Profile of secondary metabolites of Citrus hystrix DC from several solvents and its potential as an antibacterial substance

Exploration of antibacterial activity from various herbs like Citrus species can become the solution for reducing emergency resistance antimicrobial. In this study, the secondary metabolite profiles of kaffir lime leaves extract (Citrus hystrix DC) were explored, which were obtained from several solvents that have potential as antibacterial agents. Extraction of kaffir lime leaves was carried out using 96% ethanol, n-hexane, and ethyl acetate, with the comparison between simplicia and solvent was 1:5. Profile metabolites secondary observed in the study were total phenol content, total flavonoids content, saponins, tannins and antioxidants activity. Extraction of kaffir lime leaves using 96% ethanol, n-hexane, and ethyl acetate produced extract yields of 5.28%, 0.94, 1.72% respectively. The highest content of flavonoids, phenols, and tannins was from the extract of kaffir lime leaves using 96% ethanol that was which is 65.02 (mg equivalent quercetin / g extract), 4.94 (% w/w) and 3.51 (% w/w). The highest content of saponins on kaffir lime leaves extracts from hexane was 10.67 (% w/w). The best antioxidant activity was from the extract of kaffir lime leaves using 96% ethanol which was 21.81 (IC 50) (μg/mL). Kaffir lime leaves ethanol extract showed high total phenol, flavonoid, tannin content as well as high antioxidant activity.


Introduction
Secondary metabolites are metabolic intermediates or products that are formed in response to stress but are not necessary for the growth and survival of the generating plants.Instead, they are needed for the interaction of plants with their environment.The plant is shielded against diseases by their antibiotic, antifungal, and antiviral capabilities.[1] Citrus hystrix, Citrus auraria Michel, Citrus echinata Saint Lager, Citrus hyalopulpa Tanaka, and Citrus kerrii (Swingle) Tanaka are some other names for this citrus species, which is also referred to as 1292 (2024) 012018 IOP Publishing doi:10.1088/1755-1315/1292/1/012018 2 kaffir lime or makrut lime.It is a flowering shrub in the family Rutaceae that is native to tropical Southeast Asia, southern China, and northeastern India.It can reach heights of 3 to 6 meters.It produces warty, lumpy, and green fruits.Asian cuisine frequently uses the leaves and fruits as seasonings.[2] .
By using the DPPH and FRAP techniques, C. hystrix demonstrated strong antioxidant activity and significant phenolic and flavonoid content.C. hystrix looks to be a natural antioxidant source that can replace synthetic antioxidants.[3].Citrus hystrix (kaffir lime) leaves include bioactive substances as flavonoids, phenolics, tannins, and essential oils.[4].
A class of polyphenolic chemicals known as flavonoids are produced in plants as secondary metabolites.Flavonoids are abundantly distributed throughout the plant, and their antibacterial properties have drawn increased attention in order to enable their application as antibacterial agents [6].Besides having a function as an antibacterial agent, flavonoids also have a function as antioxidants [7] Factors that can affect the extraction results are the type of solvent used.The solvent used is adjusted to the polarity of the targeted compound.According to the principle of like dissolves like, a solvent will tend to dissolve compounds with the same polarity level.Polar solvents will dissolve polar compounds and vice versa.Phenol and flavonoid compounds are polar, so polar solvents are needed, including methanol, ethanol, acetone, and isopropanol.While ethyl acetate is a semi-polar solvent that may dissolve semi-polar chemicals on the cell wall, n-hexane is a sort of non-polar solvent that can dissolve non-polar compounds.
In order to conduct this study, Citrus hystrix DC leaves were macerated with ethanol, n-hexane, and ethyl acetate as solvents.The presence of secondary metabolites in the form of total phenols, total flavonoids, tannins, and saponins in Citrus hystrix DC leaves was then assessed using phytochemical assays.Furthermore, antioxidant activity testing was also carried out from Citrus hystrix DC extracts produced by these three types of solvents.

Materials and methods
This research has been conducted using experimental methods.The material used is kaffir lime leaves (Citrus hystrix DC), originating from Nguling District, Pasuruan Regency, East Java.The processing of kaffir lime leaves simplicia is by selecting fresh kaffir lime leaves which have been washed using running water.After cleaning the kaffir lime leaves, they were dried in an oven at 40°C for 48 hours, and then the particles were reduced using a grinding machine.
Kaffir lime leaves extract was obtained using three types of solvents, namely 96% ethanol, n-hexane, and ethyl acetate.Extraction was carried out by maceration on its solvent for 24 hours and stirring manually, then the resulting filtrate was then filtered and evaporated with a V-70 Butchi rotary vacuum evaporator with a pressure of 175 mbar and to remove water content with a pressure of 72 mbar.The parameters observed in this study were the yield of each solvent extract, the content of total phenols, total flavonoids, tannins, saponins, and antioxidant activity.The potency of kaffir lime leaves extract as an antibacterial agent was carried out through a literature review of the results of previous studies The yield of each extract was calculated as a percentage of the ratio of the weight of the extract obtained to the weight of the kaffir lime leaves simplicia powder in each solvent.Determination of total flavonoid content in the extract (TFC) using the colorimetric-AlCl3 method [8].Levels of flavonoids obtained were expressed in quercetin equivalent (mg/g extract).Determination of the total phenolic content in the extract (Total Phenolic Content-TPC) used the Folin-Ciocalteau (FC) colorimetric method [8], which has been based on the chemical reduction of the reagent, a mixture of tungsten and molybdenum oxide.The content of total phenolic compounds in the extract has been expressed as w/w based on the regression equation of the gallic acid calibration curve.Antioxidant activity was determined using the DPPH method [8].The total saponin content of each extract was determined using the spectrophotometric method and the results were compared with the saponin standard curve from Quilaja Bark at a wavelength of 435 nm.
The measurement of total tannins followed the Folin-Ciocalteu method with slight modifications [9].Results in units of Tannic acid equivalent (mg TA/g extract).Measurement using spectrophotometric method.A total of 10 mg of the extracted sample was dissolved in 10 mL of solvent.Then 500 µL was taken and put in a test tube.This solution is added to 2.5 mL of 10% Folin-Ciocalteu reagent.Then 2.5 mL of 7% Na2CO3 solution (0.7g/10ml) was added and incubated at room temperature for one hour.The absorbance value was measured with a UV-Vis spectrophotometer at a wavelength of 745 nm as a comparison is Tannic acid.Tannic acid solutions at 10-50 ppm concentrations were measured for their absorption at the maximum wavelength (745 nm).Then a relationship is made between the absorbance value (y-axis) and the concentration value (x-axis).

Extract yield
Extracts are preparations obtained from the separation of active compounds from medicinal plant tissues using selected solvents through standard procedures.The extraction methods that are commonly used include maceration and percolation.The extraction method to be used depends on the form and content of the material to be extracted.In addition, the selection of the extraction method is adjusted to the interests of obtaining the desired chemical content.Based on Fig. 1, the most kaffir lime leaves extract was obtained from 96% ethanol solvent, namely 5.28% compared to n hexane solvent (0.94%) and ethyl acetate solvent (1.72%).Factors that affect extraction such as material size, extraction time, extraction temperature, type and amount of solvent.96% ethanol, n hexane and ethyl acetate are organic solvents.

Figure 1. Extract yield of Kaffir lime (Citrus hystrix DC) leaves from several solvents
Organic solvents can be polar and non-polar depending on the polarity group they have.Polar solvents have a high level of polarity, suitable for extracting polar compounds from plants.Polar solvents are universal so they can be used to extract compounds with high polarity levels or compounds with lower polarity levels.Water, methanol, ethanol, acetic acid are polar solvents.Meanwhile, n hexane is a non-polar solvent and ethyl acetate is a semi-polar solvent.The high production of kaffir lime leaves extract from 96% ethanol indicates that the secondary metabolites in kaffir lime leaves are mostly polar compounds.

Total phenol content
The majority of vegetables and fruits contain phenolic compounds as secondary metabolites, as do other substances commonly identified as sources of polyphenols such phenolic acids, flavanol, flavanones, and flavones.[6,7].The phenol content found in plants has antioxidant abilities that can be useful for illness and disease prevention.Fig. 2 shows the total phenol content from kaffir lime (Citrus hystrix DC) obtained using different solvents.It was found that ethanol extract has the highest total phenol content compared to n-hexane and ethyl acetate.The concentration produced from using ethanol as a solvent double compared to the other two types of solvents.The results of this study follow those reported by Uçak [10], that the use of ethanol in the extraction of Citrus sinensis dan Citrus limon resulted in higher total phenol compared to Methanol and Acetone.The difference in total phenol content is also related to solvent characteristics (polar and non-polar).The polarity of the solvent affected type of phenolic content can be extracted based on the 'like and dislike property' [11] [12] .On kaffir lime leaves, transcinnamic, m-coumeric, and vanillic acids predominated while p-coumaric, benzoic, and sinapic acids were only found in trace levels [13].Total phenol content of kaffir lime can inhibit the growth of Streptococcus bacteria [14].Based on the Fig. 3 the flavonoid content of leaves of Citrus hystrix DC from ethanol extract is greater by 121% compared to n hexana extract, while the flavonoid content of ethyl acetate extract is 105% compared to n hexane.The content of flavonoids found in every plant is no exception in stomach oranges.Flavonoids have a role in the growth process of these plants.Flavonoids in plants play a role in giving color, taste to seeds, flowers, and fruits and aroma and protect plants from environmental influences, as antimicrobials, and protection from ultraviolet light exposure [15].Flavonoids function for the physiological survival of the plant itself, flavonoids have other benefits, among others, as antifungal agents, and traditional medicine.Flavonoids are certain chemicals found in plants, called phytonutrients, and have various health benefits.The greatest flavonol component is quercetin, which accounts for between 60 and 75 percent of all flavonoids along with its glycosides.[16].Flavonoids are widely known for their ability to fight off a variety of harmful microorganisms as antibacterial agents.Flavonoids have generated a lot of interest due to their potential as antibiotic alternatives due to the rising occurrence of untreatable diseases brought on by bacteria resistant to antibiotics.The following are the suggested antibacterial mechanisms of flavonoids: inhibition of cytoplasmic membrane function, inhibition of energy metabolism, inhibition of attachment and biofilm formation, inhibition of the porin on the cell membrane, modification of membrane permeability, and attenuation of pathogenicity [17].

Saponin
Hexane solvent extracts produced the highest levels of saponin, whilst ethanol and ethyl acetate produced lower levels (41.8 and 45.64%, respectively).The amphiphilic molecules known as saponins are known for their wide range of biological activities.Fungicidal, antimicrobial, antiviral, antiinflammatory, anticancer, antioxidant, and immunomodulatory effects have all been noted in relation to saponins [18].The sugar portion of saponins (glycosides) gives this molecule polar properties due to the presence of hydroxyl groups (-OH) which can interact with water and other polar compounds through hydrogen bonds and electrostatic attractions.As a result, saponins dissolve in polar solvents such as water and ethanol.On the other hand, the aglycone moiety of saponins is a non-polar component which gives these compounds their hydrophobic nature.This property makes saponins also soluble in non-polar solvents such as hexane.It is possible that the aglycone portion of saponins in kaffir lime is higher than that of sugar, so that the hexane solvent produces a higher value.Saponin-rich plants have the potential to increase the flow of microbial protein from the rumen, improve feed utilization, and reduce methanogenesis.They alter ruminal fermentation by selectively inhibiting certain bacteria and decreasing ruminal protozoa [19,20].Saponins are chemicals with an active surface that resemble detergent.They reduce the surface tension of bacterial cell walls and reduce membrane permeability, which prevents bacteria from surviving.Saponins readily penetrate cells and disrupt metabolism by producing denaturation of membrane proteins, which can lead to cell membrane damage and lysis when the surface tension is disrupted [21].Tannins are polyphenolic compounds with a range of molecular masses and degrees of complexity.In aqueous solution, they have the capacity to bind proteins.They form complexes predominantly with proteins and to a lesser extent with metal ions, amino acids, and polysaccharides due to their many phenolic hydroxyl groups [19] .The reverse transcriptase and DNA topoisomerase enzymes are inhibited by tannins' antibacterial effect, preventing the formation of bacterial cells.with its capacity to inhibit protein transport in the inner layer of cells, inactivate enzymes, and deactivate microbial cell adhesin.
Condensed tannins (CT) and hydrolyzable tannins (HT) are two types of complex polyphenolic molecules known as tannins [23] .Condensed tannins are made up of oligomers of flavon-3-ols, whereas hydrolyzable tannins have a carbohydrate core group and a phenolic carboxylate connected by an ester bond.[24].Condensed tannins are created when flavans condense, but hydrolysable tannins can be hydrolyzed from weak acids or weak bases to produce glucose and phenolic acids.The stronger solvent is needed to extract this type of tannin.This research shows that ethanol is better than ethyl acetate and hexane in extracting condensed tannins from kaffir lime leaves.The polarity rate of the solvent plays a major role in determining the high extraction yield.Ethanol is a polar compound, while ethyl acetate is semipolar, and hexane is a nonpolar compound.The amount of tannin extract is higher using ethanol because tannins are polar compounds.Several other studies reported similar results, extraction using ethanol solvents produced better tannin yields than other solvents [25][26][27] Low amounts of tannins added to bovine feed can improve rumen microbial function and have antioxidant and pathogenic antibacterial effects [22].Tannin have an anti-microbial effect against several types of bacteria such as of E. coli O157:H7 [28], Bacillus subtilis, Salmonella Enteritica, Clostridium perfringens, and Staphylococcus aureus [26] .Tannins have also been reported to inhibit the growth of methanogenic bacteria [20,29] However, tannins become antinutritional factors at large concentrations, decreasing the ability of nutrients, particularly proteins and carbohydrats, to be digested [30] .against DPPH radicals may be its high phenolic content in ethanol extract.Citrus fractions have the potential to donate hydrogen, which contributes to their ability to scavenge DPPH radicals.The IC50 value was obtained from several stages, namely calculating the log concentration value and probability value for each percentage of DPPH free radical inhibitor activity from kaffir lime leaves extract and quercetin.Further connect both data from the calculations obtained in 1 graph.The concentration log value is used as the X axis and the probit value is used as the Y axis.The graph of the determination of IC50 in quercetin and lime leaves extract can be seen in figure 7-10.

Antioxidant activity
Based on figure 6, it can be seen that all of Citrus hystrix DC leaves extracts from several solvents have very strong antioxidant activity, indicated by IC50 values below 50 μg/mL.Antioxidants are very strong if IC50 has a value of less than 50 μg / mL, strong if the IC50 value ranges from 50-100 μg / mL, medium if IC50 100-150 μg / mL, weak if IC50 150-200 μg / mL and very weak with IC50 more than 200 μg / mL.In testing this antioxidant activity using quercetin as a standard compound with IC50 obtained at 5.8 μg /mL [31] .Citrus hystrix DC significant antioxidant activity against DPPH radicals may be primarily due to its high phenolic content in ethanol extract.Citrus fractions' capacity to donate hydrogen is what causes them to be DPPH radical scavengers.
Ethanol extract of Citrus hystrix DC leaves had the highest antioxidant activity against DPPH radicals, followed by ethyl acetate extract and hexane extract.This is in line with the flavonoid content of the three types of Citrus hystrix DC leaves extract with different solvents (Figure 3), where Citrus hystrix DC leaves ethanol extract contains the highest flavonoids (65.02 mg / g), followed by ethyl acetate extract (56.21 mg / g) and hexan extract 53.66 mg / g).
An important source of antioxidants is dietary flavonoids.Numerous studies on the in vitro antioxidant activity of flavonoids have been published recently, and a relationship between antioxidant power and chemical structure has been evaluated [32] .It has been claimed that certain flavonoids have antioxidant action in vitro.It has been demonstrated that quercetin, rhamnetin, and isorhamnetin can increase in vivo antioxidant activity while lowering serum and liver cholesterol levels.[33].The physiological antioxidants appear to be involved differently in the in vivo antioxidant activity of both the absorbed flavonoids and their metabolites, leading to a sparing impact on R-tocopherol and âcarotene [34].The antioxidant function of flavonoid, phenolic acid, and their ester is determined by the presence of OH groups directly attached to the carbon atoms of the benzene ring.[31].Citrus hystrix lime leaves ethanol extract samples, according to different results, had an IC50 value of 228.695 g/mL, indicating that the extract had only moderate antioxidant activity.This discrepancy in the data is probably the result of different sample processing techniques that damaged the active chemicals [35].

Conclusion
An ethanol solvent-based Citrus hystrix DC or kaffir lime leaves extract with a high amount of total phenol, flavonoids, and tannin and strong antioxidant activity.Extract of kaffir lime leaves from ethanol solvent shows a high content of total phenol, flavonoids, tanin and has good antioxidant activity All of Citrus hystrix DC leaves extracts from several solvents have very strong antioxidant activity, indicated by IC50 values below 50 μg/mL and ethanol extract of Citrus hystrix DC leaves had the highest antioxidant activity against DPPH radicals

Figure 2 .
Figure 2. Total phenol content of kaffir lime (Citrus hystrix DC) leaves extract in several solvents3.3.Flavonoid contentFlavonoids are natural substances contained in plants that act as antioxidants to counteract free radicals in the body.The majority of plants include chemicals called flavonoids, which have 15 carbon atoms.More than 2000 plant-derived flavonoids have been discovered, although anthocyanins, flavonols, and flavones are the classes that are most frequently studied.Based on the Fig.3the flavonoid content of leaves of Citrus hystrix DC from ethanol extract is greater by 121% compared to n hexana extract, while the flavonoid content of ethyl acetate extract is 105% compared to n hexane.The content of flavonoids found in every plant is no exception in stomach oranges.Flavonoids have a role in the growth process of these plants.Flavonoids in plants play a role in giving color, taste to seeds, flowers, and fruits and aroma and protect plants from environmental influences, as antimicrobials, and protection from ultraviolet light exposure[15].Flavonoids function for the physiological survival of the plant itself, flavonoids have other benefits, among others, as antifungal agents, and traditional medicine.Flavonoids are certain chemicals found in plants, called phytonutrients, and have various health benefits.The greatest flavonol component is quercetin, which accounts for between 60 and 75 percent of all flavonoids along with its glycosides.[16].Flavonoids are widely known for their ability to fight off a variety of harmful microorganisms as antibacterial agents.Flavonoids have generated a lot of interest due to their potential as antibiotic alternatives due to the rising occurrence of untreatable diseases brought on by bacteria resistant to antibiotics.The following are the suggested antibacterial mechanisms of flavonoids: inhibition of cytoplasmic membrane function, inhibition of energy metabolism, inhibition of attachment and biofilm formation, inhibition of

Figure 3 .
Figure 3. Flavonoid content of kaffir lime (Citrus hystrix DC) leaves extract in several solvents hexane extract etil acetat extract

Figure 4 .Figure 5 .
Figure 4. Saponin content of kaffir lime (Citrus hystrix DC) leaves extract in several solvents 3.5.Tannin Fig.4shows that tanin from kaffir lime (Citrus hystrix DC) obtained using different solvents.It was found that ethanol extract has the highest tanin content compared to n-hexane and ethyl acetate.The results of previous studies informed that the ethanol extract of kaffir lime contained a class of flavonoid compounds, saponins, tannins, triterpenoids, alkaloids and essential oil[22]

Figure 6 .
Figure 6.Antioxidant activity of kaffir lime (Citrus hystrix DC) leaves extract in several solvents An industrial oxidizing radical called DPPH can be suppressed by antioxidants.Due to the removal of hydrogen atoms from the antioxidant molecule in this assay, DPPH's violet color was changed to a pale yellow color.The amount of DPPH decrease will increase with the amount of antioxidants present in the extract.A sample's high scavenging activity is correlated with a high decrease of DPPH.The

Figure 8
Figure 7 log Quercetin

Figure 9 Figure 10 .
Figure 9 log ethyl acetate of kaffir lime leaves