INTRODUCTION:

Plant polyphenolic secondary metabolites known for their antioxidant properties are called flavonoids, and they all have a common three-ring structure¹. Extensive research has demonstrated that gamma (γ)-benzopyran scaffolds derived from natural sources, particularly those abundant in flavonoids, exhibit strong antidiabetic properties in addition to their antioxidant properties^2,3. Research on the development of alternative synthetic molecules for antidiabetic treatment is focused⁴ on the solubility, potency, and unstable nature of natural flavonoids, as well as laborious and costly isolation and purification techniques.

Considering that benzopyrone derivatives are known to display a broad range of pharmacological actions⁵, a substantial amount of research has prompted us to alter the benzopyrone ring in order to investigate novel activities linked to this nucleus⁶. Benzopyrones with appropriate molecular modification compounds have been found to have the potential to be used as anti-diabetic agents^7,8. Since benzopyran (or benzopyran-4-one) nucleus with different substitutions on the phenyl group at position 2 are more likely to offer better lead molecules⁴, benzopyran with thiophen analogue substitutions have been chosen in this study for hypoglycemic screening. The synthetic novel flavonoid (NF) of thiophen congener has demonstrated promising free radical scavenging activity in our preliminary in vitro experiments^9,10. It has also exhibited in vitro α-glucosidase inhibitory and glucose uptake activity in the C2C12 cell line study (Data on file). Furthermore, as there are no more published data on this test compound (NF), it is necessary to screen it for hypoglycemia in experimental rats.

MATERIALS AND METHODS:

Materials:

Unless otherwise noted, the necessary reagents and chemicals were acquired from well-known commercial suppliers. Products such as 2-hydroxyacetophenone, 2-thiophen benzaldehyde were acquired from Sigma Aldrich (USA). Analytical-grade solvents, such as n-hexane, acetone, dimethylsulfoxide (DMSO), ethyl acetate, and 100% ethyl alcohol, were utilized just as is without additional purification. Metformin was the gift from Micro Labs Ltd, Bangalore, India. Vanaspati ghee, coconut oil and cholesterol powder for High fat diet preparation, were obtained from local market. Analytical graded chemicals/ reagents were used in the study.

Preparation of test compounds:

Using the methods explained by Chavan SV.,(2011)¹¹ and Algar-F-Oyamada method^12,13, the test novel flavonoid (NF) was synthesized by condensation and oxidative cyclization reactions of 2- hydroxy acetophenone with 2- thiophen carbaxaldehyde. Prior to the animals being fed, the standard metformin 100 mg/kg b.wt. and the determined amount of test NF compound were suspended in 0.5% Tween 20 with purified water solution.

Animal ethics committee approval for study protocol:

All of the study's experimental protocols have the Institutional Animal Ethics Committee (IAEC) approval (SNMC/IAEC2018-2019) in place. Wistar albino rats of both genders, weighing 150–200g body weight, were obtained from the S. N. Medical College central animal house of BVVS (Reg no 829/AC/04/CPCSEA). They were kept in polypropylene cages with standard environmental conditions (temperature of 24–26°C; relative humidity of 45–55% under a 12-hour light/dark cycle), and they were given unlimited access to water and standard rodent chow diet for a week to help them get used to the conditions of the lab.

Oral acute toxicity study:

According to OECD guidelines 425¹⁴, an oral toxicity investigation of the NF test medication was carried out on Swiss albino mice. The initial oral dose of 175mg/kg body weight was chosen because the NF compound is a novel chemical entity and its safety for animals is unknown. Dose progression factor 3.2 was then utilized to determine the subsequent dosage until the maximum dose of 2000mg/kg was reached.

This investigation was conducted using the techniques of Baghel S.S., (2011)¹⁵ and Algariri K et al., (2014)¹⁶. Oral administration of the test NF compound suspended in 0.5% Tween 20 with purified water suspension was carried out. Following oral test drug dosage, each animal was observed for four hours at first, and then once a day for the next few days, to look for any noticeable behavioral changes, such as hyperactivity, ataxia, convulsions, salivation, tremors, and sleep patterns. After the medicine was given orally, the animals were monitored for 14 days to check for any mortality. It was found to be both well-tolerated and safe up to dosages of 2000mg/kg, indicating that the LD 50 of the NF test medication seems to be safe up to the body weight limit of 2000mg.

Hence the NF compound’s 1/10th, 1/20th and 1/40th of safe limit dose that is 200mg,100mg and 50mg/kg body weight have been considered to use for glucose lowering efficacy studies.

High fat diet (HFD) preparation:

This diet was produced using a slightly modified version of the approach utilized by Asanaliyar M et al., (2021)¹⁷ and Srinivasan K et al., (2005)¹⁸. Vanaspati ghee, coconut oil, and 2% pure powdered cholesterol formed the HFD. The first two were combined in a 3:1 ratio, and then 2% pure cholesterol powder was thoroughly blended to dissolve it completely to produce a high-fat liquid diet. For eight weeks, a dose of 3ml/kg, p.o., of HFD was administered in order to produce metabolic syndrome

Administration of drugs:

The 50, 100, 200mg/kg doses of NF and 100mg/kg of standard drug (metformin) were prepared as a suspension using 5%Tween 20gel in purified water as formulation vehicle.

HFD: After 4 weeks, overnight fasted rats on HFD group were made and the above-mentioned test drugs administered daily p.o. through oral gavage.

The rats were fed either the high-fat diet (HFD) or the normal pallet diet (NPD) according to two different dietary regimens. The others were placed on a 56-day high-fat diet (HFD), while the standard control group was given a regular pallet diet (NPD).

Blood glucose estimation:

Using the glucose oxidase method, a one-touch sugar scan glucometer (Thyrocare Pvt) was used to measure blood glucose^19,20. Under light surface anesthesia using lignocaine gel, blood samples were taken from the experimental rat’ s tails at regular intervals through sterile needle pricks.

Effect of NF on Oral Glucose Tolerance Test (OGTT) in normoglycemic rats:

All animals were starved for 16 hours but allowed to free access with water before and throughout the experiment. The conventional procedures of Jarald EE,. (2008)²¹ and Acharyya S.,(2011)²²were followed when doing OGTT on non-diabetic rats. Zero minutes (0 min) was the time immediately following the fasting period. Next, five groups of six normal rats each were assigned (n=6). The OGTT²³ study design was depicted as follows.

Group I (NC) represents normal control, and it was given 5% Tween 20 vehicles (5ml/kg/o).

Group II (Met+Gl) was treated with metformin in vehicle (100mg/kg b. wt)+ oral glucose (2gm/kg) solution.

Group III (NF50+Gl), IV (NF100+Gl), and V(NF200+Gl), received oral test NF drug in vehicle at 50,100 and 200mg/kg b. wt respectively, 30 min prior to the oral glucose(2gm/kg)

After 30 minutes of single oral treatment with varying doses of the aforementioned test medications, all of the rats in the various groups received single oral glucose at a dose of 2 grams per kilogram body weight. Following oral glucose delivery, blood samples were taken from the experimental rats' tail veins at 0, 30, 60, 90, and 120 minutes in order to estimate the blood sugar level using blood glucose test strips and a glucometer.

Effect of NF on Oral Glucose Tolerance Test (OGTT) in HFD fed (before STZ induced diabetes) rats:

Based on their dietary regimen, the experimental rats were divided into two primary groups. The high fat diet (HF) groups (Groups II to VI) received oral HFD prepared locally at a dose of 3ml/kg b.wt. per day, whereas the normal control group received a vehicle (Tween 20 suspension). Rats that had been deprived for sixteen hours were split up into six groups, each with six members.

Group I animals (the normal control group) received an oral dose of 5ml/kg of a 5% Tween 20 vehicle.

Animals in Group II were solely given HFD in the vehicle.

In addition to a high-fat diet, Group III animals received the standard medication metformin (100 mg/kg), while Groups IV to VI animals received an NF test drug at doses of 50, 100, and 200mg/kg body weight, respectively. After being pretreated with the test medicines for 30 minutes, all of the experimental animals in the various groups were given an oral glucose solution at a dose of 2 grams per kilogram body weight. After the oral glucose load, blood glucose levels were measured 30, 60, 90, and 120 minutes later.

Effect of NF on blood glucose in low dose Streptozotocin-induced hyperglycemic rats fed on High fat diet:

A slightly modified version of the procedure employed by Asanaliyar M et al.¹⁷, Srinivasan K, et al.,¹⁸ and Munshi R et al.,(2014)²⁴ was utilized to develop diabetes mellitus in experimental rats. Following the ingestion of a high-fat diet for 28 days, experimental rats were fasted overnight and then intraperitoneally injected with a single dose of freshly manufactured streptozotocin (STZ 35mg/kg from Sigma-Aldrich) dissolved in ice cold citrate (pH 4.5). To avoid STZ-induced hypoglycaemic shock, the rats treated with STZ were given water containing 5% glucose and 1% saline for the next two days²⁵. FBG was measured using the glucometer strip method after these rats had a seven-day injection of STZ. The diabetic, non diabetic and normal control rats were again sub categorized in different groups (n=6) and were further treated for 21 days according to the treatment protocol besides free excess with standard rodent diet and water ad libitum as illustrated below.

Group 1 (NC), the normal control group, was given oral 5% Tween 20 vehicle once a day (5ml/kg).

Group 2 (HFC) received only oral HFD daily once at a dose of 3ml/kg/day body weight.

Group 3 (HF+D)-High fat + Diabetic group received HFD once daily for 8 weeks+STZ once on 28th day.

Group 4 (HF+D+Met)-diabetic standard group- received metformin at a dose of 100mg/kg/oral/day, after diabetes mellitus induction with STZ, along with HFD for 21days.

Group 5 (HF+D+NF50), Group 6 (HF+D+NF100) and Group 7 (HF+D+NF200) –diabetic test groups which received test flavonoid (NF) compound(after STZ induced diabetes) at low (50mg/kg), intermediate (100mg/kg) and high dose (200mg/kg) respectively once daily along with HFD for 21days.

The Glucometer device was used to measure blood sugar levels using glucose strips at several research intervals, including before, immediately after STZ, and after the diabetes induction with STZ. Table 3 displays the results of the calculation of the percentage of glucose reduction from the baseline.

Statistical analysis:

All of the data in this study were provided as Mean± SEM, and GraphPad Prism 5.0 was utilized for the statistical analysis. One way analysis of variance (ANOVA), supported by Dunnett's multiple comparison test, was employed. A p-value of less than 0.05 was deemed statistically significant.

RESULTS AND DISCUSSION:

Oral Glucose Tolerance Test in normal rats:

Table 1 displays the findings of the OGTT investigation in normoglycemic rats. After oral glucose was administered to the experimental animals for 60 minutes, the blood glucose levels quickly peaked at 30 minutes and then began to progressively decline. It was shown in rats treated with flavonoids that the oral glucose load considerably (p<0.05) inhibited the peak rise in glucose in a dose-dependent manner that was similar to that of rats given with metformin. It was found that two hours after oral therapy with metformin (100 mg/kg) and NF (200mg/kg), there was comparable significant decrease in blood glucose of 26.27% and 27.64%, respectively.

HFD-fed rats' oral glucose tolerance test (OGTT) results:

Table 2 displays the findings of the OGTT study conducted on rats fed a high-fat diet. In the control groups, the blood glucose peak rise was quickly increased from the basal blood glucose level (at 0 min) and then declined after 60 min of oral glucose loading. However, the blood glucose spike that occurs between 0 and 60 minutes following oral glucose delivery was not observed in the rats treated with metformin and test NF compounds. When compared to normal control rats, rats fed a high-fat diet together with oral glucose demonstrated a substantial rise in blood glucose from baseline levels. Because chronically fed HFD rats develop a physiological tolerance for insulin release following an oral glucose challenge, the abrupt spike in blood glucose can be explained²⁶. After a 60-minute oral glucose load, the blood glucose levels of the high-fat fed control rats decreased at a slower and less significant rate (9.35%) compared to the normal control groups (37.20%). Following an oral glucose challenge, test drug(NF) treated HFD-fed animals demonstrated an statistically significant resistance to the peak rise in blood glucose and showed a significant peak reduction of 14.73%, which is similar to groups treated with metformin (15.93%).

Blood glucose levels in the test flavonoid treated group decreased in a manner that was dosage dependent and comparable to that of the standard metformin treated groups. The highest glucose tolerance was demonstrated by NF at doses of 100 to 200mg/kg, which may be attributed to NF-mediated improved peripheral glucose consumption and insulin production in response to hyperglycemia (glucose load).

Effect of synthetic Novel flavonoid (NF) on the blood glucose in STZ induced diabetic rats fed on HFD.

Following a 3-week oral therapy, the NF compound demonstrated a dose-dependent, statistically significant (p<0.05) reduction in glucose levels in comparison to STZ diabetic (HF+D) and HF fed Control (HFC) rats. Comparing the test NF compound to the conventional metformin treated group (172%), the test NF compound demonstrated a significant (p<0.05) reduction of glucose (125%) at 100 to 200mg/kg dose. The possible lowering of blood glucose in hyperglycemic rats by NF may be attributed to its antioxidant potential, which facilitates the remodeling of body tissues for insulinomimetic activity.

CONCLUSION:

It is concluded that the synthetic flavonoid NF exhibited a potential hypoglycaemic effect in experimentally induced hyperglycemias in the study. It may be due to the presence of hydroxyl group and thiophen analogue in its chemical structure which intern provides the antioxidant activity of the test compound ^{5, 6}. Therefore, both in-vivo and in-vitro experimental models may benefit from the use of this synthetic flavonoid (NF) for antidiabetic screening.

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of BVVS’s S.N. Medical College and HSK Hospital Bagalkot for the facilities.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. De la Rosa LA, Alvarez-Parrilla E, González-Aguilar GA, editors. Fruit and vegetable phytochemicals: chemistry, nutritional value and stability. John Wiley and Sons; 2009 Oct 13.

2. Cazarolli LH, Zanatta L, Alberton EH, Reis Bonorino Figueiredo MS, Folador P, Damazio RG, Pizzolatti MG, Mena Barreto Silva FR. Flavonoids: cellular and molecular mechanism of action in glucose homeostasis. Mini Reviews in Medicinal Chemistry. 2008; Sep. 1; 8(10): 1032-8. https://doi.org/10.2174/138955708785740580

3. Havsteen BH. The biochemistry and medical significance of the flavonoids. Pharmacology and Therapeutics. 2002; Nov 1; 96 (2-3): 67-202. https://doi.org/10.1016/S0163-7258(02)00298-X

4. Nayak Y, Hillemane V, Daroji VK, Jayashree BS, Unnikrishnan MK. Antidiabetic activity of benzopyrone analogues in nicotinamide‐streptozotocin induced type 2 diabetes in rats. The Scientific World Journal. 2014; 2014(1): 854267. https://doi.org/10.1155/2014/854267

5. Khare E, Sharma A, Chowdhury A, Narwariya SS. Potential of natural products for chemoprevention of breast cancer. Research Journal of Pharmacognosy and Phytochemistry. 2023; 15(4): 305-10. doi: 10.52711/0975-4385.2023.00048

6. Som S, Jayaveera KN. Study of Synthesis and Analgesic Activity of some Benzopyrone Derivatives. Asian Journal of Research in Chemistry. 2015; 8(3): 195-200. doi: 10.5958/0974-4150.2015.00035.8

7. Pattan SR, Dighe NS, Musmade DS, Gaware VM, Jadhav SG, Khade MA, Hiremath SN. Synthesis and antidiabetic activity of some new Benzopyrone derivatives. Asian Journal of Research in Chemistry. 2010; 3(1): 126-128. Available on: https://ajrconline.org/AbstractView.aspx?PID=2010-3-1-32

8. Patel KD, Patel CN, Patel GM. Synthesis and Antidiabetic Activity of Novel 4-((2, 4-Dioxothiazolidin-5-ylidene) methyl) Substituted Benzene Sulphonamide. Asian Journal of Research in Pharmaceutical Science. 2015; 5(1): 1-7. doi: 10.5958/2231-5659.2015.00001.6

9. Bhixavatimath P, Naikawadi A, Maniyar Y, Moharir G, Daroj V. Synthesis and characterization of 2-thiophen flavonoid analogue for free radical scavenging antioxidant analysis.. Biomedicine: July. 2022; 42(3): 543-549. doi:10.51248/.v42i3.1485.

10. Bhixavatimath PS, Naikawadi A, Maniyar YA, Shalavadi M, Maher G, Daroj V. An In vitro evaluation of potential free radical scavenging antioxidant activity of selected novel synthetic flavones. Research Journal of Pharmacy and Technology. 2022; 15(9): 3947-51. doi: 10.52711/0974-360X.2022.00661

11. Chavan SV, Sawant SS, Yamgar RS. Synthesis and characterization of novel transition metal complexes of benzo-α-pyranone derivatives and their biological activities. Asian Journal of Research in Chemistry. 2011; 4(5): 834-7.

12. Algar J, Flynn JP. A new method for the synthesis of flavonols. Proceedings of the Royal Irish Academy. Section B: Biological, Geological, and Chemical Science. JSTOR. 1934; Jan 1; Vol. 42, pp. 1-8.

13. Oyamada, T. B. A New General Method for the Synthesis of the Derivatives of Flavonol. Bulletin of the Chemical Society of Japan. 1935; 10(5): 182–186. https://doi.org/10.1246/bcsj.10.182

14. OECD, Test No. 425: Acute Oral Toxicity: Up-and-Down Procedure, OECD Guidelines for the Testing of Chemicals, Section 4, OECD 2022: Publishing, Paris, https://doi.org/10.1787/9789264071049-en.

15. Baghel S.S., Dangi S., Soni P., Singh P., Shivhare Y. Acute Toxicity Study of Aqueous Extract of Coccinia indica (Roots). Asian J. Res. Pharm. Sci. 2011; 1(1): 23-25. Available on: https://ajpsonline.com/AbstractView.aspx?PID=2011-1-1-5

16. Algariri K, Atangwho IJ, Meng KY, Asmawi MZ, Sadikun A, Murugaiyah V. Antihyperglycaemic and Toxicological Evaluations of Extract and Fractions of Gynura procumbens Leaves. Trop Life Sci Res. 2014; 25(1): 75-93. PMID: 25210589; PMCID: PMC4156475.

17. Asanaliyar M and Nadig P. In - vivo Anti-diabetic Activity of Hydro-Ethanolic Seed Extract of Syzygium cumini (L.) Biomed. and Pharmacol. J. 2021; 14(1): 241-247. https://dx.doi.org/10.13005/bpj/2119

18. Srinivasan K, Viswanad B, Asrat L, Kaul CL, Ramarao PJ. Combination of high-fat diet-fed and low-dose Streptozotocin treated rat: a model for type 2 diabetes and pharmacological screaning. Pharmacological Research. 2005; 52(4): 313-20. doi:10.1016/j.phrs.2005.05.004. PMID: 15979893.

19. Trinder. P. Determination of blood glucose using an oxidaseperoxidase system with a non-carcinogenic chromogen. Journal of Clinical Pathology. 1969; 22(2): 158–61. doi: 10.1136/jcp.22.2.158. PMID: 5776547; PMCID: PMC474026.

20. Khatib NA, Patil PA. Evaluation of Garcina indica Whole Fruit Extracts For Hypoglycemic Potential in Streptozotocin Induced Hyperglycemic Rats. Research Journal of Pharmacy and Technology. 2011; 4(6): 999-1003. Available on: https://rjptonline.org/AbstractView.aspx?PID= 2011-4-6-26

21. Jarald EE, Joshi SB, Jain DC. Antidiabetic activity of flower buds of Michelia champaca Linn. Indian Journal of Pharmacology. 2008; 40(6): 256-60. http://dx.doi.org/10.4103/0253-7613.4515 1.

22. Acharyya S, Dash GK, Pattnaik S, Chhetree RR. Antihyperglycemic and antihyperlipidemic activity of Acacia suma (Roxb.) barks. Research Journal of Pharmacology and Pharmacodynamics. 2011; 3(2): 67-71. https://rjppd.org/AbstractView.aspx?PID=2011-3-2-15

23. Garud A, Vyawahare NS. Hypoglycemic and Antihyperglycemic Prospective of marketed and Herbal Resilient Mediators. Research Journal of Pharmacy and Technology. 2019; 12(6): 3012-6. doi: 10.5958/0974-360X.2019.00509.2

24. Munshi RP, Joshi SG, Rane BN. Development of an experimental diet model in rats to study hyperlipidemia and insulin resistance, markers for coronary heart disease. Indian journal of pharmacology. 2014; 46(3): 270-6. doi: 10.4103/0253-7613.132156.

25. Kaur G, Kamboj P, Kalia AN. Antidiabetic and anti-hypercholesterolemic effects of aerial parts of Sida cordifolia Linn. on Streptozotocin-induced diabetic rats. Indian Journal of Natural Products and Resources. December 2011; 2(4): 428-434. http://nopr.niscpr.res.in/handle/123456789/13342

26. Debata J, Kumar HK, Sreenivas SA. Studies on Hypoglycaemic activity of the different extracts of Solanum torvum root. Research Journal of Pharmacy and Technology. 2022; 15(9): 4088-92. doi: 10.52711/0974-360X.2022.00686

Received on 10.01.2024 Revised on 06.05.2024

Accepted on 01.08.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5768-5772.

DOI: 10.52711/0974-360X.2024.00877

Groups/Time	Blood glucose(mg/dl)					% reduction of glucose
Groups/Time	0 min	30 min	60min	90min	120min	% reduction of glucose
NC+Gl(I)	95.83±2.587	147.5±5.506	153.3±4.080	128.7±5.812	109.3±6.323	45.91
Met+Gl(II)	109.7±4.271	118.5±5.123	128.5±4.288	104.0±3.011	98.17±5.492	27.64
NF50+Gl(III)	104.5±5.932	131.7±4.169	124.2±3.781	106.7±2.679	97.67±7.509	25.38
NF100+Gl(IV)	100.7±4.248	127.7±7.509	116.5±10.66	101.2±6.575	99.17±5.845	17.20
NF200+Gl(V)	89.33±6.627	121.5±5.123	107.8±5.902	82.83±6.755	84.33±3.630	26.27

Time/ Groups(n=6)	Blood glucose(mg/dl)					% reduction of glucose
Time/ Groups(n=6)	0 min	30 min	60min	90min	120min	% reduction of glucose
NC+Gl	83.33± 2.53	138.3±5.29	149±6.12	130±8.20	118±1.71	37.20
HFC+Gl	139.2±4.40	166±4.38	172±1.98	166±2.25	159±9.34	9.35
HF+Met+Gl	144.3±5.22	158±2.17	135±2.16	128±5.62	112±0.43	15.93
HF+NF50+Gl	136.8± 5.87	162±4.82	155±3.72	144±1.53	136±2.71	13.88
HF+NF100+Gl	149.3±6.04	150±1.43	134±7.13	132±8.25	112±8.45	14.73
HF+NF200+Gl	154.0±4.99	147±3.15	137±9.31	129±2.04	139±0.24	14.19

Time/ Groups (n=6)	FBG (mg/dl) Before diabetes	FBG (mg/dl) After diabetes	FBG(mg/dl) After treatment)	% Reduction of glycaemia taken from the mean values
NC	83.33± 2.539	86.50±3.222	92.50±3.594	--7.2
HFC	149.2±4.408	137.8±4.135	146.2±2.257	-6.03
HF+D	137.2± 4.151	378.3±43.11^b	329.7±23.68^c	35.42
HF+D+Met	144.3±5.226	362.7±45.56	113.8±11.32	172.48
HF+D+NF50	146.8± 5.879	337.5±42.87	159.2±17.33	121.46
HF+D+NF100	149.3±6.042	291.3±20.07	104.5±8.555	125.11
HF+D+NF200	152.0±4.993	334.7±28.20	144.3±19.21	125.26