JCBPS; Section D; Feb. 2015 – Apr. 2015, Vol. 5, No. 2 ; 2151-2158 E- ISSN: 2249 –1929

Journal of Chemical, Biological and Physical Sciences An International Peer Review E-3 Journal of Sciences Available online at www.jcbsc.org Section D: Environmental Sciences

CODEN (USA): JCBPAT Research Article

Analysis of Bioactive Compounds in densiflorum (Lam.) SchltrPseudobulb Using UV-VIS, FTIR and GC-MS Techniques.

1Theng P. A. and 2Korpenwar A. N.

1 Shri Shivaji Science and arts college Chikhli Dist. Buldana 443201 (MS) .

2 Rashtrapita Mahatma Gandhi Science and Arts College, Nagbhid, Dist. Chandrapur 441205 (MS) India.

Received: 21 February2015; Revised: 12 March 2015; Accepted: 27 March 2015

Abstract: To characterize the bioactive constituents present in endangered terrestrial orchid Geodorum densiflorum pseudobulb extract in ethanol by using UV-VIS. FT-IR and GC-MS methods.Geodorum densiflorum is an endangered orchid belongs into family . Pseudobulb powder extract was prepared in ethanol as a solvent by Soxhlet apparatus. Ethanol extract was subjected to UV-VIS., FT-IR and GC-MS analysis. The qualitative UV-VIS analysis of ethanol extract showed peaks at 196nm, 246 nm, 376 nm and 816nm. The λ max was obtained at 376 nm. FTIR analysis proved the presence of alcohols, phenols, alkanes, aldehydes, alkenes, carboxylic acids, esters, ethers, aliphatic amines, amides, sulfides and alkyl halides compounds. The GC-MS analysis of Geodorum densiflorum revealed that presence of four compounds, these are pentane, 1, 1-diethoxy- (30.30 %), propane, 1, 1, 3-triethoxy (60.91 %), neotigogenin (4.38 %) and sarsasapogenin (4.38 %). Keywords: Phytochemical, Geodorum densiflorum, UV-VIS., FT-IR, GC-MS.

2151 J. Chem. Bio. Phy. Sci. Sec. D, February 2015 – April 2015; Vol.5, No.2; 2151-2158

Analysis … Theng and Korpenwar.

INTRODUCTION

Orchids comprise five subfamilies and approximately 870 genera occurring on all vegetated continents and even some Antarctic islands1,2. In India, family Orchidaceae is represented by 177 genera with 1195 species3 of which 400 species are endemic 4. Genus Geodorum is belongs into family Orchidaceae. Geodorum densiflorum is an endangered terrestrial orchid5. Geodorum has 20 species widely distributed in tropical and subtropical areas. The genus Geodorum (Jacks.) is represented by about 10 species distributed from tropical to and the South West Pacific Island6. Geodorum densiflorum is widely distributed in India, , Australia, , Srilanka, China, , Papua and Himalayas. It is estimated that about 1,300 species (140 genera) of orchids are found in India with Himalayas as their main home and other scattered in Eastern and Western Ghats7. In India, the genus is represented by 7 species8.Pseudobulb of Geodorum densiflorum was ethnomedicinally applied externally to cure carbuncles 9, used to regularize menstrual cycle10 andon diabetes11. There is no reference of phytochemical study of this endangered orchid so study was carried out. MATERIAL AND METHODS

Collection and processing of material: The pseudobulb of Geodorum densiflorum was collected from the Amba Barwa forest, Jalgaon Jamod tehsil, district Buldana. Pseudobulb material was thoroughly washed under tap water to remove soil particles. The pseudobulb was cut into slices, shade dried and grind into fine powder. Powder was stored in air tight container until use. Plant sample extraction: About 25 g of air dried powder of pseudobulb was extracted with 250 ml of ethanol as solvent in soxhlet assembly. This extract was subjected for further analysis. UV-VIS analysis: The ethanol extract was examined under visible and UV light for proximate analysis. For UV-VIS analysis sample was diluted to 1:10 with the same solvent. The extracts were scanned in the wavelength range from 190-1100 nm using EQUIP- TRONICS (EQ-826) and the peaks values were detected. Analysis was repeated twice for the spectrum confirmation. FTIR Spectroscopic analysis: FTIR analysis was performed using Perkin Elmer Spectrophotometer system, with wave number from 4400 to 450 cm-1. It was used to detect the characteristic peaks and their functional groups. After absorption of IR radiation, the molecules vibrates, giving rise to absorption spectrum. The peak values of the FTIR were recorded. Each analysis was repeated twice for the spectrum confirmation. GC-MS analysis: The bioactive constituents of ethanolic extract of the pseudobulb were analyzed by GC-MS. The analysis was carried out on Jeol spectrometer (Model: Accu TOF GCV). Split ratio was 20: 1. Column temperature program: Initial temperature 800C for 5 min. Ramp: 100C/min to 2600C. Again isothermal for 5 min, then ramp at 300C/min. to 2800C, isothermal for 5 min. Solvent used was ethanol. Column used was HP5, 30 m long, 0.25 mm id and 0.25 µm film thicknesses. The injector temperature was 2500C, detector temperature was 2800C. Helium was used as carrier gas at 1 mL/min. Identification of components: The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. The detection employed the NIST (National Institute of Standards and Technology) library. Interpretation of GC-MS was conducted using the database of NIST having more than 62,000 patterns. The spectrum of the unknown component was compared with the

2152 J. Chem. Bio. Phy. Sci. Sec. D, February 2015 – April 2015; Vol.5, No.2; 2151-2158

Analysis … Theng and Korpenwar.

spectrum of the known components stored in the NIST library. The name, molecular weight and structure of the components of the test materials were ascertained.

RESULT AND DISCUSSION

UV-VIS absorption: The qualitative UV-VIS spectrum analysis of ethanol extract of Geodorum densiflorum was selected at wavelength from 190-1100 nm due to sharpness of the peaks and proper baseline. The UV-VIS analysis of ethanol extract of Geodorum densiflorum showed peaks at 196 nm, 246 nm, 376 nm, 816 nm and 971 nm that showed absorption at 0.593, 0.351, 1.493, 0.452 and 0.437 respectively. The λ max was obtained at 376 nm.

Table 1: UV- VIS analysis of Geodorum densiflorum pseudobulb powder in ethanol extract. Wavelength (λ) (in nm) 196 246 376 816 971 Absorbtion 0.593 0.351 1.493 0.452 0.437

Fig.1: UV-VIS spectrum of ethanol extract of Geodorum densiflorum pseudobulb powder

FTIR Spectroscopy: IR spectrum of ethanol extract was showed in Fig. (2).The fundamental vibrations and its fraction were given in Table (2).FTIR analysis of Geodorum densiflorum pseudo bulb powder extract in ethanol proved the presence of alcohols, phenols, alkanes, aldehydes, alkenes, carboxylic acids, esters, ethers, aliphatic amines, amides, sulfides and alkyl halides compounds. Pseudo bulb powder extract in ethanol was subjected to FTIR analysis for the identification of functional constituents present in Geodorum densiflorum. FTIR spectrum was helpful to judge medicinal materials from the adulterate and even evaluates the quality of the medicinal plant materials. The FTIR is highly rapid, effective, visual and accurate analytical method for pharmaceutical research12.

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Analysis … Theng and Korpenwar.

53.2

50

781,50 701,49

45 828,47 629,46 569,46 528,46

40

35 %T 1513,36 1730,34 1363,34

1292,32 1157,32 30 1462,31 1227,30 2850,30 1614,29

25 2921,25 1073,25 1043,25

20 3368,21 18.0 4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0 cm-1

Fig.2: FT-IR spectrum of ethanol extract of Geodorum densiflorum pseudo bulb powder

Table 2: FTIR Wavelength and functional groups of ethanol extract of Geodorum densiflorum pseudobulb extract. Wave number Functional group Bond Group frequency, cm- 1 3368,21 Hydrogen bonded alcohols, phenols O-H Stretching 3600-3200 2921,25 Alkanes C-H Stretch 2970-2850 2850,30 Alkanes C-H Stretch 2970-2850 1730,34 Aliphatic Aldehydes C=O Stretch 1740-1720 1614,29 Alkenes C=C stretch 1680- 1600 1513,36 Ester C=O Stretch 1750-1730 1462,31 Alkanes C-H bend 1475-1365 1363,34 Alkanes C-H bend 1475-1365 1292,32 Anhydrides C-O stretch 1300-900 1227,30 Alcohols, carboxylic acid, esters, ethers C-O stretch 1300-1000 1157,32 Alcohols, carboxylic acid, esters, ethers C-O stretch 1300-1000 1073,25 Alcohols, carboxylic acid, esters, ethers C-O stretch 1300-1000 1043,25 Alcohols, carboxylic acid, esters, ethers C-O stretch 1300-1000 828,47 Alkenes C-H bend 1000-650 781,50 Alkenes C-H bend 1000-650 701,49 Alkenes C-H bend 1000-650 629,46 Sulfides C-S 710-570 569,46 Ring in benzene derivatives In Ring 650-510 deformation 528,46 Bromides C-Br 650-510

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Analysis … Theng and Korpenwar.

GCMS analysis: The compounds present in the ethanol extract of Geodorum densiflorum were identified by GC-MS analysis. The GC-MS analysis of Geodorum densiflorum revealed that presence of four compounds that could contribute the medicinal quality of pseudobulb. The active principle with their retention time (RT), name of compound, molecular formulae (MF), molecular weight (MW), compound nature and peak area in percentage are presented in Table(3) and figure (3 A-D). It was found that main phytoconstitut of pseudobulb are pentane, 1, 1-diethoxy- (30.30 %), propane, 1, 1, 3-triethoxy (60.91 %), neotigogenin (4.38 %) and sarsasapogenin (4.38 %).

Table 3: Phytocomponents in ethanol extract of Geodorum densiflorum pseudobulb powder by GC- MS. Peak RT Name of Compound Molecular Molecular Compound Peak area No. (min.) formula weight nature (%)

1 3.4 Pentane, 1, 1 –diethoxy- C9H20O2 160 Ether 30.30

2 7.7 Propane, 1, 1, 3-triethoxy C9H20O3 176 Ether 60.91

3 40.2 Neotigogenin C27H44O3 416 Steroid 4.38

4 40.2 Sarsasapogenin C27H44O3 416 Steroid 4.38

Fig.3 A: Mass spectrum of pentane, 1-1-diethoxy.

Fig.3 B: Mass spectrum of propane, 1, 1, 3-triethoxy.

Fig. 3 C: Mass spectrum of neotigogenin.

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Analysis … Theng and Korpenwar.

Fig. 3 D: Mass spectrum of sarsasapogenin.

Fig 3 A1: GC-MS chromatogram in ethanol extract of pseudobulb powder.

Sarsasapogenin is a bioactive component found in Geodorum densiflorum pseudobulb. Sarsasapogenin is use to regulate menstrual cycle. It was showed antidepressant activity13, apoptotic effect14,antidiabetic and improving memory15,16.Neotigogenin is a steroidal saponins showed Anticancer activity 17, antifungal activity 18.Pentane 1, 1 diethoxyand are ether compound in pseudobulb extract. Its activity was not reported till now.Propane, 1, 1, 3-triethoxy was also ether compound that showed antioxidant activity19 and use for flavor20.

CONCLUSION

The pseudobulb of Geodorum densiflorum contains many phytoconstituents in ethanol extract. UV-Vis. analysis showed 5 peaks with λ max was obtained at 376 nm wavelength. FTIR analysis proved the presence of alcohols, phenols, alkanes, aldehydes, alkenes, carboxylic acids, nitro compounds, esters, ethers, aliphatic amines, amides, sulfides and alkyl halides compounds. The GC- MS analysis revealed that presence of four compounds, these are pentane, 1, 1-diethoxy-, propane, 1, 1, 3- triethoxy, neotigogenin and sarsasapogenin. Further research work may help for the identification of new bioactive compounds in Geodorum densiflorum pseudobulb. This investigation may lead to the formulation of drug to cure various diseases. ACKNOWLEDGEMENT

The authors are grateful to the SAIF, IIT, Bombay, Powai, Mumbai, for providing the GC-MS facility. Authors are also thankful to CIL, Panjab University, Chandigarh for providing UV-VIS. and FT-IR facility.

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Corresponding author: Theng P. A.

ShriShivaji Science and arts college Chikhli Dist. Buldana 443201 (MS) India.

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