Novel Anti-Aging Benzoquinone Derivatives From Onosma Bracteatum Wall

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Abstract: The aim of this study was to investigate anti-aging molecules from Onosma bracteatum Wall, a traditional medicinal plant used in the Unani and Ayurvedic systems of medicine. During bioassay-guided isolation, two known benzoquinones,allomicrophyllone(1) and ehretiguinone (2) along with three novel benzoquinones designated as ehretiquinones B-D(3-5)were isolated from O. bracteatum. Their structures were characterized by spectroscopic analysis through 1D and 2D NMR, by MS spectroscopic analysis, and compared with those reported in the literature. sistanche The anti-aging potential of the isolated benzoquinones was evaluated through a yeast lifespan assay, and the results indicated that 1,2,4 and 5significantly extended the replicative lifespan of K6001 yeast, indicating that these benzoquinones obtained from O. bracteate have the ability to be employed as a potential therapeutic agent against age-related diseases.

Keywords: anti-aging; Onosma bracteatum; structure elucidation; benzoquinone; replicative lifespan; K6001

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1. Introduction

Aging is a natural process marked by a progressive deterioration in physiological functions and increases in mortality and is often accompanied by various human pathologies, such as cardiovascular diseases, diabetes, stroke, cancer, and neurodegenerative diseases (Parkinson's and Alzheimer's disease)[1]. Advances in public health practices, education, and medicine have not only improved life expectancy but have also increased the aged population. According to the World Health Organization (WHO), the population of people aged 60 years will double by 2050 [2]. Increasing life expectancy also increases the prevalence of age-related diseases. Thus, strategies for discovering anti-aging molecules are of great importance [3]. Natural products have been an important source of drug discovery since ancient times, and a large number of present-day drugs are derived from natural sources. In our previous studies, we reported many natural compounds with anti-aging properties by using K6001 yeast replicative life span bioassay [-6]. This assay is frequently used because yeast is inexpensive and has good reproducibility compared with other aging model organisms, such as vinegar flies, mice, and nematodes [7-10]. In 2004, Jarolim et al., described a novel bioassay system with the K6001 yeast strain to improve the lifespan assay 11]. Anti aging cistanche Onosma bracteatum Wall belongs to the family Boraginaceae. It is known as Gaozaban in the Unani system of medicine and as Sedge in the Middle East. O.bracteatum is commonly used as a demulcent, alterative, diuretic, immunity enhancer and spasmolytic and as a major constituent of various Ayurvedic formulations for the treatment of hypertension, leprosy, rheumatism, and asthma [12,13]. Pharmacological studies on O.bracteatum reported that it has antibacterial, analgesic, antioxidant, and wound healing activities [14-16]. Additionally, it has acetyl-cholinesterase and NADH oxidase inhibitory activities. It also contains carbohydrates, fatty acids, flavonoids, tannins, glycosides, and phenolic constituents[12,15]. The objective of this study was to obtain anti-aging compounds from O. bracteatum by using the K6001 yeast replicative life span bioassay. what is cistanche Arti-aging compounds (1, 2, 4, and 5) and inactive compound 3 were purified from O.bracteatum by column chromatography and then characterized by spectroscopic analysis and compared with the data described in previously published reports (Figure 1)?

2. Results

2.1. Isolation

The dried plant material of O.bracteatum was ground to uniform powder and extracted with methanol to obtain the crude extract. The crude extract was then partitioned between ethyl acetate and water. After bioassay evaluation of ethyl acetate and water layer samples, the active ethyl acetate layer sample was subjected to a series of silica gel and ODS open column chromatography under the guidance of a bioassay system and was finally purified by HPLC to yield two knowns (1 and 2) and three novels (3-5)benzoquinone derivatives. The structures of known benzoquinones were identified as allomicrophyllone (1,0.0043% of dry weight)[17,18] and ehretiquinone (2,0.0013%)[19], while structures of novel benzoquinones were elucidated and named as ehretiquinone B(3, 0.00023%), ehretiquinone C(4,0.00014%), and ehretiquinone D(5,0.00028%)(Figure 1)by spectroscopic analysis, including 1Dand 2DNMR, HR-ESI-TOF-MS, and comparison of spectroscopic data with those reported in the literature (see Supplementary Materials).

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2.2.Structure Elucidation1

Ehretiquinone B (3) was obtained as a red powder and its molecular formula(C2.H20Os)was determined by HR-ESI-TOF-MS. The'H NMR data (Table 1)showed the presence of three aromatic protons at oH6.53,6.57 and 6.61, corresponding to 1,2,4-trisubstituted benzene; two cis olefinic protons at δy 6.85 and 6.52; two trans olefinic protons at bH 6.18 and δH 5.68; three olefinic protons at H 5.13,5.31 and0H 5.64; two methylene protons at OH 2.52 and 0H 2.78; one oxygenated methylene group at bH4.20; one methine proton at bμ 3.83 and one methyl group at δH 1.69.The The13C NMR data (Table2)showed the presence of 22 carbon signals. These signals were attributed to two ketone groups (6c 195.4,193.1),a 1,2,4-trisubstituted benzene (6c114.3,114.9,117.6,127.5,144.9 and 150.0),eight olefinic carbons(6c118.2,122.5,124.6,131.9,134.0,138.7, 139.4 and 143.8),one oxygenated quaternary carbon (c 80.6), one oxymethylene (6c62.9), one quaternary carbon (6c 55.4), one methine (6c 39.3), one methylene (6c 36.2) and one methyl group (6c 22.7). cistanche benefícios The 'H-1H COSY spectra indicated the correlation signals between H-5 and H-6; H-7 and H-8; H-5'and H-6'; H-7'and H-8'. Based on these signals, a structural fragment of 3 was obtained, as shown in Figure 2. The HMBC spectrum indicated the following major'H-13C correlation: H-3 to C-4; H-5 to C-1 and C-3;H-6 to C-1 and C-4;H-7 to C-1, C-3, C-9 and C-3';H-8 to C-11;H-11 to C-8, C-9 and C-10;H-5'to C-3';H-6'to C-2'and C-4';H-7'to C-1', C-2', C-3'and C-9';H-8'to C-9'and H-11'to C-9'and C-10', On the basis of these signals, the structure fragments were connected to obtain the planar structure of 3 as described in Figure 2.

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The relative configuration of 3 was determined by NOESY correlations and coupling constants. The coupling constant (s 6=10.5 Hz) indicated the cis configuration of protons at 5' and 6'position whereas the large coupling constant (Jzg, = 16.5 Hz) and NOESY correlation between H-11'/H-7'(Figure 3)suggested the trans configuration of protons at 7' and 8' in 3. In the NOESY spectrum, the correlation between H-11/H-8 confirmed the trans configuration of the double bond, and H-10/H-7'(Figure 3) indicated the same orientation of these protons and found it to be identical to ehretiquinone (2)[19]. In addition to the same NOESY correlations, 'H NMR and 13C NMR of 3 and ehretiquinone (2), at 2',3' and 7 positions, and close to these positions, were identical which also supported the identical relative configuration of 3 and ehretiquinone (2). While comparing 3 and ehretiquinone (2), the spectroscopic data suggested that the methyl group at C-11' in ehretiquinone (2) was replaced by CH2OH in 3. The remaining structure of 3 was found to be identical to ehretiquinone (2), as shown in Figure 1. Thus, the structure of 3 was determined and named ehretiquinone B(Figure 1).

Ehretiquinone C(4)was obtained as a red powder. The molecular formula of 4(C22H20Os) was determined by HR-ESI-TOF-MS. The'H NMR and 13C NMR data of 4 (Tables 1 and 2) showed that4 has many similarities to ehretiquinone (2). The difference between the compounds was the replacement of CH3 (6H1.70,bc22.7)in2by CH2OH(6H4.05,6c 65.8) in 4 at the11position, as shown in Figure 1. The difference was confirmed by the HMBCcorrelation between H-8 to C-11 and H-11 to C-8, C-9, and C-10, as shown in Figure 2. Meanwhile, the other 'H-13C correlations and the COSY correlation were identical to 3. The NOESY correlation spectrum indicated that the relative configuration of 4 was similar to that of ehretiquinone B (3)(Figure 3). Thus, the structure of 4 was determined and named ehretiquinone C (Figure 1).

Ehretiquinone D(5)was obtained as a yellow powder. The molecular formula of 5(C, H2O)was determined by HR-ESI-TOF-MS.The 'H NMR data and 13C NMR data (Tables 1 and 2) were nearly identical to those of allomicrophyllone (1)[17,18]. The comparison of 5 and allomicrophyllone （1） indicated that CH3（OH 1.16，δc 29.8） at the 10' position in allomicrophyllone（1）was replaced by CH, OH(6H 3.41,6c69.7)in 5 which was further confirmed by HMBC correlation among the positions H-8'to C-10'; H-11'to C-10'and H-10' to C-8', as shown in Figure 2. The remaining-13CHMBCand LH-1H COSY correlations were found to be similar to those of ehretiquinone B (3) and ehretiquinone C (4)(Figure 2). The relative configuration of 5, measured by the NOESY spectrum, was found to be similar to ehretiquinone B (3). However, position 9'of the side chain, attached at 2', is the chiral center, and methods for such stereochemistry are needed to be established, and the stereochemistry of position 9'will remains to be clarified. Thus, the structure of 5 was determined and named ehretiquinone D (Figure 1). Allomicrophyllone (1) was obtained as a yellow powder. The molecular formula of1(C2.H2O5)was determined by HR-ESI-TOF-MS and identified by comparing the MS,' H NMR, and 13C NMR data with the literature [17,18]. Ehretiquinone (2) was obtained as a red powder. The molecular formula of 2(C2H2Oa) was determined by HR-ESI-TOF-MS and identified based on the comparison of MS, 1H NMR, and 13C NMR data with those in the literature [19].

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