Evaluation of Moringa Oleifera in Combination with Fluconazole as an Ointment

Moringa oleifera and Fluconazole

The previous researchers conducted a study about the possible synergistic effects of Fluconazole and Moringa
oleifera in which no other studies have been conducted yet. Their aim is intended as a response on the emergence of
antifungal resistance according to Centers for Disease Control and Prevention. The study was also intended to
improve antifungal efficacy and reduce side effects of fluconazole by reducing the concentration of the drug when in
combination with Moringa oleifera extract. The present researchers aims to formulate and create a safe and effective
dosage form for its' possible uses and further studies in the future.
Fluconazole (Brand Name: Diflucan)
Used to treat:

 Candidiasis (Fungal infection caused by the fungus Candida)

o Vaginal yeast infections
- Stopping the growth of common types of vaginal yeast (fungus)
o Oropharyngeal Candidiasis
o Esophageal Candidiasis
 Meningitis caused by the fungus Cryptococcus
Blocks the ability of fungi to reproduce/ Stops their growth

Side Effects of Fluconazole

https://medlineplus.gov/druginfo/meds/a690002.html
Side effects associated with use of Fluconazole, include the following:
 Headache
 Nausea
 Abdominal pain
 Diarrhea
 Rash
 Vomiting
Other side effects of fluconazole include:
 extreme tiredness
 unusual bruising or bleeding
 lack of energy
 loss of appetite
 pain in the upper right part of the stomach
 yellowing of the skin or eyes
 flu-like symptoms
 dark urine
 pale stools
  seizures
 blistering or peeling skin
 hives
 itching
 swelling of the face, throat, tongue, lips, eyes, hands, feet, ankles, or lower legs
 difficulty breathing or swallowing
 and more

This is where Moringa oleifera comes in:

The leaves of Moringa oleifera are particularly rich in potassium, calcium, phosphorus, iron, vitamin D, essential
amino acids, as well as known antioxidants such as carotene, vitamin C, and flavonoids (Mbikay, 2012). 
https://www.sciencedirect.com/science/article/pii/S0570178316300203

In addition to which it is packed with nutrients, packed with antioxidants, helps in lipid control and protects liver
health, it is also known to have antibacterial and antifungal properties to fight infections with very minimal or no side
effects.
Side Effects:
o Uterus contraction in pregnant women
o A concern that using Moringa might make hypothyroidism worse

Malunggay is used as an alternative treatment for wounds, burns and scratches

The antifungal activity of Moringa oleifera was studied against several fungi namely Saccharomyces cerevisiae,
Candida albicans, and Candida tropicalis. Ethanol and aqueous leaf extract showed maximum activity against
Saccharomyces cerevisiae, as well as zone inhibition in Candida tropicalis. It is also known to be biologically active
against liver toxins, tumors, viruses and other microbes. https://innovareacademics.in/journal/ijpps/Vol6Issue5/9066.pdf
Antimicrobial screening of M. oleifera results (Ethanolic extract):
 Shows antimicrobial properties on B. cereus, B. subtilis, E. coli, Staph. aureus, S. lutea and M. phlei.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3331240/pdf/ASL-14-197.pdf
Another study showed that Moringa oleifera with ethyl acetate produced inhibition against Candida albicans.
https://www.banglajol.info/index.php/CUJBS/article/view/13411

A study aqueous extract of Moringa plant inhibited the activity of these bacteria which include Bacillus
cereus; Staphylococcus aureus; Pseudomonas aeruginosa, Klebsiella pneumonia, Escherichia
coli; Enterococcus cloacae; Salmonella typhi and; Proteus vulgaris
The leaf extracts of Moringa oleifera showed varying antimicrobial activity on wide range of microorganisms.
https://www.sciencedirect.com/science/article/pii/S0570178316300203

Why ointment?
Topical skin infections commonly occur and often present therapeutic challenges to practitioners, despite the
numerous existing antimicrobial agents available today. The necessity for developing new antimicrobial means has
increased significantly due to growing concerns regarding multidrug-resistant bacterial, viral, and fungal strains.
Consequently, attention has been devoted to safe, new, and/or alternative antimicrobial materials in the field of
antimicrobial chemotherapy.

Goals of topical antifungals:

 Treat antifungal skin infections and provide the patient with symptomatic relief, successfully eliminate
infection, and prevent recurring infections
 Should relieve itching, burning, skin cracking, and scaling that are often caused by fungal skin infections.
Advantages of ointments:
 Chemically more stable than liquid dosage forms
 Facilitate application directly to the affected body part (Local effect) and avoid exposure of other body parts
to the drug
 Suitable for patients who find it difficult to take drugs via parenteral and oral routes
 Prevents passage through the liver
https://www.slideshare.net/smitachoudhary6/ointments-67551869

Formulation and Evaluation of Topical Formulations

Physical Evaluation of the Topical Formulations
1. Organoleptic Characteristics: Formulations were tested for physical appearance, color, texture, phase
separation and homogeneity. It also includes the immediate skin feel including stiffness, grittiness and
greasiness.
2. Spreadability: formulations was determined by measuring the spreading diameter of 1 g of sample between
two horizontal glass plates (10 cm × 20 cm) after one minute.
3. pH Values: One gram of each formulation (including the blank, i.e., formulation without any active
ingredients or preservatives, and drug-loaded formulation) was dispersed in 25 mL of deionized water, and
 the pH was determined using a pH meter (Mettler-Toledo Ingold Inc., Billerica, MA). Measurements were
made in triplicate. The pH meter was calibrated with standard buffer solutions (pH 4, 7, and 10) before each
use.
4. Viscosity Measurement: A Brookfield viscometer DV-I (Brookfield Engineering Laboratories, Middleboro,
MA) was used with a concentric cylinder spindle #29 to determine the viscosity of the different topical
formulations. The tests were carried out at 21°C. The spindle was rotated at 0, 0.5, 1, 2, 2.5, 4, 5, 10, 20,
50, and 100 rpm values. All measurements were made in triplicate.
In Vitro Antibacterial Activity
1. Preparation of Mueller-Hinton (MH) Agar Plates. Mueller-Hinton (MH) agar medium was prepared according
to the manufacturer’s instructions and autoclaved for 20 minutes at 20 psi.
2. Preparation of Inoculum. Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 29213) were
used to evaluate the antibacterial activity of the topical formulations. The microorganisms were subcultured
the previous day to ensure that the tested microorganisms were in their log phase of growth and to ensure
the validity of the results.
3. Inoculation of the MH Plate. 
4. Preparation of Agar Well Diffusion Assay. The dried inoculated MH agar plates prepared above were used
to perform the agar well diffusion assay. A sterile cork borer was used to make the wells by punching holes
on the inoculated MH agar plates.
Stability Study
Antibacterial activity of the ointment will be tested
Statistical Analysis
Statistical analysis of data was performed using one-way ANOVA (Tukey’s post hoc test). A difference was
considered statistically significant when p < 0.05.
https://www.hindawi.com/journals/jphar/2016/5754349/

Evaluation Tests for Ointments

 Rate of absorption
 Non-irritancy
 Rate of penetration
 Rate of drug release
 Rheological properties (viscosity)
 Content uniformity
 Preservative efficacy
https://www.slideshare.net/ShaikSana1/evaluation-of-ointments

Product quality Tests – General (USP)

 Description
 Identification
 Assay—A specific and stability-indicating test should be used to determine the strength (content) of the
drug product. See Antibiotics—Microbial Assays h81i, h621i, or Assay for Steroids h351i. In cases when the
use of nonspecific assay is justified, e.g., Titrimetry h541i, other supporting analytical procedures should be
used to achieve overall specificity. A specific procedure should be used when there is evidence of excipient
interference with the nonspecific assay
 Impurities
 Uniformity of Dosage Units
 Water Content
 Microbial Limits
 Antimicrobial Preservative Content

Conclusions need to have a back up.

Topical way of administration
Best extract
Synergistic effect
What fungi?