of G. lucidum were collected from around the delta region of Gharbia Governorate; Egypt, in the month of August 2014 and identified ¹³.

Aqueous Extract Preparation:

The fruiting bodies were dried under shade and ground to a powder using mechanical grinder. About 500 g of the powder was macerated in 2.5 L distilled water at room temperature for 24 h. It was then filtered using Whatman filter paper No.1 and filtrate evaporated to dryness in water bath at 60 °C. A yellowish brown residue about 46.3 g (9.26% yields) was obtained and kept in an airtight container at 40°C until used ¹³.

Phytochemical Evaluation of Fruiting Bodies of G. lucidum Aqueous Extract:

1-Qualitative Analysis of Secondary Metabolites: G.lucidum aqueous extract was subjected to preliminary screening for the presence of secondary metabolites (alkaloids, saponins, terpenoids, flavonoids, steroids, tannins and phenolics) using standard procedures, with some modifications ^{14, 15, 16}.

1. Test for alkaloids:

Mayer’s test:

Alkaloids give cream color precipitate with Mayer's reagent [Potassium mercuric iodide solution].

Dragendorff’s test:

Alkaloids give reddish brown precipitate with Dragendorff’s reagent [Potassium bismuth iodide solution].

Wagner's test:

Alkaloids give a reddish brown precipitate with Wagner's reagent [Solution of iodine in potassium iodide].­­­­

1. Test for Saponins:

Froth test: Five milliliters of the extract were diluted with 20 ml distilled water and shaked in a graduated cylinder for 15 minutes. Formation of 1cm layer of foam indicates the presence of saponins.

1. Test for Sterols &Triterpenoids:

Libermann- Buchard test:

Extract is treated with few drops of acetic anhydride, boil and cool, con. Sulfuric acid is added from the sides of the test tube, shows a brown ring at the junction of two layers and the upper layer turns green which shows the presence of Steroids and formation of deep red color indicates the presence of triterpenoids.

Salkowski test:

Treat extract in Chloroform with few drops of conc. Sulfuric acid, shake well and allow standing for some time, red color appears at the lower layer indicates the presence of Steroids and formation of yellow colored lower layer indicates the presence of Triterpenoids.

1. Test for Flavonoids:

Shinoda test (Magnesium Hydrochloride reduction test):

To the test Solution, add few fragments of Magnesium ribbon and add concentrated Hydrochloric acid drop wise, pink scarlet, crimson red or occasionally green to blue color appears after few minutes.

Alkaline reagent test:

To the test solution add few drops of sodium hydroxide solution; formation of an intense yellow color, which turns to Colorless on addition of few drops of dil. acid, indicates presence of Flavonoids.

E.Test for Tannins & Phenolic Compounds:

Gelatin test:

Test solution with 1 % gelatin solution containing 10% sodium Chloride gives white precipitate.

Ferric chloride test:

Test solution gives blue green color with ferric chloride.

Vanillin Hydrochloride test:

Test solution when treated with few drops of vanillin hydrochloride reagent gives purplish red color. Tannins yield bulky precipitate with phenazone especially in the presence of Sodium and phosphate.

Alkaline reagent test:

Test solution with sodium hydroxide solution gives yellow to red precipitate within short time.

Mitchell’s test:

With iron and ammonium citrate or iron and sodium tartarate. Tannins give a water-soluble iron-tannin complex,which is insoluble in solution of Ammonium acetate.

2-Quantitative Analysis of Secondary Metabolites:

a- Determination of total phenolic compounds in G. lucidum aqueous extract:

Phenolic compounds were extracted from G. lucidum aqueous extract according to the method outlined by Jindal and Singh ¹⁷. A known weight of 1 gm of powdered extract was extracted with 80 % aqueous ethanol, and centrifuged at 3000 rpm for 20 min. One ml of sample was mixed with 1 ml of Folin-Ciocalteau phenol reagent and one ml of 20% anhydrous sodium carbonate, then completed up to 5 ml with distilled water. The absorbance of the blue colour was measured after 30 min. at wavelength 650 nm against water-reagent blank. The phenolic content was obtained from a standard curve of pyrogallol, and then calculated as mg phenolic per gram dry weight.

b- Determination of total alkaloids:

Alkaloid compounds were extracted from G. lucidum aqueous extract according to Harborne ¹⁸. 5 g of the extract was weighed into a 250 ml beaker and 200 ml of 10% acetic acid in ethanol was added, covered and allowed to stand for 4 h. This was filtered and the extract was concentrated on a water bath to one-quarter of the original volume. Concentrated ammonium hydroxide was added drop wise to the extract until the precipitation was complete. The whole solution was allowed to settle and the precipitated was collected and washed with dilute ammonium hydroxide and then filtered. The residue is the alkaloid, which was dried and weighed, as mg alkaloid per 5 gram dry weight.

c- Determination of total tannins:

Tannin content in each sample was determined using insoluble polyvinyl-polypirrolidone (PVPP), which binds tannins as described by Makkar ¹⁹. Briefly, 1 ml of extract dissolved in methanol (1 mg/ml), in which the total phenolics were determined, was mixed with 100 mg PVPP, vortexed, kept for 15 min at 4°C and then centrifuged for 10 min at 3,000 rpm. In the clear supernatant the non-tannin phenolics were determined the same way as the total phenolics. Tannin content was calculated as a difference between total and non-tannin phenolic content.

Tannins showed highly presence in G. lucidum aqueous extract than non tannin contents and alkaloids. Therefore, tannin compounds were separated on thin layer chromatography and identified by high performance liquid chromatography analysis.

Thin Layer Chromatography (TLC):

The G. lucidum aqueous extract was also characterized by means of thin layer chromatography on silica gel plates (Merck) using the following developing systems (specific for tannin contents); A: acetic acid-petroleum ether-diethyl ether (1:20:80, v/v/v) ²⁰; B: Ethyl acetate : Acetic acid : Water: ethanol (8: 1: 8:1, V: V: V ) ²¹. Following developing of plates, they (A and b) were sprayed with an aqueous solution of ferric chloride to visualize tannin compounds giving purple spots compared to tannic acid as a standard then these compounds were scratched from TLC with the help of clean and dry spatula and collected in beaker containing 70% methanol and left overnight ²².

High Performance Liquid Chromatography Analysis (HPLC):

HPLC analysis of the scratching compounds was performed at Mubarak City for Scientific Research, Borg el Arab, Alexandria Governorate according to the method of Singh ²³. The HPLC system (Shimadzu Corporation, Kyoto, Japan) was used and samples were filtered through an ultra membrane filter (pore size 0.45 μm) prior to injection in the sample loop. Tannic acid was used as standard. Tannin compound present in the sample was identified by comparing chromatographic peaks with the retention time (Rt) of individual standard.

Tannic acid Extraction:

Tannic acid was extracted from aqueous extract according to the procedure of Mole and Waterman ²⁴, extraction was done by taking 30 g of G lucidium powder added 200 ml water for 24 hours, followed by filtration. Ethyl acetate was added to filtrate many times, later acetate layer was separated and evaporated by using vacuum rotary evaporator until gaining semisolid product (tannic acid) to use it in different tests. This method was repeated until we obtained the desired dose for experiment.

Antioxidant activity of extracted tannic acid (ETA) from g. Lucidum aqueous extract in vitro: (DPPH radical scavenging assay):

DPPH stable free radical scavenging activity was determined by the method of Blois ²⁵.  3 ml of aqueous extracted tannic acid were added to 1ml of 0.1 mM solution of DPPH in methanol. After incubation at 37°C for 30 min, absorbance was measured at 517nm against control using a spectrophotometer (Hitachi). The experiment was conducted in triplicate. Ascorbic acid was used as the reference materials. The percentage of inhibition was calculated by comparing the absorbance values of the different extracts with those of the controls.

The inhibition percentage (I) was calculated as radical scavenging activity as follows:

I= (Abs control – Abs sample) / Abs control X 100

Where Abs control is the absorbance of the control reaction and a sample is the absorbance in the presence of the sample of the extracts. The antioxidant activity of the G. lucidum extracts were expressed comparing with standard.

Experimental Animals:

Forty male albino rats weighing 150 – 200 g were used for this study. They were kept in standard cages at 26+1°Cwith relative humidity 55+10% and 12 h light/dark condition in the animal house of the Department of Zoology, Faculty of Science, Tanta University. The animals were acclimatized to the conditions by maintaining them at the experimental conditions for about 5 days prior to starting the experiment. The animals were fed under standard diet and water adlibitum maintained under standard laboratory conditions.  All the protocols were performed in accordance with the Institutional Animal Ethical Committee.

Experimental induction of diabetes:

Animals were injected with streptozotocin (STZ) (Sigma, UK, 65 mg/kg body weight, dissolved in citrate buffer at pH (4.5) ²⁶. Two days after the STZ injection, the diabetic state was determined by the positive response to glucose in the blood by (test strips; Glucotest, Germany). The rats were considered to be diabetic when the fasting blood glucose concentrations were higher than 200 mg/dl.

Experimental design:

The STZ induced diabetic rats were randomly assigned into four groups(1 - 4) of 10 rats (n = 10) each as follows, namely: Group 1- Control group received vehicle only (distilled water);Group 2 Diabetic control received vehicle only; Group 3 Diabetic rats received 50 mg/kg body weight of tannic acid extract; Group 4 Diabetic rats received 100 mg/kg body weight of tannic acid extract ²⁶.

Serum Preparation:

Blood samples were collected by cardiac puncture after intraperitoneal injection of thioparbital(40 mg/kg) in all groups after 8 weeks of treatment. Blood samples were centrifuged at 3000 g for 10 minutes and stored at – 70 °C for biochemical investigations.

 Biochemical Analysis:

Fasting serum glucose was determined using a glucose oxidase kit (Wako Chemicals, Japan) according to Desbuquois, et al. ²⁷. Fasting serum insulin was detected by radio-immunoassay according to Morgan and Bray ²⁸. Blood urea and serum creatinine levels were estimated according to standard methods ²⁹. Collection of 24hours urine sample for measurement of albumin excretion (microalbuminuria), each rat was transferred into cages equipped with accessory for collecting urine sample. Microalbuminuria levels were determined using ELISA kit (DRG, USA) according to Reenu, et al. ³⁰. Serum total cholesterol (TC), triglycerides (TG) and HDL-cholesterol (HDL-C) were measured by enzymatic test kits (Randox Laboratory Ltd, UK) and LDL-cholesterol (LDL-C) was calculated using Friedewald equation ³¹.

Antioxidant measurement in vivo:

In Blood:

Serum malondialdehyde (MDA) was measured as the marker of lipid peroxidation bythiobarbituric acid (TBA) test. Briefly, serum was diluted by buffered saline (1:5) and 400 μl of this mixture was added to 800 μl of trichloroacetic acid (28%) and then centrifuged in 3000 g for 30 min. The precipitation was dissolved in sulfuric acid and 600μl of this mixture was added to 150μl thio barbituric acid (1% w/v) and incubated for 15 min in boiling water. After incubation, 4 ml n-butanol was added, centrifuged and the absorption of red supernatant was recorded spectrophotometrically at 532 nm. The calibration curve was provided by using 1, 1,3,3- Tetraethoxypropan as standard ³². MDA was expressed as nmol/ml. The glutathione (GSH) content was measured according to Beutler, et al. ³³ using glutathione kits. GSH was expressed as µmol/g Hb. Glutathione peroxidase activity was determined according to the method of Paglia and Valentine ³⁴. This method was used todetermine GPx enzyme in units per gram hemoglobin.

In Renal Tissues:

Different sets of renal tissue samples were homogenized with different buffers as follows:

1. Added to 50 mM phosphate buffer (pH 7.0) containing 1 mM EDTA, for measurement of glutathione peroxidase (GPx) activity. It was expressed as nmol/minute mg protein;

2. Added to cold 1,15% KCl for measurement of malondialdhyde (MDA) or thiobarbituric acid reactive substances (TBARS). It was expressed as nmol/mg protein; and

3. Added to cold 5% trichloroacetic acid in 0.01 M HCl for non-protein sulfhydryl (NPSH) pool for reduced glutathione (GSH) measurement. It was expressed as µmol/mg protein.

Histopathological Procedures:

After 8 weeks, the rats were sacrificed under anesthesia; kidney tissues of untreated and treated rats were immediately excised according to Al-Mofleh, et al. ³⁵ and fixed in a 10% solution of formaldehyde and then dehydrated in graduated ethanol (50-100%), cleared in xylene and embedded in paraffin. The sections (4-5 µm) were examined with ordinary microscope (400x) after staining with Haematoxylin and Eosin (H&E) stain and also Periodic acid chief (PAS) to detect the thickness of glomerular basement membrane according to Bancroft, et al. ³⁶. The histological studies were carried out at pathology department, Faculty of Medicine, Tanta University.

Statistical Analysis:

Results are expressed as mean ± SD. Group means were compared with a one way analysis of variance (ANOVA) and by LSD Test. P < 0.05 was considered significant.

RESULTS:

In Vitro Results: The results of qualitative analysis of G. lucidium aqueous extract (Table 1) showed the presence of alkaloids, phenols, tannins and absence of terpenoids, flavonoids and steroids. Table 2 represented quantitative analysis of G. lucidium aqueous extract. The contents of total phenolics that were measured by Folin - Ciocalteu reagent in terms of Gallic acid equivalent were 160 mg/g, tannin contents were 135.5 mg/g. While, alkaloids were low quantitatively in G. lucidium extract as they were 1.2 mg/g.

TABLE 1: QUALITATIVE PHYTOCHEMICAL SCREENING FOR SECONDARY METABOLITES PRODUCTION IN GANODERMALUCIDIUM AQUEOUS EXTRACT:                                               

" - " : Absent

" + " :  Weak presence

" ++ " : Moderate presence

" +++ " : Strong presence

TABLE 2: QUANTITATIVE PHYTOCHEMICAL ANALYSIS OF GANODERMALUCIDIUM AQUEOUS EXTRACT:

1. Lucidium aqueous extract was loaded onto preparative silica gel thin layer plates. The chromatograms were developed in the following systems: Ethyl acetate: Acetic acid: Water: ethanol (8: 1: 8:1, V: V: V), Acetic acid-petroleum ether-diethyl ether (1:20:80, V: V: V).

The best solvent system was Ethyl acetate: Acetic acid: Water: ethanol (8: 1: 8:1, V: V: V). While, other solvent system showed a rapid tailing of the active substances in extract. By using the best solvent system, one compound was detected under the UV lamp Fig. 1. The R_f was 0.9 and compared on TLC with tannic acid as standard. The spots were developed on the TLC plate and sprayed with ferric chloride reagent in order to determine roughly the nature of the extract, it can be concluded that the obtained extract contained one tannin compound which gave purple color with ferric chloride and appeared with the same R_f with standard tannic acid.  Extracted tannin spot on the preparative silica gel plate was scratched with dry spatula and collected in beaker containing methanol and left overnight then filtered and used for the identification by HPLC.

FIG.1: THIN LAYER CHROMATOGRAPHY FOR SEPARATION, AND CHARACTERIZATION OF EXTRACTED TANNINS OF G. LUCIDIUM AQUEOUS EXTRACT:

1, 2: Extracted tannin of G. lucidium

3, 4, 5: Standard tannic acid

The HPLC ‘fingerprint’ in Fig. 2 of the  extracted tannin compound  from  G. lucidium aqueous extract showed major peak at the retention time of (1.78) min, at a wavelength of 254 nm. This showed that tannin compound is tannic acid compared with standard tannic acid.  Finally, Fig. 3 revealed the structure of tannic acid as a one of the active compounds of G. lucidium aqueous extract which was tested for its antioxidant, anti-diabetic and antihyperlimidemic activites.

FIG. 2: HPLC- ANALYSIS FOR EXTRACTED TANNINS OF G.LUCIDIUM AQUEOUS EXTRACT

A: Standard mix of tannic acid, Gallic acid, benzoic acid and salicylic acid

B: Extracted tannin "which identified as tannic acid by TLC".

 FIG.3: CHEMICAL STRUCTURE OF TANNIC ACID

It was observed from Table 3 that extracted tannic acid from G. lucidium aqueous extract at a concentration of 2 mg/ml had high equal scavenging activity compared to the same concentration of ascorbic acid. The scavenging activity of extracted tannic acid increased gradually from 38.2% to 91.5% respectively. This study showed that extracted tannic acid had the proton donating ability and could serve as free radical inhibitors or scavenging, acting possibly as primary antioxidants.

TABLE 3: ANTIOXIDANT ACTIVITY OF EXTRACTED TANNIC ACID (ETA) OF GANODERMALUCIDIUM AQUEOUS EXTRACT, IN COMPARISON TO ASCORBIC ACID (IN VITRO):

In- vivo results:

In STZ diabetic rats, a significant increase in fasting blood glucose level (p < 0.001) with a significant decrease in fasting serum insulin level (p < 0.001) was observed compared to control group Table 4. Administration of extracted tannic acid with a dose 50 mg/kg (low dose) or 100 mg/kg (high dose) to diabetic rats significantly decrease the levels of fating blood glucose (p < 0.001) and significantly increase in fasting serum insulin level (p < 0.001) after 8 weeks of treatment compared to untreated STZ diabetic rats.

Concerning the level of lipid profile in serum of normal and diabetic rats were expressed in Table 4 where streptozotocin-diabetic rats showed significant increase in the levels of serum cholesterol, triglycerides and LDL-cholesterol (P < 0.001), and significant decrease in HDL-cholesterol (P < 0.001) compared to control rats. Treatment with tannic acid with low dose in diabetic rats significantly decrease the levels of serum cholesterol, triglycerides, LDL-cholesterol (P < 0.01), and significantly increase the levels of HDL-cholesterol (P < 0.05) compared to untreated diabetic rats. While tannic acid with high dose in diabetic rats significantly decrease the levels of serum cholesterol, triglycerides, LDL-cholesterol (P < 0.001), and significantly increase the levels of HDL-cholesterol (P < 0.001) compared to STZ-induced diabetic rats.

The levels of blood urea, serum creatinine and urinary albumin excretion (microalbuminuria) in control, diabetic and diabetic treated rats were expressed in Fig. (4 A, B, C), as in STZ diabetic rats significant increase in the level of blood urea, serum creatinine and urinary excretion of albumin (P < 0.001) was observed when compared to control rats while, tannic acid-treated diabetic rats with low dose or high dose showed significant reduction in the level of blood urea, serum creatinine and urinary albumin excretion (P < 0.01 for low dose and P < 0.001 for high dose) compared to STZ-diabetic rats.

Levels of antioxidant enzymes (GSH and GPx) in red blood cells and renal tissue and lipid peroxidation products (MDA) in serum and renal tissue in control, untreated and treated diabetic rats were expressed in Fig.(5 A, B, C) and Fig. (6 A, B, C). STZ-diabetic rats showed significant decrease in GSH level as well as GPx enzyme activity in erythrocytic and renal tissues (P < 0.001) and significant increase in the level of MDA (P < 0.001) when compared to control rats. Tannic acid -treatment with low dose in diabetic rats showed significant increase in the levels of GPx activities in erythrocytes and renal tissues (P < 0.01) and non- significant (P > 0.05) changes were observed as regard GSH level in erythrocytes, but GSH level in renal tissues showed significant increase (P < 0.01) when compared with STZ-diabetic rats, while the levels of MDA significantly decreased in erythrocytes and renal tissues (P < 0.001) when compared to STZ-diabetic rats.

Tannic acid -treatment with high dose in diabetic rats showed significant increase in the levels of GSH as well as activities of GPx, in erythrocytes and renal tissues (P < 0.001) while the levels of MDA significantly decreased (P < 0.001) when compared to STZ-diabetic rats.

TABLE 4: EFFECT OF EXTRACTED TANNIC ACID 50 mg/kg (LOW DOSE) AND 100 mg/KG (HIGH DOSE) ON SERUM LEVEL OF GLUCOSE, FASTING SERUM INSULIN AND LIPID PROFILES IN mg/dl IN DIABETIC RATS.

P value statistically significant < 0.05.

(a) denotes significant differences group II vs. group I,

(b) denotes significant differences group III vs. group II.

(c) denotes significant differences group IV vs. group II

FIG. 4 : A, B, C: EFFECT OF TANNIC ACID ON STZ INDUCED DIABETIC NEPHROPATHY AS REGARD BLOOD UREA AND SERUM  CREATININE IN mg/dl AND MICOALBUMINURIA(mg/24 hours), IN DIABETIC RATS. (A) DENOTES SIGNIFICANT DIFFERENCES GROUP II VS. GROUP I, (B) DENOTES SIGNIFICANT DIFFERENCES GROUP III VS. GROUP II. (C) DENOTES SIGNIFICANT DIFFERENCES GROUP IV VS. GROUP II.

FIG. 5: A, B, C: EFFECT OF TANNIC ACID ON STZ INDUCED DIABETIC NEPHROPATHY AS REGARD SERUM (MDA) NMOL/ML AND ANTIOXIDANTS; (GSH) µMOL/G HEMOGLOBIN AND (GPX) U/G HEMOGLOBIN IN ERYTHROCYTES. AND (A) DENOTES SIGNIFICANT DIFFERENCES GROUP II VS. GROUP I, (B) DENOTES SIGNIFICANT DIFFERENCES GROUP III VS. GROUP II. (C) DENOTES SIGNIFICANT DIFFERENCES GROUP IV VS. GROUP II.

FIG. 6: A, B, C: EFFECT OF TANNIC ACID ON STZ INDUCED DIABETIC NEPHROPATHY AS REGARD (MDA) NMOL/MG PROTEIN AND ANTIOXIDANTS; (GSH) µMOL/MG PROTEIN, (GPX) NMOL/ MINUTE MG PROTEIN IN RENAL TISSUES. (A) DENOTES SIGNIFICANT DIFFERENCES GROUP II VS. GROUP I, (B) DENOTES SIGNIFICANT DIFFERENCES GROUP III VS. GROUP II. (C) DENOTES SIGNIFICANT DIFFERENCES GROUP IV VS. GROUP II.

Histopathological examination of kidney tissue (H&E and PAS stains) after treatment with extracted tannic acid showed mild glomerular atrophy and minimal tubular affection but showed noticeably dilated blood vessles (vascular congestion) compared to diabetic rats which showed markedly shrunken and atrophic glomeruli, focal glomerular sclerosis ,thickening of glomerular basement membrane and tubular vaculation (Fig.7 A, B).

FIG.7: HISTOLOGICAL SECTION OF KIDNEYS STAINED WITH H&E (A) AND PAS (B) AT 400x

Fig (A): H&E staining of kidney biopsies: (A) normal kidney (control), (B) Diabetic kidney showing shrunken glomeruus and  marked vacuolization of tubules,  (C) TA treated kidney showing normaly looking gomeruli and tubules with marked vascular congestion (x400).

Fig (B): PAS staining of kidney biopsies : (A) normal kidney (control), (B) Diabetic kidney showing thickened basement membrane with focal PAS positive sclerosis of gomeruli (arrow), (C) TA treated kidney showing  normal glomerular basement membrane with congestion of a nearby vessel (x400).

DISCUSSION: Diabetes mellitus is characterized by many chronic complications such as vascular-disorder, retinopathy, neuropathy and nephropathy ³⁷. Diabetes is often linked with abnormal lipid metabolism and is considered as a major factor for the development of atherosclerosis and cardiovascular complication ³⁸. Diabetic nephropathy is one of the most common complications of diabetes as the kidney is the main target organ affected ³⁹. Various mechanisms are implicated in the pathogenesis of diabetic nephropathy, including increased aldose reductase activity, enhanced activity of protein kinase C isoforms, and increased formation of advanced glycation end products ^{40, 41}. The rapidly increasing number of patients with diabetic nephropathy has motivated to find new safe methods to control diabetes and its complications and this has led to an increasing demand for natural products with antidiabetic activity. Therefore, the present study was aimed to assess the effect of tannic acid on hyperglycemia, lipid profile, and enzymatic and non-enzymatic antioxidants in STZ-induced diabetic rats with special concern to diabetic nephropathy.

It is the first time to evaluate the effect of extracted tannic acid against diabetic nephropathy. In our study, tannic acid was separated on TLC giving one tannin spot and this was confirmed by HPLC similar to Romani, et al. ⁴² who identified  tannic acid  from commercial plant extracts  by HPLC.

The results of the present study showed that tannic acid significantly decreased blood glucose level of STZ induced diabetic rats. In addition, fasting serum insulin levels in diabetic rats treated with tannic acid showed significant increase when compared with the untreated diabetic group. In experimental animals STZ caused partial destruction of pancreatic beta cells, and is usually used to induce diabetes mellitus in these animals through its toxic effects Kim, et al. ⁴³; Matteucci and Giampietro ⁴⁴. In the present study treatment with tannic acid reduces the blood glucose level in the diabetic rats, indicating its anti-hyperglycemic activity. The mechanism by which tannic acid reduced blood glucose level may be enhancing glucose utilization by increased insulin secretion from beta cells of pancreas as detected in our study.

The results of the present study were in accordance with Babby, et al. ⁴⁵ who suggested that tannic acid may haveanti-diabetic activity and explain the proposed mechanism of action by enhancing peripheral glucose utilization either by direct stimulation of glucose uptake or via the mediation of enhanced insulin secretion and increasing the glucose transporter activity. Also, tannic acid and Gallic acid (phenolic acids) may have the potential to become the lead compounds in the development of new types of antidiabetic pharmaceuticals that are able to reduce blood glucose levels as these compounds appear to aid in control of hyperglycemia as well as in reducing the complications associated with diabetes mellitus, Liu, et al. ⁴⁶.

In the present study, 24 hours collection of urine in STZ induced diabetic rats showed micro albuminuria which is considered as an early marker of diabetic nephropathy. It refers to the excretion of albumin in urine at a rate that exceeds normal limits, but is less than the detection level of traditional dipstick method. In the present study, microalbuminuria as well as blood urea and serum creatinine levels were significantly increased in the STZ diabetic rats. These results are in accordance with Tanwar, et al. ⁴⁷ who observed that after STZ induced diabetes; blood urea, serum creatinine and albumin excretion in urine (microalbuminuria) were elevated. However, treatment with tannic acid, in our study, caused a significant decrease in excretion of albumin in urine as well as significant reduction in the levels of blood urea and serum creatinine.

In addition, the results obtained in the current study indicate that extracted tannic acid (ETA) ameliorates hyperlipidemia caused by STZ-diabetic rats. Significant reductions were observed with ETA as regard lipid profile; serum total cholesterol (TC), triglycrides. (TG), and low density lipoprotein cholesterol (LDL-C) and significant increase in high density lipoprotein cholesterol (HDL-C) compared to STZ-diabetic rats. The rise in plasma TG levels in diabetic rats could result from decreased removal of circulating lipoproteins or from increased lipoprotein production, and both mechanisms seem to be implicated in the genesis of the hypertriglyceridemia that occurs after STZ administration.

Earlier studies have shown that in STZ-diabetic rats, hyperglycemia is associated with hyper-cholesterolemia and hyper-triglyceridemia. Abnormalities in lipid metabolism have been demonstrated in diabetic nephropathy in which the rate of free fatty acid (FFA) uptake is inversely proportional to the severity of the renal dysfunction. Elevated serum lipid levels are believed to be one of the major contributing factors in the pathogenesis of diabetic nephropathy. The elevated circulating FFA produces inhibition of glucose oxidation, abnormal high oxygen requirements during FFA metabolism, and the intracellular accumulation of potentially toxic intermediates of FFA, all of which lead to impaired renalfunctions and severe morphological changes⁴⁸.

Hyperglycemia is a main cause for elevated free radical levels, followed by production of reactive oxygen species (ROS), which can lead to increased lipid peroxidation and altered antioxidant defense and further impaired glucose metabolism in biological system ⁴⁹. Oxidative stress is a free radical mediated processes which initiates a chain of reactions that gives rise to some products like malondialdehyde (MDA). An imbalance between oxidation and antioxidant status has been shown to play an important role in mediating insulin resistance and diabetes mellitus ⁵⁰.

Overwhelming free radicals generated due to oxidative stress may develop several adverse effects commonly seen in diabetes such as vascular disorders and diabetic nephropathy ⁵¹. The major antioxidant enzymes, including SOD, CAT, and GSH-Px, are regarded as the first line of the antioxidant defense system against ROS generated in vivo during oxidative stress and act cooperatively at different sites in the metabolic pathway of free radicals ⁵².

In the current study, the kidney and serum MDA content in the STZ diabetic rats was significantly increased beside depletion of antioxidants; reduced glutathione level and glutathione peroxidase activity suggested that STZ-induced diabetic nephropathy could be a result of oxidative stress and lipo-peroxidation or suppression of the antioxidant enzymes which is agreed with earlier studies where streptozotocin induced diabetes caused lipid peroxidationas a result of increased hydrogen peroxide concentration produced in the kidney and due to the depletion of antioxidant system; reduced glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase(CAT) activity ^{53, 54}. In the present work, significant reduction in the kidney and serum MDA content and restoration of erythrocytic GSH level and GPx activities in tannic acid treated rats suggested an improvement in the in vivo antioxidant status with subsequent nephroprotective effect of tannic acid.

In the present work, histological studies indicate some characteristic changes in the kidney as there was a significant improvement of glomeruli, regeneration of tubular epithelium of kidney tissues and the renal tissue damage was minimal after administration of extracted tannic acid of G. Lucidum. These observations were in agreement with Shukla, et al. ⁵⁵ who have shown that plant extracts reverse the damage in the kidney of STZ induced diabetes.

CONCLUSION: It was revealed that tannic acid which is identified as a one of the active compounds of G. lucidum aqueous extract showed highly antioxidant, antidiabetic and antihyperlipidemic activities. Overall, the results obtained in the present study showed that extracted tannic acid can be used in treatment of diabetic nephropathy. Further studies are needed to evaluate its effectiveness in other diabetic complications and to find other mechanisms of action.

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    How to cite this article:

    Elhussainy E.M, Elzawawy NA and Shorbagy SH: A novel tannic acid from ganodermalucidum fruiting bodies extract ameliorates early diabetic nephropathy in streptozotocin induced diabetic rats. Int J Pharm Sci Res 2016; 7(1): 62-75.doi: 10.13040/IJPSR.0975-8232.7(1).62-75.

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