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Assessment of the Effects of Crude Metabolic Extracts (Leaf and Twig) of Loranthus Micranthus on Streptozotocin Induced Diabetic Rats

The antidiabetic effects of crude methanolic extracts of the leaf and twig of Loranthus micranthus was evaluated in Wister rats. To assess this, data were obtained for the determinant parameters of diabetic complications. Streptozotocin was administered for induction of diabetes; diabetic state was confirmed by persistent hyperglycemia (FBG ≥ 300mg/dl) at 72 hours post induction. Invitro inhibitory activity on αamylase and α-glucosidase was assayed. Serum insulin, TNF-α, Total cholesterol, HDL, LDL, TG, atherogenic index, liver glycogen and glycated haemoglobin were evaluated. Histology of the pancreas was assessed. Graphical Abstract Phytochemical analysis revealed the presence of unique compounds in both extracts. In-vitro assay showed inhibitory effects of both extracts on α-amylase and α-glucosidase activity. Hyperglycemia was controlled in both extract-treated groups comparable to glibenclamide. Weight loss after diabetic induction was ameliorated in extracts treated groups; serum insulin level of the extracts-treated and glibenclamide treatedgroup were higher than the diabetic control group. Serum TNF-α level of extracts-treated and glibenclamide-treated groups were significantly lower than the diabetic control group. Glycated haemoglobin levels of diabetic control group were higher than the extracts treated and glibenclamide group. Dyslipidemia observed in the diabetic control group were ameliorated in all extract-treated groups; atherogenic index of diabetic control group was higher than extracts-and-glibenclamide treated groups. Histopathological assessment showed that the leaf and twig extracts of Loranthus micranthus may possess β cell regenerating activity. Findings from this study suggest that the leaf and twig extracts of Loranthus micranthus ameliorate symptoms and complications of streptozotocin-induced diabetes in rats.

Introduction
Diabetes mellitus (DM) is a group of metabolic diseases characterized byhigh blood sugarlevels over a prolonged period (persistent hyperglycemia), due to defects in secretion of insulin or biologic action of insulin [1,2]. Prolonged diabetes causes many complications, which can be acute or chronic complications. These has been linked to severe dysfunction and damage of various body organs: the eyes, kidneys, nerves, heart, blood vessels; and results annually in more than 5% deaths worldwide [3,1]. Persistent hyperglycemia has been reported to lead to the generation of free radicals through the formation of Schiff base and amadori products, which are implicated in the end organ damage caused by diabetes mellitus [4].
In 2017, it was estimated that 451 million (age 18-99 years) are living with diabetes worldwide [5]. These figures were expected to increase to 693 million by 2045 [5]. Approximately 5 million deaths worldwide were attributable to diabetes in 2017 within 20-99 years age range [6,7]. The global healthcare expenditure on people with diabetes was estimated to be USD 850 billion in 2017 [8]. These new estimates of diabetes prevalence, deaths and healthcare expenditure present a large social, financial and health system burden across the world [5].
Diabetic patients with raised plasma glucose levels have proportionally more glycation occurring both intracellularly and extracellularly thereby necessitating the need to treat these complications as an integral part of the clinical stratification of diabetic patients [9,10]. Previous studies suggested that inhibition of the production of advanced glycated end products (AGEs) or blockade of their downstream signaling pathway to be promising targets for treatment of patients with diabetic complications [10,11,12,13].
One important complication of diabetes is the elevation of atherogenic risk, as diabetic dyslipidemia is a prominent contributor for the development of cardiovascular diseases [14]. Components of the immune system are altered in obesity and accompanying T2D, with striking changes occurring in the adipose tissue, liver, pancreatic islets, the vasculature and circulating leukocytes. These immunological changes include altered levels of specific cytokines and chemokines, changes in the number and activation state of various leukocyte populations, increased apoptosis and tissue fibrosis. Together, these changes suggest that inflammation participates in the pathogenesis of T2D [15,16].
Conventional antidiabetic drugs are both costly and associated with many adverse effects [17]. Thus, the search for safer and affordable antidiabetic drugs has led to studies investigating several plants for such activity. Among plants with potent antidiabetic principles is the hemi parasite, Loranthus micranthus (L. micranthus) which is one of the species of African mistletoe found in Nigeria [18]. Mistletoe species show varying phytochemical components and bioactivity which is dependent on the host plant. The influence of the host tree on the antidiabetic activity has been reported by a group of researchers [18,19]. Studies have also shown seasonal variations in the antidiabetic activity of this semi parasite [20,21].African mistletoe parasitic on different host trees has been used for various medicinal purposes and ethnomedically as antidiabetic, antihypertensive and antimicrobial agent [22].
The aim of the present study is to evaluate the effect of crude methanolic extracts of leaf and twig of Loranthus micranthus separately, for their antidiabetic activities and their potentials in ameliorating some parameters of diabetic complications.

Plant Materials
Fresh leaves and twigs of African mistletoe, Loranthus micranthus, were harvested during the dry season at Egbo Area in Ilaro, Ogun State, Nigeria. Plant identification was done at the Department of Plant Biology, University of Ilorin, Ilorin, Nigeria. A specimen was deposited at the herbarium and voucher number UILH/001/1260 was allocated to the sample. Plants materials were dried under the shade, at room temperature for 10days. The dried plants were separated into leaves and twigs and separately pulverized using mortar and pestle and weighed. The resulting powder was macerated in 95% methanol for 72hours.

Preparation of Extracts
Cold maceration was used for the extraction, in which 1kg each of ground dried leaves and twigs were soaked in 3.5liters of 95% aqueous methanol. The set up was intermittently shaken vigorously for 72hours. The (residue) was removed by clarification using muslin which was discarded, and later filtered through Whatman No1 filter paper. The filtrates were then dried in-vacuo using a rotary evaporator at 40 0 C. The resultant semisolid was then concentrated on a water bath shaker at 40 0 C for 60hrs.

Gas Chromatography (GC) Analysis
Gas Chromatography (GC) analyses were performed on an Orion micromat 412 double focusing gas chromatography system fitted with two capillary columns coated with Cp-Sil 5 and Cp-Sil 9 (fused silica, 25µm × 0.25mm, 0.15µm film thickness) and flame ionization detector (FID). The volume of material injected was 0.2mL, and the split ratio was 1:30. Oven temperature was programmed from 50-230 O C at 3 O C/min using helium as a carrier gas. Injection and detector temperature were maintained at 200 O C and 250 O C, respectively. Qualitative data were obtained by electronic integration of FID area percent without the use of correction factors.

Mass Spectrometry (MS) Analysis
In MS analysis, a Hewlett -Packed HP 5890A GC, interfaced with a VG analytical 70-250s double focusing mass spectrometer were used. Helium was used as the carrier gas at 1.2ml/min. The MS operating conditions were: ionization voltage 70ev, ion source 230 O C. The GC was fitted with a 25m × 0.25mm, fused silica capillary column coated with Cp -Sil 5. The film thickness was 0.15µm; the GC operating conditions were identical with those of GC analyses. The MS data were acquired and processed by on-line desktop with a computer equipped with disk memory. The percentage compositions of the compounds were computed in each case of retention indices (determined relative to the retention times of series of n-alkanes) and mass Spectra with those of authentic samples and with data from literature [23,24].

Acute toxicity study
Twenty-four female Wistar rats divided into four groups receiving L. micranthus twig and leaf extracts at doses of 100, 250, 500 and 1000mg/kg daily for a period of 2 weeks. Each group consisted of three rats for each dose employed. The animals were observed during the first hour continuously and then every hour for six hours, thereafter at 12 hours and 24 hours. Animals were then observed every 24 hours for 2 weeks for any physical signs of toxicity such as writhing, gasping, palpitation and decreased respiratory rate or mortality.

Experimental Animals
Fifty-five (55) female rats, about 8 weeks old, weighing between 100-120g were used for the study. Animals were housed in Animal House Facility in the Central Research Laboratory, University of Ilorin, Ilorin, Nigeria, for 7 days under standard conditions of appropriate ventilation, temperature of 22-25 0 C, relative humidity of 50% and 12hours day and night cycle. Animals were fed with standard rat chow and water was supplied ad libitum throughout the acclimatization period. Stressful conditions were avoided all through the experimental period.

Induction of Diabetes
Diabetes was induced in 40 rats by an intraperitoneal injection of a fresh solution of a single dose of streptozocin (STZ) Sigma Aldrich Chemicals, USA. The solution was prepared by dissolving STZ in 0.1M sodium citrate buffer, pH 4.5. Animals were administered with a dose of 65mg/kg body weight after been fasted overnight, according to the method described by Lenzen, 2008 [25]. Immediately after diabetic induction, animals were given 50% glucose solution in their drinking water ad libitum. Animals with fasting blood Glucose level (FBG) between 340 -600mg/dl after 72hrs were considered diabetic and selected for the diabetic groups. For the purpose of treatment, the rats were randomly divided into the following experimental groups:

Body Weight Evaluation
Weights of the experimental animals were obtained at the beginning of the study-day 0, day 7, day 10 and day 14 of the experiment. Weights obtained were analyzed statistically.

Plasma Glucose Level
Plasma glucose level was determined by Accucheck plus GCT Glucometer (Roche, USA). The monitor of the glucometer device uses a reflectance photometer for measuring the intensity of the color formed at the end of the reaction. This measurement uses software which translated the intensity to the corresponding glucose values. The fasting blood glucose (FBG) of all animals was measured at day zero before diabetic induction to obtain baseline glucose level for all the rats; and thereafter, following the induction of diabetes and throughout the treatment period. Plasma glucose was determined 72hours after STZ injection for confirmation of diabetes. In addition, blood glucose was measured weekly for a period of 2 weeks using the glucometer method discussed earlier, following which the animals were sacrificed.

Oral Glucose Tolerance Test
Blood Glucose level of 16 hours fasted rats was determined, and the value used as zero time of the test. At the end of the experiment, i.e. after the last dose of the vehicle, or glibenclamide or extracts, glucose solution (50%) at a dose of 2g/kg was given by oral intubation, according to the methods described by Ayala et al., (2010) [26]. Blood samples were taken at 30, 60, 90 and 120 minutes after glucose loading and blood glucose level determined with one touch glucometer (Accucheck Plus GCT, Glucometer). Plots of the values of blood glucose level (mg/dL) versus the time intervals (min) were constructed and the area under the curve (AUC) calculated using the Trapezoidal method. The AUCs foreach group was compared and tested for significance against the diabeticuntreated group and this represented the tissue glucose utilization.

α -Amylase Inhibitory Activity Assay
The effect of crude methanolic extracts of L. micranthus (leaf and twig) on α -amylase activity was determined by the method described by Ali et al., 2006 [27]. Briefly, 50µl of αamylase (5U/ml) was pre-incubated for 20 min with 50µl aliquots of extracts. The reaction was started by the addition of 50µl starch (0.5%) dissolved in 20mM phosphate buffer at pH 6.9. The reaction mixture was incubated for a further 20 min at 37 0 C and the catalytic reaction terminated by addition of 2.0 ml of DNS reagent (1% 3, 5-dinitrosalicicyclic acid (DNS) and 12% sodium potassium tartrate in 0.4 M NaOH). The reaction mixture was heated for 15 min at 100 o C. α-amylase activity was determined by spectrophotometric measurement of the absorbance at 540 nm. Percentage α-amylase inhibition was calculated according to the following formula; (A0 -A1) / A0 x 100 where, A0 was the absorbance of the control (blank, without extract) and A1 was the absorbance in the presence of the extract.

α -Glucosidase Inhibitory Activity
The inhibitory effect of L. micranthus extracts on α-glucosidase activity was determined according to the chromogenic method described by Kim et al. (2005) [28]. Briefly, α-glucosidase (5 units) was pre-incubated with 20 µg/ml of the different extracts for 15 min. Paranitrophenylglucopyranoside (PNPG) (3mM) dissolved in 20 mM phosphate buffer, pH 6.9, was added to start the reaction. The reaction mixture was further incubated at 37 o C for 20 min and stopped by addition of 2 ml of 0.1 M Na2CO3. Using a spectrophotometer, the α-glucosidase activity was determined by measuring the yellow colored p-nitrophenol released from PNPG at 400 nm. Percentage α-glucosidase inhibition was calculated according to the following; (A0 -A1) / A0 x 100 where, A0 was the absorbance of the control (blank, without extract) and A1 was the absorbance in the present extract.

Sample Collection and Handling
At the end of the 14-day treatment blood samples were drawn 3 hours after the last dose from the orbital sinus of rats, under light ether anesthesia into two different tubes (heparin and plain bottles) for each animal respectively. The samples were immediately centrifuged at 0 o C/1000g for 15min for separation of serum. The resulting serum samples was separated and kept at -20 0 C until used for required analysis.

Estimation of Plasma Insulin Level
Insulin concentration was determined by radioimmunoassay procedure using a Rat INS (Insulin) ELISA Kit (Elabscience, E-EL-R2466, 96T). This assay has a high specificity as it recognizes natural and recombinant Rat INS without significant cross-reactivity or interference between Rat INS and analogues. The ELISA kit uses Sandwich-ELISA as the method. Sample and reagent preparation was done according to the manufacturer's manual using serum samples. Spectrophotometric measurements were done at a wavelength of 450 nm ± 2 nm. Concentration of insulin in the samples was calculated by comparing the OD of the samples to the standard the curve.

Estimation of Tissue Glycogen
At the end of the experiments, livers collected were weighed and divided into 2 sample tubes, for liver glycogen assay and histopathological evaluation. Liver samples for glycogen assay were immediately frozen in phosphate buffered saline prior to the assay. Glycogen content of liver was assayed using glycogen assay kit (Sigma-Aldrich, 3050 Spruce Street, ST. Louis, MO 63103 USA). The coupled enzyme assay produces a colorimetric (570 nm) product, proportional to the glycogen concentration present in the samples.

Estimation of Plasma Lipid Profile
Blood lipid (TC, HDL and TG) levels were determined using spectrophotometric assay techniques. Assays were conducted according to the manufacturer's manual. Serums of all the animals in different groups were analyzed.

Total Cholesterol
Total cholesterol in serum was assayed using Elabscience Total Cholesterol Assay Kit. The assay is based on the enzyme driven reaction that quantifies both cholesterol esters and free cholesterol. Cholesterol esters were hydrolyzed via cholesterol esterase to cholesterol, which was subsequently oxidized by cholesterol oxidase to the ketone cholest-4-en-3-one and hydrogen peroxide. Hydrogen peroxide was detected with a high specificity colorimetric probe. Horseradish peroxidase (HRP) catalyzes the reaction between the probe and hydrogen peroxide, which was bound in a 1:1 ratio. Samples were compared to a known concentration of cholesterol standard in a 96-well microtiter plate. Samples and standards were incubated for 45 mins and then read on a standard 96-well colorimetric plate reader, at absorbance of 540 nm. The concentration of cholesterol in each sample was calculated by comparing the sample absorbance values to the cholesterol standard curve.

Serum Triglycerides Concentration
Triglycerides level in serum was assayed using GPO-PAP method (Elabscience TG Assay Kit).All reagents were ready to use and were mixed according to the manufacturer's manual. The mixture was measured after incubating at 37 0 C for 5 min. Absorbance of sample against reagent blank was read at 550nm within 60 min. Triglycerides concentration in the sample was calculated as follows: A samplex concentration of Standard = Triglycerides concentration A Stand (where Asample and Astandard: absorbance of sample and standard respectively)

High Density Lipoprotein Cholesterol (HDL-C) Assay
High Density Lipoprotein Cholesterol (HDL-C) in the serum of each sample was assayed according to the manufacturer's manual using direct method, with Elabscience High Density Lipoprotein Cholesterol (HDL-C) assay kit. 10µl of serum was mixed with reagents R1 and R2. The solutions were mixed thoroughly and incubated at 37 0 C for 5 minutes.
The absorbance was read at 546nm wavelength. HDL Cholesterol concentration in the samples was calculated according to the calculation formula in the manual.

Glycated Hemoglobin (HbA1c)
Glycated hemoglobin was assayed from the whole blood usingHbA1c ELISA kit (Elabscience) which employed the competitive enzyme immunoassay technique, utilizing a monoclonal anti-HbA1c antibody and an HbA1c-HRP conjugate. The intensity of color formed was measured spectrophotometrically at 450nm on a microplate reader. The intensity of the color was inversely proportional to the HbA1c concentration. A standard curve was plotted relating the intensity of the color (O.D.) to the concentration of standards. The HbA1c concentration in each sample was determined from this standard curve.
TNF-α was assayed using rat TNF-α ELISA Kit (Elabscience, E-EL-R0019).Standards and samples were prepared according to the manufacturer's specification. The prepared solutions were added to the appropriate micro ELISA plate wells and combined with specific antibody for TNF-α and Avidin-Horseradish Peroxidase (HRP) conjugate and were incubated. The free unbound components were washed away. Only the wells that contained TNF-α, biotinylated detection antibody and Avidin-HRP conjugate appeared blue. The enzyme-substrate reaction was terminated by addition of H2S04 which turned the blue colour yellow. The optical density (OD) was measured spectrophotometrically at a wavelength of 450nm. The OD values obtained were proportional to the concentration of TNF-α in each microplate well. The concentration of TNF-α in the samples was calculated by comparing the OD of samples was determined from the standard curve.

Histopathology
Pancreas and pancreatic tissues within the duodenal loop of each rat in all groups were taken out immediately after animalswere sacrificed, at the termination of the experiments. These were fixed in 10% phosphate buffered formaldehyde solution for at least 48 hours. The tissues were later cut into 0.5x1cm in diameter each using scalpel blade and processed using an automatic tissue processor. Processed tissues were embedded using MP3 tissue embedding center. A rotary microtome machine was used to section at 5 μm. Hematoxylin and eosin (H&E) stain was used for staining sections which were mounted using dextrin plasticizer xylene (DPX). For histopathological examination, low and high powered field of Carl Zeiss® binocular microscope was used.

Statistical Analysis
Data obtained in the present study were subjected to statistical analysis using GraphPad Prism application version 6. The means, standard errors of means and standard deviations were obtained as summary statistics. The obtained summary statistics were subjected to one-way analysis of variance (ANOVA) using the Tukey's post-hoc test to check for significant relationship between experimental groups. The data were pictorially presented as line graphs and bar charts with bars and error bars representing the means and standard errors of means respectively. Statistical significance was set at p value less than 0.05.

Phytochemical Analysis
Qualitatively, 48 and 40 compounds were found to be prominent in leaf and twig extract of L. micranthus respectively. Thirteen of the compounds were commonly found in both leaf and twig of L. * is p value < 0.05 relative to controlgroup. Data is presented as mean (bars) ± standard error of mean (error bars).

In Vitro Assay
The inhibitory activity of the leaf and twig extract of L. micranthus on α-amylase and α-glucosidase enzyme activity were evaluated in vitro at different extractconcentrations. The result obtained was analyzed and are presented in the line graphs below.
CTR= control, LML= L. micranthusleaf extract, LMT= L. micranthus twig extract, STZ= streptozotocin, GL= glibenclamide. Data is presented as mean (bars) ± standard error of mean (error bars). * is significant level of p< 0.05 relative to the STZ group.  Table 2: * and# are significant levels of difference at p<0.05 relative to control and STZ only respectively. STZ-only group showed significantly higher (p<0.05) TC compared to other groups. TG level is significantly higher (p<0.05) in STZ-only group compared to GL, LML and LMT treated groups. LML treated groups showed significantly higher HDL compared to other groups other treatment groups. LDL is significantly higher (p< 0.05) in STZ only group 3.3 ORGAN WEIGHT compared to all treatment groups. STZ-only group showed significantly higher (P<0.05) AI compared to all treatment groups. STZ+LMT500 treated group showed significantly lower (p<0.05) AI than all other groups.

FIGURE 4:
Graph A, B, C and D showing the weight of liver, kidney, heart and pancreas of experimental animals respectively. There was no significant difference in weight of liver, kidney and heart across the experimental groups. However, weight of pancreas in STZ-only, and STZ+GL groups showed significant reduction (p<0.05) compared to other groups. CTR= control, LML= L. micranthus leaf extract, LMT= L. micranthus twig extract, STZ= streptozotocin, GL= glibenclamide. * and # are p value >0.05 relative to control. Data is presented as mean (bars) ± standard error of mean (error bars).

Histopathological Observations
Following tissue microscopic examination, pancreatic tissues from all animals were evaluated and it was observed that the control group pancreas showed normal architecture, with intact acinar cell cytoplasmic and nuclear structures. In rats administered 65mg/kg body weight STZ to induce a diabetic state, it was observed that there was severe diffuse necrosis of both exocrine and endocrine pancreatic tissues. Although, the diabetic group treated with glibenclamide in a bid to reverse the destructive effect of Streptozocin treatment showed some multifocal pancreatic necrosis, overall, the animals in this group showed severe acinar (exocrine) nuclei hyperchromasia, possibly an indication of increased acinar cells' nuclei activity. The pancreas of STZ induced diabetic rats treated with L. micranthus leaf extract showed mostly normal acinar cells ( figure 1D). The pancreas of STZ induced diabetic rats treated with L. micranthus twig extract showed bridging pancreatic necrosis admixed normal acinar regions (figure 1E), implying normal pancreatic tissue admixed necrotic tissue.

DISCUSSION
Diabetes mellitus (DM) is a serious chronic disease, with heavy cost implication on human population globally [8]. This has heightened the search for alternative therapies with potentially low toxicity, and hence better safety profile, for the treatment of the disease, to improve the quality of life of diabetic patients, and/ or reverse diabetic complications.
Gas chromatographymass spectrophotometric (GC-MS) analysis showed the presence of different compounds in the twig and leaf extract at varying concentrations. This suggests that the presence of these compounds could be contributory to their unique antidiabetic activity. Leaf extract showed the presence of higher concentration of palmitic methyl ester which has been reported to possess several antidiabetic properties including activation of peroxisome proliferator-activated receptor (PPAR) family [31] and inhibition of α-amylase and αglucosidase [32]. Other components such as Thymol, linmonene, camphor, and benzoic acid derivatives, have shown good antioxidant and antidiabetic potentials [33,34,35].Thirteen compounds were found to be common in both leaf and twig extract of L. micranthus.
Acute toxicity study of both the leaf and the twig extract showed that both were safe at the maximum concentration (2000mg/kg body weight) employed. This finding supports previous safety study on L. micranthus [36]. This showed that the methanolic extracts of the both leaf and twig of L. micranthus are safe at the doses administered to the animals.
One of the mechanisms of action of antidiabetic agents include: causing reduction in glucose absorption from the gastrointestinal tract (GIT) [37]. This was assessed by evaluating the in vitro inhibitory effect of LML and LMT on starch degrading enzymes (α-amylase and αglucosidase). Clinically used antidiabetic drugs, such as Acarbose and miglitol inhibit both enzymes and reduce the rate of digestion of complex carbohydrates but this is accompanied by gastrointestinal discomforts [38]. Management of blood glucose level is a critical strategy in the control of diabetes and its complications, this makes leaf and twig extracts of L. micranthus potential candidates for controlling glucose release at GIT level. Both extracts could prevent postprandial spikes that have been implicated in cardiovascular and macro-vascular complications of diabetes [39].The twig extract showed better inhibitory effect on α-amylase and αglucosidase compared to the leaf extract.
STZ-induced hyperglycemia was established in all diabetic groups in order to evaluate the effect of LMT and LML on treatment groups. The significant weight loss (p<0.05) in all STZ-induced diabetic groups could be strongly linked to STZ-induced destruction of beta cells of the islets of langerhans of the pancreas; the attending abolition of insulin production and secretion, and resultant decreased glucose uptake and storage [40,41]. The destruction of the pancreas resulted in utilization of noncarbohydrates nutrients such as proteins and fatty acid for the synthesis of glucose. The loss of structural proteins as a result of increased gluconeogenesis together with increased glycolysis and increased synthesis of ketone bodies results in severe weight loss in diabetic animals [40,41]. L. micranthus extracts-and-glibenclamide treated groups showed similar weight gain pattern which is indicative of the ameliorating effect of the treatments on weight loss in STZ-induced diabetic rats. The effects of the extracts in ameliorating STZ-induced weight loss has been reported for borneol, cavarcol and Swertianmarine [42,43,44]. Twig and leaf extract of L. micranthus may contain principles useful in ameliorating weight loss seen in Type-1 DM. Persistent hyperglycemia is a major factor in the pathophysiology of DM. Hypoglycemic agents should possess the ability to reduce the fasting blood glucose in diabetic patients. From the presents study, the fasting blood glucose of all animals in different groups at the start of the experiment were not significantly different (p>0.05). After induction of diabetes, the diabetic groups showed significant hyperglycemia which could be attributed to destruction of pancreatic beta cells, causing reduction in insulin production and hence, poor glucose utilization. The result showed adequate control of hyperglycemia in the glibenclamidetreated group and the L. micranthus leaf and twig-extract treated groups. The hypoglycemic effect of the extracts was observed at day 7 of treatment, and was consistent up to day 14 of treatment, suggesting that the extracts could be used for management of DM-induced hyperglycemia. This is consistent with the previous studies in alloxaninduced diabetes [45,46].
The effect of glibenclamide, LML and LMT on glucose utilization assessed by the oral glucose tolerance test (OGTT) exhibited comparable glucose utilization pattern to normoglycemic group. This showed that both leaf and twig extracts of L. micranthus had similar effect on glucose utilization comparable to glibenclamide (p<0.05).
DM impairs glycogen synthesis both in the rat's liver and skeletal muscles [47]. The glycogen content of skeletal muscle and liver are markedly decreased in DM [48]. Result of the present study showed increase in the liver glycogen content of all extract-treated animals compared to the diabetic untreated group. LMT treated animal showed significantly (p<0.05) higher liver glycogen levels compared to other groups. Normal control group, LML500 and STZ-LML500 groups showed similar liver glycogen levels. This showed that the leaf and twig extracts of L. micranthus could improve glycogen storage possibly by inducing glycogenesis or reducing glycogenolysis in DM. This activity has been reported for borneol, citornellol and mytenal [42,49,50,51] which were monoterpenes derived from different plants with antidiabetic activities.
The damaging effect of streptozotocin on pancreatic beta cells resulted in reduction in the level of serum insulin of the diabetic untreated rats compared to the control, glibenclamide-treated and L. micranthus extracttreated diabetic rats. LML500 treated diabetic rats showed higher serum insulin level than glibenclamide (an insulin secretagogue. [52] and LMT 500. Thus suggesting that leaf extract of L. micranthus has better insulinsecreting activity than the twig and glibenclamide-treated group. The result correlate with the findings of Gray and Flatt [53]. LML500 and LMT500 extracts had significantly higher (p<0.05) effect at reducing haemoglobin glycation comparable to glibenclamide. Clinical studies have shown that glycated hemoglobin is higher in diabetic patients than in non-diabetic individuals [54]. In the present study, reduction in haemoglobin glycation by the extracts could also be attributed to the activity in stimulating insulin secretion, or increasing glycogenesis and glucose utilization, leading to reduced blood glucose level and reduction in the rate of haemoglobin glycation. In addition, the antioxidant effect on phytochemicals in L. micranthus extracts similar to what was reported for D-limonene and thymol in previous studies [55,56] could play a role.
The role of tumor necrotic factor-α (TNF-α), an adipocytokine in systemic inflammation has been established in DM [57,58]. LML500 and LMT500-treated diabetic group showed a significantly lower (p< 0.05) TNF-α level compared to all other groups. Results from the present study also showed that L. micranthus leaf extract exhibited better serum TNFα lowering effect than glibenclamide. The leaf and twig extracts of L. micranthus may reduce diabetic complications by exerting antiinflammatory activity and lowering levels of TNF-α in inflammatory and metabolic diseases such as DM. Similar effect has been reported in adipocytes for geniposide [59].
Dyslipidemia is a critical feature of uncontrolled DM, characterized by high total cholesterol (TC), low density lipoprotein (LDL) cholesterol and triglycerides, and a reduction in the level of high density lipoprotein (HDL) cholesterol [14]. Acute insulin deficiency initially causes an increase in free fatty acid mobilization from adipose tissue resulting in an increased production of LDL cholesterol particles and resultant dyslipidemia [60,61,62]. LML500 and LMT500 showed similar control of the TC, LDL and serum triglyceride level at all doses used. Similarly, hypercholesterolemia observed in diabetic-untreated groupwere lowered in glibenclamide, LML and LMT treated groups. In addition, HDL cholesterol level was significantly increased (p<0.05) in extract-treated animals than glibenclamide-treated group. Thus, suggesting a role for the extracts in the management of diabetic-induced dyslipidemia. Similar effect has been reported for D-Limonene and thymol [44,55] Atherogenic index is a predictor of atherosclerosis, resulting from the imbalance between HDL and total cholesterol. Atherosclerosis is a major contributor to the global morbidity and mortality, both in hypertensive and diabetic individuals [63,64]. LML-treated groups showed atherogenic index significantly lower (p<0.05) than the glibenclamide treated group. The extracts showed potent antidyslipidemic effect. The extracts may therefore be useful at reducing the risk of cardiovascular, and vascular events and death that are complications of chronic DM.
Analysis of weight of organs showed no significant difference (p>0.05) in the weight of the liver, heart and kidney of all experimental groups. Weight of pancreas for the STZ-diabetic untreated group was significantly lower (p<0.05) compared to weights of the pancreas in all other experimental groups. The increase in the weight of pancreas of LML500 treated group suggests that LML contain active phytochemicals that are capable of regenerating pancreatic beta cells. Further studies are required to confirm this finding.
The histopathological photomicrograph of the pancreas of experimental animals in this study re-enforces the destructive nature of STZ on both exocrine and endocrine pancreatic tissues, as it caused necrosis in these tissues. Also, the efficacy of glibenclamide in the treatment/management of diabetic state was shown to be partly due to increased activity of the islet and acinar cells which in the present study were observed to be hyperchromatic. This study demonstrates the ameliorating effect of L. micranthus leaf and twig extracts on the STZ-induced destruction of acinar and beta cells of the pancreas. Similar effects has been reported for menthol and palmitic acid ester [31,51,65].
In conclusion, this study showed the therapeutic benefits of L. micranthus in management of, and prevention of complications of DM via different mechanisms. Further studies are required to identify the phytochemical principles responsible for these antidiabetic actions and their possible mechanisms of action tested.


s2fieldsofstudy:
Agricultural And Food Sciences
Economics

provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
A SYSTEMS APPROACH TO MECHANIZATION TO COMMERCIALIZE CASSAVA VALUE CHAIN IN KENYA

Cassava is produced in Kenya mainly as subsistence and is the second most important food crop after maize in the Country’s Western and Coastal regions. Its production and use as human food, animal feed and industrial starch is low due to poor agronomic practices postharvest handling and processing, bulkiness and low application of mechanised operations to scale production to commercial levels. An average yield of 15.3 MT/ha on 0.2 to 1 ha household units was realised against a potential of 90 MT/ha in 2014 producing 935,089 MT of cassava valued at $ 168 million. Kenya is a low mechanized country with a mechanical power usage indicated at 30%, hand power at 50%, and animal draught power at 20%. Mechanisation models that apply a systems approach by taking into account the value chain cycle is muted as feasible a pathway for ascertaining the technical and economic sustainability of the cassava enterprise in the country. Commercialization of the cassava value chain requires household production at scales beyond 4 ha. Such scales require mechanised production and primary processing for technical feasibility. A system that will provide mechanised tillage, planting, weeding, harvesting and processing through organised delivery and technical back up support is projected as the best entry to address this limitation. This would unleash the commercial viability of the cassava value chain and grow the livelihood system of producers and processors beyond subsistence thresholds.

Cassava,
Manihot esculenta Grantz (Euphoabiaceae), is produced in Kenya mainly as a subsistence crop (KIPPRA, 2016) and is the second most important food crop after maize in the Country's Western and Coastal regions (Mwa'ngombe, 2013). It is a drought tolerant crop that grows on poor soils with 400-1700 mm rainfall (FAO, 2013, GOK, 2007. The production and use of cassava tubers for food, animal feed and starch production is low in the country due to poor agronomic practices, postharvest handling and processing. This, in turn, significantly affects the commercialization of its tubers and by-products. Cassava is a bulky crop that requires intensive labour necessitating the use of improved machinery and technologies in production activities, transportation, and processing to allow for continuous cultivation and processing to meet the demand for cassava in the country and the population's food security needs. Kenya has not yet reached its national potential yield of 90 MT/ha. National yields are at about 15.3MT/ha (Farm Concern) compared with Nigeria at 15, Malawi;21.5, Rwanda;12.3, Thailand;14 and India at 21.5 (FAO, 2013). Production rose steadily from 388, 713 MT in 2004to 935,089 MT in 2014. This was worth USD 168 million. The rise was mainly due to improved productivity growing from a low of 7 MT/ha in 2004 to 15.3MT/ha in 2014 while scale of production stabilized at 64,000 ha mainly in Western (Busia, Siaya and Homabay)  Figure 1). Household production averages 0.2 to 1 ha in Kilifi and Busia Counties.  Figure 1 shows the various by-products obtained from cassava tuber that accounts for the national consumption. Cassava products include fresh cooked, boiled, baked or fried roots used at household level, to highly processed starch as a food additive and ingredients in animal feeds and confectionaries. A greater prospect exists for increasing consumption of cassava if its industrial use in the confectionery, ethanol and paper industry were fully exploited. Commercialization of the cassava value chain hinges on increasing demand to spur household production to scales beyond 6 ha per household. Such scales require mechanised production and primary processing for technical feasibility. This paper reviewed existing practices from secondary data to underscore the need for mechanization to champion cassava commercialization in the Counties of Busia and Kilifi.

Mechanization of cassava value chain
Agricultural mechanization is the replacement of human labour sources with mechanical power or technology to perform agricultural operations to reduce drudgery and increase efficiency. In any area, mechanization is categorized into three levels: low, fair, and high. Low mechanization means that manual labour exceeds 33%, fair mechanization means that animal power use ranges from 34% to 100%, while high means that use of mechanical power ranges from 67% to 100%. Kenya is a low mechanized country because the use of mechanical power stands at 30%, hand power at 50%, and animal draught power at 20%. Mechanisation models in the country should therefore apply a systems approach by taking into account the value chain cycle to increase their technical and economic feasibility. Analysis that concentrates on segmented node approach makes mechanized services unsustainable since the scales of application in subsistence-based agriculture cannot support mechanised operations.
A system approach to operations that looks at tillage, planting and fertilizer applications, spraying, weeding, and harvesting, along with how those operations are delivered, are the necessary conditions for satisfying the need for mechanised development of the cassava value chain. The later condition cannot be underscored, most machinery suppliers in Kenya are located in major towns like Nairobi, Mombasa, Eldoret and Kisumu, Ease of access to machinery and technology suppliers becomes an additional challenge especially for smallholder producers in marginal areas like Busia and Kilifi.
Access to mechanised units and services must however be matched with a robust access to information on critical functions like markets and marketing, processing and value addition, postharvest handling and losses by the value the chain actors. With the emergence of Information Technology, a robust cassava value chain would function optimally based on the proportion of actors with access to critical information. A major constrains observed in the study area was the lack of information flow especially on critical technology issues that affect production, processing and value-addition technology, marketing and markets for the cassava commodity (Table 2). A key constraint to increasing cassava production in Kenya has been linked to the low utilization of production and processing tools. The use of cassava as an industrial crop would spur demand to levels that would require a commensurate application of appropriate tools at the farm production and primary processing levels. A contextual review of the required economic scales of production, transportation and primary processing to sustain commercial and financial viability of the cassava value chain at the rural level demands application of mechanized systems. The average household size in Kilifi and Busia Counties stood at six people in 2009 with mean household expenditure per adult equivalent of US$ 29 and US$ 21 per month for the respective counties. Within these counties 11% and 21% of the respective households depended solely on agriculture for their livelihoods. A gross margin analysis for a vertically integrated cassava value chain actor in Kilifi with production and primary processing facilities gave a return of US$ 1258 over a 14 month (Table 2) production period. Among the agricultural households this translates to USD 3 per day or USD 0.5 per capita per day. This corroborates the baseline survey information presented by Agricultural Sector Development Support Program . corroborating the current subsistence outlay of the enterprise.
Conceptually, to transition these agricultural households to surpass the World Bank threshold of living above US$ 2 per capita per day would mean increasing production units fourfold. Therefore, commercialization of cassava value chain with a commensurate livelihood improvement for the low-end producers requires expansion of the land production units to a base level of 4 ha per household. Alternatively, the net value of the produce should increase fourfold; a process that would require improved values of the primary processed products.
The level of poverty of the cassava value chain actors at the production and primary processing levels limits their capacity to save and access credit for capital investment on mechanisation equipment and technologies. Access to credit in Kilifi was in only 3% for the male-headed households, while for the female-headed households it was less than 1%. The youthheaded households had no access to credit.
In 2010, the cost of one locally fabricated cassava processing machine was US$ 600. In 2012, the cost-in-freight (CIF) for a 4-wheeled tractor at Mombasa was US$ 215,000, while that of a 2wheel tractor was about US$ 1000. This situation was compounded further when the Government of Kenya, introduced a 16% VAT levy on all imported agricultural machinery in 2013 and imposed import duty on all machinery and raw materials imported into the country. For households with annual on-farm incomes of between US$ 170-430 in Kilifi and US$ 540-970 in Busia, the cost of mechanisation equipment was not only out of reach but their meagre incomes truncated their credit worthiness as illustrated by the low 3% access statistic.
The limitations imposed by poverty, small land sizes and low access to credit have reinforced the low scales of production and application of rudimentary primary processing techniques. These have worked together to constrain commercialisation of the cassava value chan reducing it to a subsistence poverty actuating enterprise.
To expand production and processing to livelihood and commercialisation transitioning thresholds, there is need for the application of appropriate technologies and mechanization units using a systems development approach. Monk, 2015, asserts that application of mechanized systems to scale would drive major improvements in productivity across the value chain.

Harvesting operations
Harvesting is one of the most labour and costintensive field operations in cassava cultivation. It is one of the main constraints to cassava commercialisation Mechanisation of cassava harvesting is very low in Kenya due to inappropriate planting methods and scale of cultivation. Kenya's cassava production is mainly small scale covering between 0.25 to 3 acres (GoK, 2007). This coupled with the intercropping practices among most producers makes application of mechanised operations untenable. In the study undertaken by EAPP, 72% of the respondents indicated that they harvested their tubers using hand labour confirming this assertion. The field capacities of mechanical cassava harvesters range from 0.2 to 0.8 ha/hr. This is significantly different from recorded field capacities obtained by improved manual harvesting tools that range from 49.9 to 156 manh/ha with root tuber breakage of 4.32 to 19.55% and harvesting energy consumption of 470.34 to 773.72 W (Amponsah, 2017). Mechanical harvesters operate by lifting tubers and shaking them out of the soil ( Figure 5) and have been found to reduce breakage losses from 32% to 2.6% (Itodo and Daudu, 2013) and harvesting costs by 50% (Bosrotsi;et al., 2017).

Transportation
Fresh cassava roots are highly perishable under ambient conditions, becoming unmarketable in 3 days or less. However, with proper handling, fresh roots can be stored up to 30 days. Fresh roots should therefore be transported to cassava chip or starch factories on the same day of harvest to avoid losses. The bulky nature of the tubers imply that only limited amounts of the produce can be handled by un motorized transport systems. This in turn caps the scale of production constraining commercialisation prospects. The use of machinery to transport produce to the market the counties of Kilifi and Busia was low.
Only 11% of the producers interviewed utilised motor vehicles, and 20% used motor-cycle taxis to ferry their produce to the market with distance to markets being as high as 10 km in Kilifi, and 4 km for Teso South, in Busia County.
The low application of motorized transport was governed by the low production scales at household levels and lack of an organised structure for produce collection and consolidation by the small holder actors in this value chain To address this gap, Farm Concern International piloted a process in Busia where it organized producers into commercial villages (CVs) structuring them to take advantage of the cassava market within and outside Busia County. Other than market linkages, the value chain actors were provided with basic technologies for chipping and solar drying cassava. The actors were also trained on best practices that expanded their product base to include cassava planting materials and processed products.

On-farm primary processing:
Cassava processing using mechanised systems increases labour efficiency, incomes, and living standards of cassava value chain actors and consumers, as well as enhance the-shelf life of products, facilitate their transportation, increase marketing opportunities, and help improve human and livestock nutrition (FAO, ), . Various processing methods are used to detoxify cyanide in the roots and to transform the tuber into a range of products for household and industrial use. The primary processes involve peeling, washing, grating, fermentation, drying and milling into flour which can then be used in making bread, starch, beer and animal feeds among other products. Primary processing at the farm level provides the first step towards translation of cassava value chain into an industrial crop. Possibilities exist for applying it as subsidiary flour for the confectionary and bakery industry, raw material for paper manufacture, and feedstock in alcohol and ethanol production plants. It can also be used as carbohydrate concentrate in animal feeds. Such opportunities would create sufficient local market to spur its commercialization across the entire value chain.
Currently the form of processing practiced in Kilifi and Busia include the use of hand-tools and solar driers. Of the respondents interviewed, 64%, use hand tools, 31% apply unspecified tools, while 1% had improved solar driers. Majority of the producers relied on sun-drying, . confirming the subsistence nature of their actions. Commercialisation of the cassava value chain would therefore require a strategic reorganisation of the processing segment with a matched investment on acquisition and application of appropriate processing equipment and technologies.
The need for primary processing was however evident in both Kilifi and Busia with 48% of the producers interviewed in Kilifi and 88% in Busia County applying one or another form of processing. This state was illustrated in the EAPP baseline survey that found 98% of producers across seven districts engaged in some form of processing at primary activity level undertaking tasks like washing, peeling, fermenting, chopping, and peeling of the fresh tubers.
Some primary processing enterprises were however found unsustainable due to low throughputs and high costs of operations. A study in Busia observed that most processors exited the flour processing enterprise because of the high losses generated. Primary processing of tubers into flour did not translate into increased value of the product. The price of flour (at US cents 20/kg) was found to be the same as the price of dried tubers at the farm gate Above this handling and processing losses reduced the volume of flour by a factor of 3:1, meaning that 4 kg of raw tubers gave 1 kg of freshly ground cassava flour. For off-farm processors to breakeven their operations they needed raw roots priced at US cents 6.3/kg. Alternatively, they needed to increase the price of cassava flour from US cents 40/kg to US cents 95/kg and compromise its competitiveness against the highly preferred maize flour.

Proposed development pathways to commercialise cassava
Machines, equipment and technologies exist that could be applied to mechanise cassava value chain across all nodes to increase productivity with a commensurate improvement in chain competitiveness. Much focus has been put on production activities leading to a fair level of mechanization on land preparation operations. However, adoption at the other levels remains poor making the whole chain ineffective and therefore subverting cassava commercialisation and reducing to a subsistence crop. Constrains on mechanised planting and weeding, low application of value-adding technologies, lack of product differentiation and minimal use of information technologies to guide marketing and access to markets inhibit expansion and growth of cassava chain enterprises. These factors have slowed prospects for creating new and more competitive products for the market, diminished flow of capital and compromised the industrial potential of the cassava value chain. In this review; the low scale of operations, poor access to credits, inadequate and non-existent machinery support services, lack of market information, low application and use of information technology have been cited as issues of concern that when addressed could grant transformation of the cassava value chain into commercial industry. The review proposes a systems approach to mechanisation as a feasible model for unlocking the latent industrial potential of the cassava value chain. The model considers the whole value chain as a functional system whose efficiency and effectiveness are grounded on assigning tools, equipment, machinery and technologies across all the nodes. The functions must also be rolled at a scale that makes each node individually and collectively feasible in technical and economic terms, commercially viable and financially and environmentally sustainable. The model underpins financial sustainability to a household livelihood index pegged on either the World Bank's US$ 2 per capita per day basic poverty transition line or Kenya's Vision 2030 middle income aspiration daily per capita income base rate of US$ 11 (KES 1116) as illustrated in Figure 7.
Mechanization along the cassava value chain still has a long way to go before the interventions can lead to commercialization of cassava products in Kenya. The following actions are recommended to effectively improve on the levels of appropriate mechanization along the cassava value chain in Kenya:

1.
Resources should be allocated into research and development of the cassava value chain in Kenya. A collaborative effort by all the stakeholders along the value chain should be made to collect and document reliable and accurate information on the status of the value chain, which should then be made easily accessible to all actors along the chain.

2
A public-private nationwide awareness campaign is needed to promote the ascension of the cassava crop from the "poor man's crop" group, and market its multiple by-products both as a viable industrial raw material that can catalyse income generating activities in rural areas, and as a healthy food. Creating a market demand for cassava for especially non-food industries such as the animal feeds industry and the bioethanol industry would boost the market competitiveness for cassava by-products and create an incentive for local producers. 3 Collaboration between the private sector and the public sector to boost research and development of locally made labour-saving technology, as well as simple processing machinery and equipment is necessary. Capacity building for local "juakali" fabricators to not only manufacture simple and high-quality production and processing technology, but also to provide quality maintenance services especially in the rural/marginalized production areas. 4 Technology training and knowledge transfer: This can be done through demonstration farms and/or farmer field schools to help boost the knowledge transfer and adoption process of production, transport and processing technology options among producers and other key chain actors. Innovative learning and sharing platforms can be organized through extension service providers in major cassava producing areas throughout the country.

5
Access to credit through common interest groups and/or cooperatives: Producers and processors could organize themselves into Common Interest Groups. These groups can address community needs through technology support and partial financing of priority projects such as processing of cassava roots in areas where it is a priority crop. They can also help secure linkages with key players in the domestic and foreign cassava market.
These common interest groups, such as the cassava commercial village-based processing units can help meet the transportation challenges by processing and adding-value to the cassava tubers, in bulk, at or near the farms, thereby reducing losses (increasing the shelf life) and increasing the competitiveness of the cassava products in the market.

6
To help ease the challenges of the appropriateness of the available land preparation machinery in the market to small land sizes, producers owning small land sizes could contractually and temporarily consolidate their lands for purposes of a joint utilization of the available land preparation and production technology. Research into the social and economic impact of this course of action should be conducted.

7
Public and private partnerships in establishing and promoting machinery hiring and lending service groups should be promoted. Techsavvy youth groups and women's self-help groups could be empowered to engage in affordable machinery service provision especially in the areas that do not have quick and easy access to mechanization services. These groups can receive capacity building through financial support as well as technical knowledge and training of appropriate production and processing machinery.

8
Proper extension services and materials should be made readily available to stakeholders along the value chain. Access to improved, highyielding, disease and pest resistant varieties, as well as fertilizers, herbicides, etc, where needed, should be facilitated to improve on production capacities and overall productivity of the producers. This will then, in the long run, create capacities that will potentially meet the annual demand for both food and non-food uses of cassava tubers, and consequently, drive the demand for labour-saving technology in the country.

CONCLUSION
A systems approach to mechanise farm and primary processing operations complete with a structured back up support augmented by guaranteed market outlets could spur cassava production in at least 4 ha households units and thereby transform this value chain into a commercially viable and livelihood support system in Kenya. In its current state it remains a labour intensive, low yielding enterprise with untapped potential.


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Plants used as medicinal in Güémez, Tamaulipas, north-eastern Mexico

The use of medicinal plants in Mexico has a diverse and deeply-rooted tradition concerning. Plants play an important role in traditional medicine among the inhabitants of Güémez, Tamaulipas in northeastern Mexico. With the aim of obtaining quantitative information on the plants and their uses as medicinal sources, semi-structured interviews with 113 local residents were taken. In addition, Informant Consensus Factor (ICF) and Use Value (UV) were calculated. A total of 85 species of medicinal plants belonging to 44 families were identified. The most common families were Asteraceae, Lamiaceae and Euphorbiaceae. The most used part of the plant was the leaf and the most frequent preparation method was boiling, preparing as an infusion. A total of 50 medicinal uses were registered. Fever had highest ICF (0.93), whereas, according to UV, the most important plants were Artemisia ludoviciana Nutt., Mentha spicata L., Matricaria recutita L. and Achillea millefolium L. The information generated strengthens traditional knowledge, bringing new species and uses to the country ́s records for future phytochemical studies that could lead to new medicines.

Introduction
About 80% of the world's population relies on traditional medicine to satisfy primary health care needs (Bermúdez et al., 2005;Ayyanar and Ignacimuthu, 2011;Panyaphu et al., 2011). The World Health Organization has registered approximately 21,000 plant species for medicinal uses around the world (Malla et al., 2015). Medicinal plants are an important source of current drugs. About 25% of the world's prescription drugs come from plants (Bulut and Tuzlaci, 2013).

AcademicPres
Notulae Botanicae Horti Cluj-Napoca Agrobotanici 1131 Mexico has a diverse and deeply-rooted tradition concerning the use of medicinal plants (Estrada-Castillón et al., 2018). In Mexico, there are more than 23,400 vascular plants and 5,000 species are used for medicinal purposes and the most important families are Asteraceae, Lamiaceae, Fabaceae, and Euphorbiaceae (Casas et al., 2001;Camou-Guerrero et al., 2008;Alonso-Castro et al., 2012). Also is ranked sixth in the world regarding greatest cultural diversity (Mercado, 2013), as it has 62 ethnic groups (Juárez-Rosete et al., 2013). Most ethnic groups use medicinal plants because of their efficiency, tradition and low costs (Kayani et al., 2015;Tribess et al., 2015). In particular, rural areas contribute with most knowledge about medicinal plants (Kayani et al., 2015). These plants help communities to survive in remote areas (Gómez, 2012).
Nowadays, it's recognized that in Mexico and many countries around the world the transmission of knowledge regarding the use of medicinal plants from old to new generations is declining are experiencing loss of useful plants due (Quesada, 2008;Calvo et al., 2011;Hassan-Abdallah et al., 2013) as well as losing entire villages and local populations due to lack of work, education and medical care (Panyaphu et al., 2011). This fact is also a reality in rural communities because of globalization and the use of allopathic medicine (Esquivel-García et al., 2018). Therefore, it is important to rescue this knowledge to prevent the loss of ethnomedical traditions, which is a scientific and cultural heritage important for future generations.
The municipality of Güémez, located in the northeast of Mexico, covers 1.5% of the Tamaulipas state. Its economy is based on agriculture (citrus, safflower, bean, maize, henequen and sorghum), cattle raising and forestry production also take place on smaller scale. However, there are no local inhabitants selling medicinal plants. The local people live in houses of 'adobe' (building material made from soil and often organic material), wood and concrete. Health services are scarce with little medicine and few doctors. Some villages do not have drainage, clean water, electricity, so migration is commonplace due to lack of security, education and employment.
The objective of this study is to know the most commonly used medicinal plants and their uses by the inhabitants of the municipality of Güémez with the purpose to revaluing and saving this traditional knowledge in this region of Mexico.

Study area
The municipality of Güémez is located within the Sierra Madre Oriental and is part of the centralwestern region of State of Tamaulipas ( Figure 1) (24°06'-23°41'N and 99°30-98°45'W), its altitude is from 200 to 2,800 m. It covers 1,204.55 km 2 , has 43 localities with a population of 15,659 inhabitants, of which 80% live in urban areas and 20% in small villages called 'ejidos'. The climate of the region is temperate to sub-humid, in mountainous areas and semi-dry to very warm in the lowlands. The mean annual temperature ranges from 12 to 26 °C and the annual rainfall is 600 to 1,100 mm (INEGI, 2009).

Fieldwork
Wild and cultivated medicinal species in the study area were collected. Plant samples obtained were pressed and dried according to the method described by Sánchez-González and González (2007). Specimens were identified by the authors through the Manual of the Vascular Plants of Texas (Correll and Johnston, 1970), scientific names were consulted in The Plant List (http://www.theplantlist.org/). The complete collection of specimens was included in the CFNL herbarium (acronym according to Thiers, 2011). We include the common name in Spanish for all plants species registered during interviews.
The ICF adapted by Heinrich et al. (1998), is calculated according to following formula: ICF = (Nur-Nt) / (Nur -1) where Nur refers to number of citations of uses given in each category and Nt to number of species used. This method is used to test the homogeneity of information. The ICF offers a range of 0-1, where high values (about 1) are obtained, when there is a well-defined selection criterion in the community and/or if information is exchanged between informants, and the values are low (near zero) if plants are chosen at random or if there is no exchange of information about their use among informants.
The use value (Trotter and Logan, 1986), demonstrates the relative importance of locally known species and is calculated according to the following formula: UV = U / N, where U refers to number of citations per species and N to number of informants.

Richness of medicinal plants
According to the results of specimen identification, 85 species with medicinal uses, belonging to 41 families were reported by interviewees. Among them, 45 species are cultivated and 40 species are wild. The most common families of medicinal plants are Asteraceae (17.64%), Lamiaceae (11.76%), Euphorbiaceae (7.05%) and Rutaceae (4.70%). During the study, 126 samples were collected in the study area. Plants used for medicinal purposes in Güémez are presented in Appendix 1 in alphabetical order according to family and scientific name. The biological forms of the medicinal species mentioned by the interviewees were herbaceous (47 species), shrubs (21 species) and trees (17 species).
The most frequently used plant parts for the preparation of medicines were leaves (42 plants

Informant consensus factor (ICF)
There were 13 main categories of ailments based on ICF data. The ICF interval was 0.1 to 0.93. The results showed that the highest ICF was reported for the fever category with a value of 0.93, followed by ophthalmological problems (mainly eye infections) (0.75), intestinal parasites (mainly worms) (0.75), respiratory ailments (mainly colds) (0.72) and endocrine system (mainly diabetes) (0.66), gastrointestinal system (0.59) and bumps and wounds (0.59) ( Table 1).

Method of preparation of the traditional medicines
The medicinal plants used by the local inhabitants are prepared and administered in different ways. Various methods of preparation and application are presented. According to ailment, preparation differs such as being boiled (62 plants), taken raw (17 plants), fermented in water (6 plants), cooked (3 plants), fermented in alcohol (2 plants), and fried (1 plant) (Appendix 1). Boiled and crude are the most commonly used methods for preparation of remedies. As a result of the records of the interviews, 50 species of plants are prepared for consumption, 15 plants are prepared for external use, while 20 species are used to alleviate both internal and external ailments.

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The main routes of administration of the plants are oral (76%), followed by intestinal washes (24%) and poultices (4%). We do not include the exact proportions used for each medicinal plant, because there is a contradiction with respect to the exact dosage. The dose is usually measured by cups of infusion.
Most remedies are based on the use of a plant. However, Table 2 shows eleven combinations of medicinal plants reported by informants. Most are composed of six species, of which Mentha spicata is the plant most used in several combinations, followed by Psidium guajava, Ocimum basilicum, Ruta graveolens, Matricaria recutita and Poliomintha longiflora. The combination of the species Crataegus tracyi and Arctostaphylos pungens is the most recorded one found during the interviews. The main ailments treated with these mixtures are gastrointestinal diseases.  (Ghorbani et al., 2011;Bulut and Tuzlaci, 2013;Leto et al., 2013;Nawash et al., 2013;Bolson et al., 2015), probably due to the high diversity of its phytochemical constituents. Some studies have reported that this family has a wide range of biologically active compounds and has high species richness worldwide Thomas et al., 2009). Following the IUCN Red List of Threatened Species, the only species under protection status is Dalea scandens (Mill.) R.T. Clausen assessed as being of Least Concern (Groom, 2012).
The possible reasons that herbaceous plants have the highest frequency of use is because bioactive compounds can be easily extracted (Lulekal et al., 2013;Kayani et al., 2015) and their high availability (planted in gardens, wild habitat and roadsides) (Tsobou et al., 2013). Similar conclusions are drawn from other studies in other parts of the world (Ghorbani et al., 2011;Sivasankari et al., 2014;Yaseen et al., 2015).
The medicinal species best known by the population in the study area were those that were used for uncomplicated ailments, similar findings are reported by Pérez-Nicolás et al. (2017) for the state of Oaxaca in southeaster Mexico.
The most frequently used plant parts for the preparation of medicines were leaves. Several ethnomedicinal studies in Mexico, Bangladesh, China, Colombia and India have reported that leaves are the most used parts of plants (Singh and Singh, 2009;Teklehaymanot, 2009;Packer et al., 2012;Sivasankari et al., 2014;Pérez-Nicolás et al., 2017;Estrada-Castillón et al., 2018). This is because they are the most abundant part of a plant and they are easier to collect (Giday et al., 2003). It may also be due to their greater effectiveness, since leaves have increased amounts of secondary metabolites like alkaloids, tannins and inulins, which are active components of many medicines (Yemele et al., 2015). In addition, the use of leaves favors the conservation of plants, since the collection of roots can kill the plant and endanger the species (Telefo et al., 2012;Kadir et al., 2013).
In the study area the most frequently used medicinal species are Artemisia ludoviciana, Mentha spicata, Matricaria recutita and Ruta graveolens, in the same way such plants are used by the inhabitants of Santiago Camotlán, Oaxaca (Pérez-Nicolás et al., 2017).

Disease categories
Ethnobotanical studies reveal that stomach pain is the most frequent ailment treated by medicinal plants (Polat and Satil, 2012;Kayani et al., 2015;Tribess et al., 2015;Estrada-Castillón et al., 2018). Globally, gastrointestinal diseases account for 2.2 million deaths each year and are caused by viruses, bacteria or parasites (Monigatti et al., 2013). The second most notable illness was related to the kidneys, which may be due to low intake of water during the cold season and the hard-agricultural work (Kayani et al., 2015).
In the study area medicinal plants are also used for magical purposes, like to remove bad luck. This is a common practice in the Mexican tradition (Frei et al., 1998;Andrade-Cetto, 2009). Since ancient times in Mexico, people have believed that diseases were caused by evil spirits, witchcraft and magic (Gallardo-Arias, 2004). Local healers prescribe medicinal plants, through a ritual of prayers while rubbing medicinal plants on the body of sick person. Most informants mentioned that such traditional knowledge was transmitted by their parents and grandparents over the years.

Use Value (UV) and Informant Consensus Factor (ICF)
Most of the plant's species registered in this study are native to Europe and Asia and their use may have been popularized in Mexico because of their efficiency, adaptation and fast growth (Juárez-Rosete et al., 2013).
The high values of ICF could indicate that these diseases are common in the studied population, where there is a need to cure these diseases, and, thus, there is knowledge among the local population about the uses of medicinal plants (Nawash et al., 2013).

Method of preparation of the traditional medicines
Oral is the most routes of administration of plants used by the inhabitants of this study area because of their ease, similar findings have been reported in countries such as Pakistan (Kadir et al., 2012(Kadir et al., , 2013 and India (Ayyanar and Ignacimuthu, 2011).
Most remedies are based on the use of a plant and other is combinations of medicinal plants reported by informants. This can be attributed to the fact that any one plant can contain several compounds that perform different functions in the body (Focho et al., 2009). The plants are combined because people believe that the blend could improve the synergistic effect of the pharmacological effects of plants (Igoli et al., 2005;Giday et al., 2010).

Conclusions
This study reveals that for the inhabitants of the municipality of Güémez, the use of traditional medicine is frequent, and most local people still rely on plant-based remedies for common health problems. Thus, we emphasize the need to develop actions to avoid the loss of traditional knowledge of medicinal plants, not only to preserve this cultural heritage but also to record information on useful species that could be used to develop new medicines and provide other benefits, while contributing to protecting local biodiversity.


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Reuse of Residues as Substrate for Production of Eucalyptus (Eucalyptus urograndis) Seedlings

Studies about crop residue management have become essential due to the possibility of their use in forest seedlings production. This study evaluated the effect of coffee moinha (MO) on substrates composed of vermiculite (VE), coconut fiber (CF) and carbonized rice husk (CRH) in the development of Eucalyptus seedlings. The experiment was conducted in a completely randomized design, with five treatments of increasing proportions of MO/decreasing proportions of CRH (0/28, 7/21, 14/14, 21/7 and 28/0%) and fixed proportions of VE (36%) and CF (36%) in the substrate. At 85 days after planting, the following parameters were assessed: shoot height, stem diameter, root dry mass, shoot dry mass, emergence percentage and Dickson’s quality index (DQI). The results showed CRH could be fully substituted by MO (28%) in the substrate composition because the assessed variables presented lower values than the control treatment (0% MO + 28% CRH + 36% VE + 36% CF). Moreover, the maximum vegetative development in Eucalyptus seedlings was achieved when CRH was replaced by up to 20% MO. In this way, MO becomes an alternative as a substrate component for Eucalyptus seedlings production.

INTRODUCTION AND OBJECTIVES
Seedlings grown in forest nurseries provide planting stocks for implantation of forest stands, recovery of degraded areas, urban afforestation and parks, among other purposes (Walker et al., 2011). According to Zanetti (2008), Brazil has one of the largest cultivated areas of fast-growing forests, especially Pinus and Eucalyptus species. One of the most important stages in the establishment of tree plantations is the production of quality seedlings, conditioned by adequate seeds and substrates .
The production of good quality seedlings depends on the germination processes, as well as the formation of the root and shoot systems, which are directly related to the aspects that define the substrate efficiency, such as aeration, drainage, water retention and nutritional content (Caldeira et al., 2008).
The type of potting technique may additionally influence seedling quality and production costs (Kostopoulou et al., 2011). In this context, the conical plastic pot ("cone-tainer") is the most popular for forest seedling production (Wendling & Dutra, 2010).
In general, no single substrate meets all the conditions necessary to achieve a satisfactory growth of forest seedlings. So a mixture of different substrates is recommended, as they may present undesirable characteristics to the plant, when used alone (Caldeira et al., 2011).
VE is as an inert mineral of low density and variable structure, composed of tetrahedral silica sheets and iron and magnesium octahedral sheets (Caldeira et al., 2013). Coconut shell fiber has also proven useful in the production of high-quality substrates because of its longevity without deterioration of its characteristics, non-reaction with fertilizer nutrients, physical 2 -7 Monaco PAV, Junior GR, Haddade IR, Krause MR, Guisolfi LP, Almeida KM 2 properties (e.g., porosity), the possibility of sterilization, high primary quality and low cost to the producer (Carrijo et al., 2002). Additionally, CRH allows the penetration and exchange of air at the base of the roots (Saidelles et al., 2009). When evaluating the attributes of commercial substrates mixed with CRH in different proportions, Klein et al. (2002) verified that this material improved the physical and hydraulic properties of the substrates, providing better porosity. Hence, since CRH acts predominantly as a physical conditioner of the substrate, it must be used in conjunction with other materials that provide the nutrients for adequate vegetative development of the seedlings.
Thus, organic residues, as alternative substrates for forest seedlings production, have been studied extensively (Caldeira et al., 2013;Da Ros et al., 2015;Delarmelina et al., 2014;Figuerêdo et al., 2014;Silva et al., 2014). Besides possessing physical and chemical attributes that are fundamental to improving the quality of the substrate, the use of organic residues, especially if available in the cultivating area, can minimize the risk of environmental contamination resulting from improper disposal and reduce production costs.
In the mountainous region of Espírito Santo, Brazil, coffee straw stands out as one of the most generated organic residues. Also known as moinha (MO), coffee straw is a byproduct of the coffee drying process composed of plant remains, such as leaves, branches, inflorescences and poorly formed fruits, which, when dried together with the coffee cherries, are burned and released from the dryer (Meneghelli et al., 2016). When chemically characterizing this material, Meneghelli et al. (2016) verified its potential use as a fertilizer, due to high contents of phosphorus, potassium and, especially, nitrogen.
It is believed that MO, combined with VE and CF, can be a promising combination as it provides chemical and physical attributes essential for substrate quality. Also, the MO may be an alternative to rice straw, notably in regions where rice residues are not available in high amounts. In this way, it is necessary to obtain the ideal proportion of each residue that provides quality seedlings and with adequate vegetative development.
Thus, the current work aimed to evaluate the effect of the coffee processing residue (i.e., MO) on substrates composed of VE, CF and CRH in the development of Eucalyptus seedlings, and obtain the proportion of these materials in order to achieve the best vegetative development.

MATERIALS AND METHODS
The experiment was carried out at the Eucalyptus nursery sector of the Fibria Celulose S.A. company, in the Aracruz unit (Espírito Santo, Brazil), for 85 days. The Aracruz unit is in the Atlantic Forest coastal region, with geographical coordinates of 19°49'13" S and 40°16'24" W, and an altitude of 60 m. The climate is "Aw, " based on Köppen's classification, with an average annual rainfall of 1.327 mm, and an average annual temperature of 28 °C.
A completely randomized design (CRD) was used, with five treatments and six replicates, with each repetition consisting of 56 plants. In total, eight plants per replicate were evaluated. The treatments consisted of increasing levels of coffee processing residue (MO) in the substrate, mixed with CRH, VE and CF. The control treatment consisted of CRH, VE and CF in the same proportions used by Fibria Celulose S.A. The compositions of all treatments are shown in Table 1.
The MO was collected at a coffee processing unit in the municipality of Santa Teresa, ES, Brazil, while the other materials used in the composition of the substrates were donated by Fibria Celulose S.A. The chemical and physicochemical characterization of the residues used in the composition of the substrates for the production of Eucalyptus seedlings was carried out at the Laboratory of Soils and Solid Residues in the Agricultural Engineering Department of the Universidade Federal de Viçosa (Viçosa, Brazil). The physicochemical analysis consisted of the determination of the electrical conductivity, using a bench conductivity meter. The chemical analysis involved measuring of the pH, by means of a benchtop pH meter, and the quantification of the readily oxidizable organic carbon, total organic carbon, organic matter, total nitrogen, phosphorus and potassium contents, following the method described by Matos (2015). Table 2 presents the chemical and physicochemical attributes (electrical conductivity) of the CF, MO and CRH used in the experiment.
Sprouts from the hybrid clone "Ara 6791" (Eucalyptus urophylla × Eucalyptus grandis), of approximately 10 cm in length and three pairs of leaves were obtained from the mini-clonal garden of the Fibria Celulose S.A nursery. After being collected, the sprouts were stored in thermal boxes for a few minutes until transplanted to the growing container. The chosen clone presents fast development and vigorous seedlings, with satisfactory root and shoot formation.
In the sowing sector, where the air humidity was close to 100%, the cuttings were transplanted in trays with 176 cone-tainer single cells (conical tubes made of rigid plastic), with a volumetric capacity of 50 cm 3 each, filled with the different substrate mixes (treatments). In all treatments, 12 g of single superphosphate and 8.5 g of the nitrogenphosphate-potassium formulation 19-6-10 were added per tray, according to recommendations obtained by surveys conducted by Fibria Celulose S.A.  After planting, the trays were transported to the rooting greenhouse, where they remained for 23 days, under a relative humidity above 90% and temperature of 20 °C before they were transported to the growing courtyard, where they remained for 22 days, protected by a shading screen, which allowed a 50% reduction of solar radiation. Finally, the seedlings were transferred to the open courtyard, where they remained for 40 days.

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The hardening fertilization was carried out in the open courtyard. At this hardening stage, the seedling is physiologically prepared for the planting in the first weeks that follow. During the hardening process, up to three fertigation are usually carried out daily, prioritizing formulations with a low nitrogen:potassium ratio since this phase is extremely critical and directly affects the final quality of the seedlings (Alfenas et al., 2009), as well as the potential for better survival and growth on the outplanting site. In this study, the hardening fertilization was carried out three times a day, via foliar spraying, according to internal technical recommendations of Fibria Celulose S.A. The mineral fertilizers used were calcium nitrate, potassium chloride, monoammonium phosphate, magnesium sulfate, boric acid, copper sulfate and zinc sulfate.
At 85 days after planting, in order to evaluate the vegetative development of the Eucalyptus seedlings, the following parameters were assessed: shoot height (SH), stem diameter (SD), root dry mass (RDM), shoot dry mass (SDM), emergence percentage and the Dickson quality index (DQI). A ruler was used to obtain the SH, measuring from the base to the apical shoot that gave rise to the last leaf. The SD was obtained using a precision digital caliper. The SDM was estimated by cutting the seedlings close to the substrate. To obtain the RDM, the roots were carefully washed in running water over a sieve, packed in paper bags, placed in a forced air circulation oven at 65 o C until constant weight and then weighed in a 0.01 g electronic precision balance.
Seedling survival percentage (SP) was assessed by counting the seedlings at 23 days after planting the cuttings. The errors were submitted to tests of normality (Lilliefors), additivity (Tukey) and homoscedasticity (Bartlett), for the validation of their analyses of variance. Those that did not meet the requirements were transformed, according to criteria by Gravina et al. (2004), again being submitted to the same validation tests. For the errors that still did not meet the assumptions, the Kruskal-Wallis non-parametric test was applied to the data, considering the evaluation of the effects of the treatments by scores.
In the case of significant effects (p < 0.05), for the comparisons between the treatments related to the moinha level (quantitative treatments), the degrees of freedom of the treatments were adjusted in the regression models by orthogonal polynomials method. The adjusted models were chosen according to the significance of the regression coefficients, the coefficient of determination and the biological phenomenon. All the statistical analyses were performed using SAEG 9.1 software (2007).  Table 3 shows the effects of increasing levels of MO in the substrate on the variables SD, SP and DQI in E. urograndis (E. urophylla × E. grandis) by the non-parametric Kruskal-Wallis test, at 5% probability.

RESULTS AND DISCUSSION
According to Table 3, all treatments, except for T1 (7% MO), had a statistically superior SD to that of the control treatment (0% MO). The MO level of 28% provided the largest SD (3.46 mm), but the value was above that considered ideal for E. grandis seedlings, according to the studies of Lopes et al. (2007); and Wendling & Dutra (2010) that recognize a minimal thickness of 2.0 and 2.5 mm, respectively. Souza et al. (2006) described the SD as fundamental in evaluating the survival potential and growth of forest seedlings. Within the same species, the larger the stem diameter, the better their odds of survival, due to their capacity to form and grow new roots. The results obtained in the treatments with increasing levels of MO in the substrate composition were similar to those found by Silva et al. (2012), which registered SD values ranging from 3.03 to 3.58 mm in E. urophylla × E. grandis seedlings, at 90 days post-staking, grown on substrates composed of VE, CRH and CF. However, comparatively lower SD values were published by Caldeira et al. (2013), of between 2.60 and 3.25 mm in E. grandis seedlings, at 90 days after sowing, grown on substrates with different percentages of sewage sludge and VE; and da , of 1.51 and 1.90 mm in E. grandis seedlings grown in different types and combinations of organic agroindustrial-urban substrates at 90 days after sowing.
For the SP variable (Table 3), there was no statistical difference at 5% probability level, indicating that 28% MO may be an alternative option to the use of rice straw as a component of the substrate. Rocha et al. (2015) mentioned that this homogeneity and the high survival rates might be reflective of the type of propagation method used in the experiment. In the current study, the clone used has the characteristics of genetic homogeneity of the seedlings and a high percentage of survival.
As highlighted by Vidal et al. (2006), the DQI is a good indicator of seedling quality since the robustness and the balance of the biomass distribution are considered in its calculation. According to this author, the higher the DQI, the better the quality of the seedlings. Here, the highest DQI values (Table 3), 0.211 and 0.212, were obtained in treatments T1 (7% MO) and T2 (14% MO), respectively, differing statistically from the control (0% MO). It is believed that the substrate containing 14% MO and 14% CRH provided the highest DQI, due to the adequate nutrient supply (i.e., MO) and ideal porosity (i.e., CRH) presented by this combination. With increasing levels of MO in the substrates, the DQI values in the treatments (0.199 to 0.212) can be considered good results when compared with those obtained by Silva et al. (2012), who also worked with E. urograndis mini cuttings for seedlings production. The DQI obtained by these authors ranged from 0.120 to 0.190, at 90 days after planting, when evaluating the development and quality of clonal seedlings in nine different substrate compositions produced from VE, CRH and CF. The results achieved in the present work were also superior when compared with other literature data, albeit with other species and using seed propagation. Silva et al. (2014) registered DQI values ranging between 0.032 and 0.056 in E. grandis seedlings, grown on different types and combinations of urban and agroindustrial organic substrates, at 90 days after sowing. Figuerêdo et al. (2014), when studying the effect of different proportions of CRH and substrates on E. globulus seedlings, ascertained DQI values ranging between 0.0042 and 0.015, at 65 days after sowing. Steffen et al. (2011) analyzed E. grandis seedlings at 100 days after germination, and the DQI values ranged from 0.12 to 0.21, considering different vermicompost ratios in the substrate. It is acknowledged that the DQI can vary depending on the species, seedling management at the nursery, type and proportion of the substrate, container volume and, especially, the age at which the seedling was appraised (Caldeira et al., 2013).
The SH, SDM and RDM of the seedlings, are presented in Figures 1a-c, respectively, as a function of the different percentages of MO in the substrate composition. According to Figure 1, all the variables showed a quadratic polynomial behavior, as a function of the different percentages of MO in the substrate composition. It can be seen in Figure 1a that the SH increased significantly with the gradual replacement of CRH by up to 19.37% MO, providing a maximum height of 35.61 cm. Above this MO level (19.37%), the SH decreased, which may be related to the high salinity presented by this residue, evidenced by the high electrical conductivity (Table 2). This fact may have contributed to the decrease of the osmotic potential in the substrate, negatively affecting seedling growth. As Harter et al. (2014) stated, high salt concentration in the substrate is a stress factor for plants because it reduces the osmotic potential and exposes the protoplasm to ionic action. The water is osmotically retained in the saline solution so that the increased salt proportion makes it decreasingly available to plants.
Even so, the decrease in SH at MO levels above 19.37% did not reach the lowest value verified (25 cm), which was registered in the treatment with 0% MO. This observation may be associated with the low nutrient content in the CRH (especially nitrogen) when compared to the MO (Table 2). However, in all treatments, the seedling height was higher than the minimum of 15 cm recommended by Wendling & Dutra (2010) for planting. Similarly to the current work, Silva et al. (2012) registered SH values ranging between 27.1 and 37.5 cm, at 90 days after staking, in E. urophylla × E. grandis seedlings grown in substrates containing VE, CRH and CF. Likewise, de Toledo et al. (2015) determined an SH of 18.73 to 37.30 cm, in E. urograndis seedlings, at 120 days after sowing, grown on substrates composed of paper and cellulose industrial waste. On the contrary, Rocha et al. (2015) recorded SH of between 11.09 and 20.16 cm in seedlings of the same Eucalyptus species, grown on substrates composed of water treatment sludge.
As seen in Figure 1b, the SDM increased significantly as the MO level increased, up to 19.16%, providing an average 6 -7 Monaco PAV, Junior GR, Haddade IR, Krause MR, Guisolfi LP, Almeida KM 6 of 2.41 g. Above this MO level (19.16%), as verified for the variable SH, a decrease in SDM values occurred. The lowest SDM value (1.33 g), provided by the control treatment (0% MO and 28% CRH), could be associated with nitrogen limitation, a fundamental nutrient in the formation of seedlings. A nitrogen deficiency in seedlings is known to hamper development and reduce the accumulation of dry matter (Malavolta, 1980). CRH contains less nitrogen when compared with MO (Table 2), and this could have contributed to the lower SDM value. The salinity effect in substrates with MO levels above 19.16%, as noted for the SH, may have surpassed the high nutrient availability effect, providing lower SDM values.
The SDM values verified in this study were higher than those given by Silva et al. (2012) of 1.10 and 1.72 g, at 90 days after staking, in E. urophylla × E. grandis seedlings grown on substrates produced from VE, CRH and CF. Likewise, comparatively lower SDM measurements of 0.99 and 1.82 g in Eucalyptus seedlings cultivated on substrates composed of VE, CRH and CF have been established by Simões et al. (2012); and de Toledo et al. (2015), who found between 0.88 and 1.52 g in Eucalyptus seedlings grown on substrates constituted of pulp and paper-making wastes.
It is well recognized that the root system quality is a critical factor for the performance of the seedlings in the field and, hence, is a parameter of paramount importance in the definition of seedling quality (Simões et al., 2012). From Figure 1c, the RDM increased significantly with the gradual replacement of the CRH by up to 17.42% MO, providing a value of 0.67 g, but decreased at higher MO percentage, as already verified for the other variables. It is noteworthy that all MO levels provided RDM values above 0.55 g, which was consistent with previous researchers. Simões et al. (2012) found RDM values between 0.23 and 0.59 g, similar to those published by de Toledo et al. (2015), with results between 0.47 and 0.78 g; and by da Silva et al. (2012), with values between 0.35 and 0.69 g.
The results obtained in this work show that CRH can be fully substituted by MO (28%) in the substrate composition since the assessed variables presented lower values when the seedlings were submitted to the control treatment (0% MO + 28% CRH + 36% VE + 36% CF). However, maximum Eucalyptus seedlings vegetative development is achieved when replacing the CRH with up to 20% MO in the substrate. As such, MO becomes an alternative substrate component for Eucalyptus seedlings cultivation, in addition to its environmentally harmonic disposal.
Therefore, these positive agronomic results, along with the necessity to offer a suitable destination for agroindustrial wastes and byproducts, demonstrate that organic residues from the coffee drying process (i.e., MO), as a component of substrates, are a feasible alternative for the production of E. urophylla × E. grandis clonal seedlings.

CONCLUSIONS
The reuse of coffee drying process residues (i.e., MO) in the composition of substrates to produce seedlings of E. urophylla × E. grandis is a viable alternative for the final disposal of these wastes.
The MO can be used in the composition of the alternative substrate in a proportion of up to 28%, in substitution of the CRH, to produce Eucalyptus seedlings.
The use of up to 20% MO in the composition of the substrate in substitution of the CRH provides better quality seedlings of E. urophylla × E. grandis.