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Back to Journal »International Journal of Nanomedicine» Volume 16

High-load nano-suspension of Centella asiatica extract can improve skin delivery without irritation

Author Kim EA, Park JS, Kim MS, Jeong MY, Park HJ, Choi JH, Seo JH, Choi YS, Kang MJ

Published on November 3, 2021, the 2021 volume: 16 pages 7417-7432

DOI https://doi.org/10.2147/IJN.S335039

Single anonymous peer review

Editor who approved for publication: Dr. Mian Wang

Eun A Kim, Jun Soo Park, Min Seop Kim, Min Young Jeong, Hyun Jin Park, Jun Hyuk Choi, Jae Hee Seo, Yong Seok Choi, Myung Joo Kang School of Pharmacy, Dankook University, Cheonan, Chungnam, 330-714, South Korea Mailing address: Myung Joo Kang College of Pharmacy, 119 Dandae-ro, Dongnam-gu, Cheonan, 330-714, Korea Tel 82 41 550 1446 Fax 82 41 550 7899. Email [email protection] Background: The titrated extract of Centella Asiatica (CA) has attracted attention as a cosmeceutical ingredient for its anti-wrinkle effect. However, due to the low solubility and high molecular weight of pharmacologically active ingredients including Asiatic acid (AA), Asiatic acid (MA) and Asiaticoside (AS), a high payload of CA with satisfactory skin is produced Topical formulations are challenging. Absorption curve. Purpose: This study aims to design a high-load CA topical preparation using nano-crystallization technology and evaluate its skin absorption characteristics and local tolerance. Method: Using laboratory-scale bead milling technology, high-load nanocrystalline suspensions (NSs) were prepared by adjusting the type and amount of suspending agent, CA content, carrier type and grinding speed. NSs loaded with CA were characterized in terms of morphology, particle size, crystallinity, and in vitro dissolution mode. A vertical Franz diffusion cell equipped with pig skin was used to evaluate the skin absorption of CA nanocrystals. The skin irritation in vivo after topical application of high-payload NS was evaluated in normal rats. Result: The optimized NS system is composed of 10% (w/v) CA, 0.5% polyvinylpyrrolidone (PVP) K30 as a steric stabilizer and 89.5% distilled water. Its characteristics are as follows: spherical or elliptical, small in size at 200 nm, crystalline Degree is low. Under sinking conditions, the in vitro dissolution of AA or MA from NSs is significantly faster than that of raw materials. Formulated with commercially available cream (CA 1%, Madeka Cream). In addition, topical application of high-load NS is tolerable, and there is neither erythema nor edema in normal rats. Conclusion: The new NS system is expected to be a benign way to provide better skin absorption of CA without using excessive solubilizers. Keywords: Centella Asiatica, Asiatic Acid, Asiatic Acid, Madecassoside, Nanocrystalline Suspension, Bead Milling, Dissolution, Skin Absorption, Skin Irritation

The titrated extract of Centella asiatica (CA), also known as the common name of Gotu kola or Tiger Grass, is becoming more and more popular as a new type of cosmeceutical ingredient, by promoting the proliferation of fibroblasts and the 4,5 CA extract contains three The main components, namely asiaticoside (AS), asiatic acid (AA) and glycolic acid (MA) (Figure 1), as well as a variety of phytochemical components, such as flavonoids, sesquiterpenes, plant sterols, Pentacyclic triterpenoids or eugenol derivatives. 6 Local application of CA stimulates the proliferation of fibroblasts and activates the Smad signaling pathway, increases the production of type I collagen and reduces the formation of stretch marks and inflammation. 7,8 In addition, AA and MA increase blood microcirculation in the skin and prevent excessive accumulation of fat in cells. 9-11 Figure 1 The chemical structures of (A) AS, (B) AA and (C) MA. Abbreviations: AS, asiaticoside; AA, asiatic acid; MA, glycolic acid.

Figure 1 The chemical structures of (A) AS, (B) AA and (C) MA.

Abbreviations: AS, asiaticoside; AA, asiatic acid; MA, glycolic acid.

In order to initiate the tissue regeneration and wound healing activities of CA, it is a prerequisite to effectively deliver the bioactive ingredients to the relevant skin layer through the stratum corneum. The stratum corneum, the uppermost layer of the epidermis, with a thickness of 10-15 μm, is the biggest challenge for active substances to penetrate into the epidermis and dermis. 12,13 It consists of 15-20 layers of keratinocytes, which are embedded in lipid-rich interstitial spaces, mainly composed of ceramide, free fatty acids and cholesterol. The high molecular weight (959.1 Daltons) and hydrophilicity (logP value of 0.1) of 14,15 AS prevent the penetration of pentacyclic triterpenes through the stratum corneum. In addition, solubilization techniques are needed to formulate external preparations and prevent the penetration of hydrophobic aglycons into the skin layer. AA and MA are lipophilic components with poor water solubility, and their partition coefficient (logP) values ​​are 5.7 and 4.3, respectively. 16

Different pharmaceutical approaches have been implemented, such as the use of penetration enhancers, lipid nanoparticles, micelles, liposomes, and liposome vesicle systems to increase the solubility of water-insoluble ingredients and promote skin absorption into the relevant skin layer. 16-19 These methods have been implemented. Effectively increase the skin absorption of CA active ingredients and improve pharmacological activity. 16,17 However, even with these solubilization systems, the content of CA in topical preparations is controlled below 1.0% w/v because of the low solubility of AA or MA in the formulation. In addition, the previous CA skin absorption enhancement methods include high content of surfactants, fatty acids or co-solvents as dissolution or penetration enhancers, which may cause skin irritation such as itching, rash, and even local inflammation after long-term use. 20

Recently, drug nanocrystal suspensions (NS) as a promising topical and transdermal drug delivery system for lipophilic drugs and cosmetic substances have become the focus of attention. 21-23 Drug nanocrystals are colloidal dispersions of submicronized drug particles stabilized with minimal polymers or amphiphilic substances. 24,25 The decrease in particle size leads to an increase in surface area, which facilitates the adhesion of drug particles to biofilms. 26,27 In addition, the increase in dissolution rate and saturated solubility enhances the concentration gradient between the biofilm, including the stratum corneum and topical formulations, and subsequently allows higher penetration into the skin layer. 28-30 In addition, the increased curvature of particles with crystal sizes below 500 nm facilitates the penetration of intact drug particles into the skin. Hair follicles and their subsequent absorption by the surrounding hair follicles ep itherium.31 In particular, the nanosuspension system achieves high levels in topical formulations by dispersing insoluble compounds into a solid state with minimal suspension rather than dissolution in the continuous phase. Drug loading. 32,33

Here, the purpose of this study is to design a high-load NS for CA to promote the skin absorption of its three main components (AA, MA and AS). High-load NSs are prepared using laboratory-scale bead milling technology, with various formulation variables, such as the type and quantity of suspending agent, CA concentration, carrier type, and grinding speed. CA's NS is characterized by its morphology, particle size, crystallinity, and in vitro dissolution mode under water tank conditions. The in vitro skin absorption of CA nanocrystals depends on the CA content (1, 5, and 10 w/v%) or the type of carrier (distilled water (DW) or a mixture of DW and co-solvent), using pig skin installed. In addition, the skin irritation in vivo after the application of the new high-load CA system was further evaluated in normal rats.

CA dehydrated water extract, containing 36–44% AS and 54–66% AA or MA, purchased from Nanning Wellcome Pharmaceutical Technology Co., Ltd. Copolymer (Kollidon VA64), polyoxyethylene 40 hydrogenated castor oil (Kolliphor RH40) and Poloxamer 188 were purchased from Basf Co. (Ludwigshafen, Germany). With allyl sucrose or allyl pentaerythritol (Carbopol 934NF), polyoxyethylene 15 hydroxystearate (Kolliphor HS15), tyloxapol, polyoxyethylene (PEG) 40 stearate, butanediol ( BG), polysorbate 20 cross-linked acrylic acid, provided by polysorbate Co. 80. Co., Ltd. (Seoul, South Korea). Hypromellose (HPMC E50LV) was purchased from Whawon Pharm Co. Ltd. (Seoul, South Korea). The analytical standards of AS, AA and NA, sodium carboxymethylcellulose (Na.CMC), lecithin, methylcellulose and phosphate buffered saline tablets were purchased from Sigma Chemical Co. (St.Louis, MO, USA) . HPLC grade acetonitrile and methanol were purchased from JT Baker (Phillipsburg, NJ, USA). All other chemicals are of analytical grade and can be used without further refinement.

CA's NSs are manufactured by pulverizing drug powders into fine particles in an aqueous carrier using laboratory-scale bead milling technology. 34,35 Approximately 5-20 mg (0.5-2.0%, w/v) of the suspending agent listed in Table 1 is added to 1 mL of aqueous carrier (DW or a mixture of DW and BG (1:1 v/v)) Neutralize and vortex for 30 minutes to obtain a clear solution. Next, add 10-100 mg of CA and 1 g of zirconia beads (3 mm) to the solution and pre-wet using a multi-vortex for 5 minutes at room temperature. The coarse dispersion was bead-milled using ZentriMix 380R (Andreas Hettich GmbH und Co KG, Tuttlingen, Germany) at different speeds (1200, 1500 and 1800 rpm) for 4 hours. For each milling test, the cooling device was set to -10 °C to prevent thermal degradation of CA during the manufacturing process. The prepared NS was separated from the beads, placed in a scintillation vial, and stored under ambient conditions for further experiments. Table 1 The effect of suspending agent on the particle size and particle size uniformity of NSs loaded with CA

Table 1 The effect of suspending agent on the particle size and particle size uniformity of NSs loaded with CA

A scanning electron microscope (SEM, JSM-6510 type, JEOL, Tokyo, Japan) was used to examine the morphological characteristics of the raw materials and CA nanocrystals dispersed in the aqueous carrier. Approximately 20 μL of NS was loaded on an aluminum short tube by using double-sided tape and dried at room temperature for 2 hours to deplete the aqueous carrier. Next, by using an automatic sputtering coater (Model 108AUTO, Cressington, UK), the dried NS or raw material was coated with platinum at 20 mA for 10 minutes. Observe and take photomicrographs of the coating samples under an accelerating voltage of 20 kV.

Zetasizer Nano® Instruments (Malvern Instruments, UK) was used to analyze the average particle size, polydispersity index (PDI) and particle size distribution width of CA-loaded NS. 36,37 samples (100 μL) were diluted 10 times in DW, and then loaded into disposable cells. A 4 mW He-Ne laser (633 nm) was used to analyze the size distribution of NS at a scattering angle of 90° at 25 °C.

X-ray diffractometer (XRD, Model Ultima IV, Rigaku, Japan) was used to analyze the X-ray diffraction patterns of the raw materials, CA nanocrystals and carrier. The NS sample was centrifuged at 3500 × g for 10 minutes to separate the drug nanocrystals from the aqueous carrier. The collected CA nanocrystals were dried in an oven at 60°C. Load the sample on a glass plate and scan the diffraction pattern in the range of 5°–30° 2θ using Cu Kα radiation generated at 30 mA and 40 kV. The scan speed is set to 1.0 second/step, and the step size is 0.02.

A differential scanning calorimeter (DSC, model DSC 50, Shimadzu, Japan) was used to check the thermal behavior of the raw materials, the cured nanocrystals, and the carrier. The nanocrystals were collected using ultracentrifugation and a subsequent drying process as described in "X-ray powder diffraction analysis of the cross-section of CA nanocrystals". Place each solid sample (approximately 2 mg) in a standard aluminum pan and seal it with a lid. The phase change of each sample was recorded at a heating rate of 10 °C/min, and the nitrogen purge rate was 20 mL/min. An empty aluminum pan was used as a reference.

Several simultaneous quantification methods using HPLC analysis have been reported for the three CA components 38-40; however, analytical problems include low sensitivity or time-consuming gradient processes. Therefore, an HPLC analysis method for the hydrophilic component AS and another HPLC analysis method for the simultaneous determination of lipophilic compounds (AA and MA) have been established.

A Shimadzu HPLC consisting of a pump (Model 515 pump), a UV-VIS (ultraviolet-visible light) detector (Model 486) and an autosampler (Model 717 plus) was used to analyze the concentration of AS in the sample. The mobile phase (containing DW and acetonitrile in a volume ratio of 7:3) was passed through a reversed-phase C18 column (4.6 mm × 50 mm, 5.0 µm, Fortis) at a flow rate of 1.0 mL/min. The column temperature is set to 25 °C. Inject a 20 µL aliquot and monitor the column eluent at a wavelength of 210 nm. A Waters HPLC system consisting of a pump (model 515 pump), UV-VIS (ultraviolet-visible light) detector (model 486) and autosampler (model 717 plus) was used to determine the level of lipophilic compounds including MA and AA Concentration, equipped with reversed-phase C18 column (4.6 mm × 50 mm, 1.8 µm, Agilent, Santa Clara, California, USA). The mobile phase containing acetonitrile and DW in a volume ratio of 5:5 was run at a flow rate of 1.2 mL/min. The column temperature and detection wavelength were set to 25°C and 210 nm, respectively. The retention times of AA and MA are approximately 6.0 and 7.1 minutes, respectively.

The established HPLC method has been validated on a daily basis in terms of linearity, accuracy, precision, limit of detection (LOD) and limit of quantification (LOQ). For each component (AS, AA, and MA), five different concentrations were prepared by serially diluting the stock solution using the mobile phase. After analyzing the three sets of samples, the analytical accuracy of each concentration is calculated by the following formula: calculated concentration/nominal concentration × 100, and the analytical precision is calculated by the following formula: calculated concentration standard deviation/calculated average concentration × 100. The LOD and LOQ of this method are calculated based on the standard deviation (σ) of the response and the slope method. The LOD is calculated using the formula 3.3*σ/slope, and the LOQ is calculated using the formula 10*σ/slope.

The USP 2 paddle method (DT 720, Erweka) was used to compare and evaluate the in vitro dissolution profiles of AS, AA and MA from CA-loaded NS or micronized raw materials. Each formulation containing 10 mg of CA was added to 500 mL of dissolution medium maintained at 32 °C and stirred at a paddle speed of 100 rpm. In order to provide bath conditions with sufficient solubility for AS, AA and MA, 1% (w/v) sodium lauryl sulfate (SLS) was added to 10 mM phosphate buffer. Take out the dissolution medium (1 mL) through a syringe and replace it with an equal volume of preheated dissolution medium at the predetermined time (15, 30, 45, 60, 120, and 240 minutes). The taken samples were centrifuged at 13,000 rpm for 5 minutes to remove undissolved substances including drug nanocrystals. The supernatant (500 μL) was then diluted 5 times with methanol and analyzed by HPLC as described above.

The vertical Franz diffusion cell model was used to evaluate the in vitro skin penetration or retention of the three main components of CA after topical application of NSs. 41–43 One hour before the experiment, frozen pig skin tissue (thickness: 0.8–1.2 mm, Cronex Co. Ltd Gyeonggi-do, Korea) was thawed at room temperature, subcutaneous fat and muscle tissue were removed, and phosphate buffered saline (pH 7.4) Wash skin tissues. The diffusion area of ​​the Franz diffusion cell is set to 1.76 cm2. As the receptor medium, 9 mL phosphate buffer containing 1% (w/v) SLS was used to ensure the three-component tank conditions. After the temperature of the receptor medium is equilibrated at 32 ± 0.5°C, apply 250 μL of NSs (1%NS, 5%NS, 10%NS-1 and 10%NS-2) to the skin (n=4 per group) . A commercially available topical product (Madeca cream) containing 1% CA was used as a control group. At predetermined time intervals (0, 1, 2, 4, 6, 8, 10, 12, and 24 hours), remove 250 μL of receptor medium and replace with fresh receptor medium. Each sample was centrifuged at 13,000 rpm for 10 minutes, and the supernatant was diluted five times with methanol, and then analyzed by HPLC.

After 24 hours of in vitro skin penetration studies, according to previous literature, the amount of AS, AA, or MA in the skin layer was further determined. 41 Use Kim wipes to gently wipe the surface of the skin to remove the CA formula, which may adhere and remain on the skin surface. Then the collected skin was accurately weighed, cut into small pieces, and immersed in 10 mL of methanol. Use a shaking incubator at room temperature to extract the drug deposited in the skin by vigorous stirring for 24 hours. The solution was then centrifuged at 13,000 rpm for 5 minutes, and the concentration of AS, AA, and MA in the upper layer was analyzed by HPLC.

According to previous reports, the skin tolerance of high-load CA NSs was evaluated in normal rats. 44,45 This animal study was conducted after approval by the Institutional Animal Care and Use Committee (IACUC) of Dankook University (Cheonan, South Korea). (DKU-19-032, October 8, 2019) Follow the NIH Guidelines for the Care and Use of Laboratory Animals (National Academy of Sciences Press, 8th Edition, 2011). Sprague Dawley rats (male, 150-200 g, 6 weeks old) obtained from Samtako Bio Korea (Gyeonggi Province, South Korea) were raised under temperature (23±1°C) and light cycle (day/night: 12 hours)) Free access to feed and water. The acclimated rats were randomly divided into four groups (n=4 in each group). After anesthesia, chloroform inhalation was used to shave the back skin surface. After removing the hair on the back area, apply 200 µL of each sample (carrier, 10% NS-1, 10% NS-2 or Madeca cream as a positive control) topically and evenly spread on the back skin (6 cm2). This formula was applied topically every 24 hours for 5 days, and the degree of erythema or edema was evaluated every day before application. The severity of skin irritation is graded as follows: erythema score; no erythema, 0; very mild erythema, 1; clear erythema, 2; moderate to severe erythema, 3; severe erythema to eschar formation, 4; edema score, no Edema, 0; very mild edema, 1; mild edema, 2; moderate edema, 3; severe edema, 4.45,46

A one-way analysis of variance (ANOVA) was used for statistical analysis of the original data, followed by post-hoc analysis of multiple comparisons (SPSS Software 17, SPSS Inc., Chicago, IL, USA). The statistical significance was set as p-value<0.05.

In order to improve the skin absorption properties of pentacyclic triterpenes (AS, AA or MA) (CA's main pharmacologically active compounds), various carrier systems have been implemented, such as liposomes, transfer bodies, nanostructured lipid carriers or niosomes. 16-19 These drug methods can improve the penetration of the skin layer and increase the therapeutic effect, such as increasing the thickness of the dermis in animal models. However, the content of CA in these preparations is limited, ranging from 0.1% to 1.0% (w/v) because of its low solubility and/or limited load in the vesicles. Low levels of these pharmacologically active compounds cannot provide a sufficient concentration gradient between the skin and the topical formulation, thereby inhibiting the absorption of the skin layer. Therefore, as an alternative, we designed a high-load CA topical preparation using nanocrystalline technology. This alternative method is expected to promote the skin absorption of pentacyclic triterpenes by providing a high concentration gradient between the topical formulation and the biofilm, leading to an increase in the value of diffusion flux while minimizing the use of potentially harmful solubilizers, including Surfactant.

CA's NSs have been manufactured using wet grinding technology, which is a top-down method in which crude CA raw materials are crushed into sub-micron sizes in an aqueous carrier. Compared with other nano-size technologies, mechanical grinding using media grinding beads has the following advantages: low energy consumption, easy to scale up, no organic solvents, and minimal batch-to-batch variation. 47-49 In order to design CA NS, we first screened different hydrophilic polymers and surfactants as suspending agents to prevent the aggregation of drugs in the aqueous carrier or the precipitation of CA particles. The neutral hydrophilic polymer contained in the dispersion system may be adsorbed to the particle surface, thereby inhibiting crystal growth through steric hindrance. 48 Nonionic surfactants reduce the surface free energy of the dispersion system by reducing the interfacial tension between the hydrophobic drug particles and the aqueous medium. 50 When screening the suspending agent, the concentration of CA and suspending agent in the carrier were fixed at 100 mg/mL and 5 mg/mL, respectively, and the stirring speed was set to 1800 rpm. As shown in Table 1, all CA-loaded NSs prepared using hydrophilic polymers or surfactants immediately after preparation have the same size. The average particle size of CA particles is 144-347 nm, and the PDI value is an indicator of particle size uniformity, which is less than 0.35. In particular, use PVP K30 (201.4 nm), carbopol 934NF (177.7 nm), HPMC E50LV (144.6 nm) or Kolliphor HS15 (217.7 nm) to obtain smaller CA particles.

Subsequently, according to the type of suspension agent, under pressure, the aggregation or precipitation of drug particles was accelerated by centrifugation at 13,000 rpm to compare and evaluate the physical stability of the CA-loaded nanosuspension. After centrifugation, the sample was resuspended in a multi-vortex for 1 minute, and the uniformity and particle size were evaluated. Under stress conditions, the uniformity of the nanosuspension is maintained, but the crystal size increases. The particle size of NS prepared with poloxamer 188, polysorbate 20, polysorbate 80, or polyoxyethylene 40 hydrogenated castor oil increased by more than 400 nm (Table 1). On the contrary, when PVP K30 or Kolliphor HS15 is used as a dispersant in the carrier, NSs are physically stable and exhibit particle sizes of 219.7 and 215.7 nm, respectively, even after centrifugation. PVP polymer is a hydrogen bond acceptor polymer, which can be adsorbed on the surface of CA nanocrystals through hydrogen bonds and/or van der Waals interactions, which mainly contributes to the dispersion of hydrophobic nanocrystals in aqueous media without aggregation. In the case of Kolliphor HS15, the hydrophobic part of the amphiphilic stabilizer may be adsorbed to the surface of the drug crystal, effectively reducing the interfacial tension and surface free energy between the drug particle and the aqueous carrier. Among them, PVP K30 is a linear polymer of 1-vinyl-2-pyrrolidone monomer with safety in animal models, and was selected as a dispersant for CA-loaded NS. In animal studies, even with a 10% (w/v) PVP solution, no obvious signs of skin irritation or sensitization were observed51.

The particle size and uniformity of CA-loaded NS were evaluated according to component variables (PVP K30 concentration, drug concentration or carrier) or process parameters (bead milling strength). First, by adjusting the concentration of hydrophilic polymer and grinding strength to 5, 10 or 20 mg/mL and 1200, 1500 or 1800 rpm, respectively, the influence of PVP K30 concentration and bead grinding strength on crystal size was evaluated. Figure 2A). As expected, in all PVP K30 concentration tests, the particle size of CA nanocrystals decreased with the increase of mechanical grinding strength. The use of 1500 rpm or 1800 rpm milling intensity can advantageously produce uniform nanosuspensions with a size range of 190 to 230 nm, with no significant difference in PVP concentration (5, 10, or 20 mg/ml). On the other hand, when the PVP concentration is 20 mg/mL, the particle size increases to 340 nm at 1200 rpm; excessive PVP polymer (20 mg/mL) may cause drug flocculation at low grinding speed (1200 rpm) And/or gather. Therefore, in order to obtain a CA nanosuspension with a small and uniform particle size, the grinding intensity and concentration of the suspension agent were set to 1800 rpm and 5 mg/mL, respectively. Figure 2 The influence of formulation variables on the median diameter and uniformity of nanocrystals is manufactured using bead milling technology. (A) The influence of PVP K30 concentration and ball milling speed on crystal size and uniformity; (B) The influence of CA concentration and aqueous carrier (DW and BG 50 v/v% solution) on crystal size and uniformity. Abbreviations: CA, Centella asiatica; DW, distilled water; BG, butylene glycol.

Note: (A) CA concentration and grinding time are fixed at 100 mg/mL and 4 h respectively. (B) PVP K30 concentration, grinding speed and grinding time are fixed at 5 mg/mL, 1800 rpm and 4 h, respectively. Three batches of each sample were prepared, and the data were expressed as the mean ± SD (n=3).

Figure 2 The influence of formulation variables on the median diameter and uniformity of nanocrystals manufactured using bead milling technology. (A) The influence of PVP K30 concentration and ball milling speed on crystal size and uniformity; (B) The influence of CA concentration and aqueous carrier (DW and BG 50 v/v% solution) on crystal size and uniformity.

Abbreviations: CA, Centella asiatica; DW, distilled water; BG, butylene glycol.

Note: (A) CA concentration and grinding time are fixed at 100 mg/mL and 4 h respectively. (B) PVP K30 concentration, grinding speed and grinding time are fixed at 5 mg/mL, 1800 rpm and 4 h, respectively. Three batches of each sample were prepared, and the data were expressed as the mean ± SD (n=3).

Next, the influence of formulation variables (such as CA content or carrier type in the formulation) on crystal size and uniformity is shown in Figure 2B. DW and the mixture of DW and BG (1:1 v/v) were screened as dispersion media with different drug concentrations (10, 50, and 100 mg/mL). Co-solvents, such as BG, propylene glycol, or glycerin, are occasionally included in topical preparations to improve skin moisturizing effect and/or diffusion of drug nanoparticles on the skin. 52,53 The solubility mixture of AA and MA in DW and BG (1:1 v/v) is approximately 0.7 mg/mL and 0.1 mg/mL, respectively, which is higher than that of phosphate buffer (AA, 85 μg/mL; MA, 15 μg/mL, respectively) (data not shown). As shown in Figure 2B, as the CA concentration increases, the particle size tends to decrease. When DW is used as a dispersion medium, the drug particle sizes of NSs prepared with CA concentrations of 10, 50, and 100 mg/mL are estimated to be 493, 268, and 201 nm. This trend is more prominent when a mixture of DW and BG (1:1 v/v) is used; the particle size of NSs with CA content of 10, 50, and 100 mg/mL is determined to be 1178, 576, and 376 nm, respectively. Reducing the initial CA dose or increasing the solubility of the drug by adding BG leads to a decrease in solid drug particles in the carrier. Therefore, the polymer suspending agent may exceed the optimal concentration, resulting in the association and/or aggregation of CA crystals. This result is consistent with the increase in particle size with increasing PVP concentration (Figure 2A). In particular, the addition of BG may interfere with the interaction between the hydrophilic polymer and the drug particles and surface adsorption, resulting in particle aggregation and increase in particle size.

The established HPLC analytical methods used to determine the contents of AS, AA and MA have been validated on a day-to-day basis (Table 2). The assay showed excellent selectivity for retention times of 5.7, 7.6, and 8.3 minutes for AS, AA, and MA, respectively. In addition, it shows excellent linearity between the analyte concentration and the area under the peak, and the concentration ranges of AS, AA, and MA are 5–100, 10–100, and 10–100 μg/mL, respectively. The LOQs of AS, AA, and MA are estimated to be approximately 4.0, 7.0, and 5.8, respectively, ensuring the quantification of low-level active compounds in the sample (Table 2). The accuracy and precision of established HPLC methods have also been mutually verified. The accuracy of the estimation by dividing the calculated concentration by the nominal concentration. For AS, AA and MA, the range of all tested concentrations is 97.5% to 103.5%. In addition, in all concentrations of AS, AA, and MA, the relative standard deviation representing the precision of the analytical method is less than 4.0%. Therefore, the established HPLC method is used to evaluate the drug content, in vitro release profile, skin penetration and retention after topical application. Table 2 Day verification of HPLC methods for AS, AA and MA

Table 2 Day verification of HPLC methods for AS, AA and MA

Based on the above formulation research, we designed four different NS formulations, and characterized the physical and chemical properties according to appearance, drug content, particle size and crystallinity (Table 3 and Figure 3). Specifically, 0.5% PVP K30 was used as a dispersant to prepare NSs with different CA contents (1.0, 5.0, and 10% w/v) in DW (named 1% NS, 5% NS, and 10% NS-respectively). 1) ). In addition, NS composed of 10% CA and 0.5% PVP K30, in a mixture of DW and BG (1:1) (ie 10% NS-2), was used to further evaluate the effect of co-solvent on skin absorption. . Table 3 Physical and chemical properties of CA-loaded NS preparations and commercial products (Madeca Cream) Figure 3 Morphology and physical properties of CA-loaded NS. Representative micrographs of (A) CA raw material and (B) high-load NS (10%NS-1). (C) (a) Raw material, (b) CA-free carrier, (c) 10% NS-1, (d) 10% NS-2, (e) MA, (f) AS and (g) AA. (D) (a) Raw materials, (b) CA-free carrier, (c) 10% NS-1 and (d) 10% NS-2 XRD patterns. Abbreviations: CA, Centella asiatica; NS, nanocrystal suspension; DSC, differential scanning calorimeter; MA, glycolic acid; AS, asiaticoside; AA, asiatic acid; XRD, X-ray diffractometer.

Table 3 Physical and chemical properties of CA-loaded NS preparations and commercial products (Madeca Cream)

Figure 3 The morphology and physical characteristics of NS with CA. Representative micrographs of (A) CA raw material and (B) high-load NS (10%NS-1). (C) (a) Raw material, (b) CA-free carrier, (c) 10% NS-1, (d) 10% NS-2, (e) MA, (f) AS and (g) AA. (D) (a) Raw materials, (b) CA-free carrier, (c) 10% NS-1 and (d) 10% NS-2 XRD patterns.

Abbreviations: CA, Centella asiatica; NS, nanocrystal suspension; DSC, differential scanning calorimeter; MA, glycolic acid; AS, asiaticoside; AA, asiatic acid; XRD, X-ray diffractometer.

All NS preparations have uniform appearance, and the contents of AS, AA and MA in the preparations increase proportionally as the CA content increases from 1% (w/v) to 10% (w/v). The content of AS in 1% NS, 5% NS and 10% NS-1 increased proportionally to 3.49, 17.2 and 33.8 mg/mL, respectively (Table 3). In addition, the total content of aglycones such as MA or AA increased to 5.7, 25.6, and 49.1 mg/mL, respectively. This indicates that by uniformly suspending submicronized solid drug particles in an aqueous carrier, nanocrystalline technology can be used to significantly improve the loading of poorly water-soluble compounds in the aqueous carrier. As mentioned earlier, as the CA content in the carrier increases from 1% to 5% or 10%, the particle size tends to decrease; the crystal sizes in 1%NS, 5%NS, and 10%NS are determined to be 493 and 268, respectively And 201 nm. The addition of BG further increases the particle size; the particle size of 10% NS-2 is estimated to be 379.4 nm. Nevertheless, the particle size remains below 500 nm, which provides high saturation solubility and rapid diffusion compared with micronized particles. In a previous report, nanocrystals in the size range of 200-400 nm enhance the permeability of skin and mucous membranes by increasing saturated solubility, and therefore, promote the dissolution rate by reducing the diffusion distance. 54,55 Nanocrystals below 500 nm in size penetrate the skin and mucous membranes. The skin passes through the hair follicle and is subsequently absorbed by the surrounding hair follicle epithelium. 31 In addition, nanocrystals of the appropriate size (approximately 700 nm) can be deposited in hair follicles and appendages to serve as a reservoir for continuous delivery of therapeutic agents to surrounding tissues. 56

Use TEM to carefully observe the morphological characteristics of the nanocrystals suspended in the aqueous carrier. In the NS system, the nanocrystals prepared by the bead milling technology are spherical and/or elliptical (Figure 3B). Subsequently, DSC or XRD was used to evaluate the crystalline state of the nanocrystals in each formulation. In the DSC evaluation (Figure 3C), the high-purity MA, AS and AA powders showed unique endothermic peaks (representing melting points) at 270, 230, and 330 °C, respectively (Figure 3C, eg). These are consistent with the previously reported melting points of individual components. 57 These endothermic peaks are even smaller in the CA raw material (Figure 3C, a), showing that the endothermic peaks at 230 °C and 270 °C are weakened. In the CA raw material obtained by ethanol extraction, various components are fractionated together with the three main components, which may prevent the formation of a unique molecular arrangement. Part of the crystallinity of the raw materials in the NS formulation is further weakened, and there is no obvious endothermic peak in 10% NS-1 and 10% NS-2 (Figure 3C, c, and d). Similarly, XRD analysis shows that the characteristic refraction peaks of the CA raw material in the 2θ range between 12° and 17° (Figure 3D, a) are degraded in 10% NS-1 and 10% NS-2 (Figure 3D, c and d). The XRD pattern is formed by the constructive interference of X-ray beams scattered at a specific angle from the lattice plane of the crystalline material. 58 Our results show that the inherent crystallinity of the CA raw material decreases as the dispersant splits, and occasionally passes through the bead milling process with the co-solvent.

Under sinking conditions, the in vitro dissolution curves of each active ingredient of CA-loaded NSs (1% NS, 5% NS and 10% NS-1) were compared and evaluated with the in vitro dissolution curves of CA raw materials. In order to make the aglycon have sufficient solubility in aqueous media, SLS is included in phosphate buffered saline as a solubilizer. The solubility of AA or MA in 0.5% SLS solution (pH 6.8) is estimated to be 0.5 mg/mL or 0.4 mg/mL, respectively, which is approximately 50 or 76 times higher than the solubility obtained in pure PBS solution. Above the critical micelle concentration (0.5% w/v), anionic surfactants form a micellar structure containing hydrophobic compounds in the internal compartment. 59

Under water tank conditions, AA is a hydrophilic compound that dissolves rapidly in aqueous media, and the solubility exceeds 80% in 15 minutes in NS and raw materials (Figure 4A). The hydrophilicity of this compound leads to rapid dissolution and diffusion of powdered or intact CA particles. In contrast, the dissolution rate of AA or MA from NS is significantly faster than that of raw materials (Figure 4B and C). After 30 minutes, the amount of AA or MA released from the raw material was 53% and 59%, respectively. In contrast, the amount of AA and MA released from all NS formulations (1%NS, 5%NS, and 10%NS-1) within 30 minutes exceeded 80% and over 80%, respectively. This deep dissolution of NS under sink conditions can be explained by the Noyes-Whitney equation; dM/dt = k•S•Cs, where dM/dt, dissolution rate; k, rate constant; S, surface area of ​​drug particles; Cs , The solubility of the drug in the dissolution medium. The decrease in the particle size of the drug leads to a sharp increase in the surface area, thereby increasing the dissolution rate of the hydrophobic compound in the dissolution medium. 60,61 In addition, the destruction of the compact lattice exposes the internal hydrophobic surface of the crystal to the aqueous medium, increasing the saturation solubility determined by the Ostwald-Freundlich equation. 48,62 Figure 4 The in vitro dissolution profiles (Madeca cream) of (A) AS, (B) AA and (C) MA from NS loaded with CA, raw materials and commercially available cream formulations (Madeca cream) under sink conditions. Abbreviations: AS, asiaticoside; AA, asiatic acid; MA, glycolic acid; CA, Centella asiatica; NS, nanocrystal suspension; SLS, sodium lauryl sulfate.

Note: Provide tank conditions by adding 1% (w/v) SLS to 10 mM phosphate buffered saline (pH 6.8). Data are expressed as mean ± SD (n=3).

Figure 4 The in vitro dissolution profiles of (A) AS, (B) AA and (C) MA from CA-loaded NS, raw materials, and commercially available cream formula (Madeca cream) under sink conditions.

Abbreviations: AS, asiaticoside; AA, asiatic acid; MA, glycolic acid; CA, Centella asiatica; NS, nanocrystal suspension; SLS, sodium lauryl sulfate.

Note: Provide tank conditions by adding 1% (w/v) SLS to 10 mM phosphate buffered saline (pH 6.8). Data are expressed as mean ± SD (n=3).

The Franz diffusion cell installed on pig skin was used to evaluate the in vitro skin penetration and the retention of the main components of CA. In a wide range of animal models such as pigs, mice, rats, or guinea pigs, pig skin is widely used in skin absorption studies due to its high similarity to human skin and easy accessibility. 63 Although there are differences in the lipid arrangement between the two. Porcine and human stratum corneum. Porcine skin is histologically similar to human skin. The thickness (21-26 μm) and lipid composition of the stratum corneum (SC) are similar to human skin. 64-67 In addition, the average hair follicle density of pigs is 20 hair follicles/cm2 for pig ear skin, and 14-32 hair follicles/cm2 for human forehead skin. 66 To provide sinking conditions, the receptor medium contains SLS at a concentration of 0.5 w/v%. The interference or penetration enhancement effect of anionic surfactants on the skin at low concentrations is negligible. 16,68 As the test group, 1% NS, 5% NS, 10% NS-1 and 10% NS-2 were compared and evaluated with commercially available creams (Madeca Cream).

During the entire 24-hour skin penetration study under sink conditions, the main components of CA (AS, AA, and MA) were not detected in the receptor phase of all formulations including NS or commercially available products (data not shown) . AS has a high molecular weight exceeding 950 g/mol and hydrophilicity, making it difficult to penetrate into the stratum corneum. In the case of AA and MA, extreme lipophilicity (logP values ​​of 5.7 and 4.3, respectively) with a considerable molecular weight hinders skin penetration and instead is adsorbed and/or deposited in the stratum corneum. The penetration results are consistent with previous reports. Rocha et al. (2019) reported that the topical application of CA formulated into solid lipid nanoparticles resulted in more accumulation of AS in the skin layer, but AS was not detected in the receptor medium. 16 Kim et al. (2002) also reported that after topical application of the niosomal formulation of CA.17, the three components did not penetrate the excised skin of hairless mice.

After a 24-hour penetration study, the individual amount of AS, AA, or MA deposited in pig skin was determined (Figure 5). First, after applying 1% NS (AS, 3.5 mg/g; AA, 2.5 mg/ml; and MA, 3.3 mg/ml, Table 3) or Madeca cream, the amount of AS, AA or MA accumulated in the skin ( AS, 2.8 mg/g; AA, 1.7 mg/mL; and MA, 3.0 mg/mL, Table 3) contain similar amounts of active ingredients for comparison. Madeca Cream contains three dissolved ingredients through the use of solubilizers such as dipropylene glycol, caprylic/capric triglyceride, glycerin, glyceryl stearate, cetyl alcohol and vegetable oils. 69 In contrast, poorly water-soluble aglycones (AA or MA) are almost contained in the 1% NS formulation in the form of solid nanocrystals. Nevertheless, there is no significant difference in the deposition of the three components in the skin between 1% NS and Madeca cream. After topical application of 1% NS and Madeca cream, the deposition of AS was 23.5 and 30.5 µg/g, respectively. The deposits of AA (9.5 and 6.4 µg/g, respectively) or MA (13.7 and 15.3 µg/g, respectively) are also similar to 1% NS and Madeca creams. This indicates that although the hydrophobic component exists in an undissolved state in the NS, by forming a high concentration gradient between the hydrogel and the stratum corneum, the active compound is rapidly dissolved or adsorbed in the relevant skin layer. In addition, the intact CA nanocrystals and/or dissolved molecules may have penetrated into the skin through the surrounding hair follicle epithelium, exhibiting skin absorption comparable to commercially available creams, without the use of oily solubilizers or surfactants. Figure 5 (A) AS accumulation, (B) AA and (C) MA in the application load CA's NSs (1%NS, 5%NS, 10%NS-1 and 10%NS-2) or 24 hours after the market Cream formula in MA Franz diffusion cell model in pig back skin. Abbreviations: AS, asiaticoside; AA, asiatic acid; MA, glycolic acid; CA, Centella asiatica; NS, nanocrystal suspension.

Note: Data is expressed as mean ± SD (n = 4). Use one-way analysis of variance for statistical analysis. *p <0.05, compared with Madeca cream; ** p <0.05, compared with 1% NS; *** p <0.05, compared with 5% NS.

Figure 5 Application of CA loaded NSs (1% NS, 5% NS, 10% NS-1 and 10% NS-1 and 10% NS-2) or the commercially available cream formula in the Franz diffusion cell model.

Abbreviations: AS, asiaticoside; AA, asiatic acid; MA, glycolic acid; CA, Centella asiatica; NS, nanocrystal suspension.

Note: Data is expressed as mean ± SD (n = 4). Use one-way analysis of variance for statistical analysis. *p <0.05, compared with Madeca cream; ** p <0.05, compared with 1% NS; *** p <0.05, compared with 5% NS.

When the content of CA in NS preparation increases from 1% to 5% or 10% (w/v), the amount of AS, AA or MA deposited in pig skin after topical application of NS preparation increases proportionally. After topical application of 1% NS, the cumulative amounts of AS, AA, or MA deposited in the skin were 23.5, 9.5, and 13.7 µg/g, respectively, while the cumulative amounts obtained with 5% NS were 90.9, 13.3, and 60.2 µg/g, They are 3.9, 1.4 and 4.4 times higher than 1% NS, respectively. The amount of AS, AA, or MA deposited in the skin further increased with 10% NS-1. Compared with 1%, the accumulation in the biofilm increased by 6.5, 2.0, or 5.0 times NS, respectively. In addition, the amount of AS, AA, or MA accumulated in the skin with 10% NS-1 was about 1.7, 1.5, or 1.1 times higher than the amount obtained from 5% NS, respectively. This indicates that the increase in the CA load in the formulation leads to an increase in the concentration gradient between the formulation and the surface layer, leading to an increase in the value of diffusion flux, thereby promoting a higher accumulation of active ingredients in the surface layer.

Next, compare the dosages of the three ingredients after applying 10% NS-1 or 10% NS-2 to evaluate the effect of the co-solvent on the skin absorption of CA. Co-solvents, including BG, are commonly used in topical preparations and are reported to be used as humectants to moisturize the outer layer of the skin. In addition, it can dissolve the active ingredients and improve the penetration of hydrated skin. 52,53 However, in our study, there was no significant difference in skin absorption between the 10% NS-1 and 10% NS-2 groups; the AS, AA, and MA of the 10% NS-1 group were 152.9 µg/g, respectively , 19.7 µg/g and 68.1 µg/g, the 10% NS-2 group was 182 µg/gg, 21.2 µg/g and 50.0 µg/g. This indicates that the concentration gradient between the formulation and the skin layer has a greater impact on the skin absorption of the three components than the carrier type. In addition, the increase in particle size in the presence of co-solvents may hinder the dissolution and/or penetration of NSs into the skin.

According to OECD Guideline 404: Acute Skin Irritation/Corrosion 46, skin irritation with high load of NSs (10% NS-1 or 10% NS-2) was evaluated in normal rats. It is essential to assess the local tolerability or potential harm of CA high-load preparations. The negative control group received a carrier without CA, and the positive control group received a commercially available cream (Madeca cream, CA 1.0% (w/v)). As shown in Figure 6, during the 5 days of the experimental period, no skin irritation such as erythema or edema was observed in any treatment group. Therefore, when erythema and edema are scored on a scale of 0-4 according to the OECD guidelines, the scores for all groups are reported to be zero (Table 4). The tolerability of local CA is consistent with the previous report, that is, no macroscopic skin reactions attributable to allergies were observed after the dressing was sealed with 50% CA paraffin oil in guinea pigs for 24 hours. 70 Therefore, the high-payload NS system may be an effective tool to improve skin absorption by increasing the concentration gradient without causing any irritation and/or sensitization to the skin. Table 4 Apply vehicles, high-load NSs (10% NS-1 and 10% NS-2) or commercially available creams to the back of normal rats (n = 5 per group) Figure 6 Daily use of CA-free carriers, Representative image of back skin of normal rats after 5 days with high load of NSs (10% NS-1 or 10% NS-2) or commercially available cream. Abbreviations: CA, Centella asiatica; NS, nanocrystal suspension.

Table 4 Apply vehicles, high-load NSs (10% NS-1 and 10% NS-2) or commercially available cream to the back of normal rats (n = 5 per group)

Figure 6 Representative images of the back skin of normal rats after daily use of CA-free carriers, high-load NSs (10% NS-1 or 10% NS-2) or commercially available creams for 5 days.

Abbreviations: CA, Centella asiatica; NS, nanocrystal suspension.

In this study, a new type of high-load CA external preparation was successfully prepared by using nanocrystalline technology, which improved the loading capacity of the preparation and the skin absorption rate of active compounds such as AA and MA. By adjusting the type and quantity of space stabilizers, the CA content and the grinding speed, it is profitable to use wet grinding technology to prepare NS. The optimized NS system is composed of 10% CA, 0.5% PVP K30 as steric stabilizer and 89.5% DW. Its characteristics are as follows: spherical or elliptical, with a size of 200 nm and low crystallinity. Under sinking conditions, the release rate of AA and MA in NC is significantly faster than that of raw materials. With the increase of CA content in the preparation, the accumulation of hydrophilic (AS) and hydrophobic (AA or MA) ingredients in the skin layer increased significantly, providing 5, 4 and 4.5 times of AS, AA and MA, and the market Compared with selling creams. In addition, the high load system of CA is tolerable, and there is neither erythema nor edema in the normal rat model. Therefore, the new NS system can be a reasonable way to improve CA skin delivery without any skin irritation or the need for surfactants.

This research was supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF), which was funded by the Ministry of Science, Information and Communication Technology and Future Planning (NRF-2019R1C1C1004211).

The author reports that there is no conflict of interest for this work.

1. Camacho-Alonso F, Torralba-Ruiz MR, Garcia-Carillo N, Lacal-Lujan J, Martinez-Diaz F, Sanchez-Siles M. Topical application of porcine acellular bladder matrix and Centella asiatica extract for oral wound healing The effect is in the rat model. Clinical oral survey. 2019;23(5):2083-2095. doi:10.1007/s00784-018-2620-x

2. Aziz HA, Taher M, Sulaiman WMAW, Susanti D, Chowdhury SR, Zakaria ZA. In vitro and in vivo wound healing study of the methanol part of Centella asiatica extract. S Afr J Bot. 2017; 108: 163-174. doi:10.1016/j.sajb.2016.10.022

3. Yao CH, Yeh JY, Chen YS, Li MH, Huang CH. Wound healing effect of electrospun gelatin nanofibers containing Centella asiatica extract in a rat model. J Tissue Eng Regen Med. 2017; 11(3): 905–915. doi:10.1002/term.1992

4. Lu L, Ying K, Wei S, Liu Y, Lin H, Mao Y. DNA microarray analysis showed that asiaticoside induced genes related to skin fibroblasts in vitro. Br J Dermatology. 2004;151(3):571–578. doi:10.1111/j.1365-2133.2004.06146.x

5. Bylka W, Znajdek-Awiżeń P, Studzińska-Sroka E, Brzezińska M. Centella asiatica in cosmetology. Adv Dermatol Allergol. 2013; 30:46-49. doi:10.5114/pdia.2013.33378

6. Gray NE, Alcazar MA, Lak P, etc. Centella asiatica: phytochemical and neuroprotective and cognitive enhancement mechanisms. Phytochem Rev. 2017;17(1):161-194. doi:10.1007/s11101-017-9528-y

7. Lu L, Ying K, Wei S, et al. Madecassoside induces cell cycle progression, proliferation and collagen synthesis of human dermal fibroblasts. International Journal of Dermatology. 2004;43(11):801–807. doi:10.1111/j.1365-4632.2004.02047.x

8. Lee J, Jung E, Kim Y, etc. Asiaticoside induces the synthesis of human collagen I through Smad signaling that has nothing to do with TGFbeta receptor I kinase (TbetaRI kinase). Plant medicine. 2006;72(4):324–328. doi:10.1055/s-2005-916227

9. Ratz-łyko A, Arct J, Pytkowska K. Moisturizing and anti-inflammatory properties of cosmetic formulations containing Centella asiatica extract. J Pharm Sci, India. 2016;78(1):27–33. doi:10.4103/0250-474x.180247

10. Shukla A, Rasik AM, Dhawan BN. Asiaticoside-induced antioxidant levels increase in healing wounds. Phytother Res. 1999;13(1):50-54. doi:10.1002/(SICI)1099-1573(199902)13:1<50::AID-PTR368>3.0.CO;2-V

11. Flynn TC, Petros J, Clark RE, Viehman GE. Dry skin and moisturizer. Clinical Dermatology. 2001;19(4):387–392. doi:10.1016/S0738-081X(01)00199-7

12. Godin B, Touitou E. Transdermal skin delivery: human predictions from in vivo, ex vivo, and animal models. Adv Drug Deliv Rev. 2007;59(11):1152-1161. doi:10.1016/j.addr.2007.07.004

13. Ruela ALM, Perissinato AG, de Lino MES, Mudrik PS, Pereira GR. Evaluate skin absorption of drugs in topical and transdermal formulations. Braz J Pharm Sci. 2016;52(3):527–544. doi:10.1590/s1984-82502016000300018

14. Anissimov YG, Jepps OG, Dancik Y, Roberts MS. Mathematical and pharmacokinetic modeling of epidermal and dermal transport processes. Adv Drug Deliv Rev. 2013; 65: 169-190.

15. Jepps OG, Dancik Y, Anissimov YG, Roberts MS. Simulate the human skin barrier-better understand skin absorption. Advanced Drug Deliv Rev. 2013;65(2):152-168. doi:10.1016/j.addr.2012.04.003

16. da Rocha PBR, Dos Santos B, Andrade LM, etc. Lipid nanoparticles loaded with Centella asiatica enhance skin penetration of asiaticoside: the effect of extract type and stratum corneum lipid dynamics study. J Drug Deliv Sci Technol. 2019;50:305-312. doi:10.1016/j.jddst.2019.01.016

17. Kim D, Cho M, Park S, etc. Preparation and evaluation of Centella asiatica niosome/W/O system cream titration extract for site-specific targeting. J Pharm investigation. 2002;32:291-297.

18. Liu Ming, Chen Wei, Zhang X, etc. Temperature-responsive PEG-PCL-PEG copolymer modified madecassoside liposomes improve surface adhesion and wound healing. Eur J Pharm Sci. 2020;151:105373. doi:10.1016/j.ejps.2020.105373

19. Wichayapreechar P, Anuchapreeda S, Phongpradist R, Rungseevijitprapa W, Ampasavate C. Use hyaluronic acid surface-modified niosomes to target the skin of Centella asiatica extract. J liposome research. 2020;30(2):197-207. doi:10.1080/08982104.2019.1614952

20. Effendi I, Maybach HI. Surfactants and experimental irritant contact dermatitis. Contact genuine leather. 1995;33(4):217-225. doi:10.1111/j.1600-0536.1995.tb00470.x

21. Wang Y, Wang S, Xu Y, etc. Etoposide amorphous nanopowder for improving oral bioavailability: formulation development, optimization, in vitro and in vivo evaluation. Int J Nanomed. 2020; 15:7601–7613. doi:10.2147/IJN.S265817

22. Pireddu R, Sinico C, Ennas G, etc. Two novel nano-scale preparations of diclofenac polymorphs can improve local bioavailability. Eur J Pharm Sci. 2015; 77: 208-215. doi:10.1016/j.ejps.2015.06.006

23. Im S, Jung H, Ho M, etc. Montelukast nanocrystals for transdermal administration have improved chemical stability. pharmaceutics. 2019; 12(1):18. doi:10.3390/pharmaceutics12010018

24. Pawar VK, Singh Y, Meher JG, Gupta S, Chourasia MK. Engineering nanocrystal technology: in vivo fate, targeting and drug delivery applications. J Control release. 2014;183:51-66.

25. Aref ZF, Bazeed SEES, Hassan MH, etc. Clinical, biochemical and molecular evaluation of ivermectin mucoadhesion nano-suspension nasal spray in reducing mild upper respiratory tract symptoms of COVID-19. Int J Nanomed. 2021; 16: 4063-4072. doi:10.2147/IJN.S313093

26. Gao L, Zhang D, Chen M. Drug nanocrystals for the formulation of poorly soluble drugs and their application as a potential drug delivery system. J Nanopart Res. 2008;10(5):845–862. doi:10.1007/s11051-008-9357-4

27. Junyaprasert VB, Morakul B. Nanocrystals used to improve the oral bioavailability of poorly water-soluble drugs. Asia J Pharm Sci. 2015;10(1):13-23. doi:10.1016/j.ajps.2014.08.005

28. Chen Li, Wang Y, Zhang Jie, etc. Bexarotene nanocrystals-oral and parenteral formulation development, characterization and pharmacokinetic evaluation. Eur J Pharm Biopharm. 2014;87(1):160–169. doi:10.1016/j.ejpb.2013.12.005

29. Zhai X, Lademann J, Keck CM, Müller RH. Dermal nanocrystals from moderately soluble active substances-physical stability and stability of influencing parameters. Eur J Pharm Biopharm. 2014;88(1):85-91. doi:10.1016/j.ejpb.2014.07.002

30. Chen Y, Liu Y, Xie J, et al. Nasal-to-brain delivery via nano-suspension-based in situ gel breviscapine. Int J Nanomed. 2020; 15:10435–10451. doi:10.2147/IJN.S265659

31. Patzelt A, Richter H, Knorr F, etc. Selective follicle targeting by changing the particle size. J Control release. 2011;150(1):45–48. doi:10.1016/j.jconrel.2010.11.015

32. Liversidge GG, Cundy KC. Reduce the particle size to improve the oral bioavailability of hydrophobic drugs: the absolute oral bioavailability of nanocrystalline danazol in beagle dogs. Int J Pharm. 1995;125(1):91-97. doi:10.1016/0378-5173(95)00122-Y

33. Huang Tao, Wang Yan, Shen Yan, etc. Preparation of high drug-loaded celastrol nano-suspension and its anti-breast cancer activity in vitro and in vivo. Scientific Reports 2020; 10(1): 8851. doi:10.1038/s41598-020-65773-9

34. Steiner D, Bunjes H. The influence of process and formulation parameters on the preparation of solid lipid nanoparticles by double centrifugation. Int J Pharm. 2021; 3: 100085. doi:10.1016/j.ijpx.2021.100085

35. Hagedorn M, Liebich L, Bögershausen A, etc. The rapid development of API nano formulations from screening to production, combined with double centrifugation and wet agitator bead milling. Int J Pharm. 2019;565:187-198. doi:10.1016/j.ijpharm.2019.04.082

36. Erdoğar N, Akkın S, Nielsen TT, etc. Development of chitosan-polyethylene glycol coated cyclodextrin nanocapsules for oral administration of aprepitant: formulation, characterization and pharmacokinetic evaluation. J Pharm investigation. 2021;51(3):297-310. doi:10.1007/s40005-020-00511-x

37. Shamarekh KS, Gad HA, Soliman ME, etc. Monodisperse gelatin nanoparticles are produced through an improved one-step desolvation technique. J Pharm investigation. 2020;50(2):189–200. doi:10.1007/s40005-019-00455-x

38. Günther B, Wagner H. Centella Asiatica (L.) Urban extract and the quantitative determination of triterpenes in plant preparations. Plant medicine. 1996; 3(1): 59-65. doi:10.1016/S0944-7113(96)80011-0

39. Wang C, Zhao Y, Yang Rui, et al. Simultaneous analysis of five triterpenes in Centella asiatica by high performance liquid chromatography with cyclodextrin as a mobile phase additive. Science Representative 2020; 10: 18577. doi:10.1038/s41598-020-75554-z

40. Rafamantanana MH, Rozet E, Raoelison GE, etc. An improved HPLC-UV method for simultaneous quantification of triterpene glycosides and aglycons in Centella asiatica (L.) Urb (APIACEAE) leaves. J Chromatogr B Analytical technology biomedical life sciences. 2009;877(23):2396-2402. doi:10.1016/j.jchromb.2009.03.018

41. Yu Qiang, Wu Xu, Zhu Qiang, etc. Enhanced transdermal delivery of meloxicam through nanocrystals: preparation, in vitro and in vivo evaluation. Asia J Pharm Sci. 2018; 13(6): 518-526. doi:10.1016/j.ajps.2017.10.004

42. Pandey KU, Joshi A, Dalvi SV. To evaluate the efficacy of different curcumin polymorphs in transdermal administration. J Pharm investigation. 2021;51(1):75–84. doi:10.1007/s40005-020-00496-7

43. Lin C, Fang C, Al-suwayeh S, etc. Selenium-L-methionine, antioxidants and other selenium substances are absorbed through the skin in vitro and in vivo. Journal of Pharmaceuticals. 2011;32(9):1181–1190. doi:10.1038/aps.2011.89

44. Winnicka K, Wroblewska M, Sosnowska K, Car H, Kasacka I. Evaluation of skin toxicity of cationic polyamidoamine dendrimer in rat skin model. The drug Devel Ther. 2015; 9: 1367-1377. doi:10.2147/DDDT.S78336

45. Barakat NS. Evaluation of glycogen-based gel as a new carrier for topical application of naproxen. AAPS Pharmaceutical Technology. 2010;11(3):1138-1146. doi:10.1208/s12249-010-9485-x

46. ​​OECD Chemical Testing Guidelines. Part 4: Health Effects Test No. 404: Acute Skin Irritation/Corrosion. Paris: Published by the OECD; 2015.

47. Liedtke S, Wissing S, Müller RH, Mäder K. The influence of high-pressure homogenization equipment on nano-dispersion characteristics. Int J Pharm. 2000;196(2):183–185. doi:10.1016/S0378-5173(99)00417-2

48. Jacob S, Nair AB, Shah J. The emerging role of nanosuspensions in drug delivery systems. Biomaterials Research 2020;24(1):3. doi:10.1186/s40824-020-0184-8

49. Niwa T, Miura S, Danjo K. The general wet milling technology for preparing oral nanosuspensions focuses on discovery and preclinical animal research-the development of particle design methods. Int J Pharm. 2011;405(1–2):218–227. doi:10.1016/j.ijpharm.2010.12.013

50. Ziller KH, Rupprecht H. Control of crystal growth in drug suspensions: 1 Control unit design and application of acetaminophen suspensions. Drug Dev Ind Pharm. 1988;14(15–17):2341–2370. doi:10.3109/03639048809152019

51. Nair B. Final report on the safety assessment of polyvinylpyrrolidone (PVP). International Journal of Toxicology. 2017;36(2):14–58.

52. Watkinson RM, Guy RH, Hadgraft J, Lane ME. Optimization of the concentration of cosolvent for topical administration-II: The effect of propylene glycol on the penetration of ibuprofen. Skin pharmacology. 2009;22(4):225-230. doi:10.1159/000231528

53. Baba H, Takahara J, Yamashita F, Hashida M. Support vector regression and random forest are used to model and predict the influence of solvents on human skin permeability. Medical research. 2015;32(11):3604–3617. doi:10.1007/s11095-015-1720-4

54. Pireddu R, Caddeo C, Valenti D, etc. Diclofenac nanocrystals are an effective strategy to reduce skin inflammation in the body by improving the bioavailability of skin drugs. Colloidal surfing B Biological interface. 2016; 143: 64-70. doi:10.1016/j.colsurfb.2016.03.026

55. Li Y, Wang D, Lu S, et al. Pramipexole nanocrystals for transdermal penetration: characterization and its enhanced micromechanism. Eur J Pharm Sci. 2018; 124: 80-88. doi:10.1016/j.ejps.2018.08.003

56. Chogale MM, Ghodake VN, Patravale VB. Performance parameters and characterization of nanocrystals: a brief review. pharmaceutics. 2016;8(3):26. doi:10.3390/pharmaceutics8030026

57. Asian acid. Chemistry book website. Available from: https://www.chemicalbook.com/ChemicalProductProperty_EN_cb5109340.htm. Visited on October 28, 2021.

58. Bunaciu AA, Udriştioiu EG, Aboul-Enein HY. X-ray diffraction: instruments and applications. Crit Rev Anal Chem. 2015;45(4):289–299. doi:10.1080/10408347.2014.949616

59. Alizadeh MN, Shayanfar A, Jouyban A. Using sodium lauryl sulfate to dissolve drugs: experimental data and modeling. J Mol Liq. 2018; 268: 410-414. doi:10.1016/j.molliq.2018.07.065

60. Noyes AA, Whitney WR. The dissolution rate of a solid substance in its own solution. J Am Chem Soc. 1897;19(12):930-934. doi:10.1021/ja02086a003

61. Abdelghany S, Tekko IA, Vora L, Larrañeta E, Permana AD, Donnelly RF. Nanosuspension-based dissolving microneedle arrays are used for the intradermal delivery of curcumin. pharmaceutics. 2019;11(7):308. doi:10.3390/pharmaceutics11070308

62. Kesisoglou F, Panmai S, Wu Y. Nanosizing-oral formulation development and biopharmaceutical evaluation. Adv Drug Deliv Rev. 2007;59(7):631-644. doi:10.1016/j.addr.2007.05.003

63. Lademann J, Richter H, Meinke M, Sterry W, Patzelt A. Which skin model is best for studying substances applied topically to hair follicles? Skin pharmacology. 2010;23(1):47–52. doi:10.1159/000257263

64. Gray G, Yardley H. Lipid composition of cells isolated from the epidermis of pigs, humans, and rats. J Lipid Research. 1975; 16(6): 434–440. doi:10.1016/S0022-2275(20)34493-X

65. Wester RC, Melendres J, Sedik L, Maibach H, Riviere JE. Compared with humans in vivo, the transdermal absorption of salicylic acid, theophylline, 2,4-dimethylamine, diethylhexylphthalic acid and p-aminobenzoic acid in the isolated perfused pig skin flaps. Toxicol Appl Pharmacol. 1998; 151(1): 159-165. doi:10.1006/taap.1998.8434

66. Jacobi U, Kaiser M, Toll R, etc. Pig ear skin: an in vitro model of human skin. Skin repair technology. 2007;13(1):19-24. doi:10.1111/j.1600-0846.2006.00179.x

67. Wester RC, Maybach HI. In vivo method of transdermal absorption measurement. J Toxicol skin Ocul Toxicol. 2001;20(4):411–422. doi:10.1081/CUS-120001866

68. Seto JE, Polat BE, Lopez RF, Blankschtein D, Langer R. The effect of ultrasound and sodium lauryl sulfate on the transdermal delivery of hydrophilic permeate: an in vitro comparative study with full-thickness and split-thickness pig skin and human skin. J Control release. 2010;145(1):26-32. doi:10.1016/j.jconrel.2010.03.013

69. Centellian24 madeca cream strength enhancing formula. DongKook Pharmaceutical Co., Ltd. can be obtained from the following website: http://www.dkpharm.co.kr/product/view.php?idx=137. Accessed on October 28, 2021.

70. Review of cosmetic ingredients. Safety assessment of Centella asiatica-derived ingredients used in cosmetics; 2015. Available from: https://www.cir-safety.org/supplementaldoc/safety-assessment-centella-asiatica-derived-ingredients-used-cosmetics-1. Visited on October 28, 2021.

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