ABSTRACT

During the past few years, interest in the potential clinical and pharmacological
basis of the efficacy and safety of herbal medicines has increased greatly, due to
widespread domestic self-medication with these agent. Some authors have analyzed
the use of Mentha SPP. in the pharmacological industry. The essentional oil from
Mentha SPP. is used to treat discomfort of the gastrointestinal tract, decrease
symptoms of irritable bowel syndrome, myalgia and neuralgia as well as oral
mucosal inflammation. The essentional oil also contains chemical compounds that
are associated with side effects such as nausea, vomiting, allergic reactions,
flushing and headaches. Some of the benefic biological effects show that this plant
may play an important role as anti-oxidant, anti-nociceptive, anti-inflammatory,
anti-microbial, anti-carcinogenic, anti- viral, anti-allergic and anti-tumorigenic,
indicating its utility in the prevention or treatment of several diseases. Therefore,
the purpose of this review is to examine different activities shown by essential oil
of Mentha SPP and also examine what has been published on the possible clinical
and pharmacological use of essentional oils of Mentha SPP and their effective and
safe utilization in human beings.
 Chapter 1

1 Introduction

1.1 Back ground:

For many years, plants have been used as Therapeutic resources such as Herbal teas
and the empirical knowledge of the properties of many plants is the basis for their
use as home remedies as crude extract’s or as standard enriched fractions in
pharmaceutical preparations such as fluid extract, powders and pills.

The WHO estimates that 80% of people in developing countries (65% of the world
population) still rely on traditional medicine. (1,2)

Plants have been rich source of medicines because they produce a host of bioactive
molecule, most of which probably evolved as chemical defenses against infection.
Human Health are threatens by environmental pollution and this pollution can also
disturb plant and animal diversity. It causes normal activity deteriorations of bio
system. (3) Nowadays, antioxidants, which are integral part of biologically active
substances, are of great interest. They can reduce mutagenic influence, regulating
the oxidation process of free radicals. Therefore, discovery and studies of natural
sources with antioxidant activity is an urgent problem now clinical and
pharmacological basis for the efficacy and safety of herbal medicines has increased
greatly in recent years, in view of the common occurrence of domestic self-
medication with these agents. (4)

1.2 Biologically Active Substances Produce

According to literature, a number of biologically active substances which are
produce by plants and have anti-oxidant activity are known. They include ascorbic
acid (Vitamin C), B-carotene, x-tocoferol, a number of protein compounds with
enzymatic activity, polysaccharides, polyphenol compounds, terpenoids, flavonoids
and etc. (5,6,7,8,9)

1.3 Examples of some commonly used Herbal medicines

 Artichoke and several other plants reduce total serum cholesterol levels in
preliminary studies.
  , Black cohosh and other plants the contain phytoestrogens (plants molecule
with estrogen activity) have some benefits for treatment of symptoms
resulting from menopause.
 , Echinacea extracts limit the length of cold in some clinical trials, although
some studies have found it to have no effect.
 Garlic lowers total cholesterol levels, mildly reduces blood pressure,
reduces platelets aggregation and has antibacterial properties.
 St John’s Wort, though dangerous in incorrect doses, is more effective than
a placebo for the treatment of mild to moderate depression in some clinical
trials.
 Peppermint tea for problems with the digestive tract, including irritable
bowel syndrome and nausea.
 Nigella Sative (Black Cumin) is a generalist medicinal plant used for
diverse ailments such as cough, pulmonary infections, asthma, influenza,
allergy, hypertension and stomach ache. The seeds are considered
carminative (Preventing or relieving flatulence (lactation including) it is
often taken with honey seed powder or oil is externally applied for
eruptions of skin. (14)

1.4 Pharmacognosy and Phytochemicals (Phytochemistry Of Medicinal

Plants)
Pharmacognosy is the study of drugs obtaining from natural sources.
Phytochemicals (from the Greek word phyto meaning plant) are biologically active,
naturally occurring chemical compounds found in plants, which provide health
benefits for human further than those attributed to macronutrients and
micronutrients (15). They protect plants from disease and damage and contribute to
the plant’s color, aroma and flavor. In general the plant chemicals that protect plant
cells from environmental hazards such as pollution, stress, drought, un exposure
and pathogenic attack are called as phytochemicals (16,17). Recently, it is clearly
known that they have roles in the protection of human health, when their dietary
intake is significant. More than 4,000 phytochemicals have been cataloged. (18)
and are classified by protective function, physical characteristics and chemical
characteristics (19) and about 150 phytochemicals have been studied in detail. (18)
 In wide ranging dietary phytochemicals are found in fruits, vegetables, legumes,
whole grains, nuts, seeds, fungi, herbs and spices. (17) Broccoli, cabbage, carrots,
onions, garlic, whole wheat, bread, tomatoes, grapes, cherries, strawberries,
raspberries, beans, legumes and soy foods are common sources (20).

Phytochemicals accumulate in different parts of the plant such as in the roots,

stems, leaves, flowers, fruits and seeds (21) Many phytochemicals, particularly the
pigment molecules, are often concentrated in the outer layer of various plant
tissues. Levels vary from plant to plant depending upon the variety, proceessing,
cooking and growing conditions. (22). Phytochemicals are also available in
supplementary forms but evidence is lacking that they provide the same health
benefits as dietery phytochemicals. (18). These compounds are known as secondary
metabolites and have biological properties such as antioxidant activity,
antimicrobial effect, modulation of detoxification enzymes, stimulation of the
immune system, decrease of platelet aggregation and modulation of hormone
metabolism and anticancer property. There are more than thousand known and
many unknown phytochemicals (22). It is well-known that plants produce these
chemicals to protect themselves, but recent researches demonstrate that many
phytochemicals can also protect human against disease (23).

Flavonoids Glycosides Tannins Saponins Phenollics

Alkaloids Phytochemistry Terpenoids

of Plants

1.5 Biological Activities Of Phytochemicals

The phytochemicals present in plants are responsible for preventing disease and
promoting health have been studied extensively to establish their efficacy and to
understand the underlying mechanism of their Action. Such studies have included
indentification and isolation of the chemical components, establishment of their
biological potency both by in vitro and in vivo studies in experimental animals and
through epidemiological and clinical-case central studies in man. Study finding
suggest that photochemical may reduce the risk of coronary heart disease by
preventing oxidation of low density lipoprotein (LDL) cholesterol, reducing the
synthesis or absorption of cholesterol, normalizing blood pressure and clotting and
improving arterial elasticity (17,24). Phytochemicals may detoxify substances that
cause cancer. For example, according to data summarized by Meagher and
Thomson, genistein prevents the formation of new capillaries that are needed for
tumar growth and metastasis. (19). Phytochemicals have also been promoted for the
prevention and treatment of diabetes, high blood pressure and macular
degeneration. (20). While phytochemical are classified by function, an individual
compound may have more than one biological function serving as both an
antioxidant and antibacterial agent. (24).

Bio active and disease preventing phytochemicals present in plant are shown in
Table 1.

Table 1. Bioactive Photochemical in Plants

Classification Main Group of Biological Function

Compounds
NSA Cellulose, hemicelluloses, Water holding capacity delay in
(Non-starch gums mucilages, pecting, nutrients absorption, binding
Polysaccharides) lignins taxins and bile acids.
Antibacterial and Terpenoids, alkaloids Inhibitor of micro-organisms,
Antifungal phenolics. reduce the risk of fungal
infection.
Antioxidants Polyphenolic Compounds, Inhibitor of micro-organisms,
Flavonoids, reduce the risk of fungal
Carotenoids infection.
Tocopheruls,
Ascorbic acid.
Antioxidants Polyphenolic Oxygen free radical quenching,
Compounds, Inhibition of lipid peroxidation.
Flavonoids,
Carotenoids
Tocopheruls, ascorbic acid
Anticancer Carotenoids, Inhibitors of tumor,
Polyphenols, Inhibited development of lung
 Curcumine, cancer, anti-metestatic activity.
Flavonoids
Detoxifying agents Reductive Inhibiters of procarcinogen
Acids, activation, inducers of drug
Tocopherols, binding of carcisogens, inhibiters
Phenols, of tumour genesis.
Indoles,
Aromatic isothiocyanates
Coumarins, flavones,
Carotenoids, retinoids,
Cyanates, phytosterols.
Other Alkaloids, terpenoids, Neuropharmacological agents,
Volatile flavor compounds, anti-oxidant cancer
biogenic amines. chemoprevention

1.6 Classification Of Phytochemicals

Phytochemicals are classified as primary or secondary constituents, depending on

their rule in plant metabolism primary constituents include the common sugars,
aminoacids, proteins, purines and pyrimidines of nucleic acids, chlorophyll’s etc.
Secondary constituents are the remaining plant chemicals such as alkaloids,
terpenes, flavonoids, legnans, plant steroids, curcumines, saponins, phenolics
flavonoids and glucosides (25). Literature survey indicate that phenolics are the
most numerous and structurally diverse plant phytoconstituents.

Other
10%
Phenolics
Terpeno 45%
ids &
Steriods
27%
Alkalous
18%

1.7 Major Groups Of Plants Phytochemicals

Phenolic
Phenolic phytochemicals are the largest category of phytochemicals and the most
widely distributed in the plant kingdom. The three most important groups of dietary
phenolic are flavonoids, phenolic acids and polyphenols (26).

Activity of Phenolic

Varied biological activities of phenolic were reported. Increases bile secretion,

reduces blood cholesterol and lipid levels and antimicrobial activity against some
strains of bacteria such as staphylococcus aurous are some of biological activities
of phenolic acids. Phenolic possess diverse biological activities for instance
antiulcer, anti-inflammatory, antioxidant (28), Cytotoxic and antitumor,
antipasmodic and antidepressant activities. (27).

Flavonoids

Flavonoids are polyphenol compounds that are ubiquitous in nature. More

than 4,000 flavonoids have been recognised many of which occur in vegetables,
fruits and beverages like tea, coffee and fruit drinks (29).

Activity of Flavonoids

Flavonoids have gained recent attention because of their broad biological

and pharmacological activities in these order flavonoids have been reported to exert
multiple biological property including antimicrobial, cytotoxicity, anti
inflammatery as well as antitumor activities but the best-described property of
almost every group of flavonoids is their capacity to act as powerful antioxidants
which can protect the human body from free radicals and reactive oxygen species.
Antimicrobial Activity, anti-allergic activity and cytotoxic antitumor Activity. (30).

1.8 TANNIN

Tannins are a heterogeneous group of high molecular weight polyphenols

compounds with a capacity to form reversible and irreversible complexes with
proteins (mainly), polysaccharides (cellulose, hemicellulose, pectin etc), alkaloids,
nucleic acids and minerals, etc (31,32).

Activity of Tannin
In medicine, especially in Asian (Japanese and Chinese) natural healing, the tannin
containing plant extracts are used as astringents, against diarrhoea, as divetics,
against stomach and duodenal humoures. (33). And as anti inflammatory, anti
septic, antioxidant and haemostatic pharmaceuticals (34). Tannins are used in the
dyestuff industry as caustics for cationic dyes (tannin dyes), and also in the
production of risk.

Alkaloids

Alkaloids are natural product that contains heterocyclic, nitrogen dterns, are basic
in character. The name of alkaloids derives from “alkaline” and it was used to
describe any nitrogen containing base (35).

Activity of Alkaloids

Alkaloids have many pharmacological activities including antihypertensive

effective (many indole alkaloids), antiarrhythemic effect (quinidine spareien),
antimalarial activity (quinine), anticancer actions (dimeric indoles, vincristine,
visblastine) some alkaloids have stimulant property as caffeine and nicotine,
morphine are used as the analgesic and quinine as the antimalarial drug. (36).

Terpenoids

The terpenoids are a class of natural products which have been derived from five-
carbon isoprene units. Many of terpenoids are commercially interesting because of
their use as flavours and fragrances in foods and cosmetics examples menthol and
sclareal or because they are important for the quality of agricultural products, such
as the flavor of fruits and the fragrance of flowers like linalool. (37).

Activity of Terpenoids

Among plant secondary metabolites terpenoids are a structurally most diverse

group; They function as phytoalexins in plant direct defense or as signals in indirect
defense responses which involves herbivores and their natural enemies. (37).
Terpenoids can have medicinal properties such as anticarcinogenic (e.g perilla
alcohal), anti malarial (e.g artemisnin), anti-ulcer, hepaticidal, anti microbial, or
divertic (e.g glycyrrbizin) activity and the sesquiterpenoid. Antimalarial drug
artimisinin and the diterpenoid anticancer drug taxol (38,39).

Saponin

Saponin are a group of secondary metabolites found widely distributed in the plant
Kingdom. They form a stable foam in aqueous solutions such as soap, hence the
name “Saponin”.

Activity Of Saponin

Saponins may be considered a part of plants defence systems, and as such have
been included in a large group of protective molecules found in plants named
phytoanticipins or phytoprotectants. (40). Extensive research has been carried out
into the membrane permeabilising, immunostimulant, hypocholesterolaenic and
anticarcinogenic properties of sapoins and they have also been found to
significantly effect growth, feed, intake and reproduction in animals. These
structurally diverse compounds have also been observed to kill protozoans and
nolluscs, to be antioxidants, to be antioxidants, to impair the digestion of protein
and the uptake of vitamins and minerals in the gut, to cause hypoglycaemia, and to
act as antifungal and antiviral. (40,41,42).

Main Objective Of Study

The main objective of this study is to determine the probable activities of different
parts of plant. Because every part of plant have medicinal importance (leave’s stem,
root, bark, flower, fruit and seed).

1.9 Significant Plant-Derived Pharmaceutical Products

SOURCE DRUG INDICATION

 PLANT
Salix SPP Aspirin Analgesic
Anti-pyretic
Cardiovascular.
Atropa belladonna Atropine Anti-cholinergic pupil dilation
Digitalis Lanata Digoxin Cardiotonic
Mucuna deeringiana (L)-dopa Anti-parkinsonism
Dioscorea deltoidea Diosgenin Anti-fertility
Papaver Somniferum Morphine Analgesic and antitussive.
Codeine
Colchicum Colchicine Anti-tumour
autumnale Anti-gout
Cindrona ledgariana Quinine Anti-malarial
Catharnthus roseus Vincristine Anti-tumour.
Vinblastine

Every part of plant have medicinal importance. Digoxin obtained from the leaves of
digitalis lanata and from cinchona bark quine is obtained.
 Chapter 2

2 Review of Literature

2.1 Plant Introduction

Peppermint (Mentha Piperita) is a hybrid plant obtained by crossing spearmint

(Mentha Spicata L) with water mint. There are two peppermint forms: black
(M.Piperita.L. var. officinalis sole F rubescens camus) and while (M.Piperita
L.var.officinalis sole F. Pallescens camus). Black peppermint has violet stems and
leaves and white peppermint has light green deeply cut leaves. Peppermint raw
material is used in medicines, cosmetics and food industry, therefore this plant is
widely grown around the world. Black peppermint produces a large amount of
essential oils and has a better aroma than the white one, thus it is more widely
grown, especially for industrial processing. (43,44).

Among the diversity of plants, Mentha Piperita (Lamiaceae Family) is one of the
herbs most widely used worldwide, with a long history of safe use in medicinal
preparations. Its leaf is used as a remedy for common cold, inflammation of the
mouth, pharynx, liver as well as disorders in the gastrointestinal tract such as
nausea, vomiting, diarrhea, cramps, flatulence and dyspepsia. It is also used as
antioxidant, antimicrobial, antiviral, anti-inflammatory, and ant carcinogenic. (45,
46,47,48).

Plant is known for having several phytochemicals, including polyphenols that are
highly effective antioxidants and are less toxic than the synthetic ones. This
property makes it of great interest to the food industry, since the phenolic
compounds retard the oxidative degradation of lipids in proving the quality and
nutritional value of food. (49,50). Mentha piperita Linn. emend. huds. is currently
one of the most economically important aromatic crops. It is commonly known as
brandy mint, candy mint, lamb mint, balm mint, vilayati pudina or paparaminta.
The world production of peppermint oil is about 8000 tones per year.

The plant is a strongly scented, perennial, glabrous, herb 30-90 cm is height. The
square stems are usually reddish purple and smooth. The leaves are short 2.5-5 cm
long, oblong-ovate and serrate. The flowers are purple pinkish and appear in the
summer month. The leaves and flowers tops are collected as soon as flowers begin
to open and dried as crude drug for its oil and peppermint. (51).

2.2 The History Of Menthe SPP

Throughout history, a number of mint species have been used around the globe for
their various properities, both medicinal and culinary. Peppermint oil is one of the
world’s oldest herbal medicines. The gathering of dried peppermint dates back to at
least 1000 BC. In Chinese traditional medicine, peppermint is called :bo he” and its
use can be found documented in ancient Egypt, Greece and Rome, peppermint
(Mentha piperita) was not officially described untill 1696, when the English
botainst Johan Ray (1628-1705) First discovered the pepper-flavored mint.
Entering the London pharmacopoeia in 1721, peppermint has since been cultivated
for its essential oil throughout Asia, Europe and North America. (52).

After the Second World War, some of the Japanese manufacturers shifted their
manufacturing operations from Japan to Brazil. In 1987, India became an exporter
of menthol market. In Brazil, mint was available in the dense forest, where it grew
wild. When it was harvested, the field could be left as it was, to grow back later.
Brazil became one of the main source of menthol in the world. The oil is obtained
by distillation from the freshly ground leaves and ranges from colourless to
greenish-yellow. (52).

2.3 Composition Of Essential Oil

The essential oil of Mentha Piperita contain acetaldehyde, amyl alcohal, menthyl
esters, limone, phellandrene, pinene, pugelone and dimethyl sulfide, alpha pinene,
Sabinene, Ocimene, gamma-terpinene, terpinolene, alpha and beta-thujone,
citronellol, menthol (33-60%), menthone (15-32%), methofuran, menthyl acelate
(2-11%), isomenthone (2-8%) (53,54).
TABLE 2: Chemical Components (%) of the essential oils distilled from
Mentha Piperita

Compound Mentha Piperita Essential Oil

Ria % in oil
X-Pinene 939 0.32
Sabinene 975 0.26
B-Pinene 979 0.58
1,8 Cineole 1031 0.69
Cis-Sabinene Hydrate 1070 0.50
Menthone 1152 2.45
Menthofuran 1164 11.18
Neomenthol 1165 2.79
Menthol 1171 53.28
Neomenthyl acelate 1273 0.65
Menthyl acelate 1295 15.10
B-Bourbonene 1388 0.37
(Z) – Caryophyllene 1408 2.06
F-B-Farnesene 1456 0.30
Germacrene D 1485 2.01
Bicyclogermacrene 1500 0.22
Isomenthyl accelate 1305 0.61
Total 99.37

2.4 Composition of the Leaves

In the extract of the leaves of M.Piperita are present mainly flavonoids and
phenolic acids and some of the compounds are menthol, menthone caffeic acid,
acetaldehyde, amylakohal, menthyl esters, limonene, Pinene, cardial glycosides,
phellandrene, Cadinene, pugelone and dimethyl sulfide. The constituent Features
include alpha-pinene, sabinene, terpinene, Ocinene, diterpenes, gamma, terpinene,
steroids, fenchene, alpha and beta-thujone, coumarin, citronellol, carotenes,
Tocopherols, betaine, choline, saponin, tannins and other components. (55).
2.5 Clinical Uses Of The Essential Oil From Mentha SPP

Peppermint is probably best known in the field of medicine for its role in the
suppression of the symptoms of indigestion; hence the eating of mint sweets after
meals. Peppermint oil acts both to reduce spasms of the intestinal tract and to
reduce fermentation of undigested food, by encouraging a balance between oral and
intestinal microorganisms. (52).

It has been observed that peppermint relaxes the lower esophageal sphincter and is
use full as an antispasmodic agent for double-contrast barium meal examination,
and in patients with dyspepsia (56). Menthol, a calcium channel antagonist, is
thought to inhibit contraction of smooth muscle cells by blocking the inward flux of
calcium Ions, it is also believed to have an effect on the histamine,
hydroxytryptamine and cholinergic systems of the gut; the end result of these
effects is to reduce gastro duodenal motility by decreasing the number and
amplitude of contractions during the migrating motor complex. (57). Peppermint
provides a convenient and cheaper alternative to intravenous spasmolytics such as
hyoscine N- butyl bromide (Buscopan), which can some times cause systemic
upsets and anticholinergic side effects. Peppermint oil act as an antagonist at
sensory receptors involved in emesis.

Peppermint was found to inhibit spontaneous peristaltic activity; This reduces total
gastrointestinal transit or gastric emptying, decreases the basal tone in the
gastrointestinal bract, reduces the slow wave frequency in the esophagus and small
intestine (slowing peristaltic movement) and inhibit potassium depolarization-
induced responses in the intestine. Mentha SPP oil has a pain-soothing action and is
used clinically as an ingredient in many analgesic creams and in the treatment of
arthritis and other musculoskeletal conditions. Menthol relieves discomfort by
gating afferent pain impulses, as part of an astringent effect (58) and exciting thoes
nerves that recognize the sensation of coldness. This not only causes a dulling of
pain but also encourage blood flow to the treated body part. Peppermint can help
soothe headaches; moreover, applied to the gums of teething babies it can help
relieve distress and clean teeth.
2.6 Properties of M. Pipe Rita

ANTIOXIMANT ANTITUMORIGENIC

ANTI MICROBIAL

ANTI VIRAL
MENTHE PIPERITA ANTI NOCICEPTIVE

ANTI SPASMODIC ANTI CARCINOGENIC

ANTI ALLERGIC ANTI ANGIOGENIC

2.7 Antioxidant Properties

Lack of antioxidants in organism, promotes the oxidative stress due to the presence
of free radicals, which is turn causes a variety of pathological conditions.
Antioxidants, which are an integral part of biologically active substances, are of
great interest. They can reduce mutagenic influence, regulating the oxidation
process of free radicals. Mentha piperita have antioxidant proproperties due to
presence of several bioactive substances. (59)

The antioxidant properties of Mentha Piperita are important to prevent inflamation

process and dyslipdemia as well as several chronic degenerative disease as diabetes
and cardiovascular dieases.

2.8 Antimicrobial Prosperities

The constituents of the essentional oil of M.Piperita have different Modes of Action
in bacteria and eukaryotic cells. They exhibit strong bactericidal properties, and in
eukaryotic they modify apoptosis and differentiation, interfere with post translating
modification of proteins and induce or inhibit certain liver detoxifying enzymes.
(60).
Antiobacterial activity of plants may be attributed to the presence of phenolic
compounds that behave as prooxidants because they undergo high aeruginosa,
salmonella typhimurium, staphylococcus aureus, Listeria monocytogenes,
Escherichia, staphylococcus epidermidis and sccharmoyces cerevisiae. (62)

2.9 Anti-Antigenic Activity Of Mentha Piperita Leaves Extract

Angiogenesis is the development of new blood vessels arising from current

vasculature. This process is typically initiated within hypoxic tissues where
additional new blood vessels are required to maintain oxygenation and nutritional
supply. (63). As a therapeutic mode, the body is either stimulate angiogenesis, as in
perpheral arterial diease, ischemic cardic dieases, wound healing, ovulation,
furthermore in corpus luteum formation, where there is a demand to augment blood
supply.

Or lessening or stopping the angiogensis during cancer, ophthalmic problems, and

rheumatoid arthritis. So for this Menthol present in the essential oil of Mentha
Piperita is responsible for the antiangiogenic activity. Methanol and chloroform
sample extracts of Mentha Piperita leaves showed the most significant anti-
angiogenesis action together with a significant scavenging activity for the free
radical.

Oxidation, so instead of eliminating the reaction of free radical chain, they lead to
generation of superoxides and quinones. The most easily oxidized phenolics such
as quercetin and gallic acid have pro-oxidant activity but tannins, due to the high
molecular weight have little pro-oxidant activity. (61)

The bioactivity found in different compounds of plants are generally attributed to

the presence of secondary metabolites which produce physiological actions. The
extracts can be categorized into several classes among which are terpenoids,
flavonoids and phenolics that are known to be active against bacteria, viruses and
protozoa.

The antimicrobial effects of the essential oil can be attributted to their mechanism
of action within the cell membrance. The implications of this mechanism involves
lysis and loss of membrane integrity due to changes that determine the output of
ions (Hydrogen, Potassium and Calcium), causing damage in the essential cell
survival processes.

Menthol and Menthone present in the essential oil components of M.Piperita is

responsible for the antimicroblal activity.

Menthol, menthone and the essentional oil of Mentha were found to have
antibacterial activity against enterobacter. Aerogenes, Klebsiella pneumoniae,
pseudomonas.

2.10 Potential Health Risks of Mentha SPP Essential Oil

Many dangerous and lethal side effects have been reported in relation to herbal
products. These side effects may occur by several different mechanisms, including
dired toxicity, contamination and interactions with drugs or other herbs. (67). Side
effects may be due to contaminants in herbal products, such as toxic heavy metals
and metalloids, including lead, mercury and arsenic; undeclared pharmaceuticals,
intentionally and illegally added to the herbs to produce a desired effect;
microorganisms and microbial toxins and genetic factors. These plants are capable
of synthesizing a vast array of secondary metabolites as defense mechanism to
protect themselves against pathogens.

The essential oil from peppermint is associated with side effects such as heartburn,
nausea, vomiting, allergic reactions, flushing and headaches. When taken together
with “Conventional” medicine pharmacokinetic interactions may occur if the oil
affects the absorption, distribution, metabolism or excretion of the drug or there are
pharmacodynamics interactions.

Preliminary evidence suggests that peppermint oil might interact with cytochrome
P45O. ISO Forms (CYP1A2, CYP2C19,CYP2C9, CYP3A4) and therefore might
modify the levels of drugs metabolized by those cytochromes. This interaction is of
great importance in clinical practice, given that:-

 CYP isoenzymes metabolize a large number of structurally diverse drugs

and chemicals, both natural or synthetic;
  There are important genetic polymorphisms in drug disposition among
different population and
 The variability in potency and complexity of herbal medicine preparations
is hard to assess. (4)

As to suitability for applications is cosmetic formulations, Nair (2001) reported that

the topical application of the essential oil from Menthe SPP. also deserved
investigation as its use might not always be safe; For example, pulegone, a
component of this oil and recognized heptotoxin, should be limited to a
concentration of 1% being toxic at higher concentrations. Repeated intradernal
dosing with peppermint oil produced moderate and severe reactions in rabbits,
athough peppermint oil did not appear to be phototoxic. Additionally, in a
carcinogenicity study of toothpaste components no apparent difference were noted
between mice treated with peppermint oil and those treated with the toothpaste
base.

Despite the low toxicity of Menthe SPP; a few cases have demonstrated topical
sensitivity. A study carried out by Kanerva et al. in 4000 patients, however, found
no cases of skin irritation or allergy.

Exacerbation of asthma has also been associated with the use of peppermint
containing toothpaste and when taken in a non-capsulate form, the oil may
precipitate heartburn.

Thus, further scientific studies are needed to assess the safety and efficacy of the
use of the essential oil from Menthe SPP in human beings. Efforts to elucidate the
health benefits and risks of essential oils from the diverse species of Menthe should
be intensified (68).
 Chapter 3

3 Meterial and Methods

3.1 3.1, collection of plant material

The whole plant Menthe Piperita locally named as Peppermint was collected from
different part of the Kotli District particularly from Sensa, Samroor and other
surrounding areas from June to October. It was identified by Sir Tahir Zamaan and
submitted to Chemistry laboratory in University of Kotli (A.K).

3.2 PROCESSING OF PLANT SAMPLES

The whole plants part (leaves) were washed satisfactory from a
water came from piped supply, rinsed in distilled water and was let free
at shadow place and at a room temperature for 3 days so that it could dry.
By using a sterile electric blender the dried plant was crushed very well
into fine powder. Then kept within noisture free environment.
3.3 EXTRACTION OF PLANT MATERIAL
Ethanol (1.5L) was used initially to soaked fine powdered form of
plant for three days at room temperature. Filtration and descent ion was
carried out from solvent containing extracts. The solvent-containing
 extracts were than filtered with ethanol (1.5 L) the extraction was
repeated each time under reduce pressure by using rotary evaporator the
surplus solvent was evaporated to give crude concentrated extracts of
ethanol. The same methodology is repeated to get the chloroform
petroleum ether and water extracts. The water layers were freeze dried to
give water fraction. For determination for different bioassays and
phytochemical analysis these extracts were stered at 4 C.
3.4 QUALITATIVE AND QUANTITATIVE
PHYTOCHEMICAL SCREENING
3.4.1 Preliminary Phytochemical Screenings
The extracts of Mentha Piperita according to standard methods
were used to confirm the presence of tannins, Saponins, alkaloids,
glycosides, tri terpenoids, steroids, Flavonoids and carbohydrates.
3.4.1.1 Test for Alkaloids
The appearance of orange brown precipitate confirms the
presence of alkaloids, when 2 ml dilute Hydrochloric acid was mixed
with 5 ml extract then made to react with Dragondroff’s reagent.
3.4.1.2 Test for Flavonoids
Turning of yellow solution to colorless when few drops of NaoH
mixed with 2 ml of extract confirms the presence of flavonoids.
3.4.1.3 Test for Glycosides
For a few hours on a water bath the extract was hydrolyzed with dilute
hydrochloric acid. Few drops of sodium nitroprusside solution & 1ml of
pyridine were added and shacked well until they completely mixed
followed by 2-3 drops of dilute NaoH was also mixed. The presence of
glycosides indicated when pink color produced turned into red.
3.4.1.4 Test for Saponins
Formation of a stable persistent froth when extract was added to
15 ml of deionized water and shaken vigorously indicated the presence of
saponins.
3.4.1.5 Test for Steroids
 At the junction a reddish yellow color was an indicative of the
presence of steroidal ring, when 2ml of extract and 10ml of chloroform
were mixed followed by cane. H2SO4 which formed a lower layer.
3.4.1.6 Test for Tannins
The presence of yellow precipitate confirms tannins was present
when small amount of extract was added to lead acelate solution and
were mixed properly.
3.4.1.7 Test for Triterpenoids
Reddish violet color showed the presence of triterpenoids when
5ml of extract, 2ml of acetic anhydride, 2ml of chloroform and few drops
of conc. H2 SO4 were mixed.
3.4.1.8 Test for Carbohydrates
Violet ring at the junction was not formed which indicated
absence of carbohydrates, when 2ml of Molisch’s reagent and the small
amount of extract were shocked well followed by carefully addition of
2ml of concentrated H2SO4.

3.5 1,1 – diphenyl 1-2-Picrylhydrazyl (DPPH) Scavenging

Activity

O2 N NO2

Ph
N N

Ph O2N

The antioxidant activities of M.Piperita leaves extracts were

evaluated through measuring free radical scavenging activity by DPPH
method. Serial dilutions of chloroform and methanol extracts of
M.Piperita leaves in methanol were made to obtain the concentrations of
0.007, 0.015, 0.031, 0.62, 0.12, 0.5 mg/ml. 2ml of each concentration
was transferred into 96 plate wells, each concentration was repeated for
 three wells. 1ml of 0.1 MmDPPH solutions in methanol was the added.
Ascorbic acid is a Familiar antioxidant agent; it was used as positive
control in this assay. The DPPH solution (0.1 Mm) in methanol without
the leaves extract was used as the negative control and just methanol to
represent the blank. All the tests were accomplished in triplicate. After
30 min incubation, the absorbance was assessed at 490 nm by means of
ELISA plate reader. Percentage of DPPH scavenging activity (Q) was
measured through performing the formula given below (64).
Q= (AO-A) x 100%
AO
Where:
AO : The absorbance of the control.
A : The absorbance of the sample.

Chapter 4

RESULT AND DISCUSSION

4.1 RESULTS
4.1.1 Extraction Process
 Three solvents were used in the extraction of 400 gm of
M.Piperita leaves powder. These solvents are chloroform, methanol and
water. Among the three extracts, methanol extract displayed the best
yield percentage (9.59) as shown in table (3).
Table 3 : Weight and yield percentage attained from Mentha Piperita
leaves crude extracts.
Type of Extract Weight (g/400g) Yield (%)
Chloroform 24.07 6.017
Methanal 38.37 9.59
Water 27.89 6.97

400 gm of M.Piperita leaves powder used in the extraction process.

4.2 DISCUSSION
4.2.1 Extraction Process
Extraction include the separation of the Medicinally active
components of the plant tissue that is used from those that may be inert
by employing suitable solvents and the proper techniques of extraction.
In this study, grinding of the dried plant leaves aids in producing a
homogenous sample and increasing the area of sample contact with the
solvent system. The extraction process was performed consecutively
with the three solvents, beginning from the non-polar one, and ending
with highest polarity to make sure that most of the plant leaves
constituents were extracted in relation to their polarity. (65)
Methanol extract gave the greatest yield of the crude extract and
water extract came after, while chloroform gave the lowest yield of the
crude extract.
Table 4 : The percentage of DPPH free radical scavenging activity
for chloroform and menthanol extracts of Mentha Piperita leaves in
 comparison to that of ascorbic acid.
Concentration DDPH Free radical Scavenging activity (%)
(ug/ml)
Ascorbic acid Chloroform Methanol
Extract Extract
15.62 71.64±0.002 2.21±0.01 17.46±0.02
31.25 78.84±0.003 11.44±0.01 46.55±0.03
62.5 84.06±0.005 23.7±0.02 68.39±0.08
125 88.66±0.012 31.84±0.03 76.83±0.01
250 91.57±0.017 58.63±0.04 81.79±0.06
500 93.49±0.003 61.81±0.04 83.37±0.11
Ascorbic acid used as positive contral, each concentration
has been triplicated (n=3)
It shows that there are a number of factors that may affect the
difference in the yield and the constituent of photochemical in every
extract, these involveparticle size of plant leaves powder, solvent to
sample ration, mode of the extraction method, extraction process length,
water both temperature, agitation type of solvent and its PH, polarity and
concentration. It was also observed that the way for drying the part of the
plant that is used highly affects the yield as well as the composition of
the constituents in any extract which will be tested for its activity later in
research trial. The extraction process used was the cold maceration
method to avoid destruction of thermolabile compounds caused by hight
temperature. (66).
4.3 RESUITS AND DISCUSSION
4.3.1 Antioxidant and other Properties of Mentha Piperita.
Studies with Mentha piperita has demonstrated the presence of
wide variety of bioactive compounds that represent a rich resource in
phytochemicals of great interest to treat several pathologies. Some of the
beneficial biological effects show that this plant may play an important
role as anti-oxidant, anti nociceptive, anti-inflammatery, anti microbial,
anti-carcinogenic, anti-viral, anti. allergic and anti-tumorigenic,
indicating its utility in the prevention or treatment of several dieases.
Furthermore, we may say that Mentha Piperita is a promising plant that
may offer strategy for the use in Medicine and in food industry.
 Chapter 5

REFERENCES

1. Dzhambazov B, Daskalova S, Monteva A etal. Biol pharm Bull 25:499-

504m 2002.
2. Starbuck JJ. Herbal use by health care providers. In : Eskinazi D.ed.
Botanical Medicine. Mary Ann Liebert, Inc; 1999 : P.33
3. Allaby, M. (2000) Basics of environmental science 2nd edition,
Routledge London, PP.1-7.
4. Rodriguez-Fragoso L, Reyes – Esparza J, Burchil SW, Herrera – Ruiz D,
Torres E.Risks and benefits of commonly used herbal Medicine in
Mexico. Toxicol Appi Pharmacol. 2007; 227 (1): 125-35
5. Durnev, A.D; Nikitina, v.A and Vrjesinskaya, O.A. (2002). Influence of
taking vitamins on the sensitivity of peripheral blood lymphocytes to the
clastogenic action of mutagens in vitro. Bulletin of Experimental
Biology and Medicines, 134 (9), 303-307 (Russian).
6. Fridovich, 1.(1997). Superoxide anion radikal (02), Superoxide
dismutases and related matters. JBC Online, 272 (30), 18515-18517.
7. Gordienko, A.D; Komisarenko, N.F and levchenko, V.V (1987).
Antioxidant properties of natural phenols. Rep of the fifth All-Union
Symp. On Phenol compounds, 32-33 (Russian).
8. Gromovaya, V.F; Shapoval, G.S, Mironyulk, I.E and Nestyuk, N.V
(2008). Antioxidant properties of many medicinal plants.
Pharmacoceutical chemistry Journal, 42 (1), 25-28.
9. Willioms, R.J, Spencer, J.P, Rice-Evcins, C. (2004). Flavonoids:
antioxidants or signaling molecules. Free radical Biology and Medicine,
36 (7), 838-849.
10. Shirani K, Hassani FV, Azar-Khiavi KR, Moghaddam ZS, Karini G.
Determination of methanol in Iranian herbal distillates. J complement
integr Med; 2016 Jun 1; 13 (2): 123-7.
11. Roblova V, Bittova M, Kuban P, Kuban V. capillary electrophoresis
fingerprinting and spectrophotometric determination of antioxidant
 potential. Jsep Sci; 2016 Jul; 39 (14) : 2862-8.
12. Malick B, Sinha S, Roy D. Evalution of antioxidative potential of field
grown and tissue culture. Int. J. curr. Microbiol, App. Sci. 2016; 5 (3) :
382-391.
13. Sharafi SM, Rasooli I, Owlia P, Taghizadehm, Asteneh SD. Protective
effects of bioactive photochemical, with multiple health potential.
Pharmacogn.Mag; 2016; 6 : 147-153.
14. Importence of Herbs by Jack Kittredge; 2012.
15. Hasler CM, Blumbery JB. Symposium on photochemical : Bio
Chemistry and Physiology. Journal of Nutrition 1999; 129:7565-7575.
16. Gibson EL, Wardel J, Watts CJ. Fruit and Vegetable consumption,
Nutritional Knowledge and Beliefs in Mothers and Children. Appetite
1998; 31:205-228.
17. Mathai K.Nutrition in the Adult years. In Krause’s Food, Nutrition and
Diet Therapy, 10th ed, ed.L.K. Mahan and S. Escott-Stump, 2000;
271:274-275.
18. American cancer society. Photochemical. Available at
http://www.cancer.org/eprise/main/docroot/ETO/content/ETO 5 3 X
Photochemical, June 2000.
19. Meagher E, Thomsun C. Vitamin and Mineral Therapy. In Medical
Nutrition and Disease, 2nd ed; G Morrison and L Hark, Malden,
Massachusetts : Blackwell Science Inc, 1999; 33-58.
20. Moorachian ME. Photochemical : why and how? Tastings, 2000; 4-5.
21. Costa MA, Zia ZQ, Davin LB, lewis NG Chapter Four: Toward
Engineering the Metabolic Pathways of Cancer-Preventing Lignans in
cereal Grains and other crops. In recent advances in phytochemistry,
Vol.33, photochemical, in Human Health protextion, Nutrition and Plant
Defense, ed. JT Romeo, New York, 1999; 67-87.
22. King A, yong G. characteristics and occurrence of Phenolic
Photochemical. Journal of the American Dietetic Association, 1999; 24:
213-218.
23. Narasinga Rao. Bioactive photochemical in Indian Foods and their
 potential in health promotion and disease prevention. Asia pacific
Journal of clinical Nutrition, 2003: 12 (1):9-22.
24. Abuja, 2003; 1-7.
25. Hahn NI. Is phytoestrogens Nature’s cure for what Ails US? A look at
the Research. Journal of the American Dietetic Association, 1998; 98:
974-976.

26. Walton NJ, Mayer MJ, Narbad A. Molecules of Interest : Vanillin. Phyto
Chemistry, 2003; 63:505-515.
27. Ghasemzadeh, A, Jaffar, HZE, Rahmat, A.Antioxidant activities, total
phenolics and Flavonoids contents in two varieties of Malaysia Young
Ginger (Zingiber Officinale Roscoe). Molecules, 2010; 15: 4324-4333.
28. Silva EM, Souza JNS, Rogez H, Rees JF, Larondelle Y. Antioxidant
activities and polyphenolics contents of Fifteen Selected Plant Species
from the Amazonian region Food Chemistry, 2007; 101 : 1012-18.
29. Pridham JB. In : Phenolics in Plants in Health and Disease, Pergamon
Press, New York, 1960; 34-35.
30. Tapas AR, Sakarkar DM, Kakade RB. Flavonoids as Nutraceuticals : A
Review. Tropical Journal of Pharmaceutical Research, 2008; 7 : 1089-
1099.
31. Schofield P, Mbugua DM, Pell AN. Analysis of condensed tennis : a
review. Animal Feed Science Technology, 2001; 91: 21-40.
32. Vansoest PJ. Nutritional ecology of the ruminant, 2nd ed. Cornell Univ
Press. Ithaca, NY, 1994 ; 476.
33. De Bruyne T, Pieters L, Deelstra H, Vlietixk A. condensed Vegetables
Tannins : biodiversity in structure and biological activities. Biochemical
System Ecology, 1999; 27: 445-59.
34. Dolara P, Luceri C, De Filippo C, Femia AP, Giovannelli L, Carderni G,
Cecchini C, Silvi S, Orpianesi C, Cresci A. Red wine Polyphenols
influence carcinogenesis, intestinal micro flora, oxidative damage and
gene expression profiles of colonic nucosa in F344 rats. Mutation
Research, 2005; 591 : 237-46.
35. Mueller-Harvey I, McAllan AB. Tannins. Their biochemistry and
nutritional properties. In : Advances in Plant Cell Biochemistry and
Biotechnology, Vol.1 Morrison IM, ed. JAI Press Ltd, London (UK),
1992; 151-217.
36. Rao RVK, Ali N, Reddy MN. Occurrence of both Sapogenins and
alkaloid lycorine in curculigo orchioides. Indian Journal Pharma Science,
1978; 40 : 104-105.
37. Harborne JB, Tomas-Barbera FA. Ecological Chemistry and
Biochemistry of Plant Terpenoids, Clearendon, Oxford, 1991.
38. Langenhein JH. Higher plant terpenoids : A phytocentric overview of
their ecological roles. Journal of Chemical Ecology, 1994; 20 : 1223-
1280.
39. Dudareva N, Pichersky E, Gershenzon J. Biochemistry of Plant
Volatiles. Plant Physiology, 2004; 135 : 1893-1902.
40. Morrissey JP, Osbourn AE. Fungal resistance to plant antibiotics as a
mechanism of pathogenesis. Microbiological and Molecular Biological
Reviews, 1999; 63: 708-724.
41. Takechi M, Matsunami S, Nishizawa J, Uno C, Tanaka Y. Haemolytic
and anti Fungal activities of Saponins or anti-Atpase and anti-viral
activities of cardiac glycosides. Plants Medica,

42. Triode F, Faure R, Olivier E, Gasquet M, Azas N, Debrauwer L, Keita

A, Timus David P, Balansard G. Structure and antiprotozoal activity of
titerpenoid saponins from Glinus opposite folius. Planta Medica, 2000;
66: 368-371.
43. Kukreja AK, Dhawan Op, Ahuja PS et al. J Genet Breed 2000, 54 (2) :
109-15.
44. Scora RW, Chang AC Journal of Environmental Quality 1997; 26 (4) :
975-9.
45. Valente JSS, Fonseca AOS, Denardi LB, Dal Ben Vs, Falho FSM,
Baptista CT, Braga CQ, Zambrano CG, Alves SH, Botton SA, Pereira
DIB. In Vitro susceptibility of Pythium insidiosum to Melaleuca
 alternifolia, Mentha piperitaand Origanum volgare Essentional oils
combinations. Mycopathologia; 2016 Aug; 181 (7-8) : 617-22.
46. Figueroa – Perez MG, Gallegos-corona MA, Ramosgomes M,
Reyonoso-camacho R Salicylic acid elicitation during cultivation of the
peppermint plant inproves anti-diabetic effects of its infusions. Food
Funct; 2015 Jun; 6 (6) : 1865-1874.
47. David EFS, Mischan MM, Marques MOM, Boaro CSF, Physiological
indexese macro and micronutrients in plant tissues and essential oil of
Mentha Piperita L. grown in nutrient solution with variation in N,P, K
and Mg levels. Rev. Bras. PI. Med; 2014 Mar; 16 (1): 97-106.
48. Liu X, Sun ZL, Jia AR, Shi YP, Li RH, Yang PM. Extraction,
preliminary characterization and evaluation of in vitro antitumor and anti
oxidant activities of polysaccharides from Mentha Piperita Int J of Mol
Sci, 2014; 15 (9) : 16302-16319.
49. Roblova V, Bittova M, Kuban P, Kuban V. Capillary electrophoresis
Fingerprinting and spectrophotometric determination of antioxidant
potential for classification of Mentha products. J Sep; 2016 Jul; 39 (14):
2862-8.
50. Malik B, Sinha S, ROY D. Evaluation of antioxidative potential of field
grown and tissue culture derived Mentha Piperita L. Plants. Int. J. curr.
Microbial. App. Sci; 2016; 5 (3) : 382-391.
51. Fleming T, PDR for herbal medicines, Montrate, NJ : Medical
Economics Company, Inc. 1998.
52. Spirling LI, Daniels IR. Botanical perspectives on health peppermint:
more than just an after-dinner mint. JR soc health. 2001; 121 (1) : 62-63.
53. Johari NZ, Ismail IS, Solaiman MR, Abas F, Shaari K.. Acute toxicity
and netabolonics analysis of hypocholesterolemic effect of Mentha
Piperita aqueous extract in Wistar rats. International Journal of Applied
Research in Natural products; 2015; 8 (I): 1-11.
54. Badal RM, Badal D, Baddal P, Khare A, Shrivastava J, Kumare V.
Pharmacological Action of Mentha Piperita on Lipid Profile in Fructose-
Fed Rats. Iran J Pharm Res; 2011 Automn; 10 (4) : 843-848.
55. Patil SR, Patial RS, Godghate AG. Mentha Piperita Linn :
Phytochemical, anti bacterial and dipterian adulticidal approach.
International Journal of pharmacy and pharmaceutical Sciences, 2016; 8
(3) : 352-355.
56. Melzer J, Rosch W, Reichling J, Brigmoli r, Saller r, Meta-analysis;
Phytotherapy of functional dyspepsia with the herbal drug preparation
STW 5 (Iberogast). Aliment Pharmacol Ther. 2004; 20: 1270-87.
57. Beesley A, Hardcastle J, Hard castle PJ, Taylor CJ, Influence of
Peppermint Oil an absurptive and secretory proceeses in rat small
intestine. Gut 1996; 39: 214-9.
58. Mckay DL, Bumbery JB. A review of the bioactivity and potential health
benefits of peppermint tea (Mentha Piperita L). Phytother Res. 2006; 20 :
619-33.
59. Mairapetyan S, Alexanyan J, Tovmasyan A, Daryadar M, Step anian B,
Mamikonyan V. productivity, biochemical induces and antioxidant
activity of peppermint (Mintha Piperita L) and Basils (Ocimum
basilicum L) in condition of hydroponics J. Sci. Technol. Environ.
Inform; 2016 May; 3 (2) : 191-194.
60. Sharafi SM, Rasooli I, Owlia P, Taghizadeh M, Astaneh SD. Protective
effects of bioactive photochemical from Mentha Piperita with multiple
health potentials. Pharmacogn. Mag; 2010; 6 : 147-153.
61. Chakotiya As, Chawla R, Thakur P, Tanwar P, Tanwar a, Narula A,
Grover SS, Goel R, Arora R, Sharma RK. In vitro bactericidal activity of
promising nutraceuticals for targeting multidrug esistant pseudomonas
aeruginosea. Nutrition; 2016 Jul-Aug; 32 (7-8): 890-7.

62. Iscan G, Kirimer N, Kurkcuglu M, Baser KHC, Demircl F. antimicrobial

Screening of Mentha Piperita essential oils J Agric Food Chem. 2002;
50:3943-6.
63. Hayder B. Sahib, Adeeb A Al-Zubaidy, Shallal M Hussain, Ghaith ali
jassim. The Anti-Argiogenic activity of vitex agnus castus leaves
 extracts Int J pharm pharm Sci, 6, 2014, 863-869.
64. Oktay M; Gulcin I and Kofrevioglu, O.I Determination of in Vitro anti-
oxidant activity of Funnel (Foeniculum Vulgare) Seed extracts. Leb-
Wiss.U-Techn. 36, 2009, 263-271.
65. Singh D; Sachan A; Singh H; Nath r and Dixit R.K. Extraction, isolation
and characterization of photochemical. Wldj. Of pharm. Res. 4 (5), 2015,
2703-2717.
66. Shams K.A; Abdel-Azim N.S; Saleh I.A; et al.Green technology:
Economically and environmentally innovative methods for extraction of
medicinal and aromatic plants (MAP) in Eqypt. J.chem. pharm. Res.
2015; 7 (5): 1050-1074.