Study On Optimization Of Extraction Technology And Antioxidant Activity Of Polysaccharides From Yunnan Coffea Arabica Flowers

Abstract: To optimize the extraction technology and study the antioxidant activity of polysaccharides from the coffee flower (ACP), ultrasonic temperature, ultrasonic time, ratio of fluid-solid, ultrasonic power, sample immersion time and volume percentage of ethanol were investigated by using polysaccharide extraction yield as indicator. Then ultrasonic temperature, ultrasonic time and ultrasonic power were used as mainly influence factors, the extraction technology was optimized by response surface method. Antioxidant activity of ACP was investigated by DPPH· and ABTS+ · scavenging effects, and FRAP assays. The results demonstrated that the ultrasonic extraction optimum technological conditions were: Ultrasonic temperature 69.5 ℃, ultrasonic time 93 min, ultrasonic power 175 W, ratio of fluid-solid 10:1 mL/g, sample immersion time 30 min, and volume percentage of ethanol 80%. Under the the yield of polysaccharides was 2.292%. The results indicated that the value of IC50 based on DPPH· scavenging effect of ACP was 3.844 mg·mL−1, the ABTS+ · scavenging activity was 0.921 mmol Trolox/g ACP. The FRAP values of ACP by FRAP assay was 0.0565 mmol Fe2+/g ACP, which showed that ACP had weak antioxidant activity. This study would provide a theoretical basis for the comprehensive utilization and development of coffee by-products.

Keywords：coffee flowers；polysaccharide；extraction；antioxidant activity

Coffee is a plant of the Rubiaceae (Coffea) genus, mainly distributed in countries such as South America, Central America, Africa and Asia, and is grown in more than 80 countries around the world [1]. According to "Chinese Materia Medica", coffee has the effects of refreshing, diuretic and stomachic, and it is mainly used for mental fatigue and loss of appetite. It is often used as a refreshing, diuretic and stomachic medicine. Modern research has shown that coffee contains a variety of active ingredients such as alkaloids, phenolic acids, flavonoids, and terpenes, which have various pharmacological activities such as liver protection, neuroprotection, antioxidant, and antidiabetic [2-3]. my country's coffee cultivation is dominated by small-grain coffee, and more than 99% of it is distributed in Yunnan. Yunnan small-grain coffee is rich in substances, in addition to caffeine, chlorogenic acid, trigonelline and other components, it also contains ascarosides

I~II[4], paniculoside VI[4], cofaryloside I[4], villanovane I[4], caffarolides A~H[5], caffruenol A-B[6], caffruones A-D[6] and caffruolide A-B[7] and some new terpenoids. Among them, caffarolides C, D and F have been confirmed to have a certain activity of activating platelet aggregation in vitro [5]; caffruenol AB and caffruolide A-B have the effect of inhibiting lipopolysaccharide-induced NO production in 264.7 macrophages [7]. With the in-depth study of coffee, the added value of coffee continues to increase. In recent years, coffee by-products are rich in phenolic acids, flavonoids, terpenes, alkaloids and other biological

Active ingredients, which can be used as natural and sustainable sources of active ingredients such as antioxidants, liver protection and nerve protection, have made the research of coffee by-products more and more concerned by researchers [8−10]. Campa et al. reported that coffee leaves contain phenolic compounds [11]; Chen reviewed the rich chemical constituents of alkaloids, flavonoids, phenolic acids, terpenes, etc. in coffee leaves and their pharmacological activities such as antioxidant, anti-inflammatory, and antibacterial. [12], and studied the effects of coffee leaf processing methods and leaf age on its chemical composition and activity [13].

In addition, Fu Xiaoping et al. [14−15] found that the crude extract of Yunnan small coffee peel has a certain protective and recovery effect on damaged human umbilical vein endothelial cells, and also has potential antioxidant effects, and found that the main flower The cyanidins are cyanidin-3-glucoside and cyanidin-3-rutinoside.

Coffee flowers are often discarded as a major by-product in the coffee growing industry. However, existing studies have found that coffee flowers are rich in chemical components. Stashenko et al. [16] used GC-MS to analyze the volatile and semi-volatile components in small coffee flowers, and the results determined a total of 150 compounds, with the content of n-pentadecane. The highest, followed by geraniol. In addition, Nguyen et al. [17] studied the active ingredients in coffee flowers and found that coffee flowers have high content of phenolic compounds, so coffee flowers can be used as raw materials for obtaining natural antioxidant active ingredients. In addition, coffee flowers also contain caffeine and trigonelline. Caffeine is associated with a reduced risk of neurodegenerative diseases [18−19]

Trigonelline can prevent diabetes and kidney damage, and also has the effect of treating neurodegenerative diseases [20-21]. Pinheiro et al. [22] analyzed the contents of four active components of trigonelline, chlorogenic acid, gallic acid and caffeine in coffee flowers under different drying and extraction methods by HPLC, among which caffeine and trigonelline had the highest content; The antioxidant activity was evaluated by ABTS and DPPH experiments, which confirmed that coffee flower has antioxidant activity and can be used as a potential raw material for making tea beverages. At present, there are few research reports on coffee flowers, but it can be seen from the existing reports that coffee flowers have broad application prospects as a potential source of bioactive compounds.

Polysaccharides are macromolecular compounds composed of more than 10 monosaccharides bound by glycosidic bonds, and are widely found in animals, plants and microorganisms. Polysaccharides are structurally complex, with different conformations and relative molecular masses, as well as secondary structures of intrachain and interchain hydrogen bonds. Modern studies have shown that polysaccharides have pharmacological activities such as antioxidant [23-24], anti-aging [25], immune regulation [26], anti-inflammatory [27], and anti-tumor [28]. The biological activity of polysaccharides is related to its purity, chemical structure, solubility, etc. In recent years, the biological activity of polysaccharides has become a research hotspot of natural medicines, and it is also a channel for discovering new drugs and developing functional foods. Therefore, polysaccharides play an important role in the field of medicine and food. my country's Yunnan is the main producing area for coffee planting, and coffee flowers have potential development value, but there are few researches on the development of Yunnan coffee flowers, and the potential value of coffee flowers has not been tapped. Therefore, this paper takes Yunnan small-grain coffee flowers as the research object to carry out research on its active polysaccharides, aiming to deeply explore the comprehensive utilization value of Yunnan small-grain coffee.

In this paper, the polysaccharide yield was used as the evaluation index to optimize the polysaccharide extraction process and the antioxidant capacity of coffee flowers collected from Baoshan City, Yunnan Province, to provide basic data for the further development of biologically active polysaccharides. And provide reference for studying Yunnan small grain coffee and improving its added value.

1 Materials and methods

1.1 Materials and Instruments

Coffee flower Baoshan City, Yunnan Province; Anhydrous Alcohol Tianjin Chemical Reagent Co., Ltd.; Anthrone purity 98.0%, Sinopharm Chemical Reagent Co., Ltd.; concentrated sulfuric acid, hydrochloric acid Chongqing Chuandong Chemical Co., Ltd.; 1,1-diphenyl-2- Trinitrophenylhydrazine (DPPH), 2,2'-Diaza-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,4,6-Tripyridyltriazine (TPTZ), Rutin purity 98.0%, Shanghai Ruiyong Biotechnology Co., Ltd.; water-soluble vitamin E purity 98.0%, Hefei Bomei Biotechnology Co., Ltd.; ferric chloride hexahydrate analytically pure, Western

Long Science Co., Ltd.; Potassium Persulfate Analytical Grade, Tianjin Damao Chemical Reagent Factory; PBS buffer, sodium acetate buffer Xiamen Haibiao Technology Co., Ltd.

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FA2104N Electronic Balance, 722-Spectrophotometer Shanghai Qinghua Technology Co., Ltd.; KQ-250DB Ultrasonic Instrument, SHZ-D (Ⅲ) Circulating Water Vacuum Pump Gongyi Yuhua Instrument Co., Ltd.; Xuman 1000Y Multifunctional Grinder Yongkang City Boou Hardware Products Co., Ltd.; 800 Electric Centrifuge Jintan Fuhua Instrument Co., Ltd.; VFD-3000 Vacuum Freeze Dryer Beijing Bo Yikang Experimental Instrument Co., Ltd.

1.2 Experimental method

1.2.1 Extraction of coffee flower polysaccharides Refer to the method of Zheng Tingting et al. [29] with modifications. The coffee flowers collected from Baoshan City, Yunnan Province were dried in the shade at room temperature, pulverized with a pulverizer and passed through an 80-mesh sieve for use. Weigh 2.0 g of coffee flower powder, add 20.0 mL of pure water, soak for 30 min,

Ultrasonic at 100 W for 30 min at 40 °C, cooled to room temperature, and the filtrate was retained after vacuum filtration. Ethanol was added to the filtrate to a concentration of 80% for precipitation and allowed to stand for 12 h. After centrifugation at 4000 r/min for 10 min, the supernatant was discarded, and the precipitate was dissolved in water and lyophilized at −80 °C to obtain crude polysaccharide.

Prepare 5 mg·mL−1 coffee flower polysaccharide solution and set aside.

1.2.2 Preparation of polysaccharide standard curve and determination of polysaccharide content in coffee flowers The glucose standard curve was drawn with reference to the anthrone-sulfuric acid method [30]. Prepare 0.0, 25.0, 50.0, 100.0, 150.0, and 200.0 μg/mL glucose standard solutions, respectively. Accurately pipette 1.00 mL of the above glucose standard solution with different concentrations, add 1.00 mL of pure water, and place it in a 25 mL stoppered test tube. Add 5.0 mL of 2.1 mg·mL−1 anthrone-sulfuric acid solution, shake well, cool in an ice-water bath, heat in a boiling water bath for 7 min, and quickly cool to room temperature in an ice-water bath. Using deionized water as a blank control,

Absorbance A was measured colorimetrically at 625 nm. Taking the anhydrous glucose content as the abscissa (0.0, 25.0, 50.0, 100.0, 150.0, 200.0 μg), and the absorbance value as the ordinate, draw a standard curve, and the equation of the standard curve is: Y=0.0051X−0.0092 (R2=0.9970) (where: Y is the absorbance value, X is the amount of glucose, μg).

Precisely pipette a certain volume of the 5 mg/mL coffee flower polysaccharide solution prepared above into a 25 mL test tube with a stopper, and add pure water to make up to 2.00 mL. Add 5.0 mL of 2.1 mg·mL−1 anthrone-sulfuric acid solution, shake well, cool in an ice-water bath, heat in a boiling water bath for 7 min, and quickly cool to room temperature in an ice-water bath. Absorbance A was measured colorimetrically at 625 nm using deionized water as a blank control. The coffee flower polysaccharide yield was calculated according to the glucose standard curve equation, and each sample was repeated 3 times. The results were expressed as the average value, and the calculation formula was as follows:

Coffee flower polysaccharide yield (%) =X×m1×10−35×V×ms×100

In the formula: X is the polysaccharide content in the V volume of coffee flower polysaccharide solution, μg; V is the measured volume of the polysaccharide solution, mL; 5 is the concentration of the prepared coffee flower polysaccharide solution, 5 mg·mL−1; m1 is the freeze-dried coffee flower Total mass of polysaccharide, g; ms is the mass of coffee flower sample, g.

1.2.3 One-factor experiment

In the process of ultrasonic-assisted extraction of coffee flower polysaccharide, the important factors affecting the yield of polysaccharide mainly include ultrasonic temperature, ultrasonic time, liquid-material ratio, ultrasonic power, soaking time and alcohol precipitation concentration. Five levels of ultrasonic temperature were selected: 40, 50, 60, 70, and 80 °C; five levels of ultrasonic time were selected: 30, 60, 90, 120, and 150 min; : 1, 30: 1 mL/g five levels; ultrasonic power selects five levels of 100, 125, 150, 175, 200 W; immersion time selects five levels of 30, 60, 90, 120, 150 min; ethanol concentration selects five levels 75%, 80%, 85%, 90%, 95% five levels, respectively, single factor experiment. When filtering for one of the parameters

, the other factors are: ultrasonic temperature 40 °C, ultrasonic time 30 min,

Liquid to material ratio 10:1 mL/g, ultrasonic power 100 W, soaking time 30 min

and alcohol precipitation concentration of 80%.

1.2.4 Response surface optimization experiment According to the Box-Benhnken experimental design principle, with the yield of coffee flower polysaccharide as the response variable, three factors that have the greatest influence on the yield of coffee flower polysaccharide were selected from the single-factor test results, as shown in Table 1. Optimization of ultrasonic temperature with the yield of coffee flower polysaccharide as an index degree, ultrasonic time and ultrasonic power.

1.2.5 Antioxidant ability test

1.2.5.1 DPPH free radical scavenging experiments The DPPH free radical scavenging experiments were performed as described in Ref. [31]. Take 3.9 mL of 0.075 mmol/L DPPH reaction solution and mix it with 100 μL of polysaccharide solutions of different concentrations. The reaction was carried out at room temperature for 30 min in the dark, and the absorbance values were measured at 515 nm. Using rutin as the positive control, the DPPH free radical scavenging rate was calculated as: I%=[(A0–As)/A0]×100 (wherein : As is the absorbance of the sample solution; A0 is the absorbance of the solution without sample), and the antioxidant activity is expressed as 50% inhibition rate (IC50).

1.2.5.2 ABTS+ free radical scavenging experiment

ABTS+ radical scavenging experiments were performed using the method described in Ref. [32]. Take 2 mL of polysaccharide solution and add it to 2 mL of ABTS+ free radical solution respectively, after uniform mixing, react at room temperature for 6 min, and measure the UV absorption at 734 nm, and rutin is the positive control. The calculation formula of ABTS+ free radical scavenging ability is as follows: I(%)=[(A0–As)/A0]×100 (where As is the absorbance of the sample solution; A0 is the absorbance of the solution without sample) The standard curve is determined by measuring Drawing of Trolox standard solutions with different concentrations (I%=0.0247C−0.0046, R2=0.9937), the ABTS anti-

Oxidative activity is expressed as mmol Trolox/g.

1.2.5.3 FRAP method

The method described in Ref. [33] was used for the determination of FRAP antioxidant capacity. Take 5.0 mL of TPTZ, 5.0 mL of 20 mmol/L FeCl3 and 50 mL of sodium acetate buffer solution (300

mmol/L, pH 3.6) to prepare FRAP working solution; 100 μL sample was mixed with 300 μL water and 3.0 mL FRAP working solution, placed in a water bath at 37 °C for 30 min; the absorbance was measured at 595 nm. A standard curve was prepared with FeSO4 as the standard substance (A=0.572C0.008, R2=0.9974), and rutin was used as the positive control, according to the standard curve

Calculate the reducing power in mmol FeSO4/g polysaccharide.

1.3 Data processing

All experiments were repeated three times and the average value was taken. DesignExpert 8.0.6 software was used for the design and analysis of response surface experiments.

2 Results and analysis

2.1 Single factor experimental results

The results of the single factor experiment are shown in Fig. The effect of ultrasonic temperature on the yield of coffee flower polysaccharide: the ultrasonic temperature was 40-80 ℃, and the polysaccharide yield was 1.0048%-1.7982%. In the range of 40-70 ℃, the yield of coffee flower polysaccharide gradually increased with the increase of ultrasonic temperature, reached the maximum at 70 ℃, and began to decrease after 70 ℃. This may be due to the reduced polysaccharide yield due to the destruction of the structure of coffee flower polysaccharides under high temperature conditions, which has been similarly reported in the literature [29,34−35]. The sonication temperature was chosen to be 70 °C.

The effect of ultrasonic time on the yield of coffee flower polysaccharide: the ultrasonic time was 30-150 min, the polysaccharide yield was 1.0369%-1.5853%, the polysaccharide yield increased with the increase of ultrasonic time, and reached the maximum at 90 min, , the yield began to decrease with the increase of sonication time. This is because short-time ultrasonic extraction is not conducive to the full dissolution of polysaccharides, while long-time ultrasonic extraction will degrade polysaccharides and lead to a decrease in yield, which is also reported in the literature [29,34−35]. Therefore, the sonication time was chosen to be 90 min.

The effect of liquid-solid ratio on the yield of coffee flower polysaccharide: the effect of solid-liquid ratio on the polysaccharide yield is small. The polysaccharide yield increased with the increase of the liquid-to-solid ratio, and reached the maximum at 25:1 mL/g. After 25:1 mL/g, the yield decreased with the increase of the liquid-to-solid ratio. Less solvent will lead to insufficient dissolution of polysaccharide, resulting in lower yield of polysaccharide; more solvent will dissolve polysaccharide and make it difficult to precipitate out, and at the same time, the yield will be reduced due to the absorption of ultrasonic radiation by the solvent. 29,34−35] also have similar reports. Considering that the liquid-to-material ratio has little effect on the yield, in order to save the amount of reagents, the liquid-to-material ratio was selected as 10:1 mL/g.

The effect of ultrasonic power on the yield of coffee flower polysaccharide: the ultrasonic power was selected as 100-200 W, and the polysaccharide yield was 1.1185%-1.8583%. After W, the yield decreased with the increase of ultrasonic power. The increase of ultrasonic power can effectively destroy cells and tissues to dissolve polysaccharides in the solvent, so increasing ultrasonic power is beneficial to the precipitation of polysaccharides; however, the fragmentation effect and thermal effect produced by larger ultrasonic waves will also increase the dissolution of impurities in coffee flowers. , the thermal effect will destroy the polysaccharide components and cause the polysaccharide yield to decrease, which is also reported in the literature [36−37]. Therefore, the ultrasonic power was chosen to be 175 W.

The effect of soaking time on the yield of coffee flower polysaccharide: the effect of soaking time on the yield of polysaccharide was small, the soaking time was 30-150 min, and the polysaccharide yield was 1.1827%-1.4609%. In the range of 30-90 min, the polysaccharide yield increased with the increase of soaking time, and reached the maximum at 90 min. After 90 min, with the increase of soaking time, the yield decreased slightly and tended to be flat. Prolonging the soaking time can facilitate the precipitation of polysaccharides during ultrasonication and reduce energy consumption. But too long soaking can not bring higher yield, and too long soaking will also cause other components to be released and affect the polysaccharide yield. This is similar to that reported in [38]. Considering that the influence of soaking time is small, in order to save time, the soaking time was selected as 30 min.

The effect of alcohol precipitation concentration on coffee flower polysaccharide yield: alcohol precipitation concentration has little effect on polysaccharide yield, alcohol precipitation concentration is 75%~95%, polysaccharide yield is 0.9703%~1.2806%. The yield of polysaccharide increased with the increase of ethanol concentration, and reached the maximum at 85%. After 85%, the yield decreased with the increase of ethanol concentration. Water extraction and alcohol precipitation is the use of polysaccharide insoluble in alcohol to make it precipitate out. When the amount of ethanol added increases, the polysaccharide is insoluble in ethanol and precipitates out, and the yield increases. When the alcohol precipitation concentration exceeds 85%, the polysaccharide yield cannot be improved, but the reagents are wasted. This is similar to that reported in [38]. In order to simplify the operation, this paper adopts the method of directly adding ethanol to adjust the alcohol concentration for precipitation. At the same time, since the yield of 80% and 85% polysaccharide is not much different, it can save reagents and reduce waste. Therefore, the alcohol precipitation concentration was chosen to be 80%.

2.2 Response surface test results

2.2.1 Response surface test results Ultrasonic temperature, ultrasonic time and ultrasonic power have a great influence. Therefore, on the basis of the above single-factor experiments, the response surface method is optimized for the three conditions of ultrasonic temperature, ultrasonic time and ultrasonic power. The results are shown in Table 2. .

Taking the polysaccharide yield (Y) as the response index, a regression model was established with the three factors of ultrasonic temperature, ultrasonic time and ultrasonic power, and the quadratic regression equation was obtained:

Y=2.29−0.067A+0.054B−0.019C+0.34AB+0.083AC+0.011BC−0.40A2

−0.19B2−0.27C2

2.2.2 Significance test of variance

The test results are shown in Table 3.

According to the results of variance analysis in Table 3, the total model was significant (P<0.0001), and the model reached a very significant level, indicating that the difference between different factors was significant; according to the absolute value of the linear coefficient of the regression equation, it can be seen that each factor has a significant effect on the total polysaccharide yield. The order of influence is: A>B>C, that is, ultrasonic temperature> ultrasonic time> ultrasonic power. Lack of fit item P=0.5764>0.05, the lack of fit item test is not significant, indicating that unknown factors have little influence on the test results, and the residual item is mainly caused by random errors, indicating that the model selection is appropriate and correct. The influence of AB was significant (P<0.05), and the influence of A2, B2, and C2 was extremely significant (P<0.01). In the whole model, the adjustment coefficient R2Adj=0.9277 in the model, indicating that 92.77% of the response value changes can be carried out through the model. Explanation, the coefficient of determination R2 = 0.9684, indicating that the model is highly reliable, and the model fits well with the experiment, and this model can be used for analysis and prediction [39−42].

2.2.3 Response surfaces and contours

The response surface diagram of the interaction of various factors on the yield of coffee flower polysaccharide is shown in Figure 2. The interaction between ultrasonic temperature and ultrasonic time showed that the interaction between the two was significant; when the ultrasonic temperature remained unchanged, the yield of coffee flower polysaccharides first increased and then decreased with the increase of ultrasonic time; when the ultrasonic time remained unchanged, coffee The yield of flower polysaccharide first increased and then decreased with the increase of ultrasonic temperature. From the interaction between ultrasonic temperature and ultrasonic power, it can be seen that when the ultrasonic temperature is constant, the yield of coffee flower polysaccharide first increases and then decreases with the increase of ultrasonic power; when the ultrasonic power remains unchanged, the yield of coffee flower polysaccharide increases with the ultrasonic wave. The temperature increase first increases and then decreases. From the interaction between ultrasonic time and ultrasonic power, it can be seen that when the ultrasonic time is constant, the yield of coffee flower polysaccharide first increases and then decreases with the increase of ultrasonic power; when the ultrasonic power is constant, the yield of coffee flower polysaccharide increases with the increase of ultrasonic power. The increase of time increases first and then decreases.

Therefore, using the polysaccharide yield as the evaluation standard, the optimization results of the response surface method for the three conditions of ultrasonic time, ultrasonic temperature and ultrasonic power are: ultrasonic temperature 69.56 ℃, ultrasonic time 92.99 min and ultrasonic power 174.01 W, it is predicted that under this condition 2.290%. According to the actual situation, the ultrasonic temperature of 69.5 °C, the ultrasonic time of 93.00 min, the ultrasonic power of 175 W, the immersion time of 30 min, the liquid-to-material ratio of 10:1 mL/g and the ethanol concentration of 80% were selected for 4 parallel tests.

The average yield was 2.292%±0.061%. It is basically close to the theoretical value obtained by the test, indicating that there is a good fit between the predicted value and the real value, so the optimized process parameters obtained by the response surface in this study are accurate and reliable [43].

2.3 Experimental results of antioxidant capacity

DPPH test is an efficient and sensitive evaluation model for plant antioxidant capacity. The free radical scavenging capacity of the tested sample is related to its potential proton-donating capacity; ABTS test is widely used to estimate the antioxidant capacity of plant samples, which can test samples Antioxidant activity of lipophilic and hydrophilic components in the FRAP method; the reduction ability of natural products was assessed by reducing Fe3+-TPTZ to Fe2+-TPTZ [44−45]. The results of the antioxidative experiments of coffee flower polysaccharides are shown in Table 4. Coffee flower polysaccharides have certain antioxidant activities against DPPH free radicals and ABTS+ free radicals, but their antioxidant activity is lower than that of rutin.

3 Conclusion

In this experiment, Yunnan small-grain coffee flower was used as raw material, and the polysaccharide of Yunnan small-grain coffee flower was extracted by ultrasound. It was found that ultrasonic time, ultrasonic temperature and ultrasonic power have important effects on the extraction of coffee flower polysaccharides. Then, the ultrasonic time, ultrasonic temperature and ultrasonic power were optimized by response surface, and the optimal process conditions of coffee flower polysaccharide were determined as follows: ultrasonic temperature 69.5 ℃, ultrasonic time 93 min, ultrasonic power 175 W, liquid-material ratio 10:1 mL/g, The soaking time was 30 min, and the ethanol concentration was 80%. Under this condition, the polysaccharide yield was 2.292%±0.061%. The method can effectively improve the yield of coffee flower polysaccharide, while shortening the extraction time and reducing the amount of ethanol used. The results of antioxidant experiments showed that coffee flower polysaccharides showed weak antioxidant capacity. This study will provide a reference for further separation and purification of coffee flower polysaccharide and research on its activity and function, and will also provide theoretical basis and support for the further development and utilization of coffee.

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