Hydroethanolic leaf extract of Parthenium hysterophorus attenuates blood glucose in alloxan induced diabetic mice

Bashir, S., *Raza, S.A., Ahmad, M., Farhan, M., Ali, S. and William, J. Division of Science and Technology, University of Education, College Road Lahore, Pakistan Department of Chemistry, Government College University Lahore, Pakistan Department of Environmental Science, Balochistan University of Information Technology, Engineering and Management Sciences, Quetta, Pakistan Sustainable Development Study Center, Government College University Lahore, Pakistan Department of Biotechnology, School of Biological Sciences, University of Management and Technology Sialkot, Pakistan


Introduction
The production of reactive oxygen species (ROS) is a fundamental part of metabolism. The abiotic factors are also known to contribute to ROS pool of living system. The excessive production of ROS is harmful to normal cellular functions and biomolecules. The antioxidant defense system of the body maintains the homeostasis by scavenging the ROS (Giacco and Brownlee, 2010). The overproduction of ROS due to any factor results in a state of oxidative stress which is linked to the pathogenesis of many diseases including diabetes. The diabetes is expanding exponentially and type 2 is the major type prevailing in both developing and developed countries. The modern lifestyle, robotic life, consuming high caloric diets and lack of exercise collectively responsible for the development of oxidative stress which further lead to diabetes type 2 (DM-T2) (Vertuani et al., 2004). Diabetes may be defined as a metabolic disorder responsible for persistently high blood glucose levels. The DM-T2 is of keen concern to its rapid expansion and associated medical problems. The side complications of diabetes included kidney damage, blindness, cardiac problems, loss of sexual function and many more (Scheen and Paqout, 2013). The synthetic drugs to treat DM-T2 reduce the blood glucose level effectively but are also associated with complex side complications. They lack the ability to reverse the disease towards normal physiology. However, plants are known to possess such dynamic functionalities to treat DM-T2 with improvements in enhanced insulin eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources FULL PAPER production, regeneration of β-cells and activating adipose tissues to uptake glucose (Yatoo et al., 2017). Medicinal plants are the backbone of traditional medicinal systems which are based on believes and observations comprised of hundreds of years . The phytochemicals from plants are well established to counter ROS and consequent health disorders like DM-T2 (Zhang et al., 2015). The reduction in ROS and free radicals was responsible for improvements in physiological and hematological indicators, especially during diseased and stressed conditions. The phenolics and flavonoids are metabolites of medicinal significance as most of the therapeutic roles of plants are due to these functional phytochemicals (Seufi et al., 2019). The antioxidants and antidiabetic properties of plant phenolics are well established (Moukette et al., 2017). Many plants are being used to treat DM-T2 without the scientific confirmations about phytochemicals. The scientifically proved plant species must be used to treat chronic ailments for better outcomes and to avoid toxicity in some cases.
Parthenium hysterophorus (Asteraceae) is known to treat diabetes in many areas of Pakistan (Mahmood et al., 2011). P. hysterophorus was reported to have important phytochemicals like alkaloids, saponins, tannins and phenolics (Kapoor, 2016). The current work was designed to profile polyphenols and to assess invitro and in-vivo antidiabetic properties to confirm the ethnopharmacological use of P. hysterophorus in for DM -T2 management.

Extraction
The extraction of phytochemicals was performed and the yield extract was calculated. Fresh leaves were treated with liquid nitrogen and ground to a fine powder which was further freeze-dried or lyophilized. The freeze -dried powder material was suspended in 40%, 60% and 80% ethanol for 48 hrs. The extracts were ultrasonicated at 20 kHz for 30 mins and filtered. The extra solvent was removed using a rotary evaporator under vacuum. The extracts were again freeze-dried and stored at -21ᵒC for further use.

Total phenolic contents (TPC) and total flavonoid contents (TFC)
TPC was determined by following the established method with little modification (Zengin et al., 2010). 20 µL of each plant extract was added the 90 µL of Folin-Ciocalteu reagent followed by addition of 91 µL of 10% Na 2 CO 3 . Absorbance was noted on 726 nm by UV-1700 Schimadzu Japan spectrophotometer. Results were reported as mg gallic acid equivalent per gram dried extract (mg GAE/g D.E).
TFC was determined by using a previously established method based on AlCl 3 by Zishen et al. (1999). Methanol (2 mL) in 4 mL distilled water and 100 µL extract were mixed with 0.6 mL of 5% NaNO 2 solution and 0.6 mL of 10% AlCl 3 . The resultant mixture was stayed for 10 mins to complete the reaction. Then 4 mL of NaOH (1 molar) and distilled water were added to make a volume of 20 mL and stayed for 25 mins. The absorption of reaction mixture was noted at 510 nm. The results were reported as mg rutin equivalent per gram dried extract (mg RE/g D.E).

Pre-screening of antioxidant activity by TLC
The thin-layer chromatography (TLC) was used to assess the potential antioxidant activity of plant extracts before going into spectrophotometric assays. The plant extracts were suspended in methanol and 5 µL of this was applied on TLC. The TLC plate was developed by methanol in ethyl acetate (1:1). The developed plate was sprayed by 25% DPPH solution and stayed for 30 mins in dark. The bleaching of the violet color of DPPH by yellow spots was noted (Bektas et al., 2005).

In-vitro antioxidant and anti-diabetic activities
The DPPH radical scavenging by plant extracts was determined to evaluate the antioxidant activity of plant extract. The plant extracts at a concentration of 50-250 µg were used and IC 50 value was calculated (Chew et al., 2011). Simply, 25% methanolic DPPH and plant extracts in concentration (50-250 µg) were dissolved in methanol. Resultant reaction mixture stayed for 35 mins in dark at ambient conditions of temperature and humidity. The absorbance was noted at 517 nm and free radical scavenging was determined in terms of IC 50 value.
The butylated hydroxyl anisole (BHA) was used as a standard antioxidant compound. The antidiabetic activity was determined by checking the inhibition of αglucosidase on a spectrophotometer (Jabeen et al., 2013). Extracts were dissolved in 70 µL of phosphate buffer (50 Mm) of pH 6.8 and 1 unit/mL of α-glucosidase. Incubation of mixture at 37°C for 10 mins was made and the reaction started upon adding 10 µL of pnitrophenolglucopyranoside (5mM). After further incubation of 30 mins, absorbance was measured at 405 nm. All the measurements were made in triplicate. Acarbose was used as a standard reference compound and IC 50 value was computed.

In-vivo hypoglycemic activity
The 24 male albino mice (6 weeks old) having an average weight of 31.64±3.05 g were utilized and stayed for a period of 10 days to acclimatize the new environment at animal house of GC University Lahore. After adaptation, the mice were injected with alloxan monohydrate at dose 150 mg/kg body weight. The blood glucose level (BGL) was measured after 72 hrs of injection. The mice having BGL above 200 mg/dL were characterized as diabetic and a control (D0) set of mice was also run (non-diabetic) . The diabetic mice were grouped into untreated mice group (D1), metformin-treated (D2) and extract-treated group (D3). The mice were allowed ad libitum supply of water and food. The extract at a dose of 450 mg/kg body weight was administrated orally in normal saline (Raza, Chaudhary, Mumtaz, Adnan et al., 2020). The BGL levels were measured by glucometer by rupturing the vein in the tail every seven days. The monitoring remained active for 28 days.
The standard deviation (±) was applied on the experimental values. The difference of means was computed by applying Analysis of Variance (ANOVA) to see the level of significance in treatment means by using Minitab 17.0 software.

Pre-screening of antioxidant activity by TLC
All the extract fractions bleached the violet color of DPPH reagent by yellow spots indicating the qualitative assessment of antioxidant activity by plant extracts.

In-vitro antioxidant and anti-diabetic activities
The in vitro antioxidant activities were determined by measuring the DPPH free radical scavenging and antidiabetic potential was calculated by observing the αglucosidase inhibition. The IC 50 values of extracts, BHA and acarbose for DPPH radical scavenging and αglucosidase inhibition are given in Table 2. The lowest IC 50 values for DPPH scavenging and α-glucosidase inhibition were exhibited by 60% ethanol indicating the potential of 60% ethanolic leaf fraction of P. hysterophorus to encounter free radicals and to inhibit αglucosidase activity.

In-vivo hypoglycemic activity
The significant reduction in BGL of diabetic mice was observed in D3 and D2 (Figure 1). The metformin restricted the BGL to 128.12 mg/dl when compared D1 where the final value of BGL was 268.05 mg/dl. The BGL in D3 mice was also reduced gradually and became quite normal by 28 days. The final average value of BGL for D3 mice at the end of 28 days was 137.88 mg/dl. The BGL of D1 mice permanently remained high till the completion of the experiment.

Discussion
The findings revealed that solvent polarity played an FULL PAPER important role to enhance extract yields. The extract yields also influenced the TPC and TFC values. The high TPC and TFC were of immense importance due to the therapeutic role of phenolic and flavonoids, which are the most common component of plant extracts (William et al., 2019). Antioxidant activities of plants are a preliminary indication for their possible hidden medicinal potential (Raza, Chaudhary, Mumtaz, Bashir et al., 2020). The antioxidant activity of extracts was assessed by measuring the scavenging of DPPH free radical. The DPPH method is widely adopted and accepted method to evaluate the antioxidant and antiradical activity of substances. The antioxidant activities of hydroethanolic fractions obtained from leaves of P. hysterophorus were probably due to the presence of high contents of polyphenols (Arshad et al., 2019;Raza et al., 2019). The highest antioxidant activity was computed in the case of 60% ethanolic extract likely due to the polarity of solvent composition. A recent study indicated that 60% of ethanol was more efficient to improve the antioxidant activity of Conocarpus lancifolius leaf extracts (Raza, Chaudhary, Mumtaz, Adnan et al., 2020). The highest DPPH scavenging posed by 60% ethanolic extract was due to high TPC and TFC in the extract. The in vitro inhibition of αglucosidase by extracts was probably associated with phenolic and flavonoid entities. The inhibition of αglucosidase slows down the breakdown of carbohydrates and delays the glucose absorption in the brush borderline of the intestine. This restriction in absorption results in lowering of postprandial blood glucose level and is strategy is considered as an important tool to manage hyperglycemia (William et al., 2018). The structural features of secondary metabolites from plants were reported to bind the specific amino acid residues of enzyme proteins including α-glucosidases. This binding was responsible for the significant reduction in enzyme activity (Shobana et al., 2009). A recent study reported that the plant metabolites mainly polyphenols from Calotropis procera interacted with amino acid residues of α-glucosidase to inhibit the enzyme activity (Nadeem et al., 2019). Another study reported the hypoglycemic potential of Butea monosperma which was attributed to phytochemicals in the extract (Farooq et al., 2020).
The in-vivo trial indicated the potency of P. hysterophorus to reduce elevated BGL to great extent and this reduction was quite comparable with the synthetic standard drug metformin. However, it was observed from the findings that metformin reduced the BGL in comparatively rapid fashion as compared to plant extract but at the end of 28 days of the experimental protocol, there was no substantial difference. The reduction in BGL upon administering plant extract was probably due to phytochemicals present in the extract. The most probable mechanism involved in BGL reduction by plant extract was probably the antioxidant nature and α-glucosidase inhibitory properties of phytochemicals in extract (William et al., 2019). The synergic effect was proposed for this evident reduction in the glucose of diabetic mice upon consumption of plant extract. The hypoglycemic attributes of plant extract were probably due to many biochemical phenomena at the molecular level including α-glucosidase inhibition, improvement in insulin secretion, regeneration of β cells of the pancreas and enhanced glucose uptake by adipose tissues (Noor et al., 2017). The promising in-vitro and in-vivo antidiabetic potential of 60% ethanolic leaf extract of confirmed the ethnopharmacological use of P. hysterophorus to treat diabetes. Moreover, the phytochemical profiling may be expanded to develop novel antidiabetic treatments and functional foods with hypoglycemic properties.

Conclusion
The findings of the current work affirmed the ethnopharmacological use of P. hysterophorus to reduce the blood glucose concentration to treat diabetes mellitus. The 60% ethanol was the most appropriate solvent for optimum yield of polyphenols. The experimental outcomes of current work provided a potent source of polyphenols in form of P. hysterophorus which might be a useful tool for a naturopathic approach towards hyperglycemia. Further studies may be carried out to evaluate the toxicity of P. hysterophorus along with identification of effective secondary metabolites of pharmacological importance.