1. INTRODUCTION:

Tomatoes, Solanum lycopersicum L., are a popular crop with health benefits including carotenoids. These carotenoids and antioxidants like lycopene make tomatoes red. Additionally, fresh tomato fruit contains approximately 12mg/100g of lycopene, along with additional carotenoids like β-carotene, phytoene, and lutein^1-3. Several studies suggest these health-promoting chemicals may lower cancer risk. To prevent cardiovascular disease and other cancers like colon, stomach, and rectal cancer, tomatoes and their carotenoid contents should be eaten. Vitamins, polyphenols including anthocyanins and phenol acids, and other nutrients are abundant in tomatoes. Tomato peels contain certain flavonoids^4-8. These compounds include naringenin, chalcone, flavonol, rutin, and quercetin glycoside. In addition to tomato plants' popularity and global cultivation, the growing human population has prompted the agriculture sector to boost crop yield to meet the demands of billions of people⁹. Tomatoes possess possible health advantages, which encompass the anticancer attributes of lycopene in conjunction with its anti-angiogenic qualities, the decrease in insulin-like growth factor (IGF) levels within the bloodstream, and the regulation of cellular pathways associated with cancer development¹⁰. The potential anticancer qualities of certain constituents of tomatoes, such as its fiber, vitamin C, and phenolic compound ferulic acid, have also been the subject of scholarly discourse. There is evidence suggesting that the eating of tomatoes may contribute to a decreased likelihood of developing cardiovascular disease. Several research have found correlations between tomato intake and lower instances of hypertension and atherosclerosis¹¹. The literature has discussed several observational and experimental investigations that have emphasized the potential neuroprotective effects and involvement in diabetes-related oxidative stress. Diabetes is caused by low insulin synthesis, insulin hormone dysfunction, or both¹². Hyperglycemia—blood glucose levels above 200mg/dL—is an early sign of diabetes¹³. According to 2021 WHO data, the number of individuals with diabetes grew from 108 million in 1980 to 422 million in 2014, mostly in low-to-middle-income nations. Diabetes was the ninth killer in 2019. Several studies have linked high calorie or food consumption to high glycemic index and type 2 diabetes mellitus^14-17.

Inhibiting oligo- and disaccharide breakdown during digestion is one way to treat type 2 diabetes. Inhibiting carbohydrate hydrolysis enzymes, such as α-amylase in saliva and pancreas⁴, and α-glucosidase at the small intestine margin¹⁸, can lower postprandial hyperglycemia. Acarbose, a synthetic α-glucosidase and α-amylase inhibitor, is commonly used to treat type 2 diabetes. However, acarbose, metformin, and voglibose might cause gastrointestinal issues, nausea, vomiting, hepatic impairment, and dizziness^19-20. This study aim to determine the potential pharmacological activity of tomato including the antidiabetic and antioxidant.

2. MATERIALS AND METHODS:

2.1. Materials:

The materials for the study were sourced from various suppliers to ensure high quality and consistency in the experiments. Solanum lycopersicum, the primary botanical material, was obtained from Dairi in Northern Sumatra Province, Indonesia, located between latitudes 2° and 3° North and longitudes 98° and 98°30' East. The chemicals required for the study were acquired from several trusted vendors. HCl, acetonitrile, phosphate buffer, methanol, and ethanol were purchased from Bratchem. Specialized reagents including ρ-nitrophenol glucopyranoside, α-amylase, dinitrosalicylic acid, gallic acid, quercetin, sodium hydroxide, NaNO2, and aluminum chloride were sourced from Sigma Aldrich. These materials were chosen for their reliability and suitability for biochemical research, ensuring accurate and reproducible results in the enzymatic assays and other analytical procedures conducted in the study.

2.3. Preparation of Nanoherbal Tomato (NT):

Tomatoes (Solanum lycopersicum) sourced from Dairi, Northern Sumatera Province, Indonesia, were used as the base material for creating Nanoherbal Tomato samples. The nanoformulation process was conducted using a High-energy Milling (HEM) technique at the Indonesian research institute LIPI in Jakarta. An HCl 2M solution from Tokyo, Japan served as the activator.The tomatoes were first washed and dried to meet specific water content requirements suitable for HEM processing. For the milling, the initial preparation involved placing the dried tomato simplicia (the processed plant material) into a milling jar. This jar was then sequentially filled with larger and smaller diameter balls along with the tomato samples, ensuring that the combined volume did not exceed two-thirds of the jar’s capacity. Once loaded, the jar was securely sealed. The HEM process was activated for two hours to achieve the desired particle size reduction and uniform distribution. Following the milling process, the morphology and structural details of the nanoherbal tomato were analyzed using a Scanning Electron Microscope (SEM) at a magnification of 10,000×, specifically with the JSM-6390A model from Tokyo, Japan. This analysis confirmed the distribution of the particles.Further quantification of the particle size was conducted using a Particle Size Analyzer (PSA), with ethyl alcohol as the diluent to facilitate the measurement. This rigorous process of nanoformulation and analysis ensures the consistent quality and characteristics of the Nanoherbal Tomato, essential for its subsequent use in experimental and therapeutic applications.

2.3. Metabolite Profiling of NT by LC-MS/MS:

The LC-HRMS (Liquid Chromatography-High Resolution Mass Spectrometry) analysis of the Nanoherbal Tomato (NT) was meticulously carried out at the Integrated Laboratory of IPB University in Bogor, Indonesia. The instrumentation setup included an Agilent 6520 Accurate-Mass Q-TOF Mass Spectrometer, which is well-equipped with a G1311A quaternary pump, G1329A autosampler, and G1315D diode array detector. For this analysis, several specific source and scan parameters were set to optimize the detection and characterization of the NT compounds. The gas temperature was maintained at 30°C with a gas flow of 11 liters per minute, and the nebulizer pressure was set at 40 psi. Additional settings included a VCap of 3500, a fragmentor pressure of 175 psi, a skimmer1 pressure of 65.0 psi, and an octopoleRF Peak set at 750 psi.The chromatographic separation was conducted with a solvent system consisting of acetonitrile, 5mM acetate buffer, and water at a flow rate of 1.5mL/min. The gradient program started with 5% acetonitrile for the initial 0.1 minutes, increased to 30% over the next 10 minutes, peaked at 80% for 32 minutes, and then returned to the starting conditions to re-equilibrate the column. The column temperature was consistently controlled at 30 degrees Celsius throughout the elution process.After passing through the flow cell of the diode array detector, the column eluate was directed into the Q-TOF HRMS equipped with an electrospray interface. The mass spectrometry analysis was performed using positive electron spray ionization (ESI-positive mode) and covered a mass range of 100 to 2000 daltons with a scan rate of 1.03. This comprehensive setup and methodical approach ensure detailed profiling and accurate identification of metabolites within the Nanoherbal Tomato.

2.3. The α-glucosidase inhibition evaluation of NT:

100µg of Nanoherbal Tomato (NT) was dissolved in 70 µL of 50mM phosphate buffer (pH 6.8) containing 1 unit/mL of α-glucosidase. The solution was incubated at 37°C for 10 minutes. Subsequently, 10µL of 5mM ρ-nitrophenol glucopyranoside, used as the substrate, was added. The reaction was allowed to proceed for 30 minutes to ensure completion. The absorbance of the resulting ρ-nitrophenol, released from the substrate, was measured at 405nm.The acarbose was used as standard enzyme inhibitor. The % inhibition was measured by following formula:

(Absorbance of blank - Absorbance of control) x 100

% Inhibition= ----------------------------------------------

Absorbance of blank

2.4. The α-amilase inhibition evaluation of NT:

The experimental setup included a reaction mixture with α-amylase at a concentration of 0.2mg/mL and an activity of 2.6 U/mL (400 µL), combined with 800µL of test extracts or compounds at various concentrations in a 0.02 M phosphate buffer (pH 6.6). This mixture was incubated for 15 minutes at room temperature. To initiate the enzymatic reaction, 400µL of 1% starch solution was added to the mix. After a further 15-minute incubation, the reaction was halted by the addition of 300µL of dinitrosalicylic acid (DNS) reagent and 500 µL of phosphate buffer (0.02M, pH 6.9). The mixture was then heated to 100°C for 10 minutes. Upon cooling to room temperature, the absorbance was measured at 540nm using a UV-1601 Rayleigh spectrophotometer from Beijing, China. The results were expressed as IC50 values, calculated from concentration-inhibition calibration curves that included 4 to 5 data points.

2.4. Antioxidant activity of NT by DPPH scavanging activity:

To evaluate the antioxidant activity of the extracts, 1.0 mL of a 0.1M solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) was mixed with 0.9mL of a 50mM Tris-HCl buffer (pH 7.4). Then, 0.1mL of either the sample extract or deionized water (serving as the control) was added to the mixture. This mixture was thoroughly homogenized and subsequently left to incubate at room temperature for 30 minutes. After the incubation, the absorbance of the mixture was measured using a UV-Vis spectrophotometer at a wavelength of 517nm. The calculation of the DPPH scavenging activity was performed using the following formula:

Absorbance of control – Absorbance of sample

% Inhibition = --------------------------------------- × 100

Absorbance of control

2.5. Total Phenolic Compound:

The total phenolic content (TPC) was measured using the Folin–Ciocalteu method. For this procedure, a 20µL aliquot of the methanol solution of the dried sample extract (prepared at a 1:10 weight-to-volume ratio) was mixed with 1.58mL of distilled water and 100 µL of Folin-Ciocalteu reagent. Then, 300µL of a 5% sodium carbonate solution was added to the mixture. The resultant solution was then incubated at 25°C for 2 hours in a light-protected environment. Absorbance measurements were taken at 765nm using a spectrophotometer. A control was also prepared using distilled water following the same procedure to ensure accuracy. The TPC was quantified in terms of gallic acid equivalents (GAE) and expressed in milligrams per gram of dry extract. A calibration curve was established using various concentrations of gallic acid (5, 10, 20, 40, and 80 mg/L), achieving a coefficient of determination (R2) of 0.9871, indicating high reliability of the measurements.

2.6. Total Flavonoid Compound:

The total flavonoid content (TFC) of the sample was measured using a streamlined approach. In this process, 1 mL of the Nanoherbal Tomato (NT) extract was mixed with 300μL of a 5% NaNO2 solution and 300μL of a 10% aluminum chloride solution. This mixture was incubated at 25°C for 5 minutes. After the incubation, 2 mL of a 1 N sodium hydroxide solution was added. The mixture was then diluted to a final volume of 10 mL with water and thoroughly mixed using a vortex mixer to ensure complete homogenization. Absorbance was measured at 510nm using a spectrophotometer. To quantify the flavonoid content, a calibration curve was created using quercetin standards, resulting in a coefficient of determination (R2) of 0.974. The TFC was expressed in milligrams of catechin equivalents (CE) per gram of the sample on a dry weight basis. This method ensures accurate quantification of flavonoids in the sample, reflecting its potential health benefits.

2.7. Statistical Analysis:

Data in the study were reported as the mean ± standard error of the mean (SEM). All statistical analyses were performed using GraphPad Prism (version 10). For most of the data, one-way analysis of variance (ANOVA) followed by Dunnet’s post hoc test was utilized to compare the means of one group against all other groups. The correlation coefficient was determined using Microsoft Office Excel 2022. Statistical significance was established at a 5% level, meaning a p-value of ≤ 0.05 was considered significant. This approach ensures a rigorous evaluation of the data, allowing for accurate interpretation of the experimental results.

3. RESULT:

3.1. Nanoherbal Tomatao (NT) Evaluation:

Upon examination using electron microscopy, it was shown that nanoherbal tomato exhibited clots of significantly reduced dimensions, measuring 0.440 μm (Figure 1). Additionally, larger clots measuring 44 × 103 nm were observed, indicating a higher efficacy in cellular penetration. The reduction of drug molecules to the nanometer scale has the potential to enhance the effectiveness of the medicinal molecule. According to the Particle Size Analyzer (PSA), the nanoherbal tomato exhibits an average diameter distribution of 936 nm ± 112.4 (Figure 1). The cumulant result indicates a diameter of 1511.0 nm, while the polydispersity index value is 1.223. These measurements were obtained under the condition of 25°C. In addition, it is worth noting that the refractive index measured 1.3222, the viscosity in centipoise (cP) was determined to be 0.8878, and the scattering intensity in counts per second (CPS) was recorded as 11114.

3.2. Metabolite profiling of NT by LC-HRMS:

The LC-HRMS (Liquid Chromatography-High-Resolution Mass Spectrometry) profiling of NT successfully identified and quantified the presence of Ellagic acid, Hexadecanamide, Dibenzylamin, Myricetin, Kaempferol and Caretenoid. Ellagic Acid (C14H6O8, Molecular Weight: 302.006), Retention Time (RT): 7.248 min, Area (Max.): 79,509,340.55, mzCloud Best Match: 96.5. Ellagic acid is a well-known phytochemical with antioxidant properties. Its presence in the nanoherbal tomato extract suggests potential health benefits related to oxidative stress and inflammation. This compound has been linked to various health benefits, including anti-cancer and anti-inflammatory effects. Hexadecanamide (C16H33NO, Molecular Weight: 255.25), Retention Time (RT): 22.633 min, Area (Max.): 40,22,299.88, mzCloud Best Match: 99.3.

Hexadecanamide, also known as palmitamide, is a fatty acid derivative. Its identification indicates the presence of lipid compounds in the extract. Fatty acids play essential roles in various physiological processes and may contribute to the extract's hepatoprotective effects. Dibenzylamine (C14H41N, Molecular Weight: 197.12), Retention Time (RT): 7.77 min, Area (Max.): 24,123,319.45, mzCloud Best Match: 97.5 Dibenzylamine is an aromatic amine compound. Its presence suggests the existence of aromatic compounds in the nanoherbal tomato extract. Aromatic compounds can have diverse biological activities and may contribute to the extract's overall pharmacological effects. Myricetin (C15H10O8, Molecular Weight: 318.037), Retention Time (RT): 7.27 min, Area (Max.): 12,512,291.87, mzCloud Best Match: 99.5 Myricetin is a flavonoid with antioxidant and anti-inflammatory properties. Its identification is significant as it is known for its potential antidiabetic effects. Myricetin may contribute to the extract's ability to modulate blood glucose levels and improve insulin sensitivity. Kaempferol (C15H, Molecular Weight: 286.04), Retention Time (RT): 8.843 min, Area (Max.): 6,231,657.95, mzCloud Best Match: 92.3m Kaempferol is another flavonoid with antioxidant and anti-inflammatory properties. It has been studied for its potential antidiabetic effects and ability to protect against diabetic complications. Its presence adds to the extract's potential in managing diabetes and associated organ protection. The data can be shown in the Table 1.

3.3. The α-glucosidase inhibition evaluation of NT:

In the alpha-glucosidase inhibition assay, Nanoherbal Tomato demonstrated promising inhibitory activity with an IC50 value of 51.62 µg/mL. This result suggests that Nanoherbal Tomato possesses significant potential as an alpha-glucosidase inhibitor. For reference, the standard drug acarbose exhibited an IC50 value of 78.1 µg/mL, indicating that Nanoherbal Tomato's inhibitory activity is even more potent than that of acarbose in this particular assay. This finding underscores the potential therapeutic value of Nanoherbal Tomato in managing conditions related to carbohydrate metabolism, such as diabetes. Further research is warranted to elucidate the mechanisms behind this inhibitory activity and to explore its broader implications for health and disease management.

Figure 2. The α-glucosidase inhibition evaluation of Nanoherbal tomat (IC50: 51.62 µg/mL), while acarbose as drug standar (IC50: 78.1 µg/mL)

3.4. The α-amilase inhibition evaluation of NT:

In the alpha-amylase inhibition assay, Nanoherbal Tomato displayed notable inhibitory activity with an IC50 value of 62.31 μg/mL. This result suggests that Nanoherbal Tomato possesses significant potential as an alpha-amylase inhibitor, showcasing its ability to modulate carbohydrate digestion. For reference, the standard drug acarbose exhibited an IC50 value of 58.56 μg/mL, indicating that Nanoherbal Tomato's inhibitory activity is slightly higher than that of acarbose in this specific assay. These findings highlight the potential therapeutic value of Nanoherbal Tomato.

Figure 3. The α-amilase inhibition evaluation of Nanoherbal tomat (IC50 : 62.31 μg/mL), while acarbose as drug standar (IC50: 58.56 μg/mL).

3.5. Antioxidant activity of NT by DPPH scavanging activity:

In the DPPH scavenging activity assay, Nanoherbal Tomato (NT) exhibited antioxidant potential with an IC50 value of 48.281 µg/mL. While Nanoherbal Tomato's DPPH scavenging activity is substantial, it's worth noting that the standard antioxidant, vitamin C, displayed a more potent scavenging activity with an IC50 value of 12.872 µg/mL. These results indicate that vitamin C is a stronger DPPH scavenger when compared to Nanoherbal Tomato in this specific assay. However, the antioxidant activity demonstrated by Nanoherbal Tomato is still noteworthy and suggests its potential contribution to overall antioxidant defenses in biological systems. Further investigations can provide insights into the specific antioxidant compounds present in Nanoherbal Tomato and their mechanisms of action.

Figure 3. The IC50 of DPPH radical scavanging activity of NT

3.5. Total Phenol and Flavonoid Content of NT:

The Folin-Ciocalteu method was employed to estimate the total phenolic content (TPC), whereas the aluminium chloride approach was utilized to quantify the total flavonoid content (TFC). The total phenolic content (TPC) in the extracts was quantified using a regression equation of the form y = 0.007x + 0.02. Furthermore, the calculated coefficient of determination (R2) for the present regression model yielded a value of 0.9781. The quantities obtained were measured and expressed in units of gallic acid equivalency (mg GAE/g). In contrast, the quantification of the flavonoid was assessed by employing the existing standard curve of quercetin. This curve was constructed and examined using the regression equation y = 0.009x + 0.03. The determination coefficient (R2) for the regression model was computed as 0.9813. The concentration measurement was denoted as milligrams of quercetin equivalents per gram of the plant extract (mg QE/g). This study presents the results about the determination of the overall phenolic and flavonoid content in the nanoherbal tomato. The highest recorded values for the total phenolic content (TPC) and total flavonoid content (TFC) in PE were 300.21 ± 15.20 mg GAE/g and 20.78 ±1.82 mg QE/g, respectively.

4. DISCUSSION:

Nanoherbal Tomato, a product of scientific exploration and innovation, represents an intriguing intersection of traditional herbal knowledge and cutting-edge nanotechnology. This nanoscale herbal preparation has garnered significant attention due to its potential health benefits and unique properties. In this discussion, we delve into the characteristics of Nanoherbal Tomato, with a particular focus on its particle size and morphology as revealed by particle size analysis and scanning electron microscopy (SEM). The particle size of a nanomaterial is a critical parameter that can significantly influence its behavior and efficacy. In the case of Nanoherbal Tomato, particle size analysis has revealed a remarkably small average particle size of approximately 936 nm. This nanoscale size is a key feature that distinguishes it from conventional herbal extracts and has implications for its bioavailability and therapeutic potential. Nanoherbal Tomato's submicron particle size is advantageous for several reasons: Enhanced Bioavailability: Nanoparticles can more readily penetrate biological barriers, such as cell membranes, leading to improved bioavailability of the active herbal constituents. This property may enhance the absorption and distribution of the bioactive compounds in the body, potentially increasing their therapeutic effectiveness. Increased Surface Area: Nano-sized particles possess a significantly larger surface area per unit mass compared to bulk materials. This increased surface area can facilitate interactions with target cells, enzymes, or receptors, potentially enhancing the pharmacological effects of Nanoherbal Tomato^21-25. Improved Stability: The nanoscale formulation can contribute to the stability and solubility of the herbal extract. This may result in a longer shelf life and better preservation of the active compounds, ensuring product quality over time. Alpha-glucosidase and alpha-amylase inhibitors play a crucial role in the management of diabetes and metabolic disorders. These inhibitors can help regulate post-meal blood glucose levels by slowing down the digestion and absorption of carbohydrates^[26]. Nanoherbal Tomato, a product of advanced research and innovation, has demonstrated intriguing potential as an inhibitor of both alpha-glucosidase and alpha-amylase enzymes, as evidenced by its IC50 values in comparison to the standard drug acarbose. Nanoherbal Tomato exhibits a notable alpha-glucosidase inhibitory activity with an IC50 value of 51.62 µg/mL. In contrast, acarbose, the standard drug, has an IC50 value of 78.1 µg/mL. These results indicate that Nanoherbal Tomato is a more potent inhibitor of alpha-glucosidase than acarbose. This finding holds significant promise for individuals with diabetes or those at risk, as it suggests that Nanoherbal Tomato may effectively help control post-meal blood glucose levels, potentially reducing the need for synthetic pharmaceuticals. Alpha-amylase inhibition is another key mechanism for regulating carbohydrate digestion and glycemic control^[27]. Acarbose is once again employed as the standard reference compound in this context. Nanoherbal Tomato demonstrates a noteworthy alpha-amylase inhibitory activity with an IC50 value of 62.31 µg/mL, while acarbose, the standard drug, displays an IC50 value of 58.56 µg/mL. In this case, acarbose shows a slightly stronger inhibitory effect on alpha-amylase compared to Nanoherbal Tomato. However, the difference in IC50 values between the two is minimal. These findings indicate that Nanoherbal Tomato's alpha-amylase inhibitory activity is comparable to that of acarbose, a widely used pharmaceutical inhibitor²⁸. This suggests that Nanoherbal Tomato may effectively modulate carbohydrate digestion, further supporting its potential as a natural therapeutic option for individuals concerned with blood sugar management. Antioxidants play a pivotal role in maintaining health and combating oxidative stress-related conditions. Nanoherbal Tomato (NT) has emerged as a novel source of antioxidants, with potential health benefits. In this discussion, we explore the antioxidant activity of NT using the DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging assay and compare its performance to that of vitamin C, a well-known antioxidant²⁹. The DPPH scavenging assay is a widely employed method to assess the antioxidant activity of natural compounds and extracts. DPPH is a stable free radical with a purple color, which turns yellow upon reduction by an antioxidant. The degree of discoloration correlates with the antioxidant capacity of the test substance. The IC50 value represents the concentration of the test substance required to scavenge 50% of the DPPH radicals, serving as a measure of its antioxidant potency. In the DPPH scavenging activity assay, Nanoherbal Tomato (NT) demonstrated significant antioxidant potential with an IC50 value of 48.281 µg/mL. This indicates that NT possesses the ability to effectively neutralize free radicals, a hallmark of antioxidant activity^30,31. These results highlight the potential health benefits of NT in mitigating oxidative stress-related conditions and protecting cells from damage caused by free radicals. Vitamin C, or ascorbic acid, is a well-known antioxidant and an essential nutrient for human health. It serves as a benchmark for evaluating the antioxidant activity of other compounds. In the DPPH assay, vitamin C exhibited a lower IC50 value of 12.872 µg/mL compared to NT. This comparison reveals that vitamin C is a stronger DPPH scavenger than NT in this specific assay. Vitamin C is renowned for its potent antioxidant properties and its role in bolstering the immune system and promoting skin health. Its ability to effectively neutralize free radicals has been extensively documented in scientific research. The results of the DPPH scavenging activity assay indicate that NT possesses substantial antioxidant potential³¹. While vitamin C exhibits stronger DPPH scavenging activity in this particular assay, it is important to remember that the antioxidant activity of natural extracts like NT may extend beyond the scope of a single assay. In conclusion, Nanoherbal Tomato demonstrates promising antioxidant activity as evidenced by the DPPH scavenging assay. Although vitamin C surpasses it in this particular test, NT's complex mixture of bioactive compounds suggests broader potential health benefits beyond its performance in a single antioxidant assay. As we continue to unravel the mysteries of NT's antioxidant properties, it may find applications in promoting health and well-being by combating oxidative stress and its associated ailments.

CONFLICTS OF INTEREST:

The authors declare no conflict of interest.

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Received on 28.01.2024 Modified on 16.04.2024

Research J. Pharm. and Tech 2024; 17(10):4953-4960.

DOI: 10.52711/0974-360X.2024.00762

Name	Formula	Molecular Weight	RT (min)	Area (Max.)	mzCloud best Match
Ellagic acid	C₁₄H₆O₈	302.006	7.248	79,509,340.55	96.5
Hexadecanamide	C₁₆H₃₃NO	255.25	22.633	40,22,299.88	99.3
Dibenzylamin	C₁₄H₄₁N	197.12	7.77	24,123,319.45	97.5
Myricetin	C₁₅H₁₀O₈	318.037	7.27	12,512,291.87	99.5
Kaempferol	C₁₅H₁₀O₆	286.04	8.843	6,231,657.95	92.3
Caretenoid	C₄₀H₅₆	536.	11.37	52,113,211.62	99.8