Stabilization against bacterial infections
Based on the observations that people consuming microalgae on a regular basis seemed to be more resistant against infectious diseases, the first systematic research was conducted on this phenomenon in the mid-sixties in Japan. A field study with a group of about 1000 Japanese marine soldiers over a period of 95 days showed the surprising result that the soldiers in the test group who received 2 g of Chlorella vulgaris had a significantly lower risk (25%) of catching a cold (KASHIWA et al. ).
In 1973, KOJIMA et al. demonstrated the immunostimulant action of chlorella. Extracts of chlorella were injected to rats and 24 hours later charcoal particles were injected to them. KOJIMA et al. observed that the concentration of charcoal particles in the blood decreased more rapidly in the test group treated with chlorella. Examination of rat tissues showed that macrophages were much more active in the chlorella-treated rats than in the control group.
TANAKA et al. observed in 1986 that resistance to intraperitoneally inoculated Escherichia coli in mice is enhanced by intraperitoneal, intravenous or subcutaneous administration of a high molecular weight, water-soluble fraction extracted from Chlorella vulgaris (CVE). Elimination of the bacterium from the spleen of CVE-treated mice was increased, and this increased elimination was related to accelerated peroxide generation and chemokinesis in polymorphonuclear leukocytes upon CVE treatment. The ameliorative effect was detected at doses of approximately 2 mg / kg and when administered 1, 4, or 7 days before infection . Oral administration of CVE shows similar effects, which give some evidence of stimulation of non-specific cellular defense. HASEGAWA et al. fed male Fisher rats 1000 mg CVE/kg for 14 days. The rats were inoculated with 2.7.108 Escherichia coli intraperitoneally. The number of bacteria increased for 1-6 h and peaked after 6 h in both the control and CVE-administered groups. In both groups, the number of bacteria decreased to an undetectable level within 24 hours. In the group to which CVE was administered, the number of viable bacteria in each organ (spleen, liver, peritoneal cavity, and blood) was very significantly lower than in the control group, whereas the number of leukocytes, especially polymorphonuclear leukocytes in the peritoneal cavity and peripheral blood, maintained higher levels in the group to which CVE was administered . After oral administration of CVE to mice (20 mg/mouse, 10 consecutive days), resistance against intraperitoneal infection with Listeria monocytogenes was improved. The number of bacteria in the group to which CVE was administered was significantly lower in both the peritoneal cavity and the spleen than in the control group. FCM analysis revealed that g/d + Thy 1.2+ cells in the non-adherent peritoneal exudate cells (PEC) and in the spleen of mice to which CVE was administered increased more visibly in number in the early stage at day 3 or 5 post-infection compared to those of control mice. The proportion of TCR a/b + Thy1.2+ T cells in non-adherent CEP in the control group increased from 13% at day 0 to 49% in the late stage at day 10 post-infection, whereas the proportion in CVE-treated mice increased to 64% at this stage and is associated with an increased DTH response to Listeria.
The results suggest that administration of Chlorella vulgaris extract (CVE) significantly increases cell-mediated immunity to Listeria by increasing g/d + T cells in the early phase and increasing a/b + T cells in the late phase of Listeria infection (HASEGAWA et al. ).
In addition, preventive oral administration of Chlorella vulgaris biomass (CVB) shows effects on immunity. DANTAS et al. demonstrated these actions on the natural killer (NK) cell activity of mice infected with a sublethal dose of viable Listeria monocytogenes. Chlorella vulgaris treatment produced a significant increase in NK cell activity in both uninfected and infected animals compared to animals that received only placebo (water). When CVB was administered to infected mice, there was a further increase in NK cell activity that was significantly higher than that found in the infected-only group. Furthermore, CVB treatment (50 and 500 mg/kg) of infected mice with a dose of 3,105 bacteria/animal that is lethal to all untreated controls, produced a dose-response protection that resulted in a survival rate of 20% and 55%, respectively . In addition, DANTAS et al. found that this protection was due, at least in part, to an increase in granulocyte and macrophage colony-forming units in the bone marrow and an increase in serum colony-stimulating activity compared to the control group .
Organisms that have a weak immune system for example by the application of immunosuppressants can also be protected by the administration of Chlorella vulgaris or CVE. In the case of CVE administration, KONISHI et al  and HASEGAWA et al  observed accelerated recovery of polymorphic nuclear leukocytes in the peripheral blood of mice and rats rendered neutropenic by cyclophosphamide. The number of granulocyte/monocyte generating cells increased rapidly in the spleen. In contrast to mice not treated with CVE, CVE-treated animals showed increased resistance against intraperitoneal E. coli infection. It seems likely that CVE activates both mature leukocytes and hematopoietic generating cells in the bone marrow. Further studies by KONISHI et al  support this hypothesis. “Subcutaneous administration of an acidic glycoprotein prepared from CVE in 5-fluorouracil (5-FU) to treated mice showed protective effects against myelosuppression and native infections.
Administration of the glycoprotein greatly reduced the mortality of tumor-free mice given a high dose of 5-FU and was able to increase the LD50 value of 5-FU for these mice. Normally, after treatment with 5-FU, an indigenous infection develops based on the deficiency of the host defense system. The glycoprotein reduced the incidence of indigenous infection and this effect was attributable to accelerated recovery from 5-FU-induced myelosuppression. Early recovery of hematopoietic generative cells or cells responsive to interleukin-3 or granulocyte-macrophage colony-stimulating factor was observed in the bone marrow of glycoprotein-treated mice. When the glycoprotein was administered to mice with tumors during 5-FU treatment, the glycoprotein prolonged mouse survival without affecting the anti-tumor activity of 5-FU. Furthermore, the glycoprotein itself was shown to have anti-tumor activity. These results suggest that the glycoprotein may be beneficial in mitigating the side effects of cancer chemotherapy without affecting the anti-tumor activity of the chemotherapeutic agent. In terms of actions, it is logical to examine the effects of chlorella on immunocompromised hosts.
HASEGAWA et al. proposed that preventive administration of Chlorella vulgaris extract (CVE) may be effective in the treatment of opportunistic infections in patients immunocompromised due to retrovirus. He showed that oral administration of CVE restores the ability of murine acquired immunodeficiency syndrome mice (when infected with murine leukemia virus LP-BM5) to eliminate Listeria monocytogenes in association with an improvement in the diminished immune response to Listeria monocytogenes. The DTH response to Listeria monocytogenes in CVE-treated mice was significantly higher than in the control group .
The authors hypothesized that through the increase in interferon g-producing type 1 T helper cell responses, interferon g activates macrophages to produce interleukin 12 and thereby increases host defense against Listeria. Both the higher secretion of interferon g and the higher cytokine titers are detectable (HASEGAWA et al. [13, 14]).
Protection against viral infections
IBUSHUKI et al. evaluated the antiviral action of Chlorella vulgaris extract (CVE), produced via the host, against murine cytomegalovirus (MCMV) infection in ICR mice. Mice treated with 10 mg of CVE 3 days and 1 day prior to the virus challenge survived infection. The protective action of CVE was demonstrated by a decrease in replicated infectious viruses in the target organs of CVE-treated mice. CVE also protected the mice from histopathological lesions of the target organs due to MCMV infection. Both serum interferon level and 2’5′-oligo-adenylate (2-5) synthetase activity were increased and are higher than those of control mice. Natural spleen cell killing activity, which is otherwise decreased by lethal MCMV infection, was remarkably increased in CVE-treated mice. What is particularly remarkable is that neither the virulicidal nor the virostatic activity of CVE on MCMV was observed in vitro. The resistance induced by Chlorella vulgaris extract (CVE) appears to be produced via the host .
The previously cited literature shows that via administration of Chlorella vulgaris, both in the form of the alga (CVB) and the algal extract (CVE), a series of positive immunostimulatory actions are induced. It seems that via the activation of hematopoiesis and via the acceleration of the differentiation of the generative cells, the immunity due to the cells is increased, accompanied by an increasing macrophage activity. This is why the observed anti-tumor effects occur mainly via the stimulation of the body’s own defenses. However, recent studies have shown that Chlorella vulgaris also produces substances such as sterols  and glyceroglycolipids  with direct anti-tumor activity. Under both oral administration of CVB (TANAKA et al. [18, 19]) and intraperitoneal injection of CVE (KONISHI et al. ) in mice inoculated with Meth-A tumor cells, the survival time is strikingly prolonged. Mice treated with CVB and CVE show concomitant antigen-specific immunity produced through cytostatic T cells, but not through cytotoxic T cells. Natural killer cells do not appear to contribute to anti-tumor resistance in this system. NODA et al. were able to show that a high molecular weight glycoprotein that can be isolated in large quantities from Chlorella vulgaris (CVE) extract, produces the anti-tumor effect previously described.
For the screening experiments, 5 M Meth A fibrosarcoma cells induced by methylcholanthrene of BALB/c origin were inoculated subcutaneously into the right and left flanks of 8-12 week old mice. Each glycoprotein fraction (2/10/50 mg/ kg) was injected into the right flank tumor 5 times every other day starting on day 2 to assess anti-tumor activity against both tumors at 8, 10, and 12 days after tumor inoculation. Anti-tumor activity was determined as the product of the longest and shortest ellipsoidal growing tumor diameter on the skin. It was possible to identify the fraction of glycoprotein that completely inhibits tumor growth. (dose of 10 mg /kg per injection). The most active substance was found to be a glycoprotein with a molecular weight of 63,000 amu. It contains 65% carbohydrates, mainly D-galactose, and 35% protein. The protein part was determined and contained 15 amino acids. It was shown that the protein part was responsible for the anti-tumor activity . The anti-tumor activity was stable after autoclaving at 121° C for 30 min and even after treatment with 1 M HCl at 80 °C for 1 h, the anti-tumor activity did not decrease. The observed anti-tumor action was comparable to the effects of some other previously determined biological response modifiers such as OK – 432 (OKAMOTO et al., prepared from Streptococcus pyogenes ) and PSK (TSUKAGOSHI et al., prepared from Coriolus versicolor ) and sometimes stronger than that of the standard dose of OK – 432 (NODA et al.). Biological response modifiers isolated from plant tissues and bacterial products show nonspecific anti-tumor actions and produced via T cells. The action induced by glycoprotein fractions extracted from Chlorella vulgaris may depend on a mechanism produced via T cells in an antigen-specific manner [24, 19].
TANAKA et al. showed that the described glycoprotein exhibited anti-tumor action against both spontaneous and experimentally induced metastases in mice. The anti-metastatic immune potentiation was observed in euthymic but not in athymic nude mice. This fact is also an indication of a T-cell mediated mechanism. It appears that glycoprotein extract induces T-cell activation in peripheral lymph nodes of mice with tumors .
Repair of radiation injury
With regard to the aforementioned activation of hematopoietic generating cells and the effects observed in cyclophosphamide-treated rats, it is logical to study the effects of CVE/CVB on organisms with radiation injury. ROTKOVSKA et al. showed that after subcutaneous, intraperitoneal and intramuscular injection of CVE, the number of hematopoietic cells in the bone marrow and spleen of mice increased, as they do after irradiation. After irradiation with a lethal dose of gamma rays 24 hours after injection of CVE, a greater number of treated mice and rats survived compared to untreated ones. On the first day after administration, CVE protects against both the short and prolonged action of irradiation . The observed resistance to irradiation is accompanied by an increasing number of splenic colony-forming units in the bone marrow and spleen and their increasing proliferative activity. The number of granulocyte-macrophage colony-forming cells in the bone marrow increases and the colony-stimulating activity of the mouse blood serum rises very rapidly after injection of the substance. The recovery of colony-forming unit pools and granulocyte-macrophage colony-forming cells in the femoral bone marrow after irradiation occurs at a higher rate in CVE-treated animals than in the control groups (VACEK et al., , see also DANTAS ).
Comparable protection against radiation injury is also possible by oral administration of CVB. Feeding CVB (400 mg / kg) once, twice or three times a day for 28 days plus a dose not later than 0.4 h after irradiation provided significant radiation protection (SARMA et al.,).
Studies on the effect of CVB doses and administration times on protection from radiation have shown optimal results when CVB (500 mg / kg) is administered 1 h before or immediately after irradiation. The LD50/30 for mice pre- and post-treated with CVB are 8.66 and 9.0 Gy, respectively, compared to the control value of 7.8 Gy (SINGH et al., ).
The described stabilization and immune protection effects open interesting possibilities for the application of CVB / CVE as prophylactic and therapeutic treatment of malignant tumors.
In a two-year study of 20 patients with malignant gliomas, MERCHANT et al  added CVB and CVE to the patients’ diet to observe possible effects on their immune system, quality of life and survival time. CVB/CVE is given in addition to the normal treatment with radiation, chemotherapy and drugs such as anti-seizure drugs and corticosteroids. They found that the patients’ immune systems, which had been reduced by radiation, chemotherapy and drugs, returned to near-normal levels after administration of CVB/CVE.
The following articles describe some of the effects of CVB/CVE. Topical application of CVB (500 mg / kg . day) during the peri-, post or peri- and post-initialization stages of 7,12-dimethylbenz[a]anthracene-induced papilloma genesis significantly modulated the tumor burden to 5.00, 4.33 and 3.94 (control 5.88), the cumulative number of papillomas to 90, 78 and 67 (control 106) and the percentage incidence of mice with papillomas to 94, 90 and 89 (control 100). CVB as a single treatment or during the different stages of initialization significantly increased the levels of sulfhydryl- and glutathione S-transferase in liver and skin tissues (SINGH et al., ). The significant increase in hepatic sulfhydryl- and glutathione S-transferase levels is also detectable in fetal and neonatal systems after 14 days of CVB treatment of pregnant and lactating mice. The modulation of hepatic chemical metabolizing enzyme levels suggests a chemopreventive potential of CVB via perinatal passage of active constituents and/or metabolites (SINGH et al., ). For the evaluation of these results; it is also necessary to take into consideration that following the application of CVB, large amounts of chlorophyll are administered. Chlorophyll has anti-genotoxic (NEGISHI et al., ;) and anti-inflammatory properties (SINGH et al., ). Due to the small particle size of chlorella, the application of chlorophyll occurs in a very active and available manner. TANAKA et al  showed that oral administration of CVB caused clear prophylactic effects in water-immersion, stress-induced and cystamine-induced ulcer models.
MARCHANT et al  reported in a preclinical study the positive effects of CVB nutritional supplementation for patients with fibromyalgia syndrome.