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ИССЛЕДОВАНИЕ ЛИПОФИЛЬНЫХ КОМПОНЕНТОВ И БИОЛОГИЧЕСКАЯ АКТИВНОСТЬ ГЕКСАНОВОЙ ФРАКЦИИ ZYGOPHYLLUM FABAGO
Р-A. Абдул-хамид1'2, C.A. Росс34, Ж.А. Абилов1, Н.А. Султанова1
1Казахский национальный университет им. аль-Фараби,
050040, г. Алматы, пр. аль-Фараби, 71, Zharylkasyn.Abilov@kaznu.kz
2Университет Аль-Азхар,
Египет, Асьют, abozyad.reda@gmail.com
3Национальный центр натуральных продуктов исследований, Университет Миссисипи P.O. BOX 1848 • УНИВЕРСИТЕТ, MS 38677-1848,sross@olemiss.edu ^Департамент фармакогнозии,
Фармацевтическая школа, Университет Миссисипи, MS 38677, США
Методом газовой хроматографии с масс-спектрометрией в гексановой фракции Zygophyllum faba-go идентифицировали семь веществ, которые составили 51,79%. В наибольшем количестве содержался моно(2-этилгексил)фталат (18%). Результаты биологического скрининга исследуемой фракции свидетельствуют о значительной активности против лишая и грибка Leishmania do-novani и Candida albicans соответственно. Антидиабетическая активность определена методом in vitro с использованием фермента 1В протеин тирозин фосфатазы. Полученные результаты показали, что гексановая фракция обладает достаточной ингибирующей активностью на данный фермент.
Ил. 1. Табл. 4. Библиогр. 8 назв.
Ключевые слова: Zygophyllum fabago; липофильные компоненты; противогрибковая; противоли-шайниковая; антидиабетическая активность; ГХ-МС.
INVESTIGATION OF LIPOPHILIC CONTENTS AND BIOLOGICAL ACTIVITIES OF HEXANE FRACTION OF ZYGOPHYLLUM FABAGO
R.A. Abdel-Hamid1'2, S.A. Ross3'4, Zh. A. Abilov1, N.A. Sultanova1
1
Al-Farabi Kazakh National University, Faculty of Chemistry and Chemical Technology, Almaty, 050040, Al-Farabi 71, Kazakhstan, Tel.: (+7-727) 3931909
1
Al-Farabi Kazakh National University, Faculty of Chemistry and Chemical Technology, Almaty, 050040, Al-Farabi 71, Kazakhstan, Tel.: (+7-727) 3931909
2Al Azhar University, Faculty of Pharmacy, Asyut, Egypt, abozyad.reda@gmail.com 3National Center for Natural Products Research
4Department of Pharmacognosy, School of Pharmacy, the University of Mississippi, MS 38677, USA
Gas chromatography-mass spectrometry analysis of the n-Hexane fraction of Zygophyllum fabago identified seven components, which clarified about 51.79 %. The 1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester represent the major percent of the identified components by 18 %. The biological screening of this fraction revealed a significant anti-leshmanial and antifungal activities against Leishmania donovani and Candida albicans respectively. The anti-diabetic activity was assayed in vitro using protein tyrosine phosphatase 1B (PTP1B). The results showed a significant potent inhibitory activity for PTP1B enzyme, which is a key negative regulator of insulin signal transduction. 1 figure. 4 tables. 8 sources.
Keywords: Zygophyllum fabago, lipophilic constituents, antifungal, anti-leishmanial, anti-diabetic activity, GC-MS.
Zygophyllum fabago, also known as Syrian bean-caper, is a native of the region extending from the steppes of Russia southeast to Afghanistan, found widely in the Mediterranean area, Europe, part of the USSR, Caucasus, Wed Asia, Balkans, Asia Minor, Iran, Iraq, Syria being typical of the Middle East, North Africa and in the southwestern United States, where it is considered an invasive plant. It is widespread in Kazakhstan and represented by three subspecies: ssp. Typicum M. Pop.; ssp. orientale Boriss. and ssp. dolicho-carpum M. Pop. [2,3,5,8].
The plant Zygophyllum fabago is one of the important herbs with known anti-rheumatic, anthelmintic, cathartic, and anti-asthmatic properties. It is also used as a part of drug for rheumatism and gout; also used externally as poultice to cure skin diseases, external wounds, septic, and injuries. In China Z. fabago is used as antitussive, expectorant, and as antiinflammatory agent [3, 4, 6].
PLANT MATERIAL:
ZYGOPHYLLUM FABAGO
Zygophyllum fabago was collected at Almaty region (Kazakhstan) and identified by Botanist N. G. Gemedzhieva, Head of Laboratory of Plant Resources, Sc.D. of Biology, Institute of Botany and Phytointroduction. The air dried aerial parts of Zygophyllum fabago was cut into small pieces and stored at room temperature.
The air-dried aerial parts of Zygophyllum fabago pulverized separately. The dried powder (100 g) was extracted three times with 70% etha-nol (3 x 500 mL) at room temperature and mixed the extracts, then it was evaporated on a vacuum rotary evaporator. The ethanol extract (30 g) was suspended in water and extracted successively with hexane (3 x 300 mL, 25 °C) to give hexane (0,5 g).
ANALYSIS OF THE LIPOPHILIC
CONSTITUENTS:
GC-MS analysis of the n-hexane fraction was performed using Electron Impact Ionization (EI) (Gas Chromatography coupled to Mass Spectrometer), with silica capillary column coated with PES, He carrier gas, flow rate, 1mL/min, gradient column temperature (60-290 oC), injector temperature 310oC and ion source detector (EI-70eV). Identification of the components was calculated from the peak areas without using correction factors. The components were identified by comparison of their retention indices and mass spectra fragmentation patterns with those stored on the Nist147 and Wiley7 computer library built up using pure substances or with authentic compounds and confirmed by comparison of their retention indices.
The results of GC-MS analysis of the n-Hexane fraction of Zygophyllum fabago are shown in figure 1 and table 1. By using GC-MS analysis of the n-Hexane fraction identified seven components which clarified about 51,79% of the total fraction while the 48,24% contain a number of unknown compounds which can't identified by GC-MS. Therefore this findings encourage us to do further investigation and separation of the unknown components.1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester by 18% represent the major percent of the identified components, followed by 2-Pentadecanone 6,10,14-trimethyl by 8,91% and Hexadecanoic acid ethyl ester by 8,77 % and Decane by 5,60%.
BIOLOGICAL SCREENING:
ANTI-LEISHMANIAL SCREENING
The anti-leishmanial screen tests samples for their ability to inhibit Leishmania donovani, a fly-borne protozoan that causes visceral leishmania-sis. Crude extracts are initially tested in a Primary.
Fig. 1. Gas chromatogram of the lipophilic contents of Hexane extract of Zygophyllum fabago
Table 1
Component composition of lipophilic constituents of hexane fraction of Zygophyllum fabago
Name Retension time tR, min M. formula M. weight Content, % Structure
Decane 12,928 C10H22 142,2817 5,60
Tetradecane 15,703 C14H30 198,3880 2,30
2-Pentadecanone, 6,10,14-trimethyl- 25,898 C18H36O 268,4778 8,91
n-Hexadecanoic acid 27,031 C16H32O2 256,4241 4,03 О II
Phthalic acid, butyl hexyl ester 27,128 C18H26O4 306,3966 4,04 0
Hexadecanoic acid, ethyl ester 27,359 C18H36O2 284,4772 8,77 —^
1,2- Benzenedi-carboxylic acid, mono(2-ethylhexyl) ester 32,161 C16H22O4 278,3435 18,11 0 О
Known Compounds Unknown Compounds 51,79 48,24
Screen at 80^g/mL in duplicate and percent inhibitions (% inh.) are calculated relative to negative and positive controls. The drug control amphotericin B is used as positive controls.
The results obtained in the evaluation of the Anti-leishmanial activity of the n-hexane fraction of Zygophyllum fabago are shown in table 2. The results show that the n-hexane fraction shows a significant activity against Leishmania donovani with percent of inhibition about 93% compared to the standard drug Amphotericin B that shows 99% inhibition.
ANTIBACTERIAL SCREENING
Three type strains, Staphyloccocus aureus ATCC 6538, Escherichia coli ATCC 11229, Candida albicans ATCC 10231 were obtained from the National Center for Medical Culture Collections (CMCC), China. All strains were stored at - 80 °C in the appropriate medium. Antimicrobial activity was tested using the agarwell diffusion method [7]. Luria-Bertani (LB) and Sabaurauds agar (SDA) were sterilised in an autoclave and cooled to 45-50°C before be poured into 100 mm Petri dishes. The agar plates were stored at 4 °C before used.
Staphyloccocus aureus ATCC 6538 and Escherichia coli ATCC 11229 were cultured overnight at 37 °C in LB. Candida albicans ATCC10231 was cultured overnight at 37 °C in SDA. Petri dishes with 20 mL of medium were prepared, previously inoculated with 200 ^L of the culture suspension.
Anti-leishmanial test r
The wells (6 mm) were made and the sample diluted in DMSO to test concentration (100 mg/mL) was added (20 ^L/well) and the same volume (20 ^L) of DMSO was used as a control. The inoculated plates were incubated for 24/48 h. After incubation, the diameter of the inhibition zone was measured with calipers. DMSO was used as a negative control, while ampicillin and amphotericin B were used as a positive control.
The results obtained in the evaluation of the antimicrobial activity of the n-hexane fraction of Zygophyllum fabago are shown in table 3. The n-hexane fraction of Zygophyllum fabago exhibited a significant antifungal activity against Candida albicans with inhibition zones of 9,5 mm compared to positive control Ampicillin sodium with inhibition zones of 14 mm. The results obtained indicated that n-hexane fraction of Zygophyllum fabago fractions have significant antifungal activity against Candida albicans.
EVALUATION OF PROTEIN TYROSINE
PHOSPHATES1B ACTIVITY
The in vitro PTP1B activity assay was conducted based on a protocol previously described by Taghibiglou et al. [8]. During the assay, p-nitrophenyl phosphate (p-NPP) was used as substrate. p-NPP was diluted in the assay buffer (50 mM HEPES, pH 7,3, 100 mM NaCl, 0,1% BSA, and 1 mM DtT). In the evaluation of PTP1B Inhi-
Table 2
s of n-hexane fraction
Sample Name Test Concentration (^g/mL) L. donovanii % Inh.
Hexane fraction Amphotericin B 80 100 93 99
Тable 3
Antimicrobial test result of n-hexane fraction
Sample Name Sample Concentration (mM) Sample amount (ul) Inhibitory zone diameter (mm)
CA EC SA
Ampicillin sodiumm salt 10 mg/ml 5 14 — —
Ampicillin sodiumm salt 1 mg/ml 5 — 19 —
Amphotericin B 5 mg/ml 20 — — 15
Z-H 100 mg/ml 20 9,5 — —
CA : Candida albicans ATCC10231; EC : E. coli ATCC11229; SA : Staphylococcus aureus ATCC6538
Table 4
Inhibitory effect of hexane extract on PTP1B_
Sample IC50(|jg/ml)
n-Hexane fraction PTP1B Inhibitor 4,33 ± 0,17 1,46 ± 0,40
bitor, different concentrations of n-hexane frac-tionwere incubated with GST-PTP1B1-321, and the enzyme activities were detected in a 96-well microplate spectrophotometer (MD) at 30°C. The enzyme activities were determined by measuring the absorbance at 405 nm generated by the formation of product p-NPP. The PTP1B inhibition was expressed as percentage of inhibition and calculated by the following equations:
Reaction rate
(%)=([PTP1B]test/[PTP1B]Control)*100 (1) Inhibition rate
(%) = 100 - reaction rate (%) (2)
Extract concentration providing 50% inhibition (IC50) was calculated from the plot of inhibition percentage against extract concentration. All determinations were carried out in triplicate, and the results were averaged. Type II diabetes is associated with insulin resistance, possibly because of attenuated signaling from insulin receptor (IR) molecule.
Data support the concept that PTP1B is a key negative regulator of insulin signal transduction. Compounds that inhibit PTP1B have potential in therapeutic strategies for treatment of type II diabetes [8]. The n-hexane fraction was tested for their inhibitory activity on PTP1B enzyme using an in vitro assay. Inhibitory activity in series of concentrations was used to calculate the inhibitory relative concentration IC50, and the results are presented in table 4.
The results showed that the n- hexane fraction possess a significant potent inhibitory activity (IC50 value of 4,33 ^g mL-1) for PTP1B enzyme compared to the PTP1B Inhibitor control which showed activity (IC50 value of 1,46^g mL-1) these findings together with the chemical analysis of the n-hexane fraction indicate that the main components of this fraction are hydrocarbons, fatty acids and other unsaturated ester, so these findings promote us for further investigation of this fraction may lead to isolation of the main components which responsible for biological activity.
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Статья поступила в редакцию 11 июля 2014 г.
