Inositols and methylinositols in sea buckthorn (Hippophaë rhamnoides) berries

Journal of Chromatography B, 877 (2009) 1426–1432

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Inositols and methylinositols in sea buckthorn (Hippophaë rhamnoides) berries Heikki Kallio a,c,∗ , Marika Lassila a , Eila Järvenpää a , Gudmundur G. Haraldsson b , Sigridur Jonsdottir b , Baoru Yang a,d a

Department of Biochemistry and Food Chemistry, University of Turku, FI-20014 Turku, Finland Science Institute, University of Iceland, Dunhaga 3, 107 Reykjavik, Iceland c Functional Foods Forum, University of Turku, FI-20014 Turku, Finland d Department of Food Science and Engineering, Jinan University, 510632 Guangzhou, China b

a r t i c l e

i n f o

Article history: Received 8 December 2008 Accepted 8 March 2009 Available online 25 March 2009 Keywords: GC–MS HPLC 1 H and 13 C NMR Methylinositol Optical activity measurement Sea buckthorn berry

a b s t r a c t Sea buckthorn (Hippophaë rhamnoides L.) berries, especially of ssp. sinensis, contain significant quantities of an unknown, water-soluble compound, evidently a cyclitol derivative. The compound was isolated by HPLC and analyzed by GC–MS [trimethylsilyl (TMS) derivative, selected ion monitoring (SIM) and total ion chromatogram (TIC) analyses], by 1 H and 13 C NMR and by optical activity measurements. The results together with analyses of reference compound verified the unambiguous structure (−)-2-O-methyl-lchiro-inositol (l-quebrachitol). In addition, chiro-inositol and myo-inositol existing in trace amounts were identified based on reference compounds, chromatographic data and mass spectra of the TMS derivatives. Methyl-myo-inositol was tentatively identified based on chromatography and mass spectrometry. Inositols and methyl inositols are bioactive compounds essential for regulating physiological processes of plants and humans. To our knowledge, this is the first report on the presence of chiro-inositol and myo-inositol in sea buckthorn and l-quebrachitol in edible berries. The identification of the inositols and l-quebrachitol in sea buckthorn may bring new insights into the sensory properties and also mechanisms behind the health effects of the berry. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Sea buckthorn (Hippophaë rhamnoides L.) berries are known for their low sweetness due to the low sugar content and due to the abundance of fruit acids [1–7]. Also bitterness and astringency are typical characteristics of the berries [1,4] even though compounds responsible for these properties are not well defined. The only simple sugars shown to exist in sea buckthorn are glucose and fructose [5,7–10]. In addition, ethyl ␤-d-glucopyranoside has been identified based on GC, HPLC, MS and NMR analyses of the berry and reference compound synthesized [5]. The abundance of ethyl glucoside is typical to the European subspecies H. rhamnoides ssp. rhamnoides, but is present at trace level only in the Chinese berries of H. rhamnoides ssp. sinensis [7]. No sugar alcohols have been with certainty identified in the berries so far, even though some claims and suggestions concerning mannitol, sorbitol and xylitol have been published earlier [11]. The sugar/acid-ratio in the berries of sea buckthorn is typically much lower than in most of the common edibles fruits. The ratio varies widely between different sea buckthorn subspecies, varieties

∗ Corresponding author. Tel.: +358 40 5033024; fax: +358 2 3336870. E-mail address: heikki.kallio@utu.fi (H. Kallio). 1570-0232/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2009.03.027

and cultivars. The lowest values reported so far, 0.1–0.9, are from berries of ssp. rhamnoides, whereas typical values in some Russian cultivars are 1.5–3.5 [5,7]. The major acid component is the sour and bitter-tasting malic acid [4–6]. Thus, the sourness is possible to be reduced by malolactic fermentation ending up with improved sensory properties of the final product [12]. The two other fruit acids are quinic and citric acids, the latter existing typically in trace amounts only. In addition, high content of vitamin C, especially in the wild Chinese berries of ssp. sinensis, even up to 25 g/L juice [6,13–15] significantly increases the acidic taste. In addition to the previously identified sugars and sugar derivative we have found a major unknown compound in the purified sugar fraction of sea buckthorn juice, often abundant in the ssp. sinensis berries. This compound comprised even up to 10% of the sugar fraction in some berry samples. A varying number of other unknown peaks have also been found in trace amounts in the GC chromatograms of the sugar fraction [5,16]. Preliminary mass spectrometric investigations have indicated the existence of inositol derivatives. Sea buckthorn is well known as a berry with a wide range of physiological effects covering antioxidative, anti-mutagenic and chemoprotective effects and beneficial effects on skin, mucosa, cardiovascular and immune system [17–20]. So far most of the compositional studies have concentrated on the fatty acids, phenolic

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compounds, vitamins, sterols and minerals [15,17,21–26]. The mechanisms behind the widely shown health effects are still largely unknown. The biological and physiological activities of inositols and inositol derivatives have been extensively documented. As essential components of the messenger molecules responsible for signal transduction of cells, inositols participate in the regulation of most of the physiology aspects of humans [27–29]. Inositols and methylinositols are bioactive components regulating sugar metabolism and protecting cells from oxidative, cytotoxic and mutagenic damages [30–35]. In plants, inositols and their derivatives have been reported to accumulate under abiotic stress conditions, such as water deficiency or cold [36–39]. Therefore, these compounds may act as osmotic regulators and cryoprotectants. The presence of inositols and inositol derivatives may present new insights into the bioactive components and mechanisms of the health effects of sea buckthorn. The aim of this study was to identify the unknown compounds of the sugar fraction of sea buckthorn berries by chromatographic, mass spectrometric and nuclear magnetic resonance methods as well as by optical activity measurements. A special attention was paid on the possible cyclitols and their alkyl derivatives. 2. Experimental

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least overnight. For isolation of acids the columns were disconnected and acids eluted from the SAX columns by 1 mL of 15N formic acid. 2.4. Purification of the major unknown sugar compound by HPLC Ca. 0.5 mg of the dried sugar fraction was dissolved in 0.5 mL of the HPLC eluent (acetonitrile:water:methanol, 85:10:5), mixed and sonicated. The solution was filtered through a syringe filter (0.45 ␮m) and evaporated to suitable concentration for analytical or semi-preparative purposes prior to HPLC analysis (Shimadzu LC9A, Shimadzu Corp., Kyoto, Japan). Separation was achieved at the flow rate 1.2 mL/min using a Luna 5 ␮ NH2 column (250 × 4.60 mm) (Phenomenex, Torrance, CA). The detector was an evaporative light scattering detector, Sedex 55 (Sedere, Alfortville, France) operated at 45 ◦ C and 2.6 bar. To isolate the unknown compound for further analyses, the HPLC column was disconnected from the detector, and mobile phase fraction including the unknown, was collected into a vial. The chromatographic separation was repeated and effluents combined until there was enough sample for identification of the unknown compound by NMR and other analytical methods. The same HPLC method was used, when determining the purity of the isolated fraction.

2.1. Samples Frozen samples of wild sea buckthorn berries H. rhamnoides L. ssp. sinensis, from Xixian (Shanxi, P.R. China) were investigated. The berries were picked when optimally ripe, frozen immediately at −20 ◦ C, transported to Finland in dry ice and stored at −20 ◦ C until analyzed. 2.2. Reference compounds d-Glucose was from Fluka (Buchs, Switzerland) and dfructose from Sigma–Aldrich (Steinheim, Germany). Ethyl ␤-dglucopyranose was synthesized according to Goncalves et al. [40] and purified by HPLC [5]. Reference compound myo-inositol was purchased from Fluka, scyllo-inositol, d-(+)-chiro-inositol, l-(−)-chiro-inositol, muco-inositol, allo-inositol and d-pinitol (d3-O-methyl-chiro-inositol) from Sigma–Aldrich and l-quebrachitol (1L-2-O-methyl-chiro-inositol) from Alexis Corporation (Läufelfingen, Switzerland). d-(−)-Sorbitol from Fluka and L-(−)-tartaric acid from Merck (Darmstadt, Germany) were used as internal standards. 2.3. Isolation of the sugar and acid fractions Thirty grams of frozen berries were thawed in a microwave oven, crushed manually and the juice was filtered through cheese cloth in a funnel. One milliliter of the filtrate and 6 mL of 0.1N NaOH were diluted to total volume 20 mL with purified water (Millipore, Bedford, MA). The filtrate was diluted 1:20 in water and 6 mL of 0.1N NaOH were added in the total volume. The sugar fraction was isolated by a dual solid phase extraction on Isolute CH and SAX columns (International Sorbent Technology, Hengoed, UK). The upper non-polar column (cyclohexyl Isolute CH, 100 mg/1 mL) was for absorption of carotenoids and other lipophilic compounds. The lower anion exchange (Isolute SAX, quaternary amine, 200 mg/3 mL) column was for trapping of acids. Both columns were first activated separately by methanol and water and the ion exchange of the SAX columns was then performed with 1N formic acid. The columns were connected together, and 1 mL of the prepared juice sample was added. The sugar fraction was eluted by 2 mL purified water, evaporated to dryness under nitrogen flow at 40–50 ◦ C and dried in a desiccator over silica gel at

2.5. GC–FID analysis Gas chromatography was applied to the analysis of trimethylsilyl (TMS) derivatives of the combined sugar-acid fraction, the purified sugar fraction, the unknown compound isolated by HPLC, and the reference compounds. Derivatives of the dried samples were prepared as described earlier [4,5,7] using Tri-Sil reagent (HMDS/TMCS in pyridine 2:1:10, Pierce, Rockford, IL). Identity of the retention times of the TMS derivative of the HPLC-purified unknown compound with that of the unknown in the TMS-sugar fraction of sea buckthorn was verified by co-injections. The possible match with the reference compounds was studied analogously. The analysis was carried out with Varian 3300 GC equipped with a flame ionization detector (Varian, Limerick, Ireland) using a nonpolar Simplicity-1 fused silica capillary column, 30 m × 0.25 mm ID × 0.25 ␮m df (Supelco, Bellafonte, PA). One microliter sample was injected in the split mode (1:20). The injector temperature was 210 ◦ C, the detector temperature 290 ◦ C, and the temperature of the column was programmed from 90 ◦ C (hold 2 min) to 275 ◦ C at the rate of 4 ◦ C/min and held at 275 ◦ C for 10 min. The average flow of the carrier gas He was 1.0 mL/min. 2.6. GC–MS analysis The same samples of sugar TMS derivatives, as analyzed by GC–FID, were also analyzed with Shimadzu 17 A gas chromatograph with QP 5000 MSD (Shimadzu, Kyoto, Japan), controlled by Class-5000 software. A capillary column DB-1MS (30 m × 0.25 mm ID × 0.25 ␮m df , J&W Scientific/Agilent, Folsom, CA) was used. The column temperature program was the same as in the GC–FID analysis and the mass spectra were acquired at mass range m/z 40–400 with 70 eV energy. The chromatograms were recorded by either total ion chromatogram (TIC) or selected ion monitoring (SIM) modes. Wiley 229 was used as a spectral reference library. The same conditions were applied to analyze a combined sugaracid fraction. Both fractions were diluted separately with water to a volume of 3 mL, the fractions were combined, divided in three identical aliquots in dark bottles, evaporated under nitrogen and dried in a desiccator. TMS derivatives were prepared just before the GC–MS analysis.

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2.7. NMR analyses 1 H and 13 C nuclear magnetic resonance spectra were recorded on a Bruker Avance 400 (Bruker BioSpin GmbH, Rheinstetten, Germany) spectrometer in deuteriated water as a solvent at 400.12 MHz and 100.61 MHz, respectively. Chemical shifts (ı) are quoted in parts per million (ppm) and the coupling constants (J) in Hertz (Hz). The following abbreviations are used to describe the multiplicity: s, singlet; d, doublet; dd, doublet of doublets; m, multiplet. Two dimensional spectra were recorded as a HHCOSY90 standard 2D experiment for HH homonuclear correlation and a HCCORR standard 2D experiment for CH correlation. Simulations were performed using the Bruker TopSpin NMR simulator. The assignment of the spectra is based on the following numeral labeling system for the predominant chair conformation of the unknown inositol compound:

Fig. 1. GC–MS TIM chromatogram of the TMS derivatives of the combined sugar and acid fractions of Chinese sea buckthorn sample isolated by SPE. Tartaric acid (ISTA ) and sorbitol (ISSOL ) were added as internal standard. (1) Malic acid, (2) citric acid, (3) ␣-d-fructofuranose, (4) ␤-d-fructofuranose, (5) ␤-d-fructopyranose, (6) quinic acid, 7U = unknown compound (7), (8) ␣-d-glucopyranose, (9) vitamin C, (10) ␤-dglucopyranose, 11U = unknown compound (11), 12U = unknown compound (12).

1 H NMR (D O): ı 4.10 (dd, J 2 1,2

= 3.4 Hz, J1,6 = 3.8 Hz, 1H, H1 ), 3.89 (dd, J6,5 = 3.4 Hz, J6,1 = 3.8 Hz, 1H, H6 ), 3.57 (dd, J5,6 = 3.4 Hz, J5,4 = 9.7 Hz, 1H, H5 ), 3.46 (m, 1H, H3 ), 3.43 (m, 1H, H4 ), 3.29 3.29 (s, 3H, O-CH3 ) and 3.24 ppm (dd, J2,1 = 3.4 Hz, J2,3 = 9.7 Hz, 1H, H2 ). 13 C NMR (D2 O): ı 82.7 (C-2), 75.4 (C-4), 74.4 (C-3), 73.9 (C-6), 72.9 (C-5), 69.7 (C-1) and 59.4 ppm (CH3 ). 2.8. Optical activity measurements

The specific optical activity measurements were performed on an AutopolR V Automatic Polarimeter from Rudolph Research Analytical (Hackettstown, New Jersey, USA) using a 40T-2.5-100-0.7 Temp TrolTM polarimetric cell with 2.5 mm inside diameter, 100 mm optical path length and 0.7 mL volume. 3. Results and discussion

The unknown compounds 7U , 11U and 12U are according to the fractionation pattern and mass spectral information sugar alcohols or their derivatives. Mass spectra of peaks 7U and 11U analyzed according to Fig. 2 are shown in Fig. 3A and B, respectively. Profile of the TMS chromatogram (Fig. 2) represents a typical sea buckthorn sugar fraction. Glucose and fructose are always dominating sugars. Sea buckthorn juice does not contain significant quantity of sucrose. In the Chinese ssp. sinensis berries, the 7U was sufficiently abundant, as Figs. 1 and 2 indicate. In the European rhamnoides subspecies, the compound was typically only a trace component [7]. In the ssp. rhamnoides berries, ethyl ␤-dglucopyranoside is known to be a major sugar metabolite, but the berries analyzed in this study did not contain the ethyl glucoside (Figs. 1 and 2); however, traces of this compound have been reported in some berries originating from China [7].

3.1. GC–MS analysis of the combined sugar-acid fraction Analysis of sugars and acids of the Chinese sea buckthorn sample was commenced with GC–MS of the two SPE fractions collected. A TIM chromatogram of the combined sugar-acid fraction is shown in Fig. 1. Identification of the major compounds numbered was based on chromatographic retention times, reference compounds, mass spectra and earlier analyses of sugars, fruit acids and vitamin C. The three fructose peaks represent ␣- and ␤-furanose, and ␤-pyranose and the two glucose peaks are the ␣- and ␤-anomers of pyranose [4,5,7,41]. Tartaric acid (ISTA ) and sorbitol (ISSOL ) were selected as internal standards because sea buckthorn is known not to contain these metabolites. Code-numbers of the three unknown compounds of interest are marked with the letter U as subscript. Mass spectra of high or at least of sufficient quality were acquired in several repeated runs. 3.2. GC–MS analysis of the sugar fraction Fig. 2 shows a GC–MS TIM chromatogram of the purified sugar fraction of sea buckthorn juice analyzed as TMS derivatives. Mass spectra and retention times of the compounds matched with the corresponding compounds of the chromatogram in Fig. 1.

Fig. 2. GC–MS TIM chromatogram of the purified sugar fraction of wild Chinese sea buckthorn berries analyzed as TMS derivatives. Numbering of the peaks is as in Fig. 1. Traces of quinic acid and vitamin C are also visible. The arrow shows the position of ethyl ␤-d-glucopyranoside in case it would have existed in the sample.

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Fig. 4. 4A: HPLC–ELSD chromatogram of the sugar fraction of sea buckthorn juice isolated by SPE. 4B: A re-run HPLC chromatogram of the purified unknown fraction. Retention times displayed in minutes.

and GC–FID, 1 mg of the fraction was isolated for NMR analysis and optical activity measurement.

Fig. 3. Mass spectra of peaks 7U and 11U displayed in Fig. 2 are shown in Fig. 3A and B, respectively.

3.3. HPLC analysis of sugar fraction and purification of unknown compound An HPLC–ELSD chromatogram of the sugar fraction of sea buckthorn berries isolated by SPE, corresponding to the sample of Fig. 2, is shown in Fig. 4A. According to our earlier investigations [5] and verification by reference compounds, the first major peak (tR = 9.59 min) represents fructose and the third peak (tR = 13.65 min) glucose. The unknown compound, tR = 10.17 min (Fig. 4A), was isolated by HPLC. Fructose and the unknown compound eluted close to each other, with ca. 95% resolution only, making the purification a challenging task. A re-run of the HPLC chromatogram of the purified fraction (Fig. 4B) displayed >95% purity.

3.5. GC–MS analysis of the unknown fraction and reference compounds Compound represented by peak 7U (Figs. 1 and 2), was the target of the HPLC purification. The mass spectra of peak 7U (Fig. 3A) was identical with the corresponding mass spectrum of the HPLCpurified unknown analyzed separately as TMS derivative. None of the library spectra available matched with the mass spectra presented in Fig. 3A and B. The two spectra were close to each other indicating two isomeric compounds. TMS derivatives of l-quebrachitol and d-pinitol were analyzed. TMS derivative of l-quebrachitol had the same retention time as peak 7U in Fig. 2, as confirmed by co-injection. Also the mass spectra were practically identical. This verified the structure of either (+)-2-O- or (−)-2-O-methyl-chiro-inositol to the peak 7U , the mass spectrum of which is shown in Fig. 3A. According to the reten-

3.4. GC–FID analysis of the unknown HPLC fraction GC–FID analysis of the TMS derivative of the unknown fraction was confirmed to represent one compound with purity of over 98%, when calculated using peak areas without response factors (Fig. 5). Very small amounts of fructose and glucose were detected in the chromatogram. After purity checking by both HPLC–ELSD

Fig. 5. GC–FID chromatogram of the TMS derivative of the HPLC-purified unknown fraction.

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tion time, peak 11U (Figs. 2 and 3B) was not d-pinitol but it could, however, be identified as a methylinositol. TMS derivatives of six inositol isomers available as reference compounds were analyzed by GC–MS individually and as a mixture. Isomers neo-inositol, cis-inositol and epi-inositol were not available. Co-injection of the derivatized reference compounds with the sea buckthorn TMS sugar fraction was carried out to determine the retention time matches. The retention times of the TMS-chiroinositols and TMS-myo-inositol matched with two trace level peaks which existed in chromatograms of some of the berry samples (see Fig. 1). TMS-chiro-inositol and TMS-myo-inositol had the same retention times as peaks tR 35.36 min and tR 38.68 min (12U ), respectively. Mass spectra of these two trace peaks are shown in Fig. 6A and B. GC–MS analysis of TMS-chiro-inositol and TMSmyo-inositol confirmed the identity of compounds 6A and 6B, respectively. Naturally, the +/− isomerism of chiro-inositol could not be resolved. Retention time difference between methyl-chiro-inositol and chiro-inositol was 2.63 min and between compound 11U and myoinositol 2.49 min (Fig. 1). This retention analogy supports the identification of 11U to be methyl-myo-inositol.

Fig. 7. SIM chromatograms of m/z 247 (A) and m/z 260 (B). Relatively high abundance of the SIM ions compared to the TIC ions (Fig. 2.) indicates the existence of methylinositols in peaks (7U and 11U ).

The major ions in the spectra of all the inositols acquired appear also in the spectra of the methylinositols. However, the difference between inositols and methylinositols is due to the existence of one –O-CH3 group in methylinositols, which replaces one –O-Si(CH3 )3 group in its TMS derivative. Signals at m/z values 89, 133, 207, 247 and 260 were characteristic to methylinositols, showing always higher abundances than in the spectra of inositols, glucose, fructose or sorbitol. The ion m/z 247 was most clearly characteristic to methylinositols. After identification of methyl-chiro-inositol, compound 7U , and tentative identification of methyl-myo-inositol, compound 11U , a search of the possible existence of other methylinositols was carried out by GC–MS with SIM technique. GC–MS of the purified sugar fraction was run with ions m/z 133, 247 and 260. The SIM chromatograms of m/z 247 and m/z 260 are shown in Fig. 7A and B, respectively. The two compounds 7U and 11U are clearly visible and relatively more abundant in both chromatograms than other peaks in the TIM chromatogram shown in Fig. 2. No additional methylinositols were observed in this sea buckthorn sample.

Fig. 6. Mass spectra of a compound in Fig. 1 with tR 35.36 min identified as TMS-chiro-inositol (A) and the compound 12U (tR 38.68 min) identified as TMS myoinositol (B). Identifications are based on chromatography and mass spectrometry of reference compounds.

3.6. Verification of structure of the methylinositol 7U by NMR and optical activity measurements Inositol has six stereogenic centers and nine stereoisomers [42]. Seven are meso forms and two form a pair of enantiomers, i.e. (+)

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Fig. 8. The two chair conformations of 2-O-methyl-chiro-inositol.

and (−)-chiro-inositols. Thus, there exist eight diastereomers, seven of them being achiral and only one chiral. All of these compounds are known, either as synthetic or as natural products [43]. The structure of the unknown methylinositol derivative was unambiguously determined as 2-O-methyl-chiro-inositol by highresolution 1 H and 13 C NMR spectroscopy. The full assignment of the 1 H and 13 C NMR spectra is based on two-dimensional H,H- and H,CCOSY, simulations and a direct comparison with 1 H and 13 C NMR data of 2-O-methyl-chiro-inositol from a recent report [43]. The assignment was based on the assumption of a predominant chair conformation with the methoxyl group and three hydroxyl groups located in equatorial positions as illustrated in Fig. 8. That assumption was strongly supported by line-broadening when running the 1 H NMR spectroscopy at higher temperature. The spectra were identical to those obtained for a commercially available reference sample of pure l-quebrachitol. The optical activity measurements revealed that the absolute configuration of the unknown methylinositol derivative had to be of the l-configuration and thus (−)-2-O-methyl-l-chiro-inositol. This is based on the obtained (−)-sign of its optical rotation value. The unknown inositol derivative had [␣]D 20 = −112 (c = 0,05 in water), whereas the reference 2-O-methyl-l-chiro-inositol sample had [␣]D 20 = −90 (c = 0,08 in water) as compared to [␣]D 20 = −79 (c = 1 in water) according to its supplier. There is some inconsistency of these values that is believed to relate to low concentrations, but the (−)-sign of the rotation should suffice to prove the lconfiguration unequivocally. Inositols exist commonly in both the animal and plant kingdom. Of the nine isomeric structures of inositols, myo-inositol is the most abundant. Also scyllo-, muco-, neo-, (+)-chiro-, and (−)-chiroinositols have been found in plants [44]. Myo-inositol is the most common cyclitol in fruits with high content found in citrus fruits, kiwifruit, and cantaloupe [45–51]. In addition to myo-inositol, citrus fruits contain scyllo- and chiro-inositol [51]. Although myo-inositol and chiro-inositol have been detected in some berries such as bilberry, strawberry, and raspberry [45,51,52], their presence in sea buckthorn has not been reported. In the present study, we found small amounts of myo-inositol and chiroinositol in sea buckthorn berries. Inositols are bioactive compounds essential for regulating various physiological processes [27–29]. In addition, these compounds may have an effect on the sensory properties of sea buckthorn. 4. Conclusions Methylinositols are known to be present in some plant species. The presence of methylinositols and other sugar alcohols has been shown to be related to an insufficient water supply to the plants [38,53]. Sea buckthorn is a hardy plant which grows well under dry conditions. The relatively high content of l-quebrachitol in Chinese sea buckthorn berries may be a mechanism developed by the plant to fight against the long-lasting drought in the growth area. Inositols are essential component of mediator molecules such as inositol-1,4,5-triphosphate responsible for signal transduction of cells [27–29]. Furthermore, inositols and methylinositols have

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