MODIFYING FRACTION EXTRACTED FROM SESEWANUA (CLERODENDRUM FRAGRANS WILD) LEAVES IN SNEDDS PREPARATIONS: CHARACTERIZATION AND BIOAVAILABILITY TEST

Objective : This study aimed to determine the effect of compound modification using acyl chloride derivatives on n-hexane: ethyl acetate fraction of sesewanua leaves, focusing on the characteristics and pharmacokinetics profile in Self-Nano-emulsifying Drug Delivery System (SNEDDS) preparations. Methods: A quasi-experimental method was used with six SNEDDS formulas, namely F0 (without active substance), F1 (acetyl chloride fraction), F2 (propanoyl chloride fraction), F3 (butyryl chloride fraction), and F4 (pentanoyl chloride fraction) and F5 (piperine compound). The fractions were subjected to characterization tests, including particle size, polydispersity index, and zeta potential as well as determination of pharmacokinetics profile using the modified crane and Wilson method. Results: The results showed that the characterization tests of particle size using Particle Size Analyzer (PSA) for F0-F5 on gastric fluid included 15.8, 17,367, 20,367, 15.8, 28.233, and 21.533 nm. The polydispersity index values were 0.211, 0.438, 0.311, 0.383, 0.394, and 0.397, while the Zeta Potential values were-22,267,-22.2,-23.5,-24,033,-22,967, and-21.6 mV, respectively. The pharmacokinetics profile of AUC0-∞ was as follows: F0 0 μg, F1 492.83, F2 492.83, F3 245.98, F4 492.94, and F5 843.38 μg. Fraction five (F5) as a control had a higher AUC0-∞ value than compared to the fractions modified with acyl chloride derivatives. The T1/2 elimination values were F0 0 h, F1 22.5 h, F2 10.811 h, F3 35.54 h, F4 231.01 h, and F5 15.469 h. Conclusion: Based on the results, the addition of acetyl, propanoyl, butyryl, and penthanoyl chloride affected Particle Size Characterization Analysis and pharmacokinetics profile of SNEDDS preparation of n-hexane: ethyl acetate fraction. Structural modification showed the ability to alter the bioavailability of the active ingredient according to the desired therapeutic goal.


INTRODUCTION
Sesewanua (Clerodendrum fragrans Wild.) was reported to have anti-inflammatory activity, based on the study conducted by Sapiun et al., (2023).The emulgel ethanol extract of sesewanua leaves at a dose of 5% had a higher percentage of inflammatory inhibition (34%) compared to the positive control (21%).A total of four compounds have been identified in the ethanol extract of sesewanua leaves, including flavonoids, alkaloids, tannins, and saponins [1].The isolation of compounds was performed using n-hexane and ethyl acetate solvents.From the fractionation process, the most active fraction was obtained, namely 1 (9:1, 8:2, 7:3, 6:4) with very strong antioxidants, as shown by an IC50 value of 2.56033 mg/l.Based on the results, this fraction contains alkaloids with Rf value of 0.9 [2].
The oral administration of drugs accounts for more than 70% of the total dosage forms.The popularity of this route is attributed to the ease and acceptance by patients, which increases compliance [3].However, n-hexane: ethyl acetate fraction generally causes discomfort when used orally, resulting in the development of nanotechnology pharmaceutical preparations, namely the Self-Nanoemulsifying Drug Delivery System (SNEDDS).
Formulation of SNEDDS preparations can improve the bioavailability of the active substance through oral use and increase the rate of dissolution as well as absorption in the body [4][5][6].SNEDDS fractions of n-hexane: ethyl acetate have also been tested orally and shown to reduce rheumatoid arthritis index values by 44.66%.It also decreased swelling in rat legs induced with Complete Freund's adjuvant (CFA) by 79.25% [7,8].Aside from formulating active compounds in SNEDDS preparations, another strategy that can increase the ability of plant-active compounds to achieve maximum therapeutic effects is modifying the structure [9,10].
Modification of the compound structure can be achieved through an acyl nucleophilic substitution reaction (acylation reaction) [11,12].
Using acyl chloride derivatives is a method for adding structures to form ester groups that enhance the lipophilicity of compounds.The greater the lipophilicity of a compound, the easier the rate of penetrating the stomach and intestinal membranes [13,14].The analgesic efficacy of pinostrobin isolated from cat encounters has been structurally modified using acyl chloride, including acetyl, propanoyl, butyryl, and pentanoyl chloride.At a dose of 14 mg/kg body weight, the percentage of pain resistance from pinostrobin acetate was 46.57%, while those of propionate, butyrate, and pentanoate were 47.33%, 49.2%, and 55.34% respectively.For diclofenac sodium as a positive control, the percentage of pain resistance was 42.37%, and pinostrobin in the form of a negative control amounted to 33.97% [11].Based on the study by Suryadi et al. (2018), structural modifications in a compound can increase the activity characterized by an increasing percentage of pain resistance.

Ethical approval
Before conducting this study, ethical approval was received from the Ethics Commission of the Health Polytechnic of the Ministry of Health of Gorontalo (No. DP.01.01/KEPK/68/2023.

Fractionation of sesewanua leaves ethanol extract
The separation of ethanol extract to isolate alkaloid group compounds was achieved using column chromatography with nhexane: ethyl acetate mobile phases in ratios of (9:1), (8:2), (7:3), and (6:4).The first step in the chromatographic process was to insert filter paper and cotton in the lower column.Subsequently, 5 g of cleaned sand was placed inside and recoated with cotton wool.About 20 g of silica gel60 was added and mixed with ethyl acetate until the mixture was slurry-shaped.Following this, the extract (10 g) dissolved in ethanol was added to the column and allowed to descend slowly through the silica gels.It was then eluted, starting from a low polarity, and slowly increased.The fraction produced was evaporated and added with 1 g aeroside [2,18,19].
Fraction Yield Formula:

Modification of n-hexane: ethyl acetate compound with acyl chloride derivative
Modification of n-hexane: ethyl acetate compound using acetyl, propionyl, butyryl, and pentanoyl chloride was carried out by drug complexation.Initially, n-hexane: ethyl acetate fraction (0.5 g) was dissolved in 10 ml of 96% ethanol solvent (phase A).About 1 g NaCl as a complexing compound was dissolved in 1 ml each of acetic, propanoic, butyric, and pentanoic acid (phase B).Phases A and B were mixed and evaporated at a temperature of 100 °C until a powder formed [21].SNEDDS was prepared using the high-pressure homogenization (HPH) method or the method of preparing high-pressure homogenizer nanoemulsions.This method is frequently used for drug degradation of high-molecular-weight compounds.It was carried out using a hot plate with a magnetic stirrer in a gradually emulsified mixture.The initial step entailed mixing the oil phase (VCO) and the active substance (n-hexane: ethyl acetate fraction) with a magnetic stirrer at a speed of 310 rpm for 5 min.Surfactant was added, followed by stirring at the previous speed and duration, and then cosurfactant was added [22][23][24].

Characterization of SNEDDS pH
The pH of SNEDDS preparations was measured using a calibrated pH meter [25,26].

Nanoemulsified time
Nano-emulsion time testing was carried out using a magnetic stirrer.A total of 1 ml SNEDDS preparation was placed into a container filled with pH 3.5 and stirred with a magnetic stirrer at a speed of 1000 rpm.Observations were made on the time required from the beginning of SNEDDS droplets in the media until nanoemulsions were formed [19,27].

Analysis using particle size analyzer (PSA)
Particle size, polydispersity index, and zeta potential were identified using PSA Dynamic Light Scattering (DLS) method.The sample was diluted with a dispersing solution, creating a cuvette cell at a volume of 3 ml.The cuvette containing the sample was placed into the cuvette holder inside PSA device.System settings regarding sample name, dispersing media, and gate time were set up using the software.Subsequently, "Start Measurement" was clicked to start the analysis process [25].

SNEDDS bioavailability test
Bioavailability testing of SNEDDS was performed using an artificial crane and the Wilson method [28,29].In this test, 2 ml SNEDDS and 5 ml gastric fluid (pH 3.5) were enclosed in the dialysis membrane.
The dialysis membrane was created in a beaker filled with 96% ethanol-receiving medium (blood circulation) at a volume of 250 ml.Samples were made at 3 ml each for 24 h with 12 points, and then 4 points that met the peak and steady-state levels were collected.After each sampling, the receiving medium was substituted to maintain sink condition [28,30].

Determination of the maximum wavelength of piperine
Piperine (50 mg) was dissolved in 50 ml ethanol (1000 ppm) and diluted to a concentration of 500 ppm.A 500 ppm solution of piperine was diluted to 1, 5, and 10 ppm, then absorbance was recorded at a wavelength of 200-600 nm.

Piperine standard curve
Piperine weighing 50 mg was dissolved in 50 ml ethanol (1000 ppm) and diluted to a concentration of 500 ppm.Variations in the concentrations of 1, 2, 3, 4, 6, and 10 ppm were determined.Absorbance was measured using a UV-Vis spectrophotometer at λ maximum wavelength (342.50 nm).The absorbance results (y) and the concentration of the piperine raw solution (x) were plotted in a calibration curve to obtain a linear regression equation y=bx±a [31].

Determination of piperine levels
The absorbance (342.50 nm) of each sample was monitored using a UV-Vis spectrophotometer and testing was conducted in triplicate.

Data analysis
Bioavailability data of T1/2 and AUC0-∞ were statistically analyzed using Statistical Package for the Social Sciences (SPSS) program version 25.0.To determine the normality of the data, the Shapiro-Wilk test was performed.The results with a p-value<0.05were considered abnormally distributed.The Homogeneity of variance test obtained a value of>0.05,meaning that the data distribution was not homogeneous (different data variations).One-way ANOVA Post hoc Tamhane's T2 test obtained significant scores.

Yield percentage of n-hexane: ethyl acetate fraction from sesewanua leaves
As shown in table 2, the percent yield of n-hexane: ethyl acetate fraction had an excellent value, in line with the Indonesian Herbal Pharmacopoeia, which set the limit of a good extract yield at 7.2% [33].The higher the yield of the extract fraction, the greater the amount of compounds extracted between solvents [34].This was attributed to the nonpolar nature of n-hexane solvent as well as the semipolar nature of ethyl acetate, causing the withdrawal of the compound from n-hexane: ethyl acetate fraction [35].3 meet pH requirements both for SNEDDS and oral preparations.In addition, alkaloid compounds (piperine) contained in the fraction remained unaffected because SNEDDS preparations were at a stable pH>7 (alkaline) [38].This was attributed to the addition of surfactants and co-surfactants, which maintained the stability of the active substance [19].

Nanoemulsified time test
Nanoemulsified time test was performed to determine the ease with which SNEDDS formed emulsions while in the body [39].The test was carried out using gastric (pH 3.5) and intestinal fluid (pH 6.6) [40].The results of the nano-emulsified time test for SNEDDS preparation are shown in table 4.
SNEDDS emulsified time test results in table 4 meet the standard, namely less than 1 minute.Based on the results, SNEDDS was categorized as grade A, referring to a clear nanoemulsion formed quickly in 1 minute.Clear emulsions with fast emulsified times showed that SNEDDS formulas achieved emulsification easily [27,36,37,41].

Particle size of SNEDDS
Particle size is a parameter to indicate the characterization of nanoparticles in a drug delivery system [42], and the results obtained in this study are presented in table 5.  Based on table 5, the particle size test results using PSA met the acceptable SNEDDS characteristics, namely 5-100 nm in the gastrointestinal tract [43].This was consistent with a study by Mathew (2022), stating that Quartcetin SNEDDS obtained particle sizes less than 100 [44].

Polydispersity index
Polydispersity index value close to 0 shows homogeneous particle size dispersion, while values above 0.5 imply high heterogeneity [45].The results for the polydispersity index are shown in table 6.  [45].

Zeta potential
The results of the zeta potential test are shown in table 7.
A high zeta potential value is required in a formulation (positive or negative) because it shows the system stability.A good zeta potential value for emulsion preparations ranges between 20-30 mV [46].The zeta potential value in table 7 complies with the standard.

Determination of the piperine standard curve
Measurement of the standard absorbance for piperine is depicted in fig. 1.
The calibration curve in fig. 1 shows the relationship between piperine concentration and absorbance, producing a regression equation to calculate alkaloid levels in the test sample.The regression equation for the piperine standard curve was y = 0.1014x+0.0404with an r 2 value of 0.9934.An r 2 value close to 1 in the linear calibration curve shows a relationship between the concentration of the piperine solution and the absorbance value.

Concentration and bioavailability test results
Bioavailability shows the speed and amount of active substances that reach the systemic circulation.In this study, bioavailability testing of SNEDDS focused on parameters such as Cpmax, Tmax, T1/2, and AUC0-∞ [17].To the bioavailability of SNEDDS preparations, piperine levels (fig.2) Which successfully penetrated the gastric membrane, were measured.
Bioavailability parameters namely Cpmax, Tmax, T1/2, and AUC0-∞ were determined by a curve of piperine levels over time (fig.2).Cpmax refers to the maximum concentration of oral drug administration.Peak plasma levels offer insights into the pharmacological response dependent on drug levels in the body.The higher the plasma levels of the drug in the body, the better the pharmacological response [17,32].Cpmax was identified as the highest peak of each formula on the curve in fig.2, accounting for maximum concentration of SNEDDS in the body.Cpmax and Tmax values of SNEDDS preparations are presented in table 8.    Cpmax is associated with Tmax, which refers to the time required for the drug to reach maximum levels in plasma after administration.As stated in previous studies, absorption continues even after reaching the Tmax value, but the rate slower [17,32].Based on fig. 3 (A) and (B), formula 2 has the highest Cpmax in a short time, namely 6 h.This was attributed to the more acidic pKa of propanoyl chloride (2.86), making the piperine compound with a pKa of 12.22 more acidic.Meanwhile, the pKa of acetyl, butyryl, and pentanoyl is 4, 4.5, and 3.46 respectively [47,48].From the principle of "like dissolves like", a compound will dissolve in a solvent with the same properties, meaning that acidic compounds will be more soluble in acidic compounds [49].
The results in fig.3A and 3B cannot describe the total number of active compounds capable of providing therapeutic effects for the body.The Cpmax and Tmax values can be used as an initial step in determining the pharmacokinetics profile of a drug, including AUC0-∞ and T1/2 elimination [50].The results of AUC0-∞ and T1/2 elimination are presented in table 9.  Fig. 4A shows the value of AUC0-∞ obtained for n-hexane: ethyl acetate fraction with the addition of acyl chloride derivatives in the order pentanoyl>acetyl>butyryl>propanoyl chloride.Generally, the addition of acyl derivative compounds led to a change in the polarity of piperine.The addition of pentanoyl chloride caused the non-polar piperine to become even more non-polar because pentanoyl chloride has more carbon groups compared to other acyl derivatives.Non-polar compounds tend to easily penetrate the stomach membrane which is non-polar [47,54].This is attributed to the chemical bond between pentanoic acid in fat, which is formed through the reaction between the hydroxyl group on glycerol and the carboxyl group on pentanoic acid [55].The non-polar bond that occurs between pentanoyl chloride and piperine makes the drug compound more non-polar leading to the accumulation for a long time in the body with a slow rate of excretion [56].Consequently, piperine with pentanoyl chloride derivatives has a very long elimination T1/2, namely 231.01 h.The highest AUC0-∞ concentration was observed after the introduction of a new piperine pentanoate derivative.
The addition of acetyl chloride led to changes in the acidity level of piperine, resulting in polarity.However, this change did not completely replace the polarity of piperine.As stated in previous studies, pKa of piperine and acetyl chloride is 12.22 and 4, respectively [47,48].Similar to pentanoyl chloride, piperine in acetyl chloride will penetrate the stomach membrane more easily, but at a lower rate.The ability of compounds to become absorbed in body tissues is reduced because there is no direct bond between fat and acetyl chloride [55].This results in fewer acetyl chloride derivative compounds being absorbed in the body and not accumulating for an extended period due to the reduced polarity of piperine, leading to a short T1/2 elimination time.The highest AUC0-∞ concentration was observed after the introduction of butyryl chloride derivatives.
The addition of butyryl chloride causes piperine to change its polarity and become less polar, leading to fewer compounds penetrating the stomach membrane.Additionally, once in fat tissue, the piperine butyryl chloride derivative does not accumulate in the body for an extended period.Similar to acetyl chloride, butyryl chloride does not form direct bonds with fat and the polar nature of this compound translates to a short T1/2 elimination time [47,55].
The lowest AUC0-∞ concentration was observed in piperine with propanoyl chloride derivatives.
The binding of propanoyl chloride with piperine also increases polarity, primarily due to the fewer carbon groups in propanoyl chloride and a more acidic pKa value of 2.86.This results in a lower piperine propanoyl chloride derivative penetrating the non-polar stomach membrane.Moreover, propanoyl chloride cannot bind directly to fat in the body, and the polar nature leads to rapid excretion from the body [47,48,55].
The value of AUC 0-∞ from piperine without any additions was better compared to when four acyl derivatives were added.This discrepancy can be attributed to the inherent hydrophobic or nonpolar properties of piperine, restricting the solubility.Piperine in SNEDDS preparations can increase solubility because the drug dissolves in the oil phase which then forms a nanoemulsion when in contact with water in the digestive system [47,57].Due to the nonpolar nature, piperine effectively penetrates the non-polar stomach membrane.
Statistical test results for AUC0-∞ and T1/2 elimination of SNEDDS piperine compounds showed that there was a significant difference in the values of F0, F1, F2, F3, and F4 compared to F5.Specifically, AUC 0-∞ of piperine compounds was significantly influenced by the addition of acetyl, propanoyl, butyryl, and pentanoyl chloride.Therapeutic dose is the amount/measure of medication provided to obtain a healing effect, reflecting the drug level in the systemic circulation before reaching T1/2 elimination.Meanwhile, a steady state is a condition where drug usage starts to decline slowly and stabilizes when half of the dosage has been eliminated from the blood [58,59].The pharmacokinetics profile of individual SNEDDS preparations can be obtained through AUC0-∞, T1/2 elimination, therapeutic dose, and drug levels during steady state.

CONCLUSION
In conclusion, the addition of acetyl, propanoyl, butyryl, and pentanoyl chloride significantly affected the pharmacokinetics profile, specifically AUC0-∞ and T1/2 elimination of n-hexane: ethyl acetate fraction with antirheumatic effect.Structural modification provided a means to alter bioavailability of the active ingredient according to the desired therapeutic goal.However, this study was conducted on an in vitro laboratory scale, showing the need for in vivo studies and clinical trials to further prove the results.