INTRODUCTION:

Traditional medicines, especially those made from plants, are increasingly being used to assist improve the state of the public's health. Meniran is a kind of plant that has medicinal properties. The botanical name for meniran is Phylanthus niruri Linn, and it belongs to the Phyllanthus genus. In many tropical regions, P. niruri has long been used to treat a variety of illnesses, including kidney stones, chronic liver disease, diabetes, and viral infections.^1,2P. niruri has a number of pharmacological effects, including those that are hepatoprotective, diuretic, antiviral, antibacterial, and immunomodulatory.^3–5

Pyllanthus niruri L. contain secondary metabolites compounds such as lignans, coumarins, alkaloid, flavonoids, tannins, tritepenes, sterols, phytallate, lipid, and saponins.^6,7

Phyllanthin is known to have antioxidant, anti-inflammatory, anticancer, immunomodulatory, and hepatoprotective.^8–10The presence of filantin can be used as an identity compound in analyzing the condensed extract of p. niruri herb. The results of the study Alegantina et al.¹¹ stated that the level of filantin in the 70% ethanol extract of p. niruri herbs was 0.864%. The importance of p. niruri in medicine makes it necessary to develop its quality, safety, and efficacy through research.

Generally, bioactive compounds are extracted using organic solvents such as ethanol, methanol, hexane, or other solvents. Extraction using organic solvent was not environmentally friendly. Therefore, efforts to minimize, replace or even eliminate the use of volatile organic solvents (VOC) in the extraction process are continuously being developed. To reduce the negative impact due to the use of organic solvents, a safer and environmentally friendly solvent is used, namely NaDES (Natural Deep Eutectic Solvents). It has the benefits of being a more environmentally friendly resource, less expensive and toxic, better at biodegradation and biocompatibility, and more orally bioavailable.^12–15NaDES are made from natural and safe primary metabolite compounds such as amino acids, monosaccharides, disaccharides, polysaccharides, acetic acid, lactic acid, choline chloride and others. with a certain mole ratio. NaDES has gained recognition as a sustainable solvent and is frequently used in analytical and biomedical research.^16,17NaDES have been employed extensively in the extraction of numerous naturally occurring bioactive components to produce functional herbal products.^18–20

Considering the advantages that NaDES has mentioned above and the importance of using extracts of medicinal compounds that are free from chemical residues, several combinations of NaDES will be used as solvents in extracting bioactive compounds from P. niruri.Several combinations of NaDES were screened. The best combination was optimized for Ultrasonic-assisted extraction.

MATERIALS AND METHODS:

Materials:

The materials used in this study were P. niruri leaves (Yogyakarta), aquadest (Biopure®), phyllanthin (Xi’an Plant®), acetonitrile (Merck®), trifluoroacetic acid (Merck®), methanol (Merck®), choline chloride (Sigma Aldrich®), lactic acid (Bratachem®), oxalic acid (Bratachem®), citric acid (Bratachem®), ascorbic acid (Merck®), tartaric acid (Bratachem®), malic acid (Bratachem®), ethanol (Merck®).

Preparation of RP Extracts:

All the NaDES solution were prepared following the previously reported method.²¹ The compositions of NaDESs are listed in Table 1. In brief, combination of HBA, HBD and water were stirred at 80ºC until a homogenous liquid was formed.

The extract solution was obtained from 1g of dry herb powder which was weighed accurately in a centrifuge tube and extracted using 10mL of NaDES solvent or 70% ethanol. The UAE process was carried out using ultrasonic (Branson ®) for 30 minutes and a temperature of 40 ºC. The mixture was centrifuged for 15 minutes at 10,000rpm, and the supernatant was collected for analysis using HPLC. For the optimization of the extraction process, the extraction time, temperature and NaDES-water ratio were changed.

Table 1: Screening of NaDES

Code	Component	Ratio
NaDES-1	ChCl :Lactic acid	1:1
NaDES-2	ChCl : Oxalic acid	1:1
NaDES-3	ChCl : Citric acid	1:1
NaDES-4	ChCl :Ascorbic acid	1:1
NaDES-5	ChCl : Tartaric acid	1:1
NaDES-6	ChCl : Malic acid	1:1

Time (min) = 30; Temperature= 40⁰C; Ratio NaDES: Water = 70%

HPLC Analysis:

The HPLC analysis was performed on an Jasco system. The stationaryphase used was Inertsil ODS-3 (150mm x 4.6mm, 5µm, GL Science®). The mobile phase consist of 70% methanol:30% Trifluoroacetic Acetat (0.1%v/v) The flow rate was 1mL/min and the injection volume was 20µL. The chromatogram was recorded at 230nm.

Validation of HPLC Method:

The method was validated by determining its specificity, linearity, accuracy, and precision.

The specificity was evaluated by comparing the chromatograms of the standard and the samples. Linearity was determined by plotting the area versus phyllanthin concentration. The calibration curve had to have a correlation coefficient (r) of 0.995 or better.

The intra-day and inter-day precision and accuracy were measured by performing the assay of sample spiking at three concentration levels and six replicates at each concentration. The accuracy was expressed as recovery and the precision as RSD.

RESULT:

Preparation of RP Extracts:

Choline chlorides were used as HBA and the HBDs were organic acids (lactic acid, ascorbic acid, oxalic acid, citric acid, malic acid and tartaric acid). In total, 6 different NaDESs were prepared. NaDES 1 and 4 was formed crystals at room temperature and not to be used for screening process. The extract obtained for screening can be seen in Figure 1.

Figure 1: NaDES Extract

Screening of NaDESs:

Phyllanthin extraction effects from 4 different NaDESs were studied in the initial screening along with ethanol for comparison. The extraction factors were set as follows, extraction time of 30min, extraction temperature 40ºC, and the NaDES content 70%. As depicted in Figure 2, ethanol had the least impact on the extraction of phyllanthin (1.57mg/g). Higher yields of phyllanthin were achieved for all NaDESs. With a phyllanthin extraction yield of 59.80mg/g, ChCl-oxalic acid was chosen among them as the best extraction solvent.

Figure 2: Screening of NaDES

Optimization of the Extraction Factors:

Response surface methodology (RSM) is a useful approach for process optimization, particularly when numerous factors and interactions have an impact on the desired response.^22–24Evaluations were made of the effects of extraction temperature, extraction time, and NaDES ratio. In the optimization of the NaDES ratio from 50%, 70%, and 90% NaDES (v/v) was observed to have the highest extraction yields of phyllanthin. For the effect of temperature ranging from 25, 40 and 60 ºC were testified. The maximum extraction efficiency was achieved when the NaDES content was at its highest (90%), it was found that increasing the NaDES content would result in larger yields of phyllanthin. This may be due to the increasing of viscosity of the solvent. Furthermore, the likelihood of finding holes for solvent molecules or ions to travel into and the free volume are both inversely correlated with the viscosity of NADES, according to the hole theory. The ChCl/oxalic acid DES is a mixture of nonsymmetrical chloride ions and molecules of carboxylic acid that exhibits both ionic and organic solvent characteristics.²⁵

Figure 3: 3D surface at 90% ratio of NaDES

HPLC Method Validation:

The method was observed to be highly selective for phyllanthin in extract, with the retention times of phyllanthin in sample and standard at 10.958 min. The representative chromatograms of standard and sample are displayed in Figure 4.

Figure 4: Chromatogram (A) standard and (B) sample.

Calibration curve was created by plotting the peak area of phyllanthin (y) versus the standard concentration (x). From 0.5mg/L to 80mg/L in concentration, the calibration curve's peak area was linear, with a coefficient correlation (r) > 0.995 and the regression equation was y = 34021x – 10720.

The intra- and inter-day precision and accuracy were determined by analyzing the samples spikes at the concentration 10, 20, and 30mg/L.²⁶All values are listed in Table 2. All the assay results fell within the allowed range, and the method was considered accurate and precise.

Table 2: Accuracy and Precision

Analytes	Concetration (mg/L)	Measured (mg/L)	Intra-Day RSD (%)	Inter-Day RSD (%)
Phyllanthin	10	10.43 ± 0.21	2.02	4.89
	20	20.07 ± 0.21	1.04	6.09
	30	29.57 ± 0.22	0.73	2.01

DISCUSSION:

NaDES was a mixture of two solids that contained organic acid as a hydrogen bond donor (HBD) and choline chloride as a hydrogen bond acceptor (HBA). The deep eutectic system, which was developed from a liquid below 100ºC, will be created by NaDES with a specified ratio (HBA and HBD mixture).²⁷The mol ratio of 1:1 were formed liquid, except for lactic acid and ascorbic acid. ChCl-Oxalic acid was the best NaDES. The physical and chemical features of NaDES, such as hydrogen bonding, polarity, acidity, and viscosity,²⁸ are related to its ability to extract bioactive compounds from the natural product matrix. The parameters α, β, and π* indicate hydrogen bond acidity hydrogen‐bond acidity (α), hydrogen‐bond basicity (β), and dipolarity/polarizability of the solvents (π*) were employed to depict the chemical characteristics of NaDES. From a solvatochromic perspective, the Kamlet-Talf parameters for 96 percent ethanol as a solvent in typical extraction are α = 0.83, β = 0.77, and π* = 0.54.²⁹ Meanwhile, with a 1:1 (w/w) ChCl-oxalic acid ratio, these values were α = 1.90, β = 0.41, and π* = 1.07.³⁰ The hydrogen bond acidity (α=1.90) has a strong polarity and can donate and absorb hydrogen through the natural product matrix. Solvents with a high polarity have a stronger antioxidant activity.³¹Additionally, according to the hole theory, the free volume and probability that solvent molecules or ions would discover holes are directly related to the viscosity of NaDES. The ChCl-oxalic acid NaDES has both ionic and organic solvent characteristics and is made up of a lot of non-symmetrical chloride ions and carboxylic acid molecules.²⁵

This research shows that ChCl-oxalic acid NaDES-UAE methods can extract phyllanthin in p. niruri leaves. By comparing the results between ethanol-UAE and ChCl-oxalic acid NaDES-UAE methods, the ChCl-oxalic acid NaDES-UAE method showed 39-fold higher that the 70% ethanol with UAE method.³²

The optimum condition of ChCl-oxalic acid NaDES-UAE method was recommended related to RSM analysis results using equation form Design-Expert v13 as follows: Y = 0,309328+0,010971*X₁ +0,005558*X₂ – 0,005865*X₃ – 0,000021*X₁*X₂ – 0,000122*X₁*X₃+ 0,000010*X₂*X₃ – 0,000038*X₁² – 0,000080*X₂²+ 0,000088*X₃² with R² value 0.796. where Y is the yield of phyllanthin (mg/g), X₁ is temperature, X₂ is the extraction time, X₃ is the ratio of NaDES. The combination of an extraction temperature of 25ºC for 30 min and NaDES concentration of 90% resulted in the best extraction results. The extraction process more efficient at room temperature. A confirmation test was also run to verify the best extraction conditions and scale-up of the extract. As a result, a 10 g of sample was extracted using the optimum method and were obtained 61.35mg/g of phyllanthin.

The extraction time was obtained at 40min with the highest phyllanthin contents in this work. The extraction time at 40 and 30min extract the phyllanthin not significantly different (p>0.05). This indicates that the extraction can be done more faster and gives the yield equals to 40 min extraction time.

The temperature was obtained at 25ºC. The higher temperature will decrease the phyllanthin yields. Higher temperature can increase the entropy level in the NaDES matrix and thereby reduce viscosity effects.³³This indicates, the extraction process will less power consumed.

CONCLUSION:

Based on the results of the research, an effective and environmentally friendly extraction technique was developed. The most effective HBA-HBD mixture was ChCl-oxalic acid. The optimal extraction factors were as follows: extraction temperature at 25 ºC, for 30 min, and rasio of NaDES at 90%. As a result, utilizing NaDESs as a new extraction technique to extract phyllanthin from P. niruri is considerably more effective, uses less power, and yields better results than using a conventional solvent (ethanol).

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank Pakuan University for their kind support during hematological and all other lab studies.

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Received on 08.12.2022 Modified on 23.03.2023

Research J. Pharm. and Tech 2023; 16(11):5213-5217.

DOI: 10.52711/0974-360X.2023.00845