HET-19-0676 Proof Hi PDF

Human & Experimental Toxicology
A study on toxicity and anti-hyperglycemic effects of Abhrak

Bhasma in rats
Journal: Human and Experimental Toxicology
Manuscript ID HET-19-0676
Manuscript Type: Original Article

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Date Submitted by the
28-Nov-2019
Author:
Complete List of Authors: Gopinath, Harish; VIT University, Department of Chemistry School of
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Advance Sciences
Shivashankar, Murugesh; VIT University, Department of Chemistry,
School of Advance Sciences
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Streptozotocin,, Abhrak bhasma, invivo, hyperglycemic activity, blood

Keyword:
glucose, anti-hyperglycemia
Streptozotocin(STZ) is a gulcosamine-nitrosourea complex produced by

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Streptomyces achromogenes, which particularly induces DNA strand

breakage in pancreatic β-cells causing diabetes mellitus(DM). The
damage caused by STZ to pancreatic β-cells is coupled with insulin
release in the initial stage, subsequently leading to hyperglycemia owing
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to insulin deficiency. Abhrak bhasma(AB) is used for DM treatment;

however, no proper invivo investigations have been conducted to
establish the pharmacological effects of AB. In this study, the safety and
iew
effectiveness of AB with respect to invivo toxicity and STZ-induced

hyperglycemic activity in rats were examined. The anti-hyperglycemic
potential of AB in rats (40, 80, and 160mg/kg body weight (b.w.)) was
Abstract: evaluated by determining the body weight, blood glucose, organ weight,
lipid profile, and histo-morphological and histo-pathological
investigations. The highest tolerated dose of AB was 2000 mg/kg b.w.,
and sub-acute toxicity of different AB doses showed no significant
variation, when compared with the control. Interestingly, a noteworthy
reduction in blood glucose, total cholesterol, and triglycerides levels were
observed in AB-treated diabetic rats, along with a considerable increase
in body weight, when compared with those noted in the disease control
and normal control. These results suggest AB as a potential candidate of
alternate and complimentary for anti-hyperglycemia, which needs further
clinical evaluation. .
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Page 1 of 48 Human & Experimental Toxicology
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Human & Experimental Toxicology Page 2 of 48
Streptozotocin(STZ) is a gulcosamine-nitrosourea complex produced by Streptomyces

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achromogenes, which particularly induces DNA strand breakage in pancreatic β-cells causing
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5 diabetes mellitus(DM). The damage caused by STZ to pancreatic β-cells is coupled with insulin
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7 release in the initial stage, subsequently leading to hyperglycemia owing to insulin deficiency.
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9 Abhrak bhasma(AB) is used for DM treatment; however, no proper invivo investigations have been
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12 conducted to establish the pharmacological effects of AB. In this study, the safety and effectiveness
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14 of AB with respect to invivo toxicity and STZ-induced hyperglycemic activity in rats were
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16 examined. The anti-hyperglycemic potential of AB in rats (40, 80, and 160mg/kg body weight
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(b.w.)) was evaluated by determining the body weight, blood glucose, organ weight, lipid profile,
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21 and histo-morphological and histo-pathological investigations. The highest tolerated dose of AB
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23 was 2000 mg/kg b.w., and sub-acute toxicity of different AB doses showed no significant variation,
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when compared with the control. Interestingly, a noteworthy reduction in blood glucose, total
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28 cholesterol, and triglycerides levels were observed in AB-treated diabetic rats, along with a
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30 considerable increase in body weight, when compared with those noted in the disease control and
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32 normal control. These results suggest AB as a potential candidate of alternate and complimentary
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35 for anti-hyperglycemia, which needs further clinical evaluation. .
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5 1. Introduction
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7 Bhasma is a non-allopathic, natural substance used in Indian medicine to produce quality and
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safe medication, which is economically inexpensive. Owing to its hepatoprotective and
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12 immunomodulatory effects, myocardial ischemia, and diabetes. However, its applicability is
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14 limited owing to lack of scientific evidences. Abhrak bhasma(AB) is a red-colored powdery
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substance composed of oxides of iron, magnesium, calcium, silica, potassium, and
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19 aluminum. It is obtained by treating biotite (mica) with plant extract, which helps in
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21 converting the inactive material to active cellular regenerator. AB mainly comprises two types,
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23 namely, muscovite (hydrated aluminum potassium silicate [KAl2(AlSi3O10)(F, OH)2]) and
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phlogopite (potassium magnesium aluminum silicate hydroxide). Characterization studies
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28 based on scanning electron microscopy and particle size analyzer have indicated the particle
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30 size of bhasma in nano range,6 which could be the result of incineration of mica at higher
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32 temperature.
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As a result of its particle size, bhasma exhibits enhanced bioavailability and
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35 penetration capacity. Although the metallic elements present in bhasma could be useful for
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37 reducing oxidative stress induced by free radicals, inappropriate dosing of pseudo metals
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39 might disrupt the metabolic processes in the human body. Besides, improperly processed mica
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might not be safe and may cause undesirable effects owing to the presence of toxic trace
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44 elements.2, 16Hence, safety and toxicity studies on bhasma are necessary to develop appropriate
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46 standardization technique for ascertaining effective dose, route, and extent of exposure for
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treatment of acute or chronic illnesses. To formulate an effective bhasma, favorable
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51 temperature and chemical exposure are necessary, including decontamination, detoxification,
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53 incineration, and micronization performed according to Rasashastra.
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Diabetes mellitus (DM) is a chronic disease caused by the lack of secretion of endocrine insulin
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6 from β-cells of pancreas, leading to elevated blood glucose level. Failure to maintain the blood
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glucose levels could lead to this life-threatening disorder, which has been predicted to affect
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11 around 350 million individuals by 2030 according to the World Health Organization
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13 (WHO). Streptozotocin(STZ) is a gulcosamine-nitrosourea complex produced by
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Streptomyces achromogenes, which particularly induces DNA strand breakage in pancreatic β-
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18 cells causing DM. The damage caused by STZ to pancreatic β-cells is coupled with insulin
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20 release in the initial stage, subsequently leading to hyperglycemia owing to insulin deficiency.18
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Bhasma has the capability to induce insulin secretion from pancreatic β-cells, thereby reducing
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25 the blood glucose level by acting as a cellular regenerator. Thus, in the present study, the in
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27 vivo acute and sub-acute toxicity of AB was investigated in STZ-induced hyperglycemic rats,
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30 and the anti-hyperglycemic potential of AB was estimated.
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32 2. Experimental
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2.1 Chemicals
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37 AB was procured from Delta Scientific, Vijayawada, AP, India, and all other chemicals used
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39 in the study were of synthetic or analytical grade and obtained from Sisco Research
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Laboratories, India.
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44 2.2 Test animals
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46 Wistar rats (190–200g) were obtained from and investigated at Central Animal Facility,
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48 SASTRA University, India. All animal experiments were approved by IAEC,SASTRA
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51 University (IAEC Approval No:352/SASTRA/IAEC/RPP), and performed at the Central
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53 Animal Facility (RegisterNo.817/04/ac/CPCSEA; dated11.03.99) for breeding and
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55 experiments on animals according to the Control and Supervision of Experiments on Animals,
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Ministry of Forest and Environment, India. The rats were housed in ventilated cages and
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60 provided with standard rodent feed pellet (M/s. ATNT Laboratories, Mumbai, India) and
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3 reverse osmosis (RO) water ad libitum.
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7 2.3 Preparation of test substance
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9 The AB was dispersed in honey water at a ratio of 2:3 and administered at the dose of 2000
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12 mg/kg bodyweight (b.w.).19-28
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14 2.4 Acute oral toxicity assessment of AB by up-down procedure in rats
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16 Acute oral toxicity of AB was assessed according to the OECD guidelines by employing up-
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19 down procedure (UDP) (OECD 425 guidelines). After acclimation for 5 days, 5healthy female
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Wistar rats were treated with 2000mg/kg b.w. AB for 14days. Prior to AB treatment, the
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24 animals were abstained from food overnight. After AB treatment, the animals were returned to
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26 their cages immediately and feed was made available ad libitum. The body weights of the rats
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28 21
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29 were recorded before the start of the experiment. Administration of AB (2000mg/kg b.w.)was
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31 achieved through oral gavage using appropriately sized syringe and stainless steel ball-tipped
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intubation needle. After 14 days of exposure of the test substance, prominent parameters,
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36 such as percentage mortality, bodyweight, feed intake, adverse signs, and gross pathology,
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were evaluated.
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41 2.5 Sub-acute toxicity assessment of AB in rats
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43 The sub-acute toxicity of AB was examined on 6–8-week-old Wistar rats according to the
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45 OECD-407 guidelines. A total of 30rats were divided into 6 groups comprising 5 rats blinded
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48 for sex. The first group was treated with honey water and served as the vehicle control, while
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50 the other groups were treated with three different doses of AB (80, 320, and 1280 mg/kg b.w.,
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52 respectively) based on our acute toxicity assay. The satellite group was administered with high
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doses of AB to check the reversibility of AB toxicity. The test substance and vehicle were
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57 administered to respective groups for 28 days.
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3 The body weight of the rats was measured weekly during dosing and on the day of sacrifice
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6 using digital weighing balance (Sartorius AG, Germany). The manifestations of AB toxicity
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8 such as change in weekly body weight and cage side clinical observations were monitored.
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10 After 28days, all the surviving animals were allowed to fast overnight, and their blood was
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collected through retro-orbital plexus under light anesthesia. The blood collected in EDTA-
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15 treated vials was used for hematological analysis, and the serum from non-heparinized vials
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was collected to study the biochemical parameters. The experimental rats were euthanized
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20 after blood collection, and organs such as brain, heart, liver, spleen, thymus, kidneys, adrenals,
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22 testis, epididymis, and uterus were carefully dissected out, weighed and observed for the
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24 presence of any gross lesions. The relative organ weight was determined by using the
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27 f o l l o w i n g equation:23
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31 Actual organ weight (g)
Relative organ weight = × 100
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32 Rats body weight on the day of sacrifice (g)

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37 The internal organs were first fixed in 10% buffered formalin solution, and the tissues were
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39 fixed in paraffin, sectioned to a thickness of 5µm, and stained with dyes such as hematoxylin
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41 and eosin. The stained tissue sections were examined under a light microscope and clear
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43 30-34
44 photomicrographs were obtained.
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46 2.6 Hematological parameters assay
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48 The complete blood parameters were analyzed using hematology analyzer (Oxford science,
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51 USA). In addition, clotting time was ascertained manually using capillary tube method.
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3 2.7 Biochemical parameters analysis
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6 The clinical biochemical parameters such as albumin, alkaline phosphatase (ALP), total
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8 bilirubin, direct bilirubin, total cholesterol, creatinine, glucose, total protein, aspartate
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aminotransferase (AST), alanine transaminase (ALT), triglycerides (TGL), urea and uric acid
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13 were evaluated using semi auto analyzer(ErbaChem5).
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15 2.8 AB treatment and anti-hyperglycemic investigation using STZ method
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18 2.8.1 Inducing diabetes in rats using STZ
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20 Prior to STZ administration, the rats were allowed to fast overnight. Subsequently, 65 mg/kg
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22 STZ (STZ dissolved in 0.1 M glacial sodium citrate buffer at pH 4.5 and administered within
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30 min of preparation) was injected into the intra-peritoneal vein of the rats. The control rats
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27 received the vehicle alone, whereas the diabetic groups received STZ >250 mg/dL. After 48 h
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29 of STZ administration, the blood glucose level was measured using glucometer (Aspen AP
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31 6
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32 Plus).
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34 2.8.2Experimentaldesign
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36 A total of 48 rats were divided into 6 groups comprising 8 animals. Group I (control) was
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39 administered with 0.9% sodium chloride; Group II (disease control) was treated with STZ (65
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41 mg/kg b.w.); Group III (STZ-treated + standard diabetic)was administered with STZ and oral
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43 Metformin (100 mg/kg, b.w.); Group IV (STZ-treated + low dose AB) was treated with STZ
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and administered with low dose of AB (40 mg/kg b.w.); Group V (STZ-treated + medium dose
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48 AB) was treated with STZ and administered with medium dose of AB (80 mg/kg b.w); and
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50 Group VI (STZ-treated + high dose AB) was treated with STZ and administered with high dose
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of AB (160 mg/kg b.w.).
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55 The blood glucose level in all the rats was determined every week with a glucometer (Aspen
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57 AP Plus) using strip method by collecting blood from the tip of the tail. After 28 days, blood
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59 sample was obtained from retro orbital sinus under light anesthesia, and the serum total
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cholesterol and TGL levels were estimated using semi-auto analyzer (ErbaChem 5).
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6 2.8.3 Statistical analysis
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8 The results are presented as mean ± SEM. Statistical analysis was conducted by using
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10 GraphPadPrism software35, and one-way ANOVA followed by Tukey's multiple comparisons
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test was performed. The values were considered as statistically significant at P ≤0.05.
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15 2.8.4 Histopathological investigation
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17 Liver tissue from the experimental rats was dissected out and fixed in 10% neutral buffered
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formalin and processed for blind histopathological evaluations. Similarly, a partof the
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22 pancreatic tissue was dissected out and fixed in 10% neutral buffered formalin and processed
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as previously reported.
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27 3. Results and discussion
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29 3.1 Investigation of acute toxicity of AB

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31 The results of acute oral toxicity investigation revealed no considerable changes in the
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mortality of female rats treated orally with 2000mg/kg b.w. AB, when compared with those in
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36 other treatment groups. Throughout the observation period of 2weeks, the animals were
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38 evaluated for their body weight (Figure 1), feed intake (Figure 2), and vital toxicity signs which
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were all found to be generally normal, and the gross pathology was typical Although animals
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43 treated with AB showed significant changes in body weight gain on days 7 and 14, when
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45 compared with that on day 0, the daily feed intake of rats remained unaffected throughout the
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47 experimental period. No explicit signs of neuronal toxicity, such as changes in autonomic
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50 nervous system, central nervous system, and behavioral pattern, were observed during the
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4 period. All gross observations were agonal and presented no
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11 3.2 Investigation of sub-acute toxicity of AB
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13 3.2.1 Effect of AB on bodyweight
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16 The body weight of all the rats was determined on days 0, 7, 14,
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18 21,and 28 as shown in and Figures 3 and 4. The body weight of the
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20 control group at the end of day 28 was 300.2±22.54 g for male
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rats(M) and 204.86±12.58 gfor female rats (F). However, groups
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treated with 80, 320, and 1280mg/kg b.w. AB showed body weight
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27 of 301±33.29 g (M)and 192.47 ± 10.4 g (F), 298.16 ± 17.1 g (M)
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and189.99±9.77 g (F), and 288.85±30.81 g (M) and 195.51±8.1 g
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32 (F), respectively. The body weight of female rats in all AB

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36 control, but did not present any significant differences. Overall,

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39 AB did not produce any effective changes in the body weight of

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41 the experimental animals (Table 6).
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43 3.2.2 Effect of AB on relative weight of vital organs
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The relative weight of the vital organs of the experimental animals,
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48 including brain, heart, liver, spleen, thymus, kidneys(right and
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50 left), adrenals, testis (right and left), and epididymis, following
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treatment with 80, 320, and 1280mg/kgb.w. AB for 28 days was
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55 determined. In line with the total body weight, the average relative
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57 organ weight of the animals (both male and female rats) also
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3 that noted in the control (Figures 5 and 6).
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6 3.2.3 Effect of AB on hematological parameters
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8 Treatment with AB for 28 days produced no significant effect on
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eosinophil,basophil, and hematocrit),red blood cells
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15 (RBC)(hemoglobin, MCV, MCH, MCHC, RDW%, and RSD),and
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in the control. Thus, the hematological parameters were normal
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24 3.2.4 Effect of AB on biochemical parameters
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26 The biochemical parameters, including albumin, ALP, total
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31 total protein, AST, ALT, TGL, urea, and uric acid levels, exhibited
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33 no clinical changes after 28days of AB treatment, when compared
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with those noted in the control. The lack of significant increase in
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38 the levels of AST, ALT, and ALP after AB treatment indicated that
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40 the test substance had no effect on the functioning of liver. The
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values obtained for the biomarkers of liver injuries such as AST
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45 and ALT were comparable between the control and treatment
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47 groups, implying no important functional abnormalities after AB
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49 treatment. Similarly, the levels of urea, uric acid, and blood
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52 glucose in both male and female rats presented no significant
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54 changes. Billirubin is normally excreted in urine and bile, and the
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56 total bilirubin and direct bilirubin levels were within the normal
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range of <1.23 and 0.1–0.3mg/dL, respectively, in the AB-treated
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3 and control groups. The total protein concentration was <6–8g/dL
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6 and the albumin concentration was low(5.5±0.5 g/dL)in both AB-
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3.2.5 Histopathological analysis of sub-acute toxicity of AB in
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No obvious histopathological changes were noted between rats
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20 treated with 80, 320, and 1280 mg/kg b.w. AB and the control
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22 animals. The liver sections from animals treated with high dose of
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24 AB(1280 mg/kg b.w.) showed completely normal phenotypic
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27 features of hepatocytes. Furthermore, although increase in the
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29 number of Kupffer cells was observed (Figure 7) owing to local

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34 detected.25These findings indicated that normal liver architecture
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following AB treatment (Figure 7).

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3.2.6 Antihyperglycemic effect of AB in STZ-induced
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46 hyperglycemic rats
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48 The effect of AB on the blood glucose level of the experimental
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50 animals is shown in Table 15 and Figure 8. The fasting blood
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53 glucose level in the normal control and STZ-induced untreated
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55 disease control did not change during the experimental period of
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57 4weeks.In contrast, in the AB-treated groups, the glucose levels
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3 160mg/kg b.w. AB treatment, respectively, which were
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6 significantly lower than that noted in disease control
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10 group (175mg/dL). Thus, even low dosage of AB was effective in
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12 reducing the blood glucose level, which was comparable to that of
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15 Metformin standard group25.
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17 3.2.7 Effect of AB on weekly body weight of STZ-induced
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The changes in the body weight of animals in various groups were
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24 compared at the end of the study on day 28 (Figure 9). The normal
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26 control animals presented an increase in the body weight, whereas
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28 the disease control animals showed significant decrease in the body
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31 weight, which confirmed hyperglycemia and glucosuria.
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33 Treatment with 40,80, and 160mg/kgb.w. AB and Metformin
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standard (100mg/kg b.w.) substantially prevented decrease in body

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38 weight, when compared with that o b s e r v e d in disease and
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40 normal controls. While the body weight of all the hyperglycemic
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42 rats was similar at the start of the experiment, AB treatment
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significantly increased the body weight, when compared with that
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47 observed in the untreated group.
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49 3.2.8 Effect of AB on relative weight of vital organs of STZ-
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52 induced hyperglycemic rats
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54 The relative weights of vital organs, including liver and
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56 kidneys(right and left),of STZ-induced diabetic rats after 28 days
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of AB treatment (40, 80, and 160mg/kg b.w. AB) were determined.
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3 Following 28 days of AB treatment, the kidney weight of diabetic
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6 rats was found to be slightly higher(Figure10b), when compared
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8 with that of normo glycemic rats, similar to that reported in
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10 previous studies 36. In contrast, as light decrease in the liver weight
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of treated rats was noted, when compared with that of control rats
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15 (Figure10a). Nevertheless, no significant changes in the weight of
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17 vital organs were observed in the AB-treated groups, when
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compared with those in the control (Figure 10).
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26 The effect of AB on the serum lipid profile, including cholesterol
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29 and TGL levels, was assayed(Figure 11). The serum cholesterol

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31 and TG levels were found to be elevated in the disease group, when
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33 compared with those in the control. In the AB-treated group,
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considerable reduction in the serum cholesterol and TG levels was
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38 noted after 28 days of treatment. Lipid dysfunction is an associated
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40 complication of diabetes in rats, and the findings of this study
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revealed that AB could reduce the total cholesterol and TGL levels
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45 and was effective in treating hyperglycemia.
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47 3.2.10 Histopathological effects of AB on STZ-induced
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52 Histomorphological observation of endocrine pancreas, liver and
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54 kidneys of AB-treated rats showed no significant morphological
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56 changes, when compared with those observed in the normal control
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and disease control. The tissue morphology of endocrine pancreas
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3 of b o t h disease control and A B - treatedgroupswassimilar,
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6 presenting diffused and moderate islet cell atrophy/degeneration,
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8 whereas that of liver of control and AB-treated groups exhibited
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10 granuloma; multifocal, random, minimal necrosis; hepatocellular,
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multifocal, random, minimal infiltrates; and mononuclear,
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15 periportal, diffuse, minimal cells. The kidneys of both the control
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17 and AB-treated groups showed vacuolar degeneration; tubular,
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cortical, bilateral, multifocal, moderate pigment accumulation; and
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22 interstitial, multifocal, minimal cells. These results clearly

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24 demonstrated that AB had no significant toxicity on STZ-induced
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26 hyperglycemic rats (Tables 1–3).
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29 Subsequently, histopathological analysis of endocrine pancreas of

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31 the experimental rats was performed. In the control group, the
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33 islets showed no significant pathological changes, and were of
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normal size and evenly distributed throughout the pancreas. In the
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38 STZ-induced hyperglycemic groups, marked atrophy of islets was
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40 noted with respect to both size as well as number of islets. In the
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case of Metformin standard group, the islets presented moderate
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45 atrophy, whereas in the AB-treated groups, the islets showed only
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47 minimal to moderate atrophy and exhibited improvement in the
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49 number and size of islets (Figure 12).
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52 The acute oral toxicity of AB was investigated according
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54 to OECD-245 guidelines. Female rats aged 8–12 weeks were orally
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56 administered with a single dose of AB (maximum dose of
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2000mg/kg b.w.) dispersed in honey water (at a ratio of 2:3), and
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1
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3 the mortality, weekly body weight, daily feed intake, clinical
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6 parameters, and gross pathology were observed during the study
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8 period of 14 days. The results revealed no significant toxic
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10 pathological effects following AB treatment. The maximal
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tolerated dose of AB was >2000mg/kg b.w., and the mortality rate
14
15 was 0%.Besides, no visible signs of toxicity were observed, the
16
17 gross pathology was agonal in nature, and all the animals survived
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19
at the end of the acute toxicity study.
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21
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22 Sub-acute toxicity investigation was performed as per the

23
24 OECD-407 guidelines by orally administering 80, 320, and 1280
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25
26 mg/kg b.w. AB dispersed in honey water (at a ratio of 2:3) to
27
28
ee
29 Wistar rats for 28days. The results presented no clinical difference

30
31 in weekly body weight and organ weight changes during the study
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32
33 period. It must be noted that hematological system is highly
34
35
ev
36
sensitive to toxic materials and could reveal the physiological
37
38 changes in animals or human beings.36In the present study, the
iew
39
40 hematological parameters, including RBC and WBC, of AB-
41
42
treated rats were not affected even at high dose of AB (1280mg/kg
43
44
45 b.w. AB), indicating that AB is non-toxic and safe while in
46
47 circulation. Furthermore, biochemical studies revealed that the
48
49 levels of total bilirubin, direct bilirubin, and creatinine were not
50
51
52 significantly altered (P>0.05) among AB-treated groups and
53
54 control. However, a considerable increase in serum liver enzymes
55
56 (AST, ALT, and ALP), albumin, total cholesterol, glucose, total
57
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59
protein, TGL, urea, and uric acid (P<0.05) in rats administered
60
14
1
2
3 with 80, 320, and 1280 mg/kg b.w. AB was observed. Moreover,
4
5
6 histopathological studies showed improved size and number of
7
8 islets in AB-treated rats, when compared with those in normal
9
10 control and disease control, suggesting minimal to moderate
11
12
13
atrophy of islet cells. Besides, AB treatment produced no relative
14
15 changes in the levels of glucose, creatinine, AST, and ALP,
16
17 implying that AB did not affect liver and kidney tissues.
18
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20 The anti-hyperglycemic potential of AB was studied by employing
21
Fo
22 STZ-induced diabetic rat model, and the results confirmed the

23
24 dose-dependent effect of AB (40, 80, and 160mg/kg b.w.).While
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25
26
27 obvious increase in the blood glucose level was noted in disease
28
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29 control and normal control, rats treated with medium concentration

30
31 of AB showed significantly lower blood glucose level. The body
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32
33
34
weight of rats both in disease control and normal control was
35
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36 considerably increased, whereas that of rats in AB-treated group

37
38 was reduced, when compared with the initial weight.
iew
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40
Furthermore, a significant decrease in the blood glucose,
41
42
43 total cholesterol, and TG levels was observed in AB-treated
44
45 diabetic rats. The body weight of control rats presented an
46
47 significant increase, whereas the body weight of AB-treated rats
48
49
50 was lower than that of the disease control and normal control rats.
51
52 These findings suggest that AB is safe and nontoxic to rats and do
53
54 not produce any significant toxic effects. Besides, AB exhibited
55
56
57
beneficial effects as an anti-hyperglycemic agent in diabetic rats
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59 by improving various metabolic processes, such as lipid
60
15
1
2
3 metabolism, with no significant toxicity.
4
5
6 4. Conclusion
7
8 The nano-ayurvedic medicine AB showed improved cell
9
10 penetration and was effective in the treatment of hyperglycemia,
11
12
13
with no significant invivo acute and sub-acute toxicity, even at high
14
15 dosage(2000 mg/kg b.w.). The acute oral LD50of AB in Wistar rats
16
17 was>2000mg/kgb.w, and sub-acute studies showed no toxic
18
19
20
effects at >1280mg/kg b.w., along with normal histopathological,
21
Fo
22 biochemical, and hematological findings. Besides, AB exhibited

23
24 significant anti-hyperglycemic activity and high cell penetration
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25
26
efficacy in STZ-induced diabetic rats. These results confirm the
27
28
ee
29 tendency of AB to induce insulin secretion from pancreatic β-cells,

30
31 suggesting the potential use of herbo-mineral nano medicine for
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32
33 the treatment of diabetes.
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38
iew
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16
A study on toxicity and anti-hyperglycemic effects of Abhrak Bhasma in rats

1
2 Harish Gopinatha, MurugeshShivashankar*a,
3
4 Department of Chemistry, Vellore Institute of Technology, Vellore 632014, Tamil Nadu, India
5
6
Email: mshivshankar@vit.ac.in
7
8
9
10 Streptozotocin(Streptozotocin (STZ) is a gulcosamine-nitrosourea complex produced by
11
12
Streptomyces achromogenes, which particularly induces DNA strand breakage in pancreatic β-cells
13
14
15 causing diabetes mellitus(DM). The damage caused by STZ to pancreatic β-cells is coupled with
16
17 insulin release in the initial stage, subsequently leading to hyperglycemia owing to insulin
18
19 deficiency. Abhrak bhasma(AB) is used for DM treatment; however, no proper invivo
20
21
Fo
22 investigations have been conducted to establish the pharmacological effects of AB. In this study,
23
24 the safety and effectiveness of AB with respect to invivo toxicity and STZ-induced hyperglycemic
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25
26 activity in rats were examined. The anti-hyperglycemic potential of AB in rats (40, 80, and
27
28
ee
29
160mg/kg body weight (b.w.)) was evaluated by determining the body weight, blood glucose, organ
30
31 weight, lipid profile, and histo-morphological and histo-pathological investigations. The highest
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32
33 tolerated dose of AB was 2000 mg/kg b.w., and sub-acute toxicity of different AB doses showed
34
35
ev
no significant variation, when compared with the control. Interestingly, a noteworthy reduction in
36
37
38 blood glucose, total cholesterol, and triglycerides levels were observed in AB-treated diabetic rats,
iew
39
40 along with a considerable increase in body weight, when compared with those noted in the disease
41
42 control and normal control. These results suggest AB as a potential candidate of alternate and
43
44
45 complimentary for anti-hyperglycemia, which needs further clinical evaluation. .
46
47
48
49
50
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52
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60
1
1
2
3
4
5 1. Introduction
6
7 Bhasma is a non-allopathic, natural substance used in Indian medicine to produce quality and
8
9 1
safe medication, which is economically inexpensive. Owing to its hepatoprotective and
10
11 5 6
12 immunomodulatory effects, myocardial ischemia, and diabetes. However, its applicability is
13
7-8
14 limited owing to lack of scientific evidences. Abhrak bhasma(AB) is a red-colored powdery
15
16 10
substance composed of oxides of iron, magnesium, calcium, silica, potassium, and
17
18 11
19 aluminum. It is obtained by treating biotite (mica) with plant extract, which helps in
20
21 converting the inactive material to active cellular regenerator. AB mainly comprises two types,
Fo
22
23 namely, muscovite (hydrated aluminum potassium silicate [KAl2(AlSi3O10)(F, OH)2]) and
24
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25 12
26
phlogopite (potassium magnesium aluminum silicate hydroxide). Characterization studies
27
28 based on scanning electron microscopy and particle size analyzer have indicated the particle
ee
29
30 size of bhasma in nano range,6 which could be the result of incineration of mica at higher
31
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32 temperature.
11
As a result of its particle size, bhasma exhibits enhanced bioavailability and
33
34
35 penetration capacity. Although the metallic elements present in bhasma could be useful for
ev
36 14
37 reducing oxidative stress induced by free radicals, inappropriate dosing of pseudo metals
38
iew
39 might disrupt the metabolic processes in the human body. Besides, improperly processed mica
40
41
42
might not be safe and may cause undesirable effects owing to the presence of toxic trace
43
44 elements.2, 16Hence, safety and toxicity studies on bhasma are necessary to develop appropriate
45
46 standardization technique for ascertaining effective dose, route, and extent of exposure for
47
48 14,15
treatment of acute or chronic illnesses. To formulate an effective bhasma, favorable
49
50
51 temperature and chemical exposure are necessary, including decontamination, detoxification,
52
8,11
53 incineration, and micronization performed according to Rasashastra.
54
55
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60
2
1
2
3
Diabetes mellitus (DM) is a chronic disease caused by the lack of secretion of endocrine insulin
4
5
6 from β-cells of pancreas, leading to elevated blood glucose level. Failure to maintain the blood
7
8 12
glucose levels could lead to this life-threatening disorder, which has been predicted to affect
9
10
11 around 350 million individuals by 2030 according to the World Health Organization
12
17
13 (WHO). Streptozotocin(STZ) is a gulcosamine-nitrosourea complex produced by
14
15
16
Streptomyces achromogenes, which particularly induces DNA strand breakage in pancreatic β-
17
18 cells causing DM. The damage caused by STZ to pancreatic β-cells is coupled with insulin
19
20 release in the initial stage, subsequently leading to hyperglycemia owing to insulin deficiency.18
21
Fo
22
Bhasma has the capability to induce insulin secretion from pancreatic β-cells, thereby reducing
23
24
12
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25 the blood glucose level by acting as a cellular regenerator. Thus, in the present study, the in
26
27 vivo acute and sub-acute toxicity of AB was investigated in STZ-induced hyperglycemic rats,
28
ee
29
30 and the anti-hyperglycemic potential of AB was estimated.
31
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32 2. Experimental
33
34
35
2.1 Chemicals
ev
36
37 AB was procured from Delta Scientific, Vijayawada, AP, India, and all other chemicals used
38
iew
39 in the study were of synthetic or analytical grade and obtained from Sisco Research
40
41
Laboratories, India.
42
43
44 2.2 Test animals
45
46 Wistar rats (190–200g) were obtained from and investigated at Central Animal Facility,
47
48 SASTRA University, India. All animal experiments were approved by IAEC,SASTRA
49
50
51 University (IAEC Approval No:352/SASTRA/IAEC/RPP), and performed at the Central
52
53 Animal Facility (RegisterNo.817/04/ac/CPCSEA; dated11.03.99) for breeding and
54
55 experiments on animals according to the Control and Supervision of Experiments on Animals,
56
57
58
Ministry of Forest and Environment, India. The rats were housed in ventilated cages and
59
60 provided with standard rodent feed pellet (M/s. ATNT Laboratories, Mumbai, India) and
3
1
2
3 reverse osmosis (RO) water ad libitum.
4
5
6
7 2.3 Preparation of test substance
8
9 The AB was dispersed in honey water at a ratio of 2:3 and administered at the dose of 2000
10
11
12 mg/kg bodyweight (b.w.).19-28
13
14 2.4 Acute oral toxicity assessment of AB by up-down procedure in rats
15
16 Acute oral toxicity of AB was assessed according to the OECD guidelines by employing up-
17
18 20
19 down procedure (UDP) (OECD 425 guidelines). After acclimation for 5 days, 5healthy female
20
21 18
Wistar rats were treated with 2000mg/kg b.w. AB for 14days. Prior to AB treatment, the
Fo
22
23
24 animals were abstained from food overnight. After AB treatment, the animals were returned to
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25
26 their cages immediately and feed was made available ad libitum. The body weights of the rats
27
28 21
ee
29 were recorded before the start of the experiment. Administration of AB (2000mg/kg b.w.)was
30
31 achieved through oral gavage using appropriately sized syringe and stainless steel ball-tipped
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32
33 22
34
intubation needle. After 14 days of exposure of the test substance, prominent parameters,
35
ev
36 such as percentage mortality, bodyweight, feed intake, adverse signs, and gross pathology,
37
38 23
iew
were evaluated.
39
40
41 2.5 Sub-acute toxicity assessment of AB in rats
42
43 The sub-acute toxicity of AB was examined on 6–8-week-old Wistar rats according to the
44
45 OECD-407 guidelines. A total of 30rats were divided into 6 groups comprising 5 rats blinded
46
47
48 for sex. The first group was treated with honey water and served as the vehicle control, while
49
50 the other groups were treated with three different doses of AB (80, 320, and 1280 mg/kg b.w.,
51
52 respectively) based on our acute toxicity assay. The satellite group was administered with high
53
54
55
doses of AB to check the reversibility of AB toxicity. The test substance and vehicle were
56
57 administered to respective groups for 28 days.
58
59
60
4
1
2
3 The body weight of the rats was measured weekly during dosing and on the day of sacrifice
4
5
6 using digital weighing balance (Sartorius AG, Germany). The manifestations of AB toxicity
7
8 such as change in weekly body weight and cage side clinical observations were monitored.
9
10 After 28days, all the surviving animals were allowed to fast overnight, and their blood was
11
12
13
collected through retro-orbital plexus under light anesthesia. The blood collected in EDTA-
14
15 treated vials was used for hematological analysis, and the serum from non-heparinized vials
16
17 21
was collected to study the biochemical parameters. The experimental rats were euthanized
18
19
20 after blood collection, and organs such as brain, heart, liver, spleen, thymus, kidneys, adrenals,
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Fo
22 testis, epididymis, and uterus were carefully dissected out, weighed and observed for the
23
24 presence of any gross lesions. The relative organ weight was determined by using the
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26
27 f o l l o w i n g equation:23
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31 Actual organ weight (g)
Relative organ weight = × 100
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32 Rats body weight on the day of sacrifice (g)

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37 The internal organs were first fixed in 10% buffered formalin solution, and the tissues were
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39 fixed in paraffin, sectioned to a thickness of 5µm, and stained with dyes such as hematoxylin
40
41 and eosin. The stained tissue sections were examined under a light microscope and clear
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43 30-34
44 photomicrographs were obtained.
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46 2.6 Hematological parameters assay
47
48 The complete blood parameters were analyzed using hematology analyzer (Oxford science,
49
50 23
51 USA). In addition, clotting time was ascertained manually using capillary tube method.
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60
5
1
2
3 2.7 Biochemical parameters analysis
4
5
6 The clinical biochemical parameters such as albumin, alkaline phosphatase (ALP), total
7
8 bilirubin, direct bilirubin, total cholesterol, creatinine, glucose, total protein, aspartate
9
10
aminotransferase (AST), alanine transaminase (ALT), triglycerides (TGL), urea and uric acid
11
12
23
13 were evaluated using semi auto analyzer(ErbaChem5).
14
15 2.8 AB treatment and anti-hyperglycemic investigation using STZ method
16
17
18 2.8.1 Inducing diabetes in rats using STZ
19
20 Prior to STZ administration, the rats were allowed to fast overnight. Subsequently, 65 mg/kg
21
Fo
22 STZ (STZ dissolved in 0.1 M glacial sodium citrate buffer at pH 4.5 and administered within
23
24
30 min of preparation) was injected into the intra-peritoneal vein of the rats. The control rats
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26
27 received the vehicle alone, whereas the diabetic groups received STZ >250 mg/dL. After 48 h
28
ee
29 of STZ administration, the blood glucose level was measured using glucometer (Aspen AP
30
31 6
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32 Plus).
33
34 2.8.2Experimentaldesign
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36 A total of 48 rats were divided into 6 groups comprising 8 animals. Group I (control) was
37
38
iew
39 administered with 0.9% sodium chloride; Group II (disease control) was treated with STZ (65
40
41 mg/kg b.w.); Group III (STZ-treated + standard diabetic)was administered with STZ and oral
42
43 Metformin (100 mg/kg, b.w.); Group IV (STZ-treated + low dose AB) was treated with STZ
44
45
46
and administered with low dose of AB (40 mg/kg b.w.); Group V (STZ-treated + medium dose
47
48 AB) was treated with STZ and administered with medium dose of AB (80 mg/kg b.w); and
49
50 Group VI (STZ-treated + high dose AB) was treated with STZ and administered with high dose
51
52
of AB (160 mg/kg b.w.).
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55 The blood glucose level in all the rats was determined every week with a glucometer (Aspen
56
57 AP Plus) using strip method by collecting blood from the tip of the tail. After 28 days, blood
58
59 sample was obtained from retro orbital sinus under light anesthesia, and the serum total
60
6
1
2
3 25 6
cholesterol and TGL levels were estimated using semi-auto analyzer (ErbaChem 5).
4
5
6 2.8.3 Statistical analysis
7
8 The results are presented as mean ± SEM. Statistical analysis was conducted by using
9
10 GraphPadPrism software35, and one-way ANOVA followed by Tukey's multiple comparisons
11
12
13
test was performed. The values were considered as statistically significant at P ≤0.05.
14
15 2.8.4 Histopathological investigation
16
17 Liver tissue from the experimental rats was dissected out and fixed in 10% neutral buffered
18
19
formalin and processed for blind histopathological evaluations. Similarly, a partof the
20
21
Fo
22 pancreatic tissue was dissected out and fixed in 10% neutral buffered formalin and processed
23
24 30-34
as previously reported.
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25
26
27 3. Results and discussion
28
ee
29 3.1 Investigation of acute toxicity of AB

30
31 The results of acute oral toxicity investigation revealed no considerable changes in the
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32
33
34
mortality of female rats treated orally with 2000mg/kg b.w. AB, when compared with those in
35
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36 other treatment groups. Throughout the observation period of 2weeks, the animals were
37
38 evaluated for their body weight (Figure 1), feed intake (Figure 2), and vital toxicity signs which
iew
39
40
were all found to be generally normal, and the gross pathology was typical Although animals
41
42
43 treated with AB showed significant changes in body weight gain on days 7 and 14, when
44
45 compared with that on day 0, the daily feed intake of rats remained unaffected throughout the
46
47 experimental period. No explicit signs of neuronal toxicity, such as changes in autonomic
48
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50 nervous system, central nervous system, and behavioral pattern, were observed during the
51
52 entire observation
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60
7
1
2
3
4 period. All gross observations were agonal and presented no relation to AB treatment, and all
5
6 animals survived until the end of the study.
7
8
3.2 Investigation of sub-acute toxicity of AB
9
10
11 3.2.1 Effect of AB on bodyweight
12
13 The body weight of all the rats was determined on days 0, 7, 14, 21,and 28 as shown in and
14
15
16 Figures 3 and 4. The body weight of the control group at the end of day 28 was 300.2±22.54 g
17
18 for male rats(M) and 204.86±12.58 gfor female rats (F). However, groups treated with 80, 320,
19
20 and 1280mg/kg b.w. AB showed body weight of 301±33.29 g (M)and 192.47 ± 10.4 g (F),
21
Fo
22
23
298.16 ± 17.1 g (M) and189.99±9.77 g (F), and 288.85±30.81 g (M) and 195.51±8.1 g (F),
24
respectively. The body weight of female rats in all AB treatment groups remained lower, when
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25
26
27 compared with that in control, but did not present any significant differences. Overall, AB did
28
ee
29
not produce any effective changes in the body weight of the experimental animals (Table 6).
30
31
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32 3.2.2 Effect of AB on relative weight of vital organs

33
34 The relative weight of the vital organs of the experimental animals, including brain, heart, liver,
35
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36 spleen, thymus, kidneys(right and left), adrenals, testis (right and left), and epididymis,
37
38
iew
39 following treatment with 80, 320, and 1280mg/kgb.w. AB for 28 days was determined. In line
40
41 with the total body weight, the average relative organ weight of the animals (both male and
42
43 female rats) also showed no statistically significant variations, when compared with that noted
44
45
46
in the control (Figures 5 and 6).
47
48 3.2.3 Effect of AB on hematological parameters
49
50 Treatment with AB for 28 days produced no significant effect on white blood cells
51
52
(WBC)population (monocyte, eosinophil,basophil, and hematocrit),red blood cells
53
54
55 (RBC)(hemoglobin, MCV, MCH, MCHC, RDW%, and RSD),and platelet cells(MPV and
56
57 PDW%), when compared with those noted in the control. Thus, the hematological parameters
58
59 were normal without any abnormalities following AB treatment.
60
8
1
2
3 3.2.4 Effect of AB on biochemical parameters
4
5
6 The biochemical parameters, including albumin, ALP, total bilirubin, direct bilirubin, total
7
8 cholesterol, creatinine, glucose, total protein, AST, ALT, TGL, urea, and uric acid levels,
9
10 exhibited no clinical changes after 28days of AB treatment, when compared with those noted
11
12
13
in the control. The lack of significant increase in the levels of AST, ALT, and ALP after AB
14
15 treatment indicated that the test substance had no effect on the functioning of liver. The values
16
17 obtained for the biomarkers of liver injuries such as AST and ALT were comparable between
18
19
the control and treatment groups, implying no important functional abnormalities after AB
20
21
Fo
22 treatment. Similarly, the levels of urea, uric acid, and blood glucose in both male and female
23
24 rats presented no significant changes. Billirubin is normally excreted in urine and bile, and the
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25
26 total bilirubin and direct bilirubin levels were within the normal range of <1.23 and 0.1–
27
28
ee
29 0.3mg/dL, respectively, in the AB-treated and control groups. The total protein concentration
30
31 was <6–8g/dL and the albumin concentration was low(5.5±0.5 g/dL)in both AB-treated and
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32
33 control groups, suggesting normal functioning of liver in both male and female rats after AB
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36
treatment.
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38 3.2.5 Histopathological analysis of sub-acute toxicity of AB in rat liver
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40
No obvious histopathological changes were noted between rats treated with 80, 320, and 1280
41
42
43 mg/kg b.w. AB and the control animals. The liver sections from animals treated with high dose
44
45 of AB(1280 mg/kg b.w.) showed completely normal phenotypic features of hepatocytes.
46
47 Furthermore, although increase in the number of Kupffer cells was observed (Figure 7) owing
48
49
50 to local proliferation, no evidence of inflammatory lesions was detected.25These findings
51
52 indicated that normal liver architecture was maintained without any obvious pathological
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54
55
changes following AB treatment (Figure 7).
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57
58
59
60 3.2.6 Antihyperglycemic effect of AB in STZ-induced hyperglycemic rats
9
1
2
3 The effect of AB on the blood glucose level of the experimental animals is shown in Table 15
4
5
6 and Figure 8. The fasting blood glucose level in the normal control and STZ-induced untreated
7
8 disease control did not change during the experimental period of 4weeks.In contrast, in the
9
10 AB-treated groups, the glucose levels were 182.13, 220.63, and 267.25 mg/dLfollowing40, 80,
11
12
13
160mg/kg b.w. AB treatment, respectively, which were significantly lower than that noted in
14
15 disease control (318.75mg/dL) and similar to that observed in Metformin standard group
16
17 (175mg/dL). Thus, even low dosage of AB was effective in reducing the blood glucose level, which
18
19
was comparable to that of Metformin standard group25.
20
21
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22 3.2.7 Effect of AB on weekly body weight of STZ-induced hyperglycemic rats

23
24 The changes in the body weight of animals in various groups were compared at the end of the
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26 study on day 28 (Figure 9). The normal control animals presented an increase in the body
27
28
ee
29
weight, whereas the disease control animals showed significant decrease in the body weight,
30
31 which confirmed hyperglycemia and glucosuria. Treatment with 40,80, and 160mg/kgb.w. AB
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33 and Metformin standard (100mg/kg b.w.) substantially prevented decrease in body weight,
34
35
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when compared with that o b s e r v e d in disease and normal controls. While the body weight
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37
38 of all the hyperglycemic rats was similar at the start of the experiment, AB treatment
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40 significantly increased the body weight, when compared with that observed in the untreated
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42 group.
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44
45 3.2.8 Effect of AB on relative weight of vital organs of STZ-induced hyperglycemic rats
46
47 The relative weights of vital organs, including liver and kidneys(right and left),of STZ-induced
48
49
50
diabetic rats after 28 days of AB treatment (40, 80, and 160mg/kg b.w. AB) were determined.
51
52 Following 28 days of AB treatment, the kidney weight of diabetic rats was found to be slightly
53
54 higher(Figure10b), when compared with that of normo glycemic rats, similar to that reported
55
56
in previous studies 36. In contrast, as light decrease in the liver weight of treated rats was noted,
57
58
59 when compared with that of control rats (Figure10a). Nevertheless, no significant changes in
60
the weight of vital organs were observed in the AB-treated groups, when compared with those
10
1
2
3 in the control (Figure 10).
4
5
6 3.2.9 Effect of AB on serum lipid profile of STZ-induced hyperglycemic rats
7
8 The effect of AB on the serum lipid profile, including cholesterol and TGL levels, was
9
10 assayed(Figure 11). The serum cholesterol and TG levels were found to be elevated in the
11
12
13
disease group, when compared with those in the control. In the AB-treated group, considerable
14
15 reduction in the serum cholesterol and TG levels was noted after 28 days of treatment. Lipid
16
17 dysfunction is an associated complication of diabetes in rats, and the findings of this study
18
19
revealed that AB could reduce the total cholesterol and TGL levels and was effective in treating
20
21
Fo
22 hyperglycemia.
23
24 3.2.10 Histopathological effects of AB on STZ-induced hyperglycemic rats
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26 Histomorphological observation of endocrine pancreas, liver and kidneys of AB-treated rats
27
28
ee
29 showed no significant morphological changes, when compared with those observed in the
30
31 normal control and disease control. The tissue morphology of endocrine pancreas of b o t h
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32
33 disease control and A B - treatedgroupswassimilar, presenting diffused and moderate islet cell
34
35
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36
atrophy/degeneration, whereas that of liver of control and AB-treated groups exhibited
37
38 granuloma; multifocal, random, minimal necrosis; hepatocellular, multifocal, random, minimal
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40 infiltrates; and mononuclear, periportal, diffuse, minimal cells. The kidneys of both the control
41
42
and AB-treated groups showed vacuolar degeneration; tubular, cortical, bilateral, multifocal,
43
44
45 moderate pigment accumulation; and interstitial, multifocal, minimal cells. These results
46
47 clearly demonstrated that AB had no significant toxicity on STZ-induced hyperglycemic rats
48
49 (Tables 1–3).
50
51
52 Subsequently, histopathological analysis of endocrine pancreas of the experimental rats was
53
54 performed. In the control group, the islets showed no significant pathological changes, and
55
56 were of normal size and evenly distributed throughout the pancreas. In the STZ-induced
57
58
59
hyperglycemic groups, marked atrophy of islets was noted with respect to both size as well as
60
number of islets. In the case of Metformin standard group, the islets presented moderate
11
1
2
3 atrophy, whereas in the AB-treated groups, the islets showed only minimal to moderate atrophy
4
5
6 and exhibited improvement in the number and size of islets (Figure 12).
7
8 The acute oral toxicity of AB was investigated according to OECD-245 guidelines.
9
10 Female rats aged 8–12 weeks were orally administered with a single dose of AB (maximum
11
12
13
dose of 2000mg/kg b.w.) dispersed in honey water (at a ratio of 2:3), and the mortality, weekly
14
15 body weight, daily feed intake, clinical parameters, and gross pathology were observed during
16
17 the study period of 14 days. The results revealed no significant toxic pathological effects
18
19
following AB treatment. The maximal tolerated dose of AB was >2000mg/kg b.w., and the
20
21
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22 mortality rate was 0%.Besides, no visible signs of toxicity were observed, the gross pathology
23
24 was agonal in nature, and all the animals survived at the end of the acute toxicity study.
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26 Sub-acute toxicity investigation was performed as per the OECD-407 guidelines by
27
28
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29 orally administering 80, 320, and 1280 mg/kg b.w. AB dispersed in honey water (at a ratio of
30
31 2:3) to Wistar rats for 28days. The results presented no clinical difference in weekly body
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32
33 weight and organ weight changes during the study period. It must be noted that hematological
34
35
ev
36
system is highly sensitive to toxic materials and could reveal the physiological changes in
37
38 animals or human beings.36In the present study, the hematological parameters, including RBC
iew
39
40 and WBC, of AB-treated rats were not affected even at high dose of AB (1280mg/kg b.w. AB),
41
42
indicating that AB is non-toxic and safe while in circulation. Furthermore, biochemical studies
43
44
45 revealed that the levels of total bilirubin, direct bilirubin, and creatinine were not significantly
46
47 altered (P>0.05) among AB-treated groups and control. However, a considerable increase in
48
49 serum liver enzymes (AST, ALT, and ALP), albumin, total cholesterol, glucose, total protein,
50
51
52 TGL, urea, and uric acid (P<0.05) in rats administered with 80, 320, and 1280 mg/kg b.w. AB
53
54 was observed. Moreover, histopathological studies showed improved size and number of islets
55
56 in AB-treated rats, when compared with those in normal control and disease control, suggesting
57
58
59
minimal to moderate atrophy of islet cells. Besides, AB treatment produced no relative changes
60
in the levels of glucose, creatinine, AST, and ALP, implying that AB did not affect liver and
12
1
2
3 kidney tissues.
4
5
6 The anti-hyperglycemic potential of AB was studied by employing STZ-induced diabetic rat
7
8 model, and the results confirmed the dose-dependent effect of AB (40, 80, and 160mg/kg
9
10
11
b.w.).While obvious increase in the blood glucose level was noted in disease control and
12
13 normal control, rats treated with medium concentration of AB showed significantly lower
14
15 blood glucose level. The body weight of rats both in disease control and normal control was
16
17
considerably increased, whereas that of rats in AB-treated group was reduced, when compared
18
19
20 with the initial weight.
21
Fo
22 Furthermore, a significant decrease in the blood glucose, total cholesterol, and TG

23
24 levels was observed in AB-treated diabetic rats. The body weight of control rats presented an
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25
26
27 significant increase, whereas the body weight of AB-treated rats was lower than that of the
28
ee
29 disease control and normal control rats. These findings suggest that AB is safe and nontoxic to
30
31 rats and do not produce any significant toxic effects. Besides, AB exhibited beneficial effects
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32
33
34
as an anti-hyperglycemic agent in diabetic rats by improving various metabolic processes, such
35
ev
36 as lipid metabolism, with no significant toxicity.

37
38 4. Conclusion
iew
39
40
The nano-ayurvedic medicine AB showed improved cell penetration and was effective
41
42
43 in the treatment of hyperglycemia, with no significant invivo acute and sub-acute toxicity, even
44
45 at high dosage(2000 mg/kg b.w.). The acute oral LD50of AB in Wistar rats
46
47 was>2000mg/kgb.w, and sub-acute studies showed no toxic effects at >1280mg/kg b.w., along
48
49
50 with normal histopathological, biochemical, and hematological findings. Besides, AB
51
52 exhibited significant anti-hyperglycemic activity and high cell penetration efficacy in STZ-
53
54 induced diabetic rats. These results confirm the tendency of AB to induce insulin secretion
55
56
57 from pancreatic β-cells, suggesting the potential use of herbo-mineral nano medicine for the
58
59 treatment of diabetes.
60
13
1
2
3 Conflict of interest
4
5
6 The authors declare no conflict of interest.
7
8 Acknowledgments
9
10
11
The authors are thankful to VIT University, Vellore, India, for providing all facilities to
12
13 conduct this research, and to Dr. S. Panchapakesan and C. David Raj, SASTRA University,
14
15 India, for their assistance and support in performing invivo studies.
16
17
18
19
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21
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22
23
24 References
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15 germinal epithelium after heat exposure in rats. Ancient Science of Life 2012; 31(4): 171-180.
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17 29. S.B.Babita, GK.Purushottam, V.D.Jayashree et al. Abhraka Bhasma treatment ameliorates proliferation of
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germinal epithelium after heat exposure in rats. Ancient Science of Life 2012; 31(4): 171-180.
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33 32. M.A.Valentovic, A.D. Napoleon, A.C. Betts Carpenter. Streptozotocin (STZ) diabetes enhances
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37 33. Manjeshwar Shrinath.B, Ganesh Chandra.J, Ulloor.J.N. The evaluation of the acute toxicity and long term
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43 34. Aloı́sio Luiz Benedetti, Cidôni`a de LourdesVituri, Andréa Gonçalves Trentin. The effects of sub-chronic exposure
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46 35. Graphpad prism software https://www.graphpad.com/scientific-software/prism/ ( accessed 23 November 2019)
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36. Wilbur S, Abadin H, Fay M. Toxicological Profile for Chromium. Agency for Toxic Substances and Disease Registry (US),
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50 2012 Sep.
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52
53
54 Figure legends
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56
57
Figure 1 Body weight (g) of rats in Acute Toxicity Studies.
58
59 Figure 2 Daily Feed intake (g) during Acute Toxicity Studies
60 Figure 3 Body weight changes in male rats during Sub-acute toxicity studies.
16
1
2
3 Figure 4 Body weight changes in Female rats of Sub-acute toxicity studies.
4
5 Figure 5 Organ weight of treated male rats in sub-acute toxicity study
6
7 Figure 6 Organ weight of treated female rats in sub-acute toxicity study
8
9 Figure 7 Represent Liver section (Hematoxylin and eosin staining) 4X & 40X of Control and 1280mg/kg b.w
10
treated Abhrak Bhasma in Rats
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12
13
Figure 8 Effect of Blood glucose level of treated rats of hyperglycemic studies
14
15 Figure 9 Weekly body weight of rats in hyperglycaemic studies
16
Figure 10(a& b). Liver Organ weight studies of Rats and kidney Organ weight studies of Rats
17
18 Figure 11 Lipid Profile studies of rats in hyperglycemic studies
19
20 Figure 12 Represent pancreases section (Hematoxylin and eosin staining) 4X & 40X of Control, STZ induced,
21
STZ + STD adn STZ induced 160mg/kg b.w of Abhrak Bhasma
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17 Male�Control�(Honey),�4X Male�Control�(Honey),�40X
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34 Male,�Abhrak�Bhasma(1280�mg/Kg),�
Male,�Abhrak�Bhasma(1280mg/Kg),�
35 4X 40X
36
37
38 Represent�Liver�section�(Hematoxylin�and�eosin�staining)�4X�&�40X�of�
39
40
Control�and�1280mg/kg�b.w�treated�Abhrak�Bhasma�in�Rats
41
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Figure�9
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Figure�9�
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Figure�1�1
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4 Control,�4X Control,�40X
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25 STZ�induced� STZ�induced�
26 Diabetic�Rat,�4X
27
Diabetic�Rat,�40X
28
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31
Represent�pancrease�section�(Hematoxylin�and�eosin�staining)�
32 4X�&�40X�of�Control,�
33 STZ�indused,�STZ�+�STD�
and�
34 STZ�induced�160mg/kg�b.w�of�Abhrak�Bhasma
35
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Page 47 of 48 Figure�12
Human & Experimental Toxicology
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STZ�+�Control�
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16 STZ�+�Control�(Metformine�
(Metformine�100mg�mg/kg),�
17 100mg/kg),�40X
18 4X
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STZ�+Abhrak�Bhasma�High�Dose�STZ�+Abhrak�Bhasma�High�Dose�
32
(160mg/kg�b.w),�4X (160mg/kg�b.w),�40X
33
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1
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5
Table 1.Histo-Morphological observation of Endocrine Pancreas in
6 STZ induced hyperglycaemia
7 Group Morphological observation
8 Control Within normal limits
9
10
STZ induced * Islet cell atrophy / degeneration, diffuse, marked
11 STZ + STD** Islet cell atrophy / degeneration, diffuse, moderate
12 STZ + Low Dose Islet cell atrophy / degeneration, diffuse, mild
13
14
40mg/kg.B.w
15 STZ + Medium Islet cell atrophy / degeneration, diffuse, moderate
16 Dose 80mg/kg.B.w
17 STZ + High Dose Islet cell atrophy / degeneration, diffuse, moderate
18 160mg/kg.B.w
19 * 65 mg/kg 35 +30 48 hrs gap ip hyperglycemia checked by glucose, ** Metformin 100 mg/kg
20
21
28 days oral, Histo- morphological observation of Rats pancreas exposed to STZ, compared
Fo
22 with that of the standard control and bhasma treated.

23
24
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29
Table 2.Histo-morphological observation of Liver in STZ induced hyperglycaemia
30 Group Observation
31 Control Granuloma, multifocal, random, moderate, Necrosis, hepatocellular,
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32 multifocal, random, minimal, Infiltrates, mononuclear, periportal, diffuse,

33 minimal
34
STZ induced Granuloma, multifocal, random, moderate, Necrosis, hepatocellular,
35
ev
36 multifocal, random, minimal, Infiltrates, mononuclear, periportal, diffuse,

37 minimal Necrosis, hepatocellular, focal,marked
38 STZ induced + Granuloma, multifocal, random, minimal, Necrosis, hepatocellular, multifocal,
iew
39 STD random, minimal, Infiltrates, mononuclear, periportal, diffuse, minimal

40 STZ + Low Dose Granuloma, multifocal, random, minimal, Necrosis, hepatocellular, multifocal,
41
40mg/kg.B.w random, mild Infiltrates, mononuclear, periportal, diffuse, mild
42
43 STZ + Medium Granuloma, multifocal, random, minimal, Necrosis, hepatocellular, multifocal,
44 Dose 80mg/kg.B.w random, minimal Infiltrates, mononuclear, periportal, diffuse, minimal
45 STZ + High Dose Granuloma, multifocal, random, minimal, Necrosis, hepatocellular, multifocal,
46 160mg/kg.B.w random, minimal Infiltrates, mononuclear, periportal, diffuse, minimal
47 Histo-morphological observation Rats liver exposed to STZ, compared with that of the Standard
48
control and Bhasma treated.
49
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1
2
3
4
5
6
7 Table 3. Histo-morphological observation of rat Kidney in STZ induced hyperglycaemia
8 Group Observation
9 Control Infiltrates, interstitial, mononuclear, multifocal, minimal
10 STZ induced Infiltrates, interstitial / perivascular, mononuclear, multifocal, mild
11
12 STZ + STD Vacuolar degeneration, tubular, cortical, bilateral, multifocal, mild
13
14 STZ + Low Dose Vacuolar degeneration, tubular, cortical, bilateral, multifocal, minimal
15 40mg/kg.B.w
16
17
STZ + Medium Dose Vacuolar degeneration, tubular, cortical, bilateral, multifocal, mild
18 80mg/kg.B.w
19 STZ + High Dose Vacuolar degeneration, tubular, cortical, bilateral, multifocal, moderate
20 160mg/kg.B.w Pigment accumulation, interstitial, multifocal, minimal
21
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22 Histo-morphological observation of Rats kidney exposed to STZ, compared with that of the
23 standard control and bhasma treated.
24
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Human & Experimental Toxicology

A study on toxicity and anti-hyperglycemic effects of Abhrak

Journal: Human and Experimental Toxicology

Manuscript Type: Original Article

Streptozotocin,, Abhrak bhasma, invivo, hyperglycemic activity, blood

Streptozotocin(STZ) is a gulcosamine-nitrosourea complex produced by

Streptomyces achromogenes, which particularly induces DNA strand

to insulin deficiency. Abhrak bhasma(AB) is used for DM treatment;

effectiveness of AB with respect to invivo toxicity and STZ-induced

Streptozotocin(STZ) is a gulcosamine-nitrosourea complex produced by Streptomyces

32 Rats body weight on the day of sacrifice (g)

29 3.1 Investigation of acute toxicity of AB

32 (F), respectively. The body weight of female rats in all AB

36 control, but did not present any significant differences. Overall,

39 AB did not produce any effective changes in the body weight of

22 without any abnormalities following AB treatment.

29 bilirubin, direct bilirubin, total cholesterol, creatinine, glucose,

29 number of Kupffer cells was observed (Figure 7) owing to local

36 was maintained without any obvious pathological changes

following AB treatment (Figure 7).

standard (100mg/kg b.w.) substantially prevented decrease in body

22 3.2.9 Effect of AB on serum lipid profile of STZ-induced

29 and TGL levels, was assayed(Figure 11). The serum cholesterol

22 interstitial, multifocal, minimal cells. These results clearly

29 Subsequently, histopathological analysis of endocrine pancreas of

22 Sub-acute toxicity investigation was performed as per the

29 Wistar rats for 28days. The results presented no clinical difference

22 STZ-induced diabetic rat model, and the results confirmed the

29 control and normal control, rats treated with medium concentration

36 considerably increased, whereas that of rats in AB-treated group

22 biochemical, and hematological findings. Besides, AB exhibited

29 tendency of AB to induce insulin secretion from pancreatic β-cells,

A study on toxicity and anti-hyperglycemic effects of Abhrak Bhasma in rats

32 Rats body weight on the day of sacrifice (g)

29 3.1 Investigation of acute toxicity of AB

32 3.2.2 Effect of AB on relative weight of vital organs

22 3.2.7 Effect of AB on weekly body weight of STZ-induced hyperglycemic rats

22 Furthermore, a significant decrease in the blood glucose, total cholesterol, and TG

36 as lipid metabolism, with no significant toxicity.

Biomed. Nanotechnol 2011;7: 68-69.

36 Pharm Health Res 2014; 2:5 186-196.

of Biotechnology 2005; 4 (1): 72-78.

32 Interdiscip Toxicol 2014; 7(2): 60-72.

22 vulgaris: a double-blind randomized placebo controlled clinical evaluation. Journal of Ethnopharrnacology

22 with that of the standard control and bhasma treated.

32 multifocal, random, minimal, Infiltrates, mononuclear, periportal, diffuse,

36 multifocal, random, minimal, Infiltrates, mononuclear, periportal, diffuse,

39 STD random, minimal, Infiltrates, mononuclear, periportal, diffuse, minimal

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