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
Trang 1Part 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 80-90% 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:
1 To determine the acute, subacute toxicity, skin and eye irritation of the
extracts of Mallotus nanus Airy Shawin animals;
2 To evaluate the efficacy of extracts of Mallotus nanus Airy Shawin P.acnes
and acne vulgaris in animal model;
3 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
Trang 2Besides Introduction and Conclusion part, the thesisincludes 4 chapters:
- Chapter 1 Literature Review (31 pages)
- Chapter 2 Research Design (26 pages)
- Chapter 3 Research Result (32 pages)
- Chapter 4 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
Trang 3papules, 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
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
Trang 4with five known flavonoids: kaempferin (3), juglanin (4), quercitrin (5), myricitrin (6) and rhoifolin (7)
1.5.3.4 Tested Antimicrobial Activity
Chapter 2 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: 1 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 /
- 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
Trang 5112 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 4 weeks, estrogen 3 months ago, tretinoid beforr 6 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 4 hours, residual test chemical should be moved
Evaluation and scoring of erythema, oedema from 0 to 4 at 1 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 1 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 3 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,
Trang 6hematopoietic 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 6 groups, 8 mice/group
- Lot 1 (white control): injection PBS 5%
- Lot 2 (Positive con trol):P acnes: apply tetracycline
- Lot 3 (Model): P Acnes: apply alcohol solvent
- Lot 4 P acnes: apply BBL 10%
- Lot 5 P acnes: apply BBL 20%
- Lot 6 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, 8 mice/group
- Lot 1 (model group): Apply 50% oleic acid to external ear canal daily for
3 weeks, then apply 20% alcohol solvent for 2 weeks
- Lot 2 (Positive group 1: Locacid 0.05%): Apply 50% oleic acid to external ear canal daily for 3 weeks, then apply Locacid 0,05% for 2 weeks
- Lot 3 (positive control 2: Oxy-5): Apply 50% oleic acid to external ear canal daily for 3 weeks, then apply Oxy-5 for 2 weeks
- Lot 4 (test substance 1): Apply 50% oleic acid to external ear canal daily for 3 weeks, then apply BBL extract for 2 weeks
Observe the external ear canal variation Endoscopic external ear canal: before application, after 3 weeks with 50% oleic acid, and after 2 weeks drug application
Trang 72.3.2 Method of clinical studies
Clinical trials with a pre- and a post-treatment measurement: 112 patientsTreatment 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
Trang 8Causing 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 2 and rabbit No 3 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
Trang 9Timeline
Number of blood cell (T/l )𝑋" ± 𝑆𝐷 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
Control Group Experiment Group 1 st Experiment
Group 2 nd
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
Trang 10Results 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
Timeline
Number of leukocyte (G/l)𝑋" ± 𝑆𝐷
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
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
Trang 11There 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
(t- test Student) Control
Group Experiment group 1st Experiment group 2nd Before 51.90 ±11.69 56.90 ± 9.94 55.89 ± 13.92 > 0.05 After 30 days 52.40 ± 14.41 56.40 ± 8.68 53.90 ± 10.42 > 0.05
Control Group Experment
Group 1 Experment Group 2
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
Trang 12After 60 days 1.05 ± 0.05 1.05 ± 0.05 1.04 ± 0.05 > 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 > 0.05
p (pre - post) > 0.05 > 0.05 > 0.05
There is were no significant difference between group 1st and group
2ndcompairing control group
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 Concentration
(mg/mL)
Blood
MN 17.6 Not grow Not grow Not grow Not 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 Alcohl 20%
MN root extract at 8.8 mg /mL concentration inhibition growth of P acnes ATCC
Trang 133.2.1.2 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 Concentration (mg/mL) geckos Blood Chocolate KK GC
P.acnes (8.8mg/mL) Not grow Not grow Not grow Not grow
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
𝑋" ± 𝑆𝐷
After 48h 𝑋" ± 𝑆𝐷 ppre and postGroup of injection of PBS
solvent 295.34 ± 15 320.24 ± 26 > 0.05 Group of injection
3.2.2.3 Effects of MN root extract on inflammation of white mice ear
Trang 14Chart 3.1 The change of rat ear thickness after 26 days applying MN extraction
Group 1 (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 2 weeks of application
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 1 Group 3th: Locacid
0,05% hair folliclesand sebum are nomal
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, 8 and 12 weeks of treatment