Quercetin and Quercitrin from Pluchea lanceolata and Their Effect on

Chapter 6

Downloaded by NORTH CAROLINA STATE UNIV on October 26, 2012 | http://pubs.acs.org Publication Date: December 9, 1994 | doi: 10.1021/bk-1995-0582.ch006

Quercetin and Quercitrin from Pluchea lanceolata and Their Effect on Growth of Asparagus Bean Inderjit and K. M . M . Dakshini Department of Botany, University of Delhi, Delhi-110007, India

Two flavonols, quercetin and quercitrin (quercetin 3-O-rhamnoside), were isolated from the leaves of the rapidly spreading noxious perennial weed, Pluchea lanceolata. Quercitrin was also detected from the natural soils associated with the weed, while quercetin was not detected from the weed-associated soils. High performance liquid chromatography established that quercitrin concentration was higher in weed-infested soils with cultivation than in weed-infested, uncultivated soils. Aqueous extracts of 1x10 M, 5x10 M and1x10 M concentrations of quercetin and quercitrin affected the seedling growth of the legume, asparagus bean (Vigna unguiculata var. sesquipedalis). Mixture of the two flavonols also were effective inhibitors. These data indicate that quercetin and quercitrin produced by P. lanceolata may cause allelopathic interference of crops associated with this weed. -4

-4

-3

Our previous research established that the weed, Pluchea lanceolata (DC.) C. B. Clarke (Asteraceae), interfered with growth of certain plant species and physiological parameters of asparagus bean through water-soluble compounds from the leaves of the weed (1-3). Aflavanone(hesperidin), adihydroflavonol(taxifolin3-arabinoside), and an isoflavonoid (formononetin 7-O-glucoside) found in the natural soils associated with the weed, inhibit seedling growth of certain plant species (4, 5). These observations become more significant in explaining the interference potential of the weed under natural conditions as usually the above-ground parts of the weed are ploughed under in the cultivated fields prior to sowing. In view of the presence offlavonoidsin the weed-associated natural soils (4, 5), isolation and characterization of flavonoids of P. lanceolata leaves and its associated soils were undertaken to demonstrate their allelopathic potential.

0097-6156/95/0582-0086$08.00/0 © 1995 American Chemical Society In Allelopathy; Dakshini, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


6. INDERJIT & DAKSHINI

Quercetin & Quercitrin from Pluchea lanceolata

87

In spite of wide occurrence of flavonoids in seed plants (6), flavonoids have not been widely tested for their allelopathic potential (7). Stenlid (8) found that flavonoids lacking substitution on the B-ring are more potent allelochemicals. Flavonoids were reported to inhibit ATP production in mitochondria isolated from cucumber hypocotyls (8, 9). Koeppe and Miller (10) found that kaempferol inhibited phosphorylation in corn mitochondria. Tanrisever et al. (11) detected a novel flavonoid, ceratiolin, in water leachate of fresh foliage of Ceratiola ericoides. However, ceratiolin had little effect on seeds of Schizachyrium scoparium, while its degradation product, hydrocinnamic acid, cause inhibition of seed germination and root growth of Schizachyrium. The objectives of the present study were to carry out flavonoid analysis of P. lanceolata leaves and in soil associated with the plant, and to establish, if any, the allelopathic potential of flavonoids contributed by the weed. Experimental Leaves of the weed, Pluchea lanceolata, and its associated soils, were collected from the fields in and around Delhi (Long. 77.12 E; Lat. 28.38 N). Leaves were carefully cleaned and oven-dried (45 C). Soil samples were air dried and sieved (2 mm sieve). Both leaves and soil samples, were stored in paper bags until the phytochemical analysis. Extraction, Purification and Identification of Quercetin and Quercitrin. Fifteen grams of P. lanceolata leaves, and 30 g of soils associated with the plant, were soaked for 72 h in 100 ml of double distilled water (DDW) at room temperature (25 ± 3 C). Leachates were then filtered and evaporated to dryness under vacuum, and residues were extracted with 10 ml of methanol. These extracts were loaded on Whatman No. 3 (46 x 57 cm) chromatographic paper and developed through descending chromatography using n-butanol:acetic acid:water (BAW, 4:1:5, upper phase) (12). The developed chromatograms were then scanned under UV and U V+NH , and bands marked. Each band was eluted in 10 ml of methanol, concentrated under vacuum and again chromatographed on Whatman No. 3 and washed repeatedly with DDW through descending chromatography to purify these following Harborne (12). Finally each band was eluted from chromatograms with methanol and used for spectral analysis, which included acid hydrolysis and acid-base shifts (NaOH, A1C1 , A1C1/HC1, NaOAc, NaOAc/H B0 ) of parent as well as hydrolysed fractions. Fluorescence (UV and UV+NH ) and R/values of each compound were recorded in four solvent systems: DDW, BAW, 15% acetic acid and forestal (cone. HCl:acetic acid:water, 3:30:10). The aqueous layer of the hydrolysed fraction was used for the identification of sugar moiety (12). Additionally, the aglycone fraction was co-chromatographed with authentic samples of quercetin (Sigma Chemical Co., USA) and spectral data matched with that of authentic compound (13). 3

3

3

3

3

High Performance Liquid Chromatography. HPLC analysis was carried out to study the variation in concentration of isolated flavonoids from cultivated and uncultivated P. lanceolata associated natural soils. Five grams of weed-associated soils from either uncultivated areas, rarely cultivated areas, or regularly cultivated In Allelopathy; Dakshini, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

88

ALLELOPATHY: ORGANISMS, PROCESSES, AND APPLICATIONS

fields, were collected at two depths (topsoil and subsoil). The soil was shaken with 10 ml of methanol for 1 h at room temperature. Methanolic extracts of the soils and of authentic flavonoid were subjected to HPLC analysis. Reversed phase chromatorgraphy was carried out using a steel column (15 x 0.46 cm I. D.) containing a Zorbax C stationary phase and elution with methanol, the variable wavelength UV detector set at 275 nm, and a flow rate of 1 ml/min. For each analysis, 10 JLLI of compound and 30 pi of soil extracts were injected. On the basis of their retention time, flavonoid fractions were differentiated and identified (14).


]8

Test Solutions. Aqueous solutions of the identified flavonols were prepared for bioassays. Quercetin (Sigma Chemical Co.) and quercitrin (Extrasynthase, France) were initially dissolved in methanol, dried under vacuum, and dissolved in DDW to prepare the solutions of l x l 0 M, 5x10" M and l x l 0 M. Further, since the compounds cooccur in the leaf tissues, the effects of mixtures of these compounds were also investigated. For this purpose, equimolar mixed test solutions of each concentration of flavonols were prepared. 4

4

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Growth Experiments. The commonly grown legume asparagus bean, Vigna unguiculata var. sesquipedalis (L.) Walp, was selected to assess the allelopathic potential of isolated flavonoids. Earlier studies established that the growth of asparagus bean was affected by P. lanceolata under experimental conditions (3). Fifty seeds of asparagus bean were sown on filter paper (Whatman No. 1) moistened with 60 ml of DDW (served as control) or test solutions, and placed in 15cm diameter Petri plates. To maintain the uniform moisture status in the Petri plates, a cotton pad soaked in either DDW or a test solution was placed below the filter paper. During the period of investigation, a temperature regime of 30 ± 5 C and light regime of 18.8 Klux was maintained. Root and shoot lengths were recorded on the seventh day. Treatments were arranged in randomized block design and repeated and replicated three times. Comparison of growth parameters were made through one-way analysis of variance. Results and Discussion Fluorescence, R/values, and UV spectral data comparisons to the reference sample confirm the presence of flavonols, quercetin and quercitrin, in the leaves of Pluchea lanceolata, and of quercitrin in weed-associated natural soils (Table I). Quercitrin was present both in P. lanceolata leaves and its associated soils. However, quercetin was detected only from the leaves of the weed and not from its associated soils. Presence of quercitrin in the soils could be explained as it is resistant to the fungal degradation (7), whereas quercetin has been shown to be decomposed by soil fungi, especially Aspergillus (7, 15). Padron et al. (16) reported that Aspergillus, when grown in medium with quercetin as the sole carbon source, produces an enzyme which degrades quercetin immediately. However, quercetin is likely to be functional in allelopathy because of its repeated availability through aqueous leachate. Grummer (17) reported quercitrin as an allelopathic compound in the leaves of Artemisia absinthium. Quercetin was reported In Allelopathy; Dakshini, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

In Allelopathy; Dakshini, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

Br Y

Quercitrin

55

00

22

67

48

04

47

40

255 (255) 352 (350)

d

255 (255) 371 (373)

HOAc Forestal MeOH

271 (271) 331sh (330sh) 404 (399)

254 (254) 325 (325) 428 (428)

NaOH }

274 (274) 419 (423)

270 (270) 333 (333) 431 (433)

7EI

UV Absorption Spectra

274 (274) 316sh (314sh) 396 (396)

269 (268) 360sh (360sh) 431 (427)

AlCl/HCl

269 (262) 358 (359) 396 (396)

380 (380)

269 (267) 324sh

NaOAc

266 (264) 373 (370)

269 (269) 383 (386)

s

NaOAc/H B0

3

d

c

b

Quercetin was not detected in P. /a«ceo/ata-associated soils. Fluorescence key: Y , yellow; Br, brown. Solvent key: DDW, double distilled water; B A W , butanol-acetic acid-water (4:1:5, upper pahse); HOAc, 15% acetic acid; Forestal, cone. HCl-acetic acid-water (3:30:10). Values in parentheses indicate that of authentic reference compound. sh, shoulder

a

Y

Quercetin

Y

UV UV+NH,

identification

0

DDW BAW

Fluorescence* Rf values

Flavonoid

Table I. UV Absorption Spectra of Quercetin and Quercitrin Isolated from Pluchea lanceolata Leaves and its Associated Soils"


90


from ponderosa pine and associated soil which was found to have allelopathic effect against Nitrosomonas (18). Wepplo (19) identified quercetin and parasorbic acid glycoside from the leaves of American cranberry (Vaccinium macrocarpa). 1


Table II. HPLC Analysis showing Trapped Concentrations (pg g ) of Quercitrin in soils from uncultivated areas, rarely and regularly cultivated fields infested with Pluchea lanceolata 1

Soil

Trapped Concentration (jug g ) Uncultivated area

Topsoil Subsoil

8.09 5.91

Cultivated fields Rarely

Regularly

13.83 7.01

11.08 15.01

HPLC analysis of topsoil and subsoil from Pluchea lance olata-infested fields demonstrated that quercitrin concentration was higher in cultivated fields than in uncultivated areas (Table II). It is interesting that the concentration of quercitrin increased with cultivation, especially regular cultivation. Subsoils of regularly cultivated fields had higher quercitrin content than rarely cultivated fields, while this difference between cultivated sites was not found in topsoil (Table II). The fact that quercitrin concentration of topsoil of regularly cultivated fields was not higher than that of rarely cultivated fields could be due to the leaching of quercitrin from topsoil to subsoil. The higher concentration of quercitrin in the cultivated fields is probably due to more incorporation of leaves of the weed into the soil (3). Inderjit and Dakshini (20) found that in comparison to uncultivated areas, soils from P. lanceolata infested cultivated fields had higher values for total phenolics. The data suggest that concentration of quercitrin may vary in P. lanceolata soils under field conditions because of localized variations resulting from agricultural practices and irrigation. Other investigators have suggested uneven distribution of phytotoxic substances in the soil (21-23). In fact, allelopathic effects depend upon the probability of exposure of roots of affected plants to pockets of toxins in the soil. Compared to quercitrin, quercetin was a stronger inhibitor of the seedling growth of asparagus bean (Table III).This observation could be explained by the observations of Stenlid (8) that aglycone moiety of flavonoids are more active than glycosides. Quercetin significantly (P < 0.001) inhibited the root and shoot growth of asparagus bean at all the three levels of concentrations (Table III). However, quercitrin significantly (P < 0.001) inhibited the shoot growth of asparagus bean at the level of 5x10^ M concentrations, and root and shoot growth at the level of lxlO M (Table III). Furthermore, when present together in equimolar mixtures, these two flavonols inhibited the root growth at the level of 3



3

3

4

3

4

4

lxlO" M 5xlO- M Ixl0- M lxlO- M 5x1 O^M lxlO" M lxlO^M 5x1 (Htf lxl0 M

Treatment

2.7 1.7 1.0** 1-4** 2.8 1.4** 1.5** 2.8 2.1** 1.8** -48.28 -44.71 -47.84 -12.22 -09.23 -18.48 -13.56 -05.96 -41.28

± + + + + + + + + +

3.9 2.7 1.8 2.5 3.7 2.0 1.9 4.3 3.3 1.9

d

1.9 1.5** 1.3** 1.6** 2.3 2.5 1.9** 2.3* 2.2 2.3**

6.7 3.4 3.7 3.5 5.9 6.1 5.5 5.8 6.3 3.9

+ + ± ± + + ± + + +

Root

Shoot

Root

-30.65 -55.02 -36.93 -05.52 -49.49 -51.00 +07.28 -17.33 -50.25

Shoot

Percent Relative Response*

0

Seedling length (cm)

d

c

b

Seedling length on seventh day of seed sowing. Average percent increase (+) or decrease (-) in seedling length as compared to control. Each compound in equal volume. The data are means of three replicates + standard deviations of means and asterisk indicate significant differences between control and treatment values at the level of *P < 0.05; **P < 0.005.

a

Mixture

0

Quercitrin

Control Quercetin

Flavonol

Table III. Effect of Three Different Concentrations (lxlO^M, 5xl(HM and l x l 0 M ) of Quercetin and Quercitrin on Seedling Growth of Asparagus Bean

3


92


4

3

lxlO" M (P < 0.05) and lxlO M (P < 0.001) concentrations. Shoot growth was inhibited significantly (P < 0.001) by the mixture treatments of 5X10" M and lxlO* M (Table III). In contrast to observation of Williamson (24), Einhellig and Rasmussen (25), Rasmussen and Einhellig (26), Einhellig et al (27) and Williams and Hoagland (28) on synergistic activity of allelochemicals, such an effect was not apparent in the present study. The two compounds appear to have an additive inhibitory effect. However, the experiment was not designed to separate additive, synergistic, or antagonistic actions. Seedlings grown with either of the flavonols showed browning of root tip at the level of lxl0" M concentrations. Presently, it is difficult to conclude if this effect resulted from ascorbic acid reduction in xylem vessels (7) or due to oxidation of flavonols, and further study is needed. The data presented is of significance as it has established that quercetin and quercitrin can bring about allelopathic interference to the growth of asparagus bean. Furthermore, the data are relevant to field conditions. Given the perennial nature of the weed, continuous availability and periodic replenishment of these flavonols in soil will occur. Regular cultivation which is common to agricultural fields appears to enhance quercitrin in the soil. In summary, quercetin and quercitrin may play a significant role in initiating the chain of allelopathic interferences observed with crop seedling in agricultural fields infested with P. lanceolata. 4


3

3

Acknowledgments We thank Professor M. R. Parthasarthy, Department of Chemistry, University of Delhi for his help in identification of compounds. Financial assistance to one of us (Inderjit) by Department of Science and Technology is gratefully acknowledged. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Inderjit; Dakshini, K. M. M. Plant Soil 1990, 122, 298-302. Inderjit; Dakshini, K. M. M. Am. J. Bot. 1994, 81, 799-804. Inderjit; Dakshini, K. M. M. Am. J. Bot. 1992, 79, 977-981. Inderjit; Dakshini, K. M. M. J. Chem. Ecol. 1991, 17, 1585-1591. Inderjit; Dakshini, K. M. M. J. Chem. Ecol. 1992, 18, 713-718. Harborne, J. B.; Simmonds, N. W. In Biochemistry of Phenolic Compounds; Harborne J. B., Ed.; Academic Press: New York, NY, 1964, pp 77-127. Rice, E. L. Allelopathy. Academic Press: Florida, FL, 1984. Stenlid, G. Phytochemistry 1970, 9, 2251-2256. Stenlid, G. Physiol. Plant. 1968, 21, 882-894. Koeppe, D. E.; Miller, R. J. Plant Physiol. 1974, 54, 374-378. Tanrisever, N.; Fronczek, F. R.; Fischer, N. H.; Williamson, G. B. Phytochemistry 1987, 26, 175-179. Harborne, J. B. Phytochemical Methods. Chapman and Hall: New York, NY, 1973. Mabry, T. J.; Markham, K. R.; Thomas, M. B. The Systematic Identification of Flavonoids. Springer-Verlag: New York, NY, 1970. In Allelopathy; Dakshini, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

6. INDERJIT & DAKSHINI 14. 15. 16.


17. 18. 19.

20. 21. 22. 23. 24. 25. 26. 27. 28.

Quercetin & Quercitrin from Pluchea lanceolata

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Hartley, R. D.; Buchen, H. J. Chromatogr. 1979, 180, 139-143. Westlake, D. W. S.; Talbot, G.; Blakley, E. R.; Simpson, F. J. Can. J. Bot. 1959, 5, 621-629. Padron, J.; Grist, K. L.; Clark, J. B.; Wender, S. H. Biochem. Biophys. Res. Commun. 1960, 3, 412-416. Grummer G. In Mechanisms in Biological Competition; Milthorpe, F. L., Ed.; Academic Press: New York, NY, 1961; pp 219-228. Lodhi, M. A. K.; Killingbeck, K. T. Am. J. Bot. 1980, 67, 1423-1429. Wepplo, P. In Allelochemicals: Role in Agriculture and Forestry; Waller, G. R., Ed.; ACS Symposium Series 330; American Chemical Society: Washington, DC, 1987; pp 328-333. Inderjit; Dakshini, K. M. M. J. Chem. Ecol. 1994, 20, 1179-1188. Liebl, R. A.; Worsham, A. D. J. Chem. Ecol. 1983, 9, 1027-1043. Patrick, Z. A.; Toussoun, T. A.; Snyder, W. C. Phytopathology 1963, 53, 152-161. Guenzi, W. D.; McCalla, T. M. Soil Sci. Soc. Am. Proc. 1966, 30, 214216. Williamson, G. B. In Perspectives on Plant Competition, Grace, J. B.; Tilman, D., Eds.; Academic Press:New York, NY, 1990; pp 143-162. Einhellig, F. A.; Rasmussen, J. A. J. Chem. Ecol. 1978, 4, 425-436. Rasmussen, J. A.; Einhellig, F. A. Plant Sci. Lett. 1979, 14, 69-74. Einhellig, F. A.; Schon, M. K.; Rasmussen, J. A. J. Plant Growth Regul. 1982, 1, 251-258. Williams, R. D.; Hoagland, R. E. Weed Sci. 1982, 30, 206-212.

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Quercetin and Quercitrin from Pluchea lanceolata and Their Effect on

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