PHYTOCHEMICAL AND BIOLOGICAL STUDIES ON LEAVES OF Acalypha
indica Linn" ByMANJUNATH
Dissertation submitted to Rajiv Gandhi University of Health
Sciences, Karnataka, Bangalore In the partial fulfillment Of the
requirements for the degree of MASTER OF PHARMACYIn
PHARMACOGNOSY Under the guidance of MRS. USHA GAVANI Department
of Pharmacognosy The Oxford College of Pharmacy Hongasandra,
Bangalore 560068. March-2011
INTRODUCTION Anti oxidants Oxygen is essential for the survival
of all on this earth. During the process of
oxygen utilization in normal physiological and metabolic
processes approximately 5% of oxygen gets univalently reduced to
oxygen derived free radicals like superoxide anion (O2y), hydrogen
peroxide (H2O2) radical, nitric oxide (NO) radical, hydroxyl
radical (OHy) etc. These radicals known as reactive oxygen species
(ROS). This leads to a number of physiological disorders and organ
toxicities like diabetes, liver damage, nephrotoxicity, cancer,
cardiovascular disorders. There is a need to strengthen the
antioxidant system or exogenous administration of antioxidants to
overcome such challenges as a prophylactic or as curatives.
Anthelmintics
Anthelmintics are drugs that are used to treat infections with
parasitic worms. This includes both flat worms, e.g., flukes and
tapeworms and round worms, i.e., nematodes. There are several
classes of anthelmintics that impair cell structure, integrity or
metabolism. Inhibitors of tubulin polymerization-benzimidazoles and
probenzimidazoles (which are metabolized in vivo to active
benzimidazoles and thus act in the same manner). Uncouplers of
oxidative phosphorylation salicylanilides and substituted phenols.
Inhibitors of enzymes in the glycolytic pathway. Interference with
this process may occur by inhibiting the breakdown or by mimicking
or enhancing the action of neurotransmitters. The result is
paralysis of the parasite. Either spastic or flaccid paralysis of
an intestinal helminth allows it to be expelled by the normal
peristaltic action of the host.
Acalypha indica (Euphorbiaceae)A weed found throughout the
plains of India, ascending hills in Orisa.
30-100 cm in height with numerous ascending branches, leaves
2.5-7.5cm long with slender Petioled, ovate or rhombicovate, acute,
cuneate at base, Leaves and twings containe alkaloid like
acalyphine, acalyphamide, amides, quinine, sterols and cyanogenic
glycosides, Plant contains kaemperol, sitosterol, triacetonamine.
And new amides like auranthiamide and its acetate succinimide,
2methylamthraquinone, tri-o-methylellagic acid were isolated from
leaves. In folk medicine leaves and roots are known to be used in
treatment of ringworm infection, rheumatoid arthritis, scabies,
snake bite, skin diseases, constipation, ulcer, bronchitis
A. spicata or A. ciliata or A. canescana, Sanskrit
-Arittamanjarie. English -Indian acalypha. Hindi-Kuppu; Khokali.
Bengali-Muktajhuri; Sveta-basanta. Gujrati- Vanchi Kanto. Telugu -
Kuppichettu; Harita-manjiri; Tamil- Kuppivaeni; Kuppaimeni.
Kannada-Kuppigida. Malayali- Kuppamani. Konkanni-Kunkmiphal.
Uriya-Indramaris. Uses Cathartic, Anthelmintic, expectorant,
emetic, anodyne and hypnotic
AIMS AND PLAN OF WORK.The present study is aimed to carry out
phytochemical investigation, antioxidant and anthelmintic activity
of the isolated compounds/ extracts of leaves Of Acalypha indica.
Collection of authenticated plat material (Leaves of Acalypha
indica) Preparation of the extract of the drug by successive
extraction by soxhlation. Isolation of chemical constituents from
the extract by column chromatography. Identification of isolated
constituents by UV, IR and NMR spectroscopy. Antioxidant activity
of isolated compounds/extracts using in-vitro DPPH (1,1-diphenyl
-2picrylhydrazyl), Reducing power method.
Review of literature: Hexane, chloroform, ethyl acetate and
methanol extracts of leaves of Acalypha indica Linn. were evaluated
for antibacterial activity against both gram-positive and
gramnegative bacteria. All extracts shown activity against various
strains of gram positive and in gram negative only on Pseudomonas
aeruginosa.
Fresh juice of Acalypha indica Linn. leaf were evaluated for
anti-inflammatory activity on four groups of albino rats which are
pretreated orally with control, standard (indomethacin), in
combination of both Acalypha indica & Indomethacin, Acalypha
indica juice was effective in inhibition of paw volume and
oedema.
The antifungal activity of plant salts were evaluated against
the three fungal pathogenic isolates from HIV patients. The maximum
antifungal activity of Acalypha indica plant salts was shown
against the Candida albicans, Cryptococcus neoformans, followed by
Aspergillus flavus In higher concentration the leaves and roots of
the aqueous extract of Acalypha indica inhibit the growth of
Microsporum canis. Leaves, roots, stems, seeds of Acalypha indica
showed resistance to Aspergillus fumigatus, Candida albica.
Different leaf extracts viz., petroleum ether, benzene,
chloroform and acetone acetone extracts of Acalypha indica were
examined for the Daboia russelli venom neutralization potential at
intraperitoneal dose levels by in vitro and in vivo antisnake venom
studies. In vitro HRBC membrane stabilization properties of these
extracts at different concentrations revealed inhibition of
haemolysis induced by Russell s viper venom, in a concentration
dependent manner. Results are revealed that the acetone extract
possessed the most significant activity on venom-induced
lethality.
Chloroform and aqueous extract of root of Acalypha indica was
evaluated for their anti-inflammatory activity in rat using the
Carrageenan induced paw edema method. Chloroform extract exhibits
potent anti-inflammatory activity at 200 mg/kg four hours after
administration in compare with aqueous extract as well reference
standard Aspirin.
Larvicidal activity of Acalypha indica and Myristica fragrans
were assessed against 2 different types of larvae, i.e. Aedes
aegypti and Tribolium casteneum. M. fragrans has also been
evaluated for repellent activity against adult T. casteneum. The
chloroform extract of Acalypha indica appeared to be the most toxic
against both larvae. For M. fragrans, the hexane extract greatly
affected the growth and development of the larvae of T. casteneum.
However, it only shows weak toxicity against larvae of A. aegypti.
The chloroform crude extracts of M. fragrans exhibited the highest
repellency activity compared to the crude hexane and methanol
extracts. Phytochemical review. Seven cyanopyridone derivatives and
one corresponding seco compound have been isolated from a
methanolic extract of the inflorescences and leaves of Acalypha
indica Linn. The absolute configuration of the main cyanogenic
glucoside acalyphin,
(_)-(5R,6S)-5-cyano-5-b-D-glucopyranosyloxy-6-hydroxy-4-methoxy-1-methyl-2(5,6dihydro)-pyridone,
was deduced from an X-ray crystallographic study. In addition, the
6R-epimer of acalyphin, epiacalyphin, and the corresponding pair of
Ndemethylderivatives were isolated. The corresponding amide of
acalyphin and a 10,20-glucosyl-fused epiacalyphinamide were
isolated from air-dried material. Structural elucidation was
performed by means of 1H and 13C NMR-spectra, chiroptical methods
such as CD-spectroscopy and optical rotation. Two further
corresponding derivatives, an aromatized compound and an open-chain
structure, were isolated from the aqueous phase.
Four known kaempferol glycosides, mauritianin, clitorin,
nicotiflorin and biorobin, have been isolated from the flowers and
leaves of Acalypha indica. From a dried MeOH extract of
freeze-dried flowers and leaves was partitioned between CH2Cl2 and
H2O. TheH2O phase was further purified by MLCCC (800 rev./min,
n-ButOH/nPrOH/H2O 2:1:3, upper layer as mobile phase) Two
fractions, (280 300 ml, 845mg) and (301 330ml, 387mg) were
separated by MPLC (Europrep C-18).
Fresh, dried and powdered samples of leaf, stem and root of
Acalypha indica were subjected to fractional distillation in a
soxhlet apparatus using solvents such as hexane, chloroform,
acetone and methanol. The plant extracts and a synthetic antifungal
compound, Clotrimazole (authentic standard) were subjected to TLC
and HPLC analyses. The Rf (relative front) value of Clotrimazole
was 0.371. The plant s leaf, root and stem extracts also gave
distinct spots respectively at Rf value of 0.371 0.0009. In HPLC,
the TLC-separated active compound and Clotrimazole resolved at 1.90
0.2min (retention time). The amounts of active compound present in
root, leaf and stem extracts were 538, 415 and 171 g/g
respectively.
The total sugars in the leaves, roots and the root-knots of
Acalypha indica infected with the root-knot nematode were studied
and compared with similar studies on the uninfected plant. It was
observed that the sugar values were lesser in the infected plant
compared to the uninfected plant. In the infected root system
itself, the root-knots showed lesser amount of sugar compared to
the non-knotty portions adjacent to the root-knots.
An increase in nitrate reductase activity was observed with a
concomitant decrease in nitrite reductase activity in the herbicide
treated leaves of Acalypha indica Linn. And Dactyloctenium
aegyptium Bauv. This non-photosynthetic selective herbicide has an
uncoupler effect similar to that of DNP which in turn enhances the
nitrate reductase activity. The generation of NADH linked to the
glycolytic process, provides higher levels of NADH for nitrate
reduction and its accumulation in treated plants over the control
even in darkness.
METHODOLGY Collection of plant material Leaves of Acalypha
indica Linn. were collected from Gangavathi ( Karnataka) and
authenticated by Mr. Siddamallaya, Botanist, RRI Bangalore. The
leaves were dried, powdered and stored in air tight container for
further use. Extraction of plant material The method was based on
extraction of active constituents present in the drug, using
various solvents ranging from non-polar to polar. Dried and
powdered leaves of Acalypha indica were subjected to soxhlet
extraction with various solvents such as petroleum ether, benzene,
chloroform, acetone, methanol and water. Phytochemical examinations
of extracts. Detection of Carbohydrates: Molisch, benedict,
Fehling, Barfoed s Tests Detection of Glycosides : Borntrager s
Test, modified Borntrager s Test, Legal s Baljet, keddes and
Keller-kiliani Test. Detection of Saponins : Froth test. Detection
of Phytosterols : Liberman buchard s and Salkowski s test.
Detection of Alkaloids : Mayer s , wagners, Dragendorff s and Hager
s Test. Detection of flavonoids : Lead acetate, alkaline reagent,
shinoda and Vanillin hydrochloric Test. Detection of Proteins and
Amino acids : Millons, biuret and Ninhydrin Test.
Detection of Fixed Oils and Fats : Stain and soap test.
Detection of Gums and Mucilage: Alcohol Precipitate and Ruthenium
Red Test. Optimization of TLC solvent system. Isolation of
Photo-constituents by Column Chromatography. Purification of
isolated compounds. Solubility profile of the isolated compound.
Characterization of the isolated compound.
Total 400 fractions were collected from column. The details are
given in flow chart . Flow chart : Method of elution of elutes from
column. MeOH extract of AI 100% PE 95% PE in Bz 90% PE in Bz 80% PE
in Bz 70% PE in Bz
60% PE in Bz
50% PE in Bz
40% PE in Bz
30% PE in Bz
20% PE in Bz
10% PE in Bz
100% Bz
90% Bz in CHCl3
80% Bz in CHCl3
70% Bz in CHCl3
60% Bz in CHCl3
50% Bz in CHCl3
40% Bz in CHCl3
30% Bz in CHCl3
20% Bz in CHCl3
10% Bz in CHCl3
100% CHCl3
98%CHCl3 inMEOH
95%CHCl3 inMEOH
92%CHC3 inMEOH
90%CHCl3 inMEOH
85%CHCL 3 inMEOH
83%CHCl3 inMEOH
80%CHCl3 inMEOH
PE -Petroleum ether, Bz- Benzene, CHCl3- Chloroform, MeOH-
Methanol. AI-Acalypha indica.
Anthelmintic activity. Animals: Drugs : Preparation of extract
and procedure: Antioxidant activity. Total phenol content
: preparation of std, sample, reagents and procedure. Standard
drug: Gallic acid. Reagents: Preparation of Sodium carbonate (20%),
FC-Reagent (0.5ml). Procedure: method proposed by Singleton et al.,
50-250 g/ml of extracts+0.5ml FC-reagent 3 min room temp+ 2ml 20%
sodium carbonate 10 min blue color- 650nm. Total flavonoid content
: preparation of std, sample, reagents and procedure. Reagents:
Alcl3 (10%), Potassium acetate (1M)-0.98gm in 10ml. Standard drug:
Rutin. Procedure: method proposed by the Zeilshen et a 20-400 g/ml
of extracts+ 0.1ML Alcl3 + Potassium acetate+ up to 6ml water
415nm.
Reducing power assay : preparation of std, sample, reagents and
procedure. Reagents: Potassium di-hydrogen phosphate (0.2M): 0.68gm
in 25 ml water. Sodium hydroxide (0.2M) : 0.2gm in 25 ml water.
Ferric chloride (0.1%). Potassium ferricyanide (1%). Trichloro
acetic acid (10%). Procedure: method developed by Oyaizu M 2.5 ml
sample (5-100 g/ml) + 2.5 ml phosphate buffer + 2.5 ml of 1%
potassium ferricyanide this mixture was incubated for 20 min at
room Temp. then rapidly cooled + 2.5ml of trichloroacetic acid
& 2.5ml of supernatant was taken + 2.5 ml of distilled water +
0.5 ml ferric chloride mixed well allowed stand for 10 minute
absorbance was . measured at 700 nm.
DPPH Assay
:preparation of std, sample, reagents and procedure. Standard
drug: Gallic acid. Reagents: 0.1mM DPPH. Procedure: 3ml (4-125
g/ml) extract + 1ml DPPH solution absorbance measured at 517nm. The
difference in absorbances between the test and control calculated
and expressed in terms of % inhibition. The capability of %
inhibition was calculated by using following formula.
Reagent blank difference % Inhibition =
----------------------------------- x 100 Reagent blank
The antioxidant activity of the extract was expressed as
IC50.
RESULTS:
Percentage yield of Acalypha indica Linn. extracts. Extract Pet.
ether Benzene Chloroform Acetone Methanol % Yield 5.01 2.38 2.90
3.41 15.80
18.49 Aqueous Phytochemical screening of extracts of Acalypha
indica Linn.SL. NO. 1. TESTSCARBOHYDRATES
Pet.Ether
EXTRACTS Benzene Chlorofor m + + + + + + + +
Acetone
Methano l + + + + -
Aqueous
y y y y 2 y y y y 3 3.1
Molischs test Benedicts test Fehlings test Barfoeds test Mayers
test Wagners test Dragendorffs test Hagers test
+ + + +
+ + + + -
+ + + + + + + +
ALKALOIDS
GLYCOSIDESANTHRACENE GLYCOSIDES
3.2
y Mod.Brontragers y Brontragers test CARDIAC GLYCOSIDES y Keller
killani test y Legal test y Baljet test
-
-
-
-
-
-
4 5
SAPONINS
y y y 6
Froth test Salkowaski Libermann-Burchards test Ferric chloride
test Gelatin test Lead acetate test Alkaline reagent test Shinoda
test
+ +
+ +
+ +
+ +
+ -
+ -
PHYTOSTEROLS
PHENOLICS AND TANNINS
y y y y y 7
-
-
-
-
+ + + + +
+ + + + +
PROTEINS AND AMINO ACIDS
y y y 8 y y 9 y y
Millons test Biuret test Ninhydrin test Stain test Soap test
Alcohol ppt test Ruthenium red test
-
-
-
-
-
-
FIXED OILS AND FATS
GUMS AND MUCILAGE
(+) Signifies present (-)
Signifies absent
Isolated compound
% yield in mg
AC-I 6 Thin layer chromatography pattern of isolated
compound
solubility of isolated compound.Solvent Pet. ether Benzene
Chloroform Solubility + ++ +++ _ _ _
AC-I Acetone Methanol Aqueous
+++ Freely soluble, ++ partially soluble, + Sparingly soluble, -
Insoluble.
Anthelmintic activity photocopies of standard and extracts
40mg/ml
PET. ETHER 40mg/ml
BENZENE 40mg/ml
CHLOROFORM 40mg/ml
METHANOL 40mg/ml
AQUEOUS 40mg/ml
Total phenol content of standard Gallic acid. Sample
Concentration 2 (g/ml) Absorbance at 0.088 650nm 4 0.195 Gallic
acid 6 0.326 8 0.558 10 0.692 12 0.908 14 0.988 16 1.029
Total phenol content of different extracts.
Sl. Concentration Acetone extract No. (g/ml)
Absorbance at 650nm Methanol extract
Aqueous Extract
1. 2. 3. 4. 5.
50 100 150 200 250
0.066 0.131 0.250 0.321 0.399
0.078 0.140 0.210 0.288 0.368
0.245 0.475 0.729 0.880 1.101
Total flavonoid content of standard Rutin
Sample Concentration (g/ml)
Rutin 10 20 30 40 50 60 70 80 90
Absorbance at 0.236 0.439 415nm
0.672
0.924
1.068
1.338
1.540
1.704
2.312
Total flavonoid content of Acetone, Methanol and Aqueous
extracts.
Sl No. 1. 2. 3. 4. 5.
Acetone extract Concentration (g/ml) 20 40 60 80 100
Absorbance At 415nm 0.074 0.169 0.244 0.300 0.353
Methanol extract Concentration Absorbance (g/ml) At 415nm 50 100
200 400 500 0.062 0.080 0.125 0.286 0.317
Aqueous extract Concentration Absorbance (g/ml) At 415nm 50 100
200 300 400 0.058 0.120 0.170 0.270 0.321
Reducing power assay of standard Ascorbic acid. Sample Conc.
(g/ml) 2.5 5 0.198 Ascorbic Acid 7.5 0.267 10 0.381 12.5 0.449 15
0.540 17.5 0.648 20 0.733 22.5 0.790 25 0.902
Absorb0.044 ance at 700nm.
Reducing power assay of Petroleum ether, Benzene, Chloroform,
Acetone, Methanol and Aqueous extracts
Absorbance at 700nm Sl No. Concentra tion (g/ml) Petroleum
Benzene ether extract Extract 0.078 0.168 0.174 0.328 0.399 0.062
0.068 0.078 0.097
Sl. No
Concentratio n Chloroform (g/ml) extract 5 15 25 35 50 0.016
0.025 0.049 0.069 0.084
Absorbance at 700nm Acetone Extract 0.014 0.027 0.049 0.080
0.119 Methanol Extract 0.364 0.377 0.383 0.449 0.519 Aqueous
extract 0.016 0.028 0.039 0.107 0.122
1. 2. 3. 4. 5.
20 40 60 80 100
1. 2. 3. 4. 5.
0.167
DPPH assay of standard Gallic acid.
Sl. No 1 2 3 4 5
concentrations in (g/ml) 0.125 0.250 0.500 0.750 1.000
Absorbance At 517nm 0.165 0.142 0.090 0.054 0.019
% Inhibition 13.30 25.44 53.17 71.49 89.69
IC50 (g/ml)
0.4358
DPPH assay of Pet. ether and Benzene extracts.
Sl. No
Conc. (g/ml)
Pet.ether Extract Absorbance At 517nm 0.080 0.049 0.038 0.030
0.020 % Inhibition 50 69.37 76.25 81.25 87.50 IC50 (g/ml)
Benzene Extract Absorbance At 517nm 0.088 0.044 0.020 0.014
0.009 % IC50 Inhibition (g/ml) 45 72.50 87.50 91.25 94.37
1. 2. 3. 4. 5.
25 50 75 100 125
24.93
28.20
DPPH assay of Chloroform, Methanol extracts.
Sl. No.
Conc. Chloroform Extract (g/ml) Absorbance %Inhibition IC 50
(g/ml) At 517nm 4 6 8 10 20 0.087 0.081 0.077 0.045 0.041 44.23
48.76 50.64 71.87 74.37
1. 2. 3. 4. 5.
5.68
Methanol Extract Absorbance % IC50 At 517nm Inhibitio (g/ml) n
0.120 23.07 0099 36.53 10.17 0.084 46.15 0.076 57.54 0.071
60.33DPPH Assay of Aqueous extract.
DPPH assay of Acetone extract.
Sl. No
1. 2. 3. 4. 5. 6. 7. 8.
Conc. Acetone Extract (g/ml) Absorbanc % IC50 e Inhibiti (g/ml)
At 517nm on . 10 0.104 35 20 0.100 37.50 30 0097 39.37 40 0.095
40.62 69.05 50 0.087 45.62 60 0.083 48.12 70 0.079 50.62 80 0.076
52.50
Sl. No.
Conc. (g/ml)
Aqueous Extract Absorbance At 517nm 0.095 0.088 0.078 0.076
0.075 0.064 0.056 % Inhibitio n 50.00 54.16 59.37 60.41 60.93 66.66
70.83 IC50 (g/ml)
1. 2. 3. 4. 5. 6. 7.
6 8 10 20 30 40 50
4.94
Discussion, Conclusion & SummaryThe present study is an
attempt to carry out extraction, phytochemical screening isolation
and identification of phytoconstituents, anthelmintic and
antioxidant activity of crude extracts of Acalypha indica.
Extraction and isolation of phytoconstituents Characterization of
isolated compound Evaluation of anthelmintic activity Total phenol
and flavonoid content Evaluation of antioxidant activity