Phytochemical profiling and cytotoxic evaluation of Premna serratifolia L. against human liver cancer cell line.

Premna serratifolia L. (Lamiaceae) is a medicinal plant, widely distributed in the tropical and subtropical regions and commonly used in traditional medicine. The current study was focused to evaluate the cytotoxic potential of aqueous extract of root of P. serratifolia (AEPS) against human hepatoblastoma cancer cell line (Hep G2).The yield of the dried extract was 5.8% and used for further studies.Cytotoxic potential of AEPS was analyzed by MTT assay, which exhibits IC50 value 1000 µg/mL after 48 h incubation. Hoechst and AO/EtBr staining, ROS measurement, mitochondrial membrane potential, clonogenic and wound healing assays also confirmed the cytotoxic efficacy of AEPS in dose and time-dependent manner. UPLC-Q-TOF-MS/MS analysis of AEPS confirmed the presence of 12polyphenolic compounds, namely 4-hydroxy-3-methoxycinnamic acid, linarin, peonidin-3,5-O-di-beta-glucopyranoside, diosmin, trans-cinnamic acid, daidzein, saponarin, homoorietin, acacetin, sarsasapogenin, phytol and sissotrin. The cytotoxic potential of AEPS might due to presence of biologically active polyphenolic compounds.

Enhanced Cellular Internalization: A Bactericidal Mechanism More Relative to Biogenic Nanoparticles than Chemical Counterparts.

Biogenic synthesis of silver nanoparticles for enhanced antimicrobial activity has gained a lot of momentum making it an urgent need to search for a suitable biocandidate which could be utilized for efficient capping and shaping of silver nanoparticles with enhanced bactericidal activity utilizing its secondary metabolites. Current work illustrates the enhancement of antimicrobial efficacy of silver nanoparticles by reducing and modifying their surface with antimicrobial metabolites of cell free filtrate of Trichoderma viride (MTCC 5661) in comparison to citrate stabilized silver nanoparticles. Nanoparticles were characterized by visual observations, UV-visible spectroscopy, zetasizer, and transmission electron microscopy (TEM). Synthesized particles were monodispersed, spherical in shape and 10-20 nm in size. Presence of metabolites on surface of biosynthesized silver nanoparticles was observed by gas chromatography-mass spectroscopy (GC-MS), energy dispersive X-ray analysis (EDAX), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The antimicrobial activity of both silver nanoparticles was tested against Shigella sonnei, Pseudomonas aeruginosa (Gram-negative) and Staphylococcus aureus (Gram-positive) by growth inhibition curve analysis and colony formation unit assay. Further, it was noted that internalization of biosynthesized nanoparticles inside the bacterial cell was much higher as compared to citrate stabilized particles which in turn lead to higher production of reactive oxygen species. Increase in oxidative stress caused severe damage to bacterial membrane enhancing further uptake of particles and revoking other pathways for bacterial disintegration resulting in complete and rapid death of pathogens as evidenced by fluorescein diacetate/propidium iodide dual staining and TEM. Thus, study reveals that biologically synthesized silver nanoarchitecture coated with antimicrobial metabolites of T. viride was more potent than their chemical counterpart in killing of pathogenic bacteria.

Suppression in PHLPP2 induction by morin promotes Nrf2-regulated cellular defenses against oxidative injury to primary rat hepatocytes.

Recent advances indicate a possible role of phytochemicals as modulatory factors in signaling pathways. We have previously demonstrated PHLPP2-mediated suppression of Nrf2 responses during oxidant attack. The present study was designed to explore Nrf2-potentiating mechanism of morin, a flavonol, via its possible role in intervening PHLPP2-regulated Akt/GSK3ß/Fyn kinase axis. Efficacy of morin was evaluated against oxidative stress-mediated damage to primary hepatocytes by tert-butyl hydroperoxide (tBHP) and acetaminophen. The anti-cytotoxic effects of morin were found to be a consequence of fortification of Nrf2-regulated antioxidant defenses since morin failed to sustain activities of redox enzyme in Nrf2 silenced hepatocytes. Morin promoted Nrf2 stability and its nuclear retention by possibly modulating PHLPP2 activity which subdues cellular Nrf2 responses by activating Fyn kinase. Pull-down assay using morin-conjugated beads indicated the binding affinity of morin towards PHLPP2. Molecular docking also revealed the propensity of morin to occupy the active site of PHLPP2 enzyme. Thus, dietary phytochemical morin was observed to counteract oxidant-induced hepatocellular damage by promoting Nrf2-regulated transcriptional induction. The findings support the novel role of morin in potentiating Nrf2 responses by limiting PHLPP2 and hence Fyn kinase activation. Therefore, morin may be exploited in developing novel therapeutic strategy aimed at enhancing Nrf2 responses.

Morin mitigates acetaminophen-induced liver injury by potentiating Nrf2 regulated survival mechanism through molecular intervention in PHLPP2-Akt-Gsk3ß axis.

Acetaminophen (APAP) is frequently taken to relieve pain. Staggered APAP overdoses have been reported to cause acetaminophen-induced liver injury (AILI). Identification of efficacious therapeutic modalities to address complications imposed by accidental/intentional long-term APAP ingestion is needed. Morin, a plant-derived phytochemical, possesses a multitude of pharmacological properties including hepatoprotective action; however, the underlying mechanisms have been inadequately explored. Our present report demonstrates significant attenuation of APAP-mediated liver injury by morin supplementation in vivo as indicated by reduction in histological and serum markers of hepatotoxicity. Morin not only limited necroinflammation as revealed by reduced HMGB1 release, NALP3 and caspase-1 maturation, but also suppressed oxidative stress and mitochondrial dysfunction. This suggests that morin may have exerted its cytoprotective role by way of early intervention in the pathway leading to perpetuation of AILI. Morin reinforced cellular defenses by suppressing Nrf2 ubiquitination and promoting nuclear Nrf2 retention as well as ARE-Nrf2 binding affinity. The effects were observed to be a result of molecular intervention in the activity of PHLPP2, a phosphatase previously reported by us to subdue cellular Nrf2 responses via Fyn kinase activation. Morin was observed to inhibit APAP-induced increase in PHLPP2 activity ex vivo as well as its association with cellular target Akt1. As a result, morin prevented oxidative stress induced deactivation of Akt (Ser473) leading to suppression in GSK3ß and Fyn kinase activation. The study supports the inhibitory action of morin against PHLPP2-regulated Nrf2-suppression and hence indentifies Nrf2-potentiating property of morin that may be exploited in developing novel therapeutic strategy to address AILI.

Oroxylin A, a constituent of Oroxylum indicum inhibits adipogenesis and induces apoptosis in 3T3-L1 cells.

Oroxylin A (OA) is a flavonoid found in Oroxylum indicum, a medicinal plant with multiple biological activities. This study was taken up to investigate the effect of OA, on adipogenesis, lipolysis and apoptosis in 3T3 L1 cells. Pre-adipocytes were treated with 10-40 µM OA on various days of adipogenesis treatment schedule. Mature adipocytes were treated with OA for lipolysis and apoptosis studies. In maturing pre-adipocytes, 10 µM OA suppressed intracellular lipid accumulation by 42.19% which was confirmed by lipidTox imaging of cells. In addition, OA decreased the nuclear translocation of PPARγ and mRNA expression of its downstream genes (FAS and LPL) along with adiponectin secretion. In mature adipocytes, 40 µM of OA decreased cell viability by 30% of control. Annexin V/PI staining showed induction of apoptosis which was further confirmed by enhanced levels of pro-apoptotic proteins Bax, cyt c, AIF and chromatin condensation. OA enhanced TNF-α secretion, lipolysis and decreased Akt phosphorylation in mature adipocytes. Findings suggest that OA possibly exerts its anti-obesity effect by affecting adipocyte life cycle at critical points of differentiation and maturity. When we compared the potency of OA with non-methoxylated flavonoids morin, naringenin and kaempferol on adipocyte life cycle OA was far more potent. Thus, study clearly indicates a new role for oroxylin A as regulator of adipocyte life cycle. In addition, study also suggested a specific role of methoxylated group in exerting lipolysis and cytotoxic effects in mature adipocytes.

Modulation of liver function, antioxidant responses, insulin resistance and glucose transport by Oroxylum indicum stem bark in STZ induced diabetic rats.

A decoction of stem bark of Oroxylum indicum Vent. (OI) is taken (2-3 times/day) by the tribal people of Sikkim, India to treat diabetes but scientific validation of its overall potential is lacking. Present study was aimed to assess in vitro antihyperglycemic activity of standardized OI extract using inhibition of α-glucosidase, BSA glycation and enhancement of insulin sensitivity. Antidiabetic and antioxidant modulatory effects of OI extract along with the blood biomarkers of toxic response were studied in streptozotocin (STZ) induced diabetic rats. In vitro analysis showed strong antioxidant capacity of OI -and potential to inhibit BSA glycation and α-glucosidase activity which was comparable to standard counterparts. Extract also improved insulin sensitivity in mature 3T3-L1 adipocytes. In vivo effects of OI extract (oral 250 mg/kg b.wt.) on STZ induced type II diabetic rats normalized the antioxidant status (p≤0.01). Analysis of blood biomarkers of toxic response indicated its safety. Lowering of total cholesterol and HDL levels (p≤0.05) and restoration of glycated Hb (p≤0.01) were also found in OI treated diabetic rats. HOMA-IR, QUICKI analysis along with area under the curve analysis showed the capacity of OI extract to enhance the insulin sensitivity significantly (p≤0.01) which was confirmed by increased GLUT-4 translocation in skeletal muscles.

Probiotic Enterococcus lactis IITRHR1 protects against acetaminophen-induced hepatotoxicity.

OBJECTIVE: Acetaminophen (APAP), an antipyretic/analgesic drug, is reported to cause toxicity on overdose. Dietary supplements are currently being explored to decrease toxicity. In the present study, the protective effect of probiotic Enterococcus lactis IITRHR1 was evaluated at different doses (10(7), 10(8), and 10(9) colony-forming units) against APAP-induced liver damage. METHODS: Male Wistar rats were administered APAP (1 g/kg of body weight orally) for 14 d, and hepatotoxicity was assessed by marker enzymes in serum and observation of histopathologic changes. Rats were pretreated with probiotic E. lactis IITRHR1 for 7 d and modulation of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase), redox ratio, and ferric reducing antioxidant power was assessed. Oxidative damage by APAP to membrane lipids, proteins, and DNA was also observed. Involvement of Bax, Bcl2, cytochrome c (pro-/anti-apoptotic proteins), caspases, and their modulation was assessed by immunoblot analysis and reverse transcriptase polymerase chain reaction. RESULTS: The E. lactis IITRHR1 pretreatment lowered the level of biomarkers of hepatotoxicity in serum. A significant increase was observed in the level of antioxidant enzymes and redox ratio and decreased oxidative damage to membrane lipids and proteins. Probiotic E. lactis IITRHR1 also modulated key apoptotic/anti-apoptotic proteins such as cytochrome-c, Bcl2, Bax, expression of caspases, and resultant DNA damage. CONCLUSION: Probiotic strain E. lactis IITRHR1 was found to have antioxidant capacity and afforded protection against APAP-induced hepatotoxicity by modulating antioxidant status, pro-/anti-apoptotic proteins, caspases, and DNA damage.

Abrogation of nimesulide induced oxidative stress and mitochondria mediated apoptosis by Fumaria parviflora Lam. extract.

ETHNOPHARMACOLOGICAL RELEVANCE: [corrected] Fumaria parviflora Lam. is used for treating aches and pains, diarrhea, fever, influenza and other complications. The herb mixed with honey is taken to prevent vomiting as per Ayurvedic text. AIM OF THE STUDY: In vivo studies were conducted to explore the hepatoprotective potential of Fumaria parviflora Lam. Fp extract against nimesulide induced oxidative stress and regulation of critical events in mitochondria mediated apoptosis. MATERIALS AND METHODS: Group of Wistar rats were fed with nimesulide for 5 days (80 mg/kg/day, po), another group was pre-treated with Fp extract/silymarin (200mg/kg/day, po) for 5 days followed by nimesulide exposure. Liver serum biomarkers and histopathology were done to assess hepatotoxicity caused by nimesulide. Antioxidant enzymes (SOD, LPO, GPx, GR) were assessed using biochemical assays as well as gene expression by RT-PCR. GSH content and ROS generation was also evaluated using flow cytometry. Key apoptotic markers like phosphatidyl serine externalization, Bax, Bcl-2 translocation, mitochondrial membrane potential, cytochrome c release, caspases (9/3) activation and DNA damage were also observed in all the groups to confirm involvement of mitochondrial pathway. RESULTS: Pre-treatment with Fp extract for 5 days significantly reduced the impact of nimesulide induced toxicity as evident from the serum biomarkers of liver damage and histopathology. It also modulated antioxidant enzymes mRNA expression as well as activity (SOD, glutathione peroxidase, glutathione reductase) and reduced lipid peroxidation during nimesulide toxicity. Nimesulide exposure decreased GSH content (92.9%) and increased reactive oxygen species (9.29 fold) which was attenuated in Fp treated rats. Fp pre-treatment significantly altered key apoptotic events like Bcl2 and Bax translocation, inhibited mitochondrial depolarization, prevented cytochrome c release, caspase-9/caspase-3 activation and DNA damage. CONCLUSION: Our in vivo findings regarding protection accorded by Fp extract against nimesulide toxicity suggest that Fp not only reduced hepatotoxicity but attenuated critical control points of apoptotic cell death.

Involvement of mitochondria mediated pathways in hepatoprotection conferred by Fumaria parviflora Lam. extract against nimesulide induced apoptosis in vitro.

Nimesulide, a popular nonsteroidal anti-inflammatory drug, has been associated with serious hepatotoxicity. Reactive oxygen species (ROS) and mitochondrial perturbations have been implicated in drug induced hepatotoxicity, although their role in the pathway needs exploration. Study was undertaken to elucidate the effect of Fumaria parviflora Lam. (Fp) on nimesulide induced cell death in primary rat hepatocyte cultures. Fp extract treated cells showed increased viability as compared to nimesulide stressed cells as assessed by MTT assay. LDH leakage increased significantly at 500microM nimesulide, and the data suggested that apoptosis was the predominant mechanism responsible for cell death. Nimesulide induced apoptosis was further confirmed by DNA fragmentation and chromatin condensation. Nimesulide exposure increased intracellular ROS, translocation of Bax and Bcl2 followed by mitochondrial depolarization and cytochrome c (Cyt c) release along with caspase-9/-3 activity confirming involvement of mitochondria in nimesulide induced apoptosis. Events like membrane depolarization of mitochondria, expression of Bax, Bcl2, externalization of phosphatidyl serine are substantially reversed by the pre-treatment of Fp extract. Thus, the study indicates that Fp extract modulates critical events regulating pro and anti-apoptotic proteins in mitochondria dependent apoptosis induced by nimesulide.

Antihyperglycemic and antioxidant effect of Berberis aristata root extract and its role in regulating carbohydrate metabolism in diabetic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Berberis aristata DC root is used in traditional medicine for a number of ailments including metabolic disorders. AIM OF THE STUDY: The aim of the present study was to explore the antihyperglycemic and antioxidant potential of 50% aqueous ethanolic root extract of Berberis aristata (BA) in alloxan induced diabetic rats. MATERIALS AND METHODS: BA root extract (250 mg/kg) was administered to diabetic rats and standard drug glybenclamide (0.6 mg/kg) to group serving as positive control. Effect of extract on antioxidant and carbohydrate metabolism regulating enzymes of liver was studied in diabetic rats along with its safety parameters. RESULTS: The main constituents of root were identified as berberine, berbamine and palmatine through HPTLC. The extract besides being safe, lowered the blood glucose significantly without any hypoglycemic effect on their control counterparts. It increased CAT, SOD, GPx, GR activity significantly and reduced lipid peroxidation (41.6%) and protein carbonylation (30.15%). It also increased the glucokinase and glucose-6-phosphate dehydrogenase activities and decreased glucose-6-phosphatase activity in diabetic rats which play a critical role in glucose homeostasis. CONCLUSION: Thus, the extract of Berberis aristata (root) has strong potential to regulate glucose homeostasis through decreased gluconeogenesis and oxidative stress.

Bacopa monnieri modulates antioxidant responses in brain and kidney of diabetic rats.

Role of oxidative stress has been reported in various diabetic complications including neuropathy, nephropathy and cardiopathy. This study was undertaken to evaluate the protective effect of Bacopa monnieri, a medicinal plant, on tissue antioxidant defense system and lipid peroxidative status in streptozotocin-induced diabetic rats. Extract of B. monnieri was administered orally, once a day for 15 days (at doses 50, 125 and 250mg/(kgbw)) to diabetic rats. Activity of antioxidant enzymes (SOD, Catalase, and GPx), levels of GSH and lipid peroxidation were estimated in kidney, cerebrum, cerebellum and midbrain of diabetic rats and compared to reference drug, Glibenclamide. Administration of plant extract to diabetic rats showed significant reversal of disturbed antioxidant status and peroxidative damage. Significant increase in SOD, CAT, GPx activity and levels of GSH was observed in extract treated diabetic rats. The present study indicates that extract of B. monnieri modulates antioxidant activity, and enhances the defense against ROS generated damage in diabetic rats.

Molecular characterization of Desmodium species--an important ingredient of 'Dashmoola' by RAPD analysis.

Identification of medicinal plants by their molecular signature is a fast growing tool. The identification of Desmodium gangeticum (L.) DC. (Shalparni, a constituent of Ayurvedic formulation "Dashmoolarishtha") was carried out using genomic approach. Authentic samples of D. gangeticum(L.) DC., D. velutinum (Willd.) DC. and D. triflorum (L.) DC. were analyzed and compared to commercial samples of various origin. Within twenty primers used, eleven gave 223 RAPD fragments. RAPD profiles of three species showed very low similarity index (0.21-0.39), whereas market samples showed high similarity of 0.82-0.89 with authenticated D. gangeticum.

Heavy metal accumulation in medicinal plants collected from environmentally different sites.

OBJECTIVE: To estimate the heavy metal content in soil and selected medicinal plants procured from environmentally different sites of the same city. METHODS: Soil and plant samples of Abutilon indicum, Calotropis procera, Euphorbia hirta, Peristrophe bycaliculata, and Tinospora cordifolia were collected from 3 environmentally different sites of the city: heavy traffic area (HTA), industrial area (IA), and residential area (RA). Pb, Cd, Cr, and Ni were estimated in soil and plant samples by inductively coupled plasma emission spectrometry and compared. RESULTS: The level of heavy metal was higher in soil than in plant parts studied. Accumulation of heavy metals varied from plant to plant. Pb was the highest in Calotropis procera root from HTA site and the lowest in Peristrophe bycaliculata whole plant from IA site. It was also lower in residential area than in heavy traffic area. CONCLUSION: The level of heavy metal content differed in the same medicinal plant collected from environmentally different sites of the same city. Thus, it reiterates our belief that every medicinal plant sample should be tested for contaminant load before processing it further for medication.

Screening of antioxidant potential of selected barks of Indian medicinal plants by multiple in vitro assays.

OBJECTIVE: To evaluate the antioxidant potential in herbal extract barks of five therapeutically important medicinal plants native to India, i.e. Crataeva nurvala Buch.-Ham., Buchanania lanzan Spreng., Aegle marmelos Corr., Dalbergia sissoo Roxb. ex DC., and Cedrela toona Roxb. METHODS: Standardized aqueous alcoholic extracts from the selected barks having different target radicals, such as superoxide radical, nitric oxide, ABTS radical, and peroxidative decomposition of phospholipids, were prepared and screened by multiple in vitro assays. These extracts were also tested for total phenolic and tannin content and correlated with antioxidant capacity. RESULTS: Total phenolic and tannin contents were found to be the highest in C. nurvala (195 GAE mg/g and 218.3 mg/g CE). SOD mimetic activity was found to be the highest in Crataeva nurvula, although all barks showed activity more than 100 units/mg extract. Lipid peroxidation inhibitory potential was found to be the highest in Crataeva nurvala (83.4% inhibition of MDA formation/10 microg extract), and also showed a comparatively high NO quenching capacity (45.5% per 10 microg extract). The highest NO quenching potential was found in Aegle marmelos (47.3% per 10 microg extract). Cedrela toona showed the lowest LPO inhibitory potential and NO quenching capacity (50.5% and 30.5%, respectively). Buchanania lanzan, a medicinal plant extensively used for inflammatory disorders and Dalbergia sissoo also showed 72.5% and 69.1% LPO inhibitory potential/10 microg extract. Trolox equivalent antioxidant capacity ranged from 0.24 to 0.39 mmol/L TEAC/mg extract, indicating that all the barks tested had ABTS+ radical quenching capacity. CONCLUSION: Bark of Crataeva nurvula has the highest antioxidant capacity and a positive correlation between antioxidant activity and their plendic content was found.

Toxic metals and organochlorine pesticides residue in single herbal drugs used in important ayurvedic formulation - 'Dashmoola'.

Herbal formulations are getting popularity throughout the world and commercialized extensively for various medicinal properties. WHO has emphasized the need for quality assurance of herbal products, including testing of heavy metals and pesticides residues. 'Dashmoola', a popular herbal formulation, with immunomodulator and febrifugal properties, consists of ten single root drugs. In view of WHO guidelines, single herbal drugs used in 'Dashmoola', were collected from different places of India for testing heavy metals and persistent pesticides residue. Although use of roots in 'Dashmoola' is prescribed in original ayurvedic literature but now many pharmacies use stem in place of roots. Therefore, in the present study both roots and stems were selected for estimation of six heavy metals namely arsenic (As), mercury (Hg), lead (Pb), cadmium (Cd), chromium (Cr) and nickel (Ni). Apart from these, the organochlorine pesticides residue viz. different metabolites of DDT, DDE, isomers of HCH and alpha-endosulfan were checked in total 40 samples of single crude drugs. Heavy metals except Hg, were present in most of the samples. In few samples Pb and Cd concentration were beyond the WHO permissible limits. Although alpha-HCH and gamma-HCH were present in almost all the samples, but other pesticides were not detected in these samples. DDT and DDE were found only in two samples.

An evaluation of residual organochlorine pesticides in popular Indian herbal teas.

Herbal preparations are gaining popularity worldwide because of their history of use and the belief that they are free of harmful side effects. Among the most popular products are herbal teas, which are marketed extensively with emphasis on their medicinal properties. At the same time, the World Health Organization has been emphasizing the need for quality assurance of herbal products, including testing for inadvertent contamination. The authors conducted a quality-assurance evaluation of residual organochlorine pesticides in some popular brands of Indian herbal teas. Organochlorine pesticide residue build-up from agricultural or storage practices was estimated with gas-liquid chromatography. The results revealed scant presence of dichlorodiphenyltrichloroethane (DDT) or its metabolites; endosulfan--a highly toxic pesticide--was absent in all 8 brands of herbal teas studied. Hexachlorocyclohexane isomers were detected in 2 samples, but levels were below the permissible limit for pesticide residue in foods, as promulgated by the Codex Alimentarius Commission. The authors believe that all herbal preparations should be checked for toxic chemical residues to allay consumer fears of exposure to known neurotoxicant pesticides and to aid in promoting global acceptance of these products.

Cyanobacterial toxins: a growing environmental concern.

Unusual blooms of toxic cyanobacteria in water bodies have drawn attention of environmentalists world over. Major blooms of Anabaena, Microcystis and Nodularia in water storage reservoirs, rivers and lakes leading to adverse health effects have been reported from Australia, England and many other parts of the world. An overview of the morphology and taxonomy of these toxic blue-green algae; their possible sources of contamination including dietary supplements and their potential to cause hepatotoxicity and neurotoxicity is given in this review. A detailed description of different cyanotoxins, and their mode of action has also been compiled. Reports of acute and chronic exposure to these toxic algae and their health effects on unsuspecting population along with a critical evaluation of efficacy of water treatment procedures to control them is presented here.

Synergistic action of ultraviolet-B radiation and cadmium on the growth of wheat seedlings.

The increase in ground level UV-B radiation as a result of stratospheric ozone depletion may have major deleterious effects on crop photosynthesis and productivity. Plants are exposed to UV-B and other xenobiotics simultaneously in today's industrialized world. The present studies were undertaken to see the effect of dual stress of UV-B and Cd2+ exposure on the growth of wheat (Triticum aestivum L.) seedlings. The plants grown in 0.25, 0.5, 1.0, 2.5, and 5.0 ppm Cd2+-supplemented medium were exposed to UV-B for 30 min (2 J, 0.4 mW/cm(2)) per day. After 5 and 10 days of treatment the combined stress of UV-B and Cd2+ resulted in reduction of biomass yield, growth, and chlorophyll content and changes in protein, free amino acid, starch, and total reducing sugar content. These data support the assumption that UV-B may have a regulatory role besides damaging effects and that an increased UV-B environment will likely increase this regulatory influence of UV-B radiation. The results also indicate that the adverse effects of one stress may be modulated in the presence of other stresses.