Nghiên cứu độc tính và hiệu quả của dịch chiết từ rễ cây ba bét lùn (mallotusnanus airy shaw) điều trị bệnh trứng cá thông thường tiếng anh

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Nghiên cứu độc tính và hiệu quả của dịch chiết từ rễ cây ba bét lùn (mallotusnanus airy shaw) điều trị bệnh trứng cá thông thường tiếng anh

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1 Part A: INTRODUCTION OF DISSERTATION BACKGROUND Acne is a chronic inflammatory disease in pilosebaceous unitskin structures including hair follicles and their associated sebaceous glands Acne affects up to 8090% of people aged 13-25 and more than 30% of those need to be treated Clinically, it is characterized by various lesions: papules (nodules), blackheads and whiteheads (comedones), pustules Although acne tends to disappearover time, some may cause scarringleading to lifelong problems Hebal medicines for acne seem to have inhibitory sebum production, disfigure sebaceous follicles, kill P acnes, and reduce the inflammatory response However, more investigations are needed to clarify mechanisms of action, adverse effects and toxicity of these plants The Mallotus Nanus Airy Shawroots has been used by some ethnic minorities to treat acne vulgaris, so far there are no publication of material on the chemical composition, the biological effect and acne treatment effect of this plants Therefore, we conducted a study with the entitled: "Study on the toxicity and effectiveness of the extract of Mallotus Nanus Airy ShawRoot for treatment Acne vulgaris" Study’s objectives: To determine the acute, subacute toxicity, skin and eye irritation of the extracts of Mallotus nanus Airy Shawin animals; To evaluate the efficacy of extracts of Mallotus nanus Airy Shawin P.acnes and acne vulgaris in animal model; To evaluate the efficacy and undesired effects of the topical extracts of Mallotus nanus Airy Shawin acne vulgaris Practical contribution and new findings of the dissertation - This is the first time the Mallotus nanus Airy Shaw root has been studied on toxicity, bioactive and pilot study about effeciveness of acne vulgaris treatment on voluntiers - It is the first time, inducing acne on animal model (rat and rabbit) had been - The effectiveness of Mallotus nanus Airy Shaw root extract on standard strain of P acnes and 02 strains isolated from the patients - The findings of the model and clinical pilot studies suggest that the MN root extract can clear inflamatory and non-inflamatory acne lesions after 12 weeks of applying - Finally, patients had got more serious side effects so they got more than effective in pimple, dark scare and concavescar clearance Structure of the dissertation Besides Introduction and Conclusion part, the thesisincludes chapters: - Chapter Literature Review (31 pages) - Chapter Research Design (26 pages) - Chapter Research Result (32 pages) - Chapter Discussion (40 pages) The thesisincludes 27 tables, 11 charts, 32 images and 170 references (Vietnamese: 26; English: 111; Chinese: 33) Part B: CONTENTS OF DISSERTATION CHAPTER 1: LITERATURE REVIEW 1.1 Acne vulgaris: mordern medicine 1.1.1 Definition According to A.M Layton, acne is a chronic inflammatory disease ofpilosebaceous unit skin structures, characterized by increased sebum secretion, comedon formation, papules and pustules; even cysts and deep pustules; in many cases, various degrees of scarring The multifactorial pathogenesis of acne including: (i) increased sebum production, (ii) horned sebaceous gland, (iii) abnormal residence of P acnes and (iv) inflammatory 1.1.2 Pathogenesis 1.1.2.1 Genetic factors 1.1.2.2 Increases sebum secretion and the role of sebum 1.1.2.3 Hirsute capillaries of the pubic hair 1.1.2.4 Inflammatory response 1.1.2.5 The role of Propionibacterium acnes 1.1.2.6 Effects of hormones 1.1.2.7 Diet 1.1.2.8 Factors related to acne Age, gender and factors: family, weather, race, occupational, stress, endocrine diseases, drugs,… 1.1.3 Clinical manifestations of common acne vulgaris 1.1.3.1 Non-inflamatory lesions open and closed comedones 1.1.3.2 Inflammatory lesions papules, pustules, cyst formation 1.1.2.3 Distribution The distribution of acne commonly over face, shoulders, chest and back 1.1.4 Classification of the clinical level of acne vulgaris 1.1.4.1 Classification by Cunliffe et al (2003) 1.1.4.2 Classification by Hayashi et al (2008) 1.1.4.3 Current Measures for the Evaluation of Acne Severity 2008 1.1.5 Treatment 1.1.5.1 Topical treatment: retinoic acid (vitamin A), benzoyl peroxide, antibiotic, Azelaic acid (C9-dicarbonic acid), Salicylic acid, Dapson 1.1.5.2 System treatment Antibiotics, Hormones, Isotretinoin (13-cis-retinoid acid) 1.2 Acne vulgaris: traditional medicine 1.2.1 Acne vulgaris in classic literature 1.2.2 Cause and pathogenesis 1.2.3 Argumentation 1.2.4 Treatments 1.2.4.1 Treatment formulars Decotion, herbal cleansers, herbal based patches, herbal based spray mist 1.2.4.2 Non-medicated method Acupuncture, hot- Acupuncture, ear- Acupuncture 1.3 Some models cause acne in laboratory animals 1.3.1 Rabbit ear model 1.3.2 Rat ear model 1.4 The studies on acne treatment in Vietnam and in the world 1.4.1 Research on conventional acne treatment with traditional medicine in Vietnam 1.4.2 Herbal treatment for acne vulgaris in the world 1.5 Research Overview of Mallotus nanus 1.5.1 Characteristics of the herbal - Mallotus nanus (MN), the castor plant (Euphorbiacea) Two new derivatives of 2-C-beta-D-glucopyranosyl benzoic acid is called mallonanoside A (1) and B (2) were isolated from leaves of Mallotus nanus along with five known flavonoids: kaempferin (3), juglanin (4), quercitrin (5), myricitrin (6) and rhoifolin (7) 1.5.2 Chemical compositions Five clean substances have been isolated: (1) Palmitic acid, (2) Stigmast-4en-3-one, (3) β-Sitosterol, (4) Mallonanoside A, (5) Daucosterol 1.5.3 Research on biological activity 1.5.3.1 Anti-inflammatory activity 1.5.3.2 Antioxidant activity 1.5.3.3 Cellular Toxicity 1.5.3.4 Tested Antimicrobial Activity Chapter RESEARCH DESIGN 2.1 Research material 2.1.1 Materials of experimental research Root of Mallotus nanus (BBL) was dried at 55-600C, powdered, 96% ethanol extracted by Soxhlet method, distilled, the solvent was evaporated under vacuum (vacuum method) and fully extracts of the BBL root BBL root extracts: gram equivalent to 22.7 grams of dried medicinal plant 2.1.1.2 Research material for BBL extracts - BBL root extracts at concentrations of 0.2 mg / mL, 2.2 mg / mL, 4.4 mg / mL, 8.8 mg / mL, 17.6 mg / mL - Dosage: +0.05g /0,5ml (BBL 10%): extracts 4.4 mg / mL; +0.1g / 0.5mL (BBL 20%): extracts 8.8mg / mL; +0.2 g / 0.5 ml (BBL 40%): extracts17.6 mg / mL - another chemicals, tools, v.v 2.1.2 Material for clinical study - BBL10% (0.05g/0.5mL) called Dr Hoa Acne Clearer, standardized test 2.2 Research objects 2.2.1 Objectives of experimental research Swiss strain mice, Newzealand White rabbit, adult male Wistar strain rat, P acnes strain ATCC 6919 and P acnes isolates from acne patients 2.2.2 Objectives of clinical studies 112 patients were diagnosed with acne vulgaris Patient Inclusion and Exclusion Criteria from Studies - Patient Inclusion according to modern medicine Tan 2008 (Current Measures for the Evaluation of Acne Severity) - Patient Inclusion according to traditional medicine: “Wind-HeatLung channel ”and “Humidity – Heat stomach- spleen”type - Exclusion Criteria: Patients use: detergent (a week ago), alpha-hydroxy acids, topical retinoid, antibiotics, topical or systemic steroids in the previous weeks, estrogen months ago, tretinoid beforr months 2.3 Methods 2.3.1 Method of 2.3.1.1 Acute Toxicity experimental research Determination of LD50 of BBL root extract, subcutaneous injection in white mice by Litchfield - Wilcoxon method 2.3.1.2 Skin irritation OECD Guideline404 and ISO 10993-10 03 rabbits, each with two concentrations - Rabbits were clippthe dorsaland hiparea (approximately 10 cm x 15 cm), Test chemical and 0.5ml solvent was applied to small erea 2.5 cm x 2.5 cm of skin After hours, residual test chemical should be moved Evaluation and scoring of erythema, oedema from to at hour, 24, 48, 72 hours after removed test specimen chemical 2.3.1.3 Eye irritation OECD guideline405 The test chemicalis diluted0.05g/0.5 mL Three rabbits (numbered from to 3) Drop0.1 mL test substance to right eye of rabbit numbered 1,'s, nothing to the left eye Evaluation at 1h, 24h, 48h, 72h after applying test substance The rabbits should beobservedmaximum21 days 2.3.1.4 subchronic toxicity OECD guidelines 411 Rabbits were divided into groups, each of 10 rabbits The test was performed in 90 days: Control group: 20% ethanol; Treatment group 1: Apply BBL extract0.25 mL / kg / day; Treatment group 2: BBL extract0.75 mL/kg/day (3 times higher than the treatment group 1) to20% rabbit skin area Observation: body weight, hematopoietic function, liver function, kidney function, the histology of skin, kidney, liver 2.3.1.5 Effects of anti P.acnes - Sample collection - Culture and isolation of P.acnes strain - P acnes identification - Susceptibility of P acnes 2.3.1.6 Effect of BBL extract on experimental animals - TC Model inflammation on male mice ear by P acnes Pandey Chetana et al model White mice were randomly divided to groups, mice/group - Lot (white control): injection PBS 5% - Lot (Positive trol):P acnes: apply tetracycline - Lot (Model): P Acnes: apply alcohol solvent - Lot P acnes: apply BBL 10% - Lot P acnes: apply BBL 20% - Lot P acnes: apply BBL 40% Subcutaneous injection 20 µL P acnes 108/ml (right ear) Measure the thickness of the mice's ear at the first two weeks, then every 24 hours, until the rat's ear is normal Microscopic observations Acne model by oleic acid on the outer ear canal Xiao-dong and his et al model Male rabbits were randomly divided to 4groups, mice/group - Lot (model group): Apply 50% oleic acid to external ear canal daily for weeks, then apply 20% alcohol solvent for weeks - Lot (Positive group 1: Locacid 0.05%): Apply 50% oleic acid to external ear canal daily for weeks, then apply Locacid 0,05% for weeks - Lot (positive control 2: Oxy-5): Apply 50% oleic acid to external ear canal daily for weeks, then apply Oxy-5 for weeks - Lot (test substance 1): Apply 50% oleic acid to external ear canal daily for weeks, then apply BBL extract for weeks Observe the external ear canal variation Endoscopic external ear canal: before application, after weeks with 50% oleic acid, and after weeks drug application 7 2.3.2 Method of clinical studies Clinical trials with a pre- and a post-treatment measurement: 112 patients Treatment regimen: BBL Root Extract 0.05 mg/0.5mL (BBL10%) Apply 20 drops/day (equivalent 1mL) to the face, once at 9-10pm; should not be applied around eye areas Research: - Interview by medical history - Physical examination - Treat regimen Evaluate treatment results and adverse effects Compare the number of noninflammatory and inflammatory lesions a pre and a post treatment Evaluate disappearance of lesion according to Tan 2008 and side effects: redness, dryness, scarling and itching sensation 2.4 Ethics in Research Approved by Ethics Board of Hanoi Medical University at No 68B/ HĐHYHN March 25/2017 2.5 Data Analysis Software SPSS 18.0 Chapter RESULTS 3.1 Acute toxicity, skin and eye irritation, subchronic toxicityon experimental research 3.1.1 Acute toxicity LD50 of BBL extract: LD50 = 11.148 (12.753 – 7.938) g BBL subtance/kg TI =LD50/ED50 TI= [(11.148/0.2) x50]:12 = 232.25 3.1.2 Skin irritation Table 3.1 Irradiation index (PII) on rabbits’ skin at 0.05 g BBL subtances/0.5 mL Rabbit PII Rabbit 1st 0.67 Rabbit 2nd 1.33 Rabbit 3th 0.33 From Table 3.1, PII irritation index attwoconcentrations 0.05g/0.5 mL: PII = (0.67 + 1.33 + 0.33) / = 0.78 Based on the PII’s skin irritation classification, 0.05g MN/0.5 mL causes mild skin irritation Table 3.2 The irritation index (PII) on rabbits’ skin at two concentrations 0.2 g substance/0.5 mL Rabbit PII Rabbit 4th 3.0 Rabbit 5th 3.0 Rabbit 6th 3.33 From Table 3.2, the PII irritation index at 0.2g subtance/0.5 mL: (3 + + 3.33) / = 3.11 Based on PII’s skin irritation classification, ND2 (0.2g MN/0.5mL) causes moderate skin irritation 3.1.3 Eye irritation Causing eye irritation at two concentrations of 0.05g/0.5 mL in rabbit No 1, it isn’t observed corneal opacity, iris injury, red lesion, conjunctival edema at 1h, 24h, 48h and 72h after dropping - Take the same method in rabbit No and rabbit No finding same results: it isn’t observed corneal and iris lesions, conjunctivitis at 1h, 24h, 48h and 72h after dropping 3.1.4 Subchronic toxicity 3.1.4.1 General conditions During studying, three groups is normal: activities, eyes, feathers, eat, dry stool and increasing weight No difference between control and studying groups 3.1.4.2 Hematopoietic function Table 3.3 Effect of MN root extract on red blood cells Number of blood cell (T/l )𝑋" ± 𝑆𝐷 Timeline p (t- test Student) Control group Experiment group 1st Experiment group 2nd Before 4.46 ± 0.38 4.31 ± 0.45 4.38 ± 0.57 > 0.05 After 30 days 4.61 ± 0.38 4.51 ± 0.26 4.32 ± 0.50 > 0.05 > 0.05 > 0.05 > 0.05 4.45 ± 0.30 4.59 ± 0.30 4.23 ± 0.42 > 0.05 > 0.05 > 0.05 4.49 ± 0.81 4.17 ± 0.28 4.26 ± 0.37 > 0.05 > 0.05 > 0.05 p (before - after) After 60 days p(before - after) After 90 days p (before - after) > 0.05 > 0.05 There is no significant difference of the number of red blood cells between experiment and control groups (p> 0.05) Table 3.4 Effect of MN extract on hemoglobin concentration in rabbit blood Hemoglobin levels (g/dl ) Timeline p Control Group Experiment Group 1st Experiment Group 2nd (t- test Student) Before 10,80 ± 0.56 10.74 ± 1.39 10.73 ± 0.83 > 0.05 After 30 days 10.88 ± 0.53 11.05 ± 0.54 10.41 ± 1.23 > 0.05 p (before - after) > 0.05 > 0.05 > 0.05 After 60 days 11.06 ± 0.42 10.90 ± 0.41 11.00 ± 0.77 p (before - after) > 0.05 > 0.05 > 0.05 After 90 days 10.82 ± 0.39 10.62 ± 0.40 10.51 ± 0.80 p (before - after) > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 10 Results show that there is no significant difference between experiment (hemoglobin and hematocrit)and control groups (p> 0.05) Table 3.5 Effects of MN root extract on the leukocyte numbers Number of leukocyte (G/l)𝑋" ± 𝑆𝐷 Timeline p (t- test Student) Control group Experiment group 1st Experiment group 2nd Before 4.95 ± 0.89 4.70 ± 1.26 4.88 ± 0.83 > 0.05 After 30 days 5.22 ± 1.37 5.00 ± 2.08 4.82 ± 1.39 > 0.05 > 0.05 > 0.05 > 0.05 5.19 ± 1.32 4.93 ± 0.82 5.20 ± 1.15 > 0.05 > 0.05 > 0.05 4.93 ± 0.81 4.81 ± 1.16 5.20 ± 1.26 > 0.05 > 0.05 > 0.05 p (before - after) After 60 days p (before - after) After 90 days p (before - after) > 0.05 > 0.05 When using MN root extract, there is no significant difference between both groups in the number of leukocyte (p> 0.05) Table 3.6 Effect of the MN root extract on platelet Number of platelet(G/l)𝑋" ± 𝑆𝐷 Timeline p (t- test Student) Control Group Experiment group 1st Experiment group 2nd Before 318.80 ± 53.23 317.50 ± 71.92 317.33 ± 60.81 > 0.05 After 30 days 324.80 ± 58.62 313.60 ± 53.91 315.10 ± 39.68 > 0.05 p (pre - post) > 0.05 > 0.05 > 0.05 After 60 days 317.30 ± 77.42 314.50 ± 40.30 313.40 ± 31.70 p (pre - post) > 0.05 > 0.05 > 0.05 After 90 days 310.70 ± 84.79 322.20 ± 49.92 312.70 ± 44.38 p (pre - post) > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 11 There is no significant difference between experiment and control group in platelet counts(p> 0.05) and pre and post applying (p> 0.05) 3.1.4.3 Liverfunction Table 3.7 Effects of MNroot extract on ALT concentration Timeline Before After 30 days p (pre - post) After 60 days p (pre - post) After 90 days p (pre - post) ALT (UI/l) p Control Group Experiment group 1st Experiment group 2nd (t- test Student) 51.90 ±11.69 56.90 ± 9.94 55.89 ± 13.92 > 0.05 52.40 ± 14.41 56.40 ± 8.68 53.90 ± 10.42 > 0.05 > 0.05 > 0.05 > 0.05 53.10 ± 16.72 53.20 ± 10.73 54.30 ± 10.52 > 0.05 > 0.05 > 0.05 53.60 ± 9.16 52.30 ± 6.33 52.00 ± 4.92 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 There is no significant difference between control and experiment groups in AST and ALT levels in rabbit blood (p> 0.05) 3.1.4.4 Renal function Table 3.8 Effect of MN root extract on creatinine concentration Creatinin (mg/dl) P Timeline Control Group Experment Group Experment Group (t- test Student) Before 1.05 ± 0.05 1.06 ± 0.05 1.04 ± 0.05 > 0.05 After 30 days 1.05 ± 0.05 1.06 ± 0.05 1.05 ± 0.05 > 0.05 p (pre - post) > 0.05 > 0.05 > 0.05 12 After 60 days 1.05 ± 0.05 1.05 ± 0.05 1.04 ± 0.05 p (pre - post) > 0.05 > 0.05 > 0.05 After 90 days 1.05 ± 0.05 1.04 ± 0.05 1.05 ± 0.05 p (pre - post) > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 There is were no significant difference between group 1st and group compairing control group nd 3.1.4.5 Morphology of rabbit liver, kidney and skin In general, histopathology ofrabbit liver, kidney and skin arenormal before and after 90 days 3.2 Activity against P acnes and treatment acne on experimental animal models 3.2.1 Sensitivity of P acnes to MN root extract Table 3.9 Sensitivity of P acnes ATCC to MNroot extract Extract Concentrati on (mg/mL) Blood geckos Chocolate KK GC MN 0.2 Not grow Not grow Not grow Not grow MN 2.2 Not grow Not grow Not grow Not grow MN 4.4 Not grow Not grow Not grow Not grow MN 8.8 Not grow Not grow Not grow Not grow MN 17.6 Not grow Not grow Not grow Not grow MN+P acnes 0.2 grow grow grow grow MN+P acnes 2.2 grow grow grow grow MN +P.acnes 4.4 grow grow grow grow MN+P acnes 8.8 Not grow Not grow Not grow Not grow MN+P acnes 17.6 Not grow Not grow Not grow Not grow grow grow grow grow Alcohl 20% +P acnes MN root extract at 8.8 mg /mL concentration inhibition growth of P acnes ATCC 13 3.2.1.2 Sensitivity of P acnes isolated from patients with MN root extract Table 3.10 Sensitivity of P.acnes isolated from patients with MN root extract MN Extract MN10% (0.05g MN/0.5ml) Concentration (mg/mL) Blood geckos Chocolate KK GC Not grow Not grow Not grow Not grow MN 20%(0.1g MN/0.5ml) (8.8mg/mL) Not grow Not grow Not grow Not grow MN10% + P.acnes Not grow Not grow Not grow Not grow (4.4 mg/mL) MN20% + P.acnes Not grow Not grow Not grow Not grow (8.8mg/mL) (4.4 mg/mL) MN 10% root extract: 4.4 mg / mL (0.05 g MN / 0.5 ml) and MN 20%: 8.8 mg / mL (0.1 g MN/ 0.5 ml) inhibit growth of P acnes isolated from the patient 3.2.2 Effect of MNroot extract on model of white ear rats with P acnes Table 3.11 Mice’sear thickness after 48h causinginflammation No (n=48) Before 𝑋" ± 𝑆𝐷 After 48h 𝑋" ± 𝑆𝐷 ppre and post Group of injection of PBS solvent 295.34 ± 15 320.24 ± 26 > 0.05 Group of injection 293.21 ± 13 644.12 ± 95 P.acnes p 2-1 > 0.05 p 2-1 < 0.001 < 0.001 The table 3.11 shows that rabbit ear thickness of P acnes group is more 2.2 times than before injection (p 0.05) 3.2.2.3 Effects of MN root extract on inflammation of white mice ear 14 Ear thinkness µm 800 700 600 500 400 300 200 10 12 14 16 18 20 22 24 26 Days Ịnection PBS Applying tetracyclin Applying alcohol BBL 10% Chart 3.1 The change of rat ear thickness after 26 days applying MN extraction Group (PBS inoculation), there was no change of thickness day by day (p> 0.05) Group 2nd (model) daily, rat’s ear is normal after 26 days While group3th (positive control group) applied tetracycline, rat ear thickness returned to normal from day 16th Group using MN10%, rat ear returned to normal from 22nd day Using MN 20% and 40%, rat ear returned to normal after 26 days 3.2.3 Effect of MN root extraction acne model with oleic acid on rabbit external ear cannal Table 3.12 Summary of histology after weeks of application Group Rabbit ear histology (right) Group 1st: Model Hyperkeratosis of the sebaceous gland, larger sebaceous gland, acnes with stage1 Group 2nd : alcolh Hyperkeratosisof the sebaceous gland, larger sebaceous gland, acnes with stage Group 3th: Locacid hair folliclesand sebum are nomal 0,05% Group 4th: Oxy-5 hair folliclesand sebum are nomal Group 5th: MN10% hair folliclesand sebum are nomal 3.3 Therapeutic and side effects of MNroot extract 3.3.1 Patient characteristics 3.3.2 Therapeutic effect of MNroot extract Table 3.13 Changes in count of lesions after 4, and 12 weeks of treatment 15 T0 T4 T8 T12 Timeline 𝑋" ± 𝑆𝐷 𝑋" ± 𝑆𝐷 𝑋" ± 𝑆𝐷 𝑋" ± 𝑆𝐷 Noninflammatory papules 57.17±19.39 65.03±26.13 28.94±21.15 3.72±9.45 18.17±13.22 14.57 ± 12.96 5.77± 8.73 0.85 ± 3.81 Number of lessions 75.61 ± 27.25 79.6±33.19 34.71±26.79 4.57±12.81 n 109 105 105 105 Inflammatory papules The mean values of black and white heads increasing from 57.17 ± 19.39 at baseline to 65.03 ± 26.13 at week 4th and then rapidly decreasingto 28.94 ± 21.15 at week 8th and almost out of 3.72 ± 9.45 lesions at week 12th The mean values of inflammation lessions (papules and pustule) was reduced from 18.17 ± 13.22 at baseline to 14.57 ± 12.96 at week 4th and then decreased rapidly 5.77 ± 8.73 at week 8th and almost out of 0.85 ± 3.81 lesions at week 12th The mean values of inflammatory and noninflammatory lesions tend to increase slightly at the first weeks of treatment: from 75.61 ± 27.25 at baseline to 79.6 ± 33.19 at week 4th, after that decreasing rapidly to 34.71 ± 26.79 at week 8th and fewer to 4.57 ± 12.81 at week 12nd 90.5 87.6 100 81 80 79 60 40 20 12.4 T0 9.5 5.7 T4 Clean 13.3 T8 medium 20 T12 light Chart 3.2 Clearness of lesions: Current Measures for the Evaluation of Acne Severity 2008 16 On the scale of Current Measures for the Evaluation of Acne Severity in 2008,lesions were clearly seen in 79%, 20% of patients were mild (a few lesions) and only 1% was moderate 200.00 150.00 64,88±17,69 100.00 50,88±18,56 66,75±23,64 62,22±29,85 50.00 30,62±22,35 26,23±19 0.00 T0 Lung chanel hot wind T4 4,52±11,30 2,42±5,05 T8 T12 Tuần Stomach-spleen cold temperature Chart 3.3 Correlation between comedones with wind - heat Lung channel Counts of noninflammatory lesions at the first examination at“Humidity – Heat Stomach-Spleen” type was higher than that of the wind - heatLungchanel and both of them tend to increase after weeks of administration They decrease rapidly at8th week(26, 23 ± 19) and fewly at week 12th 60.00 50.00 40.00 24,92±12,31 30.00 18,65±13,81 20.00 10.00 12,67±11,32 0.00 T0 T4 Lung chanel hot wind 8,45±10,10 7,95±7,901,42±2,14 1,34±4,79 0,05±0,32 T8 T12 Tuần Stomach-spleen cold temperature Figure 3.4 Correlation between inflammatory papules wind - heat Lung channel type and “Humidity -heat Stomach-Spleen” type 17 Counts of inflammatory lesions at the first examinational the “Humidity -Heat Stomach-Spleen” type was twice than that of “wind - heat Lung channel” type Both of them tend to decrease after weeks of administration and decrease rapidly at week 8th and at week 12th, there was a few 3.3.3 The undesirable effects of MN root extract was applied to acne vulgaris patients Table 3.14 Evaluation of redness, dryness, scaly skin and pruritus T0 T4 T8 T12 Timeline 𝑋" ± 𝑆𝐷 𝑋" ± 𝑆𝐷 𝑋" ± 𝑆𝐷 𝑋" ± 𝑆𝐷 Redness skin 0.19±0.28 0.83±0.53 0.61±0.52 0.08±0.22 Dry skin 0.17±0.26 0.97±0.44 0.95±0.54 0.32±0.39 Scaly skin 0.16±0.26 0.96±0.48 0.94±0.53 0.18±0.35 Burn/pruritus 0.12±0.23 0.46±0.54 0.25±0.41 0.01±0.10 Itchy 0.1±0.22 0.66± 0.49 0.45±0.55 0.01±0.10 n 109 105 105 105 Patients were the highest level in redness, dry, scaly skin at week 4th and going to week 8th, after that decreased gradually to 12 weeks Almost all patients got mild reaction The peak burning sensation is mild level (0.46 ± 0.54) at week 4th and rapidly decreased to week 12th, most patients had no burning sensation (0.01 ± 0.10) Almost all acne suffers had mild to moderate itching reactions The highest level occurred at week 4th (0.66 ± 0.49) and then decreased gradually to week (0.45 ± 0.55), finally, decreased rapidly to week 12th (0.01 ± 0.10) most patients had no itching Chapter DISCUSSION 4.1 Safety of Mallotus nanus 4.1.1 Acute toxicity LD50 was determined to be 11.148 (12.753 - 7.938) grams of the drug extracted from MN per kg, and the treatment index of the sample was 232.25 According to the World Health Organization (WHO) guidelines for herbal medicines with a TI> 100, this drug has a low level of toxicity 4.1.2 Skin irritation 18 With sample concentration of 0.05 g MN / 0.5 mL (BBL10%), the PII rabbit skin irritation index was 0.78 (table 3.1), indicating a light degree of skin irritation With sample concentration 2, at 24 hours, 48 hours and 72 hours, erythema was clearly seen in all three rabbits In rabbits 4, and 6, erythema also spread beyond the lesion (Table 3.2) Skin edema was not observed in all rabbits PII irritation at 0.2 g / 0.5 mL: 3.11 causes moderate skin irritation This suggests that the skin irritation level of the BBL root extracts be dose dependent In other words, the higher the doses, the greater the degrees of irritation Thus, when administered in humans should start with a concentration of (0.05 g / 0.5 mL) 4.1.3 Eye irritation BBL root extracts are clinically used to treat facial acne for three months; hence, it is important to ensure that this product causes no irritation to the eyes To examine eye irritation due to BBL root extracts, 0.1 mL of a test substance was applied to the conjunctival sac of the rabbit's right eye, while the left eye was not Corneal, iridium, and conjunctival injuries were examined at hour, 24 hours, 48 hours, and 72 hours after dosing The results show that the entire observation saw no corneal lesions, iris and conjunctiva (Table 3.6) Under the OECD guidelines, it can be seen that BBL root extracts 10% did not irritate rabbits’ eyes 4.1.4 Semichronic Toxicity The WHO recommends examination of the systemic condition, body weight and hematologic indices to assess drug toxicity Blood is a body fluid of great importance as it is closely related to all body parts and organs Pathologically, blood is affected by all of those parts and organs; however, at the same time, it is affected by and reflects the particular condition of the blood-forming organs If the drug affects the hematopoietic system, the blood components will first be changed Particularly, the counts of white blood cells tend to reduce The above parameters of rabbits in both treatment groups saw no statistically significant difference when compared to those before the drug was administered and to those found in the control group at the same time BBL extracts did not cause toxicity to the systemic condition and hematopoietic organs (Table 3.3, 3.4, 3.5) and (Figure 3.1 and 3.2) The liver, another organ, has many important functions When a drug is put into the body, it can be toxic to the liver, affecting the liver’s function Therefore, when assessing drug toxicity, it is crucial to study the effects of drugs on liver function In order to identify the extent of hepatocellular injury, it is usually necessary to quantify levels of liver-derived enzymes present in the serum Their increased levels are often associated with drug toxicity because the drug causes damage to liver cells After 90 days of treatment, both A and B of ALT, AST were within normal limits (Figure 3.3, 3.4) 19 Kidneys are one of the organs of excretion that make up the excretory system Kidney tissues are highly vulnerable to endogenous and exogenous substances Therefore, when administered, the drug can be toxic to, and therefore damage kidneys, thereby affecting the kidney function Evaluation of renal function after drug administration is usually used to measure the blood creatinine level Creatinine is the most stable protein in the blood and hardly dependent on diet or physiological changes; however, its elimination largely depends on kidneys In case of glomerular damage, blood creatinine concentrations rise earlier than those of urea Creatinine is a reliable and important indicator of blood urea; it is used to evaluate and monitor kidney function The serum creatinine concentration in rabbit blood was not significantly different from that in the control group (p> 0.05) Furthermore, microscopic examination is the gold standard for assessing lesions of the two main organs responsible for metabolism and excretion Topical administration, or more specifically application to the skin, requires direct microscopic examination of the skin In all of the studied rabbits, no pathological changes were observed in the general appearance of the organs No difference in microscopic images of liver and kidney lesions was seen between treatment and experimental groups Microscopic images of the skin in the treatment groups were insignificantly different from those in the control group 4.2 Evaluation of P acnes resistance of the drug and its effectiveness in treating acne in experimental animals 4.2.1 Sensitivity of P acnes to BBL root extracts The minimum inhibitory concentration (MIC) of BBL root extracts preventing the visible growth of the P acne strain ATCC was 8.8 mg / mL The MIC of BBL root extracts in the case of P acnes isolated from patients was 4.4 mg / mL (Table 3.6, 3.7) This value was less than half the value of the BBL MIC applied to the reference strain A possible explanation is that in Vietnam, medication management is not strictly implemented; when falling sick, its citizens not seek healthcare from healthcare settings, but purchasing medications without a valid prescription In fact, almost all acne suffers used to take antimicrobial medications Besides, acne swabs may contain components that reduce bacterial virulence The experiment shows that the BBL extracts were highly active against P acnes isolates from patients stronger than those from the ACTC isolates 4.2.2 Effects of Mallotus nanus on the experimental animal model Inducing inflammation in white rats’ ear flaps with P.acnes bacteria We injected 20 µl of live bacteria with a concentration of 108 bacteria / mL into the ear flaps of the rats As can be seen in Table (3.8), P acnes showed a significantly increased inflammatory response, with in the thickness of ear flaps reached 644.12 ± 95 µm after 48 hours The difference was statistically significant compared to the time before the study (p

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