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Allelopathic substances of Citrus junos fruit waste from food processing industry

Yukitoshi Tanaka and Hisashi Kato-Noguchi

Department of Biochemistry and Food Science, Faculty of Agriculture, Kagawa University,
Email s03x608@stmail.ag.kagawa-u.ac.jp

Abstract

Growth inhibiting activity of aqueous methanol extract of peel, segment and seeds separated from C. junos waste was determined using by six plant species as test plants. The highest inhibitory activity was found in peel and followed by segment and seeds. A large amount of C. junos peel was extracted with aqueous methanol and an active compound responsible for the inhibitory effect was isolated by column chromatography, C18 Sep Pack cartridge and HPLC. The structure of the substance was determined from high-resolution MS and 1H- and 13C-NMR spectral data as abscisic acid--D-glucopyranosyl ester (ABA-GE). The concentration of ABA-GE in peel, segment and seeds separated from C. junos waste was determined. The concentration was greatest in the peel, followed by the segment and seeds; there was a good correspondence between these concentrations and the inhibitory activities of the extracts. These results suggest that C. junos waste may possess allelopathic potential and ABA-GE may be involved in the growth inhibitory effect of C. junos waste. Therefore, C. junos waste may be useful as alternative materials for biological weed control.

Media summary

We attempted to isolate the allelochemicals from C. junos fruit wastes in order to search for the possible utilization of wastes from food processing industry.

Key Words

Abscisic acid--D-glucopyranosyl ester; allelopathy; Citrus junos; fruit waste; phytotoxicity

Introduction

C. junos is one of the important crops in Shikoku island of Japan due to its agro-ecological suitability as well as its industrial value. Citrus fruits produced in this area are usually used in industries producing processed juice or fruit vinegar for its special flavor and have been marketed throughout the nation. The waste after juice extraction was mostly discarded. These accumulating wastes resulting from such fruit processing industries have recently been identified as a social problem in this area. Therefore, proper recycling method needs to be readily developed for these C. junos wastes.

C. junos peel contains a variety of biologically active or agriculturally useful compounds. The inhibitory activities of peel extracts of 13 citrus fruits were determined and C. junos was found to have greatest activity of them (Fujihara and Shimizu 2003). In this study, the allelopathic potential of C. junos waste was determined and the main allelopathic substance in C. junos was isolated.

Methods

Extraction

The fruit waste of Citrus junos after juice extraction was obtained from a local food processing company (JA-Raihoku, Tosa, Japan), freeze-dried and divided into peel, segment and seeds. These materials were separately powdered in a mortar and pestle and extracted with 20 volumes of 80 % aqueous cold methanol for three days at 4C. Then, the extract was filtered through filter paper (No. 2, Toyo, Tokyo) and the filtrate was evaporated to dryness at 40C in vacuo.

Bioassay

Six species, alfalfa (Medicago sativa L.), cress (Lepidium sativum L.), crabgrass (Digitaria sanguinalis L.), lettuce (Lactuca sativa L.), timothy (Pheleum pratense L.), and ryegrass (Lolium multiflorum Lam.) were chosen for bioassay as test plants. Samples for bioassay were dissolved in a small volume of methanol and distilled water, added to a sheet of filter paper (No.2) in a 35 mm Petri dish and dried. The filter paper in the Petri dish was moistened with 0.8 ml of 0.05% (v/v) aqueous Tween 20. After germination in the dark at 25C for 12-120 hours, ten germinated seeds were arranged on the filter paper in the Petri dish and incubated in the dark at 25C for 48 hours. The shoot and root lengths of the seedlings were then measured with ruler and the percentage length of seedlings was calculated by reference to the length of control plants treated with solution only contained 0.05% (v/v) aqueous Tween 20.

Isolation and identification of the allelochemical

Freeze-dried peel (180 g dry wt) of C. junos was powdered and extracted with 80 % aqueous cold methanol for three days at 4C. After filtration, the filtrate was concentrated at 40C in vacuo to produce an aqueous extract. The extract was partitioned five times with an equal volume of dichloromethane and all five dichloromethane phases were combined. The aqueous fraction was separated by ion exchange columns to neutral, acidic and basic fractions. The neutral fraction was purified by columns of Diaion HP-20 and Sephadex LH-20 and C18 Sep-Pak cartridge. The biological activities of the dichloromethane and aqueous extract were determined by a lettuce bioassay. The active residue was finally purified by HPLC (10 mm i.d. x 50 cm, ODS AQ-325; YMC Ltd, Kyoto, Japan) eluted at a flow rate of 2 ml min-1 with 30 % aqueous methanol, detected at 220 nm. Inhibitory activity was only found in a peak fraction eluted between 147- 151 min, yielding an active component (4.9 mg) as a colorless resin. The chemical structure of the inhibitory substance was determined from high-resolution MS and 1H- and 13C-NMR spectral data.

Quantification of ABA-GE

Freeze-dried peel, segment and seeds separated from C. junos fruit waste were powdered and extracted as described above. The extract was purified using ion exchange columns and a reverse-phase C18 Sep-Pak cartridge according to the procedure described above. Then, the sample of ABA-GE was injected onto a column for HPLC (4.6 mm i.d. x 15 cm, Hydrosphere C18; YMC Ltd, Kyoto, Japan) eluted at a flow rate of 0.8 ml min-1 with 22 % aqueous methanol, detected at 220 nm. The overall recovery rate of ABA-GE through this quantification process was about 60 %.

Results

Allelopathic potential of C. junos waste

The effects of aqueous methanol extracts of peel, segment and seeds separated from C. junos fruit waste on the root and shoot growth of six test species were determined (Figure 1). In all bioassays, the peel extract caused the greatest growth inhibition of the test plants, followed in order by the segment and seed extracts. These results shown that C. junos waste possesses allelopathic potential and the peel has the greatest activity.

Figure 1. Effects of the extracts of peel, segment and seeds separated from C. junos waste on the growth of the test plant seedlings. Shoot and root length was determined after 48 hours of incubation in the dark at 25˚C. Means SE from three independent experiments with 10 plants for each determination are shown.

Isolation and identification of the allelochemical

The growth substance causing the inhibitory effect of peel obtained from C. junos fruit was isolated by columns of Diaion HP-20 and Sephadex LH-20, C18 Sep-Pak cartridge and HPLC. The molecular formula of the inhibitory substance was determined to be C21H31O9 (m/z 427.1987; calculated for 427.1968) based on its high-resolution mass spectrum. From a comparison of these data and 1H- and 13C-NMR spectra data with those reported in the literature (Koshimizu et al. 1968; Loveys and Milborrow 1981), the substance was identified as ABA-GE (Figure 2).

Figure 2. Chemical structure of abscisic acid--D-glucopyranosyl ester

Biological activity of ABA-GE

The biological activities of ABA-GE and (+)-ABA (Sigma) were determined with lettuce seedlings (Figure 3). At concentrations greater than 0.3 and 0.1 M, respectively, ABA-GE and (+)-ABA inhibited the growth of the lettuce shoots and roots. The concentrations required for 50% inhibition of the lettuce shoots in the assay (defined as I50) were about 2.3 and 0.84 M for ABA-GE and (+)-ABA, respectively, and those of lettuce roots were about 1.4 and 0.48 M for ABA-GE and (+)-ABA, respectively. Comparing I50 values, the activity of ABA-GE on the shoots and roots of the lettuce seedlings was about one third of that of (+)-ABA

Figure 3. Effects of ABA-GE and (+)-ABA on the growth of lettuce seedlings. Shoot and root length was determined after 48 hours of incubation in the dark at 25˚C. Means SE from five independent experiments with 10 plants for each determination are shown.

Concentration of ABA-GE

The concentration of ABA-GE was determined in peel, segment and seeds separated from C. junos fruit waste (Figure 4). The peel contained ABA-GE 3.5- and 27-fold greater than the segment and seeds, respectively, on per g dry weight basis, and 12- and 133-fold greater than the segment and seeds, respectively, on per fruit basis, which indicate that most of ABA-GE in C. junos waste is in the peel.

Figure 4. Concentration and content of ABA-GE in peel, segment and seeds separated from C. junos waste. Means SE from three independent experiments with three assays for each determination are shown.

Conclusion

Aqueous methanol extracts of peel, segment and seeds separated from the fruit waste inhibited the growth of the roots and shoots of alfalfa, cress, crabgrass, lettuce, timothy and ryegrass. The inhibitory activity of peel extract was greatest (Figure 1). The growth substance causing the inhibitory effect of C. junos fruit peel was isolated and identified as ABA-GE. ABA-GE inhibited shoot and root growth of lettuce seedlings at concentrations greater than 0.3 μM, and the concentrations for 50 % inhibition of shoot and root growth were 2.3 and 1.4 μM, respectively.

The concentration of ABA-GE in peel on per g dry weight basis was 3.5- and 27-fold greater than that in segment and seeds, respectively (Figure 4). Thus, the concentration of ABA-GE in the waste and the activity of the extracts show a good correspondence. These findings together with the effectiveness of ABA-GE on growth inhibition (Figure 3) suggest that ABA-GE may be involved in the growth inhibitory effect of C. junos fruit waste. ABA-GE was known to be a physiologically inactive main conjugated ABA form in plants, and an end-product of ABA metabolism rather than a storage or transport form (Milborrow 1970 and 1978; Neill et al. 1983; Zeevaart 1983; Lehman and Vlasov 1988). However, recently an ABA-GE-cleaving enzyme, apoplastic β-D-glucosidase was found (Dietz et al. 2000). The enzyme releases free ABA from the ABA-GE, which indicates ABA-GE may not be only end-product of ABA metabolism.

As the use of chemicals increases throughout the world, agricultural weed control alternatives to the present man-made herbicide-dominated programs are now being given wide consideration. Controlling weeds through allelopathy is one strategy to reduce dependency on man-made herbicides (Putnam 1988; Rizvi and Rizvi 1992; Seigler 1996; Narwal 1999; Duke et al. 2000). It has been shown that certain plant residues and extracts may function as weed suppressive agents (Rice 1984; Putnam and Tang 1986; Einhellig 1996). It is possible that C. junos waste could be important as a weed suppressive residue or mulch in a field setting.

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