HPLC Determination of Selected Flavonoid Glycosides and their Corresponding Aglycones in Sutherlandia frutescens Materials

Mbamalu ON1*, Antunes E2, Silosini N3, Samsodien H4 and Syce J1,3 1Discipline of Pharmacology, School of Pharmacy, University of the Western Cape, Bellville 7535, South Africa 2Department of Chemistry, University of the Western Cape, Bellville 7535, South Africa 3South African Herbal Science and Medicine Institute, University of the Western Cape, Bellville 7535, South Africa 4Discipline of Pharmaceutics, School of Pharmacy, University of the Western Cape, Bellville 7535, South Africa


Introduction
Sutherlandia frutescens L., a popular plant of the family Fabaceae (pea and bean family or Leguminosae), is widely used in certain parts of Southern Africa for the treatment of various ailments [1]. Randomized placebo-controlled clinical trials have been conducted to assess its safety and efficacy in HIV patients [2,3] with preparations underway for another upcoming clinical study. While a variety of formulations from this plant are commercially available and thus can be used for the clinical trial, very little is known regarding their quality and stability profiles, as well as the content of actives in such formulations.
Flavonoid glycosides have been discovered and isolated from the plant as potential actives and possible quality control markers [4,5]. Much of the biological effects of herbal medicinal remedies have been attributed to the presence of flavonoids [6,7], which are known to exist in this plant in glycosidic and aglycone forms. These different forms have differing chemistries and, as such, can be said to be representative of the different forms and conditions under which S. frutescens actives occur in different environments. These forms are also susceptible to extreme modification and can be readily hydrolysed, oxidized, hydroxylated, methylated, glycosylated, acylated or phenylated, giving rise to the variety of compounds within a class [8,9]. As most forms of instability in medicinal products are due to oxidative and hydrolytic reactions, advantage can be taken of flavonoid susceptibility to modification, and this can be applied in the quality control of herbal drug formulations. As such, content of flavonoid glycosides and /or corresponding aglycones can be used as an indication of product quality or stability as the flavonoid glycosides may be converted to the aglycones under unstable conditions [10]. Avula et al. reported on the quantification of flavonoid and cycloartanol glycosides in commercial preparations of S. frutescens [11]. However, no method has been reported for the simultaneous determination of the flavonoid glycosides and their corresponding aglycones in S. frutescens products.
The aim of this study therefore was to develop and validate a simple HPLC method for the simultaneous estimation of nine flavonoid compounds (two flavonoid aglycones and seven corresponding flavonoid glycosides) in S. frutescens formulations, with a view to employing this in resource-constrained settings for quality control and stability studies of products derived from this plant. Four of the compounds (sutherlandins A, B, C and D) are not yet commercially available and had to be isolated from the plant material for assay development and validation. Other flavonoid compounds selected for the assay were rutin, quercitrin, kaempferol-3-O-rutinoside, quercetin and kaempferol.

Methods
Isolation of reference compounds: Sutherlandins A to D were isolated from the n-butanol soluble portion of the aqueous extract of S. frutescens spray-dried aqueous extract (SDAE) (batch number: Ferl-DST/001-1210). The crude butanol extract was subjected to reversed phase column chromatography using C 18 Strata™ solid phase extraction (SPE) cartridges for the isolation of flavonoid fractions. Conditioning of the SPE cartridge was with 100% acetonitrile followed by water (0.01% formic acid). The butanol fraction (dissolved in 50% acetonitrile: water) was applied uniformly to the SPE cartridges and the column eluted using a five step gradient elution with water: acetonitrile mixtures (0.01% formic acid) of decreasing polarity (0 to 100% acetonitrile). Six major fractions were obtained; fraction 3 was found to contain the sutherlandins, and was further fractionated using HPLC. The identity of the isolated compounds was confirmed by LC-MS/MS and NMR analyses.
Preparation of calibration standards: Stock solutions of the calibration standards (rutin, quercetin, quercitrin, kaempferol, kaempferol-3-O-rutinoside, sutherlandins A to D) were prepared separately by dissolving each standard in 50% aqueous methanol solution. Dilutions were made from these stock solutions to achieve concentrations from 0.2 to 60 µg/ml for quercitrin, 0.2 to 120 µg/ml for quercetin and kaempferol, 0.2 to 200 µg/ml for rutin and kaempferol-3-O-rutinoside, 4-180 µg/ml for sutherlandins A and D, and 4 to 200 µg/ ml for sutherlandins B and C. The calibration standards were assayed in triplicate and calibration lines constructed by linear regression of plots of peak area against concentration. The prepared standards were used to validate the developed HPLC method for linearity and range, precision, limits of detection and quantitation, and ruggedness / robustness [13,14].
Sample preparation for quantification of reference compounds in plant materials: Five samples of S. frutescens formulations, one or more of which may be utilised in a planned randomised controlled trial, were prepared using a modification of the method previously utilized by Avula et al. [11] as follows. Weighed sample of each material: LP or SDAE material was vortexed in 2 ml of 50% aqueous methanol, and sonicated for 30 minutes. This was followed by centrifugation at 3500 rpm for 15 minutes. The procedure was repeated thrice, and respective supernatants combined. The final volume was adjusted to 8 ml with 50% aqueous methanol and mixed thoroughly. Prior to injection, appropriate dilutions were made, and the solution filtered using a 0.45 µm nylon membrane filter. The first 1.0 ml was discarded and the remaining volume collected in an LC sample vial. Fifty µl of triplicate samples was injected for each plant material.
The identity of the isolated reference compounds was confirmed by LC-MS/MS and NMR. The MS data is shown in Figure 1. Highresolution electrospray ionisation mass spectrometry (HR ESI-MS) analysis provided the [M+H] + molecular ions of the respective compounds in the positive ion mode. The protonated species [M+H] + , similar to those previously reported by Avula et al. [11], were therefore observed at m/z 741.1862, 741.1857, 725.1900 and 725.1913 amu, corresponding to the identities of sutherlandins A, B, C and D, respectively ( Figure 1, Table 1).
The isolated compounds were further characterized using fragmentation patterns observed in the mass spectrum. Sutherlandins A and B showed characteristic fragments at m/z 609.1487 ([M+Hsugar] + ) and 303.0484 (quercetin), while sutherlandins C and D showed characteristic fragments at m/z 593.1472 ([M+H-sugar] + ) and 287.0554 (kaempferol). These ions reflect the loss of sugar from the flavonoid glycoside skeleton ( Figure 1, Table 1).
Final confirmation of the identity of the isolated compounds was via NMR analyses, with results comparable to that previously reported [5].

HPLC Method
A reversed phase HPLC separation method combined with PDA detection has been developed for flavonoid analysis in S. frutescens. The reference compounds were separated within 30 minutes by HPLC-   Figure 2. The flavonoid glycosides eluted between 11 and 19 minutes, and the aglycones between 26 and 29 minutes.

Method validation
Calibration curves and linearity: The linearity for each of the reference compounds was assessed over the same concentration range as the calibration curves. The calibration curves for the nine reference standards showed a linear correlation between concentration and peak area (R 2 >0.99) of the detector response for the concentration ranges studied ( Table 2).

Limit of detection (LOD) and limit of quantitation (LOQ):
The limits of detection (LOD) and quantitation (LOQ) were calculated from the calibration curve using the equations [13,14]: Where, SD and S are the standard deviation of the ordinate intercept and the slope of the calibration curve, respectively. Values for the LOD and LOQ are presented in Table 2.
Stability: Samples of the reference compounds were subjected to a variety of conditions (acid/base hydrolysis, freeze/thaw cycles, ambient light and heat exposure) and injected into the HPLC system to assess the analyte in the presence of any degradants which may arise under these conditions.

Acid/base hydrolysis
The results of acid-base hydrolysis are presented in Figure 3. The original chromatogram of each unhydrolysed compound showed only one noticeable peak (in green) in the flavonoid glycoside region for all seven flavonoid glycosides and in the aglycone region for the aglycones. On hydrolysis, the flavonoid glycosides were hydrolysed to the corresponding flavonoid aglycones (in purple), identified by comparison of their retention times and UV spectra with those of reference flavonoid aglycone compounds. This confirmed that the flavonoid glycosides are all O-glycosides, and thus prone to acid hydrolysis [15,16]. They can thus serve as references for quality control as their reactions under acid-base conditions can indicate instability.
As expected, the flavonoid aglycones were not affected by acid hydrolysis. Alkaline hydrolysis of the flavonoid glycosides led to the production of the deacylated flavonoid glycosides (navy traces, Figure 3).

Freeze-thaw cycle
The results of the freeze-thaw cycle after five consecutive freezing (at -80°C) and thawing exercises (at room temperature), are presented in Figure 4. The reference compounds showed relative stability to five successive freezing and thawing cycles, as signified by the retention times and peak sizes before and after the freeze-thaw cycle. An exception was quercetin, exhibiting instability, shown by decrease in peak size, and formation of other products. This was in contrast to   previous studies that demonstrated quercetin stability to freeze-thaw cycles, though the said studies evaluated quercetin stability in biological fluids [17,18]. Since this study utilized plant samples containing mostly flavonoid glycosides, the samples can be stored for prolonged periods in the freezer. This however may not be ideal if quercetin is to be used as a reference as it is prone to instability under freeze-thaw conditions as investigated herein, and may also be formed in cases of product instability.

Ambient light exposure
HPLC chromatograms of samples subjected to sunlight exposure for one month ( Figure 5) indicates that the flavonoid glycosides (sutherlandins A, B, C and D) showed relatively good stability with respect to the retention times (i.e., still eluted at previous retention times) while the aglycones were not stable, with additional peaks, possibly from degraded products, arising in the chromatograms for quercetin and kaempferol ( Figure 5). Retention times for the flavonoid glycosides were not affected by exposure to ambient light; however, the peak response for all the seven flavonoid glycosides decreased to between 45 and 97% of the original.
The peaks for the flavonoid aglycones, quercetin and kaempferol, were however not detected in the chromatograms following light exposure, perhaps due to the photo-oxidation of these compounds [19]. The stability of flavonoids to light is known to depend on the nature of the hydroxyl group attached to C 3 of the flavonoid structure, where the absence or glycosylation of the hydroxyl group confers some degree of stability not seen in flavonoid molecules with a hydroxyl group at C 3 [20] as is the case with quercetin and kaempferol. The seven flavonoid glycosides under study are all glycosylated at position C 3 , conferring on them some degree of photostability not obtained with the nonglycosylated flavonoid aglycones, quercetin and kaempferol. This may explain why the flavonoid glycosides, though affected by ambient light exposure which served to reduce the peak response, were affected to a lesser degree than the flavonoid aglycones of lower photostability due to the presence of a free hydroxyl group at C 3 . The 'lower-stability' flavonoid aglycones underwent degradation to such an extent that their peaks were not detected in the chromatograms obtained following ambient light exposure. The lower phtostability of the flavonoid aglycones is ascribed to a greater triplet state population cum higher singlet oxygen reactivity [20].
All the compounds exhibited instability on exposure to light; ideally, samples containing these compounds should not be exposed to ambient light conditions as tested here. This also shows that the flavonoid compounds are susceptible to light and can therefore be used as reference compounds to monitor quality and stability of S. frutescens products exposed to light.

Heat exposure
Following heat exposure (for 3 hours at 60 degrees Celsius), all the flavonoid glycosides and aglycones were still detected at their respective retention times ( Figure 6) indicating that heat did not cause exhaustive degradation of the compounds.
The peak response was however reduced to between 4 and 84% of the original for the flavonoid glycosides and aglycones under study. This is in line with a study by Manach et al. [21] which reported a 60% loss of quercetin in apple juice stored for 9 months at 25°Celsius. The flavonoid glycoside, kaempferol-3-O-rutinoside, where peak response increased to 106.8% after heat exposure was an exception from the other flavonoids in the group. However, such increase may also be viewed as being due to some form of instability or an indication of compromised quality. Ideally, samples containing these compounds should not be exposed to heat conditions as tested here. The flavonoid compounds, having shown susceptibility to heat, can therefore be used as markers for quality and stability assessment of S. frutescens products exposed to heat.

Precision and accuracy
The precision of the analytical method was determined by assaying 6 samples all at a concentration of 60 µg/ml; the highest concentration for the analyte with the lowest upper limit. Intra-day and inter-day precision were assessed by assay of six replicate samples on the same day, over three different days. The relative standard deviations (% RSD) were calculated for the standard samples at the concentrations assayed and are presented in Table 3.
The results of intraday and interday assays were consistent, with % RSD less than 1% for all reference compounds ( Table 3). The precision and accuracy data are therefore within acceptable limits for S. frutescens assay.    Table 3: Intra-and inter-day precision analyses for reference compounds at 370 nm.

Robustness and ruggedness
Evaluation of method robustness was considered in mobile phase development. The robustness and ruggedness of the method were investigated by varying chromatographic parameters such as the flow rate and temperature.
A decrease in the flow rate of the mobile phase (from 0.8 to 0.5 ml/minute) resulted in elongation of the retention times for all the reference compounds (Figure 7a), while changing the temperature of the column compartment from 40°C to room temperature did not cause significant changes in the peak sizes and retention times, showing that the developed method is robust to temperature changes (Figure 7b).

System auitability testing
System suitability parameters were calculated from the chromatograms obtained for each of the reference compounds during the study ( Table 4). Values of the capacity factor (k * ) ranged from 30 to 71, resolution (Rs) from 49 to 245 and tailing factor (T) from 1.0574 to 1.2563. The suitability of the chromatographic system in terms of column efficiency, resolution and precision, for flavonoid analysis in S. frutescens is therefore assured.

Quantification of reference compounds in plant materials
The aim of this study was to develop a simple HPLC assay for the simultaneous determination of flavonoid glycosides and their corresponding aglycones in S. frutescens formulations.    Figure 8. The flavonoid glycosides, sutherlandins A, B, C and D, represented by peaks 1, 2, 3 and 4, respectively, were detected in all the samples assayed. The flavonoid aglycones, quercetin and kaempferol, represented by peaks 5 and 6, respectively, were only detected in two of the SDAE materials and not in the LP material. Three of the nine compounds, rutin, quercitrin and kaempferol-3-O-rutinoside were not conclusively identified in the plant materials assayed. While peaks with similar retention times and UVspectra as some of these three compounds were detected in some of the S. frutescens materials, materials, they were not isolated and so their identities could not be confirmed by further analytical techniques.
The average peak area was used to quantify the reference compounds contained in each of the five S. frutescens materials. Values of the flavonoid content in the five S. frutescens materials, expressed as percentage content (w/w), and are shown in Table 5. The results show that of the two possible forms in which the flavonoids can exist in plant materials (glycoside and aglycone), the flavonoid glycosides were more abundant than the flavonoid aglycones in each of the assayed materials.
The flavonoid aglycone content (0.02 to 0.26%) was found to be lower than that of the flavonoid glycosides (0.09 to 1.97%), and in some cases, below the quantification limit. The spray-dried extracts contained 0.02 to 0.26% of the flavonoid aglycones, detected in half of the SDAE investigated while these compounds were not detected in the LP samples. This suggests that the flavonoid aglycone content may be increased by processing parameters, such as is used in the preparation of the spary-dried extracts. However, no collaboration of this was found in existing literature.
In all the plant materials assayed, sutherlandin C was found to have the highest abundance, and so can be assayed for in all the S. frutescens materials. The least abundant flavonoid glycoside was not the same in all the plant samples assayed, with different materials showing nonuniformity in the flavonoid of least abundance. For instance, in the LP and SDAE 1 materials, the flavonoid glycosides of least abundance were sutherlandin B and sutherlandin D, respectively, while in the other three spray-dried materials, the flavonoid glycoside of least abundance was sutherlandin A.
Three out of the five materials did not contain quantifiable levels of the aglycones, quercetin and kaempferol. In the two spraydried materials that contained the aglycones (SDAE 2 and SDAE 4), kaempferol content was more than quercetin content. The sum of the analysed flavonoids in the plant materials exceeds that obtained in other studies of total flavonoids [22][23][24], and suggests that the flavonoid content in the S. frutescens materials is sufficient for flavonoid use as reference marker compounds. Overall, the levels of the flavonoid glycosides and aglycones in the different S. frutescens materials assessed varied significantly (p<0.001). The assessment of flavonoid glycoside (sutherlandin A, B, C and D) content can therefore be used to differentiate between S. frutescens products.
The levels of the flavonoid aglycones (quercetin and kaempferol) in the analysed samples were quite low and in some cases, absent, thus making quantification of these compounds a challenge. The ratio of the flavonoid glycosides to the total flavonoids (glycoside + aglycone) was at least 90% in the samples studied. Therefore, the use of the flavonoid aglycones (and not the glycosides) as markers for quality control and stability assessment of S. frutescens LP or SDAE materials may not be justifiable or feasible using the specific assay employed in the present study. This however, can be expected in samples extracted using more polar solvents like methanol and water. It may well be that extraction with less polar solvents would yield more of the aglycones than the glycosides.  Each value is expressed as average percentage content (mg flavonoid x minimum purity of flavonoid reference/mg plant material) ± SD (n=3). Means within a row for each flavonoid compound are significantly different (p<0.001). Key: SA: sutherlandin A; SB: sutherlandin B; SC: sutherlandin C; sutherlandin D; Q: quercetin; K: kaempferol; ND: Not detected; <LOQ: less than LOQ.

Conclusions
The simple, validated HPLC-DAD method reported was found to be appropriate for separation and quantification of flavonoid glycosides and their corresponding aglycones in different formulations of S. frutescens. The LP and SDAE materials all contained all four marker compounds, i.e., sutherlandins A, B, C and D, with sutherlandin C being the most abundant in all the five materials. The two flavonoid aglycones, quercetin and kaempferol, were detected in two of the SDAE materials, and not in the LP material. There were more flavonoid glycosides than aglycones in all the plant materials, suggesting that the former may be more suitable as marker compounds. The use of the flavonoid glycosides as markers is therefore recommended for assessment of S. frutescens materials.
The aglycones can also be used as marker compounds because their presence, as products of flavonoid glycoside breakdown, can indicate instability of the plant materials. However, use of the flavonoid aglycones levels alone, without the flavonoid glycosides levels as well, is not recommended. Assay of all six flavonoids employed here is preferred to the assay of a single flavonoid as it gives the profiles and levels of the flavoniods in the different materials. This in turn can indicate quality status of different S. frutescens products.