Ancient Science of Life

ORIGINAL ARTICLE
Year
: 2018  |  Volume : 37  |  Issue : 4  |  Page : 188--199

Antidiabetic and nephroprotective potential of polyherbal self-fermented Ariṣṭa formulation: Evidence-based research


Amitabha Dey1, Satyajyoti Kanjilal2, Rajarshi Biswas1, Anjan Adhikari3, Satyabrata Mohapatra4, Deepa Gandhi3, Bibhuti Nath Bhatt3, Shiladitya Choudhuri5, Pallabi Chakraborty5, Tapas Kumar Sur6, Avinash Narwaria7, Chandra Kant Katiyar7,  
1 Bioassay Laboratory Medical Research, Research and Development Centre, Healthcare Division, Emami Limited, Kolkata, West Bengal, India
2 Medical Research, Healthcare Division, Research and Development Centre, Emami Limited, Kolkata, West Bengal, India
3 Department of Pharmacology, R. G. Kar Medical College and Hospital, Kolkata, West Bengal, India
4 Department of Phytochemistry, Healthcare Division, Research and Development Centre, Emami Limited, Kolkata, West Bengal, India
5 Formulation Healthcare Division, Research and Development Centre, Emami Limited, Kolkata, West Bengal, India
6 Department of Pharmacology, Institute of Post Graduate Medical Education and Research and SSKM Hospital, Kolkata, West Bengal, India
7 Healthcare Division, Research and Development Centre, Emami Limited, Kolkata, West Bengal, India

Correspondence Address:
Dr. Satyajyoti Kanjilal
Medical Research, Healthcare Division, Research and Development Centre, Emami Limited, 13, BT Road, Kolkata - 700 056, West Bengal
India

Abstract

Background: Ayurvedic Ariṣṭa preparations hold an age-old heritage in the prevention and cure of metabolic disorders and associated complications. Aim: The aim of the study was to study the antidiabetic activity of a self-fermented Ariṣṭa preparation (DB-07) and its potential in preventing or curing diabetes-induced nephropathy in experimental models. Materials and Methods: Rats with Streptozotocin-induced diabetes were used for studying the various dose effects of DB-07 (2.5, 3, and 3.5 ml/kg/day) in preventive and therapeutic treatment schedules. Metformin (500 mg/kg) was used as a standard drug. Fasting blood glucose and biochemical parameters such as blood protein, urea, uric acid, and creatinine were accessed by various biochemical tests. In vitro α-amylase and α-glucosidase inhibition assay and 2-deoxy glucose uptake assay using rat L6 myotubes were studied for determining the antidiabetic mechanism of action. Results: A dose-dependent and statistically significant effect on lowering the fasting blood glucose level was observed in both preventive and therapeutic scheduled treatments. Treatment with DB-07 significantly reversed the altered biochemical parameters and the effects were comparable to metformin-treated rats. DB-07 exhibited concentration-dependent inhibition of both α-amylase and α-glucosidase enzymes with an IC50 values of 10.93 μg/ml and 8.73 μg/ml, respectively. DB-07 also showed glucose uptake potential (ca. 70% activity at 125 μg/ml) in differentiated L6 myotubes. Conclusions: These observations have revealed the antidiabetic and nephroprotective potential of DB-07. DB-07 can be a potential herbal alternative to maintain a healthy blood glucose level and will prove useful in diabetes-associated renal complications.



How to cite this article:
Dey A, Kanjilal S, Biswas R, Adhikari A, Mohapatra S, Gandhi D, Bhatt BN, Choudhuri S, Chakraborty P, Sur TK, Narwaria A, Katiyar CK. Antidiabetic and nephroprotective potential of polyherbal self-fermented Ariṣṭa formulation: Evidence-based research.Ancient Sci Life 2018;37:188-199


How to cite this URL:
Dey A, Kanjilal S, Biswas R, Adhikari A, Mohapatra S, Gandhi D, Bhatt BN, Choudhuri S, Chakraborty P, Sur TK, Narwaria A, Katiyar CK. Antidiabetic and nephroprotective potential of polyherbal self-fermented Ariṣṭa formulation: Evidence-based research. Ancient Sci Life [serial online] 2018 [cited 2022 Jan 25 ];37:188-199
Available from: https://www.ancientscienceoflife.org/text.asp?2018/37/4/188/334722


Full Text



 Introduction



Diabetes mellitus is a slowly progressing metabolic disorder characterized by hyperglycaemia, which leads to functional disabilities of all bodily organs including those of the central and peripheral nervous system. It is an alarming health problem of all developed nations, and affects people of almost all social, cultural, economic, and genetic backgrounds.[1],[2] According to World Health Organization (WHO) and Current International Diabetes Federation, diabetes mellitus is possibly the world's fastest growing metabolic disorder, and the global prevalence of diabetes affecting all age groups is expected to increase from 422 million adults in 2014 to approximately 590 million by 2035.[3],[4],[5] More than 85% of total diabetes patients live in India, China, and other developing nations.[1] Perhaps the prevalence of diabetes in these developing countries is going to increase during the next two decades.[1],[6] The development of diabetes mellitus begins with an impairment of glucose tolerance and is usually connected with a state of insulin resistance, resulting in the persistent rise of blood glucose level.[3] In addition to high blood sugar, other factors including dyslipidemia or hyperlipidemia also plays an important role in the development of micro and macrovascular complications such as diabetic retinopathy, nephropathy, heart attack or stroke, which are the major source of mortality and morbidity.[1],[3],[4]

Although biomedicine has effective therapeutic approaches to treat different diseases, treatment of diabetes is still a big challenge. Plants have been the major source of drugs useful for the treatment of human ailments since ancient times.[7] According to Ayurveda, metabolic disturbances lead to sweet urine and are associated with abnormal body weight changes. In addition, sedentary habits and inappropriate choices of food and eating behavior have worsen the condition. However, a combination of physical exercise and the intake of herbal supplements helps in preventing diabetes and its associated vascular complications.[1],[5]

In recent decades, patient education programs have created a general awareness among the population regarding the health benefits of diet and dietary supplements. Because of that, patients are becoming more conscious regarding their diet and have started consuming vegetables or herbal supplements that are good for maintaining glucose homeostasis.[8] Herbs such as Trigonella foenum-graecum, Momordica charantia, Syzygium cumini, Azadirachta indica, Emblica officinalis, Aloe vera, Curcuma longa, and Withania somnifera are considered useful for diabetic patients of all age groups.[9],[10],[11],[12] However, the few evidences available are not enough to prove that any of these herbal supplements can actually help control diabetes or its complications.

Based on the wisdom of Ayurveda an effort was made in our laboratory to develop a polyherbal preparation intended to be used for adults to maintain glucose homeostasis. The polyherbal preparation was developed with self-fermenting Ariṣṭa technology. A total of twenty-one medicinally important Ayurvedic herbs were selected based on their traditional use with an aim to maintain glucose homeostasis. Critical analysis of currently available information on quality, efficacy, safety, and metabolic profiling of each herb strongly suggested that appropriately processed and standardized techniques apt for this polyherbal preparation could be therapeutically effective to reduce the hyperglycemia. Therefore, the validated experimental models were selected to evaluate the antihyperglycemic effect of the polyherbal blend. The results of the experiments are being described and discussed in this communication.

 Materials and Methods



Preparation of sample formulation

The Ayurvedic polyherbal Ariṣṭa formulation (coded as DB-07) was prepared in the formulation development laboratories of Healthcare Division, in the Research and Development Centre, Emami Ltd, Kolkata (Batch No.: OTC/MMR/L/100517/21; Mfg. Date: May, 2017) and was used in this study.

The polyherbal blend was prepared by using Ariṣṭa technology. A total of twenty-one selected herbs were placed in an airtight fermenter followed by anaerobic fermentation of the herbal decoction [details of the herbs used are described in [Table 1]]. Briefly, the coarsely ground raw materials were boiled together with water in a vessel, thereafter the collected herbal decoction was fermented with Dhātakī (Woodfordia fruticosa) flowers. Anaerobic fermentation was initiated and continued for 6–10 days until the desired percentage of alcohol (not more than 8% by volume) was formed. The fermentation was stopped by heating the vessels to 60°C ± 2°C and cooling immediately to room temperature. Finally, the liquid preparation was filtered and packed in amber colored glass bottles. After completion of all the developmental stages, the formulation is currently prepared in the manufacturing facilities of Emami Limited and is being marketed under the brand name of Zandu Diabrishta-21 and Diabrishta-DM.{Table 1}

Chemicals and reagents

Streptozotocin (STZ) (Sigma-Aldrich, USA), α-Glucosidase (Sigma-Aldrich, USA), α-Amylase (Sigma-Aldrich, USA), p-Nitrophenyl-α-D-glucopyranoside (Sigma-Aldrich, USA), Acarbose (Sigma-Aldrich, USA), Dinitrosalicylic acid (Sigma-Aldrich, USA), Dulbecco's Modified Eagle Medium (DMEM: Gibco, USA), Fetal Bovine Serum (HiMedia, Mumbai), MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny ltetrazolium bromide] (Sigma-Aldrich, USA), 2-Deoxy glucose (Cayman Chemical, USA), Resazurin sodium salt (HiMedia), Insulin (Sigma-Aldrich, USA), and Metformin hydrochloride (Sigma-Aldrich, USA).

All other chemicals, assay kits, buffers and reagents used were from other laboratory suppliers and of highest analytical quality available in India.

Chromatographic analysis

High-performance liquid chromatography analysis of DB-07

The sample formulation (DB-07) was analytically characterized by high-performance liquid chromatography (HPLC) system (Shimadzu LC-20, Japan). The HPLC system consisting of a quaternary pump with the vacuum degasser, solvent delivery unit (LC-20AP), photodiode array (PDA) detector (SPD M20A), auto-sampler (SIL 10AF) at 24°C, and a column oven (CTO-20A) set at ambient temperature were used. The analytical method was validated for linearity, specificity, accuracy, and range of quantification. Chromatographic data were monitored and processed using Shimadzu Lab-solution software. Separation was achieved on Enable C18, column (5 μ particle size, 150 mm × 4.6 mm, 100 A) protected by a C18 guard column (4.0 mm × 3.0 mm). DB-07 was fractionated by using three solvents of different polarities to prepare three samples. For sample preparation, 12 ml of liquid DB-07 was concentrated in a rotary evaporator (Rotavapor® R-300; Buchi, Germany) at 60°C and further freeze dried to 400 mg of residue from which 200 mg of the residue was dissolved in water (75 ml). It was partitioned subsequently in ethyl acetate and n–butanol (three times each). Ethyl acetate, n-butanol, and aqueous fractions were concentrated in the rotary evaporator at 60°C. Ethyl acetate (30 mg), n-butanol (45 mg), and aqueous (110 mg) fractions were subjected to freeze drying to obtain the dried residues and were dissolved in methanol to prepare the sample solution. The sample solution was filtered using 0.2 μ syringe filter. The mobile phase consisted of water (solvent A) and methanol (solvent B). The following gradient elution was used: 0 to 5 min, 40% B; in the next 10 min 65% B; increased in next 5 min to 80% B and maintained at 85% B for 12 min; decreased the solvent B to 40% in next 3 min. The flow rate was 0.6 ml/min, and the injection volume was 4 μl (80 ug) for each fraction. The column temperature was kept at 30°C. A PDA detector was set to monitor the wavelengths of range 200–600 nm.

High performance thin layer chromatography analysis of DB-07

For high-performance thin layer chromatography (HPTLC) analysis CAMAG Linomat 5 sample applicator, CAMAG TLC Scanner 4, and CAMAG Photodocument chamber were used. As described in the previous analysis, solvents such as ethyl acetate, n-butanol and water were used to prepare the three fractions of DB-07. Reference marker compounds were isolated and identified by Phytochemistry department, R and D Healthcare, Emami Ltd., Kolkata, India.

HPTLC analysis was performed by spotting reference standard and fractions of DB-07 on pre-coated Aluminium silica gel 60F254 TLC plates (20 cm × 10 cm, E. Merck, Darmstadt, Germany) using a Linomat V sample applicator and a 100 μl syringe. The samples, in the form of bands of length 8 mm were spotted at a constant application rate of 80 nl/s using nitrogen aspirator. Plates were developed using mobile phase chloroform: methanol: water (20: 9: 1 v/v/v). Subsequent to the development, TLC plates were dried in a current of air with the help of an air-dryer for 5 min. Then the spots were visualized by dipping the plate in anisaldehyde-sulphuric acid reagent and subsequent heated at 105°C for 5 min in a hot air oven. Slit dimension settings of length 6.00 mm × 0.30 mm and a scanning rate of 20 mm/s were employed. Densitometric scanning was performed on Camag TLC scanner 4 in absorbance mode at 366 nm and operated by win CATS planar chromatography version 1.4.9.

Animals

Male Wistar rats (150–175 g) used in this study were procured from central animal house of R. G. Kar Medical College and Hospital, Kolkata (CPCSEA Registration number: R/N 959/C/06/CPCSEA). They were randomly selected and group-housed (six animals per cage) in polypropylene cages provided with husk bed at an ambient temperature (25°C ± 1°C) and relative humidity (50% ± 10%) with a 12:12 h light/dark cycle. All the animals were acclimatized to laboratory conditions for at least 1 week before the start of the experiments. They were fed with a standard rodent diet and water ad libitum. The waste in the cages was removed daily to ensure hygienic conditions and maximum comfort for animals. Before the start of any experiment, ethical clearance for animal experimental work was obtained from the Institutional Animal Ethical Committee of the R. G. Kar Medical College and Hospital, Kolkata. The animal experiment was conducted in the department of pharmacology, R.G. Kar Medical College, Kolkata. All the experimental groups were always tested in parallel (i.e., on the same day of the experiment), and handled, weighed, and observed by a single-blinded observer and in the same laboratory environment.

Induction of diabetes in rats

Diabetes was induced in overnight fasted rats by single intraperitoneal (i.p.) injection of STZ at a dose of 45 mg/kg body weight (freshly dissolved in 0.1 M citrate buffer, pH 4.5). After 1 h of STZ injection, the animals were provided with 5% glucose solution (p.o.) for the next 2 h to prevent hypoglycemic shock. Animals showing marked hyperglycemia (fasting blood glucose ≥250 mg/dl) 48 h after STZ treatment were selected for the study. Fasting blood glucose levels were monitored weekly using glucometer.[13]

Animal grouping and drug treatment

For a single set of experiments, six groups of six animals each were used. The treatment groups were as follows: Group 1: Normal Control (Normal Saline, 0.5 ml, p.o.); Group 2: STZ Control (Normal Saline, 0.5 ml, p.o.); Group 3: STZ + metformin (500 mg/kg, p.o.); Group 4: STZ + DB-07 (2.5 ml/kg, p.o.); Group 5: STZ + DB-07 (3 ml/kg, p.o.); and Group 6: STZ + DB-07 (3.5 ml/kg, p.o.). As per human conversion rule, 30 ml/day means 0.5 ml/kg for 60 kg human (recommended human dose) and 3 ml/kg for rats (6 times higher than human dose). However, 3 ml/kg is the final dose for rat but to overcome any bias, two additional doses are considered. The selected dosages for the present study were 2.5, 3, and 3.5 ml/kg body weight.

Experimental design

For preventive study, diabetic rats were daily treated with test formulation (DB-07) at doses 2.5, 3 and 3.5 ml/kg b.w., or Metformin at a dose 500 mg/kg b.w., in the respective groups for 28 days. DB-07 or metformin was given simultaneously with the progression of disease (from day 3 to day 28 after STZ injection) to find out whether the test drugs have any role in the prevention of diabetes.

In the therapeutic schedule, treatment with DB-07 was started from day 14 after diabetes induction and lasted an additional 2 weeks. DB-07 or Metformin was given from day 14 to day 28 after the development of diabetes to find out whether the test drugs have any role in the therapeutic cure of diabetes.

Assessing antidiabetic activity

The fasting blood sugar levels were estimated on 14th and 28th day of treatment after depriving food for 16 h, with free access to drinking water. The antidiabetic (blood sugar control) activities were assessed by comparing the data from baseline as also with STZ control.

Assessing nephroprotective activity

After completion of treatment schedule, routine urinalyses particularly for glucose, protein, blood, ketone, bilirubin was done by strip test. Urinalyses were performed in a qualitative manner. On the day of the last treatment, the animals were sacrificed and blood samples were collected directly from their heart to assess blood protein, blood urea nitrogen (BUN), uric acid, and creatinine using commercial kits. Immediately after blood collection, kidney tissue histology was performed using hematoxylin and eosin staining and under a compound light microscope.

In vitro α-glucosidase inhibition assay

α-glucosidase was assayed using a method modified by Apostolidis et al.,[14] where 10 mg of freeze-dried powder sample was dissolved in 10 ml of water and ethanol mixture at a ratio of 1:1 and vortexed for 3 min. The the solution was then filtered through Whatman No. 1 filter paper. Two-fold serial dilutions of test sample concentration range from 62.50 to 0.98 μg/ml were prepared in 0.1 M phosphate buffer (pH 6.9). 20 μl of α-glucosidase solution (0.5 U/ml) was mixed well with 120 μl of 0.1 M phosphate buffer (pH 6.9) and to this 10 μl of test solution or Acarbose solution (in distilled water) was mixed, and the mixture was then incubated in 96-well plates at 37°C for 15 min. In the control wells, 10 μl of the buffer solution was used instead of test solution. After preincubation, 20 μl of 5 mM p-nitrophenyl-α-D-glucopyranoside solution in 0.1 M phosphate buffer (pH 6.9) was added to each well. Then the reaction mixtures were incubated at 37°C for 15 min. After the incubation, 80 μl of 0.2 M sodium carbonate (Na2CO3) solution was added to each well to terminate the enzyme reaction. Then the absorbance of the reaction mixtures was recorded at 415 nm using a micro-plate reader (SpectraMax i3x, Molecular Devices; USA) and compared to control which contained 10 μl of the buffer instead of the test solution. Percentage α-glucosidase inhibition was calculated as follows:

[INLINE:1]

The α-glucosidase inhibitory activity of the test sample was expressed as IC50 value (Inhibition Concentration 50%).

In vitro α-amylase inhibition assay

The inhibition of α-amylase was determined using an assay modified by Kim et al.,[15] where the sample solution was prepared as described above. A total of 100 μl of each concentration of test solution mixed with and 100 μl of 0.02 M sodium phosphate buffer (pH 6.9) containing α-amylase solution (0.1 U/ml) is incubated at 37°C for 30 min. After preincubation, 100 μl of a 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9) was added to each tube at timed intervals. The reaction mixtures were then incubated at 37°C for 20 min. The reaction was stopped with 200 μl of DNS (dinitrosalicylic acid) color reagent. The tubes containing reaction mixtures were then incubated in a boiling water bath for 5 min and cooled to room temperature. The reaction mixture was then diluted after adding 1.5 ml of distilled water. 200 μL of each reaction mixture was transferred into a 96-well plate and the absorbance was measured at 540 nm using a micro-plate reader (SpectraMax i3x, Molecular Devices; USA). The absorbance of sample blanks (buffer instead of enzyme solution) and a control (buffer in place of sample solution) was also recorded. The final extract absorbance was obtained by subtracting its corresponding sample blank reading. Acarbose was prepared in distilled water and used as positive control. Percentage α-amylase inhibition was calculated as follows:

[INLINE:2]

The α-α-amylase inhibitory activity of the test sample was expressed as IC50 value (inhibition concentration 50%).

In vitro glucose uptake assay using L6 myotubes

Rat skeletal muscle cell lines (L6 myoblasts) were procured from National Centre for Cell Sciences, Pune, India. Monolayer of L6 skeletal muscle cells were maintained at subconfluent conditions in DMEM supplemented with glucose (0.045 g/ml), 1 mM sodium pyruvate, L-glutamine, sodium bicarbonate (1.5 g/L), penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% inactivated fetal bovine serum (FBS). Cells were maintained in 25 cm2 culture flasks inside a humidified incubator maintained with 5% CO2 and temperature at 37°C.

MTT assay was performed to determine the dose range for the glucose uptake study.[16] Five concentrations of the test solution (prepared in Dulbecco's phosphate buffered saline and serially diluted with DMEM) i.e., 62.5, 125, 250, 500, and maximum 1000 μg/ml were tested. Cytotoxicity was determined by a decrease in the optical density (570 nm) compared to the control (without treatment).

Glucose uptake activity of the test sample was determined in differentiated L6 myotubes. In brief, the L6 myoblasts with 70-80% confluency were allowed to differentiate by maintaining in DMEM with 2% FBS for 7 days. During this period, the medium was changed on every 2nd day. Under the reduced serum condition, the myoblasts reach the confluence and then spontaneously differentiate into myotubes.[16] Cell differentiation was confirmed by examining the multinucleation of cells [Figure 1]. For 2-deoxy glucose uptake, differentiated L6 myotubes were seeded (3 – 5 × 104 cells/ml) in a 96-well plate using DMEM with 2% FBS. The differentiated L6 myotubes were maintained in FBS-free medium overnight and before the experiment, cells were washed twice with KRH (Krebs-Ringer-HEPES) buffer containing 0.1% bovine serum albumin (BSA) and incubated with KRH buffer for 30 min at 37°C. Cells were treated with different concentrations of test and standard insulin for 4 h along with negative controls at 37°C. After washing with KRH buffer, 2-deoxy glucose solution (2 mM 2DG in KRH buffer) was added simultaneously to each well and incubated at 37°C for 20 min. After incubation, the uptake of the glucose was terminated by removing the solution from wells and washing thrice with ice-cold KRH buffer solution. Cells were then lysed with 0.1M NaOH solution in a temperature-controlled bath (60 to 85°C for 40 to 60 min) and an aliquot of cell lysates was used to measure the cell-associated glucose. 20 μl of each cell lysate and standard 2-deoxyglucose-6-phosphate (DG6P) solution in different concentration (30, 15, 7.5, 3.75, 1.875, 0 μM) was transferred to a black fluorescence assay plate. Add the 200 μl of assay solution (containing to a final concentration of 50 mM TEA (triethanolamine buffer, 200 mM, pH 8.1), 50 mM KCl, 0.02% (w/v) BSA, 0.1 mM NADP, 0.2 units/ml diaphorase, 10 μM resazurin sodium salt, and 20 U/ml G6PDH] to each well containing samples and controls. The plate was then incubated at 37°C for 30 min and the resulting fluorescence (λex = 530–570 nm, λem = 590–620 nm) was measured using a microplate reader (SpectraMax i3x, Molecular Devices; USA). The standard curve from the fluorescence intensity of the DG6P standards was calculated, and the glucose levels in cell lysates were measured using DG6P standard curve.[17],[18]{Figure 1}

Statistical analysis

Data were expressed as mean ± standard deviation. Statistical significance between the treatment group with test and control was determined by using paired or unpaired Student's t-test or analysis of variance for repeated measures as appropriate. GraphPad Prism-5 (GraphPad software Inc., La Jolla, California, USA) was used for statistical analysis. A P < 0.05 was considered statistically significant.

 Results



Comparative high-performance liquid chromatography chemical profiling of various DB-07 fractions

The HPLC method was validated in accordance with the International Conference on Harmonization guidelines. The limit of detection and limit of quantification was estimated according to the above guidelines. In HPLC chromatograms total eight major chemical compounds were identified in three different fractions [Figure 2]. The ethyl acetate fraction detected five compounds such as gallic acid (Rt: 5.393), quercetin (Rt - 6.748), kaempferol (Rt –- 7.642), ellagic acid (Rt – 13.103), catechin (Rt – 20.175), 6-gingerol (Rt – 27.790), and piperine (Rt – 33.898). Further, n-butanol and aqueous fraction detected two compounds as gallic acid (Rt - 5.263) and diosgenin (Rt - 17.545). DB-07 was made up of 21 medicinally important herbs which contain numerous phytoconstituents in different concentrations. In ethyl acetate fraction, gallic acid was found in highest amounts (0.251% ± 0.050% [w/w]), whereas n-butanol and aqueous fractions contain 0.031% ± 0.012% and 0.022%% ± 0.010% (w/w), respectively. In ethyl acetate fraction, compounds such as quercetin (0.053% ± 0.033% w/w), kaempferol (0.005% ± 0.001% w/w), ellagic acid (0.091% ± 0.045% w/w), catechin (0.019% ± 0.010% w/w), 6-gingerol (0.01% ± 0.01% w/w), and piperine (0.001 ± 0.001% w/w) were obtained. The amount of diosgenin found in n-butanol and aqueous fraction were 0.013 ± 0.010% w/w and 0.014 ± 0.011 w/w, respectively.{Figure 2}

High performance thin layer chromatography study of various DB-07 fractions

The results of HPTLC study showed the presence of prominent four compounds in the three fractions of DB-07 [Figure 3]. In ethyl acetate fraction, withaferin-A and piperine are found in the highest amount at 0.5% w/w and 0.23% w/w respectively [Figure 4a. Protodioscin detected in n-butanol and aqueous fraction at 0.12% w/w and 0.02% w/w respectively [Figure 4]b and [Figure 4]c. Gallic acid detected in butanol and aqueous fraction at 1.3% w/w and 1.2% w/w, respectively.{Figure 3}{Figure 4}

Fasting blood glucose level

Mean changes in fasting blood glucose level on preventive aspects of the treatment groups used in this study during the course of the experiment are summarized in [Table 2]. In the preventive study, a significant increase in blood glucose level was observed in STZ administered animals of all groups when compared to normal control rats on treatment day 1. The mean blood glucose level of the normal control group was maintained continuously with standard diet, whereas that of the diabetic control group continued to increase during the course of the experiment. Such increase in blood glucose level triggered by STZ injection was decreased significantly (P < 0.05) in the test group (DB-07) and metformin-treated group on the 7th and subsequent observational days. The rate of changes in fasting blood glucose level in the DB-07 treated group (3 ml/kg/day, p.o.) during the course of the experiment was quite analogous to that of the metformin (500 mg/kg/day, p.o.) treated group. Similar effects observed with daily treatment with DB-07 at dose of 3.5 ml/kg were much higher than that of the metformin.{Table 2}

Unlike preventive study, a similar trend in the fasting blood glucose level in the DB-07 (3 or 3.5 mg/kg/day, p.o.) treated group was observed in the therapeutic experiment, and the observed effects were comparable with that of the standard metformin (500 mg/kg/day, p.o.) treated group [Table 2].

Nephroprotective activity

The results of the treatment effects on the biochemical blood parameters in the STZ injected rats are summarized in [Table 3]. In the diabetic control rats, the level of blood proteins, urea, uric acid, and creatinine was significantly high as compared to that of the normal control rats. However, continuous daily treatment with DB-07 (3 or 3.5 mg/kg/day, p.o.) or metformin (500 mg/kg/day, p.o.) significantly reduced the elevated levels of blood proteins, urea, uric acid, and creatinine as compared to diabetic control group. The results obtained in the preventive study were comparable to that of the therapeutic study. Since the activity profile of metformin group observed in this study was qualitatively as well as quantitatively quite similar to those of the DB-07 (3.5 mg/kg/day) treated group, it seems reasonable to assume also that many pharmacological targets involved in its observed activity profiles have similar functions in regulating physiological responses, or allostatic load.{Table 3}

In urinalysis, five important markers of renal dysfunctions such as glucose, protein, blood, ketone, and bilirubin were measured in rat urine by strip test after 4-week treatment in preventive study or 2-week treatment in the therapeutic study. Results have shown no traces of glucose, protein, blood, and ketone in normal rats, whereas in diabetic control rats all the parameters were observed in high amount [Table 4]. All these urine biomarkers present in diabetic control group were less severe or absent in the diabetic animals treated with DB-07 (3.5 mg/kg/day, p.o.) and metformin (500 mg/kg/day, p.o.).{Table 4}

Histopathological examination of renal tissues (kidney) of STZ induced diabetic rats showed various pathological lesions [Figure 5] such as distension of glomeruli, glomerular atrophy, loss of cellularity, and mild fibrosis. However, these signs were less severe or not present in the DB-07 (3 or 3.5 mg/kg) treated groups. Metformin also exhibited protection against glomerular damage as compared to the diabetic control group.{Figure 5}

In vitro α-glucosidase and α-amylase inhibition assay

The results obtained from the in vitro assays have revealed the potential of DB-07 in inhibiting both α-glucosidase (IC50 = 8.73μg/ml) and α-amylase (IC50 = 10.93μg/ml) enzymes in a given test system [Figure 6]. The results were comparable to that of the standard acarbose. The IC50 value of standard acarbose in both α-glucosidase and in α-amylase inhibition assays was found to be 4.53 and 4.65 μg/ml, respectively. The results indicate that the active ingredients present in the test formulation inhibit the breakdown of polysaccharides into monosaccharides within the lumen of the intestinal tract, thus contributing to reducing the postprandial hyperglycemia.{Figure 6}

In vitro glucose uptake activity

Glucose uptake activity was analyzed by measuring the rate of uptake of 2-deoxy glucose in differentiated L6 myotube cells. The results obtained from the in vitro assay reveals that the DB-07 has glucose uptake potential (ca. 70% activity at concentration 125 μg/ml) that may facilitate the utilization of glucose into skeletal muscle through GLUT4 glucose transporters [Figure 7]. A mechanism by which insulin facilitates glucose uptake into skeletal muscle cells.{Figure 7}

 Discussion



Reported observations suggest antidiabetic potential of Ariṣṭa formulation (DB-07) by suppression of enzymes responsible for starch breakdown and augmenting the physiological functions of endogenous glucose transporters. Unlike metformin, repeated daily oral doses of DB-07 (3 or 3.5 ml/kg) can actually lower the elevated glucose level in STZ challenged rats. It was also interesting to note that the antidiabetic activity of DB-07 observed in the therapeutic scheduled experiment to be almost similar to that observed with preventive scheduled experiment. This is suggestive of its potential usefulness not only in diabetics but in prevention. It is a well-known fact that, high sugar level in blood is the key factor for almost all renal pathologies associated with diabetes.[19] Diabetes-associated renal complications are widespread and yet the existing therapy does not give satisfactory outcomes[19] or is associated with adverse drug reactions. Therefore, efforts are underway in many R and D laboratories including ours to explore the ancient systems of medicine for treating diabetes and associated renal pathologies. In the present study, the significant reduction in the levels of proteins in blood, BUN, uric acid, and creatinine by DB-07 in diabetic rats suggests its nephroprotective action. This can be attributed by the direct reduction of blood glucose level.[20] At the end of treatment schedule (i.e., after 28 days), kidney tissues from each test group were subjected to histological examination, which directly measures the activity of DB-07 in the protection of kidney functions during diabetic condition. Continuous daily treatment with the polyherbal DB-07 (3.5 ml/kg) ameliorated the STZ induced kidney lesions such as distension of glomeruli, glomerular atrophy, and loss of cellularity and mild fibrosis, which indicated its beneficial effects on kidneys. It was also interesting to note that like its protective effects against STZ induced hyperglycemia, its efficacy in improving the diabetes triggered renal pathologies also increased not only with increasing doses but also with increasing the number of treatment days. In STZ induced diabetic model 3 ml/kg daily oral doses of DB-07 could afford complete protection in elevated blood glucose levels and biomarkers of renal pathologies. This experimental evidence reaffirms that the biological and pharmacokinetic interactions between structurally and functionally diverse extractable such as tannins, saponins, glycosides, amino acids, alkaloids, flavonoids, and phenolics from individual herbs are involved in their observed activity profiles.[21] In DB-07, twenty-one different herbs were used and Emblica officinalis, Syzygium cumini, Azadirachta indica, Trigonella foenum-graecum, and Withania somnifera are among the major herbs present in this formulation. During the fermentation process, the majority of the polymerised phytoconstituents present in the herbal decoctions break down to their proactive forms and help in increasing the gallic acids, ellagic acids, aglycone part of saponins and flavonoids in the final fermented formulation. Gallic acid, ellagic acid, kaempferol, diosgenin, withaferin-A, quercetin, and piperine were found in quantifiable amounts in different fractions of DB-07. It is therefore believed that the presence of these bioactive compounds in DB-07 might have contributed for its therapeutic efficacy.[22],[23],[24]

Observations of the reported in vitro experiments suggest that the DB-07 can actually improve the glycaemic load through the inhibition of the enzymatic conversion of polysaccharides to monosaccharides. The results indicated that DB-07 showed an inhibitory effect on both α-amylase and α-glucosidase enzymes with an IC50 value 10.93 and 8.73, respectively. This is less than standard acarbose. This has given us a platform to understand the mechanism of action of the product. It is well understood that α-amylase is one of the major secretory products of the pancreas and salivary glands, playing a role in the digestion of starch and glycogen, whereas α-glucosidase is present in the brush-border surface membrane of intestinal cells and activates the final step of the carbohydrate breakdown process.[25] Consequently, inhibitors of these hydrolytic enzymes suppress the influx of glucose from the intestinal tract to blood vessels resulting in a decrease in postprandial hyperglycemia.[26] Results obtained from the in vitro enzyme inhibition studies have suggested that DB-07 has shown its inhibitory activity in the carbohydrate digestion and limits carbohydrate absorption.

Results obtained during the dose range experiment in L6-myotubes using MTT reagent have clearly demonstrated that the dried powder actives DB-07 did not confer any significant lethality to the healthy L6 myotubes with an LC50 (lethal concentration 50%) value greater than 1000 μg/ml. Skeletal muscle cells are primarily involved in insulin-induced stimulation of glucose uptake.[27] Differentiated rat L6 myotubes were chosen as a validated model for glucose uptake, as L6 cells were used extensively to elucidate the mechanisms of glucose uptake in muscles via insulin-sensitive GLUT4 in response to insulin-sensitizing compounds.[17],[28],[29] In differentiated rat L6 myotubes, we have found that incubation with dried powder of DB-07 for 4 h produced a dose-dependent increase in 2- deoxyglucose uptake. These effects were analogous to the increase in 2- deoxyglucose uptake facilitated by insulin, suggesting that DB-07 increases the translocation of GLUT4 to the plasma membrane and facilitates the glucose uptake through insulin-mediated action. Presence of bioactive molecule such as saponins, alkaloids, tri-terpenoids, flavonoids, etc., in the formulation are responsible for its insulin-sensitizing action. However, more detailed studies are required to explore the molecular mechanisms and to know which metabolites act through insulin sensitizing pathway.

The present study constitutes a part of our ongoing efforts to better understand system pharmacology of Ayurvedic herbs and to obtain analytically as well as pharmacologically better standardized polyherbal preparations. This type of formula could be further developed for the prevention and cure of metabolic disorders and vascular health problems accompanying them.

Currently used oral hypoglycaemic agents which target increasing insulin secretion, improving insulin sensitivity in tissues, decreasing the rate of carbohydrate absorption from the gastrointestinal tract are only used to manage diabetes. However, these therapies also have limitations in improving the diabetes associated vascular complications. Medicinal plant biologists and Ayurvedic researchers are extensively exploring the efficacy of bioactive metabolites from natural sources such as plants, to address this issue. Observations made in this study revealing its antidiabetic and nephroprotective activities suggest that polyherbal DB-07 is well suited as a herbal alternative for prevention and cure of diabetes and its associated comorbidities.

 Conclusions



This experiment suggests that DB-07 has a potential to maintain a healthy blood glucose level. It is also effective in the prevention and maintenance of renal complications associated with diabetes. DB-07 has potential in insulin-induced stimulation of glucose uptake in skeletal muscle tissues and decreases the rate of carbohydrate absorption from the gastrointestinal tract. This multiple mode of action could be attributed to the different phytoconstituents present in the polyherbal Ariṣṭa preparation. Therefore, DB-07 could be considered as a useful herbal alternative to ameliorate diabetes and its associated nephropathies.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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