1. INTRODUCTION:
Fungal infections are contagious in nature and are
associated with high mortality.1-3 Candidiasis is acknowledged as a
global burden, with over 1 billion cases annually for superficial cutaneous
infections and approximately 130 million cases for mucosal infections.
The approximate yearly occurrence of intrusive
candidiasis is 750,000 instances, with mortality falling between 40% and 55% of
cases. It has been observed that Candida greatly affects the skin tissues
causing inflammation and itching, called as cutaneous candidiasis. Besides
skin, candida also causes infections of the nails, mouth and vaginal tissues.4
Typically, fungal infections are treated using synthetic antifungal
agents like nystatin, miconazole, fluconazole, itraconazole, ketoconazole,
clotrimazole, 5-fluorocytosine, amphotericin B, and echinocandins.5,6
Nevertheless, these synthetic drugs can induce side effects such as diarrhea,
elevated serum alkaline phosphatase levels, neutropenia, low platelet count,
transient but occasionally fatal aplastic anemia, and elevated liver enzymes
levels. Drug-resistant microbes have emerged as a result of prolonged use of
antifungal medicines.7
Given the recent surge in resistance to antifungal
agents among these fungi and the associated adverse effects of these
medications, a secure and effective substitute is desperately needed for
treating and preventing Candida infections.8
In this scenario, herbal medicines emerge as a
promising source of treatment. Since ancient times, herbal treatments have been
an essential part of traditional medical systems in India.9,10 Among
these systems, Ayurveda stands out as the widely practiced and embraced
alternative medicine system. Therefore, a wide array of herbs are readily
accessible in various forms such as fresh or dried, and are used as ingredients
in perfumes, cosmetics, and over-the-counter medications. For their medical
needs, most people on the earth rely on plants and plant extracts. Many Indian
medicinal plants are known for their varied pharmacological effects owing to
their rich array of phytochemicals. The literature review unveiled that
numerous plants distributed across the plant kingdom demonstrate anti-fungal
properties.11 Medications
derived from plant sources continue to serve as primary remedies in many
countries, especially across Africa and Asia, owing to their widespread
accessibility and comparatively lower incidence of side effects.12 A
major obstacle to the use of herbal medications is formulating effective drug
delivery systems. For skin-related disorders, a diverse range of pharmaceutical
dosage forms are available, including liquid preparations, sprays, solid
powders, ointments, creams and gels. A gel is composed of a network of
cross-linked polymers dispersed within a liquid medium, and its properties are
significantly impacted by the interplay between the solid polymer structure and
the liquid phase. Gel formulations are employed for topical drug delivery due
to their ease of application, extended duration of contact, and minimal adverse
reactions in contrast to alternative topical applications and oral intake.
Topical gel formulations offer an effective delivery system for drugs across
the skin, as they exhibit high penetration efficiency, are not oily, and can be
effortlessly wiped off the skin. Compared to creams and ointments, gel
formulations offer superior stability and application properties.13
A highly noteworthy
plant in this sector is Cassia occidentalis, an ayurvedic herb, that
typically grows as an annual or perennial, reaching heights of only 0.5 to 2.5
meters.14
It is extensively
distributed in gardens, parks, human settlements, and nearly all warmer regions
of India. Different types of
phytochemical components such as flavonoids, tannin, alkaloids, polyphenols,
steroids, glycoside and volatile oil have been reported from C. occidentalis.
The pharmacological actions such as anti-inflammatory, antifungal, antiulcer, antinociceptive,
anticancer, antidiabetic, hepatoprotective, antimicrobial, and wound healing
activities have
been attributed to the presence of diverse class of components.15,16
However, there is a
lack of substantial scientific evidences supporting the medicinal benefits of C.
occidentalis in treating skin disorders. Additionally, there have been no
systematic investigations aimed at developing an effective delivery method for C.
occidentalis to address candida-induced skin infections. Consequently, the
goal of the current investigation was to assess the anti-candida properties of C.
occidentalis extracts through in-vitro experiments. Recognizing that
the availability of an appropriate formulation is a critical factor in its
practical use, we have also formulated and assessed an in-vivo topical
gel containing C. occidentalis extract for its potential anti-candida
activity.
2. MATERIALS AND METHODS:
2.1. Materials and organisms:
Carbopol 934, poly ethylene glycol, propyl paraben,
methyl paraben and triethanolamine were obtained from Lab Fine, Mumbai. The
fungal strain C. albicans was utilized in the study. This reference
strain, MTCC 227, was collected from the National Chemical Laboratory in Pune,
India. C. occidentalis leaves were collected from the Campus of Central
Arid Zone Research Institute in Jodhpur, Rajasthan, in June 2022 and verified
by the Botanical Survey of India, Jodhpur, under the reference number 345/2022.
Wistar rats (150±20grams) were provided from the
Animal House at PBRI, Bhopal. Both male and female Wistar rats were housed
together in two groups (six rats per group) under clean polypropylene
enclosures, with males separated from females. The animals were kept in typical environmental
conditions. The animals were given one week for acclimatization to the
laboratory environment. Prior to dosage, the animals were allowed unlimited
access to drinking water and a conventional pelleted food; however, they were
required to fast for three to four hours. Under Registration number
PBRI/IAEC/16-01-23/009, the Institutional Animal Ethics Committee approved the
experimental protocol.
2.2. Extraction:
The leaves of C. occidentalis were
rinsed with purified water and naturally dried at 27±2°C.
The leaf extract was obtained using the Soxhlet extraction method. 50 grams of
dried powder were placed into a thimble and subjected to extraction with 95.5%
ethanol for 16hours. The plant extract was then filtered via Whatman number 42
filter paper. The drug: solvent ratio and extraction time were pre- optimized
using single variable at a time method for maximizing the extraction
efficiency. Following filtration, the dark brown ethanolic extract was
acquired.17
2.3. Preliminary phytochemical screening of
ethanolic extract:
A qualitative screening of the leaves
extract was screened out for the qualitative presence of major chemical
constituents according to the coloring and the precipitation reactions. The
major chemical constituents investigated were alkaloids, glycoside, tannins,
reducing sugar, phlobatannins, flavonoids, saponins, terpenoids, anthraquinone
and cardiac glycoside.
2.4. HPLC analysis of leaf extract:
The Shimadzu
system utilized a C18 Column (5μm, 150 x4.6mm) to achieve optimal HPLC
separation. Isocratic elution method was used to accomplish this with a moving
phase made up of an acetonitrile mixture and a 0.1% trifluoroacetic acid
aqueous solution in a 55:45 ratio, respectively. There was a 10μL sample
injection volume and of the mobile phase was passed through at a flow rate of
1.0mL/min. Rhein, an anthraquinone glycoside was detected at 230nm in
wavelength against the standard.20 The method was validated using
ICH Q2R1 guidelines.
2.5. Determination of anti-candida activity
of prepared extracts:
The ethanolic extract obtained from the
leaves of C. occidentalis was tested against C. albicans.
Sabauroud Dextrose broth was prepared and subsequently inoculated with the
strains of Candida. The test compound was dissolved in dimethyl sulfoxide and
dilutions were made. The tubes were placed in an incubator set at 28±2°C, and
the minimum inhibitory concentration (MIC) was noted after 72 to 96hours.17
2.6. Selection and optimization of gelling
agent:
Using a variety of gel forming agents,
including carboxy methylcellulose sodium, Carbopol 934, and hydroxyl propyl
methyl cellulose, as well as varying concentrations of viscosity enhancer, gel
formulations were prepared in order to maximize the level of concentration of
the gel forming agent and attain the desired level of consistency. Ultimately,
gel exhibiting good spreadability and consistency was chosen.18
2.7. Preparation of herbal topical gel
formulation
The formulated extract was chosen for the
preparation of the plant based topical gel preparation. To begin, 20mL of
filtered water were mixed with 1.5grams of the gelling agent, Carbopol 934, and
left to swell for half an hour before stirring to formulate a gel. In a
separate container, after being sonicated for 10minutes, one gram of the
extract was distributed in 5mL of polyethylene glycol 400. Then, 5mL of
distilled water were added, along with 0.5mL of propyl 4-hydroxy benzoate and
0.05 mL of methyl hydroxyl benzoate. Finally, the thoroughly combined materials
were continuously stirred into the Carbopol 934gel. After that, 1.5mL of
triethanolamine was added gradually, drop by drop, to bring the pH down to
6.8–7 and achieve the desired consistency of the gel.
3. EVALUATION OF FORMULATED HERBAL TOPICAL
GEL:
3.1. Physical appearance:
The prepared gel was inspected visually for
colour and the presence of any gritty particles.18
3.2. Determination of pH:
The pH of the herbal gel was measured using
a digital pH meter by completely submerging the glass electrode in the gel and
making sure it covered the electrode. Prepared gel (1gm) was mixed in distilled
water till a uniform suspension is obtained to note the pH.18
3.3. Characterization of prepared gel:
The prepared gel was evaluated for
extrudability using wood block and pulley method, consistency using cone and
rod apparatus, visual homogeneity and rheology using Brookfield viscometer as
described earlier.18
3.4. Drug release study of prepared herbal
gel formulation:
The Franz permeation cells device was
employed to carry out the non-biological release experiment. A precise quantity
of the formulation (1.0g) was evenly applied onto a membrane situated between
the donor and receptor chambers, featuring a permeation area of 59.6 ± 3.1 mm˛.
The load chamber was filled with phosphate buffer solution pH 6.8 (PBS) and a
tiny magnetic bead was used to continuously swirl the mixture at a speed of 50rpm
to guarantee consistency. A constant temperature of 37.2±0.5şC was maintained.
At various intervals time (0, 30, 60, 90, 120, 150, and 180 minutes), the
samples were obtained and replaced with an equivalent volume of PBS. Sink
conditions were consistently achieved and the sample was analyzed using the
HPLC method.
3.5. Accelerated stability study of
prepared herbal gel formulation:
An accelerated stability study was
conducted on the formulated herbal gel at 40±2°C and 75±5% RH for 180 days.
Samples of the gel were extracted at predetermined intervals of 30 days and
then evaluated to assess their physicochemical properties and drug content.18
3.6. Dermal irritation studies:
Two groups of animals were categorized:
Group I and Group II. The back skin area of 5cm x 2cm was shaved one day prior
to the commencement of the study. The gel formulation (1g) was uniformly
applied to 1 square cm area of each rat. The investigation took place for four
days. Upon its completion, the animals were observed for any indications of
skin irritation, like swelling or redness. Scoring was performed according to
the parameters outlined earlier.23 once daily at zero day, 24 hrs,
48hrs and 72hrs (post-test observation period) accordingly. If the formulation
resulted in a score of 2 or lower, it was deemed to exhibit no irritation.
3.7. Evaluation of anti-candida activity in
animals:
The groups of rats were randomly assigned
to each other.
Group-I was treated with 2mg/kg of
hydrocortisone; Group-II treated with prepared herbal gel formulation, (1g);
Group-III treated with marketed gel formulation (1 g), containing clotrimazole
1% w/w,
Experimental rats (Group II and Group III)
were subjected to immunosuppression by administering hydrocortisone 2mg/kg body
weight for three days in a row.
Twenty-four hours after the final dose of
hydrocortisone, a circular wound with an approximate radius of 1cm was
surgically created on the animal's posterior mid-dorsal side using sterile
surgical blades. Fresh C. albicans spores were collected in saline
solution to make a suspension containing 104 colony-forming units
(CFU) per mL. This suspension was topically administered to the wound in a
single dosage of 0.5mL.18 After wound creation developed gel was
applied on the wound at regular intervals of twice a day for 8 days.12
On the 9th day, material
retrieved from the dry wound, both from the treated and control groups, was
collected using a sterile swab. This material was then subjected to staining
using the gram-positive and lactophenol cotton blue staining methods, and
subsequently examined under direct microscopy. The animals were put down on the
tenth day, and samples of blood were taken for determination of the
hematological parameters and placed into glass vials containing anticoagulant
(EDTA). The total and differential count of WBC was determined using a standard
method in infected and treated groups of rats.19 One-way analysis of
variance and Tukey's multiple comparison tests were applied for statistical
analysis. Significance in statistics was defined as p-value less than 0.05.
Data is represented as mean± standard variance.19
4. RESULTS AND DISCUSSION:
4.1. Preparation of extract and
phytochemical screening:
After experimenting with different time
intervals, it was discovered that the optimal yield of the extract obtained
from C. occidentalis leaves was 5.5%. The presence of, glycosides,
terpenoids, flavonoids, saponins, and polyphenols were identified by
phytochemical screening. Scientific studies in the literature have identified
rhein in this plant, attributing anti-fungal potential to this biomolecule.20
4.2. Determination of anti-candida
activity:
The extract of C. occidentalis
exhibited strong, dose-dependent anti-candida activity. It was found that 100 µg/mL was the lowest inhibitory
concentration of the C. occidentalis extract. These findings align with
earlier study16, demonstrating the antifungal effectiveness of C.
occidentalis leaves extract against fungal strain.
4.3. Selection and optimization of gelling
agent:
The gelling agent is the main ingredient in
the gel preparation. The concentration of the gel forming agent is highly
essential since a concentration too low might produce a simple solution with
very little consistency, while a concentration too high could result in the
development of gels with high viscosity, which would cause the medicine to be
distributed unevenly and make handling the gel problematic. The best gelling
agent was identified by testing a variety of gel formers. Gels containing
cellulose sodium salt and extract from C. occidentalis leaves showed
phase separation and were thus rejected. After formulation, thin gels of C.
occidentalis containing 1.0% Carbomer 934 liquefy in 6 hours. With the
utilization of a 2.0% gel forming agent, a somewhat enhanced gelling was
achieved, yet after a full day, the liquefaction barrier remained. Similar
observations were also found with HPMC as gelling agent. A gel comprising 3.0%
Carbopol 934 formed a consistent and sleek gel that maintained stability
without liquefaction during storage. At a concentration of 4.0% Carbomer, the
gel became excessively dense and adhesive, rendering it challenging to evenly
distribute. Gels formulated with HPMC displayed inadequate consistency,
characterized by excessive thickness, as indicated by extrudability and
spreadability measurements. Therefore, a concentration of 3.0% Carbopol 934 was
chosen as the optimum level of gel forming agent. Carbopol 934 is a widely used
gelling agent in many herbal topical gels reported earlier29-33 and
is known to provide desired rheological properties and quality of formulation.
4.4. Evaluation of topical herbal gel
formulation:
It was found that the herbal gel
composition was uniform and free of coarse particles. The prepared herbal gel formulation's pH value was
found out to be 6.8 ± 0.1, suggesting a low likelihood of causing skin
irritation. The prepared
plant-based gel formulation's spreadability was observed to be 23.61±1.7gm.cm/sec.
indicating excellent spreadability. The formulated herbal gel had an
extrudability percentage of 88.67%.
The prepared herbal gel formulation's
consistency was 4.5mm which was acceptable for desired rheological behavior and
extrudability. Gels with low viscosity may flow excessively quickly, while
those with high consistency may find it difficult to extrude from the tube.
Thus, getting the right consistency is essential to extruding the gel from the
tube efficiently.23 After setting the herbal gel formulation in the
container, homogeneity was assessed through visual inspection and found as per
target quality profile of the formulation. The viscosity of the
optimized plant-based gel formulation was measured to be 7357 centipoises (cps)
at 50 revolutions per minute (rpm) using spindle number 95, with a
corresponding torque of 88.6%.
Table 1: Optimization of extraction from
leaves of Cassia occidentalis.
|
S. No.
|
Extraction method
|
Weight of powdered drug
|
Extraction solvent
|
Extraction time (hrs.)
|
Yield (% w/w)
|
|
1
|
Soxhlet
Extraction
|
50 g
|
Ethanol 95.5%
|
8
|
3.78 ± 1.04
|
|
16
|
5.5 ± 1.5
|
|
24
|
5.55 ± 0.5
|
4.4.1. HPLC analysis of leaf extract:
The HPLC analysis of C. occidentalis
leaf extract indicated the presence of the anthraquinone glycoside compound
rhein. Subsequent literature indicated that rhein possesses notable antifungal
properties.16 However it is well evident from previous studies that
agro-ecological zoning25,27, seasonal and geographical variations29,
Good Agricultural/Collection Practices37 and several others factors
while cultivation and collection of herbal drugs31-33 have
significant impact on concentrations of active constituents. Hence rhein was
established as main marker compound in this study. The HPLC method mentioned
above was validated in compliance with the quality standards outlined by the
International Council for Harmonization (ICH), while also considering recommendations
from other relevant guidelines. Validation parameters such as precision,
accuracy, linearity and stability etc. are given in Table 2 reflecting the
values within acceptable limits as per ICH Q2R1 guidelines.
Hence the developed methodology can be used to identify main active
constituents of the selected plant. Method development and validation as per
ICH guidelines confirmed the industrial acceptability of the analytical
procedure. Analytical protocols for several phyto-constituents and drug have
been established in the authorized lab in similar manner34-38 and
are widely accepted globally.
Table 2: Validation
factors as per ICH- Q2R1 guidelines.
|
S. No.
|
Parameter
|
Value
|
|
1
|
Specificity
|
Specific
|
|
2
|
Linearity
|
0.996
|
|
3
|
Accuracy
|
97.89± 0.1454
|
|
4
|
LOD (µg
/mL)
|
1.27
|
|
5
|
LOQ (µg
/mL)
|
3.87
|
|
6
|
Retention time
(min.)
|
5.3
|
|
7
|
Robustness
|
Robust
|
4.4.2. Drug release study of prepared
herbal gel formulation:
Maximally 88.67% drug in 6 hours was
released from the prepared herbal gel formulation, confirming with the expected application time for
pharmaceutical gel and was found acceptable.
4.4.3. Accelerated stability study of
prepared herbal gel formulation:
The results of accelerated stability study
of prepared herbal gel formulation were shown in Table 3.
To guarantee quality, efficacy, and safety
throughout its shelf life, the reliability of the formulated herbal gel was
evaluated following the guidelines outlined ICH. No alterations in colour,
odor, homogeneity, pH, rheological study, or total content were observed in the
topical plant-based gel formulation during stability testing conducted at 0, 1,
2, 3, 4, 5 and 6 months. The study results conclusively demonstrated stability
analysis of prepared dosage form as per ICH guidelines, which is a well adopted
method to estimate shelf life of the product39
4.4.4. Skin irritation study:
The Draize grading system was used in the
skin sensitivity study to evaluate dermal reactions. Observations were
collected at different times, including right after patch removal and again 24,
48, and 72hours later. During the preliminary examination, rats exhibited no
skin reactions following any of the three successive exposures, which occurred
at intervals of 3 minutes, 1 hour, and 4hours. In the confirmatory test, the
animals treated with the prepared herbal gel formulation showed no signs of
redness or swelling. The reaction was consistently rated as "0" at
all observation time points, as depicted in Table 4. The primary dermal
irritation index for the prepared herbal gel was recorded as zero. This
suggested that it is safe and appropriate for topical application.
Various pharmaceutical products have the
potential to cause dermal irritation by permeating through the stratum corneum
and penetrating deeper layers, often resulting in symptoms such as redness,
itching, or pain in the affected area upon contact with the product. Hence,
conducting this test is a crucial aspect of preclinical safety assessment. The
skin irritation test entails assessing local inflammation, manifested by
erythema and edema, subsequent to direct skin injury. As per the Draize scoring
system, the evaluation of the potential skin irritant effect of the formulated
herbal gel was conducted, revealing no presence of erythema or edema (Table 4).
This suggested that the formulated herbal gel preparation was deemed safe and
non-irritant.
4.4.5. Evaluation of in-vivo
anti-candida activity in animal model by using rats:
In this evaluation, the open wound of
animals infected with C. albicans was treated with prepared herbal gel
formulation for 8 days. These treated wounds were showed visible healing sign
(Fig. 2). Remarkable wound healing properties were noted upon the topical
application of the gel formulation to rats infected with C. albicans.
4.4.6. LPCB and Gram staining:
After 24-48 hours of incubation, the
isolate from rat wounds displayed colonies with a creamy to white yeasty
appearance and a smooth surface on SDA plates. LPCB staining revealed round
and unilateral budding yeast cells.
The fungus cells exhibited a Gram-positive
reaction, which is a characteristic feature of taken fungal cell species.
Swabs taken on the ninth day from the area
of the recovering wound demonstrated the presence of fungal filaments by both
the LPCB method and Gram staining (Fig. 3 and Table 5). The sample collected
from the injury area of the rats indicated the presence of C. albicans.
No fungal infection was observed in Group II, whereas in the afflicted but
untreated group (Group I), the wounds were heavily infected with C. albicans.
Staining is crucial for identifying and
classifying different types of bacteria, fungi, and other microorganisms.
Staining also helps in the diagnosis of infectious diseases by enabling the
identification of pathogens in clinical samples. For
staining and studying fungus, the LPCB staining procedure is frequently used.40
The mixture consists of three ingredients: phenol, which effectively kills
living creatures; cotton blue, which colors the chitin found in the fungal cell
walls; and lactic acid, which aids in the preservation of fungal structures. A
diagnostic procedure called Gram staining is used to find bacteria at the
location of a suspected illness, which can include the throat, lungs,
genitalia, or skin wounds.40 When the resulting herbal gel was
examined using Gram and LPCB staining, comparatively controlled fungal
infection of any kind was found.
4.4.7. Analysis of blood parameters:
White blood cells participate in immune
process. These cells combat fungal infections,
supervise the repair process, safeguard the body from toxin-induced damage, and
facilitate wound healing and tissue repair.41-46 These cells support
the body's defenses against infections and other ailments. Our body produces
more white blood cells when we are sick in order to fight against the bacteria,
viruses, or other foreign things causing the illness. This leads to an elevation
in the white blood cell count, while diseases such as cancer and HIV/AIDS
typically result in a reduction of white blood cell count. The vital role that antibodies play in the host's defense from
disseminated fungal infection was underlined by clinical observations published
earlier42. Those who are immunocompromised on the cell-mediated
front are more likely to develop superficial than widespread candidiasis.47-52
The WBC count in rat blood (103/µL) was found to be 8.06±0.571, 8.85±0.492 (C. albicans infected+untreated
group), 5.33±0.408*, 5.36±0.332* (C. albicans infected + treated with
herbal gel) and 5.95±0.151*, 6.11±0.278** (C. albicans infected+ treated
with marketed gel) on day 1 and day 2 respectively. Likewise, the notable (P<0.05) rise in antibody
levels in the immunized rats suggested that inactivated C. albicans
possesses the capability to stimulate humoral immunity, as demonstrated in this
current study.
5. CONCLUSION:
C. albicans, classified as an opportunistic pathogen, exhibits
survival capabilities of infecting various anatomical sites by biofilm
formation. The availability of antifungal agents in the current market is
restricted due to concerns regarding their toxicity, limited efficacy, chances
of resistance and the high cost associated with prolonged treatment. Hence,
there is a necessity to develop antifungal agents capable of addressing the
current scenario effectively.
Desert herbs and plants are being used as a
source of medicine from long time and it is popular in rural population. The
present study aimed to develop an antifungal herbal topical gel formulation
containing extract of leaves of arid zone plant C. occidentalis and
exhibit therapeutic evidences to its traditional use.
Based on the findings, it was determined
that the formulated herbal gel containing ethanolic extract derived from C.
occidentalis Linn. leaves exhibited optimal anti-candida activity. The
antifungal attributes of C. occidentalis may derive from its
constituents, including anthraquinone glycosides, phenols, tannins, saponins
and flavonoids. A study involving formulation and screening of physicochemical
and phytochemical properties was conducted, yielding satisfactory results. The
formulated herbal gel demonstrated promising drug content and effective release
properties. The skin irritation study conducted on rats revealed no irritancy,
indicating that the formulation is non-sensitizing to skin tissues. The
accelerated stability study demonstrated that the physicochemical properties of
the gel formulation remained unchanged over a period of six months. Hence, it
was concluded from the present examination that the leaves of C.
occidentalis particularly ethanolic extract possessed good antifungal
activity and may be explored for an alternative or supportive herbal
formulation to existing antibiotic therapy.
6. ACKNOWLEDGEMENTS:
The author extends their heartfelt
gratitude to CAZRI, Jodhpur and NCL, Pune for supplying plant material and Candida
albicans MTCC-227. We are gratefully acknowledged the PBRI, Bhopal for
providing research facility. One of the authors (Ashish Baldi) acknowledges the
‘Seed Money Grant’ (MRSPTU/SM/04) for this work.
7. ABBREVIATIONS:
mL: Milli Litre; RPM: Revolutions per
minute; MTCC: Microbial Type Culture Collection; CAZRI: Central Arid Zone
Research Institute
8. REFERENCES:
1. d’Enfert C. Persistence of opportunistic
fungal pathogens in the human host. Current Opinion in Microbiology. 2009;
12(4): 358–64.
2. Rees JR, Pinner RW, Hajjeh RA, Brandt ME,
Reingold AL. The epidemiological features of invasive mycotic infections in the
san francisco bay area 1992-1993: Results of population-based laboratory active
surveillance. Clinical Infectious Disease. 1998; 27(5): 1138–1147.
3. Schulze J, Sonnenborn U. Yeasts in the gut:
From commensals to infectious agents. Deutsches Ärzteblatt International. 2009;
106(51-52): 837-842.
4. https://www.mountsinai.org/health-library/diseases-conditions/candida-infection-of-the-skin,
accessed on 15/04/2024.
5. Enass S, Ritta J, Olavi L, Pia F.
Anti-candida activity of extracts containing ellagitannins, triterpenes and
flavonoids of terminalia brownie, a medicinal plant growing in semi-arid and
savannah woodland in Sudan. Pharmaceutics. 2022; 14(11): 2469.
6. Deborah R, Mecky M, Olipa N, Cosam J,
Zakaria M. Screening of tanzanian medicinal plants for anti-candida activity.
BMC Complementary and Alternative Medicine. 2006; 6(1): 11.
7. Mansoureh I, Monir D, Zahra R. Anticandidal
effects of aqueous and callus extracts of Artemisia aucheri. International
Journal of Molecular and Clinical Microbiology. 2014; 1: 389-397.
8. Barani K, Sunayana M, Prabhu D, Adil A,
Preethi A, Jeevika C. Antifungal activity of Morinda citrifolia (noni) extracts
against Candida albicans: An in vitro study. Indian Journal of Dental Research.
2014; 25(2): 188-190.
9. Singh PA, Bajwa N, Baldi A. Possible role of
traditional systems of medicine to manage COVID-19: A review. Israel Journal of
Plant Sciences. 2021; 68(1-2): 1-26.
10. Baldi A, Bansal PK. Traditional and herbal
medicines: Understanding and exploration (part II). Current Traditional
Medicine. 2020; 6(4):258-259.
11. Somnath D, Mangesg A, Dheeraj S, Ganesh H,
Sanjeeva Y, Pooja D, Sneha S. Formulation and evaluation of polyherbal gel
containingextracts of Azadirachta indica, Adhatoda vasica, Piper betle, Ocimum
tenuiflorum and pongamia pinnata. Journal of Research in Pharmacy. 2018; 23(1):
44-54.
12. Sousa F, Ferriera D, Reis S, Costa P.
Current insights on antifungal therapy: Novel nanotechnology approaches for
drug delivery systems and new drugs from natural sources. Pharmaceuticals.
2020; 13(9): 248
13. Krishan, Padhi S, Anju G, Prateek D, Pooja
S, Rohit B. Evolutionary aspect of antifungal topical gel-a review.
International Journal of Research and Review in Pharmacy and Applied Sciences.
2018; 8(1): 015-030.
14. Zambrana NYP, Bussmann RW, Romero C. Cassia
accidentalis L. Fabaceae. Ethaobotany of the Andes. 2020; 1: 465-470.
15. Sadiq IS, Shuaibu M, Bello AB, Tureta SG,
Izuagie T, Nasiru S, Kamaru MB. Phytochemistry and antimicrobial activities of
Cassia occidentalis used for herbal remedies. Journal of Chemical Engineering.
2012; 1(1): 38-41.
16. Arya V, Yadav S, Kumar S, Yadav JP.
Antimicrobial activity of Cassia occidentalis (leaf) against various human
pathogenic microbes. Life Sciences and Medicine Research. 2010; LSMR-9.
17. Davariya V, Vala A. Antifungal activity of
crude extracts of Cassia occidentalis. International Journal of Research in
Phytochemistry and Pharmacology. 2011; 1(2): 36-38.
18. Muruganadam AV, Bhattacharya SK. Indole and
flavonoid constituents of Wrightia tinctoria, W.tomentosa and W.coccinea.
Indian Journal of Chemistry. 2000; 39(2): 125-131.
19. Vidita V, Saluja A, Dholwani K. Detection of
heavy metal contents and proximate analysis of roots of Anogeissus latifolia.
Journal of Pharmacognosy and Phytochemistry. 2013; 1(6): 61-65.
20. Jade R. RP-HPLC method for rhein
quantification in Cassia fistula leaves. Acta Medica Philippina. 2024.
21. Khan AB, Kotta S, Ansari RH, Kumar A, Ali J.
Formulation development, optimization and evaluation of aloe vera gel for wound
healing. Pharmacognosy Magazine. 2013; 9(1): 6-10.
22. Aiyalu R, Govindarjan A, Ramasamy A.
Formulation and evaluation of topical herbal gel for the treatment of arthritis
in animal model. Brazilian Journal of Pharmaceutical Sciences. 2013;
52(3):494-507.
23. Jamadar MJ, Shaikh RH. Preparation and
evaluation of herbal gel formulation. Journal of Pharmaceutical Research and
Education. 2017; 1(2):201-224.
24. Upadhye K, Charde K, Dixit G, Bakhle S.
Formulation and evaluation of herbal gel for management of mouth ulcers. Indian
Journal of Pharmacy and Pharmacology. 2021; 8(3):226-230.
25. Wahyuni IS, Sufiawati I, Shafuria A,
Nittayananta W, Levita J. Formulation and evaluation of mucoadhesive oral care
gel containing Kaempferia galangal extract. Pharmaceutics. 2024; 16(3):1-12.
26. Sanglard D, Kuchler K, Ischer F, Pagani JL,
Monod M, Bille J. Mechanisms of resistance to azole antifungal agents in
Candida albicans isolates from aids patients involve specific multidrug
transporters. Antimicrobial Agents and Chemotherapy. 1995; 39(11):2378–86.
27. Singh PA, Sood A, Baldi A. Highlighting
agro-ecological zones to upscale the production of immunity-booster plants in
Punjab. Research Journal of Agricultural Sciences. 2021; 12(3):865-870.
28. Singh PA, Sood A, Baldi A. An
agro-ecological zoning model highlighting potential growing areas for medicinal
plants in Punjab. Indian Journal of Pharmaceutical Education and Research.
2021; 55(2):492-500.
29. Gautam VK, Datta M, Baldi A. Effects of
geographical and seasonal variations on phenolic contents and antioxidant
activity of aerial parts of Urtica diocla L. Current Traditional Medicine.
2019; 5(2):159-167.
30. Singh PA, Baldi A. Good agricultural
practices: A perquisite approach for enhancing the quality of Indian herbal
medicines. Journal of Scientific and Technical Research. 2018; 5(5):1-4.
31. Singh PA, Bajwa N, Sharma L, Baldi A.
Farm-level medicinal plants production and economic analysis in the Indian
state of Punjab. Journal of Applied Research on Medicinal and Aromatic Plants.
2023; 34(2):486-490.
32. Singh PA, Bajwa N, Chinnam S, Chandan A,
Baldi A. An overview of some important deliberations to promote medicinal
plants cultivation. Journal of Applied Research on Medicinal and Aromatic
Plants. 2022; 31(1-3): 400-405.
33. Singh PA, Sood A, Baldi A. Determining
constraints in medicinal plants adoption: A model geospatial study in the
Indian state of Punjab. Journal of Applied Research on Medicinal and Aromatic
Plants. 2021; 25(10):342-347.
34. Sharma S, Naman S, Goyal K, Baldi A.
Simultaneous estimation of atovaquone and mefloquine hydrochloride: QBD based
method development and validation. Indian Journal of Pharmaceutical Education
and Research. 2023; 57(1): 250-263.
35. Sharma S, Kushwaha RS, Naman S, Patil UK,
Baldi A. Optimized extraction of oleoresin capsicum and analytical method
validation for capsaicin using HPLC. Indian Journal of Natural Products. 2021;
35(1):38-45.
36. Singh M, Kumar D, Naman S, Madhavi N, Singh
PA, Bajwa N, Baldi A. Development and validation of HPTLC method for the
simultaneous estimation of ascorbic acid and gallic acid in amla juice
preparation. Journal of Drug Delivery and Therapeutics. 2019; 9(4):227-231.
37. Singh U, Baldi A. Simultaneous estimation of
quercetin and silymarin: Method development and validation. International
Journal of Pharmaceutical and Biological Archives. 2013; 4(3):527-531.
38. Baldi A, Sanadya J, Patidar A. Method
development and validation for estimation of quetiapine fumerate by RP-HPLC.
Asian Journal of Research in Chemistry. 2010; 3(3): 604-607.
39. Madhavi N, Kumar D, Naman S, Singh M, Singh
PA, Bajwa N, Baldi A. Formulation and evaluation of novel herbal formulations
incorporated with amla extract for improved stability. Journal of Drug Delivery
and Therapeutics. 2019; 9(4): 212-221.
40. Thomas PA, Kuriakose T, Kirupashanker MP,
Maharajan VS. Use of lactophenol cotton blue mounts of corneal scrapings as an
aid to the diagnosis of mycotic keratitis. Diagnostic Microbiology and
Infectious Disease. 1991; 14(3):219–224.
41. King W, Toler K, Woodell-May J. Role of
white blood cells in blood- and bone marrow-based autologous therapies. BioMed
Research International. 2018; 2018(9): 1-8.
42. Matthews R, Burnie J. Antifungal antibodies:
A new approach to the treatment of systemic candidiasis. Current Opinion
Investigational Drugs. 2001; 2(4): 472-476.
43. Vidya N, Shubhangi J, Sagar B, Ganesh B,
Mangesh A. Formulation and evaluation of novel herbal gel by using lemongrass
oil. Research Journal of Pharmaceutical Dosage Forms and Technology. 2019;
11(2): 67-70.
44. Baitule AW, Tawar MG, Pande SD. Formulation
and evaluation of polyherbal gel. Research Journal of Pharmacy and Technology
2023; 16(4):2013-6.
45. Kiranmayi GVN, Ricky AL, Kumar S, Lalitha
M, Vamsi MK, Sureshma MS, Vishnu M, Sai MK. Assessment of antioxidant and
antiangiogenic activities of ethanolic root extract of Cassia occidentalis.
Research Journal of Pharmacy and Technology. 2019; 12(3): 1230-1234.
46. Rani MS, Emmanuel S, Sreekanth MR, Rosemary
T. Evaluation of the antipyretic and anti-inflammatory activities of methanolic
fraction and chrysophenol of Cassia occidentalis L. Research Journal of
Pharmacy and Technology. 2010; 3(3): 888-893.
47. Rani GR, Banu N. Anti-fungal activity of
metallochlorophyllins from leucas aspera L. Research Journal of Pharmacy and
Technology. 2017; 10(2): 513-515.
48. Sondhi S, Singh N, Goyal K, Jindal S.
Development of topical herbal gel of berberine hydrochloride for the treatment
of psoriasis. Research Journal of Pharmaceutical Dosage Forms and Technology.
2021; 13(1): 12-18.
49. Sandeep DS, Prashant NJJ, Rishal RM, Sumana
DR. Formulation and evaluation of antibacterial herbal gels of Murraya
koenigii leaves extract. Research Journal of Pharmacy and Technology. 2017;
10(6): 1798-1801.
50. Gorle A, Patil S. Development and evaluation
of Rubia cordifolia gel for treatment of acne. Research Journal of Topical and
Cosmetic Sciences. 2018; 9(2): 39-43.
51. Khanpara P, Sojitra N. Formulation and
evaluation of anti-aging purple herbal gel cream. Research Journal of Topical
and Cosmetic Sciences. 2023; 14(2): 58-4.
52. Patel K, Priya S, Zaveri M. Simultaneous
quantification of pharmacological markers quercetin, berberine hcl and curcumin
using high-performance thin-layer chromatography (hptlc) from polyherbal
formulation. Research Journal of Pharmacy and Technology. 2022; 15(11): 4919-3.
|
Available online from February 27, 2025
|
|
This work is
licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0
International License. Creative Commons License.
|
|
