Isolation, characterization and antihyperlipidemic activity of secoisolariciresinol diglucoside in poloxamer-407-induced experimental hyperlipidemia

Abstract

Context: Linum usitatissimum L. (Linaceae), commonly known as flaxseed, is a good source of dietary fiber and lignans. Earlier we reported cardioprotective, antihyperlipidemic, and in vitro antioxidant activity of flax lignan concentrate (FLC) obtained from flaxseed.

Objectives: To isolate secoisolariciresinol diglucoside (SDG) from FLC and to evaluate the antihyperlipidemic activity of SDG in poloxamer-407 (P-407)-induced hyperlipidaemic mice.

Material and methods: FLC was subjected to column chromatography and further subjected to preparative HPTLC to isolate SDG. The chemical structure of the isolated compound was elucidated by UV, IR, ¹H NMR, ¹³C NMR, DEPT, COSY, HSQC, HMBC, ROESY, MS, and specific optical rotation was recorded. Further, we have investigated the antihyperlipidaemic effect of SDG (20 mg/kg) in P-407-induced hyperlipidaemic rats. Hyperlipidaemia was induced by intraperitoneal administration of P-407 (30% w/v). Serum lipid parameters such as total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C) levels were measured.

Results and discussion: The structure and stereochemistry of the isolated compound were confirmed on the basis of 1D and 2D spectral data and characterized as SDG. Finally, isolated pure SDG was screened using a P-407-induced mice model for its antihyperlipidemic action using serum lipid parameters. The isolated SDG (20 mg/kg) significantly reduced serum cholesterol, triglyceride (p < 0.001), very low-density lipoprotein (p < 0.05), and non-significantly increased HDL-C.

Conclusion: Finally, it was concluded unequivocally that SDG showed antihyperlipidaemic effects in P-407-induced hyperlipidaemic mice. Isolated pure SDG confirms that SDG is beneficial in the prevention of experimental hyperlipidemia in laboratory animals.

• Column chromatography
• flaxseed
• Linum usitatissimum

Introduction

Flaxseed, besides α-linolenic acid and mucilage, is the rich source of various types of phenolic compounds. Various bioactive phenols such as lignan, phenolic acids, and flavonoids have been reported in flaxseed (Kasote, 2013). Bakke and Klosterman (1956) reported extraction of secoisolariciresinol diglucoside (SDG) from defatted flaxseed (meal/cake) using equal parts of 95% ethanol and 1,4-dioxane. Flaxseed contains a small quantity of other lignans, namely, matairesinol, lariciresinol, hinokinin, arctigenin, divanillyl tetrahydrofuran nordihydroguaiaretic acid, isolariciresinol, and pinoresinol (Muir & Westcott, 2000). The level of SDG in flaxseed varies between 0.6 and 1.8 g/100 g or 1–4% by weight, the variability in components depends on the cultivar, the growing location, and year. It has been suggested that the lignans within flaxseed are the beneficial components within flax (Duan et al., 2006; Hosseinian et al., 2006). SDG is the major lignan found in flaxseed and plays an important role in diabetes (Prasad et al., 2000), cholesterol lowering (Zanwar et al., 2012), cardioprotection (Zanwar et al., 2011, 2013), hypercholesterolemic menopause (Lemay et al., 2002), hypertriglyceridemia, and reduction of atherogenic risks (Prasad, 1997; Prasad et al., 1998), etc.

Pure SDG and flax lignan concentrate (FLC) have a very high therapeutic potential. Several research groups have reported the isolation of SDG from flaxseed; however, there is a large variation for SDG present in the flaxseed. Eliasson et al. (2003) reported the content of (+)-SDG (11.9–25.9 mg/g), (−)-SDG (2.2–5.0 mg/g). Johnsson et al. (2000) obtained a range of 6.1–13.3 mg/g dry matter in whole flaxseeds grown in Sweden and Denmark. Westcott and Muir (1996) reported a range of 13.6–20 mg/g defatted flaxseeds in cultivars grown in Canada. Variation in flaxseed lignan concentrations largely depends on the variety, location, and crop year (Westcott & Muir, 1996). Based on a literature survey, it has been observed that dextro, i.e. (+) SDG content, was higher than that of leavo, i.e. (−) SDG, and also a large amount of variation in content is also observed. Also several research workers have isolated SDG from flaxseed, but optical rotation has not been reported. Also there is paucity of reports of appropriate data for complete chemical characterization of SDG using 1D and 2D spectral data. Previously, we have reported antioxidant, cardioprotective, antihyerlipidemic action of FLC (Zanwar et al., 2010, 2011, 2012, 2013). Hence, it was essential to isolate and carry out complete chemical characterization of SDG and conformation of retention of lipid lowering action of isolated SDG from FLC.

Hence, in the present investigation, the objective was isolation and chemical characterization of SDG followed by investigation of antihyperlipidemic activity of isolated SDG in poloxamer-407 (P-407)-induced hyperlipidemic mice.

Materials and methods

Collection and authentication of plant

Authenticated seeds of Linum usitatissimum L. (Linaceae) were obtained from Dr. P. B. Ghorpade, Principal Scientist, Punjabrao Deshmukh Krushi Vidyapeeth, College of Agriculture, Nagpur, India and voucher specimen was deposited at the institute.

Chemicals and reagents

Standard SDG was purchased from Chromadex Inc., Irvine, CA. Ethyl acetate, methanol, formic acid, chloroform (Merck, Bangalore, Karnataka, India) of GR grades were purchased from respective vendors. Sephadex LH-20 was purchased from Sigma Chemical Co. (St Louis, MO). Borosil glass column (height, 30 cm; diameter, 3 cm) was purchased from Yash Enterprises (Pune, India). Microliter syringe (Hamilton, Bonaduz, Switzerland) was obtained from Anchrome Enterprises (I) Pvt. Ltd (Mumbai, India). Precoated thin-layer chromatographic (TLC) silica gel plates (Merck, Bangalore, Karnataka, India, Kieselgel 60, F-254, 0.2 mm) and precoated glass silica gel plates were used for preparative TLC. High-performance thin-layer chromatography (HPTLC) spectra were recorded on a Linomat V (Camag, Muttenz, Switzerland).

Extraction and isolation

Preparation of FLC was carried out as described previously (Zanwar et al., 2013). Briefly, flaxseeds were subjected to cold press extraction to remove oil. Further, flaxseed cake was defatted by n-hexane to remove the residual oil. Defatted flaxseed cake was then hydrolyzed followed by extraction with alcohol. The filtrate was acidified and then dried using a Rotavac (Bharat Biotech International, Ltd., Hyderabad, India). The dry powder of hydroalcoholic extract was labeled as FLC. FLC was subjected to solvent–solvent fractionation using methanol:chloroform (9:1). Resultant filtrate was dried and subjected to pure methanol fractionation. A weighed quantity (3 g) of the methanol fraction was mixed with methanol and subjected to column chromatography (height, 30 cm; diameter, 3 cm) eluting with a mobile phase containing methanol:chloroform (50:50). Elution was carried at the flow rate of 3 ml/min. Isolation of compounds was carried out based on the molecular weight. Fractions (5 ml each) were collected in tarson tubes. Totally 18 fractions were collected and labeled as fractions 1–18. Remaining material left on the column which could not be eluted with a mobile phase was eluted with chloroform:methanol:water (75:15:10). All fractions and washing elution were analyzed by HPTLC and fractions showing similar TLC patterns were pooled together. Further, these fractions were concentrated on a Rotavac under reduced pressure. Further, these pooled fractions were again subjected to column chromatography under similar conditions using methanol:chloroform (80:20) mobile phase. A total of 70 fractions were collected and labeled as fractions 1–70 of 0.5 ml each. All fractions were analyzed by HPTLC and fractions showing similar TLC patterns were pooled together. Final drying was carried out on a Rotavac under reduced pressure and further subjected to preparative HPTLC for final purification of compound. Determination of SDG lignan content was carried out by using HPTLC as reported previously (Zanwar et al., 2011). Isolation of active constituent was carried out by preparative HPTLC.

Chemical characterization of isolated molecule

The chemical structure of the isolated compound was elucidated by UV, IR, ¹H NMR, ¹³C NMR, DEPT, COSY, HSQC, HMBC, ROESY, MS, and specific optical rotation was recorded. The 1D and 2D spectra were performed in a CD₃OD + DMSO-d₆ mixture. Optical rotation was measured in a Jasco 1022 automatic polarimeter (Merck KGaA, Darmstadt, Germany) in methanol. IR spectrum was recorded using KBr pellets on a Perkin–Elmer IR spectrometer (Perkin–Elmer, Waltham, MA).

Antihyperlipidemic activity of SDG (isolated from FLC) in P-407-induced hyperlipidemic mice

Research protocol approval

The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) constituted in accordance with the rules and guidelines of the Committee for the Purpose of Control and Supervision on Experimental Animals (CPCSEA), India.

Experimental animals

Swiss albino mice weighing 25–30 g were purchased from National Institute of Biosciences, Pune, India. They were maintained at a temperature of 25 ± 1 °C and a relative humidity of 45–55% under a 12 h light:dark cycle. The animals had free access to commercial pellet diet (Pranav Agro Industries Ltd., Sangli, India) and water was ad libitum throughout the study period.

Preparation of P-407

The P-407 solution was prepared for injection by combining the agent with saline and then refrigerating overnight to facilitate dissolution of the P-407 via the cold method.

Experimental design

Overnight fasted mice were divided into three groups containing six mice in each group. The isolated fraction, i.e., SDG was evaluated for antihyperlipidemic activity. The dose of SDG was selected based on our previous studies (Zanwar et al., 2010, 2012, 2013). The randomly selected mice were divided into the following groups:

Group I: vehicle (distilled water, 10 ml/kg), p.o

Group II: vehicle (distilled water, 10 ml/kg), p.o + P-407 (30% w/v), (i.p.)

Group III: SDG (20 mg/kg, p.o.) + P-407 (30% w/v) (i.p.)

During the study period, animals had access only to water. Blood samples were collected at 0 h (before test drug administration), and at 15 h after P-407 injection and investigated for lipid profiles, blood was withdrawn by the retro orbital plexus method under light ether anesthesia. The blood samples were centrifuged (6000 rpm/10 min at 4 °C) and serum was used for lipid analysis.

Biochemical analysis of serum

The total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C) levels were determined at 0 and 15 h after SDG/vehicle administration. The data were presented as mean ± SEM. Very low-density lipoprotein-cholesterol (VLDL-C) was calculated using Friedewald’s formula (Friedwald et al., 1972):

Statistical analysis and structure determination

Data were expressed as the mean ± SEM. Statistical analysis was carried out by a one-way ANOVA followed by post hoc Bonferroni test using graphpad prism 5.00 for Windows 7, GraphPad Software, San Diego, CA. The p value was considered significant when <0.05.

ChemDraw® Ultra, Cambridge Soft Corporation, Cambridge, MA, was used for the determination of structures of SDG, heteronuclear multiple bond correlation (HMBC), and rotating frame Overhauser enhancement spectroscopy (ROESY) correlations.

Results

Isolation of active constituent from FLC

During column chromatographic separation of the 3 g of methanol fraction of FLC, a total of 18 fractions were collected. Fractions showing close resemblance with respect to appearance of R_f and UV-λ_max in HPTLC (282 nm) with respect to SDG were pooled together. Accordingly, fractions numbers 8–11 were pooled together and concentrated. This fraction was subjected again to column chromatography and further 70 fractions were collected. These 70 fractions were again subjected individually to HPTLC analysis, based on TLC pattern fraction numbers 29–56 were pooled together and concentrated on Rotavac under reduced pressure. The pooled fraction sample was dissolved in 2 ml of methanol:water (9:1) solution and subjected to preparative chromatography for isolation of the individual compound. This recovered solution was concentrated on a Rotavac to certain extent and densitograph showed a single peak which was comparable with the standard SDG.

Characterization of isolated compound

The chemical structure of the isolated compound was elucidated by ultraviolet spectroscopy (UV), infra-red spectroscopy (IR), proton nuclear magnetic resonance (¹H NMR), carbon nuclear magnetic resonance (¹³C NMR), distortion-less enhancement by polarization transfer (DEPT), homonuclear correlation spectroscopy COSY, heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC), rotating frame Overhauser enhancement spectroscopy (ROESY), and mass spectroscopy and specific optical rotation experiment. Isolated and purified compound was obtained as a whitish colored substance. It shows single peak on HPTLC spectrum. Its ¹H NMR (Figure 1), ¹³C NMR (Figure 2), DEPT (Figure 3), COSY (Figure 4), HSQC (Figure 5), HMBC (Figure 6), ROESY (Figure 7), and ESI-MS spectra of isolated compound suggested that the sample is pure and contained only one compound. The isolated compound obtained was crystalline solid (melting point 119–120 °C). In its ESI-mass spectrum, the molecular ion peak [M−OH]⁺ at m/z 668 suggesting one of the possible molecular formula as C₃₂H₄₆O₆. Its IR spectrum showed a characteristic broad absorption band at 3446 cm⁻¹ for the hydroxyl group, it further showed broad peaks at 2922, 2819 (CH₃–O–Ar), 1594, 1383, 1352 (C_Ar–O), 1270, 1158, 1092 (C–O–C) 1045, 999 (aromatic). Its ¹³C NMR spectrum (Figure 2 and Table 1) showed 16 signals, each carbon signal had two carbon intensities meaning that the isolated compound must have 32 carbon atoms in its structure.

Figure 1. ¹H NMR spectrum of isolated compound.

Figure 2. ¹³C NMR spectrum of isolated compound.

Figure 3. DEPT spectrum of isolated compound.

Figure 4. COSY spectrum of isolated compound.

Figure 5. HSQC spectrum of isolated compound.

Figure 6. (a) HMBC spectrum and (b) HMBC correlation of the isolated compound.

Figure 7. (a) ROESY spectrum and (b). ROESY correlation of the isolated compound.

Table 1. ¹³C NMR spectral data of isolated compound.

Carbon no.	δH values (coupling constant) (present spectral data in DMSO-d6 + CD3OD)	^nJ (H,H) (Hz) (coupling constant) (reported spectral data by Chimichi et al., 1999 in CDCl3)
C-1	70.03 (t)	71.3 (t)
C-2	39.77 (d)	41.1 (d)
C-1′	34.26 (t)	35.6 (t)
C-1″	132.8 (s)	134.2 (s)
C-2″	112.5 (d)	113.7 (d)
C-3″	148.4 (s)	148.9 (s)
C-4″	143.7 (s)	145.8 (s)
C-5″	114.7 (d)	115.8 (d)
C-6″	121.8 (d)	123.1 (d)
OMe	56.3 (q)	56.4 (q)
C-1″′	103.36 (d)	104.8 (d)
C-2″′	73.78 (d)	75.3 (d)
C-3″′	76.60 (d)	78.2 (d)
C-4″′	70.14 (d)	71.7 (d)
C-5″′	76.43 (d)	77.9 (d)
C-6″′	61.22 (t)	62.8 (t)

The ¹H NMR spectrum showed the presence of two methylene groups at δ 4.12 dd, (1H, J = 5.0, 10.0 Hz), 3.53 dd (1H, J = 7.0, 10.0 Hz), and 2.75 dd (1H, J = 6, 13.0), 2.62 dd (1H, J = 9.0, 14.0 Hz) assignable to H-1′a and H-1′b groups, respectively. A signal at δ 2.11 (m) was ascribed to H-C(2) and signals at δ 6.54 dd (1H, J = 2.0, 8.0 Hz), 6.57 d (1H, J = 2.0 Hz), and 6.62 d (1H, J = 8.0 Hz) were ascribed to H-6″, H-5″, and H-2″. The ¹H NMR spectrum further showed the presence of sugar moiety at δ 4.31 d (1H, J = 8.0), 3.25 dd (1H, J = 7.9, 9.05 Hz), 3.43 t (J = 9.0 Hz), 3.36 dd (1H, J = 9.0, 9.6 Hz), 3.31 ddd (1H, J = 2.0, 5.4, 9.0 Hz), 3.72 dd (1H, J = 2.0, 12.0 Hz), and 3.86 d (1H, 5.0, 12.0 Hz). It also further showed the presence of methoxy group at δ 3.77 appeared as a singlet. The ¹H NMR spectrum exhibited, in addition to AMX spin system for the aromatic proton and the methoxy singlet at δ 3.77, a series of resolved multipletes extending from δ 4.31 to 2.10 for a total of 18 non-exchangeable protons. Taking into consideration the presence of five groups, this gives a total of 23 hydrogen atoms, thus confirming that it is a symmetrical dimer (Figure 1 and Table 2).

Table 2. ¹H NMR spectral data of compound.

Hydrogen no.	δH values (coupling constant) (present spectral data in DMSO-d6 + CD3OD)	^nJ (H,H) (Hz) (coupling constant) (reported spectral data by Chimichi et al., 1999 in CDCl3)
H-1 a	4.12 dd (J = 5.0, 10.0 Hz)	4.08 dd (J = 5.5,10.8 Hz)
H-1 b	3.53 dd (J = 7.0, 10.0 Hz)	3.48 dd (J = 6.3,10.8 Hz)
H-2	2.11 m	2.13 m
H-1′a	2.75 dd (J = 6.0, 13.0 Hz)	2.69 dd (J = 6.6, 13.8 Hz)
H-1′b	2.62 dd (J = 9.0, 14.0 Hz)	2.62 dd (J = 7.6, 13.8 Hz)
H-2″	6.57 d (J = 2.0 Hz)	6.59 d (J = 1.8 Hz)
H-5″	6.62 d (J = 8.0 Hz)	6.64 d (J = 8.2 Hz)
H-6″	6.54 dd (J = 2.0, 8.0 Hz)	6.57 dd (J = 1.8, 8.2 Hz)
H-1″′	4.31 d (J = 8.0 Hz)	4.24 d (J = 7.9 Hz)
H-2″′	3.25 t (J = 8.0 Hz)	3.21 dd (J = 7.9,9.1 Hz)
H-3″′	3.43 t (J = 9.0)	3.35 t (J = 9.1 Hz)
H-4″′	3.36 dd (J = 5.0, 9.0 Hz)	3.30 dd (J = 9.1, 9.4 Hz)
H-5″′	3.31 ddd (J = 2.0, 5.4, 9.0 Hz)	3.24 ddd (J = 2.4, 5.5, 9.4 Hz)
H-6 a″′	3.72 dd (J = 2.0, 12.0 Hz)	3.86 dd (J = 2.4, 11.9 Hz)
H-6 b″′	3.86 dd (J = 5.0, 12.0 Hz)	3.68 dd (J = 5.5, 11.9 Hz)
OCH3	3.77 s	3.74 s

¹H–¹H COSY experiment, a two-dimensional NMR experiment that represents the homonuclear correlation spectroscopy (called as COSY), shows the frequencies for a single isotope, most commonly hydrogen (¹H) along both axes. In the present study, ¹H–¹H COSY experiment (Figure 4) shows coupling of a proton at δ 2. 11 (m) with protons appearing at δ 2.62 dd (J = 9.0, 14.0 Hz), 2.75 d (J = 6.0, 13.0 Hz), 4.12 dd (J = 5.0, 10.0 Hz), and 3.53 dd (J = 7.0, 10.0 Hz) which has to be H-1′ab and H-1ab signals. Heteronuclear single quantum coherence called HMQC, which is a 2D method used to determine ¹H-¹³C connectivities (Figure 5), revealed that four signal protons appearing at δ 4.12, 3.53, 2.62, and 2.75 are on two different carbon atoms appearing at 70.0 and 34.26 which could be C-1 and C-3. The ¹³C NMR (Figure 2) spectrum exhibited 16 carbon ×2 signals in the following form: (a) three CH₂, (b) nine CH, (c) three quaternary and (d) one CH₃.

Heteronuclear multiple-bond correlation spectroscopy (called HMBC) detects heteronuclear correlations (proton–carbon couplings) over longer ranges of about 2–4 bonds. A HMBC experiment disclosed following correlations H–C(2) [2.11 m] →C-1″, C-1, C-2 and C-1′; H–C(1′) → C-1″, C-6″, C-2″, C-1 and C-2; H–C(1) → C-1′, C-2 and C-1′″; H–C(1″) → C-1, C-3′″ and C-5′″; H–C(2″) → C-3″, C-1″, C-6″, C-2″, C-1′ and C-4″ (Figure 6a and b).

COSY spectrum indicated correlation between H–C(2) δ(H) = 2.11 (m) and δ 2.62 dd, δ 2.75 dd, δ 3.53 dd, and δ 4.11 dd. It further showed the connectivity between δ 4.31 ddd and δ 3.25 t; δ 3.25 t and δ 3.43 t; δ 3.43 and δ 3.72 dd and δ 3.86 (Figure 4).

Rotating frame Overhauser effect spectroscopy (called ROESY) determines ¹H to ¹H correlations. In the present study of ROESY experiment, correlations between H-2 δ (H) 2.11 m, H-1 b (δ 3.53 dd, J = 7.0, 10.0 Hz), H-1′b (δ 2.62 dd, J = 9.0, 14.0 Hz); H-1′″ (δ 4.31 d, J = 8.0 Hz), H-1 b (δ 3.53 dd, J = 7.0, 10.0 Hz), H-3′″ (δ 3.43 t, J = 9.0 Hz), H-5′″ (δ 3.66 dd, J = 5, 9.0 Hz) (Figure 7a and b).

The UV-spectrum showed λ_max at 282 nm, which exactly matches that of the previously reported by Coran et al. (2004). Specific optical rotation was obtained as – 0.71 (CH₃–OH, c-0.7).

The structure and stereochemistry were confirmed on the basis of 1D and 2D spectral data (¹H NMR, ¹³C NMR, DEPT, COSY, ROESY, HMBC, HSQC) MS, and IR, the isolated compound was characterized as SDG. The spectral and physical data were comparable with the reported spectral data values in the literature (Chimichi et al., 1999) which confirms that the isolated molecule was characterized as 2,3-bis[(4-hydroxy-3-methoxyphenyl)-methyl]-1,4-butanediyl bis-[R–R*,R*)]-β-d-glucopyranoside (Figure 8). IUPAC name: 2,3-bis[(4-hydroxy-3-methoxyphenyl) methyl]-1,4-butanediyl bis-[R–R*,R*)]-β-d-glucopyranoside (molecular formula: C₃₂H₄₆O₁₆).

Figure 8. Structure of isolated molecule from FLC. IUPAC name: 2,3-bis[(4-hydroxy-3-methoxy phenyl)-methyl]-1,4-butanediyl bis-[R–R*,R*)]-β-d-glucopyranoside (molecular formula: C₃₂H₄₆O₁₆).

Antihyperlipidemic activity of isolated compound SDG in P-407-induced hyperlipidemic mice

Effect of SDG in P-407-induced hyperlipidemic mice on serum cholesterol at 0 and 15 h

The 0 h values were considered as basal values before treatment or induction of hyperlipidemia (Figure 9). At 15th h, results indicated a significant increase (p < 0.001) in serum cholesterol level in the P-407 alone-treated group compared with the vehicle control group. SDG (20 mg/kg) + P-407 significantly decreased serum cholesterol level (p < 0.01) as compared with the P-407 alone-treated group (Figure 9).

Figure 9. Effect of SDG in poloxamer-407-induced hyperlipidemic mice on serum cholesterol at 0 and 15 h. Values are expressed as mean ± SEM. Data were analyzed by a two-way ANOVA followed by post hoc Bonferroni test. p < 0.05 considered as significant. ###p < 0.001 compared with the control group. ***p < 0.001 compared with the poloxamer-407 group.

Effect of SDG in P-407-induced hyperlipidemic mice on serum triglyceride at 0 and 15 h

The 0 h values were considered as basal values before treatment or induction of hyperlipidemia (Figure 10). At 15th h, results indicated a significant (p < 0.001) increase in serum triglyceride level in the P-407 alone-treated group as compared with the vehicle control group. SDG (20 mg/kg) + P-407 significantly (p < 0.001) decreased serum triglyceride level as compared with only the P-407-treated group (Figure 10).

Figure 10. Effect of SDG in poloxamer-407-induced hyperlipidemic mice on serum triglyceride at 0 and 15 h. Values are expressed as mean ± SEM. Data were analyzed by a two-way ANOVA followed by post hoc Bonferroni test. p < 0.05 considered as significant. ###p < 0.001 compared with the control group. ***p < 0.001 compared with the poloxamer-407 group.

Effect of SDG in P-407-induced hyperlipidemic mice on serum HDL-C at 0 and 15 h

The 0 h values were considered as basal values before treatment or induction of hyperlipidemia (Figure 11). At the 15th h, results indicated a non-significant decrease in serum HDL-C level in the P-407 alone-treated group compared with the vehicle control group. Also SDG (20 mg/kg) + P-407 non-significantly increased serum HDL-C level compared with the P-407 alone-treated group (Figure 11).

Figure 11. Effect of SDG in poloxamer-407-induced hyperlipidemic mice on serum HDL-C at 0 and 15 h. Values are expressed as mean ± SEM. Data were analyzed by a two-way ANOVA followed by post hoc Bonferroni test. p < 0.05 considered as significant.

Effect of SDG in P-407-induced hyperlipidemic mice on serum VLDL-C at 0 and 15 h

The 0 h values were considered as basal values before treatment or induction of hyperlipidemia (Figure 12). At the 15th h, results indicated a significant (p < 0.001) increase in serum VLDL-C level in the P-407 alone-treated group compared with the vehicle control group. SDG (20 mg/kg) + P-407 significantly (p < 0.05) decreased serum VLDL-C level as compared with the P-407 alone-treated group (Figure 12).

Figure 12. Effect of SDG in poloxamer-407-induced hyperlipidemic rats on serum VLDL-C at 0 and 15 h. Values are expressed as mean ± SEM. Data were analyzed by a two-way ANOVA followed by post hoc Bonferroni test. p < 0.05 considered as significant. ###p < 0.001 compared with the control group. *p < 0.05 compared with the poloxamer-407 group.

Discussion

FLC was fractionated by column chromatography and isolation was carried out by preparative HPTLC. The single isolated compound was analyzed by physical and chemical methods to identify and determine its chemical structure. On the basis of physical properties and spectroscopic data (i.e., ¹H NMR, ¹³C NMR, DEPT, COSY, ROESY, HMBC, HSQC), MS, and IR, the isolated compound was characterized as SDG. The obtained spectral data were compared with the reported spectral data by other research groups (Chimichi et al., 1999). In the present study, the recorded-specific optical rotation using same instrument Jasco P-2000 polarimeter (Merck KGaA, Darmstadt, Germany) of isolated SDG and we confirmed the presence of leavo form ( – 0.5085, c 0.7, CH₃OH) of SDG and we also recorded the specific optical rotation of SDG purchased from Chromadex (Irvine, CA) which also recorded leavo rotation – 1.320, c 0.7, CH₃OH) under similar experimental conditions. Previously, Chimichi et al. (1999) reported that SDG has leavo rotation – 4.4, c 0.02, CH₃OH). Based on chemical characterization of SDG, the isolated compound from FLC was confirmed as a single component in the pure form. It was thought necessary to evaluate the antihyperlipidemic action of SDG in animals to ascertain the retention of antihyperlipidemic activity of FLC. The dose of SDG selected was 20 mg/kg based on the contents of SDG in FLC to be 40 mg/gram of FLC, i.e., 20 mg SDG in 500 mg of FLC.

It has been reported that intraperitoneal injection of P-407 induces hypercholesterolemia and hypertriglyceridemia for a maximum at 15 h and it is associated with alterations in HMG Co-A reductase (HMGR), lecithin cholesterol acetyltransferase (LCAT), chlolesteryl ester transfer protein (CETP), hepatic lipase (HL), and lipoprotein lipase enzymatic activities (LPL). P-407 has been utilized in the hyperlipidaemic model due to its convenience, reproducibility, and the lack of undesirable underlying biochemical changes (Johnston & Palmer, 1993; Wasan et al., 2003). Pure SDG (20 mg/kg) showed significant reduction in serum TC and non-significant increase in HDL-C. Decrease in TG and VLDL-C was also significant. These results clearly demonstrate retention of antihyperlipidemic activity by SDG. In the present study, SDG was found to improve all the lipid parameters mainly cholesterol in the P-407-induced acute hyperlipidemia model. Since P-407 induced hyperlipidemia is mainly due to inhibition of the extractable (heparin releasable) pool of lipoprotein lipase (Johnston & Palmer, 1993).

In conclusion, SDG possess significant lipid-lowering activities against experimentally induced hyperlipidemia. The predominant mechanism responsible for elevated concentrations of circulating TC and TG following administration of P-407 was inhibition of heparin-releasable LPL (Johnston & Palmer, 1993). The cholesterol-lowering effect of SDG might be attributed to decreased absorption of circulating cholesterol and increased biliary excretion in the present study. Previously, the lipid lowering effect of flax is not hepatic mediated and may be at the level of cholesterol absorption and/or bile acid reabsorption (Pellizzon et al., 2007). Additionally, SDG has improved natural antioxidant defense mechanisms and thereby decreased oxidative stress, also anti-inflammatory, anti-apoptotic, and estrogenic action may have contributed for antiatherosclerotic activity of SDG from previous studies (Zanwar et al., 2010, 2011, 2012, 2013).

Conclusion

The chemical structure of the isolated compound was determined by ¹H NMR, ¹³C NMR, DEPT, IR, MS, HSQC, HMQC, ROESY, COSY, and specific optical rotation experiment which revealed that the sample contained only single compound and characterized as SDG and it is leavo rotatory. Finally, the isolated pure SDG was screened by using P-407-induced mice model for its antihyperlipidemic action using serum lipid parameters. SDG (20 mg/kg) showed significant reduction in serum TC, TG, VLDL-C, and non-significant increase in HDL-C. It is thus concluded that hydroalcoholic extract possessed antihyperlipidemic activity mainly due to SDG. These results with isolated pure SDG and previous results with FLC confirm that SDG is beneficial in preventing hyperlipidemia in laboratory animals.

Declaration of interest

No conflict of interest to disclose. The research work was carried out as a part of Indian Council for Agriculture Research sponsored project under National Agriculture Innovation Project.

Acknowledgements

The authors would like to acknowledge Dr. S. S. Kadam, Vice-Chancellor and Dr. K. R. Mahadik, Principal, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Pune, India, for providing necessary facilities to carry out the study.