University of San Agustin

COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS

General Luna St. Iloilo City, Philippines 5000

PHARMACY DEPARTMENT

Bioautography of Golden Berry (Physalis peruviana) Active Fruit

Sub-extract against Staphylococcus aureus

Celo, M.C., Colocar, K.M., Daanoy, W.E., Delfin, D., Donasco, M.J., Fernandez, M.N.,
Gabutin, G., Ignacio, J.

Department of Pharmacy, College of Health and Allied Medical Professions,

University of San Agustin, Gen. Luna Street Iloilo City

ABSTRACT

Bacterial infections are still a major threat to the public health globally which is due
to the adaptation and uncontrolled spread of bacteria that are pathogenic to humans.
Occurrence of bacterial resistance rendered antibiotics to become less effective which
leads to the immediate discovery of potential antibacterial agents from plants. Physalis
peruviana from Solanaceae family has demonstrated antibacterial activity however class
of compounds responsible for this was not characterized. Therefore, this study aims to
determine the specific class of compounds responsible for antibacterial activity of fruit
extracts. Physalis peruviana fruit was macerated using methanol and components of the
crude fruit extract were separated according to their polarity using solvent partitioning
with water and ethyl acetate. Agar-well diffusion assay was used to screen antibacterial
activity against Staphylococcus aureus ATCC 25923. Extracts and sub-extracts were
then characterized using Thin Layer Chromatography. Bioautography of the active sub-
extracts was performed to detect the antibacterial bands from the TLC chromatogram.
Our results showed that less polar (ethyl acetate sub-extract) constituents of the crude
methanol extract of P. peruviana exhibit antibacterial activity against S. aureus ATCC
25923. Bioautography revealed that there were antibacterial bands with an Rf of 0.05 to
0.63 when viewed under 254nm and 365nm UV light. Ferric Chloride and Ninhydrin
reaction test revealed that these antibacterial bands were phenolic and amine in nature.
Aside from nutritional benefits, Golden berry (Physalis peruviana) is a good source of
antibiotics.

1
 University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
General Luna St. Iloilo City, Philippines 5000

PHARMACY DEPARTMENT

INTRODUCTION

The worldwide spread of infections is continuously alarming the global health and
considered as one of the major threats of this generation1. Infectious diseases are
usually caused by organisms such as bacteria, viruses and fungi that are easily
acquired through direct or indirect contact with the infected person2. Some of these
diseases are tuberculosis, pneumonia and hepatitis resulting to the deaths of
approximately 3.0 million people worldwide in 2016 and as of 2018, current statistics
revealed that there are about 500, 000 people across the globe suffering from difficulty
of treating bacterial infections3. Majority of these bacterial skin infections are usually
caused by Staphylococcus aureus.

Staphylococcus aureus is a versatile and virulent pathogen in humans that serves

as their natural reservoir4. It is considered to be one of the top bacterial strains in public
health concern as of 20185 and about 20% of the populations are known to be carriers,
60% are intermittent carrier and 20% are not exposed to the organism6. S. aureus is the
leading cause of mild to severe conditions, in the form of abscess that could develop to
life-threatening infection of the blood, commonly known as bacteremia. Staphylococcus
aureus bacteremia (SAB) is an alarming infection with an incidence rate ranging from 20
to 50 cases/100, 000 populations per year and about 10% to 30% of these patients died
from SAB. This accounts for a greater number of deaths than for AIDS, tuberculosis
and viral hepatitis combined7. It is undeniable that these bacteria pose a threat in the
effective prevention and cure of infections that might worsen a person’s health
conditions and hinder promotion of the quality of life.

Bacterial infections still continue to evolve at the same time the discovery of new
treatments and studies are very necessary to work into. Although, natural products of
microbial origin had been the most important source of antimicrobials, various herbs and
medicinal plants have been the source of certain medicines used to treat different
human diseases possessing different levels of defense mechanism against various
microorganisms which makes these traditional and botanical medicines a normal part of
the primary health care. Examples of these botanical medicines are potato and tomato
which both belongs to the solanaceae family and have showed to possess antibacterial
activity and has been widely used all throughout8.

The solanaceae family is known to be ethno-botanical which provides products

such as foods, spices, and drugs that are extensively used by humans. It is an important
source of Vitamin C, E, A, thiamine, niacin, pyridoxine, folacin, minerals, and dietary
fibers which play a significant role in human nutrition and helps cope with malnutrition9.
Example of plants that belong to this family and have shown antibacterial activity is
Solanum tuberosum which has pronounced antibacterial activity on gram-positive
bacteria, especially Staphylococcus aureus (MIC=0.62mg/ml). Having a great
importance in the Solanaceae family, the genus Physalis is the most developed genus
in its family10. Physalis species were reported to possess therapeutic activity like anti-
inflammatory, antioxidant and anti-bacterial properties11. Therefore, the interest in this
genus has grown in many regions of the world in recent decade and different studies

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 University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
General Luna St. Iloilo City, Philippines 5000

PHARMACY DEPARTMENT

conducted leads to prove that certain species of Physalis do have anti-bacterial activity.
In the case of Physalis angulata, chloroform extracts from epigeal parts is effective
against Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium kansaii,
Mycobacterium malmoense, and Mycobacterium intracellulare. Aqueous and ethanolic
extracts of Physalis angulata also inhibited the growth of Staphylococcus aureus and
Escherichia coli12. In addition, Physalis peruviana was also classified under this family
and genus that has been reported to have wide range of folkloric uses.

Physalis peruviana, commonly known as Tino-tino locally here in the Philippines, is

rich in different nutrient components such as minerals, vitamins and carbohydrates that
are essential in the human body. Folkloric uses of this plant are anti-hyperglycemic
agent, anti-inflammatory, and antiasthma. Moreover, its several medicinal properties
include antispasmodic, diuretic, sedative and analgesic13. P. peruviana have been
reported to have antiseptic, anticancer and antibacterial properties which its seeds and
fruits have shown to possess the most antimicrobial effect among other plant parts14.
Furthermore, it also demonstrated antibacterial effects against six bacterial strains such
as Staphylococcus aureus, Staphylococcus epidermidis, and Lactococcus lactis which
were gram-positive and Chromobacterium violaceum, Escherichia coli, and Erwinia
herbicola which are gram-negative15.

However, specific compounds responsible for the antibacterial activity were not yet
established. Therefore, the goal of this study is to determine the antibacterial activity of
P. peruviana methanolic crude fruit extract and fruit sub-extract against susceptible
Staphylococcus aureus ATCC 25923 by measuring its growth of inhibition using agar-
well diffusion method. To detect the antibacterial bands, bioautography will be used and
bands will be visually characterized using color forming reagents.

MATERIALS AND METHODS

Plant Material

P. peruviana fruits were collected last January 2019 at Nazuni, Dingle Iloilo. Plant
Sample was sent to the Department of Agriculture Region VI for authentication. Fruits
(150g) were sun-dried and grind using an industrial grinder.

Plant Extraction and Solvent Partition

P. peruviana dried fruits were extracted by maceration with 1:4 ratio by weight in
volume of methanol. It was filtered using Whatman filter paper and concentrated using
rotary evaporator at 40°C. Crude extract was partitioned using polar (water) and semi-
polar (ethyl acetate) solvents. Less polar sub-extract (ethyl acetate) was concentrated
using rotary evaporator while polar sub-extracts (aqueous) was freeze-dried16 and was
stored in bio-refrigerator at -20°C.

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 University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
General Luna St. Iloilo City, Philippines 5000

PHARMACY DEPARTMENT

Antibacterial Screening of Crude and Fruit Sub-Extracts

Agar well diffusion method was used to screen the antibacterial activity of crude and
sub-extracts of P. peruviana. Briefly, MHA and MHB were prepared and autoclaved for
15 minutes at 121°C. Pour plate method was used following CLSI guidelines17.
Treatment concentration of 150mg/mL was used for crude extract and sub-extracts and
Gentamicin of 5mg/mL was used as positive control and DMSO as negative control.
Briefly, bacterial suspension was adjusted to optical density (OD) of 0.0005 and was
plated in sterile petri dish, solidified, and wells were made using sterile borer. Extracts
were dispensed on different wells, allowed to diffuse and incubated at 37°C. After 18 –
24 hours, zone of inhibition was measured using caliper expressed in millimeter18

Thin Layer Chromatography

Thin layer chromatography was used to determine the class of compounds present
in P. peruviana active fruit sub-extract. Crude and sub-extracts were spotted in silica
plate (1” x 3”) and developed in appropriate mobile phase of 3:1.5:0.5 (hexane: ethyl
acetate: methanol). It was then dried, visualized under normal light and UV light (254nm
and 365nm wavelength). Color forming reagents were then used to detect the presence
of different functional groups. Bluish-green coloration with FeCl3 reaction indicates
presence of phenolic compounds19. Violet coloration with Ninhydrin reagent reaction
indicates presence of amine compound20 and Reddish-orange with Dragendorff reagent
reaction indicates presence of alkaloidal compounds21. Rf values of the UV active bands
was calculated using the formula:

𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑝𝑜𝑡
𝑅𝑓 =
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡

Bioautography

Identical chromatogram were made by spotting the active extract on the silica
backed TLC plate, developed in appropriate mobile phase and dried. One
chromatogram was used for bioassay and other chromatogram was for characterization
using UV light (254 nm and 365 nm). Briefly, the chromatogram used for bioassay was
overlaid with soft agar (0.08%) with an optical density (OD) of 0.00001. It was then
solidified and incubated at 37°C. After 18 – 24 hours, resazurin was used to visualize
the zone of inhibition where blue coloration indicates antibacterial activity22

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 University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
General Luna St. Iloilo City, Philippines 5000

PHARMACY DEPARTMENT

RESULTS AND DISCUSSION

Characterization of Crude Methanol P. peruviana Extract

Thin Layer Chromatography (TLC) was used to characterize constituents present in

P. peruviana fruit extracts. Optimized mobile phase was found to be 3:1.5:0.5
(Hexane:Ethyl Acetate: Methanol) for methanolic crude fruit extract and ethyl acetate
fruit sub-extract. Results showed that polar compounds in methanolic crude extract are
visible under 254nm UV light at Rf values ranging from 0.02 to 0.60 and when viewed
under 365nm UV light, it was visible at Rf values 0.02 to 0.52. In addition, less polar
compounds of methanolic crude extract were found at Rf values 0.69 to 0.73 at 254nm
UV light and Rf values 0.70 to 0.79 at 365nm UV light. Polar compounds were also
present in ethyl acetate fruit sub-extract at Rf values 0.04 to 0.55 under 254nm UV light
and Rf values 0.04 to 0.60 viewed under 365nm. Less polar compounds of ethyl acetate
fruit sub-extract were visible under 254nm UV light at Rf values of 0.66 to 0.77 and 0.68
to 0.80 Rf values under 365nm. However, aqueous sub-extract did not resolve in
3:1.5:0.5 Hexane:Ethyl Acetate:Methanol mobile phase.

A B
254nm 365nm
Rf Rf Rf Rf
0.77 0.79 0.80
0.73 0.73 0.73 0.73
0.71 0.70 0.70
0.69 0.68
0.66

0.60 0.55 0.52 0.60

0.46 0.46 0.48
0.43 0.41
0.40
0.32 0.32 0.33
0.30
0.27 0.29
0.26 0.23
0.20 0.20
0.16 0.13 0.11
0.07 0.07
0.02 7
0.04 0.04
0.02
7
1 2 3 1 2 3

Figure 1. TLC Chromatogram of Physalis peruviana. Mobile phase 3:1.5:0.5 Hexane:Ethyl

Acetate:Methanol. (A.1;B.1) Methanolic Crude Extract (A.2;B.2) Aqueous Extract (A.3;B.3) Ethyl
Acetate Fruit Sub-Extract

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 University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
General Luna St. Iloilo City, Philippines 5000

PHARMACY DEPARTMENT

Thin Layer Chromatography

Thin Layer Chromatography of P. peruviana was performed using silica back TLC
plate that subjected to different mobile phases. Silica gel is polar in nature and holds
polar compounds causing shorter distance travelled, while non-polar compounds diffuse
with the solvent and travel further distances. Based on our result, Rf values from 0.02 to
0.60 are polar bands and spots from 0.66 to 0.80 are non-polar. There were nineteen
bands visualized under 254nm indicating that these are conjugated aromatic
compounds and twenty one bands under 365nm signifies polycyclic aromatic
compounds. Therefore, these shows that the diversity of phytochemicals found in P.
peruviana could be a potential antibacterial constituent.

Antibacterial Screening of Crude and Fruit Sub-extract

Agar well diffusion method was used to screen for the antibacterial activity of P.
peruviana fruit extracts and sub-extracts against S. aureus (ATCC 25923). Our results
showed that crude methanolic extract of P. peruviana did not exhibit antibacterial activity
against S. aureus ATCC 25923 as shown in Figure 2. However, antibacterial activity
was observed with the less polar sub-extract (ethyl acetate) showing a zone of inhibition
of 11mm at 150mg/mL concentration. No antibacterial activity was observed in the polar
sub-extract (aqueous).

1
Staphylococcus aureus
ATCC 25923 N P 2
30 3
25
Zone of Inhibition (mm)

20

15

10

0
Gentamicin (5mg/mL) Ethyl Acetate Sub-extract (150mg/mL)

Treatment

Figure 2. Antibacterial Activity of P. peruviana Extracts and Sub-extracts against S.

aureus ATCC 25923. Methanolic crude fruits extract and aqueous fruit sub-extract does not
have zone of inhibition. Values are means ± Standard deviation (SD), n=3 trials

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 University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
General Luna St. Iloilo City, Philippines 5000

PHARMACY DEPARTMENT

The findings of this study showed that less polar (ethyl acetate) fruit sub-extract of
P. peruviana exhibits antibacterial activity against S. aureus ATCC 25923. Also, the
dichloromethane (non-polar) extracts of P. peruviana fruit demonstrates antibacterial
activity against S. aureus. Furthermore, less polar (chloroform, diethyl ether, ethanol,
and ethyl acetate) fruit crude extracts of P. minima had also exhibit antibacterial activity
against pathogenic microorganisms. In contrary to our result, another member of
Solanaceae family showed that the methanolic crude fruit extract and polar (aqueous)
sub-extract of P. ixocarpa was found to be active against S. aureus. However, the class
of compounds responsible for the antibacterial activity was not fully defined. Therefore,
the members of the Solanaceae family possess diverse compounds that exhibit
antibacterial activity against pathogenic microorganisms which can be used as a new
potential for antibiotics.

Antibacterial Activity of UV active bands against S. aureus

To identify specific compounds responsible for the antibacterial activity of active P.

peruviana fruit sub-extracts, bioautography assay was used. Resazurin reduction test
was then performed to visualize the zone of inhibition wherein blue to blue coloration
indicates dead cells and blue to pink coloration indicates viable organisms. Furthermore,
TLC chromatogram positive result for the presence of amine compounds is violet
colouration using Ninhydrin reagent. As seen on Figure 3 (A.4) below, chromatogram of
ethyl acetate fruit sub-extract possess amine compounds at Rf values 0.11 to 0.14 and
0.11 to 0.18 viewed under 254nm and 365nm UV light respectively which corresponds
to the zone of inhibition shown in the bioautogram. Furthermore, the presences of
phenolic compounds are indicated by bluish-green colouration using Ferric Chloride
reagent. In relation to this, TLC chromatogram as showed in Figure 3 (A.5), Rf values
0.05 to 0.55 viewed under 254nm UV light and 0.05 to 0.34 and 0.46 to 0.63 Rf values
under 365nm are phenolic constituents.

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 University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
General Luna St. Iloilo City, Philippines 5000

PHARMACY DEPARTMENT

Resazurin Ferric
Reduction Ninhydrin Chloride
Test 254nm 365nm Test Test
Rf Rf

0.63
0.61
0.55
0.50 0.50
0.48
0.46
0.39
0.36
0.34
0.30
0.25 0.23
0.20 0.18
0.14 0.14
0.11 0.11
0.05 0.05

A.1 A.2 A.3 A.4 A.5

Figure 3. Bioautography of Ethyl Acetate Fruit Sub-Extract of P. peruviana against S.

aureus Mobile Phase 3:1.5:0.5 Hexane:EA:MetOH. Blue coloration in resazurin dead plates
indicates dead cells. (A.1)Resazurin Reduction Test (A.2) TLC Chromatogram under 254nm
(A.3) TLC Chromatogram under 365nm (A.4) TLC Chromatogram with Ninhydrin Reagent (A.5)
TLC Chromatogram with Ferric Chloride Reagent

Resazurin dye was used to visualize the zone of inhibition in the bioautography
assay of P. peruviana semi-polar sub extract. The dye will be converted to resarofin and
turns pink in the presence of living or viable cells. However, zones of inhibition will be
indicated by formation of blue coloration23. Our findings showed that there were more
than one constituent (11) present in P. peruviana semi-polar sub extracts which are
found to be phenolic and amine in nature and are responsible for the antibacterial
activity against Staphylococcus aureus ATCC 25923. Steroidal content of Physalis
angulata known as Physalin B has antibacterial activity against Staphylococcus aureus
ATCC 6538P at a concentration of 100 µg/ml24. This demonstrated that P. peruviana
plant is a good source for antibiotic discovery.

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 University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
General Luna St. Iloilo City, Philippines 5000

PHARMACY DEPARTMENT

SUMMARY AND CONCLUSIONS

The result showed that the less polar constituent of P. peruviana exhibit the
antibacterial activity using Agar Well Diffusion Method. Analysis of Bioautography shows
that there were more than one antibacterial bands corresponding to Rf 0.05 to 0.63
which are active against susceptible S. aureus ATCC 25923. Characterization of these
antibacterial bands revealed that these are phenolic and amine in nature. Therefore,
Physalis peruviana fruit is a source of diverse constituents with antibacterial activities
against staphylococcal infections.

FUTURE DIRECTION

Based on the findings, there were several numbers of antibacterial bands present in
the less polar sub-extract. Further identification and isolation of active antibacterial band
is needed to determine the potency of each band and utilize P. peruviana as a source of
new antibiotics. In addition, conduct an investigation on the mode of action of the active
sub-extract against susceptible S. aureus ATCC 25923.

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 University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
General Luna St. Iloilo City, Philippines 5000

PHARMACY DEPARTMENT

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 University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
General Luna St. Iloilo City, Philippines 5000

PHARMACY DEPARTMENT

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Abubakar, A. et. al.,(2012).The Identification of Putative Antitrypanosomal
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