ATP citrate lyase

HIF Activation Suppressed by Garcinia Extract and HCA/Hydroxycitric acid Administration in Vitro [2]
The murine retinal cone cell line (661W) and the human RPE cell line (ARPE19) were used to evaluate HIF activity with a luciferase assay since photoreceptors and RPE cells significantly contribute the pathogenesis of AMD even though organoids or differentiated cells derived from iPS cells of AMD patients may be considered for better in vitro systems. Under a hypoxic condition, the activity of HIFα prolyl hydroxylase (PHD) decreases, which results in HIFα stabilization [12]. CoCl2 was added to stabilize the inhibition of PHD and to activate HIF signaling. Chetomin was used as a positive control of the HIF inhibitor. We used Garcinia extract. Table 1 lists its components showing that HCA accounts for more than half of the extract. Garcinia extract and HCA showed an HIF inhibitory effect compared with the control group in ARPE19 cells (Figure 1A) and 661W cells (Figure 1B).[2]

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Administration of Garcinia Extract and HCA/Hydroxycitric acid Downregulated Hif1a and Downstream Genes [2]
We examined how Garcinia extract and HCA affect mRNA expression of Hif1a and the downstream genes. In ARPE19 cells, Hif1a was significantly downregulated by administration of Garcinia extract regardless of the presence or absence of CoCl2 (Figure 2A). The downstream genes of HIFs such as Vegfa, Bnip3, and Pdk1, were upregulated by CoCl2 and significantly downregulated by Garcinia extract administration (Figure 2B–D). Similarly, Hif1a was downregulated by administration of Garcinia extract in 661W cells (Figure 2E). CoCl2-induced upregulation of Vegfa was also downregulated by Garcinia extract administration in 661W cells (Figure 2F). Expression of other downstream genes of HIFs showed a tendency to be downregulated as well as Vegfa (Figure 2G,H). HCA also downregulated Hif1a and the downstream genes in ARPE19 cells (Figure 3A–D) and 661W cells (Figure 3E–H). Both Garcinia extract and HCA suppressed HIF-1α protein expression increased by CoCl2 administration in ARPE19 cells (Figure 4A,B) and 661W cells (Figure 4C,D).

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Hydroxycitric acid (HCA) is one of the proven natural antiobesity agents enriched in the fruits of Garcinia gummi-gutta (L.) Roxb. (Family: Clusiaceae). The present research work was carried out to evaluate the genetic variability among 35 candidate plus trees (CPTs) using HCA estimated through HPLC and start codon targeted (SCoT) molecular markers. The association analysis between phenotypic and genotypic traits was also conducted. The selected CPTs showed an average HCA content of 29.11 mg/g and Gar 17 had the highest (48.32 mg/g) followed by Gar 6 (45.48 mg/g). SCoT marker analysis revealed that 19 primers, out of 30 yielded a total of 151 bands with 66.89% polymorphic bands. Principal coordinate analysis (PCoA) organized the CPTs into the four quadrants of a scatterplot irrespective of HCA content. Dendrogram based on neighbour joining method proved its reproducibility by its bootstrapping values, and it has three clusters. STRUCTURE analysis opened the probability of two assumed subpopulations within the selected individuals. Association analysis based on a general linear model (GLM) agreed with the strong association of SCoT 5d allele with HCA content, which also supports the promising nature of Gar 6 as per previous findings. Analysis based on HCA and SCoT markers was effective in tracing out the genetic variabilities among the CPTs and the marker-trait association. The findings are the first in G. gummi-gutta, best of our knowledge [5].

HCA/Hydroxycitric acid treatment could reduce markers of renal impairment (Blood Urea Nitrogen and serum creatinine). There was significantly less calcium oxalate crystal deposition in mice treated with HCA. Calcium oxalate crystals induced the production of reactive oxygen species and reduced the activity of antioxidant defense enzymes. HCA attenuated oxidative stress induced by calcium oxalate crystallization. HCA had inhibitory effects on calcium oxalate-induced inflammatory cytokines, such as MCP-1, IL- 1 β, and IL-6. In addition, HCA alleviated tubular injury and apoptosis caused by calcium oxalate crystals.[1]

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Administration of Hydroxycitric acid/HCA Suppressed CNV Volume in the Model Mice [2]
HCA suspended in corn oil was injected intraperitoneally at 30 mg/kg/day for a total of two weeks, and the mice were irradiated with a laser one week after beginning the injections. The volume of CNV on the seventh day of the irradiation was significantly reduced in the HCA administration group when compared with the control group (Figure 6A,B).

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Administration of Hydroxycitric acid/HCA Suppressed HIF-1α Expression in Vivo [2]
HCA suspended in corn oil was intraperitoneally administered (30 mg/kg/day) to the mice for a total of 10 days, and the mice were irradiated with a laser on the seventh day of administration. In the retina and the choroid of the mice on the third day of irradiation, HIF-1α increased due to the laser irradiation and suppressed due to the administration of HCA (Figure 7A,B) even though the signal with the RPE/choroid tissue was weak.

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A total of 135 subjects were randomized to either active Hydroxycitric acid (n = 66) or placebo (n = 69); 42 (64%) in the active hydroxycitric acid group and 42 (61%) in the placebo group completed 12 weeks of treatment (P = .74). Patients in both groups lost a significant amount of weight during the 12-week treatment period (P<.001); however, between-group weight loss differences were not statistically significant (mean [SD], 3.2 [3.3] kg vs 4.1 [3.9] kg; P = .14). There were no significant differences in estimated percentage of body fat mass loss between treatment groups, and the fraction of subject weight loss as fat was not influenced by treatment group [4].

Luciferase Assay [2]
We performed a luciferase assay using 661W and ARPE19, which were both transfected HIF-luciferase reporter gene constructs. These constructs encode the firefly luciferase gene under the control of HRE, which bind HIFs as previously described. As an internal control, these cells were co-transfected with a CMV-renilla luciferase construct. We seeded cells in 0.8 × 104 cells/well/70 μL in HTS Transwell®-96 Receiver Plate, White, TC-Treated, Sterile. At 24 h after seeding, HIF-αs were induced by 200 μM CoCl2. Garcinia extract (Garcinia Cambogia Extract 50% (Table 1)) and Hydroxycitric acid/HCA were dissolved in dimethyl sulfoxide and added into the growth medium at the same time as CoCl2. We added each compound dissolved in DMSO to the cell medium so that its concentration was 1 mg/mL considering the toxicity of the material. After the administration, cells were incubated for 24 h at 37 °C in a 5% CO2 incubator. Quantitation of the luciferase expression was performed using the Dual-Luciferase® Reporter Assay System. The fluorescent intensity was read by a microplate reader. Additionally, 100 nM of chetomin was used as a positive control for an HIF inhibitor and a DMSO-containing medium was used without CoCl2, Garcinia extract, and HCA as a vehicle control.

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Western Blot [2]
For in vitro experiments, we added 200 μM CoCl2, 1 mg/mL Garcinia extract, and Hydroxycitric acid/HCA to ARPE19 cell line while considering the toxicity of the material. Six hours after the administration, cells were collected in the RIPA buffer and mixed with protease inhibitors and MG132. Then, the cells were homogenized. Afterward, we centrifuged the samples (14,800 rpm, 4 °C, 30 min) and collected the supernatant. The protein concentration was adjusted to 75 μg/30 μL.

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Estimation of Hydroxycitric acid/HCA [5]
HCA from the dried fruit rinds of 35 CPTs of G. gummi-gutta (Babu et al., 2021; Vishnu et al., 2022) were extracted and purified according to Jayaprakasha and Sakariah (2000). The amount of HCA in 35 CPTs of G. gummi-gutta was estimated by HPLC. The Chromatography system consists of a UFLC (Shimadzu Corporation, Kyoto, Japan), SPD- 20A detector, communication bus module (CBM 20A), and Shim-pack GIST C18 column (5 μm, 4.6 ID × 250 mm). Detection of HCA was done at 0.1 AUFS sensitivity at 210 nm. The flow rate was set at 0.7 ml/ min under isocratic conditions using 0.6 mM sulfuric acid as mobile phase. HCA purified from potassium hydroxy citrate was used as the standard. Using 0.45 μm PTFE Syringe Filters (I-131 M Axiva; Sonipat, India), standard, and samples were filtered and injected into the system using a 20 μl injection syringe. A calibration curve was prepared by plotting varying HCA concentrations (200, 400, 600, 800, and 1000 mg/l) versus peak area. Concentrations of HCA in selected CPTs were estimated by the peak integration method and expressed as mg/g of sample.

Male C57BL/6J mice were divided into a control group, glyoxylate(GOX) 100 mg/kg group, a GOX+HCA 100 mg/kg group, and a GOX+HCA/Hydroxycitric acid 200 mg/kg group. Blood samples and kidney samples were collected on the eighth day of the experiment. We used Pizzolato staining and a polarized light microscope to examine crystal formation and evaluated oxidative stress via the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to detect the expression of monocyte chemotactic protein-1(MCP-1), nuclear factor-kappa B (NF κB), interleukin-1 β (IL-1 β) and interleukin-6 (IL-6) messenger RNA (mRNA). The expression of osteopontin (OPN) and a cluster of differentiation-44(CD44) were detected by immunohistochemistry and qRT-PCR. In addition, periodic acid Schiff (PAS) staining and TUNEL assay were used to evaluate renal tubular injury and apoptosis. [1]

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Administration of Garcinia Extract and Hydroxycitric acid/HCA to Mice [2]
An MF diet mixed with Garcinia extract at a concentration of 0.2% was administered to 4-week-old male mice for a total of 7 weeks while considering the toxicity of the material. The control group was administered an MF diet. The mice were irradiated with a laser 6 weeks after beginning administration. We injected 30 mg/kg/day HCA suspended in corn oil intraperitoneally to 6-week-old male mice 5 days/week for a total of 2 weeks. The control group was injected with corn oil. The laser was irradiated 1 week after the initial injection.

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Medications in vivo [3]
Hydroxycitric acid tripotassium/K-HCA and citrate acid tripotassium/K-CA were tested as inhibitors to prevent the formation of stones. We randomly divided 600 flies into 6 groups (100 flies in each group) and fed them high-oxalate (0.05% NaOx) medium. Different concentrations (0.01%, 0.1%, and 1%) of K-HCA and K-CA were added in the medium of corresponding groups. Stone formation and life span were assessed in the same way as above.

Adverse events [1] No patients withdrew from the study due to treatment-related adverse events, and there was no significant difference in the number of adverse events reported between the placebo group and the treatment group (e.g., headache, 12 and 9, respectively; upper respiratory symptoms, 13 and 16, respectively; gastrointestinal symptoms, 6 and 13, respectively).

[1]. Hydroxycitric acid inhibits renal calcium oxalate deposition by reducing oxidative stress and inflammation. Curr Mol Med. 2020;20(7):527-535.

[2]. Therapeutic Effect of Garcinia cambogia Extract and Hydroxycitric Acid Inhibiting Hypoxia-Inducible Factor in a Murine Model of Age-Related Macular Degeneration. Int J Mol Sci. 2019 Oct 11;20(20). pii: E5049.

[3]. Hydroxycitric Acid Tripotassium Inhibits Calcium Oxalate Crystal Formation in the Drosophila Melanogaster Model of Hyperoxaluria. Med Sci Monit. 2019 May 17;25:3662-3667.

[4]. Garcinia cambogia (hydroxycitric acid) as a potential antiobesity agent: a randomized controlled trial. JAMA. 1998 Nov 11;280(18):1596-600.

[5]. Start codon targeted (SCoT) variability analysis and its association with hydroxy citric acid (HCA) in Garcinia gummi-gutta (L.) Roxb. Plant Gene, Volume 34, June 2023, 100415.

Hydroxycitric acid is a carbonyl compound. It has been reported to exist in Garcinia cowa, Hibiscus sabdariffa, and Garcinia atroviridis, with relevant data available. See also: Hydroxycitric acid (note moved here). Background: Age-related macular degeneration (AMD) is a leading cause of blindness and can be classified into two types: atrophic AMD (dry AMD) and neovascular AMD (wet AMD). Dry AMD is characterized by cellular degeneration of the retinal pigment epithelium, choroidal capillaries, and photoreceptor cells. Wet AMD is characterized by the invasion of abnormal choroidal vessels. Although anti-vascular endothelial growth factor (VEGF) therapy has significant therapeutic effects on this disease, long-term potent VEGF antagonism may lead to choroidal-retinal atrophy and adverse systemic reactions. We focused on the regulation of VEGF transcription by hypoxia-inducible factor (HIF) and reported the inhibitory effect of HIF inhibitors on ocular phenotypes in animal models. Many known HIF inhibitors are classified as anticancer drugs, but their systemic side effects are a concern in clinical applications. This study explored food components with HIF inhibitory effects and validated their efficacy in an animal model of age-related macular degeneration (AMD). Methods: Food components were screened using luciferase reporter gene assay. C57BL6/J mice were administered Garcinia Cambogia extract (Garcinia Cambogia extract) and hydroxycitric acid (HCA), respectively. Choroidal neovascularization (CNV) was induced by laser irradiation. Results: Luciferase reporter gene assay showed that Garcinia Cambogia extract and HCA inhibited HIF. In the laser-induced CNV model mice, CNV volume was significantly reduced after administration of Garcinia Cambogia extract and HCA. Conclusion: Garcinia Cambogia extract and hydroxycitric acid showed therapeutic effects in a mouse model of age-related macular degeneration. Keywords: Garcinia Cambogia; age-related macular degeneration; choroid; hydroxycitric acid; hypoxia-inducible factor; laser-induced neovascularization; retina. [2]

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Background: Hydroxycitric acid (K-HCA) is a potential lithotripter for calcium oxalate (CaOx) kidney stones. This study aimed to evaluate the safety and efficacy of tripotassium hydroxycitrate (K-HCA) against CaOx crystal formation using a Drosophila hyperoxaluria model. Materials and Methods: Wild-type Drosophila were fed standard culture media supplemented with ethylene glycol or sodium oxalate to induce hyperoxaluria. Malpighian tubules were dissected and observed under a microscope every 3 days. The crystal deposition score of each Malpighian tubule was evaluated under a 200x microscope. We used the Drosophila hyperoxaluria model to investigate the inhibitory effects of K-HCA and K-HCA on calcium oxalate crystal formation and evaluated the survival rate of each group. The results showed that feeding with 0.05% sodium oxalate significantly increased the formation of calcium oxalate in Malpighian tubules, but did not shorten the lifespan of the Drosophila. Therefore, we chose the 0.05% sodium oxalate-induced hyperoxaluria model for subsequent studies. After treatment, the stone score showed that both K-CA and K-HCA significantly inhibited the formation of calcium oxalate crystals in a dose-dependent manner, and at a lower dose (0.01%), K-HCA had a better inhibitory effect than K-CA. In addition, the lifespan of each group of fruit flies did not change after K-CA or K-HCA treatment, indicating that it was safe for fruit flies. Conclusion: The fruit fly hyperoxaluria model fed with a diet of 0.05% sodium oxalate may be an effective tool for screening new drugs for the treatment of calcium oxalate stones. K-HCA may be a promising drug for the prevention of calcium oxalate stones with satisfactory efficacy and acceptable safety. [3] Background: Hydroxycitric acid is the active ingredient in Garcinia cambogia, which competitively inhibits the extramitochondrial enzyme ATP-citrate (pro-3S) lyase. As a citrate lyase that may play an important role in inhibiting de novo fat synthesis, Garcinia cambogia is thought to reduce body weight and reduce human fat mass.
Objective: To evaluate the efficacy of Garcinia Cambogia in reducing weight and body fat in overweight individuals.
Design: A 12-week randomized, double-blind, placebo-controlled trial.
Location: Outpatient Weight Control Research Center.
Participants: Overweight male and female participants (mean body mass index [weight (kg) divided by height (m) squared] approximately 32 kg/m²).
Intervention: Participants were randomly assigned to receive either an active herbal compound (1500 mg hydroxycitric acid daily) or a placebo, both groups were required to follow a high-fiber, low-energy diet. The treatment period was 12 weeks. Weight was assessed every other week, and body fat was measured at weeks 0 and 12.
Primary outcome measures: Changes in weight and body fat.
Results: A total of 135 participants were randomly assigned to the active hydroxycitric acid group (n = 66) or the placebo group (n = 69); 42 participants (64%) in the active hydroxycitric acid group and the placebo group, respectively, completed the 12-week treatment (P = 0.74). Both groups of patients experienced significant weight loss during the 12-week treatment period (P<0.001); however, there was no statistically significant difference in weight loss between the groups (mean [standard deviation], 3.2 [3.3] kg vs 4.1 [3.9] kg; P = 0.14). There was no significant difference in the estimated percentage reduction in body fat between the treatment groups, nor was the proportion of fat in the weight loss of the subjects affected by the treatment groups. Conclusion: Garcinia cambogia did not produce a more significant effect on weight loss and fat reduction than the placebo group. [4] The moderate level of genetic diversity shown by Garcinia cambogia (G. gummi-gutta) CPT supports the precise amplification of the SCoT marker and the presence of specific SCoT alleles in this species. The inter-individual interactions, regardless of hydroxycitric acid (HCA) content, are likely the result of gene migration. Gar1 is genetically distinct from other individuals, as can be seen from scatter plots and dendrograms. STRUCTURE analysis revealed that there may be two hypothetical subpopulations among the selected individuals. Gar17 had the highest HCA content. However, GLM analysis showed that germplasm Gar6 had good genetic potential, and its HCA content was significantly correlated with the SCoT 5d allele. This allele can significantly affect the HCA content of this species and should be considered when formulating breeding strategies for this species. These findings can be extended to marker-assisted selection and genetic improvement of G. gummi-gutta. [5]

C6H8O8

208.12

208.021

C, 34.63; H, 3.87; O, 61.50

6205-14-7

(-)-Hydroxycitric acid;27750-10-3;Hydroxycitric acid tripotassium hydrate;6100-05-6

123908

Typically exists as solid at room temperature

1.9±0.1 g/cm3

393.3±42.0 °C at 760 mmHg

205.8±24.4 °C

0.0±2.0 mmHg at 25°C

1.620

-1.35

5

8

5

14

271

0

[K].[K].[K].O([H])C(C(=O)O[H])(C([H])([H])C(=O)O[H])C([H])(C(=O)O[H])O[H] |^1:0,1,2|

ZMJBYMUCKBYSCP-UHFFFAOYSA-N

InChI=1S/C6H8O8/c7-2(8)1-6(14,5(12)13)3(9)4(10)11/h3,9,14H,1H2,(H,7,8)(H,10,11)(H,12,13)

1,2-dihydroxypropane-1,2,3-tricarboxylic acid

hydroxycitric acid; 6205-14-7; Hydroxycitrate; 1,2-dihydroxypropane-1,2,3-tricarboxylic acid; 1,2-Dihydroxy-1,2,3-propanetricarboxylic acid; Pentaric acid, 3-C-carboxy-2-deoxy-; 1,2-dihydroxypropane-1,2,3-tricarboxylicacid; Super CitriMax HCA 600SXS;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)