PIPER CHABA EXTRACTS WITH ANTIBIOFILM ACTIVITY INFLUENCE ANTIHYPERGLYCEMIC AND ANTIHYPERLIPIDEMIC RESPONSES IN DIABETIC WISTER RATS

Objectives: Piper chaba, native to South and Southeast Asia, has been traditionally used as a medicinal plant. Aim of this study was to evaluate the antihyperglycemic and antihyperlipidemic activities of P. chaba root extracts (RE) in streptozotocin (STZ)-induced diabetic rats along with its antimicrobial activity.
Methods: Diabetes was induced in Wister rats through the intraperitoneal administration of STZ (50 mg/kg b.w.). Antidiabetic and antilipidemic activities of the RE (in methanol, ethanol, ethyl acetate and distilled water) were evaluated by administering oral dose (200 mg/kg b.w.) for 21 days. Metformin (12.1 mg/kg b.w.) was used as a positive control. Blood samples of rats were drawn by tail vein puncture and cardiac puncture to determine the fasting blood glucose (FBG) and serum level of total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL), respectively. Standard protocols were followed to determine the antimicrobial and antibiofilm activities against two different strains of bacteria.
Results: Oral administration of P. chaba RE for 21 days resulted in a significant (p< 0.001) decrease in FBG and TC, TG, and LDL levels (p<0.001), when compared to the untreated diabetic rats. Significant (p<0.001) increase of HDL was observed when ethyl acetate and aqueous RE were administered. Out of four, two extracts showed varying antimicrobial activities, particularly against the gram-positive bacteria. 
Conclusion: It became evident for the first time that P. chaba extracts possess antimicrobial activities and can serve as biochemical compounds with great alternative therapeutic potential in the management of diabetes and hypercholesterolemia.


INTRODUCTION
In Bangladesh, four hundred and forty nine species of medicinal plants have been enlisted so far [1]. Among them, Piper chaba is a vine in the family of Piperaceae. It is found in the South Western region of Bangladesh, particularly at Jashore and Khulna regions, and people use it as an additive spice. Extracts of P. chaba possess phenolic compounds, antioxidants and showed anti-inflammatory, cytotoxic and anti-bacterial properties [2,3]. Also, phytochemicals isolated from plants of the Piper species showed anticancer properties [4,5].
Medicinal plants are of considerable interest to researchers in recent years to treat diabetes mellitus [6]. Diabetes mellitus (DM) is a group of chronic, complex metabolic disorder characterized by hyperglycemia due to defects in insulin production, insulin secretion, and insulin signaling [7]. Insulin is a potent hormone that regulates blood glucose level [8]. Globally, diabetes is the leading cause of human death. According to International Diabetes Federation (IDF), an estimated 463 million people, as of 2019, had diabetes worldwide (8.8% of the adult population), with type 2 diabetes making up about 90% of the cases [9]. According to the latest World Health Organization (WHO) data, DM-induced death reached to 28,065 or 3.61% of total deaths in Bangladesh Besides insulin, the most widely used oral hypoglycemic drugs are Insulin sensitizer, insulin secretagogues, a-glucosidase inhibitors, and dipeptidyl peptidase-4 inhibitors. But those drugs have remarkable toxicity and side effects [12]. On the contrary, the traditional medicines prepared from herbal plants containing phenolic compounds having antidiabetic activity are cost effective with fewer side effects [13]. Also, WHO has been encouraging the use of traditional medicine to treat life threatening diabetes as well as it's complications [14]. [10]. Diabetes and hyperlipidemia are two factors responsible for the development of cardiovascular diseases. They have severe effect on lipid metabolism and show atypical effect on total lipid profile. Retinopathy, cardiovascular diseases, polyurea, polyphasia result from long time uncontrolled diabetes [11].
Plants of the Piperacae family (such as P. betle, P. crocatum, P. longum, P. nigrum and P. sarmentosum) have been reported for their antidiabetic activities. Mixture extracts of P. crocatum leaves and Cinnamomum burmannii bark was reported to enhance the number of pancreatic β cells in rats, increase the rats' blood insulin levels, and reduce the rats' blood glucose levels [15]. Also, aqueous RE of P. longum was found to significantly decrease the hyperglycemic and hyperlipidemic activities in STZ-induced diabetic rats [16]. Additionally, in another study, aqueous extract of P. nigrum seeds and Vinca rosea flowers treatments lead to significant lowering of blood sugar level and reduction in serum lipids [17].
However, the literature review shows that there has been no study on the effects of P. chaba RE on STZ-induced rats to evaluate their antidiabetic and antihyperlipidemic activities. Hence, this study was undertaken to investigate these activities of P. chaba REs in induced diabetic rats.

Collection of samples and preparation of extracts
Dry roots of P. chaba were purchased from the local market of Khulna, Bangladesh. Identification and authentication of the plant material were confirmed at the Department of Botany, University of Rajshahi, Bangladesh.
The sample was powdered using mortar and pestle. 250 gm of the powder was accurately weighed and separately dissolved with methanol (MeOH), ethanol (EtOH), ethyl acetate (EtOAc) and distilled water (Aqueous, AQ), taking 1500 ml solvent and stirred for 72 h. It was then filtered and the filtrate was concentrated using a rotary evaporator (RE-401, Stuart equipment, Staffordshire, UK) at 30 °C-40 °C. The yield percentages were calculated using the following formula: Extract yield (%) = R/S × 100 (where R; weight of extracted plant residues and S; weight of the plant raw sample) [18]. The yield of EtOAc, MeOH, EtOH and AQ extracts were 28%, 12%, 20% and 25% w/w, respectively. The extracts were stored in small bottles at 4 °C.

Chemicals/reagents/diagnostic kits
The antidiabetic drug, metformin was obtained as a gift sample from Incepta Pharmaceuticals Ltd, Savar, Dhaka, Bangladesh

Experimental animals and ethical permission
. STZ (Sisco Research Laboratories Pvt. Ltd. India) and glucose standard strip/kits, and glucometer (GlucoxTD-4183 Blood glucose test strips, Germany). All other chemicals and reagents used were of analytical grade.
Three-month old healthy male Wister albino rats (180-220 gm body weight) collected from the Pharmacology Lab, Department of Pharmacy, Jahangirnagar University, Dhaka, Bangladesh. This investigation was officially recognized by the Ethical Review Committee, Varendra University, Rajshahi, Bangladesh. Rats were kept in polypropylene cages group wise, 6 animals per cage. Before the experiment, the animals were synchronized to the experimental/ laboratory condition for 7 d. The ambient temperature, humidity and 12 h day-night cycles were maintained according to the animal care and welfare guidelines [19]. All the animals were allowed to have a free access to fresh pellet diet and water.

Distribution of experimental animals in groups
For the evaluation of different extracts on the hyperglycemic and hyperlipidemic activities in normal and STZ-induced animals, adult male Wister rats were divided into 7 groups with six rats in each and treated with four different P. chaba extracts by gastric intubation via oral cavity:

Induction of diabetes and test for antidiabetic study
A single dose of 50 mg/kg b.w. of STZ was prepared with 0.01M citrate buffer (pH 4.5). Then the freshly prepared solution was administered intraperitoneally to each experimental rat. After 48 h, rats with the fasting blood glucose (FBG) level of 200 mg/dl were selected as diabetic rats and were used for the study. The extracts were orally administered for 21 d once a day. On the 21 st day, the blood sample was collected from the tail vein of rats and the FBG level was measured using one-touch glucometer (GlucoxTD-4183 Blood glucose test strips, Germany) with glucose oxidaseperoxidase reactive strips.

Measurement of serum lipid profile
At the beginning of the experiment, animals were deprived of food overnight. The animals were anaesthetized with chloroform and two milliliters of fasting blood samples was obtained from each animal of both the control and test groups via cardiac puncture. The blood sample was collected into a tube containing lithium heparin as anticoagulant. The serum obtained after centrifugation at 3500 RPM for 15 min was separated for lipid measurement. The serum concentration of total cholesterol (TC), triglycerides (TG), high density lipoprotein (HDL)-cholesterol and low density lipoprotein (LDL)-cholesterol were measured using UV-spectrophotometric methods. Laboratory kit reagents (Human, Wiesbaden, Germany) following the company's instruction were used for all biochemical analysis and the absorbance was read using a UV-Vis spectrophotometer (Shimadzu UV-1280, Kyoto, Japan).

Formation of zone of inhibition in disc diffusion assay
Evaluation of the antimicrobial activity of all four Piper chaba root extracts was determined against Bacillus cereus (gram positive) and Escherichia coli (gram negative) by disc diffusion method [20]. Each bacterial strain was subcultured overnight at 37 °C in Mueller-Hilton agar slants. The bacteria were harvested using 5 ml of sterile saline water. Its absorbance was adjusted at 580 nm by a spectrophotometer and diluted to attain a viable cell count of 10 7 CFU/ml. Filter paper discs were loaded with Piper chaba stem extract at concentrations of 200 µg/disc and 400 µg/disc. The plates were kept in the fridge at 4 °C for 2 h. to permit the diffusion of plant extract. After incubating at 37 °C for 24 h, presence of inhibition zones were measured by a transparent scale in mm and recorded as well.

Determination of antibiofilm activity
Formation of biofilm biomass was determined through the microtiter plate assay in 96-well polystyrene flat bottom plates [20]. After overnight cultures of E. coli, optical density (OD) 1.0 was determined at 600 nm. 1% test microbes was mixed with 1 ml of immediately prepared LB media with or without the ethyl acetate and methanol extracts of Piper chaba roots (60-1000 µg/ml). After incubation at 37 °C for 24 h, plates were washed with distilled water to remove the free-floating cells. The biofilm was stained with 0.4% crystal violet (Himedia, Mumbai, India) solution. After 15 min, the staining process was complete and distaining process started with 1 ml of 95% ethanol. Finally, absorbance (at 650 nm) was measured by using a multiplate ELISA reader (Biotek-ELX-800, India). Formation of biofilm was determined by using the following equation: % of reduction of OD = Absorbance of (control-test)/Absorbance of control × 100

Statistical analysis
All data reported are presented as mean±SD or ±SEM. Statistical analysis was performed by using one-way analysis of variance (ANOVA) with multiple comparison tests, where applicable. The difference between two groups was measured by the student t-test. A P-value less than<0.05 was considered statistically significant.

Determination of four different RE of P. chaba on FBG level in STZinduced diabetic rats
The effect of EtOH-, MeOH-, EtOAc-, and AQ extract of the roots of P. chaba were tested on the FBG levels in non-diabetic and diabetic rats on Day 0 and Day 21 and are shown in fig. 1. The FBG levels of the STZinduced diabetic control (DC, Gr-2) were significantly (p<0.001) higher than those of the respective non-diabetic control (NDC, Gr-1) tested both on Day 0 and Day 21 group of animals and the FBG level remained high in Gr-2 through Gr-7 tested on Day 0. However, as measured by one-way ANOVA, pretreatment of a single dose of 200 mg/kg b. w RE exhibited a significant hypoglycemic effect (p<0.001) tested on Day 21, when compared to DC. Metformin, a standard hypoglycemic drug, also had a significant (p<0.001) reduction in FBG level in Gr-3. Also, an intra-group analysis as measured by t-test, the FBG level revealed a significant (p<0.001) difference between Day 0 and Day 21 of each group (Gr-2 to Gr-7) ( fig. 1).

Evaluation of different REs of P. chaba on lipid profiles in STZinduced diabetic rats
Diabetes is also associated with altered lipid profile. The effect of EtOH-, MeOH-, EtOAc, and AQ extract of the roots of P. chaba were tested on the serum levels of LDL, HDL, TC, and TG in STZ-induced diabetic Wister rats ( fig. 2 and fig. 3).

in a day). n= number of experimental animals
The STZ-induced diabetic control (DC, Gr-2) significantly elevated the serum level of LDL (p<0.001), TC (p<0.001), and TG (p<0.001), but reduced the level of HDL (p<0.001), as compared to non-diabetic control (NDC, Gr-1, fig. 2). Metformin-treated diabetic rats (Gr-3) significantly reduced the levels of LDL (p<0.001), TC (p<0.001), and TG (p<0.001), when compared with the DC. However, as compared to DC, metformintreated diabetic rats (Gr-3) significantly (p<0.001) increased the level of HDL and showed no difference on the level of HDL, when compared with the NDC. Changes in serum lipids concentration following different REtreatments were also evaluated as shown in fig. 2 and fig. 3.

in a day). n= number of experimental animals
Treatments with extracts of EtOH, MeOH, EtOAc, and AQ extracts showed a significant (p<0.001) decrease in LDL, TC, and TG when compared with DC. On the contrary, EtOAc, and AQ extracts, but not EtOH, MeOH extracts, showed a significant (p<0.001) increase in HDL level, as compared with DC.

Determination of antimicrobial activity
Two extracts (methanol and ethyl acetate) represented varying antimicrobial activities in this study (table 1). The zone of inhibition (ZOI) against gram negative bacteria was bigger than that of gram positive bacteria. Susceptibility of these two extracts against bacteria increased with the increase in concentrations. Other two extracts (ethanol and aqueous) showed no antimicrobial activity.

Effect of Piper chaba extracts on the formation of bacterial biofilm
Ethyl acetate and methanol extracts showed antimicrobial activity against Escherichia coli (table 1). Effect of these extracts on biofilm produced by the same bacteria was also checked. It was observed that ethyl acetate and methanol extracts dose-dependently inhibited the formation of biofilm when applied in increasing concentrations (60, 125, 250, 500, 1000 and 1200 µg/ml). SEM was calculated from three independent experiments. Comparing to the control, administration of ethyl acetate and methanol extract reduced the formation of biofilm from 3 to 13% and 2 to 5%, respectively. Percentage of inhibition became highest at a concentration of 1000 µg/ml and become almost constant at 1200 µg/ml ( fig. 4).

DISCUSSION
The present investigation discusses the antidiabetic and antihyperlipidemic potential of the RE of P. chaba in STZ-induced diabetic rats. DM is a chronic metabolic disorder. Vascular complications are among the most common sequela of the condition, as cardiovascular disease accounts for 65% of mortality in DM [21]. Our study evaluated the effect of EtOH-, MeOH-, EtOAc-, and AQ extracts of the roots of P. chaba on STZ-induced diabetic rats. STZ, an N-acetyl glucosamine analogue, was used in our study to induce DM in rats. It destroys pancreatic β-cells by donating nitric oxide (NO), but has no effect on the exocrine part of pancreases [22].
Our short-term study confirmed the reducing effect of oral administration of P. chaba RE at a dose of 200 mg/kg b.w. on FBG level in diabetic rats significantly and the values of RE-treated rats after 21 d of treatment came to the level of the non-diabetic control rats. A similar outcome at the same dose after 30 d treatment of root aqueous extract was reported by P. longum [16] The results of our study was also supported by the leaf extracts of P. guineense and P. auritum administered in diabetic female and male albino Wister rats, respectively [23,24].
The recent studies have shown the increasing trends for the involvement of reactive oxygen species (ROS) and oxidative stress in the pathogenesis and development of type 2 DM [26]. A number of important phytoconstituents such as dimeric alkaloids and alkamides have been isolated from various parts of P. chaba [27]. Earlier, many researchers have shown that P. longum and P. chaba extracts from different parts of the plant contain several phytochemicals and bioactive compounds like piperine, saponin, and sesquiterpene hydrocarbons β-caryophyllene, α-humulene and germacrene D having their antioxidant properties [28,29], that possibly scavenge free radicals too. On the other hands, the protein glycation and advanced glycation end products (AGEs) formation are associated with DM, which was also inhibited by the methanolic extract of P. betle and P. auritum leaves [30].
DM has been generally seen to have strong association with hyperlipidemia. The development of marked hyperlipidemia in diabetes may be a consequence of the uninhibited actions of lipolytic hormones on the fat depots and increase in mobilization of fatty acids from fat tissue [31]. In our investigation, we have observed the diabetic hyperlipidemia as marked with enhanced TG, TC, LDL, but decreased HDL cholesterol level. However, in STZ-induced diabetic rats, all four RE of P. chaba at a dose 200 mg/kg b.w. in our findings significantly reduced the level of TG, TC, and LDL, but increased the level of HDL when treated with EtOAc and AQ extracts. The LDL and HDL are considered as more particular and sensitive biochemical markers of cardiovascular disease. A thorough literature searches on hypolipidemic effect of the extracts of other species of the Piper genus also hold up our findings. In rabbits, the ethanolic fruit extract of P. chaba (150 mg/kg) possessed significant hypolipidemic effects and lowered TC and LDL levels while showing significant effect on HDL levels as well. Whereas, P. nigrum fruit extract (250 mg/kg) significantly elevated the levels of cholesterol and HDL and lowered LDL level on albino rabbits [32]. Also, other two different studies using the methanol and ethanol leaf extracts of P. betel at the dosages of 250 mg/kg b. w and 500 mg/kg b. w, respectively, on male albino Wister rats showed significant reduction of Medicinal plants have been traditionally used for treatments against various bacteria. The antimicrobial activity of piper chaba root extracts showed dose-depended nature against Bacillus cereus (gram positive) TC, TG, LDL and VLDL and HDL level became augmented [33]. It was suggested that the piperidine alkaloids including piperine, pipernonaline and dehydropipernonaline could be responsible for producing this effect by activating AMPactivated protein kinase that regulate lipid metabolism [34]. Our results following the same trend suggest that P. chaba REs can turn down the risk of diabetes-induced cardiovascular diseases by decreasing serum TC, TG and LDL levels and increasing serum HDL levels. The increased level of HDL is very significant due to its ability to reversely transport cholesterol from peripheral tissue and to esterifies cholesterol by lecithin-cholesterol acyltransferase (LCAT) [35]. So, the effect of P. chaba RE in diabetic disorder is remarkable. and Escherichia coli (gram negative). Our result showed the highest activity against gram negative bacteria and similar results have been found in previous studies [36]. It probably happened due to the presence of biologically active compounds such as phenolic, flavonoids, polyphenols, isoprenoids, alkaloids etc. in plant extracts and their effects on the cell wall components of bacteria. Structural and compositional difference in cell wall and cell membrane of gram positive and gram negative bacteria also plays a role [37][38][39]. An earlier study discovered a relationship between the antimicrobial activity and phytochemicals present in medicinal plants extract [40].
Our result suggested that the root extract was more active against gram negative microorganisms than gram positive microorganisms. E. coli are gram negative bacteria and have the ability to form biofilm [41,42]. The antimicrobial activity of our plant (ethyl acetate and methanolic) extracts against gram negative bacteria encouraged us to investigate the properties of antibiofilm properties of these extracts. From our study it became evident that 200 µg/ml and 1000 µg/ml were the minimum and maximum concentrations of extracts to inhibit the formation of biofilm. Pronounced biofilm inhibition ability was found in ethyl acetate and also in methanol extracts. Similar result with solvent depended extracts of P. longum and P. nigrum were observed against Streptococcus pyogenes at the lowest concentration of 500 µg/ml [43]. Promising antibiofilm activity was observed in case of methanolic crude extract of P. longum against Vancomycine-resistant S. aureus-pAR1818 and S. aureus MH4 and MTCC 96 isolates whereas ethanol extracts of Piper longum fruits could reduce the cell attachment and inhibit the formation of biofilm by Staphylococcus aureus at 500 µg/ml [44,45]. Walmiki et al., found that Piper nigrum possessed antibiofilm activity against Escherichia coli and Salmonella spp [46].

CONCLUSION
The present findings, for the first time, demonstrated that the RE of P. chaba is capable of exhibiting significant antihyperglycemic and antihyperlipidemic activities in STZ-induced diabetic rats. Moreover, the extract increased the level of HDL, a protective lipid compound, in diabetic condition. Therefore, our results give scientific support for the use of this plant in traditional medicine for the management of diabetes and its associated complications though further molecular studies are required to investigate the mechanism underlying the antihyperglycemic and antihyperlipidemic effect of P. chaba REs. Moreover, marked antimicrobial and antibiofilm activities of these extracts have also been observed.

ACKNOWLEDGEMENT
We are grateful to Professor Dr. Ekramul Islam, Ex-Coordinator, Department of Pharmacy, VU, for his support to use the Laboratory at the Department of Pharmacy, VU. Also, we appreciate the efforts of Mr. Robiul Islam, Executive-Lab, Department of Pharmacy, VU, for providing technical support.

FUNDING
The research was supported by Varendra University Trust (VUT)sponsored Faculty Research Grant 2019 at the Center for Interdisciplinary Research (CIR), VU, Rajshahi, Bangladesh.

AUTHORS CONTRIBUTIONS
SR did the majority of the study and wrote the manuscript. KK, UF and SoR were involved in experimental works and data analysis. RH provided laboratory support, supervised the study and improved the manuscript through careful review and helpful suggestions.