PHYTOCHEMICAL STUDIES ON CAPSICUM FRUTESCENS

PHYTOCHEMICAL STUDIES ON CAPSICUM FRUTESCENS

Mohammad Golam Dastagir ¹, Mohammad Musarraf Husaain*², A.H.M. Masum Billah ¹, Md. Ismail ¹, Abdul Quader ¹

Department of Chemistry, University of Dhaka ¹, Dhaka-1000, Bangladesh

Department of Pharmacy, Jagannath University ², Dhaka-1100, Bangladesh

ABSTRACT

Two chemical compounds have been isolated and their structures were elucidated by spectroscopic studies including VLC, TLC and NMR. The isolated compounds are Ortho-hydroxy-N- benzyl- 16- Methyl- 11, 14- diene-octadecamide (1) and 9, 12-diene-octadecanoic acid (2).

Ortho-hydroxy-N-benzyl-16-Methyl-11, 14-diene-octadecamide  and 9, 12-diene-octadecanoic acid

INTRODUCTION: Capsicum frutescens (Family: Solanaceae) is an annual or short-lived perennial herb. It grows well all over in Bangladesh. It’s local name is morich or lanka. Probably native of the tropics, but the original habitat is obscure. The stem of Capsicum frutescens almost striate, glabrous, highiet between 1-4 feet depending on climate and growing conditions. The leaves are elliptical, slightly leathery, dark green and smooth, and measure 2^½ inches long and 1 inches wide.

The flowers are typically conical or funnel form with five petals, usually fused and color is white. The fruits are erect, ellipsoid-conical to lanceoloid, 10-20 mm long, 3-7 mm in diameter. These   fruits have range in color from green when immature to purple, red, orange or yellow when ripe ^{1, 3}. A few compounds had been isolated from C. frutescens like ester, terpenoids, noncarotenoids, lipoxygenase derivatives, carbonyls, alcohols, hydrocarbons 8, capsaicin, dihydrocapsaicin ⁴, capsiconinoid ¹¹, capsinoid ⁹. Medicinally, C. frutescens has been used to treat diabetes mellitus by traditional healers in Jamaica ¹², decrease fat intake ⁵, decrease plasma glucose level ², as antifungal agents ¹⁰, as cosmetics & food supplement, effective treatment for psoriasis ⁷.

METHODS AND MATERIALS: Collection of plant materials: Two kilogram morich bought from Anondabazar, near Dhaka university campus in March, 2006. The fruit of Capsicum frutescens was identified   by Professor Dr. Abul Hasan of the Department of Botany, University of Dhaka. The seeds of C. frutescens were separated manually from skin and dried in air. The weight of the seeds was 700 gram & seeds were ground by using a cyclotech grinding machine. A voucher specimen for this collection has been maintained in Bangladesh National Herbarium (DACB accession number 35444) Dhaka, Bangladesh.

Extraction of the powdered seeds: The powdered seeds extracted in a soxlet apparatus with petroleum ether (40^oC- 60^oC) solvent. At first the dried powder of the seeds taken in a number of precleaned cloth bags. Each bag (150 g) placed in the soxhlet apparatus and then solvent (petroleum ether) added. This was subjected to extraction by refluxing for several hours until the material extract appeared light yellowish color. The extract was filtered and concentrated under reduced pressure using a rotary vacuum evaporator.

Analysis of the Concentrated Extract: The petroleum ether extract (40^oC- 60^oC) was examined by TLC. Its TLC examination showed (solvent system: 86% petroleum ether and 14% ethyl acetate) four distinct spots along with light yellow tailing. The mixture of the compound was separated by VLC using suitable solvent.

Fractionating the Extract: Fractions obtained by VLC technique: The effluent collected in 191 (each 25ml) test tubes. The effluent of each test tube was monitored by TLC over silica gel (G-60, PL254). The effluent of similar behavior were poured into a small conical flask and thus designated as PEE1,   PEE2,   PEE3,   PEE4,   PEE5,   PEE6,   and PEE7.

Analysis of the Designated Fractions: On concentration and keeping undisturbed at room temperature the fractions  PEE_2,   PEE_3,   PEE_4,   PEE_5, gave a light yellow gummy material (63mg) & then this gummy material washed with petroleum-ether solvent. It was dissolved in chloroform and spotted on the TLC plate. The plate developed in 86% petroleum-ether and 14% ethyl acetate. When the solvent front reached just one cm below the upper edge of the plate taken out from the chamber and dried at room temperature in regular air flow until solvent was evaporated from the plate. Then, the vanillin- sulphuric acid reagent was sprayed on the plate and the plate was heated at 106^oC for 10 minutes. One spot was observed and then fractions   were concentrated by vacuum rotavapour to yield gummy mass (15 mg).

The fractions PEE_6, PEE₇ gave white residue. It was collected and washed with water, petroleum-ether, and n-hexane respectively. This material also dissolved in chloroformand spotted on TLC plate (over silica gel). The plate was developed in 86% petroleum-ether and 14% ethyl acetate. When the solvent front reached just one cm below the upper edge of the plate, the plate was taken out from the chamber and dried at room temperature in regular air flow until solvent was evaporated from the plate then the vanillin - sulphuric reagent was sprayed on the plate and then plate was heated at 106^o for 5 min. One pink spot was observed at the middle on the plate. This fraction was concentrated by hot air blow to yield oily mass (amount: 7mg).

Properties of the Isolated Compounds:

Properties of Compound -1:

Physical state: Gummy; Solubility: Sparingly soluble in petroleum-ether but highly soluble in chloroformand ethyl acetate; R_f value: 0.81 (solvent mixture: Petroleum-ether and ethylacetate).

Spectral Characteristics:

IR Spectral data (cm^-1):3400(N—H), 3000, (C-H attached to >C=C<), 1750 (-CONH₂), 1640 (>C=O), 1450-720(-CH₂-), 1100-1240 (C—O), 1360 [(CH₃)₂CH-].

¹H NMR Spectrum (400MHz, CDCl3, TMS):  δ_H value (ppm) vs indication; (7.1-6.8 , substituted aromatic ring protons), 5.3(d, J=6Hz,olefenic protons), 4.3(dd, J=10Hz,-CH₂=CH₂-); 4.14(J=6Hz,-CH₂=CH_2-), 2.8(t, -CH₂-next to>C=C<), 2.08(d, J=7Hz, -CH₃),  1.6 (s,-CH₃)_, 0.092[br, (CH₃)₂CH-].

¹³C spectrum: δ_c value (ppm) vs indication; 173(>C=O), 172(olefenic carbon or aromatic carbon or both), 86.45(quarternary carbon),68.8-62(oxygenated carbon), 13.9-34.07(>CH₃ or -CH₂-).

Properties of Compound -2:

Physical state: Oily, Solubility: Sparingly soluble in petroleum-ether but highly soluble in chloroformand ethyl acetate, R_f value: 0.83 (solvent mixture: Petroleum-ether and ethyl acetate).

Spectral Characteristics:

IR Spectral data (cm^-1): 2900 (N—H), 1700(>C=O in -COOH), 1650(>C=C<),1450 (-CH₂-or -CH₃).

¹H NMR Spectrum (400MHz, CDCl₃, TMS): δ_Hvalue (ppm) vs. indication; 5.3(dd, J=6Hz,olefenic protons), 2.6(dd, J=10Hz,-CH₂-CH₂-next to >C=C<),2.3(d, J=7Hz,-CH₂ attatched to -COOH), 1.2 -1.6(s, CH₂-),0.87(t,-CH₃ next to -CH_2-).

¹³C spectrum: δ_c value (ppm) vs. indication; 127-130(olefenic carbon), 25.5(-CH₂-joined two >C=C<), 27(-CH₂- adjacent to >C=C<), 33.8(-CH₂-adjacent to >C=O), 14(-CH₃),179(C-O), 27-31(-CH₂-).

RESULTS AND DISCUSSION:

Spectral Characteristics of Compound -1:

IR spectrum: Its IR spectrum showed a sharp peak at 3400cm^-1 indicative the presence of -NH group. Another peak at 1750cm^-1 along with a band at 1640cm^-1 was indicative of the presence of -CONH₂ and >CO groups(R-CONH-).The band at 1450cm^-1 together with the band at 720cm^-1 was suggestive of the presence of -CH₂- group. The bands at 1100 cm^{-1 to} 1240cm^-1were exhibitive of C-O stretching. The band at 1360cm^-1 along with a shoulder band 1340cm^-1 was indicative of the presence of isopropyl [-CH-(CH₃)₂] group. A sharp peak just above 3000cm^-1 was demonstrative of   H-C=C- group i.e., C-H stretching attached to a >C=C< bond.

¹H NMR spectrum: Its ¹H NMR spectrum showed multiplexes within δ_H value; 7.13-6.8 indicative of the presence of substituted aromatic ring protons, 4.1-5.3 were indicative of the presence of vinyl hydrogen, 1.6 to 2.6 were indicative of the presence of allylic hydrogen i,e hydrogen attached to a carbon adjacent to double bond ¹³. A broad singlet at δ_H value 1.2 was indicative of the methyl group, doublet at 2.07 was the indicative of -CH-CO-NH-. A 6H intensity broad singlet at0.92 was indicative of [(CH₃)₂-CH-] group.

¹³C spectrum: The ¹³C spectrum and DEPT ¹³C spectrum showed two >C=O carbons at173 and 172. The δ_c at 128.1, 128.7, 127.8, 125 were indicative of aromatic carbons. A tiny signal at 86.45 showed the presence of quaternary carbon. The signals at 68.8 and62 were indicative of oxygenated carbon. Moreover, the ¹³C spectrum showed 18 signals within δ_c value 34.07, 13.97 were indicative of the presence of either -CH₃ or -CH₂- carbon or both types of carbons.

Inference: Based on the IR, ¹H NMR and ¹³C NMR spectral data, the compound-1 has been proposed to have the following structure.

Compound-1: Ortho hydroxyl-N benzyl-16- methyl-11, 14- diene-octadecamide

Spectral Characteristics of Compound -2:

IR spectrum: Its IR spectrum showed a sharp peak at 2900cm^-1 was indicative of the presence of C-H stretching. A peak at 1700cm^-1 was suggestive of >C=O stretching in -COOH group. Another peak at 1650cm^-1 was indicative of the presence of >C=C< group. A sharp peak at 1450cm^-1 was indicative of the presence of-CH₂- or -CH₃ group.

¹H NMR spectrum: Its ¹H NMR spectrum showed doubletat 5.3 indicative of the presence of olefenic protons. Another doublet of doublet at2.6 was indicative of the presence of -CH₂- protons which joined two >C=C< moieties. A broad peak at2.0 was suggestive of -CH₂- protons next to a >C=C< moiety. A triplet at2.3 was indicative of the presence of -CH₂- protons attached to the -COOH group. Another triplet at 0.87 was suggestive of -CH₃ group next to a -CH₂- group. The peaks from 1.2-1.6 were indicative of the presence of -CH₂- protons.

¹³C NMR spectrum: The ¹³ C spectrums and DEPT spectrum showed four olefenic carbons at127.8-130.1.At25.5 was indicative of the presence of -CH₂- carbon which joined two >C=C< moieties. A signal at 27 was suggestive of -CH₂- carbon adjacent to >C=C< group. Another signal at14 was suggestive of terminal -CH₃ carbon. A tiny signal at 33.8 was indicative of the presence of -CH₂- carbon adjacent to >C=O group. Another tiny signal at 179 was suggestive of oxygenated carbon. Moreover, this ¹³C spectrum showed -CH₂- carbon at27-31.

Inference: Based on the IR, ¹H NMR and ¹³C NMR spectral data, the compound-2 has been proposed to have the following structure.

Compound-2:  9, 12-diene-octadecanoic acid ⁶

ACKNOWLEDGEMENT: Authors are highly acknowledged to Mr. Mohammad Musarraf Hussain, M. Pharm., research student, 2003, Department of Pharmaceutical Chemistry (Research Lab: Phyto-chemistry Research Laboratory), Faculty of Pharmacy, University of Dhaka, for his kind cooperation & help.

CONCLUSION: The chemical study of the stem bark of Capsicum frutescens afforded two purified compounds Ortho hydroxy- N benzyl- 16- methyl- 11, 14- diene-octadecamide (1) and 9, 12-diene-octadecanoic acid (2) whose structures were established by extensive spectroscopic studies as well as comparison with published results.

REFERENCES:

1. Abdul Ghani, Medicinal Plants of Bangladesh with chemical constituents and uses; Asiatic Society of Bangladesh, Second Edition, 2003, 300.
2. Chaiyasit K, Khovidhunkit W, Wittayalertpanya S; Pharmacokinetic and the effect of capsaicin in Capsicum frutescens on decreasing plasma glucose level; Journal of Medical Association Thailand; 2009; 92(1):108-13.
3. Kirtikar KR., Basu BD. Indian Medicinal Plants, Published by Singh B and Singh M P, India; 1980, 2nd edition, Vol-1, 800-805
4. Liu Y, Nair MG; Capsaicinoids in the hottest pepper Bhut Jolokia and its antioxidant and antiinflammatory activities; Nat Prod Commun. 2010; 5(1):91-4.
5. Mayumi Yoshioka, Makoto Imanaga, Hiromi Ueyama, Miya Yamane, Yoshiko Kubo, Andre Boivin, Jonny St- Amond, Hiroaki Tanaka, Akira Kiyonaga; Maximum tolerable dose of red pepper decreases fat intake independently of spicy sensation in the mouth; British Journal of Nutrition; 2004; 91, 991-995.
6. Pérez-Gálvez, Antonio; Garrido-Fernández, Juan; Mínguez-Mosquera, Ma.Isabel; Lozano-Ruiz, Mercedes; Montero-de-Espinosa, Vicente; Fatty acid composition of two new pepper varieties (Capsicum annuum L. cv. Jaranda and Jariza). Effect of drying process and nutritional aspects; Journal of American Oil Chemists’ Society 1999; 76 (2), 205-208.
7. Reuter J, Wölfle U, Weckesser S, Schempp C; Which plant for which skin disease? Part 1: Atopic dermatitis, psoriasis, acne, condyloma and herpes simplex; J Dtsch Dermatol Ges; 2010; 8(10):788-96.
8. Rodríguez-Burruezo A, Kollmannsberger H, González-Mas MC, Nitz S, Fernando N;  HS-SPME comparative analysis of genotypic diversity in the volatile fraction and aroma-contributing compounds of Capsicum fruits from the annuum-chinense-frutescens complex; Journal of Agricultural Food Chemistry, 2010; 58(7):4388-400.
9. Singh S, Jarret R, Russo V, Majetich G, Shimkus J, Bushway R, Perkins B; Determination of capsinoids by HPLC-DAD in capsicum species; Journal of Agricultural Food Chemistry; 2009; 57(9):3452-7.
10. Stergiopoulou T, De Lucca AJ, Meletiadis J, Sein T, Boue SM, Schaufele R, Roilides E, Ghannoum M, Walsh TJ; In vitro activity of CAY-1, a saponin from Capsicum frutescens, against Microsporum and Trichophyton species. Medical Mycology; 2008; 46(8):805-10.
11. Tolan, I; Ragoobirsing, D; Morrison, E.Y; The effect of capsaicin on blood glucose, plasma insulin levels and insulin binding in dog models; Phytotherapy Research,2001, 15(5), 391-394.
12. Tanaka Y, Hosokawa M, Otsu K, Watanabe T, Yazawa S; Assessment of capsiconinoid composition, nonpungent capsaicinoid analogues, in capsicum cultivars; Journal of  Agricultural  Food Chemistry;  2009; 57(12): 5407-12.
13. Y.R.Sharma; Elementary Organic Spectroscopy: Principles and Chemical Applications, 4th Edition, 2007; 182-190.
    

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