1

Antimicrobial activity of ​Vitex negundo ​leaf extracts as an

Antimicrobial Cream against ​Escherichia coli

Barbac, Zephanaiah Jynne C.

Dante, Ianne Lorraine L.

Montañez, Kyle Gabriel C.

Ochea, Queenie Ann G.

Paculba, Eilaine May Marie

Pintor, Jasper Kim

 2
RATIONALE

Topical skin infections are one of the infections commonly acquired by

humans. These infections are caused by either a fungi, bacteria, parasite or

virus. However the most common skin infections of hospitalized patients are

bacterial skin infections (Stulberg, Penrod & Blatny,2002). Some of the most

common bacteria associated with skin infections are ​Staphylococcus aureus,​

Streptococcus pyogenes​ and ​Coryneform ​bacteria (Maibach,1996).

Synthetic antimicrobial creams are the agents usually used to

treat these skin infections. Two studies (Lewis, 2019; Loftsson, 2014)

have concluded that the main advantage of having topical antibiotics like

cream is its low risk of systemic adverse events and drug interactions and

its polar properties as it contains a lipophilic and a hydrophilic phase, which

makes it water-washable.

At present, one of the problems we are encountering is

Bacterial Resistance. As the threat of Bacterial Resistance rapidly

increases, the demand for new drugs also increases. A lot of studies have

been conducted to find new possible drugs, and most of these studies utilize

plants or plant extracts. According to the World Health Organization (WHO),

about 80% of the world’s population depend mainly on traditional

 3
medicine and the traditional treatment involving mainly the use of plants

(Renuka, Kokilavani & Gnana, 2008).

According to Republic Act No. 8423 ​Traditional and Alternative

Medicine Act (TAMA) of 1997 Article I Section 3 ​of the Philippines, scientific

research and development of traditional and alternative health care systems

is encouraged to have a direct impact on public health care. ​Vitex negundo

have also been recognized as one of the 10 medicinal plants of the

Philippines due to the natural products it contains that can be used

as a potential drug. ​Vitex negundo Linn i​ s most commonly distributed

on roadsides and the banks of streams in the Philippines and in Asia, which

makes it more accessible and convenient to collect (Boy et. al., 2018).

In recent years, there have been studies conducted to see the

potential of Lagundi or ​Vitex negundo ​leaves extract as an

antimicrobial agent. ​Vitex negundo ​possesses many therapeutic actions

including an antimicrobial and antibacterial activity (Deogade et.

al.,2016). The leaves are useful in catarrhal fever, orchitis, syphilis,

inflammation and ulcers (Deogade et. al., 2016).

 4
To address this problem, this research will utilize V
​ itex negundo ​as an

antimicrobial cream. The researchers would like to mainly focus on

its Antimicrobial activity, although ​Vitex negundo ​has a lot of

therapeutic properties.

The primary objective of this study is to test the Antimicrobial activity

of ​Vitex negundo ​to ​Escherichia coli. ​to produce an Antimicrobial cream out

from it. Furthermore, this study would contribute a solution to the current

problem regarding Bacterial resistance and could provide a different

preference for antimicrobial products.

 5
REVIEW OF RELATED LITERATURE AND STUDIES

Related Literature

The skin is the body's outer covering, which protects against heat and

light, injury, and infection. Skin regulates body temperature and

stores water, fat, and vitamin D. The skin, which weighs about 6

pounds, is the body's largest organ. It is made up of two main layers: the

epidermis and the dermis. Intact skin serves as a wall-like barrier to

separate and protect the inside of our body from the microbial enemies of

the environment and provide a primary defense against infection. The layers

of the skin, like the outer wall and secondary inner walls surrounding a

medieval city, not only provide protection from external enemies, but

also provide niches where normal flora bacteria and fungi can live and

conduct business. When portions of the skin are damaged, broken or

disrupted microbes can have access to the “inner sanctum” and can cause

damage (Brodell & Rosenthal, 2008).

The normal flora of human skin consists of more than 200

bacterial species and a few species of eukaryotic fungi. Common normal

flora of the skin in dry areas include ​Staphylococcus epidermidis​,

Micrococcus organisms, propionibacteria, hair follicle mites, and

 6
Pityrosporon yeast. Moist areas, such as the toe webs and axilla,

harbor a greater diversity of skin flora including corynebacteria,

mycobacteria, ​Staphylococcus aureus​, and gram-negative bacteria.

These normal flora suppresses the growth of pathogenic bacteria in

several ways. First, niche occupancy and competition for nutrients limit

the growth of bacteria other than the normal flora. Second, secretion

of inhibitory metabolic products, including acetic and propionic acids,

potentiates the low pH favored by the normal flora but inhibit many

pathogenic bacteria. (Brodell & Rosenthal, 2008).

Skin and soft tissue infections (SSTI), also referred to as ​skin and skin

structure infections,​ represent a group of infections that are diverse in their

clinical presentations and degree of severity (Chahine & Sucher,

2015). SSTIs are classified as simple (uncomplicated) or complicated

(necrotizing or non-necrotizing) and can involve the skin, subcutaneous fat,

fascial layers, and musculotendinous structures, it can also be purulent and

nonpurulent. SSTIs can be classified based on their severity, presence of

comorbidities, and need for and nature of therapeutic intervention.

Simple infections confined to the skin and underlying superficial soft tissues

generally respond well to outpatient management. Common simple

SSTIs include cellulitis, erysipelas, impetigo, ecthyma, folliculitis,

furuncles, carbuncles, abscesses, and trauma-related infections.

 7
Complicated infections extending into and involving the underlying

deep tissues include deep abscesses, decubitus ulcers, necrotizing

fasciitis, Fournier gangrene, and infections from human or animal bites.

Complicated infections may present with features of systemic inflammatory

response syndrome or sepsis, and, occasionally, ischemic necrosis.

Perianal infections, diabetic foot infections, infections in patients with

significant comorbidities, and infections from resistant pathogens. Most

SSTIs occur following a breach in the protective skin barrier from trauma,

surgery, or increased tissue tension secondary to fluid stasis. The infection

may also originate from an adjacent site or from embolic spread

from a distant site. ​S. aureus ​and ​streptococci a

​ re responsible for

most simple community-acquired SSTIs. In one prospective study,

beta-hemolytic streptococcus w
​ as found to cause nearly three-fourths

of cases of diffuse cellulitis. ​S. aureus, P. aeruginosa, enterococcus, a

​ nd

Escherichia coli ​are the predominant organisms isolated from hospitalized

patients with SSTIs (Kalyanakrishnan, Salinas & Higuita, 2015).

The management of SSTIs is determined primarily by their

severity and location, and by the patient’s comorbidities. Initial

management is determined by the presence or absence of purulence,

acuity, and type of infection. Topical antibiotics (e.g., mupirocin

[Bactroban], retapamulin [Altabax]) are options in patients with impetigo

 8
and folliculitis. Beta-lactams are effective in children with nonpurulent

SSTIs, such as uncomplicated cellulitis or impetigo. In adults, mild to

moderate SSTIs respond well to beta- lactams in the absence of

suppuration. Patients who do not improve or who worsen after 48 hours

of treatment should receive antibiotics to cover possible MRSA

(Methicillin-resistant ​Staphylococcus aureus​) infections and imaging to

detect purulence. Mildly purulent SSTIs in easily accessible areas without

significant overlying cellulitis can be treated with incision and drainage

alone.

Antibiotic therapy is required for abscesses that are associated with

extensive cellulitis, rapid progression, or poor response to initial

drainage; that involve specific sites (e.g., face, hands, genitalia); and

that occur in children and older adults or in those who have significant

comorbid illness or immunosuppression. Inpatient treatment is necessary

for patients who have uncontrolled infection despite adequate outpatient

antimicrobial therapy or who cannot tolerate oral antibiotics.

Broad-spectrum antibiotics with proven effectiveness against gram-positive

and gram-negative organisms and anaerobes should be used until pathogen

specific sensitivities are available; coverage can then be narrowed.

Intravenous antibiotics should be continued until the clinical picture

 9
improves, the patient can tolerate oral intake, and drainage or debridement

is completed (Kalyanakrishnan, Salinas & Higuita, 2015).

Escherichia coli (E.coli) ​are gram negative, facultative, anaerobic, rod-

shaped organism, 2-6 micrometer long and 1.1 to 1.5 micrometer

wide. ​Escherichia coli ​are usually motile by peritrichous flagella and produce

gas from fermentable carbohydrates (Leung & Gallant, 2014). ​E.coli

is a bacteria part of the normal intestinal flora. However, some strains of

E.coli ​are pathogenic and can cause gastroenteritis, urinary tract infection

(UTI), meningitis, and wound infections (Buckle, 2015). ​E.coli st​ rains

are frequently isolated from skin and soft tissue infections (SSTI), these

strains were isolated mostly from surgical and traumatic wounds, foot

ulcers and decubitus. Results have shown that the majority of the studied

strains (65%) belonged to the B2 phylogenetic group. The most

prevalent VF was ​ompT (​ 80%), while toxin genes ​cnf1 a

​ nd ​hlyA ​were

found with prevalent of 32 and 30%, respectively. ​Escherchia coli ​was

found to be the causative agent of neonatal omphalitis, cellulitis

localized to lower or upper limbs, necrotizing fasciitis, surgical site

infections, infections after brain injuries and others. ​Echerichia coli h

​ as been

shown to be an important causative agent since it was the third most

prevalent isolated species preceded solely by ​S. aureus and ​P.

aeruginosa​ (Petkovsek, et.al., 2009).

 10
Antimicrobials are one of the main treatments for skin infections but

in an article by the Center for Disease Control and Prevention (CDC),

Antimicrobial resistance has been found in all regions of the world. Center

for Disease Control and Prevention (CDC) stated that antibiotic

resistance is responsible for around 2 million infections, more than twenty

thousand deaths and costs $55 billion each year in the United States. The

national pharmaceutical sales data on global antibiotic consumption

(2000-2010) reveals that total antibiotic consumption grew by more

than 30%. The greatest increase in antibiotics use was recorded in

Low and Middle Income Countries (LMIC). According to the Council of

Canadian Academies (2019)​, 26% of all infections are resistant to

antimicrobial treatment. By 2050, the report projects that 40% will be

resistant, directly causing 13, 700 previously preventable deaths

(Wright, 2019). ​As the increase in Antimicrobial resistance threaten

humanity, the demand for discovery of new drugs also increases

despite the numerous existing antimicrobial agents available today (Chen,

Alexander & Baki, 2016).

 11

Fig. 1. ​Vitex negundo Linn.

Lagundi (​Vitex negundo) ​is a large shrub or small tree up to

2-5 meters high, belonging to the Verbenaceae family, and is a woody,

aromatic and medicinal herb (Davi, Kokilavani & Gnana, 2008). It is

indigenous to China, India and Malay Peninsula. However, it is known

to be widely distributed in the Philippines, growing in low to medium

altitudes and even in thickets and waste places (Bautista, Cubos & Lim,

2015). It is one of the ten medicinal plants being promoted by the

Department of Health due to its medicinal properties. Lagundi ​(Vitex

negundo) h
​ as long been established for its pharmacological actions

including an anti-inflammatory action and an anti-nociceptive activity.

Among its medicinal properties are its analgesic, anthelmintic and

antiparasitic, antiandrogenic, anti-asthmatic, anti-catarrhal, antimicrobial,

appetizer, discutient, emmenagogue, hepatoprotective, larvicidal and

muscle relaxant. The leaves of the plant are generally used for fomentation
 12
of sprains, rheumatism, swollen testicles, and contusion (Jajra, et. al,

2019). As an ethnobotanical important plant, it has several bioactive

compounds extracted from leaves, seeds, roots in the form of volatile oils,

flavonoids, lignans, iridoids, terpenes, and steroids. These bioactive

compounds exhibit anti-inflammatory, antioxidant, antidiabetic,

anticarcinogenic properties (Gill, et. al, 2018). Recent studies have proven

its antiseptic, antitussive, as well as its anti-inflammatory properties. It has

been found to have antibacterial effects against ​Bacillus subtilis,​ ​Escherichia

coli​, and ​Staphylococcus aureus​ (Bautista, Cubos & Lim, 2015).

Vitex negundo ​possess many therapeutic actions some of which

are anti-inflammatory, antibacterial, moderate CNS depressant,

antifertility, antispasmodic, analgesic, hepato-protective, estrogenic,

anticonvulsant, antiarthritic, diuretic, antimicrobial, anti-parkinsonian,

antipsychotic, antidepressant, antihistamine releasing activity, mosquito

repellent activity, antifeedant, anti-malarial, juvenomimetic,

anti-androgenic. The leaves are aromatic, tonic, vermifuge and are useful

in rheumatism, arthritis, catarrhal fever, cephalalgia, sprains, orchitis,

syphilis, inflammations and ulcers (Deogade, 2016).

 13
According to the study, “​Antimicrobial Activity of Vitex Negundo Linn.

(Nirgundi) Leaves Extract,” the results of the present study of ​Vitex

negundo s
​ howed the presence of a wide spectrum of antibacterial activities

against some bacterial pathogens (Deogade, 2016). Therefore ​Vitex

negundo c
​ an be used as an antibacterial supplement and for the

development of new therapeutic agents. In an excerpt taken from the

International Journal of Recent Scientific Research, a phytochemical

analysis was conducted to ​Vitex negundo ​and ​Azadirachta indica ​to assess

the medicinal value of both plants.

Upon assay, it was seen that medically important compounds found in

both plants were alkaloids, carbohydrates, glycosides (anthraquinone

and cardiac), phenolic and tannin compounds, flavonoids, saponins and

lipids. However, ​Vitex negundo c

​ ontains terpenoids which was absent

in ​Azadirachta indica. ​This concludes that the bioactive compound makes

Vitex negundo ​have greater antimicrobial activity compared to ​Azadirachta

indica ​(Lobo, Pimpliskar, and Jadhav, 2018). Another journal article

taken from International Journal of Antimicrobial Agents stated that plant

nutraceuticals such as terpenoids, polyphenols, and thiols can act as

antimicrobial agents in food preservatives, the review summarizes the

antimicrobial activities and mechanisms of actions for three main types of

plant nutraceuticals, namely terpenoids (e.g. carnosic acid), polyphenols

(e.g. quercetin) and thiols (e.g. allicin), which are important constituents of
 14
plant essential oils with a broad range of antimicrobial effects. It is

highlighted in the article that terpenoids have interesting antibacterial

properties (del Rio, Fernandez, Lombo, 2018).

The principal constituents of ​Vitex negundo l​ eaf extract are

casticin, isoorientin, chrysophanol D, luteolin, p-hydroxy benzoic acid and

D-fructose. The phytochemical screening of ​V. negundo r​ evealed the

presence of alkaloids, tannins, hydrolysable tannins, flavonoids,

saponins, terpenoids, glycosides, and cardiac glycosides in methanol

and alkaloids, tannins, hydrolysable tannins, flavonoids, and cardiac

glycosides in aqueous leaf extract. The saponins, glycosides and

steroids are absent in aqueous leaf extract. The plant has

anti-inflammatory, antibacterial, antifungal and analgesic activity.

Today, natural products derived from ​Vitex negundo ​are being tested

for presence of new drugs with new modes of pharmacological action. A

special feature of higher plants is their capacity to produce a large number

of secondary metabolites. Plants have been major source of medicine

and the presence of plant secondary metabolites has been implicated

for most plants therapeutic activities. Phytomedicines derived from

plants have shown great promise in the treatment of intractable

infectious diseases. Also, it has been suggested that aqueous and

methanolic extracts from plants used in allopathic medicine are

 15
potential sources of antiviral, anti-tumoral and antimicrobial agents.

The higher plants as a source of medicinal compounds had continued to

play a dominant role in the maintenance of human health since ancient

times. (Kumar, 2013).

Terpenoids represent a large group of phytochemicals with promising

antimicrobial activity (Barbieri et. al., 2017). The chemical diversity of

terpenoids have led to discovery of over 40,000 structural varieties, with a

few classes serving as pharmaceutical agents, some of which include

terpenoid derived indole alkaloids (Chen et. al. 2016). There are a total of

eight different classes of terpenoids (hemiterpenoids, monoterpenoids,

sesquiterpenoids, diterpenoids, sesterpenoids, triterpenoids,

tetrapenoids, and polyterpenoids) which differ in the number of

isoprene (C5H8) units. Recently in 2017, it was reported that 67% of

potentiators belong to monoterpenes and sesquiterpenes (Zacchino et.

al., 2017). Meanwhile, specifically among the discovered potentiators

of antibacterial drugs, 75% were terpenes; these include classes of

mono-, di-, and tri-terpenes (Zacchino et. al., 2017).

Griffin et al. stated in his study that most terpenoids are able to

inhibit two crucial processes which are essential to microbial survival, this

includes oxygen uptake and oxidative phosphorylation. Aerobic

 16
microbes require oxygen in order to yield energy for their growth.

Previously, it was proven that low oxygen concentrations caused

limitation in bacterial respiration rates. Meanwhile, oxidative

phosphorylation is a crucial biochemical process which is responsible for

cellular respiration that takes place in the cytoplasmic membrane.

Thus, terpene interaction leads to alteration in cellular respiration

which later causes uncoupling of oxidative phosphorylation in the

microbe (Zengin et. al., 2014).

Additionally, carbonylation of terpenoids was believed to increase

bacteriostatic activity but not necessarily the bactericidal activity. A

bacteriostatic agent is an agent that stops or inhibits microbial

growth, while a bactericidal is responsible for killing the microbe.

Terpenoids have also been found to exhibit antiseptic potential according

to their solubility in water. Lipophilicity and/or hydrophobicity and presence

of hydroxyl groups in the terpenes are amongst the determining elements

of their antibacterial action (Zengin et. al., 2014). In skin

barrier-associated treatment, terpenes have also been reported to affect

the lipid membrane activity by interacting with lipophilic tails of

intermembrane lipid and polar head groups which, at the end, affects the

lipodial intermembrane and polar transmembrane pathways (Chen et. al.,

2016).
 17
Flavonoids are a class of compounds presented broadly in

nature. Concerns about their extensive profitable bioactive benefits,

including anti- viral/bacterial, anti-inflammatory, cardioprotective,

anti-diabetic, anti-cancer, anti-aging, have long been received great

attention and well supported by numerous studies (Wang et. al., 2018).

Flavonoids are well known as antibacterial agents against a wide

range of pathogenic microorganisms. With increasing prevalence of

untreatable infections induced by antibiotic resistance bacteria,

flavonoids have attracted much interest because of the potential to be

substitutes for antibiotics. Hydroxyls at special sites on the aromatic rings

of flavonoids improve the antibacterial activity (Yang, 2015).

Topical drug delivery system is defined as pharmaceutical

dosage forms having its application on the skin for direct treatment of the

localized condition, with the intent of confining the pharmacological or other

effect of the drug to the surface of the skin. It includes gels, creams,

ointments and pastes. Among advantages of TDDS are avoiding the

first-pass effect, possibly avoiding the deactivation by digestive and liver

enzymes and as well as reduction of doses as compared to oral dosage

forms (Verma, et. al, 2013).

 18
Creams are semisolid dosage form containing one or more drug

substances dissolved or dispersed in a suitable base and are generally

intended for external application to the skin or mucous membranes (Allen,

Popovich ​& A
​ nsel, 2014). In rheological studies conducted, it is showed that

solid and semisolid dosage forms such as creams are more stable compared

to those in liquid solutions which are easily degradable because of its

viscosity and elasticity behaviors (Kulkharni, 2016). Topical applications like

cream have its advantage on delivering drug on the surface of the

skin lowering the risk of systemic adverse events (Lewis, 2019).

Another advantage is its polar properties as it contains a hydrophilic and a

lipophilic phase (Loftsson, 2014).

Another book review stated, that topical applications, as defined

are indicated for cutaneous (dermatological) use, only have its effect is

usually confined to the surface of the skin or within the skin for a local

effect and does not require percutaneous deposition and penetration

(Shah, et. al., 2014). Pharmaceutical creams have a variety of applications

ranging from cosmetic purposes such as cleansing, beautifying, altering

appearance, moisturizing etc. to skin protection against bacterial,

fungal infections as well as healing cuts, burns, wounds on the skin. These

topical formulations are used for the localized effect for the delivery of the

drug into the underlying layer of the skin or mucous membrane.

 19
These products are designed to be used topically for the better site specific

delivery of the drug into the skin specifically to weeping or oozing surface of

skin for skin disorders. Creams are considered as a pharmaceutical product

as they are prepared based on techniques developed in the pharmaceutical

industry; unmedicated and medicated creams are highly used for the

treatment of various skin conditions or dermatoses (Rai, Poudyl & Das,

2019).

Different methods can be conducted for the extraction of ​Vitex

negundo c
​ rude drug and cream formulation, the researchers would

like to follow the method conducted by the Journal of Pharmaceutical

Sciences and Research. For the collection of plant material, a sample

of ​Vitex negundo l​ eaves were collected, subjected to washing under

tap water to remove adherent soil, dirt etc. for 2-3 times and finally

followed by ethanol wash and then allowed to shade dry at room

temperature for seven days. The leaves are then powdered to make a

coarse powder with mixer and grinder. The powder was then packed in

locked polyethylene bags, labelled and stored in the airtight container.

For the preparation of extracts, 250 g powdered leaf samples was

soaked with petroleum ether for 3 days with occasional shaking and filtered

to remove the fat contents. After filtration, the air dried marc was further
 20
soaked in 650 ml of ethanol for three days at room temperature. The

ethanol extract was then filtered and concentrated using a Rotary

Evaporator at 40°C, air dried and weighed to yield 36.6 g (15.3%) dark

green crude extract. The ethanol extract was suspended in water and

partitioned with diethyl ether and ethyl acetate successively and combined

to give a total yield of 4.7 g dark green solid (Abebayehu et al.,2016). In a

review from the ​International Journal of Advances in Pharmacy, Biology,

and Chemistry s
​ tated that essential oils and successive ethanol and ethyl

acetate extracts of ​Vitex negundo L

​ inn. showed antibacterial activity

against ​Staphylococcus aureus, Bacillus subtilis, Escherichia coli, a

​ nd

Pseudomonas aeruginosa ​bacterial strains. (Ladda and Magdum,

2012).

For the preparation of the dosage formulation, first, the aqueous

phase which consists of the water soluble components, including 9 mL

of polyethylene glycol, 27.75 mL of water, and 0.75 g of sodium lauryl

sulfate with the 34.75 ml of crude drug will be mixed in a separate container

and heat up to 80 C. In another separate container, the lipid phase

which consists of lipid soluble components including 31.25 g of beeswax,

18.75 mL of liquid paraffin, and 18.75 g of stearyl alcohol will be mixed and

heated up to 80 c. Afterwards, the lipid soluble mixture will be incorporated

with the water soluble mixture with constant stirring until cream is formed.
 21
Lastly, the mixture will then be allowed to cool in the container (Allen ​&

Ansel, 2014).

In the preparation of the subculture media for Inoculum, a loopful of

Escherichia coli was transferred from laboratory maintained culture

(agar slant) into the test tubes containing sterilized nutrient broth

medium. The tubes were incubated for 18-24 hours at 37 degrees

Celsius (Deogade, 2016).

Lastly, for the Preparation of Assay Medium and Pour plates, the sterile

Mueller Hinton Agar (MHA) was dispensed to previously sterilized Petri

dishes and allowed to cool at 40 degrees Celsius, until such duration the

medium solidifies. The Petri dish will serve as its area for growth and

cultivation. The mixture of microorganisms was lawned over the surface

solid MHA medium with sterile swak sticks. The Antimicrobial Cream

made out of the leaf extracts or the trial drug were poured onto the discs

with sterile pipette or stirring rods. The Petri dishes were kept in the

incubator for 18-24 hours at37 degrees Celsius. After 18-24 hrs the zone of

inhibition are then measured by a caliper. All the procedures were

carried out in an aseptic area (Deogade, 2016).

 22
Related Studies

Determination of primary and secondary metabolites in ​Vitex negundo

leaves for various extracts by qualitative methods. Elemental analysis was

carried out by using inductively coupled plasma mass spectrometry

(ICP-MS) technique and the functional groups have been determined by

using Fourier Transform Infrared Spectroscopy (FTIR) technique. Soluble

extractive percentage of material has been found the maximum in the

aqueous extract (6.75%) followed by methanolic extract (4.35%) and

acetone extract (1.8%). Phytochemical screening of material revealed

the presence of carbohydrates, proteins, amino acids, steroids, cardiac

and anthraquinone glycosides, saponins, flavonoids, tannins and

phenolic compounds. The elemental analysis revealed Na, Mg, K, Ca, Cr,

Mn, Fe, Co, Cu, Zn, Se, Mo, Li, B, Al, P, Cd, As, Ba, and Hg. FTIR technique

was used to identify various functional groups present in three different

extracts of the material. The FTIR study revealed the presence of

essential functional groups in three different extracts of the material. The

present investigation is most essential to discover innovative, dynamic

and novel drugs for curing various newly emerged dangerous health

problems (Kamble, Pawar, 2017).

 23
In the study, “Phytochemical Screening and Antioxidant Potency

of ​Adhatoda vasica a
​ nd ​Vitex negundo”,​ the leaf samples were

subjected to phytochemical analysis. Both the plant contained antioxidant

phytochemicals such as alkaloids, tannin, saponins, phenolics and

flavonoids; which were present in comparatively higher amount in

Vitex negundo. Tannins were recorded highest among all the

phytochemicals (93.9 ± 0.8 in Vitex negundo). Phenolics were recorded

lowest (8.1 ± 0.5 mg/g in Vitex negundo). The methanolic extracts of the

plants were also analyzed for antioxidant and reducing power

potentiality. Both plants showed strong antioxidant and reducing power

ability. The strong antioxidant and reducing power ability of the plant

underlines their use as an antioxidant supplement against diseases such

as typhoid during which antioxidant system fails; cardiovascular

diseases which are caused due to accumulation of Reactive oxygen

species; ageing related diseases, Alzheimer’s, Parkinson’s disease,

Amyotrophic lateral sclerosis, cataractogenesis and other diseases. (Kumar,

2013) Tannins, alkaloids, saponins, flavonoids and sterols have been found

to be active against pathogenic bacteria (Kennedy and Wightman,

2011). Thus the leaf of both plants can be used as effective

medicines owing to their phytochemical constituents. (Kumar, 2013).

 24
The therapeutic effects of lagundi can be attributed to the

phytochemicals produced by the plants. Secondary metabolites like

nishindaside (terpenoids), mussaenosidic acids (terpenoids), vitedoin

(polyphenols), negundin (polyphenols), and vitexin (flavonoids) are

some important bioactive agents which impart a variety of medicinal uses to

the plant (Basri et al. 2014). The antimicrobial activity of the plant

extract is strengthened in the presence of antioxidant compounds

(Ricardo et. al.,2011). It has been suggested that the antimicrobial activity

of the plant is mainly due to the presence of essential oils, flavonoids,

terpenoids, alkaloids, tannins, saponins and other natural polyphenolic

compounds or free hydroxyl groups in plant extracts (Ramkumar et.

al. 2004; Soetan,2006). Presence of flavonoids, terpenoids and tannins in

Vitex negundo have been detected in various studies (Panda et. al., 2009).

An antimicrobial susceptibility study employing a disk diffusion

method was conducted using Lagundi (​Vitex negundo)​ to ​Bacillus

cereus ​group, a multi-drug resistant bacterium employed in the study.

Results reveal that all extracts of leaves (chloroform, hexane, ethanol,

and ethyl acetate) and essential oils were responsible for antibacterial

susceptibility with inhibition zone of 29±0.7, 9±0.5, 8±0.3, 12±0.7, and

25±0.2 mm respectively. This study signifies ethanolic extract of ​Vitex

negundo L
​ . leaves as moderate antimicrobial and can be used for
 25
pharmaceutical and medicinal purposes. Therefore, isolation and

identification of bioactive compounds from this plant will be an interest for

human beings (Mahfuz-Al-Mamun, et. al., 2015).

In an experiment conducted wherein the ​Vitex negundo e

​ xtracts

were tested for in vitro antibacterial activity, the bacteria used for the

antibacterial tests were Gram (+) Staphylococcus aureus (MTCC 3160),

Bacillus subtilis (MTCC 0121) and Gram (−) Escherichia coli (MTCC 0051),

Pseudomonas aeruginosa (MTCC 0741). All the strains used for these

studies were procured from MTCC, IMTECH, Chandigarh, India. Antibacterial

potential of all three samples of essential oils and successive extracts was

evaluated by agar well diffusion method. Nutrient agar plates were swabbed

with the broth culture of the respective microorganisms (diluted to 0.5

McFarland Standard) and were kept at room temperature for 15 min for

absorption to take place. Wells of 8 mm diameter were punched into the

agar medium and filled with 100 μl each of the essential oils and extracts.

DMSO, DMF and hexane were taken as solvent blank and Ciprofloxacin was

used as positive control. The inoculated agar plates were incubated for 24 h

at 37°. All the tests were made in triplicate and diameter of the inhibition

zones was calculated in mm. The average of diameter of the inhibition

zones of each sample was taken called clearing zone (CZ) and the

antimicrobial index (AI) was computed as the clearing zone (CZ) minus the
 26
diameter of the hole divided by the diameter of the hole. All the extracts

and essential oils were found to be highly effective in inhibiting the growth

of bacteria at a minimum concentration of 30 and 60 μg/100 μl,

respectively. Each of the essential oil and extracts were found to be active

against ​B. subtilis and ​E. coli with antimicrobial index (AI) ranging from 0.3

to 1.8. Leaf essential oil inhibited ​S. aureus with maximum AI of 1.5 while

fruit essential oil showed its inhibition against ​E. coli ​and ​B. subtilis with AI

of 1.3 and 1.0, respectively. Flower oil did not show any activity against S.

aureus while leaf and fruit oils were ineffective against ​P. aeruginosa.​ Ethyl

acetate extract was found to be most potent among all the extracts tested.

Petroleum ether and aqueous extracts did not show any activity against

P.aeruginosa while all the extracts were found to be potent against ​S.

aureus.​ Ciprofloxacin was used as positive standard control and the results

of tested samples were very promising in comparison to standard drug

ciprofloxacin (Khokra, 2008).

In the research study “Antimicrobial Activity of Vitex Negundo

Linn. (Nirgundi) Leaves Extract”, the antimicrobial activity and

phytochemicals of the leaves and bark of Vitex negundo L. was evaluated

against three gram- positive bacteria viz. ​Staphylococcus epidermidis,​

Bacillus subtilis, S. aureus and three gram-negative bacteria viz. E. coli,​

Salmonella typhimurium,​ ​Pseudomonas aeruginosa​, ​Vibrio cholerae and

 27
Vibrio alginolyteus​. Both polar and non-polar extracts such as petroleum

ether, chloroform, ethanol, methanol and aqueous extracts were prepared

and studied for antibacterial activity using disc diffusion, agar cup and

broth dilution methods. Results showed promising antibacterial activity of

all the extracts of both leaf and bark against ​E. coli,​ followed by ​S. aureus.​

Ethanol and methanol extracts of the leaf showed inhibition activity

against both gram-positive and gram- negative bacteria whereas

petroleum ether and chloroform extracts of bark had better antibacterial

activity against gram-positive bacteria. Findings of the previous works

implicate the indication of the trial drug as a potent therapeutic agent

for antibacterial property (Deogade, 2016).

In a research study by Balasubramani and his colleagues, a

nanoemulsion was developed out of ​Vitex negundo L

​ . essential oil to test

its efficacy as antimicrobial, antioxidant, and larvicidal agent.

Determination of Minimum Inhibitory Concentration (MIC) of the pure

essential oil and emulsified essential oils was investigated using the

broth microdilution method using 96-well microtiter plates. For the

antimicrobial activity, bacterial pathogens ​Escherichia coli ​(MTCC-724),

Enterobacter aerogene​s (MTCC-39) and ​Enterococcus faecalis

(MTCC-2729) were the reference strains for minimum inhibitory

concentration (MIC). The results of the study shows that the Minimum
 28
Inhibitory Concentration (MIC) of the nanoemulsion has a minimum

value of 0.68 ± 0.25, obtained in ​Escherichia coli ​(MTCC-724) and a

maximum value of 19.29 ± 2.0 µg/mL obtained in ​Enterobacter

aerogenes ​(MTCC-39). In pure essential oils, the minimum MIC value is

3.28 ± 1.24 µg/mL and was obtained in Escherichia coli (MTCC724) and

the maximum MIC value is 21.26 ± 1.04 µg/mL was obtained in

Enterobacter aerogenes (​ MTCC-39). Among the test samples,

nanoemulsion showed better MIC value than the essential oil for all the

tested pathogens (Balasubramani, et. al., 2017). A study taken from the

journal Annals of Plant Sciences assayed 16 medicinal plants with methanol

to determine its constituents and as well as determine its antimicrobial

activity. Upon phytochemical analysis, it was revealed that ​Vitex

negundo i​ s positive of bioactive constituents terpenoid, alkaloid, glycoside,

cardiac glycoside, quinone, and saponins. In the determination of

antimicrobial activity, it was shown that ​Vitex negundo ​had a zone of

inhibition of 12 mm for ​Escherichia coli s

​ trains and had an average

minimum inhibitory concentration (MIC) of 100±1.50 mg/ml

(Uddandapu, P.K., 2016).

 29
Another research article taken from the International Journal of

Current Microbiology and Applied Sciences aims to evaluate the

antibacterial activity of plant extracts on antibiotic resistant bacterial

strains. Results of the study show that ​Vitex negundo h

​ as an antimicrobial

activity on ​E. coli, Salmonella typhimurium, Klebsiella pneumoniae,

Staphylococcus epidermis, Bacillus cereus, Bacillus subtilis, a

​ nd ​Bacillus

megaterium f​ rom its crude extract. From the ​Vitex negundo p

​ lant

sample, it was revealed that there is a modest antibacterial activity with a

zone of inhibition for ​E. coli ​strains of 26 mm, 16 mm for ​Salmonella

typhimurium, 2
​ 3 mm for ​Klebsiella pneumoniae, 13 mm for ​Staphylococcus

epidermis, ​19 mm for ​Bacillus cereus, ​16 mm for ​Bacillus subtilis, ​and

lastly, 18 mm for ​Bacillus megaterium b

​ acterial strains (Chandekar, 2018).
 30
RESEARCH METHODOLOGY

Study Environment

The locale of the study is found directly within the academic grounds

of Southwestern University - PHINMA located in Urgello St., Cebu City.

Room M106 of the Merlo Building is the assigned laboratory the

researchers conducted the necessary methods of its making. Merlo building

is the first establishment found directly from the university’s Main

Gate. The aforementioned laboratory, is situated at the ground floor

of the building, below right of the building's only elevated stage.

 31
Study Instruments

Vernier Caliper

A measuring device used in the experiment to measure the

zone of inhibition which is an indication for the antimicrobial activity of the

substance being studied.

 32
Study Procedure

Isolation / Preparation of Plant extract

The Vitex negundo leaves will be collected at the Southwestern

University PHINMA College of Pharmacy Botanical garden. The

Botanical garden is situated at the backside, right portion, of the College of

Dentistry building. It is directly beside the College of Pharmacy faculty

office, which is also situated behind the College of Dentistry building.

For the preparation of the plant material, soak the 250 g powdered

leaf samples of ​Vitex negundo with petroleum ether for 3 days with

occasional shaking and filtered to remove the fat contents. After filtration,

the air dried marc will be further soaked in 650 ml of ethanol for three days

at room temperature. The ethanol extract will then be filtered and

concentrated using a Rotary Evaporator at 40°C, air dried and weighed to

yield a dark green crude extract. The ethanol extract was suspended in

water and partitioned with diethyl ether and ethyl acetate successively and

combined to give a dark green solid (Abebayehu et al.,2016).

 33
Phytochemical Screening

Phytochemical Analysis will be carried out on the ethanolic extract of

the leaves of Vitex negundo Linn using standard procedures to

identify constituents as described by Singh et. al (2016)

Test for Alkaloids​: ​Dragendroff’s test

Warm the extract with 2% H2SO4 for 2 minutes then filter. To a 5 ml of

extract, a few drops of Dragendroff’s reagent will be added for the formation

of orange coloured precipitate. An orange precipitation indicates positive of

alkaloids

Test for Flavonoids​: ​Ammonium Test

Heat a small quantity of extracts with 10ml of ethyl acetate in boiling water

for 3 minutes. Filter the mixture. Discard the residue. Then shake the

filtrate with 1 ml of dilute ammonia solution (1%). Allow the layers

to separate. A yellow coloration will be observed at ammonium layer.

This indicates the presence of flavonoids.

Test for Terpenoids​: ​Salkowski Test

Mix 5 ml extract with 2ml of chloroform and 3ml concentrated H2SO4 is

carefully added to form a layer. Formation of a reddish brown coloration at

the interface indicates the presence of terpenoids.

 34
Test for Tannins: Ferric Chloride Test

Boil 10 ml of extract with 5 ml of 45% solution ethanol for 5

minutes. Each of the mixture is cooled and filtered. The filtrates will be used

to the following test.

Dilute 1ml of each filtrate with distilled water and add two drops of ferric

chloride. A transient greenish to black color indicates the presence of

Tannins. Hydrolysable tannins give blue-black color or precipitates and

Condensed tannins give brownish-green color or precipitates.

Test for Sterols​: ​Liebermann-Burchard test

To 5 ml of the extract, 5 drops of chloroform, acetic anhydride and H2SO4

was added. Observe the formation of dark pink colour to reddish brown ring

which indicates the presence of sterols.

Test for Proteins: Ninhydrin test

Add 1ml of 0.2% ninhydrin solution to the 3 ml of extract. Water bath for 5

minutes. Violet color indicates the presence of proteins.

 35
Test for Carbohydrates: Molisch’s test

To a 2 ml of the extract add 3 drops of Molisch’s reagent followed by the

addition of 1 ml concentrated H2SO4 along the sides of the test tube. Allow

the mixture to stand for 2 minutes. Formation of red or dull violet

ring at the interphase of two layers indicates the presence of carbohydrates.

Test for Glycosides​: ​Keller-Killiani test

In a 5 mL sample of the extract, add a few drops of ferric chloride

solution and mix. Then add 2- drops of sulfuric acid containing ferric

chloride solution. Two layer formation will show a reddish brown pigment

while the upper layer turns bluish green which indicates the presence of

glycosides.

Test for Saponins​: ​Foam test

To a 2ml of the extract add 2-3 drops of distilled water and shake

vigorously until persistent foam is observed. Foam formation indicates

presence of saponins.
 36
Preparation of the Dosage Form

To prepare the dosage formulation, it must start off with melting

the 13g spermaceti and 12g white wax, which both serve as stiffening

agents (Ansel, 2014) in the steam bath along with the liquid paraffin,

30ml, which serves as an emollient and cleanser (Drugster, 2018). It shall

be heated until the temperature is 70-80 degree centigrade. Then, 7g of

Sodium lauryl sulfate must be dissolved in the mixture in order to act as

the surfactant (Ansel, 2014). Next, the active ingredient which is the

extract of ​Vitex negundo,​ 25 g ​will be added to the mixture. Lastly, a

sufficient quantity of water, the solvent (Ansel, 2014) will be added to the

melted mixture and it will be stirred until congealed or desired consistency.

 37
Determination of Pharmacological effect

In determining its pharmacological effect, the process to undergo will

be as followed. Firstly, dispense the sterile Mueller Hinton Agar

(MHA) to previously sterilized Petri dishes and allow to cool at 40

degrees Celsius, until such duration the medium solidifies. The Petri dish

will serve as its area for growth and cultivation. Inoculate and swab

the Escherichia coli on the surface of the solid MHA medium with sterile

swab sticks. The Antimicrobial Cream consisted of the leaf extracts or

the trial drug will serve as the experimental group of the study. The

Vitex negundo ​leaf extracts Antimicrobial Cream will be applied onto the

sterilized filter paper discs with sterile pipette or stirring rods. The

positive control in the study is the Gentamicin antibiotic discs and

the negative control is Ethanol which is pipetted to previously sterilized

filter paper disks. The three different disks are placed in one petri dish

and are placed equidistant from each other. Prepare three petri dishes

with the same preparation to conduct three trials of the experiment. The

Petri dishes will be kept in the incubator for 18-24 hours at 37 degrees

Celsius. After 18-24 hours, the zone of inhibition are then measured by a

caliper. All the procedures will be carried out in an aseptic area

(Deogade, 2016).
 38
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