Antioxidant, anti-inflammatory and anti-hyperglycemic activity of aqueous and methanolic extract of Houttuynia cordata: an in vitro and in vivo study

Available synthetic antioxidants such as butylated hydroxyl anisole, butylated hydroxyl toluene, propyl gallate, and ascorbic acid exhibit several side effects. To curb these side effects, more effective, less toxic, and cost-effective drugs are required. Therefore, this study aims to screen and evaluate the antioxidant as well as the anti-inflammatory and antidiabetic potential of Houttuynia cordata collected from Mairang village, West Khasi Hills, Meghalaya, India using several standard methods. The aqueous and methanolic extracts of H. cordata were evaluated by screening their ability to scavenge 1,1-diphenyl-2-dipicrylhydrozyl, 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid), hydrogen peroxide, and nitric oxide. Total phenol and flavonoid content was measured by Folin-Ciocalteau and by AlCl3 colorimetric method respectively. The anti-inflammatory activity of the plant was determined using the protein denaturation method. Methanolic and aqueous extracts of Houttuynia cordata exhibit varying free radical scavenging and anti-inflammatory activity. Among the extracts used in the study, the methanolic extract of Houttuynia cordata elicited a higher activity than the aqueous extract. Houttuynia cordata also elicited a marked reduction in blood glucose level of normal and alloxan-induced diabetic mice. Flavonoids, which have been reported to possess anti-inflammatory, enzyme inhibition, antimicrobial, anticancer, antiallergy, and antioxidant properties may contribute towards the free radical scavenging and anti-inflammatory effect of Houttuynia cordata.


Introduction
Oxidative stress can lead to chronic inflammation if there is an imbalance between the production of reactive oxygen species (ROS) and their elimination [1]. It activates transcription factors which in turn alters the expression of certain genes involved in the inflammatory pathway [1]. Inflammation is a factor that is closely involved in the pathogenesis of type 2 diabetes [2]. Therefore, novel anti-inflammatory treatments for diabetes need to be tested. Natural compounds in plants like polyphenols can interact with ROS/RNS and modulate their inflammatory response [3]. Phenols and flavonoids found in all parts of plants such as leaves, fruits, seeds, roots and bark are potent free radical scavengers [4] that protect the body from various diseases by terminating the action of free radicals [5].
Oxidative stress is an important underpinning phenomenon that assists with the progression towards more severe and often fatal complications [6]. Evidence from numerous studies suggests that ROS particularly, Mitochondrial ROS play an important role in diabetes and its associated complications [7]. High blood glucose leads to production of free radicals via autoxidation of glucose and an increased flux through the polyol and hexosamine pathway [8]. Autooxidation of glucose accelerates formation of advanced glycated end products (AGEs). Cross linking AGE protein with other molecules results in cell and tissue abnormalities [9]. Natural resources have provided leads to drug discovery [10]and have drawn a lot of attention in recent years. They are the richest source of inspiration for the identification of novel scaffold structures that can serve as the basis of rational drug design [11]. In most developing countries, conventional drugs are used by 70-95 % of the population for primary health care, and 85% of these people use plants or their extracts as the active ingredient. Several epidemiological studies have suggested that plants rich in antioxidants play a role in health and against several diseases. Their consumption reduces the risks of heart diseases, stroke, hypertension, cancer, etc [12][13]. Natural antioxidants inhibit ROS generation by scavenging free radicals and altering redox potential [14].Polyphenols such as phenolic acids, and flavonoids are potent antioxidants [15]. In view of the widespread interest on medicinal plants, the present study on Houttuynia cordata provides information of its phytochemical and pharmacological activity.

Plant collection and extraction
Leaves of Houttuynia cordata (Voucher no.NEHU-11922) were collected from Mairang village, West Khasi Hills, Meghalaya. The plant material was submitted and identified by herbarium curator Dr. P.B. Gurung, Department of Botany, NEHU, Shillong. Fresh leaves were washed, dried, and powdered using an electric blender. The dried powder was then extracted in 80% methanol in the ratio of 1:10 (plant: solvent) [16]. The mixture was filtered using a muslin cloth followed by whattmann no.1. The filtrate was concentrated in a rotary evaporator (Yamato RE800) and then lyophilized (ScanvacCoolsafe) to obtain the crude powder (gm). The crude powder was weighed to calculate the percentage yield and stored at 4ᴼ for further usage.

Experimental animals
Swiss albino mice (Balb/C strains) weighing 25-30 g, procured from Pasteur Institute, Shillong, Meghalaya were used for the study. Mice were housed in a room kept under controlled conditions maintained at a temperature of 22° C on a 12 h light/dark cycle. The mice were fed with balanced mice feed obtained from Pranav Agro Ltd., New Delhi, India. All The experiments were conducted after the approval by the Institutional Ethics Committee (IEC) (Dated: 01.10.2018) of North-Eastern Hill University, Shillong, Meghalaya, India.

Preparation of diabetic mice
Healthy mice weighing 25-30 grams were administered with alloxan monohydrate prepared in 0.15M acetate buffer, pH 4.5 intravenously at a dose of 80 mg/kg body weight. Prior to induction mice were starved overnight but given water ad libitum. After 48h, mice with more than 3-4 fold increase in blood glucose (measured using glucostix; SDCheck) were considered diabetic and used for the study.

Toxicity Tests
Organization for Economic Cooperation and Development (OECD) guidelines [17] were followed in order to determine the toxicity of the plant extracts (if any). Mice provided with ad libitum were starved for 4h. For the limit test, five mice were administered with a limit dose of 2000 mg/kg b.w. plant extract. If the animal died, the main test was conducted to determine the lethal dose at which 50% of the mice died (LD50). In addition, the mice were observed for any signs of distress, convulsion, coma or death.

Total Phenolic content
Total phenolic content was determined according to Singleton [18]. The plant extract was reacted with a Folin-Ciocalteu reagent and incubated with 7.5% sodium carbonate in the dark at room temperature for 2h. Following incubation, absorbance was measured at λ 740 nm and the results were expressed as mg gallic acid equivalent (GAE)/ gm dry weight of the extract.

Total Flavonoid content
Total flavonoid content was determined according to Kosalec [19]. Plant extract or standard was reacted with 95% ethanol, 10% Aluminium chloride, and 1M potassium acetate and incubated for 30 min at room temperature. Following incubation, absorbance was measured at λ 415 nm and the results were expressed as mg Quercetin equivalent (QE)/ gm dry weight of the extract.

DPPH assay
The DPPH radical scavenging potential of the plant extracts was determined according to Brand-Williams [20]. Varying concentration of plant extract or standard was reacted with 0.004% DPPH and allowed to stand for 30 min at room temperature. Following incubation, absorbance was measured at λ 517 nm against a blank solution. Ascorbic acid was used as standard. DPPH radical scavenging activity was calculated as: Scavenging Percentage = Abs (control) -Abs (Sample) / Abs (control) x 100

Hydrogen peroxide (H2O2) assay
The hydrogen peroxide scavenging potential of the plant extracts was determined according to Jayaprakash [21]. 20 mM Hydrogen peroxide solution was reacted with different concentrations of the plant extracts or standard and incubated for 10 min. Following incubation, the absorbance was measured at λ 230 nm against a blank solution. The scavenging percentage was calculated as above.

Nitrogen oxide (N2O) assay
Nitric oxide scavenging potential of the plant extracts were examined according to Green [22] 10mM sodium nitroprusside, phosphate buffer saline, and extract or standard solution was incubated at 25ᴼC for 150 min. From the reaction mixture, 0.5ml was taken followed by the addition of 0.5ml 0.33%sulphanilic acid reagent. This was vortexed and allowed to stand for 5 min. Following the diazotization reaction, 0.1% NEDD was added and allowed to stand for 30 min in diffused light. The absorbance was then measured at 546nm against the corresponding blank solution. The scavenging percentage was calculated as above.

Total antioxidant assay
The total antioxidant capacity of the plant extracts was measured determined according to Kannan [23]. Varying concentration of plant extract or standard was reacted with reagent solution containing 28mM sodium phosphate, 4mM ammonium molybdate, and 0.6M sulfuric acid and incubated for 90 min at 95ᴼC. Following incubation, absorbance was read at 695 nm against a blank. The activity was expressed in mg ascorbic acid equivalent (AAE)/ gm dry weight of the extract. Rutin was used as a reference standard.

Trolox equivalent antioxidant (TEAC) assay
The TEAC of the plant extracts was measured using the method described by Rubio [24]. Varying concentrations of trolox or plant extract were reacted with ABTS radical cation and incubated at room temperature for 4 minutes. Following incubation, absorbance was read at 734nm against a blank solution. Inhibition of ABTS radical scavenging activity was calculated as above. The scavenging activity was expressed in mg trolox equivalent/ gm dry weight of the extract.

Anti-inflammatory assay
The anti-inflammatory potential of the plant extracts was determined according to Dey [25]. Varying concentrations of plant extract or standard were reacted with egg albumin and phosphate-buffered saline (pH 6.4). The mixture was incubated at 37°C ± 2°C for 15 min and then heated at 70°C for 5 min. The absorbance was then measured at 660 nm against the corresponding blank. Aspirin was used as a reference standard. The percentage inhibition of protein denaturation was calculated as above.

Normoglycemic and antihyperglycemic study
Normal and alloxan-induced diabetic mice were starved overnight prior to the experiment. Crude plant extract was administered via the intraperitoneal route to the test groups. The control group received only distilled water. Food was withheld during the experiment period [26].

Statistical analysis
All analyses were carried out in triplicates. Data were presented as mean ± SD. To evaluate significant relationships between experimental parameters by correlation and regression analysis, t-tests (p-value <0.001) was used.

Yield Percentage
From 10 gm crude powdered plant, the percentage yield of methanolic and aqueous extract of H. cordata was 13.80% and 5.6% respectively.

Toxicity
The LD50 and toxic dose of the plant extract was found to be 750 mg/kg b.w. and 950 mg/kg b.w. respectively.

Total Phenolic content
The total phenol content of methanolic and aqueous extract of H. cordata was found to be 256.79 ± 0.45 mg GAE/ gm of dried extract and 91.25± 0.08 mg GAE/ gm of dried extract respectively.

Total Flavonoid content
The flavonoid content of methanolic and aqueous extract of H. cordata was found to be 127.27 ± 0.33 mg QE/ gm of dried extract and 62.659 ± 0.20 mg QE/ gm of dried extract respectively.

Antioxidant assay
Methanolic and aqueous extract of H.cordata exhibited varying antioxidant scavenging activity. Methanolic extract of H. cordata was found to be more active than its aqueous counterpart in scavenging free radicals.The IC50 value for DPPH, NO2, and H2O2 was 47.94±0.60, 28.974±0.27, and 113±0.78 respectively ( Table 1). The TAC and TEAC activity was 296.23±0.67 mg ascorbic acid equivalent/gm dry weight and 383.95±2.60 mg ascorbic acid equivalent/gm dry weight respectively ( Table 2).

Aqueous extract of Houttuynia cordata
Normal mice treated with varying doses (150-450mg/kg b.w) of aqueous extract of H.cordata showed a reduction at all time intervals studied (Table 3 and figure 2). The hypoglycemic effect was observed for all doses used. However, the maximum reduction was seen at a dose of 450 mg/kg b.w. decreasing the glucose level by 46% (p<0.001), 45% (p<0.001), 56% (p<0.001) and 11% at 2, 4, 6 and 24 h respectively from that of the control.

Discussion
In this study, aqueous & methanolic extracts of H.cordata were examined and evaluated for their antioxidant, antiinflammatory, and antihyperglycemic potentials. MHC exhibited the highest antioxidant, anti-inflammatory, and antihyperglycemic activity, whilst aqueous extractwas significantly less effective. MHC was also found to possess the highest TPC and TFC. It is known that the majority of the antioxidant and anti-inflammatory activities of plants are contributed mainly by flavonoids and polyphenols [27]. According to previous researches, phenolic compounds with ortho-and para-dihydroxylation or a hydroxy and a methoxy group or both have stronger antioxidant activity than simple phenolics [28]. The presence of ketone groups as well as a conjugated double bond in the whole molecule might play different polarities in the structure of the antioxidants and can be attributed to their antioxidant activity [29]. The other factor is related to the sensitivity of Folin-Ciocalteu reagent to a broad range of phenolic compounds whereas the DPPH free radicals show different sensitivity to various antioxidants. The Folin-Ciocalteu reagent reacts to both free phenolics and bound phenolics in extracts and other samples, but the DPPH assay determines free antioxidants and phenolics [13]. Hence, the variations in the TPC content and the antioxidative potential. Greater TEAC and TAC values of MHC can be indicative of their phenolic and flavonoid content.
For classifying a plant as one with an antidiabetic potential it should be able to lower blood glucose elevation. In the present study, both aqueous & methanolic extracts of H.cordata had a significant blood-glucose-lowering effect. The effect of the extracts varied in their effects in a time and dose-dependent manner, however, the methanolic extract was more potent. The extract had a maximum reduction of 64% (p<0.01). According to Patel, plants belonging to the family Leguminoseae, Lamiaceae, Liliaceae, Curcurbitaceae, Asteraceae, Moraceae, Rosaceae, Euphorbiaceae, and Araliaceae had a potent hypoglycemic effect [30]. Incidentally, H.cordata belongs to the family Asteraceae which thereby supports its potent glucose-lowering potential in normal mice. According to studies flavonoids are known to exert hypoglycemic effects which are useful in the treatment of diabetes [31]. A prolonged antihyperglycemic effect was also observed for the MHC (Table 5 & Figure 4) which persisted even at 24h, reducing blood glucose by 63% (p<0.001) from the diabetic control. The more prolonged and increased effect of the extract may be due to the high flavonoid content. The possible explanation for the anti-hyperglycemic effect of H.cordata could be the phenol and flavonoid content of the plant. Many studies have reported the hypoglycemic effects of flavonoids on diabetes mellitus [32].

Conclusion
Present findings provide an experimental justification to the traditional use of this plant for the management of hyperglycemia. Plant extract exhibited good antioxidant and anti-inflammatory properties which may result in preventing diabetic complications that are generally attributed to excessive oxidative and inflammatory stress during hyperglycemia.