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

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:
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


2

Besides 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


3

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


4

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 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 /
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


5

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 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,


6

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 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.


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 3. 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


8

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) / 3 = 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 + 3.33)
/ 3 = 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. 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


9

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 1

Experment
Group 2

(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
2 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 <0.001). At 48 thickness of the PBS group was not
significantly difference between pre and post of 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
2

3

4

6

8

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 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

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 1

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, 8 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 4 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
0

12.4

0
T0

9.5

0

5.7

T4
Clean

13.3
T8

medium

20
1
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 4 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 4 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 8 (0.45 ±
0.55), finally, decreased rapidly to week 12th (0.01 ± 0.10) most patients had no
itching.
Chapter 4. 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 1 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, 5 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 1 (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 1 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 do 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 <0.001). This result is consistent with microscopic images


20

of anatomical pathology: The thickness of the rat helix increased markedly, the
epidermis also thickened, hair follicles of the sebaceous gland enlarged, congestive
tissues in stroma occurred, and neutrophils, lymphocytes and cytoplasm appeared at
an inflammatory site. Nevertheless, PBS injection does not cause acne inflammation
on the rats’ ear flaps.
The inflammatory response of the rats’ ear flaps to MN root extracts
The leaching residue from BBL roots was diluted in ethanol 10%, 20% and
40%. After applying tetracycline to the positive control group and the experimental
groups, the thickness of rat ears was measured once a day. The thickness was seen to

decrease between measurements. The thickness of rat ears reduced when applied with
tetracycline reduced and returned to the original size on day 16. In the experimental
group, rat ears applied with BBL extract 10% returned to the original size after 22
days, as compared to 26 days in other groups. Figure 3.6 shows that the difference
between the experimental group applied with BBL extract 10% and the experimental
group applied with alcoholic solution was statistically significant difference with p
<0.05, whereas no difference was observed in the group applied with BBL extract
20% and 40%. A possible explanation about this is the higher the concentration of
BBL is, the more irritated the skin becomes. This means it takes the skin longer to
return to the original size. It suggests BBL extract 10% be applied to human subjects.
4.2.2.3 Effects of BBL on the rabbit ear model
Endoscopic observation: When Group L1 rabbits' ears were applied with
Parafin, hair follicles and sebaceous glands presented no abnormal signs, while
Groups L2-5 had a significantly enlarged swollen follicles with black spots congested
in the skin after 3 weeks of experimentation. Two weeks after being applied to
alcoholic solution, hair follicles of Group L2 continued to enlarge with an average
point size of 3.
Groups L3, L4 and L5 were applied with Isotretinoin, Benzoyl peroxide and
MN root extract 10% daily. Visual observation of effects of the drugs on the redness
of the pores showed that the inflammatory response in the pores decreased. At the
end of the second week after application, endoscopic imaging of rabbit ears (F) was
performed, and then images prior to application were compared with rabbits’ external
ear images, The pores were no longer swollen and red, and the pore size reduced.
Although there remained some pores whose sizes had not returned to normal, red
swelling almost disappeared (equivalent to score 1).
Microscopic observation: After 2 weeks of topical drug application, Group L1
saw no change, whereas in Group L2 (applied with an alcoholic solution),
keratinocytes increased in terms of thickness and the level of keratinization, and hair
follicles remained enlarged.
The result achieved after 2 weeks of treatment with Isotretinoin, Benzoyl

Peroxide and BBL 10% in Groups 3, 4 and 5 showed a similarity between the


21

macroscopic and microscopic assessments: the hair follicles and sebaceous glands
returned to normal. This further demonstrates the obvious effects of Isotretinoin,
Benzoyl Peroxide, and BBL 10% on the dekeratinization in rabbit ears.
4.3 Evaluation of therapeutic effects and unwanted effects of BBL root
extracts on the skin of patients with acne vulgaris (or also known as common
acne)
4.3.1 Urgency of the study
A study from the United States showed that nearly 100% of juveniles had acne.
Only about 20% of them needed help from doctors. A study of adolescents in New
Zealand found that acne occurred in 91% of males and 79% of females. Severe acne
was found in 6.9% of males and only 1.1% of females.
4.3.2 General characteristics
General characteristics of study subjects included gender, age, duration of
suffering from acne (before treatment), injury sites, routine habits, type of skin, and
form of clinical lesion appearance.
4.3.3 Effects of treatment
The question raised is how BBL extracts affect the four pathogenic mechanisms
of acne. Does BBL extracts dekeratinize a hair follicle at its base? Are they able to
eradicate P. acnes? Do they contribute to the inflammatory response? In this study,
we focused only on exploring the anti-inflammatory and bactericidal effects of BBL
extracts.
From BBL root extracts, five main substances were isolated, namely
Mallonanoside A, β-sitosterol (BS), Stigmast-4-en-3-on, Daucosterol and Palmitic
acid.
Mallonanoside A has a chemical formula similar to that of salicylic acid, which

has been used clinically for the treatment of acne thanks to its anti-inflammatory and
keratin-removing effects. Besides, its formula is also similar to that of Alpha abutin,
a skin whitening and beauty product available in today’s market.
β-sitosterol (BS) is one of the most common forms of phytosterols that have
anti-cancer, antioxidant, anti-bacterial and anti-inflammatory effects.
Stigmast-4-en-3-on is a phytosterol with anti-aging effects.
Daucosterol is found in pomegranates, violet bushes and grass plants used to
heal wounds and infections.
BBL contains Phenonic compounds with antioxidant properties that help with
anti-aging by removing free radicals.
With the scientific evidence we have shown above, BBL extracts have antiinflammatory, anti-oxidant, anti-acne and keratin-removing effects.


22

In this study, 10% of BBL extracts were used as monotherapy. Evaluation of
the success rate at the end of the study was conducted after 12 weeks of treatment.
The success of the treatment included acne cleansing 79%, 20% for mild levels of
acne (some blackheads) and only 1% for moderate. Levels of acne. There saw a
statistically significant change in inflammation and non-inflammatory counts (p
<0.001) at week 12.
Grouping was based on traditional medicine of 60 patients with “Lung channel
hot wind” type and 49 patients with “stomach-spleen cold temperature” type. During
the first 4 weeks of treatment, whiteheads, and particularly blackheads, tended to
increase in number due to the drugs’ effect of skin flaking which exposes
subcutaneous acne. In Figure 3.9, the number of inflammatory lesions in the
“stomach-spleen cold temperature” type group (24.92 ± 12.31) were more than twice
as many as in the “Lung channel hot wind” type (12.67 ± 11.32). In addition, the
number of inflammatory lesions in both groups tend to decrease after 4 weeks of
treatment and minimize after 12 weeks of treatment. Thus, BBL extracts 10% prove

to be effective in patients with inflammatory acne vulgaris.
The duration of treatment in Figures 3.8 and 3.9 shows that the number of noninflammatory lessions, and especially that of inflammatory lesions returning to
normal at a faster pace than that of the “stomach-spleen cold temperature” type. At
week 8, the inflammatory lesions of “Lung chanel hot wind” type returned almost
normal (1.42 ± 2.14). Thus, BBL extracts 10% are effective in “Lung chanel hot
wind” type (8 weeks).
Clinical observation showed that the severer skin irritation, the shorter the
treatment duration, the more skin flaking and redness, and therefore, the faster the
inflammatory and non-inflammatory acne lesions return to normal. "A chronic
inflammatory lesion is very difficult to treat, but if replacing it with an acute lesion
and treating the acute lesion for 10-14 days, it is possible to remove the chronic
damage to the skin." In case of acute skin inflammation, the body will mobilize its
entire immune cells to the inflammation locus and remove that inflammation locus
as efficiently as posible.
Studies have shown that chronic inflammation produce free radicals. Acne is a
chronic inflammatory disease. Therefore, when acne treatment needs to combine the
products that are able to remove such radicals. BBL extracts have been shown to be
highly phenolic compounds (Mallonanocid A is a major source of BBL root extracts).
4.3.4 Undesired effects
In this study, most of the undesirable effects were mild to severe. Undesirable
effects include redness, dryness, pruritus, pruritus and scabs at the application area
of varying degrees. The incidence of these events in patients is 100%. Side effects
are gradually reduced in the same manner as in topical preparations containing
retinoid or belzoyl peroxide BPO.


23

In this study, we found that the more severe skin reactions, the higher the
treatment effect, the quicker the skin of the patient, the more scar tissue, the more

scar tissue. When there is a strong irritation reaction, as an inflammatory process, this
inflammatory process will accumulate more of the immune cells on the skin, clearing
out the inflammation quickly.
A question that patients may ask is whether the skin is thinned? Is it weak? Is
the skin sensitive after treatment? Our answer is no, because human skin has a layer
of basal cells called the cell layer that reproduces every cell that is lost. There are
other cells that have replaced, so when new epidermal cells form they It will be
healthier, more functional than older cells. Skin barriers or barrier functions during
irritated skin will be less effective as they are nearly destroyed. Therefore, at this
stage, the skin needs to be protected with a moisturizer and physical sunscreen. When
the epidermis skin cells have been completely replaced, the barrier function will
return to normal, the function of the epidermal cells is perfect. Patients will have a
new skin, light, color, smooth, no disease.
Thus, the task of the doctor is to protect the basal cells, control the acute
inflammation that takes place within 10-15 days enough for immune cells to complete
their work.
BBL root extracts are very effective in treating mild to moderate acne; 79% of
the patients were free of lesions; The remaining patients suffered minor injuries on a
scale of Tan in 2008. But to increase the therapeutic effect and minimize the adverse
effects of medication use should be consulted by a doctor.
4.3.5. Caviar affects the quality of life
Patient’s quality of life before treatment was 11.26 and that after treatment was
2.41 (Figure 3.12). This difference was statistically significant with p <0.001.
CONCLUSION
1. Acute toxicity, skin irritation, eyes and subchronic skin.
- Acute toxicity:
LD50 = 11.148 (12,753 - 7,938) g MN/kg
The safety zone of the BBL root extract of 0.05 mg MN / 0.5 ml has a TI> 100,
which is less toxic.
- Skin irritancy:

Irritation index of MN root extract of 0.05gMN/0.5 mL is 0.78; mild skin
irritation.
- Eye irritation:
MN root extract causes no irritation to the rabbit eyes.
- Sub chronic skin


24

In both rabbit groups, a group was applied 0.25mL/kg/day and an other group
0.75mL / kg / day ( higher 3 times ) for 90 days, results: Both doses affect no rabbit
‘weight, hematopoiesis, liver, kidney function. No difference in microscopic images
of liver and kidney lesions was seen between treatment and control groups.
2. Evaluation of P. acnes resistance of the drug and its effectiveness in treating
acne in experimental animals
MIC of total extract (MN) preventing the visible growth of the P. acne strain
ATCC was 8.8 mg / mL
MN 10% root extract (0.05 mg dose / 0.5 ml) and MN 20% (0.1 g / 0.5 ml dose)
have strong effectiveness on P. acnes isolated from patients.
MN root extract have got an anti-inflammatory effectiveness, which reduces the
duration and symptoms of inflammation caused by P. acnes in the rat ear.
Hair follicle and sebaceous glands on rabbit ear canal is normal after 2 weeks
administration, equivalent to 0.05% tretinoin and 5% benzoyl peroxide.
3. Effectiveness of MN root extract on acne patients
Effectiveness of MN extracts after 12 weeks of treatment
Counts of noninflammatory lesions decreased from (57.17 ± 19.39) to 3.72 ±
9.45. Counts of inflammatory lesions was reduced from (18.17 ± 13.22) to 0.85 ±
3.81 (0.85 ± 3.81). The total lesions (75.61 ± 27.25) were reduced to 4.57 ± 12.8. .
+ Sertivity level: In the first week, the average lesions were 87.6%, lightly
12.4%. After 12 weeks of treatment: no lesion: 79%, mild: 20% and 1% moderate.

Undesired effects
100% of patients had got redness, dry skin, scabs from mild to severe level.
Redness is the highest from week 4th (0.83 ± 0.53) to 12 th and lightly (0.08 ±
0.22).
Peaking is the highest at week 4th (0.96 ± 0.48) and at week 12th lightly skin
ablation (0.18 ± 0.35).
Burning sensation is the highest at week 4th and at mild level (0.46 ± 0.54). At
week 12th, the burning sensation is a litle (0.01 ± 0.10).
Itching sensation is the highest at week 4th (0.66 ± 0.5) and 12th week (0.01 ±
0.10), most patients have no itching.
Life quality
Prior treatment, QoL was 11.26 and after treatment QoL was 2.41. There is
statistically difference with p <0.001.