First Report of Anthracnose on Ilex cornuta caused by Colletotrichum aenigma in China.

Ilex cornuta (Aquifoliaceae) is a dark green evergreen shrub with glossy leaves that is widely distributed in China and East Asia and used as an ornamental and medicinal plant. In March 2022, typical symptoms of anthracnose were observed on I. cornuta leaves (with approximately 30% of leaves affected) in Jiangxi Academy of Forestry, Nanchang city, Jiangxi Province, China. The early symptoms were light brown spots on the edge or tip of the leaves. The spots gradually expanded to ovoid-shaped lesions and eventually become necrotic, dry, and gray with a dark brown margins. To isolate the pathogen, ten symptomatic leaves were randomly collected, the edges between diseased area and healthy area were cut into small pieces (4×4 mm), surface sterilized by dipping in 70% ethanol for 30 s and 1% sodium hypochlorite (NaClO) for 30 s, and then washed three times with sterile distilled water. Leaf pieces were then placed on potato dextrose agar (PDA) plates at 28℃ in the dark. Subsequently, six isolates were obtained using the single-spore method, five of them were similar in morphological characteristics. Colonies grown on PDA for 7 days were 75-85 mm in diameter, and were cottony, dense, and pale white on the surface and white to grayish-green on the reverse side. Conidia were single-celled, transparent and subcylindrical to clavate. The contents of the conidia were granular and 15.63-20.63 × 5.63-7.50 µm in size (=17.78 ± 1.41× 6.50 ± 0.55 µm, n = 40). The species was also identified by analysis of the internal transcribed spacer (ITS) region, actin (ACT), glyceradehyde-3-phosphate dehydrogenase (GAPDH) and ß-tubulin (TUB2), calmodulin (CAL), glutamine gynthetase (GS), DNA Lyase (APN2), intergenic spacer and partial mating type (ApMat) genes using ITS4/ITS5, ACT-512F/ACT-783R, GDF/GDR, T1/Bt2b, CL1C/CL2C, GSF/GSR, ColDL-F3/CgDL-R1 and CgDL-F6/CgMAT1F2 primers, respectively (Weir, et al. 2012; Maharachchikumbura, et al. 2014; Khodadadi, et al. 2020). The sequences were deposited in GenBank (Accession Nos. OQ600619, OQ603370, OQ603373, OQ603379, OQ974177, OQ974176, OQ974178, and OQ974175). BLASTN analysis in GenBank showed that these genes exhibited 100% similarity to the sequences of Colletotrichum aenigma (MT476812, MN525817, MN525878, MN525904, MN525836, KX620296, and MN338281) and 99% similarity to the sequence of Colletotrichum sp. strain (MT071110). Concatenated sequences of these eight genes and Colletotrichum species sequences from GenBank were then used to construct a phylogenetic tree by using the maximum likelihood method in IQtree V1.5.6. Isolate JFRL 03-1005 was grouped into a clade with C. aenigma with a high bootstrap value. Thus, the isolate was identified as C. aenigma based on morphological and molecular data. To verify Koch's postulates, pathogenicity was tested on 2-year-old healthy potted plants of I. cornuta. Ten disinfected leaves were wounded with a sterile scalpel, and then inoculated with 10 µl of conidial suspension (1 × 106 conidia/ml) from isolate JFRL 03-1005. The control leaves were inoculated with 10 µl of sterile water. Then, the potted plants were incubated at 28°C with a 12 h photoperiod and 80% humidity. After 10 days, distinct spots appeared on all inoculated leaves, whereas control leaves remained asymptotic. C. aenigma was reisolated from the spots and identified by sequencing the ITS, ACT, GAPDH, TUB2, CAL, GS, APN2 and ApMat genes. Previous studies reported that C. aenigma can caused anthracnose on the leaves of various cash crops in China, such as apple, tree peonies, mulberry, and walnut (Wang et al. 2020; Zhang et al. 2021; Wang et al. 2022; Zhu et al. 2022). To the best of our knowledge, this is the first report of C. aenigma causing anthracnose on I. cornuta in China, this report further confirmed that C. aenigma has a wide range of hosts in nature. the anthracnose on I. cornuta caused by C. aenigma has seriously affected its ornamental value. Therefore, more attention should be paid to this disease and appropriate control strategies should be formulated.

The Extract of Ilex cornuta Bark Promotes Bone Healing by Activating Adenosine A2A Receptor.

Introduction: Bone fracture is a common reason causing human disability. The delay union and nonunion rates are approximately 5-10% despite patients receiving active treatment. Currently, there is a limited number of drugs directly accelerating bone healing, especially direct extracts from plants. Moreover, the pharmacological effects of Ilex cornuta bark are still unknown. This study aimed to explore the effects and mechanisms of Ilex cornuta bark in bone healing. Methods and Results: First, the promoting effects of Ilex cornuta bark on bone healing were verified by the mice femur fracture model as Ilex cornuta bark increased the callus formation and enhanced the biomechanical stability during the bone healing process. Second, the target gene of Ilex cornuta bark in bone healing identified by bioinformatics analysis and immunofluorescence validation was ADORA2A. Third, 410 main compound compositions of Ilex cornuta bark were explored by a non-target metabolomic analysis, where 190 of them were neg ion mode, and 220 were pos ion mode. Molecular docking was used to predict the regulatory effect of the compounds on adora2a (adenosine A2A receptor), and ursonic acid had the lowest binding energy with adora2a. Finally, nfkb1 was the transcription factor (TF) of adora2a, and ursonic acid also had the lowest binding energy by bioinformatic analysis and molecular docking. Conclusion: Overall, Ilex cornuta bark water extract was a new plant extract on promoting bone healing; in addition, the mechanism of it might be activating adora2a though Nfkb1.

Isolation, Structural Analysis and Anti-Inflammatory Activity of a Polysaccharide from Ilex cornuta Fruits.

In the present study, a polysaccharide from Ilex cornuta fruits (LCFP-3) was obtained by hot water extraction, Diethyaminoethyl cellulose-52 (DEAE-52) chromatography column and Sephadex G-100 gel column purification. Its structural characteristics were further explored using high performance anion exchange chromatography (HPAEC), gas chromatography and mass spectrometry (GC/MS), scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. Monosaccharide composition analysis revealed LCFP-3 contained mainly Galactose (31.92 %), Arabinose (25.87 %) and Galacturonic acid (23.35 %) while small percentage of Rhamnose, Glucose, Mannose and Xylose. Chemical composition analysis showed that the total sugar content of LCFP-3 was 90.31 % and the protein content was 0.246 %. Gel permeation chromatography (GPC) analysis showed that its average molecular weight was 41.199 kDa. Structural analysis showed that LCFP-3 may be composed of residues, T-α-Arap, T-α-Rhap, 1,3-α-Arap, 1,4-α-Arap, T-ß-Galp, 1,4-α-GalpA(OMe), 1,4-ß-Glcp, 1,3-ß-Galp, 1,3,6-ß-Manp, 1,6-ß-Galp, 1,3,4-ß-GalpA, 1,4,6-ß-Manp, 1,3,6-ß-Glcp, 1,2,3,4-α-Xylp. The anti-inflammatory activity of LCFP-3 was evaluated using lipopolysaccharide (LPS)-induced RAW246.7 macrophages. The results showed that 1-200 µg/mL LCFP-3 could dose-dependently protect against LPS-induced toxicity and 1 µg/mL LCFP-3 could significantly inhibit LPS-induced NO production. Therefore, LCFP-3 exerted an anti-inflammatory activity and has great potential as a functional ingredient.

Triterpenoid saponins from Ilex cornuta protect H9c2 cardiomyocytes against H2O2-induced apoptosis by modulating Ezh2 phosphorylation.

ETHNOPHARMACOLOGICAL RELEVANCE: Ilex cornuta Lindl. et Paxt. (Aquifoliaceae family) belongs to the Ilex genus. The leaves of this plant are used for the popular herbal tea "Ku-Ding-Cha" in China due to their health benefits for sore throat, obesity and hypertension. Our previous studies have shown that the extract of Ilex cornuta root exerts cardioprotective effects in rat models of myocardial ischaemic injury, and several new kinds of triterpenoid saponins from Ilex cornuta (TSIC) have protective effects against hydrogen peroxide (H2O2)-induced cardiomyocyte injury. AIM OF THE STUDY: The aim of this study was to clarify the underlying mechanisms by which TSIC protect against H2O2-induced cardiomyocyte injury. MATERIALS AND METHODS: An H2O2-treated H9c2 cardiomyocyte line was used as an in vitro model of oxidation-damaged cardiomyocytes to evaluate the effects of TSIC. Apoptosis was detected with CCK-8 and annexin V assays and via analysis of the levels of apoptosis-associated proteins or genes. The underlying mechanisms related to Akt signalling, Ezh2 expression and activity, and ROS were clarified by Western blotting, quantitative PCR, flow cytometry and rescue experiments. RESULTS: TSIC protected H9c2 cells from H2O2-induced apoptosis. This effect of TSIC was attributable to inhibition of Ezh2 activity, as exhibited by attenuation of H2O2-induced Akt signalling-dependent phosphorylation of Ezh2 at serine 21 (pEzh2S21) upon TSIC pretreatment. In addition, feedback pathway between Akt-dependent Ezh2 phosphorylation and ROS was involved in TSIC-mediated protection of H9c2 cells from apoptosis. CONCLUSIONS: Our findings indicate a pivotal role of the pEzh2S21 network in TSIC-mediated protection against cardiomyocyte apoptosis, potentially providing evidence of the mechanism of TSIC in the treatment and prevention of cardiovascular diseases.

Anti-adipogenic 18,19-seco-ursane stereoisomers and oleane-type saponins from Ilex cornuta leaves.

Three undescribed seco-ursane stereoisomers, ilexcornutosides A-C, two undescribed triterpenoid saponins, ilexcornutosides D-E, and 11 known triterpenoids were isolated from the leaves of Ilex cornuta Lindl. & Paxton. Ilexcornutosides A-C and F with the same planar structures are unique 13(18)-ene-18,19-seco-ursane skeleton triterpenoids, identified as (3S,12R)-3-O-[ß-d-glucopyranosyl-(1 â†’ 2)-α-l-arabinopyranosyl]-12-hydroxyl-19-oxo-18,19-secours-13(18)-en-28,21-lactone. Among them, ilexcornutosides A and F (or ilexcornutosides B and C) are a pair of diastereomers at the C-20 position; ilexcornutosides A and C (or ilexcornutosides B and F) are a pair of diastereomers with epimerization at the C-21. Their structures were established by extensive spectroscopic (IR, 1D and 2D NMR, and HR-ESI-MS) and chemical analyses. The absolute configurations of ilexcornutosides A, B, D and F were determined by a single crystal X-ray diffraction analysis with a Cu Kα radiation. The inhibitory effect of ilexcornutosides A-F on the PPARγ expression was assessed in the 3T3-L1-Lenti-PPARγ-Luc cells using a single luciferase reporter assay. Ilexcornutosides A and C showed a comparable activity in decrease of the PPARγ expression to the positive control (T0070907) at 5 µM.

Structural characterization and biological evaluation of chemical constituents from Ilex cornuta.

One new ursane-type triterpenoid (1), one new ursane-type triterpenoid glycoside (2), and one new oleanane-type triterpenoid glycoside (3), along with 20 known compounds, were isolated from the leaves of Ilex cornuta. The structures of these natural products were elucidated on the basis of detailed spectroscopic analyses and chemical derivation. Our biological evaluation established that selective compounds showed moderate to significant antioxidant activities in the 1,1-diphenyl-2-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) methods.

Two New Ursane-type Triterpenoid Glycosides from Ilex Cornuta.

Ilex cornuta (I. cornuta) is a traditional Chinese medicine (TCM) that has been used in clinical practice for hundreds of years. In order to provide more information about the chemical basis of its pharmacological effects, phytochemical investigation on the roots of I. cornuta was conducted in this study. The roots of the plant were firstly extracted with 95% EtOH, and then the crude was partitioned with petroleum ether, EtOAc and n-butyl alcohol. Different chromatographies were employed to isolate the crude step by step and the crude was further purified by semipreparative high performance liquid chromatography (HPLC). As a result, two new triterpenoid saponins (1, 2), together with 12 known compounds (3-14), were isolated from the roots of I. cornuta. Their structures were determined based on nuclear magnetic resonance (NMR), mass spectrum (MS) technologies, chemical reactions as well as gas chromatography (GC). Compounds 4, 6, 8, 11, 12 and 13 were isolated from this genus for the first time. The structures of compounds 1 and 2 were determined as 3ß-O-α-D-xylopyranosyl-(1→3)-α-L-2-O-acetylarabinopyranosyl-(1→2)-ß-D glucopyranosyl-23-hydroxyl-20α(H)-urs-12-en-28-oic acid 28-O-ß-D-glucopyranosyl ester (1) and 3ß-O-α-D-xylopyranosly-(1→3)-α-L-2-O-acetylarabinopyranosyl-(1→2)-ß-Dglucopyranosyl- 19α,23-dihydroxyl-20α(H)-urs-12-en-28-oic acid 28-O-ß-D-glucopyranosyl ester (2).

Three new triterpenoids isolated from the aerial parts of Ilex cornuta and protective effects against H2O2-induced myocardial cell injury.

In the present study, three new triterpenoids, 23-hydroxyurs-12, 18-dien-28-oic acid 3ß-O-α-L-arabinopyranoside (1), 23-hydroxyurs-12, 18-dien-28-oic acid 3ß-O-ß-D-glucuronopyranoside-6-O-methyl ester (2), and urs-12, 18-dien-28-oic acid 3ß-O-ß-D-glucuronopyranoside-6-O-methyl ester (3), and a known triterpenoid, 3ß-hydroxy-urs-2, 18-dien-28-oic acid (4, randialic acid B), were isolated from the aerial parts of Ilex cornuta. Their structures were identified by the spectroscopic analyses (IR, ESI-MS, HR-ESI-MS, and 1D and 2D NMR) and chemical reactions. Compound 4 showed significant cell-protective effects against H2O2-induced H9c2 cardiomyocyte injury. Compounds 1-4 did not show any significant DPPH radical scavenging activity.

Antioxidant and cardioprotective effects of Ilex cornuta on myocardial ischemia injury.

Previous studies have indicated that the Ilex genus exhibits antioxidant, neuroprotective, hepatoprotective, and anti-inflammatory activities. However, the pharmacologic action and mechanisms of Ilex cornuta against cardiac diseases have not yet been explored. The present study was designed to investigate the antioxidant and cardioprotective effects of Ilex cornuta root with in vitro and in vivo models. The anti-oxidative effects of the extract of Ilex cornuta root (ICR) were measured by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging and MTT assays as well as immunoassay. Furthermore, a rat model of myocardial ischemia was established to investigate the cardioprotective effect of ICR in vivo. Eight compounds were isolated and identified from ICR and exhibited DPPH free-radical scavenging activities. They also could increase cell viability and inhibit morphological changes induced by H2O2 or Na2S2O4 in H9c2 cardiomyocytes, followed by increasing the SOD activities and decreasing the MDA and ROS levels. In addition, it could suppress the apoptosis of cardiomyocytes. In the rat model of myocardial ischemia, ICR decreased myocardial infarct size and suppressed the activities of LDH and CK. Furthermore, ICR attenuated histopathological alterations of heart tissues and the MDA levels, while increasing SOD activities in serum. In conclusion, these results suggest that ICR has cardioprotective activity and could be developed as a new food supplement for the prevention of ischemic heart disease.

Three new triterpenoids isolated from the aerial parts of Ilex cornuta and protective effects against HO-induced myocardial cell injury / 中国天然药物

In the present study, three new triterpenoids, 23-hydroxyurs-12, 18-dien-28-oic acid 3β-O-α-L-arabinopyranoside (1), 23-hydroxyurs-12, 18-dien-28-oic acid 3β-O-β-D-glucuronopyranoside-6-O-methyl ester (2), and urs-12, 18-dien-28-oic acid 3β-O-β-D-glucuronopyranoside-6-O-methyl ester (3), and a known triterpenoid, 3β-hydroxy-urs-2, 18-dien-28-oic acid (4, randialic acid B), were isolated from the aerial parts of Ilex cornuta. Their structures were identified by the spectroscopic analyses (IR, ESI-MS, HR-ESI-MS, and 1D and 2D NMR) and chemical reactions. Compound 4 showed significant cell-protective effects against HO-induced H9c2 cardiomyocyte injury. Compounds 1-4 did not show any significant DPPH radical scavenging activity.

Antioxidant and cardioprotective effects of Ilex cornuta on myocardial ischemia injury / 中国天然药物

Previous studies have indicated that the Ilex genus exhibits antioxidant, neuroprotective, hepatoprotective, and anti-inflammatory activities. However, the pharmacologic action and mechanisms of Ilex cornuta against cardiac diseases have not yet been explored. The present study was designed to investigate the antioxidant and cardioprotective effects of Ilex cornuta root with in vitro and in vivo models. The anti-oxidative effects of the extract of Ilex cornuta root (ICR) were measured by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging and MTT assays as well as immunoassay. Furthermore, a rat model of myocardial ischemia was established to investigate the cardioprotective effect of ICR in vivo. Eight compounds were isolated and identified from ICR and exhibited DPPH free-radical scavenging activities. They also could increase cell viability and inhibit morphological changes induced by HO or NaSO in H9c2 cardiomyocytes, followed by increasing the SOD activities and decreasing the MDA and ROS levels. In addition, it could suppress the apoptosis of cardiomyocytes. In the rat model of myocardial ischemia, ICR decreased myocardial infarct size and suppressed the activities of LDH and CK. Furthermore, ICR attenuated histopathological alterations of heart tissues and the MDA levels, while increasing SOD activities in serum. In conclusion, these results suggest that ICR has cardioprotective activity and could be developed as a new food supplement for the prevention of ischemic heart disease.

Chemical constituents in stems of Ilex cornuta / 中草药

Objective: To investigate the chemical constituents in the stems of Ilex cornuta and the ability of scavenging free radicals of compounds 1-9. Methods: The compounds were isolated and purified by silica gel, medium pressure column chromatography, and semi-preparative liquid chromatography, and their structures were elucidated by chemical properties and spectroscopic analyses. The antifreeradical efficiency of compounds 1-9 was evaluated by DPPH radical scavenging assay. Results: Fifteen compounds were isolated and their structures were identified as isochlorogenic acid B (1), 3,4,5-tricaffeoylquinic acid (2), 4,5-di-O-caffeoyl quinic acid methyl ester (3), 3,4-di-O-caffeoyl quinicacid methyl ester (4), 3,5-di-O-caffeoyl quinicacid methyl ester (5), 3,4,5-tri-O-caffeoyl quinic acid methyl ester (6), 3,5-dimethoxy-4-hydroxybenzaldehyde (7), ethyl gallate (8), dihydrosyringenin (9), 2,6-dimethoxy-1,4-benzoquinone (10), arctigenin (11), 1-O-(vanillic acid)-6-O-(3″, 5″-dimethoxy-galloyl)-β-D-glycoside (12), (+)-1-hydroxypinoresinol-1-O-β-D-glucopyranoside (13), (+)-(7S,8S)-syringylglycerol 8-O-β-D-glucopyranoside (14), and schaftoside (15). Compounds 1-7 had good antifreeradical efficiency. Conclusion: Compounds 6,8-10,14, and 15 are obtained from the plants of Ilex L. the first time, and compounds 2,7,11, and 12 are obtained from this plant for the first time. Compounds 1-6 have good antifreeradical efficiency.

Two new triterpenoid saponins from Ilex cornuta.

Two new triterpenoid saponins (1 and 2) were isolated from the stems of Ilex cornuta, along with two known triterpenoids (3 and 4). The structures of compounds 1 and 2 were determined as ursane-12,19-diene-28-oic acid 3ß-O-ß-D-glucuronopyranoside-6-O-methyl ester (1), 3α,23α-dihydroxy-olean-9(11),12-diene-28-oic acid 28-O-ß-D-glucopyranosyl ester (2), on the basis of hydrolysis and spectral evidence, including 1D and 2D NMR and high resolution electrospray ionization mass spectrometry (HR-ESI-MS) analyses. Protective effects of compounds 1-4 were tested against H(2)O(2)-induced H9c2 cardiomyocyte injury, and the data showed that all these compounds had no cell-protective effect.

Five new triterpenoidal saponins from the roots of Ilex cornuta and their protective effects against H2O2-induced cardiomyocytes injury.

Five new ursane-type triterpenoidal saponins (1-5), together with five known ones (6-10), were isolated from the EtOH extract of the roots of Ilex cornuta. The structures of saponins 1-5 were elucidated as 19α-hydroxyurs-12-en-28-oic acid 3ß-O-ß-D-glucuronopyranoside (1), 19α-hydroxyurs-12-en-28-oic acid 3ß-O-ß-D-glucuronopyranoside-6-O-ethyl ester (2), 19α-hydroxyurs-12-en-28-oic acid 3ß-O-α-L-arabinopyranosyl-(1→2)-ß-D-glucuronopyranoside (3), 3ß-O-[α-L-arabinopyranosyl-(1→2)-ß-D-glucuronopyranosyl]-19α-hydroxyurs-12-en-28-oic acid 28-O-ß-D-glucopyranosyl ester (4) and 3ß-O-[α-L-arabinopyranosyl-(1→2)-ß-D-glucuronopyranoside-6-O-methyl ester]-19α-hydroxyurs-12-en-28-oic acid 28-O-ß-D-glucopyranosyl ester (5), on the basis of spectroscopic analyses (IR, ESI-MS, HR-ESI-MS, 1D and 2D NMR) and chemical reactions. Protective effects of compounds 1-10 against H2O2-induced H9c2 cardiomyocyte injury were tested. Compounds 1-5, 7, and 10 showed cell-protective effects. Among them compound 5 exhibited the highest activity. No significant DPPH radical scavenging activity was observed for compounds 1-10.

New triterpenoid saponins from Ilex cornuta and their protective effects against H2O2-induced myocardial cell injury.

Five new triterpenoid saponins, 1-5, together with 10 known ones, 6-15 were isolated from the aerial parts of Ilex cornuta. The structures of compounds 1-5 were determined as 3ß-O-α-L-arabinopyranosyl-19α,23-dihydroxy-20α-urs-12-en-28-oic acid 28-O-ß-D-glucopyranosyl ester, 1; 3ß-O-ß-D-glucopyranosyl-(1→2)-α-L-arabinopyranosyl-19-hydroxy-20α-urs-12-en-28-oic acid 28-O-ß-D-glucopyranosyl ester, 2; 19α,23-dihydroxyurs-12-en-28-oic acid 3ß-O-ß-D-glucuronopyranoside-6-O-methyl ester, 3; 19α,23-dihydroxyurs-12-en-28-oic acid 3ß-O-[ß-D-glucuronopyranoside-6-O-methyl ester]-28-O-ß-D-glucopyranosyl ester, 4; and 3ß-O-[α-L-arabinopyranosyl-(1→2)-ß-D-glucuronic acid]-oleanolic acid 28-O-ß-D-glucopyranosyl ester, 5, on the basis of spectroscopic analyses (IR, ESI-MS, HR-ESI-MS, 1D and 2D NMR) and chemical reactions. Protective effects of compounds 1-15 were tested against H2O2-induced H9c2 cardiomyocyte injury, and the data showed that compounds 1, 4, 6, and 13 had significant cell-protective effects. No significant DPPH radical scavenging activity was observed for compounds 1-15.

Identification of “Gonglao Leaf” and Its Sibling Adulterants Based on ITS2 Sequence / 世界科学技术-中医药现代化

Objective: The Phenomenon of different species with the same name has created potential threats to cinical safety of medication. Because of that, two kinds of “gonglao leaf” and their sibling adulterants have been identified by molecular methods in this study, to secure its safety in medication. Methods: Ilexcornuta, Mahoniabealei, Mahoniafortuneiand their sibling adulterants total of 9 species 45 samples were collected in this experiment,Total genomic DNA was extracted from them by the method of improved CTAB, ITS2 sequences were amplified, and the PCR products were sequenced. Sequence assembly and consensus sequence generation were performed by the CodonCode Aligner V 4.2.4.. The genetic distances were computed by MEGA 6.0 in accordance with the Kimura 2-Parameter (K2P) model and the phylogenetic tree constructed by the neighbor-joining (NJ) method. Results: The analysis results of the genetic distances, variable sites and the NJ phylogenetic tree showed that Ilexcornuta, Mahoniabealei, Mahoniafortunei were seperated from their sibling adulterants obviously. Conclusion: ITS2 sequence was able to identify two kinds of “gonglao leaf” and their sibling adulterants which can provide a basis for clinical accurate medication.

Chemical constituents in roots of Ilex cornuta / 中草药

Objective: To study the chemical constituents of the roots of Ilex cornuta. Methods: The compounds were isolated and purified by silica gel, Sephadex LH-20, medium pressure column chromatography, and semi-preparative liquid chromatography, and their structures were elucidated by chemical properties and spectroscop analyses. Results: Eight compounds were isolated and their structures were identified to be β-sitosterol (1), lupeol (2), betulonic acid (3), hede-ragenin (4), 3β-acetoxy-28-hydroxyurs-12-ene (5), ursolic acid (6), 19α-hydroxy ursolic acid (7), 3β-acetoxy-ursolic acid (8), 23-hydroxyl-methyl ursolate (9), heptanoic acid (10), β-daucosterol (11). Conclusion: Compounds 4,5,8, and 9 are obtained from this genus for the first time, and compound 3 is obtained from this plant for the first time.