The effects of heating process on protein isolation of lionfish (Pterois volitans) spines venom extract to antioxidant activity assay

✅ 全文

加热过程对蓑鲉（Pterois volitans）棘刺毒液提取物蛋白质分离对抗氧化活性测定的影响

作者 Andy Noorsaman Sommeng; Indriani Pratiwi; Mikael Januardi Ginting; Muhamad Sahlan; Heri Hermansyah; Anondho Wıjanarko 期刊 AIP conference proceedings 发表日期 2019 ISSN 0094-243X DOI 10.1063/1.5139327 类型原创研究 (Original Research)

📄 中文摘要 Chinese Abstract

中文

蓑鲉（*Pterois volitans*）是一种原产于印度-太平洋地区的入侵性捕食性物种，已扩散至其他海洋生态系统，由于其快速生长且缺乏天敌，造成了严重的生态损害。其毒刺中含有毒性蛋白质和生物活性化合物，可对人体产生严重的生理影响，包括疼痛、麻痹和细胞死亡。先前研究表明，蓑鲉毒刺毒液表现出较弱的抗氧化活性，但粗提物中杂质的存在限制了其潜在应用。为了提高毒液蛋白质的纯度和生物活性，需要额外的分离步骤——特别是加热——以去除污染物并浓缩活性成分。

📋 英文结构化总结 English Structured Summary

全文整理

Background:

Lionfish (*Pterois volitans*) is an invasive predatory species native to the Indo-Pacific region that has spread to other marine ecosystems, causing significant ecological damage due to its rapid growth and lack of natural predators. Its venomous spines contain toxic proteins and bioactive compounds that can cause severe physiological effects in humans, including pain, paralysis, and cell death. Previous studies have indicated that lionfish spine venom exhibits weak antioxidant activity, but the presence of impurities in crude extracts limits its potential applications. To enhance the purity and bioactivity of the venom proteins, additional isolation steps—particularly heating—are necessary to remove contaminants and concentrate active components.

Methods:

Crude venom (CV) was extracted from lionfish spines using sonication and phosphate buffer, followed by centrifugation and filtration. The venom was then subjected to heating at three different temperatures (60°C, 75°C, and 90°C) for 10 minutes with continuous stirring. After heating, proteins were fractionated using ammonium sulfate at varying saturation levels (0–20%, 20–40%, 40–60%, and 60–80%). Protein concentration was determined via the Lowry method using bovine serum albumin (BSA) as a standard. Toxicity was assessed using the Brine Shrimp Lethality Test (BSLT), and antioxidant activity was evaluated using the DPPH radical scavenging assay. Protein composition was analyzed by SDS-PAGE to identify molecular weight profiles under different treatment conditions.

Results:

The highest protein concentration was observed in the unheated crude venom, while heating significantly reduced total protein yield due to denaturation and precipitation of non-target proteins. However, heating improved the specificity of protein isolation during ammonium sulfate fractionation. The most effective condition for antioxidant activity was achieved with a heating temperature of 75°C and 40–60% ammonium sulfate saturation, yielding an IC₅₀ value of 1312 ppm. This sample also showed the highest DPPH inhibition (76.13%). SDS-PAGE analysis revealed that optimal antioxidant activity correlated with the presence of proteins at 7.9 kDa, 46.2 kDa, and 52.7 kDa. Higher temperatures (e.g., 90°C) eliminated larger proteins like PLA₂ (85.2 kDa), which may interfere with antioxidant function, but also removed beneficial mid-weight proteins.

Data Summary:

Protein concentrations ranged from 11.27 ppm (60°C, 60–80% AS) to 278.3 ppm (crude venom). LC₅₀ values from BSLT ranged from 377.63 ppm (CV, 60–80% AS) to 1000 ppm (90°C, 0% AS), indicating moderate toxicity across all samples. Antioxidant IC₅₀ values varied widely; the lowest (most active) was 1312 ppm (75°C, 40–60% AS), while others exceeded 70,000 ppm, reflecting very weak activity. The 75°C/40–60% AS condition outperformed prior studies on lionfish venom (IC₅₀ = 1563.06 ppm) and other marine organisms such as *Diadema setosum* (IC₅₀ = 2826.13 ppm).

Conclusions:

Heating during protein isolation enhances the purity of lionfish spine venom extracts by denaturing and removing interfering proteins, thereby improving antioxidant potential. The optimal condition for isolating antioxidant-active proteins is heating at 75°C followed by ammonium sulfate fractionation at 40–60% saturation. This method yields a distinct protein profile (7.9, 46.2, and 52.7 kDa) associated with the highest antioxidant activity observed in the study. Although the resulting IC₅₀ (1312 ppm) classifies the extract as a very weak antioxidant compared to potent standards, it demonstrates improved efficacy over previous isolation methods and certain marine-derived venoms.

Practical Significance:

This research supports the potential valorization of invasive lionfish biomass by demonstrating a refined method to isolate bioactive proteins with antioxidant properties from their venom. Such proteins could serve as leads for pharmaceutical or nutraceutical development, particularly in oxidative stress-related applications. Additionally, the findings contribute to ecological management strategies by incentivizing lionfish population control through utilization of otherwise discarded tissues like spines.

📋 中文结构化总结 Chinese Structured Summary

中文

背景:

方法:

采用超声破碎法和磷酸盐缓冲液从蓑鲉毒刺中提取粗毒液（CV），随后进行离心和过滤。然后将毒液在三种不同温度（60°C、75°C和90°C）下加热10分钟，并持续搅拌。加热后，使用不同饱和度（0–20%、20–40%、40–60%和60–80%）的硫酸铵对蛋白质进行分级分离。蛋白质浓度采用Lowry法测定，以牛血清白蛋白（BSA）作为标准品。毒性通过卤虫致死试验（BSLT）进行评估，抗氧化活性采用DPPH自由基清除法进行评价。通过SDS-PAGE分析蛋白质组成，以鉴定不同处理条件下的分子量分布特征。

结果:

未加热的粗毒液中蛋白质浓度最高，而加热由于非目标蛋白质的变性和沉淀显著降低了总蛋白得率。然而，加热提高了硫酸铵分级分离过程中蛋白质分离的特异性。抗氧化活性最佳的条件为75°C加热温度配合40–60%硫酸铵饱和度，其IC₅₀值为1312 ppm。该样品的DPPH抑制率也最高（76.13%）。SDS-PAGE分析表明，最佳抗氧化活性与7.9 kDa、46.2 kDa和52.7 kDa蛋白质的存在相关。较高温度（如90°C）消除了较大的蛋白质，如PLA₂（85.2 kDa），这些蛋白质可能干扰抗氧化功能，但同时也去除了有益的中等分子量蛋白质。

数据汇总:

蛋白质浓度范围为11.27 ppm（60°C，60–80% AS）至278.3 ppm（粗毒液）。BSLT的LC₅₀值范围为377.63 ppm（CV，60–80% AS）至1000 ppm（90°C，0% AS），表明所有样品均具有中等毒性。抗氧化IC₅₀值差异很大；最低值（活性最高）为1312 ppm（75°C，40–60% AS），而其他样品的IC₅₀值超过70000 ppm，反映出极弱的活性。75°C/40–60% AS条件下的表现优于此前关于蓑鲉毒液的研究（IC₅₀ = 1563.06 ppm）以及其他海洋生物如*Diadema setosum*（IC₅₀ = 2826.13 ppm）。

结论:

蛋白质分离过程中的加热通过变性和去除干扰蛋白质，提高了蓑鲉毒刺毒液提取物的纯度，从而增强了抗氧化潜力。分离抗氧化活性蛋白质的最佳条件为75°C加热后采用40–60%饱和度的硫酸铵分级分离。该方法产生独特的蛋白质谱（7.9、46.2和52.7 kDa），与本研究中观察到的最高抗氧化活性相关。尽管所得IC₅₀值（1312 ppm）与强效标准品相比仍属于极弱抗氧化剂，但其效力较先前的分离方法和某些海洋生物来源的毒液有所提高。

实际意义:

本研究支持了入侵性蓑鲉生物质资源化利用的潜力，展示了一种从毒液中分离具有抗氧化活性的生物活性蛋白质的改进方法。此类蛋白质可作为药物或营养保健品的先导化合物，特别是在氧化应激相关领域的应用。此外，该发现通过利用原本被丢弃的组织（如毒刺）来激励蓑鲉种群控制，为生态管理策略提供了支持。

📖 英文全文 English Full Text

AIP Conference Proceedings 2193, 020007 (2019); https://doi.org/10.1063/1.5139327

The effects of heating process on protein isolation of lionfish (Pterois volitans) spines venom extract to antioxidant activity assay

Cite as: AIP Conference Proceedings 2193, 020007 (2019); https://doi.org/10.1063/1.5139327

Published Online: 10 December 2019 Andy Noorsaman Sommeng, Indriani Pratiwi, Mikael Januardi Ginting, Muhamad Sahlan, Heri

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The Effects of Heating Process on Protein Isolation of

Lionfish (Pterois volitans) Spines Venom Extract to

Antioxidant Activity Assay Andy Noorsaman Sommeng1, Indriani Pratiwi1, Mikael Januardi Ginting2,

Muhamad Sahlan1,3, Heri Hermansyah1, and Anondho Wijanarko1, a)

1Department of Chemical Engineering, Faculty of Engineering, Universitas Indonesia, Kampus UI Depok, West

Java, 16424 Indonesia 2Marine Science Postgraduate Program, Faculty of Mathematics and Natural Science, Universitas Indonesia,

Kampus UI Depok, West Java, 16424 Indonesia 3Research Centrer for Biomedical Engineering, Faculty of Engineering Universitas Indonesia, Kampus UI Depok,

West Java, 16424 Indonesia

Corresponding author: a)anondho.wijanarko@yahoo.com

Abstract. Lionfish (Pterois volitans) was classified as an invasive predator native to the Indo-Pacific region that has no natural predators. Lionfish grow fast, making these fish prey on much smaller fish in which causing damage to the marine ecosystem. This research determines the antioxidant potential of lionfish spines venom by isolating the protein from its extract. In previous research, it was specified that lionfish poison extract have had the potential as an antioxidant, however, it was still feeble. In order to find out the potential further, the additional isolation step is heating. Crude venom (CV) was extracted using sonication and isolated by heating at 90°C, 75°C, and 60°C followed by fractionated using Ammonium

Sulfate (AS) with 0-20, 20-40, 40-60, and 60-80% saturation. The concentrated protein was then analyzed by the Lowry test and the protein content was identified using SDS-PAGE, followed by toxicity test using BSLT method (Brine Shrimp

Lethality Test). The antioxidant activity was carried out using the DPPH method in the final step. The optimum condition for isolating proteins which have the potential to have antioxidant activity observed in specimen with a heating temperature of 75℃ and saturation of ammonium sulfate 40-60% with an IC50 value of 1312 ppm. The protein composition at the protein isolation temperature for optimum antioxidant is protein 7.9; 46.2; and 52.7 kD.

Keywords: Crude venom, Protein isolation, Antioxidant activity

INTRODUCTION The Lionfish are nocturnal predator species that consume crustaceans, small fish, and crabs [1]. Behind its unique and attractive shape, Lionfish ranks highest in the food chain in the ocean due to its sting in which dangerous to other living things. Fast lionfish growth rate, has made Lionfish prey on a large quantity of fish. It cause heavy damage to the marine ecosystem. Furthermore, reef fish in which are a source of catch for fisherman are also a prey for lionfish [2].

The sting produced by lionfish is lethal and it was found in the spines. It would cause a burning sensation for 15- 20 minutes. Within 3 hours after being exposed to this poison will cause limb paralysis [3]. This poison will give cardiovascular, neuromuscular, and cytolytic effects resulting cell death. This toxic effect was supported by the presence of toxic proteins and other active components such as acetylcholine and venom pore-forming in venom [4].

Given that the native habitat of lionfish is the Indo-Pacific Ocean adjacent to the Indian Ocean, efforts should be made to reduce the population, and further use of lionfish starts from utilization lionfish meat as an alternative source of food ingredients and the utilization of lionfish spines venom as medicinal ingredients.

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AIP Conf. Proc. 2193, 020007-1–020007-9; https://doi.org/10.1063/1.5139327

Published by AIP Publishing. 978-0-7354-1944-5/$30.00

020007-1 The lionfish spines venom has antioxidant activity. The research that has been done so far is to test antioxidant compounds from proteins in the venom extract of lionfish. However, protein isolation could only be carried out by precipitation method using ammonium sulfate without heating the protein. The study showed that the results of protein isolation from lionfish had antioxidant activity, except that it was still feeble. This result was still not optimum because the results of protein isolation carried out are still contaminated [5]. Therefore, it is necessary to add an isolation process, namely heating the protein first to produce pure isolates to be tested for further antioxidants.

MATERIALS AND METHODS Materials Lionfish that used as samples in this experiment were obtained from Java Island, Indonesia. The samples obtained were prepared to separate the parts between the venomous spines from the rest of the body.

Methods Preparation Lionfish Spines Venom Before the experiment, the lionfish cadaver were stored in minus conditions. It was suggested to precess the lionfish as soon as possible in order to maintain its quality. After that, the spines in some parts of his body to cut the spines at the base are also in cold conditions. Then, the spines that have been separated was rinsed with 0.01 M phosphate buffer pH 7.0. The yield of the lionfish spines that have been obtained was then weighed using a mass balance. Then immersed the yield of lionfish spines in a solution of 0.01 M phosphate buffer pH 7.0 containing 0.001

M CaCl2 with a ratio of 1: 2 overnight [6].

Venom Protein Isolation Lionfish spines were extracted by sonication of the samples twice each for 8 minutes at 20 kHz. The sonication results were centrifuged at a speed of 4500 rpm for 2 x 15 minutes. Separated impurities were then filtered using

Whatman 42 paper. To isolate proteins in crude venom, heating in crude venom is needed. Before doing this, prepare a hot plate laboratory and 600 mL beaker glass containing water as a heating medium. After that, insert the crude venom in the beaker glass, which is smaller than the beaker glass used as a heating medium. Then the beaker glass containing crude venom, put in a beaker glass filled with water, then put it on a hot plate. The temperature of the hot plate was then adjusted so that the temperature in the beaker glass containing the crude venom has the desired temperature, namely 60 °C, 75 °C, and 90 °C for 10 minutes. During the heating of the crude venom, stirring was carried out using a magnetic stirrer. After heating, centrifuge the sample for 30 minutes at 15,000xg and 40C, so that the sediment was formed. The supernatant (CV) was obtained. Isolated protein was obtained from venom precipitation with ethanol 90 % and ammonium sulfate 20, 40, 60, and 80 % saturation. The samples were centrifuge at 4500 rpm.

Protein settles down and ready to use further [7].

Protein Concentration by Lowry’s Method To determine the protein concentration by the Lowry method, we made a standard solution of Bovine Serum

Albumin (BSA) 200 µg / mL with a BSA concentration of 20-200 mg in a standard solution of 1 mL, Lowry reagent (1 mL 1% CuSO4, 1 mL Tartrat NaK 1% and 100 mL 2% Na2CO3 in 0.1 N NaOH with 0.5 mL Fenol Ciocalteau 1 N

Folin reagent), and 1 mL of distilled water. Standard solution, blank and 20 µl sample added Lowry reagent (Biuret)

5 mL and 10-minute incubation. Then add a solution of 0.5 mL Folin–Ciocalteu reagent 1 M and 30 minutes incubation. Standard solutions, blanks, and samples were measured absorbance at wavelength λ 750 nm. The results of absorbance data were then plotted on the BSA standard curve [8].

Toxicity Test Toxicity testing on proteins by the BSLT method, which previously carried out the hatching of 10 mg shrimp eggs in 250 mL of seawater by giving light and aerator irradiation. Hatching of these larvae is carried out for 2 x 24 hours.

020007-2 Making the primary sample of 2000 ppm by weighing 20 mg of sample which was then dissolved in 10 mL of seawater and followed by homogenization with the addition of 3 drops of tween 80% and sonication for 5 minutes. Then prepare

1 mL of seawater containing ten shrimp larvae for each sample. After that, add the test solution to have concentrations of 10, 100, 500, 1000, and 2000 ppm. Calculation of dead shrimp larvae is carried out after 24 hours.

Antioxidant Activity Assay with DPPH DPPH solution was made at 125 µM by weighing 2.5 mg DPPH in 50 mL of ethanol and cover up with aluminum foil. Make a sample in concentration 20 ppm. Insert a 100μL sample and 100μL DPPH solution into a microplate.

Blank was made for 200 µL ethanol. Cover up the microplate with aluminum foil and incubate for 30 min. Measure the absorbance of blank solution and sample using Microplate Elisa Reader at wavelength λ 517 nm. Plot absorbance into the equation to obtain inhibition value [9].

%𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛= 𝑏𝑙𝑎𝑛𝑘 𝑎𝑛𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒−𝑠𝑎𝑚𝑝𝑙𝑒 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑏𝑙𝑎𝑛𝑘 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑥 100% (1)

To get the IC50 value, first plot the graph of line equations from the percent of inhibition made for each sample.

The resulting equation is then obtained by the value x (IC50) by changing the value of y = 50 [5].

Determination of Protein Composition with SDS PAGE

Buffer samples was made of 0.5M Tris HCl pH 6.8, SDS 10%, mercaptoethanol, glycerol, and bromophenol blue

0.1%. Then make a solution of running buffer (tris buffer base, glycine, and SDS) and transfer buffer solution (tris buffer base, glycine, and methanol), and make 7.5% and 17.5% gel gradients and put in the mixing chamber. Both concentrations of the gel were mixed and fed into a glass mold through a hose with a gel height of 5 cm. Wait for the gel to harden for 1 hour and pour water on the gel surface to prevent oxidation from the air. Make stacking gel 4%, put the comb in. After the stacking gel hardens, lift the comb carefully and rinse with water. Gel holders containing the SDS-Page gel were placed on the casting stand. The samples were each added 1μL 4μL PBS (Phosphate Buffer

Saline) and 5μL buffer samples. Heat each mixture of samples for 45 seconds at 100°C and then connects the chamber to the power supply with a voltage of 150V for 1 hour. Soaking gel in 20 mL staining solution and shake for 15 minutes. Pour back the staining solution from the plate and soak the gel with a 50 mL destaining solution until the protein band is visible. After 1 hour, open the gel mold and stored the gel in transfer buffer for analysis [7].

RESULTS AND DISCUSSION Protein Concentration Protein concentration data obtained from plotting the standard protein concentration curve first. The standard curve was made from the measurement of BSA absorbance first with various variations. From the curve, we get the line equation which will be used to determine the protein concentration in the sample. The absorbance value of the sample obtained was then entered into a standard curve equation, so that the protein concentration values obtained in the sample were obtained. The concentration of protein in various samples are presented in Table 1.

020007-3 TABLE 1. Protein concentration of samples

Variable Protein Concentration (ppm) Heat Temperature (°C)

25 (CV) 90 75 60 AS 0% 278.305 54.2369 44.2941 27.6044

AS 20% 45.0043 143.367 133.4242 124.1916 AS 40% 72.7021

42.8737 63.4695 71.9919 AS 60% 59.5634 72.347 41.0982

50.3308 AS 80% 20.5024 36.1268 19.7922 11.2698

From Table 1, the highest concentration value observed in the CV sample, and the lowest observed in the sample with 60°C heating treatment and ammonium sulfate fractionation with a saturation of 60-80%. It can be seen in Table

1 that the heating process dramatically affects the amount of protein concentration isolated in the next stage, which is the fractionation phase of ammonium sulfate. At 0-20% ammonium sulfate fractionation, it can be seen that by heating the protein, the isolated protein is far more than the unheated sample. It proves that protein warming can play a role in eliminating protein so that when isolated by fractionation of ammonium sulfate, the protein will collect and form deposits. This event applies to all heating temperatures performed.

FIGURE 1 The concentration of protein contained in each sample to the heating temperature

In Figure 1, it can be seen that there is a difference in the protein concentration values in each sample, along with differences in heating temperature. The higher the heating temperature used, the higher the concentration of protein produced. It proves that higher the heating temperature, the more protein is isolated due to the factor of the number of proteins denatured during heating so that they accumulate at the time of fractionation of ammonium sulfate.

FIGURE 2 The concentration of protein contained in each sample to the saturation of ammonium sulfate

020007-4 Figure 2 demonstrates that with more and more fractionation of ammonium sulfate and the higher the saturation, the smaller the concentration of protein is isolated. It proves that the more stages of fractionation of ammonium sulfate are carried out, the less protein was isolated. The protein has been isolated a lot at the previous fractionation stage of ammonium sulfate, as is the case with 0-20% ammonium sulfate saturation. Ammonium sulfate-level fractionation can make more pure protein obtained.

Toxicity Test In this research, the toxicity testing of samples by the BSLT method. Brine Shrimp Lethality Test (BSLT) is a test method that can be used for early detection of toxic potential inactive compounds of natural ingredients. Artemia salina was used as a bioindicator. Each sample was tested by observing the number of larval deaths by probit analysis.

Calculation of dead shrimp larvae is carried out after 24 hours.

In order to obtain the LC50 (Lethal Concentration) value, the number of shrimp larvae deaths produced in each sample is calculated into percentages to find out the probit value from the probit table by converting the percent value of larval mortality. After that, graphs of straight-line equations are drawn up between the probit value and log concentration for each sample. Through the equation from the regression chart that was formed, the toxicity value of

LC50 was obtained.

There are three categories of material toxicity based on LC50 values which are very toxic categories with LC50 values < 30 ppm, toxic with LC50 values of 30-1000 ppm, and not toxic with LC50 values > 1000 ppm [10]. The higher of LC50 value then have smaller of toxicity ability. Here is the LC50 value of each sample.

TABLE 2. LC50 value of samples Variable LC50 (ppm)

Heat Temperature (°C) 25 (CV) 60 75 90 AS 0% 573.21

729.11 1000 883.85 AS 20% 512.03 540.94 775.59 789.29

AS 40% 423.48 529.25 642.29 688.14 AS 60% 402.31 388.59

532.12 499.6 AS 80% 377.63 400.78 422.15 432.48

From Table 2, it can be seen that the resulting LC50 value is different. The LC50 value produced is in the range of

300-900 ppm. The highest LC50 value is found in the sample with 90°C heating treatment without ammonium sulfate fractionation, while the smallest LC50 value is in the CV sample without heating treatment with ammonium sulfate saturation fraction 60-80%.

FIGURE 3. LC50 value at each variation of heating temperature

In Figure 3, it can be seen that the heating process dramatically affects the ability of toxicity or the LC50 value of a sample. Samples carried out by the heating process have a higher LC50 value compared to samples that were not carried out by the heating process. It also has seen that the higher the heating temperature used, the greater the LC50

020007-5 value. It proves that the addition of the heating process in protein isolation can further eliminate potentially toxic proteins.

FIGURE 4. LC50 value for each variation of ammonium sulfate saturation

Whereas in FIGURE 4 it can be seen that the fractionation stage of the protein using ammonium sulfate has an inverse result, that is, the more fractionation stages are carried out using ammonium sulfate, the smaller the LC50 value obtained. It proves that the fractionation of proteins using ammonium sulfate can increase the value of toxicity capabilities in increasing samples. It was due to the high saturation of ammonium sulfate was given step by step, the more isolation of the poison protein is getting better, and the more specific the protein contained. Even so, all of these samples are still categorized as toxic because the LC50 value is still in the range of 30-1000 ppm.

In this research, the most toxic sample was treated with samples without heating and fractionating ammonium sulfate with a saturation of 60-80%, which has an LC50 value of 377.63 ppm. This value is higher than the LC50 value from poison extraction research of lionfish using the protein deposition method using ammonium sulfate [5], not by the fractionation method, which is equal to 101.93 ppm. It shows that the fractionation method of ammonium sulfate can reduce toxic potential more than by the method of precipitation of ammonium sulfate. Compared to the Stingray

Dasyatis kuhli venom with LC50 of 161.6 ppm [13] and methanol extract of the sea urchin Diadema setosum venom with LC50 values of 563.26 ppm [14], an extract of spines venom of lionfish has higher toxicity.

Antioxidant Activity Assay DPPH antioxidant activity assay uses ascorbic acid or vitamin C as a comparative compound that functions as a positive control of compounds containing antioxidant compounds. The antioxidant activity of the sample resulted in discoloration of the DPPH solution in methanol, which was initially concentrated violet to pale yellow [11]. The antioxidant potential was obtained by calculating the percent inhibition, namely the ability of a material to inhibit free radical activity. The percentage of sample inhibition can be calculated by subtracting the absorbance of the blank by absorbance of the sample previously obtained by measuring using a UV-Vis spectrophotometer.

FIGURE 5. The percentage inhibition value for each heating temperature

020007-6 Based on Figure 5, it can be seen that the highest inhibition value for each variation in heating temperature has different saturation requirements of ammonium sulfate at the time of multilevel fractionation. Each heating temperature used has the effect of saturation of each ammonium sulfate. It proves that the heating temperature affects the number of saturation requirements of ammonium sulfate needed in the fractionation of ammonium sulfate. The highest inhibition percentage value was in samples with a heating temperature of 75°C and saturation of ammonium sulfate 40-60%, which was 76.13%. This value means that the potential protein has the most effective antioxidant activity isolated at a heating temperature of 75°C. Also supported by the use of ammonium sulfate-grade fractionation, the most effective isolated protein is at 40-60% ammonium saturation.

To be able to know the amount of antioxidant activity, the percentage of inhibition obtained needed to be converted to IC50 values. Antioxidant activity was expressed in IC50 value, which is an antioxidant concentration, which can cause 50% DPPH to lose a radical character or antioxidant concentration, which provides a 50% percent inhibition. A compound is said to be a powerful antioxidant if the IC50 value is less than 50 ppm, secure if the IC50 value is between

50-100 ppm, medium if the IC50 value ranges from 100-150 ppm, and weak if the IC50 value ranges from 150-200 ppm [9]. The following is the IC50 value generated from each sample.

TABLE 3. IC50 value from each sample Variable IC50 (ppm)

Heating Temperature (0C) 25 (CV) 60 75 90 AS 0% 13888.89

11627.91 4504.505 4672.897 AS 0-20% 15625 8474.576

2857.143 3731.343 AS 20-40% 10204.08 7246.377 2325.581

12820.51 AS 40-60% 8474.576 3472.222 1312.336 38461.54

AS 60-80% 3623.188 1742.16 6172.84 71428.71

In Table 3, it appears that the lowest IC50 value is 1312 ppm, which is at a heating temperature of 75°C and saturation of ammonium sulfate 40-60%. This value is the smallest IC50 value that can be interpreted as having the most significant potential for antioxidant activity among other samples. Even so, this value is still classified as a very weak antioxidant, because the IC50 value obtained is more than 200 ppm.

The IC50 value of the sample with 75°C heating and ammonium sulfate saturation was 40-60% compared to

Larasati’s research, 2018 had a lower value, which was 1563.06 ppm, so the ability of antioxidant activity was better.

Therefore, it can be concluded that the method carried out in this study produces better antioxidant activity compared to the antioxidant activity of other marine animal extracts, such as Diadema setosum sea urchins with IC50 2826.13 ppm [14] and Stichodactyla gigantea sea anemones with 2073.13 ppm [15], lionfish spines venom has better antioxidant ability. Furthermore, the antioxidant activity of the lionfish spines venom is still low. The low antioxidant activity in the sample can be due to the presence of proteins or other compounds that can reduce the ability of the antioxidant activity of the sample to be isolated with the sample. The sample comes from animals that do not consume plants at all, because plants are a source of high antioxidants, so the value of antioxidant activity produced was still relatively weak. It can be proved by comparing antioxidant activity with animals which consumes plants, such as Apis dorsata bees. The IC50 value of the extract of Apis dorsata bee venom is 139.13 ppm [16], too far away from the extract of lionfish spines venom whose antioxidant activity ability is still low.

Determination of Protein Composition In SDS-PAGE followed by electrophoresis, the rate of movement of a protein molecule depends on the density of the charge, which is the ratio between a load of protein and its molecular weight. The uniform protein content in the gel causes the movement of velocity from the negative pole (cathode) to the positive pole (anode) depending only on its molecular weight so that lower molecular weight proteins will migrate farther than more abundant molecular weight proteins. Through the SDS-PAGE test, it can be seen the optimal heating temperature in isolating potentially antioxidant proteins.

In the SDS-PAGE test, researchers used the SDS-PAGE Broad Range gel-type or gel with a wide range to find out the type of protein obtained. The SDS PAGE test this time was done to see the composition of the proteins contained in the sample seen from specific molecular weight proteins that can be isolated due to heating. In the SDS- PAGE test performed on four samples with variations in heating temperature, are 90°C, 75°C, 60°C, and without heating. Four of these samples are then injected into each well and given coloring and left to stay overnight. The results are juxtaposed with markers that already have several proteins marked with individual molecular weights.

020007-7

FIGURE 6 Determination of molecular weight by SDS-PAGE

Based on the results of the SDS PAGE test in Figure 6, using higher heating temperature resulted in the less isolated protein. It proves that warming can result in protein denaturation so that more protein was eliminated. In crude venom (CV) it appears that there are still many proteins in various molecular weights, so the samples are not pure. At temperatures of 60°C and 75°C, it appears that there is a protein with a molecular weight of 52.7; 46.7; and 7.9 kD which has the potential as an antioxidant. Even so, at 60°C, there are still other proteins outside the protein with the desired molecular weight, such as PLA2 at a protein molecule weight of 85.2 kD. The presence of PLA2 protein in the sample can inhibit antioxidant activity because PLA2 is a protein commonly found in venom and has the opposite properties of antioxidants. It supported by the many uses of antioxidant compounds as inhibitors of PLA2 activity [16].

At 90°C, there is only protein at a molecular weight of 7.9 kD. It proves that the smaller the molecular weight of a protein, the stronger the high temperature and the protein with molecular weight 52.7 and 46.7 kD will not stand the temperature of 90°C. It can be concluded that the most effective heating temperature used in obtaining a potent antioxidant is a temperature of 75°C. Even so, in proteins with a heating temperature of 75°C, there are also other proteins, namely proteins with a molecular weight of around 101 kD. This protein does not rule out the possibility of reducing the potential for antioxidant activity in the sample. Further studies were needed regarding the method of removing these proteins.

CONCLUSION Lionfish grow fast, making lionfish prey on large quantities of other fish causing damage to the marine ecosystem.

This research determines the antioxidant potential of lionfish spines venom by isolating the protein from its extract.

To find out the potential further, the additional isolation step is heating. The higher the heating temperature used, the more isolated the protein. In the BSLT toxicity test, the heating process can eliminate more proteins that are potentially toxic. Each variation in heating temperature has different saturation requirements of ammonium sulfate to achieve the optimum antioxidant activity. The optimum condition for isolating proteins that have the potential to have antioxidant activity is with a heating temperature of 75°C and saturation of ammonium sulfate 40-60% with an IC50 value of 1312 ppm. The protein composition at the protein isolation temperature for optimum antioxidant is protein 7.9, 46.2, and

52.7 kD ACKNOWLEDGMENTS This research and article’s publication supported by the Grant of Indexed International Publication (Publikasi

Internasional Terindeks - PIT 9) funded by Universitas Indonesia No. NKB-0043/UN2.R3.1/HKP.05.00/2019.

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020007-9

📖 中文全文 Chinese Full Text

中文

加热过程对狮子鱼（Pterois volitans）脊椎毒液提取物蛋白质分离及其抗氧化活性测定的影响

引用格式：AIP Conference Proceedings 2193, 020007 (2019); https://doi.org/10.1063/1.5139327 在线发布日期：2019年12月10日

作者：Andy Noorsaman Sommeng, Indriani Pratiwi, Mikael Januardi Ginting, Muhamad Sahlan, Heri Hermansyah, Anondho Wijanarko

**摘要** 狮子鱼（Pterois volitans）是一种原产于印度洋-太平洋地区的入侵性捕食者，缺乏天敌。其生长迅速，大量捕食小型鱼类，对海洋生态系统造成严重破坏。本研究旨在通过从其毒液提取物中分离蛋白质，评估狮子鱼脊椎毒液的抗氧化潜力。前期研究表明，狮子鱼毒液提取物虽具有抗氧化活性，但效果较弱。为进一步探究其潜力，本研究在分离步骤中增加了加热处理。粗毒液（CV）经超声提取后，分别在90°C、75°C和60°C下加热，随后采用硫酸铵（AS）进行分级沉淀，饱和度分别为0–20%、20–40%、40–60%和60–80%。浓缩后的蛋白质通过Lowry法测定含量，SDS-PAGE鉴定组成，并采用卤虫致死试验（BSLT）进行毒性测试。最后，利用DPPH法测定抗氧化活性。结果表明，在加热温度为75°C、硫酸铵饱和度为40–60%的条件下，所得蛋白质具有最佳抗氧化潜力，其IC50值为1312 ppm。在此最优条件下分离出的蛋白质分子量分别为7.9 kD、46.2 kD和52.7 kD。

**关键词**：粗毒液；蛋白质分离；抗氧化活性

**引言** 狮子鱼为夜行性捕食者，以甲壳类、小型鱼类和螃蟹为食。其外形独特且具吸引力，但因拥有毒刺而在海洋食物链中处于高位，对其他生物构成威胁。狮子鱼生长速度快，导致其大量捕食鱼类，严重破坏海洋生态系统。此外，作为渔民捕捞对象的礁石鱼类也成为狮子鱼的猎物。

狮子鱼产生的毒液存在于其脊椎中，具有致命性，可造成15–20分钟的灼烧感，并在接触后3小时内引发肢体麻痹。该毒液具有心血管、神经肌肉和细胞溶解效应，导致细胞死亡。这些毒性作用源于毒液中存在毒性蛋白质及其他活性成分，如乙酰胆碱和毒液成孔蛋白。鉴于狮子鱼原生于毗邻印度洋的印度洋-太平洋海域，应采取措施控制其种群数量，并进一步开发利用狮子鱼，例如将其肉类作为替代食品原料，将其脊椎毒液作为药用成分。

狮子鱼脊椎毒液具有抗氧化活性。已有研究测试了毒液提取物中蛋白质类抗氧化化合物。然而，此前仅通过硫酸铵沉淀法进行蛋白质分离，未对蛋白质进行加热处理。研究显示，经分离的狮子鱼蛋白质虽具抗氧化活性，但效果较弱，且结果未达最优，因分离产物仍受污染。因此，有必要增加加热步骤以获得更纯的分离物，用于后续抗氧化测试。

**材料与方法** **材料** 实验所用狮子鱼样本采集自印度尼西亚爪哇岛。样本经处理后，将毒刺与鱼体其余部分分离。

**方法** **狮子鱼脊椎毒液的制备** 实验前，狮子鱼尸体储存于低温条件下。建议尽快处理以保持品质。随后，在低温条件下将身体各部位的脊椎从基部切下。分离后的脊椎用0.01 M磷酸盐缓冲液（pH 7.0）冲洗。称量所得脊椎重量后，将其浸入含0.001 M CaCl₂的0.01 M磷酸盐缓冲液（pH 7.0）中，比例为1:2，浸泡过夜。

**毒液蛋白质的分离** 狮子鱼脊椎经超声提取两次，每次8分钟，频率20 kHz。提取液以4500 rpm离心2×15分钟，去除杂质后用Whatman 42滤纸过滤。为分离粗毒液中的蛋白质，需对粗毒液进行加热。实验前准备加热板和盛有600 mL水的烧杯作为加热介质。将粗毒液置于较小烧杯中，再放入盛水烧杯内，置于加热板上。调节加热板温度，使粗毒液分别达到60°C、75°C和90°C，维持10分钟，期间使用磁力搅拌器搅拌。加热后，样品在15,000×g、4°C条件下离心30分钟，形成沉淀。上清液即为粗毒液（CV）。蛋白质通过90%乙醇和20%、40%、60%、80%饱和度的硫酸铵沉淀获得。样品以4500 rpm离心，蛋白质沉降后备用。

**Lowry法测定蛋白质浓度** 采用Lowry法测定蛋白质浓度。配制200 µg/mL牛血清白蛋白（BSA）标准溶液，浓度范围为20–200 mg/mL。Lowry试剂由1 mL 1% CuSO₄、1 mL 1%酒石酸钾钠和100 mL 2% Na₂CO₃（溶于0.1 N NaOH）组成，另加0.5 mL 1 N Folin-Ciocalteu酚试剂。标准溶液、空白及20 µL样品各加5 mL Lowry试剂（双缩脲试剂），孵育10分钟。随后加入0.5 mL 1 M Folin-Ciocalteu试剂，孵育30分钟。在λ = 750 nm波长下测定吸光度，绘制BSA标准曲线。

**毒性测试** 采用BSLT法进行蛋白质毒性测试。将10 mg卤虫卵置于250 mL海水中，光照并通气孵化2×24小时。配制2000 ppm母液：称取20 mg样品溶于10 mL海水，加入3滴80% Tween-80混匀，超声5分钟。取10只卤虫幼虫加入1 mL海水，再加入测试溶液，使终浓度分别为10、100、500、1000和2000 ppm。24小时后统计死亡幼虫数。

**DPPH法测定抗氧化活性** 配制125 µM DPPH溶液：称取2.5 mg DPPH溶于50 mL乙醇，铝箔纸避光保存。样品浓度为20 ppm。在微孔板中加入100 µL样品与100 µL DPPH溶液。空白为200 µL乙醇。铝箔纸覆盖后孵育30分钟，使用微孔板酶标仪在λ = 517 nm下测定吸光度。按公式计算抑制率：

%抑制率 = (空白吸光度 − 样品吸光度) / 空白吸光度 × 100%

为获得IC50值，以抑制率为纵坐标作图，拟合线性方程，令y = 50，求得x值即为IC50。

**SDS-PAGE测定蛋白质组成** 样品缓冲液含0.5 M Tris-HCl (pH 6.8)、10% SDS、巯基乙醇、甘油和0.1%溴酚蓝。配制电泳缓冲液（Tris、甘氨酸、SDS）、转膜缓冲液（Tris、甘氨酸、甲醇），以及7.5%和17.5%梯度凝胶。将两种浓度凝胶混合注入玻璃模具，胶高5 cm。静置1小时凝固，表面加水防氧化。制备4%浓缩胶，插入梳子。凝固后小心拔出梳子，水洗。将凝胶置于电泳槽中。每孔加入1–4 µL PBS和5 µL样品缓冲液，100°C加热45秒。接通电源，150 V电泳1小时。凝胶用20 mL染色液浸泡15分钟，倾去染色液，加入50 mL脱色液直至蛋白条带清晰。1小时后开模，凝胶保存于转膜缓冲液中待分析。

**结果与讨论** **蛋白质浓度** 蛋白质浓度通过标准曲线测定。标准曲线由不同浓度BSA的吸光度绘制。将样品吸光度代入标准曲线方程，计算蛋白质浓度。各样品蛋白质浓度见表1。

表1 样品蛋白质浓度 | 变量 | 蛋白质浓度 (ppm) | |------|------------------| | 加热温度 (°C) | 25 (CV) | 90 | 75 | 60 | | AS 0% | 278.305 | 54.2369 | 44.2941 | 27.6044 | | AS 20% | 45.0043 | 143.367 | 133.4242 | 124.1916 | | AS 40% | 72.7021 | 42.8737 | 63.4695 | 71.9919 | | AS 60% | 59.5634 | 72.347 | 41.0982 | 50.3308 | | AS 80% | 20.5024 | 36.1268 | 19.7922 | 11.2698 |

由表1可见，CV样品蛋白质浓度最高，而60°C加热、60–80%硫酸铵饱和度处理的样品浓度最低。加热过程显著影响后续硫酸铵分级沉淀阶段的蛋白质量。在0–20%硫酸铵分级中，加热样品的蛋白质量远高于未加热样品，表明加热有助于去除杂质，使蛋白质在硫酸铵沉淀时更易聚集。此现象适用于所有加热温度。

图1显示，随加热温度升高，蛋白质浓度增加。这表明高温导致更多蛋白质变性，从而在硫酸铵沉淀时积累。

图2显示，随硫酸铵饱和度增加，蛋白质浓度降低。说明分级步骤越多，残留蛋白质越少，因大部分蛋白已在前期沉淀。硫酸铵分级可提高蛋白质纯度。

**毒性测试** 本研究采用BSLT法测试样品毒性。BSLT可用于天然产物中潜在毒性化合物的早期筛查。以卤虫（Artemia salina）为生物指示剂，通过概率单位分析计算幼虫死亡率。24小时后统计死亡数。

为获得LC50（半数致死浓度），将死亡率转换为概率单位值，绘制概率单位-浓度对数回归曲线，拟合直线方程，计算LC50。

根据LC50值，物质毒性分为三类：LC50 < 30 ppm为剧毒，30–1000 ppm为有毒，> 1000 ppm为无毒。LC50值越高，毒性越低。各样品LC50值见表2。

表2 样品LC50值 | 变量 | LC50 (ppm) | |------|------------| | 加热温度 (°C) | 25 (CV) | 60 | 75 | 90 | | AS 0% | 573.21 | 729.11 | 1000 | 883.85 | | AS 20% | 512.03 | 540.94 | 775.59 | 789.29 | | AS 40% | 423.48 | 529.25 | 642.29 | 688.14 | | AS 60% | 402.31 | 388.59 | 532.12 | 499.6 | | AS 80% | 377.63 | 400.78 | 422.15 | 432.48 |

表2显示，LC50值在300–900 ppm之间。最高LC50值出现在90°C加热、未硫酸铵分级样品，最低值出现在未加热、60–80%硫酸铵饱和度处理的CV样品。

图3显示，加热显著提高LC50值，表明加热可去除潜在毒性蛋白。

图4显示，随硫酸铵饱和度增加，LC50值降低，说明分级步骤越多，毒性越强。因高饱和度硫酸铵逐步沉淀出更多毒性蛋白，使产物更特异。尽管如此，所有样品仍属有毒范畴（LC50在30–1000 ppm之间）。

本研究中，毒性最强的样品为未加热、60–80%硫酸铵饱和度处理的CV样品，LC50为377.63 ppm。该值高于此前采用硫酸铵沉淀法（非分级法）提取狮子鱼毒液的LC50值（101.93 ppm），表明硫酸铵分级法比单纯沉淀法更能降低毒性。与魟鱼（Dasyatis kuhli）毒液（LC50 = 161.6 ppm）和长刺海胆（Diadema setosum）甲醇提取物（LC50 = 563.26 ppm）相比，狮子鱼脊椎毒液毒性更高。

**抗氧化活性测定** DPPH抗氧化活性测定以抗坏血酸（维生素C）为阳性对照。样品抗氧化活性表现为DPPH甲醇溶液由深紫色褪为浅黄色。抗氧化潜力通过抑制率衡量，即物质抑制自由基的能力。抑制率计算公式为：

%抑制率 = (空白吸光度 − 样品吸光度) / 空白吸光度 × 100%

图5显示，不同加热温度下，达到最高抑制率所需的硫酸铵饱和度不同。75°C加热、40–60%硫酸铵饱和度样品的抑制率最高，达76.13%，表明此条件下分离的蛋白质抗氧化活性最强。

为量化抗氧化活性，将抑制率转换为IC50值。IC50指抑制50% DPPH自由基所需的抗氧化剂浓度。根据IC50值，抗氧化活性分为：< 50 ppm为强效，50–100 ppm为安全，100–150 ppm为中等，150–200 ppm为弱。各样品IC50值见表3。

表3 样品IC50值 | 变量 | IC50 (ppm) | |------|------------| | 加热温度 (°C) | 25 (CV) | 60 | 75 | 90 | | AS 0% | 13888.89 | 11627.91 | 4504.505 | 4672.897 | | AS 0–20% | 15625 | 8474.576 | 2857.143 | 3731.343 | | AS 20–40% | 10204.08 | 7246.377 | 2325.581 | 12820.51 | | AS 40–60% | 8474.576 | 3472.222 | 1312.336 | 38461.54 | | AS 60–80% | 3623.188 | 1742.16 | 6172.84 | 71428.71 |

表3显示，最低IC50值为1312 ppm，出现在75°C加热、40–60%硫酸铵饱和度条件下，表明该样品抗氧化潜力最强。然而，该值仍属极弱抗氧化剂（IC50 > 200 ppm）。

与Larasati（2018）的研究（IC50 = 1563.06 ppm）相比，本方法所得抗氧化活性更优。此外，狮子鱼脊椎毒液的抗氧化能力强于长刺海胆（IC50 = 2826.13 ppm）和大花海葵（Stichodactyla gigantea，IC50 = 2073.13 ppm）。但其抗氧化活性仍较低，可能因样品中存在抑制抗氧化能力的蛋白质或其他化合物。由于狮子鱼为肉食性动物，不摄入植物（植物为高抗氧化物质来源），故其抗氧化活性相对较弱。相比之下，以植物为食的Apis dorsata蜜蜂毒液IC50为139.13 ppm，远高于狮子鱼毒液。

**蛋白质组成测定** SDS-PAGE结合电泳后，蛋白质迁移速率取决于电荷密度（电荷与分子量之比）。凝胶中蛋白质分布均匀，迁移仅依赖分子量，小分子量蛋白迁移更远。通过SDS-PAGE可确定分离抗氧化蛋白的最优加热温度。

本研究采用宽范围SDS-PAGE凝胶，以鉴定所获蛋白质类型。测试了90°C、75°C、60°C加热及未加热四个样品。样品上样后染色过夜，与已知分子量的标准蛋白Marker对比。

图6显示，加热温度越高，分离出的蛋白质越少，表明加热导致蛋白质变性，从而被去除。CV样品中可见多种分子量蛋白，说明样品不纯。60°C和75°C样品中可见分子量为52.7 kD、46.7 kD和7.9 kD的蛋白，具抗氧化潜力。但在60°C样品中仍存在其他蛋白，如分子量为85.2 kD的PLA2。PLA2常见于毒液，具有与抗氧化相反的性质，可能抑制抗氧化活性。已有研究使用抗氧化化合物作为PLA2抑制剂。

90°C样品仅见7.9 kD蛋白，表明小分子量蛋白更耐热，而52.7 kD和46.7 kD蛋白无法耐受90°C。因此，75°C是获得强效抗氧化蛋白的最有效温度。然而，75°C样品中仍存在约101 kD的蛋白，可能降低抗氧化潜力。需进一步研究去除该蛋白的方法。

**结论** 狮子鱼生长迅速，大量捕食其他鱼类，破坏海洋生态系统。本研究通过从毒液提取物中分离蛋白质，评估其抗氧化潜力。加热作为额外分离步骤，温度越高，蛋白质分离越多。BSLT毒性测试表明，加热可去除更多潜在毒性蛋白。不同加热温度下，达到最优抗氧化活性所需的硫酸铵饱和度不同。最优条件为：加热温度75°C，硫酸铵饱和度40–60%，IC50值为1312 ppm。在此条件下分离的蛋白质分子量为7.9 kD、46.2 kD和52.7 kD。

**致谢** 本研究及论文发表受印度尼西亚大学“国际索引出版资助（PIT 9）”支持，项目编号：NKB-0043/UN2.R3.1/HKP.05.00/2019。

**参考文献** （略）