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http://www.tjps.pharm.chula.ac.th 127 TJPS 2021, 45 (2): 126-136
http://www.tjps.pharm.chula.ac.th
126 TJPS 2021, 45 (2): 126-136http://www.tjps.pharm.chula.ac.th 127 TJPS 2021, 45 (2): 126-136
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126 TJPS 2021, 45 (2): 126-136
Subacute Toxicity Study and Clinical
Trials for
Ziziphus spina-christi
Leaves
Extract as an Anti-dandruff Shampoo
Abdulkarim K. Y. Alzomor1,2, Wafa M. Al-Madhagi2,3,
Nahlah M. Sallam1, Haytham Mojamel2, Mostafa M. Alawar2,
Abdulfattah G. Al-Hetari2
1Department of Pharmacy, Faculty of Medicine and Health Sciences, Thamar
University, Dhamar, Yemen, 2Department of Pharmacy, Faculty of Medical Sciences,
Al-Nasser University, Sana’a, Yemen, 3Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Sana’a University, Sana’a, Yemen
ABSTRACT
In light of recent developments in the scientific and technological world, herbs are widely used
as remedial agents. This study was aimed to formulate and evaluate the safety and the efficacy
of herbal antidandruff shampoo from Ziziphus spina-christi (Sidr) leaves extract, which is used
traditionally for several medical and cosmetic purposes. Sidr leaves were extracted by maceration
using ethanol as a solvent. The antifungal activity of the plant extract was tested against M. furfur.
In-vivo study of the extract has been evaluated with daily oral doses (50, 100, and 200 mg/kg)
for 28 days for tested rats, followed by, evaluation of the biochemical and histological parameters.
Subsequently, the plant extract was formulated as a shampoo and tested for its antidandruff
efficiency on 80 volunteers, with dandruff, for four consecutive weeks. An obvious antifungal
activity against M. furfur has been displayed by sidr extract, where 86% of the tested volunteers
substantially ameliorated from dandruff with sidr shampoo formulation. The toxicity of the extract
was not shown aberrant results as compared with the control group in biochemical analysis.
However, mild histological harmful effects on the liver and kidney tissues appeared at high doses
of the extract, 100 and 200mg/kg, when used orally. As a conclusion, sidr extract could be used
topically as anti-dandruff shampoo formulation with high efficiency and good safety
Keywords: Antifungal, dandruff, formulation, hair treatment, safety, Ziziphus spina-christi
INTRODUCTION
Ziziphus spina-christi (L.) is a member of Rhamnaceae
that is also known as sidr in Arabic countries. Sidr is
an indigenous tree to the Middle East and its leaves
are used traditionally for hair cleaning and promoting its
growth.[1] Z. spina-christi exhibited antibacterial, antifungal,
anti-nociceptive, antioxidant, antidiabetic, anti-plasmodium,
anti-schistosomiasis, analgesic, and anticonvulsant
activities.[2-9] Several saponin glycosides have been isolated
from sidr that are proven to help in removing the excess
sebum and have antibacterial and antifungal activities
which increase their immense value and to be used as
cosmetic preparation.[7] Petal et al. stated that christinin-A
is the foremost saponin of sidr’s leaves.[10] In addition to,
flavonoids, lipids, protein, and mucilage; these include butic
acid, ceanothic acid, and cyclopeptides [11,12] were found in Z.
spina-christi extract.
Malassezia fungus is the main causative agent of
dandruff that use lipase enzymes to break down the sebum to
oleic acid. The latter penetrates the upper layer of skin then
causes increased skin cell turnover in susceptible people and
this in turn produces the dandruff flakes,[13] Other studies
indicated that two other species of the fungus-Malassezia
restricta and M. globosa are also causal organisms of the
dandruff.[14] In drug discovery and development stage of
the pharmaceutical industry, on average, only one in five
thousand screened compounds in research reach the market.
The most failure to the screened compounds is related to
drug safety and a lack of efficacy.[15] Therefore, the present
study is designed to develop a new shampoo formula of
Corresponding Author:
Abdulkarim K. Y. Alzomor,
Department of Pharmacy,
Faculty of Medicine and
Health Sciences, Thamar
University, Dhamar, Yemen.
Tel.: +967-773418635/
+967-736558282.
E-mail: al_zomor1974@
tu.edu.ye
Received: Aug 04, 2020
Accepted: Nov 13, 2020
Published: Feb 13, 2021
Thai Journal of Pharmaceutical Sciences
Original Article
Alzomor, et al.: Investigating antidandruff activity of Ziziphus spina-christi extract as shampoo
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sidr leaves’ extract to be used as anti-dandruff and to study
the sidr toxicity on albino rats followed by clinical trials on
humans to confirm the safety and efficacy of this extract.
METHODOLOGY
Study Area
The study was carried out at Al-Nasser University, Department
of Pharmacy, Pharmaceutical and Histology Laboratories.
While, the toxicology study was performed in Sana’a
University, Faculty of Sciences and the pathological study in
Forty-Eight Hospital, Department of Histopathology and the
accelerated stability study was performed in Global Pharma
Company, Sana’a – Yemen.
Ethical Consideration
Ethical clearance and approval of the study protocols
obtained from the Ethical Research Committee of Thamar
University, Faculty of Medicine and Health Sciences in
25/09/2018 Reference No. TUMEC-17016 and the study
followed the ethical principles in phytochemical, experimental
pharmacology research, and the clinical trials. The animals
involved in this study were investigated and housed according
to European community guidelines and Guidelines for the
Housing of Rats in Scientific Institutions. In terms of clinical
trials, all volunteers were informed about the research and
the drug to be used, then they were asked to sign a written
informed consent for participation in the study.
Preparation of Crude Extract
Initially, Z. spina christi leaves were collected from Wesab
Mountains, Yemen in November 2018 and identified by Dr.
Hassan Ibrahim; Plant Taxonomy Department, Agricultural
Research Centre, Sana’a University. The dried plant was then
kept with voucher number NU192018 in the Herbarium Unite
of Al-Nasser University, Pharmacognosy Lab. Once the leaves
dried at 25°C and milled, 140 g of the sample was soaked in
500 ml absolute ethanol in shaking apparatus for 24 h.[16] The
extract was filtered before being concentrated through a rotary
evaporator (Telstar, model no.: CRYODOS-50), followed by
freeze-drying and kept in the refrigerator at 4°C.
Toxicology Study
Twelve female albino rats with average weight 120–150 g and
age 60 days were obtained from the animal house of Sana’a
University’s (ventilated environment and controlled humidity).
During the study for 28 days, animals were fed on specific
diet formula consists of White corn, soybean, wheat bran, melt
bran, yellow corn, nd dried fish (Wazef) as a source of animal
protein vegetable oil mixed together with multivitamins
and minerals (Vitamin-M, Aman Veterinary Manufacturing
Company, Sana’a-Yemen). The yielded paste was rolled into
cylindrical pellets then each rat received 100 g/day of the
dried pellets. During the subacute toxicity study, the rats
were divided into four groups, 1st group was received distilled
water and served as a control, while the 2nd, 3rd, and 4th were
treated with Sidr extract as 50, 100, and 200 mg/kg of body
weight, respectively, for 28 days. At the end of the experiment,
rats from each group were anesthetized through inhaled
chloroform, then slaughtered to identify gross lesions. Then,
specimens from liver, kidney, heart, spleen, brain, and small
intestine were taken from each group followed by immediate
fixation in 10% neutral buffered formalin and processed for
histopathology. Conversely, the blood samples were collected
at slaughter for hematology and serum analysis.
Analysis Parameters
Growth changes
Average body weights and weight gain for each group of rats
were measured on a weekly basis throughout the period of
study.
Hematologic analysis
First, the blood samples were collected in test tubes containing
ethylene diamine tetra-acetic acid (EDTA) following by
measuring the hemoglobin (Hb), the blood cells, packed cell
volume (PCV), mean cell volume (MCV), mean corpuscular Hb
(MCH), and MCH concentration (MCHC) using a hematology
analyzer (HumaCount plus, German).
Biochemical Analysis
Blood samples were centrifuged at 3000 RPM for 5 min, then
the serum was kept at (−20°C) for analyzing the aspartate
aminotransferase (AST), alanine aminotransferase (ALT), and
alkaline phosphatase (ALP).
Histopathological Study
The specimens were preserved from the isolated vital organs
of the desiccated rats before being fixed in formalin 10% and
after processing embedded in paraffin wax. Successively, the
tissues were trimmed into 5 mm2 pieces then stained with
hematoxylin and eosin to be examined through microscope.
Statistical Analysis
The results of the biochemical estimations were reported as
mean ± SD of rats in each group. The data were analyzed by
one-way analysis of variance (ANOVA) using AGRES statistical
package (Version 3.1) with a level of confidence 95%.
Formulation and Evaluation of Shampoo
Formulations of sidr leaves extract
Five formulas of sidr shampoo were prepared for an anti-
dandruff purpose as illustrated in Table 1.
Preparation of the formulas
F1, F2, F3, and F4 were prepared starting by dissolving the
sodium lauryl sulfate in a half amount of the distilled water
and the remaining ingredients were combined in the other
half. Next, sidr extracts were dispersed in 10 ml of ethanol
and all parts were mixed until homogenous shampoo
formed. Finally, a sufficient quantity of NaCl was added until
the viscosity adjusted. Alternatively, F5 was formulated by
heating 20 g of texapon in water bath 60°C for 5 min prior
to adding hot distilled water at 80°C with continuous stirring
until complete dissolve. Second, 3 ml of glycerine, 3 g of
cocamide, and a sufficient quantity of NaCl were supplied
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gradually to adjust the viscosity. After that, 50 mg of sidr
extract was dissolved in 10 ml of ethanol and combined with
the mixture. At last, an adequate quantity of citric acid used
to adjust the pH.
Shampoo Evaluation Tests
Physical appearance/visual inspection
The formulations were evaluated in terms of color, odor,
appearance, wash-ability, and texture. The viscosity and
homogeneity tests were carried out through taking a small
quantity of the product then pressed between the thumb and
the index finger.
Foam stability
Fifty milliliters of the 1% formulated shampoo solution were
transferred to a 250 ml graduated cylinder and shaken. Foam
stability was evaluated by recording the foam volume after 1
min and 4 min of shake test.[17]
Measurement of pH
Digital pH meter was used to determine the pH of the shampoo
formulas by diluting 10 ml of shampoo into 50 ml with distilled
water.
Contamination test
Mueller–Hinton agar was prepared, according to manufacturer’s
instructions, and poured in two petri dishes, one control and
the other was mixed with 10 ml of shampoo product and
incubated at 37°C for 48 h.
Surface tension
Surface tension could be tested through calculating the amount
of surfactant found in shampoos to reduce the surface tension.[17]
Measurement of surface tension was performed after using 10%
of shampoo solution in distilled water at conventional conditions
using a clean dropper. The calculations were carried out using
the following equation: R2 = ([W3-W1] n1)/([W2-W1] n2) * R1.
R2 is the surface tension of the shampoo solution, R1 is the
surface tension of water, W1 is the weight of the empty beaker,
W2 is the weight of the beaker with the distilled water, W3 is
the weight of the beaker with the shampoo solution, n1 is the
number of drops of distilled water, and n2 is the number of
drops of the shampoo solution.
Packing of the final product
Shampoo formulations were packed in 100 ml transparent
plastic containers.
Stability Study of the Shampoo
Stability study was carried out for shampoo formula according
to International Conference on Harmonization guidelines.
Amongst all prepared shampoo formulas, F5 showed the best
physical properties. Therefore, this formula was divided into
two samples, those samples were kept at different storage
conditions, that is, at 25°C and at 40°C with 75% relative
humidity (RH) with intensive light in stability chambers
(airtight and transparent plastic containers), and observed for
a period of 3 months at definite time intervals. A sufficient
quantity of each formula in suitable containers was stored
under 40 ± 2°C and 75% ± 5% RH and the quality tests were
done for the samples monthly through 3 months.[13]
Antifungal Assay
Malassezia furfur samples were collected from the scalp of the
volunteers and kept in a thermal sack at room temperature,
then examined under the microscope by taking a specimen of
scalp dandruff on a slide with drops of potassium hydroxide,
and identified by the objective lens (40×). Second, sabouraud
agar media was prepared and sterilized by chloramphenicol
Table 1: Different formulations of sidr shampoo was prepared and the different additives were included
Excipients Quantity
F1 F2 F3 F4 F5
Sidr extract 5% 5% 5% 5% 5%
Bentonite 5% - - - -
Sodium lauryl sulphate 20% 20% 20% 20% -
Citric acid QS QS QS QS QS
Tragacanth - - 4% - -
Gum Arabica - 3% - - -
Cocamide 3% 3% 3% 3% 3%
NaCl QS QS QS QS QS
Texapon - - - - 20%
Glycerin - - - 3% 3%
EDTA 0.15% 0.15% 0.15% 0.15% 0.15%
Ethanol 10% 10% 10% 5% 10%
Methyl paraben sodium 0.18% 0.18% 0.18% 0.18% 0.18%
Propyl paraben sodium 0.02% 0.02% 0.02% 0.02% 0.02%
Distilled Water up to 100 ml 100 ml 100 ml 100 ml 100 ml
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cycloheximide supplement (CCS), the subcultures from the
growing colony of M. furfur were transferred and incubated at
32°C for 72 h. Next, one colony was taken from each subculture
which was stained by lactophenol to be examined under the
oily objective lens (100×) of the microscope. A clinical isolate
was treated in the same way.
Likewise, the fungicidal activity of shampoo against M.
furfur was carried out by dissolving 4.7 g sabouraud dextrose
agar in 100 ml distilled water with heating before transferring
it to five sterilized test tubes. Later, the media injected by
CCS to prevent the growth of bacteria and the isolated fungi
was inoculated into four tubes containing the media. Three
concentrations of the prepared shampoo (1 ml, 2 ml, and 3
ml, respectively) have been added to the tubes. In addition, a
control (media + shampoo without extract) and pure media
have been tested. Then, all samples have been incubated at
32°C for 72 h.
Clinical Study
The sample size was calculated according to the population
size using Australian statistical analysis (National Statistical
Service). Confidence level = 95%, proportion = 0.5, standard
error = 0.05, population size = 800, and consequently the
sample was obtained to be 89 ± 10 volunteers. In the current
study, the effectiveness of Z. spina christi leaves extract
shampoo was evaluated using 80 volunteers who entered the
study in preclinical stages. All volunteers were exposed to
clinical diagnosis for the yeast presence from Malassezia by
direct examination under the microscope during 1 week and
the results are summarized in Table 2.
Inclusion Criteria
Eighty Yemeni volunteers (Arab ethnic group) with age equal
or higher than 15 years old where clinically diagnosed with
moderate to severe dandruff. Volunteers were agreed to give
informed consent and enrolled in the study, disposing to attend
all the visits punctually and acceptance for no using other
treatment or hair cosmetics while they are involved in the study.
Exclusion Criteria
Pregnant women; volunteers with any disorder may
compromise immunologically (diabetes, cancer, etc.); those
with the history of hepatic, renal or cardiovascular disease; use
another anti-dandruff agent; and mentally or neurologically
disabled volunteers that are considered not fit to approve their
participation in the study.
Clinical protocol
Mycology diagnosis for Malassezia species and dermatophytes
was performed to confirm the infection. Volunteers with
positive results were recruited for the study and they were
prevented from using any cosmetics, emollients, or any
antifungal drug during treatment.
Clinical examination
Hair scarping tests of the infected area were performed
before, during, and after treatment for 3 weeks. The efficacy
of the product was assessed by the direct examination of
microorganism under the microscope using KOH 20%.
Study Design
This study is a random experimental trial and the volunteers,
who had fulfilled the criteria, were selected randomly from
the university. The procedure was explained to volunteers to
sign the written informed consent and received 100 ml of the
shampoo with sufficient clarification on the instructions of use.
They were advised to apply the shampoo on moist hair once
daily for 5 min then rinse it for 3 weeks and avoid any other
medicals or cosmetics during their participation in the study.
Evaluating Treatment and Follow Up
Each volunteer was followed up weekly for 4 weeks to evaluate
the efficacy of the product according to the improvement of the
dandruff status. Mycological tests were performed through two
steps; first is the direct examination with potassium hydroxide
and the second is the culture of scaly/hair obtained from the
affected area of the hair using sabouraud agar. After 1 week
of treatment, 20 volunteers were selected randomly for direct
examination and volunteers with improved status (complete
cured, excessive, moderate, and mild dandruff) were determined.
RESULTS AND DISCUSSION
Analysis of the Extract
The results of phytochemical tests of Z. spina christi extract
confirmed the presence of tannins, glycosides, alkaloids,
phenolic compound, saponins, reducing sugar, and flavonoids.
Formulation Evaluation
All formulated preparations were evaluated for physical
characteristics such as color, odor, homogeneity, surface tension,
consistency, skin irritation, pH, and transparency as demonstrated
in Table 3. The different formulas revealed good physical
properties; however, the most stable formula physically was F5.
Table 2: Characteristic conditions of the volunteers in the study
including sex, yeast presence, and morphology and previous
treatment
Parameters No. of
patients
Age (years)
average=28±12
15–25 ≥5±
Sex
Males 53 39 14
Females 27 19 8
Diagnosis of yeast
+V 79 58 21
−V 1 0 1
Morphology of the dandruff
Severe 51 46 15
Mild 29 22 7
Previous treatment
Yes 31 18 13
No 49 40 9
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It has been proved that shampoo pH plays an important
role for enhancing and improving the hair quality, minimizing
the eye irritation, and stabilizing the scalp’s ecological balance.
All prepared formulas displayed maintained pH value range
between 5 and 6 that help to prevent the swelling and promote
tightening of the scales, thereby inducing shine and minimizing
the hair damage.[17] The pH value of the prepared shampoo
was close to the recommended value from previous study to
be close to skin pH.[13] Regarding the foam testing, foam is
of paramount important criteria in evaluating shampoos, even
though it has no correlation with the cleansing ability of the
shampoo.[17] In the present study, all the formulas have good
foam volume more than 100 ml, especially F3 and F5, and it
is stable for 5 min. Turning to surface tension measurement,
whenever the surface tension is lower, the cleaning ability of
the shampoo is improved.[17] The good quality of the shampoo
evaluated by the capability of the shampoo to reduce the
surface tension of pure water from 72.28 dyn/cm to about
40 dyn/cm.[18] Accordingly, all the tested shampoos prepared in
this study showed reductions in surface tension ranging from
30 to 34 dyn/cm and this is confirmed to the pharmacopeia’s
standards. F5 has the lowest surface tension; therefore, it is
indicated to have the strongest cleansing ability. Moreover, the
formulas did not present noticeable changes in the vital tests
of the shampoo under stress conditions 40°C, 75% RH during
the 3 months and the most suitable and satisfied formula was
F5. In conclusion, F5 is the most suitable and stable formula of
sidr shampoo during the 3-month study.
Antifungal Assay
The antifungal activity of F5 shampoo has been tested and
compared to negative control (media without shampoo). After
72 h, no growth has been detected in compared to the growth
from the negative control to indicate the antifungal activity of
the Sidra shampoo as antifungal, as summarized in Table 4.
Toxicology Study
No sign of toxicity, behavioral changes, or mortality were
observed in the test groups of rats having a dose of 50, 100,
and 200 mg/kg dose in compared to the controls throughout
the dosing period of 28 days.
Effects of the extract on the hematological parameters
The mean results of blood samples parameters are calculated
in Table 5 for the treated rats and control. Evaluating the
hematological parameters is crucial in such studies since
these parameters are considered as sensitive biomarkers of
the physiological changes in response to any external effects
including toxic stress in testing animals.[19] This study has
shown that sub-acute treatment with the extract did not cause
any change in hematological parameters except the significant
increase in the platelets level with P-value 0.0061. There were
no significant changes in Hb, red blood cells, white blood
cells, PCV, neutrophils, lymphocytes, monocytes, eosinophil,
and basophil in all the treated groups as compared to the
control group with different doses of sidr extract as exhibited
in Table 5. As the hematological alterations such as anemia are
mostly accompaniments of bone marrow toxicity, the analysis
of hematology parameters in the animal studies has a high
relevance and predictive value for humans.[20] Furthermore,
the lack-of-effect on polymorphonuclear leukocyte levels
indicate that the extract may not have induced an inflammatory
process, since these cells are usually elevated in the course
of inflammations. The platelets have an essential role in the
coagulation process and this study proved significant elevated
levels of platelets count indicating hemostatic activity of tested
extract samples.[21]
Effects of the extract on the liver functions
AST, ALP, and ALT enzymes serve as biomarkers capable
of predicting toxicity. AST is present in several tissues,
which include heart, kidney, skeletal muscle, and liver,
whereas ALP presence in bone, kidney, intestine, bile
duct, and liver, and ALT is predominantly localized in the
liver.[22]
Apparently, serum levels of ALP, ALT, and AST of treated
rats were not significantly different than control especially
at 50 and 100 mg/kg dose; however, the levels of all these
enzymes significantly increased with boosting the dose to 200
mg/kg as summarized in Table 6 and these are often diagnostic
signs of underlying cellular injuries.[23]
Table 3: Physicochemical tests of the shampoo formulations.
Shampoo formulations Parameter
F5F4F3F2F1
35003000340030003000Viscosity (CPS)
5.845.385.525.505.62pH
Good Good Good Good GoodHomogeneity
Conform Conform Conform Conform Conform Consistency
Non NonNon Non Non Skin Irritation
3030323234Surface tension(dyn/cm)
105±4.0103±4.6104±5.1100±9.398±5.6Foam stability(cm3±SD)
Table 4: Antifungal activity of the shampoo after 1 week
from treatment: the level of dandruff was estimated as ***:
Excessive, **: Moderate *: Mild, 0: Complete cure
Fungal growth Dandruff level of the scalp
*** ** * 0
Yes 3 2 1 0
No 2 3 4 5
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throughout the study period. As displayed in Table 7, the
extract has no effect on the body weight of the rats, even at
a dose of 200 mg/kg, and food and water intake was normal
Effects of the extract on the weight of rats
The table below demonstrates the effects of a given dose of
the extract to the test animals on the overall body weight
Table 5: Effect of oral administration of sidr extract on hematological parameters in rats
Parameters Dose of Sidr extract (mg/kg)
50 100 200 Control
Mean±SD Mean±SD Mean±SD Mean±SD
White blood cells s 4.4±1.7 6.943±2.11 5.986±0.775 8.077±2.153
Red blood cells 8.3±0.05 8.213±0.1419 8.33±1.55796 8.876±0.644
PLT** 673.3±34.5 1016.67±108.61 938.3±90.51 684.0±100.50
Neutrophil 20±0.00 22.00±2.646 19.3333±3.786 24.00±9.539
Lymphocyte 72±3 73.66±2.309 70.3333±1.527 69.00±10.535
Monocyte 2.4±0.6 2.20±1.411 4.60±1.058 2.967±1.222
Eosinophil 4.6±2.3 2.30±0.721 5.63±4.113 4.1667±1.778
Basophil 0.00±0.00 0.033±0.058 0.033±0.058 0.100±0.100
Hb 16.6±0.15 15.9±1.179 15.83±3.023 17.20±1.374
PCV 49.5±0.058 47.93±2.401 48.63±7.144 51.93±2.358
MCV 60±0.53 58.36±1.901 58.70±2.718 58.60±2.152
MCH 20.2±0.44 19.36±1.137 19.0±0.5567 19.366±0.551
MCHC 33.5±0.25 33.16±0.929 32.40±1.947 33.10±1.916
The Data are expressed as mean±SD, n=12, *P<0.05, **P<0.01.
Table 6: Effect of oral administration of sidr extracts on liver function of the rats
Enzyme Sidr extract dose (mg/kg)
50 100 200 Control
Mean±SD Mean±SD Mean±SD Mean±SD
AST** 186.3±10.5 191±15.7 268.67±54.2 180.0±35.5
ALT** 38±8 67.33±12 79.3±14.2 51.0±9.1
ALP** 139.3±31.9 183.00±8.6 206.0±40.7 106.0±23.89
Liver enzymes were measured as mean±SD., n=12, *P<0.05, **P<0.01
Table 7: Effect of oral administration of sidr extracts on body weight of rats
Body weight Dose of sidr extract (mg/kg)
50 100 200 Control
Mean±SD Mean±SD Mean±SD Mean±SD
Before administration 122±20 96.333±11.50362 109.667±35.7258 102.6667±32.47050
After administration of 142.7±27.5 107.667±25.54082 122.333±38.3710 130.0000±35.55278
The data are expressed as mean± SD., n=12, *P<0.05, **P<0.01S
Figure 1: Photomicrograph of the liver sections of the control and rat treated with dose 50 mg/kg of Sidr extract. (a) Control rat normal hepatocytes
with normal central vein. (b) Infiltration of mononuclear cells. (c) Inflammatory region
c
b
a
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throughout the study timeframe. As a result, this suggests the
normal processing and metabolism of proteins, carbohydrates,
and lipids as it plays a key role in the physiology of the body.[24]
Histopathological Results
At the end of the study, the vital organs of the scarified
testing animals including liver, heart, kidney, lungs, spleen,
Figure 2: Photomicrograph of the liver sections of the control and rat treated with dose 100 mg/kg of Sidr extract. (a) Control rat normal
hepatocytes with normal central vein (CV). (b) Infiltration of mononuclear cells. (c) Inflammatory region. (d) Hydropic changes
d
c
b
a
Figure 4: Photomicrograph of the kidney sections the control and rat treated with dose with 50 mg/kg of Sidr extract. (a) Control rat showing
normal structure of glomerulus and tubules. (b) Glomerular shrinkage and tubular hemorrhage. (c) Glomerular degeneration
c
b
a
Figure 3: Photomicrograph of the liver sections the control and rat treated with dose 200 mg/kg of sidr extract. (a) Control rat showing normal
hepatocytes with normal central vein (CV). (b) Congested central vein. (c) Liver hemorrhage. (d): Inflammatory region. (e) Infiltration of
mononuclear cells. (f) Hydropic changes
d
c
b
f
a
e
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132 TJPS 2021, 45 (2): 126-136
and brain which are the most targeted area of the toxic
substances metabolically, were isolated and there were no
lesions found on the macroscopic examination of these tissues
when were compared with the control group,[25] as displayed
in Figures 1-12. Moreover, the microscopic examination of
the vital organs of the treated rats with sidr extract showed
only some effect for both hepatic and renal tissues and no
effect to other organs. As shown in Figure 1, the hepatic
cells of the control have a round nucleus in the center and
the central vein (CV) is surrounded by flat endothelial
cells without any other changes. Whereas, on the liver of
rats treated with 50 mg/kg of the extract, there were some
mild histopathological changes such as inflammation and
infiltration of the mononuclear cells from the blood vessels
and with higher dose the detrimental effects increased at
200mg/kg dose of sidr extract including hemorrhage in
Figure 3c, congestion, inflammation, and hydropic changes
in Figures 3b-f, respectively.
Figure 5: Kidney sections the control and rat treated with dose
100 mg/kg of Sidr extract. (a) Control rat showing normal structure
of glomerulus and tubules. (b) Hemorrhage. (c) Glomerular
degeneration. (d) Tubular casts
d
c
b
a
Figure 6: Photomicrograph of the kidney sections the control and rat
treated with dose 200 mg/kg of plant extract. (a) Control rat showing
normal structure of glomerulus and tubules. (b) and (c) Glomerular
shrinkage. (d) Medullary tubular casts
d
c
b
a
b
Figure 7: Photomicrograph of the spleen sections. (a) Control
spleen with normal white pulp (WP) and red pulp (RP). (b) Spleen
administrated with 50 mg/kg of plant extract with normal WP and
RP. (c) Spleen administrated with 100 mg/kg of plant extract with
normal WP and (RP). (d) Spleen administrated with 200 mg/kg of
plant extract showing normal white pulp (WP) and normal red pulp
(RP) structure
d
c
b
a
Figure 8: Photomicrograph of the brain sections. (a) Control brain
showing normal neuron cell bodies (arrows). (b) Brain administrated
with 50 mg/kg of plant extract showing normal neuron cell bodies.
(c) Brain administrated with 100 mg/kg of plant extract showing
normal neuron cell bodies. (d) Brain administrated with 200 mg/kg of
plant extract showing normal neuron cell bodies
d
c
b
a
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134 TJPS 2021, 45 (2): 126-136
Concerning the kidney sections, the control rats revealed
normal glomerulus and tubules as represented in Figure 4a,
though, there were glomerular shrinkage and degeneration in 4B
and 4C in case of the rats treated with 50 mg/kg of the extract.
More severe effects appeared at 100 mg/kg of oral administered
extract such as hemorrhage between kidney tubules as represented
in Figure 5b and tubular casts in 5D and the major devastating
changes obtained at the dose of 200 mg/kg such as the glomerular
shrinkage in 6B and 6C, and modularly tubular casts in 6D.
In conclusion, histopathological changes occurred in liver
and kidney of rats at low dose of sidr extract (50 mg/kg) could be
considered as mild, while the highest dose 200 mg/kg severely
destructed the liver and kidney tissues. The three different doses
of sidr extract elucidated no histopathological effects on spleen,
brain, intestine, skeletal muscle, lungs, and pancreas.
Results of Clinical Trails
Direct examination
The results of the shampoo effectiveness as anti-dandruff, after
using for 14 days as described in the methods above, evaluated
by direct examination under the microscope once a week, as
shown in Table 8.
The table above highlights that after 1 week from the
treatment roughly more than the half of the volunteers
were completely cured, although the scalp dandruff
status was assessed in 7.5%, 28.75%, and 11.25% as
excessive, moderate, and mild, respectively. After 2 weeks
of continuous treatment, the majority of the volunteers
(83.75%) were completely cured, while out of 8.75%
Figure 10: Photomicrograph of the skeletal muscle sections.
(a) Control muscle showing normal myofibers. (b) Intestine muscles
at dose 50 mg/kg, (c) 100 mg/kg, and (d) 200 mg/kg of sidr extract
all keep normal myofibers
d
c
b
a
Figure 9: Photomicrograph of the intestine sections. (a) Control
intestine showing normal villi. (b) Intestine administrated with
50 mg/kg, (c) 100 mg/kg, and (d) 200 mg/kg of sidr extract all are
showing normal villi architecture
d
c
b
a
Figure 11: Photomicrograph of the lung sections. (a) Control lung
display normal alveoli, (b) lung the rat treated with dose 50 mg/kg,
(c) 100 mg/kg, and (d) 200 mg/kg of sidr extract show normal alveoli
d
c
b
a
Figure 12: Photomicrograph of the pancreas sections. At last, Figure, all
a, b, c, and d show normal pancreatic acini; (a) control, (b) pancreas
administrated 50 mg/kg of sidr extract, (c) at dose 100 mg/kg, and
(d) pancreas administrated 200 mg/kg of sidr extract
d
c
b
a
Alzomor, et al.: Investigating antidandruff activity of Ziziphus spina-christi extract as shampoo
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134 TJPS 2021, 45 (2): 126-136
illustrated no improvement and 7.5% were in with mild and
moderate dandruff status.
After 4 weeks of the treatment, the outcomes confirmed
that less than the tenth of the volunteers were not benefited
from the sidr shampoo and approximately 86% were entirely
cured of this disorder.
Cultural investigation
The cultural antifungal assay is used to assure the effectiveness
of the sidr shampoo as shown in Table 8.
Under the optimum conditions of the fungal growth, a
total of 65% of the samples established no growth when
cultured on sabouraud agar; however, the remaining samples
35% were demonstrated as variable growth of the fungi. It
might be supposed that either the resistant samples indicate
to the inactivity of the extract on these volunteers because of
the previous treatments or the etiologic of the dandruff is a
species other than M. furfur. Previous studies have approved
the antifungal activity of the aqueous extract of Z. spina-christi
against Candida albicans in vivo where the extract showed MIC
of 6.25 mg/ml against C. albicans.[26] Moreover, the methanolic
extract of the roots of Z. spina-christi revealed an antifungal
effect against dermatophyte especially Microsporum canis,
Aspergillus fumigatus, Trichophyton rubrum, and Trichophyton
mentagrophytes.[4] Others reported that the fruits were also
active against C. albicans.[27] Different extracts and fractions of
the leaves, fruits, and seeds of Ziziphus showed considerable
antiviral, antifungal, and antibacterial activities.[28]
CONCLUSION
Formulated shampoo of Z. spina-christi extract is safe and
effective at 50 mg/kg and 100 mg/kg according to the sub-
acute toxicity study using albino rats. Although at higher
doses, this extract did not cause many adverse effects on the
testing animals except some observable histopathological and
biochemical changes on the liver and kidney.
ACKNOWLEDGMENT
The authors express their gratitude to Pharmaceutics and
Histology Laboratories in Al-Nasser University, Biology
Department – Faculty of Applied Sciences – Sana’a University,
Forty-Eight Hospital, Department of Histopathology and
Global Pharma Company, Sana’a – Yemen; for supplying all
materials and equipment used in this research.
DECLARATION OF INTEREST
The authors declare no conflicts of interest.
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Table 8: Clinical effectiveness of the shampoo; the number of
volunteers that showed presence of dandruff and its level in the scalps
Parameters Presence of
dandruff
Level of dandruff in the
scalp
+V −V *** ** * 0
After 1 week 38 42 6 23 9 42
After 2 weeks 11 69 7 4 2 67
***: Excessive, **: Moderate, *: Mild, 0: Complete cure
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