CC BY
19
UDC 602.4+581.524.13.:549.892.1 https://doi.org/10.15407/biotech10.06.053
BIOLOGICAL ACTIVITY OF AQVEOUS SOLUTION
OF AMBER
Gryshko National botanical garden of the National Academy of Sciences of Ukraine, Kyiv Litvinenko Institute of Physical-Organic Chemistry and Coal Chemistry of the National Academy of Sciences of Ukraine, Donetsk
Institute of Physics of the National Academy of Sciences of Ukraine, Kyiv
E-mail: levchyk.n@ukr.net
Received 23.09.2017
The aim of the research was to detect the biological activity of various samples of amber from the Trancedniestria province, and to characterize their effect on the physiological processes of higher plants. The allelopathic and cytostatic effects of water solutions of amber were studied using biotesting methods in open soil conditions and in culture in vitro. The test-object was the seedlings of cucumber Cucumis sativus L. It was shown that the activity and nature of the action of the samples depend on the region of origin of the amber, the degree of shredding and concentration of the solution. According to the results, fine fractions of amber show biostimulating effects on plants. Thus, amber can be effectively used for cultivating agricultural plants in vivo and in vitro conditions. For stimulation of the increasing of the plant stem mass it is recommended to use amber B1, to increase the mass of roots — amber B4, and amber B2 — to stimulate stem growth in length.
Key words: amber, succinic acids, allelopathic and cytostatic action.
N. Ya. Levchyk A. V. Liubinska1 N. V. Skrypchenko1 O. I. Dziuba1 N. M. Kachalova2 3 O. L. Mironov2 3
1
2
3
A common challenge of our time is achieving a healthy living style. A prominent feature of the approach is the consumption of wholesome foods, grown without fertilizers or pesticides.
One of such natural organic compounds might be amber, natural organic yellow, brown and red substance containing succinic acid. It is fossilized coniferous resin of Upper Cretaceous and Paleogenic periods, 15-40 MY in age. It occurs as stalactites, drops, lens conglomerates of "resin pockets" and their chips, ranging in size from 0.02 to 50 cm (usually 2-30 cm). In Europe, amber is found in the range from Southern Sweden to the shores of the Black and Azov Seas, including the part of Ukraine to the west of the Dnieper river. The region known as "the Baltic-Dnieper amber-bearing province" [1] is the main source and provider of the stone to the world markets [2]. In Ukraine, amber occurs in Zhytomyr, Lviv, and Kharkiv regions [3]. The identification of those amber samples according to the characteristic patterns of infra-red light absorption spectra did not show substantial differences between them and the
samples from the Baltic states and Belarus, which supports their placing in the Baltic-Dnieper amber-bearing province [1, 4, 5].
The hypothesized possibility to use amber to rehabilitate and protect plants from various kinds of damage is based on the very nature of resin flow. Amber contains a stimulator of plant growth, succinic acid; terpenes with bactericide properties; organo-mineral complexes and microelements, various resinous acids with fungicide properties; and phytosteroids which are structurally similar to steroids (hormones) of the human body and have powerful anti-inflammatory and antibacterial effect.
The biological activity of succinic acid was revealed a long time ago. Nowadays, spurred by the search for human- and environment-friendly agents, it became increasing important as a natural growth and productivity enhancer and stress protectant [6-13].
Besides amber, succinic acid (HOOCCH2CHCOOH) is found free and bound in the tissues of many plants and animals, especially in unripe fruit, and in brown coal. It is a colorless crystalline powder tasting much like citric acid.
The research of Zaimenko [14] showed the possibility of managing the growth processes, physiological properties of plants of different ecological morphotypes and of different carbon metabolism by adding succinic acid. Also, the acid positively influences the biosynthesis of aminoacids in plants and can be a substrate for this biosynthesis. It was found that succinic acid content in the leaves of barley seedlings, cultured on 0.005 M solution of succinic acid, increased by 51%, while the water control increased by 39% [15].
Amber which contains succinic acid according to Elroy Rice's classification of the chemical nature of allelopathic agents is a compound of secondary posthumous origin in dead plant tissue [16]. Biogenic excretions are extraordinarily significant for the chemical interaction between organisms at different levels of their existence. They can inhibit or facilitate various vitally important functions (germination of spores and pollen, seed vitality, seedling growth etc.), affecting the life of whole biogeocoenoses and the function of balanced processes in the biosphere [17].
The aim of our work was to study the biological activity of aqueous solutions of various samples of amber from the Baltic-Dnieper province using classic techniques, and to characterize their effect on the physiological processes of higher plants.
Bearing in mind that more than 70% of amber are of value for jewelry, and there is a lot of waste when it's developed, the amber's biological properties and the possibilities of using it as an efficient and safe bioregulatory agent for cultured plants is a matter of practical importance. Research in the field of rational use of minerals obsoletes the practice of selling the raw materials and expedites the introduction of the end products into environmentally safe horticulture.
Materials and Methods
For this study, the samples of amber from Ukrainian deposits were differentiated by color into four groups: B1 darker, opaque dark-amber (or transparent, brown) color; B2 lighter, yellowish, lemon, light-amber color, translucent and transparent; B3 dark red or cherry-red, translucent and transparent; B4 mechanical mixture of all of the above. Most of the samples came from the deposits of Zhytomyr region (Olevskiy district) and Rivne regions (Klyosove and Volodymyrets) and belonged to the B1, B2 and B3 groups. Most of the B4 amber came from Volhyn region.
Fragments of B1 amber (dark-brown to reddish) were milled to 300-500 pm, lighter B2 (amber, lemon-yellow) to 100-200 pm. According to [3], dark ambers have higher ratios of resin acids (abietic acid and its derivatives) to succinic acid, and more different microelements.
The samples were analyzed using infra-red spectroscope Vertex-70, Bruker. Fragments of Ukrainian amber were milled en masse using laboratory mill Kinematica AG, model Polymix® PX-MFC 90 D to obtain particles of 50-500 pm.
Studies of the allelopathic and cytostatic activities of aqueous solutions of various amber samples were conducted after the widely used techniques of Grodzinsky and Ivanov [17, 18]. We studied the effect of the various samples on the rates of biomass growth of test plants, both above and below ground.
As the test culture, we used seedlings of cucumber (Cucumis sativus L.), sort Konkurent. Biotests to reveal allelopathic activity were replicated on 1- and 3-4-days-old seedlings, using 0.1, 0.5, 1.0 g/l concentrations of aqueous solution of amber of the B1-B4 groups. Cytostatic activity of amber was evaluated by counts of lateral roots.
To conduct in vitro tests, cucumber seeds were sterilized for 1 min in 70% ethanol, then for 2 min by 0.05% sodium merthiolate, and then planted in MS cultural medium with the addition of amber powder at the concentrations of 0.15 and 1.5 g/l. The experiment was carried out in two replicas. The results were visually evaluated by morphometry. All laboratory research was carried out at +26 °C.
The data were statistically treated using EXCEL 7.0.
Results and Discussion
Allelopathic and cytostatic activity of amber. Studying allelopathic and cytostatic activities of the four amber sample groups revealed that the aqueous solution of B2 amber, regardless of the concentration, also had significant the most effective allelopathic activity, it stimulated the growth of one-day-old cucumber seedlings in the range of 126.0-133.8%. Meanwhile, on three-days-old seedlings we saw significant depression of root growth by the solution at 0.1 g/l and notable stimulation at 0.5 g/l and 1.0 g/l. The most efficient was aqueous solution of B2 amber at 0.5 and 1.0 g/l (Fig. 1).
The solution also was high effective (131.48%) for B1 amber at 0.5 g/l. The effect was recorded during all three days of the experiment (Fig. 1).
Aqueous solution of B3 amber, regardless inhibited also the growth of four-days-old
of the concentration, exhibited an inhibitory ones. At 0.1 g/l and 0.5 g/l, we saw slight
effect on one-day-old cucumber seedlings, stimulation of the growth of four-days-old
and at the concentration of 1.0 g/l, it seedlings (Fig. 1).
H Control ■ 1 -day u 3 -day
127.69="
A
13 US* 131,12*
126:15* 12J74»
B
133,8*
C
105,5* 1019
D
93,73* 93J*
Fig. 1. Allelopathic activity of the aqueous amber solutions (% compared to control), depending on the concentration depending on the concentration
Hereafter: (% to control — 100%);1 — 0.1 g/l, 2 — 0.5 g/l, 3 — 1.0 g/l: A — sample B1; B — sample B2; C — sample B3; D — sample B4; * P < 0.05
The solution of B4 amber revealed slight inhibitory effect on the cucumber seedlings at 0.5 g/l and 1.0 g/l, while at 0.1 g/l it had a slight stimulating effect.
Functional activity of amber solutions can occur not only as the ability to have an allelopathic effect on the plant as a whole and its organs, but on the cells, too, through its ability to impact their proliferation, that is, in its cytostatic activity [19]. The cytostatic activity lies in the selective inhibition of mitosis in the main roots of the seedlings, and
in the strongly inhibited growth of lateral roots.
Studying cytostatic activity of the aqueous solution of B1 amber at different concentrations, we saw significant stimulatory effect on the cellular proliferation during the first day of the experiment, in particular, the initiation of lateral roots. Immediately after germination, the lateral roots in the experimental plants were initiated at 474.4-566.7%, compared to control (Fig. 2). However, on the third day the level of activity decreased to the level of 121.0-142.9%.
1 Control » l-dav "3-dav
A
B
100,0
110,26* 107,94*
i Control el-day h 3-day 119.2Î*
100,0
C
M Control II-day u4-day 121^67*
D
Control ■ 1-day u 4-day 153,8*
100,0 101.39
194.S*
1 2 * Fig. 2. Cytostatic activity of amber solutions depending on the concentration
B2 amber exhibited a relatively small stimulating action, 119.2% on the first day at 0.5% amber solution and 123.4% at 0.1% (Fig. 2). Significant stimulating action of the B3 amber solution in the range of 106.7-161.7% was seen on the first day of seed germination. However, on the fourth day, at 0.1 g/l and 1.0 g/l, there was a slight cytostatic action in the range of 91.1-96.8%. A significant activation of proliferation by the B4 solution in the range of 155.8-194.8%, independently of concentration was seen on the first day of seed germination, but leveled out on the fourth day.
Thus, amber and its water solutions are active stimulating substances towards plants during germination. However, only slight cytostatic activity was revealed under the influence of B3 amber. The effect's size and character depend on the amber's origin and the solution's concentration.
Testing the solution's effect on the growth and development of cucumber seedlings in vitro. Currently, urgent biotechnological research includes studying genetically transformed medicinal plants which produce their own biologically active compounds (BAC), including fructans, artemisinin and recombinant substances (interferon, micobacterial antigens) with prebiotic, hepatoprotectant, antidiabetic, antitumour, antimalarial, antioxidant, and UV-protectant properties, useful in the treatment of viral, bacterial and other diseases [20]. There are collections of such plants which produce a whole range of natural and recombinant BAC for cosmetology, medicine and veterinary. For successful culture of such plants in order to obtain industrial quantities of BAC, it's important to search for new natural growth factors to compose cultural media.
The experiments showed that B2 amber, regardless of concentration, is the most fficient factor promoting plant growth and development in vitro, compared to B1, B3 and B4. This advantage is seen in greater size, number of leaves and leaf size in experimental plants. The average values were obtained for B3 samples, and the lowest for B1 and B4.
Test plants on MS medium with B2 amber topped control plants in height by 28-38% and by 10-48% on MS medium with B1, B3 and B4 ambers (Fig. 3, 4).
Thus, our research showed that amber solutions did strongly stimulate the growth and development of test plants during early ontogenesis. The level of such stimulation depends not only on the concentration of amber in the cultural medium, but also on the region of its origin. Probably, the difference in activity is tied to the difference in composition (% of succinic acid and other components of amber), as well as to the degree of milling.
It was found that in vivo the solutions of studied samples of amber also differ in influence on the growth of above- and belowground parts of test plants, stimulating or inhibiting it (Fig. 5).
To establish the action of amber solutions we chose the parameters of growth of above-and belowground parts of test plants: mass and the increase of the relevant parts compared to dry substance,%.
Maximal effect on growth activation of the aboveground parts of the seedlings was seen in the B1 amber solutions, somewhat lower in B4. Clearly, these amber samples at a certain dilution have the optimal concentration and ratio of resin acids (abietic acid and its
13S.41*
■j| lY m<51'
90 33* ^H 100.93 100:0 ^^ ^H JjjL 100,0
mil lllli
MS+0,15 mgl MS-1.5 mg,l
Fig. 3. Morphometric characteristics of test plants in vitro depending on the concentration of ambers B1 — B4 (% to control)
derivatives) to the succinic acid and the microelements suitable to stimulate the growth of the aboveground part of the plants.
We observed a tendency, for B1 and B2 samples, independently of the amber concentration, to activate the aboveground growth of the test plants and also to significantly inhibit their root systems. It should be noted that 0.1 g/l and 0.5 g/l solutions of B3 amber strongly
inhibit both aboveground and root mass compared to control. Meanwhile, at 1.0 g/l its action is practically invisible.
All in all, the highest aboveground growth of the test plants in the range of 18-21% was observed in B1 solutions, regardless of concentration. However, the highest growth of root system of about 8% is expected in a B4 solution at 0.5 g/l.
Fig. 4. Eight-days-old cucumber seedlings on MS+amber medium (from left to right): control; 0.15 g/l; 1.50 g/l
120,0" 121,0' ' no-
I Above gi omid mass h Belowgrouud mass
110,0'
10S,0"
110.0"
9S,0
Control El: 0.1 0,5 g] 1.0 gl H
52: 0.1 0,5 gl 1.0 gl gl
33: 0.1 0_5 gl 1,0 g! gl
B 4:0,1 gl 1.0 gl Sl
Fig. 5. The growth of above- and belowground mass of test plants depending on concentrations
of the B1—B4 amber solutions
Studying the effect of water solutions of varying concentrations of different ambers on allelopathic and cytostatic activity, and also on cultural plants in vitro showed the following:
- the highest allelopathic activity was seen on a water solution of B2 amber, while its 0.1 g/l solution had an inhibitory effect;
- during the first day, the highest stimulatory activity of was seen in the B1 amber sample, regardless of the concentration;
- in vitro the highest stimulating effects were seen when we added to the MS medium a sample of B2 amber at 0.15 g/l and 1.5 g/l; if we added B1 amber at 0.15 g/l we saw the maximal inhibition of the plant life processes;
- amber solutions in vitro act differentially on the aboveground parts of the test plants and on their root systems, influencing growth parameters and the intensity of growth of the green mass of the plants.
To stimulate the growth of stem mass of the plants B1 amber should be taken, to increase the mass of the root — amber sample B4 and B2 — to stimulate the stem growth in length.
Therefore, the research demonstrated that the wastes and fine fractions of amber are a valuable source of natural complex chemical compound with growth stimulation for plants in vivo and in vitro. Amber in general, and its water solutions in particular could be very efficiently applied to plant cultivation.
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Б1ОЛОГ1ЧНА АКТИВН1СТЬ ВОДНИХ РОЗЧИН1В БУРШТИНУ
Н. Я. Левчик1 А. В. Любшська1 Н. В. Скрипченко1
А. I. Дзюба1 Н. М. Качалова2, 3 О. Л. Миронов2' 3
Нащональний боташчний сад ím. М. М. Гришка Нащонально1 академи наук Укра1ни, Ки1в 21нститут фiзико-органiчноl xímíi та вуглехiмп ím. Л. М. Литвиненка
НАН Укра1ни, Ки1в 31нститут фiзики НАН Укра1ни, Ки1в
E-mail: levchyk.n@ukr.net
Метою дослiдження було виявити бмлопч-ну активнiсть рiзних зразкiв бурштину з Придшстровсько1 провшцп й охарактеризува-ти 1х вплив на фiзiологiчнi процеси вищих рослин. Вивчали алелопатичну i цитостатичну дт бурштину методами бiотестування за умов в^критого Грунту та в культурi in vitro. Тест-об'ектом слугували проростки опрка Cucumis sativus L. Показано, що активнiсть i характер дй зразкiв залежать вiд областi похо-дження бурштину, ступеня подрiбнення й концентрацп розчину. Згiдно з результатами дрiбнi фракцп бурштину виявляють бш-стимулювальну дiю на рослини. Таким чином, бурштин може ефективно застосовуватися для культивування мльськогосподарських рослин за умов in vivo та in vitro. З метою стимулю-вання зб^ьшення маси стебла рослин дощль-но використовувати бурштин B1, для зб^ь-шення маси коренiв — бурштин В4, а бурштин B2 — для стимулювання росту стебла у довжи-ну.
Ключовi слова: бурштин, бурштинова кислота, алелопатична та цитостатична дiя.
БИОЛОГИЧЕСКАЯ АКТИВНОСТЬ ВОДНЫХ РАСТВОРОВ ЯНТАРЯ
Н. Я. Левчик1 А. В. Любинская1 Н. В. Скрипченко1
А. И. Дзюба1 Н. М. Качалова2, 3 О. Л. Миронов2, 3
Национальный ботанический сад им. Н. Н. Гришко Национальной академии наук Украины, Киев 2Институт физико-органической химии и углехимии им. Л. М. Литвиненко НАН Украины, Киев 3Институт физики НАН Украины, Киев
E-mail: levchyk.n@ukr.net
Целью исследования было выявить биологическую активность различных образцов янтаря из Приднестровской провинции и охарактеризовать их влияние на физиологические процессы высших растений. Изучали аллело-патическое и цитостатическое действие янтаря методами биотестирования в условиях открытого грунта и в культуре in vitro. Тест-объектом служили проростки огурца Cucumis sativus L. Показано, что активность и характер действия образцов зависят от области происхождения янтаря, степени измельчения и концентрации раствора. Согласно результатам мелкие фракции янтаря проявляют биостимулирующее действие на растения. Таким образом, янтарь может эффективно применяться для культивирования сельскохозяйственных растений в условиях in vivo и in vitro. С целью стимулирования увеличения массы стебля растений рекомендовано использовать янтарь B1, для увеличения массы корней — янтарь В4, а янтарь B2 — для стимулирования роста стебля в длину.
Ключевые слова: янтарь, янтарная кислота, аллелопатическое и цитостатическое действие.
