Aruna Chittamuri et al. / Journal of Pharmacy Research 2012,5(4),1827-1837 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Phytochemical and antimicrobial studies of a herbal Medicinal aspera L. Leaf extracts

Aruna Chittamuri*, Suvarnalatha Anchapakula, Alekhya Cheruku, Chaithra Dandu, Yasodamma Nimmanapalli. Meerasaheb Chittoor, Department of Botany, S.V.University, Tirupati-517502 Andhra Pradesh, . Received on:01-01-2012; Revised on: 17-02-2012; Accepted on:20-03-2012

ABSTRACT Aeschynomene aspera is administered against tuberculosis, skin infections, antidote to snake venom, menstrual disorders and small pox. Hence the phytochemical and antibacterial studies were carried out. Preliminary phytochemical studies revealed the presence of Alkaloids, Flavonoids, Phenols, Terpinoids, Anthocynadins, Indoles, Glycosides, Saponins and Tannins in Hot water, Cold Water, Alcohol and Methanol extracts. The qualitative analysis reveals the presence of 9 identified and 2 unidentified phenolic compounds along with 3 flavonoid constituents through paper chromatography. Antibacterial activity on four selected pathogens like Bacillus subtilis, Staphylococcus aureus (Gram +ve) Escherichia coli, Pseudomonas aeruginosa (Gram- ve) bacterial strains were observed with all extracts. Alcoholic extracts are more effective on all the bacteria at 10 mg concentration with an average inhibitory zone between 40 to 45 mm nearly double to that of the controls i.e., 20-25mm (Ampicillin-10 mg and Gentamycin-30mg).But Staphylococcus aureus is more resistant showing the equal inhibition to that of the controls with all the extracts. Minimum Inhibitory Concentration values in all the extracts on all the selected bacteria ranges between 0.42mg to 0.78 mg.

Key words: Phytochemical screening, Antimicrobial activity, Qualitative analysis, Aeschynomene aspera L.

INTRODUCTION Aeschynomene aspera () is a tall erect sub in swampy areas, with stout glabrous nodular stems, full of white pith. It is commonly known as Niru-jilugu (Telugu) Sola pith plant (English) Sola (Hindi) Aatrunetti (Tamil). It is used as a substitute for cork material and as a sun hats fide [1].Pith is used as helmets and floats [2]. Magico religious belief of the tribe Oraon root is used for jaundice [3]. This in North Bihar region is used as ornamental purpose and used for making berths (fish boats) also for several handicraft items like garlands, maur (head crown), Jhamp and paag which were variously used in auspicious occasions. These people called it as pith plant. In Siddha it is called as Aatrunetti where the leaves are used to cure joint pains and swellings [4-5]. In Ayurveda this species is known as Pashenabhed recommended for painful micturition and for break- ing uric acid calculi [6].It is a weed of paddies [7]. The roots are boiled in

Plant of A.aspera(Terrestrial) less quantity of water and made in to paste applied on mumps [8]. Aerial part juice is given to cure cold, cough, and fever. Dried young shoot powder with half tea spoon powdered candy is given to increase the consistency of semen; local herbalists used it for urinary troubles [9]. A.aspera is also recognized as leafy vegetable [10].

Phenolic compounds like Protocatechuic acid prevents spore germination and growth of smudge fungi and other fungi. It acts as anti amoebic [11]; modulates the cellular enzymes, acts as anti- oxidant and anti mutagenic [12]. Chemo preventive against tumor production [13]. Polymers of protocat- echuic acid acts against influenza virus [14]. Induces apoptosis in human leukemia cells and also malignant HSG cells of oral cavities [15]. Chlorogenic acid acts as anti-inflamatory and antifungal [16] anti bacterial [17]; acts as Plant of A.aspera(Sub-merged) anticoagulants [18]. Chlorogenic acid, neo-chlorogenic acid, caffeic acid along with the flavonoids shows synergic action against bronchial diseases [19]. It *Corresponding author. also prevents cardio vascular diseases [20]; Chlorogenic acid has been known C.Aruna for antioxidant due to its scavenging activity of hydroxyl ions [21] and anti Dept .of Botany, viral [22], Neo chlorogenic acid may act as anti oxidant and released in to the S.V.University, blood stream after a meal [23] Iso chlorogenic acid improves immunity [24], Tirupati-517502, along with Caffeic acid it prevents type 2 diabetes mellites [25]. Caffeic acid A.P,India. and Ferrulic acid acts as anti-inflammatory and antifungal [26-27].Caffeic acid and its derivative CAPE acts as anti-inflammatory and suppresses the in- testinal carcinogenesis’ and the estrogenic activity [28]; It also acts as anti

Journal of Pharmacy Research Vol.5 Issue 3.March 2012 1827-1837 Aruna Chittamuri et al. / Journal of Pharmacy Research 2012,5(4),1827-1837 immuno-modulatory, anti inflammatory, protects from U.V radiation ef- Qualitative estimation of phenolic compounds: fects on skin cells [29-30]. Ferrulic acid and Cinnamic acid of onion epidermal cells shows resistance in preventing fungal cell wall degradation [31]. Polyphe- Chemicals, Glassware & Equipment: nols are noticed important natural Anti-Oxidant sources from by Peroxide free Diethyl ether, Anhydrous Na2Co3 , HCl, Ethanol, Benzene, adsorbing and neutralizing free radicals [32]. Trans-p- coumaric acid acts as Acetic Acid Sodium formate, Formic Acid, Sulphanilic Acid, Sodium an anti bacterial [33]. Hydroxy Cinnamic acid amide acts against influenza Nitrite, Sodium Carbonate, Para Nitriline, Ferric Chloride, Separatory and Herpes virus [34]. Hydroxy benzoic acid, Hydroxy Cinnamic acid, and funnel, Beakers, Conical flasks, Measuring Jars, Capillary tube, Test Flavonoids like Quercetin, Catchin, Rutin acts as natural Anti-oxidants [35]. tubes, Glass tanks, Petri plates and Automizer.

Coumarins are having narcotic and sedative action also oxytocic [36] anti Extraction of phenolic compounds: inflamatory [37] antithrombotic [38]. Spasmolytic and oxytocic [39], Fresh leaves were collected and phenolic extract was collected through the vasodialatory [40]; Coumarins of Coriander decrease the content of P450 in standard method [62] .About 30 gm of the healthy and fresh, leaves were induced hepatic microsomes without a major effect on heme content [41]. macerated in approximate 100ml of 2N HCl. The homogenate was digested Oral administration of Citrus Coumarin and Iso pimpinellin blocks DNA on a boiling water bath for about 30min. The contents were cooled and adduct formation and skin tumor initiation in mice [42]. Coumarins sup- filtered through Whatmann no: 1 filter paper. The filtrate was extracted presses the expression of T3SS genes of the plant pathogen Dickeya with Peroxide free diethyl ether (solvent ether) repeatedly. The cooled dadanti, proves that plants can also defend against bacterial pathogens by extract was concentrated to a small volume and was treated thrice with manipulating the expression of the T3SS PCA (P-Coumaric Acid) which 25ml of 5% anhydrous Na2Co3 solution. The Cooled Na2Co3 solution was regulates genes through the Hrpx/y two component system [43]. adjusted to 2 PH with concentrated HCl. The acidified fraction at 2 PH was then extracted with equal volumes of fresh diethyl ether for three times and Flavonoid compounds may exhibit vast biological activities including anti- the combined ether extracts were washed with 2ml of distilled water for allergic, antiviral, anti-inflammatory and vasodialatory in human beings44. 3times to remove the traces of HCl. The ether soluble water was removed These are used in the treatment of cold [45-46] against allergy, x rays and other by freezing the extract and then the ether was evaporated to dryness on radiation injuries, hemorrhage, dermatitis and albuminuric diseases [47]. Fla- water bath at 98°C. The resulting residue was dissolved in 1ml of 95% vonoid acts as natural food antimicrobial systems [48]. Quercitin used against ethanol and was preserved at low temperature in a dark container until Urethritis and cystitis. It exerts antiseptic action on the urinary tract and used. other allergic/inflammatory mediators. In vitro studies of Quercetin shows anti tumour activities [49-50] .It is known for the treatment of capillary fragil- Separation of phenolic compounds:- ity and phlebosclerosis [51]; anti hemorrhagic [52]. It shows anti-tumor activ- 1gm of the extract was spotted on 23 × 29cm Whatmann No: 1 chromato- ity against chronic prostitis [53];Quercitin shows positive effects in prevent- graphic paper with the help of a micropipette. The origin of the spot was ing prostate cancer, heart disease, cataracts, allergies, inflammations and dried immediately with the help of hair drier. The dried sheets were run in respiratory diseases such as bronchitis and asthma [54]; It also acts as anti- bi-dimensional ascending chromatography by using rectangular chromato- inflammatory and antioxidant properties [55].Myricetin plays an important graphic glass tank. The Chambers are saturated with the chromatographic role in inhibiting the tumor growth and enhancing the diuretic and dia- solvents one day before. The development of Chromatograms has to be phoretic activities. Higher doses of Myricetin lowers the rates of prostate carried out at 22°-24°C. cancer [56].The three important Flavones (Kaempferol, Quercetin and Myricetin) reduce the risk of pancreatic cancer by 23 percent [57]. It is also Solvent I Benzene: Acetic acid: water (60: 70: 30) v/v/v (upper layer of this used against Jaundice and hepatitis [58]. Apigenin shows its anti inflamma- mixture used at first direction) tory activity [59]. Solvent II Sodium formate: Formic acid: water (10: 1: 200) v/v/v (used for second direction). MATERIALS AND METHODS: The paper after development was removed from the tank and dried at room Plant material: temperature. The dried sheet was examined under UV light and the fluores- Plant material A.aspera was collected at Mallemadugu dam along the water cent regions were marked. Then the paper was exposed to ammonia hedges near dodlamitta area of Renigunta Mandal. The botanical identity of vapors were also observed under UV light and the new fluorescent spots the plant was determined and authenticated from the literature available in were also marked. the Department of Botany and the voucher specimens (CA.27) were de- posited in the Department Herbarium. The present work was carried out in Identification of phenolic compounds using chromogenic spraying the Department of Botany, S.V.University, Tirupati. Leaf material was reagents: thoroughly washed and dried under shade at 28 ± 2°C for about 10 days. To identify the phenolic compounds separated on the chromatograms were The dried leaves were ground well into a fine powder in a mixer grinder and sprayed with Diazotized Sulphanilic acid, Paranitranilins and ferric chlo- sieved to give particle size of 50-150mm. The powder was stored in air ride reagent with the help of an automizer. The phenolic compounds were sealed polythene bags at room temperature. identified by calculating their Rf values of individual spot colors with chromogenic sprays and finally confirmed with authentic samples by co- Phytochemical analysis: chromatography.

Solubility: Preparation of Chromogenic spray reagents: The solubility was studied in eight solvents (Hot water, Cold water, Ben- Diazotized Sulphanilic acid: Sulphanilic acid Solution, 5% Sodium Nitrite zene, Hexane, Ethyl acetate, Chloroform, Methanol and Ethanol) based on Solution: 20% Anhydrous Sodium Carbonate solution, Diazotized Para polarity gradience. Nitraniline Reagent, 5%Anhydrous Sodium Nitrite solution, 10% Sodium Carbonate solution, Ferric Chloride reagent. Preliminary Phytochemical analysis of secondary metabolites: All the extracts were subjected to preliminary Phytochemical qualitative Qualitative estimation of flavonoids compounds screening for the Presence or absence of various secondary metabolites Chemicals &Glassware: - Methanol, Chloroform, Alcohol 95%, Isopro- such as alkaloids, flavonoids, phenols, Terpinoids, steroids, Anthocynadins, pyl alcohol, Ammonia, Sulphanilic acid, Concentrated HCl, Sodium Ni- anthroquinones, saponins, tannins, lignins, Indoles and glycosides were carried out by the standard methods [60-61]. Journal of Pharmacy Research Vol.5 Issue 4.April 2012 1827-1837 Aruna Chittamuri et al. / Journal of Pharmacy Research 2012,5(4),1827-1837 trate, Sodium Carbonate, Separatory funnel, Beakers, Conical flasks, Mea- Preparation of inoculums: suring jar, Capillary tube, Test tubes, Glass tanks, Petri plates, Atomizer. Stock cultures were maintained at 4°C of nutrient agar slants. Active cul- . tures for experiments were prepared by transferring a loop full of cells from Extraction of flavonoids compounds the stock cultures to test tubes of nutrient agar medium and were incubated The flavonoid compounds were extracted according to the standard meth- without agitation for 24hrs at 37°C. ods [63-64]. About 2g of dried leaf powder was taken in a boiling test tube heated up to 40°C and then 18ml of methanol and 2ml of water (9:1) was Preparation of the medium: added. Shaken well and was kept for about 24 hrs at room temperature. To prepare 1lit of nutrient agar medium 3g of beef extract, 3g of peptone, After that the upper clear solution of the extract was transferred to another 15g of agar was used. The ingredients were accurately weighed using digital test tube. To the remaining residue in the test tube, again 5ml of methanol electronic balance and dissolved in liter of distilled water before the addi- and 5ml of water (1:1) was added. Stirred well and the contents were kept tion of agar; the PH of the medium was adjusted to 7.0 by adding few drops for 24 hours and the clear extract thus obtained was cooled up with the of 0.1N NaoH/HCl using digital PH meter. Later this medium was trans- earlier sample. The combined extract was mixed well and filtered through ferred to conical flasks and plugged with nonabsorbent cotton. Medium the cotton. The filtrate was evaporated to about 1/3 of the original volume was then sterilized by autoclaving at 15lbs for 20min cooled to 4 °C and and the resultant aqueous extract was taken into a separatory funnel and used for the study. then extracted with 10ml of chloroform. This process was repeated 2or 3 times. All the chloroform extracts were combined and evaporated to dry- Agar well diffusion assay: ness under vaccume in a rotatory evaporator and the dried residue was The antibacterial activity of the leaf extracts was determined using agar well dissolved in 1ml of 95% alcohol which was stored at low temperature in the diffusion method [65] with slight modifications. Nutrient agar was inocu- dark until used. lated with the selected microorganisms by spreading the bacterial inoculums on the media. Wells (8 mm diameter) were punched in the agar and filled Separations of flavonoid compounds with plant extracts. Control wells containing pure solvents (negative con- 1 gm of the extract was spotted on 23x29cm Whatmann No: 1 chromato- trol) or standard antibiotic (positive control) viz., Gentamycin (30mg) and graphic filter paper with help of a micropipette. The origin of the spot areas Ampicillin (10 mg). The plates were incubated at 37°C for 18hrs. The was dried immediately with the shelp of hair drier. The dried sheets were antibacterial activity was assessed by measuring the diameter of the zone of run in unidimensional ascending chromatography by using rectangular chro- inhibition for the respective drug. The relative antibacterial potency was matographic glass tanks which can accommodate 4 sheets at a time. The calculated by comparing its zone of inhibition with that of the standard chromatographic chambers were saturated with any of the following sol- drug. The data of crude drugs activity is given the mean of quadruplicates vents one day before the development of the chromatograms at 22-24°C. along with the standard error. 1. Isopropyl alcohol: Ammonia (25%): Water (8: 1: 1) v/v/v. 2. n-Butanol: Acetic acd: Water (4: 1: 5) v/v/v (top layer was used) Minimum inhibitory concentration: 3. Conc.HCl: Acetic acid: water (3: 30: 10) v/v/v. MIC is defined as the lowest concentration where no visible turbidity is 4. Phenol: Water (3: 1) v/v. observed in the test tube (bacterial concentration).The method [66] modified The chromatograms after unidimensional development were removed from by Usman [67] was employed. In this method the broth dilution technique the tanks and dried at room temperature. The dried sheet was observed was used, where the leaf extract was prepared to the highest concentration under UV light and the fluorescent regions were marked. The paper while of 10mg/ml (stock concentration). By adding sterile distilled water and exposed to ammonia was also observed under UV light and the new fluores- serially diluted (two fold dilution) using the nutritive broth and later inocu- cent spots also marked. lated with 0.2ml standardized suspension of the test organisms. After 18hrs of incubation at 37°C., the test tubes were observed for turbidity .The Identification of flavonoids using chromogenic spray reagents: lowest concentration of the tube that did not show any visible growth can To identify the flavonoid compounds separated on the chromatograms be considered as the MIC. were sprayed with chromogenic spray reagents with the help of an atom- izer. The flavonoid compounds were identified by calculating their Rf val- RESULTS: ues of individual compound along with authentic samples by co-chromato- graphic studies. Solubility: Solubility with eight solvents of leaf powder revealed in increased gradience Preparation of Chromogenic sprays reagents: from hot water to alcohol (Hot water> Cold water> Benzene> Hexane> Sulphanilic acid solution. Sodium Nitrate solution, anhydrous sodium car- Ethyl acetate> Chloroform > Methanol >Ethanol). It is highest in Alcohol bonate solution. and less in Hot water respectively.

Antibacterial activity: Preliminary phytochemical screening- secondary metabolites :-( Table-1) Out of eight extracts in Methanol (8) and Alcohol (7) secondary metabo- Extracts preparation: lites were observed. In cold water (5) and Hot water (5), further Ethyl Dried leaf powder (25g) was packed in a Whatmann no.1 filter paper and acetate (3) Chloroform (1) Benzene (1) and Hexane (1). The important was extracted in a Soxlet apparatus using 100ml of solvent. Methanol, secondary metabolites are Alkaloids, Terpinoids, and Tannins along with Alcohol, Hot water and Cold water extracts were dried and stored in a few Steroids, Anthocyanidins, and Glycosides. But flavonoids are present refrigerator at 4°C. only in cold water and hot water extracts, where as Alkaloids, Phenols and glycosides present both in Alcohol and Methanol, and Anthocyanidins are Bacterial cultures: present only in Methanolic extract. The bacterial cultures Bacillus subtilis (MTCC- 441) causes Pneumonia, diarrhoea. ,Staphylococcus aureus (MTCC- 737) causes bone and joint Phenols :- (Table-2) pains, skin infections, boils; Escherichia coli (MTCC- 443) causes urinary Nearly 11 phenolic compounds were observed in the leaves of A.aspera tract infections, Pneumonia, Pseudomonas aeruginosa (MTCC- 741) causes out of which 2 are un identified. Mainly Neo-chlorogenic acid is more in urinary infections and Pneumonia. The above strains were procured from quality with bright brown colour, and Homo-protocatechuic acid in more the Department of Microbiology S.V.University Tirupati and also from the quantity spreading in larger area with buff colour. Other compounds are SVIMS Tirupati.

Journal of Pharmacy Research Vol.5 Issue 3.March 2012 1827-1837 Aruna Chittamuri et al. / Journal of Pharmacy Research 2012,5(4),1827-1837 Table.1. Preliminary phytochemical screening of Aeschynomene aspera is most susceptible with Alcoholic extracts 10mg-48 mm activity when L. leaf extracts compared to control as Ampicillin10 mg- 25mm, Gentamycin 30mg-19 mm S.no Name of the test Cold water Hot water Alcohol Methanol and S. aureus is more resistant as 10 mg -29 mm. Other pathogens as B.subtilis and E. coli also shows more susceptible with alcohol extracts 1 Alkaloids when compared to other extracts Effective inhibition of the leaf extracts is Mayers test - - + + + + + + + Wagners test - - + + + + + + + observed highest with Alcohol < Methanol

Fecl3 test + + + + + + - - MIC : (Table-5) 3 Phenols Minimum Inhibitory Concentrations of Cold water extracts are very Fecl test - - + + + + + + 3 effective as 0.53mg/ml, (E.coli); 0.61mg/ml (B.subtilis); 0.69mg/ml Ellagicacid test - - + + + + + + 4 Terpinoides (P.aeruginosa);and 0.72mg/ml (S.aureus); organism specificity is observed Liebermann + + + + + + + + + + + + + with each extract. The lowest MIC value is 0.42mg/ml S.aureus with alco- Burchards test hol extract, 0.48mg/ml Hot water extract against B.subtilis, 0.53mg/ml with 5 Steroids- cold water extracts against E.coli and 0.69mg/ml cold water against Salkowski test + + + + + + - - P.aeruginosa. Liebermann’s - - + + + + + Burchard test 6 Anthocyanidins - - - + + + DISCUSSION: 7 Saponins + + + + + + - - Phenolic compounds like Coumarins and Melilotic acid were isolated through 8 Tannins cell culture from Glyricidia sepium [68]. Vanillic acid, syringic acid, P-Hy- Gelatin test + + + + + + + + + + + + droxy benzoic acid, protocatechuic acid, Gallic acids and trans-p-coumaric Fecl test + + + + + + + + + + + + 3 acids from the major species of Rhynchosia and Polygonum glabrum. 9 Lignins - - + + + + + + [69] 10 Indols Kaempferol-3-rutinoside from Rhynchosia cyanosperma . S. grandiflora [70] [71] Enrilich test - - - - leaf contains condensed methyloleanolate , Tannins, glycosides , 11 Glycosides S.sesban leaf contains Saponins glucoronide-oleanolic acid [72]: Rhyncosia Keller-kilani test - - + + + + + + bracteata Leaves contain Isoorientin, isovitexin, vitexin and orientin and It is used for treating dysentery and Leucorrhoea [73]. From the leaflets of Table.2.Qualitative analysis of leaf phenolic compounds of A.aspera.L

Sl. No Standard Rf values Rf values in solvent UV fluorescence Colour with spray reagents Identified

I II With NH3 Without Sulphani-lic acid Para nitriline Ferric Compound

NH3 chloride

1 - - 0.04 0.18 None None Buff None None Un Identified Compound I 2 - - 0.02 0.16 None None Light brown Green None Un Identified Compound II 3 0.06 0.38 0.06 0.38 Blue Bright blue Buff Light brown Dark green Caffeic acid 4 0.02 0.58 0.02 0.48 None None Brown Brown Dark green Phloroglucinol 5 0.28 0.75 0.24 0.75 None None Dark brown Brown Reddish purple ß –Resorcyclic acid 6 0.05 0.78 0.09 0.64 Duck egg green Bright Duck egg green Brown Orange purple green *Neo-chlorogenic acid 7 0.18 0.8 0.18 0.83 None None Buff Yellow Green **Homo-Protocatechuic acid 8 0.5 0.45 0.52 0.48 Light blue Deep blue Light brown Blue None Trans-p-Coumaric acid 9 0.89 0.65 0.86 0.67 Light blue Blue Purple Blue green None Cis-Ferulic acid 10 0.77 0.31 0.7 0.23 Green Bright green Pale pink Bright green None Trans-Sinapic acid 11 0.96 0.04 0.86 0.04 None None Yellow Green None Cinnamic acid Solvent I : Benzene: Acetic Acid : Water (60:70:30 ) v/v/v,Solvent II : Sodium Formate : Formic Acid: Water (10:1:200 ) v/v/v,*Neo chlorogenic acid - showing more Colour ,**HomoProtocatechuic acid- showing more Quantity Table.3. Qualitative analysis of leaf flavonoids of A.aspera.L

SN Standard Rf values Rf values UV Flourescence Colour with spray reagents Identified compound

In Solvent With out NH3 With NH3 Sulphanilic acid 1% Alcoholic 1% Aluminium Ferric chloride chloride

1 0.07 0.07 Yellow Bright yellow Pink Olive Grey yellow *Myricetin 2 0.26 0.27 Yellow Light Bright Green Yellow **Quercetin yellow yellow 3 0.61 0.58 Reddish Reddish Brown Pale None **Apigenin brown brown green Solvent : Isopropyl Alcohol : Ammonia (25%) : Water ( 8 : 1 : 1 ) v/v/v,*Myrecitin shows more colour,**Quercitin and Apigenin shows more Quantity Caffeic acid, Phloroglucinol, ß- Resorcyclic acid, Trans-p-coumaric acid, Macroptilium atropurpureum (Siratro), leaflets and petioles of Desmodium Cis-Ferulic acid, Trans sinnapic acid and Cinnamic acid. intortum and D. tortuosum cyclitol, D (+) - Pinitol, D-glucose, D-galactose and myo-inositol, malonic acid, oxalic acid, succinic acid and Flavonoids :- (Table-3) Proanthocyanidins were isolated [74]. Whole plant of T. Purpurea, seeds of Leaves contain the three main compounds as Myricetin (pink), Apigenin Clitoria ternata, Indigofera ternata, Roots of Pseudarthria viscida; Heart (Brown) and Quercetin (Bright yellow). wood of Pterocarpus santalus,root of Mucuma pruriens, Pisum sativum are used against leprosy [75]. In about 49 representative species of Phaseolus, Antimicrobial activity : (Table-4) Vigna, Macroptilium, Strephostyles and Dysolobium were screened for their Antibacterial activity of leaf extracts with the hot water, cold water, Methanol flavonoid profiles and identified 35 compounds of flavonoids as flavonol- and Alcohol on the selected four pathogens resulted as follows. P. aeruginosa 0-glucuronides, flavone-0-glycosides, Flavonols glycosides crubinin and

Journal of Pharmacy Research Vol.5 Issue 4.April 2012 1827-1837 Aruna Chittamuri et al. / Journal of Pharmacy Research 2012,5(4),1827-1837 Table.4. Antibacterial activity of the leaf extracts(10 mg /ml) of A. aspera L. (Diameter of Zone of Inhibition in mm)

S.No Name of the organism Cold water Hot water Methanol Alcohol Controls *A-10 mg **G-30 mg

1 Bacillus subtilis 24 ± 2.1 16 ± 0.4 22 ± 1.2 40 ± 4.0 18±1.4 18±0.5 2 Staphylococcus aureus 13 ± 1.2 22 ± 1.6 15 ± 0.9 33 ± 1.6 29±1.4 22±0.4 3 Pseudomonas aeruginosa 24 ± 2.1 20 ± 0.4 28 ± 1.4 48 ± 0.4 25±1.4 19±1.6 4 Escherichia coli 25 ± 0.9 14 ± 1.8 25 ± 0.9 42 ± 0.8 20±0.4 20±0.4 Standard Deviation ± Mean of Quadruplicates

*A=Ampicillin **G=Gentamycin Table.5.Minimum inhibitory concentration of leaf extracts of A.aspera L.(mg / ml).

S.No Name of the organism Cold water Hot water Methanol Alcohol

1 Bacillus subtilis 0.61 0.48 0.78 0.78 2 Staphylococcus aureus 0.72 0.78 0.78 0.42 3 Escherichia coli 0.53 0.78 0.78 0.78 4 Pseudomonas aeruginosa 0.69 0.72 0.78 0.78

Authentic samples for flavonoids and phenols

Phenolic and flavonoid compounds of A. aspera leaf 1.Un identified

2.Unidentified

3.Caffeic acid

4.Phloroglucinol

5.b-Resorcyclic acid

6.Neo-Chlorogenic acid

7.Homo-Protocatechuic acid

8.Trans-p-Coumaric acid

9.Cis-Ferrulic acid

10.Trans-Sinapic acid

11.Cinnammic acid

Journal of Pharmacy Research Vol.5 Issue 3.March 2012 1827-1837 Aruna Chittamuri et al. / Journal of Pharmacy Research 2012,5(4),1827-1837 Antibacterial activity of A.aspera leaf extracts

P. aeruginosa E. coli P. aeruginosa E. coli

S. aureus B. subtilis S. aureus B.subtilis Alcoholic Extracts Hot Water Extracts

P. aeruginosa E. coli P. aeruginosa E.coli

S. aureus B. subtilis S. aureus B. subtilis Methanolic Extracts Cold Water Extracts [76] Rutin . Butea monosperma leaves , Dolichos biflorus, and Desmodium excessive anthelmintic maturant, demulcent useful in hydrocele [78]. [77] motorium seeds P. marsupium leaves and bark are used against diabetes . Dalberggia scisso leaves are used against bronchial disorders like asthma bispinosa seeds consists of galactose and mannose. Seeds are and pulmonary infections [79]. A.precatorius, Cassia angustifolia, C.senna, emmenagogue, stimulant, and astringent, heal chronic ulcers and remove C. Obtusifolia leaves are used for jaundice [80]. smallpox eruptions. They are useful in diseases of spleen, diarrhoea and Journal of Pharmacy Research Vol.5 Issue 4.April 2012 1827-1837 Aruna Chittamuri et al. / Journal of Pharmacy Research 2012,5(4),1827-1837

Photos showing control against gentamycin rin, maackain, pongamol. Decoction of roots given in dyspepsia, diarrhea, rheumatism, asthma and urinary bladder disorders, A.precatorius leaves are used against uterine infections and also as abotificent [83]. Flavones, flavonones, Isoflavone, chalcones and pterocarpanes from Glycirrhiza spe- cies are reviewed in respect to their structure botanical source and biological activity [84]. A. precatorius leaves and Roots contain glycyrrhizin precol, abrol, abrasive and precarine from roots. Gallic acid, abrine, hyphaphonin, alanine, serine, valine, choline, trigonelline, precatone and methyl ester, 5- B-cholamic acid, abrine A& abrine B from seeds. Leaves are used for cough, cold, pain; fresh juice is applied on skin for itching and skin diseases. Roots are diuretic, emetic alexenteric, jaundice. Seeds are purgative, aphrodisiac, abortifacient. Clitoria ternata Leaves contain Kaempferol, flowers contain Delphinine, Maldivian, Kaempferol white flower contain only Kaempferol. P. aeruginosa E. coli Seeds contain Flavonols, flavones. Leaves are useful in otalgia, hepatopathic and eruptions. Seeds are cathartic and are useful in viseralgiasic. Pongamia pinnata, Psoralea erycifolia, Tephrosia purpurea leaves are used against skin diseases [85].The study of hepato-protective activity with the aqueous and ethanolic extracts of stem bark of P.santalinus and also revealed the presence of alkaloids, phenols, saponins, glycosides, flavonoids, triterpinoids, sterols and tannins [86]. From Abrus precatorius saponins and alkaloids, Caesalpinia pulcherima tannins, saponins and alkaloids were noticed [87]. The phenolic and flavonoid constituents’ shows antioxidant activity in Pterodon emarginatus seeds [88].

Apigenin was isolated from Indigofera mysurensis, [89]: Gangetin from Desmodium gangeticus [90] and Glycyrrhizin from Glycyrrhizin glabra also [91] [92] S. aureus B. subtilis act as anti-inflammatory ; Ethanolic extracts affects diabetes in mice Anthroquinones Rheine from Cassia angustifolia, Apigenin from Trigonella Controls -ampicillin scoparia, Chalcones and deoxyflavone diadzein from Phaseolus aureus and Apigenin from Glycine max are isolated through cell culture [93]. G.uralensis was tested against the anti-tumor and immune activation activi- ties tested in human cell lines [94]: It acts as antibacterial against S.mutans [95]:Root extracts acts as estrogenic activites against MCF7-breast cancer cells [96]:Hexane/Ethanol extracts arrests DU-145 Human prostate and 4T1-Murine Mammary cancer cells [97]. New acylated Isoflavone glyco- sides from heartwood of P. santalinus were isolated and the extracts have been used in the treatment of inflammations, mental aberrations, ulcers and in diabetes. Stem bark and leaf extracts shows maximum activity against pathogenic bacteria [98]. Antibacterial potentiality of isolated Apigenin and Luteolin from Scutella barbata was tested against methilin resistant Sta- phylococcus aureus (MRSA) [99].

Clitoria ternata root methanolic extracts shows anti-inflammatory, analge- sic and antipyretic activity [100]. Pterocorpus indicus leaves, roots, stem P. aeruginosa E. coli bark with all the extracts exhibit antimicrobial activity [101]. Methanolic extracts of Peltophorum africanum root and bark; Mucuna coriaceae bark extracts and whole plant acetone extracts of Zornia minima shows inhibi- tory activity against many pathogenic bacteria [102]. Many species of Erythroxylon shows anti viral activity [103].Aqueous extracts of Vigna ra- diata acts against Staphylococcus bacteria [104]. Isoflavonones and rotenones were reported in Atyalosia and Cicer species [105].Daniellia oliver and Ficus sycamores leaf extracts acts as antidiarrhoeal [106]. Anti pneumonial activity of Erythrina senegalensis, Piliostigma thonningii and Andera innermis against streptococcus pneumonia [107]. Myricetin in 30 medicinal plants are used against Jaundice and hepatitis [108].Tephrosia purpurea seed aqueous extract acts as an anti oxidant and anti diabetic in rats [109]. Alcoholic root extracts of Piptadeniastrum africanum acts as an anti proliferative agent against human colonic cancer cells [110]. Leaf Ethanolic extract of I.tinctoria shows anti-inflamatory activity [111]. S. aureus B. subtilis Control-Gentamycin Chlorogenic acid and Kaempferol-3, 5-ß-D-diglycoside were isolated from Mucuna pruriens (Fabaceae) also called velvet bean is found in Eastern the leaves of , [81] Many Phenolic acids are isolated from Nigeria, where its seeds are used as soup thickeners. The leaves of M. Pterocarpus santalinus [82]. T.purpurea whole plant contains Pongamol, ß- pruriens are used as remedy for various diseases such as diabetes, arthritis, sitosterol, Ursolic acid, spinosterol are toxic, laxative, diuretic and dysentery, and cardiovascular diseases [112]. M. pruriens is a popular deobstrunent used in bronchitis and bilious febrile attacks also in boils, Indian medicinal plant. Roots, leaves and seeds of the plant are commonly pimples and bleeding piles, roots contain flavonoids, purpurenone, purpu- used in the treatment of impotence, snake bite, diabetes, cancer and Parkin- Journal of Pharmacy Research Vol.5 Issue 3.March 2012 1827-1837 Aruna Chittamuri et al. / Journal of Pharmacy Research 2012,5(4),1827-1837 sonism. The endocarp of Mucuna pruriens is non-toxic and is 2-3 times 6. Rajan, G Bhaskar. Irfan Alikhan. and Atiya Khanum . Strength and [113] more potent than leavodopa in controlling hyperprolactinemia , motor wealth of therapeutic medicinal plants in India. Published in Irfan [114] symptoms of Parkinson’s disease animal models . M. pruriens has also khan and Atiya khanum (Eds). Utaaz publications. 2004, Vol I. (8). P: shown to exhibit neuroprotective effect by increase brain mitochondrial 286-293. complex-I activity and significantly restoring dopamine and nor epineph- 7. Caton, B.P; M. Mortimer, J.E. Hill . A practical field guide to weds of rine levels in Parkinsonism animal model [115]. Phytochemical evaluation on rice in Asia Chem.Org.Naturist, 2004. Vol. 9. P: 240.chemistry, SVU, the seeds revealed the presence of 5-indolic compounds, especially Tpt. tryptamine and hydroxytryptamine [116], alkaloids like mucunine, 8. Rajendar, Phytotherapeutical methods used by traditional healers of mucunadine, prurine and prurienine [117]. Pueraria tuberosa is an important Eturnagaram Mandal ,Warangal, Andhra Pradesh, India, Ethnobo- and potential medicinal plant in traditional and folklore systems. The root tanical Leaflets, 2010,14:361-65. tuber is sweet, oily, cooling, tonic, and effectively used in aphrodisiac, 9. Panda and K. Mishra., Ethnobotanical Survey of some wetland plants galacatagogue and diuretic. It is also used to cure leprosy, diseases of blood of South Orissa and Their conservation. Indian Journal of Tradi- and urinary discharges. It is employed as an emetic, tonic and also believed tional Knowledge Vol. 10 (2), April 2011, Pp.296-303. to be a lactagogue [118]. In folk medicine the root tuber is applied for blood 10. Varaprasad.K.S, Integrated gene resource Management of underutilized purification and to improve sperm production. The shade dried root pow- in India, 2011. der controls overgrowth in stomach. The consumption of raw root for one 11. Markh A.J. and Lysogor, T.A. Pomogranate Polyphenols, 12V, Vyssh. month leads to sterilization in women [119]. Voheb.Zaked.Pishoh, Technol; 1973 (2), 36-B. 12. Tanaka, T., Kojima, T., Kalwamori, T., Mori, H.. Chemoprevention The genus Aeschynomene sensitive and A.indica are reported to possess of digestive organs carcinogenesis by Natural product protocatechuic insecticidal activity [120].Spermicidal activity [121-122]. From A.indica reynoutin acid, 1995, Cancer 75. P: 1433-1439. 13. Mori, H., Sugie, S., Rahman, W., Suzui, N. Chemoprevention of 2- and the aminoacid potassium aeschynomate were isolated [123]. A.mimosifolia amino-1-methyl-6-phenylimidazo (4, 5-b) pyridine induced mammary roots yielded Neoflavonoid, Mimosofoliol and Museial C-16-tricyclo- [124] carcinogenesis in rats. Cancer Lett 143, 1999. P: 195-198. heptonone derivative Mamosifolenone . 14. Lu, F.J., Tseng, S.N., Li, M.L. and Shih, S.R. In vitro anti-influenza virus activity of synthetic human’s analogues derived from protocat- So far much work was carried out with medicinal plants of Fabaceae in echuic acid. J. Arch. Virol, 2002. P: 273-284. identifying the secondary metabolites, and isolation of phenols and Fla- 15. Babich, H., Sedietcaia A.,and Kenigsberg B.. In vitro cyto toxicity of vonoid compound in the following species like Mucuna pueraria, Glyridium, protocatechuic acid to cultured human cells from oral tissue; involve- Rhyncohosia, Desmodium, Phaseolus, Macroptilium, Pterocarpus, Gly- ment in oxidative stress. Pharmacol. Toxicol, 2002, 91 (5), P: 245- cine, Indigofera, Abrus, Vigna, Strophostyles, Dysolobium, Daniella, and 253. Peptandeniastrum. 16. KUC., R. E., Henze, R. E., Ullstrup, A. J and Quackenbush, F. W. Chlorogenic acid and caffeic acids as fungistatic agents produced by Biological activities of the most of the Flavonoid and phenolic compounds potatoes in response to inoculation with Helminthosporium resulted as anti-inflammatory anti-malarial, antioxidant acts against ul- carbonum. J. Amer. Chem. Soc. 1956, Vol. 78. P: 3123-3125. cers, diabetes; skin tumors, intestinal carcinoma and rheumatism. Apige- 17. De sotillo,D.R.Hadley,M.:Wolf-Hall,C.Potato peel extract a non nin, Luteolin are used as antibacterial, antipyretic and analgesic. Crude mutagenic anti oxidant with potential anti microbial activity.Journal extracts acts against antiviral. Myricetin and quercitin are used against of Food Science,1998,63(5):907. jaundice and hepatitis. And also anti-proliferative agents against human 18. Lewis, W.H. and Lewis, P.F., ME. Medical botany plant affecting colonic cancer cells, anti-inflammatory, antihamerragic, chronic prostitis, Man’s health. John Willey and sons, Newyork (1977). pancreatic cancer, Jaundice and hepatitis. As antioxidants exerts antisep- 19. Trute, A. and Nahrstedt, A. Flavonoids in Rhynchosia species. planta tic action on urinary tract infections inhibiting the release of histamine an medica, 1997, 63,2. P: 177-179. allergic-inflammatory mediator. A.aspera is used mainly against urinary 20. 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