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Original Article

Development and evaluation of liquid oralphytoformulation of Phyllanthus amarus

John A. Avbunudiogba a,*, Christian A. Alalor a, Philip F. Builders b,Success Odozie a

aDepartment of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Delta State University,

Abraka, Delta State, NigeriabDepartment of Pharmaceutical Technology and Raw Materials Development,

National Institute for Pharmaceutical Research and Development, Abuja, Nigeria

a r t i c l e i n f o

Article history:

Received 21 May 2013

Accepted 8 August 2013

Available online 27 September 2013

Keywords:

Phyllanthus amarus

Antitussive property

Aqueous extract

Herbal syrup

Phytochemical properties

* Corresponding author. Tel.: þ234 803363333E-mail address: avbunudiogba@yahoo.co

0974-6943/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.jopr.2013.08.029

a b s t r a c t

Background: The leaves of Phyllanthus amarus (family: Euphorbiaceae) is reported to have good

medicinal values such as antitussive properties. However the extract of the plant is very

bitter, this constitutes a challenge in formulating an acceptable oral liquid dosage form.

Therefore, the aim of this study is to develop a pleasant tasting liquid preparation of the

extract by a taste masking technique as well as evaluate some physicochemical properties

of the formulation that relate to its stability.

Methods: Six formulations (AeF) of the extract were prepared. To obtain the most stable and

acceptable taste of the herbal syrup the physicochemical properties such as: colour, taste,

pH, specific gravity, as well as its antioxidant activity were evaluated.

Results and discussion: Formulation C which contains ethanol, citric acid, glycerin and syrup

BP as the taste masking agents was adjudged to have the most acceptable taste and sta-

bility. Generally formulations C showed a pH of 6.61 � 0.02 and 6.62 � 0.04, specific gravity

of 1.24 � 0.02 g/ml and 1.28 � 0.01 g/ml immediately after formulation and after storage for

10 weeks respectively.

Conclusion: Formulating P. amarus extract with ethanol, citric acid, glycerin and syrup BP

produced palatable and stable herbal syrup.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction remedies for the treatment of common illness such asmalaria

In most rural communities of many developing countries,

orthodox medicine are either not available or are expensive.

Approximate, 60e80% of the population of such countries still

relies either partly or completely on traditional medicine as

1, þ234 7088592197 (mobm (J.A. Avbunudiogba).2013, JPR Solutions; Publi

typhoid, cold, cough and catarrh.1 According to World Health

Organization (WHO), medicinal plants are the best source to

obtain the various drugs needed to combat various diseases

and it advocates the need for countries to venture into the

different aspects of traditional medicine.2 Medicinal plants

ile)

shed by Reed Elsevier India Pvt. Ltd. All rights reserved.

j o u r n a l o f p h a rm a c y r e s e a r c h 6 ( 2 0 1 3 ) 9 0 8e9 1 2 909

have been used to treat, prevent and cure diseases of humans,

plants and animals for as long as the history of man. This is

because of the diversity of phytochemicals that are synthe-

sized naturally as secondary metabolites by different plants

and are available as a cache of medicines. Many of these

phytochemicals are of immense benefit to man as therapeutic

agents. In recent times there is resurgence in the popularity of

herbs, both in the developing and developed countries alike,

this attraction could be due to the numerous benefits of the

standardized natural products as compared to the largely

synthetic orthodox medicines.3 The success of herbal prod-

ucts as a therapeutic agent is dependent upon how safe and

active their constituents are when they are ingested. For

maximum therapeutic benefits, it is important to take herbs in

the form that best capture and preserves their active constit-

uents while putting patients’ acceptability and adherence to

medication into consideration.

The oral route is the common route for administering

herbal drugs required for systemic effects. However, most

herbal medicines have unpleasant tastes which make pa-

tients’ acceptance and adherence to medication a major

problem.4

Phyllanthus amarus Schum. & Thonn. (Family Euphorbia-

ceae) is a small herb growing to less than two feet in height

with small yellow flowers, leaves and fruits. It is amotile plant

such that when the plant is picked, the feathery leaves fold in,

completely closing themselves. The plant is well known for its

medicinal properties. It is an important plant in Ayurvedic

medicine and is widely used worldwide.5 Phytochemical

studies have shown the presence of many valuable com-

pounds such as lignans, flavonoids, hydrolysable tannins

(ellagitannins), polyphenols, triterpenes, sterols and alkaloids.

The extracts and the compounds isolated from P. amarus show

a wide spectrum of pharmacological activities including

antiviral, antibacterial, antiplasmodial, antiinflammatory,

antimalarial, antimicrobial, anticancer, antidiabetic, hypo-

lipidemic, antioxidant, hepatoprotective, nephroprotective

and diurectic properties.6 Its use in cough, asthma and other

bronchial infections has also been documented.5 However,

the extracts and traditional preparations of the plant have a

bitter and astringent taste which is not acceptable by espe-

cially children and geriatrics. The aim of the present study

therefore, is to develop pleasant tasting oral liquid prepara-

tions of the aqueous ethanolic extract of P. amarus by a taste

masking technique and evaluating some important physico-

chemical properties of the formulation.

Table 1 e Formulae for preparing Liquid oral (syrup) of Phyllan

Formulation

Extract (g) Glycerine (ml) Citric acid (ml)

A 2.5 0 0

B 2.5 0 0

C 2.5 0 0

D 2.5 0.5 0.5

E 2.5 0.5 0.5

F 0 0.5 0.5

2. Materials and methods

2.1. Materials

Fresh leaves and stems of P. amarus obtained from Delta

State University environment and identified by the plant

Curator (Mr Sunday Nimehe and Victor Speaman) in the

Department of Pharmacognosy, Faculty of Pharmacy,

University of Benin, Benin city, Nigeria where a voucher

specimen was deposited for reference. Ethanol (70%), citric

acid, glycerin and 1,1-diphenyl-2-picrylhydrazil, DPPH

(Sigma Aldrich, Germany). All other chemicals used were

of analytical grade and were used without further

purification.

2.2. Method

2.2.1. Solvent extraction100 g of dried plant material was extracted with 1000 ml

aqueous ethanol using a Soxhlet extractor for 24 h. The su-

pernatant was collected and the solvent evaporated using

Rotary Evaporator (CH-9230 Flawil, Switzerland). The extract

was stored in a refrigerator in an airtight container for further

study and formulation.

2.2.2. Preparation of liquid oral (herbal syrup)To prepare liquid oral form of the extract, the following steps

were taken:

(a) Preparation of simple syrup BP: 667 g of sucrose was dis-

solved in sufficient distilled water to obtain 1000 ml of

concentrated simple syrup. The solution was filtered and

the simple syrup was used as vehicle.

(b) Preparation of final liquid oral form: Different batches of

final liquid oral of P. amaruswere prepared according to the

formulae in Table 1. The various herbal formulations were

then evaluated as per official standards.

2.2.3. Evaluation of herbal syrupThe different parameters of the various oral formulations

were assessed such as pH, physical appearance (colour, taste

and odour), and density. Stability study of the oral liquid syrup

was carried out at different temperature (i.e. at 4 �C, 27 �C(room temperature) and 47 �C).7

thus amarus extract.

Ingredients

Ethanol (ml) Distilled water (ml) to Syrup BP (ml) to

0 100 0

0 0 100

5 0 100

0 0 100

5 0 100

0 0 100

Table 2 e Taste score.

Taster Formulations

A B C D E F

1 0 3 3 3 4 5

2 0 2 3 3 4 5

3 0 1 3 3 3 5

4 0 2 2 3 4 5

5 0 2 3 3 3 5

0 e Very bitter.

1 e Bitter.

2 e Slightly bitter.

3 e Sweet.

4 e Very sweet.

5 e Extremely sweet.

A B C D E F

0

20

40

%SCAVENGING

FORMULATIONS

Fig. 2 e A chart of the in vitro assay of the scavenging

activity of DPPH.

j o u rn a l o f p h a rma c y r e s e a r c h 6 ( 2 0 1 3 ) 9 0 8e9 1 2910

2.3Qualitative DPPH test

The free radical scavenging capacity of the extracts was

determined using DPPH.8 DPPH solution (0.004% w/v) was

prepared in ethanol. The different formulations were devel-

oped in 10ml distilled water to a final concentration of 0.1mg/

ml. After adding 1ml of freshly prepared DPPH solution, it was

incubated for 20 min at 25 �C, they were read spectrophoto-

metrically at 517 nm wavelength. Vitamin C (ascorbic acid)

was used as a reference standard and developed to the same

concentration of 0.1 mg/ml. Control samplewas also prepared

containing the same volume but without any extract or

reference standard. Percentage scavenging activity of DPPH

was evaluated using Equation 1.

D% ¼ AC�ATAC

� 1001

1

where D ¼ scavenging activity of extract, AC ¼ absorbance of

control and AT ¼ absorbance of test sample.

3. Results and discussion

The formulae for the 6 formulations are presented in Table 1.

The taste score of the different formulations are presented in

6.2

6.3

6.4

6.5

6.6

6.7

6.8

6.9

7.0

7.1

AB

C

pH values of formulation s after storage

Fig. 1 e pH values of the formulations before and

Table 2. The physicochemical properties of the extract and

formulations of P. amarus such as colour, odour, taste, vis-

cosity, specific gravity and pH are shown in Figs. 1 and 2 and

Tables 3 and 4. The extract of P. amarus is brown in colour with

a characteristic odour and a bitter taste; thesewere also partly

transferred to the formulations. The development of such

herbal formulation will mark an important advancement in

developing P. amarus into an acceptable oral liquid phytome-

dicine. The bitter taste of the extract was not completely

masked in each of the formulations when compared with the

control (formulation F) in which the plant extract was absent.

However formulation E was adjudged as having the best

acceptable taste. Considering the components of the formu-

lations, the syrup served as a sweetener and vehicle for the

liquid formulation, citric acid and glycerin served to improve

the sweetening effect of the syrup while ethanol served as

sweetener and a preservative.9

Though the formulations: B, C, D. and E were sufficiently

masked, but on the basis of the taste result, formulation E can

be said to be the best masked which could be due to the

presence of glycerin, citric acid and ethanol which provides

the formulationwith extra sweet taste in addition to the sweet

taste of syrup.

Based on the physical appearance after 10 weeks of storage

it could be deduced that the plant might contains natural

preservative since formulation A did not show any sign of

spoilage after 10 weeks. This is in agreement with earlier

DE

F

after storage for 24 h ( ) and 10 weeks ( ).

Table 3 e Result of stability testing of the formulation.

Time duration 24 H 10 Weeks

Formulations Temp Turbidity/Homogeneity

Colour/odour change

Temp Turbidity/Homogeneity

Colour/odourchange

A 4 �C No turbidity No change 4 �C No turbidity No change

R.T X No change R.T X Change in odour

47 �C Homogeneity No change 47 �C Homogeneity Change in odour

B 4 �C No turbidity No change 4 �C No turbidity No change

R.T X No change R.T Turbidity with crystals No change

47 �C Homogeneity No change 47 �C Turbid No change

C 4 �C No turbidity No change 4 �C No turbidity No change

R.T X No change R.T X No change

47 �C Homogeneity No change 47 �C Homogeneity No change

D 4 �C No turbidity No change 4 �C No turbidity No change

R.T X No change R.T X No change

47 �C Homogeneity No change 47 �C Homogeneity No change

E 4 �C No turbidity No change 4 �C No turbidity No change

R.T X No change R.T Crystal present No change

47 �C Homogeneity No change 47 �C Crystal present No change

F 4 �C No turbidity No change 4 �C Crystal present No change

R.T X No change R.T Crystal present No change

47 �C Homogeneity No change 47 �C Crystal present No change

R.T ¼ Room temperature.

X ¼ No turbidity.

Table 4 e Effect of storage on specific gravity of the formulations.

Formulations A B C D E F

Specific gravity (g/ml) 24 H 1.01 � 0.01 1.21 � 0.05 1.24 � 0.02 1.19 � 0.04 1.23 � 0.03 1.34 � 0.01

1-Week 1.01 � 0.01 1.21 � 0.01 1.24 � 0.00 1.19 � 0.01 1.23 � 0.01 1.22 � 0.02

10-Weeks 1.02 � 0.03 1.29 � 0.04 1.28 � 0.01 1.29 � 0.01 1.35 � 0.02 1.34 � 0.01

j o u r n a l o f p h a rm a c y r e s e a r c h 6 ( 2 0 1 3 ) 9 0 8e9 1 2 911

work.10 However it was observed that only formulation B had

signs of microbial spoilage. This could be due to absence of

ethanol and citric acid which could have helped to augment

the natural preservative present in P. amarus.

The various formulations of P. amarus also showed in vitro

scavenging activity of DPPH radical at 0.1 mg/ml when

compared to the control that retained the violet colour of

DPPH after 20 min observation (Fig. 2).

Tastemasking is an important technique that has been used

to prevent unpalatable drugs from interacting with the taste

buds to eliminate or reduce negative sensory response such as

the bitter taste of the extracts of P. amarus.11 The formulation of

the extract as a herbal syrup is aimed at developing a liquid oral

formulation that is palatable and acceptable.

The characteristic bitter taste is producedwhen the extract

binds to G-protein coupled receptors on the surface of the

taste cell of the tongue. This then prompts the protein sub-

units of alpha, beta, and gamma to split and activate an

enzyme that converts a precursor within the cell into a sec-

ondary messenger. This secondary messenger causes the

release of calcium ions (Caþþ) from the endoplasmic reticu-

lum of the taste cell. The resulting build-up of calcium ions

within the cell leads to depolarization and neurotransmitter

release. It is this signal that is sent to the brain and is inter-

preted as a bitter taste.12

The pleasant taste of the extract in formulation C is due to

the effective blocking of the taste receptors. This has been

accomplished by the presence of the combination of ethanol

and sucrose in the formulation. Ethanol acted as a taste

masking agent by competing for the taste channel thereby

reducing the net effect of the bitter stimuli of the extract by

the characteristic burning sensation of ethanol. The sweet-

ener effect of sucrose contained in the syrup essentially aided

in completing the taste masking effect of ethanol by quickly

dissolving in saliva to provide a sweet mouth feel. Hence the

pleasant taste of formulation E.12

Based on the various physicochemical properties evaluated,

all the formulations showed physical stability even after 10

weeks of storage (Tables 2e4). Formula A contains only water

and the extract, absence of spoilage after 10 weeks of storage

despite the absence of any preservative indicate the probable

presence of a self sustaining preservative. This is in agreement

with earlier work.10 The various formulations of P. amarus also

showed in vitro scavenging activity of DPPH radical at 0.1mg/ml

when compared to the control that retained the violet colour of

DPPH after 20 min observation (Fig. 2).

4. Conclusion

On the basis of the results obtained, formulation C showed

elegance and palatability and is the most appropriate for the

preparation of a stable syrup of the extract of P. amarus, since

it exhibited high stability in terms of appearance and specific

j o u rn a l o f p h a rma c y r e s e a r c h 6 ( 2 0 1 3 ) 9 0 8e9 1 2912

gravity after 10 weeks of storage while at same time, the bitter

taste was adequately masked by the simple syrup BP and

other additives. Thus, formula C could possibly be a suitable

formulation of P. amarus for geriatrics and pediatrics.

Conflicts of interest

All authors have none to declare

Acknowledgement

The authors are grateful to the Head of Department and staff

of Pharmaceutical Chemistry, Delta State University, Abraka,

Delta State, Nigeria, for the use of their Laboratory and

equipment for the extraction process. Also to be acknowl-

edged is the Technologist in charge of Pharmaceutical

Technology Laboratory, Department of Pharmaceutics and

Industrial Pharmacy, Delta State University, Abraka, Mr. Felix

Uboh for helping to operate themachines.We are also grateful

to Dr. Matthew I. Arhewoh, Department of Pharmaceutics and

Pharmaceutical Technology, University of Benin, Benin City,

Nigeria for helping to procure the reagents and offering useful

suggestions.

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