Phytochemistry and Pharmacology of the Echinops integrifolius


U. T. Karimov1,2, Zhao Hai Qing2, H.A. Aisa2

1Institute of the Chemistry of Plant Substances AS RUz, Tashkent 100170, Uzbekistan

2Key Laboratory of Chemistry of Plant Resources in Arid Regions, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, China

*Corresponding Author E-mail:



For the first time investigated the sum of alkaloids of plant Echinops integrifolius, which totaled 0.186% of the dry weight of the plant. By column chromatography was isolated and identified eight substances: three triterpenoids, umbelliferone, isoprenoid γ-lactone-4,8,12,16- tetrametilheptadekan -4-olide, dibuthyl phthalate. There have been studies of antidiabetic properties of isolated compounds, and obtained extracts. It was investigated the antidiabetic, and antimicrobial activity, influence of umbelliferone on tyrosinase activity and melanin in the cells of the model B16 (OD475).


KEYWORDS: ethanol, extract, lupeol, lupeol acetate, umbelliferone, antidiabetic activity, tyrosinase, melanine.




The plants of the genus Echinops: (Asteraceae) perennial, biennial, rarely annual herbs with alternate, usually dissected and prickly, fluffy bottom tomentose, rarely glandular leaves. In the world there are about 120 species of the genus Echinops. They grow in Africa, Asia, Europe, and China (17 species, 5 of them endemic) 1. In the CIS countries 58 species are in Kazakhstan-18 2.


Plant of the genus Echinops spread from the Iberian Peninsula, Southern Europe and the steppe and semidesert Asia to Japan, south area captures the Mediterranean and north-east Africa2.


The Echinops integrifolius growing in dry rocky slopes, sandy slopes, open areas in forests, fields; 400-2400 m. Xinjiang (Fuyun).


According to information from the "Flora of the USSR» Echinops integrifolius grows on sandy and gravelly slopes and valleys of the foothills. In Central Asia, the plant spread to the Zaisan basin (Balkhash) and in Western Siberia (Narym Ridge Mountains Kurshim, Saur). In China in the far north Dzungaria (Junggar Kashgar). Also, the plant is common in the western territories of Mongolia.


Echinops species not have significant economic value. The spread of this genus of plant is very conducive to their low palatability due to their prickliness.


Some species have attracted the attention of honeybees, even the divorce as melliferous (Echinops bannaticus Rochel ex Shrad., Echinops ritro L., Echinops sphaerocephalus L., Echinops scaltosus). According to the beekeepers of Russia and Ukraine in productivity E. ritro successful growing conditions can reach 650-700 kg / ha. The average honey plant reaches 300-350 kg 3, 4, same data from Turkey5.


Analysis of all available reference shows that there are no data on its chemical and pharmacological studies for plants except for its botanical description.


In previous articles reported the isolation using flash chromatography of terpenes, flavonoids, coumarin6, also known about the composition of volatile compounds of different fractions of ethanolic extract of the plant Echinops integrifolius7 and the isolation of alkanes and fatty acids from plant roots8.


Were studied PTP1B inhibition effects for various ethanole extract. 93% ethanol extract (IC50=3.19±0.25) of the aerial parts have a more pronounced inhibitory activity than the roots of the plant were revealed. On the increasing content of ethanol in the extractant is enhanced inhibitory activity of the extract.


The total alkaloids of whole plant is 0.186%.


By column chromatography on silica gel eluting with different solvent systems was isolated lupeol (1), a mixture of tautomers of lupeol acetate (2a-2b), dibuthyl phthalate (3), 4,8,12,16- tetrametilheptadekan -4-olide (4) 7-hydroxycoumarin (5) β-sitosterol glucopyranoside (6). All compounds were identified by comparative analysis of 1H and 13C NMR and mass spectral data with those reported in the literature.



NMR experiments were acquired on a Varian Inova-400 and Varian Inova-600, observing 1H and 13C at 400/600 and 100/125 MHz, respectively. CDCl3 was used as solvent and TMS as internal reference.


Determination of melting point of substances carried on the device Yanaco MP-300.


Silica gel (Merck, 230-400 mesh) was used for column chromatographic separation (CC), while silica gel 60 PF254 (Merck) was used for analytical (TLC, 0.25 mm) and preparative (PTLC, 1.0 mm) thyn layer chromatography. Compounds were visualized by exposure under UV254/366 light and spraying with 5% (v/v) H2SO4 in ethanol solution, followed by heating on a hot plate.


The plant was collected in 2009 in the mountains of the Altai Territory Xinjiang during the beginning of flowering. The plant is identified by botanists Sintszyanskogo Institute of Ecology and Geography of China.


2.1. The extraction of total alkaloids:

300 g dried, powdered plant (roots and aerial parts), previously moistened with 8% ammonia solution and placed in a Soxhlet apparatus. After 2 hours, the extraction was performed with chloroform until alkaloids recovery complete. Recovery process controlled with Dragendorff reagent. The chloroform extract was treated with 5% sulfuric acid. The acidic solution was separated, alkalize by 25% ammonia solution and the alkaline solution was extracted with chloroform (4×0.3 L). There was obtained 0.56 g of total alkaloids, which is 0.186% of the dry weight of the plant.


2.2. Mixture of tautomers of lupeol acetate:

White needles, C32H52O2, m.p. 214-217 ˚C. Soluble in chloroform, acetone, hexane, partially in methanol.


In carrying out GC-MS analysis are identified two substances with RT=57.3 (63%) and RT=58.6 (37%). Mass spectrometric fragmentation of substances having a RT=57.3% 100 coincided with the published data and the NIST library, with that lup-20(29)-en-3β-ol acetate. Mass spectrometric fragmentation of the second component with RT=58.6 for the most part is not identical to that of lup-20 (29)-en-3β-ol acetate. Rupture of the ion (M-60 (OAc)), splitting of isopropenyl group in E ring, the presence of peaks with m/z 357, 297, 249, 218, 204, 189 led doubt the conclusions. A more accurate evaluation of the results was carried out a detailed study of the references where have found work on the establishment of the crystal structure of the tautomers of lupeol acetate from the plant Hieracium plumulosum 9.


Mass spectrum of the 2 and 2a contains a molecular ion m/z 468 (M+). The fragments m/z 468 and 426 indicate that a substance has lupane skeleton.


Lup-20(29)-enyl acetate (2): 1H NMR (600 МHz, CDCl3, ppm, δ, J (Hz)): 4.69 (br. d, 3.0 Hz, H-29b), 4,57 (br. dt, 1.5, 1.5, 0.8 Hz, H-29a), 4.47 (m, H-3), 2.37 (dt, 11.0, 5.8 Hz, H-19), 2.04 (s, H-2'), 1.68 (br. s, H-30), 1.03 (s, H-26), 0.94 (s, H-27), 0.86 (s, H-25), 0.85 (s, H-23), 0.84 (s, H-24), 0.79 (s, H-28).


Lup-20(29)-enyl acetate (2): 13C NMR (150 МHz, CDCl3): 171.1 (C-1'), 154.8 (C-20), 107.27 (C-29), 81,12 (C-3), 55.54 (C-5), 50.51 (C-9), 48.45 (C-18), 48.16 (C-19), 43.15 (C-17), 42.98 (C-14), 41.01 (C-8), 40.16 (C-22), 37.96 (C-4), 38.55 (C-1), 38.21 (C-13), 37.24 (C-10), 35.73 (C-16), 34.37 (C-7), 30.00 (C-21), 26.81 (C-23), 27.59 (C-15), 28.10 (C-2), 25.26 (C-12), 21.47 (C-2'), 21.10 (C-11), 19.64 (C-30), 18.37 (C-6), 18.16 (C-28), 16.65 (C-25), 16.14 (C-26), 16.34 (C-24), 14.66 (C-27).


Lup-19 (20)-en-3-ol acetate (2a) has no vinyl protons, it is evident that in the 1H NMR spectrum at 4.67 ppm we see only the signals of two vinyl protons of lup-20(29)-en-3-ol acetate. In contrast, in the 13C NMR spectrum at 150.9 ppm and 109.3 ppm we find the signals of vinyl carbon atoms C20 and C29 of the lupeol acetate, while at 154.6 ppm and 107.1 ppm there occur the signals of sp2 carbon atoms C 19 and C20 of lup- 19(20)-en-3β-ol acetate9.


On the basis 1H and 13C NMR data, m.p., mass fragmentations and the reference data the isolated compound has been identified as lup-20 (29)-en-3β-ol acetate (2) and lup-19 (20)-en-3β-ol acetate (2a).


2.3. Preparing extracts and fractions for the study of their antidiabetic activity:

a)      200g samples (aerial part, roots, whole plant) was ground to small particle size of 1-10 mm. Then boiled in for two days at a temperature of 100˚C 1 hour per day. Extracted by boiling 70% ethanol aq.. Alcoholic extract was evaporated to a thick syrupy consistency. The aqueous residue was dissolved in hot water (60-70°C). Chilled water solution is processed with petroleum ether, ethyl acetate and butanol. After distillation of solvents in vacuo gave 20 g of petroleum-ether, ethyl acetate 27g and 48 g butanol fractions.

b)      100g of roots, aerial part and whole plants was boiling in water, 93% and 70% ethanol. The plant is ground to a size of 5 mm and was conducted in boiling under reflux for 2 hours. Alcoholic extract to dryness.


For research the activities have selected samples of the ethanol extract and each fraction 1 g of each.


2.4. PTP1B inhibitory activity of Echinops integrifolius extracts and fractions:

The reagent pNPP was used as substrate for the measurement of PTP1B activity, according to PTP1B pNPP hydrolysis of the phosphate group and produces a color reaction measured the activity of PTP1B. Compounds were pre-incubated with the enzyme at room temperature for 5 min. Buffer (20 mM Hepes, 150 mM Nacl, 1 mM EDTA) 178 μL, in 96 well plates PTP1B protein solution was added 1 μL (0.115mg/mL), the test sample or positive control sample 1 μL (dubbed the sample concentration gradient) or DMSO 1 μL, mix, 10 min after adding 35mM of pNPP 20 μL 25 ºC for 30 min dark incubation, each well 10 μL 3M NaOH terminate the reaction. Then, the absorbance was determined at 405 nm wavelength. The system does not contain the enzyme solution in a blank, using a micro plate reader SpectraMax MD5 (USA Molecular Devices) absorption was measured at 405 nm. Inhibition rate=(OD405-OD405 blank sample)/OD405 blank × 100%, IC50 calculation software.


2.5. Umbelliferone influence on the activity of tyrosinase and melanin cells in the model B16 (OD475):

2.5.1. Cell culture:

Murine B16 melanoma cell lines were obtained from CAS (Chinese Academy of Sciences). The B16cells were grown in DMEM medium (GIBICO, USA) supplemented with 10% heat-inactivated fetal bovine serum (GIBICO, USA), 100U/ml penicillin and100μg/ml streptomycin (Hyclone, USA) In a humidified atmosphere with 5% CO2 at 37˚C.


2.5.2. Tyrosinase activity and melanin contents assay:

Tyrosinase activity was estimated by measuring the rate of L-DOPA oxidation as reported10. B16 cells was seeded in a 12-well plate at density of 2×105cells per well and allowed to attach for 24h.Then treated with VWI for 48h, washed with ice-cold PBS twice and Trypsin (0.25%, Hyclone, USA) was addedthen the cells were collected in EP tube, After centrifugation washed by PBS one time then 200μl of Tris-0.1% Triton-100 (pH=6.8) were added in each tube. All tubes were at-20˚C for 30 minutes after dissolved lysates were centrifuged at 12,000 rpm for 15 min to obtain the supernatant for Tyrosinase activity assay. Protein concentrations were determined by method of Bradford with bovine serum albumin (BSA) as a standard. 100μl of supernatant containing the same 10μg total proteins was added to each well in96-well plate, and then mixed with 100μl of 0.1% L-DOPA in PBS (pH6.8). After incubation at 37˚Cfor 1h, the dopachrome was monitored by measuring the absorbance at 475nm.


Total melanin in the cell pellet was dissolved in100 ml of 1MNaOH/10% DMSO for 1h at 80˚C and solubilized melanin was measured at 470nm.




Lupeol, constituting about 25% petroleum-ether fraction6 has anti-tumor, anti-hypertensive and anti-microbial properties11, inhibition laboratory models of prostate12 and the skin cancer13, 14.


The second main component with petroleum-ether fraction-lupeol acetate (about 39%6) also has anti15, antiarthritic 16, contraceptives properties17.


In literature reported the presence of bicyclic sesquiterpenes 18, sesquiterpene alcohols19 sesquiterpene lactones and20 in plants E. grijsii and E. giganteus. In papers 20, 21 also reported monoterpenoids in plants E. ritro and E. grijsii. Quinoline alkaloids found in the aerial and underground parts to more than 14 species, including E. echinatus, E. ritro and E. sphaerocephalus 21-23. In addition to the above compounds Echinops genus plants contain flavonoids, thiophenes and fatty acids24-26.


In Ethiopian traditional medicine, some types of Echinops used for diarrhea, heart pain, some forms of infections, hemorrhoids27, seizures, paresis, migraine, plexitis, myopathy28. In Chinese medicine, E. dahuricum used as an anthelmintic, lactation stimulant for the treatment of tumors, edema and gout29.


According to the author 30 plant Echinops echinatus used in the treatment of diabetes: an aqueous extract of the plant is taken twice a day. Paste from leaves taken as twice a day. The dry powder is dissolved in the milk for ingestion.


Study of inhibition of PTP1B activity of extract showed that 93% ethanol extract of the whole plant obtained by refluxing for 2 hours, has a anti-diabetic properties (Table. 1). Some studies antidiabetic activity reported previously31.


The studies were carried for the inhibition of the fractions of 93% ethanol extract of the aerial parts of the plant. Butanol and petroleum-ether fraction of extract exhibit stronger antidiabetic properties compared to extracts themselves, IC50=6.51±0.49 μM, and IC50=2.61±0.31 μM, respectively. The ethyl acetate fraction and the aqueous residue of extract have no effect in a in vitro tests.


Supposedly, this feature manifests by flavonoids in butanol and triterpenes in petroleum-ether fractions.


Lupeol inhibiting PTP1B activity in the dose IC50=5.6±0.9 μM32.


The apigenin, isolated from the plant and its derivatives have a marked antidiabetic the properties (IC50=24.76±2.17 μM.) 33, which is manifested by the presence of antidiabetic butanol fraction of extract.


The extract of the whole plant obtained by using 70% ethanol did not show of PTP1B inhibition activity.


Table 1. PTP1B inhibition activity of exctracts


IC50 (μg/mL)

whole plant boiled in water 2 hours


whole plant boiled in 93% ethanol 2 hours


whole plant boiled in 70% ethanol 2 hours

No effect

aerial part boiled in water 2 hours


aerial part boiled in 93% ethanol 2 hours


aerial part boiled in 70% ethanol 2 hours


roots boiled in water 2 hours


roots boiled in 93% ethanol 2 hours


roots boiled in 70% ethanol 2 hours


roots petroleum ether in 3 days


The authors recommended umbelliferone for the development of new drugs for the treatment of asthma 34. According to other investigation, umbelliferone has the potential to be useful as a phytogenous pesticide35. The researchers from Brazil reported that umbelliferone induces changes in structure and pharmacological activities of Bn IV, a lysine 49 secretory phospholipase A2 (sPLA2) from Bothrops neuwiedi36. Authors studied the effect of umbelliferone on haemostatic function because umbelliferone is also a coumarin derivative like the anticoagulant, warfarin. Diabetic rats had a significant decrease in prothrombin, clotting and bleeding time, and treatment with umbelliferone made these parameters almost normal37.


The research information suggest that 7-HOC interacts with sPLA2 and causes some structural modifications that lead to a sharp decrease or inhibition of the edematogenic and myotoxic activities of this enzyme, indicating its potential use to suppress inflammation induced by sPLA2 from the snake venom 38. The authors found that, a beneficial effect of UMB in reducing the blood glucose, hyperlipidemia, oxidative and Conclusion: stress and inflammatory mediators in type 2 diabetic rats39. Polish scientist investigated the comparative influence of 4-hydroxycoumarin (4-HC) and 7-hydroxycoumarin (umbelliferone; UMB) on dipalmitoylphosphatidylcholine (DPPC) membranes40.


The umbelliferone inhibited PTP1B activity in a dose-dependent manner with IC50=70.36±6.22 at concentration 500 μg/ml.


The coumarin–umbelliferone increase the tyrosinase activity and melanin amount in the model B16 (OD475) in concentration 25 μg/mL to 84.8 % and 261% respectively (Table. 2). As positive control substances were used 8-metoxypsoralen (MOP).


Table 2. The umbelliferone influence on the activity of tyrosinase and melanin cells in the model B16 (OD475)

Concentration µg/mL

Tyrosinase activity (%)

Increase the amount of melanin (%)

















According to data in Table 2 it is evident that umbelliferone at of 25 µg/ml have good activity against tyrosinase activity and melanin synthesis increases in cell models.


The umbellifeone and lupeol acetate were tested for antimicrobial activity against Escherichia coli, Candida albicans and Staphylococcus aureus. These compounds have weak antimicrobial properties and inhibited Staphylococcus aureus (zone of growth inhibition 7 mm, conc. 100 mg/mL). For this activity have been tested the petroleum-ether and butanol fractions of 70% ethanol extract of the whole plant during which found out that they did not have antimicrobial activity against the above-mentioned types of microorganisms.



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Received on 10.01.2018        Modified on 23.02.2018

Accepted on 13.04.2018       ©A&V Publications All right reserved

Res.  J. Pharmacognosy and Phytochem. 2018; 10(3): 216-220.

DOI: 10.5958/0975-4385.2018.00035.3