Liriopogons (Genera Ophiopogon and Liriope, Asparagaceae): A Critical Review of the Phytochemical and Pharmacological Research

The closely related genera Liriope and Ophiopogon (Asparagaceae), collectively known in English as liriopogons, have similar therapeutic uses in treating cough, rheumatoid arthritis, and cleaning heat. The main aim of this review is to understand the current phytochemical and pharmacological knowledge including an assessment of the quality of the scientific evidence. A literature search was conducted in line with PRISMA guidelines, by retrieving available information up to 2020 from five online resources. The bioactive metabolites of liriopogons include steroidal saponins, flavonoids, polysaccharides, organic acids, phenols. Cardiovascular protective, anti-inflammatory, anti-diabetic, anti-oxidant, anti-cancer, neuroprotective, anti-viral, anti-acute myeloid leukemia and hepatoprotective effects have been at the center of attention. From a toxicological perspective Ophiopogon japonicus seems to be safe. Some problems with the quality of the pharmacological evidence stand out including the application of excessive dose level and methodological problems in the design. Additionally, a reasonable link between local/traditional uses and pharmacological assessment is often vague or not reflected in the text. Future researches on liriopogons are required to use rigorous scientific approaches in research on evidence-based natural products for the future benefits of patients.

Effect of Paclobutrazol on the Physiology and Biochemistry of Ophiopogon japonicus

Ophiopogon japonicus is a commonly used Chinese medicine with multiple pharmacological effects. To increase the yield of O. japonicus, paclobutrazol is widely used during the cultivation, and residues of paclobutrazol cause undesired side effects of O. japonicus. In this study, the effect of different concentrations of paclobutrazol on O. japonicus was investigated, and the final residual amount of paclobutrazol in the plant sample was determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS); cell morphology was observed by transmission electron microscopy. The inhibitory effect of paclobutrazol on plant height and the stimulatory effect on root elongation were concentration-dependent from 0.6 to 11.3 g/L, reaching a maximum of about 28% and 67%, respectively. However, when the concentration was 22.5 g/L, these effects were significantly weakened, and the same trend was observed for the tuber root weight. Paclobutrazol caused the cell wall of O. japonicus to thicken, making the cells smaller and more densely arranged. Paclobutrazol also inhibited bacterial growth, irrespective of the concentration. Considering the residual concentration after application and the effects on growth, the application of 1.3 g/L or 2.8 g/L paclobutrazol can increase the accumulation of effective ingredients while promoting production, reducing application costs, and maximizing farmers’ profit.

First report of black spot on Ophiopogon japonicus caused by Alternaria alternata in Zhejiang Province, China

Ophiopogon japonicus (Linn. f.) Ker-Gawl, a traditional Chinese medicinal plant, is widely cultured in China. The root of O. japonicus, is used as the main ingredient in many presriptions. It is rich in chemical components for steroidal saponins, homoisoflavonoids and polysaccharides, which have various pharmacological activities, such as cardiovascular protection, anti-inflammation and anti-diabetes (Chen. et al. 2016). In May and July for 2018 and 2019, the symptoms of black spot on O. japonicus were observed with an incidence of 40% in Cixi County, Zhejiang Province, China. The pathogen mainly infected leaves causing severe black spots, which resulted in a 28% yield loss per acre. At the early stage of the disease, the tip of the leaf began to turn yellow, then the discoloration gradually spread to the base of the leaf and finally the whole leaf turned reddish brown with visible black spot. Symptomatic leaves were cut into small pieces (1.0 cm × 1.0 cm) and disinfected successively by submersion in 75% ethanol for 30s and 1% NaClO for 30s under aseptic conditions. After rinsing with sterile water three times and air drying, segments were placed on potato dextrose agar (PDA), and incubated at 28 ℃ in dark for a week. Then, pathogen on the PDA were transferred onto potato carrot agar (PCA), and incubated at 23 ℃ under the condition of alternation of day (12 h) and night (12 h) for a week. Colonies on PDA were dark gray in the center surrounded by white to gray on the upper side, and black with white margins on the back of the plate. Colonies on PCA were grayish with sparse hyphae. The conidia were obclavate or ellipsoid, pale brown, with 3~8 transverse septa and 1~4 longitudinal septa. Conidiophores were septate, arising singly, and measured (17.0~81.0) × (8.0~23.5) μm, Most conidia had a conical or columnar beak, approximately (0~23.5) × (2.5~9.0) μm in size. According to morphological and cultural characteristics, these isolates were preliminarily identified as Alternaria alternata. A. alternata is one of the most typical plant pathogen, more than 95% of which facultatively parasitize on plants, causing disease in numerous crops. To further confirm identification of pathogens, the internal transcribed spacer region (ITS), translation elongation factor 1-α gene (EF-1α), RNA polymerase Ⅱ second largest subunit (RPB2), major allergen Alt a 1 gene (Alt a 1), Histon 3 gene (His) and plasma membrane ATPase (ATP)were amplified with primer pairs ITS1/ITS4, EF1-728F/EF1-986R, RPB2-7cr/RPB2-5f2, Alt-for/Alt-rev, His 3-F/His 3-R, ATP-F/ATP-R (Lawrence D.P. et al. 2013; Hong, S.G., et al. 2005). BLASTN analysis of NCBI using ITS (Accession NO. MW989987), Alt a1 (Accession NO. MW995953), EF-1α (Accession NO.MW995955), ATP (Accession NO.MW995957), His (Accession NO. MW995954) and RPB2 (Accession NO. MW995956) showed 100%, 100%, 97%, 99%, 99% and 97% identity to A. alternata MN249500.1, MN304714.1, MK637432.1, MK804115.1, MK460236.1, MK605888.1, respectively. To verify pathogenicity, healthy plants (1-year-old) of O. japonicus in ten pots were spray-inoculated with conidial suspension (1 × 106 conidia/ml). Ten plants, which were treated with sterile water, were used as the control. All plants were maintained in a climatic chamber (26 ± 1 ℃, 70–80% relative humidity and a photoperiod of 16:8 [L: D] h). Fourteen days later, all inoculated plants showed typical symptoms of black spot identical to those observed in the fields. Control plants remained symptomless and healthy. The pathogenicity analysis was repeated three times. Pathogens re-isolated from symptomatic plants were identified as A. alternata by morphology observation and sequence analysis. To our knowledge, this is the first report of black spot caused by A. alternata on O. japonicus in Zhejiang, China.

First report of Meloidogyne arenaria infecting Maidong (Ophiopogon japonicus) in China

Maidong (Ophiopogon japonicus) is a perennial evergreen plant of the Asparagaceae, occurring mainly in China, Japan, Vietnam, and India. It grows in the damp place on the hillside below 2000 meters above sea level, under the forest or beside the stream;It has been widely cultivated in the Sichuan ofhina for medicinal uses; and it is included in the Chinese Pharmacopoeia. During April 2019, Maidong plants exhibiting symptoms of stunting, leaf wilting, and multiple galls in the roots associated with root-knot nematode (Meloidogyne sp.) were detected in a commercial field in near the city of Mianyang (N105°42′, E30°93′), Sichuan, China. The second-stage juveniles (J2) were collected from the soil in the root zone, and adult females were dissected from roots. Population densities of J2 ranged from 190 to 255 per 100 cm3. Subsequently, individual females (n=20) were extracted from root samples and submitted to Meloidogyne species identification by perineal pattern morphological analysis (n=20), and morphometric measurements of second stage juveniles (J2) (n = 20). The J2 showed the following morphometric characters：body length = 475.5 ± 24.2 µm, tail length = 55.2 ± 6.43µm, stylet length = 12.4 ± 1.56 µm and distance from dorsal esophageal gland opening to the stylet knot (DGO) = 2.97 ± 0.44 μm; perineal patterns of females showed a low dorsal arch, with lateral field marked by forked and broken striae, no punctate markings between anus and tail terminus were observed. These morphological characteristics are consistent with Meloidogyne arenaria (Neves et al. 2016). In addition, to confirm species identification, DNA was extracted from females (Blok, et al. 1997) and D2/D3 fragments of the 28S rRNA was amplified using the universal primers D2A/D3B. The DNA fragment obtained showed a 754 bp length (GenBank accession no. MW965614) that was sequenced and analyzed, sequences were 99.8% identical to the MH359158, KX151138 and EU364889 M. arenaria sequences. Furthermore, species-specific SCAR primers Far/Rar were used as described by Zijlstra et al. 2000. The PCR produced approximately 420 bp sequences, which was identical to that previously reported for M. arenaria (Zijlstra et al. 2000). Morphological and molecular characterization supports the identification of the isolate found on Ophiopogon japonicus as M. arenaria. To verify the nematode pathogenicity on Maidong plants, Maidong seed were planted in 20-cm diameter, 10-cm deep plastic pots containing 1000 cm3 sterilized soil and infested with 2000 M. arenaria J2 per seedling, using a sterilized micropipette. Plants were maintained at 20-25°C in a greenhouse. Control plants received sterile water, and the pathogenicity test was repeated three times. After 60 days, all inoculated plants showed reduced growth compared with control. The symptoms were similar to those observed in the field, a large number of galls (38.5 ± 2.4) and egg masses (18.5 ± 0.2) were found on each root system. Maidong was considered a good host for M. arenaria in Mianyang. M. arenaria is one of the most important plant parasitic nematode with a wide geographic distribution and causes great losses in many crops around the world (Perry et al. 2009). Through investigation, this is the first report worldwide of M. arenaria infecting Ophiopogon japonicus.