Nghiên cứu đặc điểm sinh vật học của cây ban âu (hypericum perforatum l ) và một số biện pháp kỹ thuật canh tác nâng cao năng suất, chất lượng dược liệu tại tân lạc, hòa bình ttta

i MINISTRY OF EDUCATION MINISTRY OF AGRICULTURE AND TRAINING AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES  TRAN DANH VIET STUDY ON THE MORPHOLOGY OF HYPERICUM PERFORATUM L AND SOME CULTIVATION TECHNIQUES TO IMPROVE THE YIELD AND QUALITY OF THIS MEDICINAL PLANT GROWING IN TAN LAC, HOA BINH Major: Crop Science Code: 9620110 SUMMARY OF AGRICULTURAL DOCTORAL THESIS Ha Noi, 2023 ii The thesis was completed at: VIETNAM ACADEMY OF AGRICULTURAL SCIENCES Supervisors: Prof.Dr Doan Thi Thanh Nhan Dr Nguyen Ba Hoat The thesis will be defended before the Thesis Assessment Council at the institute level in the Vietnam Academy of Agricultural Sciences (VAAS) At h, date , month , 2023 Be able to search the thesis at: National Library of Vietnam The Library of Vietnam Academy of Agricultural Sciences INTRODUCTION The necessity of the study Hypericum perforatum L is known by other names as Tipton grass, St John’s Wort H perforatum L is native to Europe, then was introduced to the US, and Australia and grows wild in many grasslands It has a natural geographical distribution by temperature in the subtropics of Europe, North America, Asia Minor, India, Russia, and China H perforatum L has been used for a long time, the stem when the flower blooms is the part used The use of this species as an herbal remedy to treat a variety of internal and external ailments dates back to the time of the ancient Greeks Since then, it has remained a popular treatment for anxiety, depression, cuts, and burns, and the plant's oil is used to heal scars, heal wounds, and relieve pain quickly Recent research suggests the effectiveness of this herb in treating other ailments, including cancer, inflammation-related disorders, and bacterial and viral diseases, and as an antioxidant and neuroprotective agent It seems to work best for mild to moderate depression, which is a common disease today, The side effects of drugs containing H perforatum L are fewer than that of some other drugs In 2006, National Institute of Medicinal Materials imported H perforatum L Into Vietnam, the plant can grow, develop well, flowered, and obtain seeds in cool climates such as Tam Dao (Vinh Phuc), Sa Pa (Lao Cai) However, to expand production, towards creating a stable growing area of H perforatum L medicinal herbs, and supplying raw materials for domestic and exporting enterprises, it is necessary to continue researching H perforatum L in Vietnam Research on the biological traits of the H perforatum L is crucial to developing planting models and selecting important farming practices Therefore, we carry out the topic “Study on the morphology of Hypericum perforatum L and some cultivation techniques to improve the yield and quality of this medicinal plant growing in Tan Lac, Hoa Binh” Research objectives 2.1 General objective Evaluation of morphology of H perforatum L., research on some key cultivation techniques contributing to build the protocol for the production of H perforatum L medicinal herbs for high yield, good quality in the cool mountainous area of Tan Lac district, Hoa Binh province 2.2 Detailed objectives - Identify the morphology of H perforatum L growing in Tan Lac- Hoa Binh - Identify some main cultivation techniques (season, density, fertilizer, and time to harvest) to create the protocol to produce medicinal material from H perforatum L growing in cool places of Tan Lac – Hoa Binh - Applying synchronously technical measures to build a farming model and evaluating of economic efficiency of planting H perforatum L in mountainous places of Tan Lac - Hoa Binh The scientific and practical significance of the study 3.1 Scientific significance The results of the study provide new scientific data on the biological characteristics of H perforatum L grown in Tan Lac (Hoa Binh) and some technical cultivation measures to improve the productivity and quality of medicinal herbs The results of the study are references for other research, teaching, and production of H perforatum L medicinal herbs in Viet Nam 3.2 Practical significance - The research results of the topic have identified some main cultivation techniques on the season, density, fertilizer, and harvest time of H perforatum L in Tan Lac (Hoa Binh) to achieve high medicinal yield, good active ingredient content - Adding a new medicinal plant to the crop structure of Tan Lac (Hoa Binh), enhancing exploitation and use of land efficiently, improving income and people's living standards in the northern mountains - Building up the H Perforatum L herbal production area to supply domestic and export enterprises Scope of study The study was conducted in Nam Son commune, Tan Lac district, Hoa Binh province Nam Son commune is one of the five highland communes of Tan Lac district, with an altitude of 850 - 900 m above sea level, a cool climate, an average annual temperature of 20 – 250C, and a rainfall of 1800 - 2000 mm which are suitable for ecological requirements of H perforatum L New contributions of the thesis The thesis is the first one in Vietnam to evaluate morphological traits including growth, development, morphology, and anatomy (stem, branches, leaves, flowers, fruits, seeds) of H perforatum L growing in the cool place of Tan Lac, Hoa Binh The thesis has researched and completed some cultivation measures such as planting season, density, fertilizer, and harvest time to serve as a scientific basis for building the process of cultivating H perforatum L in Tan Lac, Hoa Binh to achieve high yield and good active ingredient to meet the needs of mass production of H perforatum L medicinal herbs The research has analyzed and evaluated the content of hypericin, the main active ingredient in H perforatum L., reaching 0.169%, which is equivalent to the results published in Europe, America, and other countries Structure of the dissertation: The dissertation includes 117 pages: (1) Introduction on pages, (2) Charpter 1: Literature review on 40 pages, (3) chapter 2: Research materials, contents and methods on 15 pages, (4) Chapter 3: Research results and discussion on 55 pages, Conclusion and recommendations on pages, using 130 references (12 Vietnamese documents, 114 English documents, and Internet documents) CHAPTER LITERATURE REVIEW 1.1 General introduction of H perforatum L The genus Hypericum consists of 484 species distributed in many regions in the world H perforatum L is native to temperate parts of subtropical, it occurs throughout Europe, except northernmost H perforatum L followed Europeans wherever they went in the world and was introduced into North and South America, North Africa, South Africa, Asia, New Zealand, and Australia H perforatum L was imported into Vietnam in 2006, and firstly, it was called Ban Au di thuc or Ban Au It has been evaluated for adaptation in some ecological regions such as Hanoi, Tam Dao (Vinh Phuc), and Sa Pa (Lao Cai) 1.1.2 Botanical characteristics St Johnswort is an herbaceous plant, an annual or 2-3 years, when the plant matures, it becomes woody at the base It can grow to 0,3-1m Stems are straight and have many branches, there may be many stems from the base of the plant The stem can spread above the ground and give rise to roots at the site of contact Stems are bright green and often reddish Leaves are stemless, oblong, about - cm long, 0.8 - 1.0 cm wide, the leaves are opposite, and the base of the leaf blade is close to the stem or branch The leaves are green or yellow-green They are covered by translucent dots easily seen by holding the leaves up to a light; these are the perforations that give the plant its species designation "perforatum" The flowers usually bloom in the summer, and the numerous flowers (roughly 25–100 per stem) are symmetrical around a central point, determinate, in flattop clusters The short-stalked flowers are about cm in diameter, each flower has yellow to golden-yellow petals The sticky seed pod is – 10 mm in length, it is a 3-sectioned capsule with many seeds attached to the main axis of the fruit that turns deep reddish brown as it matures The small (1 mm), round, darkly colored seeds have a resinous smell like turpentine On average, a tree has about 15,000 - 33,000 seeds 1.1.3 Ecological requirements H perforatum L is most commonly found in overexploited forests and abandoned fields St John's Wort seeds germinate in spring and flower in late summer The previous studies in Iran found that altitude had a significant effect on growth and the secondary metabolism of St.John’s wort and flavonoid content decreased as altitude increased Also, the number of tannins, ascorbic acid, and carotenoids differed in a narrow range Nonetheless, 1250 meters above sea level and more than 760 mm of rainfall are the best conditions for this plant in its natural habitat, but it can grow at higher elevations as well Of course, elevations more than 1500 meters above sea level and lowlands conditions reduce seedling growth due to severe cool conditions and less than 500 mm rainfall respectively 1.1.4 Uses For centuries, the Europeans have been using it to treat a vast number of diseases, like anxiety, colds, depression, flu, hemorrhoids, womb muscle contractions during menstruation, skin infections, and wounds In reality, several of the older references to the use of this herb for hematoma, burns, wounds, and skin irritations, come from the Greek herbologist of the 1st century a.c., Dioscorides, the Roman student of the 1st century a.c., Plenius, and the Greek doctor of the 5th century b.c., the father of Medicine, Hippocrates Recent research suggests the effectiveness of this herb in treating other ailments, including cancer, inflammation-related disorders, and bacterial and viral diseases, and as an antioxidant and neuroprotective agent 1.2 Status of research on H perforatum L in the world and Viet Nam 1.2.1 Status of research in the world 1.2.1.1 Research on biological characteristics of H Perforatum L The biosynthesis of active compounds in medicinal plants in general and H Perforatum L in particular is significantly affected by environmental factors such as temperature, light and relative humidity Different environmental conditions cause variations in the quality and quantity of bioactive compounds of medicinal plants Many studies show that hypericin and pseudohypericin content are changed under the influence of light intensity, quality, and photoperiod Research on the morphology of H Perforatum L has described biological and anatomical characteristics of stem, roots, leaves, flowers, fruits, and seeds The current study was designed to evaluate the interaction of some major aerial environmental factors (CO2 and irradiance) and plant development stages of Hy-G accumulation and synthesis in the dark glands located in different organs 1.2.1.2 Research on planting techniques of H perforatum L a) Research on the sowing season of H perforatum L Seeds were sown in early or late spring, late summer and fall Seeds could be sown in a greenhouse 4-5 weeks in advance to avoid cold weather, sown in pots or jars, or sown directly into beds in the nursery They can also be sown directly on the field in the fall or spring before the last frost The tree is planted in late spring or early summer before the last cold snap of the year It grows and regenerates well after the cold winter b) Research on fertilizers of H perforatum L Pânzaru and Gille (2001) recommended manure rates are compost 10, 15, 25, 30 till 40 tons/ha; Nitrogen 30, 35, 60, 80, 100 kg N/ha; Phosphorus 20, 25, 50, 90 kg P/ha; and potassium 10, 30, 35 till 60 kg K/ A study in Iran shows that fertilizer treatments can affect herb quality and yield The highest herb yield (10,539 kg/ha) was produced in the plots that received 250 kg N and 100 kg P fertilizer as compared to the control plot (7,458 kg/ha) According to the results, NP-supply increased the number of flowering stems per plant and hypericin content There is a significant correlation between the number of flowering stems and the hypericin content of the St John’s wort herb In addition, the use of green manure as a substrate for the soil and the addition of inorganic fertilizers also affected the yield the highest dry inflorescence yield - 8,410 kg/ha in years of cropping (first year - 610 kg/ha; second year - 3,990 kg/ha and third year - 3,810 kg/ha) was obtained in the case of fertilization with 30 tons of green manure/ha applied under plowing and 45 kg N + 45 kg P2O5/ha c) Research on plant spacing of H perforatum L The study of Azizi et al (2002) used 40 x 25 cm plant spacing to conduct experiments on the effects of nitrogen and phosphorus on the flavonoid content of H perforatum L But in the other study comparing the displacement of two improved H perforatum L species compared with the wild grass species of Iranian people, Azizi et al (2011) used a sparse density of more than 30 x 40 cm d) Research on the harvest time of H perforatum L Research by Tekel'ová D and Mrlianová M (2001) shows that the time of harvesting of H perforatum L affects the content of active ingredients Specifically, In this study, only the tops of the plants were harvested (collecting the flower parts, from the pre-flowering to the fullblooming period) In the 1st year of vegetation, the herb was not harvested Harvests were made in the 2nd and 3rd years of vegetation and later plants died From the two-year old plants, a whole-year yield of 8.12-14.87 ton/ha of dry tops, and from the three-year old plants, ton/ha, was obtained The content of hypericin varied from 0.06 to 0.13%, and 60% ethanol extracted 21.7-27.9% of substances 1.2.2 Research of H perforatum L in Viet Nam 1.2.2.1 Study on the migration of H perforatum L H perforatum L was introduced into Vietnam in 2006, It has been grown in some ecological regions such as Hanoi, Tam Dao, and Sa Pa The results show that it is suitable in cool areas (such as Tam Dao, and Sapa) with average temperatures of about 25oC H perforatum L grow well, flower in May - June, fruit and seeds ripen in July – August Meanwhile, if planted in the delta (Hanoi), the tree grows poorly, and has a low survival rate and branching rate, The tree can only survive in the winter-spring season and wilt and die in the hot summer months Research on agro-biological characteristics of migratory H perforatum L in Bac Ha - Lao Cai shows that plants have grown and developed well It has a growth period from sowing to full flowering is 219 ± days, and medicinal yield is 2.82 ± 0.16 tons dry/ha The time from sowing to fruit ripening, about mid-August, is 272 ± days, and seed yield is 460.33 ± 17.79 kg/ha Seeds have a high germination rate of 90.67% 1.2.2.2 Research on planting techniques of H perforatum L Nguyen Van Thuan and his colleagues (National Institute of Medicinal Materials) have researched some technical measures to plant H perforatum L in Tam Dao The study on the effect of season on the yield and quality of H perforatum L was carried out with different seasons (15/9; 15/10; 15/11; 15/12; 15/1 and 15/2) The results show that the sowing time of 15/9 is the best for a medicinal yield of 27.08 quintals/ha Research on fertilizers on H perforatum L in Tam Dao has selected the formula (calculated on hectare): 10 tons of rotting manure + 150 kg N + 150 kg P2O5 + 120 kg K2O for the highest yield of 28.7 quintals/ha to 30.5 quintals/ha Research on the effect of planting distance on yield and quality of medicinal herbs was conducted in Tam Dao with planting distances (20 x 10 cm, 20 x 20 cm, 20 x 30 cm, and 20 x 40 cm) The results show that a planting density is 250,000 plants/ha (planting distance of 20 x 20 cm) is most appropriate, the actual yield is 27.99 quintals/ha The most suitable time to harvest H perforatum L is in June when the plants are in full bloom, this is also the time when the hypericin content and bio-accumulated productivity are highest 1.3 Overview of natural, economic, and social conditions in the research area Nam Son commune is located in the southwest of Tan Lac district It borders Lung Van commune to the east, Mai Chau district (Hoa Binh) to the west, Ba Thuoc district (Thanh Hoa) to the south, and Quyet Chien commune to the north It has an area of 2.03 km², and its population is 1,586 people, the population density reaching 78 people/km² Nam Son commune is one of the five highland communes of Tan Lac district, with an altitude of 850 - 900 m above sea level, a cool climate, an average annual temperature of 20 - 250C, and a rainfall of 1800 - 2000 mm In January 2020, the entire area and population of three communes Nam Son, Bac Son, and Lung Van were merged into Van Son commune The total area of Van Son is 5.55 km², the population is 5,346 people, and the population density is 76 people/km² The economic structure of Van Son Commune is mainly the production of Agriculture, Forestry, and Fisheries, accounting for 75.6%, so the introduction of a new plant species, H perforatum L., into the local crop structure, is appropriate It is completely suitable to increase exploitation and effective use of land, helping to increase income and improve living standards for people 1.4 Some comments drawn from the overview H perforatum L is a precious medicinal plant containing the main active ingredients such as hypericin, pseudohypericin, and hyperforin It has many effects such as anti-depressant, antiviral, anti-cancer, antibacterial, and anti-inflammatory, but the main effect is the treatment of depression, which is a common disease today Since 2001, the market for products containing H perforatum L has grown very strongly They are widely sold in many European countries, especially in Germany and Switzerland In the US, consumers are very interested in adding herbs to their diet In 2021, the herbal medicine market in the US earned about 11.26 billion dollars, of which 23.9 million dollars was due to sales of H perforatum L and its products are also in the top 40 list of herbal products at mainstream US retail stores H perforatum L was introduced into Vietnam in 2006, It has been grown in some ecological regions such as Hanoi, Tam Dao, and Sa Pa, The results show that it is suitable in cool areas with average temperatures of about 25oC, rainfall of about 1.800 - 2.200 mm/year Other research on ecological conditions in the world also demonstrates that the plant can grow and develop appropriately at the temperature range from 20oC - 30oC and under long day lighting conditions Therefore, Tan Lac, Hoa Binh, a mountainous area with a cool climate, being chosen as the experimental site is completely suitable In Vietnam, some authors have studied technical measures to plant H perforatum L in the cool mountainous area of Tam Dao, The results have determined the sowing time on September 15, planting on 15 March of the following year, the planting distance of 20 x 20 cm (corresponding to a planting density of 250,000 plants/ha) is the most suitable, and the best fertilizer formula is 10 tons of manure rotten + 150 kg N + 150 kg P2O5 + 120 kg K2O, the appropriate time to harvest medicinal herbs is in June, when the plants are in full bloom However, the mountainous area of Tam Dao mainly develops tourism, and the land used to grow crops in general and medicinal plants in particular is very limited Therefore, it is necessary to select an area with similar climatic conditions to grow H perforatum L Inheriting the research results, the thesis has researched morphological traits including growth, development, morphology and anatomy (stem, branches, leaves, flowers, fruits, seeds) and technical measures (the season, planting density, fertilizer, and harvest time) to serve as a scientific basis for building the protocol H perforatum L in Tan Lac, Hoa Binh to achieve high yield and good active ingredient content, meeting the needs of good quality H perforatum L medicinal herb for the domestic market and towards export 11 - Evaluation of some major pests and diseases on H perforatum L grown in Tan Lac, Hoa Binh Investigation on pests, diseases and prevalence was conducted according to QCVN 01-38:2010/BNNPTNT 2.4.2 Research on some farming techniques (season, density, fertilizer, and harvest time) contributing to the completion of the technical process for the production of H perforatum L herbs in Tan Lac, Hoa Binh Experiment 1: Studying the effect of season on growth, development, and yield of H perforatum L Experiment with seasons, deployed crops in 2017 - 2018 and 2018 - 2019 S1: Sowing seeds on 15/09 (control) S2: Sowing seeds on 15/10 S3: Sowing seeds on 15/11 S4: Sowing seeds on 15/12 S5: Sowing seeds on 15/01 S6: Sowing seeds on 15/02 Experimental with one factor was designed as a randomized complete block method (RCBD) with replications The area of each experimental plot is 20m2 The total area of experimental plots is 480m2 (excluding buffer row) Experiment 2: Effect of fertilizers and density on growth, development, and yield of H perforatum L The experiment was carried out in crops 2018 and 2019 The experiment was arranged in a Split-Plot style with two factors: density and rate of nitrogen Which: The main plot factor is the rate of nitrogen (large plot) with different levels: Rate of nitrogen Formula kg N1: kg N + kg P2O5 + kg K2O/ha 90kg N2: 90 kg N + 90 kg P2O5 + 60 kg K2O/ha 120kg N3: 120 kg N + 120 kg P2O5 + 80 kg K2O/ha 150kg N4: 150 kg N + 150 kg P2O5 + 120 kg K2O/ha (control) The sub-plot factor is density (small plot) with different levels: Density Formula 20 cm x 10 cm D1: Planting density of 500,000 plants/ha 20 cm x 20 cm D2: Planting density of 250,000 plants/ha 20 cm x 30 cm D3: Planting density of 166,666 plants/ha 12 The area of each experimental plot is 20 m2 The total experimental area is 960 m2 (excluding buffer row) The ratio of N:P:K is 3:3:2 with the same level of compost 15,000 kg /ha Experiment 3: Effect of harvest time on growth, development, and yield of H perforatum L The experiment was carried out in crops 2018 and 2019 The experiment was arranged in a randomized complete block design (RCBD), one factor with treatments, four replications HT1: Harvest when the tree bud HT2: Harvest when the plant blooms 10% - 20% HT3: Harvest when the plant blooms 30% - 50% HT4: Harvest when the tree is in full bloom ≥ 70% HT5: Harvest when the tree withered flowers - The area of each experimental plot is 20 m2 The total experimental area is 400 m2 (excluding buffer row) 2.4.3 Applying synchronous technical measures to build a demonstration and evaluation of the economic efficiency of planting H perforatum L in Tan Lac - Hoa Binh Applying the results of research on technical measures, select the optimal formulas in terms of season, density, fertilizer, and harvest time of H perforatum L., to deploy the demonstration of H perforatum L - Demonstration area: 5000 m2, not repeat - Deploy the demonstration in crops 2020 and 2021 2.4.4 Quantification of active ingredients in H perforatum L Hypericin in H perforatum L herbs was quantified by highperformance liquid chromatography (HPLC) method According to the Standard basis for medicinal herbs (H perforatum L.) of the Institute of Medicinal Materials issued in 2015 The results were compared with the hypericin content specified in the US pharmacopeia 2.5 Indicator - Indicators of natural conditions in H perforatum L growing area - Indicators of morphology and anatomy of H perforatum L - Indicators of growth and development time - Indicators of seedlings out of the garden - Indicators of seed - Indicators of growth when harvest medicinal herbs 13 - Indicators of pests and diseases - Factors constituting yield and yield of medicinal herbs 2.6 Statistical analysis Data were analyzed by Excel software and statistically processed by the program IRRISTAT 5.0 Chapter RESULTS AND DISCUSSION 3.1 Biological characteristics of H perforatum L grown in Tan Lac, Hoa Binh 3.1.1 Natural environment in H perforatum L growing area Natural environment including some climatic and topographical characteristics of H perforatum L growing area in Nam Son commune were collected and investigated The results show that Nam Son commune, a high mountain area of Tan Lac district, Hoa Binh province has a cool climate with an average annual temperature of 20 oC - 25 oC, and rainfall of 1800 - 2200 mm/year The maximum temperature is not more than 32oC and the minimum temperature is not less than 5oC, which is ideal for the growth and development of H perforatum L 3.1.2 Some morphological features of H perforatum L planted in Tan Lac, Hoa Binh 3.1.2.1 Characteristics of the stem The stems are upright, round, smooth, and multi-branched The plant has an underground stem or rhizome form, with latex Young stems are light green, mature are bright green, often reddish and woody at the base The average height of the tree is 66.82 ± 2.35 cm, the number of level branches/tree is 8.36 branches, the main trunk diameter is 6.81 ± 0.17 mm, and the canopy diameter is 27.03 ± 1.01 cm 3.1.2.2 Characteristics of the leaf Leaves are simple, with entire margins, greenish-gray, stemless, pointed, oblong, or elliptical shape, opposite, with rounded tips Leaves are covered with small dots, visible with a hand lens when the leaf is held up to a light, that look like tiny perforations or pin holes Leaves have an average length of 3.5 ± 0.7 cm, a width of 1.5 ± 0.3 cm, and a length/width ratio is 2.33 ± 0.5 3.1.2.3 Characteristics of the flower The tree has many flowers, (roughly 25–100 per stem), inflorescences cymose Flowers at the top of the stem, branches formed first; Flowers in leaf axils form later Flowers have striped sepals, - 14 mm long, 1.1 mm wide, pointed apex, smooth, with glands that are black spots close to and parallel to the main vein Petioles are short, smooth, mm long Each flower has bright yellow petals, spreading wide at bloom, 13.0 mm long, mm wide, oblong, obtuse tip Some glands are black spots that form parallel streaks 3.1.2.4 Characteristics of fruit and seed The fruit of H perforatum L is a capsule with shells, the old fruit is reddish brown, with traces of the calyx below the rind One Fruit has many seeds Seeds are cylindrical, glossy black brown, small size, 1mm long The number of seeds/fruit reached 222.33 ± 8.01 seeds and the number of fruits/tree reached 886.20 ± 20.07 fruits 3.1.2.5 Anatomy of root, stem and leaf - Microscopic description of root The outermost layer of the secondary root is the secondary dermal tissues consisting of corkscrewed, rectangular, flattened secondary-walled cells arranged in a tangential direction Next is a layer of soft tissue with a thick shell, This soft tissue layer is composed of round or oval cells, and thin walls, arranged in a tangential direction The secondary line is composed of small, thin-walled cells that are regularly and continuously arranged in a circle and are denser and concentrated in places corresponding to the woody branches Secondary wood occupies a large area, without internal parenchyma - Microscopic description of stem The transection of the young stem has an oval cross-section; the mature stem has a nearly circular cross-section The outermost layer of the epidermis was single-layered, cells were rectangular, flattened, and covered with a thin cuticle, often interrupted by stomata The outer surface of the epidermis has a thin serrated cuticle Under the epidermis is soft cortical tissue, consisting of - layers of cells, round or oval in shape, irregular in size, with thin primary walls The endoderm forms a continuous ring, cells are rectangular or polygonal in shape, and irregular in size below the endoderm is a layer of columnar cells which are smaller than the endoderm and have a thin primary wall Libes are arranged in clusters, small cells, and winding walls The wood zone consists of 5-7 layers of cells, arranged in a circle, with large wood veins, polygonal in shape, scattered; many wood rays The soft tissue of the pulp is round or slightly polygonal in shape, 15 irregular in size, arranged with small leads The more mature the stem, the more the soft tissue degenerates, and the trunk becomes hollow in the medulla - Microscopic description of leaf Midrib: The upper surface of the midrib is slightly concave, and epidermal cells are standing oval The sub-epidermal cells are nearly round, smaller than the upper epidermis, and both the upper and lower epidermis are covered with cutin on the periphery Soft tissue cells are large, polygonal, irregular, and arranged with small leads Wood libe towards the bottom: wood above, polygonal vein, clear wood rays; libe below Thick angular tissue is located above the wood and below the line Leaf blade: Upper epidermal cells are very large, square, oval, or polygonal with edges The stroma consists of a single layer of elongated cells Spongy tissue is oval or round cells arranged with small defects The lower epidermal cells are narrow and oval, much smaller than upper epidermis cells, have many stomata, and have very clearly raised glands 3.1.3 Results of evaluation of growth and development indicators of H Perforatum L grown in Tan Lac, Hoa Binh 3.1.3.1 Results of evaluation of growth and development indicators of H Perforatum L Bảng 3.7 Time of the growth and development stages of H Perforatum L Time from sowing to … (days) Year Sprouting Appearing Branching Appearing Floweri Blooming Fruiting Ripening true leaf bud ng profusely 2018 2019 24 ± 23 ± 34 ± 33 ± 130 ± 131 ± 201 ± 202 ± 215 ± 216 ± 226 ± 225 ± 245 ± 270 ± 246 ± 272 ± The results in 2018 and 2019 show that, the growth time of H Perforatum L was relatively stable, although there was a difference in years but it is not significant The time from sowing to sprouting was 23-24 days, to appearing true leaves was 33-34 days Flower bud initiation takes place 201 - 216 days (end of May, beginning of June) after sowing and it takes 225 - 226 days (end of June) to flowering and 245 - 246 days to fruiting The time from sowing to collecting seeds when the fruit ripens was 270 - 272 days (mid-August) Plants for harvesting medicinal herbs at the time of full flowering, the following year the plants grow poorly, so they are often replanted 16 3.1.3.3 Medicinal yield and active ingredient content of H Perforatum L Table 3.10 Medicinal yield and active ingredient content of H Perforatum L Year 2018 2019 Ratio of fresh to dry herbs 2.91 ± 0.4 2.86 ± 0.5 Criteria Individual Theoretical yield yield of dry (g/plant) herb (ton/ha) 21.23 ± 3.72 ± 0.5 22.08 ± 3.85 ± 0.3 Actual yield Hypericin of dry herb content (%) (ton/ha) 2.87 ± 0.6 0.11 2.92 ± 0.5 0.12 The yield of H Perforatum L did not significantly differ between the years 2018 and 2019, the actual yield reached an average of 2.87 tons/ha (2018) and 2.92 tons/ha (2019), hypericin content 0.11 - 0.12%, this result was higher than that of the US pharmacopeia (0.04%) The yield of H perforatum L grown in Tan Lac - Hoa Binh reached 2.70 - 3.05 tons/ha, equivalent to the yield obtained when planted in Tam Dao in 2007 3.1.3.5 Some major pests and diseases of H perforatum L grown in Tan Lac, Hoa Binh In the nursery period of years, there were not many pests, only gray worms appeared at main times, The first stage was when the temperature warmed up (February) (the plants had about 6-8 leaves), and the second stage when the tree is about to leave the garden (March) (the plant has 10 12 leaves) Worms ate leaves and gnaw stems, but not much can be handled by hand Similarly, during the nursery period, H Perforatum L nursery is not infected with many diseases Only seedling death appeared on the tree, but the damage level of the disease was less common To limit seedling death, seeds should be sown at a sparse density, not too thick, and seedling nurseries regularly check them to detect diseases in time When the disease is detected, it is necessary to remove and destroy the causative agent to prevent the disease from spreading on a large scale In the production stage of H Perforatum L., some types of pests such as worms, aphids, and gray caterpillars appear mainly when they are newly planted in the fields Wilt disease appeared in May-July But in general, the level of damage of pests and diseases is not serious and does not affect the growth, and development of plants, yield, and quality of medicinal herbs, so it is not necessary to spray pesticides 3.2 Results of research on some technical measures (seasonal, density, fertilizer, and harvest time) to complete the technical process for the production of H perforatum L herbs in Tan Lac - Hoa Binh 17 3.2.1.4 Effect of season on yield and quality of H Perforatum L medicinal herbs Table 3.18 Effect of Season on yield and quality of H Perforatum L medicinal herbs Treatment/ formula Individual Ratio of yield fresh to dry (g/plant) herbs S 15/9 S 15/10 S 15/11 S 15/12 S 15/01 S 15/02 CV (%) LSD0.05 20.17 20.57 21.20 18.33 16.50 15.50 7.60 2.15 3.17 ± 0.40 2.90 ± 0.11 2.94 ± 0.12 2.73 ± 0.18 3.17 ± 0.19 3.03 ± 0.20 - S 15/9 S 15/10 S 15/11 S 15/12 S 15/01 S 15/02 CV (%) LSD0.05 20.65 21.05 21.40 18.25 17.01 15.74 9.0 1.56 3.23 ± 0.43 3.01 ± 0.37 2.98 ± 0.32 2.70 ± 0.21 3.21 ± 0.20 3.00 ± 0.24 - Theoretical Actual Hypericin Yield of yield dry yield dry content hypericin herb ( herb (%) (kg/ha) ton/ha (ton/ha) Crop 2017 - 2018 3.53 2.75 0.156 4.29 3.60 2.81 0.158 4.43 3.71 0.161 2.85 4.58 3.21 2.43 0.156 3.79 2.89 2.10 0.155 3.25 2.71 1.88 0.152 2.85 9.10 0.34 Crop 2018 - 2019 3.61 2.78 0.160 4.44 3.68 2.85 0.161 4.58 3.75 0.168 2.88 4.83 3.19 2.45 0.157 3.84 2.98 2.17 0.155 3.36 2.75 1.91 0.151 2.88 8.2 0.19 - Research results show that: When sowing seeds at different times (from September 15 to November 15) over two crop seasons, the yield of medicinal herbs was high from 2.75 to 2.88 tons/ha, the content of active ingredient hypericin reached from 0.151 to 0.168 %, higher than that of the US Pharmacopoeia (0.04%) However, in the two seasons of September 15 and October 15, the time in the nursery is too long, so the sowing time of November 15 is the most suitable for H Perforatum L 3.2.2.2 Effect of plant density and fertilizer rate on yield and quality of H Perforatum L medicinal herbs 18 Table 3.20 Effect of plant density and fertilizer rate on yield of H Perforatum L medicinal herbs Individual yield (g/plant) Year 2018 Factor Nitrogen rates (F) Formula Average (F) Density (D) Density (D) D1 Actual yield (dry ton/ha) Theoretical yield (dry ton/ha Ratio of fresh to dry herbs Average (F) Average (F) Density (D) D1 D2 D3 D2 D3 D1 D2 D3 F1 14.31h 18.58c 17.01e 16.63ef 3.12 3.01 2.87 3.00 5.01 3.25 1.98 3.41 F2 15.03g 19.02bc 18.23d 17.43de 2.96 3.05 2.85 2.95 5.26 3.33 2.12 3.57 F3 16.36f 21.69a 20.19b 19.41b 2.91 2.97 3.03 2.97 5.73 3.80 2.35 3.96 F4 16.09f 19.13bc 18.24cd 17.82d 2.87 2.95 3.09 2.97 5.63 3.35 2.13 3.70 Average (D) 15.45fg 19.61b 2.97 3.00 2.96 5.41 3.43 2.15 18.42d D1 D2 D3 1.65i 1.75i 1.70i k 2.15e 1.86h 2.42c f 2.34c 2.89a 2.46c d 2.18e 2.54b 2.24e 14.20g 2019 Nitrogen rates (F) F2 15.13ef F3 16.26 e F4 16.00 e Average (D) 15.40e 18.61bc 21.65 18.23 bcd 19.37b 2.14f 2.56b 2.32d CV % = 6.6 LSD0.05 (F)= 0.61 LSD0.05 (D)= 0.03 LSD0.05 (F*D)= 0.06 16.07e 16.29cde 3.15 3.05 2.89 3.03 4.97 3.26 1.87 3.37 1.68hi 1.80h 1.77h 1.75h bcd 2.98 2.97 2.94 2.96 5.30 3.32 2.01 3.54 1.93g 2.35cd 2.13e 2.14e 18.98b 17.25bcd17.12 a 1.70i 2.01g 2.40c 2.14f CV % = 7.2 LSD0.05 (F)= 5.86 LSD0.05 (D)= 0.34 LSD0.05 (F*D)= 0.69 F1 Average (F) Density (D) 19.76b 19.22 18.04bcd17.42 b 2.93 bcd 17.78c 2.97 3.00 2.92 2.98 3.05 3.00 2.99 2.97 2.97 2.98 5.69 3.79 2.30 5.60 3.19 2.10 5.39 3.39 2.07 3.93 2.35 cd 3.63 2.21 de a bc 2.60b b d 2.38cd 2.92 2.52 2.63 2.31 2.04ef 2.43c 2.18e CV % = 7.89 LSD0.05 (F)= 7.90 LSD0.05 (D)= 0.46 LSD0.05 (F*D)= 0.91 CV % = 7.60 LSD0.05 (F)= 0.96 LSD0.05 (D)= 0.05 LSD0.05 (F*D)= 0.10 Table 3.21 Effect of plant density and fertilizer rate on quality of H Perforatum L medicinal herbs Content of Hypericin (%) Year 2018 2019 Factor Treatment Nitrogen rates (F) F1 F2 F3 F4 Average (D) D1 0.150 0.153 0.160 0.156 0.155 Nitrogen rates (F) F1 F2 F3 F4 Average (D) 0.157 0.156 0.161 0.158 0.158 Density (D) D2 D3 0.155 0.158 0.159 0.156 0.162 0.158 0.157 0.151 0.158 0.156 0.156 0.157 0.160 0.155 0.157 0.159 0.157 0.158 0.155 0.157 Average (F) 0.154 0.156 0.160 0.155 0.157 0.157 0.160 0.156 The results in Tables 3.20 and 3.21 show that: Yield of Hypericin (kg/ha) Density (D) D1 D2 D3 2.47 2.71 2.68 2.84 3.84 3.35 3.74 4.68 3.88 3.40 3.98 3.38 3.11 3.80 3.32 2.63 3.01 3.78 3.49 3.23 2.80 3.69 4.67 4.07 3.81 2.81 3.34 3.98 3.58 3.43 Average (F) 2.62 3.34 4.10 3.59 2.75 3.35 4.14 3.71 19 In both 2018 and 2019 crops, the two factors density and fertilizer had a clear influence on yield At the same level of Nitrogen rates, the actual yield was low at D1 thick planting and D3 thinning, the highest yield was at D2 At the same density, increasing the amount of Nitrogen rates makes the actual yield increase, specifically, the actual yield is lowest at F1 (only manure, no chemical fertilizer) and gradually increases at different levels of F2, and F3 Nitrogen rates However, when the amount of Nitrogen rates continued to increase to the F4 level, the actual yield did not increase, even tended to decrease Actual yield in the treatments ranged from 1.65 - 2.92 tons/ha, D2F3 formula (planting density 250,000 plants/ha, spacing 20cm x 20cm, fertilizer 15,000kg compost + 120kg N+120kg P2O5 + 80kg K2O/ha) had the highest net yield of 2.92 tons/ha Statistical analysis shows that there is a statistically significant difference with 95% confidence compared with the formulas The content of hypericin of H Perforatum L of the density and fertilizer formulas did not differ much, ranging from 0.150 - 0.162 %, Thus, the yield of the active ingredient hypericin of the formulations depends on the medicinal yield The highest yield of hypericin active ingredient was in the D2F3 formula at 4.68 kg/ha (2018) and 4.67 kg/ha (2019), the low hypericin yield in the F1 formulas (D1F1, D2F1 and D3F1) reached from 2.47 - 2.71 kg/ha (2018) and 2.63 - 2.81 kg/ha (2019), in which the D1F1 formula had the lowest yield of hypericin at 2.47 kg/ha (2019) 3.2.3.2 Effect of harvest time on yield and quality of H perforatum L medicinal herbs Table 3.24 Effect of harvest time on yield and quality of H perforatum L medicinal herbs Theoretical Actual Individual Ratio of Hypericin Yield of yield of dry yield of dry yield fresh to dry content hypericin herbs herbs (dry Treatment (g/plant) herbs (%) (kg/ha) (ton/ha) ton/ha) The year 2018 HT1 HT2 HT3 HT4 HT5 CV (%) LSD0.05 18.16 20.68 21.50 21.83 20.86 7.5 2.46 2.97 ± 0.26 2.70 ± 0.18 2.76 ± 0.29 2.73 ± 0.18 2.69 ± 0.16 - 3.54 4.17 4.37 4.45 4.20 - 2.15 2.61 2.85 2.93 2.73 6.8 0.27 0.052 0.074 0.096 0.162 0.042 - 1.12 1.93 2.73 4.74 1.14 - 20 HT1 HT2 HT3 HT4 HT5 CV (%) LSD0.05 18.75 21.13 21.68 22.01 21.06 9.2 0.73 2.89 ± 0.25 2.73 ± 0.17 2.84 ± 0.25 2.76 ± 0.21 2.70 ± 0.19 - The year 2019 2.17 3.56 4.12 2.58 4.26 2.80 4.47 2.89 4.23 2.67 9.4 0.15 0.058 0.076 0.090 0.167 0.040 - 1.25 1.96 2.52 4.82 1.06 - Note: HT 1: Harvest when the tree bud; HT 2: Harvest when the plant blooms 10% - 20%; HT 3: Harvest when the plant blooms 30% - 50%; HT 4: Harvest when the tree is in full bloom ≥ 70%; HT 5: Harvest when the tree withered flowers In both the 2018 and 2019 crops, the yield of European medicinal herbs and the content of active ingredients hypericin increased from treatment HT1 to HT4 The HT4 treatment when the plant is in full bloom had the highest medicinal yield and hypericin content, the medicinal yield was from 2.89 - 2.93 tons/ha, and the hypericin content was 0.162 - 0.167 % The yield of the active ingredient hypericin collected when the plant was in full bloom was 1.7 times higher than that obtained when the plant was in bloom, 30 - 50 %, and - times higher than that obtained when the plant was in bud and flowering 10 - 20 % and when the tree has withered Yield decreased when the plants had flowered, and the hypericin content was very low (0.040 - 0.042 %), even worse than that when the plants were bud (0.052 - 0.058%) Thus, the results show that harvesting when the plant is in full bloom > 70% gives a high medicinal yield, the best content of hypericin active ingredient, and the highest yield of hypericin active ingredient 3.2.4 Proposal to develop a technical process for the production of H Perforatum L herbs in Tan Lac, Hoa Binh Based on the research results, the technical process for the production of H Perforatum L herbs in Tan Lac, Hoa Binh is proposed for a medicinal yield of 2.5 - 2.9 tons dry/ha + Planting season: Sowing in 15/11, planting in March + Density: 250.000 plants/ha, planting distance: 20 x 20 cm + Fertilizers: 15.000 kg compost +120 kg N +120 kg P2O5 + 80 kg 21 K2O/ha + Harvesting time: When the plant is in full bloom (from late June to mid-July) 3.3 Result of Applying synchronous technical measures to build a planting pattern and evaluating of economic efficiency of planting H perforatum L in Tan Lac - Hoa Binh Applying the technical process of producing H perforatum L herbs in Tan Lac, Hoa Binh, to deploy the H perforatum L planting model in the two crops of 2020 and 2021 The model area is 5000 m2 3.3.2.3 Yield and content of active ingredients in H perforatum L medicinal herbs The monitoring results are presented in Table 3.30 Table 3.30 Yield and content of active ingredients in H perforatum L medicinal herbs Year 2020 2021 Ratio of fresh to dry herbs 2.70 ± 0.23 2.76 ± 0.21 Criterias Actual yield of Hypericin dry herb content (%) (ton/ha) 2.91 4.92 2.92 4.88 Yield of hypericin (kg/ha) 0.169 0.167 Table 3.30 shows that: The yield and quality of medicinal herbs in 2020 and 2021 did not differ much, the actual yield reached an average of 2.91 - 2.92 tons/ha and the hypericin content reached 0.167 - 0.169 % both higher than that in the USA Pharmacopoeia (0.04%) The yield of the active ingredient hypericin reached 4.88 - 4.92 kg/ha 3.3.3 Economic efficiency of production of H perforatum L in Tan Lac, Hoa Binh The results of comparing the economic efficiency of the H perforatum L production model with maize and peanut, which are traditional crops in the locality are presented in Table 3.31 22 Table 3.31 Economic efficiency of the H perforatum L production model compared to common crops (maize and peanut) in Tan Lac, Hoa Binh Unit: thousand dong The year 2020 No Explain I II III Total cost Seeds Fertilizes Pesticides Labor Total revenue Economic efficiency Net profit (II)(I) Marginal benefit cost ratio (MBCR) H perforatum L planting demonstration compared to peanut planting demonstration Marginal benefit-cost ratio (MBCR) H perforatum L planting demonstration compared to maize planting demonstration Year 2021 H perforatum Peanut Maize H perforatum Peanut Maize L planting planting planting l planting planting planting demonstratio demonstration demonstratio demonstration demonstrat demonstra n n ion tion 77,490 4,500 26,990 1,000 45,000 261,900 30,050 2,800 8,750 500 18,000 52,160 26,450 2,400 7,050 500 16,500 37,720 75,990 4,500 26,990 1,000 43,500 262,800 29,750 2,800 8,750 500 17,700 52,800 26,900 2,400 7,050 500 16,950 38,700 184,410 22,110 11,270 186,810 23,050 11,800 4.42 - - 4.54 - - 4.39 4.57 The results in Table 3.31 show that the H perforatum L planting demonstration brings much higher economic efficiency than that of corn and peanuts The average net profit of H perforatum L was 184.41 - 186.81 million VND/ha/year, while maize was only 11.27 - 11.80 million VND/crop/ha, (if it is planted in crops per year, the net profit of corn crop is 22.54 - 23.60 million VND/ha/year), while peanuts on average are about 21.85 - 22.11 million dong/crop/ha, (if it is planted in crop per year, the net profit of peanuts crop was 43.70 - 44.22 million dong/ha/year) The marginal benefit cost ratio (MBCR) of the H perforatum L demonstration 23 compared to the maize and peanut demonstration in 2020 and 2021 were both high from 4.42 to 4.57 times The marginal benefit cost ratio is highly accepted to develop and replicate the model H perforatum L not only gives high economic efficiency but is also very suitable for the local crop structure to help maximize the efficiency of land use Plant structure in year: winter-spring plants (all kinds of vegetables and legumes) - H perforatum L - Autumn-Winter corn From November to March, H perforatum L is sown in the nursery, the field can be arranged to plant winter-spring crops such as vegetables, cowpeas, etc H perforatum L was planted from March to July followed by the planting of winter maize Thus, it can be seen that the transformation of crop structure, from planting main crops such as corn, and peanuts to growing medicinal plants such as H perforatum L., not only brings higher economic efficiency but also represents a more suitable crop structure It achieves maximum efficiency per unit of land area and improves economic efficiency for local people CONCLUSIONS AND RECOMMENDATIONS Conclusions (1) The biological characteristics of H perforatum L were evaluated in the planting area in Nam Son commune, Tan Lac district, Hoa Binh province - The climatic, soil, and altitude conditions in the planting area in Nam Son commune, Tan Lac district, Hoa Binh province are suitable for growth and developmet of H perforatum L - Regarding Morphological indicators: Described the characteristics of stems, leaves, roots, flowers, fruits, and seeds of H perforatum L grown in Tan Lac - Hoa Binh The morphology of stems, leaves, roots, flowers, fruits, and seeds is similar to that of the tree when it was imported to Vietnam However, tap roots are not nearly as developed as H perforatum L grown in other countries, the tree mainly develops a cluster root system - Regarding growth and development indicators: The growth and development time of H perforatum L from sowing to sprouting, branching, bud, flowering, full flowering, fruit set, and ripe fruit (seed collection) has been determined The time from sowing to collecting medicinal herbs and collecting seed was 225 - 226 days, respectively When harvesting medicinal herbs, the tree reaches a height of 67.01 - 69.24 cm and has 7.89 - 8.30 branches of level 24 - In terms of yield and active ingredient content: The average yield is 2.87 - 2.92 dry tons/ha, and the content of hypericin is over 0.1%, higher than that in US pharmacopeia (0.04 %) The main farming techniques have been identified to complete the cultivation protocol of H perforatum L in the mountainous areas of Tan Lac district, Hoa Binh province The sowing season is November 15, and planting in early March The planting distance is 20 x 20 cm, equivalent to 250,000 plants/ha, combined with the formula of fertilizer, including 15,000 kg of rotting manure +120 kgN + 120 kg P2O5 + 80 kgK2O (equivalent to 15 tons’ manure + 260 kg urea + 750 kg phosphate + 134 kg potassium chloride) The most appropriate time to harvest medicinal herbs is when the flowers are in full bloom (around late June to early July) The yield of dry medicinal herbs is 2.8 - 2.9 tons/ha, and the content of hypericin is 0.16 % (3) Technical measures have been synchronously applied to build a production demonstration of H perforatum L in Tan Lac - Hoa Binh The average yield was 2.92 tons of dried medicinal herbs/ha, and the content of hypericin was 0.169 % The model achieved economic efficiency from 184.41 to 186.81 million VND/ha/year The Marginal benefit cost ratio (MBCR) of H perforatum L demonstration compared to the corn and peanut farming demonstration in 2020 and 2021 was both high from 4.42 to 4.57 times The Marginal benefit cost ratio is highly accepted to develop and replicate the model Recommendations Based on research results on farming techniques of H perforatum L., continue to improve technical measures to develop technical processes for the production of H perforatum L herbs meeting GACP - WHO standards in mountainous areas, the cool climate of Tan Lac district, Hoa Binh province, and other regions with similar soil and climate conditions Applying results of research to a pilot production project to create a H perforatum L raw material area and introduce high-quality products to the domestic pharmaceutical market and participate in export 25 PUBLISHED ARTICLES RELATED TO THE THESIS Tran Danh Viet, Đoan Thi Thanh Nhan, Nguyen Ba Hoat, Nguyen Van Dung (2022), “Study on sowing times for Hypericum perforatum L in Tan Lac district, Hoa Binh province”, Journal of Vietnam Agriculture Science and Technology, 04 (137)/2022 Tran Danh Viet, Đoan Thị Thanh Nhan, Nguyen Ba Hoat, Nguyen Van Dung (2022), “Study on biological characteristics of Hypericum perforatum L grown in Tan Lac, Hoa Binh”, Journal of Vietnam Agriculture Science and Technology, 06 (139)/2022 Tran Danh Viet, Đoan Thị Thanh Nhan, Nguyen Ba Hoat, Nguyen Van Dung (2022), “Research on harvest stage Hypericum perforatum L in Tan Lac district, Hoa Binh province”, Vietnam Journal of Agriculture and Rural Development, 18/2022 (2nd 9/2022)