2.

Review of Literature
Antimicrobial resistance is now a major challenge to clinicians for treating patients.
Hence, this short term study was undertaken to detect the incidence of multidrug-resistant
(MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) bacterial isolates in a
tertiary care hospital. In 2011, WHO declared “combat drug resistance: no action today, no cure
tomorrow” (Sharma, 2011). In recent years, strains of multidrug resistant organisms have
become quadrupled worldwide (Cohen, 2000). Presently, antimicrobial resistance (AMR) poses
a major threat to patient's treatment as it leads to increased morbidity and mortality, increased
hospital stay, and severe economic loss to the patient and nation (Rosenberger et al., 2011;
Morales et al., 2012).The clinical isolates such as Pseudomonas aeruginosa, Methicillin
Resistant Staphylococcus aureus (MRSA), Enterococci especially Vancomycin Resistant
Enterococci (VRE), and members of Family Enterobacteriaceae, for example, Klebsiella
pneumoniae, Escherichia coli, and Proteus sp., rapidly develop antibiotic resistance and spread
in the hospital environment. Actually, the health care planners have declared “Health for all by
the year 2000.”

In the last two decades, there were so much increase of infectious diseases that the
standard of public health in many parts of the world is equivalent to pre antibiotic era (Arias and
Murray, 2009). As per standardized international terminology created by European Centre for
Disease Control (ECDC) and Centre for Disease Control & Prevention (CDC), Atlanta, the
multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR)
bacteria have been well defined (Magiorakos et al., 2012). Multidrug resistant (MDR) was
defined as acquired non-susceptibility to at least one agent in three or more antimicrobial
categories. Extensively drug resistant (XDR) was defined as non-susceptibility to at least one
agent in all but two or fewer antimicrobial categories (i.e., bacterial isolates remain susceptible to
only one or two antimicrobial categories). Pandrug resistant (PDR) was defined as non-
susceptibility to all agents in all antimicrobial categories.

In the present situation, an increase in antibiotic-resistant microorganisms has become

one of the most vital threats to the healthcare sector. Multidrug-resistant bacteria (MDR) that are
deadly pathogenic are rising day by day and pose a very serious threat to human health. Earlier,
these types of antibiotic-resistant bacterial strains were rare and limited to only nosocomial-

4
acquired infections, but nowadays, they have become very common. This issue is more prevalent
among both Gram-positive and Gram-negative bacterial species, which include Escherichia
coli, P. aeruginosa, and K. pneumonia (Gram-negative), along with Enterococcus faecium,
and E. faecalis (Gram-positive). It has been found that this antibiotic resistance occurred among
these bacterial species due to the attainment of plasmids through the transfer of resistance genes
(Jubeh et al., 2020). To escape from the harmful effects of antibiotics, certain bacterial species
develop some special mechanisms like efflux pumps, less permeability of the LPS layer,
secretion of degrading enzymes, and alteration of targets (Cox and Wright, 2013). Certain factors
that are responsible for increasing this antibiotic resistance may include widespread
development, overexploitation of antibiotics, extreme use of broad-spectrum drugs, and scarcity
of target-oriented antimicrobial drugs (Mahon et al., 2014).

Medicinal plants have been playing an essential role in the development of human
culture. As a source of medicine, Medicinal plants have always been at forefront virtually all
cultures of civilizations. Medicinal plants are regarded as rich resources of traditional medicines
and from these plants many of the modern medicines are produced. For thousands of years
medicinal plants have been used to treat health disorders, to add flavor and conserve food and to
prevent diseases epidemics. The secondary metabolites produced by the plants are usually
responsible for the biological characteristics of plant species used throughout the world. The
microbial growth in diverse situations is controlled by plant derived products.

2.1. Curcuma longa

Curcuma longa Linn. syn. Curcuma domestica Valeton. (Family - Zingiberaceae) is a

perennial, erect and leafy plant with very large, lily-like leaves up to 1.2 m long. It is harvested
from February to April. The leaf blade is ovate-lanceolate, thin, entire margined and narrows to a
long sheeth-like petiole (Thomas, 2000). The leaves are large, oblong, narrowed to the base
(Chatterjee and Pakrashi, 2001; Hooker, 1990). They are elongated and borne at the top of the
non-woody underground stem, with overlapping petioles. They are light green, 30-40 cm long
and 8-12 cm wide (Ross, 2001). They are 6-10, long petioled, acuminate, dark green above,
dotted below (Anonymous, 2004). The inflorescence is cone-like, 10-15 cm long, and is attached
to a stem enclosed in a sheathing petiole. The flower has 2 pale green bracts, which are 5-6 cm
long. The covering bracts are whitish, often red tinged. The individual flowers are yellowish

5
white or yellow in colour. The flowers have a tubular, 3-lobed calyx and funnel-shaped, 3-lipped
corolla (Thomas, 2000). A stamen with short filament, broad and constricted at the apex is found
is found in the floret. The anther is versatile and usually spurred at the base. The ovaries consist
of three locules each containing two ovules. The capsules are ellipsoid (Ross, 2001). Flowering
occurs during rainy season (Chatterjee and Pakrashi, 2001). The fruit is a globular capsule
(Thomas, 2000). But it is rare (Anonymous, 2004).

Plate 1: Curcuma longa

The main rhizome is thickened to a tuber and has numerous roots. The root in turn
terminates in partially elliptical tubers (Thomas, 2000). The rhizomes are fleshy, branched, with
bright orange to yellow within (Ross, 2001). But externally, they are brownish white and scaly
(Anonymous, 2004). The primary rhizomes are ovate or pear-shaped and are known as ‘bulb’ or
‘round’ turmeric while secondary rhizomes are more cylindrical. The latter are known as
‘fingers’ and contain more yellow colouring matter than the bulb variety (Evans, 2002). All the
rhizomes are yellowish brown with stipules and appear transversely ringed when they die
(Thomas, 2000).

Turmeric (Haldi) is the dried rhizome of Curcuma longa Linn. The primary and
secondary rhizomes are dug up, steamed or boiled and dried. Turmeric has an
aromatic odour and a warm somewhat bitter taste. Large quantities of turmeric are
used in the preparation of curries and sauces (Evans, 2002). The rhizome is obtained from soil,
cut in various pieces, boiled and dried. The fresh root is poisonous in nature but after 3-4 months,
an aromatic odour develops. A part of sulphur presents in turmeric, that’s why it is called
vegetative sulphur (Aawan, 1984).

6
 Turmeric is grown generally as an annual crop. It is cultivable from sea
level up to 1200 m. Turmeric is cultivated in the hills is reported to be of a better
quality than that raised in the plains. It requires a warm and humid climate. The
process of curing the turmeric consists of boiling of the rhizome, drying, polishing
and colouring. The process gives attractive colour and characteristic aroma to the
finished product and enhances its market value. The cured and finished turmeric is brittle and has
a shining yellow colour. In pharmaceutical industry, turmeric is used as a safe colouring matter.
It also enters into the formulation of certain cosmetic soaps particularly effective in skin
problems and to remove unwanted hair (Anonymous, 2004).

Turmeric (Curcuma longa L.) a native to Asia and India, is an important spice crop in
India belonging to the family Zingiberaceae (Khan et al., 2014). India is the largest producer and
exporter of turmeric in the world to various countries like USA, UK, Middle East, Japan,
Singapore, Malaysia, South Africa, Australia and other countries, thus, India account for more
than 50% of the world trade (Chaudhary et al., 2006; APEDA, 2018). Turmeric rhizomes after
processing are used as condiments, a dye and as an aromatic stimulant in several medicines,
preparations of curry powder, coloring matter in drugs, confectionary and food industries(Khan
et al., 2014). The turmeric rhizomes contain curcumin, a phenolic compound which is used in
traditional and Ayurvedic medicines having properties of antioxidant, antibacterial, antifungal,
antiparasitic and anti-inflammatory agent besides antimutagen and anticancer properties and
induces apoptosis in a variety of cancer cells (Khanna, 1999; Hossain and Ishimine, 2005; Lin et
al., 2008; Pisano et al., 2010).

Turmeric is the native of tropical South Asia. It needs temperatures between 20º C and
30º Cand a considerable amount of annual rainfall to thrive. As a dried rhizome of an
herbaceousplant, turmeric is closely related to ginger. The spice is also sometimes called
"Indiansaffron" thanks to its yellow color. Turmeric is a spice that comes from the rootCurcuma
longa L., a member of the ginger family (Zingaberaceae). Its bright yellow pigmentis used as a
food coloring agent. It has been used for centuries as a spice and a foodpreservative, and for its
various medicinal properties (Merina Benny Antony, 2003).The extract of turmeric has many
medicinal properties including antioxidant, anti-inflammatory, antiviral, antibacterial, antifungal,
and cancer chemopreventive actions.

7
 The turmeric (Curcuma longa) plant, a perennial herb belonging to the ginger family. It is
called Zardchoubeh in Persian and Heldi in Indian (Nasri, 2014). Turmeric has been traditionally
used to treat a wide variety of disorders including indigestive and liverailments disorders, throat
infections, common colds, wound and skin sores (AqiliKhorasani, 1992).One of the most
important and active ingredients is curcumin.Curcumin was initially used as a dye, however, it
was later consumed for its medicinal and spice properties (Priyadarsini, 2014). It has high
antioxidant activity and is used for cancer, rheumatoid arthritis, degenerative diseases, diabetes,
Alzheimer’s disease, cardiovascular disease, immune and liver disorders (Nasri, 2014).

Curcumin, a yellow compound isolated from its rhizome, may be responsible for
thebioactive effects. Recent research shows that curcumin may inhibit carcinogenesis
andangiogenesis. They may have a potential to improve chronic inflammatory conditions
inobesity. Curcumin is a liposoluble compound and can be easily dissolved into organic
solventsuch as methanol, ethanol, and acetone.

Curcuminoids including curcumin, bisdemethoxycurcumin and demethoxycurcumin are

the most important components of turmeric. Curcumin is the best-studied and the most important
component of turmeric and constitutes more than 3% of the turmeric powder. Other important
components of volatile oils of turmeric include turmerone, atlantone, and zingiberene sugars,
proteins, and resins. Notably, big variation exists in curcumin content of turmeric in different
Curcuma longa.

Curcumin was first isolated in 1815 and its chemical structure was determined by
Roughley and Whiting in 1973. It has a melting point at 176–177°C; forms a reddish-brown salt
with alkali and is soluble in ethanol, alkali, ketone, acetic acid and chloroform (Chattopadhyay et
al., 2004). Curcumin (also known as curcumin I) occurs naturally in the rhizome of Curcuma
longa, which is grown commercially and sold as turmeric, a yellow–orange dye. Turmeric
contains curcumin along with other chemical constituents known as the curcuminoids.

Over the past half a century, curcumin has received growing interest in biological
(Durazzo et al., 2020), pharmacological (de Souza et al., 2020) and nutraceutical research
(Santini et al., 2018). Interestingly, it was in 1949 that its antibacterial properties were
discovered (Bagheri et al., 2020). Curcumin (C21H20O6), also named [1, 7-bis (4-hydroxy-3-

8
methoxyphenyl)-1, 6-heptadiene-3, 5- dione] (Hu et al., 2018), has a molecular weight of 368.38
g/mol and it is solid at room temperature. It is a hydrophobic molecule, mostly insoluble in water
(only 30 nM can be dissolved) and poorly soluble in hydrocarbon solvents (Bagheri et al., 2020);
however, curcumin is very soluble in polar solvents (Lopez-Malo et al., 2020). The chemical
structure of curcumin contains two aromatic ring systems with o-methoxyphenolic groups
(Lopez-Malo et al., 2020).

Concerning the benefits of curcumin, it is known to be capable of modulating growth

factors, enzymes, transcription factors, kinase, inflammatory cytokines, and proapoptotic and
antiapoptotic proteins, being a versatile molecule to treat several diseases. Specific in wound
healing, the curcumin acts in the inflammatory phase, reducing the cytokines as tumor necrosis
factor (TNF-α), interleukin-1 (IL-1), and inhibits the nuclear factor-kB (NK-B). A Concerning
similar activity is observed in cancer treatment. Curcumin reduces the inflammatory condition
using different pathways. It interacts with immune mediators and shows antioxidant activity
(Barchitta et al., 2019; Giordano and Tommonaro, 2019). Due to its easy metabolization, studies
have been conducted to enhance the stability and bioavailability of curcumin through molecular
modification (Bagheri et al., 2020; Lopez-Malo et al., 2020). In the last 20 years, a vast number
of drug delivery systems, such as micelles, liposomal vesicles, nano emulsions, phospholipid
complexes, and polymeric implants, has been developed (Bansal et al., 2011), enabling their use
for therapeutic prevention or risk reduction in the pre cancer stage or even across the blood–brain
barrier, allowing for the treatment of neurodegenerative diseases (Bagheri et al., 2020).

2.1.1. Phytochemistry

Phytochemicals (from Greek phyto, meaning "plant") are chemicals produced by plants
through primary or secondary metabolism. They generally have biological activity in the plant
host and play a role in plant growth or defense against competitors, pathogens, or predators.A
few examples of well-known phytochemicals are the flavonoids, phenolic acids, isoflavones,
curcumin, isothiocyanates, and carotenoid.

Turmeric contains about 5% of diarylheptanoid colouring materials known as

curcuminoids, the chief of which is curcumin (diferuloyl-methane), together with
smaller quantities of dicaffeoyl-methane and caffeoylferuloyl-methane. Dihydrocurcumin was

9
reported in 1980. The rhizomes of the plant contain curcuminoids, demethoxy curcumin
[feruloyl-(4-hydroxycinnamoyl)-methane],bis-demethoxy-curcumin [bis-(4- hydroxycinnamoyl)-
methane], 52 -methoxycurcumin, and dihydrocurcumin, which are found to be natural
antioxidants. Other curcuminoids identified and isolated from the rhizome are: cyclocurcumin (C
21 H20 O6), 1-(4-hydroxy-3-methoxyphenyl)-7- (3,4-dihydroxyphenyl) - 1,6-heptadiene-3,5-
dione and 1,7-bis (4-hydroxyphenyl) - 1,4,6-heptatrien-3-one (C 19 H16 O 3 ). The fresh
rhizomes contain curcumin-related phenolics viz. 1,5-bis (4-hydroxy-3 methoxyphenyl) - penta –
(1E,4E) – 1,4-dien-3- one, which possess antioxidant and anti-inflammatory activities.

2.1.2. Pharmacological Studies

A number of studies have been carried out on Curcuma longa in recent years showing the
diverse pharmacological effects. These are as follows:

2.1.2.1. Acne

Extracts of Curcuma longa was tested against acne bacterium by the total plate count
method. The hemolytic activity of RBC was tested. No hemolytic activity was observed in RBC,
which indicated the safety of extract on human beings for acne treatment and wound healings
(Rao et al., 2004).

2.1.2.2. Antiallergic

Antiallergic activities of curcumin-related compounds were assessed by measurement of

histamine release. The antioxidant activity was also assessed. The results suggest that the
hydroxyl groups of curcumin play significant role in exerting both the antioxidant and
antiallergic activity (Suzuki et al., 2005).

2.1.2.3. Antibacterial

Hexane extract of the rhizome of Curcuma longa exhibited significant activity against
Gram positive and insignificant activity against Gram negative bacteria assay as compared to
standard antibiotics. Isolated low molecular weight proteins from Curcuma longa were tested
against Escherichia coli, Staphylococcus aureus and Bacillus subtilis (Harris et al., 2005).

10
2.1.2.4. Antidepressant
A study reported that administration of ethanolic extracts of Curcuma longa exert
antidepressant activity in the rat CMS model of depression (Xia et al., 2006).

2.1.2.5. Antifertility
Turmeric possesses antifertility activity as observed with experimental animals. The
petroleum ether and aqueous extract produced 100 percent anti-implantation effect in rats at a
dose of 200 mg/kg body wt fed orally on day 1 to 7 of pregnancy (Anonymous, 2004).

2.1.2.6. Antifungal

Ethanolic extract of Curcuma longa (rhizome) tested for antifungal activity against
Aspergillus flavus, A. terreus and Mucor species. The extract in different concentrations
exhibited good fungicidal activity (Perumal et al., 2004).

2.1.2.7. Antioxidant

Turmeric and Curcumin were used to elucidate their possible interaction with conjugation
reactions. The findings indicate that both interact with conjugation reactions in the human
intestinal tract and colon (Naganuma et al., 2006). The antioxidant property of turmeric was
revealed. The aqueous and ethanol extracts of two major preparations of turmeric, corresponding
to its use in cooking and medicine, showed significant antioxidant abilities (Tilak et al., 2004).
The acetone extract of turmeric contains curcumin I, curcumin II, curcumin III, having an
antioxidant activity of 20, 9 and 8 times, respectively, stronger than that of á tocopherol.
Curcumin has also been the most abundant and most active in the methanolic extract of turmeric.
Its antioxidant activity is found to be stronger than Vit E and á-tocopherol. Turmeric exerts
significant antioxidant activity. Although, both water and fat soluble extracts are effective
antioxidants in various in vitro and in vivo models, curcumin is the most potent compound
(Khare, 2004).

2.1.2.8. Cataract

Wistar rat pups treated with curcumin before being administered with selenium showed
no opacities in the lens. The lipid peroxidation, xanthine oxidase enzyme levels in the lenses of
11
curcumin and selenium co-treated animals. Curcumin co-treatment seems to prevent oxidative
damage and found to delay the development cataract (Padmaja and Raju, 2004).

2.1.2.9. Chemopreventive
The effects of curcumin against N-nitrosodiethylamine (DENA) initiated and
Phenobarbital (PB) promoted hepatocarcinogenesis was studied in Wistar rats. Results suggest
the possible chemopreventtive effects of curcumin (Sreepriya and Bali, 2005).

2.1.2.10. Gastriculcer

A study reported that intraduodenal administration of curcumin, inhibited gastric

acid secretion in the pylorous ligated rats. Oral administration of curcumin significantly
accelerated the healing of acetic acid induced chronic gastric ulcer and promoted mucosal
regeneration in the ulcerated portion in a dose related manner. The protective effect of turmeric
extract against gastric ulcer was studied and findings suggest that Curcuma longa exhibits
gastroprotective activity by blocking H2 histamine receptors (Kim et al., 2005).

2.1.2.11. Hepatoprotective

Ethanolic extract of turmeric showed significant hepatoprotective effect (84.1%) against

CCL 4 induced hepatotoxicity (Anonymous, 2004).

2.1.2.12. Murinelymphocyte

The ethanol extract of turmeric (Curcuma longa) has been studied and found to be
stimulatory for murine lymphocytes and inhibitory for ascetic-fibro sarcoma cells (Chakravarty
et al., 2004).

2.1.2.13. Oesophagealreflex

The preventive effect of curcumin, a compound isolated from the rhizome of Curcuma
longa on experimental reflux esophagitis in rats was investigated in order to validate its potential
therapeutic use for gastroesophageal reflux disease (Mahattanadul et al., 2006).

12
2.1.2.14. P-glycoprotein

Curcuminoids from Curcuma longa was investigated for its inhibitory effect on P-
glycoprotein (P-gp) on the efflux transport of rhodamine 123 (Rho-123) in Caco-2
cells and rat ileum (Junyaprasert et al., 2006).

2.1.3. Medicinal Uses

It is therapeutically used in various diseases such as amenorrhoea, anaemia, ascitis,

asthma, boils, bruises, catarrh, chronic bronchitis, chronic dysentery, chronic fever,
conjunctivitis, cough, cystitis, dermatosis, diabetes, diarrhea, dislocation of joint, dyspepsia,
elephantiasis, eosinophilia, eye disease, facial palsy, gonorrhoea, haemoptysis, helminthiasis,
hepatitis, hysteria, impaired vision, indigestion, itching, jaundice, leech bite, leprosy,
leucorrhoea, liver disorder, loss of appetite, malignant ulcer, menstrual disorder, oedema,
ozoena, paralysis, parasitic disease, peptic ulcers, piles, pterigium, ptyriasis, ring worm infection,
scorpion sting, senility, septic puncture wound, slow lactation, small pox and chicken pox, skin
disease, tonsillitis, toothache, toxicosis, trauma, urethral discharge, urinary infection, uterine
pain, vaginal discharge, weakness of eye sight, wound and wound maggots (Aawan, 1984;
Chatterjee and Pakrashi, 2001; Chopra et al., 1956; Dey, 1988; Evans, 2002; Kantoori, 1992;
Khare, 2004; Kirtikar and Basu, 1991; Nabi, 1920; Nadkarni, 1982; Ross, 2001; Thomas, 2000;
Waring, 1982).

2.2. Syzygium aromaticum

Syzygium aromaticum (Clove) belongs to family Myrtaceace, a taxon of dicotyledon

plants is one of most valuable and second most important spice in the world trade. Various
synonymes used for the clove are Caryophyllus aromaticus, Caryophyllus silvestris, Eugenia
caryophyllus, Jambosa caryophyllus and Myrtus caryophyllus (Soh and Parnell, 2015). Clove is
commonly used in cultivation and indigenous to North Maluku Islands in Indonasia. Major
cultivator countries of clove are Pemba, Zanzibar, Indonasia, Madagascar and some of wild
clove varieties are found in Bacan, Ternate, Motir, Tidore, Makian and Western parts of Irian
Jaya. In India cultivation of clove is restricted to three states Karnataka, Tamil Nadu and Kerala.
India becomes second largest consumer of clove after the Indonesia (Board, 2010).

13
 Cloves are available throughout the year due to different harvest seasons in different
countries. The different varieties of clove tree vary in canopy shape from pyramidal to
cylindrical. The clove tree can live upto 100 years and above. The tree prefers to grow in well
drained soil with sufficient soil moisture. Clove tree requires heavy sunlight with high
atmospheric temperature (25 to 35°C), well-distributed rainfall above 150 cm and high humidity
above 70% (Danthu et al., 2014). The crop cannot withstand water logged conditions. In India
clove grows well in deep black loamy soil of humid tropics and successfully grows in the red
soils of midlands of Kerala and in the hilly terrain of Western Ghats in Karnataka and Tamil
Nadu (Byng, 2016).

Plate 2: Syzygium aromaticum

The clove is usually known as "lavang."Due to the increase in nutrition and medicinal
properties, Plants' importance in human life has increased every day. The term clove is derived
from 'clove' and 'clou' meaning 'nail.' This is used mainly in Ayurvedics (Hussain and Trak,
2009). Clove is the topical evergreen Myrtaceae family tree native to the islands of Maluka in
east Indonesia. The color is small brown, floral buds, and a spice is used. It was used as food
preservatives, flavoring agents and nutritional additives, medicinal coloring agents (Cortes-Rojas
et al., 2014). Health, cosmetic, nutritional and agricultural use clove. It is commonly used for
tooth care (Chaieb et al., 2007). This is commonly used as an expectorant for the treatment of
dental discomfort, the digestive problem, oral ulceration, mosquito repellent (Thangaselvabai et
al., 2010). Clove is a pharmacologically active medicinalplant that includes Anti-oxidant, anti-
pyretic, anti-viral, anti-microbial, anti-diabetic, anti-inflammatory, analgesic, anti-platelet, anti-
stress, anti-disease, anti-carcinogenic in cervical cancer. The clove is available in three types of
essential oil (Jain, 2019). Clove is amongst the most essential sources of Phenolic compounds,

14
including eugenol (80% -90%), eugenyl acetate (15% -17%), and beta-caryophyllene (5% -12%),
alpha-humulene (0.55%), alpha-terpenyl acetate (0.1%), and methyl eugenol (0.2%) (Yunusa,
2019).

Clove is an aromatic spice tree. Clove is conical myrtle, medium sized tree with straight
trunk which grows up to 10 to 12 m in height. The branches are semi erect, grayish in color and
dense. Leaves are large oblong to elliptic, simple obviate opposite, glabrous and possess plenty
of oil glands on the lower surface. Tree begins flowering in about 7 years and continues
flowering for 80 years or more. Flowers are small, crimson in color and are hermaphrodite
(bisexual) borne at the terminal ends of small branches. Each peduncle carries 3 to 4 stalked
flowers and inflorescence length remains between 4 to 5 cm. Initially flower buds are pale
yellow in color with glossy appearance and turn green to bright red at maturity. These are 1-2 cm
long with cylindrical thick ovary consisting of four fleshy sepals. Buds are divided into
elongated stem and a globose bulbous head which stimulates into nail. Commercially cloves used
are air-dried unopened flower buds, 2.5 cm in length and 1.25 cm wide. Fruit matures nine
months after flowering and the red ovary gradually turns to reddish purple. The fruit nearly
contains one or two seeds known as ‘mother of clove’. The cultivated trees are rarely allowed to
reach fruit stage. These are harvested when they develop dark red ellipsoid berry (Kamatou et
al., 2010; Cortes-Rojas et al., 2014).

Spices as clove, oregano, mint, thyme and cinnamon, have been employed for centuries
as food preservatives and as medicinal plants mainly due to its antioxidant and antimicrobial
activities. Nowadays, many reports confirm the antibacterial, antifungal, antiviral and
anticarcinogenic properties of spice plants. Clove in particular has attracted the attention due to
the potent antioxidant and antimicrobial activities standing out among the other spices (Shan et
al., 2005). Syzygium aromaticum (S. aromaticum) (synonym: Eugenia cariophylata) commonly
known as clove, is an median size tree ( 8 - 12 m ) from the Mirtaceae family native from the
Maluku islands in east Indonesia. For centuries the trade of clove and the search of this valuable
spice stimulated the economic development of this Asiatic region (Kamatou et al., 2012).

The clove tree is frequently cultivated in coastal areas at maximum altitudes of 200 m
above the sea level. The production of flower buds, which is the commercialized part of this tree,
starts after 4 years of plantation. Flower bud sare collected in the maturation phase before

15
flowering. In Brazil, clove is cultured in the northeast region, in the state of Bahia in the regions
of Valença, Ituberá, Taperoá, Camamu and Nilo Peçanha, where approximately 8000 hectares
are cultivated, producing near 2500 tons per year (Oliveira et al., 2009).The collection could be
done manually or chemically-mediated using a natural phyto-hormone which liberates ethylene
in the vegetal tissue, producing precocious maturation. Nowadays, the larger producer countries
of clove are Indonesia, India, Malaysia, Sri Lanka, Madagascar and Tanzania specially the
Zanzibar Island (Kamatou et al., 2012).

Clove is known by several names like Laung, Lavang, Laumg in Hindi; Lavanga,
Lavangaka, Lavangam, Bhadrasriya, Devakusuma, Haricandana, Devapuspa, Varala in Sanskrit;
Luvang in Marathi; Lavang in Gujarati; Lavanga in Bengali; Laung in Punjabi; Labanga in
Oriya; Laung, Loung in Urdu; Grampu, Karayampu, Karampu in Malayalam; Lavanga, Krambu;
Daevakusuma in Kannada; Kaaravallu, Devakusumamu, Lavangalu, Lavangamu in Telugu;
Kirampu, Kiraambu, Kirambu, Grambu, Ilavankam in Tamil (Milind and Deepa, 2011).Clove’s
side effects caused by smoking clove cigarettes include haemorrhagic pulmonary oedema,
bronchitis, pneumonia, occupational allergic contact dermatitis, and central nervous system
depression (Kaur et al., 2013). Clove oil used in low doses may have side effects like rare
allergic reactions, local irritation, and contact dermatitis. Eating or exposure to a large amount
can cause tissue injury and a syndrome of acute onset of seizures, damage to the liver and
kidneys, and coma.

2.2.1. Chemical constituents of clove essential oil

From clove species three essential oils are available: clove stem oil, clove bud oil and
clove leaf oil. Each clove essential oil differs in the chemical composition, flavour and color. In
clove essential oil amount of secondary metabolites are affected by the nature of soil, climate,
cultivation techniques and genetic factors (Verzar-Petri et al., 1985; Arslan et al., 2004).

2.2.2. Phytochemical composition of clove

Kumar et al., (2010) analysed the phtyochemical composition of dichloromethane extract

of clove bud oil which showed the presence of carbohydrates, terpenoids, glycosides, steroids,
sterols, tannins and phenolic compounds. Soni and Dahiya, (2014)revealed the presence of

16
saponins, alkaloids, flavanoids, cardiac glycosides, tannins and steroids in clove essential oil.
Jimoh et al., (2017) also revealed the presence of tannins, alkaloids, terpenoids, carbohydrates,
glycosides, ketones, aldehydes and forty-six phenolic compounds in methanol extract of clove.
Various phenolic compounds identified by using Gas Chromatography-Flame Ionization
Detector (GCFID) were gallic acid, kaempferol, rhametin, myricetin, salicyclic acid, syringic
acid, eugenin, caffeic acid, eugenitin, isohamnetin, quercetin, phenylacetic acid, isohamnetin,
protocatechuric acid and p-hydroxybenzoic acid.

Fu et al., (2007); Yang et al., (2003) and Chaieb et al., (2007) reported that antimicrobial
activity of clove oil was due to presence of eugenol, 2-heptanone, methyl salicylate,kaempferol,
gallic acid, isoeugenol and oleanolic acid. These compounds generally denatured proteins that
reacted with cell membrane and changed their permeability. Warnke et al., (2009) reported
antimicrobial activity of different essentialoils including clove bud oil against six Staphylococcus
strains including methicillin-resistant Staphylococcus aureus (MRSA), three Candida strains and
four Streptococcus strains by using agar diffusion test. The clove oil showed considerable
antimicrobial activity with diameter of inhibition zone of 12 to 20 mm. Duraipandiyan et al.,
(2006) also investigated antibacterial activity of clove oil against Enterococcus faecalis, Bacillus
subtilis, Ervinia sp, Staphylococcus epidermidis and Proteus vulgaris by using paper disc
diffusion method at different concentration of 5, 2.5 and 1.25 mg/disc. Saini et al., (2009)
reported that clove oil inhibited bacterial colonization of Klebsiella pneumonia in lungs of mice.

2.2.3. Pharmacological studies

2.2.3.1. Antimicrobial activity

Eugenol exhibits antimicrobial activity toward fungi, as wellas a widely range of gram-
positive and gram-negative bacteria. Eugenol is naturally occurring essential oil i.e,
hydroxyphenyl propene belonging to family mytraceae responsible for human infection diseases,
Oral cavity eugenol disease, food-borne pathogens eugenol demonstrates bioactive compounds
with broad range anti-microbialactivity against both plank-tonic and sessile cells carrying food
borne micro-organisms and human diseases (Marchese et al., 2017).

17
 Antibacterial activity of clove essential oil has been reported against Staphylococcus
aureus (Mishra and Kalyani, 2014) and Listeria monocytogenes in pasteurized milk (Cava et al.,
2007). Matan (2012) reported that clove oil showed strong antimicrobial resistance
against Penicillium sp., Aspergillus flavus and Staphylococcus aureus found on dried fish
(Decapterus maruadsi). Zengin and Baysal (2015) reported the antimicrobial activity of
clove oil against three gram-positive bacteria (Listeria innocua, Carnobacterium divergens and
Staphyloccous aureus) and four gram-negative bacteria (Salmonella typhimurium, Escherichia
coli, Serratia liquefaciens and Shewanella putrefacians) by broth microdilution method. The
result showed that clove essential oil inhibited the growth of all bacteria while Shewanella and
Listeria were found to beresistant to oil. Gupta et al., (2013) also found clove oil tobe effective
against food borne gram negative bacteria (Penicillium aerugenosa, Escherichia coli,
Staphylococcus chloeraesius and Yersinia enterocolitica) and gram positivebacteria (Bacillus
cereus, Listeria monocytogenes, Staphyloccous aureus and Enterococcus faecalis).

2.2.3.2. Antioxidant Activity

Antioxidant activity above essential oil separated significant eugenol compounds and its
derivative are determineology using two the free radical scavenging assays in vitro-model
involving 2, 2 – di-phenyl-1-picryl hydrazyl (DDPH) and nitric oxides. The percentage inhibition
was measured and reports were represented using ascorbic acid as normal in a term of IC50 val –
uses (Concentration when compared at which free radicals is inhibition by 50 per cent) (Kaur et
al., 2019).This activity is evaluated with DPPH. Radical scavenging activity is the very common
method to determine the activity. Capturing of free radical by eugenol is expressed as IC50,
Expresses the concentration needed to capture redicals as a medium of 50 per cent. As aresult it
shows strongly decreases anti-oxidant activity (Da Silva et al., 2018).

High antioxidant activity shown byclove oil was due the presence of phenolic compounds
like eugenol, thymol and eugenol acetate (Yadav and Bhatnagar, 2007; Dai et al., 2013 and Nam
and Kim, 2013). Eugenol present in clove oil possessed high antioxidant activity which was
comparable with the activities of synthetic antioxidants pyrogallol and BHA (Dorman et al.,
2000). Gulcin et al., (2012) observed inhibition of (97.3%) lipidperoxidation of linoleic acid
emulsion when treated with 15μg/ml of clove oil. However, standard antioxidant such astrolox,
butrylated hydroxyanisole (BHA), α-tocopherol and butylated hydroxytoluene (BHT) showed

18
inhibition of 95.6, 95.4, 84.6 and 99.7% respectively under same conditions. Abdel-Wahhab and
Aly (2005) reported that phenolic compounds in clove essential oil resulted in formation
ofepoxide aflatoxin B1 by inhibition of CYP450 enzyme andincreased the ability of liver
microsomes to catalyses aflatoxin-glutathione conjugation. Recent study showed that clove
essential oil showed ten times greater antiradicalactivity than BHT and seven times greater than
cocca butter and clove essential oil mixture (Fankem et al., 2017).

2.2.3.3. Antidiabetic Activity

There work is hypothesized to evaluate the anti-hyperglycemic ability are eugenol via
defining activity are enzymes the involved in streptozotocin (STZ)-induced glucose metabolized
in a diabetics rats. Increases in the activity of major carbohydrate metabolized enzymes such as
hexo-kinase, pyruvate-kinase, glucose-6-phosphate dehydrogenase, glucose-6-phosphatase,
fructose-1, 6-bis-phosphatase, and liver biomarkers (AST, ALT, and ALP), creatine-kinase, and
blood urea nitrogen’s inserum, and diabetes rat blood have appreciably returned to nearly normal
rates via eugenol administration. Eugenol dosing to improve body weight for diabetic rats
andhepatic glycogen quality exemplify eugenol's anti hyperglycemic ability in diabetic
rats.Current findings indicate that eugenol in experimental diabetes may potentially boost
activities of enzyme of glucose metabolized, and the prudent are extend from a scope the eugenol
used in a trial of alleviate the adverse effect of diabetes (Srinivasan et al.,2014).

Lavang and insulin influence the activity of diabetes-related genes such as a

phosphoenol-pyruvate carboxy-kinase (PEPCK) glucose 6-phosphate (G6Pase) gene,
investigating the antidiabetic effects of free and bound phenolic clove extracts against carbo-
hydrate-hydrolyzing enzyme in a similar manner. Result the both extracts blocked dose-
dependent alpha-amylase and alpha-glucosidase (Mbaveng et al., 2017).

2.2.3.4. Anticarcinogenic Activity

Clove essential oil these are demonstrate anticarcinogenic and antimutagenic ability due
to good free radical scavenging activity. Many confirmatory studies have suggested chemo-
preventive role of essential oils, showing in lung, skin and digestive cancers. Clove extract of
ethyl acetate suppresses tumor growth and helps in the arrest and apoptosis of cell cycles.

19
Eugenol behaves as a potential atom capable of obstructing multiple cell signaling pathways,
especially the NF-kB. Eugenol are combine with gemcitabine on the cervical carcinoma and
present that the combination of eugenol and gemcitabine, even in low concentration, would
inhibitgrowth of cancer cells (Mittal et al., 2014).

2.2.3.5. Analgesic Activity

Popularly used in dental practices because of its potential to soothe tooth pain. The
anesthesia effects on dental pain as well as analgesic and anti-inflammatory effect the animal
model, suppressing prostaglandins and other inflammatory mediators to weaken the sensory
receptors involved in pain perception, impair the capacity for action in sciatic nerves (Mittal et
al., 2014).

2.2.3.6. Anti-Inflammatory Activity

Essential oil open breathing channels, serving as anexpectorant to treat many of the upper
respiratory disorders including colds, eye sties, bronchitis, sinus disorders, cough and asthma. In
traditional clinical medicine, clove was used to relieve nasal hindrance and musculoskeletal
discomfort that indicates itsanti-inflammatory function and the action is attributed to COX-2
inhibition. When inhaled, the aromatic oil could help to alleviate such breathing problems such
as cough, colds, asthma, bronchitis, and sinusitis (Mittal et al., 2014).

2.2.3.7. Anticancer Activity

Showed cytotoxic effects of antimutagenic activity against cancercell lines. Clove oil
extracts demonstrated cytotoxic activity against cervical cancer Hela cells, MCF-7 cells and
MDA-MB-231 cells for breast adeno-carcinoma, DU-145cells for prostate-cancer, TE-13 cells
for oesophageal-cancer. Cytotoxicity of methanol extract from clove buds was also reported
against melanin formation, and it was found that aqueous clove infusion had a promising
function in limiting the carcinogenesis process in 9, 10- dimethyl benz (a) anthracene-convinced
skin carcinoma. There werealso studies of anti-mutagenic activities of clove seeds extract
(Mbaveng et al., 2017).

20
2.2.3.8. Antifungal Activity

Antifungal clove activity is increased if collected by isomerizing double bond or adding

nitro group on the aromatic ring. Based on the relationship between fungicides, chemical
structure and mechanism of action (MOA), activity for commercial fungicide BC-1000 is highly
documented. As a result, the compound shows potential impact and can be used to design the
natural balance (Olea et al., 2019).

2.2.3.9. Anesthetic Activity

Isoeugenol as compound to methyl isoeugenol shows similar activity and potency

between eugenol and methyl eugenol, eugenol is aallyl compound and is more active than
propenyl isomer (iso eugenol) (Dallmeier et al., 1981).

2.3. Terminaliachebula
Terminalia chebula, commonly known as black- or chebulic myrobalan, is a species of
Terminalia, native to South Asia from India and Nepal east to southwest China (Yunnan), and
south to Sri Lanka, Malaysia and Vietnam. In India, it is known as "Harad" in Hindi and Urdu,
"Kadukkai" in Tamil, "Hirada" in Marathi, "Hilikha" in Assamese and "Horitoky" in Bengali.
Terminalia chebula is found throughout South and Southeast Asia including in India, Sri Lanka,
Bhutan, Nepal, Bangladesh, Myanmar, Cambodia, Laos, Vietnam, Indonesia, Malaysia, Pakistan
and Thailand. In China, it is native in W Yunnan and cultivated in Fujian, Guangdong, Guangxi
(Nanning), and Taiwan (Nantou). In India, it is found in the Sub Himalayan region from Ravi
eastwards to West Bengal and Assam, ascending up to the altitude of 1,500 m (4,900 ft) in the
Himalayas. This tree is wild in forests of Northern India, central provinces and Bengal, common
in Madras, Mysore and in the southern part of the Bombay presidency (Bag et al., 2013). Its
habitat includes dry slopes up to 900 m (3,000 ft) in elevation. Terminalia chebula is a main
ingredient in the Ayurvedic formulation Triphala which is used for kidney and liver dysfunctions
(Tewari et al., 2017). The dried fruit is also used in Ayurveda as a purported antitussive,
cardiotonic, homeostatic, diuretic, and laxative. It is also used as a soothing agent for dry cough.

21
 It grows in India, Myanmar, Bangladesh, Iran, Egypt, Turkey, Chinaetc. In India,
Haritaki tree grows in deciduous forests and found in North India and South words toward the
Deccan table lands at 1000to 3000 ft.In Myanmar, it grows up to 5000 ft, which consists of
pericarp of a mature fruit of Terminalia chebula, whereas, a moderate sized or large tree is found
throughout India chiefly in deciduous forests and areas of light rain fall. The flowers appear from
April to August andfruits ripen from October to January (Ratha and Joshi, 2013; Baliga et al.,
2012).

Plate 3: Terminaliachebula

Halela scientifically known as Terminalia chebula Retz belongs to the family

Combretaceae, is used widely in all Indian systems of medicine such as Ayurveda, Siddha,
Unani and Homeopathy. Halela contains tannins and myrobalans and is widely used in all the
systems of Indian Medicine. Terminalia chebula Retz. traditionally used in many formulation for
anti-diabetic, anti- inflammatory, laxative, antibacterial, antifungal, cardio tonic, diuretic,
hyperlipidemic activity, jaundice etc. (Anonymous, 1999; Kirtikar and Basu, 1987; Sabu and
Kuttan, 2002; Miglani et al., 1971; Khanna et al., 1993; Malekzadeh et al., 2001). A recent study
showed that Halela has antitumour activity and another revealed its significant effect in
inhibiting the HIV virus (Juang et al., 2004). Halela is also one of the major ingredients of
several Unani compound preparations such as Itrifalat, Haboobat (Tablets), Safoofs (Powders)
etc.

Three types of Halela with distinct therapeutic activities have been described in Unani
classical literature out of which Halela Kabli, Halela Siyah and Halela Zard are commonly used
without substitution to each other. All the three varieties of Halela are the fruits of the same plant

22
but harvested at different stages of development and possess distinct therapeutic properties
(Kabeeruddin, 1948; Khan, 1926). Halela Siyah is described as a small, black and immature fruit
of Terminalia chebula, falling from the tree before the formation of the seed whereas Halela zard
is moderately larger, yellowish to dark brown ripen fruit and Halela Kabli is reddish yellow,
larger, completely ripen fruit with hard endocarp of the same plant.

Terminalia chebula plant is a native plant in India and Southeast Asia and is extensively
cultivated in Taiwan. It is a dried ripe fruit, also called as Terminalia fruit, (HuaYew et al.,
2003). T. chebula belongs to the family Combretaceae and is found throughout India especially
in deciduous forests and areas of light rainfall (Naik et al., 2004). T. chebula is a medium to
large deciduous tree, attaining a height of up to 30 m with wide spreading branches and a broad
disk-shaped crown (Chattopadhyay and Bhattacharyya, 2007). T. chebula is commonly known as
black myroblans in English and harad in Hindi. The Terminalia consists of 250 species and
widely distributed in tropical areas of the world (Ammar et al., 2002). The fruit of T. chebula is
consider as the "king of medicines" by Tibetans and second-to-none by ayurvedic apothecaries,
and also held in high regard by other folk medicinal practitioners (Karel et al., 2004). The
Sanskrit name ‛ikatiraH‟ is rich with meaning, referring to the yellowish dye (haritak) that it
contains, as well as indicating that it grows in the abode of god siva (Hari, that is the Himalayas)
and that it cures (harayet) all diseases. Its other commonly used Sanskrit name, Abhaya, refer to
the „fearlessness‟ it provides in the face of the disease (Chattopadhyay and Bhattacharyya,
2007).

T. chebula is a medium-sized deciduous tree with a height of up to 30 m, wide spreading

branches and a broad roundish crown. It grows in the altitude of 1500 to 2000 m in mostly
clayey as well as shady soils. The leaves are elliptic rhombus, with an acute tip, cordate at the
base, outskirts entire, glabrous above a yellowish pubescence below. The flowers are
monoecious, monotonous white to yellow, with a strong unlikable odor, born in terminal prickle
or short panicles. The fruit are glabrous, ellipsoids ovoid drupes, yellow to orange brown in
colour, encloses a single angle stone (Chattopadhyay and Bhattacharyya, 2007). T. chebula is of
three types - actually these are the different stages of maturity of fruits (a) small Myrobalan- the
immature fruit; (b) yellow Myrobalan- after development of seed, the mature stage of the fruit;
(c) large Myrobalan- the fully matured fruit (Chattopadhyay and Bhattacharyya, 2007).

23
 The fruit is mild laxative, stomachic, tonic, alterative, antispasmodic. It is useful in
opthalmia, hemorrhoids, dental caries, bleeding gums, ulcered oral cavity. Its paste with water is
found to be anti-inflammatory, analgesic and having purifying and healing capacity for wounds.
Its decoction is used as gargle in oral ulcers, sore throat. Its powder is a good astringent
dentifrice in loose gums, bleeding and ulceration in gums. It is good to increase appetite,
digestive aid, liver stimulant, stomachic, gastrointestinal prokinetic agent, and mild laxative. The
powder of T. chebula fruits has been used in chronic diarrhea. It is used in nervous weakness,
nervous irritability. It promotes the receiving power of five senses. It is adjuvant in hemorrhages
due to its astringent nature and good for chronic cough, chorizo, sore throat as well as asthma.
Also it is useful in renal calculi, dysurea, retention of urine and skin disorders with discharges
like allergies, urticaria and other erythematous disorders (Varier, 2002; Aslokar et al., 1992).

2.3.1. Phytochemistry

Terminalia chebula contains high phenolic content, especially hydrolyzable tannins,

anthraquinone, flavonol, carbohydrates, glucose and sorbitol. The triterpenes have been reported
which are arjun glucoside 1, arjun genin and the chebulosides 1 and 2. Other constituents
contains tannins up to 30%, chebulic acid 3-5%, chebulinic acid 30%, tannic acid 20 40%,
ellagic acid, 2,4-chebulyi–β-D-glucopyranose, gallic acid, ethyl gallate, punicalagin terflavin A,
terchebin, some purgative of the nature of anthraquinone, flavonoids like luteolin, rutins, and
quercetin etc (Jagetia et al., 2002). Ellagitannin such as punacalagin, casurarinin, corilagin and
terchebulin and others suchas chebulanin, neochebulinic acid, chebulagic acid and chebulinic
acid have been reported to be associated with the plant (Raju et al., 2009).

2.3.2. Pharmacological studies

2.3.2.1. Antibacterialactivity
Kannan et al., 2009 has investigated on two anti-bacterial compounds, Gallic acid and
ethyl ester against methicillin-resistant Staphylococcus, have been isolated from ethyl alcohol
extract of fruits of Terminalia chebula. Terminalia chebula is well effective against Helicobacter
pylori, a bacterium responsible for gastritis, ulcer and stomach cancers.

24
2.3.2.2. Antifungal activity

Shinde et al., 2011 has done on the Anti-fungal activity against a number of dermatophytes
and yeasts. Alcoholic and ethyl acetate extracts of Terminalia chebula leaves were also tested
pathogenic fungi using paper disc methodand were found effective compared to that of the
reference standard carbendazim.

2.3.2.3. Anti-amoebic and immune modulatory activities

Sohni et al., 1996 has investigated in experimental caecalamoebias is in rats to the anti-
amoebic effect of a crude drug formulation of Terminalia chebula. In immune- modulation
studies, humoral immunity was enhanced where T-cells counts remained unaffected in the
animals, but cell mediated immune response was stimulated.

2.3.2.4. Molluscicidal activity

Upadhyay et al., 2011 has examined on the molluscicidal activity of ethanolic extract of
Terminalia chebula fruit powder was studied against the vector snail Lymnaea acuminata and
was found time and concentration dependent.

2.3.2.5. Anti-helminthes activity

Kamaraj et al., 2011 The study of ovicidal and larvicidal activities of ethyl acetate,
acetone, and methanol extracts of dried leaves and seeds of Terminalia chebula were examined
by the in vitro on Haemonchuscontortus based on egg hatch and larval development assays at 50,
25, 12.5, 6.25 and 3.13mg/ml. The extract of leaves and seeds of Terminalia chebula showed
complete inhibition at 50mg/ml.

2.3.2.6. Anti-viral activity

Lin et al., 2011 the extract of fruits of Terminalia chebula showed inhibitory effects on
human immunodeficiency virus-1 reverse transcriptase. Hot water extract of Terminalia chebula
showed anti-herpes simplex virus (HSV) activity in-vivo and anti-cytomegalovirus (CMV)
activity both in-vitro and in vivo in a study. Terminalia chebula inhibited HSV-1 entry at non-

25
cytotoxic doses in A549 human lung cells by preventing binding, penetration, and cell to cell
spread, as well as secondary infection.

2.3.2.7. Antioxidant activity

Chen et al., 2011 has done on the Terminalia chebula is an excellent anti-oxidant. In a
study, 6 extracts and 4 pure compounds of Terminalia chebula exhibited in-vitroantioxidant
properties of anti-lipid peroxidation, anti-superoxide radical formation and DPPH activities at
different concentration. The results demonstrated that tri-ethyl-chebulate was a strong
antioxidant and free-radical scavenger, which might contribute to the anti-oxidative ability of
Terminalia chebula.

2.3.2.8. Anti-diabetic-activity

Murali et al., 2007 has investigated on 75% methanolic extract of Terminalia chebula
(100mg/kg body weight) reduced the blood sugar level in normal and alloxan diabetic rats
significantly within 4 hours by the oral administration. The chloroform extract of Terminalia
chebula seeds (100, 200 and 300 mg/kg body weight) produced dose- dependent reduction in
blood glucose of diabetic rats in both short term and long term study (300 mg/kg body weight for
8 weeks).

2.3.2.9. Anti-anaphylactic activity

Shin et al., 2001 has study on the Terminalia chebula along with several other medicinal
plants help to resist against a number of stressors in different ways. Terminalia chebula, when
given following anaphylactic shock, reduces the serum histamine levels showing a strong Anti-
anaphylactic activity.

2.3.2.10. Anti-mutagenic and anti-carcinogenic activities

Ponnusankar et al., 2011 has performed by the effect of 70% methanolic fruit extract
of Terminalia chebula was studied on growth of several malignant cell lines. One of the
fractionated compounds from ethanolic fruit extract of Terminalia chebula, chebulagic acid,
showed potent dual inhibition against COX and 5-LOX. It also showed anti-proliferative activity

26
against HCT-15, COLO-205, MDA-MB-231, DU-145 and K562 cell lines. A recent study has
shown the ability of triphala to inhibit cytochrome P450.

2.3.2.11. Wound healing activity

Choudhary et al., 2011 has done on the alcoholic extract of the leaves of Terminalia
chebula caused much faster healing of rat dermal wounds in-vivo due to improved rates of
contraction and a decreased period of epithelialization for the topical administration.
Biochemical studies revealed increase in total protein, DNA and collagen contents in the
granulation tissues of treated wounds.

2.3.2.12. Hepato-protective acivity

Tasduq et al., 2006 has investigated on the 95% ethanolic extract of Terminalia chebula
fruit showed hepato-protective activity against anti-tuberculosis (anti-TB) druginduced toxicity
which could be attributed to its prominentanti-oxidative and membrane stabilizing activities.

2.3.3. Traditional Uses

Fruits effectively reduce the swelling, hasten the healing and cleanse the wounds and
ulcers. Prevents accumulation of pus in skin diseases. Healing of wounds especially in burns.
Fruit is also applied in conjunctivitis for relief due toits anti-inflammatory property. Used as a
mouth rinse, is an anticancer agent. Used as anti-astringent (Rathinamoorthy and Thilagavathi,
2014).

Initially, MDR bacteria were associated with hospital-acquired infections. MDR bacteria
have spread and are now the leading cause of community-acquired infections. The spread of
multidrug-resistant (MDR) bacteria in society has resulted in an increase in morbidity, mortality,
healthcare expenditure, and antibiotic use. According to the IDSA (the Infectious Diseases
Society of America), resistance to antimicrobial compounds can be defined as “one of the
greatest threats to human health worldwide” (Spellberg et al., 2011). It has been observed that
patients infected with MDR strains of bacteria have more severe consequences than patients
infected with other vulnerable organisms (Vardakas et al., 2013; Bodi et al., 2001).

27
2.4. Methicillin resistant Staphylococcus aureus (MRSA)

The most common example of MDR bacteria is MRSA (methicillin resistant S. aureus),
i.e., transmitted efficiently from strict hospital-acquired infection to community-associated
spread. However, the epidemiology of such CA-MRSA (community-associated methicillin-
resistant S. aureus) has already been extensively evaluated (DeLeo et al., 2010; Witte, 2009).
Numerous nonlactam antibiotics were found to be effective against such CA-MRSA strains. In
the USA, a novel strain of CA-MRSA was discovered, i.e., known as the CA-MRSA-USA300
strain, that successfully substituted the previous USA400 CA-MRSA strain in the early 2000s
(DeLeo et al., 2010). It has been observed that this USA300 strain is distinguished by the
existence of staphylococcal cassette chromosome mec (SCCmec) type IV along with the genes
responsible for the secretion of Panton-Valentine leucocidin (PVL) toxins (Thurlow et al.,
2012). P. aeruginosa is well-known for causing nosocomial infections with symptoms such as
pneumonia and bloodstream illness. Favorable environments for P. aeruginosa are moist places
and can be found mostly in washing sinks, aerators, equipment like respiratory gear, and
unhygienic solutions in the hospital environment (Paterson, 2006). Moreover, fewer patients
have experienced the chronic biofilm-associated Pseudomonal establishment along with cystic
fibrosis (CF) (Hassett et al., 2009). Among such patients, repetitive applications of antibiotics
lead to the origin of MDR bacterial strains. Community-associated infections with MDR strains
of P. aeruginosa are found to be uncommon (Rodriguez et al., 2010; Anderson et al., 2014). In a
group of 60 patients suffering from community-acquired bloodstream illness due to P.
aeruginosa, 100% of isolates are meropenem susceptible, and 95% are susceptible to
ceftazidime, tazobactam, and piperacillin (Hattemer et al., 2013).

2.4.1. Vancomycin-Resistant Enterococci (VRE)

One more antibiotic-resistant bacterial strain was developed in the late 1980s, i.e., VRE
(vancomycin-resistant Enterococci), which is responsible for the major source of hospital-
acquired infections in 1990. It has also become evident in the European study that such
vancomycin-resistant Enterococci were isolated in the fecal material of healthy individuals
(Bruinsma et al., 2003). Due to this variation between Europe and the USA, avoparcin is widely
used. Avoparcin is a glycopeptide antibiotic, i.e., used to enhance the growth of animals as food

28
additives. It has become evident that avoparcin was not permitted for use in the USA or Canada,
but it was widely used in Europe until 1997 (Klare et al., 1999). When the use of avoparcin was
strictly prohibited in animal husbandry, the prevalence of vancomycin-resistant Enterococci was
decreased in both animal samples and human volunteer samples (Bruinsma et al., 2003; Klare et
al., 1999). These examples show that there is a strong link between the use of antibioticsin food
production and the rate of antimicrobial resistance in people.

The common bacteria associated with wound infection are Staphylococcus

aureus/methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes, Enterococci,
Pseudomonas aeruginosa and Clostridium perfringens (Bessa et al., 2015).Staphylococcus
aureus belongs to the family Micrococcaceae and is part of the genus Staphylococcus, which
contains more than 30 species such as S. epidermidis, S. saprophyticus and S. haemolyticus.
Among the staphylococcal species, S. aureus is by far the most virulent and pathogenic for
humans. S. aureus is a 1 μm, Gram-positive cell that in the laboratory may be observed as single
cells, in pairs or as grape-like irregular clusters. It is characterized as coagulase- and catalase
positive, non-motile, non-spore-forming and as facultative anaerobic. It grows in yellow colonies
on nutrient rich media and is referred to as the yellow Staphylococci (Winn Washington, 2006).

Staphylococcus aureus was discovered in 1880 by the surgeon Sir Alexander Ogston. He
observed grape-like clusters of bacteria when examining a purulent discharge from patients with
post-operative wounds during microscopy. He named them staphylé, the Greek expression for a
bunch of grapes. In 1884, Rosenbach succeeded in isolating yellow bacterial colonies from
abscesses and named them Staphylococcus aureus, “aureus” from the Latin word for
golden.Staphylococcus aureus has the ability to adapt to different environments and it may
colonize the human skin, nails, nares and mucus membranes and may thereby disseminate
among recipient host populations via physical contact and aerosols (Lowy, 1998). Colonization
with S. aureus is an important risk factor for subsequent S. aureus infection (Wertheim et al.,
2004; von Eiff et al., 2001).

Staphylococcus aureus causes a wide range of infections from a variety of skin, wound
and deep tissue infections to more life-threatening conditions such as pneumonia, endocarditis,
septic arthritis and septicemia. This bacterium is also one of the most common species in

29
nosocomial infections. However, little is known about the virulence factors behind all these
conditions. In addition, S. aureus may also cause food poisoning, scalded-skin syndrome and
toxic shock syndrome, through production of different toxins (Winn Washington, 2006).

2.5. Hydroyapatite

Hydroxyapatite (HAp) nanoparticles (NPs) have received considerable attention over the
last decade due to their significant biological applications (Dizaj et al., 2018; Subramanian et al.,
2020). HAp plays a vital role in biomaterial engineering due to its enhanced biocompatibility and
bioactivity (Rajabnejadkeleshteri et al., 2020; Galindo et al., 2019). The stoichiometric ratio of
calcium and phosphorous in HAp is 1.67, which resembles that of human bones, and calcium
phosphate (CaP) is the major mineral constituent of vertebrae bones and teeth (Malmberg and
Nygren, 2008; Marten et al., 2010). HAp [Ca 10 (PO 4) 6 (OH) 2] is a CaP-based biomaterial
primarily used in dental and orthopedic applications because of its structural and chemical
similarity to bones, teeth, and enamel (Sanosh et al., 2010; Vijayalakshmi et al., 2008). HAp acts
as an implantation compound for biomedical application in the form of coatings and composites
on metallic alloys due to its various properties, including osteoinduction, osteoconductivity,
osteointegrity, and non- toxicity (Anjaneyulu et al., 2016). Due to the potentiality of a rigid
control of process parameters, a sol-gel approach is a powerful method for the synthesis of
nanophase HAp. HAp NPs synthesized via the sol-gel method can enhance stability and contact
at the natural/artificial bone interfaces in both in vitro and in vivo environments. Recent studies
have also demonstrated that HAp particles prohibit the growth of many cancer cell types (Hou et
al., 2009; Li et al., 2008). Synthetic HAp may also be applied in column chromatography for the
simple and rapid fractionation of nucleic acids and proteins (Jungbauer et al., 2004). Moreover,
HAp offers convenient qualities for water treatment and remediation for heavy metal
contaminated soils (Lin et al., 2009). Among the different HAp structures, nanosized HAp with
appropriate stoichiometry, purity, and morphology have stimulated enormous interest in
elemental scientific research and various biomedical applications (Sadat-Shojai, 2010).

2.5.1. Synthesis of Hydroxyapatite nanoparticles

HAp powders were synthesized via a sol-gel process using calcium nitrate and di-
ammonium hydrogen phosphate with a molar ratio of 1.67 as precursors. First, 1 M of calcium

30
nitrate was dissolved in 50 ml of de-ionized water. Similarly, 0.6 M of di-ammonium hydrogen
phosphate solution was prepared and mixed with a calcium nitrate solution under continuous
stirring. Then, the aqueous ammonia was added dropwise to the above solution until a pH value
of 10 was obtained. The reaction mixture was stirred for 5 h then maintained undisturbed for 24
h at ambient temperature. The obtained precipitate was filtered and washed with distilled water,
followed by ethanol and dried at 100 °C for 12 h to obtain white powder. Then, the dried
substances were crushed and calcined at 500 °C for 2 h. A similar procedure was adopted for the
other three samples with 50 ml of PEG (0.1 M), CTAB (0.1 M), and CE (50 ml).

2.5.2. Characterizations of Hydroxyapatite nanoparticles

The crystallographic structural analysis was carried out using the X-ray diffraction
(XRD) method using an XPERT-PRO powder diffractometer with monochromatic CuKα
radiation (λ = 1.5406 Å) over the 2θ range of 20°–80° at a scan rate of 2 m in a Goniometer. The
operational voltage and current were 40 kV and 30 mA, respectively. The Fourier-transform
infrared (FTIR) spectrum (Bruker IFS 48) was recorded within the range of 4000-400 cm−1
using a Micro Raman Spectrometer (LabRAM HR Evolution, HORIBA, France) and was used to
identify the functional groups present in HAp. The elemental analysis was conducted using an
energy dispersive X-ray spectroscopy (EDX) Bruker detector attached to a field emission
scanning electron microscope (FE-SEM) operating at an accelerating voltage of 20 kV. The
morphology of HAp NPs was analyzed using a QUANTA 250 FEG (FE- SEM). The
microstructures of the synthesized HAp NPs were investigated using a transmission electron
microscope (TEM) JOEL model instrument 1200 EX on carbon-coated copper grids with an
accelerating voltage of 80 kV. The optical properties of the samples were studied using a JASCO
650 UV Spectrophotometer. PL studies were carried out using a photoluminescence
spectrophotometer (Varian Cary Eclipse) and the emission spectra were recorded at a scan rate of
600 nm/min in the range of 320–600 nm.

2.5.3. Antibacterial activity of Hydroxyapatite nanoparticles

The antibacterial activity of the synthesized HAp NPs was determined using the well
diffusion method. This was performed by sterilizing Mueller Hinton Agar (MHA) media. After
solidification, wells were cut on the MHA plates using a cork borer and the test bacterial

31
pathogens were swabbed onto the surfaces of the MHA plates. The samples were placed in the
wells and the plates were incubated at 37 °C for 24 h. The zones of inhibition were measured in
millimeters. Each antibacterial assay was performed in triplicate and mean values were recorded.

2.5.4. Antioxidant activity of Hydroxyapatite nanoparticles

The DPPH (2,2-diphenyl-2-picrylhydrazyl hydrate) assay was conducted according to the

methods of Brand-Williams et al., 1995 with some modifications. The scavenging and
antioxidant potentials of the samples were evaluated by the ability of the samples to scavenge the
stable free radical DPPH. Different concentrations (100-400 μg/ml) of the samples as well as the
samples with NPs were separately mixed with 3 ml of 0.1 mM DPPH and incubated in darkness
for 15 min. Rutin was used as a standard and the DPPH methanol reagent without the sample
was used as the control. The reaction mixture was mixed well and maintained in darkness at 32
°C for 30 min. The degree of decolourization from purple to yellow was measured
spectrophotometrically at 517 nm.

32