SECTION 4 PITTOSPORACEAE ANALYSIS OF THE VOLATILE COMPONENTS IN P. DASYCAULON AND P. NAPAULENSE

Transcription

1 SECTION 4 PITTOSPORACEAE ANALYSIS OF THE VOLATILE COMPONENTS IN P. DASYCAULON AND P. NAPAULENSE

2 1. INTRODUCTION Plants belonging to the family Pittosporaceae are trees, shrubs or sometimes climbers, usually with lenticels and often with spines. Several of them are with fragrant flowers and hence are cultivated in gardens. The genus Pittosporum alone is found in India, and the plants included in this genus are erect trees, shrubs or undershrubs, sometimes epiphytic, branching forked or verticillate. About 300 species of this genus is reported out of which only eleven are observed in India which are P. anamallayense, P. ceylanicum, P. dasycaulon, P. eriocarpum, P. ferrugineum, P. humile, P. napaulense, P. neelgherrense, P. podocarpum, P. tetraspermum and P. viridulum 1. Seeds of some of the species of this genus are commonly used in traditional Chinese medicine for their sedative and cough relieving effects. Phytochemical investigations on Pittosporum species have led to the identification of some physiologically active compounds such as triterpenoid saponins, carotenoids, and essential oils 2. A review submitted by Cragg and coworkers denote Pittosporaceae as hot family and the genus Pittosporum as hot genera in concern with antileukemia potential. (The term hot family refers to those families having more than 14 individual plants designated as active by the stated selection criteria, and hot genera refers to those genera containing three or more active plants) 3. The antimicrobial effects of leaves of various species of this family are reported. The presence of a number of volatile mono- and sesquiterpenes in leaves may possibly be responsible for its biological activities 4. The distribution of the Pittosporum in India is characteristic of the rain forest, occurring in the substages usually in altitudes between 500 to 2800m and is mainly concentrated in the Himalayan and Deccan regions. The species occurring in peninsular India are mainly relict species excepting P. tetraspermum and P. napaulense. The two species P. dasycaulon and P. napaulense are selected for studying their essential oils in this work. 119

3 2. ESSENTIAL OIL FROM STEM BARK AND LEAVES OF PITTOSPORUM DASYCAULON P. dasycaulon is a small tree with its young branches densely tomentose. Leaves are coriaceous, apex acute, base cuneate, margine entire; petioles 8-18 mm long, purberulous. Inflorescence is terminal or pseudoterminal, umbellate densely brownish tomentose. Sepals are 2-3 X 1 mm, petals X mm, oblong and yellow. Stamens are filaments, 6-7 mm long and anthers 2 mm long. Ovary is 3 mm long; style 4-5 mm long, glabrous. Capsules are 8-10 mm in diameter, globose and woody. Seeds may be 4-6, blackish pink. They are distributed in peninsular India and are endemic to Western Ghats. An extract of the stem bark is reported to show antibacterial and antifungal properties Present work The composition of essential oils from the stem bark and leaves of P. dasycaulon were studied. The chemical profiles of the oils were obtained from their respective GC-MS analyses Materials and Methods The leaves and stem bark of P. dasycaulon were collected from Kakkayam, Kozhikode in the month of February It was authenticated by Dr. A.K. Pradeep, Department of Botany, University of Calicut Extraction of essential oils Fresh leaves (125 g) and stem bark (125 g) were cut into very small pieces. They were then separately hydrodistiled for 4 hours in a Clevenger s apparatus according to the procedure described in the Ayurvedic Pharmacopoeia of India, part 1, volume 6 6 using diethyl ether as the collecting 120

4 solvent. The solvent was carefully removed, yielding pale yellow aromatic oil in the case leaves and a clear oil for stem bark. The oil yields (w/w) were 0.03% (leaves) and 0.05% (stem bark), all on a fresh weight-basis Sample Preparations. Stem Bark Essential Oil. Ca. 50µL (56.4 mg) of stem bark essential oil was transferred to a large neck 2mL vial. It was diluted in 450mL of n-hexane. 50µL of nonane which is used as internal stantard was also added. The concentration was ppm. This solution (20 µl) was then injected on to an apolar column with an MS detector for compound identification. Leaf Essential Oil. Ca. 50µL (60.1mg) of leaf essential oil was transferred to a large neck 2mL vial. It was diluted in 450mL of n-hexane. 50µL of nonane which is used as internal stantard was also added. The concentration was ppm. This solution (20 µl) was then injected on to an apolar column with an MS detector for compound identification Gas Chromatography-Mass Spectrometry (GC-MS) analysis For GC/MS measurements a GC-17A with QP5000 (Shimadzu) and Compaq-ProLinea data system (class 5k-software), a GC-HP5890 with HP5970-MSD (Hewlett-Packard, USA) and ChemStation software on a Pentium PC (BoÈhm, Austria), a GCQ (Finnigan-Spectronex, Germany- USA) and Gateway-2000-PS75 data system (Siemens-Nixdorf, Germany, GCQ software) were used. The carrier gas was helium; injector temperature, 250 C; interface-heating at 300 C, ion-sourceheating at 200 C, EI-mode was 70 ev, and the scan-range was 41± 450 amu. The temperature programme was: 40 C/5 min to 280 C/5 min, with a heating rate of 6 121

5 C/min. The columns were 30 m X 0.32 mm bonded FSOT-RSL-200 fused silica, with a film thickness of 0.25 mm (Biorad, Germany) and 30 m X 0.32 mm bonded Stabilwax, with a film thickness of 0.50 mm (Restek, USA). Quantification was achieved using peak area calculations, and compound identification was partly carried out using correlations between retention times 7, 8 and online Wiley, NBS and NIST library spectra. Table 4.2a Volatile components of bark essential oil Compound name RI Percent α-thujene α-pinene Camphene Sabinene β-pinene Myrcene Decane α-phellandrene α-terpinene p-cymene Limonene β-phellandrene (E)- β-ocimene γ-terpinene Terpinolene p-cymenene n-undecane (Z)-p-Menth-2-en-1-ol (E)-Nona-4,8-dimethyl-1,3,7-triene (Z)-p-Menth-2-en-1-ol Borneol Terpinen-4-ol p-cymen-8-ol

6 Compound name RI Percent α-terpineol (E)-Piperitol Thymol methyl ether Thymol Carvacrol Bicycloelemene 0.4 δ-elemene α-cubebene Ylangene α-copaene β-bourbonene (E)-Caryophyllene α-maaliene Aromadendrene Isogermacrene D Valerena-4,7(11)-diene α-humulene Epi-(E)-caryophyllene Germacrene D γ-muurolene Viridiflorene Bicyclogermacrene γ-cadinene δ-cadinene α-cadinene α-calacorene α-elemol Spathulenol Caryophyllene oxide Viridiflorol Cadin-4-en-10-ol

7 Table 4.2b Volatile components of leaf essential oil Compound name RI Percent Butyl methyl ketone 792 Composition 2.6 α-pinene Sabinene β-pinene α-terpinene p-cymene Limonene Eucalyptol γ-terpinene Terpinolene n-undecane n-nonanal Fenchyl alcohol Camphene hydrate Non-(2E)-enal Borneol Terpinen-4-ol α-terpineol Thymol methyl ether Deca-(2E,4E)-dienal δ-elemene α-copaene β-bourbonene β-elemene n-dodecanal (E)-Caryophyllene α-(e)-bergamotene α-maaliene Aromadendrene (E)-β-Farnesene

8 Compound name RI Percent α-humulene 1459 Composition Epi-(E)-Caryophyllene γ-muurolene α-muurolene γ-cadinene δ-cadinene α-cadinene α-calacorene 1544 α-elemol Spathulenol Caryophyllene oxide Viridiflorol Humulene epoxide II Epicubenol Cadin-4-en-10-ol Farnesyl acetate n-hexadecanoic acid Results and discussion The chemical compositions of the essential oil from stem bark are summarized in Table 4.2a and that of leaf essential oil are given in Table 4.2b. A total of 55 components were identified in the bark essential oil accounting for 99.65% of the total oil. The sesquiterpene hydrocarbon β- bourbonene was found to be the major component (29.4%) followed by β- pinene (15.3%), sabinene (12.6%) and bicyclogermacrene (3.5%). Spathulenol (29.5) was the major component in the case of leaf essential oil out of the 47 compounds identified. The other main constituents present are α- Terpineol (21.5%), cadin-4-en-10-ol (7.5%) and (E)-caryophyllene (3.1%). The structures of the components are given in appendix. 125

9 3. ESSENTIAL OIL COMPOSITION OF THE STEM BARK OF PITTOSPORUM NAPAULENSE Plants belonging to P. napaulense are shrubs or small trees. Leaves are 5-20 X 2-8 cm, apex acute to acuminate, margin entire or slightly wavy, glabrous; petioles 1-2 (-3) cm long. Inflorescence terminal, paniculate or compoundly corymbose-paniculate, usually brown pubescent; pedicels 7 10 mm. Flowers are 6-8 mm; sepals mm long, broadly ovate or oblong and celiate. Petals 6-7 X 2 mm, narrowly oblong, apex obtuse. Stamens: filaments 3 mm long, anthers 1.5 mm long. Ovary brownish pubescent; style glabrous. Capsules 6-8 mm, globose. Seeds 4-8 per capsule. They are distributed throughout India except Rajasthan and also in different areas of Southern Asia. The bark of the species is bitter, aromatic and valued as a medicine. The local inhabitants in the North India apply the grounded paste of the bark to inflamed and rheumatic swellings. The bark is reported to possess expectorant, febrifuge and narcotic properties and is used as a remedy for chronic bronchitis. In subtropical regions of west Himalaya, the species is also used as a fuel and fodder Present work The chemical composition of the essential oil from the stem bark of P. napaulense was studied using GC-MS analysis Materials and Methods The stem bark of P. napaulense was collected from Kakkayam, Kozhikode in the month of February It was authenticated by Dr. A.K. Pradeep, Department of Botany, University of Calicut. 126

10 4.3.3 Isolation of essential oil Fresh stem bark (125 g) was cut into very small pieces. It was then hydro distilled for 4 hours in a Clevenger s apparatus according to the procedure described in the Ayurvedic Pharmacopoeia of India, part 1, volume 6 6 using diethyl ether as the collecting solvent. The solvent was carefully removed, yielding clear aromatic oils. The oil yield (w/w) was 0.02% on a fresh weight-basis Sample Preparation Ca. 50µL of essential oil was transferred to a large neck 2mL vial. It was diluted in 450mL of n-hexane. 50µL of nonane which is used as internal stantard was also added. The concentration of the solution for analysis was ppm. 20 µl of this solution was then injected on to an apolar column with an MS detector for compound identification Gas Chromatography-Mass Spectrometry (GC-MS) analysis For GC/MS measurements a GC-17A with QP5000 (Shimadzu) and Compaq-ProLinea data system (class 5k-software), a GC-HP5890 with HP5970-MSD (Hewlett-Packard, USA) and ChemStation software on a Pentium PC (BoÈhm, Austria), a GCQ (Finnigan-Spectronex, Germany- USA) and Gateway-2000-PS75 data system (Siemens-Nixdorf, Germany, GCQ software) were used. The carrier gas was helium; injector temperature, 250 C; interface-heating at 300 C, ion-sourceheating at 200 C, EI-mode was 70 ev, and the scan-range was 41± 450 amu. The temperature programme was: 40 C/5 min to 280 C/5 min, with a heating rate of 6 C/min. The columns were 30 m X 0.32 mm bonded FSOT-RSL-200 fused silica, with a film thickness of 0.25 mm (Biorad, Germany) and 30 m X 0.32 mm bonded Stabilwax, with a film thickness of 0.50 mm (Restek, USA). Quantification was achieved using peak area calculations, and compound identification was partly carried out using correlations between retention time 7, 8 and online Wiley, NBS and NIST library spectra. 127

11 Table 4.3a Volatile components of bark essential oil Compound name LRIexp Percent Composition n-octane Butyl methyl ketone n-octanal Limonene n-undecane n-decanal Decyl alcohol n-tridecane n-undecanal Undecanol Undecanoic acid β-acoradiene Dodecanol Tridecanal n-dodecanoic acid Ethyldodecanoate Tetradec-(9Z)-enal Dodecyl acetate n-tetradecanal n-tetradecanol Tetradecanoic acid Ethyltetradecanoate n-hexadecanoic acid Results and discussion The hydrodistillation of stem bark of P. napaulense gave oil in 0.9% (w/v) yield, based on the dry weight of the plant. Twenty three components were identified representing 97.62% of the total oil. The essential oil compositions are presented in Table 4.3a where compounds are listed in order 128

12 of their elution from the column. The major constituents of the oil were n- tetradecanal (60.1%), n-dodecanoic acid (6.4%), butyl methyl ketone (4.3%) and β-acoradiene (0.3%). 129

13 4. CONCLUSION Aromatic and medicinal plants produce a wide variety of volatile terpene hydrocarbons and their corresponding oxygenated derivatives known as essential oils. The essential oils have been widely used in traditional medicine due to their antibacterial, antifungal, immunomodulatory, antiinflammatory, and antirheumatic activities. The major volatile terpenes identified from the two species studied in this section, are also recognized for their medicinal properties. The antimicrobial activity of Satureja species is accounted to the presence of compounds such as linalool, spathulenol, camphor, cis-piperitone oxide, menthone and thymol with reported antimicrobial activity 10. Also the terpenes Germacrene-D, δ-elemene and β-bourbonene are found to posess antiectoparasitic activity 11. β-bourbonene being the major essential oil component of stem bark oil of P dasycoloun, the potential for its development as a general acarine repellent for use in animal husbandry systems may be investigated. Chemical fungicides are known to be highly effective to control the post-harvest diseases in various vegetables and fruits. However, they are not considered as long-term solutions due to the concerns associated with exposure risks, health and environmental hazards, residue persistence, and development of tolerance. Essential oils are made up of many different volatile compounds that have been shown to possess antimicrobial and fungicidal properties. The compounds caryophyllene, α-humulene, β-elemene 1-octen-3-ol, β -bourbonene, α-pinene, caryophyllene oxide, camphene and limonene were found to significantly inhibit the growth many fungal organisms 11. α-pinene, camphene, sabinene, β-pinene and limonene have been reported to be typical of the defensive secretions of some Australian and 130

14 NewZealand tenebrionids 12. Also δ-cadinene, one of the components that are present in appreciable amounts in the plants studied in this section, is reported to contribute to defense of the cotton plant against herbivory and infectious disease 13. Therefore the essential oils from the two Pittosporum species, due to the presence of compounds with impressive biological activities, can find application in agriculture as biologically safe antifungal and insect repellant agents. More field research has to be undertaken in this regard. 131

15 REFERENCES 1. Nayar MP, Giri GS (1980) Fascicles of Flora of India, Facsicle 6 Pittosporaceae, Botanical Survey of India: Feng C, Li BG, Gao XP, Qi HY, Zhang GL (2010) A new triterpene and an antiarrhythmic liriodendrin from Pittosporum brevicalyx. Arch Pharm Res 33(12): Cragg GM, Newman DJ, Yang SS (2006) Natural product extracts of plant and marine origin having antileukemia potential. J Nat Prod 69: Chou TH, Chen IS, Hwang TL, Wang TC, Lee TH, Cheng LY, Chang YC, Cho JY, Chen JJ (2008) Phthalides from Pittosporum illicioides var. illicioides with inhibitory activity on superoxidegeneration and elastase release by neutrophils. J Nat Prod 71: Bhatnagar SS, Santapau H, Desa JD, Mamar AC, Ghadielly NC, Solomon MJ (1961) Biological activity of Indian medicinal plants: Part I. anti-bacterial, anti-tubercular and anti-fungal action. Indian J Med Res 49: Anonymous (2008) Ministry of health and family welfare, Department of Ayurveda, Yoga & Naturopathy, Unani,Siddha and homeopathy (AYUSH) first edition: Davies NW (1990) Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicone and Carbowax 20M phase. J Chromatogr 503: Jennings W, Shibamoto T (1980) Qualitative analysis of flavor and fragrance volatiles by glass capillary gas chromatography. Academic Press, New York 132

16 9. Dhar U, Upreti J, Bhatt ID (2000) Micropropagation of Pittosporum napaulense (DC.) Rehder & Wilson - a rare, endemic Himalayan medicinal tree. Plant Cell, Tissue and Organ Culture 63: Vagionas K, Graikou K, Ngassapa O, Runyoro D, Chinou I(2007) Composition and antimicrobial activity of the essential oils of three Satureja species growing in Tanzania. Food Chemistry 103: Birkett MA et al (2008) Antiectoparasitic activity of the gum resin, gum haggar, from the East African plant, Commiphora holtziana. Phytochemistry 69: Hossain MA, Ismail Z, Rahman A, Kang SC (2008) Chemical composition and anti-fungal properties of the essential oils and crude extracts of Orthosiphon stamineus Benth. Industrial Crops And Products 27: Wang YH, Essenberg M (2010) Inhibitor and substrate activities of sesquiterpene olefins towards (+)-d-cadinene-8-hydroxylase, a cytochrome P450 monooxygenase (CYP706B1). Phytochemistry 71 :

17 APPENDIX OH α-pinene β-pinene Borneol OH OH Camphene Camphene hydrate Fenchyl alcohol β-myrcene β-ocimene α-phyllandrene 134

18 β-phyllandrene Limonene p-cymene α -Terpinene γ-terpinene Terpenolene OH HO OH α-terpineol Terpinen-4-ol trans- Piperitol OH p-cymen-8-ol Sabinene α-thujene 135

19 OH OH OMe Carvacrol Thymol Thymol methyl ether O Eucalyptol Humulene β-caryophyllene O O Humulene epoxide II Caryophyllene oxide Valancene α-copaene Ylangene Bicycloelemene 136

20 β-elemene δ-elemene OH α-elemol trans-α-bergamotene α-santalene β-epi-santalene Curcumene β-sesquiphyllandrene α-cadinene γ-cadinene δ-cadinene α-calacorene 137

21 α-cubebene α-bisabolene β-bisabolene OH OH Epicubenol Cadin-4-en-10-ol β-bourbonene α-acoradiene β-acoradiene Valerena-4,7(11)-diene Germacrene D Isogermacrene D Bicyclogermacrene 138

22 OH α- Maalien Spathulenol Viridiflourene O H O Viridiflorol O Farnesyl acetate β-farnesene 139