Research Article
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Year 2025, Volume: 29 Issue: 4, 1601 - 1607, 05.07.2025

Fatmanur Gürbüzkol , Leyla Bitiş

https://doi.org/10.12991/jrespharm.1734601

Abstract

References

  • Semiz G, Gunal B, Dogan NM. Chemical composition and anti-biofilm activity of the essential oil from Achillea alimeana. Chemistry of Natural Compounds. 2023; 59: 984-986. https://doi.org/10.1007/s10600-023-04173-7
  • Amanpour A, Çelebi F, Kahraman IG, Çelik F. Diyet inflamatuar indeksi, inflamasyon ve beslenme. Sağlık Bilimleri ve Araştırmaları Dergisi. 2022; 5(3): 59-80. https://doi.org/10.51536/tusbad.1133247
  • Harvey RA, Lippincott’s IIIustrated Reviews Farmakoloji third eds., Nobel Tıp , İstanbul, Turkey 2009.
  • Tuzlacı E, Türkiye’nin Geleneksel Ilaç Rehberi, İstanbul Tıp Kitapevleri, İstanbul, Turkey 2009.
  • Tuzlacı E, Türkiye Bitkileri Sözlüğü, Türkçe/Latince bitki adlarının özel açıklamaları, Alfa Yayınları, İstanbul, Turkey 2006.
  • Saleki B, Selamoglu Z, Sevindik M, Fahmy NM, Al-Sayed E. Achillea spp: A comprehensive review on its ethnobotany, phytochemistry, phytopharmacology and industrial applications. Cell Mol Biol. 2020; 66(4): 78-103. https://doi.org/10.14715/cmb/2020.66.4.13
  • Dikpinar T, Bitis L. LC-MS/MS analysis and biological activities of endemic Achillea sieheana Stapf from Türkiye. Journal of Research in Pharmcy. 2022; 26(6): 1796-1804. http://dx.doi.org/10.29228/jrp.270
  • Toplan GG, Taşkın T, İşcan G, Göger F, Kürkçüoğlu M, Civas A, Ecevit-Genc G. Comparative Studies On Essential Oil And Phenolic Content With In Vitro Antioxidant, Anticholinesterase, Antimicrobial Activities Of Achillea Biebersteinii Afan. And A. Millefolium Subsp. Millefolium Afan. L. Growing In Eastern Turkey. Molecules. 2022; 27(6): 6. 10.3390/molecules27061956
  • Dikpınar T. PhD Thesis. The Active Constituents Isolation from Achillea sieheana Stapf by Bioactivity-Guided. Department of Pharmacognosy, Faculty of Pharmacy, Marmara University, Başıbüyük, İstanbul, Turkey, 2023.
  • Argentieri MP, Madeo M, Avato P, Iriti M, Vitalini S. Polyphenol content and bioactivity of Achillea moschata from the Italian and Swiss Alps. Z Für Nat C. 2020; 75(1): 57-64. https://doi.org/10.1515/znc-2019-0214
  • Kaczorová D, Karalija E, Dahija S, Bešta-Gajevi ́c R, Pari ́c A, Cavar-Zeljkovi ́c S. Influence of extraction solvent on the phenolic profile and bioactivity of two Achillea species. Molecules. 2021; 26(1): 1601. https://doi.org/10.3390/molecules26061601
  • Salomon L, Lorenz P, Bunse M, Spring O, Stintzing FC, Kammere DR. Comparison of the phenolic compound profile and antioxidant potential of Achillea atrata L. and Achillea millefolium L. Molecules. 2021; 726(1): 1530. https://doi.org/10.3390/molecules26061530
  • Khazneh E, Hribová P, Hošek J, Suchý P, Kollár P, Pražanová G, Muselík J, Hanaková Z, Václavík J, Miłek The chemical composition of Achillea wilhelmsii C. Koch and its desirable effects on hyperglycemia, inflammatory mediators and hypercholesterolemia as risk factors for cardiometabolic disease. Molecules. 2016; 21(1): 404. https://doi.org/10.3390/molecules21040404
  • Sabanoglu S, Gökbulut A, Altun ML. Characterization of phenolic compounds, total phenolic content and antioxidant activity of three Achillea species. J Res Pharm. 2019; 23(1): 567-576. https://doi.org/10.12991/jrp.2019.164
  • Gawel-Beben K, Strzepek-Gomółka M, Czop M, Sakipova Z, Głowniak K. Achillea millefolium L. And Achillea biebersteinii Afan. Hydroglycolic Extracts–Bioactive Ingredients for Cosmetic Use. Molecules. 2020; 25(15): 3368. https://doi.org/10.3390/molecules25153368
  • Todorova MN, Markova MM, Tsankova ET. Crithmifolide: A sesquiterpene lactone from Achillea crithmifolia. Phytochemistry. 1998; 49(1): 2429-2432. https://doi.org/10.1016/S0031-9422(98)00202-7
  • Hofer O, Greger H. Naturally occurring sequiterpene-coumarin ethers, VI.* new sesquiterpene-isofraxidin ethers from Achillea depressa. Monatshefte für Chemie. 1984; 115(1): 477-483.
  • Ahmed AA, Mahmoud AA, Ali ET, Tzakou O, Couladis M, Mabry TJ, Gati T, Toth G. Two highly oxygenated eudesmanes and 10 lignans from Achillea holosericea. Phytochemistry. 2002; 59(8): 851-856. https://doi.org/10.1016/S0031-9422(01)00442-3
  • Mehlfuhrer M, Troll K, Jurenitsch J, Auer H, Kubelka W. Betaines and free proline within the Achillea millefolium group. Phytochemistry. 1997; 44(6): 1067-1069. https://doi.org/10.1016/S0031-9422(96)00669-3
  • Nemeth E, Bernath J, Hethelyi E. Chemotypes and their stability in Achillea crithmifolia W. et K. populations. J Essent Oil Res. 2000; 12(1): 53-58. https://doi.org/10.1080/10412905.2000.9712041
  • Suleimenov YM, Atazhanova GA, Özek T, Demirci B, Kulyyasov AT, Adekenov S, Başer KHC. Essential oil composition of three species of Achillea from Kazakhstan. Chem Nat Compd. 2001; 37(5): 447-450. https://doi.org/10.1023/a:1014471326724
  • Unlu M, Daferera D, Donmez E, Polissiou M, Tepe B, Sokmen A. Compositions and the in vitro antimicrobial activities of the essential oils of Achillea setacea and Achillea teretifolia (Compositae). J Ethnopharmacol. 2002; 83(2): 117-120. https://doi.org/10.1016/s0378-8741(02)00218-0
  • Candan F, Ünlü M, Tepe B, Daferera D, Polissiou M, Sökmen A, Akpulat HA. Antioxidant and antimicrobial activity of the essential oil and methanol extracts of Achillea millefolium subsp millefolium Afan. (Asteraceae). J. Ethnopharmacol. 2003; 87(3): 215-220. https://doi.org/10.1016/s0378-8741(03)00149-1
  • Javidnia K, Miri R, Sadeghpour H. Composition of the volatile oil of Achillea wilhelmsii C. Koch from Iran. Daru. 2004; 12(2): 63-65.
  • Deka H, Choudhury A, Dey BK. An overview on plant derived phenolic compounds and their role in treatment and management of diabetes. Journal of pharmacopuncture. 2022; 25(3): 199-208. https://doi.org/10.3831/KPI.2022.25.3.199
  • Ge J, Liu Z, Zhong Z, Wang L, Zhuo X, Li J, Bai R. Natural terpenoids with anti-inflammatory activities: potential leads for anti-inflammatory drug discovery. Bioorganic Chemistry. 2022; 124(1): 124. https://doi.org/10.3831/KPI.2022.25.3.199
  • EMA Committee on Herbal Medicinal Products. Draft list of references supporting the assessment of Achillea millefolium L., flos. 2010. 10.1016/j.bioorg.2022.105817 (accessed on 18 May 2024).
  • Yıldırım A, Şen A, Doğan A, Bitis L. Antioxidant and anti-inflammatory activity of capitula, leaf and stem extracts of Tanacetum cilicicum (Boiss.) Grierson. International Journal of Secondary Metabolite. 2019; 6(2): 211-222. https://doi.org/10.21448/ijsm.510316
  • Gao X, Ohlander M, Jeppsson N, Björk L, Trajkovski V. Changes in Antioxidant Effects and Their Relationship to Phytonutrients in Fruits of Sea Buckthorn (Hippophae rhamnoides L.) during Maturation. Journal of Agricultural and Food Chemistry. 2000; 48(1): 1485-1490. https://doi.org/10.1021/jf991072g
  • Zhang R, Zeng Q, Deng Y, Zhang M, Wei Z, Zhang Y, Tang X. Phenolic profiles and antioxidant activity of litchipulp of different cultivars cultivated in Southern China. Food Chem. 2013; 136(1): 1169-1176. https://doi.org/10.1016/j.foodchem.2012.09.085
  • Zou Y, Chang SKC, Gu Y, Qian SY. Antioxidant Activity and Phenolic Compositions of Lentil (Lens culinaris var. Morton) Extract and Its Fractions. J. Agric. Food Chem. 2011; 59(6): 2268-2276. https://doi.org/10.1021/jf104640k
  • Ramakrishna R, Sarkar D, Schwarz P, Shetty K. Phenolic linked anti-hyperglycemic bioactives of barley (Hordeum vulgare L.) cultivars as nutraceuticals targeting type 2 diabetes. Industrial Crops and Products. 2017; 107(1): 509-517. https://doi.org/10.1016/j.indcrop.2017.03.033
  • Sen A, Kurkcuoglu M, Senkardes I, Bitis L, Baser KHC. Chemical Composition, Antidiabetic, Anti-inflammatory and Antioxidant Activity of Inula ensifolia L. Essential Oil. J. Ess. Oil Bearing Plants. 2019; 22(4): 1048-1057. https://doi.org/10.1080/0972060x.2019.1662333
  • Phosrithong N, Nuchtavorn N. Antioxidant and anti-inflammatory activites of Clerodendrum leaf extracts collected in Thailand. European J. Int. Med. 2016; 8(3): 281-285. https://doi.org/10.1016/j.eujim.2015.10.002

Comparative studies on anti-inflammatory and antidiabetic effects of some Achillea species from the Turkish flora

Year 2025, Volume: 29 Issue: 4, 1601 - 1607, 05.07.2025

Fatmanur Gürbüzkol , Leyla Bitiş

https://doi.org/10.12991/jrespharm.1734601

Abstract

This study aimed to determine antioxidant, antidiabetic and anti-inflammatory activities of methanol
extracts of Achillea crithmifolia Waldst. & Kit. (AC), A. millefolium L. (AM), A. nobilis subsp. neilreichii (A.Kern.) Velen.
(AN), A. setacea Waldst. & Kit. (AS) and A. wilhelmsii K. Koch (AW) species which grow naturally in Türkiye.
DPPH/ABTS radicals scavenging, α-glucosidase inhibition and anti 5-lipoxygenase methods were used to determine
for antioxidant, antidiabetic and anti-inflammatory activities, respectively. The total phenol content of the extracts was
calculated using Folin-Ciocalteau solution and the highest phenol content was found in AC methanol extract (ACM)
(56.71 mg GAE/g). In DPPH and ABTS radical scavenging activity assays, ACM showed the highest antioxidant activity
according to IC50 values (54.85 and 79.77 μg/mL, respectively). According to the results of α-glucosidase inhibitory
activity test, AN methanol extract (ANM) with an IC50 value of 245.50 μg/mL showed a better activity than acarbose
(261.7 μg/mL) by inhibiting the enzyme. As a result of anti-inflammatory activity, it was observed that ACM had the
highest activity (90.30 μg/mL). Therefore, ACM, which has the best antioxidant, anti 5-lipoxygenase activity and the
second best value close to the standard in terms of α-glucosidase inhibitory activity, was fractionated with 3 different
solvents (hexane-ACH, chloroform-ACC and ethyl acetate-ACEA). Biological activity studies were repeated on these
fractions. The ACH fraction showed the best anti-inflammatory activity while the ACEA fraction showed the best
antidiabetic activity. The biological activities of five Achillea species were not examined before by comparing with each
other. In addition, antidiabetic and anti-inflammatory activity studies on A. crithmifolia were conducted for the first
time.

Keywords

Achillea crithmifolia Anti 5-lipoxygenase α-glucosidase inhibition Anti-inflammatory Antidiabetic

References

  • Semiz G, Gunal B, Dogan NM. Chemical composition and anti-biofilm activity of the essential oil from Achillea alimeana. Chemistry of Natural Compounds. 2023; 59: 984-986. https://doi.org/10.1007/s10600-023-04173-7
  • Amanpour A, Çelebi F, Kahraman IG, Çelik F. Diyet inflamatuar indeksi, inflamasyon ve beslenme. Sağlık Bilimleri ve Araştırmaları Dergisi. 2022; 5(3): 59-80. https://doi.org/10.51536/tusbad.1133247
  • Harvey RA, Lippincott’s IIIustrated Reviews Farmakoloji third eds., Nobel Tıp , İstanbul, Turkey 2009.
  • Tuzlacı E, Türkiye’nin Geleneksel Ilaç Rehberi, İstanbul Tıp Kitapevleri, İstanbul, Turkey 2009.
  • Tuzlacı E, Türkiye Bitkileri Sözlüğü, Türkçe/Latince bitki adlarının özel açıklamaları, Alfa Yayınları, İstanbul, Turkey 2006.
  • Saleki B, Selamoglu Z, Sevindik M, Fahmy NM, Al-Sayed E. Achillea spp: A comprehensive review on its ethnobotany, phytochemistry, phytopharmacology and industrial applications. Cell Mol Biol. 2020; 66(4): 78-103. https://doi.org/10.14715/cmb/2020.66.4.13
  • Dikpinar T, Bitis L. LC-MS/MS analysis and biological activities of endemic Achillea sieheana Stapf from Türkiye. Journal of Research in Pharmcy. 2022; 26(6): 1796-1804. http://dx.doi.org/10.29228/jrp.270
  • Toplan GG, Taşkın T, İşcan G, Göger F, Kürkçüoğlu M, Civas A, Ecevit-Genc G. Comparative Studies On Essential Oil And Phenolic Content With In Vitro Antioxidant, Anticholinesterase, Antimicrobial Activities Of Achillea Biebersteinii Afan. And A. Millefolium Subsp. Millefolium Afan. L. Growing In Eastern Turkey. Molecules. 2022; 27(6): 6. 10.3390/molecules27061956
  • Dikpınar T. PhD Thesis. The Active Constituents Isolation from Achillea sieheana Stapf by Bioactivity-Guided. Department of Pharmacognosy, Faculty of Pharmacy, Marmara University, Başıbüyük, İstanbul, Turkey, 2023.
  • Argentieri MP, Madeo M, Avato P, Iriti M, Vitalini S. Polyphenol content and bioactivity of Achillea moschata from the Italian and Swiss Alps. Z Für Nat C. 2020; 75(1): 57-64. https://doi.org/10.1515/znc-2019-0214
  • Kaczorová D, Karalija E, Dahija S, Bešta-Gajevi ́c R, Pari ́c A, Cavar-Zeljkovi ́c S. Influence of extraction solvent on the phenolic profile and bioactivity of two Achillea species. Molecules. 2021; 26(1): 1601. https://doi.org/10.3390/molecules26061601
  • Salomon L, Lorenz P, Bunse M, Spring O, Stintzing FC, Kammere DR. Comparison of the phenolic compound profile and antioxidant potential of Achillea atrata L. and Achillea millefolium L. Molecules. 2021; 726(1): 1530. https://doi.org/10.3390/molecules26061530
  • Khazneh E, Hribová P, Hošek J, Suchý P, Kollár P, Pražanová G, Muselík J, Hanaková Z, Václavík J, Miłek The chemical composition of Achillea wilhelmsii C. Koch and its desirable effects on hyperglycemia, inflammatory mediators and hypercholesterolemia as risk factors for cardiometabolic disease. Molecules. 2016; 21(1): 404. https://doi.org/10.3390/molecules21040404
  • Sabanoglu S, Gökbulut A, Altun ML. Characterization of phenolic compounds, total phenolic content and antioxidant activity of three Achillea species. J Res Pharm. 2019; 23(1): 567-576. https://doi.org/10.12991/jrp.2019.164
  • Gawel-Beben K, Strzepek-Gomółka M, Czop M, Sakipova Z, Głowniak K. Achillea millefolium L. And Achillea biebersteinii Afan. Hydroglycolic Extracts–Bioactive Ingredients for Cosmetic Use. Molecules. 2020; 25(15): 3368. https://doi.org/10.3390/molecules25153368
  • Todorova MN, Markova MM, Tsankova ET. Crithmifolide: A sesquiterpene lactone from Achillea crithmifolia. Phytochemistry. 1998; 49(1): 2429-2432. https://doi.org/10.1016/S0031-9422(98)00202-7
  • Hofer O, Greger H. Naturally occurring sequiterpene-coumarin ethers, VI.* new sesquiterpene-isofraxidin ethers from Achillea depressa. Monatshefte für Chemie. 1984; 115(1): 477-483.
  • Ahmed AA, Mahmoud AA, Ali ET, Tzakou O, Couladis M, Mabry TJ, Gati T, Toth G. Two highly oxygenated eudesmanes and 10 lignans from Achillea holosericea. Phytochemistry. 2002; 59(8): 851-856. https://doi.org/10.1016/S0031-9422(01)00442-3
  • Mehlfuhrer M, Troll K, Jurenitsch J, Auer H, Kubelka W. Betaines and free proline within the Achillea millefolium group. Phytochemistry. 1997; 44(6): 1067-1069. https://doi.org/10.1016/S0031-9422(96)00669-3
  • Nemeth E, Bernath J, Hethelyi E. Chemotypes and their stability in Achillea crithmifolia W. et K. populations. J Essent Oil Res. 2000; 12(1): 53-58. https://doi.org/10.1080/10412905.2000.9712041
  • Suleimenov YM, Atazhanova GA, Özek T, Demirci B, Kulyyasov AT, Adekenov S, Başer KHC. Essential oil composition of three species of Achillea from Kazakhstan. Chem Nat Compd. 2001; 37(5): 447-450. https://doi.org/10.1023/a:1014471326724
  • Unlu M, Daferera D, Donmez E, Polissiou M, Tepe B, Sokmen A. Compositions and the in vitro antimicrobial activities of the essential oils of Achillea setacea and Achillea teretifolia (Compositae). J Ethnopharmacol. 2002; 83(2): 117-120. https://doi.org/10.1016/s0378-8741(02)00218-0
  • Candan F, Ünlü M, Tepe B, Daferera D, Polissiou M, Sökmen A, Akpulat HA. Antioxidant and antimicrobial activity of the essential oil and methanol extracts of Achillea millefolium subsp millefolium Afan. (Asteraceae). J. Ethnopharmacol. 2003; 87(3): 215-220. https://doi.org/10.1016/s0378-8741(03)00149-1
  • Javidnia K, Miri R, Sadeghpour H. Composition of the volatile oil of Achillea wilhelmsii C. Koch from Iran. Daru. 2004; 12(2): 63-65.
  • Deka H, Choudhury A, Dey BK. An overview on plant derived phenolic compounds and their role in treatment and management of diabetes. Journal of pharmacopuncture. 2022; 25(3): 199-208. https://doi.org/10.3831/KPI.2022.25.3.199
  • Ge J, Liu Z, Zhong Z, Wang L, Zhuo X, Li J, Bai R. Natural terpenoids with anti-inflammatory activities: potential leads for anti-inflammatory drug discovery. Bioorganic Chemistry. 2022; 124(1): 124. https://doi.org/10.3831/KPI.2022.25.3.199
  • EMA Committee on Herbal Medicinal Products. Draft list of references supporting the assessment of Achillea millefolium L., flos. 2010. 10.1016/j.bioorg.2022.105817 (accessed on 18 May 2024).
  • Yıldırım A, Şen A, Doğan A, Bitis L. Antioxidant and anti-inflammatory activity of capitula, leaf and stem extracts of Tanacetum cilicicum (Boiss.) Grierson. International Journal of Secondary Metabolite. 2019; 6(2): 211-222. https://doi.org/10.21448/ijsm.510316
  • Gao X, Ohlander M, Jeppsson N, Björk L, Trajkovski V. Changes in Antioxidant Effects and Their Relationship to Phytonutrients in Fruits of Sea Buckthorn (Hippophae rhamnoides L.) during Maturation. Journal of Agricultural and Food Chemistry. 2000; 48(1): 1485-1490. https://doi.org/10.1021/jf991072g
  • Zhang R, Zeng Q, Deng Y, Zhang M, Wei Z, Zhang Y, Tang X. Phenolic profiles and antioxidant activity of litchipulp of different cultivars cultivated in Southern China. Food Chem. 2013; 136(1): 1169-1176. https://doi.org/10.1016/j.foodchem.2012.09.085
  • Zou Y, Chang SKC, Gu Y, Qian SY. Antioxidant Activity and Phenolic Compositions of Lentil (Lens culinaris var. Morton) Extract and Its Fractions. J. Agric. Food Chem. 2011; 59(6): 2268-2276. https://doi.org/10.1021/jf104640k
  • Ramakrishna R, Sarkar D, Schwarz P, Shetty K. Phenolic linked anti-hyperglycemic bioactives of barley (Hordeum vulgare L.) cultivars as nutraceuticals targeting type 2 diabetes. Industrial Crops and Products. 2017; 107(1): 509-517. https://doi.org/10.1016/j.indcrop.2017.03.033
  • Sen A, Kurkcuoglu M, Senkardes I, Bitis L, Baser KHC. Chemical Composition, Antidiabetic, Anti-inflammatory and Antioxidant Activity of Inula ensifolia L. Essential Oil. J. Ess. Oil Bearing Plants. 2019; 22(4): 1048-1057. https://doi.org/10.1080/0972060x.2019.1662333
  • Phosrithong N, Nuchtavorn N. Antioxidant and anti-inflammatory activites of Clerodendrum leaf extracts collected in Thailand. European J. Int. Med. 2016; 8(3): 281-285. https://doi.org/10.1016/j.eujim.2015.10.002
There are 34 citations in total.

Details

Primary Language English
Subjects Pharmacognosy
Journal Section Articles
Authors

Fatmanur Gürbüzkol

Leyla Bitiş

Publication Date July 5, 2025
Submission Date June 19, 2024
Acceptance Date July 1, 2024
Published in Issue Year 2025 Volume: 29 Issue: 4

Cite

APA Gürbüzkol, F., & Bitiş, L. (2025). Comparative studies on anti-inflammatory and antidiabetic effects of some Achillea species from the Turkish flora. Journal of Research in Pharmacy, 29(4), 1601-1607. https://doi.org/10.12991/jrespharm.1734601
AMA Gürbüzkol F, Bitiş L. Comparative studies on anti-inflammatory and antidiabetic effects of some Achillea species from the Turkish flora. J. Res. Pharm. July 2025;29(4):1601-1607. doi:10.12991/jrespharm.1734601
Chicago Gürbüzkol, Fatmanur, and Leyla Bitiş. “Comparative Studies on Anti-Inflammatory and Antidiabetic Effects of Some Achillea Species from the Turkish Flora”. Journal of Research in Pharmacy 29, no. 4 (July 2025): 1601-7. https://doi.org/10.12991/jrespharm.1734601.
EndNote Gürbüzkol F, Bitiş L (July 1, 2025) Comparative studies on anti-inflammatory and antidiabetic effects of some Achillea species from the Turkish flora. Journal of Research in Pharmacy 29 4 1601–1607.
IEEE F. Gürbüzkol and L. Bitiş, “Comparative studies on anti-inflammatory and antidiabetic effects of some Achillea species from the Turkish flora”, J. Res. Pharm., vol. 29, no. 4, pp. 1601–1607, 2025, doi: 10.12991/jrespharm.1734601.
ISNAD Gürbüzkol, Fatmanur - Bitiş, Leyla. “Comparative Studies on Anti-Inflammatory and Antidiabetic Effects of Some Achillea Species from the Turkish Flora”. Journal of Research in Pharmacy 29/4 (July 2025), 1601-1607. https://doi.org/10.12991/jrespharm.1734601.
JAMA Gürbüzkol F, Bitiş L. Comparative studies on anti-inflammatory and antidiabetic effects of some Achillea species from the Turkish flora. J. Res. Pharm. 2025;29:1601–1607.
MLA Gürbüzkol, Fatmanur and Leyla Bitiş. “Comparative Studies on Anti-Inflammatory and Antidiabetic Effects of Some Achillea Species from the Turkish Flora”. Journal of Research in Pharmacy, vol. 29, no. 4, 2025, pp. 1601-7, doi:10.12991/jrespharm.1734601.
Vancouver Gürbüzkol F, Bitiş L. Comparative studies on anti-inflammatory and antidiabetic effects of some Achillea species from the Turkish flora. J. Res. Pharm. 2025;29(4):1601-7.