The document summarizes research conducted on the anti-inflammatory potential of Plumeria pudica leaf extracts. Leaves were extracted using different solvents, with methanol providing the highest yield. Phytochemical analysis revealed various anti-inflammatory compounds. The methanolic leaf extract was tested in a carrageenan-induced rat paw edema model and showed a dose-dependent reduction in inflammation, though not as significant as the ibuprofen control. The research concluded P. pudica leaves contain anti-inflammatory phytoconstituents and represent a readily available natural anti-inflammatory source.

1. INFLAMMATION
Inflammation is a highly dynamic process, which can be characterized as the first
protective response of body’s immune system.
Inflammation is a normal, protective response to tissue injury caused by physical
trauma, noxious chemicals or microbiological agents. Inflammation is essential for
the survival of the host, and is accompanied by its classical signs i.e. redness, heat,
swelling, pain and loss of function, which are the main cause of patient discomfort,
especially after surgical procedures.
There are mainly two types of inflammation which are as follows:-
Acute inflammation: Acute inflammation has a rapid onset of minutes or hours,
usually resolves in a few days, has classic signs and symptoms, and has cellular
infiltrate primarily composed of neutrophils. It is associated with increased vascular
permeability, capillary infiltration and emigration of leukocytes.

2. Chronic inflammation: Chronic inflammation has a slow onset of days, a long duration of
years, less prominent classical signs and symptoms, and cellular infiltrate primarily
composed of monocytes/macrophages and lymphocytes.
Causes of Inflammation:-
- Infective Agents like Bacteria, Viruses and their toxins, Fungi, Parasites.
- Immunological Agents like Cell-mediated and Antigen-Antibody reactions.
- Physical Agents like Heat, Cold, Radiation, Mechanical Trauma.
- Chemical Agents like Organic and Inorganic Poisons.
- Inert Material such as Foreign Bodies.

4. Pathophysiology of Inflammation
All inflammatory diseases have almost a common pathway of generation of disease
which involves generation of various inflammatory mediators at various stages due to
initial stimulation by one or various etiological factors which may be an infection, an
injury or even an allergic stimulus.
The etiological agent causes increased vascular permeability after initial vasodilation
and increased blood flow in the area due to release of various substances including
Histamine from the mast cells in the areas.
The increase in vascular permeability may be due to formation of endothelial gaps
under the influence of Histamine, Leukotrienes, Bradykinins, or it may also be
because of transcytosis (transcellular transport) which is due to intracellular formation
of vesiculovacuolar organelles across the endothelial cells.

5. After extravasation from the blood vessels into the tissue fluid, the leucocytes are
further attracted to the site of inflammation by various chemotactic agents, which
include endogenous substances, e.g. chemokines as well as bacterial products acting as
chemotactic substances.
Anti inflammatory drugs:-
The main anti-inflammatory drugs are either
Steroidal e.g., Betamethasone, Prednisolone, and Dexamethasone
Or
Nonsteroidal (NSAIDs) e.g. Aspirin, Diclofenac, Ibuprofen, Indomethacin. Naproxen,
Nimesulide, and Celecoxib.
Used to treat both acute inflammatory condition and chronic inflammatory diseases such as
osteoarthritis and rheumatoid arthritis. Of these, the NSAIDs are the most commonly prescribed
classes of medication for pain and inflammation. They are responsible for approximately 5-10%
of all medications prescribed each year.

6. In Vivo models for anti inflammatory activity
1. Paw edema in rats (various modifications and various
irritants)
Male or female Sprague-Dawley rats with a body weight between 100 and 150 g are
used. The animals are starved overnight. To insure uniform hydration, the rats receive
5 ml of water by stomach tube (controls) or the test drug dissolved or suspended in
the same volume. Thirty minutes later, the rats are challenged by a subcutaneous
injection of 0.05 ml of 1% solution of carrageenan into the plantar side of the left
hind paw.
The paw is marked with ink at the level of the lateral malleolus and immersed in
mercury up to this mark. The paw volume is measured plethysmographically
immediately after injection, again 3hr and 6hr, and eventually 24 hr after challenge.

7. 2. Croton-oil ear edema in rats and mice
A total of 15µl of an acetonic solution containing 75µg of croton oil is applied to the
inner surface of right ear of each mouse. Left ear remains untreated. Control animals
receives only the irritant while indomethacin (100µg/ear) serves as reference. Varying
dose levels of test drug are applied to the inner surface of right ear of each mouse by
dissolving them in inflammation inducing solution. Animals are sacrificed by cervical
dislocation 6 hr later and a plug (6 mm in diameter) is removed from both the treated
and untreated ear. The difference in weight between the two plugs is taken as measure
of edematous response.

8. Plumeria pudica
Plumeria pudica is used as medicinal plant native to Mexico, Central America, the Caribbean
and South America spreaded throughout the tropics. About 155 genera and 2000 species are
distributed primarily in the tropical and subtropical region. About eight species are known in
India and of which Plumeria acuminata and Plumeria rubra are commonly grown. They are
commonly known as "Temple tree" or "Champa" in India. Depending on location many other
common names also exists like "Kembang kamboja" in Indonesia."Kalachuchi" in the
Phillipines, Champa in laos and Dead man's fingers in Australia.
PLANT PROFILE

9. Morphology
It is small tree, 3 to 7 m high, stem smooth
and shinning succulent with abundant white
latex, easily breaks.
Scientific Classification
Kingdom - Plantae
Family - Apocynaceae
Genus - Plumeria
Species - P. Pudica

10. Leaves
Leaves are dark green and unique fiddle-
shaped, or spoon-shaped. Maximum
Leaves length - 29cm, Maximum leaves
width 11cm.
Flowers
The flowers are bisexual, fragrant, the
upper portion whitish, while the inner
lower portion yellow, 5-6cm long.

11. Objective
From the literature it was evident that the Plumeria species especially Plumeria
acuminata has being studied widely for its pharmacological potential. It was also
found that the antioxidant potential, the related anti inflammatory and antidiabetic
property of the plant has also being scientifically explored by researchers.
But all the research was directed either towards leaves or the flowers of the plant.
Some studies of other species of Plumeria were also found but no study on leaf
extracts of Plumeria pudica was revealed in the literature.
It was found that the latex proteins from Plumeria pudica possessed anti-
inflammatory, antidiarrheal activities and also has protective actions against
ulcerative colitis.
It was therefore envisioned that extracting the leaves of the Plumeria pudica in
various solvent and studying the anti-inflammatory potential of the various extracts.
RESEARCH ENVISAGED

12. The objective of the study was therefore twofold:
1. To extract the shade dried leaves of Plumeria pudica using various solvents.
2. To determine the anti-inflammatory action of the leaf extracts.
Plan of Work
The work would be divided into the following stages and performed
accordingly:-
a. Literature review.
b. Collection and authentication of the plant material.
c. Successive solvent extraction of the dried powder of leaves of the plant.
d. Phytochemical screening of the extracts.
e. Evaluation of the in vivo anti-inflammatory action of the highest yield
extract in carrageenan induced rat paw edema in rats.

13. Review of literature

14. Selection of the Plant
Plumeria pudica is planted at large in households for its decorative purpose. The
perennial flowering capability of the plant and the ease of propagation have been the
prime factors for the wide use of the plant. This ornamental plant was therefore
considered as a potential candidate for the study.
Collection and identification of the plant material
The leaves of Plumeria pudica were collected from the local surrounding of Bhopal,
Madhya Pradesh in the month of January and authenticated at Saifia Science College,
Bhopal.
EXPERIMENTAL

15. Preparation of the plant material
The collected plant leaves after authentication was washed with distilled water and was dried
under shade. The completely dried leaves were converted to fine powder form using a blender at
low speed. The powdered leaves were stored in air tight container till taken for further processes
and investigation.
Extraction of leaves
The leaves powder prepared using the above procedure was used for extraction process. Hot
continuous extraction was performed for extracting out the phytochemicals from the leaf powder.
Briefly, 500 g of the leaf powder was evenly packed in the extractor of the soxhlet apparatus and
extracted successively with various solvents of increasing polarity. The solvents used for
extraction included benzene, chloroform, methanol and water. The extraction process was carried
out for about 13 h for each solvent.

16. The extracts were filtered while hot through Whatman filter paper to remove any un-
dissolved material (debris or impurities). The extracts were concentrated by distillation
to reduce the volume to one tenth. The concentrated extracts were then transferred to
100 ml beaker and the remaining solvents were evaporated on thermostatically heated
water bath. The extracts were collected and placed in desiccators to remove the
excessive moisture. The dried extracts were stored in desiccators until used for further
investigational procedures.
Extraction Yields
The extraction yield of the leaf using different solvents is presented in Figure. The
extraction abilities of different solvents for recovering extractable components from
leaves followed the order: methanol>water>chloroform>benzene. No similar study was
found in any of the scientific reports made on the plant species.

18. Preliminary phytochemical testing
All the extracts were subjected to qualitative phytochemical testing procedures for
identifying the presence or absence of usual plant secondary metabolites. The test was
performed for alkaloids, triterpenes/steroids, glycosides, tannins, flavonoids, saponins,
and phenolic acids. The color, intensity of color or the precipitate formation was used as
observational responses to the reactions occurring in these tests.
Alkaloids
The testing for presence of alkaloids was done by re-dissoving the extracts in 5 ml of 1%
HCl solution. This extract solution was then subjected to various tests of alkaloids as
under.
• Mayer’s test: To a few ml of plant sample extract, two drops of Mayer’s reagent was
added along the sides of test tube. The formation of a cream colored precipitate was
taken as indicator of a positive response to the test

19. Glycosides
Saponin glycosides
• Froth test: 1 ml solution of the extract was boiled in distilled water and filtered. To
the filtrate was added 3 ml distilled water, shaken vigorously and heated. The
samples were observed for the persistence appearance of foam lasting for at least 15
min was taken as confirmation for the presence of saponins.
Tannins and phenolic compounds
• Ferric chloride test: To the extract is added a freshly prepared solution of ferric
chloride. Development of blue-green color is taken as indication for the presence of
tannins and phenolics.
Sterols and triterpenoids
• Libermann Burchard test: Extract was treated with few drops of acetic anhydride,
boiled and cooled. Concentrated sulphuric acid was added from the sides of the test
tube. Change in color from violet to blue or green indicates the presence of steroids.

20. Chemical
Tests
Observation Benzene
extract
Chloroform
extract
Methanolic
extract
Aqueous
extract
Alkaloid
Mayer's
Reagent
Cream color
PPt
- - - +
Glycosides
Froth Test Frothing is
seen
- + + -
Tannins and Phenols
Ferric
Chloride Test
Blue Green
Color
+ + + +
Sterols and Triterpenoids
Liberman-
Burchard
Test
Brown ring
at junction
Upper layer
turns green
+ - - -

21. Anti-inflammatory action using carrageenan induced rat paw
edema method
Animals
Healthy Wistar rats of either sex, weighing 180-250g were used for the study. The animals
were housed in cages during the course of experimental period and maintained at 12 day
and night schedule with a temperature [17-26°C] maintained at standard experimental
condition. The animals were fed with standard rodent pellet feed and water ad libitum.
The animals were fasted 12 hours before the experiment with free access to only water.
The protocol was approved by the Institutional Ethical Committee.
Carrageenan induced rat paw edema method
The carrageenan induced rat paw edema method was used for evaluating the anti-
inflammatory activity of the Methanolic leaf extract of Plumeria pudica (MLEPP)

22. Paw edema was induced by subcutaneous injection of 0.1mL (1% solution) of Carrageenan
into the plantar surface of the right hind paw of the rat. The test sample was administered in
dose of 10 mg/kg in different groups of animals, 30 min prior to carrageenan injection.
Ibuprofen (10 mg/kg IP) was used as a standard anti-inflammatory drug which was
administered 30 min prior to carrageenan injection.
Animals were divided into 4 groups (n = 6) as follows :-
Group -- I - Control - treated with vehicle (normal saline)
Group -- II - Standard drug – Ibuprofen
Group – III– MLEPP was administered in dose of 100 mg/kg.
Group – IV– MLEPP was administered in dose of 200 mg/kg.
Paw diameters were measured immediately before the administration of the Carrageenan and
thereafter at 1, 2, 4 and 6hr using Vernier Caliper. The results obtained were compared with
control group.

23. Group Change in Paw Thickness (mm)
1hr 2hr 4hr 6hr
Normal Saline 1.674 ± 0.025 3.02 ± 0.072 3.28 ± 0.086 4.10 ± 0.047
Ibuprofen 0.84 ± 0.007 1.26 ± 0.01 1.19 ± 0.014 1.03 ± 0.025
MLEPP (100
mg/kg)
1.33 ± 0.059 2.59 ± 0.110 2.73 ± 0.076 2.92 ± 0.063
MLEPP (200
mg/kg)
1.19 ± 0.025 2.14 ± 0.014 2.19 ± 0.003 2.01 ± 0.025
Results are reported as mean ± SD (n=6)

24. The percentage inhibition of paw inflammation exhibited by each group was calculated by using
following formula:
% inhibition = C-T/ C x 100
C= Paw volume (mm) in vehicle treated group (control)
T= Paw volume (mm) in drug treated group

25. Comparison of anti-inflammatory effect of Ibuprofen and MLEPP
Carrageenan-induced acute inflammation is one of the most suitable test procedures to
screen anti-inflammatory agents. As shown in the table, MLEPP was not able to inhibit
edema significantly in the early hours and in low dose but was able to inhibit edema
considerably at 6hr when the dose of 200 mg/kg was administered. The anti-
inflammatory effect of MLEPP was not very significant as compared to Ibuprofen.

26. Summary And Conclusion
The leaves of Plumeria pudica grow whirling around the stem and have a sessile base. The leaves are
light green in color and the shape of the blade is spoon like, the apex is sharp.
The extraction abilities of different solvents for recovering extractable components from leaves
followed the order:
Methanol > Water > Chloroform > Benzene.
The results suggest the presence of alkaloids, saponin glycosides, phenolics, terpenoids, sterols,
proteins and flavonoids in the leaf of the plant.
The Methanolic Leaf Extract of Plumeria pudica (MLEPP) was able to reduce the inflammation in a
dose dependent manner. The maximum inhibition of edema by MLEPP at 100 mg/kg dose was
28.78% at the end of the 6th hour while that with 200 mg/kg dose was 50.4%.

27. Conclusion
The objective of the present study was to assess the anti-inflammatory potential
of different leaf extract of Plumeria pudica using the in vivo model.
The results obtained led to the conclusion that Plumeria pudica leaves contain
high amounts of potential phyto-constitutents that lead to anti-inflammatory
activity.
The ease of availability of the plant and easy adaptability to all climatic
conditions make the evergreen flowering plant a good source of natural anti-
inflammatory compounds.