ISOLATION AND CHARACTERIZATION OF n-DOCOSANE FROM HEARTWOOD OF BERBERIS ARISTATAHTML Full Text
ISOLATION AND CHARACTERIZATION OF n-DOCOSANE FROM HEARTWOOD OF BERBERIS ARISTATA
Deepti Katiyar*1, Ramesh Kr. Singh 1, Shobha Singh 1, Divyash Singh 2 and Vijender Singh3
Deptartment of Pharmacy, Rameshwaram Institute of Technology & Management, Sitapur Road, Lucknow (UP), India
Sherwood College of Pharmacy, Barabanki (UP), India
- V. Northland Institute, Greater Noida (UP), India
A considerable number of studies have already been performed involving the roots and stems of Berberis aristata and thus the following investigation has been carried out for the phytochemical study of ethanolic extract of heartwood of Berberis aristata. The heartwood (1.8 kg) of Berberis aristata DC. Var aristata (Berberidaceae) was exhaustively extracted in 95% ethanol using Soxhlet apparatus. The column was prepared for isolating various phytoconstituents using solvents of increasing polarity. On the basis of various spectral data analysis i.e., IR, 1HNMR, 13CNMR and +ve ion FAB MS, one of the isolated phytoconstituents was characterized as an aliphatic hydrocarbon, n-docosane.
INTRODUCTION: Berberis aristata DC var. aristata (Berberidaceae), is an erect, glabrous, spinescent, deciduous shrub, 3-6m in height 1 with obovate to elliptic, subcute to obtuse, entire or toothed leaves, yellow flowers in corymbose racemes and oblong-ovoid to ovoid, bright-red berries 2, found in Nepal, grown in Nilgiris at an altitude of 1000-2400m and all our temperate Himalayas at an altitude of 1000-3000 m 3.
Berberine (responsible for hepatoprotective activity), the chief alkaloidal constituent and others including berbamine, aromoline, palmatine, oxycanthine and oxyberberine have also been isolated from roots and stem bark 1, 4. Calumbamine, umballiatine, jatrorrhizine and hydrastine have been studied in Berberis aristata roots and bark. Fruits contain citric acid and malic acid.
E-caffeic acid, quercetin, chlorogenic acid, meratin, rutin have been isolated from the flowers of Berberis aristata 5, 6. New protoberberine alkaloids- karachine and taxilamine have been isolated and characterized 7. Anti-carcinogenic 3, anti-diarrhoeal 7, anti-hepatotoxic 8, 9, anti-inflammatory 8, anti-microbial 10, anti-pyretic11, anti-hyperglycaemic 12, antioxidant 12, 13, anti-malarial 14, immunomodulatory15 and tuberculostatic 16 activities have been studied on various parts of Berberis aristata DC. Most of the studies have been done on roots and stems of Berberis aristata, so the present study has been carried out on the phytochemical investigation of ethanolic extract of heartwood of Berberis aristata.
MATERIAL AND METHOD: All melting points were determined in Centigrade scale in one- end open capillary on Perfit melting point apparatus and are uncorrected. IR spectra were recorded on Perkin Elmer spectrum RX 1 model. 1H and 13C-NMR spectra were scanned on Bruker DRX-300 NMR (300MHz) instrument in CDCl3 and D2O using Tetramethylsilane (TMS) and CDCl3 as the internal standard and coupling constants (J values) are expressed in hertz (Hz).Mass spectra were recorded by affecting electron impact ionization at 70 eV on a Jeol SX-102 (FAB) mass spectrometer equipped with direct inlet prob system. The m/z values of the more intense peaks are mentioned and the figures in bracket attached to each m/z values indicated relative intensities with respect to the base peak.
The solvents used were of Qualigens LR grade. Silica gel (Qualigen 60-120 μm mesh) was used for column chromatography. TLC was performed on plates coated with silica gel G (Qualigen). Anhydrous sodium sulphate was used for drying all the solvents used during the research work.
Plant Material: The plant material was procured from AIMIL Pharmaceuticals, New Delhi. It was authenticated as Berberis aristata by Dr. M.P. Sharma, Reader, Department of Botany, Jamia Hamdard, New Delhi and a voucher specimen is preserved in the herbarium section of Department of Pharmacognosy, R.I.T., Greater Noida, Uttar Pradesh.
Extraction: The plant material (1.8kg) was air dried crushed to smaller pieces, redried, coarsely powdered and was then exhaustively extracted with ethanol (95%) in a Soxhlet Appratus for 72 hours. The ethanolic extract was dried and dark brown mass, 50gm (2.77%) was obtained.
Preparation of Slurry: The concentrated extract of the drug was taken in a china dish and heated continuously on a water bath, gradually adding methanol in small portions with constant stirring till desired consistency was obtained. Weighed quantity of silica gel (60-120 mesh) was added slowly with mixing with a stainless steel spatula until a desired consistency was obtained. It was dried in air; the larger lumps were broken-up and finally passed through a sieve (No. 8) to get a uniform particle size.
Packing of Column: The lower end of a clean dry column was plugged with adsorbent cotton. The column was then half filled with petroleum ether. Silica gel was added in small proportions and allowed to settle down gently until the necessary length of the column was attained. All the air bubbles were allowed to escape by running the column blank thrice with solvent. The dried silica gel slurry of the extract was packed in the column and plugged with the adsorbent cotton and then eluted successively in the order of increasing polarity with different solvents.
The development and elution of the column was carried out with successive series of solvents in various combinations, viz., petroleum ether, chloroform in petroleum ether (0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%) chloroform(100%), and methanol in chloroform. The fractions collected were subjected to thin layer chromatography. Chromatographically identical fractions were combined and concentrated.
Isolation of Phytoconstituents: Elution of the column with chloroform afforded a colourless mass of a compound which was recrystallized from acetone: methanol (1:1), yield 100mg (0.20%).
Rf: 0.92 (Benzene: Chloroform, 1:1)
Melting point: 1200 C-1210C
IR υmax (KBr): 3020, 2925, 2854, 2360, 1437, 1215, 1039, 929, 762 cm-1.
1HNMR (CDCl3): δ 1.55(4H, brs, 2 × CH2), 1.33(2H, brs, CH2), 1.30(2H, brs, CH2), 1.28(2H, brs, CH2), 1.25(30H, brs, 15 × CH2), 0.89(3H, t, J=6.4 Hz, Me-1), 0.86(3H, t, J=6.8Hz, Me-22).
13C NMR (CDCl3): δ 32.81(CH2), +ve ion FAB MS m/z(rel. int.): [M]+ (C22H46) (21.3), 253 (32.8), 239 (33.2), 225 (16.7), 211 (22.4), 197 (23.7), 183 (24.5), 169 (26.9), 155 (33.6), 141(35.8), 127 (61.0), 113 (63.5), 99 (40.1), 85 (73.2), 29.69(17×CH2), 29.36(CH2), 22.36(CH2), 14.17(CH3), 14.16(CH3).
RESULT AND DISCUSSION: The compound, an aliphatic hydrocarbon, was obtained as a colourless mass from chloroform eluants. It did not decolorize bromine water and there was no reaction of the acetylating and oxidizing agent indicating saturated nature of the molecule devoid of any alcoholic group. Its IR spectrum exhibited absorption band for long chain hydrocarbon at 762 cm-1.
The absence of any distinctive band in the range 3500-3200 cm-1 and 1750-1600 cm-1 suggested the saturated nature of the molecule without hydroxyl and carbonyl groups. The mass spectrum had a molecular ion peak at m/z 310 consistent with the molecular formula of saturated hydrocarbon, C22H46, a large number of ion fragments recorded in its mass spectrum with a uniform difference of 14 mass units supported the presence of a long aliphatic chain of the molecule. The absence of [M- Me]+ ion peak indicated the straight chain nature of the molecule.
The mass spectrum had ion fragments relating to CnH2n+1, CnH2n and CnH2n-1 and in higher abundance for lower fragments indicating long chain of the molecule. The most prominent ion fragments relating to CnH2n+1 indicated saturated nature of the molecule. On the basis of the spectral data analysis and chemical reaction the structure of the compound has been established as n-docosane. The results are shown in different figures as spectras of IR, 1HNMR, 13C NMR and Mass spectra n-Docosane.
- MASS SPECTRA OF n-DOCOSANEREFERENCES:
- Sharma PC, Yelne MB, and Dennis TJ: Database on Medicinal Plants used in Ayurveda 1, Central Council for Research in Ayurveda and Siddha, New Delhi 2002:120-122.
- Anonymous: The Wealth of India 2: B, Council of Scientific and industrial Research, New Delhi 1998: 114-118.
- Anonymous: Quality Standards of Indian Medicinal Plants 3. Indian Council of Medical Research, New Delhi 2005:78-87.
- Jha NK: Berberis aristata: Daruharidra: Indian Barberry. Phytopharm 2004:5:3-11.
- Rastogi RP, Mehrotra BN: Compendium of Indian Medicinal Plants 5, Central Drug Research Institute, Lucknow and National institute of Science communication, New Delhi 2005:126-128.
- Anonymous: Reviews on Indian Medicinal Plants 4, Indian Council of Medical research, New Delhi 2004:150.
- Rastogi RP, Mehrotra BN: Compendium of Indian Medicinal Plants 5, Central Drug Research Institute, Lucknow and National Institute of Science communication, New Delhi 2005:126-28.
- Fukuda K et al: Inhibition by Berberine of cyclooxygenase-2 transcriptional activity in human colon cancer cells. Journal of Ethnopharmacology 1999:66:227-33.
- Janbaz KH et al: Studies on preventive and curative effects of Berberine. Fitoterapia 2000:71: 25-33.
- Freile ML et al: Antimicrobial activity of aqueous extracts and of Berberine isolated from Berberis heterophylla. Fitoterapia 2003:74:702-05.
- Yesilada E et al: Berberis crataegina Root exhibits potent anti-inflammatory, analgesic and febrifuge effects in mice and rats. Journal of Ethnopharmacology 2002:79:237-248.
- Singh J, Kakkar P: Antihyperglycemic and antioxidant effect of Berberis aristata root extract and its role in regulating carbohydrate metabolism in diabetic rats. Journal of Ethnopharmacology 2009:123:22-26.
- Tiwari BK, Khosa RL: Evaluation of the hepatoprotective and antioxidant effect of Berberis asiatica against experimentally induced liver injury in rats. International Journal of Pharmacy and Pharmaceutical Sciences 2010:2:92-97.
- Chopra RN, Nayar SL, Chopra IC: Glossary of Indian Medicinal plants, National institute of Science communication and Information Resources New Delhi 2002: 36.
- Sohni YR, Bhatt RM: Activity of a crude extract formulation in experimental hepatic amoebiasis and in immunomodulation studies. Journal of Ethnopharmacology 1996:54:119-24.
- Soffar SA et al: Evaluation of the effect of a plant alkaloid (Berberine derived from Berberis aristata) on Trichomonas vaginalis In-Vitro. Journal of Egypt Soc Parasitol 2001:31:893-904.