Microencapsulation of Asiaticoside and Ginger Extract for Oral Application of Anti cellulite by ISERP ISERP

Athanasia Amanda Septevani et al. / (IJAEST) INTERNATIONAL JOURNAL OF ADVANCED ENGINEERING SCIENCES AND TECHNOLOGIES Vol No. 1, Issue No. 1, 043 - 048

Microencapsulation of Asiaticoside and Ginger Extract for Oral Application of Anti-cellulite Active Agent Athanasia Amanda Septevani (*), Dewi Sondari, Agus Haryono, Sri Budi Harmami Indonesian Institute of Sciences Polymer Chemistry Group, Research Center for Chemistry Kawasan Puspiptek Serpong, Tangerang 15314 - INDONESIA fani.manda@yahoo.com

primarily afflicts on thighs, buttocks and may also be present on stomach and uppers arms. This condition is frequently described as “orange peel skin” or the cottage cheese effect [1]. Cellulite is more visible in woman mainly because women’s connective tissue is inflexible so as female gain weight, their fats cells expand and tend to bulge upwards towards the surface of skin giving the classic orange peel appearance of cellulite. Men usually have less fat on their thighs and thicker outer skin which obscures any exceeding fat below [2]. Nowadays, there are many kind of anti-cellulite treatment, but have not been given satisfied solution yet. Some kinds of anticellulite treatment such as eating healthy, low fat foods such as fruits, vegetables, and fiber may help one to avoid cellulite. Similarly, exercising regularly, maintaining a healthy weight, and reducing stress are recommended to prevent cellulite. In addition wearing thongs, boy shorts, or looser fitting undergarments can prevent cellulite that might form due to tight elastic. Centella comprises some 50 species, inhabiting tropical and sub-tropical regions. This genus belongs to the plant family Apiaceae (Umbelliferae) and includes the most ubiquitous species Centella asiatica. Centella asiatica can stimulate collagen biosynthesis and increase the incorporation of proline, thus modulating the metabolism of connective tissue. The pharmacological activity of Centella asiatica is due to several saponin constituents including asiaticoside, asiatic acid and madecassic. Asiaticoside, pure triterpene from Centella asiatica, is known to stimulate vascular and perivascular with specific activity on collagen type I. It works by significantly reducing aging and increasing the tensile strength of newly formed skin [3]. Ginger (Zingiber officinale) is known to have antioxidant properties which help to neutralize free radicals in human body. Antioxidant compound in a sufficient amount, substantially inhibit the activity of collagenase and elastase in the skin [4]. Thus, asiaticoside extract and ginger extract can reduce or eliminate the appearance of cellulite. Microencapsulation is defined as technology of packaging solids, liquids, or gaseous materials in miniature, sealed capsules that can release their contents at controlled rates

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ABSTRACT: We studied microencapsulation of anti-cellulite active agent containing asiaticoside from pegagan herbs extract (Centella asiatica) and ginger herbs extract (Zingiber officinale) in maltodextrin and gum arabic using spray-drying. Asiaticoside, triterpene compound from pegagan (Centella asiatica), had already utilized as traditional medicines either in the form of fresh or processed ingredients (jamu). It had already utilized as an effective medication to solve cellulite problem known as “orange-peel” skin. Ginger (Zingiber officinale) is known to have antioxidant properties that can neutralize free radicals in human body and reduce or eliminate the appearance of cellulite. In an attempt to control time release, enhance the stability and obtain a product which is more effectively and holistic, the microencapsulation of asiaticoside and ginger (Zingiber officinale) extract for oral application of anti-cellulite active agent was studied. In this study, we produced microencapsulation of asiaticoside and ginger (Zingiber officinale) extract through an economical and simple process of spray drying and utilizing two types of covering materials of starch derivate. The morphology of microcapsules was observed by scanning electron microscopy whose analysis showed in spherical form and a tendency to form several shrinkages at the outer surface. Particle size analysis showed a main mode in intermediate range 0.4-0.8 μm. Safety of anti-cellulite active agent product microencapsulated for human oral application was determinated by toxicity acute test with the value of 14 days LD50 is more than 20,583mg/kg weight. Keywords: Microencapsulation, spray drying, cellulite, Centella asiatica, Zingiber officinale

I. INTRODUCTION

Cellulite is composed from combination of fat, toxins (cellular wastes), and water that form gel-like masses, trapped in connective tissue under the skin surface. This connective tissue will become thicker and harder. Cellulite is a term used to describe the dimpled appearance of skin caused by fat deposits that are just below the surface of the skin. Cellulite

*Corresponding author .Tel. Phone : +62-21-7560929, Fax:+62-21-7560549 Email address: fani.manda@yahoo.com (A.A. Septevani)

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Athanasia Amanda Septevani et al. / (IJAEST) INTERNATIONAL JOURNAL OF ADVANCED ENGINEERING SCIENCES AND TECHNOLOGIES Vol No. 1, Issue No. 1, 043 - 048

combining the core material with ethanol and stirred to homogeneity with Ultra-Turrax homogenizer X120. 2.2 Wall Solutions Wall solutions consisting of maltodextrin (Merck) and Gum arabic (Merck) were prepared in destiled water then stirred to homogeneity with Ultra-Turrax homogenizer X120. The wall solution was prepared in varied ratio between maltodextrin to gum arabic of 1:1; 4:1; 9:1 and 100 % of malto dextrin. The core material to wall materials weight ratio is 1:4.

2.3 Preparation of Feed Solution Maltodextrin and gum arabic wall solution were mixed with the anti-cellulite active agents core material containing asiaticoside and ginger extract (10 % solid contents). Around 20% of wall solutions was slowly poured into active agents solution and stirred to homogeneity with Ultra-Turrax homogenizer X120 for Âą30 minutes. Then, the remaining wall solution was slowly added until reached 10 % solid contents and stirred to homogeneity with Ultra-Turrax homogenizer X120 for Âą15 minutes. For spray drying process, 500-1000 ml of feed solutions were prepared. 2.4 Spray Drying The feed solutions were spray dried in spray drying chamber. The operating condition was at inlet air temperature of 1600C 2000C and outlet air temperature of 800C -1200C. Feed solutions temperature was 250C.

under specific conditions [5,6]. Microencapsulation permit time release of the nutrients, enhance the stability to extremes in temperature and moisture [5,7]. Wall material used to encapsulate active ingredients in food industries is usually starch derivates [8]. Maltodextrin is a water soluble material. When used as capsule wall material, it can protect encapsulated ingredients from oxidation [9]. Maltodextrin can also reduce the problem of stickiness and agglomeration during storage, thereby improving product stability [10]. Gum arabic, a complex heteropolysaccharide [11], has been used as an encapsulating material in microencapsulation by spray drying, mainly because of its good emulsifying capacity and low viscosity in aqueous solution, which aids the spray drying process [10]. In this research, maltodextrins and gum arabis was studied as wall material to encapsulate anti-cellulite active agent content of asiaticoside from pegagan herbs extract (Centella asiatica) and ginger herbs extract (Zingiber officinale) using spray drying. Spray drying is the most commonly used encapsulation method in food industry. The process is economical and flexible, using equipment that is readily available and produces particle of good quality [12, 14, 15]. Spray drying is one of the oldest processes to encapsulate active agent. Spray drying of active agent is commonly achieved by dissolving, emulsifying, or dispersing the active in an aqueous solution of carrier material (wall material), followed by atomization and spraying of the mixture into hot chamber [16]. The objective of this study was to produce microcapsules of anti-cellulite active agent containing asiaticoside from pegagan herbs extract (Centella asiatica) and ginger herbs extract (Zingiber officinale) in maltodextrin and gum arabic using spray-drying and to determine the effect of different ratio of wall material used (maltodextrin and gum arabic) and also to ensure the safety of anti cellulite product for human oral application by using toxicity acute test.

2.5 Scanning Electron Microscopy (SEM) Particle structure of the microcapsule powder was characterized by using Scanning Electron Microscopy JEOL JSM-5600LV SEM Instrument, in BATAN, Indonesia's National Nuclear Agency.

2.1 Active Agent Solutions Materials Samples of Centella asiatica (L.) Urban were obtained from plantation area in Solo, Indonesia. Species determination of C. asiatica was confirmed by Research Center for Biology, Indonesian Institute of Sciences. Ginger herbs extract (Zingiber officinale) was received from PT Matina Berto, Indonesia.. The core material was containing asiaticoside extract from pegagan herbs (Centella asiatica) and ginger herbs extract (Zingiber officinale). Asiaticoside extract and ginger extract (Zingiber officinale) was extracted in Division of Natural Products, Food and Pharmacy, Research Center for Chemistry, Indonesia Institute of Sciences. The core material was containing 2.5% (w/w) asiaticoside and 1% (w/w) ginger extract of (solid contents). In all case, total solids content (active agent material and wall material) of feed solution was 10% (w/w). The active agent solution was prepared by

2.7 Acute Oral Toxicity Test Acute oral toxicity test of microcapsule product was carried out to obtain safety doses of 14 days LD50 using mice (Mus musculus). Acute oral toxicity test was conducted by Hanabio, Environmental Toxicology Laboratory, Indonesia. Environment condition during testing was at 27-290C with 7075% humidity. The mice were grown at 12 hours light and 12 hours dark condition. Mice used were around 3 month age with average weight of 23.1 g/mice at the beginning of the test. Microcapsule powder was given through oral administration directly into the stomach to 5 groups dose, each consist of 20 mice (10 male mice and 10 female mice) at 5 dose levels of 0 (control); 1.088; 2.346; 4.907; 9.876; and 20.583 mg/kg body weight. Mice were not feed for maximum of 12 hours before sample administration and returned to its cage after sample administration.

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2.6 Particle Size Analyzer (PSA) Particle size analysis of the microcapsule was conducted using Coulter LS Particle Size Analyzer in Research Center for Chemistry, Indonesian Institute of Sciences.

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Evaluation conducted were mortality rate of mice, clinical signs observation, and anatomic pathology observation. Mortality rate observation of mice and clinical signs observation were conducted around 1, 2, and 3 hours after initial sample administration (0 day) then 2 times per day in the morning and afternoon on the next days (1st until 14th day). Anatomic pathology observation was conducted on major organ system of the thorax cavity and abdominal cavity.

The outer surface of the spray dried microcapsules characterized by the presence of these shrinkage were attributed to the shrinkage of the particle during spray drying and cooling. Similar shrinkages were observed in the study of milk powder microcapsule (Rosenberg et al, 1985) and ascorbic acid microcapsule (Priscilla et al, 2005). 3.2 Particle Size Analyzer (PSA)

III. RESULT AND DISCUSSION 3.1 Scanning Electron Microscopy (SEM) The morphology of microcapsules can be observed in Fig 1

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Figure 2. Particle size analysis: (A) maltodextrin: gum = 1:1 (B) maltodextrin : gum = 4:1 (C) maltodextrin : gum = 9:1 (D) 100% maltodextrin

Figure 1. Structure of microcapsule: (A) maltodextrin : gum = 1:1 (B) maltodextrin : gum = 4:1 (C) maltodextrin : gum = 9:1 (D) 100% maltodextrin

One reason for using SEM in this research of microencapsulation is the need to determine the encapsulating ability of various wall material compositions. Morphological analysis showed the shape and the common aspects of the microcapsule prepared from different wall material composition were in spherical form. It was also observed that microcapsules tend to form several shrinkages at the outer surface. The microcapsule product gave spherical shape but most of them had irregularities (some shrinkage) in their surface. Although the outer surface of the capsule had irregularities (some shrinkages), there were no observed pores or crack in the microcapsule surfaces. The presence of these shrinkages has an adverse effect on the flow properties of microencapsulated product powders, but they do not affect core material stability (asiaticoside and ginger extract).

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Table 1. Particle size distribution Microcapsule diameter (Âľm) Sampel % < 10

%< 25

% < 50

% < 75

% < 90

maltodextrin : gum arabic = 1:1

0.414

0.462

0.548

0.680

0.855

maltodextrin : gum arabic = 4:1

0.412

0.458

0.540

0.659

0.817

maltodextrin : gum arabic = 9:1

0.413

0.460

0.542

0.664

0.824

100% maltodextrin

0.415

0.466

0.555

0.593

0.875

Particle size analysis showed a main mode in intermediate range 0.4-0.8 Îźm. The Particle size analyzer result was given in Fig 2. Table 1 shows the particle size distribution of microencapsulated powder product as anti-cellulite oral treatment.

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The smallest particle size was produced by microencapsulation with maltodextrin to gum ratio of 9:1 with mean diameter of 0.593 μm. The biggest particle was produced by microencapsulation with maltodextrin to gum arabic ratio of 1:1 with mean diameter of 0.613 μm. Microcapsules prepared from maltodextrin to gum arabic ratio of 9:1 showed the narrowest size distribution, exactly what we wish because narrower particle distribution means more homogenous particle. This wall composition particle can obtain better stability and improve release accuracy. 3.3 Acute Oral Toxicity Test The purpose of acute oral toxicity test is to determine acute lethal dose for oral administration of microencapsulated powder product as anti-cellulite oral treatment. No

Doses

Life/ death

(mg/Kgwei ght)

2nd

llife

death

llife

death

2.346

llife

death

4.907

9,876

20.583 Doses

24th

1st

2nd

3rd

4th

llife

death

llife

death

llife

Quantity of mice until 0 0 0 0

14th

Life/

(mg/Kg

death death

weight )

Continue of Table 2. Doses (mg/ kg weight) of microencapsulated product powder and mortality of mice at 0, 1st , 2nd, 3rd hours and at 1st until 14th days after administration sample.

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0 (Control)

1.088

2.346

4.907

9,876

20.583

Days

3rd

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1st

0 (Control)

1.088

Hours 0

Quantity of Mice until

Table 2. Doses (mg/kg weight) of microencapsulated product powder and mortality of mice at 0, 1st , 2nd, 3rd hours and at 1st until 14th days after administration sample.

Generally, acute oral toxicity test was referred to “OECD Guidelines for testing of chemicals, Acute Oral Toxicity-Up– and-Down Procedure OECD 425” (OECD, 2001a) [17]. Mice mortality rate observation showed that there were no deaths of mice as shown in Table 2. Table 3 shows the mice mortality percentage 14 days after administration of microencapsulated product powder sample.

0 6

life

death

life

death

life

death

life

death

life

death

life

death

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Athanasia Amanda Septevani et al. / (IJAEST) INTERNATIONAL JOURNAL OF ADVANCED ENGINEERING SCIENCES AND TECHNOLOGIES Vol No. 1, Issue No. 1, 043 - 048 Doses

Quantity of mice at beginning

Quantity of death mice

Quantity of life mice

Percentage of Mortality (%)

(1)

(2)

(3)

(4)

(5)

0 (Control)

0/20x100% =

120

(mg/Kg weight)

1 2

1.088

2.346

4.907

9,876

20.583 Total quantity of mice

Table 4. Clinical signs observation of mices

Doses

Skin and fur

Eyes

Mucous membrane

(1)

(2)

(3)

(4)

0 (control)

good

1.088

good

2.346

good

4.907

9,876

20.583

Breathing

Salivation

Tremors and convulsions

Strange behavior

(5)

(6)

(7)

(8)

good

not found

good

not found

good

not found

good

not found

good

not found

good

not found

(mg/Kg

weight)

Table 3. Mortality rate observed at day 14th from initial sample administration of microencapsulated product powder.

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Clinical signs observation showed that there was no sickness observed on mice that were found as shown in Table 4. Anatomic pathology observation in Picture 1 showed that there was no obvious change on color and organ size (liver, lymph, kidney, lungs, heart and intestine) between treated and control mice. Based on mortality rate, clinical signs observation, and anatomic pathology observation in this acute oral toxicity test, microencapsulated product is safe for human oral administration. The value of 14 days LD50 of microencapsulated product powder as anti-cellulite treatment is more than 20,583mg/kg weight.

Picture 1. anatomic pathology observation of control mices and treated mices Noted: 1.liver; 2.lymph; 3.kidney; 4.heart ; 5.lungs

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Athanasia Amanda Septevani et al. / (IJAEST) INTERNATIONAL JOURNAL OF ADVANCED ENGINEERING SCIENCES AND TECHNOLOGIES Vol No. 1, Issue No. 1, 043 - 048

Based on the result obtained in the present study, it can be recommended the use of microencapsules containing 2.5% (w/w) asiaticoside and 1% w/w ginger extract (Zingiber officinale) encapsulated by mixture of maltodextrin and gum arabic of 9:1 for oral application of cellulite treatment. This sample presented the optimum result. It was observed that the smallest average particles size (0.593 μm) also gave narrowest particle size distribution so the particles were more homogenous. The morphology of microcapsules was observed by scanning electron microscopy whose analysis showed in spherical form and a tendency to form several shrinkages at the outer surface. Based on mortality rate, clinical signs observation, and anatomic pathology observation in this acute oral toxicity test, microencapsulated product is safe for human oral administration. The value of 14 days LD50 of microencapsulated product powder as anti-cellulite treatment is more than 20,583mg/kg weight. REFERENCES

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[6]

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IV. CONCLUSION

[7] [8] [9]

[10]

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