Assessment of Antioxidant and Anticancer Activities of Microgreen Alga Chlorella vulgaris and Its Blend with Different Vitamins

There is a very vital antioxidant extracted from microgreen alga. Chlorella vulgaris has major advantages and requires high yield worldwide. Some microalgae require vitamins for their growth promotion. This study was held to determine the impact of different vitamins including Thiamine (B1), Riboflavin (B2), Pyridoxine (B6), and Ascorbic acid (c) at concentrations of 0.02, 0.04, 0.06, and 0.08 mg/L of each. Each vitamin was added to the BG11 growth medium to determine the effect on growth, total carbohydrate, total protein, pigments content, antioxidant activities of Chlorella vulgaris. Moreover, antitumor effects of methanol extract of C. vulgaris without and with the supplement of thiamine against Human prostate cancer (PC-3), Hepatocellular carcinoma (HEPG-2), Colorectal carcinoma (HCT-116) and Epitheliod Carcinoma (Hela) was estimated in vitro. C. vulgaris supplemented with various vitamins showed a significant increase in biomass, pigment content, total protein, and total carbohydrates in comparison to the control. Thiamine was the best vitamin influencing as an antioxidant. C. vulgaris supplemented with thiamine had high antitumor effects in vitro. So, it’s necessary to add vitamins to BG11 media for enhancement of the growth and metabolites.


Introduction
Cancer lingers to be one of the principal sources of death in the Earth. Researchers sustained searching for harmless and further actual chemoprevention and care is required for the enhancement of the proficiency and to lower the cost for cancer treatments [1]. Microalgae have usually become part of the daily diet, specifically in East Asian countries; algae are an attractive research topic because of their active contents used to treat cancer [2]. Microalgae are usually used as an additive in food to enrich the nutrient of food and/or mend the health of beings due to phytochemical contents and bioactive molecules. The protein composition of microalgae is the chief goal for the untraditional source of protein [3]. Moreover, the microalgae which could synthesize vital amino acids to humans and animals compares beneficially with that of other sources of proteins [4]. In recent years, many researchers studied the assembly of extracellular antibiotic metabolites by marine algae, and the researchers discovered that a large number of antifungal agents are present in green microalgae extracts [5]. Ultimately, microalgae are almost an available supply of natural antioxidants due to their huge biodiversity, more varied than plants.
was increased within three days of inoculation with all tested concentrations of Pyridoxal (vitamin B6). The best concentrations were 0.08 µg/L of Pyridoxal followed by 0.06 µg/L. Meanwhile the control C. vulgaris (without vitamin supplementation) produced the least amount of biomass compared with the other concentrations of Pyridoxal. Figure 1b demonstrated the effect of different concentrations of Ascorbic acid (vitamin C) on C. vulgaris growth within 18 days of incubation. Moderate growth was obtained at the first three days of incubation. The maximum growth was obtained with the highest concentrations (0.08 µg/L) until the 14 days of incubations, but after 14 days the elevations of growth were obtained by the following Ascorbic acid (vitamin C) concentrations 0.02, 0.04, 0.08, and 0.06 µg/L and control, respectively. The elevation of C. vulgaris growth was observed in the first three days of incubation with all the tested concentrations of Thiamine. From three to seven days the stationary phase of C. vulgaris occurred with 0.06 µg/L of Thiamine. Moderate elevations of C. vulgaris growth occurred with the following concentrations, 0.02, 0.04, and 0.08 µg/L of Thiamine at 3 to 7 days of incubations, meanwhile, the elevations of growth were obtained after seven days of C. vulgaris incubation until 16th day of incubation with 0.08, 0.08 µg/L and control, respectively, Figure 1c. BG 11 media when supplemented with riboflavin (vitamin B2), the growth of C. vulgaris was increased, Figure 1d. High elevations of C. vulgaris growth were obtained in the first three days of cultivations with all concentrations of riboflavin. The 0.02 µg/L was the best concentration of riboflavin that caused high elevation of growth at 10 to 20 days followed by 0.08, 0.04 and 0.06 µg/L, respectively, in compared with control.

The Effect of Vitamins Supplementations on the Photosynthetic Pigments of C. vulgaris
The present results indicated that there were significant differences in chlorophyll-a, chlorophyll-b and carotenoid pigments at the different sampling days except for chlorophyll-b in the control group showed non-significant differences. Moreover, significant differences were observed in the pigments contents for different

The Effect of Vitamins Supplementations on the Photosynthetic Pigments of C. vulgaris
The present results indicated that there were significant differences in chlorophyll-a, chlorophyll-b and carotenoid pigments at the different sampling days except for chlorophyll-b in the control group showed non-significant differences. Moreover, significant differences were observed in the pigments contents for different concentrations of Pyridoxal at different experimental days being at range from 0.19 (0.08 µg/L concentrate after 3 days) to 3.06 (control group after 11 days) for chlorophyll-a and from 0.84 (0.08 µg/L concentrate after 3 days) to 6.42 (0.02 µg/L concentrate after 16 days) for chlorophyll-b, while they varied from 5.20 (0.08 µg/L concentrate after 3 days) to 24.12 (0.02 µg/L concentrate after 11 days) for Carotenoid (Table 1).
It appears that the sampling time had no significant influence on chlorophyll-a content at 0.06 and 0.08 concentrations of ascorbic acid; chlorophyll-b for the control group and 0.06 ascorbic acid concentration; Carotenoid for 0.06 and 0.08 µg/L ascorbic acid concentrations. In contrast, a significant effect of sampling time was observed on all pigments at 0.02 and 0.04 µg/L ascorbic acid concentration; chlorophyll-b at 0.08 µg/L concentration; chlorophyll-b and Carotenoid for the control group. Furthermore, significant differences appeared in the pigments' contents for different concentrations of ascorbic acid at different experimental days varied from 1.10 to 5.56 for chlorophyll-a, from 0.36 to 4.56 for chlorophyll-b, and from 5.36 to 22.91 for Carotenoid as presented in Table 2. Table 3 demonstrates the effect of different concentrations of thiamine and days of incbation on the pigments content of C. vulgaris. As presented in Table 1, no-significant effects of sampling time were observed on chlorophyll-b content for control group, 0.04 and 0.08 µg/L Thiamine concentrations; Carotenoid at 0.02 and 0.06 µg/L concentrations, while significant differences were recorded for chlorophyll-a content at different concentrations; chlorophyll-b at 0.02, 0.06 µg/L concentrations; Carotenoid at 0.04 and 0.08 µg/L concentrations. In addition, significant differences were observed in the pigments' contents for different concentrations of Thiamine at different experimental days except 0.02 and 0.04 µg/L concentrations after 3 and 16 days; concentration 0.06 µg/L after 11 and 16 days. Minimum estimates were 0.03, 0.08, and 3.77 µg/L, while the maximum estimates were 4.05, 4.26, and 29.27 µg/L for chlorophyll-a, chlorophyll-b, and carotenoids, respectively.
Results in Table 4 indicate that there was a significant effect of sampling time from day 3 to day 20 post the beginning of the experiment on chlorophyll-a, chlorophyll-b, and Carotenoid pigments except for chlorophyll-b in the control group showed non-significant differences. In addition, non-significant differences were observed in the pigments' contents for different concentrations of riboflavin at different experimental days except control group at different days, 0.02 µg/L concentration after 3, 11, and 20 days, 0.06 after 3 and 16 days and 0.08 µg/L concentrations after 9 and 16 days. Minimum estimates were 0.04, 0.40, and 0.04 µg/L, while the maximum estimates were 5.30, 4.48, and 17.48 for chlorophyll-a, chlorophyll-b and carotenoids, respectively. Table 5 indicated that there were highly significant differences in carbohydrates and proteins contents under the effect of the different concentrations of the tested vitamins. The best concentration of vitamins for enhancement of carbohydrate content of C. vulgaris was 0.08 µg/L for all the tested vitamins. Meanwhile, the best concentration was different among vitamins concerning their effect on proteins content as the best concentration was 0.08 µg/L Thiamine, 0.06 Riboflavin, and Pyridoxal. Meanwhile, Ascorbic acids diminished the protein content of C. vulgaris. concerning protein values, minimum values were coupled with 0.04 concentration for all vitamins, while the maximum values were observed at 0.08 concentration for Thiamine, 0.02 for Ascorbic acid; 0.06 for Riboflavin and Pyridoxal.

Antitumor Effect of C. vulgaris and C. vulgaris Supplemented with Different Concentrations of Thiamine (Vit. B1)
The effects of different concentrations of C. vulgaris and C. vulgaris blended with 0.08 µg/mL of Thiamine on the inhibition of various cancer cells were summarized in Table 6. Results revealed that methanol extracts of C. vulgaris and C. vulgaris supplemented with Thiamine at different concentrations (100, 200, 400, 600 and 800 µg/mL) affect inhibition of various cancer cells (HEPG-2, HCT-116, Hela and PC-3). However, C. vulgaris supplemented with Thiamine gives better results compared with C. vulgaris without Thiamine supplementation. The higher concentrations were most effective against cancer cells

FT-IR
The FT-IR analyzes of C. vulgaris biomass (control) and blended with thiamin embodied various absorption peaks changed to 2959 and 2923 in C. vulgaris (control), and C. vulgaris to blend with Thiamine The peaks range from 3300-2500 cm −1 are representing O-H stretching vibration occasion of carboxylic acids. The band 1655 was moved to 1646 and 1045 changed to 1076. The band 2426 was showed only with C. vulgaris blended with Thiamine, and the two bands 1549 and 1048 were showed only with C. vulgaris (control), these results denoted the change in chemical structure of C. vulgaris blended with Thiamine

Discussion
Vitamins play vital roles in the development of the contents of microalgae [24]. Algae required various nutrients in addition to carbon dioxide, light, and water for growth. Meanwhile, for faster growth, numerous microalgae require the supply of vitamins such as thiamine, biotin, or cobalamine because they cannot synthesize them [25,26]. Certain types of microalgae grow fast and efficiently in the presence of prokaryotes that produce vitamin B that is absorbed by microalgae [27]. Vitamins perform a vital role in the promotion of the primary productivity of the marine ecosystem and induction of microalgae bloom [28,29]. Vitamin B1 was the first cofactor that affects in microalgae growth and also plays a major role in carbohydrate and protein metabolism [30][31][32]. A large number of microalgae demand an external supply of thiamine [24]. Total chlorophyll-a of Cyclotella nana increased with all concentrations of vitamin B12 [23]. Moreover, Menzel and Spaeth [33] reported that vitamins have a vital role in marine ecology not only in the capability to impact in yields but also in affecting in growth rate. When Cyclotella nana grew in different concentrations of vitamin B12, the cell numbers were little changed during the first 2 days, the best concentration of vitamin B12 was 3 ng [23]. However, Algae were significantly increased when vitamins were found in the growth media [33]. Total chlorophyll-a was elevated daily when Cyclotella nana grew at various concentrations of vitamin B12 but in Monochrysis lutheri, chlorophyll-a, elevated in the first 3 days of incubations [23]. Furthermore, Lwoff and Dusi [34] investigated that some cryptophyta demands vitamins such as thiamine as a growth factor. Menzel and Spaeth [33] showed that diatom blooms grow moderately when cobalamine was supplemented at the highest concentrations. In addition, several studies correlate algal yields and vitamin concentrations [35,36]. Harmful algal blooms were increased with vitamins concentration in water in a similar manner as amino acids and protein [37]. More than 70% of microalgae need supplementations of one or more vitamins [38]. Microgreen algae Volvox aureus and Pandorina morum required thiamine and vitamin B12, Desmidium swartzii required vitamin B12, Synura petersenii required vitamin B12, and biotin for growth [38,39]. Supplementation of Chu 10 D medium with vitamins B1, B6, B7 and B12 produced higher biomasses of Chlorella vulgaris that were possibly used as feedstock for the productions of biofuels [40]. Vitamin B6 enhances the growth of Chaetoceros calcitrans [41]. The growth rate of Haematococcus was increased when the growth media were supplemented with 50 mg/L vitamin B12 [42]. Desouky [43] reported that the total carbohydrate contents of C. vulgaris were significantly elevated when supplemented with 200 ppm of riboflavin (vitamin B2). Vitamins are essential for the continued growth of some algae and also an adverse effect of some heavy metals [43][44][45]. Thiochrome (a natural metabolite of thiamine) can enhance the reproduction of Chlorella vulgaris [46]. Total carbohydrate content was significantly elevated when 200 ppm of ascorbic (vitamin C) acid or thiamine (vitamin B1) were added for different levels of either CoCl 2 or NiCl 2 to Chlorella vulgaris [47]. The Chlorella vulgaris cell number, dry weight, total pigments content, and total carbohydrates were significantly increased when inoculated in different levels of Pb +2 and supplemented with 200 ppm riboflavin [43]. Total carbohydrates and total proteins increased significantly when Chlamydomonas reinhardtii was supplemented with thiamine and different concentrations (25, 50 and 75 mM) of NaCl [48]. The free amino acid contents of Scenedesmus obliquus were significantly increased when supplemented with 200 ppm pyridoxine and riboflavin [49]. C. vulgaris supplemented with Thiamine is a potential source of natural antioxidants [50]. Chlorella vulgaris has antioxidant activities and anti-cholesterol [51]. The addition of Thiamine promoted the oxidative damage of Cu +2 on C. vulgaris growth and improved growth, pigment contents [52]. The antioxidant enzymes of Chlorella vulgaris were increased when exposed to UV-B [53]. The higher antioxidant activities were present in Chlorella methanolic extract [54]. Magnetic fields enhance the growth and antioxidant activities of Chlorella vulgaris [55]. Chlorella vulgaris has antioxidant and anticancer activities [56]. Hot water extract of Chlorella vulgaris decreased the number of viable HepG2 cells due to increased DNA damage and apoptosis [57]. Nanocellulose and the Au/cellulose nanocomposite derived from Chlorella vulgaris posses' significant cytotoxicity against lung cancer cells (A549) due to improve the relative expression of p53 gene, while reduced that of the Raf-1 gene [58]. In the FT-IR spectroscopy analysis of C. vulgaris biomass and supplemented with Thiamine there was the change in the peak position and also there were new peaks and disappear other peaks in the alga supplemented with thiamine, that denoted that modification of algal structure after treatments and hence effect in properties and metabolites [59]. Structural changes particular components were investigated using vibrational spectroscopy [60]. Fourier transform infrared (FTIR) spectra measured the change of macromolecular composition of Microcystis aeruginosa and Protoceratium reticulatum in response to the treatments [61].

Tested Alga
Chlorella vulgaris was attained from our lab (GEBRI), University of Sadat City, Egypt, and used as a test organism. The medium was BG11 (Table 7) nutritive culture for the enrichment and growing of the tested alga. Chlorella vulgaris culture subject (control) and four different vitamins concentration were supplemented to BG11 medium, Thiamine (B1), Riboflavin (B2), Pyridoxine (B 6 ), Ascorbic acid (c). Vitamin concentrations were adjusted to 0.02, 0.04, 0.06 and 0.08 µg/L. Two. Each vitamin was added to the culture media after the medium sterilization in autoclave and the vitamin sterilize by Millipore filter 45 µ pore, due to the vitamins may be decompose high temperature in autoclave. The cultures were put at a west-facing window receiving natural daylight at a temperature 30 ± 2 • C and shaken moderately thrice a day to prevent clumping. Culture growth was estimated by optical density at 660 nm by using Shanghai Unico UV-2000 spectrophotometer.

Preparation of Algal Extracts
Algal cultures, C. vulgaris, and C. vulgaris supplemented with different concentrations of the tested vitamins were put in the previously mentioned conditions. In a log phase, algal biomass was collected, washing and dried. Equal to one g of dried alga was mixed with 100 mL of methanol for 4 h in magnetic stirrer, after 4 h the algal mixture was filtrated through Whatman No.1 filter paper, and then dried by vacuum at 50 • C using a rotary evaporator.

Estimation of Pigments Content of C. vulgaris
Five mL of homogenized C. vulgaris suspension were centrifuged for 10 min at 4000 rpm. The extractions of pigments were obtained by using 5 mL 90% acetone from the pellets. The tubes were placed in the dark for 24 h. After that sample was centrifuged for 15 min at 5000 rpm and the supernatant was collected. The absorbance was read at 662, 645, and 470 nm (A662) against 90% acetone as blank by using Shanghai Unico UV-2000 spectrophotometer. The calculation of pigments according to the following equations: Chlorophyll-a (µg·mL −1 ) = 11.75·A662 − 2. Protein contents of C. vulgaris supplemented with various concentrations of the tested vitamins were determined using Folin phenol reagent according to the method adopted by Lowry et al. [62], carbohydrate contents were determined by the Anthrone method of Hedge and Hofreiter [63]. The antioxidant activity of C. vulgaris extracts and C, vulgaris extracts supplemented with the tested vitamins at various concentrations (0.02, 0.04, 0.06, and 0.08 µg/L) were tested by 2, 2,2-diphenyl-1-picrylhydrazyl (DPPH) test. One ml of 0.03 g DPPH in 50 mL methanol was mixed to 1 mL of methanol extract C. vulgaris culture subject (control), and along with four different vitamins concentration and incubated for 30 min at room temperature, the absorbance was measured at 517 nm by using Unico UV-2000 spectrophotometer. The antioxidant activity was calculated according to the following equation.

MTT Assay
The cell lines declared above were used to investigate the suppression effects of methanol extracts of C. vulgaris and C. vulgaris blended with 0.08 µg/L of thiamine on cell growth using the MTT assay [64,65]. (The methanol extracts of C. vulgaris and C. vulgaris blended with 0.08 µg/L of thiamine were evaporated by Rotary evaporator, followed by dissolved in Dimethyl sulfoxide (DMSO), to prepare the following concentration (100, 200, 400, 600, and 800 µg/mL). The colorimetric assay is measured at absorbance of 570 nm using a plate reader (EXL 800, New York, NY, USA). The relative cell viability in percentage was calculated as (A570 of treated samples/A570 of untreated sample) × 100.

FT-IR Analysis
Fourier transform infrared (FTIR) spectroscopy was used to estimate the presences and change of the functional groups in C. vulgaris and C. vulgaris blended with Thiamine [66].

Statistical Analysis
Fixed effects included vitamin concentrate, pigment, and time were investigated according to Proc ANOVA using a statistical analysis system (SAS, 2012), and a p-value of ≤0.05 was considered to indicate statistical significance. The differences between means were detected by Duncan's Multiple Range Test [67].

Conclusions
C. vulgaris has applications in food, antibiotics, pharmaceutical compounds and is also used in biofuels such as biodiesel and bioethanol. In this work, we have tested the effects of adding various vitamins at different concentrations to C. vulgaris growth media on the C. vulgaris growth criteria (Optical density, total photosynthetic pigments, antioxidant (DPPH) activity and total protein, carbohydrate contents) during 18 days of cultivation compared with control C. vulgaris (without vitamins supplementations). The results confirmed that all the supplemented vitamins have a significant effect on alga growth (increasing C. vulgaris biomass) and the best vitamin was thiamine at a concentration of 0.08 µg/L. In addition, C. vulgaris without and with thiamine have anticancer properties when tested in vitro however, C. vulgaris supplemented with thiamine was more effective.

Conflicts of Interest:
The authors declare that they have no known competing interest.
Sample Availability: Samples of the compounds are available by authors.