versión impresa ISSN 0102-695X
Rev. bras. farmacogn. vol.22 no.5 Curitiba sept./oct. 2012 Epub 03-Abr-2012
Manas Mathur; Raka Kamal*
Laboratory of Medicinal Plant Biotechnology, University of Rajasthan, India
Trigonelline (N-methylnicotinate) biosynthesized from nicotinate is one of the metabolically active pyridine alkaloid, widely distributed in plant kingdom. In the present study trigonelline has been isolated from various plant parts and callus cultures of Moringa oleifera Lam., Moringaceae, and was identified using TLC, GLC, GC-MS, which was comparable to that of the standard trigonelline. The trigonelline recovery was found to be maximum in the pods and minimum in flowers. In order to enhance the production of trigonelline in vitro grown cultures, different treatment doses of nicotinic acid (250, 500 and 750 mg L-1) were supplemented in the medium as precursor. Maximum increase (up to 1.10 fold) was observed in the treatment dose of 500 mg L-1 of nicotinic acid.
Keywords: callus culture; GC-MS; Moringa oleifera; nicotinic acid; TLC; trigonelline
Plants are regarded as molecular factory, as they have capacity to synthesize enormous diversity of metabolites termed as bioactive compounds. Alkaloids have gained attention of the biochemists because of their diversified physiological and pharmacological actions (Verpoorte et al., 1991). Trigonelline (N-methylnicotinate) (1), a pyridine alkaloid mostly reported from family Fabaceae, is biosynthesized from nicotinate (Joshi & Handler, 1960; Kogan et al., 1953; Taguchi et al., 1986). Trigonelline is metabolically active as hypocholesterolemic agent (Evans et al., 1979) and its nutritional benefits have also been well documented (Viani & Horman, 1974; Taguchi et al., 1986). It has been reported it also possess glucose lowering activity (Olthof et al., 2011).
Biosynthetic pathway of any bioactives is regarded as the key point for industrial production of any pharmaceutical, which may lead to a drastic effect at a commercial level by use of precursor (Patel, 1998). Exogenous supply of a precursor to culture medium has been advocated to increase the yield of the desired product in cell cultures (Staba, 1985). Successful attempts have been made to increase the production of metabolites by supplying precursor or intermediate compounds in vitro (Moreno et al., 1993; Whitmer et al., 1998; Silvestrini et al., 2002; Namdeo et al., 2007).
Moringa oleifera Lam. commonly called as drum stick belongs to family Moringaceae, which bears fourteen species among which M. oleifera is most commonly found. The plant is native to northern India where it was noted as medicinal plant around 2000 BC. The pods and leaves are rich in protein and minerals and cooked as vegetable (Verma et al., 1976), posses certain bioactivities such as antiurolithiatic (Karadi et al., 2008), anti-inflammatory (Sulaiman et al., 2008), antiasthmatic (Agrawal & Mehta, 2008), antioxidant and hepatoprotective (Fakurazi et al., 2008) have been reported. In literature, the presence of trigonelline was not previously recorded in M. oleifera.
The present study deals with the isolation, identification and quantitative determination of trigonelline (1) in various plant parts and tissue cultures of M. oleifera. The regulation of its production by nicotinic acid (precursor) feeding at different treatment doses was also attempted. The present investigation is the first report of trigonelline in M. oleifera.
Material and Methods
Moringa oleifera Lam., Moringaceae, (flowers, leaves, stem, pods and roots) was collected from fields at Jaipur and authenticated by Dr. N J Sarna, Department of Botany, University of Rajasthan. Plant parts were cleaned and oven dried at 35 ºC for 30 min and then at 25 ºC till constant weight was achieved and then powdered. The voucher (RUBL No. 20393, Rajasthan University Botanical Library) of experimental plant was deposited in Herbarium at Department of Botany, University of Rajasthan, Jaipur).
Standard trigonelline used were of analytical grade and obtained from Sigma- Aldrich (Germany)
Surface sterilization and inoculation
Nodal segments were surface sterilized with mercuric chloride (HgCl2) solution (0.1% w/v) for 2 min and subsequently rinsed several times with sterile distilled water and treated with antibiotic (ciprofloxacin, 250 mg L-1) prior to inoculation in order to remove any kind of microbial interactions. Surface sterilization was done in a horizontal laminar flow hood fitted with ultraviolet light. Nodal segments were inoculated in the flasks containing culture medium aseptically having combination of BAP:IBA (3:3 mg L-1), BAP: NAA (1:0.2 mg L-1)
Cultured flasks were incubated in culture chamber at the temperature 25±1 ºC and light of intensity (1200 lux) provided from fluorescent tubes and incandescent bulbs (40 watt) for 16 h. The cultures were observed and examined every week and at final morphogenetic data were recorded.
The maintained calli were harvested regularly at the transfer age of 2, 4, 6, 8 weeks. Each of the callus samples was harvested and their growth indices (GI) calculated on fresh weight basis
Five such replicates were examined in each case and average values calculated.
Powdered and weighed plant materials (flowers, leaves, stem, pods and roots) along with callus tissue were taken in 100 mL Erlenmeyer flasks added 50 mL distilled water for each g of the dried plant/tissue material (v/w ) and 5 mL of 0.05 N sulphuric acid was added to it. Mixture was macerated for 3-4 h and boiled gently for 25 min. Heavy magnesium oxide (2.5 g/g) was added to the mixture and again boiled gently for 20 min. It was cooled at room temperature and 20 mL of volume was completed to 100 mL with distilled water 10 mL of alcohol was added to remove the mucus. Mixture was filtered through Whatman filter (having diameter-12.5 cm) paper 1 (Kogan et al., 1953). Filtrate was evaporated to dryness in vacuo, reconstituted in distilled water for further analysis.
Thin layer chromatography
Extracted sample were dissolved in distilled water and applied on activated TLC plates, along with authentic samples of trigonelline. These plates were developed in presaturated chromatographic chamber containing solvent mixture of butanol:acetone:water (4:1:5) (Johnson & Lin, 1953) and isobutanol and water (85:15) of (Kogan et al., 1953). Plates were visualized under UV light and fluorescent spots were marked and then were sprayed with Dragendorff's reagent to develop coloured spot co comparable with authentic sample applied simultaneously.
PTLC (Preparative thin layer chromatography)
Preparative thin layer chromatography of isolated trigonelline samples was also carried out as above on thick Silica Gel G coated plates (500 µm thick) by spotting extract along with the trigonelline. The developed plates were compared with authentic samples under UV light. Spot coinciding with standard compound was marked and collected by scrapping silica gel G, dried, weighed (mg) and crystallized in methanol and water. Crystals were subjected to GLC and GC-MS analyses.
Gas Liquid Chromatography (GLC)
The extract and the standard samples were analyzed by GLC. A Perkin Elmer OV-17 gas chromatograph equipped with a flame ionization detector on gas Chrom Q. The operating temperature used for analysis was 260 ºC. Nitrogen was used as carrier gas at a flow rate 0.6 mL/min, chart speed 0.28 cm/min.
Gas Chromatography and Mass Spectroscopy (GC-MS)
The extract and the standard samples were analyzed by GC-MS of Hewlett-Packard 6890/5973 operating at 1000 eV ionization energy, equipped with using Agilent 7890A/5975C GC HP-5. Capillary column (phenyl methyl siloxane, 25 m×0.25 mm i.d) with Helium (He) was used as the carrier gas with split ratio 1:5. Oven temperature was 100 ºC (3 min) to 280 ºC at 1 to 40 ºC/min; detector temperature, 250 to 280ºC; carrier gas, He (0.9 mL/min). Retention indices were determined by using retention times of samples that were injected under the same chromatographic conditions. The components of the standard and plant samples were identified by comparison of their mass spectra and retention time with those given in literature and by comparison with the mass spectra of the Wiley library or with the published mass spectra.
Effect of precursor feeding
Six months old maintained calli as suspension cultures were transferred in the fresh medium supplemented with various dose treatment of nicotinic acid (250, 500 and 750 mg L-1, separately) as precursor. A control was run in parallel without supplementation of nicotinic acid. The cultures were harvested at different time intervals of 2, 4, 6 and 8 weeks of precursor treatment, cells cultures were harvested, filtered and kept at 100 ºC for 20 min to inactivate the enzymes and then at 60 ºC till constant weight was achieved and were analyzed for trigonelline content.
Data were subjected to one-way analysis of variance (ANOVA) using SPSS version 10. The significance level was determined at p=0.05. When there were significant differences, the means were separated using Duncan's multiple range test (DMRT). Differences are denoted in tables and figures by different letters. The standard error was also noted where appropriate.
Nodal segments were used for the tissue culture studies. The GI was found to be minimum in two weeks (0.75) and maximum in six weeks old tissue (3.06). Thereafter, the GI steadily declined thus showing a characteristic sigmoid curve of growth (Figure 1)
The qualitative and quantitative analysis of trigonelline from various plant parts and callus cultures of M. oleifera was carried out.
Isolated alkaloid gave single fluorescent spot on thin layer chromatographic plates developed in solvent system of butanol:acetone:water (4:1:5) under UV lamp. On spraying with Dragendorff's reagent, brick red coloured spot was observed at the Rf 0.09 which coincided with standard trigonelline.
Among various plant parts maximum trigonelline content was observed in pods (3.55 mg/g) followed by leaves (2.60 mg/g), roots (2.15 mg/g), stem (1.90 mg/g) and minimum in flowers (1.60 mg/g). Callus cultures had lower (2.38 mg/g) content than pods and leaves but higher than other plant parts (Figure 2).
The GC-MS studied showed that the retention time and peaks of the isolated trigonelline was comparable with that of standard. The active principles with their retention time (RT) and concentration (%) in the standard and sample are presented. Compounds found in standard are methyl pyridine-3-carboxylate (retention time-7.457* and 69.20% area), 1H-pyrrole-3-carboxylic acid (retention time-10.100 and 2.42% area), 1H-indole, 1,3-dimethyl (retention time-10.382 and 0.66%), 1H-pyrrole-3-carboxylic acid (retention time-10.758 and 21.90% area), hydroxy(4-nitrophenyl) acetic acid (retention time-12.034% and 5.82%) and 49 compounds are identified in alkaloid extract isolated from pods of M. oleifera along with their biological activity. Phytocompounds and its biological activities obtained through the GC-MS study are presented in Table 1 and 2.
The GLC studied showed that the retention time and peaks of the isolated trigonelline was comparable with that of standard (Figure 3).
Effect of precursor feeding
The quantification data of trigonelline obtained from different treatment doses of nicotinic acid gave maximum content of 3.27 mg/g in callus obtained from 50 mg% nicotinic acid supplementation dose, which was significantly superior to all other treatments and minimum content of 2.12 mg/gdw (gram dry weight) was observed in callus of control, which was at Par with callus of 25 mg% nicotinic acid treatment dose (2.14 mg/g). The marginal mean content in relation to time showed maximum content in six weeks old callus (2.97 mg/g), which was significantly superior to other treatments and minimum (2.12 mg/g) was in two weeks old callus (Table 3).
In the present investigation callus was raised from nodal segments on each of the combination hormonal doses, the best response was observed in combination doses of BAP: IBA (3 mgL-1, each). The presence of auxins and cytokinin in the culture medium regulates various aspects of dedifferentiation and differentiation (Woodward & Bartel, 2005; Wang et al., 2008) at cellular levels in a particular dose.
There is no uniform and clear definition of growth of plant cell cultures and dry weight or fresh weight methods have been in use for determining GI, because of its preciseness, accuracy in observing variation (Grossmann, 1988). Several workers have observed the sigmoid growth pattern of the callus culture (Staba, 1980; Endress, 1994). In the present study a sigmoid pattern of growth curve was observed in M. oleifera with maximum GI at the 6th week of subculture indicated the exponential growth phase and minimum at 2nd week of subculture. An increase in GI after supplementation of various growth regulators finds support that the growth of tissue some time depends upon the culture medium and also controlled by other factors like pH, treatment dose and combinations of growth regulators used (Barz et al., 1977; Heble, 1985; Schripsema et al., 1990).
In many plant species alkaloids get accumulated in seeds in relatively high concentration as a chemical defense. During germination alkaloids get metabolized and their nitrogen is reused for seedlings metabolism (Wink & Witte, 1985). Preliminary reports on presence of alkaloids in M. oleifera have been shown (Ramchandran et al., 1980; Dangi et al., 2002; Patel et al., 2008).
Trigonelline, serves as a storage form of nicotinic acid (Blaim & Wanner, 1960) has been reported from other plants also. Trigonelline from seeds of T. foenum-graecum (0.38%) was reported by Kuhn & Gerhard (1943). Samola & Gerhard (1983) reported (0.8% to 1.1%) trigonelline from green coffee. Kaushik & Khanna (1990) reported 0.014% from Dolichos lablab seeds and 0.0068% of trigonelline from its callus cultures.
Present study is the first report of presence of trigonelline in M. oleifera. The maximum content was found in pods (3.55 mg/g). The content in callus cultures was lower than pods and leaves and higher than rest of the plant parts. In tissue culture trigonelline has been reported from Trigonella foenum-graecum (Antony et al., 1975; Willeke et al., 1970, Radwan & Kokate, 1980), Pisum sativum and Glycine max (Tramontano et al., 1985), Trigonella polycerata (Kamal et al., 1996), Dolichos lablab (Kaushik & Khanna, 1990).
Use of precursor in the culture medium enhance end product have been advocated (Staba, 1980). Nicotinic acid acts as precursor in the biosynthesis of trigonelline (Johnson & Lin, 1953). Khanna & Jain (1972) observed an increase in growth and trigonelline in tissue cultures of Trigonella foenum-graecum fed with nicotinic acid in 8 week old callus cultures. Later, Khanna et al. (1989) reported 2 fold increase in six weeks old callus obtained from 500 mg L-1 nicotinic fed medium in Allium cepa callus cultures. In the present studies, similar results were observed in M. oleifera callus cultures, where the trigonelline content increased (1.10 fold) in six weeks old tissue fed with nicotinic acid (Figure 2 ), whereas the growth of callus decreased remarkably, which may be due to enhanced trigonelline content which checks cell proliferation by cell arrest as suggested by Lynn et al., (1984).
The present investigation is the first report of trigonelline from M. oleifera confirmed by GLC and GC-MS analyses. The biosynthesis of trigonelline was enhanced in M. oleifera by feeding the precursor nicotinic acid in its cell cultures. Our results confirm that specific plant metabolites could be modulated by exogenous precursor supplemented according to their specific metabolite pathway.
Authors are thankful to University Grant Commission, New Delhi, India for providing fellowship to one of the author (MM).
Agrawal B, Mehta A 2008. Antiasthmatic activity of Moringa oleifera:a clinical study. Indian J Pharmacol 40:28-31. [ Links ]
Antony A, Gopinathan KP, Vidyanathan CS 1975. Biosynthesis of trigonelline in root callus cultures of fenugreek (Trigonella foenum-graceum). Indian J Exp Biol 13:39-41. [ Links ]
Barz W, Reinhard E, Zenk MH (Eds.) 1977. Plant Tissue Culture and its Biotechnological Application. Springer Verlag. Berlin, Germany. [ Links ]
Blaim K, Wanner H 1960. Presence of trigonelline in seed and seedling of Melilotus officinales Naturwissenchaften 47:331-332. [ Links ]
Dangi SY, Jolly CI, Narayanan I 2002. Antihypertensive activity of the total alkaloids from leaves of Moringa oleifera. Pharm Biol 40:144-148. [ Links ]
Endress R 1994. Plant Cell Biotechnology. Springer Verlag. Germany. [ Links ]
Evans LS, Almeida MS, Lynn DG, Nakanishi K 1979. Chemical characterization of a hormone that promotes cell arrest in G2 in complex tissues. Science 203:1122-1123. [ Links ]
Fakurazi S, Nanthini U, Hairuszah I 2008. Hepatoprotective and antioxidant action of Moringa oleifera Lam. against acetaminophen induced hepatotoxicity in rats. Int J Pharmacol 4:270-275. [ Links ]
Grossmann K 1988. Plant cell suspensions for screening and studying the mode of action of plant growth retardants. Adv Cell Cult 6:89-136. [ Links ]
Heble MR 1985. Multiple shoot cultures. A viable alternative in vitro system for the production of known and new biological active plants constituents. In: Primary and Secondary Metabolism of Plant Cell Cultures. Neumann KH, Barz W and Reinard F (Eds.). Springer Verlag. Berlin, Heidelberg, New York, USA. p. 281-292. [ Links ]
Johnson BC, Lin PH 1953. Nicotinic acid metabolism I. The use of paper chromatography in the study of nicotinic acid metabolism. J Am Chem Soc 75:2971-2973. [ Links ]
Joshi IG, Handler P 1960. Biosynthesis of trigonelline. J Biol Chem 235:2981-2983. [ Links ]
Kamal R, Mehra P, Yadav R 1996. In vitro influence of nicotinic acid on production of trigonelline from Trigonella polycerata. Indian J Exp Biol 34:1147-1149. [ Links ]
Karadi RV, Palkar MB, Gaviraj EN, Gadge NB, Mannur VS, Alagawadi KR 2008. Antiurolithiatic property of Moringa oleifera root bark. Pharma Biol 46:861-865. [ Links ]
Kaushik P, Khanna P 1990. Trigonelline, a pyridine alkaloid from Dolichos lablab L in vivo and in vitro tissue cultures. Ind Drugs 28:67-69. [ Links ]
Khanna P, Jain SC 1972. Effect of nicotinic acid on growth and production of trigonelline by Trigonella foenum-graecum L. tissue cultures. Indian J Exp Biol 10:248-249. [ Links ]
Khanna P, Sharma A, Kaushik P, Chaturvedi P 1989. Trigonelline from three plant species in vivo and in vitro tissue cultures. Invest New Drug 26:334-336. [ Links ]
Kogan L, Dicarb J, Maynard WE 1953. Determination of coffee by paper chromatography. Anal Chem 25:1118-1120. [ Links ]
Kuhn A, Gerhard H 1943. The trigonelline and nicotinic acid content of fenugreek. Arch Pharm 281:378-379. [ Links ]
Lynn DG, Lewis DH, Tramontano WA, Evans S 1984. Induction of cell arrest in G2: structural specificity of trigonelline. Phytochemistry 23:1225-1228. [ Links ]
Moreno PRH, Van der Heijden R, Verpoorte R 1993. Effect of terpenoid precursor feeding and elicitation on formation of indole alkaloids in cell suspension cultures of Catharanthus roseus. Plant Cell Rep 12:702-705. [ Links ]
Namdeo AG, Jadhav TS, Rai PK, Gavali S, Mahadik KR 2007. Precursor feeding for enhanced production of secondary metabolites. Phcog Rev 1:227-231. [ Links ]
Olthof MR, van Dijk AE, Deacon CF, Heine RJ, van Dam RM 2011. Acute effects of decaffeinated coffee and the major coffee components chlorogenic acid and trigonelline on incretin hormones. Nutr Metab 8:10. [ Links ]
Patel RN 1998. Tour de paclitaxel: biocatalysis for semisynthesis, Annu Rev Microbiol 98:361-395. [ Links ]
Patel RK, Patel MM, Patel MP, Kanzaria NR, Vaghela KR, Patel NJ 2008. Hepatoprotective activity of Moringa oleifera Lam. fruit on isolated rat hepatocytes. Phcog Mag 4:118-123. [ Links ]
Radwan SS, Kokate CK 1980. Production of higher levels of trigonelline by cell cultures of Trigonella foenum-graceum then by differentiated plant. Planta Med 147:340-344. [ Links ]
Ramachandran C, Peter KV, Gopalakrishnan PK 1980. Drumstick (Moringa oleifera): a multipurposeIndian vegetable. Econ Bot 34:276-283. [ Links ]
Schripsema J, Meijer AH, van Iren F, ten Hoopen HJG, Verpoorte R 1990. Dissimilation cures as a simple method for the characterization of growth of plant cell suspension cultures. Plant Cell Tiss Org 22:55-64. [ Links ]
Silvestrini A, Pasqua G, Botta B, Monacelli B, van der Heijden R, Verpoorte R 2002. Effect of alkaloid precursor feeding on a Camptotheca acuminata cell line. Plant Physiol Bioch 40:749-753. [ Links ]
Samola V, Gerhard S 1983. Polarographic determination of trigonelline in coffee. Z Lebensm Unters Forsch 177:114-116. [ Links ]
Staba EJ 1980. Secondary Metabolism and Biotransformation in plant tissue. Plant Tissue Culture as a source of Biochemical's. CRC Press, Proca Raton, Florida, USA, p. 59-67. [ Links ]
Staba EJ 1985. Milestones in plant tissue culture systems for the production of secondary products. J Nat Prod 48:203-209. [ Links ]
Sulaiman MR, Zakaria ZA, Bujarimin AS, Somchit MN, Israf DA, Moin S 2008. Evaluation of Moringa oleifera aqueous extract for antinociceptive and anti-inflammatory activities in animal models. Pharm Biol 46:838-845. [ Links ]
Taguchi H, Muneto S, Shimabayashi Y 1986. Content of quinolic acid trigonelline and M-methyl nicotinamide in various food and thermal conversion of acid and nicotinamide. Betamin 60:537-546. [ Links ]
Tramontano WA, Evans LS, Gingley PA 1985. Effects of cytokinins on promotion of cell arrest in G2 by trigonelline and trigonelline concentration in cultured roots of Pisum sativum and Glycine max. Environ Exp Bot 25:83-88. [ Links ]
Verma SC, Banerji R, Misra G, Nigam SK 1976. Nutritional value of Moringa. Curr Sci India 45:769-770. [ Links ]
Verpoorte R, Van der Heijden R, Van Gulik WM, Hooken GJH 1991. Plant biotechnology for production of alkaloids: Present status and prospects. In: The alkaloids. Brossi A (Ed.). Academic Press. San Diego, USA. p. 1-8. [ Links ]
Viani R, Horman I 1974. Thermal behavior of trigonelline. J Food Sci 39:1216-1217. [ Links ]
Wang W, Zhao X, Zhuang G, Wang S, Chen F 2008. Simple hormonal regulation of somatic embryogenesis and/or shoot organogenesis in caryopsis cultures of Pogonatherum paniceum (Poaceae). Plant Cell Tiss Org 95:57-67. [ Links ]
Whitmer S, Canel C, Hallard D, Goncalves C, Verpoorte R 1998. Influence of precursor availability on alkaloid accumulation by transgenic cell lines of Catharanthus roseus. Plant Physiol 116:853-857. [ Links ]
Willeke U, Hefger V, Neise N, Neuhana H, Schind-elemiser I, Vordemfelde K, Barz W 1970. Mutually exclusive occurrence and metabolism of trigonelline and nicotinic acid arabinoside in plant celll cultures. Phytochemistry 18:105-110. [ Links ]
Wink M, Witte L 1985. Quizalizidine alkaloids as nitrogen source for lupine seedlings and cell suspension cultures. Z Naturforsch C 40:767-775. [ Links ]
Woodward AW, Bartel B 2005. Auxin: regulation, action, and interaction. Ann Bot-London 95:707-735. [ Links ]
Received 13 Jun 2011
Accepted 26 Sep 2011