EXPERIMENTAL INVESTIGATIONS

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1 64 EXPERIMENTAL INVESTIGATIONS :64-7 Growth Dynamics of Perilla L. Species and Their Varieties During the Vegetation Period in Middle Lithuania Lina Raudonė 1, Valdimaras Janulis 1, Ona Ragažinskienė 2, Almantas Ražukas 3 1 Department of Pharmacognosy, Medical Academy, Lithuanian University of Health Sciences, 2 Kaunas Botanical Garden, Vytautas Magnus University, 3 Vokė Branch, Lithuanian Institute of Agriculture, Lithuania Key words: Perilla L.; medicinal plants; growth; medicinal raw material. Summary. The aim of the study was to investigate and determine the regularities of growth and development of the terraneous part (height and relative projection area) of the species and varieties of the genus Perilla L., namely Perilla frutescens (L.) Britton var. crispa f. viridis, Perilla ocymoides L. var. bicolorlaciniata, Perilla frutescens var. nankinensis laciniata, and Perilla frutescens (L.) Britton and to compare the dynamics of growth rate with that of Perilla frutescens (L.) Britton during their vegetation period under the climatic conditions of Middle Lithuania. Materials and Methods. The study was carried out in the medicinal plant collection and trial area at Kaunas Botanical Garden of Vytautas Magnus University in Meteorological factors of vegetation period the sum of average circadian temperatures and the amount of precipitation were taken from agrometeorological bulletins. Phenological observation and biometric measurements of the terraneous part of model plants (1 repeats) were performed during the vegetation period. Results. It was determined that Perilla frutescens (L.) Britton var. crispa f. viridis plants were higher, and their terraneous part developed best (P<.5) as compared with other plants of the genus Perilla L. Under the conditions of Lithuanian climate, P. frutescens var. crispa provided the greatest amount of vegetative raw material (herb). Perilla frutescens (L.) Britton var. crispa f. viridis, Perilla ocymoides L. var. bicolorlaciniata, and Perilla frutescens var. nankinensis laciniata are the new plants that have been cultivated under the conditions of Lithuanian climate for the first time. Conclusions. After performing further research on their introduction, the species and varieties of Perilla L. will enrich the diversity of medicinal plants and national genetic resources. Introduction In the recent years, more attention has been paid to research on biological characteristics, cultivation, preparation of medicinal raw material of foreign and local flora plants, and their phytochemical research (1). According to the requirements of the World Health Organization and Good Manufacture Practice, the quality of herbal raw materials must fulfill the parameters of safety, efficiency, and stability. Medicinal herbal raw materials from cultivated plants are of higher quality, which can be guaranteed by carrying out research on biological characteristics of cultivated plants, standardizing cultivation conditions, setting up optimal time for herbal raw material preparation, drying, and other conditions. It is necessary to investigate the biological characteristics of foreign plants, expand their assortment, and increase biological diversity and accumulation of national genetic resources (2, 3). For this reason, research on the biological characteristics of the genus Perilla L. plants was carried out under the conditions of Lithuanian climate. Herbal raw material and phytopreparations of Perilla L. species are used in the treatment of food poisoning, digestive problems, cold, asthmatic cough, and also in relieving or laxative preparations (4 7). Many scientific studies have been carried out, which have determined antimicrobial, immunostimulating, antiallergic, antioxidant, and antitumor effects of Perilla frutescens L. preparations (8 12). In Lithuania, research on the biological characteristics of Perilla frutescens (L.) Britton has been carried out as well. It has been determined that this species, growing in Lithuania, follows full vegetation process to fruit ripeness. The influence of meteorological factors on the growth and vegetation rhythmics of Perilla has been determined, and multiannual increment dynamics of the terraneous part during different vegetation periods has been analyzed (13, 14). Having researched the quality, antioxidant, an- Correspondence to L. Raudonė, Department of Pharmacognosy, Medical Academy, Lithuanian University of Health Sciences, A. Mickevičiaus 9, 4437 Kaunas, Lithuania raudone.lina@gmail.com Adresas susirašinėti: L. Raudonė, LSMU MA Farmakognozijos katedra, A. Mickevičiaus 9, 4437 Kaunas El. paštas: raudone.lina@gmail.com

2 Growth Dynamics of Perilla L. Species and Their Varieties During the Vegetation Period in Middle Lithuania 65 timicrobial, and immunomodulatory characteristics of the raw material of perilla cultivated in Lithuania, the possibilities of its wider practical application have been evaluated (15 17). Plants of Perilla frutescens L. varieties differ in their chemical composition and morphological features: herbal raw material (leaves, herb, fruits) used in Southeast Asian medicine depends on the variety of the cultivated plant (4, 5). Expanding the assortment of valuable medicinal vegetative raw material, biological characteristics of the genus Perilla L. plants Perilla frutescens (L.) Britton var. crispa f. viridis, Perilla ocymoides L. var. bicolorlaciniata, and Perilla frutescens var. nankinensis laciniata have been investigated, and productive species providing high-quality medicinal vegetative raw material used in medical practice and food industries have been selected. The aim of this study was to investigate and determine the regularities of growth and development of the terraneous part (height and relative projection area) of the species and varieties of the genus Perilla L., namely Perilla frutescens (L.) Britton var. crispa f. viridis, Perilla ocymoides L. var. bicolorlaciniata, Perilla frutescens var. nankinensis laciniata, and Perilla frutescens (L.) Britton and to compare the dynamics of growth rate with that of Perilla frutescens (L.) Britton during their vegetation period under the climatic conditions of Middle Lithuania. Material and Methods The following varieties of the genus Perilla L., Lamiaceae (dead nettle) family, were used in the study: Perilla frutescens (L.) Britton, Perilla frutescens (L.) Britton var. crispa f. viridis, Perilla ocymoides (L.) var. bicolorlaciniata, and Perilla frutescens (L.) var. nankinensis laciniata. The study on Perilla frutescens (L.) Britton (P. frutescens), Perilla frutescens (L.) Britton var. crispa f. viridis (P. frutescens var. crispa), Perilla ocymoides (L.) var. bicolorlaciniata (P. ocymoides), and Perilla frutescens (L.) var. nankinensis laciniata (P. frutescens var. nankinensis) was carried out in the medicinal plant collection and trial area at Kaunas Botanical Garden of Vytautas Magnus University in Meteorological factors of vegetation period the sum of average circadian temperatures and the amount of precipitation were taken from agrometeorological bulletins (18). The hydrothermal coefficient (HTC) was calculated according to the formula: HTC=H/.1ΣΤ, where H is the amount of precipitation in mm throughout the analyzed period; and ΣΤ is the sum of average circadian temperatures higher than 1 C of the same period (19). Plants were planted from sprouts with 3 leaves in rows of the trial area. The distance among plants were 5 cm and among rows 7 cm. Phenological observation and biometric measurements of the terraneous part of model plants (1 repeats) were performed during the vegetation period (2). Plant observation and biometric data were analyzed using Statistical Package for Social Sciences (SPSS, version 11.) and Microsoft Excel. The nonparametric Mann-Whitney criterion for independent samples was applied in order to compare morphometric data and mass quantities of plants during the vegetation. Significance level was chosen α=.5 (21). Results After analyzing P. frutescens growth under the conditions of Lithuanian climate, it was determined that the average maximum height of 77.2 cm was reached on day 229 of the year at the beginning of mass blossoming (in August) (14). When comparing the characteristics of dynamics of the height and relative projection area of the terraneous part of P. frutescens var. crispa and P. ocymoides with P. frutescens, the same regularities of growth were determined (Figs. 1 and 2). The highest relative growth rate of the relative projection area of the terraneous part was determined at the beginning of the vegetation (in June) (1% to 14% per day), and it stabilized (5% to 13% per day) during intense growth. P. ocymoides demonstrated the most intensive relative growth rate of the terraneous part at the beginning of the vegetation (18% per day), meanwhile P. frutescens showed the lowest relative growth rate (1% per day). On day 216 of the year (beginning of budding), the relative growth rate of all plants studied was 3% per day. During the vegetation period to day 229 of the year (at the end of August), there were no significant differences in the height and relative projection area of the terraneous part of different plants. The mean height of P. frutescens var. crispa and P. ocymoides was 87.4 cm and 7.8 cm, respectively (Table). At the end of August, the height and relative projection Table. Relative Projection Area and Height of the Terraneous Part of Species and Varieties of the Genus Perilla L. in Perilla L. variety S, cm 2 SE h, cm SE Perilla frutescens (L.) Britton Perilla frutescens (L.) Britton var. crispa f. viridis Perilla ocymoides (L.) var. bicolorlaciniata S, relative projection area; h, height; SE, standard error.

3 66 Lina Raudonė, Valdimaras Janulis, Ona Ragažinskienė, Almantas Ražukas 1 P. frutescens - h P. frutescens var. crispa - h P. ocymoides - h P. frutescens - V P. frutescens var. crispa - V P. ocymoides - V 25 Height, cm Day Growth Rate, % per Day Fig. 1. Dynamics of height (h) and its relative growth rate (V) of the terraneous part of Perilla L. species and varieties studied during the vegetation period (June October, 27 28) Relative Projection Area, cm P. frutescens - S P. frutescens var. crispa - S P. ocymoides - S P. frutescens - V P. frutescens var. crispa - V P. ocymoides - V Day Growth Rate, % per Day Fig. 2. Dynamics of relative projection area (S) and its relative growth rate (V) of the terraneous part of Perilla L. plants during the vegetation period (June October, 27 28) area of the terraneous part of P. frutescens var. crispa were significantly greater in comparison with other Perilla L. plants (P<.5). Herb, fruits, and leaves are vegetative raw material of Perilla used in medical practice (22, 23). After analysis of the dynamics of leaves (pieces) of the species and varieties of the genus Perilla L., the tendency of the number of leaves to increase was observed from the beginning of vegetation to day 23 of the year when the differences became significant. The number of P. frutescens var. crispa leaves was the greatest as compared with other Perilla L. plants (P<.5), and P. frutescens had the least number of leaves among all Perilla L. plants studied (Fig. 3). Analysis of raw material quantity in and dynamics of medicinal raw material (herb and leaves) quantity throughout vegetation periods as well as continuous growth of raw material mass of all perillas from the beginning of vegetation to day 23 of the year in August was performed. The comparative analysis of the increase in raw material quantity demonstrated no significant differences among all Perilla L. species and varieties from the beginning of vegetation to the beginning of blossoming. The growth of P. frutescens decreased during blossoming, whereas the growth rate of P. frutescens var. crispa did not decrease even at the end of vegetation. P. frutescens var. crispa provided the greatest quantity of herbal raw material (P<.5). There was no significant difference in the maximum amount of herbal raw material comparing P. ocymoides with P. frutescens, but the increment of both tended to decrease to the minimum at the end of vegetation. After evaluation of fresh and air-dried mass quantities of one model plant, it was determined that at the end of vegetation, P. frutescens var. crispa provided the maximum

4 Growth Dynamics of Perilla L. Species and Their Varieties During the Vegetation Period in Middle Lithuania 67 Number of Leaves P. frutescens P. frutescens var. crispa P. ocymoides Day Fig. 3. Number of leaves of the analyzed Perilla L. species and varieties during the vegetation period in June October, Mass, g P. frutescens var. crispa P. ocymoides P. frutescens Fresh material Dried material Fig. 4. Fresh and air-dried mass of Perilla L. model plants, September amount of fresh herb mass quantity (11±18.3 g fresh and 38±42.6 g air-dried), and P. frutescens provided the minimum quantity (398±63.8 g fresh and 113±16.3 g air-dried material) (Fig. 4). During the study in different years, the moisture content in the raw materials varied significantly, the maximum and minimum amounts of fresh and air-dried mass varied likewise. A correlation between HTC and the loss on drying (R 2 =.971; R=.985; P<.1) and between the dry mass part and HTC (R 2 =.97; R=.985; P<.5) was found. Perilla L. is a typical short-day plant, which blossoms when the length of the night is optimal for the species (24). In Lithuania, P. frutescens starts blossoming at the end of August; meanwhile, when cultivated in Egypt, it starts blossoming in July (13, 25). P. frutescens var. crispa and P. ocymoides that were grown in Lithuania in did not follow through all vegetation periods, and the seeds did not ripen. In 28, under the conditions of Lithuanian climate, the periods of plant budding and beginning of blossoming of P. frutescens var. nankinensis were observed at the beginning of October. The analyzed P. frutescens var. nankinensis did not effectively ripen seeds. Discussion The adaptation of the introduced plants to the new environmental conditions depends on their characteristics related to the growth rate, the nutrient use efficiency, resistance to herbivores, and seed maturation. The moisture index is one of the most important agrometerological indices, which characterize the conditions of the plant vegetation period. Most often, this agrometerological index is defined as a hydrothermal coefficient, proposed by Seleninovas (19). If the HTC is 1.6, it is considered excessive moisture; if , optimal moisture; if.9 to.8, mild drought; if.7 to.6, average drought; if.5 to.4, strong drought; and if <.4, very strong drought. The findings of this study revealed that the growth of P. frutescens depended on meteorological factors (13). The relative projection area of the terraneous part reached its maximum values in the presence of excessive humidity when the HTC was The evaluation of the relative projection area of the terraneous part of P. ocymoides revealed that the relative projection area was significantly greater in 28 than 27 (P<.5). This difference confirms the dependency of the relative projection area

5 68 Lina Raudonė, Valdimaras Janulis, Ona Ragažinskienė, Almantas Ražukas on the HTC. The relative projection area of P. frutescens var. crispa was also greater in 28 than 27; however, no statistically significant difference between the years was determined. The amount of P. frutescens dry raw material is greater, when the HTC is in the interval of the excess moisture. This is explained by the fact that the growth of the aboveground part of P. frutescens is most intensive before the bud formation and the start of the flowering period. It was found that the higher HTC during the intensive growth is, the later the bud formation occurs (correlation between the bud formation period and HTC, R 2 =.815; R=.93; P=.1). A similar tendency was found between the HTC and the start of the flowering period (R 2 =.494; R=.73; P<.5). The later the plants pass through the bud formation period, the more time is given for the vegetative growth and the plant grows the greater mass of the medicinal herbal raw material. It was found that Perilla L. species and varieties grow the most of the herbal raw material (herb) mass during the intense growth period. Separate varieties of Perilla L. species differ in the way they react to day length (26). Under the conditions of Lithuanian climate, only P. frutescens follows all vegetation periods to seed ripening (13). At the time when night length becomes optimal for plant growing and blossom formation, P. frutescens var. crispa, P. ocymoides, and P. frutescens var. nankinensis shift to the end of vegetation period. Due to too low temperatures during the day, the vitality of plants decreases; part of leaves and stems die and fall down. For the purposes of the perspectives of Lithuanian medicinal herb cultivation development, it is important to select the species and of Perilla L., which flower during vegetation, mature the seeds, and are suitable for reproduction. Seed maturity period of P. frutescens var. nankinensis laciniata plants has also been registered; however, it is not effective and meets the end of the growing season. The collected amount of seeds is not sufficient for the successful cultivation of P. frutescens var. nankinensis laciniata plants in Lithuania. The third stage of ontogeny has not been detected in P. ocymoides var. bicolorlaciniata and P. frutescens var. crispa f. viridis; therefore, their cultivation under the Lithuanian climate conditions is not effective. The ontogeny of the investigated Perilla L. species and varieties affect the plant growth characteristics during the vegetation period. The ability to mature the seeds and to pass through all the development stages is one of the main characteristics when introducing new species. The genetic origin of the plant, the quantity of growth regulators, and the ecologic factors determine the periods of plant flowering and maturity. P. frutescens plants are the ones that most effectively go through all development cycles under the Lithuanian climatic conditions. Their seed maturation period covers 95% to 1% of plants and ends when the environmental conditions are still favorable for growth. The beginning and duration of the seed maturation depends on the sum of active air temperatures (R 2 =.865; R=.93; P<.5). The seed maturation begins earlier, when the sum of active air temperatures is higher. Dependence between the seed maturation and the flowering periods has been determined (R 2 =.628; R=.792; P=.6). After the evaluation of growth dynamics during the vegetation and dependency of blossoming on day and night length and hydrothermal coefficient of the terraneous part (relative projection area, height, and number of leaves) of the genus Perilla L. plants, namely Perilla frutescens L. Britton var. crispa f. viridis, Perilla ocymoides L. var. bicolorlaciniata, Perilla frutescens var. nankinensis laciniata, and Perilla frutescens (L.) Britton, characteristics of growth and development of plants studied were determined for the climatic conditions in Middle Lithuania. The comparison of growth and developmental characteristics of new varieties of Perilla frutescens (L.), i.e., Perilla frutescens L. Britton var. crispa f. viridis, Perilla ocymoides L. var. bicolorlaciniata, and Perilla frutescens var. nankinensis laciniate, grown in Lithuania for the first time revealed the superiority of Perilla frutescens L. Britton over the abovementioned varieties and its better adaptation to Lithuanian climatic conditions. The field tests determined that not all of the Perilla L. species and varieties are suitable for cultivation under the Lithuanian climatic conditions. Studies on the productivity of medicinal plants under the certain cultivation conditions are relevant for the herbal cultivation development perspectives. It is important to cultivate and prepare the highquality medicinal herbal raw materials, which can be applied in producing phytopreparations. Productivity analysis was carried out in order to assess the quantity of Perilla L. species and varieties herbal raw materials. It is important to assess the relation between the increment of the aboveground medicinal herbal raw material and the meteorological factors throughout the different years in Lithuania. The data patterns of these investigations support the productivity forecasts of Perilla L. species and varieties aboveground part raw materials. Conclusions Research on the biological characteristics of Perilla frutescens (L.) Britton var. crispa f. viridis, Perilla ocymoides L. var. bicolorlaciniata, and Perilla frutescens var. nankinensis laciniata cultivated under

6 Growth Dynamics of Perilla L. Species and Their Varieties During the Vegetation Period in Middle Lithuania 69 the conditions of Lithuanian climate was performed. It was determined that not all Perilla L. species and varieties followed a complete developmental cycle to seed maturity. The morphometric research on the terraneous part of the plants demonstrated that Perilla frutescens (L.) Britton var. crispa f. viridis plants were higher, and their terraneous part was developed best as compared with other plants of the genus Perilla L, although P. frutescens had better adaptation under the Lithuanian climatic conditions. After performing further research on their introduction, the species and varieties of Perilla L. will enrich the diversity of medicinal plants and national genetic resources. Acknowledgments This study was supported by the Scientific Foundation, Lithuanian University of Health Sciences (former Kaunas University of Medicine). Statement of Conflict of Interest The authors state no conflict of interest. Perilla L. genties rūšių ir jų varietetų augimo dinamika vegetacijos laikotarpiu Vidurio Lietuvoje Lina Raudonė 1, Valdimaras Janulis 1, Ona Ragažinskienė 2, Almantas Ražukas 3 1 Lietuvos sveikatos mokslų universiteto Medicinos akademijos Farmakognozijos katedra, 2 Vytauto Didžiojo universiteto Kauno botanikos sodas, 3 Lietuvos žemės ūkio instituto Vokės skyrius Raktažodžiai: Perilla (L.), augimas, vaistinė augalinė žaliava. Santrauka. Tyrimo tikslas. Ištirti ir nustatyti augimo ir vystymosi dėsningumus Perilla L. genties rūšių ir varietetų: Perilla frutescens (L.) Britton var. crispa f. viridis, Perilla ocymoides L. var. bicolorlaciniata, Perilla frutescens var. nankinensis laciniata ir Perilla frutescens (L.) Britton antžeminės dalies (aukščio, santykinio projekcinio ploto, antžeminės dalies masės) bei augimo greičio kitimą lyginant su Perilla frutescens (L.) Britton vegetacijos laikotarpiu Vidurio Lietuvos klimato sąlygomis. Medžiaga ir metodai. Tyrimai atlikti m. vegetacijos laikotarpiu (gegužės spalio mėn.) Vytauto Didžiojo universiteto Kauno botanikos sodo vaistinių augalų kolekcijoje ir bandymų plote. Vegetacijos laikotarpio meteorologiniai veiksniai, t. y. vidutinė paros oro temperatūrų suma ir kritulių kiekis, naudoti iš agrometeorologinių biuletenių. Fenologiniai stebėjimai bei modelinio augalo (1 pakartojimų) antžeminės dalies biometriniai matavimai atlikti vegetacijos tarpsniais. Rezultatai. Tirtų augalų antžeminė dalis geriausiai išsivysto iki žydėjimo tarpsnio, kai yra perteklinis drėgmės kiekis (HTC=1,95). Atliekant morfometrinius augalų antžeminės dalies tyrimus, nustatyta, kad Perilla frutescens (L.) Britton var. crispa f. viridis augalai yra aukštesni ir jų antžeminė dalis geriausiai (p<,5) išsivysčiusi lyginant su kitais tiriamais Perilla L. genties augalais. Nustatyta, kad Lietuvos klimatinėmis sąlygomis daugiausia augalinės žaliavos (žolės) teikia P. frutescens var. crispa. Perilla frutescens (L.) Britton var. crispa f. viridis, Perilla ocymoides L. var. bicolorlaciniata, Perilla frutescens var. nankinensis laciniata tai nauji augalai pirmą kartą pradėti auginti Lietuvos klimato sąlygomis. Išvados. Atlikus tolesnius introdukcijos tyrimus, Perilla L. rūšys ir varietetai praturtins vaistinių augalų įvairovę ir nacionalinius genetinius išteklius. References 1. Briskin PD. Medicinal plants and phytomedicines. Linking plant biochemistry and physiology to human health. Plant Physiology 2;124: Radušienė J, Janulis V. Vaistinių ir aromatinių augalų įvairovės tyrimo, jų panaudojimo bei išsaugojimo tendencijos. (Improvement of diversity, trade and conservation of medicinal and aromatic plants.) Medicina (Kaunas) 24; 4(8): Motiekaitytė V. Conservation diversity of vascular plants and their communities in situ, applying the conception of ecosystem pool. Ekologija 26;2: Brenner DM. Perilla: botany, uses, and genetic resources, In: Janick J, Simon JE, editors. NY: New Crops, John Wiley and Sons; p He-Ci-Yu, Kosuna K, Haga M. The genus Perilla. Netherlands Overseas Publishers: Association; p Honda G, Koezuka Y, Kamisako W, Tabata M. Isolation of sedative principles from Perilla frutescens. Chem Pharm Bull 1986;34(4): Ueda H, Yamazaki C, Yamazaki M. Liuteolin as an antiinflammatory and anti-allergic constituent of Perilla frutescens. Biol Pharm Bull 22;25(9): Makino T, Furuta Y, Fuji H, Nakagawa T, Wakushima H, Saito K, et al. Effect of oral treatment of Perilla frutescens and its constituents on type-i allergy in mice. Biol Pharm Bull 21;24(1): Kang R, Helms R, Stout MJ, Jaber H, Chen Z, Nakatsu T. Antimicrobial activity of the volatile constituents of Perilla frutescens and its synergistic effects with polygodial. J Agric Food Chem 1992;4: Xu M, Floy HS, Greth SM, Chang WL, Lohman K, Stoyanova R, et al. Perillyl alcohol-mediated inhibition of lung

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