MATERIALS AND METHODS:
PLANT PROFILE:
Scientific name- Acalypha Indica
Scientific classification:
Kingdom- Plantae
Phylum- Magnoliophyta
Class- Dicotyledonae
Order- Euphorbiales
Family- Euphorbiaceae
Genus- Acalypha
Species- Acalypha Indica
Common Name - Copper leaf, Indian Acalypha
Other Scientific Names - Acalypha spicata Forssk.
Cupamenis indica (L.)
Local Common Names :
Sanskrit : Arittamanjarie.
English : Indian acalypha.
Hindi : Kuppu; Khokali.
Telugu : Kuppichettu;Harita-manjiri;
Kuppinta or Muripindi.
International Common Names:
Alcalifa : Brazil
Tie Xian : China
Baro , Berbere : Ethopia
Kuppimeni : India
Ricinela : Spain
Kuppameniya : Sri Lanka
EPPO code - ACCIN (Acalypha indica)
PLANT MATERIAL:
The leaves of Acalypha indica were procured from the surroundings of S.K.University Campus and
were authenticated by Prof. B. Ravi Prasad Rao Head and Director, S.K.U(Herbarium)Department
of Botany.
ACALYPHA INDICA:
Acalypha indica is one of weed plants that contain important medicinal values for human
health applications.
It grows in disturbed places such as waste lands, road sides, crevices in walls.
It also grows in rocky hillside, foret edges and river banks.
It prefer moist and shaded places.
It grows from sea level upto 1350 m altitude.
ACALYPHA INDICA
Extraction Procedure:
• Phytoextraction using the Soxhlet apparatus.
• Evaluation of antidiarrheal activity in rats/mice by Acalypha indica leaves extraction.
• Collect the leaves of the plants, wash and dry in the shade for 2 weeks.
• Dissolve the leaves in different solvents such as methanol, ethanol, water, hexane,acetone...etc.
• Once the solvent is found, the extraction process begins.
• Soxhlet apparatus was consist of Condenser, cooling water, extractor, sample, heating mantle,
solvent holder
• Water flow must be continuous throughout the extraction.
• Temp 60-80⁰C.
• Collect the extract from the round bottom flask.
• After the extraction is complete, the extract is dried to obtain the final extract.
Preliminary phytochemical Study:
The plants may be considered as biosynthetic laboratories for human consumable chemical
compounds [carbohydrates, proteins, lipids] which were taken in the form of food and Detection
of Glycosides: therapeutically active chemical compounds [like glycosides, alkaloids, volatile oils,
tannins which exerts physiological effect]. The compounds which are responsible for therapeutic
effects are usyally considered as cellular secondary metabolites.
Qualitative Chemical Tests:
The Coleus blumei extract was subjected to following qualitative tests for identification of
chemical groups present in the extract.
• Alkaloids
• Flavonoids
• Glycosides
• Proteins and Amino acids
• Phytosterols
• Saponins
• Phenolic compounds
• Fixed oils
• Gums and Mucilage
• Aromatic acids
• Terpenoids
Detection of Alkaloids:
A small quantity of extract is treated with few drops of dilute HCl and filtered. The filtrate
was used for the following tests.
A) Mayer’s test:
To detect the presence of alkaloids, a few drops of Mayer’s reagent is added in solvent
free extracts. Alkaloids solution produces cream colored precipitate in presence of Mayer’s reagent.
B) Hager’s test:
Solvent free extract 50mg is stirred with few ml of dilute HCl and filtered take few ml of
filtrate and add 1 or 2 ml of Hager’s reagent. A prominent yellow precipitate indicates the
presence of Alkaloids.
Detection of Flavonoids:
Shinoda test:
The extract was treated with one gram Magnesium turnings and few drops of concentrated
HCl and boiled for 5 minutes. Formation of orange colour shows presence of flavonoids. Small
portion of each is dissolved in alkali, yellow colour was produced.
Detection of glycosides:
Test for Cardiac Glycosides:
A)BORNTRAGER’S TEST:
50mg of extract is hydrolysed with concentrated hydrochloric acid for 2 hours on a water
bath, filtered, to 2ml of filtered 3ml of chloroform is added and shaken, chloroform layer is
separated and 10% ammonia is added to it. Pink colour indicates the presence of glycosides.
B)BROWN RING TESt:
To test the cardiac glycoside phytochemicals presence, in a test tube 5 ml of extract was treated
with 2 ml of glacial acetic acid containing a drop of ferric chloride (FeCl3) solution. Afterwards it was
underplayed with 1 ml concentrated sulphuric acid (H2SO4). A brown ring of the interface indicates a
de-oxy sugar characteristic of cardenolites.
Test for Anthraquinone Glycosides:
A)Borntrager’s test:
About 1 ml extract was boiled with dilute sulphuric acid and filtered. The filtrate was
extracted with chloroform. The chloroform layer was separated and equal quantity of dilute
ammonia was added. Formation of pink or red colour in organic layer indicates presence of
glycosides.
B)Bromine test:
A little amount of solution was treated with 5N Sodium hyposulphite. Appearance of red colour
indicates the presence of glycosides.
Detection of Carbohydrates:
A minimum of various extracts was taken and dissolved in respective solvents and treated with
HCl to detect the presence of Carbohydrates by following tests.
A)Fehling’s (A and B) test:
Reducing sugars two methods were used to test for reducing sugars. First, the ethanol extract
(1 ml) was added to 1ml of water and 20 drops of boiling Fehling’s solution (A and B) in a
test tube was added too. The formation of a precipitate red-brick in the bottom of the tube
indicates the presence of reducing sugars. Second, added to 2 ml of aqueous solution, 5-8 drops
of boiling Fehling's solution. A red-brick precipitate showed the presence of reducing sugars.
B)BENEDICT’S TEST:
The extract (100mg) is dissolved in 5ml of water and filtered. To 0.5ml of filtrate, 0.5ml of
Benedict’s reagent is added. The mixture is heated on boiling water bath for 2 minutes. A
characteristic coloured precipitate indicates the presence of sugars.
C)IODINE TEST:
The aqueous extract 5ml was treated with the reagent of the starch (iodine). Any shift to blue
violet indicates the presence of starch.
Detection of Proteins and Amino acids:
A)Million's test:
Crude extract when mixed with 2ml of million’s reagent, white precipitate appeared which
turned red upon gentle heating that confirmed the presence of protein.
B)Biuret test:
An aqueous sample is treated with an equal volume of 1% strong base like
sodiumhydroxide (or) potassium hydroxide followed by drops of aqueous copper(II) sulfate. If
the solution turns purple, protein is present.
C)Ninhydrin test:
Crude extract when boiled with 2 ml of Ninhydrin, violet colour appeared suggesting the
presence of amino acids and proteins.
Detection of Phytosterols:
A small quantity of extract was dissolved in 5ml chloroform. The solution is subjected to tests
like
A) Salkowski’s test:
To 1ml solution few drops of concentrated sulphuric acid is added. Brown colour was produced.
B) Libermann-Buchards test:
The prepared chloroform solution was treated with 2 drops of concentrated sulphuric acid
followed by 3 drops of acetic anhydride. Emerald green colour formed.
C) Zark’s test:
To 1 ml solution mixture of glacial acetic acid, ferric chloride and concentrated sulphuric acid
was added. Purple colour is produced.
Detection of Saponins:
To test the saponin phytochemicals presence in various extract, the extract was diluted with
20 ml distilled water and was agitated in a graduated cylinder for 15 minutes. The formation of
1cm layer of foam indicates the presence of saponin.
Detection of Phenolic compounds:
A)Lead acetate test:
The extract (50mg) is dissolved in distilled water and to this 3ml of 10% lead acetate
solution is added. A bulky white precipitate indicates the presence of phenolic compounds.
B)Test for the Tannins:
To test the tannin phytochemical presence, in a test tube 1 ml of 5% ferric chloride added
to solvent free extract. The presence of tannin is indicated by the formation of bluish black or
greenish black precipitate.
Detection of Fixed oils:
• Between the two filter papers, small quantity of extracted solution was pressed
• It was treated with Sudan red 3 (or) Tincture of Alkana, which produces red colour.
Detection of Gums and Mucilage:
a. To the extract, the Rheuthenium red solution was added.
b. If the pink colour is formed, this indicates the presence of mucilage in the extract.
c. If treated with Methylene blue produces the deep blue colour.
d. If treated with Iodine and sulphuric acid produces the violet colour.
Detection of Aromatic acids:
• Tothe extractedsolution (small quantity), Neutral Ferric Chloride was added.
• If the aromatic acid present, a buff colour precipitate was formed.
Test for Triterpenoids:
Salkowski's test:
The substance was warmed with tin and thionyl chloride. Pink color indicates the presence
of Triterpenoids.
Hirschohn reaction:
When substance is heated with trichloroacetic acid, red to purple colouration is observed at
110c.
DRUG PROFILE:
LOPERAMIDE:
Loperamide is an anti-diarrheal agent that is available as various over-the-
counter products for treating diarrhea.The drug was first synthesized in 1969 and used medically
in 1976.It is a highly lipophilic synthetic phenylpiperidine opioid that is structurally similar to
opiate receptor agonists such as diphenoxylate and haloperidol.Due to pharmacological properties,
loperamide has been misused and abused to self-manage opioid withdrawal symptoms and to
induce euphoria.However, loperamide is associated with a risk for experiencing a range of adverse
effects, often life-threatening, if taking for non-therapeutic reasons or at doses higher than the
recommended dose.
STRUCTURE:
IUPAC name : 4-[4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl]-N,N-dimethyl-2,2-
diphenylbutanamide
CAS number : 53179-11-6
Chemical Formula : C29H33ClN2O2
Mechanism of action :
Enteric neurons synthesize and release endogenous opioid peptides and other
neurotransmitters, such as acetylcholine and substance P. Endogenous opioids bind to opioid
receptors expressed on these neurons to regulate gastrointestinal signalling, motility, and balance
of fluids and electrolytes.
Loperamide acts on the mu-opioid receptor expressed on the circular and longitudinal
intestinal muscle. Receptor binding leads to the recruitment of G-protein receptor kinases and the
activation of downstream molecular cascades that inhibit enteric nerve activity.By inhibiting the
excitability of enteric neurons, loperamide suppresses neurotransmitter release, pre-synaptic and
post-synaptic inhibition of transmission of excitatory and inhibitory motor pathways, and
secretomotor pathways. Loperamide inhibits the release of acetylcholine and prostaglandins,
thereby reducing propulsive peristalsis and increasing intestinal transit time. Loperamide
stimulates the intestinal absorption of water and electrolytes by inhibiting calmodulin. Loperamide
can bind to and hyperpolarize submucosal secretomotor neurons, promoting dry, hard stools.
Pharmacokinetic data :
Protein binding: 97%
Metabolism: hepatic Half life: 9.1 to 14.4 hours
Dose:
The usual adult dose is 4mg initially followed by 2mg after each subsequent loose stool, up
to 16md per day. Recommended maximum daily doses for children are 3mg for ages 2 to 5
years, 4mg for ages 6 to 8 years and 6mg for ages 8 to 12yrs. Loperamide should not be used
in children younger than 2 yrs of age.
Adverse Effects:
•Abdominal cramps and rashes are the common side effects.
•Paralytic ileus, toxic mega colon with abdominal distension is a serious complication in young
children.
•Fatatilties have occurred, probably due to absorption of toxins from the intestines.
•Contraindicated in children less than 4 years.
•Loperamide appears to be the most effective and most suitable of the anti motility drugs.
Contraindications:
Treatment should be avoided in the presence of fever or if the stool is
bloody (dysentery). It is of no value in diarrhea caused by cholera, Shigella or Campylobacter.
Treatment is not recommended for patients that could suffer detrimental effects from rebound
constipation. If there is a suspicion of diarrhea associated with organisms that can penetrate the
intestinal walls, such as E. coli or salmonella, loperamide is contraindicated.
Clinical Uses:
Loperamide has been shown to be effective against traveler's diarrhea, used
either alone (or) in combination with anti microbial agents (trimethoprim, sulfamethoxazole or a
fluoroquinolone). If clinical improvement in acute diarrhea does not occur within 48 hrs, 29
Loperamide should be discontinued. However, loperamide also has been used as adjunct
treatment in almost all forms of chronic diarrheal disease.
CASTOR OIL:
Castor oil is a vegetable oil obtained from the castor beari technically castor
seed as the castor plant, Ricinus communis, is not a member of the bean family) Castor oil is a
colorless to very pale-yellow liquid with mild or no odor or taste. Its boiling point is 313 °C (595
°F) and its density is 961 kg/m³. It is a triglyceride in which approximately ninety percent of
fatty acid chains are ricinoleic acid. Oleic and linoleic acids are the other significant components.
STRUCTURE :
CAS number : 8001-79-4
Mechanism of action :
Castor oil is a mix of triglycerides consisting of mainly ricinolein, linoleic acid,
oleic acid, palmitic acid, stearic acid, dihydroxystearic acid, and traces of other fatty acids .
The main pharmacodynamic effects of castor oil is mediated by ricinoleic acid, a hydroxylated
fatty acid released from castor oil by intestinal lipases. It was believed that ricinoleic acid acts
as an anionic surfactant that reduces net absorption of fluid and electrolytes, and stimulates
intestinal peristalsis. However, a recent study suggests that ricinoleic acid interacts with EP3
prostanoid receptors expressed on intestinal and uterine smooth muscles. Via activating EP3
prostanoid receptors on intestinal and uterine smooth muscle cells, ricinoleic acid promotes
laxation and uterus contraction, respectively. EP3 receptor act as the major prostanoid receptor in
the intestine mediating propulsive effects on gut motility, and activation of EP3 receptors has
been demonstrated to evoke contraction of uterine smooth muscle.
USES :
Castor oil in food :
In the food industry, castor oil (food grade) is used in food additives, flavorings,
candy (eg, chocolate), as a mold inhibitor, and in packaging. Polyoxyethylated castor oil (eg,
Cremophor Eld is also used in the foodstuff industries.
Medicinal use of castor oil :
The United States Food and Drug Administration (FDA) has categorized castor
oil as "generally recognized as safe and effective" GRASE] for over-the-counter use as a laxative,
with its major site of action the small intestine. However, although it may be used for
constipation, it is not a preferred treatment. Undecylenic acid, a castor oil derivative, is also
FDA-approved for over-the-counter use on skin disorders or skin problems.
Castor oil penetrates deep into the skin thanks to its molecular weight, which
is low enough to penetrate into the stratum corneum. Castor Isostearate Succinate is a polymeric
mixture of esters with Isostearic Acid and Succinic Acid used for skin conditioning, such as in
shampoo, lipstick and lip balm. Ricinoleic acid is the main component of castor oil and it exerts
anti inflammatory effects. One study has found that castor oil decreased pain more than
ultrasound gel or Vaseline during extracorpsreal shockwave application.
Therapeutically, modern drugs are rarely given in a pure chemical state, so
most active ingredients are combined with excipients or additives. Castor oil or a castor oil
derivative such as Cremophor El olyethoxylated castor oil, a nonienie surfactant), is added to
many modern drugs, including.
•Miconazole, an anti-fungal agent.
•Paclitaxel, a mitotic inhibitor used in cancer chemotherapy,
•Sandimmune (cyclosporine injection, USP) immunosuppressant drug widely used in connection
with organ transplant to reduce the activity of the patient's immune system;
•Nelfinavir mesylate, an HIV protease inhibitor;
•Saperconazole, a triazole antifungal agent (contains Emulphor EL-719P, a castor oil derivative);
•Tacrolimus, an immunosuppressive drug (contains HCO 60, polyoxyl 60 hydrogenated castor oil)
•Xenaderm ointment, a topical treatment for skin ulcers is a combination of Peru balsam, castor
oil, and trypsin; 31
•Aci-Jel (composed of ricinoleic acid from castor oil, with acetic acid and oxyquinoline), used to
maintain the acidity of the vagina.
