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 patient was removed from the study protocol for a treatment-related adverse event, and the number of reported adverse events was not significantly different between the placebo and treatment groups (eg, headache, 12 vs 9, respectively; upper respiratory tract symptoms, 13 vs 16, respectively; and gastrointestinal tract symptoms, 6 vs 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.
Hydroxycitric acid has been reported in Garcinia cowa, Hibiscus sabdariffa, and Garcinia atroviridis with data available.
See also: Hydroxycitric acid (annotation moved to).

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Background: Age-related macular degeneration (AMD) is the leading cause of blindness and can be classified into two types called atrophic AMD (dry AMD) and neovascular AMD (wet AMD). Dry AMD is characterized by cellular degeneration of the retinal pigment epithelium, choriocapillaris, and photoreceptors. Wet AMD is characterized by the invasion of abnormal vessels from the choroid. Although anti-vascular endothelial growth factor (VEGF) therapy has a potent therapeutic effect against the disease, there is a possibility of chorio-retinal atrophy and adverse systemic events due to long-term robust VEGF antagonism. We focused on hypoxia-inducible factor (HIF) regulation of VEGF transcription, and report the suppressive effects of HIF inhibition against ocular phenotypes in animal models. Many of the known HIF inhibitors are categorized as anti-cancer drugs, and their systemic side effects are cause for concern in clinical use. In this study, we explored food ingredients that have HIF inhibitory effects and verified their effects in an animal model of AMD.
Methods: Food ingredients were screened using a luciferase assay. C57BL6/J mice were administered the Garcinia cambogia extract (Garcinia extract) and Hydroxycitric acid (HCA). Choroidal neovascularization (CNV) was induced by laser irradiation.
Results: Garcinia extract and HCA showed inhibitory effects on HIF in the luciferase assay. The laser CNV model mice showed significant reduction of CNV volume by administering Garcinia extract and HCA. Conclusions: Garcinia extract and HCA showed therapeutic effects in a murine AMD model.
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 is a potential lithontriptic agent for calcium oxalate (CaOx) stones in the kidneys. This study aimed to evaluate the safety and efficiency of hydroxycitric acid tripotassium (K-HCA) against CaOx crystal formation using Drosophila melanogaster hyperoxaluria models. MATERIAL AND METHODS Wild-type D. melanogaster were fed standard medium with ethylene glycol or sodium oxalate added to induce hyperoxaluria. Their Malpighian tubules were dissected and observed under a microscope every 3 days. Crystal deposit score of each Malpighian tubule were evaluated under a magnification of ×200. Using hyperoxaluria Drosophila models, we investigated the inhibitory efficiency of hydroxycitrate acid tripotassium and citric acid tripotassium (K-CA) against CaOx crystal formation. The survival rate of each group was also assessed. RESULTS When fed with 0.05% NaOx, the CaOx formation in Malpighian tubules increased significantly, without reduction of life span. Therefore, we selected 0.05% NaOx-induced hyperoxaluria models for the further investigations. After treatment, the stone scores showed that K-CA and K-HCA both significantly inhibit the formation of CaOx crystals in a dose-dependent manner, and with smaller dosage (0.01%), K-HCA was more efficient than K-CA. Moreover, after treatment of K-CA or K-HCA, the life span in different groups did not change, reflecting the safety to life. CONCLUSIONS The hyperoxaluria Drosophila models fed on 0.05% NaOx diet might be a useful tool to screen novel agents for the management of CaOx stones. K-HCA may be a promising agent for the prevention CaOx stones, with satisfying efficiency and acceptable safety. [3]

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Context: Hydroxycitric acid, the active ingredient in the herbal compound Garcinia cambogia, competitively inhibits the extramitochondrial enzyme adenosine triphosphate-citrate (pro-3S)-lyase. As a citrate cleavage enzyme that may play an essential role in de novo lipogenesis inhibition, G cambogia is claimed to lower body weight and reduce fat mass in humans.
Objective: To evaluate the efficacy of G cambogia for body weight and fat mass loss in overweight human subjects.
Design: Twelve-week randomized, double-blind, placebo-controlled trial.
Setting: Outpatient weight control research unit.
Participants: Overweight men and women subjects (mean body mass index [weight in kilograms divided by the square of height in meters], approximately 32 kg/m2).
Intervention: Subjects were randomized to receive either active herbal compound (1500 mg of hydroxycitric acid per day) or placebo, and both groups were prescribed a high-fiber, low-energy diet. The treatment period was 12 weeks. Body weight was evaluated every other week and fat mass was measured at weeks 0 and 12.
Main outcome measures: Body weight change and fat mass change.
Results: 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.
Conclusions: Garcinia cambogia failed to produce significant weight loss and fat mass loss beyond that observed with placebo. [4]

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The moderate level of genetic diversity exhibited by the CPTs of G. gummi-gutta supports the precise amplification nature of SCoT markers and the availability of specific SCoT alleles within the species. Interrelationships shown by the individuals irrespective of Hydroxycitric acid/HCA content may be the result of gene flow through migrations. Gar1 was genetically distinct, obviously seen from the scatterplot and dendrogram. STRUCTURE analysis discovered the probability of two assumed subpopulations within the selected individuals. The highest concentration of HCA content was measured in Gar17. However, GLM analysis revealed the promising nature of accession Gar6 as evident by the strong association of its HCA content with SCoT 5d allele. The corresponding allele can significantly contribute to HCA content in the species, which could be taken into consideration while implementing breeding strategies in the species. The findings can be extended for 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.)