INTRODUCTION:

Diabetes a chronic group of metabolic diseases leads to the glycation of body protein, which may further cause severe associated complications¹. According to the Diabetes atlas of 2019, 77 million Indians are suffering from diabetes which will rise up to 101 million by 2030 and 135million by 2045. At global level about 463 million people are having diabetes and every year about 4.2 million deaths are directly credited to diabetes. The numbers of diabetics are gradually increasing over the last few decades² and it is becoming uncontrollable as well as the third most deadly disease worldwide. Hence, looking at the current situation there is a global need to bring to a halt the progression of diabetes and obesity by 2025³.

Hyperglycemia is associated with increased thirst followed by increased urinary output, ketonemia, and ketonuria, related with abnormalities in the metabolism of carbohydrates, fat, and protein. If ketoacidosis happen, it can lead to diabetes-associated complications⁴. In diabetes beta cells don’t produce enough insulin or body doesn’t respond to the insulin, glucose get builds up in the blood instead of being absorbed by cells in the body⁵. Diabetes can lead to many complications affecting different body parts, thus increasing the overall risk of dying prematurely. Possible complication includes kidney failure, liver problem, hyperlipidemia, vision loss; 2-3 fold increased risk of heart attacks and strokes⁶. Apart from that, uncontrolled diabetes during pregnancy may put the fetal life at risk and may cause associated complications in mother as well as new born⁷. Decreased blood flow in diabetes, leads to nerve damage in the feet, foot ulcers and if not treated can lead to the amputation of the limb⁸. Some of the most prevalent complications reported to be associated with diabetes is diabetic dyslipidemia⁹, which may be responsible for increased risk of issues associated with cardiovascular disease or coronary artery disease. It has been reported that, if one has both diabetes and high cholesterol, a person is at higher risk for heart disease and stroke¹⁰. As per the study, at least 68 percent of people of 65 or older with diabetes die from some form of heart disease; and 16% die of stroke¹¹. Another consequence associated is diabetes-induced anemia, which may further develop complications associated with the eye and nervous system; it can further worsen kidney, heart, and artery-related diseases. If a person becomes anemic, it can be an early signal of a problem related to kidneys¹². Diabetes can result in inflamed blood vessels; which can obstruct bone marrow from getting an indication essential to produce RBC, even some antidiabetic medications may also drop the hemoglobin levels. Different research has revealed that about 25-30 % of people with diabetes eventually develop anemia and this ratio may increase with the addition of renal impairment¹³.

Different studies and research revealed, that allopathic system for diabetes therapy have shown various adverse effects on hematological parameters, lipid profile and other biochemical parameters. Hence, research has been conducted to develop antidiabetic polyherbal formulations that would be efficacious, comparatively less toxic with fewer side effects and patients feel more comfortable compared to synthetic drugs^14,15. It has also been noted that compared to single herb, polyherbal enhance the therapeutic action and reduce the concentrations of single herbs, thus reducing adverse events associated. In research, pharmacodynamic synergisms have been considered. Where, active constituents with similar therapeutic activities are targeted to a similar physiological system^16,17. Hence, polyherbal formulations have been developed by using four different plant parts having antidiabetic potential, including leaves of Annona squamosa, calyx of Hibiscus sabdariffa, leaves of Aegle marmelos, and stem bark of Ficus religiosa. The polyherbal formulations have been developed by geometrical dilution methods, to get the best possible formulations with optimum results. Research planning has been done to develop and assess the polyherbal formulations having high efficiency and therapeutic potential for the management and treatment of diabetes and its complications associated with kidney, liver and blood.

MATERIAL AND METHODS:

Instruments and chemicals:

Grinder, soxhlet apparatus, vacuum distillation, glucometer (Accu-Check active; Roche Diagnostic India Pvt. Ltd), centrifuge, automated hematology analyzer (BC-5000; ASPEN Diagnostics), chemistry analyzer (BS-120) (Mindray Medical, India), spectrophotometer (Shimadzu UV-1800), prism graph pad version 5.0. petroleum ether, 50% ethanol, animals feed (Hindustan Lever Ltd.), glibenclamide (Ozone International, Mumbai), D-glucose (S.D. Fine-Chem. Ltd, Mumbai), streptozotocin (Sigma Chemical Co., Bangalore, India.), nicotinamide (Aster Pharmaceuticals India), glucose-oxidase peroxidase kit (Merck, India) Carboxy methyl cellulose, mild ether anesthesia, EDTA, rat/mouse ELISA kit, (Sigma-Aldrich Chemical Private Ltd), diagnostic kit for lipid analysis and all other chemicals of analytical grade.

Collection and identification of plant materials:

Three plant’s parts selected for research study i.e., leaves of Annona squamosa, Linn, leaves of Aegle marmelos, (L.) Corr, stem bark of Ficus religiosa, Roxb, were collected from the Khajuri kalan village, Bhopal, Madhya Pradesh and fourth plant i.e., calyx of Hibiscus sabdariffa, Linn. (HSC) was purchased from local market of Baikunthpur, Dist. Koriya, Chhattisgarh in the month of November 2018. Plant materials were identified and authenticated by Dr. S.N. Dwivedi, Department of Botany, Janata PG College, APS University, Rewa M.P. India. Herbarium specimens of each were prepared and deposited with voucher specimen number JC/B/PAN 482, for future reference.

Extraction of plant material:

The plant’s parts collected were washed in tap water and rinsed by using distilled water. Then they were shade dried for 8–10 days and were coarsely pulverized using electrical grinder. Powdered drugs were defatted with petroleum ether and finally were subjected for complete soxhlet extraction using hydroalcoholic solvent (50% ethanol v/v). All four extracts were concentrated using vacuum distillation and were dried using desiccators. Physical characteristic of different extracts obtained from different plants were observed and noted in terms of nature (non-sticky, oily or sticky), color and odor.

Designing of polyherbal formulations:

To develop therapeutically effective polyherbal formulations, hydroalcoholic extracts of calyx of Hibiscus sabdariffa (H) leaves of Aegle marmelos (A), stem bark of Ficus religiosa (F) and leaves of Annona squamosa (A) were mixed in different proportion by changing ratio i.e., 25:50:75:100 of HAFA extracts following geometrical dilution methods and were coded as HAFA 1 to HAFA 24. To identify the effective formulations out of the twenty-four, they were subjected to the in-vivo oral glucose tolerance test (OGTT) in normal rats.

Preparation of standard and test suspension:

Total 24 formulations i.e., HAFA 1 to HAFA 24 were used to develop 24 different test suspension respectively. These test suspensions were developed by dissolving 5gm/100ml 0.5% carboxy methyl cellulose, which will give 50mg/ml concentration. Standard drug solution was prepared by dissolving crushed 10 tablets of glibenclamide 5mg each in 100ml distilled water, which gave 1ml = 0.5mg of glibenclamide. These formulations and standard drug were administered orally by using an intragastric tube.

Animals and experimental setup:

OGTT, hypoglycemic and antihyperglycemic studies were conducted by using 8 – 10 weeks old albino rats of Wistar strain of 180-250 g each. For OGTT total 27 groups of 6 animals each was selected, followed by 6 groups of 6 animals each for hypoglycemic and 7 groups of 6 animals (n=6) for antihyperglycemic (including both male and female). All the female animals chosen for the study were nulliparous and non-pregnant. Animals’ were housed at room temperature 25±2° with relative humidity 30-60 percent and animals were subjected to controlled 12hours light/dark cycle. For acclimatization before starting of the study, animals were housed in cages for 7 days. All the animals were fed with customary animals feed and water ad libitum. All the experimental steps performed were approved and was executed in accordance with guidelines of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals), for the use of laboratory animals, at RKDF University, Bhopal, India (Registration number: 1693/PO/Re/S/13/CPCSEA). Experimentation was performed on overnight fasted animals.

Evaluation of HAFA using oral glucose tolerance test (OGTT) in normal rats:

OGTT was performed tomeasure the body’s ability to use glucose effectively^18,19. During experimentation the overnight (12 hours) fasted animals were loaded with 2.5g/kg, P.O., D-glucose solution after 30 minutes of standard drug solution and 24 polyherbal formulation administration except group I, i.e., normal control group. Animals were grouped as group 1 as normal control, group 2 as glucose control, group 3 as standard treated and group 4 to group 27 as HAFA 1 to 24. Blood glucose concentration was noted at the beginning of the study. After administrating (at 0 minutes) normal saline, standard drug Glibenclamide (0.5mg/kg b.w.) and test formulations (HAFA 1 to HAFA 24), the blood glucose concentrations of the animals were evaluated at 30, 60, 90 and 180 minutes after glucose loading. The blood samples were collected aseptically by pricking the rat’s tail and the blood glucose was measured using the glucometer as per the glucose oxidase method²⁰.

Evaluation of hypoglycemic potential of HAFA in normal rats:

Hypoglycemic effect was studied using 6 groups having six animals in each (n=6). This study was performed in normal rats to evaluate most effective four formulations HAFA 2, 4, 8 and 10, out of the 24 HAFA, in comparison with standard drug Glibenclamide²⁰. Standard drug and HAFA were given orally using intragastric tube and blood glucose concentrations were measured at the beginning of the experiment and were repeated at 1, 3, 5 and 7 hours by tail pricking method^{21, 22}.

Evaluation of anti-hyperglycemic activity of HAFA in diabetic rats:

Non-insulin dependent diabetes mellitus²³ was induced to the overnight fasted rats, by using a single intraperitoneal (i.p.) injection of streptozotocin 60 mg/kg body weight (b.w.), prepared freshly by using 0.1 M cold citrate buffer pH 4.5²⁴. After 15 minutes, 120 mg/kg b.w. of nicotinamide (NA) was administered intraperitoneally²⁵ dissolved in normal saline. Hyperglycemia was confirmed using a glucometer by the raised glucose levels in plasma, determined at 72 hours and then on day 7, after STZ injection. The rats having fasting blood glucose concentrations between 300-350mg/dl were considered permanent and severe diabetic²⁶. To prevent STZ-induced hypoglycemia and mortality due to hypoglycemic shock, 10% dextrose solution was given at regular intervals for the next 24 hours²⁷. Only those rats that were found to have permanent NIDDM were used for the anti-hyperglycemic activity and rest were rejected and replaced. Acute and sub-acute anti-hyperglycemic studies were performed separately.

Acute and sub acute anti-hyperglycemic study:

Total 30-30 diabetic animals were taken separately for both study and segregated into seven groups having six rats in each. Standard drug and HAFA formulations were administered orally once daily till the end of the experiment.

Grouping of animals

Group I	Normal control	Received normal saline
Group II	Diabetic control	Diabetic rats received normal saline
Group III	Standard treated	Diabetic rats treated with Glibenclamide 0.5 mg/kg b.w.
Group IV	HAFA 2 treated	Diabetic rats treated with HAFA 2 formulations 250 mg/kg b.w.
Group V	HAFA 4 treated	Diabetic rats treated with HAFA 4 formulations 250 mg/kg b.w.
Group VI	HAFA 8 treated	Diabetic rats treated with HAFA 8 formulations 250 mg/kg b.w.
Group VII	HAFA 10 treated	Diabetic rats treated with HAFA 10 formulations 250 mg/kg b.w.

Assessment of acute and sub acute anti-hyperglycemic activity:

After treatment, blood samples were analyzed at 0, 1, 3, 5, and 7 hours for the blood glucose level in acute antihyperglycemic study. Whereas, in sub-acute antihyperglycemic study, fasting blood glucose level were studied at every seven days interval i.e., on 0, 7, 14, 21, and 28th day. Blood was collected by the tails prick method taking aseptic precautions and blood glucose levels were determined by using a glucometer²⁸. Diabetes caused alteration in body weight as well as food and water intake; hence these all were noted as percentage change at the end of 28 days of treatment^29,30.

Effect of HAFA on different biochemical parameters:

Collection of blood and separation of plasma and serum:

After the experimental regimen of 28 days, animals were sacrificed by cervical dislocation under mild ether anesthesia and blood was collected from the arterial jugular with EDTA. Plasma and serum were separated by centrifugation at 3000rpm for 10 minutes at 30° C and were analyzed for various biochemical parameters associated with diabetes.

Determination of plasma insulin, HOMA-IR and glycosylated hemoglobin levels:

Insulin content was determined by rat/mouse ELISA (Enzyme-Linked Immunosorbent Assay) kit, using rat insulin as standard³¹, HOMA-IR³²,glycosylated hemoglobin (HbA1c) was estimated by the method of Nayak and Pattabiraman (1981)³³ with modification according to Bannon, (1982)³⁴.

Determination of hematological parameters:

Total 13 hematology parameters were studied, red blood cell (RBC) and white blood cell (WBC) count was estimated according to the visual method of Dacie and Lewis (1991)³⁵. Hemoglobin (Hb) was determined by the cyanmethemoglobin method of Drabkin and Austin, (1932)³⁶. According to the hematocrit method Alexander and Grifiths, (1993)³⁷the percentage packed cell volume (PCV) was determined. Mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC) and mean corpuscular hemoglobin (MCH) were calculated as described by Dacie and Lewis (1991)³⁵. Percentages of differential leukocyte count (DLC) granulocytes (neutrophils, eosinophils, and basophils) monocytes and lymphocytes (T cells and B cells) were estimated using the method of Osim et al., (2004)³⁸. Platelet (PLT) count and Mean platelet volume (MPV) were also estimated. CBC was estimated by using an automated hematology analyzer.

Estimation of lipid profile:

Complete lipid profile³⁹ estimations were done by using commercially available diagnostic kit using chemistry analyzer (BS-120). Total cholesterol was estimated by the enzymatic method described by Allain, et al., (1974)⁴⁰. Triglycerides by using diagnostic kit based on the enzymatic method described by McGowan, et al., (1983)⁴¹. HDL-cholesterol by using diagnostic kit, based on the enzymatic method as described by Izzo, et al., (1981)⁴². VLDL and LDL were calculated using the formula given by Friedwald, et al., (1972)⁴³. VLDL cholesterol = TG/5; LDL cholesterol = TC- (HDL cholesterol + VLDL Cholesterol).

Statistical analysis:

All the results were expressed as mean±SD for six rats in each experimental group. Statistical analysis was performed using prism graph pad version 5.0. The data were evaluated using a one-way analysis of variance (ANOVA) followed by Dunnett’s Test. P-values < 0.05 were considered as statistically significant, P< 0.01 as very significant and P< 0.001 as highly significant.

RESULTS AND DISCUSSION:

Extraction and physical characteristics of extracts:

Plants extract yield (in percentage) of leaves of Annona squamosa, leaves of Aegle marmelos, stem bark of Ficus religiosa and calyx of Hibiscus sabdariffa was found to be 12.95%, 12.43%, 16.75% and 20.82% respectively. It was noted for all extracts were non-sticky, colours were found to be blackish-green, dark green, reddish-brown and red respectively, whereas the odor was noted to be offensive, odorless, and characteristics and like floral berry.

Evaluation of HAFA by oral glucose tolerance test in normal rats:

Twenty four different formulations named as HAFA 1 to HAFA 24 were developed and subjected to in-vivo oral glucose tolerance test to identify the most effective formulations. By result it was noted that, average blood glucose which was found to be normal and rose in 30 minutes after administering 2.5g/kg b.w. glucose orally, decreased by approx 19% in first 30 minutes and finally decreased by approx 30% in next 150 minutes after administration of HAFA. Glibenclamide treated group, decreased by approx 38% in 150 minutes. It was further noted that, among all 24 formulations, HAFA 2, 4, 8 and 10 numbered formulations showed comparatively better oral glucose control. HAFA 2 reduced the elevated blood glucose by approx 37%, followed by 43% by HAFA 4, 35% by HAFA 8 and approx 40% by HAFA 10 in 150 minutes, which was highly significant. It was observed that HAFA formulations which had higher ratios of the Hibiscus sabdariffa and Ficus religiosa followed by Aegle marmelos, and Annona squamosa, showed significant reduction. After OGTT, four selected formulations were subjected to hypoglycemic and anti-hyperglycemic studies with detailed biochemical analysis.

Evaluation of hypoglycemic potential of HAFA in normal rats:

Hypoglycemic study data has been summarized in, table 1. It has been noted that after 7 h of a treatment with HAFA 2, 4, 8 and 10, blood glucose reduction was observed approx 6% only and doesn’t caused hypoglycemia, whereas standard drug showed 15.13% reduction from 80.5 to 59.83mg/dL (i.e., to below normal) and caused hypoglycemia in normal rats.

Table 1: Hypoglycemic effects of HAFA formulations in normal rats

Groups name	Blood glucose level (mg/dL) at different time intervals (in hours)
Groups name	0	1	3	5	7
Group I Normal control	79.67±1.37	79.83±1.47	80.00±0.63	79.50±0.55	79.17±1.17
Group II Standard treated	80.50±1.05	79.67±1.03	76.50±1.38	64.17±1.17	59.83±1.17
Group III HAFA 2 treated	80.33±1.21	78.67±1.21	76.00±1.26	75.17±1.17	75.50±1.05*
Group IV HAFA 4 treated	81.00±1.79	80.17±1.72	79.00±1.10	77.83±1.17	77.83±1.47**
Group V HAFA 8 treated	80.67±1.63	79.50±1.05	77.67±1.21	75.33±1.21	75.67±1.05*
Group VI HAFA 10 treated	80.67±1.63	79.83±1.94	78.33±1.63	75.83±1.47	76.50±1.38**

Values are expressed as mean ± SD (n=6); * (p< 0.05) significant; ** (p < 0.01) very significant; *** (p < 0.001) highly significant compared with normal control

Table 2: Acute anti-hyperglycemic study of HAFA formulations on STZ-induced diabetic rats

Groups name	Blood glucose level (mg/dL) at different time intervals (in hours)
Groups name	0	1	3	5	7
Group I Normal control	76.50±4.55	76.17±4.26	76.00±4.94	76.00±5.06	75.83±4.58
Group II Diabetic control	321.67±5.16	323.50±3.78	324.17±3.06	326.67±3.98	325.83±4.79
Group III Standard group	344.17±3.31	299.50±3.02	261.50±3.78	227.67±1.97**	196.50±4.23**
Group IVHAFA 2 treated	340.67±5.89	313.67±3.50	273.00±5.18	240.83±3.06*	200.67±1.97*
Group V HAFA 4 treated	343.17±3.66	310.83±4.79	253.33±4.55	212.33±3.14**	175.17±3.25**
Group VI HAFA 8 treated	339.00±3.74	319.33±2.07	280.50±3.27	243.33±3.20*	211.17±2.79*
Group VII HAFA 10 treated	342.00±3.10	314.67±4.63	269.50±5.47	226.50±4.04**	190.17±4.07**

Values are expressed as mean ± SD (n=6); * (p< 0.05) significant; ** (p < 0.01) very significant; *** (p < 0.001) highly significant compared with normal control

Table 3: Sub-acute anti-hyperglycemic study of HAFA formulations on STZ-induced diabetic rats

Groups name	Blood glucose level (mg/dL) at different time intervals (in days)
Groups name	0th Day	7th Day	14th Day	21th Day	28th Day
Group I Normal control	76.67±2.07	77.17±1.94	76.83±1.47	77.33±1.51	77.00±1.41
Group II Diabetic control	328.83±3.43	331.00±3.41	331.17±2.86	331.67±2.88	331.83±2.14
Group III Standard group	333.83±3.92	244.50±2.95	189.83±3.87*	140.50±4.28**	87.67±1.86***
Group IV HAFA 2 treated	340.67±3.14	293.17±3.43	228.67±2.50	180.17±3.13*	95.50±3.21**
Group V HAFA 4 treated	343.33±3.72	266.50±3.78	199.00±3.58*	141.50±4.59**	84.67±1.86***
Group VI HAFA 8 treated	339.00±3.74	294.17±3.54	232.83±2.56	193.83±1.94*	100.00±2.61**
Group VII HAFA 10 treated	341.17±3.06	273.17±3.82	219.67±2.42	169.50±3.21*	90.50±1.87***

Values are expressed as mean ± SD (n=6); * (p< 0.05) significant; ** (p < 0.01) very significant; *** (p < 0.001) highly significant compared with normal control

Table 4: Effect of HAFA formulations on body weight of STZ-induced diabetic rats

Groups name	Mean body weight changes(STZ induces hyperglycemia)
Groups name	0th Day	7th Day	14th Day	21th Day	28th Day
Group I Normal control	224.00±3.52	210.33±4.72	209.00±4.34	228.33±3.88	230.67±4.76
Group II Diabetic control	171.67±2.42	170.17±3.19	169.33±2.34	169.83±2.71	168.83±2.71
Group III Standard group	176.00±1.26	185.33±2.73	192.67±3.01	202.00±2.53**	212.00±4.73***
Group IV HAFA 2	175.83±3.19	182.83±1.83	190.67±3.08	196.17±3.76*	205.00±2.90**
Group V HAFA 4	179.83±2.93	192.17±3.06	197.67±1.86	214.17±2.79**	223.00±3.35***
Group VI HAFA 8	181.00±2.37	185.00±2.83	191.00±2.00	194.17±2.48*	200.17±2.48**
Group VII HAFA 10	180.83±1.94	186.00±3.52	192.50±2.43	205.17±2.99**	214.83±2.32***

Values are expressed as mean ± SD (n=6); * (p< 0.05) significant; ** (p < 0.01) very significant; *** (p < 0.001) highly significant compared with normal control

Table 5: Effect of HAFA formulations on food and water intake of STZ-induced diabetic rats before and after treatment

Groups name	Food intake (g/24 hours)		Water intake (ml/24 hours)
Groups name	Initial	Final	Initial	Final
Group I Normal control (15-20 g/day)	15.00±0.89	16.00±0.63	11.33±0.82	11.67±0.52
Group II Diabetic control	24.83±1.17	28.00±0.89	41.50±2.17	44.83±2.56
Group III Standard group	28.67±2.07	15.83±1.60***	43.33±2.16	18.33±1.63***
Group IV HAFA 2 treated	27.50±1.87	19.83±1.72***	46.00±2.53	22.50±1.87**
Group V HAFA 4 treated	29.50±1.05	16.00±1.90***	44.50±2.07	15.83±1.60***
Group VI HAFA 8 treated	27.33±1.21	21.67±1.21**	45.50±1.87	23.17±1.94**
Group VII HAFA 10 treated	26.17±1.72	17.67±1.97***	45.33±1.03	20.00±1.41**

Values are expressed as mean ± SD (n=6); * (p< 0.05) significant; ** (p < 0.01) very significant; *** (p < 0.001) highly significant compared with normal control

Table 6: Effect of HAFA formulations on plasma insulin, HOMA-IR and HbA1Clevel of STZ-induced diabetic rats

Groups	Biochemical analysis
Groups	Plasma insulin (µU/mL)	HOMA-IR	HbA1c (%)
Group I Normal control	14.73±1.05	2.80±0.22	4.90±0.22
Group II Diabetic control	6.12±0.27	5.02±0.24	11.70±0.24
Group III Standard group	12.65±0.55***	2.74±0.14***	5.80±0.20***
Group IV HAFA 2	13.22±0.99***	3.12±0.26***	5.50±0.17***
Group V HAFA 4	14.00±0.49***	2.93±0.14***	4.90±0.40***
Group VI HAFA 8	12.91±0.60***	3.19±0.22***	5.70±0.37***
Group VII HAFA 10	13.75±0.87***	3.07±0.22***	5.20±0.20***

Values are expressed as mean ± SD (n=6); * (p< 0.05) significant; ** (p < 0.01) very significant; *** (p < 0.001) highly significant compared with normal control

Table 7.Effect of HAFA formulations on hematological parameters (RBC, Hb, PCV, MCV, MCH, MCHC) of STZ-induced diabetic rats

Groups	Serum profile
Groups	RBC (10⁶/mm³)	Hb (g/dL)	PCV %	MCV (fl)	MCH (pg.)	MCHC (g/dL)
Group I Normal control	7.18±0.36	13.78±0.29	42.64±0.95	51.05±0.24	21.76±0.58	37.02±0.91
Group II Diabetic control	5.14±0.09	10.13±0.14	26.78±0.63	48.73±1.22	18.03±0.58	30.64±0.58
Group III Standard group	6.77±1.05**	12.27±0.11**	40.80±1.14**	55.05±2.68**	20.68±0.54**	34.71±0.34**
Group IV HAFA 2 treated	6.52±0.24*	12.12±0.32*	42.69±0.37*	55.22±2.91*	20.26±0.60*	34.27±1.10*
Group V HAFA 4 treated	7.01±0.12**	13.43±0.24**	46.81±0.32**	59.99±1.04**	20.71±1.41**	35.61±1.33**
Group VI HAFA 8 treated	6.07±0.41*	12.03±0.24*	41.40±0.66**	54.37±1.90*	18.53 ±0.54*	33.30±0.75*
Group VII HAFA 10 treated	6.69±0.20**	13.11±0.24**	43.55±0.52**	57.72±1.64**	20.67±0.46**	34.68±1.45**

Values are expressed as mean ± SD (n=6); * (p< 0.05) significant; ** (p < 0.01) very significant; *** (p < 0.001) highly significant compared with normal control

Table 8: Effect of HAFA formulations on hematological parameters like WBC, Platelet and differential leukocyte count of STZ-induced diabetic rats

Groups

WBC

(x10³/mm³)

Serum profile (DLC in Percentage)

Platelet

(x10³/mm³)

Lymphocytes

Monocytes

Neutrophils

Esinophils

Basophils

Group I Normal control

4.72±0.06

69.67±3.14

6.43±0.48

25.67±2.42

2.65±0.46

0.69±0.06

323.33±10.01

Group II Diabetic control

2.48±

0.08

53.00±

2.37

8.24±

0.28

41.33 ±

2.80

1.00±

0.17

0.78±

0.11

562.83±

15.98

Group III Standard group

3.43±

0.12***

70.67±

4.80**

6.51±

0.10***

32.83±

3.54*

2.22±

0.23**

0.40±

0.03**

397.17±

8.84**

Group IV HAFA 2 treated

3.99±

1.44*

69.00±

3.16***

7.54±

0.16**

33.33±

2.16*

2.28±

0.12**

0.64±

0.03**

376.83±

6.08**

Group V HAFA 4 treated

4.65±

0.07***

72.50±

2.74**

6.82±

0.11***

27.00±

2.94**

2.46±

0.08***

0.32±

0.03***

390.83±

7.36**

Group VI HAFA 8 treated

3.45±

0.16*

67.50±

2.17**

7.73±

0.19**

34.14±

4.26*

2.03±

0.17**

0.62±

0.05**

373.67±

8.12**

Group VII HAFA 10 treated

4.22±

0.15**

69.50±

1.64***

6.97±

0.39***

28.67±

2.07**

2.34±

0.06**

0.45±

0.05**

388.17±

5.12**

Values are expressed as mean ± SD (n=6); * (p< 0.05) significant; ** (p < 0.01) very significant; *** (p < 0.001) highly significant compared with normal control

Effect of HAFA formulations on hematological parameters:

STZ induced diabetes caused a significant decrease in the levels of complete blood count except platelet which increased, when compared with the normal control. Oral administration of HAFA for 28 days significantly improved hematological parameters to near normal. Out of four formulations, HAFA 4 showed most significant activity, than HAFA 2, 8 and 10 summarized in table 7 and 9. It was comparatively significant as standard drug’s reversing effect. It has been reported that the occurrence of disturbed CBC in diabetes is due to the increased non-enzymatic glycosylation of red blood cells (RBC) membrane proteins, oxidation of proteins and hyperglycemia caused an increase in the production of lipid peroxides that lead to hemolysis of RBC^52,53. HAFA corrected diabetes-induced alteration in CBC which may be due to the presence of antioxidants like phenols, glycosides in formulations. HAFA might have worked by stimulating the formation or secretion of the hormone erythropoietin from the kidney, as evidenced by the increased level of RBC. The reduced level of WBC may contribute to various complications associated with diabetes^53,54 treatment with HAFA raised it to near normal. It has been reported that diabetes directly contributes to greater platelet reactivity by promoting glycation of platelet protein. Hyper-triglyceridemia, insulin resistance and insulin deficiency also increases the platelet reactivity as insulin responsible to antagonize the activation of platelets was absent or reduced. Thus relative deficiency of insulin would be expected to increase platelet reactivity⁵⁵. This increase in long term may lead to the development of unwanted and unnecessary blood clotting throughout the body or bleeding or stroke. Treatment with HAFA reversed the elevated platelet significantly as well as prevented from other complications. These all effect might have resulted due to the presence of micronutrients and antioxidant in HAFA formulations. Thus, from the study, it was revealed that HAFA has the potential to correct diabetes-induced anemia and correct hematological parameters which might have disturbed due to diabetes and also doesn’t have any adverse effect on hematological parameters⁵⁶.

Effect of HAFA formulations on lipid profile:

In diabetes, insulin resistance possibly leads to dyslipidemia or cause disturbed lipid profile in rats, which might increase the risk factor for coronary heart disease. In diabetes mobilization of free fatty acid from adipose tissue occur when reduction in insulin takes place which may lead to increased production of LDL and dyslipidemia, whereas the level of HDL significantly reduced. In case of insulin deficiency lipoprotein lipase that hydrolyses triglycerides, is not activated which results in hypertriglyceridemia⁵⁷. In study STZ induced diabetes caused a significant rise of total cholesterol, triglycerides, LDL and VLDL whereas the level of HDL significantly decreased. On treatment with four HAFA formulations for 28 days, the level of different lipid profile significantly got reversed and was brought to near normal. HAFA formulations might have acted by inhibiting lipogenic enzymes and HMG-CoA reductase in the liver or might have increased the uptake of cholesterol by the liver through stimulation of LDL receptor binding. This might directly increase biliary and fecal excretion of cholesterol⁵⁸, thus preventing from hypercholesterolemia. Thus a number of metabolic abnormalities that occur sequentially in uncontrolled diabetes lead to the progression of hypertriglyceridemia and hypercholesterolemia⁵⁹ were also controlled by HAFA treatment. HAFA had shown activity may be due to the presence of antioxidant activity that may reduce the complications of lipid profile⁶⁰. It was revealed that after 28 days of treatment with four different HAFA formulations, HAFA 4 comparatively showed better activity in managing dyslipidemia (P<0.001) than HAFA 2, 8, 10 and standard GLB. HAFA 4 reduced TC by 32.53%, TGL by 40.57%, VLDL by 40.57% and LDL by 47.64% which was significantly better than other HAFA formulations, thus HAFA formulations showed significantly better antihyperlipidemic effect. Protective action against hyperlipidemia may be shown due to the presence of high flavonoids and polyphenol compounds.

Atherogenic index (AI) and coronary risk index (CRI) represented by LDL-c/HDL-c and TC/HDL-c ratios. High non HDL-c and Low HDL/LDL ratio a reliable predictor of cardiovascular risk were analyzed. In the present study diabetes raised the TC/HDL-c ratio, LDL/HDL-c ratio and non HDL-c ratio were significantly reversed, whereas HDL/LDL-c ratio decreased in diabetic rats were significantly elevated after treatment with HAFA formulations. This reduction might be due to the presence of high antioxidant phytochemical in the herbs present in the formulations. Thus HAFA formulations may play an important role in preventing or reversing development of cardiovascular risk⁶¹. The supplementation of HAFA with beneficial phytoconstituents especially, polyphenols compounds might have induced an inhibitory effect on intestinal dietary fat/cholesterol absorption that lead to decreased triglycerides accumulation and hence ameliorate dyslipidemia. Polyphenols available in HAFA might have increased the formation reaction of bile acids of cholesterol and then excreted it through fesses⁶².

Table 9: Effect of HAFA formulations on lipid profile of STZ-induced diabetic rats

Groups

Lipid profile in mg/dL

TC/HDL-c Ratio

LDL/HDL-c Ratio

HDL/LDL-c Ratio

Non HDL-c

TGL

HDL

VLDL

LDL

Group I Normal control

92.67

±2.94

91.00

±3.22

41.50

±1.05

18.20

±0.64

52.97

±3.10

2.23

±0.06

1.28

±0.07

0.79

±0.04

51.17

±2.56

Group II Diabetic control

193.67

±4.50

215.67

±5.32

34.50

±1.52

43.13

±1.06

106.07

±4.55

5.62

±0.22

3.08

±0.18

0.33

±0.02

159.17

±4.07

Group III Standard group

151.67

±4.46

144.50

±4.64

42.83

±2.32

28.90

±0.93**

79.93

±5.62***

3.55

±0.27*

1.88

±0.22

0.54

±0.07

108.83

±6.24

Group IV HAFA 2 treated

145.50

±4.64***

140.83

±4.31***

44.17

±2.86***

28.17

±0.86**

73.17

±4.83***

3.30

±0.23

1.66

±0.18

0.61

±0.06

101.33

±4.89

Group V HAFA 4 treated

130.67

±5.32***

128.17

±5.08***

49.50

±2.74***

25.63

±1.02***

55.53

±6.75***

2.65

±0.20

1.13

±0.18

0.91

±0.14

81.17

±6.37

Group VI HAFA 8 treated

149.83

±2.93***

144.17

±2.64***

43.00

±2.45***

28.83

±0.53**

78.00

±4.22***

3.49

±0.23

1.82

±0.18

0.55

±0.06

106.83

±4.31

Group VII HAFA 10 treated

139.50

±2.88***

134.17

±3.76***

49.67

±3.14***

26.83

±0.75***

63.00

±3.11***

2.82

±0.17

1.27

±0.13

0.79

±0.08

89.83

±3.71

Values are expressed as mean ± SD (n=6); * (p< 0.05) significant; ** (p < 0.01) very significant; *** (p < 0.001) highly significant compared with normal control

CONCLUSION:

By the present study it was concluded that in comparison to HAFA 2, 8 and 10, HAFA 4 significantly improved glucose tolerance and controlled blood glucose without causing hypoglycemia. HAFA 4 has proven to be effective anti-hyperglycemic in all parameters. It significantly improved mean body weight, normalized food and water intake. HAFA 4 doesn’t have any adverse effect on hematological parameters and has significant potential to correct diabetes-induced anemia and alteration in CBC. HAFA 4 significantly reversed the altered lipid profile to near normal after 28 days of continuous treatment and potentially minimize the possible cardiovascular risk. Thus HAFA polyherbal formulation can be breakthrough in the treatment of diabetes and associated complications.

ACKNOWLEDGEMENT:

The authors are greatly thankful to the research cell, Vedica college of B.Pharmacy, RKDF University, for providing necessary facilities.

CONFLICT OF INTEREST:

There are no conflicts of interest.

ABBREVIATIONS AND SYMBOLS:

· TG - Triglycerides

· TC- Total cholesterol

· HDL- High Density Lipoprotien

· LDL- Low Density Lipoprotien

· VLDL- Very Low Density Lipoprotien

· HAFA- Hibiscus sabdariffa, Annona squamosa, Ficus religiosa, Aegle marmelos

· GLB- Glibenclamide

· EDTA – Ethylenediaminetetraacetic acid

· HOMA-IR – Homeostasis model assessment-estimated insulin resistance

· HMG-CoA- reductase β-hydroxy β-methylglutaryl-CoA reductase

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Received on 06.05.2022 Modified on 03.07.2022

Research J. Pharm. and Tech 2023; 16(4):1607-1616.

DOI: 10.52711/0974-360X.2023.00263