Novel Anti-Aging Benzoquinone Derivatives From Onosma Bracteatum Wall Part 2

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2.3. Anti-Aging Activity in K6001 Yeast Strain

Anti-aging activities of isolated benzoquinones (1-5) were evaluated by using a K6001 yeast replicative lifespan bioassay system. Resveratrol (Res), a well-known anti-aging substance, was used as a positive control to check the reliability of the bioassay system. The most effective concentration of Res was 10 μM in the yeast replicative lifespan bioassay system as reported in our previous publications 【4-6】. cistanche herba As shown in Figure 4， allomicrophyllone（1）at 3 μM， ehretiquinone （2） and ehretiquinone C（4） at 1 and 3 μM, and ehretiquinone D （5） at 1 μM， exhibited a significant increase in the replicative lifespan of the K6001 yeast strain, while ehretiquinone B (3) was inactive. This result indicated the anti-aging potential of benzoquinones (1, 2, 4, and 5) isolated from O. bracteatum.

3. Discussion

In the context of global aging, research and development of anti-aging drugs to improve the quality of life of aged people and reduce the prevalence of age-related diseases, are of great importance. cistanche penis growth This study reports the isolation, structure elucidation, biological activity, and brief SAR study of anti-aging benzoquinone derivatives from O.bracteatum1.

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In this study, two known(1 and 2) and three novel (3-5) benzoquinone derivatives were isolated from an ethyl acetate layer samples of O.bracteatum under the guidance of a K6001 yeast replicative lifespan bioassay. The structures of novel benzoquinones (3-5)were elucidated by extensive spectroscopic analysis. The known benzoquinones, allomicrophyllone(1), and ehretiquinone(2) were purified and identified by comparing spectroscopic data with those reported in the literature [17-19]. Previously, both compounds were reported to have anti-allergic and anti-inflammatory activity, respectively[17,19]. To the best of our knowledge, no anti-aging-related study was reported for these compounds. These isolated compounds shared the same benzoquinone skeleton and their differences were in the modifications at C-11 and C-11 positions.

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The biological activities evaluation was performed for the study of the structure-activity relationship. cistanche salsa benefits Results in Figure 4 indicated that isolated allomicrophyllone (1), ehretiquinone(2), and ehretiquinones C-D (4-5) significantly prolonged the replicative lifespan of yeast. However, ehretiquinone B (3)which possessed the same benzoquinone skeleton but the different modifications were inactive. According to the structure-activity relationship (SAR) study within these isolated compounds, we concluded that the presence of methyl and exomethylene groups at C-9'is important for anti-aging activity as in ehretiquinone (2) and ehretiquinone C(4). The modification like the hydroxylation of the C-11/methyl group as in ehretiquinone B (3) resulted in the loss of activity. Furthermore, a comparison of ehretiquinone (2) and ehretiquinone C(4)with better activity than those of allomicrophyllone (1) and ehretiquinone D (5)showed that the presence of the exomethylene group at C-9' played an important role in anti-aging activity for these molecules. Ehretiquinone C(4) exhibited better anti-aging activity than that of resveratrol because the mean lifespan value of 4 at 1 uMis was comparable with resveratrol at 10 uM which showed the same value. cistanche tubulosa dosage reddit Therefore, ehretiquinone C(4)can be considered a promising lead compound, and its anti-aging mechanism of action should be further intensively studied. In addition, these results also provide important structural information to design and synthesize novel anti-aging drugs. This study will make a contribution to the prevention and treatment of age-related diseases. The results also suggested that O.bracteatum enriched with anti-aging ingredients can be used as medicine or as a food supplement.

4. Materials and Methods

4.1.General

A JASCO P-1030 digital polarimeter was used for optical rotation measurements. Preparative high-performance liquid chromatography (HPLC) with a system equipped with ELITE P-230 pumps and a UV detector was used. High-resolution mass spectra(HR-ESI-TOF-MS)were obtained with an Agilent Technologies 6224A accurate mass TOF LC/MS system. NMR spectra were observed with Bruker AVII-500 spectrometer and NMR chemical shifts inδ（ppm）were referenced to solvent peaks of bc 77.0 and6H7.26 for CDCl. Column chromatography was performed by using silica gel (200-300 mesh, Yantai Chemical Industry Research Institute, Yantai, China) or reversed phase C18 (Octadecylsilyl, ODS) silica gel(Cosmosil 75C18-OPN, Nacalai Tesque, Kyoto, Japan).

4.2. Plant Material

The plant material was purchased from Mansehra, Khyber Pakhtunkhwa, Pakistan, and identified as Onosma bracteatum Wall by Dr. Zafar Ulah Zafar, Associate Professor, Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan. The voucher specimen (20170220) of the plant was deposited at the Institute of Materia Medica, Zhejiang University.

4.3. Extraction1 and Isolation1

Dried plant material(1.5 kg)was ground to powder and extracted with methanol (CH3OH) for 3 days along with continuous shaking at room temperature. The obtained crude extract (120 g)was partitioned between ethylacetate (EtOAc) and water(H,O).The active ethylacetate layer sample (30 g)was chromatographed on a silica gel open column and eluted with n-hexane/CH,Cl(100:0,80:20, 50:50,0:100) and CH,Cl/CH3OH (98:2,95:5,90:10 and 0:100).Six fractions were obtained. Fraction4 (8g)that was eluted with CH,Cl,/CH3OH (95:5, 90:10) was further processed by ODSopen column. CH3OH/H,O(40:60,50:50,55:45,60:40,70:30,90:10 and 100:0)was used as solvent.Of the seven fractions (Fr.1-Fr.7) obtained, Fr.2-Fr.4, were further separated as follows:

Fr.4 (500 mg) was separated by silica gel open column and eluted with CH2Cl2/CH3OH (100:0, 99:1,98:2,97:3,95:5,90:10 and0:100).Nine samples (Fr.4-1-Fr.4-9)were obtained.Sample Fr.4-3 was further purified by HPLC【C30-UG-5（φ10× 250 mm， Nomura Chemical）， mobile phase∶ acetonitrile (CH3CN)/H2O(40:60), flow rate:3 mL/min, and detector:210nm] and yielded compound (1) (65 mg, tR =21.2 min).

Fr.3(113 mg) was separated by using ODS open column and eluted with CH;OH/H2O(40:60, 45:55,50:50,55:45,60:40,80:20 and 100:0).Ten fractions(Fr.3-1-Fr.3-10)were obtained, and Fr.3-7 was further purified by HPLC【C30-UG-5（φ10×250 mm， Nomura Chemical）， mobile phase∶methanol (CH3OH)/HO(68:32), flow rate: 3 mL/min, and detector:210nm] and yielded compound (2) (20 mg, IR =20.3 min).

Fr.2(450 mg) was subjected to silica gel open column and eluted with CH2Cl2:CH3OH (100:0, 99:1,98:2,97:3,95:5,90:10,0:100).Nine fractions (Fr.2-1-Fr.2-9)were obtained,and Fr.2-4(20 mg)was subjected to ODS open column chromatography with CH3OH/H2O(30:70, 32:68,35:65,40:60 and 100∶O），and the fifth fraction （6.5 mg）was purified by HPLC 【C30-UG-5（φ10×250 mm， Nomura Chemical), mobile phase:acetonitrile(CH3CN)/H,O(35:65), flow rate: 3 mL/min, and detector:210 nm] to yield compound (3)(3.5 mg, t = 31.0). Fr.2-5(70 mg) was separated by ODS open column using CH3OH/H2O(25:75,27:73,30:70, 32:68,34:66,36:64,40:60 and 100:0) and the seventh fraction （11 mg）was purified by HPLC 【C30-UG-5（φ10×250 mm， Nomura Chemical）， mobile phase∶acetonitrile (CH,CN)/HO(32:68), flow rate:3 mL/min, and detector: 210 nm] to yield compound (4)(2.1 mg, tg=43.3).Fr.2-8(75 mg)was separated by ODS open column using CH3OH/H2O(20:80, 22:78.24:76.25:75.26:74,30:Z0.35:65.40:60 and 100:0) and the seventh fraction(10 mg) was purified by HPLC 【C30-UG-5 （φ10×250 mm， Nomura Chemical），mobile phase∶ acetonitrile（CH3CN）/H2O (30:70),flow rate:3 mL/min,and detector: 210 nml to vield compound (5)(4.3 mg, tn=22.3). Allomicrophyllone (1). Yellow powder; [α]6+0.02 (c 0.57, CHCl3);High-resolution ESI-TOF-MS m/z 389.1359， calculated for C2H22O5Na （M+ Na）+ 389.1359;'H NMR （500 MHz， CDCl3）∶bμ=6.83 （1H，d.I=10.5 Hz,H-5^),6.61(1H,d,J=8.6 Hz,H-6),6.56(1H,d, J=2.8Hz,H-3),6.51(1H, dd, J=2.8, 8.6 Hz,H-5),6.51(1H,d, J=10.5 Hz, H-6'),5.76(1H, d,J=15.9 Hz,H-8'),5.67 (1H, d, J=15.9 Hz, H-7),5.65(1H,d,J=7.1Hz,H-8),3.77(1H,d,J=7.1Hz,H-7),2.73(1H,d, J=19.2Hz,Hb-10),2.50 (1H,d,J=19.2 Hz,Ha-10),1.69 (3H,s, H-11),1.22(6H,s, H-10'& H-11');13CNMR(125 MHz,CDCl3):6c=195.8(C-1'),193.1(C-4'),150.0(C-4),145.0(C-1),143.3(C-8'),139.4(C-5'),138.6(C-6'),131.8(C-9), 127.7(C-2), 122.5(C-8),122.0(C-7'),117.6(C-6),114.9(C-5),114.4(C-3),80.7(C-3'),71.0(C-9),54.9 (C-2'),39.1(C-7), 36.2(C-10),29.8(C-10'),29.8(C-11'),22.6(C-11).The structure was identified through comparison of MS, 'H NMR and 13C NMR spectra as well as specific rotation with literatures [17,18]. Ehretiquinone (2). Red powder;[α]16+1.01(c 0.12, CH3OH); high-resolution ESI-TOF-MS m/z 371.1254， calculated for C2.H20OANa （M+ Na）+ 371.1252;lH NMR（500MHz， CDCl3）∶δμ=6.83 (1H,d,J=10.5Hz,H-5'),6.62(1H,d, J=8.6Hz,H-6),6.58(1H,d,J=2.2 Hz,H-3),6.53(1H,m,H-5), 6.52(1H,d, J=10.5 Hz,H-6'),6.25(1H,d,J=16.0Hz,H-8),5.65(1H,d,J=6.2Hz,H-8),5.50(1H,d, J=16.0Hz,H-7),5.04(1H,s,Hb-10),4.97(1H,s,Ha-10'),3.83(1H,d, J=6.2Hz,H-7),2.78(1H,d, I=19.1Hz,Hb-10),2.52(1H,d,J=19.1 Hz,Ha-10),1.73(3H,s,H-11'),1.70(3H,s,H-11);13CNMR (125MHz,CDCl):6c=195.4(C-1),193.2(C-4),150.0(C-4),145.1(C-1),140.8(C-9),139.2(C-5),138.8 (C-6'),137.5(C-8'),131.9(C-9),127.7(C-2),124.1(C-7'),122.7(C-8),119.3(C-10'),117.6(C-6),114.9 (C-5),114.4(C-3),80.7(C-3'),55.3(C-2'),39.4(C-7),36.3(C-10),22.7(C-11),18.3(C-11).The structure was identified through comparison of MS,'H NMR and 13C NMR spectra as well as specific rotation with literature [19].

Ehretiquinone B (3).Red powder; [α|6+0.99(c 0.31, CH3OH); high-resolution ESI-TOF-MS m/z 387.1208, calculated for C22H20OsNa (M+ Na)+ 387.1203; Data for 1H NMR and 13C NMR are described in Tables 1 and 2 respectively.

Ehretiquinone C(4). Red powder; [α] 16+0.37 (c 0.12, CH,OH); high-resolution ESI-TOF-MS m/z 387.1174, calculated for C2H20O;Na (M+ Na)+ 387.1174; Data for 1H NMR and 13C NMR are described in Tables 1 and 2 respectively.

Ehretiquinone D (5). Yellow powder; [αl5+0.51(c 0.41, CHCl3); high-resolution ESI-TOF-MS m/z 405.1300,calculated for C2,H,O,Na (M+Na)+405.1300;Data for HNMR and 13CNMR are described in Tables 1 and 2 respectively.

4.4. Lifespan AsSay/

The bioassay method used for the anti-aging study was previously described [4]. K6001 yeast strain was cultured on a YPGalactose or YPGlucose medium. YPGalactose medium was prepared with 3% galactose, 2% hipolypeptone and 1% yeast extract while YPGlucose medium, contained 2%glucose instead of galactose. cistanche แอ ม เว ย์ To prepare agar plates,2% agar was added to the medium. To evaluate samples through bioassay, the K6001 yeast strain was inoculated and incubated for 24 h in the yeast peptone galactose medium along with shaking. After24 h, the medium containing the yeast strain was centrifuged. The resulting yeast pellets were washed with phosphate buffer solution (PBS) three times and diluted. After dilution, the cells were calculated with a hemocytometer, and around 4000 cells were inoculated on agar plates containing samples with various concentrations. Then, the plates were placed in an incubator at 28°C for 2 days. The yeast cells in the agar plate were observed under a microscope after 48 h. A total of 40 microcolonies from each plate were selected randomly, and the number of daughter cells was counted and analyzed.

4.5. Statistical Analysis

The significant differences among groups were determined by one-way ANOVA with Dunnett's multiple comparison test. GraphPad Prism 5.0 software was used. Ap value of <0.05 was considered statistically significant.

5. Conclusions

Three new and two known benzoquinones were isolated and characterized from O. bracteatum under the guidance of the K6001 yeast replicative lifespan assay. The structure of the new benzoquinones (3-5) was elucidated by spectroscopic analysis, and the known ones (1-2) were identified by comparing the spectroscopic data with those in the literature. All the isolated benzoquinones were evaluated for anti-aging activity by yeast replicative lifespan assay and 1,2,4 and 5exhibited significant anti-aging activity. how much cistanche to take Ehretiquinone C(4) showed the most promising anti-aging activity and can thus be considered a potential lead compound. Its anti-aging mechanism should be further studied.

Supplementary Materials: The 1D and 2D NMR spectra are available online at http://www.mdpi.com/1420-3049/24/7/1428/s1.

Author Contributions: U.F. performed the chemical study and drafted the manuscript. YP performed the replicative lifespan assays. D.D.participated in the identification of compounds. J.Q.designed and supervised this study and revised the manuscript.

Funding: This work was financially supported by the National Key R&D Program of China [No.2017YFE0117200]and NSFC [Grant Nos.21661140001,21877098, and 21572204].

Acknowledgments: We are grateful to Michael Breitenbach of Salzburg University, Austria for the gift of the S. cerevisiae yeast K6001 strain. U.F. also acknowledges the support for doctoral study from the Higher Education Commission Pakistan.

Conflicts of Interest: The authors declare no conflict of interest.

This article is extracted from Molecules 2019, 24, 1428; doi:10.3390/molecules24071428 www.mdpi.com/journal/molecules