1. INTRODUCTION:

All food basically provides the energy and nutrients needed to maintain human survival. Functional foods are foods that have been scientifically proven to provide health benefits regardless of how much nutritional value they contain¹. Fermentation is a method to preserve food at a low cost but is able to guarantee the quality and safety of the food².

Currently, there are various fermented foods or drinks that provide benefits for the human body, one of which is Kombucha. Kombucha (Figure 1.) is a beverage obtained from fermented tea, especially black tea with added sugar as a substrate for fermentation using symbiotic cultures of acetic acid bacteria (such as Acetobacter xylinum, Acetobacter xylinoides, or Bacterium gluconikum) and yeast (such as Schizosaccharomyces pombe, Saccharomyces ludwigii, Zygosaccharomyces rouxii Candida)¹.

Figure 1. Kombucha

Modern society tends to have a lifestyle that prioritizes safe products for health. Herbal health products are currently widely used by the public to prevent or treat diseases because they are considered safe from toxic chemicals to the body. In addition, sometimes expensive drug prices trigger people to try alternative treatments in maintaining health through herbal products³. Many claims have been reported regarding the beneficial effects of Kombucha, such as reduced inflammation and arthritis, cancer prevention and increased antioxidant activity⁴. Kombucha is also said to prevent cardiovascular disease, improve digestion and stimulate immunity. During the fermentation process of making Kombucha, the contained bacteria and yeast are able to metabolize sucrose into a number of organic acids such as acetic acid and glucuronic acid, amino acids, antibiotics and various micronutrients⁵.

Most plants have antioxidant compounds that have a wide variety and chemical properties. Characterization of the antioxidant activity of vegetables can yield many insights into the benefits of these plants^6,7. Natural antioxidants are preferred over synthetic ones (butylated hydroxyanisole and butylated hydroxytoluene) since they are considered less toxic and more robust than synthetic antioxidants. Natural antioxidants such as vitamin C, tocopherols, flavonoids, and other phenolic compounds are present in certain plants^8,9,10. Phenol compounds have beneficial effects related to antioxidant activity¹¹. Sauropus androgynus (L.) Merr., or locally known as Katuk, is one of the most popular vegetables in Indonesia as traditional medicine, especially for increasing the milk of breastfeeding women. The leaves' content is rich in vitamins so that many call it a 'multivitamin plant'. The antioxidants present in katuk can prevent cell and tissue damage^11,7. Then another natural ingredient that can be used as an antioxidant is kelor (Moringa oleifera Lam)^12,13,14,15. It is an ornamental plant native to the tropics and subtropics and is commonly cultivated throughout Indonesia as a vegetable for cooking purposes. All parts of the kelor plant have various biological activities such as reducing hyperglycemia, anti-inflammatory, anti-diabetic, antimicrobial, anticancer and antioxidant^16,17,18,19. Katuk leaves and kelor leaves (Figure 2.) are natural sources of antioxidants. Therefore, the purpose of this study was to determine the antioxidant activity, and compounds identification by LCMS/MS of a single and mixture of katuk and kelor leaves infusions and after being fermented with Kombucha.

2. MATERIALS AND METHODS:

2.1 Kombucha Culture Starter:

The starter culture of Kombucha or SCOBY (symbiotic cultures of bacterial and yeast) was obtained from the Fermentation House, Indonesia. The starter culture used in this study was stored in a refrigerator (4ºC) and consisted of a liquid component (sour broth) and a layer of cellulose on the surface.

Figure 2. Katuk (Sauropus androgynus (L.) Merr.)(A) and Kelor (Moringa oleifera Lam) (B) Leaves

2.2 Plant Materials:

Fresh leaves of katuk (Sauropus androgynous (L.) Merr) and kelor (Moringa oleifera Lam) were collected around Serang, Banten Province, Indonesia. The samples were sent to the Herbarium Bogoriense, Research Centre for Biology, Indonesian Institute of Sciences, Bogor, Indonesia, to determine their scientific name. In addition, the leaves were air-dried not direct to the sun for 7 days, and used directly for the materials of this study.

2.3 Sample preparation:

In one liter of water, eight grams of samples (Table 1.) and 10 g of commercial sucrose were added and boiled for 10 minutes, then filtered and cooled down for 30 minutes, half was used for unfermented samples, and the other half was added with kombucha starter (specially prepared by Fermentation House for 500 ml fermentation solution) and fermented in the dark for 7 days at room temperature.

Table 1. List of katuk and kelor unfermented and fermented samples tested.

No.	Code	Sample
1	Kt	Unfermented katuk
2	Kl	Unfermented kelor
3	KtKl 1:3	Unfermented mixture of katuk and kelor in ratio 1:3 w/w
4	KtKl 3:1	Unfermented mixture of katuk and kelor in ratio 3:1 w/w
5	KoKt	Kombucha Fermented katuk
6	KoKl	Kombucha Fermented kelor
7	KoKtKl 1:3	Kombucha Fermented mixture of katuk and kelor in ratio 1:3 w/w
8	KoKtKl 3:1	Kombucha Fermented mixture of katuk and kelor in ratio 3:1 w/w

2.4 pH Measurement:

The pH measurement using Eutech benchtop pH meter was conducted for all unfermented and fermented samples.

2.5 Antioxidant Activity:

DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity was conducted according to Yen and Chen with some modifications ^20,21. Sample 10-50 ul was directly used, methanol was added to give a final volume of 200ul, then methanolic 1 mM DPPH solution (40 µL) was added. The solution was stored at room temperature in the dark for 30 min. Absorbance was measured at 515 nm using a Thermo Scientific Varioskan® Flash. Inhibition of DPPH radical scavenger activities was assessed using the following formula:

% Inhibition = (Abs. Blank – Abs. sample x100%)/Abs.blank

Information:

Abs Blank: Abs of DPPH without sample

Abs Sample: Abs of DPPH with sample

Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)

TPC was determined with the Folin-Ciocalteu reagent using gallic acid as the standard^22,23. Absorbance was measured at 750 nm using the spectrophotometer with the same mixture, except the sample extract was replaced by methanol as the blank. TPC was expressed as mg GAE (Gallic Acid Equivalent)/L. TFC was determined according to aluminium trichloride method using quercetin as the reference compound ^22,24.

2.6 LC-MS/MS Analysis:

LCMS/MS was performed according to the method described in Sinaga et al., ²⁵. Mass spectrometry was performed on an LC-MS/MS Xevo, G2-XS QTof (Waters MS Technologies). The ionization type is ESI. The scan range was from 100 to 1200 m/z. The capillary and cone voltage was set at 0.8 kV and 30 kV, respectively, and was used in positive electron spray mode. The desolvation gas was set to 1000 L/h at a temperature of 500 °C and the cone gas was set to 50 L/h and the source temperature was set to 120 °C. The UPLC analysis was performed using a Waters Acquity Ultra Performance LC system. Chromatographic separation was carried out on an ACQUITY UPLC HSS T3 column (100 mm x 2.1 mm, 1.7 µm) at a column temperature of 40 °C. The mobile phase consisted of solvent A (0.1% formic acid in the water, v/v) and solvent B (0.1% formic acid in acetonitrile), with gradient polarity from 95:0.5 (A: B) to 0.5:95 (A: B). The flow rate was set at 0.3 ml/min. The column and autosampler were maintained at 40 °C and 20 °C, respectively. The injection volume was 1 µl. The data acquisition and processing were performed using UNIFI. The parameter used was retention time (RT) in the range of 1-15 minutes.

3. RESULTS AND DISCUSSION:

3.1 pH value:

Table 2. shows the results of pH measurement of unfermented and fermented samples. The results show that all unfermented samples have pH values above 7.0, whereas all the fermented samples have around 4.0. The pH value of all samples that have not been fermented is not yet in the acidic category. However, the sample changed to acid after being fermented for 7 days. The increasing amount of acid in samples fermented with Kombucha indicates that Kombucha bacteria undergo a growth phase. During the fermentation process, bacteria and yeast metabolize sucrose into a number of organic acids, such as acetic acid and glucuronic acid. Increasing the concentration of organic acids produced during fermentation can reduce the pH of kombucha rapidly²⁶. The higher the organic acid contained in Kombucha, the higher the total acid produced, thereby lowering the pH in kombucha²⁷. The low pH can make the acid in Kombucha has the ability to limit pathogenic bacteria from various other microorganisms, including contaminants that may be present in the growing medium³.

Table 2: pH Values of Unfermented and Fermented Katuk and Kelor Samples*

Condition	Sample	pH
Unfermented	Kt	7.42
	Kl	7.27
	KtKl 1:3	7.60
	KtKl 3:1	7.34
Fermented	KoKt	4.07
	KoKl	4.02
	KoKtKl 1:3	4.05
	KoKtKl 3:1	4.03

Note: * The pH of unfermented and fermented samples were measured at the same time, unfermented samples were frozen before taking measurements

3.2 Total Phenolic Content (TPC) and Total Flavonoid Content (TFC):

TPC and TFC of unfermented and fermented samples of katuk and kelor leaves are shown in Table 3. The results show better fermented samples of TPC and TFC values than unfermented samples. The fermentation process increased total phenol. For example, the total phenol value in the Kl sample was 372 µg GA/ml, then increased in the KoKl sample to 445 µg GAE/ml. The increase in total phenol was due to the complex phenolic compounds in kombucha fermentation being degraded by enzymes released by bacteria and yeast during fermentation. The degradation of polyphenol complexes into small molecules ultimately increases total phenolic compounds²⁶. The high increase in the TPC of the kombucha samples during fermentation was due to the presence of enzymes produced by the kombucha consortium, which produced organic acids²⁸.

Table 3 showed an increase in TFC of the sample before fermentation compared to the sample after 7 days of fermentation. The fermentation process increased flavonoids such as total flavonoids in the sample Kl 2,208 µg QE/ml then increased in the sample KoKl to 5,438 µg Q/ml. The increase in the total flavonoid content may be due to some microorganisms' activity in SCOBY²⁹. Based on the table above, it can be seen that the TFC of all samples showed higher results than the TPC of all samples, in accordance with Zubaidah et al. in their research³⁰.

Notes: TPC presented as µg Galic Acid Equivalent/ml TFC presented as µg Quercetin Equivalent/ml

Figure 3. Total Phenolic Content (A) and Total Flavonoid Content (B) of Unfermented and Fermented Katuk and Kelor Samples

3.3 Antioxidant Activity

Analysis of antioxidant activity was conducted using the DPPH method, the results shown in Table 4. All samples showed antioxidant activity, whereas fermented samples with kombucha showed higher antioxidant activity than unfermented samples. The highest antioxidant activity was observed at KoKtKl 1:3.

Table 4. Antioxidant Activity of unfermented and fermented Katuk and Kelor Samples

Condition	Sample	% Inhibition to DPPH
Condition	Sample	10 µl	20 µl	30 µl	40 µl	50 µl
Unfermented	Kt	21.8	30.7	38.5	46.1	49.0
	Kl	32.8	41.8	47.3	50.2	56.8
	KtKl 1:3	15.1	30.1	39.8	49.3	56.1
	KtKl 3:1	15.2	28.6	37.1	45.0	52.8
Fermented	KoKt	26.1	40.1	47.7	52.1	55.1
	KoKl	40.4	52.4	61.3	67.4	75.4
	KoKtKl 1:3	41.4	54.6	67.6	77.0	80.5
	KoKtKl 3:1	33.8	46.7	59.5	69.9	73.0

Measurements were made at a volume of 10-50 µl to determine the trend of antioxidant activity due to volume differences which determine as % inhibition. From the results in the table above, it can be seen that that increasing the sample volume increases the inhibitory activity for all samples. The results also shown that antioxidant activity increased after the fermentation process for single or mixed samples. For example, in the KtKl 1:3 at a volume of 50µl, the value of % inhibition was 56.1%. In the KoKtKl 1:3 sample, the inhibition increased to 80.5%, indicating that the 7-day fermentation can increase antioxidant activity. The highest antioxidant activity was achieved from a mixture of fermented katuk and kelor. However, the highest polyphenols and flavonoids came from fermented kelor. It seems that there is an interaction between the compounds contained in katuk and kelor when mixed, which in terms of antioxidant activity occurs synergistically, but causes a decrease in the total phenol and total flavonoid content compared to kelor only. Further research is needed to understand the occurrence of this phenomenon. In medicinal plants, other than the active substance as the most influential main component, other compounds may affect the expected response³¹. Antioxidant ability is determined by extracellular enzymes involved in structural changes of compounds during kombucha fermentation. Therefore, samples that have been fermented with kombucha have the potential to have higher antioxidant activity than samples without fermentation³². With the presence of enzymes secreted by the kombucha microbial consortium, the polyphenol compounds decompose so that the antioxidant activity increases³³.

3.4 Identification of Chemical Content:

LCMS/MS analysis (Figure 3,4 and Table 5.) on unfermented and kombucha fermented katuk and kelor samples identified five known compounds (kaempferol-3-O-rutinoside; phenyl propionic acid; trichosanic acid; kaempferol-3-O-β-D-glucopyranoside and quercimeritrin) and eight compounds only as predicted mass. The five compounds were reported to have various biological properties, particularly antioxidants (Table 6). The mixture of katuk and kelor leaves before and after fermentation produces active compounds that dominantly act as antioxidants. Meanwhile, only Trichosanic acid has cardiovascular properties. Kaempferol-3-O-rutinoside was proven to have antioxidant activity using the TEAC (Trolox equivalent antioxidant capacity) method found in green beans³⁴. In addition, kaempferol-3-O-rutinoside can also act as an antihypertensive because it has been proven in vivo to reduce systolic, diastolic and mean arterial blood pressure³⁵. Phenylpropionic acid has in vivo demonstrated its role as an anti-inflammatory properties³⁶. Trichosanic acid is an unsaturated fatty acid that has been used for thrombotic cardiovascular disease as ischemic heart disease, which has an inhibitory effect on platelets³⁷. Kaempferol-3-O-β-D-glucopyranoside is a flavonoid compound with antioxidant activity and an antibiotic reported in studies on Brassica nigra³⁸. Flavonoids have been shown to exhibit a range of biological effects, among which are prominent in the inhibition of lipid peroxidation and platelet aggregation due to their antioxidant properties and their ability to scavenge free radicals. And the last one is Quercimetrin, a flavonoid group that is proven to have very strong antioxidant activity and can work as an anti-inflammatory^39,40.

Katuk (Kt)

Kombucha Katuk (KoKt)

Kelor (Kl)

Kombucha Kelor (KoKl)

Retention Time [min]

Katuk : Kelor 1:3 (KtKl 1:3)

Kombucha Katuk : Kelor (KoKtKl 1:3)

Katuk : Kelor (KtKl 3:1)

Kombucha Katuk : Kelor 3:1 (KoKtKl 3:1)

Retention Time [min]

Figure 3: LC Chromatogram of katuk and kelor unfermented and Kombucha fermented samples

Table 5: Chemical compounds of katuk and kelor leaves samples analysed by LC-MS/MS

		Unfermented				Fermented
No.	Compounds	*Katuk*	*Kelor*	*Katuk Kelor* (1:3)	*Katuk Kelor* (3:1)	*Katuk*	*Kelor*	*Katuk Kelor* (1:3)	*Katuk Kelor* (3:1)
1.	Kaempferol-3-O-rutinoside	-	+ 245, 516	+ 259, 627	+ 126, 498	-	+ 463, 886	+ 446 ,432	+ 91, 543
2.	Phenylpropionic acid	-	-	+ 50, 978	+ 31, 319	-	-	-	-
3.	Trichosanic acid	-	-	-	-	+ 110, 146	-	-	-
4.	Kaempferol-3-O-β-D-glucopyranoside	-	-	-	-	-	+ 65, 464	+ 76, 423	-
5.	Quercimeritrin	-	-	-	-	-	+ 107, 309	+ 100, 898	-
6.	Candidate Mass C₁₄H₁₇N₃O	+ 274, 697	+ 288, 337	+ 144, 706	+ 195,395	+ 152, 640	-	-	+ 139, 834
7.	Candidate Mass C₁₇H₂₁N₃O	+ 245, 497	-	-	-	+ 113, 254	-	-	-
8.	Candidate Mass C₁₁H₁₇NO₆	+ 213, 945	-	-	+ 117,690	+ 185, 351	-	-	+ 312, 570
9.	Candidate Mass C₂₀H₃₄O₈	+ 163, 501	-	+ 129, 137	-	+ 134, 753	-	+ 144, 933	+ 133, 523
10.	Candidate Mass C₁₅H₁₉NO₆	+ 131, 739	+ 432, 840	-	-	-	-	-
11.	Candidate Mass C₁₉H₂₉NO₁₀	-	+ 1, 311, 243	+ 396, 087	+ 229, 462	-	+ 804, 708	+ 781, 658	+ 204, 602
12.	Candidate Mass C₂₅H₃₉NO₁₅	-	+ 708, 915	-	-	-	-	-	-
13.	Candidate Mass C₁₇H₂₄O₁₃	-	-	-	-	-	+ 151,006	-	-

Note: (-) not present; (+) present; (number) detector count as an indication of compound abundant in the sample

Kaemferol-3-O-rutinoside

Kaemferol-3-O-β-D-glucopyranoside

Quercimeritrin

Phenylpropionic acid

Trichosanic acid

Figure 4: Chemical structures of identified compounds in katuk and kelor unfermented and fermented kombucha samples

Table 6: Reported bioactivities of identified compounds in katuk and kelor unfermented and fermented kombucha samples

No	Chemical Compounds	Bioactivity
1.	Kaempferol-3-O-rutinoside	Antioxidant³⁴ Antihypertensive³⁵
2.	Phenylpropionic acid	Anti-inflammatory³⁶
3.	Trichosanic acid	Cardiovascular³⁷
4.	Kaempferol-3-O-β-D- glucopyranoside	Antioxidants and Antibiotics³⁸
5.	Quercimeritrin	Antioxidant³⁹Anti-inflammatory⁴⁰

CONCLUSION:

Katuk and kelor leaves showed antioxidant activity, either as a single preparation or a mixture, as well as unfermented or Kombucha fermented preparations. Fermentation with lombucha for 7 days reduced pH but increased the antioxidant activity, TPC and TFC of katuk and kelor samples. The results showed that the kombucha fermented katuk and kelor mixture (KoKtKl 3:1) at 50µl sample had the highest antioxidant activity (80.5% inhibition of DPPH). In contrast, at the same condition, the unfermented sample only has 56.1% inhibition of DPPH. Sample with the highest antioxidant activity (KoKtKl 3:1) was identified as containing kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucopyranoside and quercimetrin, which are flavonoid glycosides that might be responsible for the antioxidant activity. This study showed that kombucha fermented of katuk and kelor leaves have the potential to be studied further as a functional beverage for antioxidants.

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Received on 22.10.2021 Modified on 19.01.2022

Research J. Pharm. and Tech 2022; 15(11):5184-5191.

DOI: 10.52711/0974-360X.2022.00873