Page 1

Volume 1 Issue 2, Year 2022

ISSN: 2158-8155 (Online)
American Journal of Multidisciplinary DOI: https://doi.org/10.54536/ajmri.v1i2.230
Research and Innovation (AJMRI) https://journals.e-palli.com/home/index.php/ajmri

Phytochemical Screening and Antimicrobial Assay of Selected Plant Extracts with

Ethnopharmacological Potential for Wound Healing
Junar Cano1*, Joey F. Pescuela1, Dr. Dorothy A. Antesa1, Vinge Lyssa S. Mortejo1,
Chrisabelle P. Aguirre1, Sophia Alyannah P. Gallardo1

Article Information ABSTRACT

Bacteria usually colonize wounds, and their low level is beneficial to the wound healing
process. Thus, antibacterial agents are usually applied to wounds. In the Philippines, many
Received: April 23, 2022 herbal plants are used to speed up the wound healing process. With their number, literature
Accepted: April 27, 2022 revealed no scientific study yet on which among these plants could well inhibit wound bac-
teria. Hence, this study focused on the phytochemical screening and antibacterial activity of
Published: April 29, 2022 the ethanolic extracts of six herbal plants, namely: Caesalpinia sappan Linn. (Sibukao), Jatropha
curcas Linn. (Tuba-tuba), Lantana camara Linn. (Kantutay), Mimosa pudica Linn. (Makahiya),
Keywords Moringa oleifera Lam. (Malunggay), and Psidium guajava Linn. (Guava). Betadine was used as
the positive control. Qualitative phytochemical analysis and antimicrobial assay on wound
Antibacterial assay, Herbal bacteria using the agar well diffusion method were employed from the six extracts. Results
revealed that phytochemicals, namely alkaloids, saponins, flavonoids, and tannins, were pres-
plants, Phytochemical screening, ent in the plant extracts except for anthraquinones. Furthermore, the plant extracts showed
Plant extracts, Wound healing antibacterial activity on wound bacteria, and P. guajava leaf extract exhibited the most sig-
nificant antibacterial effect among the treatments and is greater than the positive control
(Betadine solution). On the other hand, all the plant extracts have a lower antibiotic activity
index when compared with Amikacin 30mcg and Erythromycin 15 mcg. However, except
for Lantana camara extract, the five plant extracts have a remarkably higher antibiotic activity
index when compared with Penicillin 10 U. It is concluded that the plants studied have vary-
ing levels of antibacterial activities that could promote wound healing.

INTRODUCTION healing process is more likely to be impeded among

A wound is defined as a loss of cellular and functional diabetic individuals (Sharma et al., 2013).
continuity of underlying living tissues in organisms. It The basic principles governing an effective wound healing
can damage the tissues to varying degrees (Udobre et process are highly dependent on the rapid and complete
al., 2012). However, in favorable conditions, wounds restoration of injured tissues and the suppression of
undergo a normal healing process. Nayak & Pinto- exponential bacterial growth at the wound site. While
Pereira (2006) posited that after an injury, the body these principles are highly critical, they are claimed to
initiates an inflammatory response that can cause the be challenging to enforce entirely in wound care clinical
cells below the dermis (the deepest skin layer) to start practice. Sabath (2006) posited that the techniques used
and increase collagen production. The collage produced to treat wounds are vital in preventing bacterial invasion.
results in the regeneration of the epithelial tissues of Bacterial growth at the wound site must be appropriately
the skin. Meanwhile, the present estimates indicate that managed to avoid infection and promote a rapid wound
there are nearly 6 million people across the world that healing process. In recent years, herbal medicines have
suffer from chronic wounds. Unhealed wounds inflict gained considerable interest as good sources of organic
pain by constantly producing inflammatory mediators compounds for treating a variety of diseases in many
that can increase the pain and swelling at the wound site parts of the world. Similarly, these herbal medicines have
(Kumar et al., 2007). One of the reasons for the delay been time-tested for their relative safety for human use,
in the wound healing process is infection. The infection affordability, environmental friendliness, and accessibility.
of the wounds happens when the body’s immune For instance, in the Philippines, many herbal plants are
defenses cannot control the average bacterial growth. used to speed up wound healing. Among these herbal
In most cases, infected wounds are caused by bacteria medicines are Caesalpinia sappan Linn., Jatropha curcas
that arise from the skin and other areas of the body Linn., Lantana camara Linn., Mimosa pudica Linn., Moringa
and the surrounding environment. According to Alam oleifera Lam., and Psidium guajava Linn.
et al. (2011), the most prevalent forms of bacteria that Caesalpinia sappan Linn. is a little thorny tree that grows
cause wound infection are Staphylococcus aureus and other to a height of 6-9 meters and is found in the Philippines,
staphylococci bacteria. Similarly, unsanitary facilities and India, Peru, and Malaya. In English, it is referred to as
Page 8

inadequate wound dressing practices may raise the risk of sappan wood or Brazil wood. Historically, the wood
infection. In addition, a person’s underlying health issues was used in calico printing of cotton, wool, and silk but
may prolong the healing process. For instance, the wound was eventually phased out in favor of synthetic colors.
1
Notre Dame of Marbel University, Philippines
*
Corresponding author’s e-mail: [email protected]
 Am. J. Multidis. Res. Innov. 1(2) 8-17, 2022

It has been historically used to treat a broad range inhibit wound bacteria. This prompted the researchers to
of diseases and is said to possess a wide variety of investigate the phytochemical screening and antimicrobial
therapeutic characteristics. It has been found to possess activity of selected herbal plants used in folk medicine
anticonvulsant, anti-inflammatory, anti-proliferative, on wound healing. If proven effective, these plants could
antibacterial, anticoagulant, antiviral, immunostimulant, be processed and could be cheap alternative sources of
and antioxidant properties (Sathya, 2010). drugs to cure wound infections and probably hasten the
Jatropha curcas Linn. is native to the American tropics, epithelial repair of injured tissues. This study aimed to
most likely Mexico and Central America. It is a deadly, determine the phytochemicals and antimicrobial activities
semi-evergreen shrub that grows up to 6 meters in of selected plant extracts with ethnopharmacological
height. It is a versatile tree with economic value due to potential for wound healing. Specifically, this sought to
its many industrial and medical applications. In Africa, (1) determine the presence of secondary metabolites in
Asia, and Latin America, Jatropha species are used to the selected plant extracts; (2) determine if the selected
treat various diseases (Arekemase et al., 2011). They are plant extracts are effective in inhibiting the growth of
utilized as antibacterial agents, and various studies have wound bacteria; (3) determine which of the selected plant
been conducted to ascertain their scientific foundation extracts are significantly effective in inhibiting the growth
(Kalimuthu et al., 2010). of wound bacteria; and (4) determine the antibiotic
Lantana camara Linn. is a flowering shrub native to activity indexes of the plant selected plant extracts.
tropical America used as an ornamental plant (Agrawal
et al., 2012). Various portions of the plant are utilized LITERATURE REVIEW
in folklore remedies and traditional medical systems to Wound Bacteria
cure various human diseases. The leaves are used to cure Infected wounds are wounds in which bacteria or other
itches, wounds, ulcers, swellings, bilious fever, eczema, microorganisms have colonized, causing either a delay in
and rheumatoid arthritis. Numerous pharmacological wound healing or wound deterioration. Bacteria typically
studies have shown that extracts of the leaves possess contaminate most wounds. However, infected wounds
antibacterial activity (Agrawal et al., 2012). Mimosa pudica result when the body’s immune defenses are overwhelmed
Linn. is a creeping perennial plant often cultivated for or cannot cope with average bacterial growth. Infection
its value. It is a Brazilian native plant that has become of wounds caused by surgery is a severe health risk, as
a pantropical weed. Young plants have an upright stem, studies have shown that 70 percent of the deaths of
but the stem becomes creeping or trailing as they mature. patients who have undergone surgery are caused by
The plant reaches a maximum height of 1.5 meters. Its surgical site infections (Udobre et al., 2012). Most infected
leaves are compound bi-pinnately, with one or two pinnae wounds are caused by bacteria originating either from the
pairs and ten to twenty-six leaflets per pinna. Its petioles skin, other body parts, or the outside environment. The
are likewise thorny, and a study reveals that the top half skin contains bacteria (normal flora) which are generally
of the floret petals are red, while the filaments are pink harmless if the skin is intact. However, the protective
to lavender. The fruit comprises clusters of two to eight barrier formed by the skin is disrupted when there is a
pods measuring between one and two centimeters in wound, and this normal flora can colonize the injured
length and spiky on the borders (Gandhiraja et al., 2009). area. This results in further tissue damage and may
Moringa oleifera Lam. is a highly coveted plant that grows prolong wound healing by promoting more inflammation,
in various tropical and subtropical regions. It has a which prolongs the process of wound healing. The most
broad variety of high nutritional value and therapeutic common bacteria causing wound infection is Staphylococcus
applications. Its different components include a variety aureus and other groups of staphylococci (Alam et al.,
of essential minerals, vitamins, proteins, and other 2011). Contamination from other parts of the body
phenolic compounds (Farooq et al., 2007). The plant may also cause wound infection. Poor wound dressing
leaves, seeds, roots, bark, flowers, fruits, and immature techniques and unhygienic conditions may increase the
pods all work as cardiac and circulatory stimulants and are risk of wound infection (Sharma et al., 2013).
said to have anti-inflammatory, antitumor, antiepileptic, Wound Healing
antipyretic, and antiulcer properties (Farooq et al., A wound can be defined as a break in the continuity
2007). Psidium guajava Linn. is a tropical American native of the soft tissues like skin, mucous membranes, and
evergreen shrub that grows in tropical and subtropical the tissue surface. An external wound is a wound with
regions. It is a phytotherapeutic plant that has been used a varying degree of damage to the tissue, including the
in folk medicine for centuries. It is thought to have active skin. An internal wound damages the underlying tissue
ingredients that aid in the treatment and management to a varying degree, leaving the skin intact (Udobre et
of various ailments. Numerous plant components have al., 2012). According to Nayak and Pinto-Pereira (2006),
traditionally been used to cure several ailments, including wound healing is the process of repair that follows
vomiting, malaria, gastroenteritis, wounds, ulcers, coughs, injury to the skin and other soft tissues. After an injury,
toothache, and sore throats (Biswas et al., 2013). an inflammatory response occurs, and the cells below
Page 9

With these number of herbal plants, literature revealed no the dermis (the deepest skin layer) increase collagen
scientific study yet on which among these plants could well (connective tissue) production. Later, the epithelial tissue

https://journals.e-palli.com/home/index.php/ajmri
 Am. J. Multidis. Res. Innov. 1(2) 8-17, 2022

(the outer skin layer) is regenerated. There are three Different parts of the plant are used in folklore remedies
stages to wound healing: inflammation, proliferation, and and traditional medicine systems to treat various human
remodeling. The proliferative phase is characterized by ailments. The leaves are used to treat itches, cuts, ulcers,
angiogenesis, collagen deposition, epithelialization, and swellings, bilious fever, eczema, and rheumatism. Many
wound contraction. pharmacological investigations indicated that extracts of
Delay in the Wound Healing Process the leaves exhibit antibacterial properties (Agrawal, 2012).
Wound healing is impaired in diabetic patients with In the study of Agrawal (2012), the extract of L. camara
infection or hyperglycemia (Sharma et al., 2013). The Linn. leaves in methanol exhibited good antibacterial and
guiding principles for wound healing include the rapid and inhibited the growth of all the isolates used. The aqueous
complete repair of the created defect and preventing the extract was effective against all the bacterial cultures
bacterial invasion during the period the natural barriers except Pseudomonas aeruginosa and Staphylococcus aureus.
are defective. Although these principles appear to be Mimosa pudica Linn.
individual goals, they are impossible to attain separately Mimosa pudica L. is a creeping annual or perennial herb
in the clinical care of wounds. In practice, the maneuvers often grown for its curiosity value, as the compound
employed to promote rapid wound healing are intimately leaves fold inward and droop when touched and
related to preventing bacterial invasion (Sabath, 2006). reopen within minutes. It belongs to the Fabaceae
Caesalpinia sappan Linn. family. Mimosa pudica is native to Brazil but is now a
Caesalpinia sappan Linn. is a small thorny tree, 6-9 m pantropical weed. The other names given to this plant
high, in the Philippines, India, and Peru. In the study of are Humble plant, Shame plant, Touch me not, Sleeping
Sathya (2010), a preliminary phytochemical analysis of grass, Prayer plant. The species epithet “pudica” is a Latin
the aqueous extract revealed the presence of flavonoids, equivalent for “Bashful” or “Shrinking” because of its
saponins, sterols, glycosides, triterpenoids, and tannins. curious nature and easy procreation. The stem is erect in
Ethanol showed a positive test for flavonoids, saponins, young plants but becomes creeping or trailing with age.
glycosides, triterpenoids, and tannins. It is being used The plant grows to a height of 1.5m (5 ft). The leaves
traditionally for many ailments and is reported to have a are bipinnately compound, with one or two pinnae pairs
wide variety of medicinal properties. Its anticonvulsant, and 10-26 leaflets per pinna. The petioles are also prickly,
anti-inflammatory, anti-proliferative, antimicrobial, and on close examination, it is seen that the floret petals
anticoagulant, antiviral, immunostimulant, and are red in their upper part, and the filaments are pink to
antioxidant activities have been reported. According to lavender. The fruit consists of clusters of 2-8 pods of
Ayurveda, the heartwood is valuable in vitiated conditions 1-2cm long each, prickly on the margins (Gandhiraja et
of Pitta, burning sensation, wounds, ulcers, leprosy, skin al., 2009).
diseases, diarrhea, dysentery, and diabetes. A decoction Moringa oleifera Lam.
of the heartwood is commonly used in Kerala, India, for Moringa oleifera Lam. is a highly valued plant distributed in
its anti-thirst, blood purifying, antidiabetic properties, and many countries of the tropics and subtropics. It has an
the plant is one of the ingredients in many traditional impressive range of medicinal uses with high nutritional
Ayurvedic formulations (Sathya, 2010). value. Different parts of this plant contain essential
Jatropha curcas Linn. minerals and are a good source of protein, vitamins, β
Jatropha curcas Linn. (Euphorbiaceae) is native to the – carotene, amino acids, and various phenolics (Farooq
American tropics, most likely Mexico and Central et al., 2007). The Moringa plant provides a rich and rare
America. It is a multipurpose tree of commercial combination of zeatin, quercetin, kaempferol, and many
significance because of its several industrial and other phytochemicals. It is essential for its medicinal value.
medicinal uses. Jatropha species are used in traditional Various parts of the plant, such as the leaves, roots, seed,
folklore medicine to cure ailments in Africa, Asia, and bark, fruit, flowers, and immature pods, act as cardiac
Latin America (Arekemase et al., 2011). They are used and circulatory stimulants, possess antitumor, antipyretic,
as antimicrobial agents, and scientists have carried out antiepileptic, anti-inflammatory, antiulcer (Farooq et
several works to find out their scientific basis (Kalimuthu al., 2007). Other critical medicinal properties of the
et al., 2010). Preparations of all parts of this plant in the plant include antispasmodic, diuretic, antihypertensive,
form of the decoction are used in traditional medicine cholesterol-lowering, antioxidant, antidiabetic,
and for veterinary purposes. The decoction of leaves is hepatoprotective, antibacterial, and antifungal activities
used against cough and as an antiseptic after birth. Latex (Nickon et al., 2003).
has antimicrobial properties against many species. The The phytochemical screening indicated the presence
oil of this plant is used traditionally for the treatment of of phenolics, flavonoids, tannins, and glycosides in the
sciatica dropsy, paralysis, rheumatism, dysentery, diarrhea, extracts. The leaves of M. oleifera contain alkaloids, which
and certain skin diseases (Kalimuthu et al., 2010). showed potential antimicrobial properties by intercalating
Lantana camara Linn. with bacterial DNA. It is believed that the higher
Lantana camara Linn. is commonly known as wild sage. reducing power of the aqueous extract could be due
Page 10

It is a flowering shrub native to tropical America and is to the better solubility of the antioxidant components
cultivated throughout the world as an ornamental plant. in water, whereas the principal antibacterial activity in

https://journals.e-palli.com/home/index.php/ajmri
 Am. J. Multidis. Res. Innov. 1(2) 8-17, 2022

organic solvent extracts as compared to aqueous extracts wide array of microorganisms (Roy et al., 2006). Alkaloids
indicates that the active components responsible for the isolated from the plant are commonly found to have
bactericidal activity are more soluble in organic solvents. antibacterial properties (Biswas et al., 2013).
These studies provide evidence to support the traditional
medicinal uses of the plant (Vinoth et al., 2012). MATERIALS AND METHODS
Psidium guajava Linn. Collection of Plant Sample
Psidium guajava Linn. is an evergreen shrub native to Healthy, intact, and young leaves of Jatropha curcas,
tropical America that has neutralized South East Asia. It Lantana camara, Mimosa pudica, Moringa oleifera, and Psidium
is an important food crop and medicinal plant available in guajava were collected in the vicinities of the researchers’
tropical and subtropical countries. It is a phytotherapeutic residences. The Caesalpinia sappan bark was purchased
plant used in folk medicine that is believed to have active in the public market of Koronadal City, Philippines. The
components that help to treat and manage various leaves were thoroughly washed with running water to
diseases. It contains essential phytoconstituents such remove dirt on the plants and dried with a clean cloth.
as tannins, triterpenes, flavonoid: quercetin, pentacyclic Ethanolic Extraction
triterpenoid: guajanoic acid, saponins, carotenoids, The leaves of the plants were cut into small pieces using
lectins, leucocyanidin, ellagic acid, amritoside, beta- scissors. The bark of C. sappan was chopped into small
sitosterol, uvaol, oleanolic acid, and ursolic acid. Many pieces. The plant parts were separately placed in a beaker
pharmacological studies have demonstrated the ability and were soaked in ninety-five percent (95%) ethanol
of this plant to exhibit antioxidant, hepatoprotective, for 24 hours. Filtration was done on each sample, and
anti-allergy, antimicrobial, antigenotoxic, antiplasmodial, the filtrates were concentrated in a rotary evaporator at
cytotoxic, antispasmodic, cardioactive, anticough, a temperature of 45oC. The concentration of the extract
antidiabetic, anti-inflammatory, and antinociceptive was computed and expressed as grams of plant parts per
activities, supporting its traditional uses. It suggests mL of extract. The concentrated ethanolic crude extracts
a wide range of clinical applications. The many parts were placed in vials with labels and were stored in the
of the plant have been used in traditional medicine to refrigerator to prevent the growth of microorganisms.
manage conditions like malaria, gastroenteritis, vomiting, Phytochemical Screening
diarrhea, dysentery, wounds, ulcers, toothache, coughs, The phytochemical screening of the plant extracts was
sore throat, inflamed gums, and several other conditions carried out based on the procedures of Guevarra (2005).
(Biswas et al., 2013). Test for Alkaloids
P. guajava contains several chemical constituents, which An equivalent of twenty (20) grams of plant material
possess antibacterial, antidiarrheal, antimycobacterial, from the stock plant extract was placed in an evaporating
antihyperglycemic, antimalarial, cytotoxic, and antioxidant dish. This was evaporated to a syrupy consistency over a
activities (Roy et al., 2006). A recent study showed that steam bath. Five (5) mL of 2M hydrochloric acid (HCl)
the P. guajava aqueous extract possessed antibacterial was added and stirred for about 5 minutes and was
activity against Salmonella typhi and Klebsiella pneumoniae. allowed to cool. One (1) mL of the filtrate was tested
However, no effect on the growth of Escherichia coli, with 2 to 3 drops of Dragendorff ’s reagent. Another one
Staphylococcus aureus, and Streptococcus fecalis organisms (1) mL of the filtrate was tested with 2 to 3 drops of
(Geidam et al., 2010). Meyer’s Reagent. The relative amount of precipitation
Phytochemicals in Antibacterial Activity was observed as follows:
The medicinal and pharmacological actions of medicinal (+) slight turbidity, (++) definite turbidity,
herbs are often dependent on the presence of bioactive (+++) heavy precipitation
compounds called secondary herbal metabolites (Kumar Confirmatory test: Three (3) mL of the crude extract was
et al., 2007). Saponin and tannins are reported to possess obtained, and 28% ammonia solution was added until
antibacterial activities. Phytochemical analysis of the the solution turned alkaline to litmus paper. The alkaline
methanol and distilled water extracts of Lantana camara solution was extracted three times with small portions
showed the presence of saponin, tannin, and flavonoid, of less than ten (10) mL of chloroform. The lower
which could be the active principle. The secondary chloroform extract was collected, and the upper aqueous
metabolites like tannin, flavonoids, and steroids showed layer was reserved for the quarternary and amine oxide
antibacterial activities (Udobre et al., 2012). test. The chloroform extract was evaporated to dryness
The mode of action of antibacterial effects of saponins over a water bath. Five (5) mL of 2M HCl was added,
seems to involve membranolytic properties rather than stirred over a water bath for about 2 minutes, and allowed
simply altering the surface tension of the extracellular to cool. The solution was then filtered, and the filtrate was
medium, thus being influenced by microbial population separated into two portions. One (1) portion was tested
density (Usman & Osuji, 2007). Flavonoids are phenolic with Mayer’s reagent and the other with Dragendorff ’s
structures containing one carbonyl group; since plants reagent. The result was recorded as (+), (++), and (+++).
synthesize them in response to microbial infection, it Positive results indicated the presence of primary,
Page 11

should not be surprising that they have been found in secondary, or tertiary alkaloids.
vitro to be effective antimicrobial substances against a Test for quarternary bases and amine oxide: The alkaline

https://journals.e-palli.com/home/index.php/ajmri
 Am. J. Multidis. Res. Innov. 1(2) 8-17, 2022

aqueous layer obtained in the confirmatory test was Test for Tannins
acidified with 2M HCl. The solution was then filtered, An equivalent of ten (10) g of plant material was taken
and the filtrate was divided into two portions. One from the stock plant extract and was evaporated to
portion was tested with Mayer’s reagent and the other incipient dryness over a steam bath. Twenty (20) mL
portion with Dragendorff ’s reagent. The results were of hot distilled water and five (5) drops of 10% sodium
observed and recorded as above. A score of (++) or chloride solution were added to salt out undesirable
(+++) in both Mayer’s and Dragendorff ’s tests was constituents. The solution was filtered, and the filtrate
taken to indicate the presence of both quarternary and was divided into three (3) test tubes. One (1) test tube
amine oxide bases. A (+) score was recorded as the served as blank, and an aqueous solution of tannic acid
result of incomplete chloroform extraction and thus was served as the reference standard.
considered negative for quarternary bases. Gelatin Test: Three (3) drops of gelatin salt reagent were
Test for Saponins (Froth Test) added to another test tube. The same was done for the
A volume of the plant extract equivalent to two (2) grams aqueous solution of tannic acid. The formation of any
was transferred into a test tube. In a separate test tube, precipitate indicated the presence of tannins.
one (1) mL of “gogo” extract was placed, and this served Ferric Chloride Test: The third test tube was treated with
as the standard. Ten (10) mL distilled water was added three (3) drops of ferric chloride solution. The same
to each test tube, stoppered, and shaken vigorously for was done to the reference standard. A blue-black color
30 seconds. The solutions in the test tubes were allowed indicated the presence of hydrolyzable tannins, while
to stand for ten (10) minutes, and the formation of a a brownish green color indicated the presence of
“honeycomb” froth was evaluated. The result for the condensed tannins.
plant extract was compared with that of the standard. Test for Anthraquinones (Borntrager’s Test)
Positive result: If the “honeycomb” froth is greater than A portion of crude extract equivalent to one (1) g of
two (2) cm in height from the surface of the liquid and plant material was evaporated to incipient dryness over
persists after ten (10) minutes, the sample was considered a water bath. The residue was taken with ten (10) mL
positive for saponins. For plant extracts with poor frothing of distilled water and filtered. The filtrate was treated
effects, a small amount of 5% sodium carbonate solution twice with five (5) mL portions of benzene. The benzene
was added to basify the extract. The formation of a stable extract was divided into two (2) portions. One (1) portion
and dense froth indicated the presence of free fatty acids. served as the control. The other portion was treated with
Test for Flavonoids five (5) mL of ammonia solution, shaken, and compared
An equivalent of ten (10) g of plant material from the with the control. A red coloration in the lower alkaline
stock plant extract was allowed to evaporate in incipient layer indicated the presence of anthraquinones.
dryness over a steam bath and allowed to cool at room In Vitro Antimicrobial Screening of the Plant
temperature. The residue was defatted with nine (9) mL Extracts on Wound Bacteria
petroleum ether. Petroleum ether was then discarded. Isolation of Wound Bacteria
The defatted aqueous layer was diluted with ten (10) mL Three days after wound induction on Mesocricetus auratus
of 80% ethyl alcohol, filtered, and the filtrate was divided (hamster), bacteria in the wound were aseptically inoculated
into three (3) test tubes. One (1) portion was taken as in the sterile nutrient broth using a cotton swab. The
control. nutrient broth was prepared by mixing 1 gram peptone,
Test for leucoanthocyanins by Bate-Smith and Metcalf method: 0.6 grams beef extract, and 200 mL distilled water. The
One (1) portion of the alcoholic filtrate was treated with culture was incubated for 24 hours at 37oC.
0.5 mL concentrated HCl (12M). A change in color was Antimicrobial Screening of the Extracts by Agar Well
observed. The solution was warmed for 15 minutes in a Diffusion Assay (Guevarra 2005)
water bath. Any further change in color observed within Muller Hinton Agar (MHA) was used for the antimicrobial
an hour was recorded and compared with the control. activity test. MHA was prepared by weighing 38 grams of
Positive result: A strong red or violet color indicates the the powdered agar into 1000 mL of distilled water in a
presence of leucoanthocyanins. clean conical flask. It was cooked in low flame, and 15 mL
Test for γ-benzopyrone nucleus: Wilstatter “cyanidin” of the medium was poured into test tubes and autoclaved
test: The other portion of the alcoholic filtrate was at 121oC, 15 psi pressure for 15 minutes. Petri dishes,
treated with 0.5 mL concentrated HCl (12M). Three (3) pipettes, culture tubes, steel cylinders (borer), and forceps
to four (4) pieces of magnesium turnings were added. were individually wrapped in paper and were sterilized
Any color change observed within ten (10) minutes was in the autoclave for 15-20 minutes at 121 oC and 15 psi
recorded and compared with the control tube. If definite together with the previously prepared culture media. 0.5
coloration was observed, an equal volume of water and mL of the incubated wound bacteria were inoculated
one (1) mL of octyl alcohol were added. The mixture was on a sterile test tube containing nutrient broth. After
shaken and was allowed to stand. The color of each layer which, the inoculum density of the test organisms was
was noted. compared with the 0.5 McFarland Nephelometer barium
Page 12

Positive result: Colors ranging from orange to red, to sulfate turbidity standard containing approximately 1.5
crimson and magenta, and occasionally to green or blue. x 108 CFU / mL of the test organism. Wound bacteria

https://journals.e-palli.com/home/index.php/ajmri
 Am. J. Multidis. Res. Innov. 1(2) 8-17, 2022

was added if the turbidity was lesser than the McFarland Erythromycin 15 mcg, and Penicillin 10 U were used as
standard. positive controls. The discs were spaced far enough to
The base plate was prepared by transferring 15 mL avoid reflection waves from the edges of the Petri dishes
of the sterile MHA on a sterilized Petri dish and was and overlapping rings of inhibition.
allowed to solidify. The seeded top agar was prepared Ethical Consideration
by transferring 1mL of the standardized test organism The researchers conducted this study in complete
to 99 mL of sterilized MHA. Only 5 mL of the seeded accordance with established research protocols. The
top agar was added to each solidified plate. There were researcher ensured that potentially hazardous biological
eight treatments in this study, namely: Treatment A agents risk assessment form and human and vertebrae
- Caesalpinia sappan extract, Treatment B - Jatropha curcas animal tissue form were approved prior to the conduct
extract, Treatment C - Lantana camara extract, Treatment of the study.
D - Mimosa pudica extract, Treatment E - Moringa oleifera Data Gathering
extract, Treatment F - Psidium guajava extract, Treatment After all the plates were incubated, they were examined
G – Betadine solution (positive control), and Treatment for the presence of zones of inhibition. The diameter
H – Sterile distilled water (negative control). of the zone of inhibition for both in the agar well and
After the top agar had solidified, four 6mm holes were in the discs was measured using a plastic ruler in mm.
aseptically bored into the seeded plate. The agar plugs The activity index of the ethanolic plant extracts was
were removed inside the cork borer by pushing them calculated as follows:
out with the help of a metal rod and were discarded Activity index (A.I.) = Mean of zone of inhibition of
aseptically. With a marking pen, the underside of the Petri the extract / Zone of inhibition obtained for standard
dishes under each well was labeled specifically with letters antibiotic drug.
to identify treatments to be delivered. Two (2) drops Used agars and microbial cultures were decontaminated
(approximately 0.1 mL) of each of the prepared plant in the autoclave prior to disposal.
extracts were delivered to the agar wells using a sterile Statistical Analysis
Beral pipette. When all the treatments were delivered on The antimicrobial activity was determined by measuring
the agar, the plates were inverted and wrapped individually the diameter of the zone of inhibition and the mean of
with clean paper. These were then placed in the incubator triplicates ± Standard Deviation (S.D.) of three replicates.
for 24 hours at 36.5oC. One-way Analysis of Variance (ANOVA) was used to
Determination of Antibiotic Activity Index of the determine if there was a significant difference among the
Plant Extracts (Disc Diffusion Method) treatment means.
The antibiotic activity index of the extracts was
determined by the Kirby-Bauer agar diffusion method RESULTS AND DISCUSSION
based on the National Committee for Clinical Laboratory Phytochemical Screening
Standards. The base plate was prepared by transferring 15 Phytochemicals are non-nutritive plant compounds that
mL of the sterile MHA on a sterilized Petri dish and was provide the plant with disease-preventative characteristics.
allowed to solidify. The seeded top agar was prepared by Qualitative analysis was performed on the phytochemical
transferring 1mL of the standardized test organism to 99 components, including alkaloids, flavonoids, saponins,
mL of sterilized MHA. Only 5 mL of the seeded top agar tannins, and anthraquinones. Table 1 summarizes
was added to each solidified plate. The sterile discs made the results of the phytochemical screening. Note. CS
from Whatman #1 filter paper (diameter 6mm) were - Ceasalpinia sappan Linn. (Sibukao); JC - Jatropha curcas
soaked in plant extracts and were placed on the MHA Linn. (Tuba-tuba); LC - Lantana camara Linn. (Kantutay);
agar surface with flamed forceps and gently pressed down MP- Mimosa pudica Linn. (Makahiya); MO - Moringa oleifera
to ensure contact with the agar surface. Amikacin 30 mcg, Lam. (Malunggay); PG - Psidium guajava Linn. (Guava);
Table 1. Results of Phytochemical Screening
Phytochemicals Tests Results
Alkaloids C.S. JC LC MP MO PG
Mayer’s Test - + + + + +
Dragendorff ’s Test - + + + + +
Confirmatory Test Mayer’s Test - ++ + - + ++
Dragendorff ’s Test - + + - + +++
Quarternary Bases and Mayer’s Test - +++ + ++ + ++
amine oxides Dragendorff ’s Test - +++ + + + ++
Saponins Froth Test - + + + + +
Flavonoids Leucoanthocyanins “Bate-Smith and Metcalf Method + + - + - +
y-benzopyrene nucleus Wilstatter “cyanidin” Test + + + - - +
Page 13

Tannins Gelatin Test - + - + - +

Ferric Chloride Test + + + + + +
Anthraquinones Borntrager’s Test - - - - - -

https://journals.e-palli.com/home/index.php/ajmri
 Am. J. Multidis. Res. Innov. 1(2) 8-17, 2022

Alkaloid Mayer’s and Dragendorff ’s Test: (+) slight turbidity; very active. Table 2 showed that Psidium guajava was very
(++) definite turbidity; (+++) heavy precipitation; active among the eight treatments, followed by Mimosa
Confirmatory Test: (+) primary alkaloid; (++) secondary pudica and betadine as active, Caesalpinia sappan,
alkaloid; (+++) tertiary alkaloid; Test for quarternary Jatropha curcas, and Lantana camara as partially active,
bases and/or amine oxide: (+) absent; (++) or (+++) Table 2. Treatment means + S.D. and their
present; Test for Saponins, Tannins, and Anthraquinones: (+) interpretations based on 6 mm test discs.
present; (-) absent. Treatments Mean + S.D. Interpretation
The phytochemical analysis of the plant extracts revealed (Guevarra, 2005)
the presence of the following secondary metabolites: Caesalpinia 12.30 + 1.530 c Partially Active
saponins, flavonoids, alkaloids, and tannins. According to sappan Bark
Biswas et al. (2013), the different secondary metabolites Extract
such as saponins, flavonoids, alkaloids, and tannins Jatropha curcas 11.30 + 0.577 c Partially Active
account for the majority of plants’ antibacterial activity. Leaf Extract
Saponins’ antibacterial effect is due to their capacity to Lantana camara 11.00 + 1.000 c Partially Active
inhibit the membranolytic characteristics of bacteria Leaf Extract
rather than just modifying the surface tension of the Mimosa pudica 14.67+ 1.150 b Active
extracellular medium (Usman & Osuji, 2007). Likewise, Leaf Extract
flavonoids are phenolic compounds with a single Moringa oleifera 9.33 + 1.15d Inactive
carbonyl group generated by plants in response to Leaf Extract
microbial infection. Hence, it is unsurprising that they Psidium guajava 23.00 + 1.000 a Very Active
are efficient antibacterial agents against a diverse array of Leaf Extract
microorganisms in vitro (Roy et al., 2006). Betadine Solution 15.000 + 0.000 b Active
Meanwhile, the antibacterial activities of alkaloids derived Distilled Water 6.00 + 0.000 e Inactive
from plants are frequently reported (Biswas et al., 2013). Values with different superscripts on the same column
Similarly, research using various plant extracts has shown have a different interpretation of zones of inhibition
that phytochemicals such as flavonoids and tannins are based on Guevara (2005) in the 6 mm test disc.
known to aid the wound healing process. This is mainly Table 3. One-way analysis of variance (ANOVA) on
due to their astringent and antibacterial qualities, which the zones of inhibition of the treatments.
contribute to wound contraction and epithelialization Source df SS MS F (Comp.) F (Tab.)
rate increase (Usman & Osuji, 2007). Treatments 7 528.70 75.52 82.39** 2.66
Antimicrobial Activity Screening (p<0.05)
Wound bacteria isolated from an incised Mesocricetus 4.03
auratus (hamster) were treated with ethanolic extracts (p<0.01)
from leaves of Jatropha curcas, Lantana camara, Mimosa Error 16 14.67 0.92
pudica, Moringa oleifera, and Psidium guajava, and the Total 23
Note. ** significant at 0.05 and 0.01 levels of
significance.
and Moringa oleifera and distilled water as inactive.
One-way analysis of variance (Table 3) revealed a highly
significant difference among treatment means at 0.05 and
0.01 levels of significance with a computed F value of
82.39.
The comparison between treatment means revealed
that P. guajava leaf extract with a mean Z.I. of 23.00
+ 1.000 was the most significantly effective treatment
against wound bacteria. On the other hand, among the
plant extracts, M. oleifera leaf extract with a mean Z.I.
of 9.33 + 1.15 was the least effective in inhibiting wound
Figure 1. Zones of inhibition of the treatments on bacteria. Distilled water did not inhibit wound bacterial
wound bacteria
growth based on the 6mm agar well.
bark of Caesalpinia sappan. Figure 1 showed the zones The broad antibacterial activity of plant extracts may
of inhibition of the treatments on wound bacteria in the be due to the presence of secondary metabolites from
different replicates. As shown in Figure 1, P. guajava exhibit the plants (alkaloids, flavonoids, tannins, and saponins).
the highest zone of inhibition among the treatments. Previous studies have demonstrated that tannins have
Furthermore, Guevarra, 2005 made an inference on the antibacterial activity because of their ability to infer
Page 14

results of the Zones of Inhibition produced on the test bacterial protein synthesis (Akiyama et al., 2001; Sanches
discs. <10 mm may be expressed as inactive, 10-13 mm et al., 2005; Min et al., 2008). Usman and Osuji (2007)
as partially active, 14-19 mm as active, and >19 mm as posited that tannins had been commonly used topically

https://journals.e-palli.com/home/index.php/ajmri
 Am. J. Multidis. Res. Innov. 1(2) 8-17, 2022

to superficial wounds. Consequently, it is possible that Table 5. Antibiotic activity index (A.I.) of the plant
the extract’ tannins and other phenolics in plants were extracts when compared with antibiotics
responsible for these broad antibacterial activities. On the Plant Extracts Antibiotics
other hand, flavonoids and saponins can inhibit bacterial
growth by forming complexes with extracellular and Amikacin Erythromycin Penicillin
soluble proteins and bacterial cell walls (Min et al., 2008). 30 mcg 15mcg 10 U
Accordingly, the antibacterial activity of P. guajava was Caesalpinia 0.45 0.47 1.46
consistent with Roy et al.’s (2006) research, which revealed sappan Bark
that P. guajava contains several chemical compounds with Extract
antibacterial, antimycobacterial, cytotoxic, antidiarrheal, Jatropha curcas 0.32 0.34 1.05
antimalarial, antihyperglycemic, and antioxidant Leaf Extract
properties. Begum et al. (2004) reported the presence Lantana camara 0.28 0.30 0.91
of over twenty chemicals in the leaves of P. guajava. Leaf Extract
Tannins are present in the aqueous leaf extract, whereas
Mimosa pudica 0.37 0.38 1.18
the ethanolic extract is rich in anthocyanins, alkaloids,
Leaf Extract
flavonoids, tannins, and steroids/terpenoids.
Antibiotic Activity Index Moringa oleifera 0.32 0.33 1.02
The antibiotic activity of the six (6) selected ethanolic Leaf Extract
plant extracts were compared with the three (3) Psidium guajava 0.76 0.79 2.43
antibiotics, namely: Amikacin 30 mcg, Erythromycin 15 Leaf Extract
mcg, and Penicillin 10 U. As shown in Table 4, the mean Note. Activity index (A.I.) = Mean of zone of
zones of inhibition for the plant extracts were greatest in inhibition of the extract / Zone of inhibition
Psidium guajava Leaf Extract with a mean of 16.32 mm obtained for standard antibiotic drug.
and least in Lantana camara Leaf Extract with a mean of
Penicillin 10 U, ranged from 2.43 to 0.91, with the highest
inhibition of 6.12 mm. The zone of inhibition for the
activity observed in Psidium guajava leaf extract and the
antibiotics was greatest in Amikacin 30 mcg with a mean
least A.I. was observed in Lantana camara leaf extract.
of 21.60 mm, followed by Erythromycin 15 mcg with a
The antibiotic activity index reading of 1.00 means that
Table 4. Zones of inhibition of the plant extracts and the plant extract has the same antibiotic activity compared
antibiotics on wound bacteria. to a specific antibiotic. The study results showed that
Treatments Mean Zone Of Inhibition when the A.I. of the plant extracts were compared with
(mm) Amikacin 30 mcg and Erythromycin 15 mcg, they had A.I.
Caesalpinia sappan Bark 9.77 ± 0.37 values lesser than 1.00, which means that they have lower
Extract A.I. than Amikacin 30 mcg and Erythromycin 15 mcg.
Jatropha curcas Leaf Extract 7.05 ± 1.41 However, the A.I. of the plant extracts, when compared
Lantana camara Leaf 6.12 ± 0.10 with Penicillin 10 U, showed remarkable results. Among
Extract the six plant extracts, only Lantana camara leaf extract had
Mimosa pudica Leaf Extract 7.90 ± 0.45 an A.I. value lower than 1. This means that the five plant
Moringa oleifera Leaf Extract 6.85 ± 0.68 extracts have higher antibiotic activity than Penicillin 10
U in inhibiting the growth of wound bacteria.
Psidium guajava Leaf Extract 16.32 ± 1.96
The antibacterial activity of Psidium guajava suggests that
Amikacin 30 mcg 21.60 ± 0.53 it has a broad variety of therapeutic uses. Numerous
Erythromycin 15 mcg 20.63 ± 2.71 components of the plant have been used in traditional
Penicillin 10 U 6.70 ± 1.13 medicine to treat various diseases, including wounds,
Note. Values are mean ± standard deviation. gastroenteritis, vomiting, dysentery, malaria, diarrhea,
coughs, sore throats (Biswas et al., 2013).
mean of 20.63 mm, and with Penicillin 10 U with a mean
of 6.70 mm. CONCLUSIONS
Meanwhile, the antibiotic activity index of the plant The phytochemical components, namely alkaloids,
extracts (Table 5), when compared with Amikacin 30 saponins, flavonoids, and tannins, were present in the
mcg, ranged from 0.76 to 0.32, with the highest activity ethanolic plant extracts. Anthraquinones were absent in
observed in Psidium guajava leaf extract, the least A.I. the ethanolic extract of all plants evaluated. More so,
was observed in Jatropha curcas leaf extract and Moringa Caesalpinia sappan Linn., Jatropha curcas Linn. , Lantana
oleifera leaf extract.The antibiotic activity index of the camara Linn., Mimosa pudica Linn., Moringa oleifera Lam.,
plant extracts, when compared with Erythromycin 15 and Psidium guajava Linn. were effective in inhibiting the
mcg, ranged from 0.79 to 0.30, with the highest activity growth of wound bacteria when compared with betadine
Page 15

observed in Psidium guajava leaf extract and the least A.I. solution. All the six plant extracts exhibited antibacterial
was observed in Lantana camara leaf extract. The antibiotic activity, and that Psidium guajava Linn. was the most
activity index of the plant extracts, when compared with effective plant extract, which is more effective than

https://journals.e-palli.com/home/index.php/ajmri
 Am. J. Multidis. Res. Innov. 1(2) 8-17, 2022

betadine in inhibiting wound bacteria. Further, all the G. (2007). Moringa oleifera: a food plant with multiple
plant extracts have a lower antibiotic activity index when medicinal uses. Phytotherapy Research, 21(1), 17 – 25.
compared with Amikacin 30mcg and Erythromycin 15 https://doi.org/10.1002/ptr.2023.
mcg. However, except for Lantana camara extract, the five Gandhiraja, N., Sriram, S., Meenaa, V., Srilakshmi, J.,
plant extracts have remarkably higher antibiotic activity Sasikumar, C., & Rajeswari, R. (2009). Phytochemical
index when compared with Penicillin 10 U. The findings Screening and Antimicrobial Activity of the Plant
of this research corroborate the traditional uses of the Extracts of Mimosa pudica L. Against Selected
plants tested and indicate that some of the plant extracts Microbes. Ethnobotanical Leaflets, 5(8). https://
contain antibacterial components that might be used as opensiuc.lib.siu.edu/ebl/vol2009/iss5/8.
antimicrobial agents. Geidam, Y. A., Ambali, A. G., & Onyeyili, P. A. (2010).
On that note, the researchers recommended to determine Preliminary phytochemical and antibacterial
the bioactive substances from the plant extracts; to carry evaluation of crude aqueous extract of Psidium
out more pharmacological evaluation on Psidium guajava guajava leaf. http://www.aseanbiodiversity.info/
to maximize its use as an antimicrobial agent; bacteria abstract/51007124.pdf
from the wound of hamster be isolated and identified; Guevarra, B.Q. (Ed). (2005). A guidebook to plant
test the selected plants on in vivo wound healing activity; screening: Phytochemical & Biological. Philippines: UST
and test other plants for their phytochemicals that can be Publishing House.
isolated and purified and employed in the formulation of Kalimuthu, K., Vihayakumar, S., & Senthilkumar, R.
novel antimicrobial agents for the treatment of wounds (2010). Antimicrobial activity of the diesel plant, J.
antibacterial activity on wound bacteria. curcas. International Journal of Pharma and Bio Sciences, 1
Acknowledgment (3). https://rb.gy/zeqenh
The researcher would like to thank and acknowledge the Kumar, B., Vinaykumar, M., Govindarajan, R., &
Notre Dame of Marbel University-IBED for allowing Pushpangadan, P. (2007). Ethanopharmacological
them to conduct this study; to their families and friends approaches to wound healing exploring medicinal
for their undying support; and above all, to the Almighty plants of India, J. Ethanopharmacol, 114(2), 103-113.
Father, for giving them the courage and strength to https://doi.org/10.1016/j.jep.2007.08.010.
complete this study. Min, B.R., Pinchak, W.E., Merkel, R., Walker, S., Tomita, G.,
& Anderson, R. C. (2008). Comparative antimicrobial
REFERENCES activity of tannin extracts from perennial plants on
Agrawal, A., Milin, K., Surendra, V., & Goyal. (2012). mastitis pathogens. Scientific Research and Essays, 3(2),
Antibacterial screening of extract of the leaves of 66-73. http://www.academicjournals.org/SRE.
Lantana camara. Indian Journal of Life Sciences, 1(2), Nayak, B., & Pinto-Pereira, L.M. (2006). Catharanthus
97+. https://rb.gy/u1pxh9 roseus flower extract has wound-healing activity in
Akiyama, H., Fujii, K., Oono, T., & Iwatsuri, K. (2001). Sprague Dawley rats. BMC Complement Altern Med
Antibacterial action of several tannins against 6(41). https://doi.org/10.1186/1472-6882-6-41.
Staphylococcus aureus. Journal of Antimicrobial Nickon, F., Saud, Z. A., Rehman, M.H., & Haque, M.
Chemotheraphy, 48(4), 487-491. https://doi. E. (2003). In vitro antimicrobial activity of
org/10.1093/jac/48.4.487.PMID:11581226. the compound isolated from chloroform extract of
Alam, G., Singh, M. P., & Singh, A. (2011). Wound healing M. oleifera Lam. Pak. J. Biol. Sci. 22, 1888– 1890.
potential of some medicinal plants. International Journal Roy, C. K., Kamath, J. V., & Asad, M. (2006).
of Pharmaceutical Sciences Review and Research, 9(5), 136- Hepatoprotective activity of Psidium guajava Linn.
145. https://rb.gy/fjy2fo leaf extract. Indian journal of experimental biology,
Arekemase, M. O., Kayode, R. M. O., & Ajiboye, A. E. 44(4), 305–311. https://pubmed.ncbi.nlm.nih.
(2011). Antimicrobial Activity and Phytochemical gov/16629373/.
Analysis of Jatropha curcas Plant against Some Sabath, H. (2006). A survey of plants used in wound
Selected Microorganisms. International Journal of healing in animals. Online Veterinary Journal. https://
Biology, 3(3), 52-59. https://doi.org/10.5539/ijb. fjps.springeropen.com/articles/10.1186/s43094-
v3n3p52. 021-00202-w.
Begum, S., Hassan, S. I., Ali, S. N., & Siddiqui, B. S. Sanchez, N.R., Cortez, D. A. G.,Schiavini, M. S.,
(2004). Chemical constituents from the leaves of Nakamura, C. V. & Filho, B. P. D. (2005). An
Psidium guajava. Natural product research, 18(2), 135–140. Evaluation of antibacterial activity of Psidium
https://doi.org/10.1080/14786410310001608019. guajava. Brazilian Archives of Biology and Technology,
Biswas, B., Rogers, K., McLaughlin, F., Daniels, D., & Yaday, 48(3), 429-436. https://doi.org/10.1590/S1516-
A. (2013). Antimicrobial activities of leaf extracts of 89132005000300014.
guava (Psidium guajava L.) on two gram-negative Sharma, Y., Jeyabalan, G., Singh, R., & Semwal, A. (2013).
and gram-Positive Bacteria. International Journal of Current Aspects of Wound Healing Agents From
Page 16

Microbiology. https://doi.org/10.1155/2013/746165. Medicinal Plants: A Review. Journal of Medicinal Plants

Farooq, A., Sajid, L., Muhammad, A., & Anwarul Hassan, Studies, 1(3). https://rb.gy/pfnli1

https://journals.e-palli.com/home/index.php/ajmri
 Am. J. Multidis. Res. Innov. 1(2) 8-17, 2022

Srilakshmi, V. S., Vijayan, P., Raj, P. V., Dhanaraj, S. A., 136-139. https://rb.gy/54yjp1
& Chandrashekhar, H. R. (2010). Hepatoprotective Usman, H., & Osuji, J. C. (2007). Phytochemical and in vitro
properties of Caesalpinia sappan Linn. heartwood on antimicrobial assay of the leaf extract of Newbouldia
carbon tetrachloride induced toxicity. Indian journal of laevis. African journal of traditional, complementary, and
experimental biology, 48(9), 905–910. https://pubmed. alternative medicines : AJTCAM, 4(4), 476–480. https://
ncbi.nlm.nih.gov/21506498/. pubmed.ncbi.nlm.nih.gov/20161915/.
Udobre, A. S., Usifoh, C. O., Eseyin, A. O., Udoh, A. E., Vinoth, B., Manivasagaperumal, R, & Balamurugan, S.
Awofisayo, A. O., Akpan, A. E. (2012). The wound (2012). Phytochemical analysis and antibacterial activity of
healing activity of methanol extract of the stem bark Moringa oleifera. http://www.urpjournals.com.
of Nauclea latifolia. Int J Pharm Biomed Sci. 3(3),

Page 17

https://journals.e-palli.com/home/index.php/ajmri