Moringa oleifera L. Microgreens and their Antioxidant Activity

. . This research aims to study the growth and antioxidant activity of Moringa oleifa L. microgreens. The microgreen phase occurred at 7 d after germination with a height of 36.72 cm ± 4.20 cm, fresh weight of 7.30 g ± 0.92 g 10 plants –1 , and the proportion of leaf to shoot is 16.01 % ± 1.31 %. M. oleifera microgreen leaves have a chlorophyll a, b, and carotenoid concentration respectively of 1 180.7 µg g –1 ± 329.0 µg g –1 of fresh weight, 631.7 µg g –1 ± 35.1 µg g –1 of fresh weight and 458.3 µg g –1 ± 52.0 1 µg g –1 of fresh weight. Examination of the antioxidant activity (DPPH) showed the antioxidant activity of the leaves is greater than that of the stem, with the IC50 of the leaves at 4.7 g L –1 and the IC50 of the stem at 10.1 g L –1 . Generally, M. oleifera microgreens can be harvested 7 d after germination with over twice the antioxidant activity in the leaves compared to the stems.


Introduction
Microgreens (tiny vegetable green or veggies) are green vegetables that are harvested when the cotyledons are fully opened or the true leaves are fully formed [1,2].Microgreens have gained much traction in recent years due to the short time needed between planting and harvesting, and their relatively high nutrient concentrations relative to their mature counterparts.Vitamins such as vitamin C, vitamin E, vitamin E, and beta-carotenes were found in general to be much higher in microgreens than the mature plants.Essential amino acids were also found in higher concentrations than their mature counterparts [3].With their high nutrient content, microgreens have a significant potential to be used in preventing malnutrition, improving health, or even reducing the risk of degenerative diseases.
Microgreens are plants that are in the growth phase directly after sprouts [2].Sprouts themselves are commonly consumed by many people in Indonesia [4].In contrast to sprouts, microgreen consumption in Indonesia is still very rare and only limited to some people.There are at least two factors that might affect this, first is that microgreens are not very well known to the general population and the second is that microgreens tend to be relatively expensive [5].The majority of microgreens in Indonesia are produced from seeds of vegetables that are imported from abroad and this is a reason why microgreens tend to be more expensive.Utilizing local vegetable seeds to produce microgreens would lower the cost of microgreens so that the general population would be able to consume nutrient-rich food at an affordable price in the form of microgreens.
Indonesia has a diverse array of vegetables, one of which is kelor or merunggai or limaran (Moringa oleifera L.) is a nutrient-rich plant that grows in tropical regions such as Indonesia [6].M. oleifera is known to contain high levels of antioxidants which can counteract the activity of free radicals [6][7][8].An excessive amount of free radicals can destroy several molecules such as protein and DNA which cause cancer and several degenerative diseases such as cardiovascular diseases and asthma [9].The objective of this research is to study the characteristics of M. oleifera microgreens and their antioxidant activity.

Materials and methods
M. oleifera seeds used in this experiment were obtained from online shops.Based on the author's preliminary data, seeds were soaked in tap water for 6 h and then spread evenly on the rockwool in plastic containers.The seeds were then covered by rice husks and kept until germination.The M. oleifera was cultivated in a greenhouse under natural conditions and was regularly irrigated with tap water until the microgreen phase without the addition of fertilizer.
The cultivation was stopped when the M. oleifera produced full open true leaves, and then 100 plants were randomly selected to measure the shoot height.To measure the shoot, stem, and leaf fresh weight, every 10 th plant was randomly selected and cut at the junction between stem and remaining seed.Leaves and shoots were separated and both fresh weights were measured using an electronic balance with an accuracy of four decimal points.Pigment content in the leaves was measured according to [10,11].An amount of 100 mg of leaves was ground into powder and then suspended in 5 mL of 95.5 % acetone followed by incubation at 4 °C for 2 d.The pigment concentrations were measured using UV-vis Spectrophotometer in three different wavelengths: 662 nm, 644 nm, and 470 nm, respectively.The concentration of each pigment was measured by Formula (1) to Formula (3): Antioxidant activity in the microgreens was examined using the DPPH scavenging assay.Antioxidants were extracted using the method detailed by [12].Amount of 200 mg of stem or leaf powder was suspended in 20 mL of 80 % (V V -1 ) methanol and homogenized for 30 s.A volume of 1 mL of aliquots was mixed with 0.4 mL of hexane and then homogenized for 30 s.The mixtures were centrifuged at 3 000 g for 10 min at 25 ℃ and then the supernatant was discharged from the tube followed by washing with hexane twice.The methanol extract was then filtered with 0.45 m millipore mesh before being used for the DPPH scavenging assay.
Antioxidant activity was measured by obtaining the IC 50 (50 % inhibitory concentration) in the DPPH scavenging assay.The plant extract was dissolved to produce solutions with concentrations between 100 mg L -1 to 1 300 mg L -1 .A total of 50 μL of 0.15 mM DPPH was mixed with 30 μL of M. oleifera leaf or stem extract.The solution was then incubated in darkness for 30 min before the absorbance was measured using a spectrophotometer at a wavelength of 517 nm.As a control, 4.9 mL of DPPH solution was mixed with 0.1 mL of methanol and the absorbance was measured at the same wavelength was measured.Percent inhibition was calculated using the Formula (4):

Results and discussions
The vegetables were considered to be in the microgreen phase when the cotyledons had fully opened or when the true leaves were formed.Usually, the vegetables take 7 d to 14 d to reach the microgreen phase [1,13].In this research, M. oleifera was determined to be in the microgreen phase when one true leaf that has fully opened was formed because the cotyledons of moringa remain in the seed during germination [14,15].M. oleifera was found to reach microgreen phase 7 d after germination (Figure 1) with a height of 36.7 cm ± 4.2 cm, a shoot fresh weight of 8.6 mg ± 0 .8mg 10 plants -1 , and a leaf proportion of 16.0 % ± 1.3 %.M. oleifera microgreens are roughly three times taller than most commercial microgreens which usually have and height of approximately 5 cm to 10 cm [13].The leaves are rich in a variety of pigments which play a role in photosynthesis.These pigments include chlorophyll a, b, and carotenoids [16].When consumed, these pigments can act as non-enzymatic antioxidants which protect the body from free radicals produced in the body [17].Leaves from the M. oleifera microgreens have a chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid content respectively of (1 180.7 ± 329.0) µg g -1 FW, (631.7 ± 35.1) µg g -1 FW, (1812.4 ± 340.4) µg g -1 FW, and (458.3 ± 52.01) µg g -1 FW.The total chlorophyll content of the microgreen leaves in his study was lower than the average content of mature leaves (> 2 000 µg g -1 FW) reported by [7].The results of this research were consistent with the study done by [18] using Butterhead lettuce (Lactuca sativa L. var.capitate).The chlorophyll content of the mature lettuce leaves was roughly twice that of the lettuce microgreen leaves.Compared to other vegetable microgreens such as lettuce (L.sativa) [18], radish (Raphanus sativus L.), spinach (Spinacia oleracea L.) , 00018 (2023) https://doi.org/10.1051/e3sconf/202337400018E3S Web of Conferences 374 3 r d NRLS basil (Ocimum basilicum L.), garden pea (Pisum sativum L.), and broccoli (Brassica oleracea L.) [19], M. oleifera microgreens have a higher chlorophyll content.
However, the total chlorophyll content of the microgreen leaves in his study was lower than the average content of mature leaves (> 2 000 µg g -1 FW) reported by [7].The results of this research were consistent with the study done by [18] using butterhead lettuce (L.sativa).The chlorophyll content of the mature lettuce leaves was roughly twice that of the lettuce microgreen leaves.
Currently, a study using artificial light showed that light quality can improve chlorophyll content in microgreens as reported by [22].A study on the effect of artificial light on lettuce microgreen showed that LED light with red and blue (R/B) rations equal to three produce induced higher chlorophyll content compared to other rations, and white light was produced by fluorescence light.In the future, exploration of different light qualities should be investigated to improve the chlorophyll content of M. oleifera microgreen  The DPPH scavenging assay shows that the antioxidant activity of M. oleifera microgreen leaves is over two times greater than the stem (Fig 3).The leaves had an IC 50 of 4.7 g L -1 FW (4.7 mg mL -1 FW) while the stem had an IC 50 of 10 g L -1 FW (10 mg mL -1 FW).These results are consistent with the study done by [20] on L. camara L. plants.The leaf extract of L. camara had three times the antioxidant activity as the stem.Antioxidant activity in L. camara had a positive correlation with the phenol content of the extract.The same pattern was observed in a study done by [21] on mature M. oleifera plants.[21] reported that the antioxidant activity of the leaves was greater than that of the stem with leave and stem extract IC was 387 mg L -1 FW and 1.116 mg L -1 FW respectively.If compared to the result from [20], the antioxidant activity of the M. oleifera microgreens in this study was less than that of the mature M. oleifera.This difference could be natural as a result of chemical changing during plant growth and development or might be the effect of environmental conditions.However, to confirm this further research needs to be carried out.
The current study showed that M. oleifera microgreen has lower chlorophyll and antioxidant activity compared to mature leave reported by [7] and [20], respectively.However, chlorophyll and antioxidant are not the only nutrient content in microgreens.Other nutrients such as vitamin, macro, and important trance elements need to be investigated in the future in order to explore the potential of application of M. oleifera microgreen.Moreover, the investigation of the effect of an environmental condition such as light quality can be explored to improve the nutrient value of M. oleifera microgreen.

Conclusions
Generally, M. oleifera microgreens can be harvested 7 d after germination with over twice the antioxidant activity in the leaves compared to the stems.The microgreen stage occurred at 7 d after germination with a height of 36.72 cm ± 4.20 cm, fresh weight of 7.30 g ± 0.92 g 10 plants -1 , and the proportion of leaf to shoot is 16.01 % ± 1.31 %.The chlorophyll a, b, and carotenoid content in the leaves of M. oleifera microgreen were 1 180.7 µg g -1 ± 329.0 µg g -1 of fresh weight, 631.7 µg g -1 ± 35.1 µg g -1 of fresh weight, and 458.3 µg g -1 ± 52.0 1 µg g -1 of fresh weight, respectively.The antioxidant activity test using DPPH showed the antioxidant activity of the leaves is greater than that of the stem, with the IC50 of the leaves at 4.7 g L -1 and the IC50 of the stem at 10.1 g L -1 .

Fig. 1 .
Fig. 1.Growth and development of M. oleifera microgreen. A. Microgreen growth from the day of germination to the microgreen phase.B. Microgreen showing shoot and true leaf.D day after germination

Fig 2 .Fig 3 .
Fig 2. Chlorophyll a, b, total carotenoid, and total chlorophyll content in the leaf of M. oleifera microgreens.Data are shown as average ± SD with three replications.

Table 1 .
Shoot height, shoot, stem and leaf fresh weight and leaf proportion of M. oleifera microgreen.Data present as average ± SD.
* Measurement were done on one hundred plant # Ten replications