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Improvisation of a spectrophotometric method to quantify hydroxycitric acid
Analytical Biochemistry 586 (2019) 113412
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Improvisation of a spectrophotometric method to quantify hydroxycitric acid
T
Disha Patel, Aditi Buch∗ Department of Biological Sciences, P D Patel Institute of Applied Sciences, Charotar University of Science and Technology, CHARUSAT Campus, Changa, 388 421, Dist. Anand, Gujarat, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Hydroxycitric acid Improvised spectrophotometric method Hydroxycitric acid-producing microbes
Existing spectrophotometric method to quantify hydroxycitric acid (HCA), although is specific and sensitive; finds limited use owing to poor stability of HCA-metavanadate complex. Present study describes improvisation of this method with respect to source of HCA standard and assay parameters. Assay system consisting of HCA and metavanadate reagent was modified to include 1 M NaOH to neutralize excess acidity. Resulting complex showed λmax at 485 nm, obeying Beer-Lambert's law within concentration range of 33–677 μg/ml, with linear calibration curve showing a good coefficient of determination (R2 = 0.998). Moreover, HCA-metavanadate complex showed enhanced stability retaining up to 70% absorbance even after 60 min of its formation. Similar consistency in scaled-down assay system renders the method suitable for high-throughput screening of HCAproducing microbes. Of the tested metabolites and media components, only tartrate interfered with the spectrophotometric estimation of HCA; a correction factor to eliminate which was also established. Accordingly measured HCA level in the culture supernatant of a bacterial isolate IT6 was comparable to that determined using the standardized HPLC method. The proposed procedure therefore is a convenient, sensitive, accurate and high-throughput method suitable for primary screening of HCA producing microbes; the only ecofriendly alternative source of optically pure HCA.
1. Introduction Hydroxycitric acid (HCA, 1, 2-dihydroxypropan-1,2,3-tricarboxylic acid) is rapidly gaining importance as herbal therapeutic and dietary supplement [1]. Two of four possible stereoisomers of HCA, (2S, 3S) and (2S, 3R), occur naturally and are biologically active, while their corresponding lactones or methyl derivatives are less potent [2]. (2S, 3S)-HCA is principally accumulated in fruit rinds of Garcinia plant species (~10–30% by weight) predominantly surviving in India and SriLanka [3] while (2S, 3R)-HCA is enriched in calyxes and leaves of Hibiscus subdariffa (Roselle), H. rosa-sinensis and H. cannabinus cultivated in several tropical and semitropical countries [2]. HCA has gained more attention in recent years on account of its ability to mediate unique regulatory effect on fatty acid synthesis, lipogenesis and carbohydrate metabolism leading to controlled appetite and weight loss, with no serious side-effects and toxicity as monitored in animal and human systems [3–6]. Hence, Garcinia fruit extract and thence derived stable HCA salts are currently used as a safe and efficacious ingredient in a number of over-the-counter weight loss products worldwide [2]. Moreover, its distinct chiral structure similar to γ-butyrolactone, makes
∗
HCA an attractive inexpensive starting point for preparation of a range of chiral synthons and ligands for catalysts [2,7]. Unfortunately, availability of natural HCA is restricted due to geoclimatically limited cultivation of source plants, complications in clonal propagation of their elite genotype and difficult stereoselective organic synthesis of bioactive isomers [8–10]. Therefore, microbial production of HCA is being considered as an economical and convenient option for bulk production of natural optically pure HCA. While only few bacterial species are reported to produce (2S, 3R)-HCA [11], fermentation technologies and genome shuffling did not greatly enhance their HCA production [2,8]. Thus, screening of HCA producing bacteria becomes essential, which is usually performed by monitoring the presence of HCA in culture supernatant of bacteria grown in defined minimal medium containing probable HCA biosynthetic precursors like glucose, mannitol, α-ketoglutarate, etc. as sole carbon sources [11]. Qualitatively HCA can be identified by characteristic absorbance maxima at 214 nm; FTIR and HPTLC analysis, IR and 1H NMR spectroscopy as well as paper and thin layer chromatography [12–15]. An existing method to quantify HCA involves acid-base microtitration, which gives the total acidity and hence is non-specific [16]. Most
Corresponding author. E-mail addresses: [email protected] (D. Patel), [email protected] (A. Buch).
https://doi.org/10.1016/j.ab.2019.113412 Received 7 June 2019; Received in revised form 18 August 2019; Accepted 28 August 2019 Available online 29 August 2019 0003-2697/ © 2019 Elsevier Inc. All rights reserved.
Analytical Biochemistry 586 (2019) 113412
D. Patel and A. Buch
(0.4 ml). AS-2: HCA in 0.05 N H2SO4 (1 ml) +1 N NaHCO3 (1 ml) 2.5% sodium metavanadate (0.4 ml). AS-3: HCA in 0.05 N H2SO4 (1 ml) + 1 N NaOH (1 ml) + 2.5% sodium metavanadate (0.4 ml). HCA extract in 0.05 N H2SO4 as described in section 2.1, was used as the source of HCA throughout all the experiments. Optimization with respect to metavanadate reagent was done by preparing the reagent in 6 N and 3 N H2SO4. One milliliter of 1 N NaHCO3 and 1 N NaOH were separately incorporated in the assay system to neutralize the acidity of the metavanadate reagent. Effect of modified assay system on absorbance maxima of the orange-coloured product was monitored by spectrum analysis of the assay mixtures containing 0.2, 0.4, 0.6 and 0.8 ml of HCA extract, respectively, between 400 and 800 nm using UV–Visible spectrophotometer (UV-1800, Shimadzu), immediately after addition of metavanadate reagent. Assay mixture containing 0.4 ml aliquot of HCA extract was subjected to time-scan analysis immediately after addition of metavanadate reagent, at identified λmax in order to determine stability of HCA-metavanadate complex. The absorbance was monitored at interval of 10 s for 60 min and the change in absorbance with time was used to predict the stability of the HCAmetavanadate complex. Sensitivity of the method was determined by using variable aliquots of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 1.0 ml of HCA extract in appropriate assay system (as discussed subsequently) to generate a standard calibration curve. These measurements were carried out across three independent experiments each using HCA extracted from different capsule. Each experimental set consisted of two replicate measurements each performed in triplicates.
explored method that qualitatively and quantitatively measures HCA from source plant extracts, commercial samples as well as from bacterial culture supernatants, is high-performance liquid chromatography (HPLC) [11,13,17]. However, screening of HCA producing bacteria would require HCA analysis from large number of samples, more so because HCA is relatively a rarely encountered metabolite unlike other structurally related common metabolic intermediates. Besides, HCA-producing bacteria could also produce other organic acids and metabolic by-products depending on the carbon source used; which may interfere with HCA estimation. Therefore, a convenient yet specific and accurate method for quantitative determination of HCA becomes essential to facilitate high-throughput screening of HCA-producing bacteria. A suitable spectrophotometric method based on formation of coloured complex between HCA and sodium metavanadate has been reported previously [18]; however, the method is not widely adopted. The present work describes improvisations in this spectrophotometric method to make it more suitable for use during microbial screening procedures. This work also optimizes the use of commercially available G. cambogia extract as more economic source of HCA standard. 2. Materials and methods 2.1. Extraction of HCA from Himalaya Vrikshamla capsules HCA from one capsule of Himalaya Vrikshamla (containing 350 mg dried Garcinia extract as described by the manufacturer) was extracted in a total volume of 200 ml 0.05 N H2SO4. The extraction was initiated by suspending 350 mg of capsule content in 80 ml of 0.05 N H2SO4 in a screw-capped 250 ml flask and subjecting it to continuous stirring for 30 min at room temperature, using a magnetic stirrer. Subsequently, the suspended particles in the flask were allowed to settle and the supernatant was gradually decanted and filtered through Whatman filter paper #1. The residual particles were similarly treated for re-extraction in 3 steps using 40, 40 and 20 ml 0.05 N H2SO4 respectively. The filtrates collected after all the four steps were pooled and the volume was made up to 200 ml using 0.05 N H2SO4. Thus extracted HCA was used as the source of standard HCA in the spectrophotometric estimations. The residues were finally washed with 2 ml of 0.05 N H2SO4 and the filtrate was subjected to spectrophotometric estimation of HCA so as to confirm complete extraction of HCA. This HCA extract was prepared independently from three capsules similarly processed and were used to obtain replicate observations for generation of HCA standard curve to determine the range of the method.
2.4. Interference by media components and structurally similar biomolecules In order to demonstrate the specificity of the method towards measuring HCA, the assay system was setup to include varying concentrations of probable interfering agents including structurally similar organic acids, commonly used sugars in microbial growth medium and standard microbial growth media. Organic acids and by-product metabolites like citrate, succinate, acetate, malate, tartrate and acetoin were used at maximum concentration of 10 mM; sugars like glucose, sucrose and mannose were used at final concentrations of 10, 20, 40, 60, 80 and 100 mM; while microbial growth media like Luria broth (LB), Nutrient broth (NB) and 1X M9 salt solution (prepared as per the manufacturers’ recommendations) were used in the aliquots of 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 ml; in an otherwise constant assay system composition. Various above probable interfering agents were accommodated within 1 ml of sample volume in the assay system; with volume made up using 0.05 N H2SO4. Subsequently, absorbance of assay mixture at appropriate λmax was measured to indicate successful interaction with metavanadate reagent. A correction procedure was applied to derive correct spectrophotometric measurement of HCA in presence of the interfering agent.
2.2. Detection and quantitation of HCA using HPLC HCA extracted in 0.05 N H2SO4 (section 2.1) was filtered using 0.22 μ nylon membrane and subjected to HPLC analysis (obtained as outsourced service from Vasu Research Center, Vadodara, Gujarat, India) using C18 column. Column was maintained at ambient temperature and HCA was eluted in isocratic system using 0.01 M potassium dihydrogen phosphate as mobile phase at a flow rate of 1 ml/min and detected at 215 nm using PDA detector. HCA levels in this extract were quantified with reference to the area under the peak generated by 74.2% pure, 0.1 mg/ml Ca2+-HCA standard prepared in 3% orthophosphoric acid.
2.5. Spectrophotometric determination of HCA levels in bacterial culture supernatants A locally isolated Gram positive, endospore forming bacterial isolate IT6 was inoculated in 3 ml of nutrient broth and allowed to grow with overnight shaking at 37 °C with 150 rpm. A 100 μl of this fresh inoculum was added in a 30 ml M9 minimal broth supplemented with 100 mM glucose as sole carbon source and the culture was allowed to grow under shaking conditions (37 °C, 150 rpm). After 24 h of growth, the culture was harvested by centrifugation at 10,000 rpm for 5 min and the resultant supernatant was stored at −20 °C until further analysis. One milliliter of the supernatant was used to quantify HCA levels spectrophotometrically as in section 2.3 along with the necessary corrections applied as well as through HPLC analysis as in section 2.2.
2.3. Optimization of spectrophotometric method for estimation of HCA The method is largely based on colour complex formation by HCA in presence of metavanadate [18]. Optimization of this method with respect to reagent preparation, composition of assay system, incubation time and absorbance maxima of the coloured complex was carried out by introducing several modifications as per following assay system (AS) combinations. AS-1: HCA in 0.05 N H2SO4 (1 ml) + 2.5% sodium metavanadate 2
Analytical Biochemistry 586 (2019) 113412
D. Patel and A. Buch
Fig. 1. Quantitation of HCA levels in extracts of Himalaya Vrikshamla using HPLC. (a) Chromatogram of Ca2+-HCA standard; inset image shows the molecular structure of HCA (b) Representative chromatogram of HCA extract in 0.05 N H2SO4.
3. Result and discussion
and 0.2 ml of 5% sodium metavanadate. Formation of reddish orange coloured complex exactly after 20 min was measured as absorbance at 467 nm, against a blank similarly prepared without HCA. The modifications introduced in the present work are as follows: (i) HCA standard was extracted and prepared in 0.05 N H2SO4 (ii) sodium metavanadate reagent was prepared in 6 N H2SO4 initially to ensure its complete solubility and (iii) assay volume was reduced drastically to maintain proportionate concentration of metavanadate reagent as indicated in primary assay system AS-1. One milliliter of HCA extract used in AS-1, although formed a typical red-orange coloration at first instance, it rapidly disappeared upon incubation, converting into a blue coloured complex with absorbance maxima of ~760 nm almost completely at the end of 40 min (Fig. 2a). This could be probably due to rapid change in the oxidation states of vanadium due to excess acidity of the assay system caused by metavanadate reagent prepared in 6 N H2SO4 [19]. Use of metavanadate reagent prepared in 3 N H2SO4 in AS-1 could alleviate this instability to certain extent however could not maintain the stability of HCA-metavanadate complex for even 20 min as suggested originally [18]. Since reagent preparation could not be further altered owing to poor solubility of sodium metavanadate, assay system was further amended with a neutralizing agent to overcome the effect of excess acidity. Accordingly, constituting the assay system AS-2 with additional 1 ml sodium bicarbonate could stabilize the orange-red coloration however monitoring the absorbance became difficult due to constant effervescence. Substituting NaHCO3 by NaOH of equivalent
3.1. Extraction of HCA from Himalaya Vrikshamla capsule and optimization of its use as HCA standard EDTA salt of HCA used to generate calibration curve in existing method for spectrophotometric quantitation of HCA, is poorly available commercially. While K+ and Ca2+ salts of HCA are expensive, extraction and preparation of these salts from appropriate plant source is an elaborate process. Therefore, developing a relatively easily accessible and economic source of standard HCA to facilitate the calibration process, could be useful. Commercially available Himalaya Vrikshamla capsules were utilized to extract the Garcinia-type HCA using 0.05 N H2SO4 in a simple method based on the extraction procedure described by Hida et al. [11]. A total of 200 ml of 0.05 N H2SO4 in a stepwise procedure could extract 677 ± 64 μg/ml HCA as averaged from extracts from three capsules, as determined by HPLC analysis (Fig. 1); while ensuring complete recovery as evident by no HCA detected in the recovery filtrate. 3.2. Improvisation of spectrophotometric method for quantitation of HCA with respect to spectral properties and analytical variables The assay system as per Antony et al. [18] primarily consisted of 1 ml of pure HCA-EDTA in 0.1 N H2SO4 made up to 100 ml total volume 3
Analytical Biochemistry 586 (2019) 113412
D. Patel and A. Buch
Fig. 2. Spectral properties of HCA-metavanadate complex. (a) Instability of HCA-metavanadate complex in presence of excess acidity (b) Absorbance spectrum of orange-red HCA-metavanadate complex as obtained using various aliquots with increasing amount of HCA (c) Time scan of HCA-metavanadate complex over 60 min immediately after the addition of the reagent.
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Analytical Biochemistry 586 (2019) 113412
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Fig. 3. Calibration curves for HCA quantitation Graphs generated using (a) modified assay system AS-3 and (b) scaled-down assay system. Results are expressed as Mean ± SD of (a) eighteen and six independent measurements at 485 nm (as explained in Section 2.3) and 467 nm, respectively (b) three independent measurements each performed in duplicates.
485 nm after an incubation of 30 min at room temperature.
volume and concentration to constitute assay system AS-3, stabilized the HCA-metavanadate complex enabling monitoring of stable absorbance even at the end of 20 min as monitored visually. This modification of the assay system shifted the absorbance maxima of the HCAmetavanadate complex from 467 nm to 485 ± 1.63 nm, as monitored by scanning assay mixtures containing varying HCA concentrations between 400 and 800 nm (Fig. 2b). Subsequently, time-scan analysis of the HCA-metavanadate complex formed in AS-3, at 485 nm suggested that the orange-red complex required initial 30 min to stabilize quickly losing up to 22.1% of initial absorbance (Fig. 2c). For 30 min of further incubation, the complex remained relatively stable retaining 93% of its absorbance recorded after first 30 min; implying extended stability of the HCA-metavanadate complex. Thus, unlike the original method where measuring the absorbance exactly at 20 min was crucial; this method showed improved stability leading to more convenient application. Accordingly, a revised procedure was prescribed based on AS-3 which consisted of HCA standard in 1 ml, 1 ml of 1 M NaOH and 0.4 ml of 2.5% sodium metavanadate reagent; with resultant orange-red complex formed to be measured at
3.3. Validation of the proposed spectrophotometric method Sensitivity of the modified method was determined by measuring the absorbance of HCA-metavanadate complex formed with various amounts of HCA at 485 nm. The resultant calibration curve followed Lambert-Beer's law between 33 μg/ml to around 677 μg/ml of HCA, with a linear curve equation of y = 0.0024x-0.0422 and R2 value of 0.998. On the contrary, at 467 nm, the linearity of the curve was relatively maintained only up to 450 μg/ml as per the modified method (Fig. 3a). The sensitivity of the original method has been demonstrated to be 5–50 μg/ml [18]. The revised method apparently possessed extended sensitivity for HCA estimation towards higher concentrations; however, the sensitivity at concentrations lower than 33 μg/ml was unclear. Also, a high-throughput method requiring lower sample volumes as well as reduced assay system would be useful, especially with respect to screening of large number of bacteria for HCA production. To address 5
Analytical Biochemistry 586 (2019) 113412
D. Patel and A. Buch
Fig. 4. Specificity of HCA quantitation from microbial cultures with respect to interference from growth media components. MM: Minimal medium; Results are expressed as Mean ± SD of three independent measurements. Table 1 Comparative analysis of HCA measured using optimized spectrophotometric method and HPLC Results are expressed as Mean ± SD of three independent measurements; ns non-significant when compared between spectrophotometric and HPLC measurements. Both HCA and tartrate levels were normalized with respect to bacterial growth measured as OD600nm. Bacterial isolate
HCA measurements (μg/ml/OD600nm) Spectrophotometric method (with correction)
IT-6
100.13 ± 35.18
ns
Tartrate measurements (μg/ml/OD600nm) HPLC
Spectrophotometric method (with correction)
88.28 ± 36.90
329.88 ± 36.88
ns
HPLC 459.61 ± 89.39
the medium lead to maximum reduction in the absorbance, suggesting that PO43− was possibly responsible for the interference observed. Phosphate ions are known to form stable complex with oxovanadium (IV) cations [20]. Since phosphate cannot be omitted from bacterial growth media being an essential nutrient, a blank consisting of plain unspent growth medium becomes essential while using the method for HCA from bacterial culture supernatants. Sugars like glucose, sucrose and mannose showed negligible interference as indicated by maximum OD485nm of about 0.1 even at concentrations as high as 100 mM (Supplementary Material-Fig. S1). Organic acids like succinate, acetate, malate and common metabolic byproduct acetoin did not significantly interfere with the estimation (Supplementary Material-Fig. S2). In fact, malate and citrate marginally reduced the intensity of the metavanadate reagent as suggested by negative absorbance values. On the contrary, tartrate showed significant absorbance in the spectrophotometric assay when measured at 485 nm (Supplementary Material-Fig. S2); although the sensitivity of the method towards tartrate was relatively poor as compared to that for HCA. However, spectrophotometric quantitation of HCA using metavanadate reagent at 485 nm would be significantly influenced by presence of tartrate. In order to take into consideration this bias, a correction procedure was applied as described by Dumas et al. [21]. Conventionally, spectrophotometric determination of tartaric acid frequently used in wine analysis involves a similar method where tartratemetavanadate complex is measured at 530 nm [22]. Therefore, different concentrations of tartaric acid within the range of the method were subjected to reaction with metavanadate and absorbance of the resultant complex was additionally measured at 530 nm. This calibration curve followed Lambert-Beer's law between the tested concentrations of 141 μg/ml to 1410 μg/ml of tartrate, with a linear curve equation of y = 0.0004x-0.0725 and R2 value of 0.991 (Supplementary Material-Fig. S2). Subsequently, actual absorbances
this, the modified assay system was proportionately scaled down 10 folds with respect to HCA sample, NaOH and metavanadate reagent volumes. Resulting assay system of total 0.24 ml was set up in a 96-well plate with all other operational parameters maintained same, to obtain the calibration curve by measuring absorbance at 485 nm. The scaleddown method obeyed Lambert-Beer's law between 67 and 542 μg/ml of HCA concentrations tested, with the linear curve equation of y = 0.0016x - 0.0593 and R2 value of 0.981 (Fig. 3b). This linear equation was almost identical to that derived for the calibration curve generated using AS-3; thus, suggesting that the scaled-down method could as well be successfully used to quantitate HCA using lower sample volumes.
3.4. Specificity of the proposed method for HCA quantitation: emphasis on possible interference from microbial cultures The convenient and high-throughput nature of the proposed spectrophotometric method makes it suitably applicable in screening HCAproducing microbes. Considering microbial HCA production, structurally related di- and tri-carboxylic acids either used as growth supplement or produced as by-products of microbial growth as well as other growth media components are likely to interfere. Selected of these metabolites and components were analyzed for their individual ability to contribute to absorbance at 485 nm when incorporated in AS-3 instead of HCA sample. Commonly used rich growth media like Luria broth and nutrient broth, contributed to the absorbance at 485 nm at low yet linear consistency showing maximum OD485nm of about 0.4 (Fig. 4). On the other hand, 1X M9 salt solution showed significantly high interference, showing OD485nm of about 1.2 at maximum aliquot of 1 ml (Fig. 4). In order to decipher the specific media component(s) causing the interference, 1X M9 salt solution devoid of one component at a time was used in the assay. Omission of Na2HPO4 and KH2PO4 from 6
Analytical Biochemistry 586 (2019) 113412
D. Patel and A. Buch
doi.org/10.1016/j.ab.2019.113412.
measured at 485 nm for HCA concentrations within this range were used to compute the proportion of tartrate measured as OD485nm relative to OD530nm (Supplementary Material-Fig. S3); a value found to be ranging between 17.7% and 42.3%, which significantly increased with higher absorbance values (Pearson's product-moment correlation: r = 0.999). The resulting linear function of y = 1.1574x + 0.2183 (Supplementary Material-Fig. S3) could be used to derive unbiased estimate of HCA. Conversely, to rule out the possible interference of HCA while quantifying tartrate levels, absorbance of HCA-metavanadate complex was also measured at 530 nm which generated a linear curve over the tested concentration range of 203–677 μg/ml, with equation of y = 0.001x-0.1717 (Supplementary Material-Fig. S4). Relative average absorbance of tartrate at 530 nm was 35.09 ± 6.05% of that of the HCA across the entire concentration range.
Declaration of interest None. References [1] J.W. Yun, Possible anti-obesity therapeutics from nature – a review, Phytochemistry (Oxf.) 71 (2010) 1625–1641 https://doi.org/10.1016/j.phytochem.2010.07.011. [2] T. Yamada, H. Hida, Y. Yamada, Chemistry, physiological properties, and microbial production of hydroxycitric acid, Appl. Microbiol. Biotechnol. 75 (2007) 977–982 https://doi.org/10.1007/s00253-007-0962-4. [3] B.S. Jena, G.K. Jayaprakasha, R.P. Singh, K.K. Sakariah, Chemistry and biochemistry of (−)-hydroxycitric acid from Garcinia, J. Agric. Food Chem. 50 (2002) 10–22 https://doi.org/10.1021/jf010753k. [4] C. Hansawasdi, J. Kawabata, T. Kasai, Alpha-amylase inhibitors from roselle (Hibiscus sabdariffa Linn.) tea, Biosci. Biotechnol. Biochem. 64 (2000) 1041–1043 https://doi.org/10.1271/bbb.64.1041. [5] I. Onakpoya, S.K. Hung, R. Perry, B. Wider, E. Ernst, The use of Garcinia extract (hydroxycitric acid) as a weight loss supplement: a systematic review and metaanalysis of randomised clinical trials, J. Obes. (2011) 509038 https://doi.org/10. 1155/2011/509038. [6] F. Márquez, N. Babio, M. Bulló, J. Salas-Salvadó, Evaluation of the safety and efficacy of hydroxycitric acid or Garcinia cambogia extracts in humans, Crit. Rev. Food Sci. Nutr. 52 (2012) 585–594 https://doi.org/10.1080/10408398.2010.500551. [7] I. Ibnusaud, P.T. Thomas, M. Rani, P.V. Sasi, T. Beena, A. Hisham, Chiral γ-butyrolactones related to optically active 2-hydroxycitric acids, Tetrahedron 58 (2002) 4887–4892 https://doi.org/10.1016/S0040-4020(02)00431-3. [8] H. Hida, T. Yamada, Y. Yamada, Genome shuffling of Streptomyces sp . U121 for improved production of hydroxycitric acid, Appl. Microbiol. Biotechnol. 73 (2007) 1387–1393 https://doi.org/10.1007/s00253-006-0613-1. [9] J. Govinden-Soulange, N. Boodia, C. Dussooa, R. Gunowa, S. Deensah, S. Facknath, B. Rajkoma, Vegetative propagation and tissue culture regeneration of Hibiscus sabdariffa L. (Roselle), World J. Agric. Sci. 5 (2009) 651–661. [10] R.P. Tembe, M.A. Deodhar, Clonal propagation and hydroxycitric acid production from in vitro shoot cultures of Garcinia indica using root suckers as explant, in Vitro Cell, Dev. Biol. Plant 47 (2011) 399–409 https://doi.org/10.1007/s11627-0119349-4. [11] H.H. Hida, T.Y. Amada, Y.Y. Amada, Production of hydroxycitric acid by microorganisms, Biosci. Biotechnol. Biochem. 69 (2005) 1555–1561. [12] G.K. Jayaprakasha, K.K. Sakariah, Determination of organic acids in leaves and rinds of Garcinia indica (Desr.) by LC, J. Pharm. Biomed. Anal. 28 (2002) 379–384 https://doi.org/10.1016/S0731-7085(01)00623-9. [13] R. Ravikumar, B.V. Ranganathan, S. Vinothkumar, S.R. Yamuna, P. Yasodha, P. Priyanka, R.V. Rasikaa, Y. Sownisha, Extraction and characterization of hydroxy citric acid from Garcinia combogia cultivated at two different locations of Malabar and Srilanka, Int. J. Pharm. Chem. 07 (2017) 100–102 https://doi.org/10.7439/ ijpc.v7i7.4298. [14] M.G. Soni, G.A. Burdock, H.G. Preuss, S.J. Stohs, S.E. Ohia, D. Bagchi, Safety assessment of (-)-hydroxycitric acid and super CitriMax, a novel calcium/potassium salt 42 (2004), pp. 1513–1529 https://doi.org/10.1016/j.fct.2004.04.014. [15] L.C. Bainto, E.I. Dizon, A.C. Laurena, K.A.T. Castillo-Israel, Isolation and quantification of hydroxycitric acid from batuan [Garcinia binucao (Blanco) Choisy] fruit, Int. J. Food Res. 25 (2018) 706–711. [16] C. Vijay, P. Chary, A novel method of microtitration and rapid extraction of (-) hydroxycitric acid for identification of elite chemotypes of Garcinia cambogia (Gaertn.) Desr. (Clusiaceae), J. Res. 37 (2009) 7–13. ANGRAU. [17] A. Gogoi, N. Gogoi, B. Neog, Estimation of (-)-hydroxycitric acid (hca) in Garcinia lanceaefolia roxb. using novel HPLC methodology, Int. J. Pharm. Sci. Res. 5 (2014) 4993–4997 https://doi.org/10.13040/IJPSR.0975-8232.5(11).4993-97. [18] B. Antony, W. Varghese, M.B. Elisa, Spectrophotometric determination of hydroxycitric acid, Indian J. Pharm. Sci. 61 (1999) 316–317. [19] S.K. Sinha, A study of reduction of vanadate, Int. J. Adv. Res. Innov. Ideas Educ. 3 (2017) 206–212. [20] D.C. Crans, Fifteen years of dancing with vanadium, Pure Appl. Chem. 77 (2005) 1497–1527 https://doi.org/10.1351/pac200577091497. [21] Z. Dumas, A. Ross-Gillespie, R. Kümmerli, Switching between apparently redundant iron-uptake mechanisms benefits bacteria in changeable environments, Proc. Biol. Sci. 280 (2013), https://doi.org/10.1098/rspb.2013.1055 20131055. [22] M.G.F. Sales, C.E.L. Amaral, C.M. Delerue Matos, Determination of tartaric acid in wines by FIA with tubular tartrate-selective electrodes, Fresenius’ J. Anal. Chem. 369 (2001) 446–450.
3.5. Application of proposed spectrophotometric method to quantitate HCA from bacterial culture supernatant To validate the applicability of the proposed method, HCA levels in the culture supernatants of bacterial isolate IT6 grown on glucose as sole carbon source (as described in section 2.5), determined spectrophotometrically were verified against those determined using HPLC. HPLC profile of the culture supernatant revealed the presence of tartrate in addition to HCA (Table 1). For spectrophotometric measurements, 1 ml of culture supernatant was reacted with metavanadate reagent as in AS-3 and the absorbance of the resultant complex was measured at both 485 and 530 nm, to quantify HCA and tartrate, respectively. Our results revealed that HCA levels measured through proposed spectrophotometric method after application of correction procedures, were similar to those determined using HPLC (Table 1). To determine the tartrate levels, 35% of the absorbance measured at 530 nm was used to calculate tartrate concentrations using the applicable linear equation (y = 0.0004x-0.0725), which were comparable with those determined through HPLC (Table 1). Collectively, the advantage of the proposed improvised method is that it could utilize a conveniently available HCA standard as well as possessed enhanced stability and improved sensitivity. Moreover, the method could be scaled down successfully to reduce the assay volume thus being suitable for quick, accurate and simultaneous analysis of large number of samples, which could be encountered especially while screening HCA producing bacteria. Furthermore, the correction procedure introduced allows precise measurement of HCA from microbial culture supernatant, rendering the method largely applicable. Nevertheless, owing to significant interference by phosphate, this method may not be useful for optimizing HCA production by microbes grown on different minimal media and in such conditions use of HPLC becomes necessary. Acknowledgements This work was supported by Science and Engineering Research Board, Department of Science & Technology, Government of India [Grant numbers: SR/FT/LS-92/2012; EMR/2016/003524]. Appendix A. Supplementary data Supplementary data to this article can be found online at https://
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Improvisation of a spectrophotometric method to quantify hydroxycitric acid
T
Disha Patel, Aditi Buch∗ Department of Biological Sciences, P D Patel Institute of Applied Sciences, Charotar University of Science and Technology, CHARUSAT Campus, Changa, 388 421, Dist. Anand, Gujarat, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Hydroxycitric acid Improvised spectrophotometric method Hydroxycitric acid-producing microbes
Existing spectrophotometric method to quantify hydroxycitric acid (HCA), although is specific and sensitive; finds limited use owing to poor stability of HCA-metavanadate complex. Present study describes improvisation of this method with respect to source of HCA standard and assay parameters. Assay system consisting of HCA and metavanadate reagent was modified to include 1 M NaOH to neutralize excess acidity. Resulting complex showed λmax at 485 nm, obeying Beer-Lambert's law within concentration range of 33–677 μg/ml, with linear calibration curve showing a good coefficient of determination (R2 = 0.998). Moreover, HCA-metavanadate complex showed enhanced stability retaining up to 70% absorbance even after 60 min of its formation. Similar consistency in scaled-down assay system renders the method suitable for high-throughput screening of HCAproducing microbes. Of the tested metabolites and media components, only tartrate interfered with the spectrophotometric estimation of HCA; a correction factor to eliminate which was also established. Accordingly measured HCA level in the culture supernatant of a bacterial isolate IT6 was comparable to that determined using the standardized HPLC method. The proposed procedure therefore is a convenient, sensitive, accurate and high-throughput method suitable for primary screening of HCA producing microbes; the only ecofriendly alternative source of optically pure HCA.
1. Introduction Hydroxycitric acid (HCA, 1, 2-dihydroxypropan-1,2,3-tricarboxylic acid) is rapidly gaining importance as herbal therapeutic and dietary supplement [1]. Two of four possible stereoisomers of HCA, (2S, 3S) and (2S, 3R), occur naturally and are biologically active, while their corresponding lactones or methyl derivatives are less potent [2]. (2S, 3S)-HCA is principally accumulated in fruit rinds of Garcinia plant species (~10–30% by weight) predominantly surviving in India and SriLanka [3] while (2S, 3R)-HCA is enriched in calyxes and leaves of Hibiscus subdariffa (Roselle), H. rosa-sinensis and H. cannabinus cultivated in several tropical and semitropical countries [2]. HCA has gained more attention in recent years on account of its ability to mediate unique regulatory effect on fatty acid synthesis, lipogenesis and carbohydrate metabolism leading to controlled appetite and weight loss, with no serious side-effects and toxicity as monitored in animal and human systems [3–6]. Hence, Garcinia fruit extract and thence derived stable HCA salts are currently used as a safe and efficacious ingredient in a number of over-the-counter weight loss products worldwide [2]. Moreover, its distinct chiral structure similar to γ-butyrolactone, makes
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HCA an attractive inexpensive starting point for preparation of a range of chiral synthons and ligands for catalysts [2,7]. Unfortunately, availability of natural HCA is restricted due to geoclimatically limited cultivation of source plants, complications in clonal propagation of their elite genotype and difficult stereoselective organic synthesis of bioactive isomers [8–10]. Therefore, microbial production of HCA is being considered as an economical and convenient option for bulk production of natural optically pure HCA. While only few bacterial species are reported to produce (2S, 3R)-HCA [11], fermentation technologies and genome shuffling did not greatly enhance their HCA production [2,8]. Thus, screening of HCA producing bacteria becomes essential, which is usually performed by monitoring the presence of HCA in culture supernatant of bacteria grown in defined minimal medium containing probable HCA biosynthetic precursors like glucose, mannitol, α-ketoglutarate, etc. as sole carbon sources [11]. Qualitatively HCA can be identified by characteristic absorbance maxima at 214 nm; FTIR and HPTLC analysis, IR and 1H NMR spectroscopy as well as paper and thin layer chromatography [12–15]. An existing method to quantify HCA involves acid-base microtitration, which gives the total acidity and hence is non-specific [16]. Most
Corresponding author. E-mail addresses: [email protected] (D. Patel), [email protected] (A. Buch).
https://doi.org/10.1016/j.ab.2019.113412 Received 7 June 2019; Received in revised form 18 August 2019; Accepted 28 August 2019 Available online 29 August 2019 0003-2697/ © 2019 Elsevier Inc. All rights reserved.
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(0.4 ml). AS-2: HCA in 0.05 N H2SO4 (1 ml) +1 N NaHCO3 (1 ml) 2.5% sodium metavanadate (0.4 ml). AS-3: HCA in 0.05 N H2SO4 (1 ml) + 1 N NaOH (1 ml) + 2.5% sodium metavanadate (0.4 ml). HCA extract in 0.05 N H2SO4 as described in section 2.1, was used as the source of HCA throughout all the experiments. Optimization with respect to metavanadate reagent was done by preparing the reagent in 6 N and 3 N H2SO4. One milliliter of 1 N NaHCO3 and 1 N NaOH were separately incorporated in the assay system to neutralize the acidity of the metavanadate reagent. Effect of modified assay system on absorbance maxima of the orange-coloured product was monitored by spectrum analysis of the assay mixtures containing 0.2, 0.4, 0.6 and 0.8 ml of HCA extract, respectively, between 400 and 800 nm using UV–Visible spectrophotometer (UV-1800, Shimadzu), immediately after addition of metavanadate reagent. Assay mixture containing 0.4 ml aliquot of HCA extract was subjected to time-scan analysis immediately after addition of metavanadate reagent, at identified λmax in order to determine stability of HCA-metavanadate complex. The absorbance was monitored at interval of 10 s for 60 min and the change in absorbance with time was used to predict the stability of the HCAmetavanadate complex. Sensitivity of the method was determined by using variable aliquots of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 1.0 ml of HCA extract in appropriate assay system (as discussed subsequently) to generate a standard calibration curve. These measurements were carried out across three independent experiments each using HCA extracted from different capsule. Each experimental set consisted of two replicate measurements each performed in triplicates.
explored method that qualitatively and quantitatively measures HCA from source plant extracts, commercial samples as well as from bacterial culture supernatants, is high-performance liquid chromatography (HPLC) [11,13,17]. However, screening of HCA producing bacteria would require HCA analysis from large number of samples, more so because HCA is relatively a rarely encountered metabolite unlike other structurally related common metabolic intermediates. Besides, HCA-producing bacteria could also produce other organic acids and metabolic by-products depending on the carbon source used; which may interfere with HCA estimation. Therefore, a convenient yet specific and accurate method for quantitative determination of HCA becomes essential to facilitate high-throughput screening of HCA-producing bacteria. A suitable spectrophotometric method based on formation of coloured complex between HCA and sodium metavanadate has been reported previously [18]; however, the method is not widely adopted. The present work describes improvisations in this spectrophotometric method to make it more suitable for use during microbial screening procedures. This work also optimizes the use of commercially available G. cambogia extract as more economic source of HCA standard. 2. Materials and methods 2.1. Extraction of HCA from Himalaya Vrikshamla capsules HCA from one capsule of Himalaya Vrikshamla (containing 350 mg dried Garcinia extract as described by the manufacturer) was extracted in a total volume of 200 ml 0.05 N H2SO4. The extraction was initiated by suspending 350 mg of capsule content in 80 ml of 0.05 N H2SO4 in a screw-capped 250 ml flask and subjecting it to continuous stirring for 30 min at room temperature, using a magnetic stirrer. Subsequently, the suspended particles in the flask were allowed to settle and the supernatant was gradually decanted and filtered through Whatman filter paper #1. The residual particles were similarly treated for re-extraction in 3 steps using 40, 40 and 20 ml 0.05 N H2SO4 respectively. The filtrates collected after all the four steps were pooled and the volume was made up to 200 ml using 0.05 N H2SO4. Thus extracted HCA was used as the source of standard HCA in the spectrophotometric estimations. The residues were finally washed with 2 ml of 0.05 N H2SO4 and the filtrate was subjected to spectrophotometric estimation of HCA so as to confirm complete extraction of HCA. This HCA extract was prepared independently from three capsules similarly processed and were used to obtain replicate observations for generation of HCA standard curve to determine the range of the method.
2.4. Interference by media components and structurally similar biomolecules In order to demonstrate the specificity of the method towards measuring HCA, the assay system was setup to include varying concentrations of probable interfering agents including structurally similar organic acids, commonly used sugars in microbial growth medium and standard microbial growth media. Organic acids and by-product metabolites like citrate, succinate, acetate, malate, tartrate and acetoin were used at maximum concentration of 10 mM; sugars like glucose, sucrose and mannose were used at final concentrations of 10, 20, 40, 60, 80 and 100 mM; while microbial growth media like Luria broth (LB), Nutrient broth (NB) and 1X M9 salt solution (prepared as per the manufacturers’ recommendations) were used in the aliquots of 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 ml; in an otherwise constant assay system composition. Various above probable interfering agents were accommodated within 1 ml of sample volume in the assay system; with volume made up using 0.05 N H2SO4. Subsequently, absorbance of assay mixture at appropriate λmax was measured to indicate successful interaction with metavanadate reagent. A correction procedure was applied to derive correct spectrophotometric measurement of HCA in presence of the interfering agent.
2.2. Detection and quantitation of HCA using HPLC HCA extracted in 0.05 N H2SO4 (section 2.1) was filtered using 0.22 μ nylon membrane and subjected to HPLC analysis (obtained as outsourced service from Vasu Research Center, Vadodara, Gujarat, India) using C18 column. Column was maintained at ambient temperature and HCA was eluted in isocratic system using 0.01 M potassium dihydrogen phosphate as mobile phase at a flow rate of 1 ml/min and detected at 215 nm using PDA detector. HCA levels in this extract were quantified with reference to the area under the peak generated by 74.2% pure, 0.1 mg/ml Ca2+-HCA standard prepared in 3% orthophosphoric acid.
2.5. Spectrophotometric determination of HCA levels in bacterial culture supernatants A locally isolated Gram positive, endospore forming bacterial isolate IT6 was inoculated in 3 ml of nutrient broth and allowed to grow with overnight shaking at 37 °C with 150 rpm. A 100 μl of this fresh inoculum was added in a 30 ml M9 minimal broth supplemented with 100 mM glucose as sole carbon source and the culture was allowed to grow under shaking conditions (37 °C, 150 rpm). After 24 h of growth, the culture was harvested by centrifugation at 10,000 rpm for 5 min and the resultant supernatant was stored at −20 °C until further analysis. One milliliter of the supernatant was used to quantify HCA levels spectrophotometrically as in section 2.3 along with the necessary corrections applied as well as through HPLC analysis as in section 2.2.
2.3. Optimization of spectrophotometric method for estimation of HCA The method is largely based on colour complex formation by HCA in presence of metavanadate [18]. Optimization of this method with respect to reagent preparation, composition of assay system, incubation time and absorbance maxima of the coloured complex was carried out by introducing several modifications as per following assay system (AS) combinations. AS-1: HCA in 0.05 N H2SO4 (1 ml) + 2.5% sodium metavanadate 2
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Fig. 1. Quantitation of HCA levels in extracts of Himalaya Vrikshamla using HPLC. (a) Chromatogram of Ca2+-HCA standard; inset image shows the molecular structure of HCA (b) Representative chromatogram of HCA extract in 0.05 N H2SO4.
3. Result and discussion
and 0.2 ml of 5% sodium metavanadate. Formation of reddish orange coloured complex exactly after 20 min was measured as absorbance at 467 nm, against a blank similarly prepared without HCA. The modifications introduced in the present work are as follows: (i) HCA standard was extracted and prepared in 0.05 N H2SO4 (ii) sodium metavanadate reagent was prepared in 6 N H2SO4 initially to ensure its complete solubility and (iii) assay volume was reduced drastically to maintain proportionate concentration of metavanadate reagent as indicated in primary assay system AS-1. One milliliter of HCA extract used in AS-1, although formed a typical red-orange coloration at first instance, it rapidly disappeared upon incubation, converting into a blue coloured complex with absorbance maxima of ~760 nm almost completely at the end of 40 min (Fig. 2a). This could be probably due to rapid change in the oxidation states of vanadium due to excess acidity of the assay system caused by metavanadate reagent prepared in 6 N H2SO4 [19]. Use of metavanadate reagent prepared in 3 N H2SO4 in AS-1 could alleviate this instability to certain extent however could not maintain the stability of HCA-metavanadate complex for even 20 min as suggested originally [18]. Since reagent preparation could not be further altered owing to poor solubility of sodium metavanadate, assay system was further amended with a neutralizing agent to overcome the effect of excess acidity. Accordingly, constituting the assay system AS-2 with additional 1 ml sodium bicarbonate could stabilize the orange-red coloration however monitoring the absorbance became difficult due to constant effervescence. Substituting NaHCO3 by NaOH of equivalent
3.1. Extraction of HCA from Himalaya Vrikshamla capsule and optimization of its use as HCA standard EDTA salt of HCA used to generate calibration curve in existing method for spectrophotometric quantitation of HCA, is poorly available commercially. While K+ and Ca2+ salts of HCA are expensive, extraction and preparation of these salts from appropriate plant source is an elaborate process. Therefore, developing a relatively easily accessible and economic source of standard HCA to facilitate the calibration process, could be useful. Commercially available Himalaya Vrikshamla capsules were utilized to extract the Garcinia-type HCA using 0.05 N H2SO4 in a simple method based on the extraction procedure described by Hida et al. [11]. A total of 200 ml of 0.05 N H2SO4 in a stepwise procedure could extract 677 ± 64 μg/ml HCA as averaged from extracts from three capsules, as determined by HPLC analysis (Fig. 1); while ensuring complete recovery as evident by no HCA detected in the recovery filtrate. 3.2. Improvisation of spectrophotometric method for quantitation of HCA with respect to spectral properties and analytical variables The assay system as per Antony et al. [18] primarily consisted of 1 ml of pure HCA-EDTA in 0.1 N H2SO4 made up to 100 ml total volume 3
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Fig. 2. Spectral properties of HCA-metavanadate complex. (a) Instability of HCA-metavanadate complex in presence of excess acidity (b) Absorbance spectrum of orange-red HCA-metavanadate complex as obtained using various aliquots with increasing amount of HCA (c) Time scan of HCA-metavanadate complex over 60 min immediately after the addition of the reagent.
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Fig. 3. Calibration curves for HCA quantitation Graphs generated using (a) modified assay system AS-3 and (b) scaled-down assay system. Results are expressed as Mean ± SD of (a) eighteen and six independent measurements at 485 nm (as explained in Section 2.3) and 467 nm, respectively (b) three independent measurements each performed in duplicates.
485 nm after an incubation of 30 min at room temperature.
volume and concentration to constitute assay system AS-3, stabilized the HCA-metavanadate complex enabling monitoring of stable absorbance even at the end of 20 min as monitored visually. This modification of the assay system shifted the absorbance maxima of the HCAmetavanadate complex from 467 nm to 485 ± 1.63 nm, as monitored by scanning assay mixtures containing varying HCA concentrations between 400 and 800 nm (Fig. 2b). Subsequently, time-scan analysis of the HCA-metavanadate complex formed in AS-3, at 485 nm suggested that the orange-red complex required initial 30 min to stabilize quickly losing up to 22.1% of initial absorbance (Fig. 2c). For 30 min of further incubation, the complex remained relatively stable retaining 93% of its absorbance recorded after first 30 min; implying extended stability of the HCA-metavanadate complex. Thus, unlike the original method where measuring the absorbance exactly at 20 min was crucial; this method showed improved stability leading to more convenient application. Accordingly, a revised procedure was prescribed based on AS-3 which consisted of HCA standard in 1 ml, 1 ml of 1 M NaOH and 0.4 ml of 2.5% sodium metavanadate reagent; with resultant orange-red complex formed to be measured at
3.3. Validation of the proposed spectrophotometric method Sensitivity of the modified method was determined by measuring the absorbance of HCA-metavanadate complex formed with various amounts of HCA at 485 nm. The resultant calibration curve followed Lambert-Beer's law between 33 μg/ml to around 677 μg/ml of HCA, with a linear curve equation of y = 0.0024x-0.0422 and R2 value of 0.998. On the contrary, at 467 nm, the linearity of the curve was relatively maintained only up to 450 μg/ml as per the modified method (Fig. 3a). The sensitivity of the original method has been demonstrated to be 5–50 μg/ml [18]. The revised method apparently possessed extended sensitivity for HCA estimation towards higher concentrations; however, the sensitivity at concentrations lower than 33 μg/ml was unclear. Also, a high-throughput method requiring lower sample volumes as well as reduced assay system would be useful, especially with respect to screening of large number of bacteria for HCA production. To address 5
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Fig. 4. Specificity of HCA quantitation from microbial cultures with respect to interference from growth media components. MM: Minimal medium; Results are expressed as Mean ± SD of three independent measurements. Table 1 Comparative analysis of HCA measured using optimized spectrophotometric method and HPLC Results are expressed as Mean ± SD of three independent measurements; ns non-significant when compared between spectrophotometric and HPLC measurements. Both HCA and tartrate levels were normalized with respect to bacterial growth measured as OD600nm. Bacterial isolate
HCA measurements (μg/ml/OD600nm) Spectrophotometric method (with correction)
IT-6
100.13 ± 35.18
ns
Tartrate measurements (μg/ml/OD600nm) HPLC
Spectrophotometric method (with correction)
88.28 ± 36.90
329.88 ± 36.88
ns
HPLC 459.61 ± 89.39
the medium lead to maximum reduction in the absorbance, suggesting that PO43− was possibly responsible for the interference observed. Phosphate ions are known to form stable complex with oxovanadium (IV) cations [20]. Since phosphate cannot be omitted from bacterial growth media being an essential nutrient, a blank consisting of plain unspent growth medium becomes essential while using the method for HCA from bacterial culture supernatants. Sugars like glucose, sucrose and mannose showed negligible interference as indicated by maximum OD485nm of about 0.1 even at concentrations as high as 100 mM (Supplementary Material-Fig. S1). Organic acids like succinate, acetate, malate and common metabolic byproduct acetoin did not significantly interfere with the estimation (Supplementary Material-Fig. S2). In fact, malate and citrate marginally reduced the intensity of the metavanadate reagent as suggested by negative absorbance values. On the contrary, tartrate showed significant absorbance in the spectrophotometric assay when measured at 485 nm (Supplementary Material-Fig. S2); although the sensitivity of the method towards tartrate was relatively poor as compared to that for HCA. However, spectrophotometric quantitation of HCA using metavanadate reagent at 485 nm would be significantly influenced by presence of tartrate. In order to take into consideration this bias, a correction procedure was applied as described by Dumas et al. [21]. Conventionally, spectrophotometric determination of tartaric acid frequently used in wine analysis involves a similar method where tartratemetavanadate complex is measured at 530 nm [22]. Therefore, different concentrations of tartaric acid within the range of the method were subjected to reaction with metavanadate and absorbance of the resultant complex was additionally measured at 530 nm. This calibration curve followed Lambert-Beer's law between the tested concentrations of 141 μg/ml to 1410 μg/ml of tartrate, with a linear curve equation of y = 0.0004x-0.0725 and R2 value of 0.991 (Supplementary Material-Fig. S2). Subsequently, actual absorbances
this, the modified assay system was proportionately scaled down 10 folds with respect to HCA sample, NaOH and metavanadate reagent volumes. Resulting assay system of total 0.24 ml was set up in a 96-well plate with all other operational parameters maintained same, to obtain the calibration curve by measuring absorbance at 485 nm. The scaleddown method obeyed Lambert-Beer's law between 67 and 542 μg/ml of HCA concentrations tested, with the linear curve equation of y = 0.0016x - 0.0593 and R2 value of 0.981 (Fig. 3b). This linear equation was almost identical to that derived for the calibration curve generated using AS-3; thus, suggesting that the scaled-down method could as well be successfully used to quantitate HCA using lower sample volumes.
3.4. Specificity of the proposed method for HCA quantitation: emphasis on possible interference from microbial cultures The convenient and high-throughput nature of the proposed spectrophotometric method makes it suitably applicable in screening HCAproducing microbes. Considering microbial HCA production, structurally related di- and tri-carboxylic acids either used as growth supplement or produced as by-products of microbial growth as well as other growth media components are likely to interfere. Selected of these metabolites and components were analyzed for their individual ability to contribute to absorbance at 485 nm when incorporated in AS-3 instead of HCA sample. Commonly used rich growth media like Luria broth and nutrient broth, contributed to the absorbance at 485 nm at low yet linear consistency showing maximum OD485nm of about 0.4 (Fig. 4). On the other hand, 1X M9 salt solution showed significantly high interference, showing OD485nm of about 1.2 at maximum aliquot of 1 ml (Fig. 4). In order to decipher the specific media component(s) causing the interference, 1X M9 salt solution devoid of one component at a time was used in the assay. Omission of Na2HPO4 and KH2PO4 from 6
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doi.org/10.1016/j.ab.2019.113412.
measured at 485 nm for HCA concentrations within this range were used to compute the proportion of tartrate measured as OD485nm relative to OD530nm (Supplementary Material-Fig. S3); a value found to be ranging between 17.7% and 42.3%, which significantly increased with higher absorbance values (Pearson's product-moment correlation: r = 0.999). The resulting linear function of y = 1.1574x + 0.2183 (Supplementary Material-Fig. S3) could be used to derive unbiased estimate of HCA. Conversely, to rule out the possible interference of HCA while quantifying tartrate levels, absorbance of HCA-metavanadate complex was also measured at 530 nm which generated a linear curve over the tested concentration range of 203–677 μg/ml, with equation of y = 0.001x-0.1717 (Supplementary Material-Fig. S4). Relative average absorbance of tartrate at 530 nm was 35.09 ± 6.05% of that of the HCA across the entire concentration range.
Declaration of interest None. References [1] J.W. Yun, Possible anti-obesity therapeutics from nature – a review, Phytochemistry (Oxf.) 71 (2010) 1625–1641 https://doi.org/10.1016/j.phytochem.2010.07.011. [2] T. Yamada, H. Hida, Y. Yamada, Chemistry, physiological properties, and microbial production of hydroxycitric acid, Appl. Microbiol. Biotechnol. 75 (2007) 977–982 https://doi.org/10.1007/s00253-007-0962-4. [3] B.S. Jena, G.K. Jayaprakasha, R.P. Singh, K.K. Sakariah, Chemistry and biochemistry of (−)-hydroxycitric acid from Garcinia, J. Agric. Food Chem. 50 (2002) 10–22 https://doi.org/10.1021/jf010753k. [4] C. Hansawasdi, J. Kawabata, T. Kasai, Alpha-amylase inhibitors from roselle (Hibiscus sabdariffa Linn.) tea, Biosci. Biotechnol. Biochem. 64 (2000) 1041–1043 https://doi.org/10.1271/bbb.64.1041. [5] I. Onakpoya, S.K. Hung, R. Perry, B. Wider, E. Ernst, The use of Garcinia extract (hydroxycitric acid) as a weight loss supplement: a systematic review and metaanalysis of randomised clinical trials, J. Obes. (2011) 509038 https://doi.org/10. 1155/2011/509038. [6] F. Márquez, N. Babio, M. Bulló, J. Salas-Salvadó, Evaluation of the safety and efficacy of hydroxycitric acid or Garcinia cambogia extracts in humans, Crit. Rev. Food Sci. Nutr. 52 (2012) 585–594 https://doi.org/10.1080/10408398.2010.500551. [7] I. Ibnusaud, P.T. Thomas, M. Rani, P.V. Sasi, T. Beena, A. Hisham, Chiral γ-butyrolactones related to optically active 2-hydroxycitric acids, Tetrahedron 58 (2002) 4887–4892 https://doi.org/10.1016/S0040-4020(02)00431-3. [8] H. Hida, T. Yamada, Y. Yamada, Genome shuffling of Streptomyces sp . U121 for improved production of hydroxycitric acid, Appl. Microbiol. Biotechnol. 73 (2007) 1387–1393 https://doi.org/10.1007/s00253-006-0613-1. [9] J. Govinden-Soulange, N. Boodia, C. Dussooa, R. Gunowa, S. Deensah, S. Facknath, B. Rajkoma, Vegetative propagation and tissue culture regeneration of Hibiscus sabdariffa L. (Roselle), World J. Agric. Sci. 5 (2009) 651–661. [10] R.P. Tembe, M.A. Deodhar, Clonal propagation and hydroxycitric acid production from in vitro shoot cultures of Garcinia indica using root suckers as explant, in Vitro Cell, Dev. Biol. Plant 47 (2011) 399–409 https://doi.org/10.1007/s11627-0119349-4. [11] H.H. Hida, T.Y. Amada, Y.Y. Amada, Production of hydroxycitric acid by microorganisms, Biosci. Biotechnol. Biochem. 69 (2005) 1555–1561. [12] G.K. Jayaprakasha, K.K. Sakariah, Determination of organic acids in leaves and rinds of Garcinia indica (Desr.) by LC, J. Pharm. Biomed. Anal. 28 (2002) 379–384 https://doi.org/10.1016/S0731-7085(01)00623-9. [13] R. Ravikumar, B.V. Ranganathan, S. Vinothkumar, S.R. Yamuna, P. Yasodha, P. Priyanka, R.V. Rasikaa, Y. Sownisha, Extraction and characterization of hydroxy citric acid from Garcinia combogia cultivated at two different locations of Malabar and Srilanka, Int. J. Pharm. Chem. 07 (2017) 100–102 https://doi.org/10.7439/ ijpc.v7i7.4298. [14] M.G. Soni, G.A. Burdock, H.G. Preuss, S.J. Stohs, S.E. Ohia, D. Bagchi, Safety assessment of (-)-hydroxycitric acid and super CitriMax, a novel calcium/potassium salt 42 (2004), pp. 1513–1529 https://doi.org/10.1016/j.fct.2004.04.014. [15] L.C. Bainto, E.I. Dizon, A.C. Laurena, K.A.T. Castillo-Israel, Isolation and quantification of hydroxycitric acid from batuan [Garcinia binucao (Blanco) Choisy] fruit, Int. J. Food Res. 25 (2018) 706–711. [16] C. Vijay, P. Chary, A novel method of microtitration and rapid extraction of (-) hydroxycitric acid for identification of elite chemotypes of Garcinia cambogia (Gaertn.) Desr. (Clusiaceae), J. Res. 37 (2009) 7–13. ANGRAU. [17] A. Gogoi, N. Gogoi, B. Neog, Estimation of (-)-hydroxycitric acid (hca) in Garcinia lanceaefolia roxb. using novel HPLC methodology, Int. J. Pharm. Sci. Res. 5 (2014) 4993–4997 https://doi.org/10.13040/IJPSR.0975-8232.5(11).4993-97. [18] B. Antony, W. Varghese, M.B. Elisa, Spectrophotometric determination of hydroxycitric acid, Indian J. Pharm. Sci. 61 (1999) 316–317. [19] S.K. Sinha, A study of reduction of vanadate, Int. J. Adv. Res. Innov. Ideas Educ. 3 (2017) 206–212. [20] D.C. Crans, Fifteen years of dancing with vanadium, Pure Appl. Chem. 77 (2005) 1497–1527 https://doi.org/10.1351/pac200577091497. [21] Z. Dumas, A. Ross-Gillespie, R. Kümmerli, Switching between apparently redundant iron-uptake mechanisms benefits bacteria in changeable environments, Proc. Biol. Sci. 280 (2013), https://doi.org/10.1098/rspb.2013.1055 20131055. [22] M.G.F. Sales, C.E.L. Amaral, C.M. Delerue Matos, Determination of tartaric acid in wines by FIA with tubular tartrate-selective electrodes, Fresenius’ J. Anal. Chem. 369 (2001) 446–450.
3.5. Application of proposed spectrophotometric method to quantitate HCA from bacterial culture supernatant To validate the applicability of the proposed method, HCA levels in the culture supernatants of bacterial isolate IT6 grown on glucose as sole carbon source (as described in section 2.5), determined spectrophotometrically were verified against those determined using HPLC. HPLC profile of the culture supernatant revealed the presence of tartrate in addition to HCA (Table 1). For spectrophotometric measurements, 1 ml of culture supernatant was reacted with metavanadate reagent as in AS-3 and the absorbance of the resultant complex was measured at both 485 and 530 nm, to quantify HCA and tartrate, respectively. Our results revealed that HCA levels measured through proposed spectrophotometric method after application of correction procedures, were similar to those determined using HPLC (Table 1). To determine the tartrate levels, 35% of the absorbance measured at 530 nm was used to calculate tartrate concentrations using the applicable linear equation (y = 0.0004x-0.0725), which were comparable with those determined through HPLC (Table 1). Collectively, the advantage of the proposed improvised method is that it could utilize a conveniently available HCA standard as well as possessed enhanced stability and improved sensitivity. Moreover, the method could be scaled down successfully to reduce the assay volume thus being suitable for quick, accurate and simultaneous analysis of large number of samples, which could be encountered especially while screening HCA producing bacteria. Furthermore, the correction procedure introduced allows precise measurement of HCA from microbial culture supernatant, rendering the method largely applicable. Nevertheless, owing to significant interference by phosphate, this method may not be useful for optimizing HCA production by microbes grown on different minimal media and in such conditions use of HPLC becomes necessary. Acknowledgements This work was supported by Science and Engineering Research Board, Department of Science & Technology, Government of India [Grant numbers: SR/FT/LS-92/2012; EMR/2016/003524]. Appendix A. Supplementary data Supplementary data to this article can be found online at https://
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