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Procedia Chemistry 14 (2015) 367 372

2nd Humboldt Kolleg in conjunction with International Conference on Natural Sciences,

HK-ICONS 2014

Potential of Ciplukan (Physalis angulata L.) as

Source of Functional Ingredient
RetnoWindya Kusumaningtyasa*, Noer Lailya, Putri Limandhaa
a

Laboratory for Development of Agroindustry and Biomedical Technology, The Agency of the Assessment and Application Technology,
LAPTIAB - BPPT; Kawasan PUSPIPTEK , Building No. 611 Serpong, Tangerang Selatan, 15314, Indonesia

Abstract
The aims of the study was to obtain a comprehensive data of the ciplukan as functional ingredients, whose efficacy acts as
immunomodulator and antioxidant. The extraction of ciplukan was performed using ethanol and water as solvent. The total phenol content and
antioxidant activity of ethanol ciplukan extract was higher than that of using water. The ciplukan leave extracts at all concentrations observed
induced an increased lymphocyte cell proliferation, however, all concentrations tested showed lower stimulation index compared to the
mitogen.

2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
2015 R.W. Kusumaningtyas, N. Laily, P. Limandha. Published by Elsevier B.V.
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the Scientific Committee of HK-ICONS 2014.
Peer-review under responsibility of the Scientific Committee of HK-ICONS 2014
Keywords: Antioxidant; ciplukan (Physalis angulata L.); lymphocyte proliferation; stimulation index.

Nomenclature
DMSO dimethyl sulfoxide
CFU
colony-forming unit
BHA 3-tert-butyl-4-hydroxyanisole
* Corresponding author. Tel. +62 21 756 0536; +62 21 7560 729 ext. 130; Fax. +62 21 756 6922; cell phone +62 812 999 4571
E-mail address: rwindya@yahoo.com.

1876-6196 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the Scientific Committee of HK-ICONS 2014
doi:10.1016/j.proche.2015.03.050

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Retno Windya Kusumaningtyas et al. / Procedia Chemistry 14 (2015) 367 372

1. Introduction
The plant kingdom is vast and unique use of plants to treat various diseases in human, however, they have not well known
yet. There are a considerable number of natural products used in the traditional medical systems in many countries as alternative
ones for the treatment of various diseases1. One of these plants, Physalis angulata, known in Indonesia as ciplukan or
ceplukan, is a branched annual shrub that belongs to the Solanaceae family.
This plant is widely distributed throughout tropical and subtropical regions of the world. Its extracts or infusions have been
used in many countries as popular medicine for treatments of varieties of diseases such as malaria, asthma, hepatitis, dermatitis
and rheumatism24. Some in vitro studies showed that purified compounds of P. angulata such as physalins (A, B, D and F) and
glycosides (e.g. Myricetin-3-O-neohesperidoside) exhibited antitumoral activities on HA22T (hepatoma), HeLa (cervix uteri),
leukemia, lung adenocarcinoma and epidermoid carcinoma of the nasopharynx KB-16 cell lines2,4,5. The number of evidences
related to the biological effects of plant extracts have been constantly increasing.
A number of herbal medicines and products were claimed to modify or boost immunity without scientific support. In addition,
plants cultivated in different parts of the world may not have pharmacological activities as previously reported6. Therefore,
attempts were made to verify P. angulata plants for in vitro stimulating human lymphocyte activity.
In the present study the possibility of P. angulata extract used as functional food ingredient and the effectiveness of extraction
process were evaluated. The extraction process was conducted using polar solvents such as water and ethanol. Bioactive
compounds in the plant extract were evaluated using determination of total phenolic content and the antioxidant activity.
Immunomodulatory effect was also evaluated in vitro using human lymphocytes cells.

2. Materials and methods

2.1. Chemicals
Standards of phenolic acids (gallic acid) and 2,2-dyphenyl-1-picrylhydrazyl (DPPH) were obtained from Sigma Chemicals
Co., St Louis, MO, USA. The Folin-Ciocalteus phenol reagent and 3-tert-butyl-4-hydroxyanisole (BHA) were from Fluka
Chemie AG, Buchs. All other solvents and chemicals used in the extraction process were analytical grade. Medium used in
lymphocyte cell proliferation assay were RPMI basal, complete RPMI (45 mL RPMI basal containing five mL serum FBS and
0.5 mL antibiotic penicillin-streptomysin), Ficoll-hystopaque, mitogen concanavalin A, LPS, tripan blue, 10 % EDTA, 0.5 %
MTT and 0.04 N HCl-isopropanol.
2.2. Plant material
A ciplukan plant (Physalis angulata) was collected from trees growing wild in the garden around the Laboratory for
Development of Agroindustrial and Biomedical Technology in Serpong, Tangerang. This plant has been identified and collected
by the Herbarium of the Center of Pharmacy and Medical Technology, Agency for the Assessment and Application of
Technology (BPPT) with specimen number : PMT 908. The plant material was separated according to their parts (leaves or fruit)
prior to use in the experiment.
2.3. Extraction
Preparation of extraction processes were conducted using 17.375 g of fresh ciplukan leaves or 32.58 g of fresh ciplukan fruits
(equivalent to 9 g dry weight). The water content of ciplukan leaves and fruits were 48.27 % and 72.44 % respectively. To
prepare water extract, each sample was grounded finely with a mortar, then put them in a 250 mL erlenmeyer and was added
with 100 mL of aquades. Then the samples were heated to boil and allowed to stand for 5 min. The sample was centrifuged at 6
000 rpm (60 rpm = 1 hertz) for 15 min. The filtrate was evaporated using a rotary vacuum evaporator at 80 C until dry. Then
the dried extract was added with DMSO until the final volume was 3 mL. Thus extract with a concentration of 9 g dry weight per
3 mL was obtained.
To prepare ethanol extract, each sample was added with 100 mL of ethanol, then macerated using a shaker at 240 rpm for 25
min at room temperature. The filtrate was filtered and stored in the refrigerator. The residue was macerated again with 100 mL
ethanol, then shaken and filtered. This stage was repeated until the filtrate collected a total of 300 mL. The filtrate was
concentrated by evaporation at 45 C until dry. Then the dried extract was added with DMSO until the final volume was 3 mL.
Thus extract with a concentration of 9 g dry weight per 3 mL was obtained. Subsequently the filtrate was transferred in a brown
bottle, then stored in a freezer.
2.4. Determination of total phenolic content in the plant extracts
The concentration of phenolics in plant extracts was determined using spectrophotometric method 7. The ethanol and aqueous
extract in the concentration of 1 mg mL1 were used in the analysis. The reaction mixture was prepared by mixing 0.5 mL of

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369

extract, 2.5 mL of 10 % Folin-Ciocalteus reagent dissolved in water and 2.5 mL 7.5 % NaHCO3. Blank was concomitantly
prepared, containing 0.5 mL ethanol or aquades, 2.5 mL 10 % Folin-Ciocalteus reagent and 2.5 mL of 7.5 % of NaHCO3. The
samples were incubated in a thermostat at 45 C for 45 min. The absorbance was determined using spectrophotometer at max =
765 nm. The samples were prepared in triplicate for each analysis and the mean value of absorbance was calculated. The same
procedure was repeated for the standard solution of gallic acid and the calibration line was construed. Based on the measured
absorbance, the concentration of phenolics was read (mg mL1 ) from the calibration line; then the content of phenolics in
extracts was expressed in terms of gallic acid equivalent (mg of GA g1 of extract).
2.5. Evaluation of antioxidant activity in the plant extracts
Evaluation of antioxidant activity was conducted according to the method of Mimura 8. Briefly, 0.2 mM DPPH solution was
prepared by dissolving 19.7 mg 1,1-diphenyl-2-picrylhydrozyl (DPPH) in 250 mL of absolute ethanol and freshly prepared
every time prior to using. 100 mM Tris-HCl buffer solution was prepared by dissolving 12.1 g of Tris in 800 mL of distilled
water and adjusting the pH to 7.4 with HCl 1 N, then filled up a volume to 1 000 mL. 5 mM BHA stock solution was prepared by
dissolving 90 mg of 3-tert-butyl-4-hydroxyanisole in 100 mL of absolute ethanol. Standard solutions in the range of
concentration 50 M to 500 M were prepared by diluting stock solution step by step using ethanol.
The reaction mixture was prepared by mixing 200 L of sample solution (or BHA standard), 800 L of 100 mM Tris-HCL
buffer and one mL of 0.2 mM DPPH solution. After standing at room temperature for 20 min in dark place, the absorbance was
determined using spectrophotometer at max = 517 nm. The antioxidant activities of plant extracts were plotted against the values
of BHA as the standard substance. The activity was revealed as percentage according to the following formula:
Antioxidant activity (%)

AB1  ( AS  AB 2 )
u 100
AB1

(1)

In which AS is absorbance of sample, AB1 is absorbance of first blank and AB2 is absorbance of second blank.
2.6. Isolation of human peripheral lymphocyte cells
Blood was withdrawn from a healthy male respondent aseptically by a paramedic. Blood sample was placed in a sterile tube
containing anticoagulant 10 % EDTA. Lymphocytes was isolated from cellular components using centrifugation at 1 500 rpm for
5 min at room temperature. Buffy coat layer that mainly contains lymphocyte cells, then was separated using ficoll-hystopaque
solution with centrifugation at 2 500 rpm for 30 min. Isolated lymphocytes were counted using hemacytometer. Lymphocyte
suspension with the number of living cells over 95 % was available (cells number at least 6 106 CFU mL1).
2.7. MTT assay for lymphocyte cells proliferative response.
The plants extracts stock solution was prepared at the concentration 1 g mL1 in DMSO solution. These extracts were
sterilized using 0.22 m and 0.45 m membrane filters. The serial dilution was made to prepare samples at the end concentration
of 1 mg 100 L1, 0.75 mg 100 L1, and 0.5 mg 100 L1.
The reaction mixture was prepared by mixing 50 L lymphocyte cells, 10 L sample extract and 40 L RPMI basal medium
into micro-well plate. The negative control was prepared by mixing 50 L lymphocyte cells, 10 L RPMI basal containing serum
+ antibiotic and 40 L RPMI basal medium. The positive control was prepared by replacing extract sample with mitogen LPS
and concanavalin A in the concentration of 2 10-6 mg 100 L1 and 1 10-6 mg 100 L1. The samples were incubated in a
CO2 incubator. After 68 h, the mixtures were added with MTT and isopropanol solution. The absorbance was determined using a
microplate ELISA reader at a wavelength of 570 nm. Immunostimulant activity was expressed in terms of percentage stimulation
index according to the following formula:
Stimulation Index (%) = sample absorbance/control absorbance 100 %

(2)

3. Result and discussion

3.1. Total phenolic content in the plant extracts
The total phenolic contents in the examined plant extracts using the Folin-Ciocalteus reagent is expressed in terms of gallic
acid equivalent (the standard curve equation: y = 0.001x 0.0418, r2 = 0.9982). The values obtained for the concentration of total
phenols are expressed as mg of GA g1 of extract (Table 1). The total phenolic contents in the examined extracts ranged from
(0.46 to 0.51) mg GA g1 of extract. The concentration of phenols measured in ethanolic extract was higher than in water

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extract. Water extract contains considerably smaller concentration of phenols. The total phenolic content in the water fruit extract
was smaller than the water leave extract. In addition, the concentration of phenols in the ethanolic fruit extract was higher than
the ethanolic leave extract. The sample extraction process using water solvent at the boiling point could affect the stability of
phenol compound, so that there was a possibility that the total phenolic content measured was lower than the reality. The result of
Chen and Lin9 research for yam sample showed that the total phenolic content declined in line with rising temperature of heating.
Maitsuthisakul et.al10 categorized the phenolic compound content into three categories (low 10 mg, medium = 10 to 20, high
40 mg GA g1 of extract), so that the extract of ciplukan leaves and fruit had low total phenolic compound content (0.46 mg to
0.51 mg GA g1 extract).
The total phenolic contents in plant extracts of the species Physalis angulata depended on the type of extract, i.e. the polarity
of solvent used in extraction and parts of the plant. Different parts of the plant such as leaf, fruit or root, contained different types
of phytochemical compounds. High solubility of phenols in polar solvents provides high concentration of these compounds in the
,12
extracts obtained using polar solvents for the extraction 11 .
Table 1. Total phenolic contents in the plant extracts expressed in terms of gallic acid equivalent (mg of GA g1 of
extract)
Total phenolic contents (mg of GA g1 of extract)
Water extract of ciplukan fruit
Ethanolic extract of ciplukan fruit
Water extract of ciplukan leaves
Ethanolic extract of ciplukan leaves

0.46
0.51
0.47
0.49

3.2. The antioxidant activity of the plant extracts

The antioxidant activity of different plant extracts from Physalis angulata was determined using a methanol solution of
DPPH reagent. DPPH is very stable free radical. Unlike in vitro generated free radicals such as the hydroxyl radical and
superoxide anion, DPPH has the advantage of being unaffected by certain side reactions, such as metal ion chelation and enzyme
inhibition. A freshly prepared DPPH solution exhibits a deep purple colour with an absorption maximum at 517 nm. This purple
colour generally fades when antioxidant molecules quench DPPH free radicals (i.e. by providing hydrogen atoms or by electron
donation, conceivably via a free-radical attack on the DPPH molecule) and convert them into a colourless or bleached product
such as 2,2-diphenyl-1-hydrazine, or a substituted analogous hydrazine, resulting in a decrease in absorbance at 517 nm band 13.
The antioxidant activity of four different extracts from the species Physalis angulata was stated in mg g1 BHA equivalent
(Table 2). The antioxidant activities of plant extracts were plotted against the values of BHA as the standard substance. The
examination of antioxidant activities of plant extracts from Physalis angulata showed different values. The obtained values
varied from 1 575 mg g1 to 4 311 mg g1 BHA. The largest capacity to neutralize DPPH radicals was found for ethanolic
extract of ciplukan leaves, at the concentration of 4 311 mg g1 BHA. The minutest capacity to inhibit DPPH radicals was
determined for water extract of ciplukan fruit at the concentration of 1 575 mg g1 BHA. The extraction of ciplukan fruit and
leaves using ethanol solvent showed higher antioxidant activity than using water solvent. Due to low activity of water extract,
the extraction of antioxidant substances of different chemical structure, was achieved using solvents of different polarity.
Numerous investigations of qualitative composition of plant extracts revealed the presence of high concentrations of phenols in
the extracts obtained 68 using polar solvents14. The extracts that perform the highest antioxidant activity have the highest
concentration of phenols. Phenols are very important plant constituents because of their scavenging ability on free radicals due to
their hydroxyl groups.
Table 2. Antioxidant activity in the plant extracts expressed in terms of BHA equivalent
Antioxidant activity (mg g1 BHA)
Water extract of ciplukan fruit
Ethanolic extract of ciplukan fruit
Water extract of ciplukan leaves
Ethanolic extract of ciplukan leaves

1 575
2 258
2 875
4 311

3.3. Stimulation of the lymphocyte cells proliferation

Furthermore, the assay on the activity of extract to stimulate the lymphocyte cells proliferation was carried out in vitro.
Proliferation is an essential biological function of lymphocytes, namely the process of differentiation and division (mitotic) cells.
Lymphocytes are single cells that survive better when cultured in simple media, and they consistently remain in the silent phase
and do not divide until the addition of mitogen. In the lymphocyte cell proliferation assay, the number of produced and survived
cells after the addition of mitogen in the culture was measured. Mitogen is a molecule that stimulates proliferation. Mitogen
concanavalin A (Con A) used in this research is good to stimulate T cells, while lipopolysaccharide (LPS) to stimulate B cells.

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371

Lymphocytes cells were isolated from healthy human blood. The results of stimulation indexes on the cell proliferation of the
extracts was shown in Figure 1.
Data of the response proliferation of lymphocyte showed that the ethanolic extract of ciplukan fruit at the concentrations of
0.5 mg 100 L1, 0.75 mg 100 L1 and 1 mg 100 L1 were 176.21 %, 140.00 % and 127.58 % of stimulation index
values respectively. The ethanol extract of ciplukan leaf at the concentrations of 0.5 mg 100 L1, 0.75 mg 100 L1 and
1 mg 100 L1 showed stimulation index values of 156.36 %, 200.15 % and 204.09 % respectively. The stimulation index
values of water fruit extracts at the concentrations of 0.5 mg 100 L1, 0.75 mg 100 L1 and 1 mg 100 L1 were 136.82 %,
147.12 % and 154.39 % respectively. The stimulation index values of water leaf extracts at the concentrations of 0.5 mg 100
L1, 0.75 mg 100 L1 and 1 mg 100 L1 were 113.18 %, 117.27 % and 145.45 % respectively. Although the concentrations
of extracts were used in nearly a thousand times higher than the concentration of mitogen, the ciplukan extract at all
concentrations tested showed lower stimulation index compared to LPS and ConA at a the concentration of 2 10-6 mg 100
L1 or 1 10-6 mg 100 L1.

Fig.1. The data of response of lymphocyte cell proliferation to the plant extract was stated as percentage of stimulation index

In the lymphocyte cell proliferation assay, MTT (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) solution was
added in the end of the steps and the living cells in the culture absorbed the colour of the reagent. There was a linear correlation
between absorbance value and the number of living cells in the culture. Determination of the activity of the plant extracts to
stimulate proliferation of lymphocyte cell was carried out in various concentrations. The water extract of P. angulata leaves or
fruit and the ethanolic extract of leaves, assessed in this study induced an increase in cell proliferation (Figure 1) in a dosedependent response. But significant alteration induced by the ethanolic extract of ciplukan fruit was not dose-dependent. This
may possibly be due to variation in chemical constituents responsible for plant activities. Increased lymphocyte proliferation
response was influenced by several factors such as the nature of the plant phenol compounds that were bound to proteins to form

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complexes through hydrogen bonds. This allows the binding of phenolic compounds in the extracts to the receptor proteins of
lymphocyte membrane so that finally they activate membrane enzyme systems that play a role in cell proliferation15.
Another factor is the nature of antioxidative phenols that can protect lymphocytes from reactive oxygen molecules, and the
active component that is soluble in the polar solvent induces the proliferation of white blood cells. Wagner16 also reported that
the class of phenolic compounds, such as flavonoids, tannins derivatives, phenilpropan and other simple phenolic compounds
had immunomodulatory activity either by complement system or intracellular biochemical reactions. The results suggested that
the extracts of P. angulata have stimulating activity on human lymphocytes and could be clinically useful for modulating
immune functions of the body.
4. Conclusion
In this study, ciplukan extraction process using ethanol and water solvent was evaluated. These solvents are usually used in
the extraction process in which the extract was applied in food products. Extraction process using ethanol have been proved to be
effective to get the extract with high total phenolic content and antioxidant activity. Although the residual ethanol in the extract
should be analyzed further when used as a food ingredient.
Generally, the water and ethanol extracts of ciplukan leaves at all concentrations observed induced an increase in lymphocyte
cell proliferation, however, all concentrations tested showed lower stimulation index compared to the mitogen. Thus ciplukan
Physalis angulata extracts have potential as functional food ingredient whose efficacy acts as immunomodulator. Further
development of the extraction process should be done to get a more efficient extraction process with higher active compounds
and better immunomodulatory activity of the extract. In addition, the toxicity test and the organoleptic test should be done for its
application in the food or beverage formula.

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