Structurally modified Cyclovirobuxine-D Buxus alkaloids as effective analgesic agents through Cav3.2 T-Type calcium channel inhibition.

Cyclovirobuxine-D (CVB-D) is a Buxus alkaloid and a major active constituent in the Chinese medicinal herb Buxus microphylls. Traditionally, the natural alkaloid cyclovirobuxine-D has a long history of use as a traditional Chinese medicine for cardiovascular diseases as well as to treat a wide variety of medical conditions. As we found that CVB-D inhibited T-type calcium channels, we designed and synthesized a variety of fragments and analogues and evaluated them for the first time as new Cav3.2 inhibitors. Compounds 2-7 exhibited potency against Cav 3.2 channels, and two of them were more active than their parent molecules. As a result of the in vivo experiments, both compounds 3 and 4 showed significantly reduced writhes in the acetic acid-induced writhing test. Studies of molecular modeling have identified possible mechanism(s) of Cav3.2 binding. Moreover, the relationship between structure and activity was studied in a preliminary manner. Our results indicated that compounds 3 and 4 could play an important role in the discovery and development of novel analgesics.

Structurally diverse alkaloids from Buxus sempervirens with cardioprotective activity.

Extensive phytochemical study of the methanol extract of twigs and leaves of Buxus sempervirens resulted in the identification of 17 Buxus alkaloids, including 12 new ones, namely buxusemines A-L (1-12). Their structures were delineated by detailed analysis of the HRESIMS and NMR data, as well as quantum chemical NMR calculations. Buxusemine A (1) represents the second Buxus alkaloid with a unique spiro[4.6]undecatriene moiety, buxusemines B-C (2-3) are a rarely occurring class of Buxus alkaloids featured with an additional five-membered ring through the ether or lactone linkage between C-10 and C-23, and buxusemines D-F (4-6) are another rare type of Buxus alkaloids with an epoxy motif. In the assessment of their bioactivities, buxusemine F (6) and buxanoldine (17) displayed more potent protective effects than the positive control cyclovirobuxinum D in the doxorubicin-induced cardiac injury model.

The Alkaloid-Enriched Fraction of Pachysandra terminalis (Buxaceae) Shows Prominent Activity against Trypanosoma brucei rhodesiense.

In the course of our studies on antiprotozoal natural products and following our recent discovery that certain aminosteroids and aminocycloartanoid compounds from Holarrhena africana A. DC. (Apocynaceae) and Buxus sempervirens L. (Buxaceae), respectively, are strong and selective antitrypanosomal agents, we have extended these studies to another plant, related to the latter-namely, Pachysandra terminalis Sieb. and Zucc. (Buxaceae). This species is known to contain aminosteroids similar to those of Holarrhena and structurally related to the aminocycloartanoids of Buxus. The dicholoromethane extract obtained from aerial parts of P. terminalis and, in particular, its alkaloid fraction obtained by acid-base partitioning showed prominent activity against Trypanosoma brucei rhodesiense (Tbr). Activity-guided fractionation along with extended UHPLC-(+)ESI QTOF MS analyses coupled with partial least squares (PLS) regression modelling relating the analytical profiles of various fractions with their bioactivity against Tbr highlighted eighteen constituents likely responsible for the antitrypanosomal activity. Detailed analysis of their (+)ESI mass spectral fragmentation allowed identification of four known constituents of P. terminalis as well as structural characterization of ten further amino-/amidosteroids not previously reported from this plant.

Investigation of the Variability of Alkaloids in Buxus sempervirens L. Using Multivariate Data Analysis of LC/MS Profiles.

Buxus sempervirens L. is a common ornamental plant in southern and central Europe, and has been used ethopharmacologically against a wide variety of diseases due to it containing nor-triterpene alkaloids of the nor-cycloartane type. Recently, we demonstrated the interesting antiprotozoal potential of some of these compounds. To characterize the temporal variability in the alkaloid profile of two different varieties and their leaves and twigs, 30 different extracts of B. sempervirens were evaluated by Ultra High Performance Liquid Chromatography/positive Mode-Electrospray Ionization Quadrupole Time-of-Flight-Tandem Mass Spectrometry (UHPLC/+ESI-QqTOF-MS/MS). The analytical profiles were thoroughly investigated by various methods of multivariate data analysis (MVDA). A principal component analysis (PCA) model elucidates the seasonal variation in the phytochemical composition of B. sempervirens var. arborescens and suffruticosa along with differences between the varieties. Analysis of a volcano plot illustrated the differences between the two organs, the leaf and twig. Eighteen compounds were highlighted by the models as constituents of the plant characteristic for a season, variety or organ. These compounds were dereplicated based on their chromatographic and +ESI-QqTOF-MS and -MS/MS data. In addition, mass spectral fragmentation pathways for already known alkaloids as well as new natural products could be postulated for the first time. In conclusion, the MVDA models give detailed information on the temporal variability in the alkaloid profile of two different varieties and their organs (leaf vs. twig) of B. sempervirens. Thus, the results of this study allow, e.g., the identification of characteristic compounds for the different varieties, plant organs, seasons, and the optimal harvesting time for the isolation of particular Buxus-alkaloids of interest for subsequent studies.

Target-Guided Isolation of O-tigloylcyclovirobuxeine -B from Buxus sempervirens L. by Centrifugal Partition Chromatography.

The increasing drug resistance of malaria parasites challenges the treatment of this life-threatening disease. Consequently, the development of innovative and effective antimalarial drugs is inevitable. O-tigloylcyclovirobuxeine-B, a nor-cycloartane alkaloid from Buxussempervirens L., has shown promising and selective in vitro activity in previous studies against Plasmodiumfalciparum (Pf), causative agent of Malaria tropica. For further investigations, it is indispensable to develop an advanced and efficient isolation procedure of this valuable natural product. Accordingly, we used liquid-liquid chromatography including centrifugal partition chromatography (CPC) to obtain the pure alkaloid on a semi-preparative scale. Identification and characterization of the target compound was accomplished by UHPLC/+ESI-QqTOF-MS/MS, 1H NMR and 13C NMR. In conclusion, this work provides a new and efficient method to obtain O-tigloylcyclovirobuxeine-B, a valuable natural product, as a promising antiplasmodial lead structure for the development of innovative and safe medicinal agents.

Cyclovirobuxine D inhibits cell proliferation and migration and induces apoptosis in human glioblastoma multiforme and low­grade glioma.

Gliomas are the most common neoplasm of the human central nervous system. Glioblastoma multiforme (GBM) is one of the most serious types of gliomas. Although considerable progress has been made in the development of cancer therapeutic agents, several antineoplastic drugs fail to penetrate the blood­brain barrier (BBB), resulting in a low survival rate of glioma patients. Recent studies have revealed that the traditional Chinese medicine Buxus microphylla contains the main active component Cyclovirobuxine D (CVB­D), which can cross the BBB with a novel delivery system. However, it remains unclear whether CVB­D exerts anticancer effects against GBM and low­grade glioma (LGG). The aim of the present study was to explore the feasibility of CVB­D as a new effective agent in the treatment of GBM and LGG. The ability of CVB­D to inhibit GBM and LGG cell proliferation was detected by CCK8 assay. Flow cytometry was used to detect cell cycle progression and apoptosis induction by Annexin V­FITC/PI assay. The expression levels of the apoptosis­associated proteins, namely cleaved caspase­3 and Bax/Bcl­2, were detected by western blot analysis. The mitochondrial membrane potential (ΔΨm) was detected by Rh123 dyed fluorescence micrograph. Hoechst staining was used to observe the morphological characteristics of the apoptotic cells. The scratch test was used to evaluate the migration of GBM and LGG cells. The results indicated that CVB­D reduced cell viability of T98G and Hs683 cells. Flow cytometry demonstrated that CVB­D­treated cells were arrested at the S phase of their cell cycle. The expression levels of the apoptosis­associated proteins were increased in CVB­D­treated cells. Rh123 and Hoechst staining indicated morphological changes and mitochondrial membrane potential changes of the cells undergoing apoptosis. The data confirmed that CVB­D inhibited cell proliferation by arresting the cell cycle of GBM and LLG cells and that it promoted the induction of cell apoptosis by altering the mitochondrial membrane potential. The findings of the present study indicate the potential value of CVB­D in the treatment of glioma.

Buxus alkaloid compound destabilizes mutant p53 through inhibition of the HSF1 chaperone axis.

BACKGROUND: P53 is the most frequently mutated gene in most tumour types, and the mutant p53 protein accumulates at high levels in tumours to promote tumour development and progression. Thus, targeting mutant p53 for degradation is one of the therapeutic strategies used to manage tumours that depend on mutant p53 for survival. Buxus alkaloids are traditionally used in the treatment of cardiovascular diseases. We found that triterpenoid alkaloids extracted from Buxus sinica found in the Yunnan Province exhibit anticancer activity by depleting mutant p53 levels in colon cancer cells. PURPOSE: To explore the anticancer mechanism of action of the triterpenoid alkaloid KBA01 compound by targeting mutant p53 degradation. STUDY DESIGN AND METHODS: Different mutant p53 cell lines were used to evaluate the anticancer activity of KBA01. MTT assay, colony formation assay and cell cycle analysis were performed to examine the effect of KBA01 on cancer cell proliferation. Western blotting and qPCR were used to investigate effects of depleting mutant p53, and a ubiquitination assay was used to determine mutant p53 ubiquitin levels after cells were treated with the compound. Co-IP and small interfering RNA assays were used to explore the effects of KBA01 on the interaction of Hsp90 with mutant p53. RESULTS: The triterpenoid alkaloid KBA01 can induce G2/M cell cycle arrest and the apoptosis of HT29 colon cancer cells. KBA01 decreases the stability of DNA contact mutant p53 proteins through the proteasomal pathway with minimal effects on p53 mutant protein conformation. Moreover, KBA01 enhances the interaction of mutant p53 with Hsp70, CHIP and MDM2, and knocking down CHIP and MDM2 stabilizes mutant p53 levels in KBA01-treated cells. In addition, KBA01 disrupts the HSF1-mutant p53-Hsp90 complex and releases mutant p53 to enable its MDM2- and CHIP-mediated degradation. CONCLUSION: Our study reveals that KBA01 depletes mutant p53 protein in a chaperone-assisted ubiquitin/proteasome degradation pathway in cancer cells, providing insights into potential strategies to target mutant p53 tumours.

Investigations into the therapeutic effects of aerial and stem parts of Buxus papillosa C.K. Schneid.: In vitro chemical, biological and toxicological perspectives.

In this study, different solvent extracts (methanol, dichloromethane and n-hexane) from aerial and stem parts of Buxus papillosa C.K. Schneid (Buxaceae) were investigated for a panoply of bioassays. Biological profiles were established by determining antioxidant and enzyme inhibition profiles. Toxicity was tested using MTT cell viability assay on five different human cancer cell lines i.e, MCF-7, MDA-MB-231, CaSki, DU-145 and SW-480. For chemical fingerprinting, total bioactive contents and UHPLC-MS secondary metabolites profile were determined. Generally, both aerial and stem methanol extracts had highest total bioactive contents, radical scavenging and reducing power potential. DCM and n-hexane extracts were found to be most active for total antioxidant and metal chelating activity. The UHPLC-MS analysis of methanol extracts revealed the presence of several phenolic, flavonoid, alkaloid, saponin and depsipeptide derivatives. All the extracts were significantly active against butyrylcholinesterase, whereas moderate inhibition was observed for acetylcholinesterase, α-glucosidase and urease. Similarly, a considerable level of cytotoxicity was observed against all the tested cell lines with IC50 values ranging from 26 to 225.9 µg/mL. Aerial methanol and stem n-hexane extracts were found to be most cytotoxic. Principal component analysis was also performed to find any possible correlation between biological activities and total bioactive contents. On the basis of our findings, B. papillosa may be considered as promising source of bioactive molecules.

Buxus sempervirens L. Improves Lipid Profile in Diabetic Rats.

INTRODUCTION: Buxus sempervirens L. is used in Morocco as a plant with some medicinal properties including treatment of diabetes and cardiovascular diseases. METHODS: The objective of the study was to assess the effect of the aqueous extract of Buxus sempervirens L. (Boxwood) leaves on the blood lipid parameters including total cholesterol, triglycerides, High-Density Lipoprotein Cholesterol (HDL-C), Low-Density Lipoprotein Cholesterol (LDL-C) and Very Low-Density Lipoprotein Cholesterol (VLDL-C) in normal and streptozotocininduced diabetic rats. In addition, antioxidant activity of aqueous Buxus sempervirens extract was evaluated by DPPH and FRAP assays. RESULTS: The results indicated a significant decrease in serum total cholesterol and LDL-C levels, while, no change was observed on triglycerides and VLDL-C values in diabetic rats. Lipid profile of normal animals did not reveal any modification. In addition the aqueous extract of Buxus sempervirens exerted a significant antioxidant activity. Therefore, the results suggest that the aqueous extract of Buxus sempervirens exhibits lipid lowering activity which could be due partially to its antioxidant ability. CONCLUSION: In conclusion, Buxus sempervirens leaf extract may be helpful in controlling the development of hyperlipidemia as well as atherosclerosis in diabetic animals.

Buxus sempervirens L Improves Streptozotocin-induced Diabetes Mellitus in Rats.

BACKGROUND: Buxus sempervirens L. (Boxwood) is a medicinal plant used in Moroccan traditional medicine for diabetes treatment in Morocco. However; there is no experimental support of this ethnopharmacological use. METHODS: The present investigation is aimed at studying the antidiabetic and the preliminary phytochemical of the aqueous extract of the leaves of Buxus sempervirens (AELBS). AELBS (5 mg/kg) was administered by gavage to normal and streptozotocin-induced diabetic rats and blood glucose levels were measured for 6 hours (acute study) and 15 days (chronic study). Oral glucose test tolerance (OGTT) and histopathological examination of liver and pancreas were carried out using standard methods. Furthermore, preliminary phytochemical screening and quantification of phenolic and flavonoid contents were also realized. RESULTS: AELBS reduced the blood glucose of both healthy and diabetic rats. This extract was also able to improve oral glucose tolerance in diabetic rats and it ameliorated hepatic histology, however, no enhancement was noticed in pancreatic histology. Preliminary phytochemical study revealed that AELBS possesses many chemical compounds such as alkaloids, phenolic compounds (flavonoids, tannins, and cyanidins), carbohydrates, reducing sugars and mucilage. Additionally, this extract is rich in phenolic compounds including flavonoids. CONCLUSION: According to all these findings, we conclude that AELBS at a low dose demonstrated a potent antihyperglycemic effect in streptozotocin rats and further investigations should be necessary for supporting the use of this plant in the management of diabetes by the Moroccan population.

Antispasmodic, bronchodilator, vasorelaxant and cardiosuppressant effects of Buxus papillosa.

BACKGROUND: The present research was carried out to investigate pharmacological properties of Buxus papillosa C.K. Schneid. (Buxaceae). METHODS: Buxus papillosa extracts of leaves (BpL), stem (BpS), roots (BpR) and BpL fractions: hexane (BpL-H), aqueous (BpL-A) also plant constituent, cyclomicrobuxine effect were studied in jejunum, atria, aorta and tracheal preparations from rabbit and guine-peg. RESULTS: Ca++ antagonistic effect of BpS, BpR, BpL-H, BpL-A and cyclomicrobuxine were conclusively suggested, when spontaneous contractions of rabbit jejunal preparation was relaxed along with subsequent relaxation of potassium chloride (80 mM) induced contractions. Ca++ antagonistic effect was further confirmed, when a prominent right shift like that of verapamil was observed in Ca++ concentration-response curves, drawn in a tissue pretreated with BpL (0.3-1.0 mg/mL). In rabbit tracheal tissues BpL, BpS, BpR, BpL-H and BpL-A produced a prominent relaxation in contractions induced by potassium chloride (80 mM) and carbachol (1 µm). When tested in rabbit aortic rings, BpL, BpS, BpR, BpL-H and BpL-A showed concentration-dependent (0.1-3.0 mg/mL) vasorelaxant effect against phenylephrine (1 µM) and high K+-induced contractions. In isolated guinea-pig right atria, BpL, BpS, BpR, BpL-H and BpL-A suppressed atrial force of spontaneous contractions, with BpL-A being most potent. CONCLUSIONS: Our results reveal that Buxus papillosa possesses gut, airways and cardiovascular inhibitory actions.

Buxusoside, a Flavonoid Disaccharide from Buxus sinica.

A new flavonoid diglycoside named buxusoside (1), together with its aglycone chrysosplenol-D (2), as well as 4', 5-dihydroxy-3,6,7-trimethoxyflavone (3) and 3',4',5-tiihydroxy-3,6,7-trimethoxyflavone (4), were isolated from the 70% EtOH extract of the air-dried plant of Buxus sinica. Its structure was elucidated mainly by iD and 2D NMR spectra.

[SCREENING OF WILD SPREAD AND CULTIVATED OF BUXUS SPECIES GROWING IN GEORGIA ON THE CONTENT OF ALKALOIDS AND BIOLOGICAL ACTIVITY].

Georgian flora is represented by about 4150 plant species. Many important alkaloid-containing plant species and among of them are species Buxus L. of genus in Adjara. The aims of the research were: sequential screening of the plants for the consistence of alkaloids; Study of anatomical characteristics of Buxus colchica Pojark. and revealing of specific pharmacological activity of steroidal alkaloids. The objects of research were B. colchica, B. balearika and B. sempervirens, growing in Adjara (Georgia), collected in active phase of flowering of the plants. There were revealed 370 species of alkaloid containing plants. Sum of alkaloids and crude aqueous extract have spasmolitic and antihistaminic activity. Experimental anatomical research of diagnostic characteristics of the bines showed the existence of monocyclic transient system with fiber like tracheids, dorsoventral mesophyll of the leaves; the structure for the upper part of epidermis is linear and the lower part is curved, type of stomata is paracitic.

New Bioactive Lupane Triterpene Coumaroyl Esters Isolated from Buxus cochinchinensis.

Five new lupane triterpene coumaroyl esters (1-5), together with betulin (6) and a known Buxus alkaloid, N-3-benzoyldihydrocyclomicrophylline F (7), were isolated from a CHCl3-soluble partition of a methanol extract of Buxus cochinchinensis Pierre ex Gagnep. (Buxaceae) collected in Vietnam. Isolation work was monitored using human colon cancer cells (HT-29). The structures of the new compounds (1-5) were determined on the basis of spectroscopic data interpretation. In addition to their cytotoxicity against HT-29 cells and nuclear factor-kappa B (p65) inhibitory activity in an enzyme-linked immunosorbent assay, all isolates as well as two semisynthetic compounds derived from betulin and 5, respectively, were also evaluated for their in vitro antiplasmodial activities against the drug-resistant Dd2 strain of Plasmodium falciparum and antifungal effects on the growth of the pathogenic yeast Candida albicans. The new lupane triterpene coumaroyl esters (1-5), along with a betulin derivative and the known Buxus alkaloid, were found to show significant in vitro antimalarial activities, with IC50 values ranging from 0.26 to 2.07 µM.

Quantification of steroidal alkaloids in Buxus papillosa using electrospray ionization liquid chromatography-triple quadrupole mass spectrometry.

Buxus papillosa is one of the most extensively studied species of the genus Buxus known to possess steroidal alkaloids which can be used for assessing the various pharmacological activities of this plant. This paper describes the liquid chromatography-electrospray ionization triple quadrupole mass spectrometry (LC-ESI-QQQ-MS) method for the quantification of six steroidal alkaloids as chemical markers in the extracts of leaves, roots and stems of B. papillosa. Quantitative MS/MS analysis was carried out by optimization of the most sensitive transition for each analyte. This has yielded detection and quantification limits of 0.486-8.08 ng/mL and 1.473-24.268 ng/mL, respectively for all analytes. Linearity of response was also achieved and the regression coefficient found to be >0.99 for all analyzed compounds. The newly developed MRM (Multiple Reaction Monitoring) method showed excellent sensitivity for the quantification of steroidal alkaloids within 15 min run time. This paper describes the application of LC-QQQ-MS technique for steroidal alkaloids analysis in plant samples.

Molecular characteristics of woody extracts of Buxus microphylla.

As one of famous shrubs in China, Buxus microphylla is considered as the important wattle. However, the constituents of Buxus microphylla stem extracts aren't used effectively. Therefore, the molecules of stem extracts in Buxus microphylla are analyzed to further utilize the resources. The results show that the optimal extraction time of ethanol/methanol extraction, petroleum ether/ benzene extraction, and benzene/alcohol extraction are 7h, 7h, and 5h, respectively. The HK-61, HK-63, HK-73, HK-81, HK-82 stem extracts are obtained 1, 9, 1, 27, and 1 components, respectively. The stem extracts of Buxus microphylla is rich in drug and biomedical activities. Buxus microphylla stem is fit to extract 1,5-hexadien-3-yne, squalene, and dibutyl phthalate.

Antiprotozoal activity of Buxus sempervirens and activity-guided isolation of O-tigloylcyclovirobuxeine-B as the main constituent active against Plasmodium falciparum.

Buxus sempervirens L. (European Box, Buxaceae) has been used in ethnomedicine to treat malaria. In the course of our screening of plant extracts for antiprotozoal activity, a CH2Cl2 extract from leaves of B. sempervirens showed selective in vitro activity against Plasmodium falciparum (IC50 = 2.79 vs. 20.2 µg/mL for cytotoxicity against L6 rat cells). Separation of the extract by acid/base extraction into a basic and a neutral non-polar fraction led to a much more active and even more selective fraction with alkaloids while the fraction of non-polar neutral constituents was markedly less active than the crude extract. Thus, the activity of the crude extract could clearly be attributed to alkaloid constituents. Identification of the main triterpene-alkaloids and characterization of the complex pattern of this alkaloid fraction was performed by UHPLC/+ESI-QTOF-MS analyses. ESI-MS/MS target-guided larger scale preparative separation of the alkaloid fraction was performed by 'spiral coil-countercurrent chromatography'. From the most active subfraction, the cycloartane alkaloid O-tigloylcyclovirobuxeine-B was isolated and evaluated for antiplasmodial activity which yielded an IC50 of 0.455 µg/mL (cytotoxicity against L6 rat cells: IC50 = 9.38 µg/mL). O-tigloylcyclovirobuxeine-B is thus most significantly responsible for the high potency of the crude extract.

Structure-fragmentation relationship and rapid dereplication of Buxus steroidal alkaloids by electrospray ionization-quadrupole time-of-flight mass spectrometry.

RATIONALE: Tandem mass spectrometric studies of natural products revealed the identification of key fragments which can be helpful for their rapid dereplication in plant extracts utilizing a liquid chromatography/tandem mass spectrometry (LC/MS/MS) approach, particularly for the thermally labile compounds. The knowledge of the collision-induced dissociation (CID) fragmentation pattern of the molecule is essentially required prior to the analysis by LC/MS/MS. METHODS: The fragmentation patterns of eleven Buxus steroidal alkaloids were studied by using a positive ion electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QqTOF-MS/MS) hybrid instrument. Chromatographic separation of a Buxus papillosa extract was achieved using a capillary HPLC system coupled with the mass instrument. RESULTS: ESI-QqTOF-MS (positive ion mode) showed the presence of several characteristic fragments which can be used to rapidly identify various classes of Buxus steroidal alkaloids. The presence of a cyclopropane ring in the cycloartenol skeleton and the hydroxyl group at C-10 was found to effect on the fragmentation pattern and afford characteristic peaks. This study distinguishes between different types of Buxus steroidal alkaloids based on logical fragmentation pathways. This strategy was successfully applied in LC/ESI-QqTOF-MS/MS analysis of Buxus papillosa extract to investigate and characterize Buxus steroidal alkaloids and 14 compounds were identified as steroidal alkaloids. CONCLUSIONS: The knowledge of the fragmentation pattern was used for the rapid identification of this bioactive group of biosynthetically unique steroidal alkaloids in complex plant extracts of Buxus species, especially in the absence of any reference material, by combining key fragments, exact mass measurements and relative abundances of diagnostic fragment ions.

Patterns of spatio-temporal distribution of winter chronic photoinhibition in leaves of three evergreen Mediterranean species with contrasting acclimation responses.

High irradiance and relatively low temperature, which characterize Mediterranean winters, cause chilling stress in plants. Downregulation of photosynthetic efficiency is a mechanism that allows plants to survive these conditions. This study aims to address whether this process shows a regular spatial pattern across leaf surface or not. Three species (Buxus sempervirens, Cistus albidus and Arctostaphylos uva-ursi) with contrasting responses to winter stress were studied. During 7 days, macro and micro Fv/Fm spatial patterns were monitored by the use of chlorophyll fluorescence imaging techniques. In the field, the strongest photoinhibition was found in B. sempervirens, while there was almost no chronic photoinhibition in C. albidus. In leaves of the first species, Fv/Fm decreased from base to tip while in C. albidus it was uniform over the leaf lamina. An intermediate behavior is shown by A. uva-ursi leaves. Spatial heterogeneity distribution of Fv/Fm was found inside the leaves, resulting in greater Fv/Fm values in the inner layers than in the outer ones. Neither xanthophyll-linked downregulation of Fv/Fm nor protein remobilization were the reasons for such spatial patterns since pigment composition and nitrogen content did not reveal tip-base differences. During recovery from winter, photoinhibition changes occurred in Fv/Fm, pigments and chloroplast ultrastructure. This work shows for the first time that irrespective of physiological mechanisms responsible for development of winter photoinhibition, there is an acclimation response with strong spatio-temporal variability at leaf level in some species. This observation should be taken into account when modeling or scaling up photosynthetic responses.