Antidiabetic and hypolipidemic potential of Campomanesia xanthocarpa seed extract obtained by supercritical CO 2

Campomanesia xanthocarpa, a plant belonging to the Myrtaceae family, is popularly known as gabiroba. Leaves of gabiroba has been popularly used to treat various diseases, including inflammatory, renal, and digestive, among others. Additionally, studies have shown an effect to reduce blood cholesterol levels. The aim of this study was to evaluate the antihyperglycemic and hypolipidemic effects of Campomanesia xanthocarpa seed extract in hyperglycemic rats. The results showed that 400 mg/kg of seed extract was able to decrease blood glucose levels and to increase the muscular and hepatic glycogen content as well as to inhibit the sucrase and maltase activity. At doses of 200 mg/kg and 800 mg/kg, the activity of these enzymes was also reduced. In the lipid profile 400 mg/kg produced a decrease in total and LDL cholesterol serum levels; and with 200 mg/kg there was an increase in HDL cholesterol levels. The extract did not present hepatic and renal toxic effects at the different doses tested. The results suggest that the treatment with Campomanesia xanthocarpa seeds extract is useful in reducing glycemia, total cholesterol and LDL levels with potential adjuvant therapeutic in the treatment of diabetes and hypercholesterolemia, however, additional pharmacological and toxicological studies are still required.


Introduction
Diabetes mellitus (DM) is a serious global health problem affecting about 425 million people worldwide (WHO, 2018). DM is a chronic disease characterized by an elevated blood sugar level (hyperglycemia) which is due to carbohydrate, protein, and lipid metabolism disturbance caused by an absolute or relative deficiency of insulin or by insulin resistance at the cellular level (WHO, 2018). It is well established that hyperglycemia and diabetic dyslipidemia associated lead to several comorbidities including macro-and microvascular damage (Naveen and Baskaran, 2018). For a significant number of patients, the treatment of Type 2 DM (T2DM) must include pharmacological agents in order to reach satisfactory glycemic control as well as multifactorial risk reduction (Khavandi et al., 2017). In this context, many synthetic drugs have been widely used for the treatment of diabetes and dyslipidemia, but herbal medicines still remain a popular choice. In addition, the use of plant-derived drugs for diabetes has been approved by the World Health Organization (WHO, 2002).
Among the compounds found in the leaves (Pereira et al., 2012) and fruits (Sant'Anna et al., 2017) of the plant are the phenolic compounds (gallic acid, chlorogenic acid and quercetin) while the seeds are rich in terpenoids, flavonoids and alkaloids (Capeletto et al., 2016). Flavonoids and triterpenes are secondary metabolites present in several plant species in variable amount and possess various therapeutic properties described, among them the antidiabetic (Alkhalidy et al., 2018;Hussain et al., 2017;Leonidas et al., 2017). Conventionally, natural compounds have been extracted from plant materials by using different solvents, but these procedures result in contaminating the target compounds/extracts with extraction solvents (Brglez Mojzer et al., 2016).
Supercritical fluid extraction (SFE) is an alternative method considered as a technological innovation because it uses supercritical fluids as solvents and its use in industrial processes is increasing due to the environmental and quality factors involved (Silva et al., 2016a;Paula et al., 2016). It is a process free of toxic waste, which does not cause the thermal degradation of the extracts and active principles. There is a wide range of compounds that can be used as supercritical fluids. From the environmental point of view, the most widely used is carbon dioxide due to its characteristics, such as being non-toxic, non-reactive, environmentally acceptable and non-flammable, which makes it a very attractive solvent for the preparation of extracts and oils with pharmaceutical application (Knez et al., 2014;Rodríguez-Pérez et al., 2016).
Considering the relevance of C. xanthocarpa extracts to treat several diceases, especially T2DM (Tangvarasittichai, 2015), this study aimed at evaluating the hypoglycemic and hypolipidemic effects in rodents of C. xanthocarpa seed extracts obtained from SFE-CO 2 fluid technique.

Plant material and extract production
The fruits of Campomanesia xanthocarpa Berg were collected from native plants at Quilombo city, Southern Brazil (26°47'23.6"S, 52°45'42.41"W). The voucher specimen was identified and deposited in the Herbarium at Universidade Comunitária de Chapecó (Herbarium Unochapecó, SC, Brazil) under the access number 3153. The extraction from the seeds was carried out by supercritical extraction technique according to Capeletto (2015). Approximately 30 g of the dried and ground seeds were placed into extraction compartment of the apparatus. CO 2 was pumped at a constant flow rate (2 mL/min) and kept in contact with the seeds for at least 30 minutes to stabilize the system. Extraction took place for 150 minutes at 40 °C and 250 bar of pressure. After that, the extract was collected and the amount obtained was weighed. This procedure was performed until no further changes in the weight of the extract were observed (Capeletto et al., 2016). The extract was then stored at -20 °C for further use in the biological assays.

Chemical characterization of the extract
The extract was analyzed by Agilent GC/MS (7890B) gas chromatography coupled to a quadripolar mass spectrometer (5977A) (Agilent Technologies, Palo Alto, CA, USA). The experimental conditions of the GC/MS system were described by (Scapinello et al., 2018) with some adjustments. Briefly, the system conditions were as follows: Agilent 19091S capillary column, dimension: 30 m × 250 μm × 0.25 μm. The mobile phase flow (carrier gas: He) was adjusted to 1.0 mL min −1 . The GC temperature program was 40.0 °C at 4.0 min to 240.0 °C at a rate of 10 °C min −1 and up to 300.0 °C at a rate of 40.0 °C min −1 (maintained for 5 min). The injector temperature was 280.0 °C, sample injection volume 1 μL, split ratio 1:20, the extract was solubilized in dichloromethane 10 mg ml -1 . The MS transferline temperature was set to 150.0 °C and the source of ions temperature was set at 230.0 °C. For GC-MS detection, an electron ionization system was used with ionization energy set at 70 eV, and mass range atm/z 40-400. The chemical components present in the extracts were identified by comparison with the equipment library (Agilent P/N G1033A). The relative amounts of each individual component were calculated using their respective peak areas in the chromatogram.

Experimental animals
The International Guidelines for Care and Use of Laboratory Animals were followed for all experiments, and the experimental protocol was approved by the local Ethics Committee on Animal Use (CEUA-Unochapeco Number 006/2016). Male Wistar rats weighing 160-200 g (50-55 day-old) from the Central Animal House-Unochapecó were used. They were housed in plastic cages and fed on pellets (Biobase -Biotec © ) with free access to tap water. Room temperature was controlled at 22 ± 2 °C with a 12 h light:12 h dark cycle (lights on at 07:00 am and off at 07:00 pm) and minimal noise. Animals described as fasted were deprived of food for 12 h but it had free access to water.

Studies of Campomanesia xanthocarpa seeds extract on the oral glucose tolerance curve
Rats were divided into groups of six animals. Group I: normal rats that received 1% tween 80 solution in saline (0.5 mL/100 g bw); Group II: hyperglycemic rats that received glucose solution (4 g/kg; 8.9 M); Group III, IV and V: hyperglycemic rats that received glucose solution plus Campomanesia xanthocarpa seeds extract solution (200, 400 or 800 mg/kg, respectively) (Zanatta et al., 2008) and Group VI: hyperglycemic rats treated with glibenclamide (10 mg/kg) + glucose solution, by gavage. Glucose (4 g/kg) was administrated 30 min after the rats received the treatment (zero time) (Frederico et al., 2012). Blood samples from the tail vein were collected just before (zero time), and at 60, 90 and 210 min after treatment and the serum glucose levels (mg/dL) were assayed by a glucometer (Accu-Chek® Performa) (Nicolau et al., 2009). At the end of the experimental period, the animals were anesthetized by a mixture of lidocaine and sodium thiopental (10 and 150 mg/kg, respectively). Blood aliquots were collected by cardiac puncture for biochemical analyses, and the animals were then euthanized by exsanguination (CONCEA, 2013). Liver and soleus muscle were collected, weighed, and immediately homogenized for the evaluation of glycogen content. A segment of the small intestine was removed for disaccharidases activity assay.

Glycogen content measurements
Glycogen was isolated from the rat liver and soleus muscle, and measured after 210 min of treatment as described by Krisman with minor modifications (Zanatta et al., 2008).

Disaccharidases isolation and assay
A segment of the small intestine was removed, washed, dried, weighed, trimmed and homogenized with 0.9% NaCl. The resulting extract was centrifuged (8,000 rpm/8 min) (Pereira et al., 2012). Maltase (EC 3.2.1.20), lactase (EC 3.2.1.23) and sucrase (EC 3.2.1.48) activities were determined using a glucose diagnosis kit based on the reagent glucose oxidase. For determination of the disaccharidase activity 10 μl of supernatant were incubated at 37 °C for 60 min with 10 μl of the substrate (Dahlqvist, 1984). One enzyme unit (U) was defined as the amount of enzyme that catalyzed the release of 1 μmol of glucose per min under the assay conditions. The specific activity was defined as enzyme activity (U) per mg of protein.
Protein was determined according to method previously described by Peterson (1979).

Biochemical analysis of blood samples
Serum total cholesterol (TC), LDL, HDL, triglycerides (TG), creatinine and aspartate aminotransferase (AST) levels were determined by enzymatic colorimetric methods (UV/Vis) using commercial Labtest® kits according to the manufacturer's instructions.

Insulin serum measurement
The plasma insulin levels were measured by ELISA according to the manufacture's instructions. Insulin levels were estimated by means of colorimetric measurements at 450 and 550 nm through interpolation from a standard curve. Samples were analysed in duplicate and results were expressed as ng insulin/ml serum (Castro et al., 2018). The sensitivity of the assay was 5 µUI/mL.

Statistical analysis
The results are means ± SEM. One-way or two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test were used to identify significant differences between groups. Differences were considered significant at p ≤ 0.05.

Chemical constituents of Campomanesia xanthocarpa seeds extract
The critical CO 2 pressure is 73.8 bar at 31.1 °C, which is relatively easy to achieve and allows for industrial scale use without much energy expenditure (Ruttarattanamongkol et al., 2014). The pressure used in this experiment was 250 bar (density 879 kg/m 3 ). The polarity of the supercritical CO 2 in these conditions is similar to that of hexane or others non polar solvents commonly used in extraction processes with the advantage of being easily separated from the extract without changing its chemical composition (Shi et al., 2018). Thus, low polarity causes that mainly apolar compounds such as terpenes, fatty acids and carotenoids are extracted. Table 1 shows the compounds identified by chromatographic analysis. Among them it can be highlighted the higher concentration of caryophyllene (11.80%), α-Eudesmol (11.64%), Guaiol (9.52%), α-Selinene (9.20%) and β-cadinene (7.87%).
The presence of a variety of compounds in the extract as well as the synergy between them may have been responsible for the biochemical results verified in this work. Natural products represent a source of biologically active molecules that have several important physiological, preventive and curative roles. The α-Eudesmol, one of the major compounds of C. xanthocarpa extract, is a sesquiterpene known to reduce cell proliferation and to induce tumour cell death (Bomfim et al., 2013) besides the anti-neurogenic inflammation action (Asakura et al., 2000).
Similarly, guaiol has been shown to have good action as a promising drug against Leishmania amazonenses (Garcia et al., 2018) and for antitumor treatment in lung neoplasms (Yang et al., 2016). Caryophyllene is a natural sesquiterpene that presents a wide range of biological activities, including anesthetic, antioxidant, anti-inflammatory, anticancer and anti-diabetic actions (Pant et al., 2014;Basha and Sankaranarayanan, 2014). Others have also reported that sesquiterpenes and other terpenoids have an anti-diabetic potential by stimulating insulin secretion and glucose uptake, improving glycogen synthesis and inhibiting α-glucosidase (Zhao et al., 2012;Naveen and Baskaran, 2018). In this context, the effects observed with C. xanthocarpa may be due to the presence of sesquiterpenes such as caryophyllene.

Effect of Campomanesia xanthocarpa seeds extract on oral glucose tolerance curve and insulin secretion
The effects of different doses of the extract from C. xanthocarpa seeds on the glucose tolerance curve are demonstrated in Figure 1. By 30, 60 and 180 min after starting the glucose tolerance test (which correspond to times 60, 90 and 210 min, respectively), blood glucose concentration was significantly higher than at zero. A single oral administration of the extract (400 and 800 mg/kg) caused a significant antihyperglycemic (-13.24% and -6,85%) effect after 60 min of treatment when compared to the respective hyperglycemic group (p<0.0001 and 0.001, respectively). Also, 400 mg/kg of extract significantly reduced the glycemia (-9,30%) after 90 min (p<0.0001). Additionally, 10 mg/kg glibenclamide, an insulin secretagogue, showed a typical hypoglycemic effect from 60 to 210 min after treatment, producing lower glucose levels compared to the hyperglycemic group (p<0.0001 at all times). Finally, at all studied times no change was observed in insulin levels with the extract treatment (data not shown) suggesting that the effect of extract on glycemia is not due to increased pancreatic insulin secretion. Among the possible targets of action of natural products in glycemic control are the increased of glucose uptake by tissues, inhibition of intestinal glucose absorption, increased glycogen synthesis, inhibition of dipeptidyl peptidase-IV (DPP-IV), among others (Naveen and Baskaran, 2018). Our results are in accordance with the findings of the studies carried out with the leaves of C. xanthocarpa in diabetic rats (Vinagre et al., 2010) and in rats subject to a hypercaloric diet (Biavatti et al., 2004) that demonstrated the antihyperglycemic potential of this species. Apparently, the reduction in blood glucose was not as intense, however other pharmacological studies with medicinal plants with hypoglycemic properties consider 12% to 20% reduction in blood glucose levels a significant reduction (Sánchez-Salgado et al., 2007).
Several plants rich in terpenes, such as sesquiterpenes found in C. xanthocarpa have been shown an effect on blood glucose levels (Alam et al., 2018;Belhadj et al., 2018). Considering the presence of terpenoids in the extract, among them the caryophyllene that previously demonstrated anti-diabetic activity (Basha and Sankaranarayanan, 2014), the observed effect on glycemia may be due to the presence of these compounds. Furthermore, it is important to highlight that this is the first time that the antihyperglycemic potential of the seeds of C. xanthocarpa has been demonstrated evidencing the possibility of a better exploitation of the plant. Figure 2A shows that the muscle glycogen content was significantly increased (+70.3% and +57.44%) 210 min after the administration of 400 and 800 mg/kg of extract, respectively, compared with hyperglycemic rats (p<0.05). Moreover, the glycogen content in liver of rats treated with 400 mg/kg of extract enhanced (+107.86%) when compared with hyperglycemic group (p<0.05; Figure 2B).

Glycogen content after treatment with Campomanesia xanthocarpa extract
Assessment of glycogen content is used as a marker for the evaluation of the antidiabetic activity of natural products (Kang et al., 2008). In mammals, glycogen accumulation is a physiological response to the increase in blood glucose concentration that occurs after a meal, with skeletal muscle and liver as the major storage sites. Glycogen metabolism is regulated by insulin/glucagon through activation and/or inhibition of several enzymes and proteins (Ferrer et al., 2003). The demonstration of the stimulatory effect of the C. xanthocarpa extract on glycogen content in the skeletal muscle and liver suggests an additional and expressive role in managing glucose in order to ameliorate the glucose postprandial state.
Our findings are in agreement with the study of Vinagre et al. (2010) that demonstrated the oral administration of decoction of C. xanthocarpa leaves restored the levels of glycogen in diabetic rats. Simillary Basha and Sankaranarayanan (2014) observed that carryophilene (sesquiterpene presentes in C. xanthocarpa) restored the levels of glycogen. Terpenoids and plant extracts with proven antihyperglycemic effect have been shown to influence glycogen deposition in different tissues as well as to interact with key enzymes of the glycolytic route (Sridhar et al. 2005;Henriques, 2017;Leonidas et al., 2017).

Inhibitory effect of Campomanesia xanthocarpa extract on disaccharidases activity
The role of the C. xanthocarpa extract in disaccharidase assays are showed in Figure 3. A significant inhibitory effect on maltase (-50.16, 60.88 and 57.50%; p<0.05, p<0.01 and p<0.01, respectively, Figure 3A) and sucrase (79.59, 83,65 and 51.44; p<0.01, p<0.01 and p<0.05, respectively, Figure 3B) activity was observed after 210 min of treatment with all doses (200, 400 and 800 mg/kg) of the extract in hyperglycemic rats. No significant changes were observed on lactase activity ( Figure 3C). The intestine plays an important role in glucose homeostasis. Inhibition of the activity of the carbohydrate-hydrolyzing enzymes, such as α-glucosidase, generates a delay in glucose absorption and is an important therapeutic approach in the control of postprandial glycemia (Melo and Carvalho, 2006). Some plants with properties similar to antidiabetic α-glucosidase inhibitor drugs, such as acarbose, were identified (Kumar et al., 2011;Henriques, 2017). Additionally, studies have shown that sesquiterpenes, present in large quantities in the extract of C. xanthocarpa, have the ability to inhibit α-glycosidases improving glucose metabolism (Alam et al., 2018) as well as other terpenes (Ding et al., 2018). Figure 4 presents the lipid profile of hyperglycemic rats treated with C. xanthocarpa extract. The 400 mg/kg C. xanthocarpa-treated group presented significantly lower serum total cholesterol (TC) and LDL cholesterol concentrations than hyperglycemic group ( Figure 4A and D, respectively). The reduction on TC levels was about 26%, and the level of LDLwas reduced by around 49% in rats treated with 400 mg/kg of extract. All the doses of the extract were found to be ineffective in reducing both serum triglycerides, VLDL and HDL cholesterol.

Effect of Campomanesia xanthocarpa extract on lipid profile
Our results are in accordance with previous studies with humans demonstrating that oral administration of capsules of leaf from C. xanthocarpa reduced blood total cholesterol and LDL levels without changing the triglyceride, VLDL or HDL levels in hypercholesterolemic individuals (Klafke et al., 2010;Viecili et al., 2014). Also, Espindola et al. (2016) demonstrated that C. adamantium root aqueous extract displayed a reduction in serum levels of total cholesterol and triglycerides in rats. However, this hypocholesterolemic effect was not observed in rats treated with infusion (Biavatti et al., 2004) or decoction (Vinagre et al., 2010) of the leaves of C. xanthocarpa. These differences observed on C. xanthocarpa effect could be due to the different method of leaf preparation (trituration, infusion, decoction) which could be influencing in the amount and profile of bioactive compounds (Rodrigues et al., 2015). The presence of some phytochemicals like terpenes and flavonoids in C. xanthocarpa (Capeletto et al., 2016) could also be the cause of the decrease of total and LDL cholesterol since bioactive compounds act as reactive oxygen species scavengers (Klafke et al.,2010) or therefore, the possible mechanism involved in cholesterol reduction could be inhibition of HMG-CoA reductase, as demonstrated by Klafke et al. (2010). Baldissera et al. (2017) found that β-caryophyllene, a sesquiterpene presents in C. xanthocarpa extract seeds, has hypolipidemic effect via inhibition of the hepatic HMG-CoA reductase and Youssef et al. (2019) demonstrated that caryophillene improved glycemic parameters, dyslipidemia, and vascular oxidative stress in rats subject to diet-induced dyslipidemia.
It is estimated that 30-60% of patients with T2DM have dyslipidemia. Besides hyperglycemia, hyperlipidemia is another condition that presents a key role in the development of the micro-and macrovascular complications of diabete that may result in cardiovascular events like myocardial infarction and stroke (Feingold and Grunfeld, 2000). Thus, T2DM patients with an increased risk for cardiovascular disease commonly use a combination therapy consisting of the anti-diabetic and a cholesterol-lowering drugs (van Stee et al., 2018). Therefore, the use of a single drug capable to control serum glucose and lipid levels is extremely advantageous. Figure 5 shows that there was no significant change on creatinine and AST levels in hyperglycemic rats treated with different doses of C. xanthocarpa, suggesting that acute administration does not cause damage to the liver and kidneys. Similarly, Viecili et al. (2014) demonstrated there was no significant change in the AST, ALT, urea and creatinine levels in hypercholesterolemic individuals after 90 days of treatment with C. xanthocarpa leaves. Additionally, da Silva et al. (2016b) demonstrated that animals exposed to C. xanthocarpa, did not exhibit clinical signs of acute toxicity for the administered doses.

Conclusions
In summary, our study showed for the first-time the in vivo beneficial role of C. xanthocarpa extract seeds, obtained by a green extraction technique, in glycemia and lipid profile on hyperglycemic rats. These data provide evidence to justify the use of C. xanthocarpa in the folk medicine to treat diabetes and hypercholesterolemia. Despite these findings, additional studies must be performed to define the mechanism of action and possible molecular target.