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- Rockrose land management : contribution of periodic harvesting to increase value and to control Cistus ladanifer L. shrublandsPublication . Frazão, David F.; Gonçalves, José Carlos; Silva, Amélia M.; Delgado, F.M.G.: Cistus ladanifer L. (Cistaceae) occupies extensive areas as a dominant species (shrublands) or is associated to other major forest typologies in the Iberian Peninsula. Cistus ladanifer shrublands are mostly present in oligotrophic lands with little valorisation and management and as they develop over the years (up to 20-years-old) they promote the ignition and perpetuation of fire. To contribute to the proper management and valorisation of such systems, a 5-year-old dense shrubland was evaluated for its labdanum resin, seeds, and biomass productivity using different non-destructive harvest periodicities (annual and biennial) and seasons (early, mid-, and late summer), in a two-year case-study. Annual harvest modality maximized labdanum resin productivity (reaching 230 ± 50 kg·ha−1 ·2 years−1 at late summer) and photosynthetic biomass productivity. In contrast, a biennial harvest yielded significant amounts of more diversified products. It maximized seeds productivity (reaching 75 ± 41 kg·ha−1 ·2 years−1 independently of the summer season) and lignified biomass. However, it also reached a labdanum resin productivity of 134 ± 20 kg·ha−1 ·2 yearrs−1 at late summer and a photosynthetic biomass productivity around two times lower than the annual harvest. In this study, we propose two modalities of periodic harvest to be considered as proper long cycle management practices of rockrose lands. It intends to minimize fire risks, break the vegetation auto-succession mechanism, and increase profit from nonproductive lands based on three direct outputs with a myriad of applications and valorisation pathways.
- Labdanum resin from Cistus ladanifer L. : evaluation of residual water vs. extraction yieldPublication . Frazão, David F.; Mesquita, Maria da Conceição; Silva, Helena; Silva, Amélia M.; Gonçalves, José Carlos; APH- Associação Portuguesa de HorticulturaCistus ladanifer L. (Cistaceae) is an endemic and abundant resource in the Iberian Peninsula and North Africa. This plant exudes an aromatic resin nowadays valued in the perfumery and fragrance industry. Traditional processes for the extraction and isolation of such resin use boiling water or alkaline water followed by acidic precipitation. However, a concern arises about the effluents resulting from these extraction processes. To overcome this concern, labdanum resin was extracted with Na2CO3 solution (25 g/L) at 60 oC and precipitated with sulphuric acid (5 M). The residual water was evaluated regarding total phenolic content, suspended solids, electric conductivity, and sulphate, sodium, magnesium, and calcium content. The effluent was characterized by a total phenolic content of 1245 ± 455 mgGAeq/L, 1338 ± 101 mg/L of suspended solids, pH of approximately 2, electric conductivity of 34.8 ± 0.7 mS/cm, 22284 ± 710 mg/L of sulphate, 9696 ± 1072 mg/L of sodium, 3.97 ± 0.24 mg/L of magnesium, 3.52 ± 0.80 mg/L of calcium, and a Sodium Adsorption Ratio of 876 ± 112. Because the values were far from the limit values set by Portugal decree-law 236/98 for residual waters discharged and irrigation waters, it was concluded that efforts should be made to optimize the extraction process. In that regard, a factorial designed experiment was done to evaluate the effect of Na2CO3 concentration (0; 2.5; and 25 g/L), extraction temperature (60 and 100 oC) and acidification extent (pH 2, neutralization, and no acidification) on the residual water quality and on the yield of labdanum resin extraction. Alkalinization and acidification are important to obtain high resin extraction yields (Andalusian vs. Zamorean process), but mostly alkalinization may be reduced to meet sulphate criteria for discharge without significantly affecting resin extraction yields. Despite that, to meet salinity criteria for irrigation waters a higher reduction in alkalinization is needed for Andalusian processes. Phenolic content, although lower for extractions done at 60 oC, was far from the limit values for discharge, regardless experimental conditions. Given the high phenolic content the residual water from labdanum extraction by both traditional processes must be treated before discharge. If separated, phenolic compounds may be valorized as a by-product.
- Cistus ladanifer L. tissue culture from leaf and stem explants.Publication . Frazão, David F.; Barroca, Celina; Silva, Amélia M.; Delgado, F.M.G.; Gonçalves, José CarlosCistus ladanifer L. exudes a phenolic and terpenoid resin with interesting bioactive and aromatic properties. Despite its high abundance in the wild, this plant can be cultivated to advantage on oligotrophic and trace-elements contaminated soils. Plant tissue culture may be used to produce specific metabolites or for clonal propagation of specific genotypes for plantation. From a biotechnological perspective this is the second study that has attempted in vitro propagation of C. ladanifer from adult plant material. Its goal was to evaluate the potential of leaf and internodal stem explants from C. ladanifer for in vitro tissue culture. Three plant growth regulators were tested: 2,4-Dichlorophenoxyacetic acid (2,4-D), 6-Benzylaminopurine (BAP), and 1-Naphthaleneacetic acid (NAA). From both explants, shoots were regenerated under the influence of BAP (38%) and two types of compact calli were induced: dark green calli were induced under the influence of BAP (above 70%) and light green calli were induced under the influence of 2,4-D with or without BAP (100%). Light green calli grew between 558 and 708% during subsequent subcultures and showed rhizogenic capacity when the amounts of BAP were lower than of 2.4-D, but they showed low potential for shoot organogenesis. Dark green calli were associated with shoot organogenesis. The suitability of the two calli lines to produce metabolites and their transposition to liquid cultures is worth further study in comparison to organ in vitro cultures.
- Labdanum resin from Cistus ladanifer L. as a source of compounds with anti-diabetic, neuroprotective and anti-proliferative activityPublication . Frazão, David F.; Gomes, Carlos Martins; Sosa Diaz, Teresa; Delgado, F.M.G.; Gonçalves, José Carlos; Silva, Amélia M.Labdanum resin or “gum” can be obtained from Cistus ladanifer L. by two different extraction methods: the Zamorean and the Andalusian processes. Although its main use is in the fragrance and perfumery sectors, ethnobotanical reports describe its use for medicinal purposes in managing hyperglycemia and mental illnesses. However, data concerning the bioactivities and pharmacological applications are scarce. In this work, it was found that the yield of labdanum resin extracted by the Andalusian process was 25-fold higher than the Zamorean one. Both resins were purified as absolutes, and the Andalusian absolute was purified into diterpenoid and flavonoid fractions. GC-EI-MS analysis confirmed the presence of phenylpropanoids, labdane-type diterpenoids, and methylated flavonoids, which are already described in the literature, but revealed other compounds, and showed that the different extracts presented distinct chemical profile. The potential antidiabetic activity, by inhibition of α-amylase and α-glucosidase, and the potential neuroprotective activity, by inhibition of acetylcholinesterase, were investigated. Diterpenoid fraction produced the higher α-amylase inhibitory effect (~30% and ~40% at 0.5 and 1 mg/mL, respectively). Zamorean absolute showed the highest α-glucosidase inhibitory effect (~14% and ~24%, at 0.5 and 1 mg/mL, respectively). Andalusian absolute showed the highest acetylcholinesterase inhibitory effect (~70% and ~75%, at 0.5 and 1 mg/mL, respectively). Using Caco-2 and HepG2 cell lines, Andalusian absolute and its purified fractions showed moderate cytotoxic/anti-proliferative activity at 24 h exposure (IC50 = 45–70 µg/mL, for Caco-2; IC50 = 60–80 µg/mL, for HepG2), whereas Zamorean absolute did not produce cytotoxicity (IC50 ≥ 200.00 µg/mL). Here we show, for the first time, that labdanum resin obtained by the Andalusian process, and its fractions, are composed of phytochemicals with anti-diabetic, neuroprotective and anti-proliferative potential, which are worth investigating for the pharmaceutical industry. However, toxic side-effects must also be addressed when using these products by ingestion, as done traditionally.