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Republic of the Philippines DE LA SALLE UNIVERSITY - DASMARIÑAS Dasmariñas, Cavite College of Science BS Biology major in Human Biology

SCIENTIFIC ARTICLE

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BIOL101c General Biology Panoncillo, Dawn Mitzerl V HUB 11 Ms. Causaren

Plant Anatomy and Chemistry” Érica Weinstein Teixeira1. but not in shoot apices.A. Brazil and 4Viçosa Federal University. Meira. Shoot apices of Baccharis dracunculifolia (alecrim plant. Viçosa.“Plant Origin of Green Propolis: Bee Behavior. including anthracene derivatives. resin masses and green propolis. Differences were detected between male and female apices and. Fragments were detected in propolis and identified as alecrim vestiges by detection of alecrim structures. Institute of Biosciences. and resin and propolis were microscopically analyzed by inclusion in methacrylate. has gained popularity as a food and alternative medicine. manipulate and place the resulting mass in the corbiculae. University of São Paulo. Renata M. Probably. São Paulo. Compounds so far unreported for propolis were identified. Bees cut small fragments from alecrim apices. such as anti-microbial. anti-inflammatory and anticancer. Its constituents have been shown to exert pharmacological effects. The present work aimed (i) to observe the collecting behavior of bees.S. SP. Department of Botany. . MG. Some compounds were found in propolis and resin mass. Bee behavior was observed. Asteraceae) have been pointed out as sources of resin for green propolis. Prenylated and non-prenylated phenylpropanoids. Department of Plant Biology. MG. Brazil Abstract: Propolis. a honeybee product. (ii) to test the efficacy of histological analysis in studies of propolis botanical origin and (iii) to compare the chemistries of alecrim apices. Dejair Message and Antonio Salatino Agência Paulista de Tecnologia dos Agronegócios/SAA-SP. Viçosa. resin and propolis. Pindamonhangaba. Ethanol extracts of shoot apices. green propolis composition is more complex and unpredictable than previously assumed. Chemical composition of alecrim apices seems to vary independently of season and phenology. Department of Animal Biology. Alecrim apices are resin sources for green propolis. SP. resin and propolis were analyzed by gas chromatography/mass spectroscopy. terpenoids and compounds from other classes were identified. among apices. Viçosa Federal University. Giuseppina Negri.

from shrubs in Paula Cândido municipality (state of Minas Gerais. including the United States of America. (ii) freshly collected resin mass and (iii) propolis of nearby hives. Materials and Methods Plant Material Shoot apices of Baccharis dracuncufolia DC (alecrim) were collected in the rainy season (January). as a means to search propolis botanical origin. southeast Brazil. W42°54′) in a period of high availability of plant material. with areas altered by anthropic influence at their . containing material collected from buds or exudates of plants (resin). The present study aims to raise new data about collection of resin by bees visiting apices of alecrim plants and to verify the anatomical characteristics of alecrim vestiges in resin and propolis. It aims also to compare (i) the chemical composition of alecrim apices.Keywords: africanized Apis mellifera – anthracene derivatives – Baccharis dracunculifolia – dehydrocostus lactone – prenylated phenylpropanoids Introduction Propolis is a chemically complex resinous bee product. Elsewhere in this volume information is given about uses and properties of propolis and an account about difficulties dealing with aspects of origin and variation in chemical composition of green propolis. volatile substances and beeswax (1–3). the European Union and Japan. with the purpose of evaluating the degree of chemical congruence among the three materials. Propolis has gained popularity as an alternative medicine or food for health amelioration and disease prevention in various parts of the world. S20°49′. namely Brazilian propolis derived mainly or exclusively from alecrim plants (Baccharis dracunculifolia). Fragments of mountainous semi-deciduous seasonal forest characterize the local predominant vegetation.

From some bees. The behavior of the insects was registered from arrival at the plant and along work on the apex. Masses of propolis were also used for histological study. alcohol. Apices were collected separately from female and male individuals. The wooden box housing the hives had slits 3 cm wide on both lateral sides. to stimulate propolis production.borders. resin masses and propolis was carried out over a 7-day period. A sample pooled from five hives was used for chemical analysis. A sample of the propolis used in this investigation is maintained for reference in a freezer at Viçosa Federal University. 50% acetic acid) for histological analysis of the resin mass. The whole process of collection of shoot apices. according to an unpublished procedure (patent PI 0306421-2. Only propolis recently produced was collected for the present study. Soon after introducing the masses of resin into the corbiculae. samples of plant material collected by the bees were fixed in FAA 50 for histological analysis. resin masses were taken out of the corbiculae and introduced in Eppendorf tubes for chemical analysis. The process of resin collection and time required was also registered. Bee Behavior and Collection of Material for Histology and Chemical Analysis Observations of bee behavior were visually monitored. Plants were aluminum tagged as female and male during the previous flowering season. Voucher specimens are deposited in the Herbarium of the Viçosa Federal University (UFV). Serial slices 12 μm thick were placed on histological slides and stained . Propolis was collected for analysis from hives growing in the same site. The methacrylate inclusion was sliced with a Leica RM2155 rotatory microtome with automatic advance and glass razor. which undergo a process of early succession. recorded with a Panasonic NV/M30 video camera and photographed with a digital Mavica/MVC FD88 and Leica MZ6 stereomicroscope. Along the observations. Brazil). Resin masses and propolis were included in methacrylate. to flight back to the hive. resin masses and propolis. some bees were caught with a pincer and placed in a vial containing FAA50 (formaldehyde. Histology Slides were prepared for histological analysis from plants.

followed by an isothermal period of 30 min. Injector and detector temperature was 300°C. 10 female apices (0. The software Image Pro Plus was used in the process of collecting images. Purification and Isolation of Compounds Propolis sample (5 g). filtration. The methanol extracts of apices and resin masses were concentrated under reduced pressure and transferred to glass vials with a small volume of methanol. The residues were dissolved in ethyl ether at concentrations of 1000 p.m.0332 g dry weight) were treated with hexane for 3 h in Soxhlet. concentration of the filtrate and dissolution of the residue in 5 ml of methanol.0119 dry weight) and 18 resin masses (0.p. Gas Chromatography (GC)/Electron Ionization Mass Spectrometry (EIMS) and Identification of Compounds Part of the wax-free methanol extracts was treated with diazomethane for methylation of carboxylic acids. The mass limit was 40–500 m/z. 10 male apices (0. filming camera and video. Oven temperatures ranged from 100 to 310°C at 10°C/min. A second extraction in Soxhlet followed. for 3 h. Anatomical characteristics of fragments detected in the slides were compared with anatomical structures of alecrim histological preparations from a reference slide collection made from material of the same area. (apices). The solvent was evaporated and the residue weighed. the extract having not been used in this investigation.25 mm internal diameter.p.25 μm film thickness). helium as carrier gas with flux 1. A DBS fused silica capillary column (30 m × 0.0 for 18 min at room temperature.m. Anatomical observations were made with an Olympus AX70 photomicroscope. 0. with methanol. (resin mass and propolis) and 2000 p.5 ml/min and splitless mode were used. The final ether solutions were kept in freezer. equipped with U-Photo system. equipped with an auto injector AOC-5000 and mass selective detector.with toluidine pH 4.0138 g dry weight). The dried slides were mounted with Permount. Waxes from the propolis extract were eliminated by three consecutive steps of cooling in freezer. . Extraction. Ether solutions (1 μl) of diazomethane treated and non treated extracts were injected into a Shimadzu GCMS-QP5050A 17A ChemStation System Mass Spectrometer operating with the EI mode at 70 eV.

Some details are relevant as diagnostic of the resin plant source. which shows a fragment of a young alecrim leaf from a resin mass. Only known compounds are here reported. the bees move the resin mass to the median legs and then to the opposite corbicula (Fig. Histology of Resin Masses and Propolis Evidences the high degree of fragmentation of the material in the resin mass. leaf primordia and young leaves). 7 min. The time spent from the beginning of the collecting process to the deposit of the resin mass in the corbiculae was. Using the first pair of legs. Resiniferous ducts are conspicuous in histological sections of leaves of Baccharis. The bees manipulate the fragments. a mass of resinous material being the final product.Identification of the substances followed computer searches over library Wiley 229L. on average. Several ducts are apparent in. 1). depending on several factors. glandular and non-glandular trichomes are also visible. among which was the hive's demand for propolis. and solutions of some reference compounds were injected in order to assist in the identification. several leaf . Very rarely the material is transferred directly from the first pair of legs to the corbiculae. Results Bee Behavior Numerous honeybees were seen on alecrim plants fragmenting vegetative apices (bud. The resin masses may be transferred to the corbicula of the same side. Another leaf fragment from a resin mass with a resiniferous duct in cross section is shown in. indicating liberation of resinous substances from trichomes and ducts. The frequency of visits varied. The fragments in the mandibles have sticky aspect from the beginning of the collecting process. In.

to waxy substances.fragments in the residue of the propolis sample are seen. Viewed with polarized light. some of them common in propolis. Hence the strategy of choosing a time of intense vegetative alecrim growth. Discussion Our results support previous observations that Africanized bees in Brazil have a preference for alecrim plants as sources of resin propolis. A total of 64 substances were identified. most substances corresponding to phenolics. A higher score of coincidences is observed comparing resin masses and propolis (23 matches). Slides prepared from the propolis samples showed no fragments of plants other than alecrim.6) and B. 13 comparing propolis/apices and 12 comparing all three sources. and to compounds from other classes. Glandular and non-glandular trichomes of alecrim leaves were commonly seen. most with conspicuous ducts. because the chlorenchyma in all cases is still undifferentiated. observed that Africanized . 42 were detected in male and 33 in female apices. Park et al. 29 in the resin masses and 34 in the propolis sample. no palisade and spongy parenchyma being apparent. No exact match is observed in the distribution of compounds among the four resin sources. but other compounds (probably unknown) were also detected. which correspond to starch grains and were detected in histological preparations of green propolis evidence that the fragments correspond to young plant leaves. Among the identified substances. alecrim glandular trichomes show brilliant contents. in the resin masses and in the pooled propolis sample. female or both apices. the use of hive boxes with slits to promote propolis production and the restriction of such production to a short time was successful to attain a product from a single plant source. Refers to phenolic compounds.dracunculifolia. Prenylated and non-prenylated (compounds 1–9) cinnamic acid derivatives were often detected. There are 17 matches comparing resin masses either with male. The observed collecting behavior is similar to literature reports regarding visits of bees to apices of Populus (5. to terpenoids. Chemical Analyses List the substances identified in shoot apices of female and male alecrim plants. such as derivatives of cinnamic acids.

The ramification of alecrim plants after a process of intense resin collecting activity is very common and results from breakage of the apical dominance due to destruction of vegetative buds. either from resiniferous ducts or glandular trichomes. but no foliar fragments with resiniferous ducts in the residue of green propolis. observing bees is even more difficult. This fact can be indicative that. Oliveira and Bastos found glandular and non-glandular trichomes. The present investigation gives no support to this conclusion. not to plant parts. in such cases. Central and North America. Baccharis is a huge (more than 500 species) and cosmopolite genus distributed in Oceania. as in the present investigation. the bees destroy shoot apices of alecrim plants.1%) plants (unpublished results). the resin source corresponds to exudates. In fact. Dense populations of Baccharis species are often found in field vegetation in Brazil. Volatile substances. are probably effective at attracting bees for resin collection. In tropical regions. . In previous observations. It is known that bee antennas have high olfactory capacity and that Baccharis plants produce volatile oils. Liberation of such substances probably triggers bee attraction.9%) over male (44. The bees were seen to probe the apices with their antenna for a few seconds and then move to another plant. Integer and robust apices were often rejected for similar apices of other plants. which is in agreement with our observations. we verified a significant preference for female (55. but rarely adult leaves. Observation of bees in the field is not easy. Preferences for plants independently of sex were also noted in the present study. Identification of fragments of plant material is a powerful means for assignment of propolis origin. because of the small percentage of individuals collecting propolis. because of the greater plant diversity. Absence of plant vestiges in propolis samples is sometimes verified. A procedure for assigning propolis botanical origin is the anatomical study to detect fragments of plants for comparison with reference to histological slides of likely plant sources. South. Absence of differentiated chlorenchyma is coherent with behavioral observations of this work and that of Park et al. although bee visits may become numerous.honeybees collect alecrim leaf-buds and unexpanded leaves. concluding that bees collect resin stemming from surface trichomes but not from internal ducts.

Kumazawa et al.4.dracunculifolia. This guaianolide lactone has been shown to exert several activities. alecrim apices are probably also sources of other classes of terpenoids such as sesquiterpenes. In a study by HPLC (high performance liquid chromatography).dracunculifolia and green propolis. farnesol. Such is the case for cis-3-methoxy-4-hydroxycinnamic acid. 2-hydroxy-7. although not in the shoot apices. Anthracenes and anthraquinones are probably rare.5-diprenyl cinnamic acid). 1compound farnesol.7-trimethyl-3phenyl-2-oxabicyclo deca-3. in propolis. was found again as a relevant constituent. found identical composition in apices of alecrim plants and propolis from associated hives. but was detected in propolis. 17-pentatriacontene. Labdane diterpenes identical or similar to compounds 28–30 were reported from Brazilian propolis and B.4a. dehydrocostus lactone. In addition. the list of substances they reported is distinct from that of For example. compound 50. hydroxy-2-(1-methoxyethyl)-3-methoxyanthraquinone. 1. they detected chlorogenic acid and other caffeoylquinic acids. compound 29. . a sesquiterpene reported from an unusual sample of propolis.8-dimethyl-5hydrindacene. simple phenylpropanoids and . A prenylated phenylpropanoid recently reported as a novel compound. but never in a typical green propolis. compound 1. Artepillin C (4-hydroxy-3. chromene derivatives and allyl-3-prenylcinnamate are not listed in their paper. compound 64. Some compounds detected in the present investigation have so far not been reported in propolis. 26.12-octadecadienoid acid. trans-3-methoxy-4compound 19.9. acid. 9. reported the same composition of alecrim shoot apices and a sample of green propolis. Also Park et al. a compound from Brazilian propolis with anti-tumor activity. compound 59. isomaturnin. compound 61. not only in propolis but also in alecrim apices. triterpenes and steroids.10- hexahydrophenanthrene. allyl-3-prenylcinnamate.3. The main compounds were prenylated and non-prenylated cinnamic acids. was not found in apices and resin mass. viridiflorol. possibly so far unreported. Dehydrocostus lactone. and 4.4aß-dimethyl-7-isopropyl-2.12-dimethylanthracene. 4-ethyloctane. among several substances not detected in the present work. among them antimycobacterial and against Trypanosoma cruzi . 5-diene. Artepillin C. was detected in the propolis sample and resin mass. Other phenolics from Brazilian propolis possess not only cytotoxic but also hepatoprotective activity. hydroxycinnamic isomaturnin. However. compound 55. whereas terpenoids.7.Prenylated phenylpropanoids are characteristic of B. an important cardiac parasite in rural areas of Brazil. 18.

is an interesting substance. Coherently. On the other hand. suggest that other factors probably influence propolis composition. it is no wonder that green propolis may co-exist with other propolis types. the reverse holds. with the unusual cisgeometry of the extra-ring double bond. growing side by side (as is probably the case in this study). season and phenology (factors often assumed to affect propolis chemistry). insofar as they indicate alecrim as main source of green propolis resin. 32. 28.flavonoids were the reported compounds. 60 and 63. was not detected in apices. 9. 29. a green propolis substance with high notoriety for its cytotoxicity. undetected in female plants. In fact. growing side by side. a conviction grows that green propolis composition is more complex and unpredictable than previously assumed. 24. Compounds 60 and 62 are volatile and hence probably contribute to the plant odor. Even artepillin C. In addition to flora. As new chemical data accumulate. at the same phenologic status. Possibly the integer apices collected for chemical analyses were chemically different from the apices visited by the bees and then converted into resin masses. 38. 27. . 59 and 61 were detected in propolis and not in apices. for compounds 6. 55. claimed that herbivores make distinction among individuals of different sex and age in the same taxon. Compound 5. probably because of differences in chemical composition. for example. 26. the latter had compositions closer to the propolis sample. Compounds 2. Our results agree with the papers of Kumazawa et al. On the other hand. 11. 31. Dannel et al. With so many factors affecting its composition. 51. from which they collected material. 45. 20. 15–17. The higher score of matches in the comparison resin masses/propolis is not surprising. it was commented above that bees were noticed probing the apex of a plant and then moving to another. since material from resin is undergoing incorporation into the propolis mass. have long been known. 21 . no mention was made of prenylated compounds and terpenoids. For example. Different compositions occur between apices of female and male plants. and Park et al. It seems that apices of male alecrim plants are chemically more complex than those of female plants. 23. The differential preference of bees observed in this study suggests different chemical contents of plants of the same species. 19. Chemical differences between individuals of the same species. 50. they disagree from those papers because our results indicate differences between compositions of apices and propolis.

This is of great interest for the basic science and application of products from honeybees and the broader context of agriculture and CAM. reporting a phenomenon perhaps akin to a population crash. Seoul Korea. The jolting news concerns honeybees and a phenomenon referred to as Colony Collapse Disorder (CCD). thinks he has rediscovered an old weapon that could be useful in the fight against these nasties. such recognition strengthens the relevance of propolis chemistry and pharmacology in the process of raising inventories of active natural products. especially those derived from marine and terrestrial animals. 'Hospitals do more than house sick patients while they are treated. an outbreak of a disease that spreads rapidly through a community. It is still used in the Antipodes. One doctor.the superbugs that are endemic in many places. From the inception of eCAM there has been a conscious effort to capture or focus on the beneficial effects of natural products. Add a sprinkling of antibiotics and drug-resistant strains emerge -. It sounded like a pandemic in that it occurs over a large area of the world -not an epidemic. They also provide convenient havens for dangerous bacteria. 2007).Under such circumstance. without which interesting pharmacologically active substances would hardly be uncovered. however. On the other hand. can offer. a disorder in which disturbing numbers of bees are disappearing from their colonies. an Australian company makes a product called Medihoney . Honey was commonly used in medicine before antibiotics became widespread. June 20-25. The news caught my immediate attention since the affected population particularly concerns my initiative to advance bioprospecting and more specifically biotherapy (I lectured at the VII Biotherapy Congress. SUMMARY: Very recently some surprising and puzzling news appeared in the media: television and newspapers. Cramming infirm people into one place creates the ideal breeding ground for disease. attempts to establish patterns of Brazilian propolis according to geographic areas and chemical composition may be frustrated. honey. In the companion article the thrust is more towards what the bees' product. It is honey.

The investigation will involve 150 patients in several countries including Britain. Green propolis derives mainly from from vegetative alternative apices resin of Baccharis sources. This formulation is a certified medicine in Europe. Around a third of them were also given some antibiotics at the same time as having their wounds dressed with honey. antimicrobial and anti-inflammatory) effects. Probably few plant species contribute as major resin sources.for medicinal use. samples investigated. Propolis has been used as an antiseptic and wound healer since ancient times and interest for the product has increased recently. and who failed to respond to other drugs. was free of this superbug within 48 hours of receiving the honey treatment. However. They investigated whether Brazilian green propolis exerts neuroprotective effects in the retina in vitro and/or in vivo. Propolis has gained popularity as a food and alternative medicine. but has not been much used there because doctors developed a taste for prescribing conventional antibiotics. wide variation detected in the chemical composition suggests contributions plant Predominant components of the resin of green propolis are cinnamic acids. Propolis is a hive product containing chiefly beeswax and plant-derived substances such as resin and volatile compounds. such as sesqui. Propolis research has uncovered potentialities of substances previously isolated from plants and has detected constituents of plant origin that would hardly be known otherwise. chiefly compounds bearing prenyl groups. which left their wounds from surgery vulnerable to infection. The patients were often children whose immune systems had been weakened by chemotherapy. One patient. but not all. Its constituents have been shown to exert pharmacological (anticancer. whose wounds had become infected by the potentially fatal strain of Staphylococcus aureusthat is resistant to the antibiotic methicillin (MRSA). Dr Simon has already used honey on 150 patients who were not responding to treatment.' This excerpt has now been expanded. di and pentacyclic triterpenoids. Germany and Australia. with some promising results. Arne Simon of Bonn University Children's Clinic in Germany is now leading an international study to compare honey with existing drugs. have been detected in many.Propolis inhibited the neurotoxicity and apoptosis . Analyses also focus on various assays designed to test the efficacy especially viewing in vitro models. dracunculifolia (alecrim plants). Terpenoid compounds. Certain studies merit more attention as they focus on the nervous and immune systems.

' Those myriad aspects of honey. which the hive is called 'the oldest laboratory of the world. Giuseppina Negri.induced in cultured retinal ganglion cells. “Plant Origin of Green Propolis: Bee Behavior. Renata M. bee venom. bee bread.and that a propolis-induced inhibition of oxidative stress may be partly responsible for these neuroprotective effects. Meira. This article interests me a lot because it includes the study of the life of bees. bee wax and bee larvae are linked to treatments of all the major . REFLECTION/REACTION: The name of the article I chose is.A. Also it can cure life. Dejair. royal jelly. These findings indicate that Brazilian green propolis has neuroprotective effects against retinal damage both in vitro and in vivo. Antonio Salatino. Plant Anatomy and Chemistry” it was conducted by: Érica Weinstein Teixeira. propolis. Medihoney was created for medical use. according to one doctor honey was commonly used as an antibiotic. To give you an idea of what the world would lose with the disappearance of the bee.S.

Of course. all these treatments do not have an evidence base but imagine how fertile the hive and its products are for evidence-based research! In this time of crisis.pathologies from nutritional problems to arthritis. . I hope that scientists all over the world turn their brilliant minds to the mystery of CCD and that researchers continue to seek an evidence base for the miraculous honey bee. rheumatism and 23 other areas.