Evaluation of Physical Quality and Antioxidant Activity of Ethanol Extract of Moringa Leaves (Moringa oleifera LAM) Formulated in Emulgel Preparation

 

Benni Iskandar1,4, Gressy Novita1, Fiona Fitri Annisa1, Leny2*, Ihsanul Hafiz2,

Meircurius D.C. Surboyo3, Ching-Kuo Lee4

1Department of Pharmaceutical Technology, Sekolah Tinggi Ilmu Farmasi Riau, Pekanbaru, Indonesia.

2Faculty of Pharmacy and Health, Institut Kesehatan Helvetia, Medan, Indonesia.

3Department of Oral Medicine, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia.

4School of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan.

*Corresponding Author E-mail: leny@helvetia.ac.id

 

ABSTRACT:

Moringa leaves contains flavonoids which has the activity of free radical scavenging or as an antioxidant. The purpose of this study was to formulate the water insoluble extract of Moringa leaves into emulgel dosage forms in various concentrations to produce a stable preparation during storage and have antioxidant activity. The formulation of the emulgel preparation was made by varying the concentration of the ethanol extract of Moringa leaves at 0%(F0), 0.5%(FI), 1%(FII), and 2%(FIII). The study examined the emulgel preparation including organoleptic test, irritation test, pH test, homogeneity test, emulsion type test, spreadability test, freeze and thaw stability and antioxidant properties by using DPPH method. The results showed that all emulgel preparations were stable during storage, did not irritate, pH value between 5.50-6.04, homogeneous preparation, emulsion type was oil on water phase (o/w), spreadability between 3.2-3.9cm, IC50 from ethanol extract of Moringa leaves was 135.539ppm. For emulgel preparation, FIII formulation showed best activity compared to another formula in its capacity which was 192.48 ppm for 1st week storage and 292.94ppm for the 8th week storage. The conclusion of this study shows that emulgel containing Moringa leaf extract has antioxidant activity and stable in physical properties during storage.

 

KEYWORDS: Antioxidant, emulgel, extract, Moringa oleifera.

 

 


INTRODUCTION:

Moringa oleifera contains a lot of free radical inhibitor molecules, ascorbic acid, β-carotene, tocopherols, flavonoids, phenolics, hydroxynnamic acid          derivatives1-4. Moringa leaves contain bioactive compounds such as quercetin whose antioxidant power is 4-5 times higher than vitamin C and vitamin E5-8.

 

Ethanol extract of Moringa leaves does not dissolve in water9, to overcome this problem it is made into an emulgel dosage form to increase dispersion and facilitate application in pharmaceutical dosage forms.

 

 

Emulgel is an emulsion preparation mixed with a gelling agent so that it is thermodynamically stable, transparent, isotropic, and has a high absorption and diffusion rate. Emulgel helps mix hydrophobic drugs into the oil phase and then the oil globules are dispersed in the water phase by mixing them on a gel base10-12.

 

The purpose of this study was to formulate a water-insoluble ethanol extract of Moringa leaves to increase dyspersion and can be penetrated better and applied in pharmaceutical dosage forms. This gel preparation was made in various concentrations of the ethanol extract of Moringa leaves and then tested the antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) trapping method. Besides determining the antioxidant activity of extract and emulgel containing various concentration of extract, emulgel preparations were tested for its characteristics including organoleptic test, irritation test, pH test, homogeneity test, emulsion type test, spreadability test, and stability test using the freeze and thaw method.

 

MATERIALS AND METHODS:

Materials and Chemicals:

The tools used in this study were a rotary evaporator, a microplate reader (Epoch BioTek), analytical balance (Shimadzu), a hot plate (Thermo Scientific), a burette (Pyrex), pH meter, a cup, a mortar and a stamfer, watch glass, erlenmeyer, object glass and cover glass. The materials used in this study were the ethanol extract of Moringa leaves, carbopol 940, TEA, Tween 80, Span 80, sodium metabisulfite, liquid paraffin, propylene glycol, Oleum rosae and distilled water.

 

Moringa Leaves Extraction:

The sample was washed with running water until clean, then drained. Furthermore, it is dried using a drying cabinet at 45°C for 3 days until the sample dry. Dried leaves were then ground into powder with milling. The refined sample was put into a dark bottle then immersed in 96% ethanol solvent for 5 days while stirring it occasionally. The liquid extract was then filtered with filter paper. Soaking was done for 3 times and the filtrate is transferred into a closed vessel. The filtrates were concentrated using a rotary evaporator to remove the solvent and obtain a crude extract of Moringa leaves. The extract stored in a dark bottle and stored in a place protected from light until it is used13-14.

 

Emulgel Formulation of Moringa Leaves Extract:

Span 80 and Moringa leaf ethanol extract is dissolved in liquid paraffin for the oil phase. Then the aqueous phase was prepared by dissolving Tween 80 in distilled water. Sodium metabisulfite is dissolved into propylene glycol. The whole mixture was then stirred using a magnetic stirrer at 300rpm for 10 minutes until an emulsion was formed (mixture 1). Carbopol 940 is mixed into hot distilled water (80 - 90°C) for 30 minutes, then added with TEA, mixed homogeneously, to form a gel preparation (mixture 2). Add mixture 2 to mixture 1 then add drops of Oleum rosae as the fragrance. Mix it inside hot mortar to form a homogeneous emulgel13-16. The emulgel formula can be seen in Table 1.

 

Table 1: Emulgel Formula of Moringa Leaf Extract

Materials

Amount (%)

F0

FI

FII

FIII

Moringa Leaf Extract

-

0.5

1

2

Carbopol 940

1

1

1

1

TEA

0.5

0.5

0.5

0.5

Sodium Metabisulfite

0.01

0.01

0.01

0.01

Tween 80

1

1

1

1

Span 80

1.5

1.5

1.5

1.5

Paraffin Liquid

7.5

7.5

7.5

7.5

Propylene glycol

5

5

5

5

Oleum Rosae

3 drops

3 drops

3 drops

3 drops

Distilled Water (up to)

100 ml

100 ml

100 ml

100 ml

Evaluation of Emulgel Formula:

The evaluation on the emulgel formula includes an organoleptic test that is visually observed the physical characteristics of the preparation. The parameters observed in the organoleptic test were the consistency, color and odor of the preparations. In addition, the pH test was carried out using a pH-meter, emulsion type test, spreadability and homogeneity17-19. The stability test was carried out by using the freeze and thaw method, all preparations were stored at refrigerator (4°C) for 24 hours and oven (40°C) for 24 hours. After 48 hours, it is called as one cycle. Emulgel preparations were kept for 6 cycles (12 days). In each cycle, emulgel preparations was observed for its changing19-21.

 

Irritation Test:

The test was carried out with the patch test method on 12 panelists divided into 3 panelists for one formula, carried out by applying 0.1gram of emulgel preparation on the inner arm with a diameter of 2cm and then covered with waterproof plaster gauze for 24 hours. After 24 hours taped on skin, gauze was opened and skin was observed for the reactions of erythema and edema22-23.

 

Antioxidant Activity Test:

The antioxidant activity test was carried out using the DPPH radical scavenging method. Among in vitro assays, the DPPH method is the most popular one due to its simplicity and speed24. The relatively stable DPPH radical had been used widely to test the ability of compounds to act as free radical scavengers or hydrogen donors. This capability made DPPH was choosen in evaluating antioxidant activity15. DPPH was first dissolved using methanol to obtain a concentration of 80 µg/mL. As a positive comparison in this study used vitamin c (ascorbate acid) dissolved in methanol with a concentration of 3.125, 6.25, 12.5, 25, 50 and 100µg/ mL. In this antioxidant activity test, the samples used were moringa leaves extract emulgel preparation. The antioxidant activity test on the extract and emulgel preparation of each formula was made in various concentrations of 31.25, 62.5, 125, 250, 500 and 1000µg /mL25-28.

 

The antioxidant activity test was carried out using a microplate reader and a UV-vis spectrophotometer with a wavelength of 517nm. The comparison used in the test was methanol solution as a negative control and ascorbic acid as a positive control. The DPPH concentration used in the measurement was 40µg/mL29-31. The antioxidant activity was determined from the inhibition percentage value by calculating the absorbance ratio of the sample to the negative control. The inhibition percentage value of 50% was calculated using a linear regression equation to obtain the IC50 value32-33. The percent of reduction of DPPH was calculated by the formulation:

 

% DPPH reduction = (1 − As/Ac) × 100

Where, As = absorbance of sample,

          Ac = absorbance of a control34.

 

RESULTS AND DISCUSSION:

Moringa Leaf Extract and Emulgel:

Moringa leaves were extracted using ethanol 96% as the solvent. The amount of material used and the resulting extract can be seen in Table 2.

 

Table 2. The amount of sample and extract obtained

Fresh Leaves (g)

Dried Leaves (g)

Ratio (%)

The Number of Dry Leaves Extracted (g)

Extract Obtained (g)

Ratio (%)

2000

750

37.5

450

90.98

20.22

 

The extract obtained from maceration was then formulated in the form of an emulgel with various concentration of Moringa leaf extract. F0 or blank emulgel does not contain any extract, FI with 0.5% moringa leaf extract, FII with 1% and FIII with 2% moringa leaf extract. The resulting emulgel preparations can be seen in Figure 1.

 

 

Figure 1: Emulgel preparation

 

Evaluation Result of the Emulgel Formula Stability:

The characteristics of the emulgel throughout the organoleptic test are semi-solid form, blank emulgel (F0) is white in color. For emulgel containing moringa leaf extract, it is cream, beige and dark beige respectively as shown in Figure 1. The higher the concentration, the darker was the emulgel color (FI-FIII). The emulgel appeared stable and there was no phase separation. The homogeneity test also showed that the preparations were homogenous and stable during storage. All emulgel preparations showed a homogeneous arrangement, this was indicated by the color of the emulgel was well distributed, there was no visible coarse particles in each emulgel preparation35. The type of emulsion was oil in water (o/w) and there was no phase inversion after 8 weeks storage. The stability test using the freeze and thaw method also showed good stability with no changes in each emulgel formula. The skin irritation test on the panelists also showed no signs of irritation on the area where the emulgel was applied.

 

The results of the spreadability test for each formula showed that there was no significant difference in testing the weight variation (0, 10, 20, 50, 100 and 200 g) for three measurements (1st week, 4th week and 8th week). The data from the measurement of the spreadibility diameter can be seen in Table 3. Blank emulgel and moringa leaves extract of emulgel have fulfill the requirement of emulgel spreadability which is 3-5 cm 36.

Measurement of the pH value of the emulgel preparation was observed every week with no significant differences in each formulation. The results of pH measurements can be seen in table 4.


 

 

Table 3. Result of Spreadability Test

Observation Time (week)

Heavy Load (g)

Spreadability Diameter (cm)

Anova Test Value (p)

Homogeneity of Variance (p)

F0 (0%)

FI (0,5%)

FII (1%)

FIII (2%)

1st

0

1.4

1.8

1.6

1.7

0.999

0.995

10

2.2

2.3

2.4

2.4

20

2.5

2.7

2.7

2.8

50

3.0

3.1

3.2

3.2

100

3.1

3.6

3.6

3.5

200

4.0

3.9

3.9

3.9

4th

0

1.9

1.6

1.4

1.6

10

2.2

2.1

1.9

1.8

20

2.4

2.2

2.3

2.2

50

2.7

2.7

2.6

2.5

100

3.1

3.1

3.0

2.7

200

3.6

3.4

3.4

3.3

8th

0

1.3

1.2

1.2

1.1

10

1.75

1.7

1.8

1.7

20

2.1

2.2

2.2

2.1

50

2.5

2.4

2.5

2.6

100

2.9

2.8

2.9

2.9

200

34

3.2

3.3

3.4

 

Table 4. pH of Emulgel

S. No

Formula

Week

pH-Value ±Std. Error

Significance of Difference

1

2

3

4

5

6

7

8

1

F0

7.09

7.07

7.04

7.05

7.04

7.03

7.03

7.03

7.0475±0.07

1.00

2

FI

5.88

5.89

5.87

5.88

5.87

5.86

5.87

5.86

5.8775±0.05

1.00

3

FII

5.70

5.68

5.66

5.66

5.67

5.66

5.64

5.65

5.6650±0.05

1.00

4

FIII

5.66

5.50

5.59

5.64

5.67

5.62

5.63

5.62

5.6075±0.06

1.00

 

 

Acorbic Acid (Positive Control)

 

Moringa Leaf Extract

 

F0 (1st Week)

 

F0 (8th Week)

 

FI (1st Week)

 

FI (8th Week)

 

FII (1st Week)

 

FII (8th Week)

 

FIII (1st Week)

 

FIII (8th Week)

Figure 2. Regression Equation Curve for Moringa Leaf Extract Emulgel Antioxidant Activity

 


These pH value of all emulgel are in accordance with the skin pH requirements, which is between 4.5-6.537. After 8 weeks of storage, there was a decrease in the pH of the preparations in the four formulas. This can be caused by the ethanol extract of Moringa leaves experiencing decomposition of fatty acids due to hydrolysis, oxidation in the presence of oxygen and light. In addition, the ethanol extract of Moringa leaves has an acidic pH so that the higher the concentration of the Moringa leaf ethanol extract used, the lower the pH of the preparation38-41.

 

Antioxidant Activity Results Study:

Antioxidant activity can be monitored using the scavenging effect of radicals on DPPH40. The antioxidant activity of ascorbic acid (positive control), moringa leaves extract, blank emulgel and emulgel containing the moringa leaf extract are described in the form of a linear regression activity curve which can be seen in Figure 2. Antioxidant activity was measured in each formula (F0-FIII) in 2 measurements, namely the first and eighth week. The IC50 value of each sample in each measurement can be seen in Table 5.

 

Table 5. The IC50 Value of Moringa Leaf Extract and Emulgel on Antioxidant Activity

S. No

Samples

IC50 (µg/ml)

1st Week

8th Week

1

Acorbic Acid (Positive Control)

14.58

2

Moringa Leaf Ethanol Extract

135.639

3

F0 (Blank)

428.37

578.24

4

FI (0.5 %)

307.90

376.154

5

FII (1%)

281.46

327.01

6

FIII (2%)

192.48

292.94

 

Based on the value of antioxidant activity possessed by moringa leaf extract, it shows stronger antioxidant activity than in the emulgel dosage form. According to Jun et.al 2003, antioxidant activity is classified into 5 groups: highly active, active, moderate, weak and inactive42. For more detail can be seen in Table 6.

 

Table 6.  The Power levels of antioxidant by DPPH method

S. No

Intensity

IC50 values (µg/ml)

1

Highly active

< 50

2

Active

50-100

3

Moderate

101-250

4

Weak

251-500

5

Inactive

>500

 

This is due to the small concentration of extract in the preparation, the antioxidant activity provided by the extract and emulgel preparation comes from the content of polyphenols including vitamin A, vitamin C, vitamin E, vitamin K, vitamin B (Choline), vitamin B1 (Thiamin), vitamin B2 (Riboflavin), vitamin B3 (Niacin), vitamin B6, alanine, alpha-carotene, arginine, beta-carotene, beta-sitosterol, Caffeoilcuinic acid, campesterol, carotenoids, chlorophyll, chromium, delta-5-avenasterol, delta-7-avenasterol, glutathione, histidine, indole acetic acid, indoleasetonitril, kaempferal, leucine, lutein, methionine, myristic acid, palmitic acid, prolamine, proline, quercetin, routine, selenium, threonine, tryptophan, xanthine, xanthophyll, zeatin, zeasantin, and zinc43-44.

 

CONCLUSION:

This study concluded that emulgel containing the ethanol extract of Moringa leaves has good characteristics and physical stability during storage. Both moringa leaf extract itself and emulgel containing Moringa leaf extract show moderate level of antioxidant intensity, but higher result was shown by the antioxidant activity of Moringa leaf ethanol extract which is 135.639 µg/mL.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest. 

 

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Received on 18.04.2021           Modified on 17.07.2021

Accepted on 20.10.2021         © RJPT All right reserved

Research J. Pharm. and Tech. 2022; 15(6):2703-2708.

DOI: 10.52711/0974-360X.2022.00452