|
 
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| ORIGINAL ARTICLE |
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| Year : 2015 | Volume
: 9
| Issue : 4 | Page : 240-248 |
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Protective effect of Gymnema sylvestre L. against advanced glycation end-product, sorbitol accumulation and aldose reductase activity in Homoeopathic Formulation
Lalit Kishore, Randhir Singh
Department of Pharmacology, Maharishi Markandeshwar College of Pharmacy, Maharishi Markandeshwar University, Mullana-Ambala, Haryana, India
| Date of Submission | 10-Jun-2015 |
| Date of Acceptance | 03-Dec-2015 |
| Date of Web Publication | 30-Dec-2015 |
Correspondence Address:
Randhir Singh
Maharishi Markandeshwar College of Pharmacy, Maharishi Markandeshwar University, Mullana-Ambala 133 207, Haryana
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/0974-7168.172866
Background: Oxidative stress ensues due to the imbalance between the production and elimination of reactive oxygen species. Chronic hyperglycemia along with oxidative stress plays major role in aggravation of chronic disorders such as diabetes and its related complications.
Objective: This study was designed to evaluate the protective effect of Gymnema sylvestre L. against oxidative stress.
Materials and Methods: Potencies of G. Sylvestre were procured from Dr. Willmar Schwabe India Pvt. Ltd. In vitro antioxidative potential of G. sylvestre was evaluated by employing various in vitro antioxidant methods.
Results: The total phenol content was found to be 2124, 998 and 546 mg/g Gallic Acid Equivalents in Mother tincture, 6C and 30C of G. sylvestre and total antioxidant capacity was found to be 2940,802 and 559 μM/g ascorbic acid equivalents respectively. Mother tincture, 6C and 30C of G. sylvestre were found to have strong reducing power, 2,2-diphenyl-1-picrylhydrazyl radical, hydrogen peroxide, nitric oxide and superoxide radical scavenging activity. Percentage inhibition of advanced glycation end-products formation by Mother tincture, 6C and 30C of G. sylvestre (10-50 μl) was found to be 38.66 to 95.80%, 30.93 to 81.48% and 31.34 to 60.92% respectively. Mother tincture, 6C and 30C of G. sylvestre showed an inhibitory effect against sorbitol accumulation with inhibitory concentration (IC50) value 27.55 μl, 197.96 μl and 1.009 ml respectively whereas in aldose reductase inhibition assay, the IC50value of 28.10 μl, 159.71 μl and 0.82 ml respectively.
Conclusion: These results suggested that Homoeopathic preparations of G. Sylvestre had potent antioxidant and antiglycation activity. Keywords: Advanced glycation end-products, Aldose reductase inhibition, Diabetic complications, G. sylvestre, Homoeopathic preparations, Oxidative stress, Sorbitol accumulation
How to cite this article:
Kishore L, Singh R. Protective effect of Gymnema sylvestre L. against advanced glycation end-product, sorbitol accumulation and aldose reductase activity in Homoeopathic Formulation. Indian J Res Homoeopathy 2015;9:240-8 |
How to cite this URL:
Kishore L, Singh R. Protective effect of Gymnema sylvestre L. against advanced glycation end-product, sorbitol accumulation and aldose reductase activity in Homoeopathic Formulation. Indian J Res Homoeopathy [serial online] 2015 [cited 2023 Apr 2];9:240-8. Available from: https://www.ijrh.org/text.asp?2015/9/4/240/172866 |
| Introduction | |  |
Overproduction of reactive oxygen species (ROS) have been entailed as the causative factor for a variety of pathological effects viz., DNA damage, cellular degeneration inducing various diseases such as cancer, diabetes and its complications, atherosclerosis and aging.[1] Various synthetic and natural antioxidants are being used for attenuating the production of ROS. Toxicity and carcinogenesis caused by synthetic antioxidants are the leading cause for their declining use. Therefore, it is necessary to evolve natural antioxidants to produce effective and non-toxic amelioration of ROS.[2]G. sylvestre commonly known as Gurmar, Gurmabooti, is a traditional medicinal plant, with reported use as a remedy for diabetes mellitus, stomachic and diuretic problems. The plant extracts are also used in folk, Ayurvedic and Homoeopathic systems of medicine.[3]G. sylvestre has been proved to treat diabetes as it showed tendency to reduce serum glucose concentrations and also improves glucose tolerance.[4],[5] Besides antidiabetic potential, G. sylvestre has the ability to lower triglyceride and total cholesterol in serum and its anti-atherosclerotic potential were almost similar to that of a standard lipid lowering agent-clofibrate.[6] In Homoeopathy, G. sylvestre in different potencies is used to abolish taste of bitter things, sense of altered taste and snake bite.[7]
In this study in vitro antioxidative potential of Mother tincture, 6C and 30C potencies of G. sylvestre were evaluated by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging assay, nitric oxide (NO) scavenging assay, hydrogen peroxide (H2O2) scavenging assay, reducing power, superoxide radical scavenging activity, total phenolic content by Folin-Ciocalteau method and total antioxidant activity by phosphomolybdenum method.
| Materials and Methods | | |
Materials and Reagents
Potencies (Mother tincture, 6C and 30C) of G. sylvestre L. were procured from Dr. Willmar Schwabe India Pvt. Ltd. 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Bovine serum albumin (BSA), Nitroblue tetrazolium (NBT), Folin-Ciocalteu reagent and N-(1-Naphthyl) ethylenediamide dihydrochloride were purchased from Molychem Pvt. Ltd., India. All other chemicals and reagents used were of analytical grade.
In Vitro Antioxidant Activity
Determination of total phenol content
Total phenolic content in the Mother tincture, 6C and 30C of G. sylvestre were determined with Folin-Ciocalteu reagent using gallic acid as a standard phenolic compound. The sample was diluted appropriately to obtain absorbance in the range of calibration curve. An aliquot of 1 ml of sample solution was mixed with 1 ml of Folin–Ciocalteu reagent. Three min later 3.0 ml of 2% sodium carbonate was added and the mixture was allowed to stand for 3 h with intermittent shaking. The absorbance of the blue color that developed was measured at 760 nm. The concentration of total phenolic compounds in the sample was obtained as mg of gallic acid equivalent (GAE) per gram dry weight.[8]
Total Antioxidant Capacity
An aliquot of 0.3 ml of Mother tincture, 6C and 30C of G. sylvestre were mixed with 3 ml of the reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). In case of blank 0.3 ml of water was used instead of sample. The tubes were capped with aluminium foil and incubated in boiling water bath at 95°C for 90 min. After the samples had cooled to room temperature, the absorbance was measured at 695 nm against a blank. Ascorbic acid was used as a standard. Total antioxidant capacity (TAOC) was expressed as equivalents of ascorbic acid (μmol/g).[9]
2,2-Diphenyl-1-Picrylhydrazyl Scavenging Activity
The DPPH radical scavenging ability of Mother tincture, 6C and 30C of G. sylvestre was evaluated according to the method given in literature with slight modification.[10] The different concentrations in each reaction set, were mixed with 1.0 ml of 0.1 mM of DPPH in ethanol. The mixture was incubated in the dark for 30 min at room temperature. Degree of inhibition of DPPH by monitoring the decrease in absorbance measured at 517 nm. Ascorbic acid was used as positive control. Radical scavenging activity was expressed as inhibition percentage of free radical by the sample and was calculated using the following formula:
Where A0 was the absorbance of control (blank without sample) and At was the absorbance in the presence of sample. All the tests were performed in triplicate and the graph was plotted with mean values.
Hydrogen Peroxide Scavenging Activity
H2O2 scavenging activity was evaluated as described previously.[11] An aliquot of 40 mM H2O2 solution (0.6 ml) was mixed with various concentrations of Mother tincture, 6C and 30C of G. sylvestre. To the mixture 2.4 ml of phosphate buffer (0.1 M, pH 7.4) was added and the mixture was shaken vigorously and incubated at room temperature for 10 min. Then, the absorbance of the reaction mixture was determined at 230 nm. Ascorbic acid was used as positive control. The H2O2 scavenging activity was calculated as follows:
Where A0 is the absorbance of the control (water instead of sample), A1 is the absorbance of the sample and A2 is the absorbance of the sample only (phosphate buffer instead of H2O2 solution). The inhibitory concentration 50% (IC50) value represented the concentration of the compounds that caused 50% inhibition of H2O2.
Reducing Power Assay
The Fe 3+-reducing power of Mother tincture, 6C and 30C of G. sylvestre was determined according to the method described in literature.[12] Different concentrations of samples (2.5 ml) were mixed with 2.5 ml of 0.2M sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide and incubated at 50°C for 20 min. After incubation, 2.5 ml of 10% trichloroacetic acid (w/v) was added and the mixture centrifuged at 1000 rpm for 8 min. The supernatant (5 ml) was mixed with 5 ml of distilled water and 1 ml of 0.1% of ferric chloride, and the absorbance was measured spectrophotometrically at 700 nm. The assay was carried out in triplicate and the results expressed as mean values α standard deviations. Ascorbic acid was used as positive control. The sample concentration providing 0.5 of absorbance (EC50) was calculated from the graph plotted between absorbance at 700 nm against sample concentration.
Nitric Oxide Scavenging Activity
At physiological pH, an aqueous solution of sodium nitroprusside spontaneously generates NO [13] which interacts with oxygen to produce nitric ions that can be estimated using Griess reagent. Scavengers of NO compete with oxygen leading to reduce the production of NO. The reaction mixture of 5mM Sodium nitroprusside in phosphate buffer saline (PBS) and 3.0 ml of different concentrations of the Mother tincture, 6C and 30C of G. sylvestre was incubated at 25°C for 150 min. After incubation, the samples were added to Griess reagent (1% sulfanilamide, 2% H3 PO4 and 0.1% napthylethylenediamine dihydrochloride). The pink chromophore generated during the diazotization of nitrite with sulfanilamide and subsequent coupling with napthyl-ethylenediamine was measured at 546 nm. Ascorbic acid was used as positive control. The percentage of inhibition was measured by the following formula:
Where A0 was the absorbance of the control (blank, without sample) and At was the absorbance in the presence of the sample. All the tests were performed in triplicate and the graph was plotted with the mean values.
Superoxide Radical Scavenging Activity
The activity was measured by the reduction of NBT (NBT reagent) method as described by Shukla et al., 2009.[14] The method is based on the generation of superoxide radical (O 2-) by auto-oxidation of hydroxylamine hydrochloride in the presence of NBT, which gets reduced to nitrite. Nitrite in the presence of ethylene diamine tetra acetic acid (EDTA) gives a color that was measured at 560 nm. Different concentrations of Mother tincture, 6C and 30C of G. sylvestre were taken in a test tube. To this, a reaction mixture consisting of 1 ml of (50 mM) sodium carbonate, 0.4 ml of (24 mM) NBT and 0.2 ml of 0.1 mM EDTA solutions were added to the test tube and immediate reading were taken at 560 nm. After incubating the reaction mixture at 25°C for 15 min, about 0.4 ml of (1 mM) of hydroxylamine hydrochloride was added to initiate the reaction and reduction of NBT was measured at 560 nm. Ascorbic acid was used as the positive control. Decreased absorbance of the reaction mixture indicates increased superoxide anion scavenging activity. The percentage of inhibition was calculated according to the following equation:
Where A0 was the absorbance of the control (blank, without sample) and At was the absorbance in the presence of the samples. All the tests were performed in triplicate and the graph was plotted with the mean values.
Anti-glycation Activity
In vitro antiglycation activity of Mother tincture, 6C and 30C of G. sylvestre was examined by testing their ability to inhibit the fluorescence of BSA in accordance with a previous method.[15] The reaction mixture of BSA (10 mg/ml), 1.1 M fructose in 0.1 M PBS, pH 7.4 containing 0.02% sodium azide with or without sample (Mother tincture, 6C and 30C of G. sylvestre dissolved in PBS) was incubated in darkness at 37°C for 1, 2, 3, and 4 weeks. Advanced glycation end-product (AGE) formation was measured by fluorescent intensity at an excitation wavelength 355 nm and emission wavelength 460 nm using Elico-SLI74 Spectrofluorometer fitted with Xenon Lamp (Elico, India, Pvt Ltd.). Aminoguanidine (AG; 500 μg/ml) was used as a positive control for this study.
Erythrocyte Sorbitol Accumulation Inhibition
Five-mililitre ml of Heparinized human blood was collected from overnight fasted healthy male volunteer and erythrocytes were separated from the plasma by centrifugation at 3000 g for 30 min. The cells were washed 3 times with isotonic saline at 4°C and in the final washing, the cells were centrifuged at 1500 gfor 15 min to obtain a consistently packed cell preparation. The packed cells (1 mL) were then incubated in Krebs-Ringer bicarbonate buffer (pH 7.4) (4 mL) containing 55 mM glucose in the presence or absence of samples (Mother tincture, 6C and 30C of G. sylvestre) at 37°C for 3 h. The erythrocytes were washed with cold saline by centrifugation at 2000 g for 5 min, precipitated by adding 6% of cold perchloric acid (3 mL), and centrifuged again at 2000 g for 10 min. The supernatant was neutralized with 2.5 M K2 CO3 at 4°C and used for sorbitol determination.[16] The relative fluorescence due to NADH was measured by a fluorescence spectrometer (Elico-SLI74 Spectrofluorometer fitted with Xenon Lamp, Elico, India) at an excitation wavelength of 366 nm and an emission wavelength of 452 nm. The experiments were performed in triplicates.
Aldose Reductase Enzyme Inhibition
Partial purification of Aldose reductase 1 (ALR1) from rat kidney (IAEC Protocol No.: MMCP/IAEC/13/07) was carried out following the previously described methods.[17] Isolated kidney was homogenized in 3 volumes of 10 mM sodium phosphate buffer, pH 7.2 containing 0.25 M sucrose, 2.0 mM EDTA, and 2.5 mM 2-mercaptoethanol. The homogenate was centrifuged at 10,000 g for 20 min and the supernatant was subjected to ammonium sulfate precipitation. Precipitate obtained between 45% and 75% saturation was dissolved in the above buffer. The supernatant was used as the source of ALR1. The activity of ALR1 was measured spectrophotometrically by monitoring the oxidation of NADPH at 340 nm as a function of time at 37°C using glyceraldehyde as substrate. The assay mixture in 1 ml contained 50 mM sodium phosphate buffer of pH 7.2, 0.2 M ammonium sulfate, 10 mM DL-glyceraldehyde, 5 mM β-mercaptoethanol and 0.1 mM NADPH.
Various concentrations of Mother tincture, 6C and 30C of G. sylvestre were added to assay mixtures of ALR1 and incubated for 5 min before initiating the reaction by NADPH as described above. The percentage inhibition was calculated considering the activity in the absence of Mother tincture, 6C and 30C of G. sylvestre as 100%. The IC50 values were determined by linear regression analysis of the plot of percent inhibition versus inhibitor concentration.
| Results and Discussion | | |
Total Phenol Content
Phenols are very important plant constituents because of their scavenging ability owing to their hydroxyl groups.[18] The phenolic compounds may contribute directly to antioxidative action. It is known that polyphenolic compounds have inhibitory effects on mutagenesis and carcinogenesis in humans when ingested up to 1 g daily from a diet rich in fruits and vegetables.[19] Natural antioxidants may be useful in preventing deleterious effects of oxidative stress. Results obtained in this study revealed a considerable level of phenols in the Mother tincture, 6C and 30C of G. sylvestre. Total phenol content present in Mother tincture, 6C and 30C of G. sylvestre was found to be 2124, 998 and 546 mg/g GAE respectively.
Total Antioxidant Capacity
TAOC reflects the total antioxidant defence system. The phosphomolybdenum method used in this study involves the reduction of molybdenum VI (Mo 6+) to a green Mo 5+ complex in acidic medium. TAOC of Mother tincture, 6C and 30C of G. sylvestre was found to be 2940, 802 and 559 μM/g ascorbic acid equivalent respectively.
2,2-Diphenyl-1-Picrylhydrazyl Scavenging Activity
DPPH free radical scavenging method has been widely adopted as a tool for estimating the free radical scavenging activities of lipophilic radicals. A chain of lipophilic radicals is initiated by the lipid auto-oxidation.[20] Radical scavenging activity is very important due to the deleterious role of free radicals in foods and in biological systems. Diverse methods are currently used to assess the antioxidant activity of plant phenolic compounds. Chemical assays are based on their ability to scavenge synthetic free radicals, using a variety of radical-generating systems and methods for detection of the oxidation end-point. DPPH radical-scavenging method is a common Spectrophotometric procedure for determining the antioxidant capacities of components.[21] The reduction capacity of DPPH was determined by the decrease in absorbance at 517 nm. On the DPPH radical, Mother tincture, 6C and 30C of G. sylvestre had a significant effect with increasing concentration from 10-50 μl. The IC50 value of Mother tincture, 6C and 30C of G. sylvestre was 33.88, 43.62 and 46.88 µl respectively [Figure 1] and that of ascorbic acid was found to be 1.43 μg/ml. A higher DPPH radical scavenging activity is associated with lower IC50 value. | Figure 1:Effect of Gymnema sylvestre on 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activities in homoeopathic formulation. Values are mean ± standard deviation for n = 3
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Hydrogen Peroxide Scavenging Activity
H2O2 is generated in vivo by several oxidase enzymes. H2O2 via OH · acts as a messenger molecule in the synthesis and activation of inflammatory mediators.[22] The loss of H2O2 can be measured at 230 nm when it is incubated with a scavenger. [Figure 2] show the scavenging ability of Mother tincture, 6C and 30C of G. sylvestre on H2O2 at different concentrations. The IC50 value of Mother tincture, 6C and 30C of G. sylvestre was found to be 25.82 μl, 0.2 ml and 0.2 ml respectively. IC50 value of ascorbic was found to be 80 μg/ml. | Figure 2: Effect of Gymnema sylvestre on hydrogen peroxide radical-scavenging activities in homoeopathic formulation. Values are mean ± standard deviation for n = 3
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Reducing Power Assay
Reducing power may serve as a significant indicator of potential antioxidant activity. In this method reducing power was determined based on the ability of the sample to reduce ferric ions/ferricyanide complex to ferrous ion complex. The formation of ferrous ion complex was observed by the formation of Perl's Prussian blue color at 700 nm [Figure 3]. EC50 (effective concentration at which the absorbance is 0.5) was calculated from the calibration curve and was found to be 39 µl, 0.22 ml and 1.01 ml for Mother tincture, 6C and 30C of G. sylvestre respectively and 21.42 μg/ml for ascorbic acid. | Figure 3: Effect of Gymnema sylvestre on reducing power in homoeopathic formulation. Values are mean ± standard deviation for n = 3
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Nitric Oxide Scavenging Activity
NO is an essential regulating molecule required for several physiological processes such as vasodilation, nerve transmission and control of blood pressure.[23] Excess generation of NO leads to a chain of reactions via formation of peroxynitrite ions that are extremely toxic and can be detrimental to human health.[24] The plant products have the property to counteract the effect of excessive NO generation.
The various concentrations of Mother tincture, 6C and 30C of G. sylvestre showed significant inhibition against NO radical in a dose dependent manner. The concentration of Mother tincture, 6C and 30C of G. sylvestre required for IC50 was found to be 24.83 µl, 0.15 ml and 0.82 ml respectively [Figure 4]. IC50 of ascorbic acid was found to be 63.55 µg/ml. | Figure 4: Effect of Gymnema sylvestre on nitric oxide radical-scavenging activities in homoeopathic formulation. Values are mean ± standard deviation for n = 3
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Superoxide Scavenging Activity
Superoxide radical is a harmful free radical acting as a precursor of more reactive oxidative species such as oxygen and hydroxyl radicals that contributes to tissue damage and various other pathological conditions.[23] Mother tincture, 6C and 30C of G. sylvestre had significant activity against superoxide radicals in a dose dependent manner [Figure 5]. IC50 of ascorbic acid was found to be 27.96 µg/ml and that of Mother tincture, 6C and 30C of G. sylvestre was found to be 45.50 µl, 0.16 ml and 0.707 ml respectively. | Figure 5: Effect of Gymnema sylvestre on superoxide dismutase radical-scavenging activities in homoeopathic formulation. Values are mean ± standard deviation for n = 3
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Advanced Glycation End-Products Inhibition Activity
Accumulation of AGEs in the tissues is a significant contributor to diabetic complications such as nephropathy, neuropathy and retinopathy.[25] Scientific studies reveal that the inhibition of AGEs reflects the attenuation of diabetic complications. AGEs by acting on their receptors AGE modify the plasma protein leading to the excessive formation of free radicals.[26] Aminoguanidine inhibits the formation of AGEs by trapping the intermediate Amadori products. In this study, the formation of AGEs was monitored weekly by measuring the fluorescence intensity of the BSA-fructose solutions for 4 weeks. A significant inhibition of AGEs formation (93.37%) was observed in fructose-induced glycated BSA plus aminoguanidine (500 μg/ml). At 4th week of incubation, the percentage inhibitions of AGEs formation by G. sylvestre Mother tincture (10-50 μl) was 38.66 to 95.80%, respectively; 6C (10-50 μl) was 30.93 to 81.48% and for 30C (10-50 μl) was found to be 31.34 to 60.92% respectively [Figure 6],[Figure 7],[Figure 8]. | Figure 6: The effects of Gymnema sylvestre Mother tincture on the formation of fluorescent advanced glycation end-products in Bovine serum albumin incubated with fructose. Values are mean ± standard deviation for n = 3 Aminoguanidine
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 | Figure 7: The effects of Gymnema sylvestre 6C on the formation of fluorescent advanced glycation end-products in Bovine serum albumin incubated with fructose. Values are mean ± standard deviation for n = 3 Aminoguanidine
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 | Figure 8: The effects of Gymnema sylvestre 30C on the formation of fluorescent advanced glycation end-products in Bovine serum albumin incubated with fructose. Values are mean ± standard deviation for n = 3 Aminoguanidine
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Erythrocyte Sorbitol Accumulation Inhibition
In diabetes, activation of sorbitol pathway leads to accumulation of sorbitol in cells. This leads to cellular injury, formation of ROS, oxidative stress and eventually diabetic complications. Prevention of intracellular sorbitol accumulation would ameliorate the diabetic complications. This study revealed the significant inhibitory effect of Mother tincture, 6C and 30C of G. sylvestre on sorbitol accumulation [Figure 9]. IC50 of Ascorbic acid was found to be 186.08 µg/ml and that of Mother tincture, 6C and 30C of G. sylvestre was found to be 27.55 µl, 197.96 µl and 1.009 ml respectively. | Figure 9: Effect of Gymnema sylvestre on erythrocyte sorbitol accumulation inhibition assay in homoeopathic formulation. Values are mean ± standard deviation for n = 3
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Aldose Reductase Inhibitory Activity
In chronic hyperglycemia, ALR converts excess glucose to sorbitol using NADPH and accumulation of excess sorbitol leads to cellular and organ damage.[26] Agents inhibiting the ALR activity would lead to the attenuation of diabetic complications. ALR1 was partially purified from kidney of Wistar rat and the activity of ALR1was measured spectrophotometrically by monitoring the oxidation of NADPH at 340 nm. IC50 of quercetin (standard) was found to be 5.17µg/ml and that of Mother tincture, 6C and 30C of G. sylvestre was found to be 28.10 μl, 159.71 μl and 0.82 ml respectively [Figure 10]. | Figure 10: Effect of Gymnema sylvestre on aldose reductase inhibitory assay in homoeopathic formulation. Values are mean ± standard deviation for n = 3
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| Conclusion | | |
Results obtained in this study suggested that Mother tincture, 6C and 30C of G. sylvestre have a higher amount of phenols, which are known to scavenge free radicals. G. sylvestre was also effective in DPPH, H2O2, NO and superoxide dismutase scavenging activity. Further, the study reflects the ability of G. sylvestre to inhibit the formation of AGEs and sorbitol accumulation. This profound protective effect of G. sylvestre may suggest its extensive use as antioxidant and future studies are directed for their in vivo evaluation in diabetic complications.
Financial Support and Sponsorship
Financial Assistance received from Ministry of AYUSH, Govt. of India, under EMR scheme.
Conflicts of Interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]
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