Nanoparticles of Muntingia calabura L. Ethanol extract:

Preparation and Anti-inflammatory activity

Tengku Ismanelly Hanum^1,2, Hetty Lendora Maha¹*, Henny Sri Wahyuni³, Rahmad Fadli¹, Indah Yuliasari Saragih¹

¹Department of Pharmaceutical Technology, Faculty of Pharmacy, Universitas Sumatera Utara,

Medan, 20155, Indonesia.

²Nanomedicine Innovation Center, Universitas Sumatera Utara, Medan, 20155, Indonesia.

³Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Sumatera Utara,

Medan, 20155, Indonesia.

*Corresponding Author E-mail: hetty_maha03@usu.ac.id

ABSTRACT:

Kersen (Muntingiaa calabura L.) is one of the plants empirically used by the public as an anti-inflammatory drug. Muntingiaa calabura is a flowering plant belonging to the Elaocarpaceae family. Kersen contains flavonoids, saponins, and tannins. The flavonoids that are contained in Kersen are flavones, flavanones, flavanones, and biflavan. Flavonoids have received much attention because of their antibacterial, anti-inflammatory, and antioxidant properties. Nanotechnology is intended to increase the capability activities of Kersen leaves phytochemical compounds. In nano size, the surface contact area of particles becomes more extensive, which can increase the number of active substances isolated, thus increasing anti-inflammatory activity. This study aimed to prepare and determine the anti-inflammatory activity of nanoparticles of ethanol extract kersen leaves (NEEKL) (Muntingia calabura L.). Preparation of NEEKL by ionic gelation method using variations of chitosan and sodium tripolyphosphate (TPP-S), then characterized for its particle size and morphology. The anti-inflammatory activity of NEEKL was evaluated using the carrageenan-induced rat paw edema method. It was performed in six different groups. Each group consisted of 4 rats. The negative control was given 0.5% CMC-Na suspension; the positive control was given diclofenac sodium 4.5mg/kg body weight; the 3rd and 4th were given NEEKL as much 100, 300mg/kg body weight; 5th groups were given ethanolic extract kersen leaves (EEKL), and 6th group was given 1% chitosan-TPP-S. The measurements were done in 6 hours with intervals of 60 minutes. All the data obtained were statistically analyzed. The NEEKL with variation chitosan: TPP-S (1.5:1) had the smallest particle size (94.21nm). Based on the percentage of inflammation, there was a significant difference between negative control and no significant difference between positive control and other groups. The percentage of anti-inflammatory activity of positive control, NEEKL (100mg/kg body weight), NEEKL (300mg/kg body weight), EEKL (500mg/kg body weight), and chitosan-TPPS complex were 86.10±0.90%; 81.16±0.55%; 84.47±0.85%; 85.51±1.56%, and 77.44±1.83% respectively. EEKL can be made in the form of nanoparticles using the ionic gelation method to fulfill the nanoparticle requirements. NEEKL has effective anti-inflammatory activity at a dose of 300mg/kg body weight.

KEYWORDS: Nanoparticles, Kersen Leaves, Muntingia calabura, Anti-Inflammatory, Ionic Gelation.

INTRODUCTION:

Inflammation is a reaction of living tissues towards injury, comprising systemic and local responses¹. Inflammation is a biological response in the form of a vascular reaction that causes the channeling of fluids, dissolved substances, and cells from the blood circulation to the interstitial tissues around an injury. Inflammation is divided into two, namely acute and chronic². Several chemical drugs are used to prevent inflammation. Modern medicine is commonly used as an anti-inflammatory drug, i.e., non-steroidal anti-inflammatory drugs (NSAIDs)³. NSAIDs and corticosteroid drugs are used to treat inflammation. However, long-term use of NSAIDs can cause various side effects such as ulcers and gastrointestinal bleeding, nephrotoxicity, and hepatotoxicity^4,5. Steroids can suppress the immune system and trigger erectile dysfunction, manic depression, hypertension, cramps, dizziness, the emergence of active diabetes, skin atrophy, decreased bone density, heartburn with possible perforation of the stomach wall, irregular menstruation, vision, allergy problems, and reduce wound healing⁶.Therefore, it is necessary to look for alternative treatments to fight and control pain and inflammation with fewer side effects, for example, drugs derived from plants.

One of the plants empirically used by the public as an anti-inflammatory drug is Kersen (Muntingiaa calabura L). Muntingiaa calabura is a flowering plant belonging to the Elaocarpaceae family. Kersen leaves have cardioprotective, antipyretic, antioxidant, anti-inflammatory, anti-diabetic, antibacterial, and antiulcer effects⁷. Besides that, Kersen also has pharmacological effects as an antiplatelet and cytotoxic activity. Kersen contains flavonoids, saponins, and tannins⁸. Based on a previous study report, Flavonoids like Quercetin, Rutin, and Kaempferol are reported to exhibit anti-inflammatory action. Various monoterpenes like geraniol, linalool, Eugenol, 1,8 cineole, alpha-pinene, and alpha-terpineol have anti-inflammatory activity⁹. The flavonoids contained in Kersen are flavones, flavanones, flavanones, and biflavan. Flavonoids have received much attention because they have antibacterial, anti-inflammatory, and antioxidant properties¹⁰.

The medicinal plants used to treat infectious diseases are generally still conducted conventionally. People usually use dry ingredients (simplicia) by powdering and then brewing. Developments in science and technology, such as nanoparticle technology, allow simplicia to be powdered with a smaller size (<1μm)^11,34. Nanotechnology is intended to increase the capability activities of Kersen leaves phytochemical compounds. In nano size, the surface contact area of particles becomes more extensive, which can increase the number of active substances isolated, thus increasing anti-inflammatory activity^12,35. In the 2010-2020 period, there will be a tremendous acceleration in the application of nanotechnology in the industrialized world. This indicates that the world is heading for a nanotechnology revolution. Nanotechnology is the technology for designing, manufacturing, and applying structures/materials with nanometer dimensions¹³. One of the nanotechnology developments in nanoparticle synthesis. Nanoparticle synthesis is increasing because it can be widely used in the environment, electronics, optics, and biomedicine¹⁴. The number of drugs in the blood during systemic delivery will also increase the risk of side effects and adverse events and the risk of reaching a toxic limit¹⁵. In many cases, an increase in blood levels of the drug is necessary for the drug to have a pharmacological effect. Therefore, nanoparticles provide a suitable solution because they can provide pharmacological effects at smaller (efficient) doses^16,36.

Based on the description above, further research is needed to obtain a preferable anti-inflammatory effect from Kersen leaves. The present study was conducted to determine the anti-inflammatory effectiveness of ethanol extract nanoparticles (orally) from Kersen leaves against λ-carrageenan induced white male rats.

MATERIAL AND METHODS:

Materials:

Fresh leaves of Kersen (Muntingia calabura) were collected (in the morning) from Binjai Regency, Sumatera Utara Province, Indonesia. Muntingia calabura was identified in Herbarium Medanese (MEDA) (letter number: 5149/MEDA/2020), Universitas Sumatera Utara, Medan, Indonesia. Chitosan (medium molecular weight) was purchased from Merk Indonesia, and other ingredients used were Analytical grade.

Methods:

Preparation of Muntingia calabura leaves ethanol extract:

Extraction was performed by maceration method using ethanol (96%) as a solvent. The fresh leaves of M. calabura were cleaned and then washed with water flow several times. The cleaned leaves were drained, spread on the parchment, and then aerated (to obtain water absorbed). Afterward, the leaves were dried at 40°C (in a drying cabinet) to obtain leaves brittle. Next, the simplicia was pounded into a fine powder and stored in a plastic container (to prevent the influence of moisture and other impurities). The fine powder of leaves (one part) was put into a dark container, and then 75 parts of ethanol (96%) was added. Afterward, it was covered and left for five days (protected from light). After that, it was stirred (occasionally) and filtered (using filter paper) to obtain macerates. After this step, 25 parts of ethanol (96%) were added to the residue, left for two days, and filtered to obtain macerates. All the macerates were combined, and then the rotary evaporator was used at ±400°C to obtain the ethanol extract from kersen leaves (EEKL). Afterward, the ethanol extract was concentrated in the water bath to obtain a thick texture (as a crude extract)¹⁷.

Preparation of nanoparticles Muntingia calabura leaves ethanol extract:

Ethanol extract nanoparticles from Kersen leaves were carried out using ionic gelation^18,19 by adding TPP-S as a crosslinking agent with chitosan. The EEKL (5g) was dissolved with an admixture (50mL) of ethanol: water (70:30). Next, 100mL of chitosan solution (0.05%) was added, then diluted with distilled water to 250mL. Afterward, 350mL of TPP-S solution (0.01%) was dropped (gradually) and stirred at 3000rpm for 6hours. Then, it was sonicated for 1hour to obtain ethanol extract nanoparticles from Kersen leaves (NEEKL). The NEEKL were separated by centrifuge. After that, the residue was frozen in a freezer (Chest Freezer AB-108-R) for 24hours. Next, the residue was dried by heating at 40°C to obtain dry powder. The dry powder was crushed utilizing mortar for 1 hour.

Nanoparticle characterization:

Nanoparticle characterization was conducted utilizing a Particle Size Analyzer²⁰ (Beckman ulter) and Scanning Electron Microscopy (SEM)²¹. Particle Size Analyzer was used to measure the particle size, and SEM was used to determine the powder’s morphological condition. SEM was measured at 1000x and 2500x magnifications.

Animals:

Male Wistar rats (Rattus Copernicus L.) with average weights of 150–200g were used as experimental animals¹⁸ with as many as 24 heads.

Preparation of experimental materials:

Ethanol extract from Kersen leaves (dosage of 500mg/ kg body weight), ethanol extract nanoparticles from Kersen leaves (dosage of 100mg/kg body weight and 300mg/ kg body weight), and TPP-S chitosan were used as experimental materials. Diclofenac-Na suspension was used as a positive control with an 18mg/kg bb dosage. CMC-Na suspension (0.5%) was used as a negative control. Carrageenan (1%) was used as an inducer.

Preparation of CMC-Na (0.5%) suspension:

CMC-Na (0.5g) was sprinkled in a mortar filled with hot distilled water and left for 15 minutes. Afterward, CMC-Na was crushed to obtain transparent mass and continued to be crushed to obtain homogeneity. After this step, CMC-Na was diluted with distilled water, homogenized, and put into a flask (100mL). Afterward, the distilled water was added to the obtained volume of 100mL.

Preparation of EEKL and NEEKL suspensions:

The ethanol extract (500mg) and ethanol extract nanoparticles (100mg and 300mg) from Kersen leaves were weighed and put into the mortar, then were added with CMC-Na (0.5%) suspension gradually while crushed to obtain homogeneity. Afterward, the volume was added to obtain 10mL.

Preparation of diclofenac-Na suspension:

The diclofenac-Na tablet (20 tablets) was weighed and crushed, and the total weight was weighed. The active ingredient, diclofenac-Na, weighed 25mg (in 1 tablet of diclofenac-Na), and the total active ingredient was 500 mg for 20 tablets. Afterward, the tablet weighed 2.25 mg/kg and was put into a mortar. Next, CMC-Na (0.5% w/v) suspension was added gradually while crushed to obtain homogeneity. After that, the volume was added to obtain 10 mL.

Preparation of λ-carrageenan (1%):

The λ-carrageenan (100mg) was weighed and crushed (with 0.9% NaCl solution) to obtain homogeneity. Next, it was put into a flask (10mL). Afterward, NaCl solution (0.9%) was added to obtain 10mL, then incubated at 37°C for 24hours²².

Preparation of TPP-S chitosan (1%):

The TPP-S chitosan (0.1g) was weighed. Next, the chitosan was dissolved with acetic acid. Afterward, the chitosan and TPP-S were put into the mortar. After that, CMC-Na (0.5%) suspension was added gradually and crushed to obtain homogeneity. Afterward, the volume was added to obtain 10mL.

Anti-inflammatory activity test:

The anti-inflammatory activity was studied on nanoparticles of Muntingia calabura leaves on the Carrageenan-induced rat paw edema model in Wistar albino rats using a plethysmometer^9,23. The rats were fasted for 18hours before testing (water was still being given to drink). Rats were grouped into six groups (each group consisting of 4 male rats), i.e., negative control group (CMC-Sodium suspension), positive control group (diclofenac-Na), NEEKL group (dosage 100 and 300mg/kg body weight), EEKL group (dosage 500mg/ kg body weight), TPP-S chitosan group:

Group I : CMC-Na suspension (0.5%)

Group II : Diclofenac Sodium suspension (4.5mg/kg body weight)

Group III: NEEKL suspension (100mg/kg body weight)

Group IV: NEEKL suspension (300mg/kg body weight)

Group V : EEKL suspension (500mg/kg body weight)

Group VI: TPP-S chitosan (1%)

a) Each animal was weighed (on an experimental day) according to their respective groups and marked on the tail and left paw.

b) Afterward, the rat's left paw was inserted into the cell containing a particular liquid on the plethysmometer to obtain a liquid rise to achieve the upper limit line. Next, the pedal was held, and the number on the monitor was recorded as initial volume (V0), i.e., the paw's volume before treatment of positive control (diclofenac Sodium) and inducer (carrageenan 1%).

c) Next, each animal was given an orally suspended suspension of experimental materials according to its group.

d) After that (60 minutes later), each rat's left paw was injected intraplantarally with 0.1 mL solution of λ-carrageenan (1%).

e) Next (30 minutes later), the rat's left paw was dipped into a plethysmometer cell (for measurement) containing a special liquid to obtain the limit line, and then, the pedal was held.

f) Afterward, the numbers on the plethysmometer monitor were recorded. The changes in fluid volume were recorded as rat's paw volume at a particular time (V_t). Measurements were conducted every 30 minutes for 6 hours, and each treatment was repeated three times (to determine the effectiveness of each treatment). The inflammatory volume was calculated based on the number of differences in the volume of the rats' paws after and before the inducer (carrageenan) was injected²⁴.

For each measurement: the cell fluid was added to the limit line (red line on top of the cell) to obtain the same fluid volume, the zero buttons were pressed on the main menu of the plethysmometer monitor, and the rat's left paw was dried beforehand²⁰, besides, the boundary markings on the rat's paw must be clear, and the rat's paw must be immersed to the limit specified. The ethical committee of Animal Research Ethics Committees, Universitas Sumatera Utara, Medan, Indonesia, approved all experimental procedures performed in the current study involving animals.

Inflammation percentage and inflammation inhibition percentage:

The inflammation percentage (%R) was calculated using the following formula.

Vt-V0

(%R) = ------ x 100

Information:

V_t = rat's paw volume at t time

V₀ = rat's paw initial volume

The inflammation inhibition percentage (%IR) was performed utilizing the following formula:

a-b

(%IR) = ----- x 100

Description:

a: average inflammation percentage of the control group

b: average inflammation percentage of the treatment group that received the experimental materials or comparative drug^24,25.

Statistical analysis:

The research data were analyzed using analysis of variance (ANOVA) with Statistical Product and Service Solutions (SPSS) software version 18 (confidence level of 95%). Then, they were continued by Tukey's test to determine which groups had the same or significantly different effects from one another.

RESULTS AND DISCUSSION:

Plant identification:

The identification of plants that have been done showed that the Kersen leaves have the species name Muntingia calabura L. and a family of Elaeocarpaceae.

Ethanol extract from Kersen (Muntingia calabura) leaves:

The fresh Kersen leaves weighed 12 kg; then the weight after dried and crushed into powder was 1318 g. The crude extract (macerate) weight was 104.12 g. The yield of extract from Kersen leaves powder and crude extract of Kersen leaves was 7.89%.

Nanoparticle characterization:

The size of nanoparticles was determined using a Particle Size Analyzer (PSA). Particle measurement data, as shown in Table 1, exhibited that the particle size was influenced by concentration, volume ratio of chitosan, and preparation method used. The particle size increase was directly proportional to the concentration increase of chitosan and the volume increase of the chitosan solution.

The effect of chitosan and TPP-S concentrations on the formation of nanoparticles was performed by providing a variety of chitosan concentrations (0.05%, 0.075%, 0.1%, 0.2%, 0.3%, 0.4%) with the concentration of TPP-S (0.01%). As presented in Figure 1, the nanoparticle size was formed at a specific chitosan concentration. It was caused by high chitosan concentration or a high ratio of chitosan to TPP-S. The higher the chitosan concentration, the higher the size of the extracted nanoparticles. At a chitosan concentration of 0.05%, the establishment of nanoparticles was relatively more accessible, and micro-sized particles formed much less than in other chitosan concentrations. Meanwhile, the chitosan concentration of 0.4% formed more micro-size particles than other concentrations. This result aligns with previous work, showing that the average particle size can also be reliably increased by raising the chitosan concentration^26,27.

Figure 1. Effect of chitosan variation on nanoparticle size

The effect of TPP-S concentration was decreased with the lower chitosan concentration. This occurred because the number of polycations of chitosan that would react with poly-anions from TPP-S was minimal. Thus, nanoparticle formation only depends on chitosan concentration^19,26.

The chitosan concentration with a fixed amount of TPP-S that is greater would also increase the size of the nanoparticles because of a tendency to agglomerate. The particle's high concentration was formed from the reaction between chitosan and TPP-S, which is very dense, established clusters into aggregate into micro-sized particles. The surface morphology of nanoparticles with unflat surfaces and forming loose aggregates was observed utilizing an SEM instrument. SEM analysis was performed on F1 with a magnification of 1000x and 2500x, as shown in Figure 2. The previous study also reported that the aggregation is formed because of a lack of cross-link bonding between chitosan and TPP-S²⁸.

(a)

(b)

Figure 2. Nanoparticle surface morphology with a magnification of 1000x (a); 2500x (b)

Anti-inflammatory effect test

Oral anti-inflammatory effects of nanoparticles were performed by inducing inflammation in rats' paws (intraplantar) with λ-carrageenan (1%) solution. Inflammation measurements were conducted utilizing a digital plethysmometer (UGO Basile). This anti-inflammatory effect measurement was based on Archimedes' law, i.e., when a solid object was put into a liquid, it would give an upward force or pressure equal to the moved volume. Carrageenan was chosen as an inducer because it leads to edema formation in an inflammatory model; carrageenan can release prostaglandins after being injected into experimental animals. Therefore, carrageenan can irritate experimental methods to find anti-inflammatory drugs^24,29.

Inflammation Percentage and Inflammation Inhibition Percentage:

The average inflammation percentage was calculated based on the rat's paw volume changes. The average inflammation percentage results for each group are presented in Table 2. The inflammation group with a lower percentage value than the negative control group indicated that an experimental formula could suppress inflammation induced by λ-carrageenan. The negative control group treated with CMC-Na exhibited a significant increase in paw volume among other groups.

Table 2: Average inflammation percentage of rat’s paw and standard error (Mean ± SEM)

Time (min)	CMC-Na (0.5%)	Diclofenac-Na (4.5 mg/ kg body weight)	NEEKL (100 mg/ kg body weight)	NEEKL (300 mg/ kg body weight)	EEKL (500 mg/ kg body weight)	Chitosan and TPP-S
30	7.38±0.38^b	4.43±0.21^a	6.38±0.14^b	5.75±0.15^a	5.37±0.39^a	5.99±0.47^b
60	11.95±0.40^b	7.46±0.52^a	10.18±0.53^b	9.19±0.15^a	8.89±0.86^a	10.04±0.75
90	18.22±0.59^b	12.12±0.63^a	14.99±0.79	15.26±0.96	13.16±0.84^a	16.01±0.90^b
120	24.29±0.98^b	16.93±0.68^a	21.24±0.85^b	19.69±0.94^a	17.74±0.51^a	21.39±0.40^b
180	30.27±0.7^b	21.46±0.6^a	26.77±1.69^b	24.98±0.95^a	23.43±1.02^a	26.60±1.20^b
240	34.74±1.25^b	16.08±1.06^a	19.98±0.73^a	18.10±0.69^a	16.69±1.20^a	20.37±0.67^a
300	39.84±1.67^b	9.68±0.62^a	13.49±0.28^a	11.92±1.13^a	10.53±0.90^a	14.89±0.76^ab
360	43.11±1.52^b	6.02±0.53^a	8.13±0.46^a	6.73±0.59^a	6.22±0.65^a	9.78±1.02^a

a: Different compared to negative control (CMC-Na)

b: Different compared to positive control (diclofenac-Na)

Table 3: Average inflammation inhibition percentage of rat’s paw and standard error

Time (minute)	Diclofenac-Na (4.5 mg/ kg body weight)	NEEKL (100 mg/ kg body weight)	NEEKL (300 mg/ kg body weight)	EEKL (500 mg/ kg body weight)	Chitosan and TPP-S
30	39.47±4.51	12.79±5.20	21.73±2.67	26.22+8.00	17.52±9.35
60	37.33±4.96	14.50±5.61	22.74±3.34	25.07±8.19	15.71±6.53
90	33.52±2.14	17.40±5.54	16.32±4.31	27.24±6.66	11.91±5.46
120	29.74±4.92	12.01±5.56	18.91±2.68	26.41±4.71	11.36±4.85
180	28.57±2.79	11.29±6.61	17.56±1.24	22.23±5.14	11.90±4.99
240	53.49±3.69	47.73±4.11	47.85±1.51	51.51±4.73	41.07±1.21
300	75.64±1.50	66.03±1.01^a	69.95±1.14	73.39±2.48	62.62±1.30^a
360	86.10±0.90	81.16±0.55	84.47±0.85	85.51±1.56	77.44±1.83^a

a: Different compared to positive control (diclofenac-Na)

Statistical analysis also showed that the NEEKL (dosage of 300 mg/ kg body weight) was not significantly different from the EEKL group (dosage of 500 mg/ kg body weight) at minutes of 30 to 360, p = 0.66 (P> 0.05), indicating that NEEKL (dosage of 300 mg/ kg body weight) had equal effect to EEKL (dosage of 500 mg/ kg body weight). These results represented that extract nanoparticles from Kersen leaves were enabled as an anti-inflammatory with a low dosage, and this extract nanoparticle had an equal effect to a high dosage of ethanol extract from Kersen leaves. This finding showed that nanoparticles in low dosages had anti-inflammatory effects, and effects were equal to those in high extracts. In addition, the low dosage of nanoparticles provided preferable anti-inflammatory effects compared to TPP-S chitosan treatment. These results were obtained because the nanoparticles were smaller than other formulations, thus providing a preferable impact^24,32. The size and particle form become factors that affect drug effectiveness²² due to particle size being very influential towards solubility, absorption, and drug distribution²⁹. A previous study reported that the protein denaturation inhibition potential of the extract was lower than that of the extracted nanoparticles at an equal dosage, and the extract nanoparticles had a protein denaturation inhibition potential lower than that of the positive control solution³³.

Based on the percentage decrease in edema volume, anti-inflammatory activity was produced. This finding is due to the possible presence of flavonoid compounds in Kersen leaves that have been known to play an essential role in inhibiting prostaglandins (PGE) and lipoxygenase (LOX). The flavonoid mechanism to suppress inflammation pathway was known in two ways: i.e. capillary permeability inhibited, arachidonic acid metabolism blockade, and lysosomal enzymes secretion from neutrophil and endothelial cells. Flavonoids primarily act on the microvascular endothelium to reduce permeability and inflammation. Several flavonoids can inhibit arachidonic acid release and lysosomal enzyme secretion from the membrane through cyclooxygenase pathway blockade. Cyclooxygenase pathway inhibition has a broad effect due to the cyclooxygenase reaction becoming the first step leading to eicosanoid hormones such as prostaglandins and thromboxane^3,25,29.

CONCLUSION:

The ethanol extract nanoparticles from Kersen (Muntingia calabura) leaves exhibited an anti-inflammatory effect (at a low dosage of 100 mg/ kg body weight) against λ-carrageenan induced rat, and it had an equal effect as high-dosage ethanol extracts (500 mg/ kg body weight). This finding might emphasize the potency of ethanol extract nanoparticles from Kersen leaves (NEEKL) as an alternative preferable anti-inflammatory formulation from a natural source.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

Acknowledgment:

The authors of this study are thanks to Research Institutions and the Faculty of Pharmacy, Universitas Sumatera Utara, Indonesia.

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Received on 27.07.2022 Modified on 08.09.2023

Research J. Pharm. and Tech 2024; 17(10):4677-4684.

DOI: 10.52711/0974-360X.2024.00721

Formula	Chitosan Concentration (%)	TPP-S Concentration (%)	Comparison of TPP-S and Chitosan	Particle Average Size
F1	0.05	0.01	5:1	94.21 nm
F2	0.075	0.01	7.5:1	167.49 nm
F3	0.1	0.01	10:1	397.1 nm
F4	0.2	0.01	20:1	428.6 nm
F5	0.3	0.01	30:1	17.12 µm
F6	0.4	0.01	40:1	18.47 µm