Isolation, purification and characterization of bioactive compounds from banana rhizome var. Nanjanagudu Rasbale

Transcription

1 Chapter 3: Isolation, purification and characterization of bioactive compounds from banana rhizome var. Nanjanagudu Rasbale 59

2 INTRODUCTION Extensive use of plant extracts in alleviating various human health diseases and disorders are in practice since, time immemorial in Ayurveda, Unani and other traditional medicinal systems throughout the world. The search for bioactive components with multiple bioactivities from plant source has gained increasing importance in recent time, due to growing worldwide concern about alarming increase in the rate of infection by antibiotic-resistant microorganisms and carcinogenicity of synthetic compounds. Natural products continue to provide greater structural diversity than standard combinatorial chemistry and they offer great opportunities for finding novel bioactive compounds with potential bioactivities. Thus plant extracts offer a significant role in the discovering of bioactive compounds of our interest. However, it is important to establish the scientific rationale to defend their use in nutraceutical and functional foods and as potential source of drug. In this regard, potentiality of banana pseudostem and rhizome has been explored as a source of bioactive compound. The earlier study indicated that pseudostem of banana var. Nanjanagudu Rasbale as a highest source of polyphenols among all the eight commercial varieties screened. Interestingly, highest source of polyphenolic content was observed in banana rhizome in comparison with pseudostem. Consequently, the banana rhizome of var. Nanjanagudu Rasbale has been focused for isolation and characterization of bioactive compounds. The detail work carried out is presented in this chapter. 60

3 Materials and Methods Preparation of extracts The extraction of banana rhizome powder using serial extraction process with increasing polarity of solvents was followed as discussed in the chapter 1 (materials and methods). Isolation and purification of bioactive compounds from banana rhizome var. Nanjanagudu Rasbale extracts The isolation and purification of bioactive compounds were carried out using repeated silica gel column chromatography and thin layer chromatography (TLC). The purified bioactive compounds were characterized by subjected to UV, IR, LC-MS and NMR spectroscopy studies. Isolation and purification of antioxidant compound from banana rhizome var. Nanjanagudu Rasbale The acetone extract of banana rhizome showed high total phenolic and total flavonoid content and multiple antioxidant activity in all the in vitro tested models. Hence, antioxidant activity (DPPH radical scavenging method) guided fractionation was employed to isolate and purify antioxidant compound from acetone extract of banana rhizome. Fractionation of the crude acetone extract To isolate antioxidant compound from acetone extract of banana rhizome, activated silica gel ( mesh) was packed onto a glass column (450 mm x 40 mm) using n-hexane solvent and 20 g of crude acetone extract was loaded on the top of silica gel. The column was eluted stepwise at a flow rate of 1 ml min -1 with 500 ml of hexane and linear gradient of 2000 ml of hexane : chloroform (75 : 25 to 0 : 100 v/v), 2600 ml 61

4 of chloroform : ethyl acetate (75 : 25 to 0 : 100 v/v), 8000 ml of ethyl acetate : acetone (75 : 25 to 0 : 100 v/v) and 3500 ml of acetone : methanol (75 : 25 to 0 : 100 v/v). In this column chromatographic separation, about 83 fractions measuring 200 ml each were collected and concentrated by using the rotary evaporator. Weight of each fraction was measured. Thin-layer chromatography (TLC) An aliquot of all the concentrated fractions were loaded on the activated silica gel TLC plates (20 cm x 20 cm). The plates were developed in ascending direction from 18 to 19 cm of height with different proportions of chloroform and methanol as mobile phase. After air-drying, the spots on the plate were located by exposure to iodine. Fractions were pooled based on the spotting pattern and R f values on the TLC plate. The pooled fractions were numbered (Fr.1'-Fr.18'). All the eighteen pooled fractions were tested for antioxidant activity using DPPH radical scavenging method as discussed in chapter 1 (materials and methods). Purification of bio-active fraction Since, fraction nine (Fr.9') obtained from first step of column chromatography (Fig. 3.1) showed high antioxidant activity and yield, it was selected for further purification. About 2 g of bioactive Fr.9' were further purified using silica gel ( mesh) column (450 mm x 40 mm). The column with Fr.9' was eluted stepwise at a flow rate of 1 ml min -1 with 100 ml of hexane, followed by linear gradient of 200 ml of hexane : chloroform (90 : 10 to 0 : 100 v/v), 1100 ml of chloroform : ethyl acetate (90 : 10 to 0 : 100 v/v), 900 ml of ethyl acetate : acetone (90 : 10 to 0 : 100 v/v) and 400 ml of acetone : methanol (90 : 10 to 0 : 100 v/v). In this second column chromatographic separation, about 27 fractions from Fr.9' measuring 100 ml each were collected and concentrated by using the rotary evaporator. Weight of each fraction was measured. An aliquot of all the fractions were loaded on the TLC plate, fractions having similar R f values and spotting pattern were pooled and numbered into sub-fractions (Fr.9.1''- 62

5 Fr.9.5''). These sub-fractions were tested for antioxidant activity using DPPH radical scavenging method. Figure 3.1: Schematic representation of isolation and purification of antioxidant compound from acetone extract of banana rhizome var. Nanjanagudu Rasbale Sub-fraction three (Fr.9.3'') obtained from second step column chromatography (Fig. 3.1) showed high antioxidant activity, hence selected for further purification. About 800 mg of bioactive sub-fraction three Fr.9.3'' was further purified on a silica gel ( mesh) column (600 mm x 15 mm). The column was eluted stepwise at a flow rate of 1 ml min -1 with linear gradient of 100 ml of hexane: chloroform (90: 10 to 0: 100 v/v), 400 ml of chloroform: ethyl acetate (95: 05 to 0: 100 v/v), 400 ml of ethyl acetate: 63

6 acetone (95: 05 to 0: 100 v/v) and 200 ml of acetone: methanol (90: 10 to 0: 100 v/v). In this third column chromatographic separation, about 22 fractions measuring 50 ml each were collected and concentrated. Weight of each fraction was measured. Fractions with similar R f values and spotting patterns on the TLC plate were pooled and numbered (Fr.9.3.1'''-Fr.9.3.3'''). Among these, sub-fraction two (Fr.9.3.2''') obtained from the third step column chromatography showed purity in the TLC profile. This pure compound was subjected to various spectroscopic techniques for elucidation of the structure. The chromatographic separation of sub-fraction two (Fr.9.3.2''') afforded one pure compound. This pure compound was subjected to various spectroscopic techniques to elucidate the structure and studied for their antioxidant activity using different in vitro models in detail. The entire process of purification is shown in figure 3.1. High performance liquid chromatography (HPLC) These purified compound was tested for their purity by using HPLC, on C-18 column (model LC-10A, Shimadzu Corporation, Japan), with ultraviolet (UV) detection using a diode array detector (DAD) operating at 220, 280 and 320 nm. An isocratic solvent system, consisting of methanol: water: trifluoro acetic acid (89.5:10:0.5), was used as a mobile phase at a flow rate of 1 ml min -1. Ultraviolet (UV) detection was carried out with a diode array detector (Shimadzu). Isolation and purification of antimicrobial compounds from banana rhizome var. Nanjanagudu Rasbale Chloroform extract showed high antimicrobial activity against wide spectrum of bacterial (Gram +ve and Gram -ve bacteria) and fungal strains. Hence, antimicrobial compounds were aimed to isolate from chloroform extract by antimicrobial activity guided fractionation method. 64

7 Fractionation of the crude chloroform extract Figure 3.2: Schematic representation of isolation and purification of antimicrobial compounds from chloroform extract of banana rhizome var. Nanjanagudu Rasbale The crude chloroform extract from serial extraction of banana rhizome powder of about 10 g (Fig. 3.2) was subjected to column (450 mm x 40 mm) chromatography using silica gel ( mesh) and eluted stepwise with linear gradient of 1000 ml of hexane : Ethyl acetate (95:5 to 75:25 v/v), 2000 ml of chloroform : ethyl acetate (90:10 to 0:100 v/v), 800 ml of ethyl acetate : acetone (90:10 to 0:100 v/v) and 400 ml of acetone : methanol (90:10 to 0:100 v/v). About 42 fractions, measuring 100 ml, were collected, concentrated by using the rotary evaporator. Weight of each fraction was measured. 65

8 Thin layer chromatography (TLC) An aliquot of column fraction was spotted on to a silica gel TLC plate (20 cm x 20 cm). The plates were developed in ascending direction from 18 to 19 cm of height with different proportions of hexane and ethyl acetate as mobile phase. After air-drying, the spots on the plate were located by exposure to iodine. Fractions were pooled based on the spotting pattern and R f values on the TLC plate. The pooled fractions were numbered (Fr.1'-Fr.8'). All the eight pooled fractions were tested for antimicrobial activity as discussed in chapter 1 (materials and methods) Purification of bio-active fraction Since fraction three (Fr.3 ), obtained from first step column chromatography showed high antimicrobial activity and yield, it was selected for further purification (Fig. 3.2). About 1.7 g of bioactive Fr.3' was further purified using silica gel column (450 mm x 40 mm) chromatography and eluted with linear gradient of 1700 ml of hexane and ethyl acetate (95:5 to 5:95 v/v) and with 500 ml of ethyl acetate and methanol (90:10 to 50:50 v/v). About 22 fractions measuring 100 ml were collected, concentrated by distillation and after TLC analysis, based on R f value and spotting pattern, fractions were pooled into five sub-fractions (Fr.3.1''-3.5''). Weight of each fraction was measured and assayed for their antimicrobial activity. Sub-fraction (Fr.3.2'') obtained from the second step column chromatography (Fig. 3.2) showed high antimicrobial activity and yield, hence selected for further purification. About 900 mg of bioactive sub-fraction two (Fr. 3.2'') was further purified on a silica gel ( mesh) column (600 mm x 15 mm). The column was eluted stepwise at a flow rate of 1 ml min -1 with linear gradient of 600 ml of hexane: chloroform (90: 10 to 0: 100 v/v), 400 ml of hexane: ethyl acetate (95: 05 to 0: 100 v/v) and 200 ml of ethyl acetate: acetone (95:05 to 0:100 v/v). In this third column 66

9 chromatographic separation, about 24 fractions measuring 50 ml each were collected and concentrated. Weight of each fraction was measured. Fractions having similar R f value and spotting pattern on the TLC plate were pooled and numbered (Fr '''- Fr '''). Among these, sub-fraction one and two (Fr.3.2.1''' and 3.2.3''') obtained from the third step column chromatography showed purity in the TLC profile. These pure compounds were subjected to various spectroscopic techniques to elucidate the structure and studied for their antimicrobial activity in detail. The entire process of purification is shown in fig 3.2. High performance liquid chromatography (HPLC) These purified compounds were tested for their purity by using HPLC, on C-18 column (model LC-10A, Shimadzu Corporation, Japan), with ultraviolet (UV) detection using a diode array detector (DAD) operating at 220, 280 and 320 nm. An isocratic solvent system, consisting of acetonitrile: water: trifluoro acetic acid (89.5:10:0.5), was used as a mobile phase at a flow rate of 1 ml min -1. Ultraviolet (UV) detection was carried out with a diode array detector (Shimadzu). Acetylation of purified compounds For acetylation of purified compounds, about 20 mg of compound, dissolved in 0.5 ml anhydrous pyridine, to which 0.5 ml anhydrous acetic anhydride was added and stirred in a stoppered conical flask overnight at room temperature. Reaction mixture was then poured into ice-cold water ( 50 ml) with constant stirring and left for 1 h. The insoluble acetylated compound was separated by filtration, using a Whatman filter paper No

10 NaBH 4 reaction Reduction of acetylated pure fraction was carried out using NaBH 4 at ice cold condition in methanol. In a typical reaction, 100 mg of fraction was taken in 5 ml methanol and 0.5 equivalent of NaBH 4 was added. The reaction mixture was stirred at ice cold condition for 1 hour. Characterization of bioactive compounds from banana rhizome var. Nanjanagudu Rasbale extracts UV-Visible Spectroscopy UV-Visible spectrum of the isolated compound was recorded on a UV-Visible spectrometer (Shimadzu UV-160A, Singapore) at room temperature. About 1 mg of compound dissolved in 20 ml of acetone/chloroform and was used to record the spectrum in the wavelength range of (λ) 200 to 800 nm. Infra red (IR) Spectroscopy IR spectrum of isolated compound was recorded on a Perkin-Elmer FT-IR Spectrometer (Spectrum 2000) at room temperature. About 1 mg of isolated compound was mixed with spectroscopic grade KBr and well ground before preparing the pellet. The IR spectrum was taken in the frequency range (ν) of 4000 cm cm -1. Liquid chromatography-mass spectrometry (LC-MS) Mass spectrum of the compound was recorded on instrument HP 1100 MSD series (Palo Alto, CA) by electro spray ionization (ESI) technique with a flow rate of 0.2 ml min -1 on C-18 column and total run time of 40 min. The sample used for recording 68

11 the mass spectrum was prepared by dissolving 0.1 mg of compound in 10 ml of methanol/acetonitrile. NMR Spectroscopy The 1 H NMR was recorded for the isolated compound (5mg in 600µL DMSO-D6) at 500MHz on BRUKER AQS NMR spectrometer, Bruker biospin AG, Switzerland. The J-modulated spin-echo for 13 C -nuclei coupled to proton to determine number of attached protons (SEFT) was recorded at 125 MHz. The spectral width for 1 H NMR was 0-12 ppm and ppm for 13 C NMR. Bioactive properties of isolated compounds The isolated pure compounds from acetone (chlorogenic acid) and chloroform (4- epicyclomusalenone and cycloeucalenol acetate) extracts were used to study their bioactive properties viz., antioxidant activity, antimicrobial activity, platelet aggregation inhibition activity and cytotoxicity in detail. Antioxidant activity The antioxidant activities of the isolated compounds ( µl from 1 mg/ ml of stock) were investigated by using eight different in vitro assays Viz., DPPH radical scavenging activity (DPPH RSA), superoxide radical scavenging activity (SRSA), β- carotene bleaching inhibition (βcbi) assay, anti-lipid peroxidation (ALPO) activity, metal chelating activity (MCA), hydrogen peroxide scavenging activity (HPSA), nitric oxide scavenging activity (NOSA) and total reducing power (TRP) assay. BHT, EDTA, ascorbic acid and curcumin were used as standard antioxidants. Results were expressed as EC 50 value, which represents the sample concentration require to show 50% antioxidant activity. The methodology for all the antioxidant assays followed as described in chapter 1 (materials and methods section), except for total reducing power (TRP) assay, which is followed as described in chapter 2 (materials and methods section). 69

12 Antimicrobial activity The minimum inhibitory concentrations (MIC) of the isolated compounds against bacterial and fungal strains were determined as the procedure discussed in chapter 2 (materials and methods) Determination of minimum bactericidal concentration (MBC) Minimum bactericidal concentration was determined according to the method of Smith-Palmer et al., (1998). Test tubes containing nutrient broth with different concentrations ( ppm) of isolated compounds were inoculated with 100 µl of the bacterial suspension (10 5 CFU ml -1 ) Inoculated tubes were incubated for 24 h at 37ºC and growth was observed both visually and by measuring OD at 600 nm. About 100 µl from the tubes not showing growth were plated on nutrient agar as described earlier. MBC is the concentration at which bacteria failed to grow in nutrient broth and nutrient agar inoculated with 100 µl of suspension. Triplicate sets of tubes were maintained for each concentration of the test sample. Determination of minimum fungicidal concentration (MFC) The minimum fungicidal concentration (MFC) was determined using the method of Rotimi et al. (1988). The mixtures of the fungus and isolated compounds in MIC studies which showed no visible growth after 7 days of incubation were subcultured onto a potato dextrose agar (PDA) plate using an inoculum of 10 µl. The plates were incubated at 27 C for 72 h. The MFC was regarded as the lowest concentration that prevented the growth of any fungal colony on the solid medium. Platelet-aggregation inhibitory activity The platelet aggregation inhibition activity of the purified compounds was studied by the methodology followed as described in chapter 1 (materials and methods). 70

13 Cytotoxicity Cytotoxicity of the purified compounds was studied by using HepG-2 cell lines. The methodology followed as described in chapter 1 (materials and methods). Results and discussion Characterization of Bioactive Compounds Elucidation of structure of antioxidant compound from acetone extract of banana rhizome var. Nanjanagudu Rasbale Table 3.1: UV, FTIR and LC-MS spectral data of chlorogenic acid Spectra Chlorogenic acid UV 340 nm 810 cm - 1 (alkane = C-H stretching) 1107 cm - 1 (ester C-O stretching) 1449 cm - 1 (aromatic C=C stretching) 1771 cm -1 (ester carbonyl C=O stretching) FTIR 2726 cm -1 (alkane C-H stretching ) 2963 cm -1 (OH-carboxylic stretching) 3452 cm -1 (O-H stretching) Mass (M +1 ) The pure compound was subjected to various spectroscopic analysis viz. UV, FTIR, LC-MS and NMR to elucidate the structure. The compound exhibited UV λ maxima at 340 nm corresponding to π- π* transition of C=C double bonds. FTIR spectral data showed characteristic stretching frequencies corresponding to carbon skeleton present in the molecule as follows; 3452 cm -1 OH stretching, 2963 cm -1 Broad, carboxylic acid OH stretching, 2726 cm -1 Sharp, CH 2, 1771 cm -1 ester carbonyl C=O stretching, 71

14 1449 cm -1 aromatic C=C stretching, 1107 cm -1 ester CO stretching and 810 cm -1 Sharp, alkane CH stretching. LC-MS data showed major parent molecular ion [M +1 ] peak at m/z (100%). The elemental analysis of this compound reveals the presence of C %, H-5.12% and O-40.64%. The elemental analysis and mass spectrum indicated the chemical formula as C 16 H 18 O 9. The characteristic signals corresponding to cyclohexanyl moiety integrating for four protons appear at ppm. The related carbon signals (36.51, 37.37) appear with negative intensity in 13 C-SEFT experiment confirming the presence of two CH 2 groups. Two vicinal protons attached to hydroxyl group at position 4 and 5 of cyclohexane ring appeared at 3.57 and 3.93 ppm, the related 13 C signals appeared as positive signals indicating the presence of two CH s. Proton signal for ester attached cyclohexane ring was appeared at 5.07 ppm and related carbon signal appeared at 71.01ppm confirming the ester linkage to the cyclohexane ring. α,β-unsaturated double bond attached protons respectively appeared at 6.15 and 7.42 as doublet with J value of Hz, related olefinic methine carbons appeared at & ppm with positive intensity. The two negative quaternary carbon signals appearing at & ppm indicated presence of two carbonyl groups. The presence of broad signal integrating to single proton in the 1 H experiment at ppm leads to the conclusion of presence of carboxyl group. The aromatic region of the spectrum presented signals integrating for three protons as doublet (J = 8 Hz), double doublet (J 1 = 8 Hz, J 2 = 1.9 Hz) and doublet (J = 1.9 Hz) appeared at 6.77, 6.98 & 7.04 ppm respectively. Two broad singlets corresponding to phenolic hydroxyl appear at 9.16 & 9.59 ppm. All these spectral features indicated the molecule has close resemblance with the standard chlorogenic acid spectral characteristics. The spectral assignments were again confirmed by the two dimensional NMR experiments (HSQC, HMBC & COSY) comparison with that of standard chlorogenic acid. 72

15 Table 3.2: The NMR Spectral data of chlorogenic acid C 1 H J (Hz) 13 C SEFT 1 H- 1 H 2D COSY Correlated H 13 C- 1 H 2D HMBC Correlated C C m (dd, 12.8, 7.80) CH m CH m CH m CH m (dd, 13.83, 3.42) CH 2 H2 (1.94), H2' (2.03), H5 (3.93) H2' (2.03), H4 (3.57) H3 (5.07), H5 (3.93) H2 (1.79), H4 (3.57) H6 (1.79), H3 (5.07) H6' (2.01) C1 (73.69), C3 (71.01) C4 (70.64), C5 (68.33) C7 (175.04) C1 (73.69), C3 (71.01) C4 (70.64) C3 (71.01) - C1 (73.69), C3 (71.01) C4 (70.64), C5 (68.33) C7 (175.04) CO CO (d, 15.85) CH H10 (7.42) (d, 15.85) CH H9 (6.15) C8 (165.88), C11 (125.78) C8 (165.88), C12 (114.93), C16 (121.46) C (d, 1.90) CH H16 (6.98) C16 (121.46), C10(145.06), C13 (148.48), C14 (145.72) C C (d, 8.08 Hz) (dd, 8.08, 1.92) CH H16 (6.98) CH H15 (6.77) C11 (125.78), C13 (148.48) C14 (145.72) C12 (114.93), C13 (148.48) C10 (145.06), C14 (145.72) 73

16 Based on all these spectral data and comparison with standard chlorogenic acid, the structure was elucidated to be a (1S, 3R, 4R, 5R)-3-((E)-3-(3, 4-Dihydroxyphenyl) acryloyloxy)-1, 4, 5-trihydroxycyclohexanecarboxylic acid and designated it as a Chorogenic acid (Fig 3.3). This is the first report of isolation and characterization of chlorogenic acid from acetone extract of banana rhizome. O HO HO OH OH O O OH OH Figure 3.3 Structure of chlorogenic acid Yield of purified chlorogenic acid The yield of compound chlorogenic acid is 350 mg/10 g of crude extract, 35 mg/kg of dried banana rhizome powder and 6.3 mg/kg of fresh banana rhizome of var. Nanjanagudu Rasbale. Elucidation of structure of antimicrobial compounds from chloroform extract of banana rhizome var. Nanjanagudu Rasbale 74

17 Elucidation of structure of compound 1 Table 3.4: UV, FTIR and LC-MS spectral data of cycloeucalenol acetate Spectra 4-Epicyclomusalenone UV 243 nm 763, 887 cm -1 (alkane =C-H stretching) 1376 cm -1 (alkane C-H stretching) 1453 cm -1 (aromatic C=C stretching) FTIR 1711 cm -1 (aliphatic carbonyl C=O stretching) 2868 cm -1 (alkane C-H stretching) cm -1 (alkane C-H stretching) Mass [M +1 ] The structure of the isolated bioactive compound was characterized by analyzing UV, FTIR, LC-MS and NMR spectral data. UV spectra of the compound displayed characteristic absorption maxima at 243 nm corresponds to π- π* transition indicating the presence of C=C double bonds (Table 3.3). IR spectral data showed alkane C-H stretching at 2935 and 2868 cm -1, aliphatic carbonyl C=O stretching at 1711 cm -1, C=C stretching at 1453 cm -1, alkane C-H at 1375 cm -1, alkane =C-H stretchings at 959 and 887 cm -1 indicating the presence of aliphatic carbonyl and olefinic double bond groups. LC-MS spectrum showed major parent molecular ion [M +1 ] peak at m/z (100%). The elemental analysis of this compound reveals the presence of C-84.84%, H-11.39% and O-3.77%. The elemental analysis and mass spectrum indicated the chemical formula as C 30 H 48 O. This compound also showed positive reaction for liebermann-burchard reaction indicating it to be a triterpeniod. Proton NMR spectrum (Table.) showed the presence of six methyl groups at δ (ppm) 0.89 (3H), 0.92 (3H), 1.01 (3H), 1.02 (3H), 1.03 (3H) and 1.64 (3H) attached at C-20, 14, 13, 24, 4 and 25 respectively. It also indicated the presence of protons of olefinic methylene group at δ (ppm) 4.69 (bs, 2H) on C-27 attached to C-24. Another methylene group in a trimembered (C-9, 10 and 19) ring at C- 75

18 19 is showed the signals at δ (ppm) 0.41 (d, 4.0 Hz, 1H) and 0.64 (d, 4.0 Hz, 1H). Signals at δ (ppm) 2.44 (m, 2H) and 2.24 (m, 1H) indicated the presence of methylene and methyne groups on the adjacent carbons of carbonyl at C-3. The downfield shift of these signals is due to anisotropic effect of carbonyl group. All the remaining protons on methylene and methine carbons are resonated between δ (ppm) Carbon NMR spectrum (Table..) showed the signals at δ (ppm) 213, and for C-3, C-25 and C-27 respectively, confirmed the presence of carbonyl group at C-3 and double bond between C-25and C-27. All other methyl, methylene, methyne and quaternary carbons resonated between ppm. Assignments are confirmed with the help of SEFT, HSQC and HMBC spectra (table ). All these signals are comparable with that of the reported values for 4-Epicyclomusalenone (Akihisa et al., 1997). The structure of the isolated compound is elucidated as 4-Epicyclomusalenone. Based on all these spectral data the structure was elucidated to be a (3aS, 3bS, 5aS, 6R, 9 1 R, 10 1 S, 12aR) ((2R, 5R) 5, 6 - dimethylhept en - 2- yl) - 3a, 6, 12a - trimethyltetradecahydrocyclopenta (a) cyclopropa (e) phenanthren - 7 (3bH) one, and designated it as a 4-Epicyclomusalenone (Fig 2.2). This is the first report of tritepene compound 4-Epicyclomusalenone from chloroform extract of banana rhizome. H O H Figure 3.4 Structure of 4-Epicyclomusalenone 76

19 Table 3.3: NMR spectral data of 4-Epicyclomusalenone H/C 1 H Multiplicity JH-H (Hz) 13 C δ (ppm) HMBC δ (ppm) 1-CH m 1.60 m C-5,19 2- CH m C-1,3 3-C=O CH 2.24 m C-29 5-CH 2.11 m C-1,6,7,10 6-CH m 1.37 m CH m 1.71 m CH 1.66 m C C CH m 1.25 m CH m C C CH m CH m 1.91 m C CH 1.60 m CH s CH d d CH 1.39 m CH d CH m 1.37 m CH m 1.46 m CH 2.11 m C C CH s CH bs CH d CH d CH s C-24, 25,26 77

20 Yield of purified compound The yield of compound 4-epicyclomusalenone is 420 mg/10 g of crude extract, 42 mg/kg of dried banana rhizome powder and 7.6 mg/kg of fresh banana rhizome. Elucidation of structure of compound 2 Table 3.5: UV, FTIR and LC-MS spectral data of cycloeucalenol acetate Spectra Cycloeucalenol acetate UV 244 nm 886 cm - 1 (alkane =C-H stretching) 959 cm - 1 (alkane =C-H stretching) 1166 cm - 1 (ester C-O stretching) 1248 cm -1 (ether C-O stretching) 1458 cm -1 (aromatic C=C stretching) FTIR 1735 cm -1 (ester carbonyl C=O stretching) 2859 cm -1 (alkane C-H stretching) 2925 cm -1 (alkane C-H stretching) 3448 cm -1 (O-H stretching) Mass (M +1 ) and 409 The pure compound was subjected to various spectroscopic analysis viz. UV, FTIR, LC-MS and NMR to elucidate the structure (Table 3.5). The compound exhibited UV λ maxima at 244 nm corresponding to π- π* transition of C=C double bonds. FTIR spectral data showed O-H stretching at 3448 cm -1, alkane C-H stretching at 2925 and 2859 cm -1, ester carbonyl C=O stretching at 1736 cm -1, C=C stretching at 1458 cm -1, alkane C-H at 1375 cm -1, ester C-O stretchings at 1249 and 1166 cm -1, alkane =C-H stretchings at 959 and 887 cm -1 indicating the presence of ester carbonyl and olefinic 78

21 double bond groups. LC-MS data showed major parent molecular ion [M +1 ] peak at m/z (100%) and along with other fragment m/z 409 (M + - CH 3 COO, 20%). Fragment m/z 409 indicated the presence of acetoxyl group in the molecule. The elemental analysis of this compound reveals the presence of C-81.99%, H-11.18% and O-6.83%. The elemental analysis and mass spectrum indicated the chemical formula as C 32 H 52 O 2. Compound showed positive reaction for liebermann-burchard reaction indicating it to be a triterpeniod. Proton NMR spectrum (Table 3.5) showed the presence of six methyl groups at δ (ppm) 0.86 (3H), 0.90(3H), 0.91(6H), 1.01(3H) and 1.02(3H) attached at C-14, 13, 4, 20, 25 and 26 respectively. It also indicated the presence of protons of olefinic methylene group at δ (ppm) 4.69 (bs, 1H) and 4.74(bs, 1H) on C-28 attached to C-24. Protons on adjacent carbons C-23 and 25 showed the signals at δ (ppm) 2.04 (m, 2H) and 2.24 (m, 1H) respectively due to the anisotropic effect as well as electron withdrawing effect of double bond. Another methylene group in a trimembered (C-9, 10 and 19) ring at C-19 is showed the signals at δ (ppm) 0.17 (bs, 1H) and 0.41(bs, 1H). Signals at δ (ppm) 2.08 (S, 3H) and 4.52 (dt, 10.5, 4.5 Hz, 1H) indicated the presence of acetoxyl group and it attachment at C-3. The signals at δ (ppm) 2.02 (m, 1H) and 1.45 (m, 1H) indicated the two protons on C-2. One proton is shifted to downfield due the anisotropic effect of carbonyl group on C-3. All the remaining protons on methylene and methine carbons are resonated between δ (ppm) Carbon NMR spectrum (Table 3.6) showed the signals at δ (ppm) and for C-28 and C-24 double bond carbon respectively. The signals at δ (ppm) 78.5, and 21.2 for C-3, acetoxyl carbonyl and methyl carbons confirmed the presence of acetoxyl group on C-3. All other methyl, methylene, methyne and quaternary carbons resonated between ppm. Assignments are confirmed with the help of SEFT, HSQC and HMBC spectra (table 3.6). All these signals are comparable with that of the reported values for cycloeucaloenol acetate (Kikuchi et al. 1986). The structure of the isolated compound is elucidated as cycloeucaloenol acetate. 79

22 Table 3.5: NMR spectral data of cycloeucalenol acetate H/C 1 H JH-H C HMBC Multiplicity δ (ppm) (Hz) δ (ppm) 1-CH m 1.45 m CH m 1.45 m CH 4.52 dt CH 1.41 m CH 1.28 m CH m 0.61 m CH m 1.32 m CH 1.60 m C C CH m 1.22 m C-9,10,12,19 12-CH m C-10,11,13,14,18 13-C C CH m CH m C CH 1.59 m CH s C-13,14 19-CH bs 0.41 bs CH 1.39 m CH d CH m CH m C CH 2.24 m C-26,27 26-CH d CH d CH bs C bs C-23 OCOCH s OCOCH 3 30-CH s OCOCH CH s

23 Based on all these spectral data and comparison with literature, the structure was elucidated to be a ((3aS, 6S, 7S, 9 1 R, 10 1 S, 12aR)-3a, 6, 12a-trimethyl-1-((R)-6-methyl-5- methyleneheptan-2-yl) hexa decahydrocyclopenta [a] cyclopropa [e] phenanthren-7-yl acetate), and designated it as a Cycloeucalenol acetate (Fig 3.5). This is the first report of tritepene compound cycloeucalenol acetate is isolated and characterized from chloroform extract of banana rhizome. O O Figure 3.5 Structure of cycloeucalenol acetate Yield of purified compound The yield of compound cycloeucalenol acetate is 400 mg/10 g of crude extract, 40 mg/kg of dried banana rhizome powder and 7.2 mg/kg of fresh banana rhizome of var. Nanjanagudu Rasbale. 81

24 Bioactive properties of isolated compounds In vitro antioxidant activities of isolated compounds Table 3.6: Antioxidant activity (EC 50 value in µg/ml) of isolated compounds and standard antioxidants using different in vitro assays Sl. Antioxidant Chlorogenic Cycloeucalenol 4-Epicyclomusalenone No assays acid acetate Standards 1 DPPH RSA 12±0.3 a 260±5.0 b 296±5.3 c BHT <10 2 SRSA 22±1.2 a - - BHT <10 3 βcbi 62±2.2 a 170±3.0 c 152±3.2 b BHT <10 4 ALPO 26±1.2 a 60±1.8 b 80±2.4 c BHT <10 5 MCA 90±2.5 a 110±2.0 c 102±2.2 b EDTA <10 6 HPSA 44±1.6 a 180±2.5 b 196±3.8 c Asc. acid <10 7 NOSA 28±1.5 a 206±3.6 b 242±4.6 c Curcumin <10 8 TRP 4.5±0.2 a 44±1.2 c 35±1.0 b Asc. acid 24±2.0 Mean values in a column with different superscripts differ significantly at p<0.05. EC 50: Effective concentration of the sample to show 50% of antioxidant activity; DPPH RSA-1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, SRSA-superoxide radical scavenging activity, βcbi-β-carotene bleaching inhibition assay, ALPO-Anti-lipid peroxidation activity, MCA-metal chelating activity, HPSA-hydrogen peroxide scavenging activity, NOSA- Nitric oxide scavenging activity and TRP-total reducing power assay; Asc.acid-ascorbic acid. sample concentration to get 0.5 of absorbance at 700 nm DPPH radical scavenging activity (DPPH RSA) Any molecules, which donate an electron or hydrogen to a reaction mixture, can react with and bleach DPPH. Further, DPPH is reduced from a purple compound to a light yellow compound by electrons from oxidant compounds. Substances which are able to perform this reaction can be considered as antioxidants and therefore radical scavengers (Brand-Williams et al., 1995). The reaction of DPPH with hydroxyl groups involves a hemolytic substitution of one the DPPH phenyl rings, which yields 2-(4- hydroxyphenyl)-2-phenyl-1-picryl hydrazine as a major product, and 2-(4-nitrophenyl)-2- phenyl-1-picrylhydrazine via a series of secondary processes. Table 3.6 explains the effect of purified compounds on DPPH radical scavenging activity. The compound chlorogenic acid showed highest activity (EC 50 of 12±0.3 µg/ ml) followed by 4- epicyclomusalenone (EC 50 of 260±5.0 µg/ml) and cycloeucalenol acetate (EC 50 of 82

25 296±5.3 µg/ml). The DPPH radical scavenging activity of chlorogenic acid was comparable to the standard antioxidant BHT (EC 50 of <10 µg/ml). Superoxide radical scavenging activity (SRSA) Superoxide radical is known to be very harmful to cellular components as a precursor of more reactive oxygen species (Soares et al., 1997). This radical is a powerful oxidizing agent that can react with biological membranes and induce tissue damage (Castelluccio et al., 1996). It may also decompose to singlet oxygen, hydroxyl radical, or hydrogen peroxide (Halliwell and Gutteridge, 1985). Effect of purified compounds on superoxide radical is shown in table 3.6. Compound chlorogenic acid showed high superoxide radical scavenging activity (EC 50 value of 22 µg/ml). However, triterpenoid compounds both cycloeucalenol acetate and 4-epicyclomusalenone did not demonstrated EC 50 value upto the concentration of 300 µg/ml. β -carotene bleaching inhibition (βcbi) assay In this model, β-carotene undergoes rapid discoloration in the absence of an antioxidant. The presence of an antioxidant can hinder the extent of β-carotene destruction by neutralizing the linoleate free radical and any other free radicals formed within the system (Kamath and Rajini, 2007). In term of β-carotene bleaching effect, the purified compounds exhibited in the following order: BHT > chlorogenic acid > cycloeucalenol acetate > 4-epicyclomusalenone. Chlorogenic acid exhibited a marked antioxidant activity (EC 50 of 62±2.2 µg/ml) followed by triterpenoid compounds cycloeucalenol acetate (EC 50 of 152±3.2 µg/ml) and 4-epicyclomusalenone (EC 50 of 170±3.0 µg/ml). While, standard BHT showed EC 50 <10 µg/ml (table 3.6). It was clear that hydroperoxides formed in this system may be decomposed by the purified compounds of banana rhizome. Hence, the degradation rate of β-carotene depends on the antioxidant activity of the purified compounds. This suggests that the purified compounds may have potential use as antioxidative preservatives in emulsion-type systems. 83

26 Anti-lipid peroxidation (ALPO) activity Lipid peroxidation can inactivate cellular components and plays an important role in oxidative stress of biological systems. Furthermore, several toxic byproducts from the peroxidation can damage other bio-molecules (Box and Maccubbin, 1997). Lipid peroxidation causes destabilization and disintegration of the cell membrane, leading to liver injury, atherosclerosis, kidney damage, aging, and susceptibility to cancer (Rice- Evans and Burdon, 1993). It is well established that transition of metal ions, such as iron and copper, can stimulate lipid peroxidation through various mechanisms. They may either promote the generation of hydroxyl radicals to initiate the lipid peroxidation process or propagate the chain process via decomposition of lipid hydroperoxides (Braughler et al., 1987). Lipid peroxidation is a free radical mediated propagation of oxidative insult to polyunsaturated fatty acids involving several types of free radicals. Its termination occurs in biological system through enzymatic means or by radical scavenging activity by antioxidants (Heim et al., 2002). In this study, high ALPO activity with EC 50 value of 26±1.2 µg/ml was shown by chlorogenic acid followed by 4- epicyclomusalenone (60±1.8 µg/ml) and cycloeucalenol acetate (80±2.4 µg/ml) (table 3.6). Metal chelating activity (MCA) Iron is known to generate free radicals through the Fenton and Haber-Weiss reaction (Halliwell and Gutteridge, 1990). Ferrous ions can stimulate lipid peroxidation by Fenton reaction, and also accelerates peroxidation by decomposing lipid hydro peroxides into peroxyl and alkoxyl (Halliwell, 1991; Gulcin et al., 2003). Metal ion chelating activity of an antioxidant molecule prevents oxyradical generation and the consequent oxidative damage. Metal ion chelating capacity plays a significant role in antioxidant mechanism since it reduces the concentration of the catalyzing transition metal in lipid peroxidation (Duh et al., 1999). The compound chlorogenic acid was found to be more potential antioxidant activity with an EC 50 value of 90±2.5 µg/ml followed by cycloeucalenol acetate and 4-epicyclomusalenone with an EC 50 value of 102±2.2 and 84

27 110±2.2 µg/ml respectively (Table 3.6). The action of these compounds as antioxidant may be related to its iron-binding capacity. It was reported that chelating agents, which form σ-bonds with a metal, are effective as secondary antioxidants because they reduce the redox potential thereby stabilizing the oxidised form of the metal ion (Gordon, 1990). High chelating ability of these compounds may be beneficial as a most effective proxidants in the food system. Hydrogen Peroxide Scavenging Activity (HPSA) The compound chlorogenic acid was found to be more potent in scavenging hydrogen peroxide (44±1.6 µg/ml), followed by 4-epicyclomusalenone (180±2.5 µg/ml) and cycloeucalenol acetate (196±3.8 µg/ml) (Table 3.6). The H 2 O 2, formed by the twoelectron reduction of O 2 is not a free radical, but is an oxidizing agent. In the presence of O 2 and transition metal ions especially iron and copper, H 2 O 2 can generate OH. via the Fenton reaction (Shahidi, 1992). H 2 O 2 can cross membranes and may slowly oxidize a number of biomolecules and compounds. H 2 O 2 is formed in vivo when superoxide dismutates and also by many oxidase enzymes. H 2 O 2 at micromolar levels is poorly reactive. However, higher levels of H 2 O 2 can attack some energy-producing systems. H 2 O 2 inactivates the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. Hence, metal chelating and H 2 O 2 scavenging processes are important for living organisms (Halliwell and Gutteridge, 1985; Aruoma, 1998; Marcocci et al., 1994). Nitric oxide scavenging activity (NOSA) In the present study, nitric oxide radical generated from sodium nitroprusside at physiological ph was found to be inhibited by purified compounds chlorogenic acid, cycloeucalenol acetate and 4-epicyclomusalenone (Table 3.6). Incubation of sodium nitroprusside with purified compounds from banana rhizome revealed that the inhibition of nitrite production was highest in compound chlorogenic acid (EC 50 of 28±1.5 µg/ ml) whereas, 4-epicyclomusalenone and cycloeucalenol acetate showed moderate inhibition of nitrite production and exhibited EC 50 of 206±3.6 and 242±4.6 µg/ ml respectively. In addition to reactive oxygen species, nitric oxide is also implicated in inflammation, 85

28 cancer and other pathological conditions (Moncada et al., 1991). The plant/plant products may have the property to counteract the effect of NO formation and in turn may be of considerable interest in preventing the ill effects of excessive NO generation in the human body. Further, the scavenging activity may also help to arrest the chain of reactions initiated by excess generation of NO that are detrimental to human health. Total reducing power (TRP) assay The reducing capacity of a compound from Fe 3+ /ferricyanide complex to the ferrous form may serve as a significant indicator of its antioxidant capacity (Meir et al., 1995). The compounds viz., chlorogenic acid, cycloeucalenol acetate and 4- epicyclomusalenone were showed high reducing power with EC 50 value of 4.5±0.2, 35±1.0 and 44±1.2 µg/ml respectively (Table 3.6). The activity of chlorogenic acid was found to be higher than the standard ascorbic acid (24±2.0). The result revealed that these compounds are the potential electron donor and also could react with free radicals, converting them to more stable products and terminating the radical chain reaction. Multiple antioxidant activities of the isolated compounds and their structure activity relationship Chlorogenic acid, 4-epicyclomusalenone and cycloeucalenol acetate were exhibited multiple antioxidant activities in all the in vitro models tested. The activity shown by chlorogenic acid in some of the in vitro antioxidant assays viz., total reducing power (4.5±0.2 µg/ml), DPPH radical scavenging activity (12±0.3 µg/ml), superoxide radical scavenging activity (22±1.2 µg/ml), anti-lipid peroxidation activity (26±1.2 µg/ml) and nitric oxide scavenging activity (28±1.5 µg/ml) were comparable to the standard antioxidants viz., BHT, curcumin and ascorbic acid. Whereas, cycloeucalenol acetate and 4-epicyclomusalenone were showed high antioxidant activity with low EC 50 value in total reducing power assay (EC 50 of 35±1.0 and 44±1.2 µg/ ml, respectively), anti-lipid peroxidation activity (EC 50 of 80±2.4 and 60±1.8 µg/ ml, respectively) and metal chelating activity (EC 50 of 102±2.2 and 110±2.0 µg/ml, respectively). 86

29 DPPH radical scavenging by antioxidants has been attributed to their hydrogendonating ability of -OH and -CH 3 groups. The antioxidant activity of the compound may be attributed to the presence of -OH and C=O groups (Chen and Ho, 1995; Nikolaos, et al., 2003). The compound chlorogenic acid possess -OH and C=O groups, whereas, compound 4-epicyclomusalenone and cycloeucalenol acetate possess -CH 3 and C=O groups. Lipid peroxidation inhibitory activity was mainly attributed to the number of hydroxyl groups of the compounds (Sopheak and Betty, 2002). Presence of six hydroxyl and two methylene groups in chlorogenic acid and methyl group in teriterpenoid compounds (4-epicyclomusalenone and cycloeucalenol acetate) may be responsible for high lipid peroxidation inhibitory activity. It was reported that the structures containing two or more of the following functional groups: -OH, -SH, -COOH, -PO 3 H 2, C=O, -NR2, -S- and -O- in a favorable structure-function configuration is responsible for metal chelating activity (Lindsay, 1996; Yuan et al., 2005). Chlorogenic acid, which is having - OH, -COOH, C=O and -O- functional groups might have contributed for the metal chelating activity. Whereas, 4-epicyclomusalenone and cycloeucalenol acetate have only C=O may explain the less activity, when compare to the compound chlorogenic acid. The solubility and hydrophobicity of these compounds plays important role in their antioxidant activity (Sopheak and Betty, 2002). High antioxidant activities of chlorogenic acid than triterpenoid compounds (4- epicyclomusalenone and cycloeucalenol acetate), due to their possessing an O-dihydroxy B-ring structure, which conferred higher stability in the radical form and participated in electron delocalisation. This conclusion was consistent with those reported in the literature (Pietta, 2000). This also depends on the hydrogen-donating ability of antioxidants. Studies on oxidation potentials and redox reactions between and transition metal ions have shown that the o-dihydroxyl feature is a crucial factor for the reducing efficiency (Makris and Kefalas, 2005). Antioxidant activity of phenolics increased when there were more hydroxyl groups in the molecule (Kumaran and Karunakran, 2006). Presence of two phenolic hydroxyl groups apart from four other hydroxyl groups in the compound chlorogenic acid explains the antioxidant potential. Moreover, presence of 87

30 trihydroxy cyclohexane and α hydroxy carboxylic acid might have contributed the antioxidant activity of the compound chlorogenic acid. In vitro antioxidant properties of chlorogenic acid with multiple mechanisms involving free radical scavenging, metal ion chelation, and inhibitory effect on specific enzymes responsible for free radical and hydroperoxide formation were reported earlier (Chen and Ho, 1997; Zhou and Zheng, 1991; Kono et al., 1997). Triterpenes, which are known to have diverse physiological and pharmacological activities (Consolacion et al., 2007 Fernandes et al., 2003; Harmand et al., 2003). The antioxidant activity of terpenoids was well documented (Chae et al., 2009; D Abrosca et al., 2006). Antibacterial activity of isolated compounds Minimum inhibitory concentration (MIC) The triterpenoid compounds 4-epicyclomusalenone and cycloeucalenol acetate isolated from chloroform extract were exhibited higher antimicrobial activity when compared to chlorogenic acid isolated from acetone extract of banana rhizome (table 3.7). Further, these compounds showed antibacterial and antifungal activities against wide spectrum of bacterial and fungal species and were more effective against Gram +ve bacteria (high activity with low MIC value) than Gram -ve bacteria. Highest activity with lowest MIC value were observed against Gram +ve bacteria of 60 ppm was observed in 4-epicyclomusalenone against M. luteus followed by cycloeucalenol acetate against M. luteus (90 ppm). While, chlorogenic acid (90 ppm), and 4-epicyclomusalenone (90 ppm) against B. subtilis. Among Gram -ve bacteria high activity with low MIC value of 160 ppm was shown by cycloeucalenol acetate against K. pneumoniae, followed by 180 ppm and 220 ppm were shown by 4-epicyclomusalenone and cycloeucalenol acetate respectively against S. typhi. Interestingly, chlorogenic acid showed MIC value only for two Gram -ve bacteria viz., P. aeruginosa (320 ppm) and K. pneumoniae (770 ppm). 88

31 Table 3.7: Antimicrobial activity (MIC and bactericidal and fungicidal) in ppm concentration of isolated compounds Microbial strains Bacterial strains Chlorogenic acid 4-Epicyclomusalenone Cycloeucalenol acetate Bactericidal Bactericidal Bactericidal MIC MIC MIC activity activity activity Gram +ve bacteria M. luteus 100 Yes 60 Yes 90 Yes S. aureus 260 Yes 270 No 550 No E. fecalis 380 No 100 Yes 190 Yes B. cereus 150 Yes 120 Yes 150 Yes B. subtilis 90 Yes 90 Yes 170 Yes L. monocytogenes 110 Yes 180 Yes 110 Yes Gram -ve bacteria P. aeruginosa 320 Yes Yes E. coli S. typhi Yes 220 Yes K. pneumoniae 770 No 240 Yes 160 Yes E. aerogenes No 790 No P. mirabilis Y. enterocolitica Fungal strains Fungal strains MIC Fungicidal Fungicidal Fungicidal MIC MIC activity activity activity A. niger 260 Yes 280 Yes 210 Yes A. flavus 340 No 410 No 560 No A. fumigatus 180 Yes 320 Yes 450 Yes A. parasiticus 220 Yes 400 No 650 No P. rubrum No 850 No F. moniliforme 300 No 780 No 900 No *Each value represents mean of three different observations Determination of bactericidal effect The isolated compounds viz., chlorogenic acid, 4-epicyclomusalenone and cycloeucalenol acetate were found to be bactericidal against wide range of bacterial species tested (table 3.7). All the three compounds showed bactericidal effect against all 89

32 the Gram +ve bacteria tested, except against S. aureus (by 4-epicyclomusalenone and cycloeucalenol acetate) and E. fecalis (by chlorogenic acid). Among Gram -ve bacteria, 4-epicyclomusalenone showed bactericidal effect against S. typhi and K. pneumoniae, and cycloeucalenol acetate showed bactericidal effect against P. aeruginosa, S. typhi and K. pneumoniae. Interestingly, chlorogenic acid showed bactericidal effect against only one Gram -ve bacteria (P. aeruginosa). It appeared that effective MIC also represents the effective bactericidal concentration for the bacteria tested. The sensitivity difference between Gram +ve and Gram ve bacteria towards the tested pure compounds could be ascribed to the morphological differences between these microorganisms. The structures of cell envelope, including cytoplasmic membrane and cell wall component are different between Gram +ve and gram -ve bacteria. Gram -ve bacteria possess an outer membrane surrounding the cell wall, which restricts diffusion of hydrophobic compounds through its lipopolysaccharide covering. Without outer membrane, the cell wall of Gram +ve bacteria can be permeated more easily and polyphenols can disturb the cytoplasmic membrane, disrupt the proton motive force (PMF), electron flow, active transport and coagulation of cell contents (Burt, 2004). Antifungal activity All the three isolated compounds were found to be effective against tested fungal strains (table 3.7). Unlike bacterial strains, fungal strains were found to be more susceptible to chlorogenic acid, followed by 4-epicyclomusalenone and cycloeucalenol acetate. Triterpene compounds showed antifungal activity (MIC value) against all the fungal strains tested. Whereas, chlorogenic acid failed to inhibit P. rubrum. High antifungal activity with low MIC values were observed in chlorogenic acid against A. fumigatus (180 ppm), cycloeucalenol acetate against A. niger (210 ppm) and chlorogenic acid against A. parasiticus (220 ppm). The isolated compounds viz., chlorogenic acid, 4- epicyclomusalenone and cycloeucalenol acetate were found to be fungicidal against wide range of fungal species tested. Compound chlorogenic acid showed fungicidal effect against A. niger, A. fumigatus and A. parasiticus. Whereas, compounds 4-90