Pharmacognosy Research

: 2020  |  Volume : 12  |  Issue : 4  |  Page : 342--351

Gas Chromatography-High-Resolution Mass Spectrometry Elucidation and in vitro Cell Line Studies (Sulforhodamine B) on Niosomal Gel of Benincasa hispida

Gaurav Mahesh Doshi1, Akanksha Dilip Badgujar2, Ishan Sudhir Mathurvaishya3,  
1 Department of Pharmacology, SVKM'S Dr. Bhanuben Nanavati College of Pharmacy, Mithibai Campus, Mumbai, Maharashtra, India
2 Department of Quality Assurance, Vivekanand Education, Society's College of Pharmacy, Mumbai, Maharashtra, India
3 Department of Pharmaceutics, Dr. L.H. Hiranandhani College of Pharmacy, Thane, Maharashtra, India

Correspondence Address:
Dr. Gaurav Mahesh Doshi
Department of Pharmacology, SVKM'S Dr. Bhanuben Nanavati College of Pharmacy, Mithibai Campus, Vile Parle (W), Mumbai - 400 056, Maharashtra


Background: The Cucurbitaceae family has been known for its presence of phytoconstituents, namely, lupeol, β-sitosterol, terpenoids, phenols, and proteins with therapeutic potential. Objectives: The research paper tries to focus on major therapeutic field of people demographic area of type of cancer. Soxhlet extraction of Benincasa hispida (BH) was carried out and crude extract was converted to powder. Niosomal gel was formulated after the optimization of drug concentration from the extract of Niosomal solution. Solution with maximum entrapment was selected for the preparation of gel. Gel was prepared by using different Carbopol grades and with comparison of different parameters. Gel was studied for gas chromatography-high-resolution mass spectrometry (GC-HRMS) evaluation for check toxicity. Biologic activity was analyzed by SRB (Sulforhodamine B) assay with in vitro cell lines (human oral cancer cell line KB and Mus Musculus skin melanoma B16-F10). Results: Solution which was clear and white was selected for gel development. Carbopol 934 (1') as gelling agent showed good formulation properties. It was non-gritty, easily spreadable, and washable. The gel with maximum entrapment efficiency was evaluated further. GC-HRMS revealed 1.2-ethyl 2-Hexen-1-ol; 1,8-(3-Octyl-2-oxiranyl)-1-octanol; Dodecanoic acid, 2-penten-1-yl ester; Cholestane, 4, 5-epoxy-,(4α,5α); Cyclopentaneundecanoic acid; 17-octadecynoic acid; Octadecanoic acid, methyl ester; Methyl oleate; Tridecanoic acid, methyl ester; and Silane, dimethyl (2,3,6, tricholrophenoxy) heptadicycloxy. The selected cell lines did not show satisfactory activity at different concentrations in SRB assay. Conclusion: This research paper tried to witness the probable potential of BH for cancer with analytical base. Future studies may be undertaken at various stages and levels to further explore the potential of these families of plants.

How to cite this article:
Doshi GM, Badgujar AD, Mathurvaishya IS. Gas Chromatography-High-Resolution Mass Spectrometry Elucidation and in vitro Cell Line Studies (Sulforhodamine B) on Niosomal Gel of Benincasa hispida.Phcog Res 2020;12:342-351

How to cite this URL:
Doshi GM, Badgujar AD, Mathurvaishya IS. Gas Chromatography-High-Resolution Mass Spectrometry Elucidation and in vitro Cell Line Studies (Sulforhodamine B) on Niosomal Gel of Benincasa hispida. Phcog Res [serial online] 2020 [cited 2021 Feb 26 ];12:342-351
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Full Text


The authors have tried to screen the potential of Benincasa hispida gel formulation (PEBH) for using vitro cell lines (human oral cancer cell line KB and Mus Musculus skin melanoma B16-F10). Gel was studied for gas chromatography-high-resolution mass spectrometry in order to study the toxic degraded components in the formulation.


Abbreviations Used: GC-HRMS: Gas chromatography-high-resolution mass spectrometry, LC-MS: Liquid chromatography and mass spectroscopy, PEBH: Petroleum ether extract of Benincasa hispida, TEM: Transmission electron microscopy, BCC: Basal cell carcinoma, SCC: Squamous cell carcinoma, SRB: Sulforhodamine B assay, Tz: Time zero, C: Control growth.


Natural products research have known to witness upsurge in the 21st century.[1] The literature cited in herbal pharmacopoeia have proved to provide evidence-based research studies. These secondary metabolites have been gaining importance among the world population as “drug-likeness and biological friendliness than totally synthetic molecules.” Photochemistry branch is evergreen bridge that always tries to fill the gap between botany and chemistry. The entire world has ample resources which needs to be explored.[2] There is co-existence of vast no. of species in various families of plants. Cucurbitaceae is one such family comprises of 965 species in 95 genera. Benincasa hispida (BH) is popularly known as vegetable crop which has medicinal and nutritional importance. Phytochemical analysis showed presence of vital constituents in fruits such as volatile oils, uronic acid, flavonoids, β-sitosterin, glycosides, minerals, sacchrides, proteins, vitamins, and carotenes. The therapeutic potential includes central nervous effects (anxiolytic, muscle relaxant, antidepressant, Alzheimer's disease), antioxidant, antimicrobial, antidiabetic, nephroprotective, diuretic, antiasthmatic, anti-inflammatory effects.[3]

The skin mainly comprises of three layers, namely, epidermis (protective skin layer with external environment), dermis (mechanical resistance and elasticity), and hypodermis (deepest layer of the skin). There has been upinterest in the society for transdermal delivery. Nanocarriers have been as a powerful replacement for hydrophilic and lipophilic drugs. They are effective in various treatment remedies by their unique property of penetrating through the stratum corneum.[4] These nanocarriers (nanoparticles, liposomes, dentrimers, and ethosomes) can be made by the incorporation of various different materials, which are unique in their chemical and structural nature. Niosomes are spherical and consist of microscopic lamellar (unilamellar or multilamellar) structures. The bilayer is formed by nonionic surfactants, with or without cholesterol and a charge inducer. Examples of surfactants include sorbitan fatty acid esters and polyoxyethylene fatty acid esters.[5] They are too small to be detected by the immune system and effective in delivering the drug in the target organ using lower drug doses in order to reduce side effects. Carbomers have been used extensively in topical liquid preparations. Carbomers are considered to be non-toxic and non-irritant materials, no hypersensitivity reactions. Taking the above concepts idea, an attempt has been made to formulate Niosomal gel for studies.[6],[7]

Melanoma comprises of 2' of skin cancer which is of malignant type and are fatal. More than 2 million cases of skin cancer have been reported in the USA in 2010.[8],[9] These cancers include basal cell carcinoma (BCC) which is common slow growing, locally invasive and rarely metastasizes whereas squamous cell carcinoma (SCC) is non-melanomatous skin cancer (approx. 20' to 30').[10],[11] There are four major subtypes of invasive cutaneous melanoma that are grouped for their distinct histologic patterns: Superficial, nodular, lentigomaligna and acral lentiginous. Once a diagnosis has been made by biopsy of the lesion, pathologic staging must be completed to determine prognosis and treatment. India has been witnessing substantially arise in SCC cases.[12] B16 melanoma has been used as research model for human skin cancers which is considered as boon to chemotherapy regimens.[13],[14],[15]

Cucurbitaceae family of plants have revealed immense therapeutic potential in different ailments. Some plants have been fully explored to its utility in the major fields of the research, hence further studies needs to be undertaken. Our research paper is collective effort of plant based topical formulation followed by gas chromatography-high resolution mass spectrometry (GC-HRMS) studies of the components. The developed formulation was been exponentially studied at specific concentration using SRB (Sulforhodamine B) assay. The research paper tries to emphasize on the role of one such family of plant which would prove its evidence in cancer and explore future research.

 Materials and Methods

Collection, authentication and extraction

Dried seeds of BH were obtained from local market, Mumbai. They were previously authenticated by Agharkar Research Institute, Pune (authentication no. 13-083). The dried seeds were again sun dried at 30°C–35°C. The seeds powder was subjected to continuous Soxhlet extraction apparatus with petroleum ether (65°C) for 3–4 days till colorless liquid was obtained in the thimble (PEBH). The concentrate was then evaporated to complete dryness at 40°C using Rotary Evaporator. The crude extract was collected and stored in air-tight containers under refrigeration at 5°C.

Formulation development[16],[17],[18],[19],[20]

The niosomes were prepared by film hydration method using ROTAVAP (Rotary Evaporator) with following steps in details as follows:

Step 1 – The surfactant and cholesterol was dissolved in chloroform and drug was added to it Step 2 – The homogenous solution was introduced in RBF and the film was formed under vacuum at 70°C ± 2 Step 3 – It was then hydrated with phosphate buffer for 30 min Step 4 – The solution was ultrasonicated for 20 minStep 5 – It was then subjected to centrifugation on 10,000 rpm for 10 min for removal of unentrapped drug.

The Niosomal formulation was developed by using different excipient and excipient ratios. The cholesterol was added in the formulation to increase the efficiency of the drug entrapment. Various trials were conducted to optimize and obtain the high entrapment of the drug. Final Niosomal solution (F 14) was used for preparation of Niosomal gel (NG 1). The selected excipients for formulation of the gel were Carbopol 934, menthol, triethanolamine, methyl and propyl paraben, and distilled water. Carbopol 934 was added to small quantity of distilled water and soaked for a period of 24 h. After 24 h, the extract along with other excipients were added and stirred on a magnetic stirrer until a gel like consistency was obtained. Finally, triethanolamine was added to adjust the pH of the formulation.

Evaluation of the formulations[16],[17],[18],[19],[20]

Niosomal solution

The solution was physically checked for homogeneity and color (white). The pH was measured using a digital pH meter. The microscopy was performed to determine the size of niosomes formed using optical microscopy at ×45 and transmission electron microscopy (TEM). The entrapment efficiency of different solutions was evaluated and the optimized formulation was selected for preparation of Niosomal gel.

Niosomal gel

The gel was physically checked for homogeneity and color (white). The pH was measured using a digital pH meter. The viscosity of gel was determined using Brookfield viscometer with spindle S96 at 3 rpm. The microscopy was performed to determine the size of niosomes using optical microscopy at ×45. The spreadability of the gel formulations was determined by measuring the spreading diameter of 1 g of gel between two horizontal plates (10 cm × 25 cm). The standard weight used (200 g) was placed above it for 30 s. After 30 s, the upper tile was removed, and the formulations were found to spread on the tile. The total amount of formulations spread on the tile was measured with the help of a scale, by measuring the diameter. These measurements were taken thrice and the average values obtained by formula S = M × L/T. The ease of removal of gel formulation applied was examined by washing the applied part with tap water. After the gel formulations have been set in container, testing for visual inspection, appearance, homogeneity, and presence of aggregates was done. The grittiness and uniformity of particles in the gels was checked microscopically. About 1 g of formulation was weighed and added to a 100 ml volumetric flask and volume was made up with phosphate buffer of 5.5 pH. The solution was filtered to remove any solid particles. Further dilutions were made and estimated spectrophotometrically using ultraviolet (UV)/visible spectrophotometer. The formulations were kept for a period of time, alternatively in the refrigerator, and at room temperature to observe the bleeding of liquid. It is tested in the form of a “patch test.” Formulation is applied on 1 cm2 patch of skin and observed for inflammation or rashes on skin.

In vitro drug release studies

The in-vitro drug release of gel was carried out by using Franz diffusion method. The diffusion of the drug from the semi-solid product throughout the membrane was regulated by assaying the receptor substrate with sequentially selected samples. UV spectroscopy was used to retrieve the aliquot of the sample from the receptor compartment for drug content analysis at determined time points. After each sampling, the receptor compartment was topped with a fresh medium.[16],[17],[18],[19],[20]

Stability studies

The stability studies were performed as per ICH guidelines at 30°C ± 2°C/65' relative humidity ±5' RH and 40°C ± 2°C/75' RH ± 5' RH.[20],[21],[22],[23],[24]

Gas chromatography-high-resolution mass spectrometry

Gel studies were undertaken at the Sophisticated Analytical Instrument Facility (SAIF), Indian Institute of Technology (IIT), Powai, Mumbai. The following are the details furnished with instrument specifications used during the experiment:

Extracts: PEBHChemical (solvent): Methanol

EI-MS spectrum was scanned at -70 eV with instrument details was:

Model of MS: Joel Model: Accu time-of-flight analyzer (TOF) GCV Specification: Mass range – 10–2000 amu and resolution - 6000. Make of GC: Agilent 7890 Detector: Flame ionization detector (FID)Run time: 40 min.

Gas chromatography-mass spectrometric (GC-MS) analysis was performed by splitless injection of 1.0 μL of the sample in methanol on a Hewlett Packard 6890 (USA) gas chromatograph fitted with a cross-linked 5' phenyl methyl siloxane HP-5 MS capillary column (30 m × 0.32 mm × 0.25 mm coating thickness), coupled with a mass detector. GC-MS operating conditions were as follows: injector temperature 215°C, transfer line 280°C, oven temperature program 80°C–280°C with ramping 5°C min,[1] carrier gas: helium at 1.5 mL min,[1] mass spectra: electron impact (EI+), individual components were identified by NIST MS 2.0 f structural library which was used for identification and interpretation of components.[21],[22],[23],[24]

In vitro cell-line studies

The Niosomal gel was subjected to in-vitro cell-lines screening related to skin. Cell lines were developed in 1640 RPMI medium consisting 10' fetal bovine serum and 2 mM L-glutamine. In the current screening experiment, the cells were inoculated to 96-well microtiter plates in 100 μL at plating densities as described in the summary of the report above, based on the doubling time of individual cell lines. Upon cell inoculation, the microtiter plates were incubated for 24 h prior to the introduction of experimental drugs at 37°C, 5' CO2, 95' air and 100' relative humidity. Gels were initially solubilized at 100 mg/ml in dimethyl sulfoxide and diluted with water to 1 mg/ml and preserved in frozen form before use. A frozen concentrate aliquot (1 mg/ml) with complete medium containing test article was thawed and diluted to 100, 200, 400, and 800 μg/mL at the time of drug introduction. Aliquots of 10 μl of these various drug dilutions were introduced to the suitable microtiter wells containing 90 μl of medium, resulting in the appropriate final concentrations of the drug i.e., 10, 20, 40, and 80 μg/ml. After the addition of gels, the plates were incubated for 48 h under standard conditions and the assay was completed by adding cold TCA. Cells were fixed in situ by adding 50 μl cold 30' (w/v) TCA (final concentration, 10' TCA) and incubated at 4°C for 60 min. The supernatant was discarded; with tap water, the plates were washed five times and air dried. Sulforhodamine B (SRB) solution (50 μl) was introduced to each of the wells at 0.4 per ' (w/v) in 1 per ' acetic acid and plates were incubated at room temperature for 20 min. Unbound dye was retrieved after staining, and the residual dye was eliminated by washing five times with 1' acetic acid. The plates were air dried. Bound stain was subsequently eluted with 10 mM Trizma base and the absorbance was read on a plate reader at a wavelength of 540 nm with 690 nm reference wavelength.

Percent growth was calculated on a plate-by-plate basis for test wells relative to control wells. Percent growth was expressed as the ratio of average absorbance of the test well to the average absorbance of the control wells ×100. Using the six absorbance measurements (time zero [Tz], control growth© and test growth in the presence of drug at the four concentration levels [Ti]), the percentage growth was calculated at each of the drug concentration levels.

Percentage growth inhibition was calculated as: (Ti/C) × 100'.[25],[26]

 Results and Discussion

The Cucurbitaceae family of plants has been well known as gourd family. Some of them with well-known local names and useful for human consumption are Cucurbita (squash, pumpkin, zucchini etc.), Lagenaria (calabash), Citrullus-watermelon (C. lanatus, C. colocynthis), Cucumis – cucumber (C. sativus), and Momordica – bitter melon. Our selected PEBH yield was found to be 9.1'.

Natural products play an important role as nutraceuticals and treatment modules ranging from arthritis to multiple body supplements. The drug discovery process from natural products has its base in structural skeletons of carbon units. Due to the occurrence of secondary metabolites, the phytoconstituents elucidated from plants have gained interest.[1]

For targeting drugs, carriers such as serum proteins, synthetic polymers, immunoglobulin, microspheres, niosomes, and liposome microspheres erythrocytes have been used.[27] Out of all of these carriers, niosomes is regarded as the best among all. The probable mechanism is their concentration in the tissues with proper localization at the site.[28] They are capable of enabling maximum efficacy of the medication without affecting surrounding tissues.[29] As per the designed study protocol, different trials were undertaken with surfactant grades for Niosomal solution [Table 1]. The ratio of cholesterol was varied to obtain and evaluate maximum entrapment efficiency. The trial batches revealed F14 (Formulation 14) to be considered as optimized solution based on the evaluation results. The solution was homogenous and no drug leakage. The F 14 [Figure 1]a obtained after centrifugation with maximum entrapment efficacy [Figure 1]b was selected for the preparation of the Niosomal gel.{Table 1}{Figure 1}

Gels are considered to be two component semisolid system consisting of dispersion made up of either small inorganic particles or large organic molecules enclosing and interpenetrated by liquids.[30] The application is done directly on the mucous membrane of the eye or on the skin. They are known as drugs which act for a long duration of time when injected intramuscularly. Carbomers have been used in liquid or semi-solid pharmaceutical formulations as rheology modifiers. Carbomer copolymers act as emulsifying agents in the preparation of oil-in-water emulsions for external administration. Carbomer formulations are effective in improving moderately to severe symptoms of dry eye syndrome.[31]

In contrast to the conventional drug delivery like injection and oral drug administration, drug delivery by transdermal route proves to be an effective alternative. Not only the transdermal drug delivery provides convenience to the patient, but also is noninvasive and provides delivery of drug for a period of days. It offers multiple sites to avoid local irritation and toxicity, yet it can also offer the option of concentrating drugs at local areas to avoid undesirable systemic effects.[32] At present, drug delivery by transdermal route is limited as there are only few drugs which can be viable by transdermal route. The advantages of surfactant-based niosomes are:

Non-immunogenic, biodegradable, and biocompatible They help to release drugs in a controlled manner through their closed bilayer structure They are found to be effective in providing improved response by mechanism of reduced clearance and specific targeting.They have studied for transdermal, parenteral, and ophthalmic route.[33]

NG1 was selected as the optimized batch [Figure 1]c as investigated under trial batches [Table 2]. It was found to have clear white appearance. NG1 was found to have appealing smooth texture and consistency. NG1 gel was evaluated by tanning electronic microscopy [Figure 2]a. The formulations were found to have pH in the range of 5.5–7.1 and had drug content ranging from 95' to 98' which suffices the specified limits as per ICH guidelines. Gel was observed to be non-gritty, easily spreadable, and washable [Figure 2]b,[Figure 2]c,[Figure 2]d,[Figure 2]e. Physical mixtures of drug and excipients kept at specified conditions of temperature and humidity revealed the compatibility of excipients with PEBH extract [Table 3]. The Niosomal gel was prepared by using different Carbopol grades and with comparison of different parameters, and optimized Niosomal gel was determined based on evaluation results [Table 3]. It was observed that increasing the polymer concentration in gel directly increased the viscosity of the gel. In vitro drug release showed consistency with Carbopol 934 [Figure 2]f. Stability studies were performed of the optimized gel. There were no significant changes observed in the formulations. It was concluded obtained optimized gel was found to be stable over a period of 6 months [Table 4].{Table 2}{Table 3}{Table 4}{Figure 2}

Chromatography is hyphenated technique which offers critical evaluation of phytoconstituents. The coupling of hyphenated technique has offered the entire world with interest in plant-related ailments. Analytical chemists have been able to best elucidate plant related constituents according to their area of interest related to therapeutic potential. The phytoconstituents elucidated has gained importance in to day-by-day affairs by offering their advantage over synthetic available treatments. Techniques such as liquid chromatography and mass spectroscopy (LC-MS) and GC-MS are considered important due as they are used in the identification of volatile and nonvolatile constituents.[1]

Niosomal PEBH gel on GC-HRMS was found to elucidate ten components [Figure 3] and [Table 5]. Component 1 [Figure 4]a, 1.2-ethyl 2-Hexen-1-ol, 28'] with m/z 128 and fragment ions 55, 67, 85, 95, 110 containg hyroxy groups have been used as antibacterial agents.[34] Component 2 [Figure 4]b, 1, Cis-9, 10-Epoxyoctadecan-1-ol, 35'] with m/z 171 and fragment ions 55, 69, 81, 95, 111, 123, 143, 155, 171 has been confirmed.[35] Component 3 [[Figure 4]c, Dodecanoic acid, 2-penten-1-yl ester, 33'] was found to be fatty acid with m/z 200 and fragment ions 57, 69, 85, 95, 109, 128, 141, 163, 183.[36] Component 4 [[Figure 4]d, Cholestane, 4, 5-epoxy-, [4α, 5α], 31'] with m/z 386 and fragment ions 55, 67, 81, 95, 107, 124, 137, 175, 203, 231, 246, 259, 273, 315, 371.[37] Component 5 [[Figure 5]a, cyclopentaneundecanoic acid, 36'] with m/z 268 and fragment ions 55, 74, 87, 97, 129, 143, 157, 158, 199, 225, 268.[38] Component 6 [[Figure 5]b, 17-octadecynoic acid, 34'] with m/z 265 and fragment ions 55, 67, 81, 95, 109, 123, 137, 177, 195, 223, 247.[39] Octadec-17-ynoic (monounsaturated fatty acid) acid has been discovered as acetylenic fatty acid which has been doubly dehydrogenated at positions 17 and 18 to give respective alkynoic acid. It has been well evaluated as a P450 inhibitor, leukotriene-B420-monooxygenase inhibitor and an alkane 1-monooxygenase inhibitor.[40],[41] Component 7 [[Figure 5]c, Octadecanoic acid, methyl ester, 29'] with m/z 298 and fragment ions 55, 74, 87, 97,111, 129, 143, 157, 185, 199 has used as anti-foaming agent, nutrient additive and cosmetic industries.[42] Component 8 [[Figure 5]d, 9-Octadecadienoic acid (Z)-, methyl ester, 29'] with m/z 278 and fragment ions 55, 74, 81, 95, 123, 179, 180, 222, 264 has been known as hazardous but limitation puts its usage as functional additive.[43] Component 9 [[Figure 6]a, Tridecanoic acid, methyl ester, 29'] with m/z 228 and fragment ions 55, 74, 143, 185, 228 is a methyl ester of fatty acid.[44] It is known as metabolite of plant, flavoring agent and adds fragrance. Component 10 [[Figure 6]b, Silane, dimethyl [2, 3, 6 trichlorophenoxy] heptadecyloxy, 27'][45] with m/z 495 and fragment ions 55, 75, 97, 159, 219, 255, 313.The components present in the gel PEBH extract were predominantly found to be saturated and unsaturated fatty acids and their esters, diterpenes and methylated phenols.{Figure 3}{Figure 4}{Figure 5}{Figure 6}{Table 5}

Melanoma is an aggressive malignant neoplasm derived from melanocytes. Malignant non-melanoma skin cancers originate from keratinized epithelial cells. BCC is caused by cell mutations induced by UV radiation and commonly noticed on sun-exposed areas (nose, ears, face and backs of hands). Cutaneous SCC arises from malignant, uncontrolled proliferation of epidermal keratinocytes.[46],[47] Drug–delivery systems using niosomes as alternative therapy has been the focus of this research paper. There is recent interest among the researchers to search for complementary medicine for the treatment of cancer. Many of the existing drugs have not proved to be effective against different types of cancer. Some of the well-known plants used in chemotherapy of cancer Rauwolfia serpentia, Panax ginseng etc. India has been witnessing substantially arise in SCC cases.[13],[14] B16 have been used for metastasis and solid tumor formation.[15] Our initial screening for human oral cancer cell line KB [Table 6] and Mus Musculus skin melanoma B16-F10 [Table 7] has shown unsatisfactory results at the selected concentrations as indicated by graphs [Figure 7]a,[Figure 7]b,[Figure 7]c,[Figure 7]d,[Figure 7]e,[Figure 7]f,[Figure 7]h. The article will help to further explore research endeavors in exploring plant-based treatment strategies. Nevertheless, the earth has ample resources dearth lies in the correct identification of the phytoconstituents. Proper phytoconstituent prospective studies help to explore many plants from various families for different therapeutic categories.{Table 6}{Table 7}{Figure 7}


The researchers have attempted to evaluate potential of BH niosomal gel for in vitro cell lines of cancer with detailed physiochemical and analytical evaluation. Future studies may be undertaken at various stages and levels to further explore the potential of these family of plants in depth related to growth of multiple therapeutic areas.


We would like to acknowledge the help and guidance received from the SAIF Department, IIT, Mumbai. We also wish to acknowledge Hemen S Ved and Ami P Thakkar (Research Scholars, Department of Pharmacology, SVKM's Dr. Bhanuben Nanavati College of Pharmacy), for their help and support in the compilation of the article.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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