Potential Lipolytic Effect of Panduratin A Loaded Microspicule Serum as a Transdermal Delivery Approach for Subcutaneous Fat Reduction

Abstract

Boesenbergia rotunda (L.) Mansf contained a potent anti-obesity agent. The objectives of this study were to investigate the anti-adipogenesis and lipolysis effects of panduratin A from B. rotunda extract and develop extract-loaded lipolytic body microspicule (MS) serum. Panduratin A that was separated from the ethanolic extract of B. rotunda in fraction 3 (BP-3) were studied the bioactivity of 3T3-L1 preadipocyte cells. The extract-loaded MS serum was formulated and evaluated for safety and efficacy. The BP-3 extract containing panduratin A at 0.29 g per g of the extract was not toxic to the cells at concentrations lower than 10 µg/mL, and the antiadipogenesis and lipolysis effects of the BP-3 extract were strong at 10 µg/mL. To deliver bioactive panduratin A into and through the skin, MS serum was successfully formulated. Application of BP-3 extract-loaded MS serum to the human thigh for 14 d reduced the thigh circumference and increased skin hydration and firmness. Although the skin erythema was increased, no severe redness or pain was found. In conclusion, BP-3 extract acts as a potent bioactive compound to inhibit adipocyte cells, and the antiadipogenesis and lipolysis effects of BP-3 extract in MS serum might play an important role as a potential lipolytic body product for reducing human subcutaneous fat mass.

INTRODUCTION

Boesenbergia rotunda (L.) Mansf. (Family: Zingiberaceae) is commonly known as a medicinal plant, and many bioactive compounds, such as pinostrobin, cardamonin, boesenbergin, 5,7-dihydox-yflavone, 1,8-cineole, and panduratin A, have been found in this extract.¹⁾ The anti-obesity activity of B. rotunda has been reported as a potent anti-obesity agent for high-fat diet-induced obesity in mice by activating AMP-activated protein kinase (AMPK) and regulating lipid metabolism. Oral administration of B. rotunda extract (200 mg/kg/d for 8 weeks) significantly reduced body weight gain, serum levels (total cholesterol, low-density lipoprotein cholesterol, and triglyceride) and fat pad masses by reducing adipocyte size. Active panduratin A (Fig. 1) has biological activities, including anti-inflammatory, antioxidative, antibacterial, and antiobesity activities.²⁾ Moreover, B. rotunda extract and its active compound panduratin A are potent nutraceuticals that enhance skin hydration and barrier function based on their cornified envelope formation and filaggrin processing.³⁾ However, the effect of panduratin A isolated from B. rotunda on topical lipolytic body products has not yet been clarified.

Fig. 1. Chemical Structure of Panduratin A

Topical fat loss formulations have been developed to affect thigh circumference, skinfold thickness, and fat mass. Several active ingredients, such as aminophylline, l-carnitine, gotu kola, yohimbe, and caffeine, have been used in this product to improve body composition.⁴⁾ Transdermal delivery of active compounds is effective in therapeutic administration without side effects and hepatic first-pass metabolism by the gastrointestinal system, which is a problem of oral drug delivery. In addition, the transdermal delivery system has several advantages over hypodermic injection, such as noninvasiveness, painlessness and patient self-administration.⁵⁾ However, the permeability barrier of the skin is a major limitation to deliver drugs into the skin.⁶⁾ To overcome the skin barrier, microneedles are efficient physical enhancers that bypass the stratum corneum barrier and deliver active compounds through the skin.⁷⁾ Moreover, microdermabrasion can also be used to increase skin permeability by damaging or removing the top layer of the skin stratum corneum.⁸⁾

Microspicules (MSs) are needle-like structures of sponge spicules with tips growing out from two opposite sites.^9,10) The skeleton structure is controlled by the precipitation of calcium carbonate or silicon dioxide. MS has been used as a natural alternative approach to dermabrasiveness and to treat skin problems, such as hyperpigmentation of different etiologies, fine wrinkles, sun-damaged skin, superficial scarring, comedones, enlarged facial pores, and dull skin. Upon massaging Spongillia spicules onto the skin, mechanical separation of the epidermal layers has been reported, leading to reduced keratinocyte cohesion, increased stratum corneum sloughing, and removal of loosened keratinocytes. Moreover, the skin erythema and tingling sensation normally disappear within 12–24 h.¹¹⁾ Therefore, panduratin A isolated from B. rotunda in topical MS serum might be an effective subcutaneous adipose tissue delivery system for a potential lipolytic body product.

The aims of this study were to investigate the antiadipogenic and lipolytic effects of panduratin A from B. rotunda extract and develop extract-loaded lipolytic body MS serum. Panduratin A was isolated from the ethanolic extract of B. rotunda. Bioactivity studies were performed in 3T3-L1 preadipocyte cells. MS was mixed with a serum base to deliver panduratin A from the extract into the skin. The in vitro skin permeation and deposition were determined by Franz-type diffusion cells. The in vivo human study was also evaluated after applying the formulation.

MATERIALS AND METHODS

Materials

Rhizomes of B. rotunda were collected from a local market in Ubon Ratchathani Province, Thailand and identified by Asst. Prof. Dr. Kusuma Jitsaeng from the Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmaceutical Sciences, Ubon Ratchathani University. Mouse 3T3-L1 preadipocytes, Dulbecco’s modified Eagle’s medium (DMEM), and bovine calf serum were obtained from the American-Type Culture Collection (ATCC, Rockville, MD, U.S.A.). Fetal bovine serum (FBS), trypsin–ethylenediaminetetraacetic acid (EDTA), L-glutamine (Glutamax™) nonessential amino acids, and penicillin–streptomycin were purchased from Gibco BRL, Rockville, MD, U.S.A. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and polydimethylacrylamide (PDMA) were purchased from Sigma-Aldrich, MO, U.S.A. Hexane and ethyl acetate were purchased from RCI Labscan Limited, Thailand. All other chemical agents were analytical reagent grade.

Extraction of B. rotunda

The dried B. rotunda rhizomes were ground and extracted with 95% ethanol at a ratio of 1 : 5 for 7 d. After that, the extract was filtered, followed by solvent evaporation (yield 5.90%). The dried extract was further fractionated with gradient elution of a hexane and ethyl acetate solvent system through column chromatography containing silica gels (SiliaFlash^®G60, SiliCycle, Inc., Canada). The six fractions were collected and identified by TLC and compared to the panduratin A standard (>98% purity). The collected fraction-3 containing panduratin A (BP-3), which was carried out at a ratio of hexane and ethyl acetate of 90 : 10, was dried and confirmed by HPLC.

HPLC Analysis

Panduratin A was a major bioactive compound in this study. Samples were dissolved in ethanol. An injection volume of 50 µL was applied for each sample, and the eluent was monitored at 285 nm with a UV detector in an HPLC system (Thermo Scientific™ Dionex™ UltiMate 3000, Germering, Germany) equipped with a C18 HPLC column (VertiSep™ GES, 4.6 × 250 mm, 5 µm pore size, Vertical Chromatography Co., Ltd., Bangkok, Thailand). The flow rate was 1.5 mL/min. The solvent system was acetonitrile and phosphoric acid (0.1% (v/v)), which was subsequently mixed using a linear gradient starting with 20% acetonitrile for 0.5 min. The gradient was gradually decreased to 30% (over 5 min) and held for 12.5 min and then to 50% (over 20 min) and held for 32.5 min and finally to 80% (over 14 min). Each run was 60 min.

Cytotoxicity Study of BP-3 Extracts on 3T3-L1 Cells

Mouse 3T3-L1 preadipocyte cells were cultured in preadipocyte medium (DMEM with 10% bovine calf serum, 100 U/mL penicillin and 100 µg/mL streptomycin) and incubated under a humidified atmosphere (5% CO₂, 95% air, 37 °C) for 48 h until 70–80% cell confluency. The 3T3-L1 cells (2 × 10³ cells/well) were seeded into a 96-well plate and incubated until confluent. After washing the cells with phosphate buffer saline (PBS) at pH 7.4, differentiation medium (DMEM with 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1.0 µM dexamethasone and 1.0 µg/mL insulin) was added to the cells and incubated at 37 °C, 5% CO₂, and 95% relative humidity (RH) for 48 h. After removing the differentiation medium, fresh adipocyte maintenance medium (DMEM with 10% FBS and 1.0 µg/mL insulin) was added to the cells every 48–72 h until completing lipid droplet formation for 8 d.

For the cytotoxicity study of the pretreated BP-3 extract, the extract was diluted to obtain various concentrations (0–50 µg/mL). After removing the preadipocyte medium from the cell plates, each concentration of BP-3 extract was added and performed as described above. In the case of posttreated BP-3 extract, the lipid droplets of 3T3-L1 adipocyte cells were treated with different concentrations of diluted extract for 48 h. Both pre- and post-treated 3T3-L1 cells were determined for cell viability by MTT assay. The treated cells were washed with PBS at pH 7.4, mixed with an MTT solution (0.5 mg/mL) and incubated for 3 h. After that, the medium was removed, and 100 µL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals that formed in the living cells. The absorbance was analyzed by a microplate reader (VICTOR Nivo™ Multimode Plate Reader, PerkinElmer, Inc., Germany) at 550 nm. The percent cell viability was calculated using Eq. 1.

  

(1)

Antiadipogenesis Evaluation by Oil Red O Staining Assay

After the cells were treated with the BP-3 extract for 8 d, lipid droplets were stained with Oil Red O solution (Sigma-Aldrich, MO, U.S.A.). Briefly, the differentiated adipocytes were washed with PBS at pH 7.4 and fixed with 10% formalin for 30 min. After washing the cells, 60% isopropanol was added to the cells and incubated for 5 min. Then, 60% isopropanol was removed from the cells, and 0.5% (w/v) Oil Red O solution was incubated with the cells at 37 °C and 5% CO₂ for 1 h. The stained cells were photographed using a microscope (Nikon ECLIPSE TE2000-S, Tokyo, Japan). The retained dye in the adipocytes was extracted with 100% isopropanol, and the absorbance was determined at 510 nm using a microplate reader.

Lipolysis Effects by Using a Glycerol Assay

The differentiated 3T3-L1 adipocytes were treated with BP-3 extract. Isoproterenol (100 mM) was used as a positive control. After incubation for 2 h, cell lipolysis was examined by a lipolysis (3T3-L1) Colorimetric Assay Kit (Sigma-Aldrich). Briefly, the treated cells were washed with lipolysis wash buffer, and then, lipolysis assay buffer was added. Fifty microliters of cell lipolysis were mixed with 50 µL of reaction mixes (glycerol assay buffer, glycerol enzyme mix and glycerol probe) for 30 min at room temperature. The absorbance at 570 nm was measured using a microplate reader.

Formulation of BP-3 Extract-Loaded MS Serum

BP-3 extract (0.1% (w/w)) was dissolved in ethanol (3% (w/w)), polyethylene glycol 400 (PEG400; 3% (w/w)), glycerin (4% (w/w)), and Polysorbate 20 (Tween 20; 4% (w/w)), and an ultrasonic bath was used for 15 min. After that, the serum base containing distilled water, polyacrylate crosspolymer-6 (Sepimax™ Zen), microcare PHC and ethylene diamine tetra acetic acid disodium salt (EDTA 2Na) was mixed with a BP-3 extract solution. For the MS serum, 0.5% (w/w) MS (Sponge spicule extract power; 98% plus spicule; Hunan Sunshine Bio-Tech Co., Ltd., Hunan, China) was added to the serum formulation. The physical properties of the formulation were evaluated, such as appearance, panduratin A content and MS shape using a microscope (Nikon DS-Ri2, Nikon Corporation, Tokyo, Japan).

In Vitro Skin Permeation Study

Abdominal porcine skins were taken from intrapartum stillborn animals from a local farm in Sisaket Province, Thailand. This study was approved by an Investigational Review Board (04/2565/IACUC, Animal Experimentation Ethics Committee, Ubon Ratchathani University). Removal of the subcutaneous layers was performed with medical scissors. The skins were 600–700-µm thick and kept in a refrigerator at −20 °C before use.

A skin permeation study was performed by vertical Franz-type diffusion cells. Approximately 12 mL of PBS at pH 7.4 with polysorbate 20 (5 g/L):ethanol at a ratio of 1 : 1 was used as a receptor medium and continuously stirred using a magnetic stirrer. The temperature was maintained at 32 ± 0.5 °C. The formulation was applied on the skin with a gentle massage for 2 min (approximately 160 rubbing times/2.01 cm² of skin area) by using the forefinger with a medical glove. Each treated skin was inserted between the donor and receptor compartments. Five hundred microliters of receiver medium were collected at 1, 2, 4, 6, 8 and 24 h for HPLC analysis.

After 24 h of skin permeation, the treated skins were washed, cut into small pieces, and then extracted with ethanol and a probe sonicator for 20 min in an ice bath. The amount of panduratin A deposited into the skin was analyzed by HPLC. Each sample was analyzed in triplicate.

In Vivo Human Skin Study

The study involved 13 healthy human volunteers (between 22 and 37 years old) who have a body mass index (BMI) more than 25, high percent body fat analyzed by body composition analyzer (Tanita corporation, Tokyo, Japan), and agreed to participate in a clinical trial. This study was approved by an Investigational Review Board (UBU-REC-93/2565, Human Studies Ethics Committee, Ubon Ratchathani University). At least 12 h before the experiments, the skin was not treated with moisturizer products. BP-3 extract was applied to the thigh skin in MS serum with gentle massage. Erythema was evaluated after applying the formulation for 5 min. The applied skins were measured at 7 and 14 d. The DermaLab^® series (SkinLab Combo; Cortex Technology, Hadsund, Denmark) was used to evaluate the skin in terms of erythema, hydration, viscoelasticity and ultrasound imaging. Moreover, thigh circumference was also determined, and the percent change was calculated using Eq. 2.

  

(2)

where TC_t is the thigh circumference of treated skin (cm) and TC is the thigh circumference of untreated skin (cm).

The effect of formulations on erythema (vascularity) was determined by generating the value of the percent change in erythema index (% EI), in which vascularity equated to 100%, as shown in Eq. 3. The percent changes in skin hydration and elasticity were calculated using Eq. 4 and 5, respectively.

  

(3)

where E_t is the erythema value of treated skin and E is the erythema value of untreated skin.

  

(4)

where H_t is the hydration value of treated skin and H is the hydration value of untreated skin.

  

(5)

where VE_t is the viscoelasticity of treated skin (mPa) and VE is the viscoelasticity of untreated skin (mPa).

Data Analysis

The data are presented as the mean ± standard deviation (S.D.). A statistically significant difference was analyzed by one-way ANOVA, followed by Tukey’s post hoc test. For the in vivo human study, the Wilcoxon signed rank test was evaluated. The significance level was set at p < 0.05.

RESULTS

Physicochemical Properties of the BP-3 Extract

The BP-3 extract exhibited a yellowish color and odor of B. rotunda. The percent yield of the BP-3 extract was 10.30% of the crude extract. Panduratin A was found as 0.29 g/g of BP-3 extract. This extract was used as an active compound for bioactivity studies on adipocytes.

Cytotoxicity of BP-3 Extracts on 3T3-L1 Cells

As shown in Fig. 2, BP-3 extract representing panduratin A was not toxic to 3T3-L1 cells at both the preadipocyte stage and mature adipocyte stage at concentrations of 1–10 µg/mL, and the percent cell viability ranged from 92.25 to 102.37% of the control at these tested concentrations. Thus, this concentration range was safe for further experiments.

Fig. 2. Percentages of Cell Viability of 3T3-L1 Pre-adipocyte Cells and Mature-Adipocyte Cells after Treatment with Various Concentrations of BP-3 Extract

Data are presented as the mean ± S.D. (n = 3). * indicates a significant difference from the control group (untreated cells) (p < 0.05).

Anti-adipogenesis and Lipolysis Effect of BP-3 Extract

Lipid accumulation in adipocytes was observed by staining the solution with Oil red O, representing triglyceride accumulation in adipocyte cells. As shown in Fig. 3, 3T3-L1 preadipocytes were the (+) control, and differentiated 3T3-L1 cells stained with Oil Red O were the (−) control. The 3T3-L1 preadipocytes exhibited a fibroblast-like appearance before adipogenic stimulation. After adipocyte differentiation, the number of Oil Red O-positive cells and lipid droplets showed size enlargement and round-shape cells.¹²⁾ For the anti-adipogenesis effect of BP-3 extract, the treated cells had smaller size and lower number of lipid droplets than differentiated cells ((−) control). The accumulation of lipids was significantly reduced by BP-3 extract concentrations of 1–10 µg/mL, with lipid contents ranging from 47.53 to 50.63% of the (−) control. This indicated that treatment of 3T3-L1 adipocytes with BP-3 extract during adipogenesis markedly decreased triglyceride accumulation without cytotoxicity, indicating that the anti-obesity potential of BP-3 extract can reduce adipogenesis and lipid accumulation.²⁾

Fig. 3. Anti-adipogenesis Effect of BP-3 Extract on 3T3-L1 Cells: (A) Oil Red O Staining of Oil Droplets and (B) the Percentage of Lipid Accumulation (% of (−) Control) after Treatment with BP-3 Extract for 8 d

Each data point represents an average ± S.D. (N = 3). * indicates a significant difference from the (−) control) (p < 0.05).

Cells treated with isoproterenol had significantly higher glycerol release than untreated adipocytes. For the lipolysis effect on 3T3-L1 adipocytes after treatment with BP-3 extract, lipolysis induction by the increase in released glycerol content in a dose-dependent manner was found, and the concentration of 10 µg/mL BP-3 extract was not significantly different from isoproterenol (Fig. 4). This suggested the lipolysis effect of the BP-3 extract on 3T3-L1 adipocytes.

Fig. 4. Lipolysis Effect on 3T3-L1 Adipocytes after Treatment with Different Concentrations of BP-3 Extract

Each data point represents an average ± S.D. (N = 3). * indicates a significant difference from the isoproterenol ((+) control) (p < 0.05).

BP-3 Extract-Loaded MS Serum

The BP-3 extract was completely mixed with a general hydrophilic cosmetic serum. MSs were added into serum-based BP-3 extract, in which microneedling shapes of MSs were a tip growing out from two opposite sites with a 17.00 ± 2.65-µm middle width and 239.00 ± 22.52-µm spicule length (Fig. 5). The formulation exhibited a good appearance and smooth texture upon application onto the skin.

Fig. 5. The Images Show (1) Visual Appearance and (2) Optical Microscope Imagine of BP-3 Extract in MS Serum (10× Objective Lens)

Skin Permeation and Deposition of BP-3 Extracts

After skin permeation for 24 h, BP-3 extract-loaded MS serum exhibited panduratin A permeation through the skin at 4 to 24 h, while the studies in serum and oil solution were unable to detect the panduratin A in the receiver compartment (Fig. 6(A)). This indicated that panduratin A in serum and oil solution did not pass through the skin. As shown in Fig. 6(B), the skin treated with MS serum for 24 h had the highest amount of panduratin A permeated through and deposited into the skin compared to the serum and oil formulation, indicating that MS serum increased the skin permeability of panduratin A.

Fig. 6. (A) Cumulative Panduratin A Permeation vs. Time Profile and (B) Comparison of Panduratin A Remaining on the Skin and Permeated into the Receiver Compartment from the BP-3 Extract in Oil, Serum and MS Serum after a 24 h in Vitro Skin Permeation Study

Each value represents the mean ± S.D. (n = 3). * indicates a significant difference from the BP-3 extract in oil (p < 0.05).

In Vivo Human Skin Study

After 14 d of BP-3 extract in MS serum applications, all subjects were monitored for changes in thigh circumference and skin erythema, elasticity, and hydration, as shown in Fig. 7. The thigh circumferences of all volunteers were changed in the range of −7.00 to +2.50 cm at Day 7 and −7.00 to +4.00 cm at Day 14, and the mean values of thigh circumference change were −1.30 ± 2.55 cm at Day 7 and −3.25 ± 3.12 cm at Day 14 (data not shown). For the percent change of thigh circumference, BP-3 extract in MS serum reduced the thigh circumference after 7 d of application, and significantly reduced the thigh circumference on Day 14, compared with Day 7.

Fig. 7. (A) The Percent Change in Thigh Circumference, (B) the Percent Erythema Index (%EI), (C) the Percent Hydration, and (D) the Percent Viscoelasticity after the BP-3 Extract in MS Serum Was Applied to the Skin of Healthy Volunteers at Days 7 and 14

The data are presented as the mean ± S.D. (n = 13). * indicates a significant difference from Day 7 (p < 0.05).

The erythema value refers to irritant and allergic reactions. The %EI of skin treated with BP-3 extract in MS serum for 5 min was slightly decreased, indicating that this formulation did not induce redness and irritation in the skin (data not shown). After applying MS serum onto the skin for 7 and 14 d, the %EI of treated skins increased more than 100%, but there was no significant difference from the initial day. Moreover, no pain or severe redness was found.

The hydration of skin treated with BP-3 extract in MS serum showed an increase in the hydration percentage, and the treated skin on Day 14 seemed to increase skin hydration more than that on Day 7 (Fig. 7(C)). For skin elasticity that was also measured as shown in Fig. 7(D). The value of skin viscoelasticity was basically between 2 and 15 MPa (megaPascals), in which a higher MPa value refers to a higher vacuum strength needed to lift the skin and indicates a better firmness of skin.¹³⁾ The percent change in viscoelasticity after treatment with BP-3 extract in MS serum showed an increase in the mean value at Days 7 and 14, indicating the firmness of skin treated with BP-3 extract in MS serum.

Ultrasonography evaluates the acoustic signal recorded for a digital sound wave reflected from biological tissues, providing direct visualization of epidermal, dermal and adipose tissue thickness, and changes in fibrosclerotic tissue could also affect the appearance of cellulite.¹⁴⁾ As shown in Fig. 8, untreated skin showed fat cells and lymphatic fluid. After 7 and 14 d of application, the skin tissues became measurably more compact, representing a strengthening of the connective tissue and reduction of echo-free interspace (black area).¹⁵⁾ Moreover, ultrasound images can be used to evaluate visually detectable changes in tissue structures. The results showed that the network of collagen/elastic fibers in the dermis and subcutis became denser and measurably firmer. The effect of BP-3 extracts on MS serum improved skin firmness, suggesting the stimulation of lipolysis and inhibition of lipid synthesis by the extract.

Fig. 8. Ultrasonography of the Human Thigh Area

Images are of the dermis (green), hypodermis (black), and dermal–hypodermal interface exhibiting fat herniations into the dermis at the initial day (A) and after application of BP-3 extract in MS serum for 7 d (B) and 14 d (C).

DISCUSSION

Panduratin A from B. rotunda extract had the bioactivity on adipocytes and no cytotoxic to 3T3-L1 cells at concentrations of 1–10 µg/mL. According to Thongnuanjan et al., panduratin A derivatives do not significantly affect cell viability but also protect cells from the cytotoxicity of cis-diamminedichloroplatinum at concentrations of 5 and 10 µM.¹⁶⁾ Thus, this concentration range was safe for further experiments.

Lipid homeostasis is regulated by the fine-tuning of adipogenesis and lipolysis, which are regulated by lipid regulatory enzymes in peripheral tissues, such as adipose, liver, and muscle tissues.¹⁷⁾ Adipogenesis is the transformation process from preadipocytes to mature adipocytes via intercellular adipogenesis.¹⁸⁾ Lipid in adipocytes was represented by the accumulation of triglyceride in the cells. The anti-adipogenesis effect of BP-3 extract exhibited small size and low number of lipid droplets, referring to reduce the lipid contents in the adipocytes.²⁾ However, the concentration-dependent effects of BP-3 extract on the lipid content were not clearly observed at the concentration range of 1 to 10 µg/mL. According to Rungsa et al., isopanduratin A suppresses adipogenesis in human preadipocytes in a dose-dependent manner at 1, 5, and 10 µM (or 0.41, 2.03 and 4.01 µg/mL), but in differentiated 3T3-L1 cells were strongly suppressed by 5–10 µM isopanduratin A. This indicated that low concentration of isopanduratin A could reduce cellular fat accumulation in human preadipocytes.¹⁹⁾ Therefore, treatment of 3T3-L1 adipocytes with BP-3 extract at the concentrations of 1 to 10 µg/mL during adipogenesis markedly decreased triglyceride accumulation without cytotoxicity, indicating that the anti-obesity potential of BP-3 extract can reduce adipogenesis and lipid accumulation.

Adipocyte lipolysis is a catabolic process that leads to the breakdown of the triglycerides stored in fat cells into fatty acids and glycerol. The stimulation of lipolysis by the catecholamine isoproterenol provides glycerol release by mature adipocytes as a result of triglyceride hydrolysis.²⁰⁾ The lipolysis effect on 3T3-L1 adipocytes after treatment with BP-3 extract exhibited the increase in released glycerol content in a dose-dependent manner, demonstrating the lipolytic effect. According to Kim et al., isolated panduratin A from B. rotunda was used as an active compound to stimulate anti-obesity mechanisms for high-fat diet-induced obesity in mice. Panduratin A stimulated AMPK signaling, leading to increased lipid catabolism and decreased lipid accumulation.²⁾ Additionally, the mechanism of the lipolytic action of isopanduratin A have been reported as the suppressive effect on 3T3-L1 adipogenesis by downregulating adipogenic effectors (fatty acid synthase (FAS), perilipin 1 (PLIN1), lipoprotein lipase (LPL), and adiponectin) and adipogenic transcription factors (sterol regulatory element-binding protein 1c (SREBP-1c), peroxisome proliferator-activated receptor γ (PPARγ), and CCAAT/enhancer binding protein α (C/EBPα)), which these lipid metabolism proteins are essential for maintaining cellular lipid homeostasis and associated with metabolic conditions such as hyperlipidemia, insulin resistance, atherosclerosis, and obesity.¹⁹⁾

BP-3 extract-loaded MS serum was successfully formulated with a good appearance and smooth texture upon application onto the skin. MS serum strongly increased the skin permeability of panduratin A, because microneedling can create transportation routes for many compounds through the skin. The needle-like structure of MS exhibited a tip growing out of two opposite sites with micrometer-scale spicule length. According to Udompataikul et al., puncture resembling mesotherapy is the mechanism of action of MS, in which the micron scale and sharp thorn-like nature of the spicules make deep punctures into the skin, delivering hydrogen peroxide across the skin.²¹⁾ The insertion of MS into the skin creates a possible route to transport active compounds into and through the skin, bypassing the tightly packed stratum corneum barriers (10–20 µm thickness) and improving the ability to deliver active compounds into the skin.^22,23) Therefore, MS in serum formulation importantly enhanced the permeation of panduratin A from BP-3 extract into and across the skin as a minimally invasive technique. This indicated that MS serum was a suitable formulation as a delivery system for the BP-3 extract.

For in vivo human study, the thigh circumferences of all volunteers were reduced after the application of BP-3 extract in MS serum for 7 d and significantly reduced the thigh circumference on Day 14. For irritant and allergic reactions, %EI of treated skins was increased, but no significant difference from the initial day. In a previous study, MS consisting mainly of calcium carbonate or silica and collagen presented a safe and effective method for the delivery of bioactive compounds through the skin.²⁴⁾ Physical skin disruption depended on a dose-dependent manner, and the accumulation of MS in the skin occurred over at least 72 h. Although increasing the applied dose of MS, massage time and intensity increased the amounts of spicules penetrating into the skin and skin permeability of many compounds, the skin irritation and pain should be considered.^9,25) In this study, the self-massage with MS serum was performed by our own participations in experimentation. Some irritation was found following the strong hand massage when applying the formulation. However, no pain or severe redness was found.

BP-3 extract in MS serum can increase the skin hydration. According to Woo et al., an ethanol extract of B. rotunda and panduratin A improved skin hydration and barrier function through cornified envelope formation and filaggrin processing, in which the cornified envelope prevents the loss of water from the body by forming several proteins cross-links, and the degradation of filaggrin is an important process to produce natural moisturizing factors.³⁾ Moreover, skin elasticity after treatment with BP-3 extract in MS serum showed an increase in the mean value at Days 7 and 14. Therefore, the skin treated with BP-3 extract promoted an increase in skin hydration and firmness.

Ultrasonography of the skin treated with BP-3 extracts in MS serum showed that the network of collagen/elastic fibers in the dermis and subcutis became denser and measurably firmer, suggesting the stimulation of lipolysis and inhibition of lipid synthesis by the extract. Adipose tissue is a loose connective tissue mostly composed of adipocytes, and its location beneath the skin is called subcutaneous adipose tissue. Several health risks, such as insulin resistance, hepatic steatosis, metabolic syndrome and hypertension, are associated with high amounts of total body fat, visceral adipose tissue, and subcutaneous adipose tissue. Many options have been used for treating subcutaneous fat, and liposuction is also an option for the removal of excess subcutaneous adipose tissue.²⁶⁾ However, the inherent limitations and safety issues related to liposuction must always be respected for serious complications and unfavorable results.²⁷⁾ The effectiveness and safety of topical body fat loss products have been widely developed. Active panduratin A from BP-3 extract that exhibited bioactivity to inhibit lipid synthesis and stimulate lipolysis of 3T3-L1 adipocytes was successfully formulated with MS serum, resulting in improved skin permeability, effectiveness in reducing subcutaneous fat and safe use. Therefore, the BP-3 extract in MS serum plays an important role as a potential lipolytic body product.

CONCLUSION

BP-3 extract exhibited a high content of panduratin A and no cytotoxicity on 3T3-L1 cells at concentrations below 10 µg/mL, and the antiadipogenesis and lipolysis effects of the BP-3 extract were strong at 10 µg/mL. To deliver bioactive panduratin A into and through the skin, MS serum was successfully formulated. Applying BP-3 extract-loaded MS serum to the human thigh for 14 d reduced the thigh circumference and increased skin hydration and firmness. Although the skin erythema was increased, no severe redness of pain was found. In conclusion, BP-3 extract acts as a potent bioactive compound in adipocyte cells, and the highest bioactive potential of BP-3 extract-loaded MS serum might play an important role in antiadipogenesis and lipolysis in lipolytic body product.

Acknowledgments

This research was funded by the agricultural research development agency (public organization) (ARDA; Grant No. CRP6505030260). The authors would like to acknowledge the facility support from the Faculty of Pharmaceutical Sciences, Ubon Ratchathani University, and Faculty of Pharmacy, Silpakorn University.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1) Kim MS, Pyun HB, Hwang JK. Effects of orally administered fingerroot (Boesenbergia pandurata) extract on oxazolone-induced atopic dermatitis-like skin lesions in hairless mice. Food Sci. Biotechnol., 22 (Suppl. 1), 257–264 (2013).
• 2) Kim DY, Kim MS, Sa BK, Kim MB, Hwang JK. Boesenbergia pandurata attenuates diet-induced obesity by activating AMP-activated protein kinase and regulating lipid metabolism. Int. J. Mol. Sci., 13, 994–1005 (2012).
• 3) Woo SW, Rhim DB, Kim C, Hwang JK. Effect of standardized Boesenbergia pandurata extract and its active compound panduratin A on skin hydration and barrier function in human epidermal keratinocytes. Prev. Nutr. Food Sci., 20, 15–21 (2015).
• 4) Escalante G, Bryan P, Rodriguez J. Effects of a topical lotion containing aminophylline, caffeine, yohimbe, L-carnitine, and gotu kola on thigh circumference, skinfold thickness, and fat mass in sedentary females. J. Cosmet. Dermatol., 18, 1037–1043 (2019).
• 5) Prausnitz MR, Langer R. Transdermal drug delivery. Nat. Biotechnol., 26, 1261–1268 (2008).
• 6) Menon GK, Lee SH, Roberts M. Ultrastructural effects of some solvents and vehicles on the stratum corneum and other skin components: evidence for an “extended mosaic partitioning model of the skin barrier”. Dermal absorption and toxicity assessment. (Roberts MS, Walters KA, eds) Marcel Dekker, New York, p. 727 (1998).
• 7) Petchsangsai M, Rojanarata T, Opanasopit P, Ngawhirunpat T. The combination of microneedles with electroporation and sonophoresis to enhance hydrophilic macromolecule skin penetration. Biol. Pharm. Bull., 37, 1373–1382 (2014).
• 8) Andrews SN, Zarnitsyn V, Bondy B, Prausnitz MR. Optimization of microdermabrasion for controlled removal of stratum corneum. Int. J. Pharm., 407, 95–104 (2011).
• 9) Villani M. U.S. Patent application US20100080853A1 (2010).
• 10) Sethmann I, Wörheide G. Structure and composition of calcareous sponge spicules: a review and comparison to structurally related biominerals. Micron, 39, 209–228 (2008).
• 11) Ha JM, Lim C, Han K, Ha JC, Lee HE, Lee Y, Seo YJ, Kim CD, Lee JH, Im M. The effect of micro-spicule containing epidermal growth factor on periocular wrinkles. Ann. Dermatol, 29, 187–193 (2017).
• 12) Nobusue H, Endo T, Kano K. Establishment of a preadipocyte cell line derived from mature adipocytes of GFP transgenic mice and formation of adipose tissue. Cell Tissue Res., 332, 435–446 (2008).
• 13) Sulaiman W, Azad AK, Daud NA, Sunzida NK. Evaluation of skin elasticity after used different seaweed containing products by DermaLab^® Combo. Asian J. Pharm. Pharmacol., 3, 72–76 (2016).
• 14) Young VL, DiBernardo BE. Comparison of cellulite severity scales and imaging methods. Aesthet. Surg. J., 41, NP521–NP537 (2021).
• 15) Christ C, Brenke R, Sattler G, Siems W, Novak P, Daser A. Improvement in skin elasticity in the treatment of cellulite and connective tissue weakness by means of extracorporeal pulse activation therapy. Aesthet. Surg. J., 28, 538–544 (2008).
• 16) Thongnuanjan P, Soodvilai S, Fongsupa S, Thipboonchoo N, Chabang N, Munyoo B, Tuchinda P, Soodvilai S. Panduratin A derivative protects against cisplatin-induced apoptosis of renal proximal tubular cells and kidney injury in mice. Molecules, 26, 6642 (2021).
• 17) Choi H, Eo H, Park K, Jin M, Park EJ, Kim SH, Park JE, Kim S. A water-soluble extract from Cucurbita moschata shows anti-obesity effects by controlling lipid metabolism in a high fat diet-induced obesity mouse model. Biochem. Biophys. Res. Commun., 359, 419–425 (2007).
• 18) Chumchoochart W, Sutthanut K. Anti-obesity potential of glutinous black rice bran extract: anti-adipogenesis and lipolysis induction in 3T3-L1 adipocyte model. Songklanakarin J. Sci. Technol., 42, 284–291 (2020).
• 19) Rungsa P, San HT, Sritularak B, Böttcher C, Prompetchara E, Chaotham C, Likhitwitayawuid K. Inhibitory effect of isopanduratin A on adipogenesis: a study of possible mechanisms. Foods, 12, 1014 (2023).
• 20) Langin D. Adipose tissue lipolysis as a metabolic pathway to define pharmacological strategies against obesity and the metabolic syndrome. Pharmacol. Res., 53, 482–491 (2006).
• 21) Udompataikul M, Wongniraspai M, Showpittapornchai U, Jariyapongsakul A. The study on effects and safety of spongilla lacustris in 3% hydrogen peroxide solution on rat skin. J. Med. Assoc. Thai., 95 (Suppl. 12), S15–S20 (2012).
• 22) Sahle FF, Gebre-Mariam T, Dobner B, Wohlrab J, Neubert RHH. Skin diseases associated with the depletion of stratum corneum lipids and stratum corneum lipid substitution therapy. Skin Pharmacol. Physiol., 28, 42–55 (2015).
• 23) Tansathien K, Chareanputtakhun P, Ngawhirunpat T, Opanasopit P, Rangsimawong W. Hair growth promoting effect of bioactive extract from deer antler velvet-loaded niosomes and microspicules serum. Int. J. Pharm., 597, 120352 (2021).
• 24) Tansathien K, Suriyaaumporn P, Charoenputtakhun P, Ngawhirunpat T, Opanasopit P, Rangsimawong W. Development of sponge microspicule cream as a transdermal delivery system for protein and growth factors from deer antler velvet extract. Biol. Pharm. Bull., 42, 1207–1215 (2019).
• 25) Zhang S, Ou H, Liu C, Zhang Y, Mitragotri S, Wang D, Chen M. Skin delivery of hydrophilic biomacromolecules using marine sponge spicules. Mol. Pharm., 14, 3188–3200 (2017).
• 26) Mittal B. Subcutaneous adipose tissue & visceral adipose tissue. Indian J. Med. Res., 149, 571–573 (2019).
• 27) Dixit VV, Wagh MS. Unfavourable outcomes of liposuction and their management. Indian J. Plast. Surg., 46, 377–392 (2013).