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