Developmental and ultrastructural effect of Uncaria tomentosa (Willd.) DC extract on the paprika Capsicum annuum L.

Industrial Crops and Products 19 (2004) 59–67

Developmental and ultrastructural effect of Uncaria tomentosa (Willd.) DC extract on the paprika Capsicum annuum L. Krzysztof Gulewicz a,∗ , Teresa Tykarska b , Wojciech Wysocki a , c , Mieczysław Kura´s b ˙ Julita Augustynowicz b , Katarzyna Zurowska a

Laboratory of Phytochemistry, Institute of Bioorganic Chemistry PAS, Z. Noskowski str. 12/14, 61-704 Poznan, Poland b Department of Plant Morphogenesis, Warsaw University, Miecznikowa str. l, 02-096 Warsaw, Poland c A-Z Medica Spółka z.o.o., Czy´zewskiego 6/1 A, 81-706 Sopot, Poland Received 25 May 2002; accepted 25 July 2003

Abstract The effect of liana (Uncaria tomentosa L.) extract on paprika (Capsicum annuum L.) cv. Ko´zlak. development, leaf ultrastructure and yielding were studied under greenhouse conditions. Uncaria extract was supplied before flowering of plants by two manners: to the soil or leaves. It is shown that the effect of Uncaria extract on paprika was dependent on the concentration and the way of supply of the preparation to plant. In the case of extract supply to the soil, distinct growth inhibition of plants and ultrastructural manifestations of senescence were observed. Simultaneously, acceleration of flowering and fruiting were noticed. The phenomena mentioned above caused the lower yield of paprika fruits compared to the control. In the case of extract supply to leaves, high acceleration of flowering and fruits maturation of paprika were observed. Spraying of leaves with Uncaria extract resulted in an increase of fruits yield compared to the control. © 2003 Elsevier B.V. All rights reserved. Keywords: Uncaria tomentosa L.; Paprika; Development; Flowering; Fruiting; Leaves ultrastructure; Senescence

1. Introduction Uncaria tomentosa Wild. DC. of family Rubiaceae is a hispid liana growing in jungles of South and Middle America (Peru, Colombia, Ecuador, Guiana, Venezuela, Trinidad; Suriname, Costa Rica, Panama). Recently, this plant has been of tremendous interest to scientists. It is a result of unique pharmacologi∗ Corresponding author. Tel.: +48-61-852-85-03; fax: +48-61-852-05-32. E-mail address: [email protected] (K. Gulewicz).

cal properties of this plant and its wide use in folk medicine. It is named: ‘Una de gato’, ‘Vilcacora’, ‘Cat’s claw’ and so on. Pharmacological properties of natural compounds present in Uncaria species extract are very well known and described in numerous papers (e.g. Harada et al., 1974; Nakazawa et al., 1991; Yano et al., 1991; Konno et al., 1997; Mimaki et al., 1997; Takayama et al., 1997; Masumiya et al., 1999). Also, the chemical composition of various Uncaria species extracts is well known and widely described in literature. The main compounds of

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extract are: tetra- and pentacyclic indole and oxindole alkaloids (Raymond-Hamet, 1952; Hemingway and Philipson, 1972, 1974; Philipson and Hemingway, 1973, 1975; Philipson et al., 1978; Montenegro de Matta et al., 1976; Wagner et al., 1985a,b; Aquino et al., 1991; Seki et al., 1993; Laus et al., 1996, 1997; Kepłinger et al., 1999), tannins and procyanidines (Montenegro de Matta et al., 1976; Wirth and Wagner, 1997; Kohlmunzer, 1998), sterols, mainly p-citosterol (Senatore et al., 1989), quinovic acid glycosides (Ceni et al., 1988; Aquino et al., 1991; Yepez et al., 1991) and triterpenes, as well as their derivatives (Aquino et al., 1990, 1991, 1997; Kitajima et al., 2000). Up to now, studies on biological activity of Uncaria species have been performed mainly on animal organisms. In this work, we would like to answer the following question: Does the U. tomentosa L. extract influence the development and yield of cultivable plants, and if so, is such influence observed also in their ultrastructure. This subject is not new for us because it is closely referred to our perennial interest in biological activity of lupin alkaloid-rich extracts (e.g. Gulewicz and Trojanowska, 1995; Gulewicz et al., 1996; Wyrostkiewicz et al., 1997; Sas-Piotrowska et al., 1997; Wysocki et al., 2001). In our present work, we extended it by adding ultrastructural observations.

2. Materials and methods 2.1. Characteristic of preparation The object of our studies was water extract of U. tomentosa L. bark. The preparation was obtained from Laboratiorios Indu¸auimica Lima, Peru in the form of fine homogenous powder with brick-red colour and acrid-bitter taste. The total of alkaloids expressed as content of mitrafiline was on the level 1.17%. 2.2. Green house experiments Biological activity of U. tomentosa extract was determined by its effect on growth and yield of paprika (Capsicum annuum L.) cv. Ko´zlak. The experiments were carried out in greenhouse in determined conditions within a period of March–December, 2001 us-

ing garden soil containing: 320 mg/l N; 50 mg/l P.; 380 mg/l K, 2870 mg/l Ca and micronutrients like Cu, Fe, Mn, Mo, Zn, Mg, B. The seeds of paprika were sowed to the soil on March 26, The seedlings of paprika were transplanted to 12 cm diameter pots on May 18. After 17 days the paprika was transplanted once again to 20 cm diameter and 5 l capacity pots. The experiments with paprika were performed according to the following scheme: 1. Uncaria extract was added to the soil in doses: 80.0 and 320.0 mg per pot per plant. 2. Uncaria extract was applied to the leaves in doses: 8.0 and 80 mg per plant∗ . 3. The control combinations were treated with water only. Each combination was done with six plants simultaneously. One pot containing one plant constituted one repetition. As distinguished from combination (1), where Uncaria extract was added to the soil, in combination (2) 10 g of dry weight of horse manure was added to each pot. The Uncaria extracts were supported in 200 ml doses per pot in the case (1) and 10 ml ones per plant in the case (2). Simultaneously, the control samples (3) were treated with 200 and 10 ml of water respectively. In order to avoid to get Uncaria extract into the soil during spraying of paprika leaves (2) the soil in pots was covered with blotting paper. The treatment of paprika with U. tomentosa extract was carried out on June 6, short time before flowering of plants. Additionally, during vegetation period the plants were fed twice on May 13 and September 14, during flowering and fruiting, with 200 ml 0.3% Azofoska fertiliser (13.6% N; 6.4% P2 O5 , 19.1% K2 O, 4.4% MgO and micronutrients) per pot. The experiments were finished on December 21. The fruits of paprika from each particular combination were collected, weighed and yields were calculated. Additionally, the yield of dry weight of paprika vegetative parts (roots, stalk and leaves) was determined. 2.3. Observation of development of paprika During vegetation time of paprika, the observations of development of plants were performed.

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These observations referred to: (a) (b) (c) (d)

development of plants; colour and falling of leaves; time of flowering; time of fruiting.

2.4. Ultrastructural observations Ultrastructural observations were carried out in control plant leaves and in leaves of plant after administering of U. tomentosa extract into soil, in the amount of 320 mg per pot (each holding one plant). Leaves were collected on June 28, 2001, 21 days after administering the extract, when morphological difference in leaves of both types of plants was distinct. The leaves were picked from two levels of a plant: the upper one (third leaf from the top)—a young leaf, and the bottom level (the lowest leaf of the plant)—an old leaf. Squares of side length l mm were cut from their middle part (between the main nerve and the second and third side nerve) and fixed for 2 h in 2.5% glutaraldehyde, at pH 7.2 (0.1 M cacodylate buffer) with 1% caffeine added, and subsequently post-fixed for 12 h in OsO4 . After desiccation in ethanol, the material was embedded through propylate oxide in the Epon/Spurr mixture. The embedded leaves were cut into cross sections with ultramicrotome (produced by LKB company). Ultrathin sections, 60–90 ␮m thick, were contrasted with uranyl acetate and lead citrate according to Reynollds (1983), and finally observed. Electronograms were taken in transmission electron microscope produced by TESLA. 2.5. Statistical analysis Statistical calculations were done with support of one way analysis of variance of balanced design according to the method Elandt (1964).

3. Results and discussion Observation on development of paprika after treatment with Uncaria extract showed dependence of plant reaction on support manner of preparation. After 10 days of treatment of paprika with Uncaria extract (added to the soil) distinct inhibition of growth of

Scheme 1. Influence of U. tomentosa extract supplied to soil on paprika. (1, 2); control, (3); dose 80 mg per plant, (4); dose 320 mg per plant.

plants in comparison to control was noticed. Moreover, in this combination about 2 weeks acceleration of paprika flowering compared to control was observed. Unfortunately, among twelve treated plants in this experiment only four fruited (two at dose 80.0 mg per plant and two at dose 320.0 mg per plant). Small, not complete maturated fruits were dropping down. Also, in this combination yellowing and dropping down of leaves was observed. (Scheme 1). Similar behaviour of plants is very often observed in nature when they are in stress condition (food deficiency, lack of water and so on). Contrary to the experiment mentioned above spraying of paprika leaves with Uncaria did not have any optical effect on plants development in the first 10 days after treatment. During this time, no differences and disturbances in development of paprika plants treated with extract compared to the controls were visible. The differences between treated plants and controls in this combination appeared later (about 3 weeks after spraying). The paprika sprayed with Uncaria showed distinct flowering acceleration in comparison to control. Finally, earlier maturation of fruits was observed (Scheme 2). These observations are compatible and confirmed by the data presented in Tables 1 and 2. Table 1 shows the effect of Uncaria extract supplied to the soil on average yield of paprika. As can be seen, both doses of extract used (80.0 and 320.0 mg per plant), influence significantly fruit paprika yielding.

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Scheme 2. Influence of U. tomentosa extract fed onto leaves of paprika. (1) Control; (2) dose 8 mg per plant; (3) dose 80 mg per plant.

to control was noticed for both doses and equalled 79 and 87%, respectively. The difference in yield of fruits results from their size but their amount. Uncaria extract doses used did not have any influence on yield of vegetative part. The higher yield of vegetative part in comparison to experiment presented in Table 1, was an effect of the addition of horse manure. Unfortunately, the considerable increase of vegetative part of plant compared to previous experiment (Table 1) caused lower yield of paprika fruits. However, the comparison of Uncaria extract effect on yield of fruits and biomass of paprika in both experiments is difficult due to presence of horse manure in the experiment 2. Nevertheless, we can determine the influence of horse

Decrease in paprika fruit yield (about 95%) is noticed. It is worthy of note that a very low yield of paprika was not only due to distinct smaller number of fruits, but also due to their size. Also, significant decrease (about 50%) of vegetative part of paprika was observed. The effect of U. tomentosa extract on average yield of paprika (supplied to leaves) is shown in Table 2. Contrary to the experiment mentioned above we used the Uncaria doses considerably lower, 8.0 and 80.0 mg per plant, respectively. The highest dose (80.0 mg per plant) in this experiment corresponded to the lowest dose in that above mentioned. Significant increase in paprika fruit yield in comparison

Table 1 Average yield of vegetative and generative part of paprika (g) at different doses of U. tomentosa fed to soil Part of plant

Doses (mg per plant) 0.0 Fresh mass

Vegetative part Leaves Stalk Roots Total Generative part Fruits Total yield Number of fruits

24.15 19.66 1.85 45.86a 86.83a 520.96a 7

80.0

320.0

Dry mass

Fresh mass

Dry mass

Fresh mass

Dry mass

4.17 4.34 1.71 10.22A

12.40 10.30 1.15 23.85b

1.58 1.83 1.03 4.44B

14.72 12.17 1.56 28.35b

1.73 2.34 1.41 5.48B

5.01b 30.03b 2

4.89b 29.35b 2

Different letters ‘a and b’ (for fresh mass), and ‘A and B’ (for dry mass) indicate statistical significance at P < 0, 05.

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Table 2 Average yield of vegetative and generative part of paprika (g) at different doses of U. tomentosa fed to leaves Part of plant

Doses (mg per plant) 0.0 Fresh mass

A. vegetative part Leaves Stalk Roots Total B. generative part Fruits Total yield Number of fruits

48.42 45.85 5.07 99.34a

8.0

80.0

Dry mass

Fresh mass

Dry mass

Fresh mass

Dry mass

50.59 8.58 3.58 17.75A

46.56 49.09 5.70 101.35a

5.56 8.76 3.91 18.23A

48.60 48.38 6.29 103.27a

6.12 8.69 4.02 18.83A

39.48a 236.88a 9

70.58b 423.48b 10

73.85b 443.10b 10

Different letters ‘a and b’ (for fresh mass), and ‘A and B’ (for dry mass) indicate statistical significance at P < 0, 05.

manure on yield of biomass and fruits of paprika by comparison of control groups in both experiments. As we can see (Tables 1 and 2) horse manure affected mainly increase of biomass of paprika (99.34 g – control sample exp. 2, Table 2 in comparison to control sample exp. 1, Table 1 45.86 g).Unfortunately, increase of biomass was in correlation with decrease of yield of fruits (Tables 1 and 2) Against the background of horse manure action, an effect of Uncaria extract on yield vegetative and generative part of paprika in both experiments was distinct. As we can see, the manner of Uncaria extract supply: to soil or leaves, have an essential effect on the development and yield of paprika. In the case of Uncaria supply to the soil, the inhibition of paprika plant development and essential decrease in fruits yield are observed. Different doses of extract: 80.0 and 320 mg per pot per plant used in this experiment did not have any significant influence on this result. Simultaneously, observation of the behaviour of whole paprika plant (distinct flowering, fruiting acceleration, yellowing and falling of leaves) supports an evidence that addition of Uncaria extract to the soil creates disadvantageous, stress condition for plant development. The observed shortening of ontogenetic development of plants treated with the extract has its source in changes appearing in leaf cell ultrastructure. Although only leaf ultrastructure was investigated, similar changes, though of different intensity, must have appeared in whole plant.

Leaves of control plants had typical structure: mesophyll cells and strongly vacuolised epidermis, and cellular organelles located adjacent to cell walls in a narrow band of cytoplasm. The youngest leaves showed large chloroplasts, containing numerous grana and large starch grains, scarce small plastoglobules (Fig. 1); mitochondria with electron-dense matrix and distinct cristae, nucleus with dispersed euchromatin and distinct electron-dense ball-shaped aggregations in vacuoles (Fig. 2). The oldest leaves contained less starch, but in many chloroplasts, they had larger (Figs. 3 and 4) and more numerous plastoglobules. This is a commonly known feature of ageing leaves (Burton, 1966; Dodge, 1970; Hurkman, 1979; Nii et al., 1988). Also slight condensation of nuclear euchromatin was observed (Fig. 4), which again corresponds with the senescence program. In some plants, e.g. Nicotiana tabacum and Ornithogalum vivens, symptoms of natural senescence of leaves are much more intensive (Simeonova et al., 2000). The observations showed that in old leaves of control plants the amount of aggregations in the central cell vacuole was higher than in the youngest leaves. This indicates specific type of metabolism in paprika cells. Numerous biochemical investigations, carried out mainly on fruit cells due to usability of paprika, showed very wide range of substances deposited there (Somos, 1984). Many of them, like capsaicine and its derivatives typical for paprika, have phenolic origin.

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Figs. 1-4. Control plant: ultrastructure of young (1 and 2) and old (3 and 4) leaves. (1) Chloroplast of palisade parenchyma cell. Large starch grains (S). small plastoglobules (pg), properly formed grana (g) and stromal thylacoids (th) are visible; bar: l ␮m. (2) A fragment of bottom epidermis and spongy parenchyma. Large. round. electron-dense deposits (d) are located in vacuoles; bar: 2 ␮m. (3) A fragment of palisade parenchyma cells. Large plastoglobules (pg) and dilated thylacoids (arrow) are visible in a chloroplast; bar: 1 ␮m. (4) A nucleus of a palisade parenchyma cell. Chromatin condensations are visible (arrows); bar: l ␮m.

This may also apply to deposits described in this work. After application of Uncaria cortex extract into soil, significant differences appear as early as in the youngest leaves, as compared to the control plants. Electron-dense deposits almost totally disappear (Fig. 5). Disappearance of large black structures indicate possible variation in quality and quantity of natural products.

Another symptom of influence of extract of Uncaria are acceleration and intensification of ageing of leaves. Chloroplasts, which are the most vulnerable to this process, contained slightly broadened thylacoids even in the youngest leaves (Fig. 6). In the chloroplasts of the oldest leaves, the thylacoid system was disintegrated, grana were on the decline (Fig. 7), and plastoglobules were usually more numerous and extremely large in size. Due to condensation of euchromatin, the

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Figs. 5-8. A plant treated with U. tomentosa extract: ultrastructure of young (5 and 6) and old (7 and 8) leaves. (5) A fragment of bottom epidermis and spongy parenchyma. There are no deposits in the cells. in contrary to vacuoles of control plants (see (2)); bar: 2 ␮m. (6) A chloroplast of palisade parenchyma cell. Note dilatation of thylacoids (arrows); bar: 1 ␮m. (7) A fragment of spongy parenchyma cell. Large plastoglobules are visible in chloroplast matrix. Strongly disturbed structure of grana and thylacoids. The arrows show strongly dilated lunen of thylacoids; bar: 1 ␮m. (8) A fragment of palisade parenchyma cells. In the cell nucleus (N) large fragments of condensed chromatin (arrows) are visible. Chloroplasts (Ch) contain numerous large plastoglobules (pg); bar: 1 ␮m.

cell nucleus looked like mosaic (Fig. 8), which limits the transcription ability of such cells. Such condensation of particular transcription areas indicates strong influence of the extract on the plant metabolism. On the other hand, the spraying of leaves with Uncaria extract caused completely different behaviour of paprika plant. We did not observe any particular ef-

fect on plants development in the first days after extract treatment. The differences appeared in about 3 weeks after spraying and referred to flowering acceleration and maturation of paprika fruits. Finally, the spraying of paprika leaves resulted in significant increase of fruits yield. In spite of essential differences of Uncaria doses of 8.0 and 80 mg per plant in this

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experiment we did not observe any effect on paprika yield between them. Horse manure addition to the soil caused only distinct increase of vegetative part of plants, but decrease of yield of paprika fruits. It is very visible when we compare vegetative and generative parts of controls in both experiments (see Tables 1 and 2). Although different doses for both experiments were used, one dose, i.e. 80 mg per pot per plant was the same for both cases. Consequently, we can say that manner of Uncaria extract supply has an essential influence on development and yield of plants. We observed the same effect in the case of spinach (Spinacia oleraceae L.) where Uncaria extract was added to the soil or onto leaves (non published data). The explanation that manner of supply of the same preparation may have different influence on this same plant is not easy. Firstly, the chemical compounds of Uncaria extract added to the soil can undergo a change under the influence of different microbial enzymes present in the soil before absorption by roots of plant. Besides, leaf and root have different ways of absorption of applied substances. Thus, plant would be supported by Uncaria compounds metabolites. Such changes of chemical composition of Uncaria extract can have negative influence on development of plants. Second, the mechanisms of nutrients uptake by plants via the roots or the leaves are principally different. Hence, quite different compounds can be supplied to the plant cells dependently on the manner of their delivery. As mentioned in the Introduction, up to now the studies on U. tomentosa L. were directed towards their pharmacological properties. Therefore, we did not have an opportunity to compare and discuss our results with others. The results obtained for Uncaria extract presented in this work bear a resemblance to studies performed on biological activity of alkaloid-rich lupin extract (Kahnt and Hijazi, 1987, 1991; Gulewicz and Trojanowska, 1995; Gulewicz et al., 1996). These extracts of different genus of plants origin do not contain typical plant hormones like auxins, cytokinins, or giberelins. Both extracts from Lupinus and Uncaria show similarly distinct effects on plant development. Ultrastructural observations show its inhibitory properties after application of the extract into soil. The above supports an evidence mat extract–plant in-

teraction is very complex and probably based on the synergy of various components. Similar investigation on plants in the second experiment will allow further conclusions. An appropriate method for extract fractionation, able to separate groups of chemical compounds may significantly simplify this problem.

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