Puerarin-A Promising Flavonoid: Biosynthesis, Extraction Methods, Analytical Techniques, and Biological Effects.

Flavonoids, a variety of plant secondary metabolites, are known for their diverse biological activities. Isoflavones are a subgroup of flavonoids that have gained attention for their potential health benefits. Puerarin is one of the bioactive isoflavones found in the Kudzu root and Pueraria genus, which is widely used in alternative Chinese medicine, and has been found to be effective in treating chronic conditions like cardiovascular diseases, liver diseases, gastric diseases, respiratory diseases, diabetes, Alzheimer's disease, and cancer. Puerarin has been extensively researched and used in both scientific and clinical studies over the past few years. The purpose of this review is to provide an up-to-date exploration of puerarin biosynthesis, the most common extraction methods, analytical techniques, and biological effects, which have the potential to provide a new perspective for medical and pharmaceutical research and development.

Isoflavones isolated from the fruits of Ficus altissima and their anti-proliferative activities.

Ficus altissima, also known as lofty fig, is a monoecious plant from the Moraceae family commonly found in southern China. In this study, we isolated and identified one new isoflavone (1), three new hydroxycoumaronochromones (2a, 2b and 3a) and 12 known compounds from the fruits of F. altissima. Their chemical structures were determined using spectroscopic analysis methods. We also tested all the isolated compounds for their anti-proliferative activities against eight human tumour cell lines (A-549, AGS, K562, K562/ADR, HepG2, HeLa, SPC-A-1 and CNE2) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Our experiments showed that compound 6 exhibited obvious anti-proliferative activity against the K562 cell line with an IC50 value of 1.55 µM. Additionally, compounds 8 and 9 showed significant anti-proliferative activities against the AGS and K562 cell lines, respectively. Moreover, compound 6 induced apoptosis in K562 cells through the caspase family signalling pathway.

Antiviral Rotenoids and Isoflavones Isolated from Millettia oblata ssp. teitensis.

Three new (1-3) and six known rotenoids (5-10), along with three known isoflavones (11-13), were isolated from the leaves of Millettia oblata ssp. teitensis. A new glycosylated isoflavone (4), four known isoflavones (14-18), and one known chalcone (19) were isolated from the root wood extract of the same plant. The structures were elucidated by NMR and mass spectrometric analyses. The absolute configuration of the chiral compounds was established by a comparison of experimental ECD and VCD data with those calculated for the possible stereoisomers. This is the first report on the use of VCD to assign the absolute configuration of rotenoids. The crude leaves and root wood extracts displayed anti-RSV (human respiratory syncytial virus) activity with IC50 values of 0.7 and 3.4 µg/mL, respectively. Compounds 6, 8, 10, 11, and 14 showed anti-RSV activity with IC50 values of 0.4-10 µM, while compound 3 exhibited anti-HRV-2 (human rhinovirus 2) activity with an IC50 of 4.2 µM. Most of the compounds showed low cytotoxicity for laryngeal carcinoma (HEp-2) cells; however compounds 3, 11, and 14 exhibited low cytotoxicity also in primary lung fibroblasts. This is the first report on rotenoids showing antiviral activity against RSV and HRV viruses.

Auriculasin enhances ROS generation to regulate colorectal cancer cell apoptosis, ferroptosis, oxeiptosis, invasion and colony formation.

Colorectal cancer (CRC) is one of the most common malignant tumors in the digestive system, and Chinese herbal medicine plays an important role in tumor treatment. The in-depth study of auriculasin isolated from Flemingia philippinensis showed that auriculasin promoted reactive oxygen species (ROS) generation in a concentration-dependent manner; when ROS scavenger NAC was added, the effects of auriculasin in promoting ROS generation and inhibiting cell viability were blocked. Auriculasin induced CRC cell apoptosis, led to mitochondrial shrinkage, and increased the intracellular accumulation of Fe2+ and MDA. When auriculasin and NAC were added simultaneously, the levels of apoptosis, Fe2+ and MDA returned to the control group levels, indicating that auriculasin activated apoptosis and ferroptosis by inducing ROS generation. In addition, auriculasin promoted the expression of Keap1 and AIFM1, but significantly reduced the phosphorylation level of AIFM1, while NAC significantly blocked the regulation of Keap1 and AIFM1 by auriculasin, which indicates that auriculasin can also induce oxeiptosis through ROS. When Z-VAD-FMK, Ferrostatin-1, Keap1 siRNA, PGAM5 siRNA and AIFM1 siRNA were added respectively, the inhibitory effect of auriculasin on cell viability was significantly weakened, indicating that auriculasin inhibits cell viability by inducing apoptosis, ferroptosis and oxeiptosis. Auriculasin also inhibited the invasion and clone forming ability of CRC cells, while NAC blocked the above effects of auriculasin. Therefore, auriculasin can promote CRC cell apoptosis, ferroptosis and oxeiptosis by inducing ROS generation, thereby inhibiting cell viability, invasion and clone formation, indicating that auriculasin has a significant antitumor effect.

Antioxidant Potential and Cytotoxic Effect of Isoflavones Extract from Thai Fermented Soybean (Thua-Nao).

Thua-nao, or Thai fermented soybeans, is a traditional Lanna fermented food in Northern Thailand. It is produced by using a specific bacterial species called Bacillus subtilis var. Thua-nao. We investigated the antioxidant activity and cytotoxic effect of isoflavones from Thua-nao. The phenolic compound contents and total flavonoid contents were determined by spectrophotometry. The antioxidant activity was examined using the ABTS, FRAP, and DPPH assays. The isoflavone contents and phenolic compositions were examined by the high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) techniques. The ability of isoflavones to inhibit human cancer cell growth was assessed by the MTT assay. The total phenolic content, total flavonoid content, and antioxidant activities of the isoflavones were 49.00 ± 0.51 mg GAE/g of dry extract (DE), 10.76 ± 0.82 mg QE/g of DE, 61.03 ± 0.97 µmol Trolox/g of DE, 66.54 ± 3.97 µM FeSO4/g of DE, and 22.47 ± 1.92% of DPPH inhibition, respectively. Additionally, the isoflavone extracts from Thua-nao had high isoflavone contents and polyphenolic compound compositions, especially daidzein and genistein. The isoflavone demonstrated a weak inhibition of MCF-7 and HEK293 cancer cell growth. It has a high antioxidant component, which is beneficial and can be developed for new therapeutic uses. However, further studies on the benefits of Thua-nao should be performed for realizing better and more effective uses soon.

Antibacterial Activities of Prenylated Isoflavones from Maclura tricuspidata against Fish Pathogenic Streptococcus: Their Structure-Activity Relationships and Extraction Optimization.

Streptococcus zoonotic bacteria cause serious problems in aquaculture with clinical effects on humans. A structure-antibacterial activity relationships analysis of 22 isoflavones isolated from M. tricuspidata (leaves, ripe fruits, and unripe fruits) against S. iniae revealed that prenylation of the isoflavone skeleton was an important key for their antibacterial activities (minimum inhibitory concentrations: 1.95-500 µg/mL). Through principal component analysis, characteristic prenylated isoflavones such as 6,8-diprenlygenistein (4) were identified as pivotal compounds that largely determine each part's antibacterial activities. M. tiricuspidata ripe fruits (MTF), which showed the highest antibacterial activity among the parts tested, were optimized for high antibacterial activity and low cytotoxicity on fathead minnow cells using Box-Behnken design. Optimized extraction conditions were deduced to be 50%/80 °C/7.5 h for ethanol concentration/extraction temperature/time, and OE-MTF showed contents of 6,8-diprenlygenistein (4), 2.09% with a MIC of 40 µg/mL. These results suggest that OE-MTF and its active isoflavones have promising potential as eco-friendly antibacterial agents against streptococcosis in aquaculture.

Calycosin-7-O-ß-Glucoside Isolated from Astragalus membranaceus Promotes Osteogenesis and Mineralization in Human Mesenchymal Stem Cells.

Stem cells have received attention in various diseases, such as inflammatory, cancer, and bone diseases. Mesenchymal stem cells (MSCs) are multipotent stem cells that are critical for forming and repairing bone tissues. Herein, we isolated calycosin-7-O-ß-glucoside (Caly) from the roots of Astragalus membranaceus, which is one of the most famous medicinal herbs, and investigated the osteogenic activities of Caly in MSCs. Caly did not affect cytotoxicity against MSCs, whereas Caly enhanced cell migration during the osteogenesis of MSCs. Caly increased the expression and enzymatic activities of ALP and the formation of mineralized nodules during the osteogenesis of MSCs. The osteogenesis and bone-forming activities of Caly are mediated by bone morphogenetic protein 2 (BMP2), phospho-Smad1/5/8, Wnt3a, phospho-GSK3ß, and phospho-AKT, inducing the expression of runt-related transcription factor 2 (RUNX2). In addition, Caly-mediated osteogenesis and RUNX2 expression were attenuated by noggin and wortmannin. Moreover, the effects were validated in pre-osteoblasts committed to the osteoblast lineages from MSCs. Overall, our results provide novel evidence that Caly stimulates osteoblast lineage commitment of MSCs by triggering RUNX2 expression, suggesting Caly as a potential anabolic drug to prevent bone diseases.

Potential therapeutic interventions of plant-derived isoflavones against acute lung injury.

Acute lung injury (ALI) is a life-threatening syndrome that possibly leads to high morbidity and mortality as no therapy exists. Several natural ingredients with negligible adverse effects have recently been investigated to possibly inhibit the inflammatory pathways associated with ALI at the molecular level. Isoflavones, as phytoestrogenic compounds, are naturally occurring bioactive compounds that represent the most abundant category of plant polyphenols (Leguminosae family). A broad range of therapeutic activities of isoflavones, including antioxidants, chemopreventive, anti-inflammatory, antiallergic and antibacterial potentials, have been extensively documented in the literature. Our review exclusively focuses on the possible anti-inflammatory, antioxidant role of botanicals'-derived isoflavones against ALI and their immunomodulatory effect in experimentally induced ALI. Despite the limited scope covering their molecular mechanisms, isoflavones substantially contributed to protecting from ALI via inhibiting toll-like receptor 4 (TLR4)/Myd88/NF-κB pathway and subsequent cytokines, chemokines, and adherent proteins. Nonetheless, future research is suggested to fill the gap in elucidating the protective roles of isoflavones to alleviate ALI concerning antioxidant potentials, inhibition of the inflammatory pathways, and associated molecular mechanisms.

Fungicidal Activity and Mechanism of Action of Glabridin from Glycyrrhiza glabra L.

Glycyrrhiza glabra (Licorice) belongs to the Fabaceae family and its extracts have exhibited significant fungicidal activity against phytopathogenic fungi, which has mainly been attributed to the presence of phenolic compounds such as flavonoids, isoflavonoids and chalcones. In this study, a series of licorice flavonoids, isoflavonoids and chalcones were evaluated for their fungicidal activity against phytopathogenic fungi. Among them, glabridin exhibited significant fungicidal activity against ten kinds of phytopathogenic fungi. Notably, glabridin displayed the most active against Sclerotinia sclerotiorum with an EC50 value of 6.78 µg/mL and was 8-fold more potent than azoxystrobin (EC50, 57.39 µg/mL). Moreover, the in vivo bioassay also demonstrated that glabridin could effectively control S. sclerotiorum. The mechanism studies revealed that glabridin could induce reactive oxygen species accumulation, the loss of mitochondrial membrane potential and cell membrane destruction through effecting the expression levels of phosphatidylserine decarboxylase that exerted its fungicidal activity. These findings indicated that glabridin exhibited pronounced fungicidal activities against S. sclerotiorum and could be served as a potential fungicidal candidate.

Prenylated isoflavones from the roots of Flemingia philippinensis as potential inhibitors of ß-amyloid aggregation.

In our efforts to find potential agents for the treatment of Alzheimer's disease in naturally occurring compounds, a systematic investigation for the active constituents of Flemingia philippinensis was carried out. Four new prenylated isoflavones, philippinone A-D (1-4), together with six known analogues (5-10), were obtained from the roots of Flemingia philippinensis. Their structures were established through extensive physical and spectroscopic data analysis. All the isolates were evaluated for their inhibitory effect of self-induced Aß aggregation among which compound 5 showed significant Aß aggregation inhibitory ability with the inhibitory rate of 70.56%. The results of molecular docking experiment for compounds 1 and 6 corresponded to that of the thioflavin-T assay. Moreover, the results further clarified the effects of different substituent group of tested compounds on the Aß aggregation inhibition. A preliminary structure-activity relationship is further discussed. Our results suggested that the isoflavonoids may mitigate the progression of AD and compounds 2 and 5 may be a candidate in the treatment of AD.

Cytotoxicity of isoflavones from Millettia dura.

The first phytochemical investigation of the flowers of Millettia dura resulted in the isolation of seven isoflavones, a flavonol and a chalcone. Eleven isoflavones and a flavonol isolated from various plant parts from this plant were tested for cytotoxicity against a panel of cell lines, and six of these showed good activity with IC50 values of 6-14 µM. Durmillone was the most active with IC50 values of 6.6 µM against A549 adenocarcinomic human alveolar basal epithelial cancer cell line with low cytotoxicity against the non-cancerous cell lines BEAS-2B (IC50 = 58.4 µM), LO2 hepatocytes (IC50 78.7 µM) and CCD19Lu fibroblasts (IC50 >100 µM).

Soy isoflavones recover pancreatic islet function and prevent metabolic dysfunction in male rats.

We tested whether chronic supplementation with soy isoflavones could modulate insulin secretion levels and subsequent recovery of pancreatic islet function as well as prevent metabolic dysfunction induced by early overfeeding in adult male rats. Wistar rats raised in small litters (SL, three pups/dam) and normal litters (NL, nine pups/dam) were used as models of early overfeeding and normal feeding, respectively. At 30 to 90 days old, animals in the SL and NL groups received either soy isoflavones extract (ISO) or water (W) gavage serving as controls. At 90 days old, body weight, visceral fat deposits, glycemia, insulinemia were evaluated. Glucose-insulin homeostasis and pancreatic-islet insulinotropic response were also determined. The early life overnutrition induced by small litter displayed metabolic dysfunction, glucose, and insulin homeostasis disruption in adult rats. However, adult SL rats treated with soy isoflavones showed improvement in glucose tolerance, insulin sensitivity, insulinemia, fat tissue accretion, and body weight gain, compared with the SL-W group. Pancreatic-islet response to cholinergic, adrenergic, and glucose stimuli was improved in both isoflavone-treated groups. In addition, different isoflavone concentrations increased glucose-stimulated insulin secretion in islets of all groups with higher magnitude in both NL and SL isoflavone-treated groups. These results indicate that long-term treatment with soy isoflavones inhibits early overfeeding-induced metabolic dysfunction in adult rats and modulated the process of insulin secretion in pancreatic islets.

A new isoflavone glucoside and other compounds from Poiretia bahiana C. Mueller: Chemophenetics, fragmentation pattern and biogenetic implications.

The genus Poiretia belongs to the Fabaceae (Leguminosae) family and it encompasses twelve species of flowering plants. The chemistry of this genus is scarcely investigated, although some studies have demonstrated the potential of Poiretia species to produce important bioactive compounds. Herein, we describe the phytochemical investigation of P. bahiana C. Mueller leaves. A new isoflavone glucoside named as 2',4',5'-trimethoxyisoflavone-7-O-ß-D-glucopyranoside (1), along with six known isoflavones (2-7), two rotenones (8-9), cyclitol 3-O-methyl-chiro-inositol (10), the amino acid proline (11), a mixture of sitosterol (12) and stigmasterol (13), and a mixture of the triterpenes lupeol (14) and ß-amirine (15) were obtained from P. bahiana leaves. The structures were established by extensive analysis of their spectroscopic data, which included 1H NMR, 13C NMR, DEPT, and 2D-NMR (13C1H HETCOR and 13C1H COLOC). Two isoflavones (3 and 5) and two rotenones (8-9) exhibited antifungal activity against the plant pathogenic fungus Cladosporium sphaerospermum. Furthermore, the biogenetic implications of the oxygenation pattern of the B-ring of the isoflavones, and the chemophenetics and fragmentation pattern of the isoflavones and rotenones are discussed.

Anti-proliferative and anti-inflammatory prenylated isoflavones and coumaronochromones from the fruits of Ficus altissima.

Ficus altissima, an evergreen arbor belonging to the Moraceae family, is mainly cultivated in the tropics and subtropics of South and Southeast Asia with the characteristic of exuberant vitality and luxuriant foliage. In this article, four new prenylated isoflavones (1-4), along with ten previously described isoflavones (5-14) and two known prenylated coumaronochromones (15 and 16) were firstly obtained from the fruits of F. altissima. Their structures were identified by various spectroscopic techniques including specific optical rotation, HR-ESI-MS and NMR. The isolated products were evaluated for their anti-proliferative activities against three human tumor cell lines (HepG2, MCF-7 and MDA-MB-231) through MTT assay. Compounds 2, 3 and 16 exhibited obvious anti-proliferative activities against MDA-MB-231 cell line and compounds 3, 13 and 16 showed effective cytotoxic effects on HepG2 cell line in a concentration-dependent manner, as verified by the colony formation assay, cell and nucleus morphological assessment and apoptosis assay. Meanwhile, compounds 5 and 12 exhibited significant inhibition activities on NO production in LPS-stimulated RAW 264.7 cell line compared with positive control indometacin. The phytochemical investigation of the fruits of F. altissima in this study could provide the evidence for the discovery of lead compounds.

Isoangustone A induces autophagic cell death in colorectal cancer cells by activating AMPK signaling.

Phytochemicals, especially flavonoids, have been widely investigated for their diversified pharmacological activities including anticancer activities. Previously we identified isoangustone A from licorice-derived compounds as a potent inducer of cell death. In the present study, the exact mechanism by which isoangustone A induced cell death was further investigated, with autophagy as an indispensible part of this process. Isoangustone A treatment activated autophagic signaling and induced a complete autophagic flux in colorectal cancer cells. Knockdown of ATG5 or pre-treatment with autophagy inhibitors significantly reversed isoangustone A-induced apoptotic signaling and loss of cell viability, suggesting autophagy plays an important role in isoangustone A-induced cell death. Isoangustone A inhibited Akt/mTOR signaling, and overexpressing of a constitutively activated Akt mildly suppressed isoangustone A-induced cell death. More importantly, isoangustone A inhibited cellular ATP level and activated AMPK, and pre-treatment with AMPK inhibitor or overexpression of dominant negative AMPKα2 significantly reversed isoangustone A-induced autophagy and cell death. Further study shows isoangustone A dose-dependently inhibited mitochondrial respiration, which could be responsible for isoangustone A-induced activation of AMPK. Finally, isoangustone A at a dosage of 10 mg/kg potently activated AMPK and autophagic signaling in and inhibited the growth of SW480 human colorectal xenograft in vivo. Taken together, induction of autophagy through activation of AMPK is an important mechanism by which isoangustone A inhibits tumor growth, and isoangustone A deserves further investigation as a promising anti-cancer agent.

Utilization of Isoflavones in Soybeans for Women with Menopausal Syndrome: An Overview.

Based on their nutrient composition, soybeans and related foods have been considered to be nutritious and healthy for humans. Particularly, the biological activity and subsequent benefits of soy products may be associated with the presence of isoflavone in soybeans. As an alternative treatment for menopause-related symptoms, isoflavone has gained much popularity for postmenopausal women who have concerns related to undergoing hormone replacement therapy. However, current research has still not reached a consensus on the effects of isoflavone on humans. This overview is a summary of the current literature about the processing of soybeans and isoflavone types (daidzein, genistein, and S-equol) and supplements and their extraction and analysis as well as information about the utilization of isoflavones in soybeans. The processes of preparation (cleaning, drying, crushing and dehulling) and extraction of soybeans are implemented to produce refined soy oil, soy lecithin, free fatty acids, glycerol and soybean meal. The remaining components consist of inorganic constituents (minerals) and the minor components of biologically interesting small molecules. Regarding the preventive effects on diseases or cancers, a higher intake of isoflavones is associated with a moderately lower risk of developing coronary heart disease. It may also reduce the risks of breast and colorectal cancer as well as the incidence of breast cancer recurrence. Consumption of isoflavones or soy foods is associated with reduced risks of endometrial and bladder cancer. Regarding the therapeutic effects on menopausal syndrome or other diseases, isoflavones have been found to alleviate vasomotor syndromes even after considering placebo effects, reduce bone loss in the spine and ameliorate hypertension and in vitro glycemic control. They may also alleviate depressive symptoms during pregnancy. On the other hand, isoflavones have not shown definitive effects regarding improving cognition and urogenital symptoms. Because of lacking standardization in the study designs, such as the ingredients and doses of isoflavones and the durations and outcomes of trials, it currently remains difficult to draw overall conclusions for all aspects of isoflavones. These limitations warrant further investigations of isoflavone use for women's health.

A new anti-austerity agent, 4'-O-methylgrynullarin from Derris scandens induces PANC-1 human pancreatic cancer cell death under nutrition starvation via inhibition of Akt/mTOR pathway.

An ethanolic extract of Derris scandens flowers showed potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrient-deprived condition, with a PC50 value of 0.7 µg/mL. Phytochemical investigation of this active extract led to the isolation of four prenylated isoflavones (1-4) including a new compound named 4'-O-methylgrynullarin (1). The structure elucidation of the new compound was achieved by HRFABMS and NMR spectroscopic analysis. The isolated compounds exhibited potent anti-austerity activity against four different human pancreatic cancer cell lines under nutrient-deprived conditions. The new compound 4'-O-methylgrynullarin (1) was also found to inhibit PANC-1 cell migration and colony formation under nutrient-rich condition. Mechanistically, compound 1 inhibited key survival proteins in the Akt/mTOR signaling pathway. Therefore, 4'-O-methylgrynullarin (1) can be considered as a potential lead compound for the anticancer drug development based on the anti-austerity strategy.

Preparative Isolation and Purification of Calycosin and Formononetin from Astragali Radix using Hydrolytic Extraction Combined with High Speed Countercurrent Chromatography.

Calycosin and formononetin were efficiently extracted from Astragali Radix and purified by high-speed countercurrent chromatography. Calycosin and formononetin could be hydrolyzed from calycosin-7-glucoside and ononin, respectively. The best extraction conditions were realized by single factor and orthogonal experiments, which were 100% ethanol, 2.5 mol/L hydrochloric acid, 1:40 ratio of solid to liquid, extracted 2 h and one time. The two-phase solvent system of n-hexane-ethyl acetate-ethanol-water (3:5:3:5, v/v) was selected for the purification of calycosin, and 1.3 mg calycosin (the purity was 95.8% and the recovery was 85.9%) was obtained from 264.9-mg crude extraction. The two-phase solvent system of n-hexane-ethyl acetate-ethanol-water (4:5:4:5, v/v) was selected for the purification of formononetin, and 2.0 mg formononetin (the purity was 98.9% and the recovery was 84.4%) was obtained from 248.9-mg crude extraction. Their structures were identified by HPLC, melting points, UV, FTIR, ESI-MS, 1H NMR and 13C NMR spectrum. According to the antioxidant activity assay, the scavenging abilities of calycosin to 1,1-diphenyl-2-picrylhydrazyl and hydroxyl free radicals (·OH) were stronger. The scavenging effect of formononetin was not demonstrated.

Enhancement of developmentally regulated daidzein secretion from soybean roots in field conditions as compared with hydroponic culture.

Analyses of metabolite secretions by field-grown plants remain scarce. We analyzed daidzein secretion by field-grown soybean. Daidzein secretion was higher during early vegetative stages than reproductive stages, a trend that was also seen for hydroponically grown soybean. Daidzein secretion was up to 10 000-fold higher under field conditions than hydroponic conditions, leading to a more accurate simulation of rhizosphere daidzein content.

6,7,4[Formula: see text]-Trihydroxyflavanone Prevents Methamphetamine-Induced T Cell Deactivation by Protecting the Activated T Cells from Apoptosis.

Methamphetamine (METH) is an extremely addictive drug that has raised serious public health concerns recently. METH addiction not only results in neuronal cytotoxicity, but it also affects immune cell activity, including T lymphocytes. 6,4,7[Formula: see text]-trihydroxyflavanone (THF), isolated from Dalbergia odorifera, has been studied for its antibacterial activity, but evidence for whether THF has an anti-cytotoxic and protective effect on T cell activation exposed to METH is lacking. In this study, results showed that treatment with THF was not cytotoxic to Jurkat T cells but dose-dependently mitigated the cytotoxicity induced by exposure to METH. The Western blot results demonstrating pre-treatment with THF maintained the expression of anti-apoptotic proteins and phosphorylation of PI3K/Akt/mTOR downregulated by treatment with METH. Furthermore, we found that decreased expression of IL-2 and CD69 by METH exposure was partially restored, and viability was significantly prevented by pre-treatment with THF in activated T cells. These findings were involved in re-elevated expression of anti-apoptotic proteins as well as recovered pathways including MAPK/PI3K/Akt/mTOR in activated T cells pre-exposed to METH. Our results suggest beneficial effects of THF against the cytotoxic and immune-modulating effect of METH on T cells and therapeutic potential of THF for patients with immunodeficiency caused by METH addiction.