Bioprospecting antiobesity and anticholesterol activity of ethanolic extract of Clidemia hirta leaves grown at Merapi Mountain National Park in a high fat diet-induced obese Swiss Webster mice

Clidemia hirta is one of the invasive species. Its presence is undesirable in conservation areas such as Merapi Mount National Park. Bioprospecting its antiobesity activity could not only be the alternative solution to solve obesity problems but also to control their presence in Merapi Mount National Park. This study aims to determine the antiobesity activity of the ethanolic extract of C. hirta against the Swiss Webster strain male mice induced high-fat diet. Dried leaves were extracted using ethanol 96% with the maceration method for 48 hours. The extract was investigated for its secondary metabolites’ quantification for flavonoid, tannin, alkaloid, and total polyphenol content. Then the antiobesity test was carried out by dividing six treatment groups consisting of 4 mice. The treatment group was divided into normal control, negative control, positive control (orlistat 60 mg/kg body weight), and extract doses of 250, 500, and 750 mg/kg body weight. The parameter observed was the activity of mice, feces, feed residue, body weight, and total blood cholesterol levels for each group of mice during the treatment for 28 days. The results showed that extracts contained 35.56 ± 1.99 mg QE/g, 2.651 ± 5.10 mg TAE/g, 4.500 ± 0.49 mg GAE/g, 0.305 ± 0.85 mg AE/g. Ethanolic extract of C. hirta leaves showed antiobesity and anticholesterol activity compared to positive control. Clidemia hirta is a potential plant to be developed as a product for antiobesity and anticholesterol.


Introduction
Obesity is an increase in body fat tissue mass due to an imbalance between nutrient intake and energy use [1].According to the 2018 National Basic Health Research Report,the prevalence of overweight and obesity is 16.0% in adolescents aged 13-15 years and 13.5% in adolescents aged 16-18 years [2].Based on a 2014 Global Nutrition Report, Indonesia is one of the countries with nutritional problems, obesity, and overweight (overweight) [3].One of the efforts to reduce the prevalence of obesity to prevent the risk of degenerative diseases is the drug orlistat.Orlistat is a reversible gastrointestinal lipase inhibitor that prevents reabsorption by 30% and inactivates hydrolysis of dietary fat, thereby reducing caloric intake in obese patients [4].The use of orlistat is still limited because it is relatively expensive and has some side effects on the gastrointestinal tract, kidney function, and liver [5].Noticing the side effects caused, other alternative therapies are needed to minimize side effects by utilizing natural ingredients or switching to herbs that have medicinal properties.The use of natural ingredients is considered safer for consumption by the community.It is also believed to prevent, reduce or treat disease.One alternative medicinal plant is Harendong Bulu (Clidemia hirta (L.) D. Don).
Clidemia hirta is one of the invasive species.Its presence is undesirable in conservation areas such as Merapi Mount National Park.Bioprospecting its antiobesity activity could not only be the alternative solution to solve obesity problems but also to control their presence in Merapi Mount National Park.The content of secondary metabolites of C. hirta are tannins, flavonoids, and steroids/triterpenoids.Metabolic compounds such as flavonoids, tannins, saponins, and ellagic acid have 1255 (2023) 012069 IOP Publishing doi:10.1088/1755-1315/1255/1/012069 2 antioxidant activity by reducing adipocyte oxidative stress to prevent obesity [6].Based on the properties possessed by C. hirta leaves, it is necessary to research the antiobesity and anticholesterolemia activity of C. hirta leaves to provide information on natural ingredients for antiobesity for the community.

Plant materials
Fresh leaves of C. hirta were taken from the Turgo area of Mount Merapi National Park.The preparation of dried leaves consists of a sampling process, dry sorting, washing, wet sorting, and drying.Leaf retrieval was taken manually in the field with the criteria for fresh green leaves measuring 5 to 15 cm.Drying the leaves using an oven at 40℃ for 1x24 hours.The dried C. hirta leaves were mashed using a blender and grinder and then sieved with mesh 61.The finely sifted powder was put into a plastic clip, weighed, labeled, and then stored in a closed container [7].

Animal test methods
The research was conducted according to Regulations of The Drug and Food Control Agency Number

Extraction
Dried leaves of 100 g were macerated for 2x24 hours using 500 mL ethanol 96% as solvent.The extract was filtered and evaporated using a vacuum rotary evaporator.The yield of the extract was measured.The concentrated extract was then preserved in the refrigerator and used as the sample.

Total flavonoid content
The total flavonoid content method was carried out by Stankovic (2011) with modification [9].Samples of harendong bulu leaf extract were taken as much as 100 mg, then put into a test tube, and added 2 mL of 96% ethanol.Then the solution was homogenized with vortex so that a solution with a concentration of 50.000 ppm was obtained.The diluted solution is taken as much as 1 mL, put into a test tube, and then added 96% ethanol as much as 9 mL until a solution with a concentration of 5.000 ppm is obtained.The solution was taken as much as 0.5 mL and then added 0.1 mL of 10% AlCl3 and 0.1 mL of K-Acetate 1 M, and 4.3 mL of aqueous in 3 different test tubes (3x repeats).The solution is then incubated in a dark room for 30 minutes.The sample solution is then absorbed using a spectrophotometer at a wavelength of 415 nm.Flavonoid levels are calculated by the formula:

Total alkaloid content
Samples of C. hirta leaf extract were taken as much as 10 mg, then put into a test tube, and added 96% ethanol as much as 1 mL.The solution was homogenized with a vortex to obtain a concentration of 10.000 ppm.The diluted solution is taken as much as 1 mL and put into a test tube, added with 9 mL 96% ethanol until a solution with a concentration of 1.000 ppm is obtained.A solution of 1 mL was taken, and 0.5 mL of phosphate buffer, 0.5 mL of BCG, and 2 mL of chloroform were added.The solution is then separated by a vortex with an interval of 20-30 seconds.The chloroform phase was then taken and added with as much as 1 mL chloroform.The absorption is measured using a spectrophotometer at a wavelength of 430 nm.The total alkaloid contents were calculated from linear regression of a standard curve prepared with atropine sulfate.The content of total phenolic compounds is expressed as mg AE (atropine equivalent/ g extract).Each sample was done in triplicate.

Total tannin content
A total of 1 mL of 100 ppm concentration was taken and poured into three different test tubes.In each test tube, Folin-Ciocalteu reagent was added to as much as 0.5 mL, then added 20% Na2CO3 as much as 2 mL and covered with aluminum foil.Next, incubated for 30 minutes inside a dark room.The absorbance is calculated with a wavelength of 500 nm, and the number of tannins in the sample is calculated.The total tannin contents were calculated from linear regression of a standard curve prepared with tannic acid.The content of total phenolic compounds is expressed as mg TAE (tannic acid equivalent/g extract).Each sample was done in triplicate.

Total phenolic content
The total phenolic content of the C. hirta extract was analyzed by using the Folin-Ciocalteau method [10] with some modifications.Gallic acid was used as a reference standard for plotting the calibration curve.A volume of 0.4 mL of the plant extract was mixed with 0.4 mL of the Folin-Ciocalteau reagen, and added 4 mL of sodium carbonate solution (7 %).The reaction mixture was incubated at room temperature for 1 hour.The absorbance was measured at 750 nm using a UV-VIS spectrophotometer.The total phenolic contents were calculated from linear regression of a standard curve prepared with gallic acid.The content of total phenolic compounds is expressed as mg GAE (gallic acid equivalent/ g extract).Each sample was done in triplicate.

Antiobesity activity test
Swiss Webster strain male mice were acclimatized for 7 days under laboratory conditions at 27 C.
During the acclimatization period, the mice were given a standard feed of 3-5 g/day and water ad libitum, and the cage was cleaned every 3 days and a 12-hour light and dark cycle.Mice in the treatment group, negative control, and positive control were given a high-fat diet of 5 g/day and water ad libitum, while the normal group was given a standard diet of 5 g/day.Induction was carried out for 21 days.The comparison drug orlistat with a dose of 60 mg/kg body weight (BW) and extract of C. hirta leaves was weighed according to the treatment at doses of 250, 500, and 750 mg/kg body weight dissolved in 0.5% carboxymethyl cellulose (CMC) administered to mice using an oral needle.Tests were carried out every day during the treatment period for 28 days after the induction of obesity.Mice activity, feed weight, remaining feed, mouse weight, and fecal weight were weighed daily [11].

Measurement of cholesterol levels in mice
Cholesterol levels were measured after the obesity-inducing period (treatment of harendong bulu extract) on days 0, 7, 14, 21, and 28.Mice measured cholesterol levels will be satisfied for 18 hours first, then measured cholesterol levels of mice taken from blood samples through capillary blood vessels by cutting the tail of mice about 1 mm, previously cleaned first with 70% alcohol using scissors.The first drop of blood is removed then cholesterol levels are measured.Mouse blood cholesterol levels are normally between 26.0 to 82.4 mg/dL [12].

Results and Discussions
Simplisia is an extract of raw material that must meet monographic requirements and specific and nonspecific standard parameters [13].Standardization is carried out to ensure the quality of the raw materials used to maintain stability, safety, and consistency of the active compound content in the raw materials to be used by referring to specific and non-specific parameters [14].The results of the standardization of C. hirta leaf simplisia can be seen in Table 1.The specific parameters tested were water soluble and ethanol levels, while the non-specific parameters tested were total ash content and insoluble acid ash, drying loss, and water content.All the parameters were appropriate based on the standard.Thus, the simplisia could be used as raw material for the extraction process.
The extracts were then carried to the analysis of phytochemical screening.The qualitative phytochemical screening was tested for alkaloid, triterpenoid, steroid, flavonoid, tannin, and saponin content.Table 2. shows that C. hirta leaves contain flavonoids, steroids, tannin, saponin, and alkaloids.Triterpenoid was not found in C. hirta leaves.This result is in accordance with Tuginah et al. (2020) that in C. hirta leaves, there are no triterpenoid compounds [15].The differences might be due to the different growth places since biosynthesis of the secondary metabolite is affected by growth place [16].Ethanolic extracts of C. hirta leaves then be investigated for the quantitative analysis of secondary metabolites and the extraction yield.Ethanol solvents were used because they include GRAS (Generally Recognized as Safe) solvents by the FDA (Food and Drug Administration) [17].Ethanol solvents can enter plant tissues so that many compounds can be extracted [18].In addition, ethanol solvents can extract many compounds from plants.The result of extraction yield and quantitative analysis are shown in Table 3.The maceration process with optimal time results in contact between the sample and the solvent more often, increasing the yield until it reaches the saturation point of the solution.Contact of the sample with the solvent is assisted by stirring so that the sample and solvent experience more frequent contact and an optimal maceration process occurs.The high % yield (Table 3) can be influenced by the extraction time carried out for 48 hours.In addition, the % yield produced shows that 96% ethanol solvent is suitable for extracting C. hirta leaves.The yield of 37.98% shows a good yield result because it has a > value of 10% [19].The total flavonoid levels of ethanol extract of C. hirta leaves in this study (Table 3) were 35.56 ± 1.99 mg QE/g extract.The polarity of the solvent used in the extraction process can affect total flavonoid levels.the polarity of ethanol would be higher or increase inversely proportional to the decrease in concentration in water [20].A mixture of water with ethanol will produce extracts with high quality and quantity of tannins because the polarity of water with ethanol is in accordance with the polarity of the tannin compound and polyphenols.Flavonoids are antioxidants with several functions or activities related to weight loss and cholesterol [21].In addition, flavonoids also function as cholesterol-lowering by protecting cells (outside and inside cells) [15].Alkaloids inhibit the activity of lipase enzymes that break down fat so that fat is removed from the body through feces [22].In addition, this compound can inhibit the activity of pancreatic lipase enzymes, thereby increasing fat secretion through feces [23].
Antiobesity and anticholesterolemia activity of C. hirta leaves was carried out for 28 days by giving the extract once a day, and checking total cholesterol levels was carried out at the beginning before treatment and at the end of each treatment week, on the 0th, 7th, 14th, 21st, and 28th days with induction of high-fat feed by 13.98% for the previous 21 days.The result is shown in Figure 1.Body weight measurement was carried out to determine the development of mouse weight during the treatment of adding high-fat feed with ethanol extract of C. hirta leaves with concentrations of 250, 500, and 750 mg/kg body weight for 28 days.The higher intake of mouse feed can induce weight gain and increase body weight because mice experience increased energy intake [24].The treatment showed a weight loss trend in the positive control group (orlistat 60 mg/kg body weight) and the administration of ethanol extract doses of C. hirta leaves of 250, 500, and 750 mg/kg body weight group.In comparison, the negative control showed weight gain.Furthermore, negative control treatment increased from day 0 to day 28 with an initial body weight of 29.75 ± 2.22 g and a final treatment weight of 33.75 ± 2.99 g, an increase of 4 g (Table 3).The treatment dose of 250 mg/kg body weight decreased by 5 g from the initial body weight of 30 ± 6.27 g to the final body weight of 24 ± 2.83 g.The treatment dose of 500 mg/kg body weight decreased from day 0 to day 28 with an initial body weight of 29.75 ± 2.63 g and a final body weight of treatment of 23.25 ± 1.50 g, which decreased by 6.5 g.
Cholesterol is the main fat or lipid molecule present in the bloodstream.Total cholesterol levels were measured to determine the development of total cholesterol levels of mice during the treatment of ethanol extract of C. hirta leaves with concentrations of 250, 500, and 750 mg/kg body weight for 28 days.Total cholesterol measurement was carried out from the beginning after the obesity feeds induction process on the last day, then at the end of every week (days 7, 14, 21, and 28) during the 28-day extract treatment period.It calculated the difference to find the result.Based on the research that has been done, the average results of total cholesterol are obtained in Table 4.Total cholesterol levels in 6 treatment groups on day 0 (initial) were measured after the induction treatment of high-fat artificial feed, which was 14%.Initial total cholesterol levels (day 0) show total cholesterol levels with a range of 124 to 143 mg/dL, which is already in the range of abnormal total cholesterol levels of mice, namely in the range of 40-130 mg/dL.This high total cholesterol condition falls into the hypercholesterol category, where total cholesterol levels exceed the maximum standard level of 130 mg/dL [25].This proves that the induction period of 21 days can generate hypercholesterolemia in mice.Based on the data in Table 4. showed a decrease in total cholesterol levels in the normal control group, positive control (orlistat 60 mg/kg body weight), and at the dose of ethanol extract of C. hirta leaves of 250, 500, and 750 mg/kg body weight.In contrast, the negative control showed an increase in total cholesterol levels.
Normal control treatment decreased from day 0 to day 28 with initial cholesterol levels of 124 ± 5.72 mg/dL and final body weight of treatment of 121.75 ± 4.65 mg/dL, which decreased by 2.25 mg/dL.Negative control treatment increased from day 0 to day 28 with initial cholesterol levels of 124 ± 6.68 mg/dL and final treatment weight of 133 ± 2.22 mg/dL, an increase of 9.25 mg/dL.Negative control treatment increased from day 0 to day 28 with initial cholesterol levels of 124 ± 6.68 mg/dL and final treatment weight of 133 ± 2.22 mg/dL, an increase of 9.25 mg/dL.The decrease in total cholesterol levels per dose given is not significant.This can be influenced by the concentration level of an extract so that body weight will have different concentrations at the concentration of doses given from 250, 500, and 750 mg/kg.

Figure 2. Total cholesterol level of high fat diet-induced obese Swiss Webster mice
This decrease in total cholesterol levels (Figure 2) shows that with ethanol extract of C. hirta leaves dose of 250 mg/kg body weight which is not too concentrated, can be well absorbed so that existing chemical compounds can reach the target location because they are perfectly penetrated and leads to the decrease of total blood cholesterol levels.The tannin compounds in the intestinal organs will shrink the intestine so that peristalsis becomes reduced and inhibits pancreatic lipase enzymes [26].At a dose of 750 mg/kg body weight decreased, although not significantly, to 14.67 mg/dL.This can occur due to the influence of too concentrated extracts, so the chemicals contained in the ethanol extract of C. hirta leaves rise and cause interactions with insignificant effects with low doses.The effects of each of these components are synergistic.
There was an increase in total cholesterol levels in the negative control treatment group because mice were left obese with high-fat artificial feed without treatment.This happens because of the consumption of feed containing high fat and cholesterol contained in artificial feed [27].The amount of fat content in artificial feed is 14%.The positive control treatment (orlistat 60 mg/kg body weight)  cholesterol levels significantly at all doses given concentrations.In addition to a decrease in total cholesterol levels, there is also weight loss and antiobesity activity.Flavonoids as antioxidants can work as pancreatic lipase inhibitors and reduce appetite.Inhibition of triglyceride hydrolysis through lipase enzyme inhibition can reduce and prevent obesity.Flavonoid compounds can reduce glucose levels in the blood and regulate the activity of enzyme secretion involved in carbohydrate metabolism [28].Flavonoids have activity in inhibiting the work of lipase enzymes.In the body, lipase enzymes can hydrolyze oil into saturated fatty acids [29].Besides being able to reduce appetite, flavonoid compounds can also reduce cholesterol levels in the blood by inhibiting the work of the enzyme 3-hydroxy-3-methylglutaroyl-coenzyme A (HMG-CoA) and acetyl-coenzyme A acetyltransferase (ACAT) so that cholesterol synthesis in the liver becomes inhibited [30].Cholesterol transported to the liver from the intestine, the enzyme HMG-CoA reductase will convert acetyl-coA into mevalonate in the process of cholesterol synthesis so that the results of the cholesterol synthesis process produced by the liver will decrease [31].
Tannin compounds play a role in preventing obesity by reducing body mass and fat, whereas tannin compounds play a role in contracting the intestine so that peristalsis is reduced [32].tannins have an inhibitory mechanism of pancreatic lipase enzymes so that less fat is absorbed by the body [33].In addition, tannins function in inhibiting fat absorption in the intestinal organs, namely by the reaction between mucosal proteins and intestinal epithelial cells, so that fat levels that cause obesity can decrease.Tannins will also precipitate protein mucosa on the surface of the small intestine so that the effectiveness of cholesterol and fat absorption will be reduced.Alkaloid compounds inhibit the activity of lipase enzymes, such as the mechanism of action of orlistat.Alkaloids work by inhibiting lipase enzymes in the digestive tract so that the fat absorption process derived from triglyceride hydrolysis will be inhibited.In addition to flavonoids, tannins, and alkaloids, saponin compounds also play a role in antiobesity activity, losing weight by inhibiting pancreatic lipase activity that binds to cholesterol.Saponins will enter the gastrointestinal tract and are not absorbed by the digestive tract so that saponins bound to cholesterol can come out of the gastrointestinal tract [33].Secondary metabolite compounds in C. hirta leaves reduce body weight and total cholesterol levels of obese mice.

Conclusion
Ethanolic extracts of C. hirta leaves have been able to reduce body weight and reduce total cholesterol levels.It can be concluded that the C. hirta leaves have the ability as an antiobesity and anticholesterolemia with the presence of secondary metabolite compounds flavonoids, tannins, polyphenols, and alkaloids.Clidemia hirta leaves could be further investigated for bioprospection as the product for antiobesity and anticholesterolemia.
=       C = Concentration of compound in sample solution (mg/L) V = Volume of sample solution (mL) Fp = Dilution factor W = Sample weight (mg)

Figure 1 .
Figure 1.The body weight of high fat diet-induced obese Swiss Webster mice for 28 days with various treatment 18 of 2021 about "Preclinical Pharmacodynamic Tests Guidelines Traditional Medicine" and method animal test was established by Health Research Ethics Committee, Faculty of Medicine, Duta Wacana Christian University with ethical clearance No. 1407/C.16/FK/2022.Permission of the ethical commission is available.

Table 1 .
The result of simplisia standardization of C. hirta leaves

Table 2 .
Qualitative phytochemistry of C. hirta leaves

Table 3 .
Extraction yield and quantitative analysis of secondary metabolites

Table 4 .
Body weight of high fat diet-induced obese Swiss Webster mice

Table 5 .
Total cholesterol level of high fat diet-induced obese Swiss Webster mice for 28 days showed weight loss from day 0 to day 28 treatment with an initial body weight of 136.5 ± 9.57 mg / dL decreased to 114.25 ± 1.7 mg/dL, decreased by 22.25 mg/dL.This suggests that orlistat plays a role in reducing total cholesterol levels in mice by inhibiting fat absorption.A treatment dose of 250 mg/kg body weight decreased the total cholesterol levels of mice by 28.25 mg/dL from initial levels of 143.75 ± 27.4 mg/dL to final levels of 115.5 ± 0.7 mg/dL.A treatment dose of 500 mg/kg body weight decreased total cholesterol levels from day 0 to day 28 treatment with an initial body weight of 138.5 ± 3.87 mg/dL and final total cholesterol levels of treatment of 112 ± 1.63 mg/dL, decreased by 26.5 mg/dL.The treatment dose of 750 mg/kg body weight decreased total cholesterol levels by 14.67 mg/dL from initial levels of 126 ± 8.04 mg/dL to final body weight of 111.33 ± 2.31 mg/dL.Treatment of ethanol extract of harendong bulu leaves by 250, 500, and 750 mg/kg body weight for 28 days can reduce total