Clinical Application of Chamomilla Recutita in Phlebitis : Dose Response Curve Study 1

This experimental and dose-response curve study aimed to carry out the quality control of the Chamomilla recutita sample, as well as to estimate the ideal dose, for anti-inflammatory effect, of the extract of its capitula, in patients with phlebitis due to peripheral intravenous infusion of antineoplastic chemotherapy and to evaluate the toxicity of this extract in human beings. The therapeutic efficacy, concerning the anti-inflammatory potential, of different doses of Chamomilla recutita extract were analyzed and compared in 25 patients. The time of regression of phlebitis was shorter for groups with 2.5% concentration (mean=29.2h, standard deviation = 8.98) and 5% concentration (mean = 38.8h, standard deviation = 17.47). Local toxicity was almost not observed. This research contributes to the innovation of the nursing clinical practice, since it suggests an alternative for the treatment of phlebitis through the clinical use of phytotherapeutic drugs. (ClinicalTrials.gov Identifier: NCT 00989599).


Introduction
Phlebitis can be considered a temporary or permanent limiting factor for treatment continuity in cancer patients, as, after its occurrence, the peripheral venous catheter should be immediately removed (1) .
Depending on the extent of the inflammatory process established at the venipuncture site, the vascular endothelium suffers irreversible injuries, such as phlebosclerosis, so that the venous segment cannot be used for new intravenous infusion punctures, nor even for simple blood collection (2) .Thus, phlebitis prevention, control of its evolution after its occurrence and reversal of its characteristic inflammatory signs are needed, mainly in patients whose venous network is already very sensitive as a result of antineoplastic chemotherapy.
Conventionally, as a nursing intervention for phlebitis treatment, topical lukewarm compresses are indicated to reduce the local inflammatory process.
Depending on the phlebitis intensity, however, applying Reis PED, Carvalho EC, Bueno PCP, Bastos JK. compresses alone is not enough to improve the inflammatory process, leading to the need to for medical prescription of systemic anti-inflammatory agents, which constitutes an additional factor for the immune system commitment of cancer patients.Therefore, it is fundamental for Brazilian nursing to start to research, within its professional competency area, on alternative and more effective phlebitis treatment forms, using phytotherapy for example.The Federal Nursing Council establishes and recognizes this practice, through COFEN Resolution 197/1997 (3) , as a specialty and/or qualification for nursing professionals.
According to the "Guia para a realização de estudos de toxicidade pré-clínica de fitoterápicos" (4) [Guide for preclinical phytotherapeutic drug toxicity studies], any and all phytotherapeutic or vegetal drugs under analysis should obligatorily be submitted to toxicological tests.
Toxicity studies need to be conducted with standardized samples of the phytotherapeutic or vegetal drug based on which it is produced.According to the same standard, toxicity should be assessed after the user's exposure to a single or fractionated dose, which should be administered to the patient within 24 hours.

The Lista de Registro Simplificado [Simplified
Registry] by the Brazilian Health Surveillance Agency (5) already contains Chamomilla recutita (L.) Rauschert (Asteraceae), so that proving efficacy and safety is not The physical-chemical evaluation of C. recutita inflorescences was done in accordance with the methods described in Farmacopéia Brasileira (6) and USP 28 (7) .A sample of about 500.0 g was grinded and submitted to physical-chemical characterization, including identification, purity and integrity tests and marker dosage.This dosage consisted in the quantification of total flavonoids in the floral capitula and α-bisabolol in essential oil.

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Total flavonoid content in the floral capitula of chamomile was determined by 425 nm spectrophotometry, according to an adapted method described in literature (8)(9) .Total flavonoid concentration (expressed in quercetin) was calculated per 100.0 g of the sample.The same procedure was applied to determine total flavonoid contents in the four infusion dosages (Table 1), with a view to better characterization and comparison with future study data.
α-bisabolol content was determined by gas chromatography with flame ionization detector (GC-FID).
Therefore, the essential C. recutita oil was previously extracted, using hydrodistillation with a Clevenger apparatus.Triple analysis was performed, using an analytic α-bisabolol curve ranging from 200.0 to 1000.0 µg/mL, using piperonal as internal standard.
Finally, the main constituent elements present in the essential oil were identified, using gas chromatography with mass detector (GC-MD).effects obtained during the clinical application (10) .
The research was carried out at a public hospital in the Federal District, which is a tertiary referral institution for cancer care and offers 18 hematology and clinical oncology beds.The sample comprised 25 patients who formally agreed to participate by signing the Informed Consent Term (ICT).All patients had been diagnosed with degree-2 phlebitis according to the staging proposed by the Infusion Nursing Society (11) and were between 20 and 30 years old.Thirteen participants were women and 12 men, whose white blood cell count showed adequate normality levels for neutrophil (2000 -7500/µl) and monocyte (100 -800/µl) (12) counts, medically diagnosed recutita infusion when applied in the trial groups (Table 1) or with lukewarm water in the control group.In all groups, temperature was set at 38 °C.
The graphs showing the dose-response curve are semi-logarithmic (10) , with the dose axis showing the drug concentration in exponential progress.Hence, in this study, the researchers took care to establish the following dose concentrations, specified in Table 1.Compress application time was set at 20 minutes for all groups and compresses were changed every 5 minutes.
Only the main researcher applied the intervention in all patient groups.After the phlebitis diagnosis, the application time of the first compress was registered.
The treatment site was assessed daily at three distinct times: at 8, 13 and 19 hours, with a view to uniform readings.The intervention was continued until the complete disappearance of the erythema, considered the main outcome.
The erythema was chosen as the parameter to assess the inflammatory regression as this is a classical sign of any inflammation and an objective data, present in degree-2 phlebitis (local pain, erythema and/or edema), according to the staging proposed by the Infusion Nursing Society (11) .Thus, the erythema regression time constituted a safe parameter to monitor the inflammation, offering the advantage that nursing professionals know and identify clinical phlebitis staging criteria very well, entailing additional security for assessment precision.To measure to erythema, transparent paper ruled in one-centimeter squares was used, based on which the erythema area was calculated very precisely, so that any alteration in the dependent variable could be identified. .
One-way variance analysis (ANOVA) was used for statistical analysis of the results, followed by multiple comparison test -Bonferroni test (13)(14) , using Graph Pad Prism* software, demo version, 5.0 for Windows.
Significance was set at 5% in all tests.Approval for this research project was obtained from the Institutional Review Board of the Federal District Health Secretary, opinion number 062/2004.

Results
Intergroup comparison showed a statistically significant difference in the groups that received 2.5% and 5% infusions (i.e.corresponding to total flavonoid contents of 0.04 and 0.08 mg/mL, respectively) regarding the phlebitis regression time in comparison with the other groups (concentrations of 1.25% and 10%) and the control group (Figure 1) Erythema regression time in the study sample ranged between 19 and 120 hours for different dose concentrations used.The group in which the 2.5% concentration compress was used showed the shortest regression times: 19 and 24 hours (Table 3).The doseresponse curve (Figure 2) evidences that the average phlebitis regression time was shorter for the 2.5%

Dose (%) Regression time (h) Dose-response curve
As for toxicity, moderate to severe itching was reported on the left forearm of one of the patients allocated in trial group C, whose compress had been applied on the anterior front forearm.As the itching expanded, the transparent PVC film used on the entire forearm could have caused this.The subject was forwarded to the medical team, medicated with an antihistaminic drug and showed complete regression of the itching within two hours.Local treatment was continued with warm water compresses at 38° C until the complete regression of the erythema.
With regard to toxicity evaluation in subjects without phlebitis (n=4), no manifestations of hypersensitivity reactions occurred, nor reports of burning, itching or any other symptoms related with possible hypersensitivity to the drug.

Discussion
It is fundamental to determine the total flavonoid contents when assessing a plant's quality, especially in studies that use flavonoids for therapeutic purposes (15) .Tests involving about 100 samples of 12 chamomile varieties cultivate in identical conditions showed flavonoid contents ranging between 1.0 and 2.6%, while twenty other samples of different origins showed total flavonoid content levels varying between 0.3 and 3.0% (15) .The sample used in this study showed 2.5% m/m of total flavonoids, in line with previously found results.As for α-bisabolol content in essential oil, the level identified in the sample (10.9% m/m) surpassed literature recommendations according to the used temperature, which is 7% (15) .Qualitative analysis of the plant's active principles confirmed available literature data about the chemical composition of C. recutita, describing terpenes (α-bisabolol, bisabolol oxide A and B, camazulene and sesquiterpenes), flavonoids (apigenin-7-glucoside, luteolin, quercetin), coumarins (umbelliferone) and steroids (16) .It should be reminded that these elements, terpenes, steroids and sesquiterpenes exert antiinflammatory effects, generally inhibiting the classical route of the complement system, interfering, in turn, in arachidonic acid metabolism (17) .
With regard to the clinical phase, research subjects in all groups were comparable, as the main confounding factors, including age, sex, white blood cell count and baseline disease were similar.Among the 25  recutita -the compress could not be totally moistened with the quantity of solvent used.This quantity did not need to be adjusted though, as lower doses had already demonstrated an excellent effect in terms of regression time of the inflammatory process.
Other clinical trials have confirmed the antiinflammatory effect of C. recutita in radiation dermatitis, through the use of ointments with chamomile extract (Kamilosan ® ) (18) , in mucositis, through oral solution (Kamilosan ® Solução Oral) (19) , in contact dermatitis and eczema, through ethanolic extract (20)(21) , showing even superior efficacy results when compared with steroidal and non-steroidal anti-inflammatory drugs (21) .
Researchers (22) have assessed the efficacy of medical As for the dose, literature recommends concentrations between 3 and 10% for external use in compresses (23)(24) .It was observed, however, that with 2.5% concentrations, results were as satisfactory as with 5% concentrations, to the extent that the shorted regression time of the inflammation process was obtained in two patients, i.e. 19 and 24 hours.
Both concentrations showed statistically significant results when compared mutually (p<0.05) and with the control group (p<0.001).When compared with the other concentrations (1.25% and 10%), the 2.5% dose showed a statistically significant difference (p<0.05), while the 5% dose showed no statistically significant difference with the other doses.
Therefore, the researchers decided to choose the in 250 mL of water (25) , i.e. 2.4%, which is basically the same standard dose found in this research phase.The same source highlights that concentrations between 3 and 10% are indicated for ointments and gels.
It is also important to highlight that using less grams of floral capitula with better results in terms of time for phlebitis regression generates a better cost-benefit relation and, hence, greater advantage for consumers, due to the obvious economy of the main resource.
Itching was reported in one of the patients allocated in trial group C, with a 5% infusion concentration.The reported hypersensitivity reaction -itching -occurred across the subject's left forearm, who classified it as moderate to intense when the researcher asked about the intensity, although the compress had only been applied on the anterior front middle third of the left forearm.
With regard to this episode, although allergic reactions to C. recutita are quite rare, these can happen, to the extent that one of the exclusion criteria was exactly the patient's affirmative response as to any adverse reaction to chamomile or any plant from the Asteraceae or Compositae family.
It should be clarified that the sites where the compresses were applied, in the trial as well as in the toxicity control group, were assessed for an additional two days after the application, with a view to investigating late signs and symptoms of toxicity.This was not verified in any of the other subjects who accepted to participate in the research.As for the itching in one of the sample subjects, the symptom regressed within 72 hours, without the need for any medication intervention.

Conclusion
The standardization of vegetal raw material, ranging from the selection of the species, seeding, cultivation, harvesting, drying, storage and quality assessment is fundamental, mainly when used for therapeutic purposes, like in the case of this research.
Based on the results, it can be inferred that, at This research contributes to the innovation of clinical nursing practice, as it suggests a treatment alternative for phlebitis deriving from peripheral intravenous infusion during antineoplastic chemotherapy.Moreover, theoretical support is provided regarding the methods to adopt for the clinical use of phytotherapeutic drugs.
needed.Infusion is not considered a pharmaceutical form yet in Brazil, however -as opposed to what happens in other countries like Germany, which considers C. recutita infusion a pharmaceutical form.This research aimed to estimate the ideal dose, for anti-inflammatory purposes, of C. Recutita floral capitula infusion, in patients with phlebitis due to peripheral intravenous infusion of antineoplastic chemotherapy, as well as to assess the toxicity of this infusion in human beings.Besides, the vegetal species used was especially grown, standardized and characterized with a view to certification for medicinal use and later validation of its therapeutic efficacy and safety, as a secondary study goal.Method Cultivation, standardization and characterization of C. recutita "Cultivar Mandirituba" C. recutita seeds were used for planting, donated by Empresa de Assistência Técnica e Extensão Rural do Paraná (EMATER-PR) [Paraná State Rural Technical Assistance and Service Company].Organic planting was followed, in an aviary bed, located in a nursery in the Medicinal Plant Garden of the Federal University of Mato Grosso do Sul (HPM-UFMS).Sowing occurred in May 2005.During the cultivation cycle, weed control, performed with the help of a hoe, and irrigations through sprinklers were done whenever necessary.No pesticides were used directly on the plants to control plagues or diseases.Manual harvesting occurred in September 2005 by technicians from HPM-UFMS.The drying of the collected floral capitula was done in a forced-air circulation glasshouse with temperature ranging between 36º ± 2ºC, to constant mass, resulting in 56.57 million ha -1 , equivalent to 8kg of floral capitula of C. recutita.After drying, the floral capitula of C. recutita were stored in lidded glass pots and kept refrigerated.

α
-bisabolol content in the essential oil could be quantified through the equation of the straight line obtained from the analytic curve with internal standardization (R = 0.9993), corresponding to 10.9% m/m.According to GC-MD analysis of the essential oil, five major peaks of the plant's main active principles could be clearly identified, which should be highlighted: α-bisabolol oxide B (19.6%); α-bisabolone oxide (5.2%); α-bisabolol (9.0%); camazulene (1,3%); α-bisabolol oxide A (40.7%), besides spathulenol, β-elemene, limonene oxide, β-farnesene and d-nerolidol.Clinical Phase A dose response curve trial was carried out, in which the therapeutic efficacy of different C. recutita infusion doses was analyzed and compared in cancer patients with phlebitis deriving from peripheral intravenous infusion of antineoplastic chemotherapy, looking at the anti-inflammatory potential.Dose response curve studies demonstrate the relation between the dose (concentration) of an administered drug and the produced tissue response or effect, permitting knowledge on the adequate dose based on the wanted and unwanted

with
Acute Myeloid Leukemia (AML), submitted to the first, second or third cycle of the chemotherapy protocol IDA + ARA-C (idarubicin and cytarabine), through peripheral intravenous infusion.The exclusion criteria were the patients' affirmative response when asked about previous adverse reactions to chamomile or any plant in the Asteraceae or Compositae family, medical prescription of systemic or topical antiinflammatory drug on the phlebitis site and refusal to continue participating in the study.The primary researcher established the criterion to allocate the subjects to the groups as follows: the first selected patient was automatically allocated to trial group A, the second to group B, the third to group C, the fourth to group D and the fifth to the control group.This process was repeated until 25 patients had been allocated.Data were collected between September and December 2005.Patients who complied with the selection criteria were allocated in five groups, one of which was the control group.A 20 cm 2 cotton compress was used for the intervention, moistened with the C.
plant infusions (chamomile, salvia and calendula) for topical application in the treatment of Hand-Foot Syndrome resulting from intravenous capecitabine infusion, an antineoplastic chemotherapy drug used in breast cancer patients.The sample comprised 11 patients who immersed their hands and feet into the infusion daily.Significant regression of the inflammatory process was found in all cases.

2 .
5% concentration (10 g/400 mL) for floral capitula infusions of as the standard dose for this study, although literature recommends using concentrations between 3 and 10% for compresses.This small difference of -0.5% between the result obtained in the dose-response curve and the concentration recommended in literature (at least 3%) can be attributed to the excellent quality of the test sample, of Brazilian origins.Besides, it should be highlighted that literature itself shows different variations in terms of quantity definitions and even measurement units.The US Pharmacopeia, for example, indicates the use of two dessert spoons for external use, equivalent to approximately 6g of dry floral capitula of C. recutita

α
= 5%, the C. recutita infusion presents minimal or almost zero toxicity for topical application.This study also demonstrated that the 2.5% concentration for floral capitula infusion of C. recutita, when applied for antiinflammatory purposes in case of phlebitis deriving from peripheral intravenous chemotherapy infusion, is as effective as the concentration suggested by literature, i.e. between 3 and 10%.www.eerp.usp.br/rlaeRev. Latino-Am.Enfermagem 2011 Jan-Feb;19(1):3-10.

Table 1 -
Physical-chemical characterization of C. Recutita inflorescences

Table 2 -
Distribution of patient groups according to infusion dose day (morning, afternoon and night), according to the dose established in the group the patient had been allocated to, i.e. trial group A, B, C, D or control.As soon as the compress had been applied, the limb was wrapped in transparent PVC film to preserve local heat.

Table 3 -
Regression time, in hours, of phlebitis according to infusion dose group of C. recutita floral capitula Figure 2 -Dose-response curve according to mean regression times, in hours and infusion concentrations of 1.25%, 2.5%, 5% and 10%, respectively, of C. Recutita floral capitula, obtained through ANOVA with Bonferroni post-test