K.divya mgr thesis (1)

ANTI-INFLAMMATORY ACTIVITY OF CHRYSOPOGON
ZIZANIOIDES HYDROALCOHOLIC EXTRACT
K. DIVYA

ABSTRACT
Chrysopogon zizanioides which have long been known to be avery important source of
pharmaceutical properties. The present study we examined the anti inflammatoryeffect of
Chrysopogon zizanioidesextract by albumin denaturation and proteinase inhibition assay. The
anti-inflammatory properties of Chrysopogon zizanioideswere studied at 500, 250, 100, 50 and 5
μg/mL using an albumindenaturation assay and proteinase inhibition assay. The results showed
that the anti-inflammatory activity shows a strong inhibition at 500 µg/ml and the IC 50 value
was found to be 101 μg/ml, and proteinase 156.2 μg/ml respectively. This study provides an
evidence that the Chrysopogon zizanioidespossesses anti-inflammatory activity in ivtro asnalysis.
CONTENTS

S.NO TITLE PAGE NO
1 INTRODUCTION
2 OBJECTIVE OF THE WORK
3 REVIEW OF LITERATURE
4 METHODOLOGY
5 RESULTS AND DISCUSSION
6 BIBLIOGRAPHY
1. INTRODUCTION

Inflammation is a normal, protective response to tissue injury caused by physical trauma,
noxiouschemicals or microbiological agents. There aremainly two types of inflammation which
are asfollowsacute inflammationIt is associated with increased vascularpermeability, capillary
infiltration and emigrationof leukocytes.Chronic inflammationit is associated with infiltration of
mononuclearimmune cells, macrophages, monocytes,neutrophils, fibroblast activation,
proliferation(angiogenesis) and fibrosis.Inflammation is a common clinical conditions
andrheumatoid arthiritis (RA) is a chronic debilitatingautoimmune disorder (Nadkarni 2000),
that affects about 1% of thepopulation in developed countries (Cardinali and Esquifino
2003).The classicsigns of inflammation are local redness, swelling,pain, heat and loss of function
(Pervical 1999). Nitric oxide (NO) implicated as a mediator of inflammation andmodulation of
biosynthesis or activity of NO results in amelioration of acute inflammation andexperimental
arthiritis model (Daniel 1998, Zumora and Billar 2000). NO is generatedvia the oxidation of the
terminal guanidine nitrogenatom of L-arginine by the enzyme Nitric OxideSynthase (NOS).
Three major isoforms of NitricOxide Synthase (NOS) have been identified.Twoexpressed
constitutively, are calcium/calmodulindependentand are classified together asconstitutive NOS
isoforms (cNOS). The third is cytokine-inducible, calcium/calmodulinindependentisoform of
NOS (iNOS) is regulatedinthe gene by a variety of inflammatory mediators6.Increased NOS
activity or NO releasehave beendemonstrated in both acute and chronic models ofinflammation
(Mederos 1995). Further, administration of Largininea precursor for NO synthesis increased
thepaw swelling in adjuvant arthiritis (Corbett 1991). NSAIDS areamong the most commonly
used drugs worldwide.They are prescribed for orthopaedic conditionssuch as osteoarthritis, soft-
tissue injuries andfractures etc (Malizos 2009).

The magnitude of global plant diversity is estimated at more than 500,000 species
(Corlett 20116, Lauranceet al., 2012) and thevariety and complexity of plant metabolites
represent a challenge when considering exploration of thechemical repertoire odder. From this
point of view, the Plant Kingdom has been pragmatic, especiallywhen these molecules are
reported as substances with the high medicinal potential to treat diseasesthat affect living beings
(Souzaet al., 2013).Medicinal plants continue to be an interesting source of natural products for
treating varioushealth conditions. It is estimated that more than 150,000 plant species have been
studied, many ofwhich contain valuable therapeutic agents, and the applications of novel
compounds from plants forpharmaceutical purposes have been gradually increasing in recent
years (Shazhniet al., 2018, Caoet al., 2018).
Free radicals produced fromoxygen to form reactive oxygen speciessuch as the singlet
oxygen, superoxide,peroxyl, hydroxyl and peroxynitriteradicals, are constantly produced
withinliving cells for specific metabolicpurposes (Waffo-Teguoet al., 2008). Living cells have
complexmechanisms that act as antioxidantsystems to counteract the damaging effectsof reactive
species.
Oxygen radicals induceoxidative stress that is believed to be aprimary factor in various
diseases as wellas normal process of ageing. However;there have been concerns about
syntheticantioxidants such as butylatedhydroxyanisole (BHA) and butylated hydroxytoluene
(BHT) because of theirpossible activity as promoters ofcarcinogenesis (Atiqur Rahmanet al.,
2011). Ionidiumsuffruticosum(Ging.) (Syn: Hybanthus enneaspermus) itbelongs to the family
Violaceae knownsas Orilaitamarai, Amburuha, Charati. It is animportant plant in the Indian
system ofmedicine.

It is well known how the composition of a plant extract can vary considerably according
to several factors, including the conservation procedure. In this regard, it appears that the work
by Gabbianelli et al. on Nigella sativa oil obtained from a cultivar produced in the region Marche
is of particular interest. The authors demonstrated that its thymoquinone content is higher than
that from other cultivars in the Mediterranean area, Asia, and Indonesia. More interestingly, they
showed how storage may affect thymoquinone content as well as the anti-inflammatory and
antioxidant effects of the oil. Finally, they also demonstrated that both the antioxidant and anti-
inflammatory effects of the oil does not simply rely on thymoquinone content, but also on other
oil components that may exert synergistic effects.
A novel chemical investigation came from the work of Kukula-Koch et al. on the
flavonoid constituents of Crataegus almaatensis leaves. The authors found that the three main
flavonoids (hyperoside, quercitrin, and afzelin) optimized their isolation technique and evaluated
extracts for their vascular, anti-nociceptive, and anti-inflammatory effects, in vitro and in vivo.
In light of the current interest on the alternative use of vegetal wastes, the study from
Catoi et al. investigated 10 tomato varieties in terms of carotenoids content, phenolic
composition, and their related antioxidant and antimicrobial activities. Taking into account the
results obtained, the authors concluded that tomato industrial by-products may represent a source
of natural bioactive molecules with applicability in the nutraceuticals and food industries.
Notwithstanding its nutritional role and properties, meat has been recently linked to an
increased incidence of non-communicable diseases, such as colorectal cancer. In the study from
Nieto et al., authors aimed to evaluate the inflammatory role of processed meat and develop a
functional meat product enriched in natural extracts with antioxidant and anti-inflammatory
properties. The authors found that cooked ham that is low in fat and salt had antioxidant and anti-

inflammatory properties related to the inhibition of substances involved in gut inflammation,
such as reactive oxygen species, nitric oxide, and cytokines produced by macrophages. These
health-promoting inhibitory properties were significantly increased when the cooked ham was
enriched with bioactive phytochemicals. Therefore, the authors proposed that incorporating
botanical extracts rich in phenolic compounds (chlorogenic acid, catechins, rosmarinic acid, and
hydroxytyrosol) in cooked ham is a good strategy to produce a healthy functional meat product.
Studies, on the health-promoting effects of green tea have typically focused on catechins.
Unlike catechins, green tea flavonols have received little attention with respect to their usage and
functionality. In their paper, Kim et al. investigated the antioxidative, anti-inflammatory, and
antiproliferative effects of flavonol glycoside (FLG)- and flavonol aglycone (FLA)-enriched
fractions isolated from green tea extract. These fractions contained 16 and 13 derivatives,
respectively, including apigenin, kaempferol, myricetin, and quercetin. FLA exhibited higher
radical-scavenging activity than that of FLG. Interestingly, both attenuated intracellular oxidative
stress in neuron-like PC-12 cells significantly reduced inflammatory genes in murine
macrophages and inhibited proliferation of both the colon adenoma cell line DLD-1 and the
breast cancer cell line E0771. Moreover, treatment with FLG or FLA combined with paclitaxel
had synergistic anticancer effects on the DLD-1 cell line. The authors concluded that flavonols
from green tea may exert beneficial health effects that may be superior to those of flavan-3-ols.
In light of the, so far, underexplored properties of Salvia apiana and Salvia farinacea,
particular interest comes from the work by Cardoso et al., which demonstrates significant
antioxidative and anti-inflammatory effects of these two Salvia extracts, which also exert
cytotoxic and antimicrobial effects.

The accumulation and concentration of phytochemicals in different parts and organs of
plants can considerably vary with regard to the function of these compounds in a plant’s lifecycle
and growth phase. The work of Maslennikov et al. has investigated the accumulation of phenolic
compounds and the antioxidant activity of extracts from different parts of Rumex
crispus and Rumex obtusifolius (roots, stems, leaves, and reproductive organs), collected at both
the flowering and fruiting stages. The maximum number of phytochemicals was found in the
reproductive organs, both in the flowering and fruiting period. On the contrary, stems showed a
minimum content of active compounds [17].
The valorisation of agrochemical wastes has been widely recognized as a crucial way to
reduce the health spending and improve the accessibility of bioactive natural compounds. In line
with this, Aiello et al. have investigated the chemical composition of three Glycyrrhiza
glabra leaf extracts, as well as the anti-oxidative and anti-inflammatory profile of their three
main components, i.e., pinocembrin, glabranin, and licoflavanone. Interestingly, the authors
demonstrated a modulation of NF-κB/MAPK pathways by licoflavanone, highlighting the
potential of this natural compound as a new scaffold in anti-inflammatory drug research [18].
Hyperglycemia-induced oxidative stress fosters severe vascular damage and induces an
inflammatory state strictly implicated in the etiopathogenesis of atherosclerosis. In the work by
Kim et al., Carpinus turczaninowii extract has been demonstrated to attenuate high glucose-
induced inflammation and arterial damage. Relevantly, authors provided mechanistic insights of
the protective effects observed, as well as the identification of 15 types of phenolic compounds
present in the extract (including quercetin, myricitrin, and ellagic acid) that exhibit antioxidant
and anti-inflammatory properties.

Anji white tea is a unique variety of green tea rich in polyphenols. In the work by Zhao et
al., the effect of Anji white tea polyphenols on the prevention of carbon tetrachloride-induced
liver injury was investigated. Interestingly, the extract effectively counteracted liver injury
through a redox-, anti-inflammatory-, NF-κB-dependent mechanism with an efficacy comparable
to that of silymarin.
In light of the previously reported data on the antioxidant and anti-inflammatory
properties of a crude oil extracted from the seeds of Nigella sativa produced in the Marche
region, this second work from Gabbianelli et al. has evaluated its analgesic and anti-
inflammatory potential in a rat, adjuvant-induced arthritis model. Interestingly, at the same time,
the authors evaluated its safety profile in vivo. Results showed that the extract was effective in
the control of acute phase inflammation and prevented the development of rheumatoid arthritis
(RA). On the other hand, it was not able to ameliorate the severity of arthritis in the chronic
phase. Interestingly, despite the positive effects on the clinical signs of RA, the extract did not
reduce plasma IL-6 levels, whereas it decreased plasma total cholesterol levels, strongly
suggesting a potential control of cholesterol metabolism.
Ionidium suffruticosum Ging (Violaceae) is a sporadic, rare, ephemeral, ethnomedicinal
herb (Deshpande 2006) widely used in traditional healers to treat the diseases such as jaundice,
male sterility (Kheraro & Bouquet 1950; Senthil Kumar et al. 2013), diabetes (Sarita et al. 2004),
malaria (Soh & Benoit-Vical 2007), urinary tract infections and water retention (Pushpangadan
& Atal 1984) and also used as a tonic. The fruits are antidote for scorpion-sting. The leaves are
sub-sessile, liner to oblanceolate, 1.5–2.0 0.08–0.3 inches, entire or serrate, flowers solitary,
axillary, red, spurred, fruit a small sub globose capsule containing ellipsoid, longitudinally
striate, yellowish white seeds. The leaves and tender stalks are demulcent and used as a

decoction or electuary, in conjunction with oil, employed in preparing cooling liniment for the
head. The plant is a seasonal, perennial herb and is widely distributed in Africa, Madagascar,
Srilanka, China, New Guinea, tropical Australia and India. In nature, the plants appear for only a
few months from June to September (monsoon season). The aim of the present study is to
investigate the anti-inflammatory effect ofchrysopogon zizanioideshydroalcoholic extract by
albumin denaturation assay.
Work flow of the present study

2. OBJECTIVE OF THE WORK
Collectionof Chrysopogon zizanioides plant
Extractionof Chrysopogon zizanioides by
hydroalcoholic extractionusing Soxhlet
apparatus
Anti-inflammatory analysis of Chrysopogon
zizanioideshydroalcoholicextract by albumin
denaturation assay
Anti-inflammatory analysis of
Chrysopogon zizanioides hydroalcoholic
extract by protease inhibition assay
Statisticalanalysis using GraphPadPrism
software

• The aim of the present study was to analyze the anti-inflammatory effect of chrysopogon
zizanioidesextract using the albumin denaturation and proteinase inhibition assays
Objectives
1. Collection of chrysopogon zizanioides
2. Soxhlet extraction of chrysopogon zizanioides
3. Anti-inflammatory effect of chrysopogon zizanioidesby albumin denaturation assay
4. Anti-inflammatory analysis of chrysopogon zizanioidesextract by proteinase inhibition
assay
5. Statistical analysis of anti-inflammatory activity using GraphPad Prism software
3. REVIEW OF LITERATURE
3.1.Inflammation

Inflammation, which means, “to set on fire” is a body’s natural response against harmful
pathogen and stimuli that occurs in two stages namely, acute and chronic inflammation. Acute
inflammation is a part of innate immunity initiated by the immune cells that persists only for a
short time. However, if the inflammation continues, the second stage of inflammation called
chronic inflammation commences which instigates various kinds of chronic diseases, including
arthritis, cancer, cardiovascular diseases, diabetes, and neurological diseases via dysregulation
of various signaling pathways such as nuclear factor kappa-B (NF-κB), signal transducer and
activator of transcription 3 (STAT3) etc. Hence, targeting the inflammatory pathways has high
potential in preventing and eradicating these deadly diseases. However, most of the drugs
developed till today for the treatment of chronic diseases are highly expensive and associated
with adverse side effects. Therefore, there is an urgent need to develop novel, safe, affordable,
and highly efficacious agents for the management of these diseasesPavord et al. 1999; Little et
al. 2000.
Congregate evidence suggests that a diet rich in plant-based agents including spices has
the ability to prevent most of the chronic diseases. The earliest evidence of the use of spices by
humans dates back to 5000 B.C., and till today their biological activities have been extensively
studied. “Spice” originates from the Latin word, “species”, which means a commodity of special
distinction or value. Spices have been extensively used since ancient times as means of remedy,
coloring agent, flavoring agent, and preservative. Subsequently, tremendous studies have shown
that nutraceuticals derived from spices such as clove, coriander, garlic, ginger, onion, pepper,
turmeric, etc., remarkably prevent and cure various chronic diseases by targeting inflammatory
pathways. This review emphasizes the association between inflammation and chronic diseases
and the benefits of spices in warding off these global major health issues.

3.2.Chronic diseases with inflammation
Chronic diseases are the leading cause of mortality in the world accounting for
approximately 60% of all deaths. Aforementioned, various inflammatory biomarkers are altered
in chronic diseases such as transcription factors (NF-κB, STAT3) and their downstream products
such as inflammatory cytokines (TNF-α, IL-1, IL-6, IL-8) and pro-inflammatory enzymes such
as COX-2, MMP-9, cell adhesion molecules (CAM), vascular endothelial growth factor (VEGF)
etc.
Amongst the chronic diseases, cancer is one of the major diseases caused by chronic
inflammation. In 2009, Colotta et al. proposed inflammation as the seventh hallmark of cancer.
Both inflammation and cancer are linked through intrinsic and extrinsic pathways i.e. oncogenes
regulate the inflammatory microenvironment intrinsically, whilst the inflammatory
microenvironment facilitates the development and progression of cancer extrinsically.
Specifically, the inflammatory response positively aids in tumor development and increases the
risk of malignancy. Approximately 15% of the cancer cases are caused by persistent infection
and chronic inflammation. It has been well established that NF-κB is constitutively activated in
various cancers such as cancers of the breast, colon, liver, lung, pancreas etc. in response to
carcinogens such as tobacco, alcohol, and exposure to radiation etc. Upregulation of NF-κB
subsequently activates hundreds of pro-inflammatory gene products including TNF-α, IL-1, IL-6,
chemokines, MMP-9, 5-LOX, VEGF, and COX-2. These pro-inflammatory cytokines play
a vital role in inflammation-induced cancer cell proliferation, angiogenesis, invasion, metastasis,
and suppression of apoptosis. In addition, even in cancers that are not instigated by
inflammation, inflammatory cells enter the tumor stroma and consequently induce cancer
development. More importantly, an in vivo study has illustrated that NF-κB activation via the

IκB kinase (IKK) complex acts as a molecular link between inflammation and cancer. Moreover,
NF-κB activation also leads to radioresistance and chemoresistance. These observations suggest
that NF-κB plays an important role in inflammation and cancer. Therefore, anti-inflammatory
agents that target NF-κB and its regulated products may have high efficacy in both the
prevention and treatment of cancers.
Aforesaid, unresolved inflammation of the pancreas, pancreatitis leads to pancreatic
cancer. It has been demonstrated that O-GlcNAc transferase (OGT)—mediated O-
GlcNAcylation activated NF-κB signaling pathway and inflammation in pancreatic acinar cells,
ultimately leading to the progression of acute pancreatitis. T helper cell-mediated inflammation
also has been found to be associated with pancreatic β-cell dysfunction and leads to chronic
pancreatitis. COPD is an epidemic chronic inflammatory disease of the lung. Interleukin-33
enhances the production of the inflammatory cytokine such as IL-6 and IL-8 in chronic airway
inflammation, thus contributing to COPD development. It has also been reported that
inflammatory responses in COPD promote lung tumor initiation and progression. Another
inflammation induced chronic disease is rheumatoid arthritis (RA) which is an autoimmune
disease characterized by the production of the pro-inflammatory cytokine IL-17. Studies
suggested that pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α also play pathological
roles in the development of RA. In addition, it has been demonstrated that STAT3 also caused
chronic inflammation and joint destruction in RA. Hence, targeting inflammatory pathways can
be used for the prevention and treatment of RA.
In Alzheimer’s disease (AD), which is the prevalent chronic neurodegenerative disease,
inflammation has an essential role in the disease pathogenesis. Studies have indicated that
microRNAs, astrocytes, microglia, and infiltrating immune cells from the peripheral region

might affect the development of neuroinflammation and neurodegeneration in AD patients [37].
Accumulated evidence has depicted that deposition of extracellular amyloid beta (Aβ) in AD
leads to upregulation of pro-inflammatory mediators IL-1β, IL-6 and TNF-α, by the activated
immune cells, which promote additional inflammatory pathways via instigation of COX-2 and
NF-κB.
Inflammatory bowel disease (IBD) is a group of inflammatory disorders of the digestive
tract, which mainly includes Crohn’s disease and ulcerative colitis. Studies have shown that IBD
patients have high susceptibility to develop colorectal cancer. Inflammatory mediators including
cytokines (TNF-α, IL-1β, IL-6, IL-17, and IL-21), eicosanoids, and reactive oxygen metabolites
play a vital role in causing the chronic inflammatory condition in IBD. In addition, activation of
STAT3 signaling pathway is associated with colitis and colorectal cancer.
Allergic asthma is an airway inflammatory disease caused due to exposure to allergens
causing bronchoconstriction. Asthma is characterized by an imbalance between the T helper type
1 (Th1) and T helper type 2 (Th2) responses and excessive production of reactive oxygen species
(ROS). Th2 cells release several cytokines such as IL-4 and IL-13 that in turn produces
immunoglobulin, IgE resulting in allergic response. Numerous studies also indicate that
attenuation of the Type 2 inflammatory pathway caused a clinically substantial reduction in
asthma exacerbations. Thus, it is now evident that type 2 inflammation is an imperative
mechanism of susceptibility to asthma exacerbation.
Diabetes mellitus (DM) is a predominant metabolic chronic disease that affects more than
170 million people globally. Type 1 DM is induced by the chronic inflammation of pancreatic
islets, while type 2 DM is associated with insulin resistance resulting in elevated production of

inflammatory markers such as C-reactive protein (CRP), IL-6, and TNF-α. Patients with type 2
diabetes have a higher chance of developing atherosclerosis, which is a disease wherein plaque
accumulates in arteries. Arachidonic acid derived eicosanoids such as prostaglandin E2 (PGE2)
and leukotriene B4 (LTB4) are the potential pro-inflammatory mediators in atherosclerosis and
are regulated by NF-κB.
Collectively, it is apparent that dysregulation of inflammatory pathways is the underlying
mechanism of various chronic diseases. Therefore, many drugs have been developed that target
inflammatory pathways for the management of these diseases. However, most of these drugs
developed so far are highly expensive and are not devoid of adverse side effects. Hence, there is
an urgent need to develop safe, affordable, and efficacious drugs for the prevention and treatment
of these chronic diseases. It has been well established that the population who consume spices
are less susceptible to the development of chronic diseases. The components present in these
spices have the ability to inhibit inflammatory pathways that lead to chronic inflammation, which
contributes to the biological properties of these spices.
3.3. Anti-inflammatory effect of herbal plants
Mother nature has bestowed us with a profuse source of remedies to treat various kinds of
ailments. Since time immemorial, phytochemicals, both in their natural as well as synthetic
forms have been used for the treatment of various chronic diseases. The root, leaf, bud, seed,
bark, berry, stigma of a plant or flower used for the culinary purpose are generally called as
spices. Spices not only add flavor and taste to food, but also exhibit tremendous health benefits.
Numerous results from preclinical and clinical studies over the past several decades have
ascertained the efficacious role of spices and their active components in preventing and

combating various diseases including arthritis, asthma, cancer, cardiovascular
diseases, diabetes, and neurodegenerative diseases. The most commonly used spices for culinary
purpose that shows biological activities are black pepper, cardamom, cinnamon, clove, cumin,
fenugreek, fennel, garlic, ginger, onion, rosemary, turmeric etc.
Turmeric (Curcuma longa) is the most commonly used spice in the world. Curcumin, the
main component of turmeric (2–5%), obtained from rhizomes of this plant, is a yellow colored
compound, which gives the golden color to turmeric, was first isolated by Vogel in 1842. In
1910, the structure of curcumin was determined as diferuloylmethane and later synthesized and
cocrystallized with 5-LOX in 2003. This ‘golden spice’ is recognized for its anti-inflammatory,
antimicrobial, insecticidal, antimutagenic, radioprotective, and anticancer properties. Over ten
thousand studies have been reported in the literature about the biological activities of this
compound including more than 120 clinical trials.
Besides curcumin, the other active components of turmeric include demethoxycurcumin,
bisdemethoxycurcumin, sesquiterpenes, diterpenes, triterpenoids. Black pepper (Piper nigrum),
another commonly used spice is widely known for its immunomodulatory, anti-oxidant, anti-
asthmatic, anti-carcinogenic, anti-inflammatory and anti-ulcer properties. Other than its main
component piperine, black pepper also contains β-caryophyllene, limonene, δ-3-carene, α-
pinene, β-pinene, α-phellandrene, myrcene, terpinolene, etc.. Another extensively used spice,
ginger (Zingiber officinale) is reported to have different biological properties such as antioxidant,
anti-inflammatory and antiproliferative properties. 6-gingerol is the main component of this
spice, which is responsible for its biological properties. Other than gingerol, ginger also contains
6-paradol, 6-gingerdiol, gingerdione, shogoal, zingiberene, citral (neral and geranial), bisabolene,
cineol, α-farnesene, β-phellandrene, zingerone etc.. The most commonly used spice for

cardiovascular diseases in the ancient system of medicine is garlic (Allium sativum). It also
possesses anti-inflammatory, gastroprotective and anti-cancer properties due to the presence of
phytochemicals such as diallyl sulfides, diallyl disulfides, ajoene, allicin, alliin, diallyl trisulfide,
S-allylcysteine, methiin, isoalliin, cycloalliin, S-allylmercaptocysteine.
Another spice that is widely used all over the world to enhance the spice level of dishes is
red pepper (Capsicum). Apart from capsaicin, red pepper also contains β-carotene, zeaxanthin,
lutein, caffeic acid and capsanthin. The other commonly used spices and their active components
include cardamom (1,8-cineole, α-terpinyl acetate, limonene, linalool, linalyl acetate, terpinolene
and myrcene); cinnamon (cinnamaldehyde, cinnamyl acetate, cineole, coumarin, ethyl
cinnamate, linalool, humulene, β-caryophyllene, τ-cadinol); clove (eugenol); fenugreek
(diosgenin, yamogenin, choline, resins, trigonelline); black cumin (thymoquinone,
cuminaldehyde, γ-terpinene, β-pinene, p-mentha-1, 3-diene-7-al, p-mentha-1, 4-dien-7-al, p-
cymene); kokum (garcinol, xanthochymol, isoxanthochymol, 1,2-dihydroxypropane-1,2,3-
tricarboxylic acid); rosemary [bornyl acetate, rosmarinic acid, carnosol, carnosic acid, camphor,
limonene, camphene, borneol, cineole, α-pinene, (Z)-linalool oxide]; saffron (crocetin and
crocin); star anise (estragole, trans-anethole, limonene) etc. Hence, it is evident that spices
contain a diverse range of active components that provide tremendous health benefits
Senthil kumar and vijaya kumar 2017 reported that the antimicrobial and antioxidant
activity of Cycas circinalis (Cc) and Ionidium suffruticosum (Is). The Cc showed 0.7 mm of the
zone of inhibition for Staphylococcus aureus in culture, whereas Is showed 1 mm and 0.4 mm of
the zone of inhibition for Escherichia coli and Salmonella typhi, respectively. The rest of all
species were nonreactive for both the herbal extracts. The IC50 values of Is at various
concentrations were 95.6, 96.0, and 97.6, respectively, whereas, for Cc, it was 78.7, 85.9, and

87.5, respectively. The IC50 value of IS shows, that the herb has more antioxidant property when
compared to that of Cc and standard (ascorbic acid).
The antioxidant and anti-inflammatory potential (inhibitoryeffect on pro-inflammatory
enzymes) of two hydroalcoholic extracts of F. triandra obtained by macerationand soxhlet
extraction. Both extracts showed similar biological activity and composition (mainly
polyphenoliccompounds, flavonoids and phenolic acids). They exhibited an important
antioxidant activity by scavengingABTS+ and nitrite radicals, by inhibiting β-carotene bleaching
and reducing Fe3+. Antioxidant properties arerelated to anti-inflammatory capacity, and both
extracts showed a significant inhibition of hyaluronidase andtwo enzymes of arachidonic acid
pathway (cyclooxygenase-2 and lipoxygenase) (Torres Carroet al., 2016).
Chenet al., 2016 reported that the ability of C. asiatica ethanol extract (CAE) to protect
PC12 and IMR32 cells from Ab1e40-induced production ofreactive oxygen species (ROS) and
concomitant neurotoxicity. Aggregated Ab1e40 treatment resulted inreduced cell viability, which
can be reversed by cotreatment with 25, 50, and 100 mg/mL CAE. Moreover,CAE eliminated the
Ab1e40-mediated increase in ROS production. Thus, CAE-mediated protection
againstaggregated Ab1e40-induced neurotoxicity is attributable to modulation of the
antioxidative defensesystem in cells, including the activities of superoxide dismutase, catalase,
glutathione peroxidase,glutathione reductase, and levels of glutathione and glutathione disulfide
by CAE. This emphasizes thepotential therapeutic and preventive value of CAE in the treatment
of AD.
The therapeutic anti-inflammatory action of NSAIDs is produced by the inhibition of
COX-2, while the undesired side effects arise from inhibition of COX-1 activity. Thus, it was

though those more selective COX-2 inhibitors would have reduced side effects. Based upon a
number of selective COX-2 inhibitors (Rofecoxib, Celecoxib etc.) were developed as safer
NSAIDs with improved gastric safety profile. Several newer applications like prophylaxis of
stroke with aspirin are now common place. Use of these drugs for the prophylaxis of conditions
like Alzheimer’s disease and colorectal cancer is being evaluated. Unfortunately, they have
several toxicities ranging from minor heartburn to severe gastrointestinal haemorrhage and
perforation. Therefore, newer NSAIDs has been introduced in recent years to circumvent this
problem (Catella-Lawsonet al., 2001).
Shaikhet al., 2016 reported that the sequentially extracted plant samples as,Cissus
quadrangularis, Plumbago zeylanica, Terminalia bellarica and Terminalia chebula in water,
ethanoland hexane were evaluated in-vitro for COX-1 and 2 inhibitory and antioxidant activities.
The in vivoanti-inflammatory activity of selected samples showing promising COX-2 inhibition
was assessed usingcarrageenan and Phorbol Myristate Acetate (PMA) induced mice edema
animal model. The results obtainedreveals that most of the plants were found to inhibit COX-2
activity as compared to COX-1. It wasobserved that the extracts of T. bellarica (73.34 %) and T.
chebula (74.81 %) showed significant COX-2selective inhibition as compared to other samples.
The ethanol extract of the selected plants demonstratedeffective DPPH, OH and superoxide
radical scavenging activity. In vivo anti-inflammatory studyshows that, T. bellarica and T.
chebulla had a significant impact on inhibition of edema formation. Thecytotoxicity evaluation
study of ethanolic fraction of selected medicinal plants indicates that the selectedsamples have
no effect on cell viability. HPTLC fingerprint of flavonoids of the selected samples was
alsoprepared as a measure of quality control.

To compare the pre-emptive use of a cyclooxygenase-2 (COX-2) inhibitor with a well
establishedsteroidal anti-inflammatory drug for pain and edema relief following periodontal
surgery for crown lengthening. Thirty patients requiringperiodontal surgery were randomly
assigned to receive one of the following medications: selective COX-2 inhibitor or steroidal anti-
inflammatory drug, 60 min before the surgical procedure. To examine patient anxiety, a Corah’s
dental anxiety scale was appliedbefore surgery. Using a visual analog scale, the extent of
pain/discomfort during the trans-operative period and immediately after thesurgery was
measured. Additionally, intensity of pain/discomfort and edema were examined 4, 8, 12 and 24 h
postoperatively. Withregard to anxiety, no statistical differences between the groups were
observed (p>0.05). With respect to the extent of pain/discomfortduring the trans-operative,
immediate and late postoperative period, data demonstrated no significant differences (p>0.05)
between theCOX-2 inhibitor and steroidal groups. With regard to edema, intragroup analysis did
not reveal any statistically significant difference(p>0.05) during the 24 h following surgery in
either group (Mariaet al., 2012).
3.4.Types of inflammation
Onget al., 2007 reported that the Non-steroidal anti-inflammatory drugs (NSAIDs)
including both traditional non-selective NSAIDs and the selective cyclooxygenase (COX)-2
inhibitors, are widely used for their anti-inflammatory and analgesic effects. NSAIDs are a
necessary choice in pain management because of the integrated role of the COX path way in the
generation of inflammation and in the biochemical recognition of pain. NSAIDs are the
competitive inhibitors of cyclooxygenase (COX), the enzyme which mediates the bioconversion
of arachidonic acid to inflammatory prostaglandins (PGs). Their use is associated with the side
effects such as gastrointestinal and renal toxicity. They are the most commonly employed first

line drugs for all these conditions and many others-like musculoskeletal trauma, minor aches and
pains, and dysmenorrhoea. The therapeutic anti-inflammatory action of NSAIDs is produced by
the inhibition of COX-2, while the undesired side effects arise from inhibition of COX-1 activity.
Unfortunately, they have several toxicities ranging from minor heartburn to severe
gastrointestinal haemorrhage and perforation.
Krishnanet al., 2020 reported that the medicinal plants and theirimportant constituents
like phytochemial compounds for arthritis treatment beyond thecommercial use of modern
medicines. This review highlights the importance of various medicinalplants like Withania
somnifera, Boswellia serrata, Curcuma longa, Piper nigrum, Commiphoramukul, Zingiber
officinale, Pongamia pinnata, Betula platyphylla and its therapeutic applications.Active
compounds present in medicinal plants used for treating arthritis, its mode of action on cells and
proteins and the role of nanocarriers for delivery these active phyto compounds into the target
site.
Muniappan Ayyanar and Savarimuthu Ignacimuthu 2009 reported that the present
communication constitutes a review on the medicinal properties, ethnomedicinal uses and
pharmacological activities of five common medicinal plants (Acalypha indica L., Achyranthes
aspera L., Adhatodavasica Medicus, Coriandrum saticumandCentella asiatica) used in Indian
traditional medicine. These plants are known to contain various active principles of therapeutic
value and to possess biological activity against a number of diseases.
3.5. Causes of anti inflammation
Punchardet al., 2004 reported that the Inflammation covers the full range of underlying
cellular and molecular mechanisms involved, not only in the production of the inflammatory

responses but, more importantly in clinical terms, in the healing process as well. This includes
molecular, cellular, animal and clinical studies related to the study of inflammatory conditions
and responses, and all related aspects of pharmacology, such as anti-inflammatory drug
development, trials and therapeutic developments.
Five cardinal signs, there is actually just one specific macroscopic sign of inflammation,
namely localized edema.Further, the driving force for tissue fluid accumulation is defined in
biochemical terms and as such taken for the definitionof the inflammatory process. Inflammation
may be defined as a degenerative process which is intense enough to causelocal accumulation of
low molecular weight catabolic products, which in turn elevates tissue osmotic pressure that
attractsextra fluid, with or without heat release sufficient for significant elevation of tissue
temperature. This process is in a sharpcontrast to the pathogenesis of burns, where externally
applied heat causes a process that is in essence opposite toinflammation, bearing only some
superficial similarities with the latte (Srdan and Stankov 2012).
Inflammation is a biological response of the immune system that can be triggered by a
variety of factors, including pathogens, damaged cells and toxic compounds. These factors may
induce acute and/or chronic inflammatory responses in the heart, pancreas, liver, kidney, lung,
brain, intestinal tract and reproductive system, potentially leading to tissue damage or disease.
Both infectious and non-infectious agents and cell damage activate inflammatory cells and
trigger inflammatory signaling pathways, most commonly the NF-κB, MAPK, and JAK-STAT
pathways (Chenet al., 2017).
3.6. Types of inflammatory mechanism

The therapeutic anti-inflammatory action of NSAIDs is produced by the inhibition of
COX-2, while the undesired side effects arise from inhibition of COX-1 activity. Thus, it was
though those more selective COX-2 inhibitors would have reduced side effects. Based upon a
number of selective COX-2 inhibitors (Rofecoxib, Celecoxib etc.) were developed as safer
NSAIDs with improved gastric safety profile. Several newer applications like prophylaxis of
stroke with aspirin are now common place. Use of these drugs for the prophylaxis of conditions
like Alzheimer’s disease and colorectal cancer is being evaluated. Unfortunately, they have
several toxicities ranging from minor heartburn to severe gastrointestinal haemorrhage and
perforation (Jahnaviet al., 2019).
Pundet al., 2010 reported that to inhibit COX-2 activity as compared to COX-1. It
wasobserved that the extracts of T. bellarica (73.34 %) and T. chebula (74.81 %) showed
significant COX-2selective inhibition as compared to other samples. The ethanol extract of the
selected plants demonstratedeffective DPPH, OH and superoxide radical scavenging activity. In
vivo anti-inflammatory studyshows that, T. bellarica and T. chebulla had a significant impact on
inhibition of edema formation. Thecytotoxicity evaluation study of ethanolic fraction of selected
medicinal plants indicates that the selectedsamples have no effect on cell viability. HPTLC
fingerprint of flavonoids of the selected samples was alsoprepared as a measure of quality
control.
Human autoimmune diseases (AD) occur frequently (affecting in aggregate more than
5% of the population worldwide), and impose a significant burden of morbidity and mortality on
the human population. AD are defined as diseases in which immune responses to specific self-
antigens contribute to the ongoing tissue damage that occurs in that disease. ADs may be either
tissue-specific (e.g., thyroid, β-cells of the pancreas), where unique tissue-specific antigens are

targeted, or may be more systemic, in which multiple tissues are affected, and a variety of
apparently ubiquitously expressed autoantigens are targeted. The development of autoimmune
diseases depends on a combination of genetic and environmental factors. Most autoimmune
diseases are thought to be polygenic, involving more than one gene. For clinicians, autoimmune
diseases appear to be either systemic (e.g. systemic lupus erythematosus) or organspecific (e.g.
Type 1 diabetes mellitus. A more useful division distinguishes between diseases in which there is
a general alteration in the selection, regulation or death of T cells or B cells and those in which
an aberrant response to a particular antigen, self or foreign, causes autoimmunity. Antigens are
taken up by antigen presenting cells (APC) such as dendritic cells (DC) and processed into
peptides which are loaded onto MHC molecules for presentation to T cells via clonotypic T cell
receptors (TCR) (Shashi Pratab Singhet al., 2016).
3.7. Controlling methods of inflammation
Jarvandiet al., 2012 reported that the type 2 diabetes is associated with low-grade
systemic inflammation, increasing the risk for various adverse health outcomes. Stratified
logistic regression showed that for both men and women, BMI was a strong predictor of elevated
CRP after adjusting for age, energy intake, race/ethnicity, medications, diabetes duration, and
glycosylated hemoglobin. However, among men, but not among women, the likelihood of
elevated CRP increased with lower diet quality and physical inactivity.
Van et al., 2008 reported that the inflammatory response to injury or another trigger is
necessary, chronic diseases, such as coronary heart disease and diabetes, may develop because of
unchecked inflammatory responses that have maladapted over decades. For example, the earliest
changes in atherosclerosis occur in the endothelium, leading to a cascade of inflammatory

responses, such as accumulation of monocytes and T cells, migration of leukocytes into the
intima, monocyte differentiation and proliferation, and lesion and fibrous cap development.
Inflammatory markers, such as C-reactive protein, may allow clinical insight into these decades-
long processes, adding value to predictive measures of disease outcomes. Anti-inflammatory
factors, such as adiponectin, may provide further understanding of the inflammatory pathways
involved.
The consumption of probiotic bacteria such as those found in yogurt and other fermented
milk products can beneficially alter the composition of the gut microbiome, which in turn
changes the host metabolism. Obesity, insulin resistance, fatty liver disease, and lowgrade
peripheral inflammation are more prevalent in patients with low a diversity in the gut
microbiome than they are in patients with high diversity. Fermented milk products, such as
yogurt, deliver a large number of lactic acid bacteria to the gastrointestinal tract. Theymay
modify the intestinal environment, including inhibiting lipopolysaccharide production and
increasing the tight junctions of gut epithelia cells (Wen and Duffy 2017).
3.8. Available treatment methods for inflammation
Anti-inflammatory drugs can interfere in the pathophysiological process of inflammation,
to minimize tissue damage and provide greater comfort to the patient. Therefore, it is important
to note that due to the existence of a large number of species available for research, the
successful development of new naturally occurring anti-inflammatory drugs depends mainly on a
multidisciplinary effort to find new molecules (Clara et al., 2020).
The biochemical pathways of inflammatory pain, the potentially serious side effects of
nonsteroidal drugs and commonly used and clinically studied natural alternative anti-

inflammatory supplements. Although nonsteroidal medications can be effective, herbs and
dietary supplements may offer a safer, and often an effective, alternative treatment for pain
relief, especially for long-term use (Josephet al., 2010).
The plant is used as an anti-helminthic, anti-ulcer, hypotensive, anti-diarrheal,
phytoremediation, antitumor etc., The present study aims to evaluate the anticancer activity of
ethanolic extract of Catharanthus roseus leaves on the human hepatocellular carcinoma cell line,
antimicrobial properties and the phytochemical analysis of the extract. A phytochemical
screening of the major constituents of the aforesaid plant revealed the presence of tannins,
flavonoid, alkaloid, quinone, terpenoid, coumarins. Further, the ethanolic extract was tested for
its anti-microbial activities where the maximum zone of inhibition (21mm) was observed in
Escherichia coli at a concentration of 5mg/ml and 16mmzone of inhibition against Bacillus
subtillus at a concentration of 5mg/ml. MTT assay confirmed the anticancer activity of the
ethanolic extract. Thus antimicrobial and anticancer potency of the leaves of Catharanthus
roseus was proved (Stessy Ann et al., 2017).
Monika Sain and Vandana Sharma 2013 reported that the alkaloids like Vinblastine and
vincristine are mainly present in aerial parts of C. roseus, which are used in treatment of various
human cancers, so it is considered as mile stone in cancer chemotherapy. Apart from this it
shows anti-diabetic, anti-bacterial, anti-oxidant, anti-ulcer and anti-diarrheal properties. Stems
and leaves of C.roseus have enormous amount of phytochemical constituents.
Dietary plant anti inflammation have been considered beneficial to human health.
Antioxidants can eliminate free radicals and other reactive oxygen and nitrogen species, and
these reactive species contribute to most chronic diseases. Dietary plants contain variable

chemical families and amounts of antioxidants. Vegetables provide the body, an added source of
antioxidants to fight against free radicals. Without the necessary intake of healthy vegetables,
free radicals can spread and eventually lead to various types of cancer (Anoopet al., 2013).
3.9. Drawback of available treatment methods
Lisowskaet al., 2018 reported that the healthy bone tissue instead of a scar. Many factors
can inhibit or impair the bone healing process, and their influence is critical during the stages of
inflammation and angiogenesis and finally on the clinical outcome. Nonsteroidal anti-
inflammatory drugs (NSAIDs) play an essential role associated with their analgesic potency and
anti-inflammatory effects. NSAIDs are also the most often used drugs in patients who require
pain control and inflammation reduction due to musculoskeletal diseases or injures. Although
their analgesic effect is well documented, NSAIDs also interfere with bone healing; therefore,
the relative benefits and disadvantages connected with their administration should be taken into
consideration.
Germane De cosmo and Elisabetta congedo 2015 reported that the NSAIDs are commonly
used as single analgesics in minor surgery or as component of multimodal analgesia associated
with opioids or locoregional techniques in the postoperative period to assure a better analgesia
and reduce the dose of opioids. The analgesic potency evaluated as number needed to treat
(NNT) is not very different between the traditional non selective NSAIDs and the selective
cyclo-oxygenase-2-inhibitors (Coxibs). The effectiveness as analgesics is unquestionable also if
these drugs are not devoid of risks. There is debate in literature about the possible side effects
when administered in the perioperative period: anastomotic leakage, reduced ossification,

bleeding and acute renal failure. Recent data underline as the Coxibs but also traditional NSAIDs
can induce cardiac toxicity even if they are utilized for few days.
Acetaminophen is the safest medicine as analgesics for nociceptive pain and antipyretics
in childhood and pregnancy. Fever and pain during pregnancy and in childhood themselves are
probably associated with adverse gestational and childhood outcomes. Acetaminophen should be
used at the lowest effective dosage and for the shortest time. It is reasonable to judge that
acetaminophen>2,000 mg/day causes upper gastrointestinal complications. If
acetaminophen>2,000 mg/day is administered, gastroprotective agent is probably necessary.
Acetaminophen 2,000 mg/day is a gray zone. Proton pump inhibitors cause many serious adverse
effects. If proton pump inhibitors are administered with acetaminophen, the advantages of
acetaminophen that acetaminophen provides slight and mild adverse effects disappear. Nobody
knows which gastroprotective agent is optimal in combination with acetaminophen(>2,000
mg/day). It is reasonable to judge that acetaminophen is ineffective for low back pain and pain
due to osteoarthritis. It is true that acetaminophen causes various adverse effects including
serious adverse effects. However, it is also true that acetaminophen (<2,000 mg/day) is safer than
non-steroidal anti-inflammatory drugs (NSAIDs). If analgesic effects of acetaminophen (<2,000
mg/day) and NSAIDs are the same, NSAID should not be administered (Katsuhiro 2018).
3.10. Scope of Indian Traditional Medicine for the Treatment of Inflammation
Ghasemianet al., 2016 reported that the Inflammation is a pathologic condition that
includes a wide range of diseases such as rheumatic and immune-mediated
conditions,diabetes,cardiovascular accident, and etcetera.Anti-inflammatory effects have been

evaluatedin clinical and experimental studies. Curcuma longa, Zingiber officinale, Rosmarinus
officinalis, Borago officinalis, evening primrose,and Devil’s claw are some of the introduced
medicinal herbs.
Ali Mobasheri et al., 2012 reported that the Ethnopharmacology, Ayurvedic medicine,
and nutrigenomics before discussing the scientific and mechanistic rationale for targeting
inflammatory signalling pathways in OA by use of herbal medicines. Special attention is drawn
to the conceptual and practical difficulties associated with translating data from in-vitro
experiments to in-vivo studies.
Ghizlane Hajja and Aziz Bahlouli 2018 reported thatRheumatoid arthritis is a chronic,
inflammatory disorder that may affect many tissues and organs, but principally attacks flexible
(synovial) joints. The process produces an inflammatory responses of the capsule around the
joints, secondary swelling of the synovial cells.A total of 11 plant species have been identified as
active or promising sources of phytochemicals with anti–arthritic properties.
Last three decades has confirmed these observations and identified the molecular basis
for most chronic diseases and for the associated inflammation. The transcription factor, Nuclear
Factor-kappaB (NF-κB) that controls over 500 different gene products, has emerged as major
mediator of inflammation. Thus agents that can inhibit NF-κB and diminish chronic
inflammation have potential to prevent or delay the onset of the chronic diseases (Bharatet al.,
2011).
Sen and Chakraborty2017 reported that the Traditional medicine particularly herbal
medicine consideredas a major healthcare provider around the globe particularly in rural and
remote areas. A large section ofpeople depends on such medicine for their primary healthcare

mainly in underdeveloped or developingcountries. Indian traditional medicinal systems like
Ayurveda, Siddha and Unani has a very rich history oftheir effectiveness; modern research also
acknowledged the importance of such medicine. Indiantraditional medicine or medicinal plants
are also considered as a vital source of new drugs.
3.11.Chrysopogon zizanioides
It is a miraculous grass native to India first developed for soil and water conservation by
the World Bank during mid 1980s. The plant has a unique characteristic of being xerophyte
(tolerates prolonged drought) but it survive under long seasonal flooding; it tolerates extreme
temperature and grows over a wide range of soil pH. A perennial aromatic grass grows up to 2
meter in height. The plant has small stout rhizomatous stolen which gives rise to spongy, fibrous,
dense root system. Roots have aromatic properties and grow 20-30 cm deep in medium textured
marginal soils under cultivation. The leaves are linear, narrow, erect, grassy, and glabrous with
scabrid margins. Inflorescence is a panicle upto 15 to 45 cm long, bearing numerous racemes in a
whorl on a central axis. Spikelets grey in a panicle of numerous racemes. One floret in spike is
bisexual and sessile, fruits oblong grains. The other floret is pedicelled and staminate. The lower
spikelet are reduced to lamina. There is considerable diversity in pattern of growth, orientation
and thickness of roots as well as occurrence of secondary roots. Java vetiver is non flowering
type has a broader leaves (1.1mm) medium thick stems, bushy growth bearing with high pollen
sterlitiy the plants give out more branching roots with higher oil contents and oil is dextro-
rotatory in nature. Khas (Vetiver) grass is tall, stout and perennial herb with oblong penicle over
30 cm long which has whorled branches bearing spikeletes 5-6 mm long, with a few tubercle-
based short bristles. The species under cultivation do not flower. Vetiver roots are stout and
contain oil which provide its fragrance

This tufted grass grows throughout the plains of India ascending up to an elevation of
1200 m. Having wide ecological amplitude, this grass grows in a wide variety of ecological
habitats covering all bio-geographic provinces of India. Khas grass grows wild in many states,
namely Haryana, Uttar Pradesh, Rajasthan, Gujarat, Bihar, Orissa and Madhya Pradesh and
throughout South India. It is systematically cultivated in the North Indian states of Rajasthan,
Uttar Pradesh and Punjab and in the South Indian states of Kerala, Tamil Nadu, Karnataka and
Andhra Pradesh. The bulk of the roots used for cooling purposes and for the extraction of the oil
are obtained from the wild
The grass grows luxuriantly in areas with an annual rainfall of 800 -2000 mm and
temperature ranging from 22 to 400 C. Marshy riverbeds with sandy loam are best suited for this
grass. It is commercially cultivated as large population of Khas (Vetiver) grass in wet and damp
environments over marshy places and riverbanks. However the yield from the cultivated crops,
however, meets only a very small percentage of the requirements of the country.
3.12.Phytoremediation Research
on various aspects of vetiver make it an excellent plant describing many characteristics
including phytoremediation, water purification, eachate and effluent disposal, utilizing
wastewater, removing nitrogen and phosphorus etc.
3.13. Vetiver oil
Vetiver oil is one of the most valuable and important raw materials in perfumery,
extensive applications in the soap and cosmetic industries, for pharmaceutical companies and as

antimicrobial and anti-fungal agent (Singh et al. 1978; Dikshit and Husain 1984). These
properties may open new possibilities to the utilization of vetiver extracts in the pharmaceutical
industry. Vetiver oil is being used in Ayurvedic system of medicine in India (Lavania, 2003).
Vetiver oil is prescribed to relief rheumatism, lumbago, headache, sprain (Anon., 1976), and
vetiver drink is prescribed for fever, inflammation and irritability of stomach. Pure vetiver
(Khus) root oil known in trade as “Ruh – Khus” and its use in scents since ancient time. It is the
major source of the well known vetiver oil with world-wide demand of 250 metric tonnes
annually. The ‘Sunshine’ cultivar of vetiver (Vetiveria zizanioides (L.) Nash, =Chrysopogon
zizanioides (L.) Roberty) show fungicidal and bactericidal properties. Some biological activities
are also attributed to the vetiver root extracts such as antifungal (Sridhar et al., 2003) antioxidant
(Kim et al., 2005) and antiinflammatory (Jagtap et al., 2004).
The vetiver oil can also be used for nursing care residents with dementia-related
behaviours, in order to increase mental alertness and cognitive function (Bowles et al., 2002).
World market the demand for vetiver oil is increasing day by day due to its unique odour, for
which it is used in both flavour and fragrance industries. Moreover, this oil cannot be substituted
with reconstituted oil and cannot be made through synthetically. Vetiver perfumes give pleasing
aroma and has slow evaporation rate. Its major constituents in oil extract are Vetivone, Zinanal
and Epizizizanal. Zinanal and Epizizizanal also have insect repellent activity. It is widely used in
perfumes and cosmetics and for scenting soaps. Popularly known as ‘KHUS’, it is the major
source of the well-known oil of vetiver, which is used in medicine, cosmetics and in perfumery
making agarbattis, soaps, soft drinks, pan masala. It blends well with the oils of sandalwood
patchouli and rose. The oil is reported to be used as carminative in flatulence colic and obstinate

vomiting. Major constituents of the essential oil of Khas Khas are vetiselineol and khusimol
which render the herb its pharmacological properties.
Vetiver oil has potent antibacterial, drug-resistant modifying, hydroxyl radical
scavenging, anticancer, antihepatotoxic and antioxidant activity in intact and spent root of
vetiver. The essential oil of vetiver has extensive applications in toiletries and cosmetics,
possesses sedative property and has traditionally been used in aromatherapy for relieving stress,
anxiety, nervous tension and insomnia. The plant roots are steam distilled to obtain Vetiver oil.
The oil is finest oriental perfume with persistent fragrance. The blended perfume oil of vetiver
acts as an excellent fixative for volatile compounds. It is known for its cooling properties.
Vetiver oil is the basis of the Indian perfume ‘Majmua’ and is the major ingredient in some 36%
of all western perfumes (e.g. Caleche, Chanel No. 5, Dioressence, Parure, Opium) and 20% of all
men’s Perfume.
3.14. Traditional medicinal properties
Chrysopogon zizanioidesplays an important role in the socioeconomic life of rural India.
In Madhya Pradesh and Maharashtra, the plant is used as anthelmintic for children. The plant is
used as a tonic for weakness; the Lodhas of West Bengal use the root paste for headache,
rheumatism and sprain, and a stem decoction for urinary tract infection; the Mandla and Bastar
tribes of Madhya Pradesh use the leaf juice as anthelmintic; the tribes of the Varanasi district
inhale the root vapour for malarial fever. The root ash is given to patients for acidity by the
Oraon tribe. Likewise, there are very many different applications of the plant for different
ailments among different ethnic tribes (Jain 1991; Singh & Maheshwari 1983).Root is also
important in traditional medicine as a carminative, stimulant and diaphoretic. The decoction of

leaves is recommended as a diaphoretic. A decoction When applied locally applied in
rheumatism lumbago and sprain, it is a good ambrocatoin and affords relief in. In Madhya
Pradesh the plant is used as an anthelmintic for children. Formulations containing oil and/or
extracts of vetiver have been reported to treat inflammatory bowel disease, urinary tract
infection, and in making insect repellents. It is one of the most promising aromatic plants known
to possess antimicrobial, antioxidant and germicidal properties. However, the plant has not been
studied exclusively for other pharmacological activities and there is a lack of scientific evidence
to prove these effects.
3.15. Desert coolers
Its roots are used for desert coolers in summer in North India. In India, since ancient
times, the roots have been used for making screens, mats, hand fans, and baskets. The screens are
hung like curtains in the houses and when sprinkled with water, impart a fragrant coolness to the
air; they are in great demand during the summer. In Kerala, the roots are woven along with
bamboo splits and made into flat mattresses for use as under-beds to give a cooling effect. The
roots have found increased use in electric room-coolers.
3.16. Soft drinks and perfumery products
It is also used in in perfumery, cosmetics and soaps and for flavoring sherbets (Indian
cool drinks). Roots are also used for preparing Sherbet or soft drink during summer, especially in
North India. Both vetiverols and acetates have softer odours and fixative qualities, and are used
as blender with highclass perfumery products. They blend well with ionone, linalool, cinnamic
alcohol, oakmoss, vanila, sandalwood, patchouli and rose bases, and are frequently used in
western type of fragrances having chypre, fougere, rose, violet and amber aldehyde base, and

oriental fragrances and floral compounds. In addition to its direct perfumery applications, vetiver
oil in its diluted form is extensively used in after-shave lotions, air freshners and bathing
purposes, as well as flavoring syrups, ice cream, cosmetic and food preservation. Khus essence is
used in cool drinks, and for reducing pungency of chewing tobacco preparations, providing
sweet note to other masticatories and incense sticks. 3.5 Chemical constituents The chemical
constituents present in the plant are Vetiverol, Vetivone, Khusimone, Khusimol, Vetivene,
Khositone, Terpenes, Benzoic acid,Tripene-4-ol, ß-Humulene, Epizizianal, vetivenyl vetivenate,
iso khusimol, ß-vetivone, vetivazulene. In the roots, the main component was valencene
(30.36%), while in the shoots and leaves, they were 9-octadecenamide (33.50%),
2,6,10,15,19,23- hexamethyl-2,6,10,14,18,22- tetracosahexaene (27.46%), and 1,2-
benzendicarboxylic acid, diisooctyl ester(18.29%). The results showed that there were many
terpenoids in the volatils. In shoot volatiles, there existed 3 monoterpenes, 2 sequiterpenes and 1
triterpene. Most of the volatiles in roots were sesquiterpenes.
3.17. Medicinal properties
It is also used for boils, burns, epilepsy, fever, scorpion sting, snakebite, and sores in the
mouth. Root extract is used for headache and toothache. Vetiver oil is regarded as stimulant,
diaphoretic and refrigerant. Local application of leaf paste for rheumatism, lumbago and sprain
gives good relief. The dried roots are also used to perfume the linen clothes. The rachis is used in
the manufacture of moodas, sirkies, etc. 5. Commercial Applications The commercial
applications of the grass mainly pertain to the extraction of vetiver oil through distillation of the
roots. Over 150 compounds have been isolated and characterized from vetiver oil so far. A major
portion of oil consists of sesquiterpene alcohol (Thakur et al. 1989). 6. Additional uses Besides
these medicinal properties of the plant, the dried culms of the plant are used as brooms and to

thatch roofs. Pulp of the plant is used to prepare straw boards and paper. In India, the roots have
been used for making screens, mats, hand fans, and baskets. The young leaves are browsed by
cattle and sheep. Dried culms are used for making brooms and thatching of huts. Apart from the
medicinal uses, the culms along with the panicles form a good broom for sweeping. The culms
and leaves are also extensively used by the tribes and villagers for thatching their huts, mud
walls, etc. Some tribes (in Kerala) use the mats of the roots and leaves as bed for a cooling effect.
Recommendations Several villagers and rural folk collect the roots of the grass in large quantities
from Lucknow, Delhi, Kanpur, etc. Three to four months of livelihood of several rural families is
sustained extensively by the sale of roots of this grass, which thus plays a significant role in the
socio-economic lives of village and rural folk. In view of its tremendous use and also in view of
its declining wild populations, the authors strongly recommend the large-scale systematic
cultivation of this grass in the plains of North India. Counting both the grass and the root system,
vetiver can produce up to 100 tons/hectare of biomass (dry weight) within 8 months. If the grass
is only harvested as a biomass fuel, and the perennial vetiver grass is left in the ground and
allowed to grow back, year after year lasting for ever with only minimum care and some
fertilizer (TVN, 2008).
4. MATERIALS AND METHODS

4.1.Collection of plant materials
The Chrysopogon zizanioideswere collected from Hosur, Tamil Nadu. The plant material
was identified and confirmed by the botanist.
Figure 1. Collection of Chrysopogon zizanioides
4.2.Chrysopogon zizanioides(Soxhlet apparatus)
The crude powders (10 g) of Chrysopogon zizanioideswere defated with 100 ml of
petroleum ether (60°- 80°c) using soxhlet apparatus. After defating, the extraction was
carried out using 100 mL of 50% ethyl alcohol and 50% distilled water (75.0°c) for 4 h. After
extraction, the samples were evaporated on the rotary evaporator to remain with important
ingredients.
4.3.Anti-inflammatory activity - Inhibition of albumin denaturation
Principle
Protein denaturation is a process in which proteins lose their tertiary structure and
secondary structure by application of external stress or compound, such as strong acid or base, a
concentrated inorganic salt, an organic solvent or heat. Most biological proteins lose their

biological function when denatured. Denaturation of proteins is a well documented cause of
inflammation. As part of the investigation on the mechanism of the anti-inflammation activity,
ability of plant extract to inhibit protein denaturation was studied. It was effective in inhibiting
heat induced albumin denaturation.
Material Required
Acetyl salicylic acid, BSA was purchased from Sigma Aldrich, USA. 10X PBS was
purchased from Himedia, India.
Procedure
Denaturation of proteins is the main cause of inflammation. Inhibition of protein
denaturation was evaluated by the method of Mizushima and Kobayashi and Sakat et al. with
slight modification. 500 μL of 1% bovine serum albumin was added to Chrysopogon zizanioides
(500, 250, 100, 50, 10 and 0.5 μg/mL) of test sample. This mixture was kept at room temperature
for 10 minutes, followed by heating at 51°C for 20 minutes. The resulting solution was cooled
down to room temperature and absorbance was recorded at 660 nm. Acetyl salicylic acid was
taken as a positive control. The experiment was carried out in triplicates and percent inhibition
for protein denaturation was calculated using the following formula and the statistical analysis
was carried out using GraphPad Prism Software 6.0, USA.
% Inhibition=100−((A1-A2)/A0)*100)
Where A1 is the absorbance of the sample, A2 is the absorbance of the product control and A0 is
the absorbance of the positive control.

A dose response curve was plotted to determine the IC50 values. IC50 is defined as the
concentration sufficient to obtain 50% of a maximum scavenging capacity. All tests and analyses
were run in triplicate and averaged.
4.4. Proteinase inhibitory assay
The proteinase inhibitory assay was performed following the method modified by
Oyedepo and Femurewa [34]. The reaction mixture (2 ml) contained 0.06 mg trypsin, 1 ml Tris-
HCl buffer (20 mM, pH 7.4) and 1 ml test plant extract sample at different concentrations. The
reaction mixture was incubated at 37 °C for 5 min and then 1 ml of 0.8% (w/v) casein was added.
The mixture was incubated for an additional 20 min. Perchloric acid (2 ml of 70%) was added to
stop the reaction. The cloudy suspension was centrifuged and the absorbance of the supernatant
was measured at 210 nm against Tris-HCl buffer as blank. The experiment was performed in
triplicate.
4.5. Statistical analysis
The difference in estimated parameters between the groups was analyzed using one-way
ANOVA with Bonferroni’s test. Data expressed as mean ± SD. All parameters were analyzed at
95% confidence intervals and P value of <0.05 was considered to be statistically significant.
Statistical analysis of the data was performed using Graphpad Prism version 6.00 for Windows,
GraphPad Software, San Diego California USA.

5. RESULTS AND DISCUSSION
Inflammation is an adaptive response triggered by noxious stimuli and conditions such as
infection and tissue injury. It has extensively been demonstrated that strong and complex
interconnections occur between oxidative stress and the inflammatory response. Alterations of
the endocellular redox state play a key role in the activation and/or dysfunction of immune cells.
Along these lines, the plant kingdom contains an immense variety of secondary metabolites,
named phytochemicals (PhC), with significant redox-modulating properties that have been
recently shown to effectively modulate the inflammatory response.
Chrysopogon zizanioidesis a seasonal multipotent and an important medicinal herb
belongs to the family Violaceae. The plant claims highest medicinal values and used traditionally
to treat diabetes, jaundice, male sterility, urinary tract infections, gonorrhea, bowel complaints,
urinary problems and various ailments. No detailed pharmacognistical works are available,
hence, in the present study, we evaluated the anti-inflammatory potential of Chrysopogon
zizanioides hydroalcoholic extract using the albumin denatuaration assay.
4.1. Soxhlet extraction of Chrysopogon zizanioides
Chrysopogon zizanioideswere cleaned with distilled water and dried at shade for 2-3
days. As shown in figure 2 the dried plant materials were grounded and 50g of plant powdered
samples are used for extraction by soxhlet apparatus at a boiling temperature. Solvent systems
used for extraction was hydro alcoholic. The crude extract obtained was 1.32g.

Figure 2. Soxhlet extraction of Chrysopogon zizanioides
5.2. Albumin denaturation assay
Inflammation is usually associated with the denaturation of proteins. Results from the
present study revealed that IS significantly inhibited protein/albumin denaturation. IS had the
highest anti-inflammatory potential (strong inhibition of protein denaturation) at the
concentration of 500 μg/ml with 35 % of inhibition. The IC50 value of the albumin
denaturation assay was found to be 101 μg/ml.

Figure 3. Anti-inflmmatory effect of Chrysopogon zizanioidesextract by albumin
denaturation assay
A. OD Value at 660 nm
Control Mean OD value: 0.393
S.
No
Tested sample
concentration
(μg/ml)
OD Value at 660 nm (in
duplicates)
1. Control 0.433 0.419 0.329
2. 500 μg/ml 0.248 0.259 0.257
3. 250 μg/ml 0.276 0.265 0.302
4. 100 μg/ml 0.311 0.316 0.319
5. 50 μg/ml 0.358 0.378 0.388
6. 10 μg/ml 0.394 0.390 0.389
500 250 100 50 10 C

Cz  g /m l
O
D
a
t
6
6
0
n
m
0 2 0 0 4 0 0 6 0 0
0 .2 0
0 .2 5
0 .3 0
0 .3 5
0 .4 0
0 .4 5
0 .5 0
C o n tr o l
Cz  g /m l
B. Inhibition percentage of albumin denaturation (%)
S. No Tested sample
concentration
(μg/ml)
Inhibition percentage
albumin denaturation (%)
(in duplicates)
Mean
Value
(%)
1. Control 100 100 100 100
2. 500 μg/ml 36.89 34.09 34.60 35.19
3. 250 μg/ml 29.77 32.56 23.15 28.49
4. 100 μg/ml 20.86 19.59 18.82 19.75
5. 50 μg/ml 8.90 3.81 1.27 4.66
6. 10 μg/ml 0.50 0.76 1.01 0.75

Cz  g /m l
I
n
h
i
b
it
i
o
n
p
e
r
c
e
n
t
a
g
e
o
f
a
lb
u
m
in
d
e
n
a
t
u
r
a
t
io
n
%
C
o
n
tro
l
5
0
0

g
/m
l
2
5
0

g
/m
l
1
0
0

g
/m
l
5
0

g
/m
l
1
0

g
/m
l
0
5 0
1 0 0
1 5 0
C. IC50 Value of testedsample: 101.0 μg/ml
log(inhibitor) vs. normalized response --
Variable slope
Best-fit values
LogIC50 2.004
HillSlope -2.195
IC50 101.0
Std. Error
LogIC50 0.03400
HillSlope 0.3594
95% Confidence Intervals
LogIC50 1.931 to 2.078
HillSlope -2.971 to -1.419
IC50 85.28 to 119.6
Goodness of Fit
Degrees of Freedom 13
R square 0.9524
Absolute Sum of Squares 1092
Sy.x 9.164
Number of points
Analyzed 3 15

5.3.Proteinase inhibitory assay
Proteinases have been implicated in arthritic reactions. Neutrophils are reported to be a
rich source of serine proteinases, which are localised in lysosomal granules. Leukocyte
proteinases are involved in the development of tissue damage during inflammatory reactions and
proteinase inhibitors provide substantial protection against this effect. Methanol extracts of O.
corniculata have been reported to have significant antiproteinase activity. In the present study,
IS hydroalcoholic extract showed high correlation coefficients values of anti-proteinase
inhibition activity at the concentration of 500 μg/ml and the IC50 value of protease inhibition
assay was found to be 57.63 μg/ml.
Figure 4. Anti-inflmmatory effect of Chrysopogon zizanioidesextract by protease
inhibition assay
A. OD Value at 517 nm
Control Mean OD value: 3.471

Cz  g /m l
O
D
a
t
5
1
7
n
m
0 2 0 0 4 0 0 6 0 0
0
1
2
3
4
C o n tro l
C z  g /m l
S. No Tested sample
concentration
(μg/ml)
OD Value at 517 nm (in
triplicates)
1. Control 3.488 3.349 3.578
2. 500 μg/ml 0.698 0.727 0.780
3. 250 μg/ml 0.894 0.915 0.922
4. 100 μg/ml 1.096 1.392 1.015
5. 50 μg/ml 2.172 2.243 2.080
6. 10 μg/ml 3.187 3.120 3.109

B.Percentage of inhibition
Cz  g /m l
P
e
r
c
e
n
t
a
g
e
o
f
i
n
h
i
b
i
t
i
o
n
C
o
n
tro
l
5
0
0

g
/m
l
2
5
0

g
/m
l
1
0
0

g
/m
l
5
0

g
/m
l
1
0

g
/m
l
0
5 0
1 0 0
1 5 0
S.
No
Tested sample
concentration
(μg/ml)
Percentage of inhibition
(in triplicates)
Mean
value (%)
1. Control 100 100 100 100
2. 500 μg/ml 79.89 79.05 77.52 78.82
3. 250 μg/ml 74.24 73.63 73.43 73.76
4. 100 μg/ml 68.42 59.89 70.75 66.35
5. 50 μg/ml 37.42 35.37 40.07 37.62
6. 10 μg/ml 8.18 10.11 10.42 9.57

C. IC50 Value of testedsample: 57.63μg/ml
log(inhibitor) vs. normalized response --
Variable slope
Best-fit values
LogIC50 1.761
HillSlope -2.581
IC50 57.63
Std. Error
LogIC50 0.01534
HillSlope 0.2629
95% Confidence Intervals
LogIC50 1.728 to 1.794
HillSlope -3.148 to -2.013
IC50 53.40 to 62.20
Goodness of Fit
Degrees of Freedom 13
R square 0.9879
Absolute Sum of Squares 260.3
Sy.x 4.475
Number of points
Analyzed 3 15

6. Conclusion
Chrysopogon zizanioidesis an important medicinal plant widely used as a herbal
traditional medicine in Ayurveda for the treatment of infertility. Currently, little
pharmacological information is available on anti-inflammation. Results from this study
revealed that Chrysopogon zizanioidesextracts has the anti-inflammatory and anti-proteinase
activities. These extracts have potential to be used to prevent food spoilage and to treat
inflammation as well as skin irritations. Future research work will be focused on the use of
Chrysopogon zizanioidesplant extract to protect against peroxidative damage related to
carcinogenesis.

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