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Isolation, structural elucidation and chemistry of an allelopathic compound from a medicinal plant.

Shobha Sondhia

National Research Centre for Weed Science 1Jabalpur-482004, India, Department of Post Graduate Studies and Research in Chemistry2 R.D. University, Jabalpur-482001, India
Email:shobhasondia@yahoo.com

Abstract

Tagetes patula, commonly known, as French marigold is an ornamental medicinal plant belonging to the Compositeae family. It is a native of Mexico and South America and found in all parts of India. It is an annual herb and the whole plant is used medicinally. The butanolic fraction of this plant possesses very strong allelopathic activity against various weeds. The active principle is a novel pentacyclic triterpene saponin. The structure of this saponin molecules was determined by detailed spectroscopic analysis viz 1 HNMR, 13 C NMR, FAB-MS and IR spectroscopy and several chemical degradation and chemical colour reactions methods. The pentacyclic saponin was composed of the disaccharide side chain at C-3, namely one glucose unit and one galactose unit. Data on the in vitro allelopathic activity of this plant will be presented.

Media Summary

A bioactive saponin molecule has been isolated from Tagetes patula plant. Tagetes patula showed very strong inhibitory effect on Ischaemum rogusum, Vicia sativa and Echinochloa colona weeds. The active principle is found to be a novel pentacyclic triterpene saponin isolated from this plant.

Key words

Tagetes patula, allelopathic activity, pentacyclic triterpene saponin

Introduction

Allelopathy can be defined as biochemical interaction among plants, algae and microorganisms. The mechanism of allelochemicals at molecular level and their influence on soil and other plants plays a vital role in management of natural and agricultural ecosystem. Tagetes patula is an ornamental and medicinal plant belonging to compositeae family and is a native of Mexico and South America. It is commonly known as French marigold or Genda. This plant blooms throughout the summer and fall and the longer lasting flowers are great for cut flowers. T. patula is an annual herb and the whole plant is used for medicinal purpose of western MP tribals. It is used for fungal infections, skin infections, cuts, sprains, wounds, and as an antiseptic.

The saponin are a pharmacodynamic group of natural product with a wide spectrum of biological activities and widely distributed in nature, being present in more than ninety plant families and among them triterpenoid saponin constituted the major group. In the present paper pentacyclic saponin molecule was isolated, identified and characterized and its allelopathic activity against Ischaemum rogusum, Vicia sativa and Echinochloa colona weeds.

Methods

Plant material

Whole mature plant of Tagetes patula were collected from the National Research Centre for Weed Science, Jabalpur, India. It was identified by the subject expert and was dried under shade and powdered.

Extraction and isolation

Triterpene saponin molecule was isolated as described by Sondhia and Saxena, (2002). The process of isolation and purification yielded a white amorphous powder having Rf value (0.88) on TLC and it was further confirmed to be a triterpene by the Libermann-Burchard chemical test.

Hydrolysis of saponin

Hydrolysis was done with 10 % H2SO4 at 100 oC for 6 hours. Hydrolyzed saponin (aglycone) was methylated and methanolysed with 3 % methanolic hydrochloride at 80 o C for six hours followed by neutrilization with silver carbonate. The ethyl acetate fraction comprises of pure aglycone was designated as compound I (aglycone fraction), whereas the aqueous fraction comprises of the sugars hydrolyzed from saponin and was designated as compound II (glycone fraction).

Analysis of compound 1 by various spectroscopic methods

The saponin molecule was elucidated by various spectroscopic methods such as IR (Shimadzu 8201 PC instrument having a range of 4000-350 cm–1), 1H NMR, 13C NMR (Bruker DRX 300 MHz FT- NMR) and Mass Spectroscopy (JEOL SX-102 /DA-6000 FAB-MS/data system using Argon/Xenon (6 KV) as the FAB gas).

Allelopathic activity of T. patula

Allelopathic activity of the pentacyclic triterpene saponin isolated from T. patula was evaluated against Ischaemum rogusum, Vicia sativa and Echinochloa colona weeds.

Preparation of saponin extract dilutions

Isolated crude saponin (0.92 g) was dissolved in 2 ml methanol and 20%, 10 %, 5%, 2.5 % and 1.25 % concentrate were prepared by adding distilled water and distilled water served as control.

Bioassay

Twenty seeds of E. colona, I. rogusum or V. sativa were placed on a Whatman filter paper No. 5 in a hundred mm diameter petridish supported by a thin layer of sterilized absorbent cotton. Ten ml of desired concentration of crude saponin extracts, viz. 10%, 5% 2.5% and 1.25 % were added in each petridish and petridishes were kept in seed germinator for 72 hours bioassay at + 25 C and at 80 % humidity. The data on germination, root elongation and shoot growth were taken thereafter. The experiment was replicated thrice.

Results

Structural elucidation by various spectral studies

The saponin molecule had a melting point of 260 0C. The IR peak at 3600-3250 cm-1 and 1050 cm-1 indicating hydroxyl group and one absorption bands at 1650 cm-1 indicating double bonds. Further it showed an absorption band at 3600-3400 cm-1 due to the carbohydrate moiety. The prominent peak of 3200-3400 cm-1 (for carbonyl OH) and strong signal at 1720 cm-1 due to carbonyl absorption, 2950, 1640 for (C=C) and 1465, 1380, 1050 cm-1 for (C-C, C-H) tallied well with the olefinic acid ring structure. The number of anomeric carbon in a saponin are determined by 13C NMR and the number of individual sugar residue present by comparing the chemical shift with appropriate model sugars (Seo et al. 2002). The 13C NMR spectra of saponin molecule showed signal at 89.3 shifted downfield by 10 ppm as compared to oleanoic acid due to glycosylation shift, indicating the C-3 sugar linkage and suggesting that sugar is linked through C-3 hydroxyl of aglycone. Similarly 1H NMR spectra showed the proton signal of ethylene proton at 5.5 ppm as a rather broad singlet while the anomeric proton of the monosaccaride residue appeared at 4.4 ppm. The 13C NMR values of saponin molecule obtained in pyridine were 38.4, 27.6, 90.5, 38.8, 18.1, 32.6, 39.2, 47.8, 36.8, 23.5, 122.7, 144.1, 42.1, 25.8, 23.1, 46.5, 41.0, 45.9, 30.6, 33.8, 32.5, 27.6, 15.3, 16.2, 17.1, 25.8, 180.0, 33.2, 23.5 (C1-C30), Sugars 103.3, 73.3, 78.9, 68.7, 71.4, 61.4, 61.7 (C1-C6), 100.9, 71.6, 72.7, 68.2, 71.0, 62.3 (C1′-C6′).

1H NMR values of saponin molecule obtained in D2O, δ ppm were viz 0.81 (9H, s), 0.85 (3H, s), 1.08 (3H, s), 1.15 (3H, s), 1.25 (3H, s) 5.5 (1 H, b, s, CH=C-), and 4.4 (1 H, d, glu) together with 13 C NMR indicated that the aglycone belongs to the oleanolic type pentacyclic triterpene ring (Doddrell et al 1974). Consequently the remaining 1H and 13C NMR signals corresponding to the carbinolic hydrogen and carbon atoms were used to identify a beta –D-glucopyranosyl moiety at C-3. Further 1 H NMR spectrum indicated the presence of three anomeric protons in the sugar moiety at d 4.32(1H), 5.01 (1H) and 4.42 (1H). The D- configuration of glucose and galactose were determined by physical data (co-preparative TLC) and confirmed by comparing with authentic sample of D-glucose and D-galactose as well as by comparing the chemical shift with values from the literature (Doddrell et al. 1974) The interglycosidic linkage between sugars was deduced to be Glu (1→3) Gal from the deshielding of one of the CH units of this moiety in the 13C NMR spectrum of compound 1 (δ 78.9).

The positive ion Fast Atom Bombardment- Mass Spectra (FAB-MS) of the whole saponin molecule exhibited the following fragmentation pattern. The fragment ion m/e 248 as the base peak in mass spectrum. Characteristic peaks at m/z 248 and 208 are the results by the fragmentation of ring C, and are the most common peak of oleanane ring skeleton and indicated that the double bond is present in ring C at 12-13 positions. There was a subsequent loss of methyl group (m/z 233) reported. Peak at 179 obtained due to the loss of hexose moiety and peak at 553 showed a rearrangement type of ion. This pattern of peaks indicated that one carboxylic group is present in ring E (Rahman et al. 2000). The positive ion peak of FAB-MS of compound 1 revealed quasimolecular ion at m/z 781 (M+H) correlate with the formula C42H68O13 and peak at 618 and 456 attributed to the fragment loss of one glucose and one galactose units respectively. This structure can be theoretically named and considered as 3-O- [beta-D-glucopyranosyl- (1→3) –beta-D-galactopyranosyl (3 beta)-3-hydroxy olean-12-en-28-oic acid and molecular weight and formula 780.

Allelopathic activity of Tagetes patula

Allelopathic potential is also a characteristic feature of saponin isolated from plants (Sondhia and Saxena 2002). Alfalfa saponins possess high allelopathic potential against plants, fungi and microorganism (Oleszek, 1996). Our bioassays show that T. patula has highest allelopathic potential on E. colona seedlings. All the saponin dilutions bioassays showed significant differences at probability 0.05 level as compared with control. Further, in E. colona root growth was affected more severely as compared to shoot growth and almost 100 % reduction was observed at 10 % saponin concentration. It was seen that 10 % saponin was very toxic to E. colona. Similar results were obtained by Waller et al. (1995) where Soya saponin I, and II inhibited the growth of mungbean that was growing for 72 hours. The present results clearly demonstrate that the T. patula saponin significantly inhibited in vitro, root and shoot growth of E. colona and I. rogusum.

Figure 1: Effect of T. patula crude saponin on the shoot growth of E. colona, I. Rogusum and V. sativa.

The effect of T. patula saponin on E. colona, I. rogusum and V. sativa are presented in Fig 1 and 2. Significant results were obtained at all saponin concentration. In all the weed seedlings decreasing trend in shoot growth and root growth were observed and it was higher at higher concentration as might be expected. From fig 1 and 2 it clearly appeared that all the concentration of saponin extracts inhibited the seedling of E. colona, I. rogusum and V. sativa however, 10 % (w/v) saponin completely inhibited shoot growth of E. colona. In E. colona, 20 % saponin completely inhibited seedling growth. Root growth was affected more severely than shoot growth (Fig 1 and 2). Further, 20 % crude saponin of T. patula completely inhibited the growth of I. rogusum and V. sativa seedlings (root and shoot) as compared to control. The order of sensitivity among all tested weeds were I. rogusum > E. colona> V. sativa. The allelopathic activity of pentacyclic saponin isolated from the T. patula showed that they have potential to completely inhibit the growth of E. colona, I. rogusum and V. sativa at 20 % saponin extract, respectively and may be used as a potential herbicide template.

Figure 2: Effect of T. patula crude saponin on the root growth of E. colona, I. rogusum and V. sativa.

Conclusion

T. patula saponin strongly inhibited the growth of I. rogusum and E. colona and moderately inhibited the growth of V. sativa. This weed selectivity is comparable with other candidate allelochemicals (Fujii et al. 1991, Sondhia and Saxena 2003). In spite of strong herbicidal activity of T. patula crude saponin on E. colonum, V. sativa and I. rogusum seedlings, much multidisciplinary research is required to evaluate its herbicidal activity in field trials as well as on other weeds. The phytotoxicity of T. patula to the crop is another issue. The fact that saponin is a natural product with potent herbicidal activity makes this topic more worthy of future investigation. We have explored the allelopathic activity of crude pentacyclic saponin since these compounds appear to have a role in plant defense system and play a regulatory role in interactions of plants with other plants. Thus T. patula may play an important role in controlling weeds in crop in future.

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