Introduction

Changes in people diets, which contain more fat and calories especially during industrial growth may increase the risk of free radical exposure in the body.  It is caused by the body’s lack of antioxidants, so additional antioxidant intake from external sources is needed. Many antioxidants are developed from herbal ingredients because they are safer than synthetic ones. One herbal ingredient that has antioxidant activity is sambiloto leaf (Andrographide paniculata)¹.  In Indonesian society, sambiloto herbs are used for brewing dry ingredients. However, the drying methods can cause a decline in the amount and activity of functional components, for example, phenolics ², flavonoids ³, chlorophyll ⁴, and andrographolide compounds⁵. One process known to reduce chlorophyll degradation is blanching with a ZnCl₂ solution⁶. However, the research results of Usman et al. ⁷ showed that the blanching process decreased total phenolic and chlorophyll content because much of it was dissolved in the blanching media. This is because the shape of the sambiloto leaf is thin and small. Therefore, this research carried out the metallochlorophyll formation process on simplicia sambiloto powder by mixing the ZnCl₂ solution with the material and continuing with the heating process.

There are several types of heating equipment, including ovens and autoclaves which are normally used to produce dry simplicia sambiloto. The temperature and heating time can be controlled in the oven heating process. The heat conduction medium in the oven is dry air and conduction from the container to the material. In autoclave heating, heat is delivered through the saturated steam and factors that control temperature, time, and water vapor pressure. The different heating principles will affect the level of degradation of functional components such as phenolics and their antioxidant activity⁸. Therefore, research has been carried out on the process of making sambiloto simplicia powder by forming a Zn-chlorophyll complex at various reagent concentrations and using two heating methods, namely the oven method and the autoclave method on the dried sambiloto leaf, to obtain sambiloto simplicia powder, which has high antioxidant activity. This research will be important especially to evaluate the influence of ZnCl₂ concentration and heating methods on the chemical properties and antioxidant activity of Simplicia sambiloto powder and to produce simplicia sambiloto powder rich in Zn, which has high antioxidant activity.

Materials and Methods

Materials

The material used in this research was dried simplicia from the Tawangmangu Medicinal Plant Research Institute. The chemical for forming metallochlorophyll complex is ZnCl₂ (Sigma, Aldrich). Chemicals for testing phenolic, flavonoid, and andrographolide content and antioxidant activity are gallic acid and quercetin standard, Folin-Ciocalteu reagent, linoleic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and butylated hydroxytoluene (BHT) from Merck with standard specifications for analysis (pro analysis).

Methods

Sample Preparation

The simplicia sambiloto was purchased from Tawangmanggu Medicinal Plant Research Institute. Sambiloto simplicia powder is made through sorting stages to remove the tough leaf stalks, grinding with a dry blender for 1 minute at three times, sieving (60 mesh), and crushing materials that do not pass through the sieve again. Intermittent grinding is carried out to avoid heat accumulation, which can cause degradation of the functional components.

Metallochlorophyll complex formation

The metallochlorophyll complex is formed by mixing simplicia powder with the reagent, continued by stirring, heating, and drying in a cabinet drier⁹. The reagent used is ZnCl₂ with concentrations 0, 200, 300, 400, and 500 ppm. The ratio between ingredients and reagents is 50:15 (w/v). Mixing is done with a spray and stirring to ensure homogeneity. The mixture was then heated using two methods. These are heated with an oven (Memert) and an autoclave (All American on electric pressure, model 1925X) for 10 minutes at 110 °C. The sambiloto simplisia powder was dried again using a cabinet dryer at 50 °C for 3 hours and stored in plastic wrapped in aluminum foil. 

Total chlorophyll

Total chlorophyll content (TCC) was measured using the Rababah et al., (2015)³. A 5 g of sambiloto simplicia powder was weighed, extracted with 80% acetone (20 ml), homogenized, centrifuged at 8000 rpm for 15 minutes, and filtered with Whatman paper no. 1 and 42. The filtrate obtained was added with 80% acetone until the volume reached 25 ml in a volumetric flask. The extract obtained was measured for absorbance at 663 and 645 nm wavelengths using a UV-Vis spectrophotometer. The total chlorophyll content is determined by equation 1.

A₆₆₃ means absorbance at 663 and A₆₄₅ means absorbance at 645, then FP means dilution factor. 

Zn content

The sambiloto simplicia powder was weighed (2 g) and ashed at a temperature 600 °C for an hour. After ashing, it was dissolved in 10 ml of 6 N HCl, and then the solution was evaporated by heating in an acid chamber until dry. The residue was dissolved in 0.1 N HNO₃ 5 ml and then analyzed by atomic absorption spectrophotometer (AAS, Contr, AA300, Analytic Jena).

Total Phenolic

Total phenolic content (TPC) was determined by the Folin Ciocalteu method¹³ used gallic acid as a standard to determine TPC. Acetone extract obtained from analyzing chlorophyll levels was also used to analyze phenolic and flavonoid groups. Sample 50 µl, add 250 µl of Folin-Ciocalteu solution, then leave for 1 minute and add 750 µl of 20% NaCO₃, then homogenized with a vortex and distilled water to a volume of 5 ml. After incubation for 5 minutes at room temperature, the absorbance was measured at λ 760 nm. Gallic acid was used as a standard, and a calibration curve was prepared with gallic acid. The TPC calculation result is mg Gallic Acid Equivalent (EAG) per one gram of dry extract.

Total Flavonoid Content

Total flavonoid levels (TFC) were determined using the Wariyah and Riyanto (2020) method. 50 µl extract plus 4 ml distilled water and 0.3 ml 10% NaNO₂. After leaving it for 6 minutes, add 0.3 ml of 10% AlCl₃.6H₂O, leave it for 5 minutes, then add 4 ml of 10% NaOH. Next, distilled water was added (until the total volume was 10 ml), homogenized with a vortex for 1 minute, and left for 15 minutes. Absorbance was measured at a wavelength of 510 nm. The blank used is distilled water. TFC was calculated using a quercetin standard with a concentration of 1.25-80 mg/L and calculated as mg quercetin equivalent (EK)/g dry based.

Androghapolide Content (AP)

Determination of andrographolide content (APC) using Thin Layer Chromatography (TLC). Using a Microliter syringe, the samples were spotted 1 cm from the bottom with an 8 cm elusion distance on pre-coated silica gel aluminum plate 60F-254 (10 x 10 cm) (E. Merck, Germany) and a Linomat 3 sample applicator. The isolated andrographolide was then subjected to TLC analysis with the reference standard of andrographolide, where a mixture of chloroform and methanol at a ratio of 9:1 was utilized. To ensure the purity of andrographolide, three different mobile phases, including ethyl acetate and acetone 7:3, chloroform and methanol 9:1, and chloroform and acetone 7:3 were used¹³. 

Antioxidant activity

Antioxidant activity was determined with lipid peroxidation inhibition (LPI) and radical scavenging activity (RSA). Determination of lipid peroxidation inhibition (LPI) activity using a linoleic acid emulsion system and the FTC method Suryani et al., 2018²⁸. The sambiloto extract was made by weighing a 0.5 g sample and adding 10 ml of 80% methanol, homogenizing, filtering using Whatman filter paper no. 42, and adjusting the volume to reach 25 ml. Methanol extract of sambiloto simplicia powder or standard BHT (33.3 μg/ml) was mixed with linoleic acid emulsion made by 1 ml of 2.5% linoleic acid mixed with 0.1 ml of Tween 20. A 4 ml of sambiloto simplicia extract sample and 0.02 M potassium phosphate buffer was added to reach a volume of 10 ml. The emulsion was incubated at 37 ± 1⁰ C in the dark for six days, and every day, a 0.1 ml aliquot was taken for analysis. The degree of oxidation was measured by 0.1 ml aliquot in  5 mL ethanol (75% v/v), 0.1 mL ammonium thiocyanate (30% w/v), and 0.1 mL FeCl₂ (0.02 M in 3.5% HCl v/v), then homogenized. After homogenization, the absorbance was measured at 500 nm and repeated thrice. The solution without extract was used as a blank, and BHT was used as a comparison at the same concentration of 100 ppm. The percentage of inhibition of lipid peroxidation was determined using equation 2 with Ao absorbance of the control and A1 absorbance of the sample.

The DPPH free radical scavenging activity (RSA) was determined using the method of Kang et al. (2018)¹⁵. This method was chosen because the sample volume tested was smaller than the volume of the DPPH solution, so the effect of chlorophyll color during measurements could be minimized. A sample of 0.2 ml methanol extract of sambiloto simplicia powder, added to  3.8 ml of 0.1 mM DPPH solution in methanol solvent, was mixed using a vortex for 1 minute; the resulting filtrate was incubated in the dark a room temperature for 30 minutes. Controls were made using methanol as a sample substitute and Butylated Hydroxytoluene (BHT) as a comparison at a concentration of 100 ppm. After incubation, the filtrate was measured for absorbance using UV-Vis spectrophotometry (Shimadzu) at a wavelength of 515 nm. The data obtained are A₀: absorbance of DPPH without sample, A_S: absorbance of model added with DPPH, and A_b: absorbance of extract sample without DPPH. Radical Scavenging Activity is expressed in percentage. The RSA value shows the ability of the sample to bleach DPPH violet and is calculated using Equation 3 (Kang et al., 2018)¹⁵.

Data Analysis

The research data was analyzed with ANOVA and the Pearson correlation test with the SPSS software. The data was obtained from two treatment batches and three replicate analyses.

Results

TCC and Zn Content

Figure 1 (a) shows that the high ZnCl₂ concentration promotes the high number of TCC. However, the effect is insignificant if the heating oven is more than 200 ppm. When heating with an autoclave at more than 300 ppm, the chlorophyll content was decreased because the residual chlorine causes chlorophyll oxidation¹⁶.

Androghapolide Content (AP), Total Phenolic Content (TPC), and Total Flavonoid Content (TFC)

Figure 2 (a) shows that AP content increased with increasing ZnCl₂ concentrations. However, in the oven heating method, it is insignificant, whereas in the autoclave, it expands to 300 ppm and decreases again at a concentration of 500 ppm. Autoclave heating can better maintain TPC and TFC as with chlorophyll and andrographolide content. This is because the phenolic and flavonoid components are very susceptible to heat.

Antioxidant activity

Figure 2 (d) shows that increasing concentration of Zn²⁺ will be followed by the higher number of LPI when heated in an autoclave. The simplicia sambiloto powder has RSA 36.46-77.52%. The higher the Zn²⁺ concentration, up to 400 ppm, the higher the RSA.

Correlation

Data in Table 1 shows that the lipid peroxidation inhibitory activity of sambiloto simplicia powder is correlated positively with an andrographolide content (P<0.01) and negatively with bound Zn levels and associated with total phenolic groups but not associated with total flavonoids and total chlorophyll levels (P>0.05). However, it was not related to total flavonoids and total chlorophyll content (P>0.05). Meanwhile, DPPH radical scavenging capacity was positively correlated (P<0.01) with TPC, TFC, Zn content, and TCC. Moreover, it is positively correlated with andrographolide content (P<0.05).

Figure 1: TCC (a) and Zn content (b) of sambiloto simplicia powder in various heating methods and Zn concentrations in the formation of the Zn-chlorophyll complex. Click here to view Figure

Table 1: Correlation between TPC, TFC, APC, Zn content, and TCC on the antioxidant activity of sambiloto simplicia powder

Note: Value in blanket means p value

*Significant in P<0.05

**Highly Significant in P<0.01.

Figure 2: AP content (a), TPC (b), TFC (c), and antioxidant activity (d) of sambiloto simplicia powder in various heating methods and Zn concentrations in the formation of the Zn-chlorophyll complex. Click here to view Figure

Discussion

TCC and Zn Content

Figure 1 shows that the higher the ZnCl₂ concentration, the higher the total chlorophyll content; however, if the oven is heated with a reagent concentration of more than 200 ppm, the effect of increasing chlorophyll levels is not significant. Meanwhile, when heating with an autoclave, the total chlorophyll level still increases to 300 ppm. At 400 ppm and 500 ppm, the chlorophyll content was decreased. The chlorine residue is suspected to be more significant, resulting in chlorophyll oxidation, which reduces chlorophyll content¹⁶. On heating with an oven, the formation of metallochlorophyll complexes is ineffective. The heat conductor medium in oven heating is dry air so that the reaction could be more optimal. Higher ZnCl₂ concentrations will result in Cl₂ residues triggering chlorophyll degradation, which has not yet formed a Zn-chlorophyll complex, so chlorophyll levels are lower⁹.

Data in Figure 1 (b) shows that the higher the ZnCl₂ concentration in oven and autoclave heating, the higher the Zn content, but it is insignificant in oven heating with more than 300 ppm. The higher the concentration of ZnCl₂, the more Zn is bound by chlorophyll and other components such as phenolics and flavonoids. Zn binding is less effective in oven heating because medium heating is dry air, whereas autoclave heating is under water vapor pressure; thus, damage in oven heating is very high. Elshaafi et al.¹⁷ state that increasing the oven heating temperature by 10 °C has resulted in high levels of phenolic and flavonoid component damage. The Zn binding ability of the sambiloto simplicia powder is theoretically higher than the Zn critical ability of the chlorophyll molecules¹⁸ in the sambiloto simplicia powder. This is caused by other components that can also bind to Zn²⁺, namely aromatic compounds such as 2-acetyl-1-pyrroline¹⁹, phenolics ²⁰, and flavonoids²¹.

AP content, TPC, and TFC

An essential component in sambiloto is andrographolide, and this compound plays many roles such as an antioxidant³, anti-inflammatory²², hepatoprotective²³, anticancer, antiviral, antihyperglycemic, antibacterial, and antifertility²⁴. Figure 2 (a) showed that the interaction between varied ZnCl₂ concentration and heating methods significantly affected the AP content of sambiloto simplicia powder. As in the results of the TCC analysis, AP content also increased in line with increasing ZnCl₂ concentrations. However, in the oven heating method, it is insignificant, whereas in the autoclave, it expands to 300 ppm and decreases again at a concentration of 500 ppm. According to Chokthaweepanich et al.²⁵, the optimum temperature for drying sambiloto in a hot air oven that retains andrographolide is 65 °C. However, in this study, at a temperature of 110°C with autoclave heating, it could prevent the degradation of andrographolide in Simplicia Sambiloto powder. Figure 2 (b) shows that the autoclave heating method can better maintain phenolic levels as with chlorophyll and andrographolide content. This is because the phenolic component is very susceptible to heat. Kessy et al.² state that steam blanching can maintain phenolic components compared to oven heating. The results of total flavonoid levels also had the same trend as total phenolic levels. The data from Figure 2 (c) show that the autoclave heating method can better maintain total flavonoid levels. At the same temperature (110 °C), in autoclave heating, the stability of flavonoids is reported to be higher than oven heating²⁶. 

Antioxidant activity

Figure 2 (d) shows that increasing concentration of Zn²⁺ contributes the higher LPI when heated in an autoclave. This is because the total phenolic and flavonoid content, andrographolide, and chlorophyll content of sambiloto simplicia powder with oven heating tend to be smaller, and Zn content is higher. The effectiveness of inhibiting linoleic acid peroxidation from chlorophyll extract is also influenced by the presence of phenolic and flavonoid components and total carotenoids in the chlorophyll extract²⁷. Sheeja et al.²⁸ also stated that andrographolide extract has lipid peroxidation inhibitory activity of up to 80%. The LPI was compared with BHT, and it was found that simplicia sambiloto powder had an LPI reaching 97.56% of BHT.

DPPH radical scavenging activity is one of the most widely used methods to measure the antioxidant activity of a material because it is relatively simple and fast, with results that can be compared with standards. The ability to capture DPPH radicals is influenced by the molecular structure of chlorophyll and the presence of phenolic, flavonoid, and andrographolide compounds. Figure 2 (c) shows that simplicia sambiloto powder has RSA 36.46-77.52%. The higher the Zn²⁺ concentration, up to 400 ppm, the higher the RSA. This is related to increased TCC, TPC, TFC, and AP content. Previous research discovered that blanching fresh sambiloto leaves in a Zn solution could increase the antioxidant activity of the sambiloto simplicia powder¹¹. Adam et al.³ stated that androghapolide extract has moderate DPPH radical scavenging power with an IC₅₀ of 0.883 ± 1.597 mg/ml and 0.514 ± 0.285 mg/ml compared to vitamin C 0.048 ± 0.004 mg/ml. In this study, the DPPH radical capture capacity was compared with BHT. The RSA of simplicia sambiloto powder can reach 94.25% compared to BHT.

Correlation

Data in Table 1 shows that the lipid peroxidation inhibitory activity of sambiloto simplicia powder is positively correlated with an andrographolide content(P<0.01) and correlated negatively with bound Zn content (P<0.01) and associated with total phenolic content but not related to total flavonoids and total chlorophyll content (P>0.05). Meanwhile, DPPH radical scavenging capacity was highly positively correlated (P<0.01) with TPC, TFC, Zn content, and TCC and positively correlated with andrographolide content (P<0.05). TCC was not associated with LPI because the higher levels of Zn in the material can act as a cofactor for lipid oxidation. However, Zn is a cofactor for enzymes that act as antioxidants in the body, so Zinc deficiency can result in reduced cell immunity, which can cause inflammation and cell death²⁹. Therefore, it is essential to develop food or drink products rich in Zinc that can increase zinc intake to meet the body’s needs.

Conclusion 

Based on the research results, it can be concluded that the concentration of ZnCl₂ as a reagent for the formation of Zn-chlorophyll and the heating method has a significant effect on chlorophyll content, Zn content, total phenolic content, total flavonoid content, andrographolide content and antioxidant activity of sambiloto simplicia powder. The higher the concentration of ZnCl₂ up to 300 ppm, the greater the chlorophyll content, phenolic and flavonoid content, Zn content, and andrographolide content.

Acknowledgement

The authors are grateful to all research team whom without their participation this research would not be conducted.

Funding Sources

This work was supported by University of Mercu Buana Yogyakarta

Conflict of Interest

The author(s) declares no conflict of interest.

Authors’ Contributions

Chatarina Lilis Suryani and FX Suwarta conceived and designed the experiments; Ichlasia Ainul Fitri performed the experiments and analyzed the data; Chatarina Lilis Suryani contributed reagents/materials/analysis tools; Chatarina Lilis Suryani wrote the initial draft; Ichlasia Ainul Fitri and FX Suwarta revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

This statement does not apply to this article.

Ethic Approval Statement

This research did not involve human participants, animal, subjects, or any material that requires ethical approval

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