Chemical composition and in vitro biological activities of essential oil fromAllochrusa gypsophiloides

Rano Z.Mamadalieva,Farukh S.Sharopov,Alidjan A.Ibragimov,Shavkat V.Abdullaev,Vahobjon U.Khujaev

1Kokand State Pedagogical Institute,Kokand 713000,Turon str.23,Uzbekistan; 2Research Institution “Chinese-Tajik Innovation Center for Natural Products”,National Academy of Sciences of the Republic of Tajikistan,Ayni 299/2,Dushanbe 734063,Tajikistan; 3Fergana State University,Fergana 150100,Murabbiylar Str 19,Uzbekistan; 4Namangan State University,Namangan 160136,Uychi Str 216,Uzbekistan.

Abstract Background: Allochrusa gypsophiloides Rgl (Caryophyllaceae) is the best known endemic saponin-bearing plant of Central Asia,being widely used in traditional medicine and different branches of industry.In traditional medicine,the decoction of the roots of this plant has been used as an expectorant for bronchitis,similar to other drugs with saponins.Methods: For the first time,the chemical composition of the volatile secondary metabolites obtained from the aerial parts of A.gypsophiloides was evaluated using GC-MS method.In vitro antioxidant activity of the essential oil was evaluated using DPPH and ABTS assays.The cytotoxic activities were carried out by MTT assay.Results: A total of 59 components were identified in this species representing 89.8% of the whole oil.The A.gypsophiloides essential oil was rich in oxygenated monoterpenoids,whereas pulegone (32.9%),trans-p-menthan-3-one (6.2%),hexadecanoic acid (3.6%),trans-verbenol (3.2%),phytol (3.2%),2-isopropyl-5-methylcyclohexanone (2.7%),isopiperitenon (2.1%) were predominant in the oil.The weak activity was found for the oil with IC50 values 456.4 ±5.39 µg/ml for DPPH and 745.0 ± 6.87 µg/ml for ABTS assay.The essential oil showed moderate cytotoxic effects against prostate cancer (PC-3) (IC50: 89.9 ±2.01 µg/ml) and colon adenocarcinoma (HT-29) cells (IC50: 43.6 ±2.38 µg/ml).Conclusion: The cytotoxic activity of A.gypsophiloides essential oil against HT-29 (colon) cancer cells is consistent with antitumor effects of some species from Allochrusa genus and their traditional use to treat cancer diseases.

Key words: Allochrusa gypsophiloides,essential oils,pulegone,antioxidant,cytotoxicity

Background

Flora of Central Asia is rich in diversity of plant species [1].Turkestan soap root (local name Beh,Bekhdoru,Etmak) or Allochrusa gypsophiloides(Regel) Ovcz.et Czuk.(syn.Acanthophyllum gypsophiloides Rgl.from Caryophyllaceae family),is the best known endemic saponin-bearing plant of Central Asia.This plant is distributed in Kazakhstan,Kyrgyzstan,Tajikistan,and Uzbekistan [2].Roots of A.gypsophiloides contain up to 30% saponins with a hemolytic index of 1:1000 or 1:2860 and aboveground parts of the plant produce saponins with an index of 1:240.The underground part of the plant is represented natural resource of secondary metabolites [3,4].Many plant metabolites such as polysaccharides,triterpene glucosides have been isolated from underground parts of A.gypsophiloides [3-5].

Moreover A.gypsophiloides is widely used in traditional medicine and different branches of industry.In traditional medicine,the decoction of the roots of this plant has been used as an expectorant for bronchitis,similar to other drugs with saponins.Local people of Central Asia are used roots of A.gypsophiloides in large quantities for the preparation of special sweet delicacy,"nishollo" or “nisholda”- a thick foamy white liquid with tonic effect [6].In addition,the A.gypsophiloides saponins are utilized in manufacturing sweets or natural washing aids [2].Saponins isolated from the roots of A.gypsophiloides were exhibited immunoadjuvant properties on female Swiss mice [3].Although,there are some reports about antitumor and antioxidant effects of some species from this genus,biological studies have not yet been conducted on A.gypsophiloides.No reports are available on the presence of other metabolites such as volatile compounds.Therefore,the objectives of this study were to study chemical composition of volatile metabolites of A.gypsophiloides and to evaluate the corresponding antioxidant and cytotoxic activities (in vitro).

Materials and methods

Plant material.

The aerial parts of A.gypsophiloides were collected from Tashkent regions of Uzbekistan during the flowering phase.Voucher specimens (QDPI 20192051)were identified by Dr.R.N.Muminova and deposited at the Department of Botany (Kokand State Pedagogical Institute,Uzbekistan).

Essential oil isolation.

Fresh aerial parts (stems,flowers,and leaves) of the A.gypsophiloides (200 g) were hydrodistilled for 3 h using a Clevenger-type apparatus.As only small amounts of essential oil were present,the oils were trapped in dichloromethane,which were dried over anhydrous sodium sulphate and left over night in a hood to remove the dichloromethane solvent till the weights were fixed stored at -4°C in the dark until use.

GC-MS analysis.

GC-MS characterization of A.gypsophiloides oil was carried out on a Agilent 7890B gas chromatograph equipped with a VF-Wax CP 9205 fused silica column(100% polyethylene glycol,30 m × 0.25 mm,film thickness 0.25 μm,Agilent Technologies,Netherlands),interfaced with an Agilent mass selective detector 5977A (Agilent Technologies).Interface temperature:280°C; MS source temperature: 230°C; ionization energy: 70 eV; scan range: 45-950 atomic mass units.The sample (0.5 μl) was injected automatically into the chromatograph using a GC auto sampler.The oven temperature was kept at 50°C for 5 min,then rising from 50°C to 280°C at 5°C/min,and finally held isothermally at 280°C for 15 min; injector temperature 250°C; detector temperature 270°C; carrier gas helium(0.9 ml/min); with split mode (split ratio,1:20).C7-C40 Saturated alkanes standard was purchased from Sigma-Aldrich (Germany).Enhanced ChemStation software,version MSD F.01.01.2317(Agilent Technologies) was used for recording and integrating the chromatograms.The compounds were identified by comparison of their mass-spectral data and retention indices (RIs) with those of the Wiley Registry of Mass Spectral Data (9th Ed.),NIST Mass Spectral Library (2011),and with Laboratory database.

Antioxidant assay.

DPPH radical-scavenging assay.In the 2,2-diphenyl-1-picrylhydrazyl (DPPH)radical-scavenging assay,100 μL of essential oil solution (diluted in methanol to a concentration range of 7.8 to 1000 µg/ml) was mixed with 100 μL 0.2 mM DPPH in methanol in wells of a 96-well plate.The plate was kept in the dark for 15 min,after which the absorbance of the solution was measured at 515 nm in a microplate reader.Appropriate blanks (methanol) and standards (ascorbic acid in methanol) were assessed simultaneously.This method follows closely that used by previous workers [7,8].

ABTS radical-scavenging assay.

The 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical-scavenging activity was determined using the method as described by Brangoulo and Molan [9].38 mg of ABTS reagent and 6.5 mg of potassium persulphate were dissolved in 10 mL of deionised purified water.The solution was kept in the dark at room temperature for 16 h to form the stable ABTS•+radical cation.The ABTS•+solution was further diluted with deionized purified water to an absorbance value between 2.0 and 2.4 at 645 nm.Trolox standard (5 mg/mL) was prepared in absolute ethanol a few minutes prior to analysis.A 96-well microplate was loaded with 100 μL essential oil solution (with concentration range 5.0-0.005 mg/ml) or trolox or water blank and mixed with 100 μL ABTS•+solution.Absorbance was read at 645 nm using a spectrophotometer.

Cytotoxicity assay.

MTT assay.Cytotoxicity was determined in human tumor PC-3 (prostate cancer) and HT-29 (colon adenocarcinoma) cells by the MTT assay.The cells were seeded at a density of 2×104cells/well (PC-3)and 4×104cells/well (HT-29).The essential oil was dissolved in 0.5% DMSO in complete culture medium and then,intended dilutions (200,100,50,25,and 12.5µg/ml) were prepared and 100 μl liquid of each concentration was applied to the wells of a 96 well plates.Cells were incubated with the samples for 48 h before the medium was removed and replaces with fresh medium containing 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).The formazan crystals were dissolved in DMSO 4 h later.The absorbance was measured with an automated microplate reader at 540 nm with a reference wavelength of 670 nm.

Data evaluation.All experiments were carried out three times.The obtained results were expressed as mean ± standard deviation (SD) and statistically evaluated by one-way ANOVA (by Tukey test,p <0.05).The IC50was determined as the drug concentration which resulted in a 50 % reduction in viability or inhibition of the biological activity.

Results and discussion

GC-MS analysis.The chemical composition of the essential oil of A.gypsophiloides was qualitatively and quantitatively analyzed by GC-MS and the yield of oil was estimated by 0.008% (v/w) for the species.Fifty-nine compounds were identified in total,which accounts for 89.8% of the oil (Table 1).Pulegone(32.9%),trans-p-menthan-3-one (6.2%),hexadecanoic acid (3.6%),trans-verbenol (3.2%),phytol (3.2%),2-isopropyl-5-methylcyclohexanone (2.7%),isopiperitenon (2.1%) were the main constitutes of the essential oil examined.According to GC-MS analysis significant content of fatty acids such as hexadecanoic acid (3.6%),tetradecanoic acid (1.1%),octadecanoic acid (0.5%),linoleic acid (0.2%) were found in A.gypsophiloides essential oil.The results showed that oxygen containing monoterpenes constituted the main fraction in the essential oil of A.gypsophiloides(Figure 1).

Figure 1.The chemical structure of the main volatile secondary metabolites of A.gypsophiloides

Table 1 Chemical composition of the hydro-distilled oil obtained from air-dried aerial parts of Allochrusa gypsophiloides

Antioxidant activities.

The DPPH and ABTS assays are a simple,rapid and sensitive method for evaluating free radical scavenging ability.The results showed that the DPPH free radical scavenging ability of essential oil of A.gypsophiloides was moderate (IC50456.4 ±5.39 µg/ml).According to the IC50value (745.0 ± 6.87 µg/ml),a weak ABTS scavenging activity were observed for the essential oil.

Cytotoxic activity.A cytotoxicity screening of the essential oil of A.gypsophiloides was carried out in prostate cancer (PC-3) and colon adenocarcinoma(HT-29) cells.Results showed that oil exhibited the moderate level of cytotoxicity with IC50: 89.9 ±2.01µg/ml and IC50: 43.6 ±2.38 µg/ml in PC-3 and HT-29 cell lines,respectively.Numerous reports have shown cytotoxic properties of plant steroidal and triterpenoid against various human cancer cell lines.A previous investigation by Khatuntseva et al.[3] showed that A.gypsophiloides might be a potential source of immunoactive agents.In another report triterpene saponins from the Acanthophyllum sordidum,A.elatius and A.lilacinum showed a moderate cytotoxicity against two human colon cancer cell lines(HT-29 and HCT116) [10].In our study,the MTT assay of the essential oil of A.gypsophiloides showed a substantial cytotoxicity against HT-29 cells.It is likely that the monoterpenes from this oil cause cell death in a synergistic way,as shown for other plants with terpenes.

Conclusions

The current study reports for the first time the chemical content and in vitro biological effects of the essential oil from A.gypsophiloides.The results showed that oxygen containing monoterpenes constituted the main fraction in the essential oil.Essential oil showed weak antioxidant effects in DPPH and ABTS assays,but in MTT assay it showed better cytotoxic effects against colon adenocarcinoma cells.Further chemical analyses of other metabolites and pharmacological experiments will complete the information about this important endemic species of Central Asian flora.