THE EFFECT OF NANOENCAPSULATED CENTELLA ASIATICA L AND ZINGIBER OFFICINALE ROSC. VAR. RUBRUM COMBINATION TO PROMOTECOLLAGEN SYNTHESIS AND DECREASE THE DIAMETER OF ADIPOCYTE CELLS IN FEMALE WISTAR RATS

THE EFFECT OF NANOENCAPSULATED CENTELLA ASIATICA L AND ZINGIBER OFFICINALE ROSC. VAR. RUBRUM COMBINATION TO PROMOTECOLLAGEN SYNTHESIS AND DECREASE THE DIAMETER OF ADIPOCYTE CELLS IN FEMALE WISTAR RATS

Zullies Ikawati ^*1, Retno Murwanti ¹, Yenny Meliana ² and Witta Kartika ²

Depr of Pharmacology ¹, Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Indonesia

Indonesian Institute of Science ², Jakarta, Indonesia

ABSTRACT: Cellulite is a normal condition judging from the medical aspect, but from aesthetics aspect, cellulite deserves more attention, especially for women. Centella asiatica is reported to promote both fibronectin and collagen synthesis. Meanwhile, Zingiber officinale is reported to have lipolysis activity. Combination of the two herbals is assumed to have complementary effect as anti cellulite agents. The combination of the herbal extracts is prepared in nanoemulsion form to enhance the bioavailability. The study aimed to determine the effect of nano emulsion of C. asiatica and Z. officinale combination (proportion of 5:1) to stimulate skin collagen synthesis and decrease the diameter of adipocyte cells using histological parameters as an indicator of lipolysis activity. Twenty female Wistar rats weighing 120-140 g were fed a high-fat diet for 30 days. The animals were divided into 4 groups, with the 3 groups received the nanoemulsion of herbal combination with the dose of 50, 100, and 200 mg/kg BW for 30 days, and 1 group received CMC Na 0, 5% as negative control. On the day 31, rats were sacrified and skin samples of 1.0 cm², including fatty tissue, were obtained from the subjects.  One part of the tissue sample was used for collagen assay, while another was used for hematoxylin-eosin staining. The amount of collagen in skin tissue was assayed using Sirius Red Collagen Detection Kit (Chondrex). The collagen thickness was also measured histologically using Sirius red staining. The diameter of adipocyte cells were measured under light microscope to represent lipolysis activity. Results showed that the amount of skin collagen was increased with the increase of extract doses, however the lowest dose showed no significant different with the normal animal. The diameter of adipocyte cells was also decreased in dose-dependent manner. Results indicate that the combination of C. asiatica and Z. officinale can be developed as herbal medicine for anti cellulite agent

cellulite, collagen synthesis, adipocytes cells diameter,

C. asiatica, Z. officinale

INTRODUCTION: Cellulite is an appearance changing of the skin that resembles an orange peel. Women aged 20 years old or more, 90% of them have cellulite experience with varying degree of severity, meanwhile men had a smaller number (2%) ⁶.

Cellulite is a normal condition judging from the medical aspect, but from aesthetics aspect, cellulite deserves more attention, especially for women. It is not specific to overweight women although increased adipogenicity will exacerbate the condition.

 It is difficult to pinpointitsaetiology and physiology/ pathophysiology of cellulite, as thereare many factors that are involved it, affect it, and many processes that contribute simultaneously and sequentially ². Cellulite occurs due to a microcirculation diminishing, infiltration of the interstitial fluid (edema), hypertrophy on local adipose tissue, oxidative stress, inflammation mild persistent, and changes in the extracellular matrix ^{6, 11, 16}. Several mechanisms can reduce the appearance of cellulite, such as increasing the synthesis of collagen, stimulate lipolysis activity, using PDE inhibitors, increasing blood flow to smooth microcirculation, laser, etc.

 In traditional Asian medicine, the herb of Centela asiatica has been used for hundreds of years, especially in dermatological conditions, to improve small wounds, scratches, burns, hypertrophic wounds healing, and as an anti-inflammatory agent, particularly in eczema ³. C. asiatica, which containsasiatic acid, madecassic acid, and asiaticoside is reported to stimulate human collagen synthesis ⁴, that often used in skin care products. Besides that, it reported that C. asiatica could increase microcirculation and capillary permeability effects on the skin. Another activity shown by C. asiatica was lipolysis and antioxidant that affect in reducing cellulite ⁸.

 Ginger, the rhizome of the perennial plant Zingiber officinale Roscoe, is used as a flavoring agent for food, mostly in a powdered and candied form. In addition, ginger is widely used as a herbal medicine for a number of conditions including those affecting the digestive tract, headaches and motion sickness⁵. The characteristic pungent taste of ginger is attributed to the gingerols (6-gingerol, 8-gingerol and zingerone). Ginger was reported that it could stimulate the lipolysis activity, which is the process of triglyceride hydrolysis into glycerol and free fatty acids. This was indicated that ginger could be used to reduce lipid pile, so that it could reduce cellulite appearance ^{7, 15}.

 This recent study investigates the effect of combination of C. asiatica and Z. officinale extracts in nanoemulsion form to stimulate collagen synthesis and decrease diameter of adipocyte cells which indicate the lipolytic action of the fatty cells. This study is the first study for this combination to develop herbal product for anti cellulite agent.

MATERIALS AND METHODS:

Experimental Animal and Materials: Twenty-five non-pregnant female Wistar rats (BW: 140-160 g, aged: 8 weeks) were used in this study. The tested extract prepared from nanoemulsion containing 5% of C. asiatica herbs extract and 1 % Z. officinale rhizome extract which mixed with malt dextrin as the carrier. The tested extract was dissolved with 0,5% CMC-Na before administered to the animals. Sirius Red Collagen Kit Assay was purchased from Chondrex. All other reagents used were of analytical grade.

Experimental Design:

Twenty female Wistar rats weighing 120-140 g were fed a high-fat diet for 30 days. The animals were divided into 4 groups, with the 3 groups received the nanoemulsion of herbal combination with the dose of 50, 100, and 200 mg/kg BW for 30 days, and 1 group received CMC Na 0,5%  as negative control. One group of rats (n=5) was fed normal diet and served as normal control. On the day 31, rats were sacrified and skin samples of 1.0 cm², including fatty tissue, were obtained from the subjects.  One part of the tissue sample was used for collagen assay, while another was used for hematoxylin-eosin staining. The amount of collagen in skin tissue was assayed using Sirius Red Collagen Detection Kit (Chondrex). The collagen thickness was also measured histologically using Sirius red staining. The diameter of adipocyte cells were measured under light microscope to represent lipolysis activity.

Procedures:

Combination extract of C. asiatica and Red ginger was dissolved with 0,5% CMC-Na. Stock solutions test were prepared every 3 days to maintain the stability of the solution.

Treatment Applied: The rats were injected with stock solutions test and lard for 30 days. The solution volume was determined based on BW and treatment groups. Testedrats weighed every 3 days to determine the BW gain. Took the rats skin to measure the concentration and thickness of collagen after 30 days of treatment.

Skin Sample Preparation and Reading of Collagen Concentration:

Organ preparat made and painted by Sirius Red which gave red color to collagen fibers. Meanwhile, skin that have been taken subsequently weighed and then cut into small pieces. NaCl physiological saline added in skin slices as much as 2 mL then crushed using homogenizer. Put the skin solution into5 mL conical flask, then 1 mL NaCl physiological solution added. Skin that been destroyed was centrifuged for 90 minutes at 2000 rpm. The supernatant discarded and the sediment was being taken. Acetic acid solution 1 mL (0.05 M) mixed with sediment then homogenized using vortex. Afterthat the solution was re-centrifuge for 90 minutes, then the supernatant was taken (1 mL) using micropipette. Supernatant stored in tubes kept in refrigerator. Meanwhile, prepared the collagen kit assay, started with making collagen standard solution. The collagen standard solution made by certain level solution to make regression equation. The equation made to determine collagen concentration in the sample.

Fill the acetic acid solution (0.05 M) into 8 tubes in amount of 250 µL each. Take 250 µL of collagen standard solution and fill into the first tube, then homogenized using vortex. After that, 250 mL solution taken from the first tube and put itinto the second tube then re-homogenized using vortex. Dilution carried out up to the seventh tube. The eighth tube was used as blank. When all the solution was already prepared, took 100 µL solution from standard, sample and blank solution thenfillit into another tube. Added 500 mL Sirius Red on each tube and homogenized using vortex, then incubated for 20 minutes in room temperature. The solvent that was incubated subsequently centrifuged at a speed of 10000 rpm for 3 minutes. The supernatant removed carefully to get the sediment. After the sediment was obtained, then added 500 µL washing solution. Re-vortex and centrifuged at the same speed and time. Supernatant re-discarded to obtain the sediment. The sediment was added by 250 µL buffer extract solution. It was re-vortex to dissolve the sediment then was analyzed using micro plate reader. The solvent taken as much as 200 µL and placed on 96-well plate then read the absorbance at OD 550 nm.

Statistical Analyses: The collagen concentration data obtained was converted into milligrams within skin-tested milligrams. All of the data were statistically analyzed using One-Way ANOVA test with 95% confidence level.

RESULTS AND DISCUSSION:

Results:

Collagen Concentration:

The collagen concentration levels in C, D and E groups are 0.0752 mg/mg skin; 0.1637 mg/mg skin; 0.1785 mg/mg skin, respectively. It showed that increasing dose of solution increases collagen concentration levels in skin. Meanwhile, the collagen concentration levels in groups A and B are 0.1407 mg/mg skin and 0.0611 mg/mg skin. The result also showed that combination extract of C. asiatica and red ginger was affect collagen concentration significantly on group C towards D and E groups (p=0.045 and p=0.022). Whereas, the treatment group D and E was not different (p> 0.05). If control group was compared to the treatment group, the result showed differe