Exploring Therapeutic Efficacy of Coriandrum sativum and Allium 1 sativum Aggregate in Alloxan-Induced Diabetic mice

13 Diabetes mellitus (DM) is a global health problem with 422 million individuals around the 14 world suffering from diabetes. Coriandrum sativum and Allium sativum possess numerous bioactive 15 compounds which are of therapeutical significance. This study was undertaken to explore the 16 combined potential of the two herbs in managing diabetes using diabetic albino mice as model. 17 Extracts of both herbs were prepared and were orally administered in 10% Alloxan monohydrate 18 (alloxan) induced diabetic albino mice over 28 days (in four doses with 7 day interval) to determine 19 the optimal therapeutic and lethal doses. Safe dose limit of both extracts was deduced to be below 20 600 mg/kg (<600 mg/kg). Diabetic mice were given extracts (200 mg/kg and 400 mg/kg) over 56 21 days at 7 day intervals, and biological parameters were evaluated, at each interval, including Glucose 22 level (mg/dl), HbA1C (%), Hepatic and Renal biomarkers, and Lipid profile. Garlic-coriander 23 combination (at 400 mg/kg) was able to reduce glucose level (i.e.194.5 mg/dl) (P<0.05). Also, it 24 showed a worthwhile effect on biological parameters as well as in Hepatic and renal tissues. 25 Histological examination of hepatic and renal tissues indicated a restoration of normal tissue 26 architecture.

level (mg/dl), HbA1C (%), Hepatic and Renal biomarkers, and Lipid profile. Garlic-coriander 23 combination (at 400 mg/kg) was able to reduce glucose level (i.e.194.5 mg/dl) (P<0.05). Also, it 24 showed a worthwhile effect on biological parameters as well as in Hepatic and renal tissues. 25 Histological examination of hepatic and renal tissues indicated a restoration of normal tissue 26 architecture. Diabetes mellitus is considered as one of the most common causes of death worldwide and is 38 existing as a global health problem 1,2 . As per World Health Organization (WHO), about 422 million 39 individuals around the world are suffering from diabetes and a significant fraction of nationals is 40 from low and middle income countries. About 1.6 million deaths occur due to diabetes each year and 41 a steady rise in the number of cases and prevalence has been observed. Worldwide, diabetes has 42 become the fourth leading cause of non-communicable diseases (NCDs) while the prevalence has 43 risen from 4.7% to 8.5% 3 . 44 Diabetes mellitus exists as type 1 diabetes, which is caused by failure of pancreas to produce 45 sufficient insulin for the body, and type II diabetes which results from the inability of the body to 46 efficiently utilize insulin leading to enhanced blood glucose concentration in body; complications can 47 result including hypertension, kidney disorder, eye damage, nerve damage, damage to blood vessels, 48 lower lib amputation, heart damage, stroke and blindness 3,4 . 49 At present, for controlling glucose level, the modern medicines recommended for diabetes are 50 classified into six categories (i.e. biguanides (metformin), alpha-glucosidase inhibitors, meglitinides 51 (glinides), sulfonylureas, thiazolidinediones (glitazones), and DPP-4 inhibitors) and two types of 52 injections 4 . However, several side and adverse effects of such medications are known including 53 gastrointestinal disorder, nausea, diarrhea, compromised renal function, de-compensated heart 54 failure, liver disease, weight gain, cardiovascular risks and increased risk of heart attack 5 . 55 People worldwide are more concerned and conscious about the side effect and the cost 56 effectiveness of drugs and, as a complementary or alternative approach, herbal therapies (being used 57 by about one-third of the patients for managing diabetes) are getting increasing attention, which are 58 comparatively less exploited for their medicinal qualities 6 . Herbal drugs having anti diabetic, anti-59 hyperglycemic as well as insulin sensitizing activities are increasingly sought after by diabetic 60 patients and healthcare professionals 7 . Hence, there is need to explore therapeutic effects of 61 medicinal plants against diabetes and associated conditions 8 . 62 Coriandrum sativum (coriander) possesses a variety of bioactive molecules which can have 63 anticancer, antioxidant, neuroprotective, analgesic, anxiolytic, anticonvulsant, hypolipidemic, 64 hypoglycemic, hypotensive, antimicrobial, and anti-inflammatory activities 9 . Allium sativum (garlic) 65 is also reported to have many therapeutic benefits due to presence of sulphur-containing compounds 66 (e.g. S-allylcysteine sulphoxide) and other effective molecules that can have anti-thrombotic, 67 antioxidant, antibiotic, hypocholesterolaemic, hypoglycaemic and hypotensive activities 10 68 In the present research, effect of garlic and coriander dose combinations were administered 69 in alloxan induced diabetic mice and estimation of positive effects on metabolism including blood 70 Glucose level, Cholesterol, HbA1C, Cholesterol, Hepatic and Renal biomarkers as well as histological 71 examination of Hepatic and Renal tissues. 72 116 After completing blood analysis, mice were dissected and small section of heart was cut from 167 all groups. Heart tissues were dried in filter paper and washed with normal saline to remove blood 168 and preserved in 4% formalin. By microtoming, slices of tissues were obtained and observed under 169 light microscope. The slices were fixed by using gelatin on slide and were placed in oven for 10 hours 170 at 58°C, followed by Hematoxylin and Eosin staining 22 .

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Statistical Analysis 173 Statistical analysis of data was done by performing two-way Analysis of Variance (ANOVA) with 174 Graph Pad Prism v. 6.0, (Graph Pad Software, San Diego, California, USA). A p-value of ≤ 0.05 was 175 interpreted as result being statistically significant. 176 177 178

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Percentage Yields of Herb Extracts 181 Highest percentage yield was produced with extracts prepared in Ethanol (21.5% for garlic and 182 23% for coriander) followed by methanol, ethyl acetate, petroleum ether, chloroform and finally 183 water (  Lethal doses of both herbs was evaluated by administering extract at doses 600 mg/kg, 800 201 mg/kg and 1000 mg/kg for 28 days, and track of mortality of mice was monitored along the time 202 period. From Table 3, it could be seen that the mortality of animals increased with increasing the 203 concentrations of each of the herb type. By 28 th day, only one out of six (1/6) animals was alive with 204 1000 mg/kg garlic extract; while with coriander extract of 800 mg/kg 1/6 animals were alive by Day 205 21 st indicating greater toxicity of coriander extract as compare to garlic extract. Safe dose limit of 206 both extracts was henceforth deduced to be below 600 mg/kg (<600 mg/kg).    Effects on HbA1C (%) 252 Effect of various doses of garlic and coriander extract on HbA1C (%) of alloxan (130mg/kg) 253 induced diabetic rats was done with garlic and coriander administration alone and in combination 254 (with eight dose replications). The values of HbA1C (%) for garlic, coriander and garlic-coriander 255 combination (400 mg) were quite similar and comparable with the standard control i.e. 8.75% 256 HbA1C, but non-significant as showed by the p-values for coriander (p=0.566) and garlic-coriander 257 combination (p=0.133), while for garlic extract was p=0.000 (Figure 3a-c).  Figure 1). 297 Effects on Tissue Hepatic Biomarkers 329

Figure 3 a) Effects of Medicinal herbs on HbA1c (%) in diabetic mice(s). b) Estimation of comparative effectiveness of extracts on HbA1c (%) over two dose levels. c) Estimation of comparative effectiveness of extracts on HbA1c (%) over eight equal time intervals.
Analysis of tissue Hepatic biomarkers of alloxan (130mg/kg) induced diabetic rats indicated 330 that garlic + coriander (400mg) (administered with dose replications) had mean SOD level 205.63 331 U/g tissue (p=0.000) while that for disease control group was 178 U/g tissue (i.e. higher values as 332 compared to the disease control group) (Figure 5a-c). Individual effects of herbs (at 200mg and 400 333

Figure 4a): Effects of Medicinal herbs on AST (1U/L) in diabetic mice(s). b) Estimation of comparative effectiveness of extracts on AST (1U/L) over two dose levels. c) Estimation of comparative effectiveness of extracts on AST (1U/L) over eight equal time intervals.
a b c mg) were almost similar, however slightly lesser values of mean SOD (U/g tissue) as compared with 334 the combined effect (at 400mg). 335 Mean CAT (U/g tissue) and GSH (mg/g tissue) values indicated a similar trend with the dose 336 types and levels of garlic and coriander as observed with mean SOD level (U/g tissue) 337 ( Supplementary Figures 5 and 8). Mean CAT (U/g tissue) of garlic-coriander (400mg) indicated mean 338 CAT level of 914.25 U/g tissue (p=0.000) while that for disease control group was 742 U/g tissue. 339 Mean GSH (mg/g tissue) of garlic-coriander (400mg) indicated level of 3.81 mg/g tissue 340 while that for disease control group was 1.8 mg/g tissue (p=0.000).

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The values for mean MDA (nmol/L P*g tissue) (p=0.312) and LPO (nmol of MDA formed/L P*g 342 tissue) (p=0.000) were reduced compared to disease control groups ( Supplementary Figures 6 and  343  7). Mean MDA (nmol/L P*g tissue) of garlic-coriander (400mg) showed value of 42.25 nmol/L P*g 344 tissue (P=0.312) and 52 nmol/L P*g tissue for disease control group; however difference was not 345 statistically significant as per the P value. 346 Mean LPO (nmol of MDA formed/L P*g tissue) of garlic-coriander (400mg) showed value of 347 135.75 nmol of MDA formed/L P*g tissue (P=0.000) and 162 nmol of MDA formed/L P*g tissue for 348 disease control group. Estimation of mean Urea level (mg/dl) of diabetic mice showed that garlic-coriander extract 403 (400mg) was able to reduce the level to 28.25 mg/dl (P=0.872); but due to statistical insignificant 404 result indicated by P-value, the evidence could not be convincing enough. However, garlic alone (at 405 both concentrations) indicated promising result with 28.38 mg/dl at 200mg (P=0.000) and 29.50 406 mg/dl at 400 mg (P=0.000), while disease control group had 35 mg/dl urea concentration (Figure 7  407 a-c). 408 Result of uric acid concentration (mg/dl) showed garlic-coriander (at 400mg) were able to Estimation of Renal tissue biomarkers showed that mean SOD level (U/g tissue) increased 436 with increasing garlic and coriander dose levels and using combination made a positive effect as 437 compared to the tissues from disease control mice. 438 Garlic-coriander (400 mg) combination showed a mean SOD level 265.13 U/g tissue 439 (P=0.000) while disease control group SOD level was 231 U/g tissue (Figure 8 a-c). 440 Similar effects were observed with mean CAT (537.13 U/g tissue for 400 mg combination, 441 P=0.000 and 417 U/g tissue for disease control) and GSH (5.11 mg/g tissue for 400 mg combination, 442 P=0.004, 2.88 mg/g tissue for disease control) ( Supplementary Figures 11 and 14).

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The values for mean MDA (nmol/L P*g tissue) and LPO (nmol of MDA formed/L P*g tissue) were 444 reduced compared to disease control group. Garlic-coriander extract (400 mg/kg) indicated mean 445 MDA (nmol/L P*g tissue) of 47.88 nmol/L P*g tissue (p=0.064) compared to disease control 52 446 nmol/L P*g tissue, while the mean LPO (nmol of MDA formed/L P*g tissue) 126.13 (p=0.035) 447 Effect of extract on Lipid Profile in alloxan (130mg/kg) induced diabetic rats after eight weeks 484 treatment was evaluated. Significant reduction in mean Cholesterol level (mg/dl) was observed with 485 Garlic and Garlic Coriander combination as compared to disease control mice. Administration of 486 Garlic Coriander extract at 400mg/kg concentration decreased the mean Cholesterol level (mg/dl) 487 to 137.50 mg/dl (P=0.242) in comparison with disease control which was at 256 mg/dl ( Figure 10); 488 however, a high P-value making it difficult to reach to a valid conclusion.

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The results of Triglycerides (mg/dl) values indicated that Garlic Coriander extract at 400mg/kg 490 concentration decreased the level to 147.50 (P=0.000) as compared to disease control which was at 491 235 mg/dl ( Figure 11) and LDL-cholesterol values (mg/dl) level were reduced to 51.38 (P=0.000) 492 as compared to disease control which was at 93.5 mg/dl (Supplementary Figure 15) while the values 493 for HDL-cholesterol ("good" Cholesterol) were raised to 31mg/dl with Garlic Coriander extract 494 (400mg/kg) as compared to disease control i.e. 15.5 mg/dl (P= 0.001) (Supplementary Figure 16). 495 496 Pakistan has 27.4 million cases of diabetes (≥20 years). Data reveals that 415.03% increase in cases 517 of diabetes in Pakistan from 5.32 to 27.4 million 23 . Pakistan, being a third world country, under strong 518 economic burden, and is already allocating a considerable share in dealing with communicable 519 diseases (e.g. (e.g. HIV, Hepatitis B and C), practices of managing a healthy lifestyle by adopting 520 natural remedies for non-communicable disease e.g. diabetes can help to circumscribe the economic 521 burden in health sector. 522 Use of modern medicines have seeming benefits in diabetic patients, the usefulness is 523 accompanied by various side effects 25 , and while some studies have shown anti-diabetic and 524 involving the combined effect of both herbs are scarce 34 . 526 This study was conducted to show the aggregate effect of garlic-coriander 527 combination in adjusting levels of key biochemical indicators in diabetic mice to manageable levels. 528 While the results for certain indicators (i.e. HbA1C, Tissue Hepatic Mean MDA, Serum Renal Urea 529 level and total cholesterol), could not be within limit of statistical significance (i.e. p≤ 0.05), other 530 biochemical parameters were well managed by combined dose (i.e. 400 mg/kg) of garlic-coriander 531 and within the limit of statistical significance.