Bioactive Compounds from Okra Seeds: Potential ... - ACS Publications

Chapter 16

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Bioactive Compounds from Okra Seeds: Potential Inhibitors of Advanced Glycation End Products Bishambar Dayal,*,1,2 Vineela Reddy Yannamreddy,2 Ajay P. Singh,3 Michael Lea,2 and Norman H. Ertel1 1Department

of Medicine, UMDNJ-New Jersey Medical School, Newark, New Jersey 07103 2Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, Newark, New Jersey 07103 3Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 08901 *E-mail: [email protected]

Advanced Glycation End Products (AGEs) have been associated with the micro-vascular complications in diabetes and other age-related neurodegenerative diseases. We have investigated the effect of glycosylation of bovine serum albumin (BSA) in the presence of okra seed extracts. The degree of protein glycation with glucose was assessed by tryptophan AGE, AGE-induced cross-linking by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and NanoDrop spectrophotometry. Fluorescence spectra (excitation at 360 nm and read at 460nm) of BSA solution incubated for 90 days with okra seed extracts showed significant inhibitory potential (45-50%) at 0.1mg/ml concentration in a dose dependent manner. Intensity of fluorescence spectra combined with densitometry measurements exhibited 50% inhibition of glycation of BSA. We propose that the fluorescence emission spectra were altered by glycation when incubated with okra seed extracts and thus inhibited the advanced glycation end products. Further studies are needed to understand the bioactive compounds present in okra seed extracts in in vivo models.

© 2012 American Chemical Society In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Introduction Recent studies by Huang et al. (1) have predicted that people inflicted with diabetes will double and the treatment costs will triple in the next 25 years. This study further highlighted and reinforced the importance of public health measures to educate and bring awareness in people about this epidemic. The number of people becoming obese in United States and also in India has been rising steadily for many years (2). The data shows that one in five people with Type-2 diabetes are morbidly obese while 1 in 3 African-American adults with diabetes, are morbidly obese or approximately 100 pounds overweight (1–3). The major cause and control of Type-2 diabetes is not clear but recent studies have focused on dietary factors which may be involved in the regulation and control of this devastating disease. The accumulation of Advanced AGEs due to non enzymatic glycation of proteins has been implicated in both Type 2- diabetes and cardiovascular disease (4–6). This reaction takes place between reducing sugar and the free amino group of the protein, ε-amino group of lysine and the guanidine group of arginine forming an Amadori product) (7–9). The formation of complex glycoxidation products, carboxylated methyl lysines (CMLys), brown pigments, protein cross-links, antigenic AGEs and pentosidine have been shown to accumulate irreversibly in several human and animal tissues during aging (10, 11). This process the so called the Maillard reaction or advanced glycation occurs slowly in vivo causing damage to proteins. Further studies have illustrated the role of AGEs in the development of several pathophysiological associated age-related neurodegenerative diseases such as Alzheimer’s, arthritis, end-stage renal disease, nephropathy and neuropathy and cataract formation (10–16). Oxygen radicals are implicated in Maillard reaction damage to proteins (14). Since obesity is a major environmental risk factor for diabetes (1–3), lifestyle changes have been recommended to reduce obesity. Low-fat low-calorie vegetarian fruit diet rich in polyphenols and antioxidants may be helpful in the prevention and treatment of various age-related neurodegenerative diseases. Studies have indicated that a high fat diet supplemented with flavonoids may reverse the progression of diabetes, heart disease and cancer (17–22). In the present study okra seed (Fig. 1) extracts were examined to see their beneficial inhibitory effects on AGEs formation and antioxidant activity.

Experimental Procedures Microwave Extraction A rapid microwave-assisted enhancement reaction chemistry procedure (23–27) was utilized for the extraction of flavonol glycosides using methanol or ethanol solvents. Briefly, 5g of powered okra seeds was extracted with methanol (5ml) in the microwave oven (MW) at low power setting. After filtration each extract was evaporated under nitrogen to dryness and used for chemical analysis and bioassays. 288 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 1. Okra: Abelmoschus esculentus (L.) Moench.

Determination of Total Phenolics and DPPH Radical Scavenging Activity Assays were performed with methanolic extracts. Phenolics were measured using the Folin- Ciocalteau reagent and expressed as mg cholorogenic acid equivalent per gram wet weight. Anti-oxidant activity was determined using a 2, 2-diphenyl-1-picrylhydrazyl assay and is expressed as mg trolox equivalent per gram wet weight (34).

Identification of Bioactives by LC-MS/MS Flavonoids with different sugar moieties (Fig. 2) were identified by high-performance liquid chromatography coupled with mass spectrometry (HPLC–MS–MS) (28, 29). The HPLC system (Shimadzu Co., 10VP Series, Columbia, MD, USA) employed a HypersilGold C18 (3 µm particle size; 150 mm length × 3.0 mm ID; Thermo Electron Co., Bellefonte, PA). Five microlitre was injected onto the column and a gradient elution was used for separations. Solvent A consisted of 10% MeOH in H2O adjusted to pH 3.5 with formic acid. Solvent B consisted of 20% H2O (pH 3.5), 20% MeOH, and 60% acetonitrile. At a flow rate of 0.3 mL min−1, the following gradient was used: 0 min, 100% A; 10 min 20% A; 20 min, 40% A; 40 min, 0 % A; held at 0% A for 15 min. Five minutes of equilibration at 100% A was performed before and after each injection. Effluent from the column was introduced into a triple-quadrupole mass spectrometer (Micromass Inc., Beverly, MA, USA) equipped with a pneumatically-assisted electrospray ionization source (ESI). Mass spectra were acquired in the negative ion mode under the following parameters: capillary voltage, 3 kV; source block 289 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

temperature, 120 °C; desolvation gas temperature, 400 °C. Nitrogen was used as the drying and nebulizing gas at flow rates of approximately 50 and 450 L/h. For full-scan HPLC–ESI-MS analysis, spectra were scanned in the range of 50–1200 m/z. Data acquisition and processing were performed using a Mass-Lynx NT 3.5 data system (Micromass Inc., Beverly, MA, USA).


NMR Spectroscopy of Okra Seed Extract One- Dimensional Proton, 13C NMR spectra were recorded on a Bruker 500 and 125 MHz respectively at room temperature using 3 mm tubes. Samples (3 mg) were dissolved in CD3OD. Attached-Proton-Test (APT) spectra were recorded to identify "multiplicity" (quaternary, CH, CH2 or CH3) of peaks in a 13C spectrum as described previously (30, 31).

Glycation of BSA BSA (0.1g/ml), was incubated at 37 °C with 1M glucose in 0.4M phosphate buffer, pH 7.4 containing 0.02% sodium azide to prevent degradation (8, 13). After thorough mixing okra seed extract (0.1g/ml) was added. A corresponding control without the okra seed extract was also prepared and the mixture kept in the same incubation chamber for a period of 30-90 days.

SDS-PAGE Analysis Effect of Okra seed extracts on glucose-induced glycation of BSA in the absence and presence of aqueous okra seed extracts was resolved and analyzed via SDS-PAGE analysis (Fig. 4). Soluble protein and molecular weight standards (kDa) were loaded onto 8% polyacrylamide gel and were visualized via Commassie blue staining procedure.

Densitometry Measurements Densitometry plots were generated with Image J software of the sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) of glycated BSA with and without okra seed extract. The sample concentrations (1A,1B and1C, 0.1mg/ml) without okra seed and (2A, 2B and -2C, 0.1mg/ml) with okra seed extract treated BSA samples are shown in (Fig. 5).

290 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Advanced Glycation End Products (AGE) Analysis Fluorescence of the samples was measured at the excitation and emission maxima of 360 and 460 nm, respectively (13). An un-incubated blank containing BSA, glucose and okra seed extract as an inhibitor was also prepared and measured (Fig. 6).


Protein Concentration via NanoDrop Spectrophotomter and Fluorospectrometer The individual variation in BSA concentrations in the presence and absence of okra seed extracts were determined utilizing Nano Drop spectrophotometer and fluorospectrometer which has the ability to analyze multiple emission profiles from a single sample (Fig. 7).

Results and Discussion The okra seeds (5 g) were treated with methanol (5ml) and heated using a microwave oven. The extracts/aliquots were used for the identification of major flavonoids as described in the experimental section. Results of liquid chromatography-mass spectrometry data as presented in Table 1 revealed five major flavonoids. A representative LC-MS spectra of quercetin-diglucoside is described in Fig. 3. From the analysis of the LC-MS/MS data in the negative ion mode five flavonoids exhibited fragment ions [M-162]+ after the loss of the respective sugar moiety (glucose, rhamnose or galactose) for glycosides and [M-132]+ for arabinosides (Fig. 2). The LC-MS/MS data presented in Table 1 is based on LC-MS mass fragmentation pattern and also comparison with authentic standards.

Table 1. Identified Flavonoids from Okra Using LC-MS-MS [M-H]-

Name of The Compound

Retention Time (min)

433

3,5,7,3′,4′-pentahydroxyflavonol-3O-α-L-arabinofuranoside

22.27

447

Kaempferol-3-O-glucoside

25.76

609

Quercetin-3-O-rutinoside

20.02

463

Quercetin-3-O-glucoside

20.95

625

Quercetin-3-O-diglucoside

16.76



Figure 2. Chemical structures of flavonoids identified from okra seed extract.

Figure 3. Typical HPLC analysis top panel (Rt=16.76 min) and Electrospray-ionization mass spectra of quercetin-3-O-diglucoside bottom panel illustrates [M-H]-= 625 based on LC/MS/MS fragmentation pattern in the negative ion mode. As illustrated in Figure 2, the compounds present in okra seed extract were flavonoids with different sugar moieties. Their structural identity was determined using ESI-MS. 292 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Discussion The okra (Abelmoschus esculentus) vegetable is a valuable source of micronutrients (33) and is a major source of flavonoids (Fig. 2). Polyphenolics present in okra seed have potent antioxidant properties (Table 2) and thus inhibit generation of free radicals and cell death (33–35).


Table 2. Distribution of Phenolics and Antioxidant Activity in Okra Okra parts

Phenolic (mg/g)

Antioxidant Activity

Skin

0.20

0.22

Skeleton

0.09

0.23

Seed

2.85

7.55

Stem

0.13

0.59

Assays were performed with methanolic extracts. Phenolics were measured using Folin- Ciocalteau reagent and expressed as mg chlorogenic acid equivalent per gram wet weight. Anti-oxidant activity was determined using a 2, 2-diphenyl1-picrylhydrazyl assay and is expressed as mg trolox equivalent per gram wet weight. Values are means of duplicate determinations. Since oxidative stress is clearly associated with cellular dysfunction such as in diabetes, the bio-active compounds present in okra seed may play a key role in the prevention and treatment of diabetes. Upon administering okra juice to volunteers, its soluble fiber helped to lower their serum cholesterol and reduced blood sugar levels. It has been suggested that In vitro binding of bile acids with the phenolics present in okra is the highest (16%) in comparison to other vegetables (36). The faecal excretion of bile acids has been hypothesized as a possible mechanism by which soluble okra fiber lowers cholesterol (36). The mucilage or the insoluble portion helps to maintain intestinal tract healthy and may be helpful in alleviating inflammation in ulcerative colitis/colon cancer and inflammatory bowel disease syndromes . In the dietary context, the most important compounds present in okra seeds are polyphenolic flavonols namely, quercetin and kaempferol, in addition to cinnamic acid derivatives (Fig. 2). The flavonols present in okra seeds are in glycosylated form, with one, or two, sugar moieties (hexoses, pentoses, rhamnoses) attached to flavonol hydroxyl groups (30–32). Further studies have shown that flavonols have anti carcinogenic and antioxidant properties (32–37). More recent studies have pointed out the role of such small molecules as sirtuin activators and a good target for diseases of aging such as diabetes, cancer, metabolic diseases, inflammation and neurodegenerative diseases (38–41). We have investigated the effect of glycosylation of bovine serum albumin (BSA) in the presence of okra seed extracts. The glycosylated and non-glycosylated protein bands were resolved on SDS- polyacrylamide gel 293 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


electrophoresis (SDS-PAGE). The degree of protein glycation with glucose was assessed by tryptophan AGE, AGE-induced cross-linking by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and NanoDrop spectrophotometry. The Fig. 4 presents effect of okra seed extracts on glucose-induced glycation of BSA in the absence and presence of aqueous okra seed extracts by SDS-PAGE analysis. Soluble protein was loaded onto 8% polyacrylamide gel.

Figure 4. *Effect of okra seed extracts on glucose-induced glycation of BSA in the absence and presence of aqueous okra seed extracts by SDS-PAGE analysis. Soluble protein was loaded onto 8% polyacrylamide gel. Molecular weight standards (kDa) are indicated alongside of the gel. Lane PM- Molecular weight markers; Lane 1- BSA standard 0.1mg/mL; Lane 2- Sample 1A; Lane 3Sample 1B; Lane 4- Sample 1C; Lane 6- Sample 2A; Lane 7- Sample 2B; Lane 8- Sample 2C.

The protein bands in this SDS gel were resolved and visualized by Coomassie blue staining. The positions of molecular mass markers (M) in (kilodaltons) are shown to the left of the gels in panels treated without/with okra seed extracts) The results of SDS Page combined with densitometric plots as presented in (Fig. 5) with BSA (0.1mg/ml, 1A-1C and 2A-2C) without and with okraseed extract show the inhibition of glycation and AGE. Fluorescence measurements at the 294 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


excitation and emission of 360 and 460 nm also show the inhibition of glycation and AGE with okra seed extracts versus BSA and glucose alone when incubated for 90 days (Fig 6). This was further substantiated with protein concentration measurements via nanodrop spectrophotometer (Fig. 7) showing inhibition at different concentrations in comparison to no inhibitor added at the physiological concentrations. The degree of protein glycation as assessed by tryptophan AGE (Fig. 6), AGE-induced cross-linking by SDS-PAGE showed significant inhibitory potential (45-50%) at 0.1mg/ml concentration in a dose dependent manner. Intensity of fluorescence spectra combined with densitometry measurements exhibited 50% inhibition of glycation of BSA with glucose (Figs. 4-7).

Figure 5. Densitometry plots generated with Image J software of the sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) of glycated BSA without okra seed extract (1A-1C) and (2A-2C) with okra seed extract treated samples.

We believe fluorescence emission spectra were altered upon glycation when incubated with okra seed extracts in vitro and thus inhibited the advanced glycation end products (13–16, 42–49).The glycosylated BSA protein concentration was also decreased as a result of okra seed treatment as measured via nanodrop spectrophotometer (middle curve Fig. 7) . Glycosylation of BSA in the presence of okra seed extracts leads to a change in the conformation of the protein probably 295 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


due to an increased exposure of tryptophan residues (42–48). Thus the glycation of BSA in the absence and presence of aqueous okra seed extracts by SDS-PAGE analysis. Soluble protein was loaded onto 8% polyacrylamide gel. Molecular weight standards (kDa) are indicated alongside of the gel. Lane PM- Molecular weight markers; Lane 1- BSA standard 0.1mg/mL; Lane 2- Sample 1A; Lane 3Sample 1B; Lane 4- Sample 1C; Lane 6- Sample 2A; Lane 7- Sample 2B; Lane 8- Sample 2C. bioactive compounds present in okra seed extracts may serve the same mechanisms of inhibition in vivo as well.

Potential Inhibition of Nonenzymatic Glycosylation of HDL Apolipoprotein A-1 Cholesterol Levels with Okra Juice Hyperglycemia in diabetes type-2 is caused by the post-translational nonenzymatic glycosylation of plasma and cellular proteins (49). As illustrated in earlier the reaction of glucose with protein alpha and (ε-amino group of lysine and the guanidine group of arginine forming an Amadori product) occurs in vivo (7, 9, 11, 12). This reaction leads to the loss of charged groups on lysine residues causing conformational changes in proteins. We recently elucidated HDL apolipoprotein A-1 cholesterol levels (50) in severely controlled Type-2 diabetes patients and found increased levels of glycosylated HDL apolipoprotein A-1 Cholesterol. We also carried out studies to measure inflammatory molecular markers of coronary risk such as high-sensitivity C-Reactive Protein (hs-CRP) and interleukin-6 (IL-6) and found that elevated levels of IL-6 accelerates the hs-CRP Pathway (50–54).

Figure 6. Fluorescence spectra exhibiting glycation of BSA with (lower curve) and without okra seed extract (upper curve).



Figure 7. Evaluation of BSA protein concentration in the presence and absence of okra seed extract using NanoDrop Spectrophotometer.

Since in diabetes patients hyperglycemia results in the nonenzymatic glycosylation of many proteins (glycation is the attachment of sugar to proteins), a basal level of HDL apolipoprotein A-1, a cardiovascular biomarker and a major carrier protein in High Density Lipoprotein cholesterol undergoes glycation in normal individuals. But there is a nearly 400% increase in the level of HDL-apoA-1 glycation in diabetic patients (49). The level of glycation has been positively correlated with the degree of hyperglycemia and such a functional abnormality in HDL-apoA-1 is responsible for the accelerated development of atherosclerosis in diabetic patients (49). Since there is 16 % binding of bile acids with okra juice (36) food fractions prevent their reabsorption and stimulate plasma and liver cholesterol transformation to synthesize more bile acids thus lowering cholesterol. Since advanced glycation end products and atherogenic glycosylated or oxidized LDL is formed in vivo in diabetic patients, we believe the therapeutic mediation of inhibiting protein glycation by Okra juice would not only prevent the progression of diabetes but also cardiovascular disease as well. Furthermore the levels of inflammatory molecular markers of coronary risk such as high-sensitivity C-Reactive Protein (hs-CRP) and interleukin-6 (IL-6) may also be lowered (50–55).



Supersized LDL and HDL Cholesterol Carriers Key to Longevity of Life and The Potential Role of Okra Juice Potential inhibition of glycosylated or oxidized LDL by Okra seed extract may suggest an extremely valuable antidiabetic and antiatherogenic therapy. Recent studies have shown that smaller sized LDL-cholesterol particles get stuck or clog the arteries much faster than their supersized LDL-particles in some individuals. Studies by Barzilai et al. indicated that supersized cholesterol carriers (large particles of both HDL cholesterol and LDL cholesterol) may protect against heart disease and thus lead to exceptional longevity (56). These studies attributed enhanced longevity with specific biological genetic factors. It is known that oxidized or glycosylated LDL is more atherogenic than native LDL and AGE and atherogenic LDL are both present in vivo in diabetic patients. Therefore, the inhibition of glycation and oxidation by the antioxidant polyphenolics in Okra juice may play a role in preventing oxidation as well as formation of glycosylated atherogenetic LDL. This process will then increase the size of LDL particles. By using SELDI ProteinChip MS technology as a rapid method for the identification of HDL-apoA1 and high sensitivity C-Reactive Protein (hs-CRP) in diabetic patients who have cardiovascular disease, a comparative molecular characterization and expression levels of super sized HDL and LDL can be achieved. Further development of these techniques may facilitate both epidemiological and therapeutic trials in assessing the role of hs-CRPand HDL apolipoprotein A-1 and their glycosylated products in atherosclerosis. Our long term goal is to find a natural product which can therapeutically prevent protein glycation and stop Amadori Products (AP) in the initial stages of advanved glycation end products. Although a well-studied AGEs inhibitor aminoguanidine reacts with AP and stops the progression of diabetic complications but an inexpensive and non-toxic natural product would be ideal for the prevention and treatment of diabetic complications. Future studies are in progress to evaluate the inhibitory nature of bioactives present in okra seeds to prevent the oxidation and glycosylation of LDL, a cardiovascular biomarker. A recent Iranian publication Grasas Y Aceities, (57) reported that the methanol extract of okra seeds had a remarkable antihypoxic effect in both models of circulatory and haemic hypoxia. The seed extract had protective effect against hypoxia-induced lethality in mice. The present studies presented have highlighted the potential benefits and importance of highly antioxidant nature of flavonoids present in Okra Seeds. Further okra research along these lines may have potential implications on diabetes and cardiovascular biomarkers such as HDL-apoA1, hs-CRP, Il-6 protein expression profiles and blood-brain barrier chemistry.


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