Scientific And Reasonable Exploitation And Utilization Of Zanthoxylum Nitidum, The Essential Raw Materials Of Sanjiu Weitai Granules

Zanthoxyli Radix(两面针,Liangmianzhen, Shinyleaf Pricklyash Root) is one of the herbal medicine that commonly used in Lingnan area. Original plant of the herb varies in the Quality Standards promulgated by different provinces; roots of various plants are used as Zanthoxyli Radix in different folk medicine, which leads to the disunity of the herb in clinical use. Officially it is the dried root of Zanthoxylum nitidum (Roxb.) DC. (Fam. Rutaceae) in the Pharmacopoeia of People’s Republic of China,2010. To meet the national standard, the plant has to grown more than four years. However, excavating officinal part of the herb results in severe injury of the plant itself and serious damage of the ecological environment. Hence, the feasibility studies of expanding medicinal parts of Z. nitidumby using the whole plant is demanded, which should be conducted by finely study of root and other parts of Z. nitidum. In recent years, the commercial herb is motley due to lack of undomesticated grown plants and the fast growing market demand. Serious shortage of raw materials has become restriction factor in the proprietary Chinese medicines and functional cosmetic products industry using Zanthoxyli Radix as essential material. To ensure sustainability of the plant resource, the domesticating and good agricultural practice (GAP) of Z. nitidum cultivation are problems demanding prompt solution.The present thesis focuses on the Zanthoxyli Radix, the essential raw material of Sanjiu Weitai Granules, one of the key products of China Resources Sanjiu Medical & Pharmaceutical Co., Ltd., specifically on the resource investigation and collating, herbal quality standard research, and furthermore, studies of engaging parts of the plant other than root as officinal used parts has been attempted and the GAP of domesticating the plant has been carried out in a trial size. The experience of the research inspired the thought of a new way of protecting and sustainable use of herbal plants. Some new thinking-ways of essential raw herbal material exploitation and utilization for proprietary Chinese medicine enterprises are also explored.The achievements of this thesis are as follows:1. Resources investigation and collating of Z. nitidum and adulterants. The original plant species, the sexual status, blooming period, plant morphological characters, host plants, distribution of Liangmianzhen (Z. nitidum) and adulterant of Zanthoxyli Radix was finely studied by herbarium sheet consulting, field survey and expert consultation. The official original plant of Liangmianzhen, Radix Zanthoxyli, is Z.nitidum (Roxb.) DC., of which flowers are unisexual; the blooming period observed is ahead of time recorded in literatures. The climbing feature of the plant converted to upright shrubby in some of the individuals after domesticating is also observed. The plant distributes widely in Lingnan area as an adaptable, but not endangered species. However the wild resource is now significantly reduced due to the huge market demanding. The species of host plants for Z. nitidum are highly similar in Guangdong and Guangxi province (region). The plant taxonomy is investigated in the current study. Field survey showed the Z. nitidum plant individuals that with prickles on only one side of the leaves were taken for Danmianzhen in some area, of which (Danmianzhen) the original plant should be Z. dissitum Hemsl., instead. The plant Maoye-Liangmianzhen is recognized as a varietas of Z. nitidum. The so called Mao-Liangmianzhen, which used to be named Z. nitidum DC. var. neglectum How (1956), or Z. nitidumf. fastuosumHow ex Huang (1974), has been re-classified to the species Z. nitidum (1987). It is improper, therefore, to distinguish Z. nitidum plants with short hair as Z. ni tidum DC. var. neglectum How, or Z. nitidum f. fastuosum How ex Huang, in some research papers. Also in the Pharmacopoeia of the People’s Republic of China (2010), identifying Z. nitidum f. fastuosum How ex Huang by detection of toddalolactone is then improper, either, from the view of plant taxonomy. Intensive study of identifying Liangmianzhen and Mao-Liangmianzhen using TLC, molecular biology and HPLC will be further conducted. The Maoye-Liangmianzhen(Zanthoylum nitidun var. tomentosum Huang) also needs in-depth study for the feasibility of being the officinal plant origin of the Zanthoxyli Radix.2. Perfecting and improving the quality standard of Zanthoxyli Radix. Microscopic observation of transverse section and powder of Z. nitidum showed characteristics of the cork layer, phloem and xylem; the prism of calcium oxalate are individually scattered or gathered in parenchyma cells; cork cells are neat arranged or appear rectangular, polygonal, or irregular shape, with brownish red or yellow substance inside, scattered with brown lumps. Stone cells are class circular or irregular; reticulate or bordered pit vessels are commonly observed. The DNA barcoding technology is proved to be an effective molecular method for distinguishing Z. nitidum and its adulterants. The results showed that psbA-trnH and ITS2 should be employed complementarily, supported by the highest the second highest identification efficiency, respectively.Referring to the method for Thin Layer Chromatography (TLC) proposed by Pharmacopoeia of People’s Republic of China,2010 (Appendix VI B), TLC methods were developed for analysis of the marker compounds of Radix Zanthoxyli, nitidine chloride and ethoxychelerythrine, respectively. For analysis of nitidine chloride, High Silica gel G pre-coated plates for TLC was used as stationary phase and chloroform, methanol and concentrated ammonium hydroxide (30:1:0.2) as mobile phase. After developing, the plate was examined under ultraviolet light at 365nm.For ethoxychelerythrine, dichloromethane and methanol (40:1) was used as mobile phase, instead.Characteristic fingerprints of LC/UV and LC/MS of Z. nitidumroot was established, respectively, which may be used for authentication and identification of the herb. The variation of chromatographic fingerprints among different sampling batches was observed. Samples from different growing regions varied from each other, implying that growing region plays an important role in the entire compositions of Radix Zanthoxyli. The characteristic fingerprints could be used to distinguish Zanthoxyli Radix and its adulterants, including Toddalia asiatica (L.)Lam., Z. Planispini Sieb. et Zucc., Z. dissitum Hemsl.Referring to the regulation method in Pharmacopoeia of People’s Republic of China 2010, nitidine chloride content was determined in roots, stems and leaves of Z. nitidum (Roxb.) DC., respectively. The results showed that the content differed along with growing regions and tissues. Nitidine chloride level in roots was the highest among three tissues, varying from 0.3838 to 4.7268 mg/g among 14 batches. While stems of Z. nitidum growing in Guangxi Province contained nitidine chloride, those in Guangdong Province had little or no nitidine chloride. The differences between the samples from two Provinces might depend on plant age, germplasma or growing environment, which require further studied. No nitidine chloride was detected in leaves of Z. nitidum.Characteristic fingerprints comparison of three tissues of Z. nitidum revealed that chemical compositions of roots and stems were similar, yet the content of each composition varied greatly. Moreover, leaves of Z. nitidum were greatly different from roots and stems in chemical compositions. Chromatographic peaks with the retention time between 15-20 min found in the chromatograms of leaves remained unidentified. The fact that the similarity of different batches of Z. nitidum stems was lower than that of roots may be resulted from the different sampling positions along stems. The contents of magnoflorine, chelerythrine and neoherculin in Z. nitidumwere determined simultaneously. The values were between 0.52-4.08 mg/g,0.55-6.41mg/g and 0.21-3.65 mg/g, respectively. Their contents in corresponding stems were much lower, particularly chelerythrine. Compared with stems of Z. nitidum growing in Guangxi, neoherculin content level similar to that of roots was detected from those growing in Guangdong. No magnoflorine, nitidine chloride and chelerythrine were detected in leaves of Z. nitidum, and neoherculin content was comparatively lower.3. Study on expanding officinal parts other than root of Z. nitidum. Gastric mucosal injury in rats induced hydrochloric acid alcohol and body torsion pain in mice induced by acetic acid were used as models to evaluate the pharmacological effect of extracts from different parts of Z. nitidum, including root, stem, leaves, and aerial part. For the formal model, leaf extract showed the best protective activity, the root extract showed less, and the stem and aerial part extracts were similar to each other. For the latter model, the stem and aerial part extracts showed the best analgesic activity, the root extract took the second place, and the stem extract was the worst. LC/UV and LC/MS were engaged to study chemical compounds and fingerprints of different parts of Z. nitidum from the perspective of chemistry. There are common compounds in the root and stem of the plant, including nitidine chloride, chelerythrine, magnoflorine, neoherculin, and some unknown compounds. Pharmacological experiments showed that leaf extract had the best activity among different parts of the plant. The root and stem of Z. nitidum are used as officinal parts of Rudijinniu in Chinese herbal medicine standard of Guangdong Province (2010). Root and stem, or the whole plant were used as officinal parts of Liangmianzhen in the 1990 and 1996 editions of Chinese herbal medicine standard of the Guangxi Zhuang Autonomous Region. The whole plant is widely used in folk medicine. To protect natural plant resource, expanding medicinal parts of Z. nitidum by using the whole plant is proposed. The future study will search the chemical compound(s) that is (are) responsible for the pharmacological activity(s) in the plant, establish the fingerprint chromatograms or characteristic spectrums of Z. nitidum, elevate or perfect the current edition of pharmacopoeia, provide experimental data to support the next edition of pharmacopoeia, and further, ensure the sustainability use of Z. nitidumresource.4. Establishing the SOP of GAP of Z. nitidum cultivation and setting up of demonstration bases.Linwu village located in suburb of Nanning, Guangxi was chosen as an experimental field of Z. nitidum after fine survey had been done among several cultivated fields. Plant size, leaf size, growth speed, single plant yield, resistivity to disease and pest, pharmacological active compound content, etc., were used to screen the fine varieties. Sandy loam with a pH of 5.0-7.0, or black loam with rich humus, was determined to be the best for Z. nitidum growth. The seed collecting and storage, sowing, cuttage, young plant management was thoroughly studied, and seminal propagation was chosen to be used in scale up growing of Z. nitidum. The cultivation management measures were also studied by plant experiments and pest control study, the approaches were standardized for better growth and higher yield of Z. nitidum, and the best harvest time evaluated by nitidine chloride content was found to be 4-5 years or longer cultivation. The standard operating procedures (SOPs) were then established for large-scale Z. nitidum cultivation. Several demonstration bases for Z. nitidumGAP cultivation have been set up in suburb of Nanning, Guangxi, and Pingyuan, Guangdong. A systematic model for exploitation and utilization and sustainable usage of essential raw herbal material for Proprietary Chinese medicine enterprise is enlightened by the fine study on Z. nitidum as presented above. The enterprises here should act as the principle part when proceeding exploitation and sustainable utilization of essential raw herbal material in production of Proprietary Chinese medicine. The actual production requirement should be centered on when approaches such as officinal plant resource survey, wild medicinal plant domestication, chemistry, standard arid pharmacology studies, standard and large-scale GAP base of high-quality medicinal materials development, etc., are used to supply and ensure safe, effective, and quality controllable herbal materials for Chinese Medicine clinical use and proprietary Chinese medicines production. Dynamically monitoring the cultivation and production of raw herbal material should be carried out by setting up an observation system and relational database of the medicinal plant resources.