Studies On Good Agricultural Practice Of Herba Nerviliae Fordii

Herba Nerviliae Fordii is the dried herba or folium of Nervilia fordii (Hance) Schitr. It is one of the famous Southen drugs in China, and is considered to be good in clearing away heat from the lung to arrest cough, activating spleen to improve digestion, inducing sedation and relieving pain, clearing away heat, detoxicating, dissolving lumps and treating carbuncles. It is used in phthisic, cough and hemoptysis, furuncles, injuries from falls,infantile dyspepsia and malnutrition, psychopathy, mouth inflammation, urgent faucitis. It is especially good in curing wean pneumonia.It mainly distributes in Guangdong, Guangxi and Hainan provinces. It is commonly used in Hongkong, Macao and southeast Asia.Nervilia fordii grows mainly in wildness. After years of rooted out, its resource was profoundly damaged, species were indengered. To ensure a sustainable development of Nervilia fordii resource, we established a standardized plant basement for Nervilia fordii in Pingyuan county Guangdong province, to domesticate its wild breed. According to the Quality Control Standard on Production of Medicinal Herb (Draft), a Good Agricultural Practices (abbreviated as GAP) of Nervilia fordii was studied in detail.The whole study included 4 aspects: selecting plant basement and analyzing environmental factors; species research; standardized cultivation technique research and quality control research. Based on the research we established the Quality Standard and Standardized Operation Practice (SOP) of Herba Nerviliae Fordii.Ⅰ Selecting Cultivation Basement and Analyzing Environmental Factors 1 .Investigating of Environment Conditions of Cultivation BasementThe investigation showed that Pingyuan county was rich in plant and animal resources and had a long history of Traditional Chinese Herb Medicine planting. The local government strongly supported its development.An investigation on the environment condition showed that the geography location, weather condition and soil of this area fitted well to the growth requirement of Nervilia fordii.2.Analysis of Air, Soil and WaterThe samples of air, soil and water of Pingyuan were analyzed, the results showed that their qualities were much better than the national standard setted by the Quality Control Standard on Production of Medicinal Herb (Draft). Therefor, Pingyuan forest farmland was chosen as planting basement of Nervilia fordii.Ⅱ .Study on Species and Resouce of Nervilia fordii.1.Determining the original plant species and scientific nameAfter investigating its original plant species and scientific name, confusions over source of Herba Nerviliae Fordii were clarified. Its origin was Nervilia fordii (Hance) Schitr. (Orchidaceae, Polychondreae, Nervilieae, Nervilia Comm. ex Gaudich.), which distributed mainly Guangdong, Guangxi, Hainan, Yunnan provinces of China. The quality of Guangdong province was good, the quantity of Gungxi province was big.2.Breed types of Nervilia Fordii17 samples were collected in Guangdong, Guangxi and Hainan provinces. Dayie, Zhongyie, Xiaoyie were the same, their origins were Nervilia fordii.In the light of a comparative study on the morphological charatristics, scanning electromicroscopy characteristics of leaf surface, the breeds of Nervilia Fordii were classified into 3 types, the results showed that the breed which leaf diameter below 3.5 cm is Xiaoyie, 3.5~5cm is Zhongyie, exceed 7 cm is Dayie.3.Substitute, promiscuous and fake species of Herba Nervilia fordiiThe species source of Nervilia fordii was complicated and mixed. According to textual research and folk use, we considered Nervilia plicata (Andr.) Schitr. to be thesubstitute of Nervilia fordii.The promiscuous species of Nervilia fordii were:Semiaquilegia adoxoides (DC.) Makino; Begonia fimbristimpulata Hance;The fake species of Nervilia fordii were:Plantago asiatica L.(Plantago major L. van asiatica (L.) Decne.) ;Plantago major L.; Centella asiatica (L.) Urb.; Pinellia ternata (Thunb) Breit; trphonium divarication (L.)o4. Research on Botanic Characteristics of Nervilia fordiiWe studied the morphological, microscopy and scanning electromicroscopy characteristics of the pollen, fruit and seed of Nervilia fordii. The results showed that the shape of pollen was oval and rotundity. The type of fruit was capsule. Seeds were very tiny and abundant, their structures were very simple, they had no albumen, the fresh seeds were vigor and could be dyed by TTC, but their vigor would loss within 1 month if they were stored in the common environment.Ⅲ.Study on the Standardized Cultivation of Nervilia fordii 1.Selecting plant terraSelecting fertile flat where water resource was plenty and western exposure time was short. Built cover shed to keep conceal rate within 60% and 70%. Dug fine barrel-drain. Built farmland 80cm wide and 20cm high. Applied enough basic fertilizer, mixed it with soil and splashed above the farmland.2.Study on breeding methods and management1)Seed breedingSeeds of Nervilia fordii were tiny and powder like. Their clean degrees were 99.2%, One thousand seeds weighted 4 mg. Burgeon rate was 0%, burgeon potential was 0%.2)Tuber breedingSeeds' vigor losted easily. Tuber breeding had a high livability and was used as a common breeding method. Collecting tuber and wild plants on the middle ten days of May. Preserved them with fresh wit river sand in cool place. Planted them in fine days.The survival rate of tuber was above 97%, of plant was 99% 1 week later, 95% 1 month later. Retransplanted the survival plants on the middle ten days of August, the survival rate was 100% 1 week later, 98% 1 month later.3)Bio-technology breeding (l)Rhizoma reducingEffects on indusement of Rhizoma and Plant regeneration of different implants, density of hormones, additives were studied. Results showed that the best explant was tuber. Effect of 6-BA2mgL-1 were better than 6-BA1 mg·L-1 on rhizoma reducing.(2)Optimization of rhizoma multiplication medium.Effects of medium (MS, B2, White) and hormon (2,4-D, 6-BA, NAA) on rhizoma freshweight increasing rate were studied by orthogonal design. Effects of sucrose content (10,20, 30, 40, 50, 60 g·L-1) and crude extractive (potato, apple, banana, tomato, bean sprout, watermelon juice, coconut juice) by mono-factorial design .The experiments showed that the best medium for rhizoma multiplication was 1/2MS+6-BA1mg· L-1 + 20%Suc+10 g · L-1 tomato or apple。(3)Plant regeneration and transplantationThe white rhizoma could be induced to tubers and regeneration plants on 1/2MS + 1‰ active carbon. The green rhizoma could be induced directerly to regeneration plants on 1/2MS+6-BA2+NAA2.The tube seedings could be lively transplanted after trainning.(4)Protoplast cultureProtoplasts were obtain from leaf treated with enzyme for 4h. After seperating and purifying, protoplasts were recultured on 1/2MS+6-BA2+Suc 3%+10% coconut juice. 1 month later, 1mm size white tissue culture were formed, when it grew to 2mm, transplanted it on fresh mediem.3. Tissue cultures cryopreservationTo preserve tissue cultures of Nervilia fordii using cryopreservation method, suitable factors in cryopreservation (-196℃) were studied by monofactorial and uniformdesign. The experiments showed that five-weeked tissue cultures were the best material for cryopreservation. Tissue culures treated for 12days at 4℃ were the best cryotolerant. The optimal cyroprectants was 12.5%DMSO+0.25%LH. The best freezing procedure was to reduce the temperature from 0℃ to -18℃,and stay at that temperature for 1 hour, then drop them in liquid nitrogen(-196℃) to be preserved. 20 ℃ was good for melting the frozen material. Unfrozen tissue cultures survived when they were recutured. Tissue cultures of Nervilia fordii can be preserved by cryopreservation method.4.Fertilization studyTo find out a suitable fertilizer for Nervilia fordii, a comparative study of different fertilizers (biological-organic, compond chemical, farmers' home-produced) was carried out by monofactorial design.Results showed that all the 3 fertilizations can significantly increase the leaf and whole plant growth, with biological-organic fertilizer being superior.5.Investigation and prevention of harmful insects and diseaes of Nervilia fordiiInvestigation of diseases and harmful insects showed that speckle disease was the commonest disease, while Sitobion avenae (Fabricius), Spodoptera litura Fabricius and Helix graminum H. were the commonest harmful insects. Their symptoms and ways of onset were detected and recorded.Methods of prevention include agricultural, biological and chemical preventions. To find out the effects of pesticide on plants of Nervilia fordii, a comparative study of different pesticides (biological-pesticide A, biological-pesticide B, chemical pesticide) was carried out by monofactorial design.The results showed that all these 3 pesticides did harm to plants, their degrees to upgroud parts were: biological-pesticide A> biological-pesticide B> chemical pesticide, to underground parts were: biological-pesticide B> chemical pesticide > biological-pesticide A.The results also showed that all 3 pesticide increased the heavy metal content in the drug, the increasing degrees were: biological-pesticide B> chemical pesticide > biological-pesticide A. Apart from Hg, other heavy metal contents all exceeded thestandards. So we suggest that no pesticide used in planting. 6.CoUection study (1)Time of collectingThe study showed : considering appearance and leaf or whole plant weight, the end of July was in order; considering dry substance, the end of September was in order.Dayie was the high yield species considering leaf weight or whole plant weight, while Xiaoyie was the high tuber reproduction species.(2)Part of collectingNervilia fordii was indengered species, in order to protect its resouce, we suggest using its leaf or herb which wipe off the baby tuber.Ⅳ .Study on quality criteria of Nervilia fordii1. Measurement of water, dried material, extractions, heavy metals.To set up quality standards of Herba Nerviliae Fordii through determination of the contents of moisture, total ashes, ashes imdissolvable in acid, water extracts (cool water) and ethanol extracts in this herb. These indexes were determined based on procedureds recorded in appendix ⅨH, ⅨK and ⅩA of Chinese pharmacopeoia, published in 2000, for monsture, ashes and extracts determination respectively. Results showed that the moisture in Herba Nerviliae was less than 11.75% , with the total ashes content within 29.85%, ashes imdissolvable in acid not higher than 19.02%, the content of water extracts (cool water) higher than 2.63%, that of 70% ethanol extracts not less than 0.38%. These data are quantitatively referencable for making quality standards of Herba Nerviliae Fordii.2. Study on the Constituents of Volatile Oil from Nervilia fordiiTo study the constituents of volatile oil from Nervilia fordii, GC-MS technique was used. Results showed that eighty-nine components were seperated. Among them, fifty-three ones were identified and their relative contents in percentage were identified and their relative contents in percentage were calculated. The main constituents were 2-Pentadecanone,6,10,14-trimethyl(13.55%), 4-ethyl-trans-3-thiobicyclo[4,4,0]decane(6.54%), Benzenesulfonamide,4-methyl-N-(2-oxo-2phenylethl)(6.33%), Phytol(6.32%), d-Carinol(4.54%), β- Ionone (4.43%), Caryophyllene oxide(4.13%), which account for 45.84%.3. Experimental Study on Antitussive and Antiasthmatic Action of Nervilia fordiiEffects of antiasthmatic and antitussive of Nervilia fordii were studied. Cough induced by spraying ammonium hydrsxide in mice, asthma induced by spraying mixture of histamine- acetylcholine in guinean pig were adopted for observing the related pharmacological effect of different doses of Nervilia fordii. Results showed that Extractions from Nervilia fordii markedly prolonged the latent period of cough by ammonium hydrsxide in mice, and decreased the cough frequency, significantly prolonged the latent period of asthmatic attack by mixture of histamine- acetylcholine in guinean pig. Nervilia fordii had significant antitussive and antiasthma action.4. Study on antiviral action of Nervilia fordii against influenza virusAntiviral actions of Nervilia fordii against influenza virus in vitro were studied. Cell culture technique was used in MDCK cells to observe antiviral action of abstracts of Nervilia fordii against influenza virus A (FM1 strain) and B (Kun 70B strain). Results showed that Water soluber part of Nervilia fordii can inhibit the proliferation of influenza virus A (FM1 strain) in MDCK cells. Nervilia fordii is effective for influenza virus A in vitro.Based on the research, the quality standard of Herba Nerviliae Fordii was established. Nervilia fordii (Hance) Schltr. was identified as the original plant of Herba Nerviliae Fordii.According to the Quality Control Standard on Production of Medicinal Herb (Draft), A Standardized Operation Practice (SOP) of Nervilia fordii was established on the basis of our study and planting practice. The SOP can be divided into the following aspects:(1)Natural conditions of planting field(2)Species and its breeds...