[Study on optimal harvesting period and rational medicinal parts of Zanthoxylum nitidum based on statistics of main effective components].

To determine the optimal harvesting period and rational medicinal parts of Zanthoxylum nitidum, the main effective components of cultivated Z. nitidum samples, which originate from various growth years, harvesting months, and different parts were analyzed and compared with the wild samples. HPLC was performed on a Kinetex C18 column(4. 6 mm×100 mm, 2. 6 µm) with the gradient elution of 0. 3% phosphoric acid solution-acetonitrile(80 ∶ 20) containing 0. 2% triethylamine. The flow rate was 1. 0 m L·min-1, and the detection wavelength was 273 nm. The column temperature was 30 ℃. Nitidine chloride and chelerythrine, the main effective components, were determined as the markers. The results showed there was no significant difference in the contents of the main effective components among the roots of wild and cultivated Z. nitidum, as well as the roots and roots + stems of cultivated Z. nitidum. The statistical results of HCA and PCA indicated that the roots and stems could be clearly distinguished, but no distinction could be made between wild and cultivated products, which was consistent with the results of the significance analysis. The total contents of nitidine chloride and chelerythrine in roots and stems of Z. nitidum of 1-6 years old were 0. 114%-0. 256% and 0. 030%-0. 133%, respectively. These results suggested a positive correlation between the content of the main effective components and the growth years. No significant difference was observed between the contents of samples harvested in different seasons, indicating that the harvest season had no effect on the content of the main effective components of the Z. nitidum samples. The total contents of nitidine chloride and chelerythrine of the dried Z. nitidum samples(excluding branches) from three plantation bases were 0. 308%±0. 123% in Yunfu, 0. 192%±0. 025% in Maoming, and 0. 197%±0. 052% in Nanning, respectively, and they were all not less than 0. 15%, or in other words, the roots(including fibrous roots, taproots, and underground stems) and stems(aboveground stems) of Z. nitidum transplanted for more than 2. 5 years can meet the medical requirements. This study demonstrates that the cultivated Z. nitidum could be used as a valid substitute for the wild Z. nitidum, which provides a guarantee for the sustainable development and the application of Z. nitidum resources. The stems and roots could be considered medicinal parts of Z. nitidum. It is recommended to revise the medicinal parts of Z. nitidum to dried roots and stems in the next edition of Chinese Pharmacopoeia, and the medicinal parts can be harvested all year round. In order to ensure the content of effective components and clinical effectiveness, the root and stem should be harvested for medical use after the seedlings of Z. nitidum have been transplanted for more than three years.

[Comparison on volatile components between Artemisiae Verlotori Folium and Artemisiae Argyi Folium based on GC-MS and chemometrics].

Artemisiae Argyi Folium is commonly used in clinical practice. Artemisiae Verlotori Folium, the dried leaves of Artemisia verlotorum, is often used as a folk substitute for Artemisiae Argyi Folium in Lingnan area. In this study, gas chromatography-triple quadrupole mass spectrometry(GC-MS) was used to detect the volatile oil components of 27 samples of Artemisiae Verlotori Folium and 13 samples of Artemisiae Argyi Folium, and the volatile components were compared between the two species. The internal standard method was combined with multi-reaction monitoring mode(MRM) to determine the content of six major volatile components. Hierarchical clustering analysis(HCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were carried out for the content data. The results showed that the Artemisiae Argyi Folium samples had higher content and more abundant volatile oils than the Artemisiae Verlotori Folium samples. Artemisiae Argyi Folium mainly had the components with lower boiling points, while Artemisiae Verlotori Folium mainly had the components with higher boiling points. Terpenoids were the main volatile components in Artemisiae Verlotori Folium(mainly sesquiterpenoids) and Artemisiae Argyi Folium(monoterpenoids). In addition, Artemisiae Argyi Folium had higher content of oxygen-containing derivatives than Artemisiae Verlotori Folium. Furthermore, the stoichiometric analysis showed that the two species could be distinguished by both HCA and OPLS-DA, indicating that the volatile components of the two were significantly different. This study can provide a scientific basis for the quality evaluation and data support for the local rational application of Artemisiae Verlotori Folium in Lingnan.

[Textual research on Bungarus Parvus].

Bungarus Parvus, a precious animal Chinese medicinal material used in clinical practice, is believed to be first recorded in Ying Pian Xin Can published in 1936. This study was carried out to analyze the names, geographical distribution, morphological characteristics, ecological habits, poisonousness, and medicinal parts by consulting ancient Chinese medical books and local chronicles, Chinese Pharmacopeia, different processing standards of trditional Chinese medicine(TCM) decoction pieces, and modern literatures. The results showed that the earliest medicinal record of Bungarus Parvus was traced to 1894. In 1930, this medicinal material was used in the formulation of Annao Pills. The original animal, Bungarus multicinctus, was recorded by the name of "Bojijia" in 1521. The morphological characteristics, ecological habits, and poisonousness of the original animal are the same in ancient and modern records. The geographical distribution is similar between the ancient records and modern documents such as China Medicinal Animal Fauna. The dried body of young B. multicinctus is used as Bungarus Parvus, which lack detailed references. As a matter of fact, it is still inconclusive whether there are differences between young snakes and adult snakes in terms of active ingredients, pharmacological effects, and clinical applications. This study clarified the medicinal history and present situation of Bungarus Parvus. On the basis of the results, it is suggested that systematic comparison on young and adult B. multicinctus should be carried out to provide references for revising the medicinal parts of B. multicinctus.

[Mechanism of moistening process of Rehmanniae Radix based on kinetic process].

The moistening process of Rehmanniae Radix was characterized quantitatively by moisture phase, texture properties, and component content based on water absorption kinetics and expansion kinetics. Non-linear fitting of water absorption kinetics and expansion kinetics in the moistening process of Rehmanniae Radix was carried out. Low-field nuclear magnetic resonance and imaging(LF-NMR/MRI) technology was used to investigate the phase state and distribution changes of water during the moistening process. The Texture Analyzer was used for the determination of texture properties. The correlations between water absorption rate, expansion rate, water phase state, hardness, and compression cycle work of Rehmanniae Radix at different moistening time were analyzed. The results showed that the water absorption kinetics and expansion kinetics of Rehmanniae Radix were in accordance with the first-order kinetics. Moreover, the water absorption rate and expansion rate increased with the increase in temperature but decreased with the increase in the size of the medicinal materials.In the moistening process, the moisture was transferred from the outside to the inside, and the proportion of the moisture phase changed significantly.Within 16 hours, free water increased from 0.825% to 97.7%,while bound water decreased from 99.2% to 2.33%.Within 28 hours, the texture properties, such as hardness and compression cycle work, decreased gradually with the prolongation in moistening time.At 32 hours, water was evenly distributed throughout the whole medicinal material, and the texture properties also tended to be stable.Pearson correlation bivariate analysis showed that moistening time, water absorption rate, expansion rate, the relative content of free water and bound water, hardness, and compression cycle work were significantly correlated, suggesting that water absorption kinetics and expansion kinetics, LF-NMR/MRI,and Texture Analyzer could directly and quantitatively characterize the moistening process.This study is expected to provide a scientific basis for clarifying the scientific connotation of the moistening process of Rehmanniae Radix.

Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment.

BACKGROUND: Steroid drugs are essential for disease prevention and clinical treatment. However, due to intricated steroid structure, traditional chemical methods are rarely implemented into the whole synthetic process for generating steroid intermediates. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) is a novel steroid drug precursor, suitable for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain. RESULTS: The Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. Hsd4A, encoding a ß-hydroxyacyl-CoA dehydrogenase, and fadA5, encoding an acyl-CoA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains were able to accumulate 0.59 g L-1 and 0.47 g L-1 9-OH-4-HP from 1 g L-1 phytosterols, respectively. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 deficient strain was 11.9% higher than that of the Hsd4A deficient strain and 40.4% higher than that of the strain with FadA5 deficiency strain, respectively. The purity of 9-OH-4-HP obtained from the Hsd4A and FadA5 deficient strain has reached 94.9%. Subsequently, the catalase katE from Mycobacterium neoaurum and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment, leading to a higher 9-OH-4-HP production. Ultimately, 9-OH-4-HP production reached 3.58 g L-1 from 5 g L-1 phytosterols, and the purity of 9-OH-4-HP improved to 97%. The final 9-OH-4-HP production strain showed the best molar yield of 85.5%, compared with the previous reported strain with 30% molar yield of 9-OH-4-HP. CONCLUSION: KstD, Hsd4A, and FadA5 are key enzymes for phytosterol side-chain degradation in the C19 pathway. Double deletion of hsd4A and fadA5 contributes to the blockage of the C19 pathway. Improving the intracellular environment of Mycobacterium neoaurum during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

[Textual research of Schizonepetae Herba and Schizonepetae Spica].

Schizonepetae Herba and Schizonepetae Spica are well-known Chinese herbal medicines for wind dispersing and exterior releasing. Through textual research on Schizonepetae Herba and Schizonepetae Spica, the discrimination of their medicinal parts in history was clarified, and the processing, the property(nature and flavor), meridian tropism, functions, indications, usage, dosage, and the selection of decoction pieces were compared to provide the basis for clinical application. As a result, the whole herb of Schizonepeta tenuifolia was used as medicine in the early records. The aerial part and the dried spike of S. tenuifolia were used as medicines separately in the Song Dynasty, which was recorded in the Atlas of Materia Medica(Ben Cao Tu Jing). Some ancient classics emphasized that only the dried spike could be used as medicine. The separation of Schizonepetae Herba and Schizonepetae Spica meets the different needs of clinical medication and supports the concept of rational development and utilization of Chinese medicine resources. About ten processing methods for Schizonepetae Herba and Schizonepetae Spica have been recorded since ancient times, and raw and charred drugs were the major products. Raw Schizonepetae Herba is required to be used in sections, whereas raw Schizonepetae Spica in clean preparation. Both charred products should avoid scorching. Schizonepetae Herba and Schizonepetae Spica are similar in the property(pungent, bitter, and warm), meridian tropism(lung and liver meridians, as well as qi and blood aspects), and functions(releasing exterior, dispersing wind, regulating and stopping blood, promoting eruption, dispelling sores, promoting digestion, eliminating alcohol effect, etc.), but Schizonepetae Spica is superior in efficacy. For Schizonepetae Herba and Schizonepetae Spica in traditional Chinese medicinal prescriptions, the raw and charred products are similar in usage and dosage, while their focuses in clinical compatibility vary. The raw and charred products of Schizonepetae Herba and Schizonepetae Spica are widely applied clinically. Decoction pieces of different specifications can result in different efficacies and clinical applications, so medication should be performed with caution.

[Herbalogical study on Liangmianzhen (Zanthoxyli Radix)].

Liangmianzhen(Zanthoxyli Radix) has long been used as medicine. The current medicinal parts are different from those in the ancient. As recorded in the Chinese Pharmacopeia, the medicinal part is root. However, in ancient works, the medicinal parts include root, stem, leaf, and fruit. In an attempt to find the historical basis that stem is a reasonable medicinal part, the herbalogical study was carried out on this medicinal based on the formal names, synonyms, original plant, medicinal parts, habitat of the medicinal plant, producing area, processing and preparation methods, efficacy, and indications recorded in ancient Chinese materia medica and local gazetteers. The results showed that Liangmianzhen was firstly recorded as a medicinal in Shennong's Classic of Materia Medica with the formal name of "Manjiao". "Manjiao" was adopted from the Han Dynasty to the Qing Dynasty when it was changed to "Rudijinniu", the name originating from the folk in the south of the Five Ridges. Now, the formal name is "Liangmianzhen", which was firstly recorded in Wuxuan County Gazetteer in 1914 and then as a synonym in the Updated Records of Picking Herbs in the South of the Five Ridges. According to the formal names, synonyms, and the descriptions of the original plant, the medicinal plants of Liang-mianzhen have the characteristics of shrub-like young seedlings, vine adult seedlings, corymbiform thyrsus, stems with thorns, amphitropous golden-yellow roots with horn-like branches, and thorns on both sides of the leaves. Thus, "Manjiao", "Rudijinniu", and "Liangmianzhen" were from the same species of Zanthoxylum nitidum(Rutaceae), which was also verified based on the growth environment, habitat, processing and preparation methods, efficacy, and indications. In ancient times, the stem and root were the main medicinal parts and leaves and fruits were also used. However, in the Chinese Pharmacopeia, root is recorded as the only medicinal part, which is obviously inconsistent with the records in the ancient classics. In light of the limited medicinal resources for Liang-mianzhen, other medicinal parts of Z. nitidum is recommended. This study clarified the medicinal parts of Z. nitidum in history. It is recommended that the stem be added to the medicinal parts of Z. nitidum in the next edition of Chinese Pharmacopeia.

[Herbalogical study on historical evolution of collection, processing and efficacy of Citri Exocarpium Rubrum and Citri Grandis Exocarpium].

In ancient times, the original plants of Citri Exocarpium Rubrum and Citri Grandis Exocarpium had experienced succession and change, including tangerine(Citrus reticulata), pomelo(C. grandis), and Huazhou pomelo(C. grandis 'Tomentosa'), a specific cultivar of C. grandis produced in Huazhou, Guangdong. Before the Qing Dynasty, tangerine was the main original plant, while Huazhou pomelo came to the fore in the Qing Dynasty. In the 1950 s and 1960 s, the producing area of Huazhou pomelo was destroyed, and thus it had to be supplemented with pomelo. From then on, C. grandis 'Tomentosa' and C. grandis were both listed as the original plants of Citri Grandis Exocarpium in the Chinese Pharmacopoeia. This paper reviewed the historical evolution of the collection, processing, and efficacy of Citri Exocarpium Rubrum and Citri Grandis Exocarpium. The research showed that:(1)The harvest time of the original plants of Citri Grandis Exocarpium and Citri Grandis Exocarpium had changed from maturity to immaturity. The collection and processing of Citri Exocarpium Rubrum was first recorded in the Illustrated Classics of Materia Medica in the Song Dynasty. During the Ming and Qing Dynasties, the mesocarp of Citri Exocarpium Rubrum needed to be removed completely, and Citri Grandis Exocarpium from C. grandis 'Tomentosa' was processed into different specifications such as seven-piece, five-piece, and single piece. Furthermore, processed young fruits of Huazhou pomelo appeared.(2)Citri Exocarpium Rubrum and Citri Grandis Exocarpium were processed with carp skin for the first time in the Master Lei's Discourse on Medicinal Processing. It was suggested that carp skin might be helpful for eliminating bones stuck in throat. During the Song, Jin, and Yuan Dynasties, some other processing methods such as ba-king, stir-frying, and salt-processing appeared. Honey, soil, ginger juice, and alum were firstly used as adjuvants for the processing in the Ming and Qing Dynasties. Citri Exocarpium Rubrum was mainly prepared with salt in order to improve the effect of lowering Qi, while it was unnecessary for Citri Grandis Exocarpium from C. grandis 'Tomentosa' because of its obvious effect of lowering Qi and eliminating phlegm. The stir-frying and honey-frying methods helped reduce the strong effect of Citri Grandis Exocarpium from C. grandis 'Tomentosa'.(3)According to the application of Citri Exocarpium Rubrum and Citri Grandis Exocarpium in history, their medicinal use began in Han and Tang Dynasties, developed in Song, Jin, and Yuan Dynasties, and matured in Ming and Qing Dynasties. Citri Grandis Exocarpium from C. grandis 'Tomentosa' was originally applied in Ming and Qing Dynasties, and it still plays an important in role treating COVID-19 nowadays. Moreover, Citri Grandis Exocarpium from C. grandis had cold medicinal property, while Citri Grandis Exocarpium from C. grandis 'Tomentosa' had warm medicinal property, and thus they should not be treated the same. At present, Huazhou pomelo has a certain production scale. Therefore, it is recommended that in the next edition of Chinese Pharmacopoeia, only C. grandis 'Tomentosa' should be included as the original plant of Citri Grandis Exocarpium, and C. grandis should be deleted. The results are conducive to the further development and utilization of Citri Exocarpium Rubrum and Citri Grandis Exocarpium, and support the rational use of Citri Grandis Exocarpium and its processed products.

[Herbalogical analysis of Aconiti Carmichaeli Radix].

Tianxiong has been used as a Chinese medicinal in China for thousands of years, and the earliest record can be traced back to the Shennong's Classic of Materia Medica. It is effective in dispersing wind, dissipating cold, and replenishing fire to streng-then yang. To clarify the origin of Tianxiong, the present herbalogical study reviewed the ancient and modern literature from the origin, processing, and clinical efficacy. Before the Tang Dynasty, although the description of Tianxiong was quite superficial, an apparent difference between Tianxiong and Fuzi was recognized. In the Tang and Song Dynasties, Tianxiong and Fuzi were mistakenly recognized to be prepared from a same plant since their raw materials came from artificial cultivation. Medical literature in the Ming and Qing Dynasties mostly followed the previous records, with the origin of Tianxiong remaining controversial. There were three mainstream views about the origin of Tianxiong according the ancient medical books. First, Tianxiong was a kind of Aconiti Radix(Chuanwu) without attachment of Fuzi. Second, Tianxiong was the large Fuzi. Third, Tianxiong derived from Aconiti Kusnezoffii Radix(Caowu) about 10 cm in length. By contrast, Fuzi in a large size was simply regarded as Tianxiong in modern times. The processing methods were diversified in the ancient times, and the fire-processing was continuously applied. With the deepening of the research on the efficacy and detoxification mechanism, more methods were discovered, such as processing with ginger juice, child's urine and alcohol. As for modern times, the processing of Tianxiong has not been nearly passed down. The characteristic processing of Tianxiong only handed down in Sichuan province and Lingnan area, which can be discriminated by the last step. The efficacies of Tianxiong can be directly understood from its literal name, including dispersing wind, dissipating cold, and replenishing fire to assist yang. Nowadays, Tianxiong is mostly used to strengthen yang.

[Herbalogical study on historical evolution of Juhong and Huajuhong].

In ancient times, there were two types of "Juhong" came from the tangerines(Citrus reticulata) and the pomelos(C. grandis and its cultivars), which corresponded to Juhong and Huajuhong recorded in the Chinese Pharmacopoeia respectively. In different periods, Juhong basically came from the same species and the same medicinal parts, but there were also some differences. This article sorted out the ancient and modern literature, under the guidance of "Succession theory of Medicinal materials varieties" and "Change theory of Medicinal materials varieties"(XIE Zong-wan), and combined with field investigation, the evolution and reasons of the original plants and medicinal parts of Juhong were analyzed. In the Han Dynasty and before, the peel of tangerines and pomelos were both used as medicine. In the Southern and Northern Dynasties, the way tangerine peel was used was dried and aged, and then "soaked in hot water and scraped off the mesocarp", which had the essence of only using exocarp as medicine of Juhong already, and its original plant was C. reticalata. In the Song Dynasty, the name of "Juhong" and its medicinal usage were recorded in book on materia medica, and the species and medicinal parts of tangerine were inherited from the previous dynasties. The way tangerine peel was used was only dried and aged without removing the mesocarp. The medicinal material obtained by the way was called Chenpi(dried and aged tangerine peel). The item "Juhong" listing as a separate medicinal material was first recorded in the Collected Discussions from Materia Medica(Bencao Huiyan) in the Ming Dynasty. In the Ming Dynasty, the Dao-di habitat of Juhong was recorded as Guangdong province in most books on materia medica, and the original plants probably were C. reticalata and C. grandis 'Tomentosa'(Huazhou pomelo, a special cultivated species of C. grandis produced in Huazhou, Guangdong, which was recorded in the Chinese Pharmacopoeia as "Huajuhong"), according to the records in the local chronicles. During the Qing Dynasty and the Republic of China, the original plants of Juhong were C. reticalata and C. grandis 'Tomentosa'. Of the two, the latter one was considered as the better. As far the medicinal part, it was still the exocarp, while the whole young fruit of C. grandis 'Tomentosa' began to be used as medicine. After the founding of The People's Republic of China, the exocarps of Citrus reticalata, C. grandis and C. grandis 'Tomentosa' were listed in the Chinese Pharmacopoeia under "Juhong". From the Northern and Southern Dynasties to the Republic of China, C. grandis exocarp was a fake of Juhong. Therefore, it was contradictory to historical records that C. grandis exocarp was listed in the Chinese Pharmacopoeia as Huajuhong. Juhong had been divided into two types as "Juhong" and "Huajuhong" since 1985. The medicinal part of Huajuhong was only the exocarp of immature and nearly mature fruits, but not the whole young fruit, the actual mainstream medicinal part of Huajuhong. The results are helpful to clarify the historical evolution of species and medicinal parts of Juhong and Huajuhong. It is suggested that in the next edition of Chinese Pharmacopoeia, only C. grandis 'Tomentosa' should be included as the original plant of Huajuhong, and C. grandis should be deleted, and the young fruit should be added in the medicinal parts besides the exocarp of immature and nearly mature fruit.