The preparation of a boronate affinity-based controlled oriented imprinting coating on a silica nanoparticle surface for the separation and purification of shikimic acid in herbal medicine.

Shikimic acid (SA) is one of the most effective drugs against the A (H1N1) virus and has high medicinal value. Additionally, it has the ability to generate non-toxic herbicides and antimicrobial medications. The extraction from plants has proven to be the main route of production of SA with economic benefits and environmental efficiency. Therefore, it is necessary to perform purification of SA from these herbal medicines before quantifying it. In this study, researchers employed a boronate affinity-based controlled oriented surface imprinting technique to produce molecularly imprinted polymers (MIPs) as highly effective solid phase extraction (SPE) adsorbents for the isolation and purification of SA. 3-Fluoro-4-formylphenylboronic acid functionalized silica nanoparticles were used as supporting materials for immobilizing SA. Poly(2-anilinoethanol) with a higher hydrophilic domain can be used as an effective imprinting coating. The prepared SA-imprinted silica nanoparticles exhibited several significant results, such as good specificity, high binding capacity (39.06 ± 2.24 mg g-1), moderate binding constant (6.61 × 10-4 M-1), fast kinetics (8 min) and low binding pH (pH 5.0) toward SA. The replication of SA-imprinted silica nanoparticles was deemed satisfactory. The SA-imprinted silica nanoparticles could be still reused after seven adsorption-desorption cycles, which indicated high chemical stability. In addition, the recoveries of the proposed method for SA at three spiked level analysis in star aniseed and meadow cranesbill were 96.2% to 109.0% and 91.6% to 103.5%, respectively. The SA-imprinted silica nanoparticles that have been prepared are capable of identifying the target SA in real herbal medicines. Our approach makes sample pre-preparation simple, fast, selective and efficient.

A simple and effective method for the preparation of high-purity shikimic acid from chromatography wash effluent of Ginkgo biloba leaf extract by macroporous resin considering the effect of varying feed solution compositions.

OBJECTIVES: The present study investigated the feasibility of preparing high-purity shikimic acid (SA) from the chromatography wash effluent of Ginkgo biloba leaf extract by macroporous resin. METHODS: First, static/dynamic adsorption and desorption were conducted to screen out the optimal resin. Second, the key parameters of the chromatographic process were optimised with face-centred central composite design (CCD). Third, wash effluent indices were measured, different batches of wash effluent were used to prepare SA under the optimised parameters, and the effect of varying feed solution compositions on final products was investigated. KEY FINDINGS: It was found that the final purity and recovery rate of SA prepared with ADS-21 resin were not lower than 70 and 60%, respectively, when the purity of SA in the wash effluent was higher than 21.4%. The quality of the final product can be predicted based on the properties of wash effluent. CONCLUSIONS: The proposed method could not only provide a simple, green and promising approach for the large-scale purification of SA from wash effluent but also be used to develop process intermediate quality standards for other natural products.

Mangiterpenes A-C and 2',3'-seco-manginoid C, four sesquiterpene/monoterpene-shikimate-conjugated spirocyclic meroterpenoids from Guignardia mangiferae.

Mangiterpenes A-C and 2',3'-seco-manginoid C, four undescribed sesquiterpene/monoterpene-shikimate-conjugated meroterpenoids with spiro ring systems, were isolated from Guignardia mangiferae. The structures and absolute configurations of these compounds were established by comprehensive spectroscopic analyses and electronic circular dichroism (ECD) calculations. Mangiterpenes A-C represent the first examples of sesquiterpene-shikimate-conjugated spirocyclic meroterpenoids, and 2',3'-seco-manginoid C features an unexpected 2',3'-seco-manginoids skeleton. Mangiterpene C strongly inhibited the production of NO inducted by LPS, with an IC50 value of 5.97 µM. It showed an anti-inflammatory effect by means of blocking in the NF-κB signaling pathway and decreasing the expression of inflammatory mediators.

Interspecies Developmental Differences in Metabonomic Phenotypes of Lycium ruthenicum and L. barbarum Fruits.

Fruits of Lycium ruthenicum (LR) and L. barbarum (LB) in Solanaceae family contain abundant bioactive metabolites used widely as functional food and natural medicine. To characterize the fruit developmental molecular phenotypes, we comprehensively analyzed metabolite composition of both Lycium fruits at three developmental stages using the combined NMR, liquid chromatography-tandem mass spectrometry, and gas chromatography-flame ionization detector/mass spectrometry methods. The metabonomes of these fruits were dominated by over 90 metabolites including sugars, amino acids, tricarboxylic acid (TCA) cycle intermediates, fatty acids, choline metabolites, and shikimate-mediated plant secondary metabolites. Metabolic phenotypes of two species differed significantly at all three developmental stages; LB fruits contained significantly more sugars and amino acids but less TCA cycle intermediates, fatty acids, and secondary metabolites than LR. Interspecies differences for fatty acid levels were much greater after color-breaking than precolor-breaking. Furthermore, LR fruits contained more osmolytes than LB fruits indicating different osmoregulation requirements for these fruits during development. Significant differences were also present in biosynthesis of shikimate-mediated plant secondary metabolites in LR and LB. These findings provided essential metabolic information for plant physiology of these  Lycium species and their utilizations demonstrating the usefulness of this metabonomic phenotyping approach for studying fundamental biochemistry of the plant development.

Monitoring in vivo metabolic flux with a designed whole-cell metabolite biosensor of shikimic acid.

Knowledge of intracellular metabolite levels is important for the understanding of metabolic flux distributions. Whole-cell biosensors of key metabolites are ideal for the monitoring of carbon flow in important metabolic pathways, thus guiding metabolic engineering for microbial improvement. However, lack of biosensors for metabolites of interests has limited their applications. In this study, a genetically encoded whole-cell biosensor specifically responding to shikimic acid has been developed by screening a site-saturation mutagenesis library of the binding pocket of a uric acid-responsive regulatory protein. This biosensor has been successfully applied in analyzing and engineering metabolic flux in the shikimic acid pathway, through genome-wide screening of gene targets critical for the pathway flux, and by improving the specific activity of pathway key enzyme, AroG. This work demonstrates the feasibility of monitoring metabolic flux with the aid of whole-cell biosensors designed for key metabolites.

Tentative Characterization of Polyphenolic Compounds in the Male Flowers of Phoenix dactylifera by Liquid Chromatography Coupled with Mass Spectrometry and DFT.

Phoenix dacylifera is an ancient palm species rich in (poly)phenols. These phenolic compounds were tentatively identified by using liquid chromatography coupled with ion spray mass spectrometry in tandem mode (LC/MS/MS) with negative ion detection. Negative identification of the compounds was based on their retention times and mass spectra in full scan mode (MS), and in different MS/MS modes. For the first time, complete hypothesis, and routs for both p-coumaroylshikimic acids (CoSA) and caffeoylshikimic acids (CSA) were suggested and confirmed by Density Fonctional Theory (DFT) study. Notably, of the 53 compounds characterized, 19 hydroxycinnamates derivatives were tentativelycharacterized in male flowers of date palm and 15 of them were recorded for the first time. In addition, five organic acids, six B-type proanthocyanidins, two anthocyanidin and 21 flavonoid derivatives have been tentatively characterized. Identification of B-type proanthocyanidins were based on the diagnostic ions resulting from heterocyclic ring fission (HRF) and retro-Diels-Alder (RDA) reaction of flavan-3-ol provided information on the hydroxylation pattern and the type of inter-flavan bond proanthocyanidins. The sequence of proanthocyanidins was detected through ions extracted from quinone methide (QM) cleavage of the inter-flavan bond.

Polar extracts from the berry-like fruits of Hypericum androsaemum L. as a promising ingredient in skin care formulations.

ETHNOPHARMACOLOGICAL RELEVANCE: The top flowering aerial parts of the Hypericum species are traditionally used to prepare ointments to heal cuts and burns. Sometimes even the fruits are used for these purposes. Hypericum androsaemum L., commonly known as tutsan or shrubby St. John's Wort, is a Mediterranean medicinal plant which has been traditionally used to prepare an ointment for treating cuts and wounds. AIM OF THE STUDY: To evaluate the extracts obtained from H. androsaemum red berries as functional ingredients for skin care formulations. MATERIALS AND METHODS: The methanolic extract was obtained by Soxhlet extraction while the aqueous extract was prepared by decoction; their composition was determined by HPLC analysis. Their biological activities were measured in terms of proliferation and migration of human fibroblasts, inhibition of collagenase activity, and immunomodulatory effects on human peripheral blood mononuclear cells (PBMCs). In addition, we evaluated their photostability by UV spectroscopy and their protective effects against APPH-induced hemolysis in red blood cells (RBC). RESULTS: The polar extracts contained significant amounts of shikimic (108,143.7-115,901.3mg/kg) and chlorogenic acids (45,781.1-57,002.7mg/kg). The main components of these extracts made an important contribution to a significant increase in human fibroblast migration. Both extracts were also active as collagenase inhibitors, with the aqueous one showing a greater inhibitory capacity (IC50 value of 88.1µg/mL), similar to that of chlorogenic acid. The kinetic parameters determined for the enzymatic reaction revealed for both aqueous extract and chlorogenic acid an uncompetitive mechanism of inhibition. The methanolic extract showed important effects on PBMCs by modulating IL-6. Both extracts proved to be photostable in the UVA/B range and protected RBC against peroxidation at low concentrations. CONCLUSIONS: H. androsaemum red berries were proven to contain phytochemicals that improve skin regeneration, hence potentially employable in skin care formulations.

Phytochemical and biological studies on Alnus glutinosa subsp. glutinosa, A. orientalis var. orientalis and A. orientalis var. pubescens leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Alnus species have been used for the treatment of rheumatism, hemorrhoids and for wound healing in folk medicine. MATERIALS AND METHODS: Aqueous and methanol extracts of A. glutinosa (L.) Gaertner subsp. glutinosa, A. orientalis Decne. var. orientalis and A. orientalis var. pubescens Dippel leaves were evaluated for their wound healing, anti-inflammatory and antioxidant activities. In vivo wound models of linear incision and circular excision were performed. "Inhibition of acetic acid-induced capillary permeability", "carrageenan-induced hind paw edema" and T"PA-induced ear edema" assays were applied to determine the anti-inflammatory effects. For the antioxidant activity assessment, DPPH and ABTS radicals scavenging effect, reducing power and denaturation of nonspecific hydroxyl radical-targeted 2-deoxyribose were used. In vitro inhibitory effects on enzymes hyaluronidase, collagenase and elastase were evaluated. The methanol extract of the leaves of A. glutinosa subsp. glutinosa (MB), the most potent extract, was fractionated by bioassay-guided fractionation technique. The structure of the isolated compound was determined as shikimic acid by using NMR and IR analyses. RESULTS: MB increased the wound tension by 42.79% value and provided a contraction by 51.58%. Wound tension, contraction capacity and tissue hydroxyproline levels were increased with the application of the fraction EtOAc: MeOH (Fr. D), subfraction D27-38 and shikimic acid. In the "inhibition of acetic acid-induced capillary permeability" assay, MB, Fr. D, subfraction D27-38 and shikimic acid inhibited the permeability with significant inhibition values of 30.22%, 32.46%, 38.24% and 27.19%, respectively. In carrageenan-induced hind paw edema model, MB displayed 29.1% inhibition. Likewise, Fr. D, subfraction D27-38, and shikimic acid were found to exhibit remarkable anti-inflammatory and antioxidant effects. Shikimic acid exhibited significant inhibitory effect (38.24%) on the hyaluronidase enzyme. CONCLUSIONS: This is the first and unique study that investigates wound healing, anti-inflammatory and antioxidant effects of some Alnus taxons growing in Turkey. According to the results, shikimic acid was found to be the major compound responsible from the activity.

Investigating strain dependency in the production of aromatic compounds in Saccharomyces cerevisiae.

Although Saccharomyces cerevisiae is the most highly domesticated yeast, strain dependency in biotechnological processes still remains as a common, yet poorly understood phenomenon. To investigate this, the entrance to the aromatic amino acid biosynthetic pathway was compared in four commonly used S. cerevisiae laboratory strains. The strains were engineered to accumulate shikimate by overexpressing a mutant version of the pentafunctional ARO1 enzyme with disrupted activity in the shikimate kinase subunit. Carbon tracing and 13 C metabolic flux analysis combined with quantitative PCR, revealed that precursor availability and shikimate production were dramatically different in the four equally engineered strains, which were found to be correlated with the strains' capacity to deal with protein overexpression burden. By implementing a strain-dependent approach, the genetic platform was reformulated, leading to an increase in yield and titer in all strains. The highest producing strain, INVSc1-SA3, produced 358 mg L-1 of shikimate with a yield of 17.9 mg g-1glucose. These results underline the importance of strain selection in developing biological manufacturing processes, demonstrate the first case of high production of shikimate in yeast, and provide an appropriate platform for strain selection for future production of aromatic compounds. Biotechnol. Bioeng. 2016;113: 2676-2685. © 2016 Wiley Periodicals, Inc.

Site-specific integration and constitutive expression of key genes into Escherichia coli chromosome increases shikimic acid yields.

As the key starting material for the chemical synthesis of Oseltamivir, shikimic acid (SA) has captured worldwide attention. Many researchers have tried to improve SA production by metabolic engineering, yet expression plasmids were used generally. In recent years, site-specific integration of key genes into chromosome to increase the yield of metabolites showed considerable advantages. The genes could maintain stably and express constitutively without induction. Herein, crucial genes aroG, aroB, tktA, aroE (encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, dehydroquinate synthase, transketolase and shikimate dehydrogenase, respectively) of SA pathway and glk, galP (encoding glucokinase and galactose permease) were integrated into the locus of ptsHIcrr (phosphoenolpyruvate: carbohydrate phosphotransferase system operon) in a shikimate kinase genetic defect strain Escherichia coli BW25113 (ΔaroL/aroK, DE3). Furthermore, another key gene ppsA (encoding phosphoenolpyruvate synthase) was integrated into tyrR (encoding Tyr regulator protein). As a result, SA production of the recombinant (SA5/pGBAE) reached to 4.14 g/L in shake flask and 27.41 g/L in a 5-L bioreactor. These data suggested that integration of key genes increased SA yields effectively. This strategy is environmentally friendly for no antibiotic is added, simple to handle without induction, and suitable for industrial production.

New method for the rapid extraction of natural products: efficient isolation of shikimic acid from star anise.

A new, practical, rapid, and high-yielding process for the pressurized hot water extraction (PHWE) of multigram quantities of shikimic acid from star anise (Illicium verum) using an unmodified household espresso machine has been developed. This operationally simple and inexpensive method enables the efficient and straightforward isolation of shikimic acid and the facile preparation of a range of its synthetic derivatives.

Antibacterial and antioxidant constituents of Acalypha wilkesiana.

This study was aimed at characterising the secondary metabolites responsible for antibacterial and antioxidant activities of Acalypha wilkesiana. Purification of the defatted methanol leaves extract was guided by the DPPH free radical scavenging assay as well as by evaluation of the antibacterial activity against four bacterial strains. As a result, geraniin, corilagin, quadrangularic acid M and shikimic acid were purified and isolated. Shikimic acid, reported for the first time from this plant, proved to be the major metabolite of the extract. All the four isolated compounds showed bactericidal activity against extended spectrum beta-lactamase-producing Klebsiella pneumoniae (700603), while corilagin was the single compound to exhibit antioxidant activity (IC50 53 µg/mL).

5-O-caffeoylshikimic acid from Solanum somalense leaves: advantage of centrifugal partition chromatography over conventional column chromatography.

Solanum somalense leaves, used in Djibouti for their medicinal properties, were extracted by MeOH. Because of the high polyphenol and flavonoid contents of the extract, respectively, determined at 80.80 ± 2.13 mg gallic acid equivalent/g dry weight and 24.4 ± 1.01 mg quercetin equivalent/g dry weight, the isolation and purification of the main polyphenols were carried out by silica gel column chromatography and centrifugal partition chromatography. Column chromatography led to 11 enriched fractions requiring further purification, while centrifugal partition chromatography allowed the easy recovery of the main compound of the extract. In a solvent system composed of CHCl3/MeOH/H2O (9.5:10:5), 21.8 mg of this compound at 97% purity was obtained leading to a yield of 2.63%. Its structure was established as 5-O-caffeoylshikimic acid by mass spectrometry and NMR spectroscopy. This work shows that S. somalense leaves contain very high level of 5-O-caffeoylshikimic acid (0.74% dry weight), making it a potential source of production of this secondary metabolite that is not commonly found in nature but could be partly responsible of the medicinal properties of S. somalense leaves.

Protective effect of coconut water concentrate and its active component shikimic acid against hydroperoxide mediated oxidative stress through suppression of NF-κB and activation of Nrf2 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Conventionally coconut water has been used as an 'excellent hydrating' drink that maintain the electrolyte balance and help in treating diverse ailments related to oxidative stress including liver function. The present study was aimed to elucidate whether and how the coconut water concentrate (CWC) and its major active phytoconstituent shikimic acid (SA) can effectively protect murine hepatocytes from the deleterious effect of hydroperoxide-mediated oxidative stress. MATERIALS AND METHODS: Bioactivity guided fractionation of CWC resulted in the isolation of a couple of known compounds. Freshly isolated murine hepatocytes were exposed to hydrogen peroxide (H2O2) (1 and 3mM) in the presence or absence of CWC (200 and 400 µg/ml) and SA (40 µM) for the determination of antioxidative, DNA protective, cellular ROS level by modern methods, including immunoblot and flowcytometry to find out the possible mechanism of action. RESULTS: Pre-treatment of hepatocyte with CWC and SA showed significant prevention of H2O2-induced intracellular ROS generation, nuclear DNA damage along with the formation of hepatic TBARS and cellular nitrite. Further, the H2O2 induced cell death was arrested in the presence of CWC through the inhibition of CDC42 mediated SAPK/JNK pathways and activation of other molecules of apoptotic pathways, including Bax and caspase3. Moreover, CWC and SA help in maintaining the GSH level and endogenous antioxidants like Mn-SOD, to support intracellular defense mechanisms, probably through the transcriptional activation of Nrf2; and inhibition of nuclear translocation of NF-κB. CONCLUSION: CWC and its active components SA reversed the H2O2 induced oxidative damage in hepatocytes, probably through the inhibition of NF-κB, with the activation of PI3K/Akt/Nrf2 pathway and reduction of apoptosis by interfering the SAPK/JNK/Bax pathway.

The volatile profiles of a rare apple (Malus domestica Borkh.) honey: shikimic acid-pathway derivatives, terpenes, and others.

The volatile profiles of rare Malus domestica Borkh. honey were investigated for the first time. Two representative samples from Poland (sample I) and Spain (sample II) were selected by pollen analysis (44-45% of Malus spp. pollen) and investigated by GC/FID/MS after headspace solid-phase microextraction (HS-SPME) and ultrasonic solvent extraction (USE). The apple honey is characterized by high percentage of shikimic acid-pathway derivatives, as well as terpenes, norisoprenoids, and some other compounds such as coumaran and methyl 1H-indole-3-acetate. The main compounds of the honey headspace were (sample I; sample II): benzaldehyde (9.4%; 32.1%), benzyl alcohol (0.3%; 14.4%), hotrienol (26.0%, 6.2%), and lilac aldehyde isomers (26.3%; 1.7%), but only Spanish sample contained car-2-en-4-one (10.2%). CH2 Cl2 and pentane/Et2 O 1 : 2 (v/v) were used for USE. The most relevant compounds identified in the extracts were: benzaldehyde (0.9-3.9%), benzoic acid (2.0-11.2%), terpendiol I (0.3-7.4%), coumaran (0.0-2.8%), 2-phenylacetic acid (2.0-26.4%), methyl syringate (3.9-13.1%), vomifoliol (5.0-31.8%), and methyl 1H-indole-3-acetate (1.9-10.2%). Apple honey contained also benzyl alcohol, 2-phenylethanol, (E)-cinnamaldehyde, (E)-cinnamyl alcohol, eugenol, vanillin, and linalool that have been found previously in apple flowers, thus disclosing similarity of both volatile profiles.

Deletion of the aroK gene is essential for high shikimic acid accumulation through the shikimate pathway in E. coli.

Shikimic acid (SA) is an important metabolic intermediate with diverse commercial applications. In this work, antisense RNA interference and gene deletion were carried out to inactivate the aroK gene in an SA-producing Escherichia coli strain, DHPYA-T7. In this strain, the aroL, ptsHIcrr and ydiB genes are deleted, and the tktA, glk, aroE and aroB genes are overexpressed. Flask cultivations of the DHPYA-T7 derivative strains showed that the accumulation of SA increased 2.69-fold after aroK gene deletion (DHPYAAS-T7) and 1.29-fold after antisense RNA interference (DHPYAS-T7). Furthermore, the activity of shikimate kinase in DHPYAAS-T7 was 0.21-fold of that in strain DHPYAS-T7. In a 10-L fermentation, SA accumulation increased to 1850 mg L(-1) in strain DHPYAAS-T7, which is a 1.5-fold increase over that in strain DHPYAS-T7. These results demonstrate that aroK gene inactivation in DHPYA-T7 leads to high SA accumulation, especially when this inactivation is caused by chromosomal deletion.

Antinociceptive activity of aqueous extract and isolated compounds of Lithrea molleoides.

ETHNOPHARMACOLOGICAL RELEVANCE: Lithrea molleoides (Vell.) Engl. (Anacardiaceae) is a medicinal plant commonly used in traditional medicine in South America. AIM OF THE STUDY: In the present study, the in vivo antinociceptive effect of L. molleoides' aqueous extract and its isolated compounds has been investigated. MATERIALS AND METHODS: Antinociceptive activity was evaluated through writhing, formalin and hot plate tests in mice. The phytochemical analysis was performed. RESULTS: The extract produced significant inhibition on nociception induced by acetic acid (ED50: 8.7 mg/kg, i.p.) and formalin (ED50: 7.7 mg/kg, i.p.) administered intraperitoneally and also orally. Yohimbine diminished the activity of the extract in the acetic acid test meanwhile haloperidol enhanced its effect. Two majority compounds, shikimic and vanillic acid were active in chemical nociceptive models used in this work, producing the highest inhibition of the writhing response at a dose of 30 mg/kg i.p. (55.4% and 57.1%, respectively) meanwhile at 100 mg/kg p.o. produced a slight response (23.3% and 23.9%, respectively). CONCLUSIONS: These results suggest that L. molleoides' aqueous extract produced antinociception possibly related to the presence of shikimic and vanillic acid. The adrenergic and dopaminergic systems seem to be involved in the mechanism of antinociception of the extract.

Identification of hypoglycaemic compounds from berries of Juniperus oxycedrus subsp. oxycedrus through bioactivity guided isolation technique.

ETHNOPHARMACOLOGICAL RELEVANCE: Decoction of Juniperus oxycedrus subsp. oxycedrus L. (Cupressaceae) berries is used internally as tea and pounded fruits are consumed to lower blood glucose levels in Turkey. AIM OF THE STUDY: To evaluate hypoglycaemic and antidiabetic activity of J. oxycedrus subsp. oxycedrus berries and to identify active compounds through bioactivity guided isolation technique. MATERIAL AND METHODS: Hypoglycaemic effect of J. oxycedrus subsp. oxycedrus (Joso) berry extracts on oral administration was studied using in vivo models in normal, glucose-hyperglycaemic rats. Streptozotocin induced diabetic rats were used to examine antidiabetic activity of Joso extracts, subextracts, fractions, subfractions and shikimic acid (SA). RESULTS: Through in vivo bioactivity-guided fractionation processes, shikimic acid, 4-O-ß-d-glucopyranosyl ferulic acid and oleuropeic acid-8-O-ß-d-glucopyranoside were isolated from the n-butanol subextract by silica gel and reverse phase column chromatography as the main active ingredient of the active subfraction. After 8 days administration of the major compound shikimic acid, blood glucose levels (24%), malondialdehyde levels in kidney tissues (63-64%) and liver enzymes (AST, ALT, ALP) of diabetic rats were decreased. CONCLUSION: Results indicated that Joso berry extract and its active constituents might be beneficial for diabetes and its complications.

Extraction and isolation of shikimic acid from Ginkgo biloba leaves utilizing an ionic liquid that dissolves cellulose.

Shikimic acid, the starting material in the commercial synthesis of oseltamivir phosphate (Tamiflu®), was efficiently extracted and isolated from Ginkgo biloba leaves utilizing the ionic liquid 1-butyl-3-methylimidazolium chloride ([bmim]Cl), which dissolves cellulose.

[Study on the chemical constituents of Codonopsis lanceolata].

OBJECTIVE: To study the chemical constituents of Codonopsis lanceolata. METHODS: Chemical constituents were separated with the column chromatographic, and their structures were identified by chemical and spectroscopic methods. RESULTS: Six compounds were isolated and identified as syringin (1), shikimic acid (2), friedelin (3), alpha-spinasterol (4), stigmasterol (5), stigmasta-7-dien-3beta-ol (6). CONCLUSION: Compounds 3-6 are isolated from this plant for the first time.