cinnamomum verum research study

1. [1]
1.1 Introduction:
The plant kingdom is a virtual goldmine of potential drug targets and other active
molecules waiting to be discovered. Plants have been important source of
medicine for thousands of years. It has been estimated that only 10-15% of
7,50,000 existing species of plants have been surveyed for medicinal uses
(Bramwell, 2002). Because of instinctive urge, intuition and accumulated
knowledge, the human being uses the natural resources to cure common ailments.
It is estimated that approximately one quarter of prescribed drugs contain plant
extracts or active ingredients obtained from plant substance (Vermaand
Kuriakose, 2010). The plant kingdom thus represents an enormous reservoir of
pharmacologically valuable molecules still waiting to be discovered
(Hostettmannet al., 2000). Traditional medicine, making use of herbs in different
preparation greatly relied upon especially by rural dwellers, for the treatment of
various ailments, traditional practitioners or healers are the dispensers of such
concoctions (Nuhuand Aliyu, 2008).
Medicinal plants, since time immemorial, have been extensively used as a source
of medicine (Hoareauand Dasilva, 1999; Kumar, 2004; Patwardhanet al. 2005,
Vermaand Baksh, 2009). Uses of plants as a cure for human ailments are a
tradition as old as human civilization. Ancient traditions of medicine viz.
Ayurveda, Unani, Siddha, Homeopathy and now even Allopathy derive many of
their curative tools from plants. The wide usage of herbal remedies and health care
preparations, as those describalin ancient text such as Vedas, Samhitasand Bible
are obtained from commonly used traditional herbs and medicinal plants which
has been traced to the occurrence of natural product with medicinal properties
(Fomumet al. 1986; Vermaand Dahake, 2011).

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1.2 Cinnamon verum
The bark of various cinnamon species is one of the most important and popular
spices used worldwide not only for cooking but also in traditional and modern
medicines. Overall, approximately 250 species have been identified among the
cinnamon genus, with trees being scattered all over the world [Sangal, A., 2011].
Botanical Classification
Kingdom – Plantae
Sub kingdom - Tracheophytes
Super division - Angiosperms
Division - Magnoliids
Class - Magnoliopsida
Order – Laurales
Family - Lauraceae
Genus - Cinnamomum
Species - verum
(Ref: Rawat, I., et al., 2019)
In addition to being used as a spice and flavoring agent, cinnamon is also added
to flavor chewing gums due to its mouth refreshing effects and ability to remove
bad breath. Cinnamon can also improve the health of the colon, thereby reducing
the risk of colon cancer [Wondrak, G.T., et al., 2010].
Cinnamon is a coagulant and prevents bleeding. Cinnamon also increases the
blood circulation in the uterus and advances tissue regeneration. This plant plays
a vital role as a spice, but its essential oils and other constituents also have

3. [3]
important activities, including antimicrobial, antifungal, antioxidant, and ant
diabetic [Rao, P.V. and Gan, S.H., 2014].
1.3 Phytochemicals
The potential of higher plants as source for new drugs, is still largely unexplored.
Among the estimated 250,000-500,000 plant species, only a small percentage has
been investigated phytochemically and the fraction submitted for biologically or
pharmacologically screening is even smaller. Medicinal plants are now getting
more attention than ever because they have potential of myriad benefits to society
or indeed to all mankind, especially in the lien of medicine and pharmacological
studies. The medicinal value of these plants lies in bioactive phytochemical
constituents that produce definite physiological action on the human body
(Akinmoladun et al., 2007). Some of the most important bioactive phytochemical
constituents are alkaloids, essential oils, flavonoids, tannins, terpenoid, saponins,
phenolic compounds and many more (Edeoga et al., 2005).
Due to their specialized biochemical capabilities, plants are able to synthesize
and accumulate a vast array of primary and secondary chemicals useful for the
plant itself as protecting against environmental stress factors. These compounds
have made many plants useful also for humans for instance as spices and
medicines etc. (Verma et al., 2010a, 2011).
The phytochemical studies on medicinal plants has served the dual purpose of
bringing up new therapeutic agents and providing useful leads for
chemotherapeutics studies, directed towards the synthesis of drugs, modeled on
the chemical structure of natural products. In addition, the knowledge of chemical

4. [4]
constituents of plants would further be valuable in discovering the actual value of
folklore remedies (Verma et al., 2010b).
Secondary Metabolites
Phytochemicals are divided into groups, which are primary and secondary
constituents, according to their functions in plant metabolism. Primary metabolites
are responsible for the basic functions of life and comprise common sugars, amino
acids, proteins, lipids and chlorophyll. Secondary metabolites are not responsible
for basic functions of life yet they are important for other important activities such
as defence. Secondary metabolites consist of a variety of compounds i.e.
alkaloids, terpenoids and phenolic compounds (Krishnaiah et al., 2007) and many
more such as flavonoids, tannins and so on. The strong structure-function
relationship is observed with these secondary metabolites, which enables the
scientists to predict about the biological functions of such metabolites. Hence, a
phytochemical screening of any given plant makes an indispensible tool for
biological research (Verma and Baksh, 2010).
Phenolic compounds are secondary metabolites in plants that are involved in a
number of metabolic pathways and are essential for plant growth and
reproduction, and as protecting agents against pathogens. Phenolic compounds
may play an important role in preventing chronic illnesses such as cardiovascular
disease, certain. type of cancers, neurodegenerative disease, and diabetes (Scalbert
et al., 2005). In plants, these metabolites and their derivatives play an important
role in cell wall integrity and defence against pathogens (Faulds and Williamson,
1999).
Flavonoids and other polyphenols belong to the recently popular phytochemicals,
chemicals derived from plant material with potentially beneficial effects on human

5. [5]
health. The antioxidant activity of flavonoids is efficient in trapping superoxide
anion (O₂), hydroxyl (OH), peroxyl (ROO) and alcohoxyl (RO) radicals (Repetto
and Llesuy, 2002).
Tannin is a general descriptive name for a group of polymeric phenolic
substances. They are divided into two groups, hydrolyzable and condensed
tannins. Alternatively, tannins may be formed by polymerization of quinone units
(Geissman, 1963). One of their molecular actions is to complex with proteins, thus
mode of antimicrobial action may be related to their ability to inactivate microbial
adhesions, enzymes, cell envelope transport proteins etc. (Ya et al., 1988)
C. verum elaborates different classes of organic compounds of medicinal
important including Carbohydrate, protein, alkaloid, flavonoids, steroids, phenol ,
terpenoids, saponins, tannin and terpenoids.
1.4 Pharmacological activities
Antioxidants
The term antioxidant is generally used for those compounds that scavenge the
free radicals or reactive oxygen species formed in the human body. The human
body has a defense mechanism against free radicals, but excessive production of
free radicals can cause oxidative damage in cells (Silalahi, 2001). It can also be
described as substances capable of counteracting the damaging effects of
oxidation in body tissues. Antioxidants are of great importance in terms of
reducing oxidative stress that is thought to cause damage to biological molecules
(Bektas et al., 2005).

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Antioxidants can inhibit or delay the oxidation of an oxidizable substrate in a
chain reaction, therefore, appear to be very important in prevention of many
diseases (Halliwell et al., 1992). Thus synthetic antioxidants are widely used in
the food industry. However, because of their toxic and carcinogenic effects, their
use is being restricted. Thereby, interest in finding natural antioxidants, without
undesirable side effects, has increased greatly (Rechner et al., 2002).
Natural antioxidants from plant extracts provide a measure of production that
slows the process of oxidative damage (Bergman et al., 2001). Antioxidant
compounds can scavenge free radicals and increase shelf life by retarding the
process of lipid peroxidation, which is one of the major reasons for deterioration
of food products during processing and storage (Halliwell and Gutteridge, 1999).
Antioxidants neutralize highly unstable and extremely reactive molecules, called
free radicals, which attack the cells of human body every day (Stauth, 2007). Free
radical damage is believed to contribute to a variety of health problems, including
cancer, heart disease and ageing (Stauth, 2007).
Antioxidants based drugs/ formulation for the prevention and treatment of
complex diseases like atherosclerosis; stroke, diabetes, Alzheimer's disease, and
cancer have appeared during the last 3 decades (Devasagayam et al., 2004). This
has attracted a great deal of research interest in natural antioxidants.
The number of antioxidant compounds synthesized by plants as secondary
products, mainly phenolics, serving in plant defense mechanisms to counteract
ROS in order to survive, is currently estimated to be between 4000 and 6000
(Havsteen, 2002). Several studies have described the antioxidant properties of
medicinal plant rich in phenolic compounds (Tsao and Deng 2004; Nijveldt et al.,
2001). Medicinal plants represent a constant interest as sources of new antioxidant
substances. The large majority of substances isolated from plant with antioxidant
activities are flavonoids (Nijveldt et al., 2001).

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1.5 The Present Study
In view of the various studies dealing with C. verum, the present study assesses
the potential of C. verum in relation to its traditional uses and in terms of findings
based on modern bio scientific research. The link between conventional remedies
and recent research in various areas has been well established in other plants
which facilitate to determine effective mode of action of plant derived products.
The plant is known to contain several pharmacological important bio molecules,
whose efficacy is well established by several biochemical and pharmacological
studies. However, there are several missing links in the modern knowledge. The
present study is an attempt to make a comprehensive study which will deal with
all possible missing links related to C. verum. The objectives of the study were:
1. To validate the traditional claims of selected plant as free radical scavenger.
2. To evaluate botanical and physicochemical parameters of crude drug as per
WHO guidelines.
3. To evaluate the secondary metabolites in cinnamon bark powder.
4. To find out the antioxidant activity of C. verum.
5. To find out the antimicrobial activity of C. verum.
6. To find out the anti-diabetic activity of C. verum.

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2.1 Historical perspectives
Cinnamon is native to Sri Lanka (formerly Ceylon), the neigh bouring Malabar
Coast of India, and Myanmar (Burma) and is also cultivated in South America
and the West Indies. The spice, consisting of the dried inner bark, is brown in
colour and has a delicately fragrant aroma and a warm sweet flavour. Cinnamon
has been known from remote antiquity [Sangal, A., 2011]. It was imported to
Egypt as early as 2000 BC, but those who reported that it had come from China
had confused it with cinnamon cassia, a related species. In Ancient Egypt,
cinnamon was used to embalm mummies. From the Ptolemaic Kingdom onward,
Ancient Egyptian recipes for kyphi, an aromatic used for burning, included
cinnamon and cassia. The gifts of Hellenistic rulers to temples
sometimes included cassia and cinnamon. During the 1500s, Ferdinand Magellan
was searching for spices on behalf of Spain, and in the Philippines found
Cinnamomum mindanaense, which was closely related to C. verum the cinnamon
found in Sri Lanka [Jakhetia, V., et al, 2010.].
Use of cinnamon can be dated back to almost 2800 BC where it was initially
referred to as “Kwai” in Chinese language. It was a component of the anointing
oil used by Moses for the purpose of anointment (to make a person holy) as
mentioned in the Bible. The Romans used it for its medicinal properties for
ailment of the digestive and respiratory tract. It was also used in Roman funerals
in order to fend off the odor of dead bodies. It was used in Egypt for embalming
of mummies as well as for its fragrance and flavoring properties. However being
very expensive and highly treasured, the quest for cinnamon led to a world
exploration in the15th century. It was the motivation behind Christopher
Columbus’s voyage which led to the discovery of the new world and for Vasco da
Gama’s exploration of South India and Sri Lanka. The native of true cinnamon or
Ceylon cinnamon was then found to be in Sri Lanka (also known as Ceylon). Thus

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it became evident, that any country which could hold that area captive had a
control over the world trade of cinnamon and would ultimately reap immense
profits. Thus over years, initially the Portuguese ruled who were later
overpowered by the Dutch, followed by the British in 1815. Now it’s cultivated in
Sri Lanka along the coastal belt from Negombo to Matara (Kawatra, P. and
Rajagopalan, R., 2015.).
2.2 Botanical description, Biography and Ecology:
The C. verum tree is evergreen, grows to around 10 m (30 ft). Its branches are
strong and are smooth and yellowish in colour. It has leathery leaves, 11 to 16 cm
(4.5 to 6.25 in) long, with pointed tips. The leaves are dark green on top and
light green at the bottom. The inconspicuous yellow flowers with a
disagreeable odour, which are tubular with 6 lobes, grow in panicles (clusters)
that are as long as the leaves. The fruit is a small, fleshy berry, 1 to 1.5 cm (0.25
to 0.5 in) long, that ripens to black, partly surrounded by a cup-like perianth
(developed from the outer parts of the flower). The spice form of cinnamon is
obtained by removing the outer bark of the tree, and scraping the inner bark,
which is dried and ground into powder. Cultivated trees may also be coppiced (cut
back to encourage shoot development), so that the coppiced shoots can be
harvested. Cinnamon oil is steam distilled from the leaves and twigs (Rawat, I., et
al., 2019).
Cinnamomum is adapted to a wide range of climatic conditions. Cinnamon
requires a warm and humid climate with a well distributed annual rainfall of
around 2000-2500 mm, and average temperatures of about 27°C. Wild cinnamon
trees are adapted to tropical evergreen rainforests. It grows best at low altitudes,
and is usually grown without shade, but being essentially a forest tree, light shade
is tolerated. It grows well on different soils in the tropics, but soil type has a

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pronounced effect on bark quality. Fine sandy and lateritic gravelly soils rather
than rocky and stony substrates are best in Sri Lanka and India, but in the
Seychelles and Madagascar more loamy soils are preferred. Cinnamon is
considered susceptible to salinity, and a bitter product results from waterlogged
and marshy conditions (Rawat, I., et al., 2019).
2.3 Phytochemical composition of Cinnamon
The phytochemical analysis shows that cinnamon contains a variety of
compounds such as alkaloids, tannins, flavonoids, phenols etc. Previews study
showed the present of 21 chemical components; the major components were
cinnamaldehyde (85.50%), stigmasterol (3.69%), cadinene (1.37%), (E)-
cinnamaldehyde (1.35%), αamorphene (1.33%), hydrocinnamaldehyde (1.28%),
α-cubebene (1.25) and ergosterol (1.09%). This result are in agreement to those2.4
Pharmacological activity found by Raeisi et al. ; where cinnamaldehyde was
dominant major compound (79.74%) in hydro-distillated essential oil, other
components were similar too. Also Huang et al.; results of C. cassia the
cinnamaldehyde (68.52%) was found to be the major compound was also agreed
our result. Francisco et al. ; results of C. zeylanicum and C. cassia essential oil
was also proved that cinnamaldehyde was the major component, where it's agreed
our obtained results. Ainane et al.; showed that in their result the major
component of the essential oil of C. verum bark cinnamaldehyde (89.31%), which
also in agreement with our result. Also Valizadeh et al. and Al-fekaiki et al. ;
their study result on the essential oil of the Cinnamon was shown the major
component was cinnamaldehyde (69.15%), cinnamaldehyde (57.83%)
respectively which similar to our result. Variation of cinnamon essential oil
composition may be affected by geographical location and harvesting time
(Ahmed, H.M., et al., 2020.).

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Antioxidant/free radical scavenging activity
Cinnamon have antioxidant compounds : polyphenols,phenolic acid and
flavonoids give the health benefits for cinnamon as antioxidants, and prevent
oxidative stress in the body by its respond to free radicals and reduce damage
from metabolic diseases in the body . (Abeysekera et al.2019)Many antioxidants
that have been indicated in cinnamon: camphene, eugenol, salicylic acid, and
epicatechin. In contemporary time, natural antioxidants are the concentrate of
major interesting different studies that indicated it’s affected. And fixed how it
can be using there as effective foods and can block oxidative damage in the
organism (Mahdi et al. 2018). The Extracted oil and eugenolshowedvery powerful
activities (Rao and Gan 2014). Other study found that limit nitric oxide build-up
in the blood and block fat per oxidation, the free radicals and nitric oxide can raise
the danger of cardiovascular disease , brain disorders, carcinoma (Abeysekera et
al. 2019). Studies showed the many antioxidants find in this herb help to stop
harmful free radicals in the organism and blocked oxidative stress
Antimicrobial activity
To date, several antimicrobial activities of cinnamon and its oils have been
reported in various studies [Prabuseenivasan, S., et al., 2006]. For example, Matan
et al. reported the effects of cinnamon oils on different bacterial
(Pediococcushalophilus and Staphylococcus aurous), fungal (Aspergillusflavus,
Mucorplumbeus, Penicilliumroqueforti, and Eurotium sp.), and yeast species
(Candida lipolytica, Pichiamembranaefaciens, Debaryomyceshansenii, and
Zygosaccharomycesrouxii) [Matan, N., et al., 2006.], indicating that cinnamon is a
natural antimicrobial agent.

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Goñi et al. described the antibacterial activity of a combination of cinnamon and
cinnamon oils against Gram-positive organisms (Listeria monocytogenes,
Enterococcus faecalis, Staphylococcus aurous, and Bacillus cereus), as well as
against Gram-negative bacteria (Salmonella choleraesuis, Escherichia coli,
Pseudomonas aeruginosa, and Yersinia enterocolitica) [Goñi, P., et al., 2009.]. A
study from Hili et al. indicated that cinnamon oils have potential action against
various bacteria (Pseudomonas aeruginosa, Staphylococcus aurous, and
Escherichia coli) and yeast (Torulopsisutilis, Schizosaccharomycespombe,
Candida albicans, and Saccharomyces cerevisiae) [Hili, P., et al., 1997]. A recent
study reported the activity of the aqueous extract of cinnamon and other plants
against oral micro flora. Overall, the essential oil from cinnamon is more potent
than other tested plant extracts, such as Azadirachtaindica and Cinnamomum
verum (Rao, P.V. and Gan, S.H., 2014).
Anti-Diabetic activity
A substance from cinnamon has been isolated and coined as “insulin-potentiating
factor” (IPF) [Khan, Aet al., 1990], while the ant diabetic effects of cinnamon
bark have been shown in streptozotocin-induced diabetic rats [Onderoglu, S., et
al., 1999.]. Several studies have also revealed that cinnamon extracts lower not
only blood glucose but also cholesterol levels [Rao, P.V. and Gan, S.H., 2014].
A study comparing the insulin-potentiating effects of many spices revealed that
the aqueous extract of cinnamon was 20-fold higher than the other spices.
Methylhydroxychalcone polymer (MHCP) is the purified polymer of
hydroxychalcone with the ability to stimulate glucose oxidation. Anderson et al.
isolated and characterized the polyphenol type-A polymers from cinnamon and
found that these substances act as insulin-like molecules [Anderson, R.A., et al,

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2004]. Following this characterization, a new compound from hydroxycinnamic
acid derivatives named naphthalene methyl ester, which has blood glucose-
lowering effects, has been identified, further confirming cinnamon's ant diabetic
effects (Rao, P.V. and Gan, S.H., 2014).
Several polyphenols have been isolated from cinnamon. These polyphenols
include rutin (90.0672%), catechin (1.9%), quercetin (0.172%), kaempferol
(0.016%), and isorhamnetin (0.103%). Cao et al. (2007) demonstrated that the
aqueous extract of cinnamon containing polyphenols purified by high
performance liquid chromatography (HPLC) showed insulin-like activity. The
aqueous extract of cinnamon markedly decreased the absorption of alanine in the
rat intestine. Alanine plays a vital role in gluconeogenesis, is altered back to
pyruvate in the liver, and is utilized as a substrate for gluconeogenesis
[Kreydiyyeh, S.I., Usta, J. and Copti, R., 2000]. However, another study
conducted on diabetic postmenopausal women supplemented with cinnamon
showed poor glycemic control, even though cinnamon is generally believed to be
useful for diabetes. However, it is plausible that differences in the dose of
cinnamon used, as well as baseline glucose and lipid levels, have led to these
variations (Rao, P.V. and Gan, S.H., 2014).
In a recent study, suitable doses of cinnamon (5, 10, and 20 mg/kg) of the linalool
chemo type were found to help with glycemic control in diabetics due to enhanced
insulin secretion. It is plausible that the amelioration of oxidative stress and the
proinflammatory environment in the pancreas may confer protection to pancreatic
β cells [Lee, S.C., et al., 2013], which should be further investigated.
2.5 Final perspectives of literature Reviewed

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Medicinal plants are the richest bioresource of drugs for traditional systems of
medicine, modern medicines, Nutraceuticals, food supplements, folk medicines,
pharmaceutical intermediates and chemical entities for synthetic drugs. Aromatic
plants are a source of fragrances, flavours, cosmeceuticals, health beverages and
chemical terpenes.Medicinal plants are important for pharmacological research
and drug development. Over three-quarters of the world population relies mainly
on plants and plant extracts for health care. One fifth of all the plants found in
India are used for medicinal purpose. Out of these the bark of Cinnamon is widely
used as a spice due to its distinct odour of different compounds. The detailed
information as presented in this review on its Phytochemistry and various
pharmacognistic and pharmacological properties of the spice. Moreover the
mechanisms of some compounds are identified so far. Hence extensive research is
required to find out the mechanism of action of other compounds in cinnamon and
exploit their therapeutic potential to combat various diseases. Therefore,
Cinnamon plays an important role in modern system of medicine as a
multipurpose medicinal spice.
On the basis of biological activities of Cinnamomum verum, extract and derived
phytochemicals and their uses as pharmacological agents in traditional and
modern research are possible but will first require more clinical trials and product
development. The current evidence is largely limited to correlation between
identified phytochemicals and mode of action for any pharmacological activity.
Mechanism of action studies are expected to lead the way in the discovery of new
agents with improved and intriguing pharmacological properties. This could be
achieved by molecular modeling studies involving interaction of bioactive
phytochemicals from Cinnamomum verum with their respective molecular targets
and the extract of Cinnamomum verum could be further explored in the future as a
source of useful phytochemicals for the pharmaceutical industry.

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3.1 Identification and Collection of plant material of C. Verum
According to the US National Plant Germplasm System, C. verum, commonly
known as Cinnamon, belongs to the family Lauraceae. The dried powder of bark
of C. Verum was provided by Excellent Bio Research Solutions PVT. Limited for
the present study.
3.2 Phytochemical Studies
Phytochemical screening: (Trease and Evans, 1983; Harborne, 1998: Thimmaiah,
2004)
Phytochemical screening is an initial set of experiments to gather first hand
information about any plant in order to identify the possible potential use of the
plant for a variety of applications. In a narrower sense the terms are often used to
describe the large number of secondary metabolic compounds found in plants.
Phytochemicals are usually divided into primary and secondary metabolites.
The primary metabolites are essential for growth and survival of producer plant
and include carbohydrates, amino acids, proteins, lipids (fatty acids and fats) and
fibers (Alstone and Irwin, 1961). Secondary metabolites are synthesized by
secondary metabolic pathways, which are almost independent of
primarymetabolic pathway. Though not involved in primary metabolism of the
plants, these secondary metabolites have a role in defense and other important
activities.
Due to the different chemical nature of phytochemicals, a systematic
phytochemical extraction procedure has to be followed involving solvents of

16. [16]
different polarity. The usual technique involves extraction of phytochemicals by
polar solvent directing towards non-polar solvents. The dried plant part powders
were extracted as given below.
A. Aqueous extract:
For first extraction of phytochemicals from C. verum, cold percolation method
was used (Harborne, 1998). For this, 10 g of dried plant sample was suspended in
250 ml of cold distilled water. The suspension was stirred continuously in cold for
48 h. After 48 h, the suspension was filtered through a multi-layered muslin cloth
and the filtrate was kept refrigerated till use. The remaining powder was extracted
two more times in a similar fashion to ensure complete extraction. All the filtrates
were pooled and pooled extract was concentrated by keeping in a wide Petri plate
at 40°C in a hot air oven (Jindal, India). The volume was reduced to 20 ml,
filtered with Whatman No. 1 filter paper and was kept refrigerated until use. The
residue after cold percolation was dried and used for further extraction.
B. Methanol extract:
The residue obtained after the aqueous extraction was air dried. The dried
material was placed in a thimble prepared by rolling the filter paper sheet and
sealing one end. The thimble was placed in the extractor arm of the Soxhlet
extractor (ASGI, India). The reservoir of the extractor was filled with 250 ml of
methanol (Qualigens, India). The reservoir was heated at a constant temperature
of 45°C via a heating mantle. The water cooled condenser condenses the vapors of
methanol which drips into the extraction unit containing thimble loaded with plant
material. The methanol extracts the phytochemicals and comes out of the thimble
which is made from a filter paper. Once the extraction arm is full, the methanol
with extracted plant material goes down to the solvent reservoir via a looped

17. [17]
leveler arm from extraction unit to the reservoir. It completes one cycle. The
methanol continues to evaporate, condense and dripping down on plant material
completing the extraction.
The plant material was extracted with methanol for ten complete Soxhlet cycles
(this means the plant material was extracted with 2 L of methanol as one cycle
uses 200 ml of methanol). After extraction, the Soxhlet unit was dismantled and
the extracted plant material was recovered from the solvent reservoir. The volume
of the extract was reduced to 20 ml, filtered and refrigerated till use. The thimble
with plant residue was removed from the extractor arm and kept at 40°C for
drying in an oven. Since, the chlorophyll is extracted with methanol from the
greener parts viz. leaf, the extracts carry very dark green color for such plant parts.
Such extracts were clarified using activated charcoal. For this, 1 g of activated
charcoal (Qualigens, India) was added to the extract and stirred for 15 min at RT.
Afterwards; the suspension was filtered through Whatman No. 1 filter paper to
obtain a clarified extract.
C. Ethyl acetate extract:
The dried plant material after methanol extraction along with thimble was used to
extract phytochemicals with ethyl acetate. For this, 10 cycles of Soxhlet extraction
was used as described above with the ethyl acetate as solvent in the solvent
reservoir. The temperature of the heating mantle was kept to 40°C. The residue
obtained was further dried as described above. The extract was collected from the
reservoir and concentrated to 20 ml as described above.
Qualitative Tests:

18. [18]
Qualitative tests for major phytochemicals were performed in different plant parts
extracted sequentially with four solvents. The volume of each extract was 20 ml.
1. Alkaloid:
Alkaloids are a group of naturally occurring heterocyclic compound containing
nitrogen, with an alkaline pH and marked physiological action on animal
physiology.
Qualitative tests for Alkaloids:
One ml of each plant extract was mixed with 2 ml of 1.5% HCl and filtered
through filter paper. The filtrate was divided into 3 parts and following tests were
performed with them:
a. Mayer's test:
Preparation of Mayer's Reagent: Dissolved 1.358 g of HgCl₂ in 60 ml of water
and pour into a solution of 5 g of KI in 10 ml of Distilled water. Sufficient water
was added to make the volume up to 100 ml.
Test: To 1 ml of plant sample 1ml of Mayer's reagent was added. Formation of
white precipitate indicated the presence of alkaloids.
b. Wagner's test:
Preparation of Wagner' reagent: Dissolved 2 g of iodine and 6 g of potassium
iodide (KI) in 100 ml of water.

19. [19]
Test: To 1 ml of plant sample 1ml of Wagner's reagent was added. Appearance of
brown precipitate indicated the presence of alkaloids.
c. Dragendorff's test:
Preparation of reagent:
Solution A: Bismuth nitrate (0.17g) in acetic acid (2 ml) and H₂O (8ml)
Solution B: KI (4g) in acetic acid (10ml) and H₂O (20ml)
Mixed solutions A and B and diluted to 100 ml with H₂O.
Test: To 1 ml of plant extract, added 2 ml of Dragendorff's reagent. Formation of
orange-white precipitate indicates the presence of alkaloids.
2. Saponins:
Saponins are amphipathic glycosides grouped, in terms of phenomenology, by
the soap-like foaming they produce when shaken in aqueous solutions, and, in
terms of structure, by their composition of one or more hydrophilic glycosides
moieties combined with a lipophilic triterpene derivative.
Qualitative test for saponins:
Foam test is a usual way to test presence of saponins. 1 ml of plant extract was
taken in a test tube with small amount of water. Sodium bicarbonate was added to
it and shaken vigorously for 5min. Formation of foam indicated the presence of
saponins.

20. [20]
3. Flavonoids:
Flavonoids are polyphenolic compounds that are ubiquitous in nature and
categorized, according to chemical structure into flavonols, flavones, flavonones,
is flavones, catechins, anthocyanides and chalcones. They are water soluble with
various colors i.e. red, crimson purple or yellow. They have been reported to have
antiviral, anti-allergic, anti-platelet, anti-inflammatory, anti-tumor and antioxidant
activities.
Flavonoids contain chemical structural elements that may be responsible for their
antioxidant activities. Flavonoids are important for their antioxidant and free
radical scavenging activities.
Qualitative test for Flavonoids:
To 0.5 ml of plant extract 5 ml diluted ammonia was added followed by 1ml of
concentrated sulphuric acid. A yellow coloration that disappears on standing
indicated the presence of flavonoids.
4. Resins:
The resin produced by most plants is a viscous liquid, composed mainly of
volatile fluid terpenes, with lesser components of dissolved non-volatile solids
which make resin thick and sticky.
Resin is a hydrocarbon secretion of many plants. It is valued for its chemical
constituents and use, such as varnishes and adhesives, as an important source of
raw materials for organic synthesis, or for incense and perfume.

21. [21]
Qualitative test for Resins:
In a dry test tube, 2 ml of acetic acid and 2 drops of concentrated sulphuric acid
were added to 0.5 ml of plant extract. A purple color which changes to violet
within 10 min indicated the presence of Resin.
5. Tannins:
Tannins are polyphenolic compounds widely spread in the nature and present
nearly in all plant parts. Tannins are of two types; hydrolysable tannin and
condensed tannin. Hydrolysable tannin consist a polyhydric alcohol. Condensed
tannins are mostly flavonoids and cannot be hydrolyzed to simple compounds.
Qualitative test for Tannins:
For the qualitative tests of tannins, 10 ml of all the plant extracts were dried. The
residue was dissolved in 10 ml of water and filtered. The tests were performed
using this filtrate.
a. Gelatin solution test:
To the little of filtrate, 1% gelatin solution was added. Formation of curdy white
precipitate indicated the presence of tannin.
b. Lead acetate test:

22. [22]
To the filtrate, 5 ml of 10% lead acetate solution was added. Formation of white
precipitate indicated the presence of tannin.
c. Ferric chloride test:
To the filtrate, 5 drops of 5% ferric chloride solution was added. Formation of
blue green coloration indicated the presence of tannin.
6. Sterols:
Sterols are a class of lipids and important class of organic molecules. They occur
naturally in plants, animals, and fungi, with the most familiar type of animal sterol
being cholesterol. Sterols of plants are called phytotsterols. Phytotsterols include
campesterol, sitosterol and stigma sterol.
Qualitative test for Sterols:
a. Salkowski test:
To 0.5 ml of the extract, 2 ml of chloroform and 2 ml of concentrated sulphuric
acid was added from the side of the test tube. The test tube was shaken for few
minutes. The development of red color in the chloroform layer indicated the
presence of sterols
7. Cardiac Glycosides:
Cardiac glycosides represent a group of triterpenoids, also known as cardinolides.
There are many members of this group present with complex mixture in the same

23. [23]
plant. The most typical example is oleandrin; the toxin for Oleander leaves.
Cardiac glycosides are generally toxic and exhibit pharmacological activity,
especially on heart.
Qualitative test for Cardiac Glycosides:
Keller Killiani test:
5 ml of plant extract was evaporated to get the residue. A few milligram of
residue was diluted to 5 ml with water. 2 ml of glacial acetic acid containing one
drop of ferric chloride solution was added to it. This solution was underplayed
with 1 ml of concentrated Sulphuric acid. A brown ring at the interface indicates
presence of deoxy sugar characteristic of cardiac glycosides.
8. Triterpenes:
Few mg of plant extract residue was mixed with 5 ml of chloroform and warmed
for 30 min (40°C). Few drops of concentrated sulphuric acid were added and
mixed well. The appearance of red color indicated presence of Triterpenes.
9. Coumerins:
Coumerins are double ring phenolic compounds having distinct sweet smell.
They are also known as anti coagulating agents. Coumerins are used as blood
thinners and rodent poisons
Quantitative test for coumerin:
A small amount of plant extract residue was taken in a test tube and covered with
a filter paper moistened with dilute sodium hydroxide solution. The covered test

24. [24]
tube was placed in a boiling water bath for several minutes. The paper was
removed and exposed to Ultra Violet light. Appearance of yellowish green
fluorescence indicated the presence of coumerin in sample.
10. Anthraquinone:
Anthraquinone are colored pigments that range from pale yellow to almost black.
These phenolic compounds are widespread in plants and show a variety of
pharmacological activities.
Quantitative test for Anthraquinone:
A small amount of plant extract residue was dissolved in 5 ml of 10% Sulphuric
acid, boiled for few minutes and filtered while hot. The filtrate was cooled and
shaken with 3 ml of benzene. The benzene layer was separated and shaken with
half of its volume of 10% ammonia. The ammonical layer acquiring pink color
indicated the presence of anthraquinone
Antimicrobial or Antibacterial activity
The different extracts of C. verum were screened for antibacterial activity against
Staphylococcus aurous ATCC 25923 bacteria. These bacteria were procured from
American Type Culture Collection (ATCC), USA through Microbiologics®,
USA. The agar well diffusion method was used to determine the antibacterial
activity using Bauer-Kirby method (Bauer et al., 1966).
For antibacterial activity, Muller Hinton agar (HiMedia, India) was used. The
composition of Muller Hinton agar was
Composition**
Ingredients g/ Litre

25. [25]
Beef, infusion from 300.000
Casein acid hydrolysate 17.500
Starch 1.500
Agar 17.000
Final pH ( at 25°C) 7.3±0.1
**Formula adjusted, standardized to suit performance parameters
For preparation of the media, 38.00 g of dehydrated powder was suspended in 1
L of distilled water and the suspension was placed for 2 min in a microwave oven,
so that agar is melted. The media was autoclaved at 121°C and 15 lbs. pressure for
15 min. the media was allowed to cool till 35°C and was poured into sterile Petri
plates under laminar air flow. The plates were allowed to solidify before use.
A sterile non-toxic cotton swab was dipped into this standardized inoculum. The
excess media was removed from the swab by pressing the swab by the side of the
culture tube. The inoculums on swab was streaked onto entire surface of Muller
Hinton agar plate (HiMedia, India). The process of streaking was repeated three
times, turning the plate at 60° each time to obtain a confluent growth on
Petriplate. The plate was allowed to dry for 15 min at RT.
For agar well preparation, 6 mm of agar wells were punched using the well punch
onto the plate prepared above. The media from the well was removed using fine
forceps. 25 μl of the plant extract (aqueous, methanol, ethyl acetate and petroleum
ether extracts of different plant parts equivalent to 500 mg per ml. concentration in
terms of plant dry weight) was placed into this well. Ampicillin (10 µg) was used
as a control antibiotic for Gram negative bacteria while amikacin (30 µg) was
used as control antibiotic for Gram positive bacteria. The plates were allowed to
stand for 30 min and carefully placed into incubator without spilling the extracts.
The plates were incubated for 37± 1°C for 48 h. After 48 h the results were

26. [26]
expressed as diameter of the clearing zone around the well measured in
millimeters using a zone measurement scale (HiMedia, India).
3.4.3 Antioxidant Activities
Free radical scavenging activity of any molecule is an important pharmacological
activity useful to eradicate free oxygen radicals made during metabolism. Such
free radicals show a variety of side effects as they tend to bind to a majority of
molecules due to their hyperactive ionic nature. Antioxidants bind to such free
radicals and help removing them from the body. On the nature of free oxygen
radicals i.e. peroxide, superoxide molecules, the antioxidant activity can be
assessed using various techniques.
DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity
DPPH assay is an rapid and sensitive method for the antioxidant screening of
plant extracts is free radical scavenging assay using 2,2,diphenyl-1-picrylhydrazyl
(DPPH) stable free radical spectrophotometrically (modified method of
Matsushing, 1996; Kumar et al, 2008). DPPH free radical is dark violet in color
which in the presence of an antioxidant obtains one more electron to become
DPPH and the absorbance decreases at 517 nm (Koleva et al, 2002). The resulting
de-colorization is stoichiometric with respect to number of electrons captured.
DPPH absorbance assay:
DPPH solution:

27. [27]
For DPPH stock solution, 2.366 mg of DPPH free radical (Sigma, USA) was
dissolved in solution. 100 ml of absolute ethanol to obtain 60 µM DPPH free
radical
Sample preparation:
All the extracts from different plant parts of P. amarus were dried as described in
FTC method. 25 mg of dried extract was dissolved in 25 ml of absolute ethanol
and kept in dark.
Positive control:
Ascorbic acid served as a positive control. 25 mg of ascorbic acid (SRL, India)
was dissolved in 25 ml of absolute ethanol to get 1 mg per ml concentration and
kept in dark.
Procedure:
The scavenging effect of plant samples as well as ascorbic acid was carried out
according to Kumar et al, (2008) and Matsushinge, (1996). The sample solution of
each tested material (500µl) was mixed with the same volume of DPPH solution.
and allowed to stand for one and a half hour at room temperature in dark (or until
stable absorption values were obtained).The absorbance was then measured at 517
nm using a spectrophotometer. Ethanol served as negative control.
The percentage scavenging effect was determined by comparing the absorbance
of the solution containing the test sample to that of negative control solution
(ethanol) without test sample taking corresponding blanks. The absorbance was

28. [28]
taken three times each after 30min till the stable readings are achieved. The mean
of three measured values for each sample were taken.
Calculation:
% antioxidant activity for DPPH = (A-Ax)/A x 100
Where
A- Absorbance of DPPH solution with ethanol.
Ax- Absorbance of DPPH solution with test solution
Antidiabetic activity
There are various methods to assess the antidiabetic property of any given plant
extract. Inhibition of activity of enzyme a-amylase, which is responsible for
conversion of starch in to sugars, is one of the most promising in vitro method
(Bhutkar and Bhise, 2013).
The a-amylase inhibition assay was performed using the 3,5dinitrosalicylic acid
(DNSA) method (Wickramaratne et al., 2016). The a amylase inhibitory activity
was expressed as percent inhibition of starch hydrolysis. The amount of starch
remained in the reaction mixture was calculated using the standard curve of
starch.
Reagents
Plant extract: The extracts from C. verumwere dissolved in 10% DMSO and was
further dissolved in phosphate buffer (0.02 M with 6 mMNaCl, pH 6.9) to give a
concentration of 1 mg ml¹.

29. [29]
α-amylase solution: Commercially available enzyme powder (2 units/ml, SRL,
India) was dissolved in 0.02 M with 6 mMNaCl, pH 6.9. The enzyme solution
was prepared fresh.
Starch Solution: 1 g of potato starch powder (SRL, India) was suspended in 0.02
M phosphate buffer, pH 6.5. The solution is kept in a water bath at 65°C for 30
min to facilitate complete dissolution of starch. Cooled at RT.
Dinitro Salicylic Acid (DNSA) reagent: 12g of sodium potassium tartrate
tetrahydrate (Qualigens, India) was dissolved in 8.0 ml of 2 M NaOH and 20 ml
of 96 mM of 3,5-dinitrosalicylic acid solution (SRL, India).
Standard curve of starch
For preparation of standard curve of starch, 100 mg of potato starch was
dissolved in 10 ml of 0.02 M phosphate buffer, pH 6.5 under warm conditions.
Various amounts of this solution were taken in order to get 1, 2,3, 4, 5, 6, 7, 8, 9
and 10 mg starch in different test tubes. The volume of the tubes was made upto 1
ml with phosphate buffer. To this, added 1 ml of DNSA reagent and the reaction
mixture was boiled for 10 min in a water bath at 85-90 °C. The mixture was
cooled to ambient temperature and was diluted with 5 ml of distilled water, and
the absorbance was measured at 540 nm using a UV-Visible spectrophotometer
(El, India) against a blank which does not receive the starch solution.
The absorbance of the different starch concentrations were plotted against
concentration using Microsoft Excel®. A linear regression line was plotted using
the software and the standard equation as well as correlation coefficient (R) value
was obtained. The standard curve was used for calculation only if the R² value
exceeds 0.95 (95% confidence limit).

30. [30]
a-amylase inhibitory activity assay
For the test, 200 μl of a-amylase solution was mixed with 200 μl of the plant
extract and was incubated for 10 min at 37 "C. To this reaction mixture, 200 μl of
the starch solution was added and incubated for 3 min. The reaction was
terminated by the addition of 200 μl DNSA reagent and was boiled for 10 min in a
water bath at 85-90 °C. The mixture was cooled to ambient temperature and was
diluted with 5 ml of distilled water, and the absorbance was measured at 540 nm
using a UV-Visible spectrophotometer (El, India). A blank reaction was prepared
using the plant extract at each concentration in the absence of the enzyme
solution. The negative control with 100% enzyme activity was prepared by
replacing the plant extract with 200 μl of buffer. A positive control sample was
prepared using Acarbose (100 µg/ml-2 µg/ml) and the reaction was performed
similarly to the reaction with plant extract as mentioned above.
The absorbance of the reaction mixtures were converted to amount of residual
starch left unhydrolyzed. The present inhibition of the a-amylase activity was
calculated by comparing the values with respect to the Acarbose.

31. [31]
Phytochemical Studies
Extraction of plant material:
Ten grams of powder of plant part (cinnamon bark) were extracted first with
distilled water using cold percolation method for 48 hours, then stir it properly
with the help of magnetic stirrer. The extract was filtered, concentrated under
vacuum and the volume was finally reduced to 20 ml. The residue after aqueous
extraction was extracted with methanol , ethyl acetate respectively using Soxhlet
extractor and the final volumes were reduced to 20 ml for each extract.
Result
The phytochemical screening showed presence of various phytochemicals in
different extracts of plant. Table4.1 Shows tha phytochemicals present in aqueous
extract, methanol extract and ethyl acetate extract of Cinnamomum verum. The
alkaloids were found to be present in methanol extract as Mayer, Dragendroff's
and Wagner's tests for alkaloids were positive. Ethyl acetate extract showed
presence of saponins with positive foam test. Presence of flavonoids was found in
methanol and aqueous extracts. Resins were found absent in all extracts. Among
tannins, Gelatin test, Lead acetate test and Ferric chloride test were positive with
methanol and aqueous extracts. Sterols was found in methanol and aqueous
extracts shows by positive Salkowaski test. Cardiac glycoside were not found in
all extracts of Cinnamomum verum. Triterpenes were found in methanol extract of
Cinnamomum verum. Caumerins were found absent in all extracts as are
anthraquinone.

32. [32]
Pharmacological activities
4.4.1 Antibacterial activities
The different extracts from different plant parts of Cinnamomumverum were
screened for antibacterial activity against Gram positive bacteria Staphylococcus
aureus ATCC 25923. These bacteria were procured from American Type Culture
Collection (ATCC), USA. The agar well diffusion method was used to determine
the antibacterial activity and the results were expressed as diameter of the clearing
zone around the well measured in millimetres. Gentamicin gen50mcg was used as
control antibiotic for Gram positive bacteria. Fig2 represents the effect of solvents
and control antibiotics on the growth of test bacteria. No organic solvent could
produce a zone of inhibition towards any of the test bacterium.
Table 2 shows the antibacterial activity of different extracts of
Cinnamomumverum. Gentamicin gen50mcg showed zone diameters of 30mm
against Gram positive bacteria Staphylococcus aureus ATCC 25923.The aqueous
extract and methanol extract showed inhibition towards Staphylococcus aureus
ATCC 25923.
The aqueous extract showed the zone of inhibition diameters of 15mm and
methanol extract showed the zone of inhibition diameters of 20mm against Gram
positive bacteria Staphylococcus aureus ATCC 25923.Ethyl acetate extract failed
to inhibit towards bacteria.

33. [33]
4.4.2Antioxidant activity
The antioxidant activity is judged by the potential of any compound to scavenge
free radicals. The antioxidant potentials of different extracts of Cinnamomum
verum were assessed using DPPH free radical scavenging method.
Antioxidant activity using DPPH free radical:
DPPH is a free radical showing absorbance at 517 nm. When the free radical
obtains one more electron (donated by the antioxidant compound), it is converted
into DPPH- reduced form and the absorbance at 517 nm decreases. The decrease
in absorbance corresponds to the potential of antioxidant activity by the given
compound.
During the present study, the antioxidant potential of Cinnamomum verum
extracted with different solvents was tested for their antioxidant potential against
DPPH free radical. For the assay, 10 ml of the extracts (corresponding to 5 t
material) prepared using different solvents were dried off completely and
redissolved in 1 ml methanol. Negative control was placed which received 1 g dry
plant ml of methanol. Positive control received ascorbic acid (4 mg ml) in the
reaction mixture. The absorbance of the reaction mixture was taken after 1 hour
and every half an hour thereof till the absorbance of the reaction mixture is stable.
The ascorbic acid showed 98.44% reduction of absorbance as compared with
negative control after 1h 30min.

34. [34]
Table 3 shows that different extracts of C. Verum with different solvents showed
varied antioxidant potential. Ethyl acetate extract showed 58.54% reductionin
absorbance against negative control, while aqueous and methanol fraction showed
better antioxidant activity by reducing the absorbance of DPPH free radical by
87.04and 96.89% respectively.
4.4.3Antidiabetic
Table 4.4.3.1 shows the readings of standard curve of glucose and Fig 4.2.3
shows the standard curve of glucose.
Table 4.4.3.2 shows that antidiabetic activity of different extracts of
Cinnamon, while the positive control (antidiabetic drug; acarbose) was able to
inhibit 97.40% activity of a-amylase, the Aqueous extract was ableto
show comparable activity by showing 96.71% inhibition of the enzyme.
Methanol and ethyl acetate shows 68.21 and 59.77%.
This means, the Cinnamon has a potential to reduce the conversion rate of
starch into glucose and thereby maintaining blood glucose level to the
normal level.

35. [35]
Discussion
The development of functional foods should be a priority to respond to global
health problems.Functional foods may be designed by supplementation with an
active ingredient that is known for its health benefits. The long list of beneficial
physiological effects of cinnamon suggests that it might be considered as an
essential part of human diet. The use of cinnamon in an innovative functional food
product is strategically and technologically feasible; however, the route to achieve
that goal is lengthy and challenging. A series of investigations in natural product
chemistry, in vitro bioassay, biopharmacy, animal experiments, and clinical
studies are still needed. The information obtained from those studies is required to
create functional foods that promote human health. Considering the route of the
research, a collaboration between scientists from various research areas is needed
to build a valid methodology and to avoid an incorrect assessment.
Phytochemicals occur in various parts of plants. Their functions are diverse
which include provision of strength to plants, attraction of insects for pollination
and feeding, defense against predators, provision of color, while some are simply
waste products (Ibegbulem et al., 2003). These secondary plant metabolites
exhibit varied biochemical and pharmacological actions in animals when ingested
(Trease and Evans, 1983). Cinnamon contains a large number of phytochemicals
including triterpenoid, alkaloids, glycosides, tannins, flavonoids ,saponins and
coumerin. (M. B. Saeed et. al. 2020)

36. [36]
Our results show that aqueous and methanol solvents were able to extract
different phytochemicals, whereas ethyl acetate fails to extract none of them.
Alkaloids, tannins, sterols and triterpenes were found in methanolic extract.
Saponins and tannins were found in aqueous extract. Tannins were found in both
aqueous and methanolic extract.
Cinnamon essential oil showed strong antimicrobial activity against selected
pathogens in vitro and the minimum inhibitory concentration values against all
tested microbes were detrmined as 0.5 microlitres except for S. aurues for which
the essential oil was not effective in tested concentration. In our study, only
methanolic extract showed inhibitory zone against tested bacteria, whereas
aqueous and ethyl acetate extracts were failed to show inhibitory zone. The study
clearly indicates that the phytochemicals responsible for the antibacterial activity
were extracted in methanolic solvent only.
The antioxidant potential of Cinnamon extracts was investigated in the search for
new bioactive compounds from natural resources by J Mancini- Filhoet. Al.
1998). The percentage free radical scavenging activity of methanol, ether and
aqueoes extracts were 95.5, 68 and 87.5 %. (J Mancini- Filhoet. Al. 1998).
In our study, methanol extract showed highest degree of potential to scavenge
DPPH free radical i.e., 96.89% Aqueous showed 87.04% and Ethyl acetate
showed 58.54%. It is clear from the result that cinnamon bark has potential to be
an good Antioxidant agent.
According to the in vitro studies suggest cinnamon exerts antidiabetic effects
through inhibiting gastro-intestinal enzymes, modulating insulin response and
sensitivity, improving glucose uptake, inhibiting gluconeogenesis and increasing
glycogen synthesis. Human studies assessing in vivo effects of cinnamon have

37. [37]
shown mixed results. Therefore, there is yet no clear consensus on the antidiabetic
potential of cinnamon ,and this highlights the importance of further
research.(Nicholas J. Hayward 2019).
Our study shows that Aqueous extract was found to be more potent than other
extracts with 96.71 % inhibition of amylase activity. Ethyl acetate shows 59.77%
and methanol extract shows 68.21 % inhibition of amylase activity. By the results
it is clear that cinnamon is good for diabetes.

38. [38]
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