Abstract
Antibiotic resistance in the context of treating malarial infections is a major challenge in India. Home remedies such as thulasi leaves (Ocimum tenuiflorum), black pepper seeds (Piper nigrum), clove buds (Syzygium aromaticum), cinnamon bark (Cinnamomum verum), and nilavembu whole plant powder (Andrographis paniculata) were taken to explore antimalarial and methicillin-resistant Staphylococcus aureus (MRSA) activity. Among the five extracts, the best two extracts, C. verum and P. nigrum extract, showed the presence of Quercetin. Phytoniosomes were prepared by simple probe sonication with the two extracts and the resultant vesicles were in the size range of (319.7 nm). They showed significant (P < 0.001) antimalarial potency IC50 at 5.25 µg/ml against P. falciparum 3D7. In addition, their cytotoxicity (TC50) against Vero cell line was found to be > 100 µg/ml. The therapeutic index was found to be > 32 µg/ml. Phytoniosomes were converted to a capsule dosage form by lyophilization and this capsule was stable up to 90 days.
Keywords: Methicillin-resistant Staphylococcus aureus, Phytoniosome, Plasmodium falciparum, Antimalarial activity, Traditional medicine
Introduction
Malaria is a one of the major health problems of our country. India reports 1 million cases annually (Tunsirikongkon et al. 2013). Malaria is curable if effective treatment strategies are followed. Plants or plant extracts or their active constituents have been used for the treatment for malaria. Artemisia species are widely used for the treatment of diseases such as malaria, hepatitis, inflammation and infections by fungi, bacteria, and viruses (Tunsirikongkon et al. 2013; Sachan and Gupta 2014; Aronsson et al. 1981).
Literature reports state that Artemisia-based products have begun to show resistance to malaria in recent years (Gunes et al. 2017; Bagavan et al. 2011). Hence, there is a need for identifying new plant-based products for treating malaria backed with scientific evidence (Brewer et al. 1994; Carvalho and Krettli 1991). After detailed literature review, it was observed that no scientific evidence-based reports have been published for the combined extracts of thulasi leaves (Ocimum tenuiflorum), black pepper seeds (Piper nigrum), clove buds (Syzygium aromaticum), cinnamon bark (Cinnamomum verum), and nilavembu whole plant powder (Andrographis paniculata) for antimalarial property. These selected plants have a broad spectrum of bioactivity, owing to the presence of several active components and secondary metabolites.
The selected plants have traditionally been used for antimalarial, appetizer, antispasmodic, antiparasitic, and methicilin-resistant S. aureus activity (Didem et al. 2016; Dondorp et al. 2009). Different literature study reports proving the effects of plants on malaria have been investigated using alcoholic or aqueous extracts (Hong et al. 2009; Jude et al. 2017). Reports also state that ethanol, hexane, and aqueous extracts of the individual plants were found to be active in in vitro against the malaria parasite (Kamaraj et al. 2012).
A previously reported study has shown that O. tenuiflorum (Banyal et al. 2015), P. nigrum (Samuel et al. 2016), S. aromaticum (Batiha et al. 2020a), C. verum (Batiha et al. 2020b), and A. paniculata (Mishra et al. 2009) have antimalarial activity. A study done on the combination of A. paniculata and H. corymbosa showed substantial enhancement in their antimalarial activity. Combinatorial effect of each of these with curcumin also revealed antimalarial effect. Additive interaction between the plant extracts and their individual synergism with curcumin was evident from this study. Increased in vivo potency has also observed with the combination of plant extracts over the individual extracts and curcumin (Mishra et al. 2009).
In this study, the combined extracts of A. paniculata, P. nigrum, S. aromaticum, O. tenuiflorum, and C. verum and extracts loaded phytoniosomes were evaluated for antibacterial activity against methicillin-resistant S. aureus and in vitro antimalarial activity on CQ-sensitive strain 3D7 of P. falciparum. So far, no study has been reported for the development of phytoniosomes with the above selected plants neither individually nor combined.
Materials and methods
Materials
Stearic acid, myristic acid, cholesterol, glycerol mono-stearate, tween 80, and talc were purchased from Loba chemicals, Mumbai. Palmitic acid, Tween 60, Potassium dihydrogen phosphate, Mannitol, Magnesium stearate, Quercetin, and Sodium hydroxide were purchased from Merck laboratories, Bangalore.
Plant material
Five different plant parts were purchased from Pooja herbal store, Peelamedu Coimbatore. They were authenticated by the national institute of siddha, Chennai. Authentication number: O. tenuiflorum (Thulasi)—NISMB3132018, A. paniculata (Nilavembu)—NISMB3132018, C. verum (Cinnamon)—NISMB3132018, S. aromaticum (Clove)—NISMB3132018, and P. nigrum (Black pepper)—NISMB3132018.
Powder microscopy
The powder samples of O. tenuiflorum, A. paniculata, C. verum, S. aromaticum, and P. nigrum were treated with concentrated hydrochloric acid and phloroglucinol. The powder characteristics of structural details of the plant drugs were identified (Kayano et al. 2011).
Preliminary phytochemical screening and physicochemical evaluation of the extracts
The various extracts obtained after extraction were subjected for phytochemical screening to determine the presence of chemical constituents such as alkaloids, glycosides, tannins, flavonoids, saponins, steroids, and phenols (Lohar and Ravindra 2008).
Determination of ash values
The ash values are determined to assure the exclusion of low-grade products, exhaused drugs, and sandy or earthy matter (Jude et al. 2017).
Total ash value
Accurately 2 g of air-dried powder was weighed in a tarred silica crucible and incinerated at a temperature not exceeding 450 °C until free from carbon then it was cooled and weighed. Further, the percentage of total ash with reference to the air-dried powdered drug was calculated.
Acid insoluble ash
The ash obtained in the above method was boiled for 5 min with 25 ml of diluted HCl. The residue was collected on ash-less filter paper and washed with hot water, ignited and weighed. The percentage of acid insoluble ash was calculated with reference to the air-dried drug.
Water-soluble ash
The ash obtained in total ash was boiled for 5 min with 25 ml of water. The insoluble matter was collected on an ash-less filter paper, further it was washed with hot water and ignited to constant weight at a low temperature. The weight of insoluble matter was subtracted from the weight of the ash. The difference in weights represents the water-soluble ash. The percentage of water-soluble ash with reference to the air-dried drug was calculated.
Soxhlet extraction
About 50 g of dried Aerial part of A. paniculata powder and 25 g of dried bud powder of S. aromaticum were subjected for aqueous extraction using soxhlet apparatus with the temperature maintained at 60–70 °C. About 25 g of dried fruit powder of P. nigrum was subjected to soxhlation using ethanol at temperature 40–50 °C. Hexane extract of O. tenuiflorum and C. verum was obtained separately using Soxhlet apparatus with 25 g of leaves and 25 g of dried bark at the temperature of 50–60 °C. The extracts were obtained after complete evaporation on water bath by placing it on evaporating dish (Al-Owaisi et al. 2014).
Determination of heavy metals
The test was designed to determine the content of metallic impurities (such as lead and arsenic) that are colored by sulfide ion, under specified conditions. The limit for heavy metals is indicated in the individual monographs in terms of the parts of lead per million parts of the substance (by weight), and it was determined by visual comparison of the color produced by the substance with that of a control prepared from a standard lead solution.
High-performance thin layer chromatography (HPTLC)
About 4 mg of quercetin standard was taken and dissolved in 10-ml methanol. Ethyl acetate, methanol, glacial acetic acid, and water in the ratio 10:1.1:1.1:2.6 was used as the solvent system. Different volumes of the standard (1, 2, 3, 4, and 5 µl) and extracts (O. tenuiflorum, A. paniculata, C. verum, S. aromaticum, P. nigrum and our formulation) were applied on different tracks in a precoated TLC plates using Linomat 5 (Kayano et al. 2011; Lohar and Ravindra 2008).
Determination of total flavonoid content (TFC) by aluminum chloride method
Total flavonoid content in crude extracts of C. verum, S. aromaticum, A. paniculata, P. nigrum, and O. tenuiflorum was determined using quercetin as a standard. Briefly, 10 mg of quercetin was dissolved in 80% ethanol and then diluted to 20, 40, 60, 80, and 100 μg/ml. The diluted standard solutions of quercetin or plant extracts (0.5 ml) of different concentration were separately mixed with 1.5 ml of 95% ethanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 mol/l potassium acetate, and 2.8 ml of distilled water in a test tube. The test tubes were incubated for 30 min at room temperature to complete the reaction. The absorbance of the reaction mixture was measured at 415 nm with an UV–Vis spectrophotometer against a blank. The amount of flavonoid was calculated from linear regression equation obtained from the quercetin calibration curve. The flavonoid content was calculated as mean ± SD (n = 3) and expressed as mg/g of quercetin equivalent (QE) of dry extract (Jude et al. 2017).
Determination of total phenolic content (TPC) by Folin–Ciocalteu reagent method
Folin–Ciocalteu reagent was used to determine the total phenolic content (TPC) of C. verum, S. aromaticum, A. paniculata, P. nigrum, and P. tenuiflorum crude extracts. Gallic acid was used as a reference standard (20–100 μg/ml) for plotting calibration curve. A volume of 0.5 ml of the plant extract was mixed with 1.5 ml of Folin–Ciocalteu reagent (diluted 1:10 with de-ionized water) and was neutralized with 3 ml of sodium carbonate solution (7.5%, w/v). The reaction mixture was kept in dark at room temperature for 30 min with intermittent shaking for color development. The absorbance of the resulting blue color was measured using UV–Vis spectrophotometer at fixed wavelength of 765 nm. The TPCs were determined using linear regression equation obtained from the standard plot of gallic acid. The content of total phenolic compounds was calculated as mean ± SD (n = 3) and expressed as mg/g gallic acid equivalent (GAE) of dry extract (Jude et al. 2017).
Formulation of phytoniosomes
Phytoniosomes were prepared by film hydration method following hydration and bath sonication. Initially, Tween 60, mannitol, and cholesterol were dissolved in chloroform in 1:1:1 molar ratio. Plant extract 1.0 mg/ml was mixed appropriately in a round bottom flask. Afterwards, chloroform was removed under constant rotation at 55 °C using a rotary evaporator to obtain a thin film on the surface of the flask. The dried film was hydrated with phosphate-buffered saline (PBS) by agitation in a water bath at 55 °C for 2 h. The resulting solution was then subjected to bath sonication for 20 min to obtain finer vesicles. The prepared phyto vesicles were stored at 4 °C for further chromatographic analysis and in vitro cell culture experiments. The synthesis of blank (unsonicated) phytoniosomes was carried out in the same way without using plant extract. Lyophilization of the prepared phytoniosomes was done by freezing the suspensions at − 80 °C overnight, and then the samples were transferred to a freeze-dryer for 72 h, to get phytoniosome powders (Brewer et al. 1994; Tunsirikongkon et al. 2013).
Particle size and zeta potential analysis
The size distribution and zeta potentials of phytoniosomes were assessed by measuring their dynamic light scattering and electrophoretic mobility with a Malvern Zetasizer.
Microscopical analysis
The shapes of the prepared phytoniosomes were analyzed using atomic force microscopy.
In vitro cultivation of P. falciparum and of antimalarial activity assessment
Chloroquine (CQ)-sensitive strain 3D7 of P. falciparum was used for in vitro blood stage culture to test the antimalarial efficacy of different extracts/formulations. P. falciparum 3D7 cultures were maintained in fresh O+ve human erythrocytes suspended at 4% hematocrit in RPMI 1640 (Sigma) containing 0.2% sodium bicarbonate, 0.5% albumax, 45 μg/l hypoxanthine, and 50 μg/ml gentamicin and incubated at 37 °C under a gas mixture 5% O2, 5% CO2, and 90% N2. Every day, infected erythrocytes were transferred into fresh complete medium to propagate the culture. For P. falciparum, albumax was replaced by 10% pooled human serum.
Drug dilutions
Stock solutions of Chloroquine were prepared in water (milli-Q grade) and test formulations/extracts were made in dimethyl sulfoxide (DMSO). All stocks were then diluted with culture medium to achieve the required concentrations (in all cases except CQ)—the final solution contained 0.4% DMSO, which was found to be non-toxic to the parasite. CQ and test formulations were then placed in 96-well flat-bottom tissue culture grade plates.
Evaluation of anti-plasmodial activity
The formulations/extracts were evaluated for their antimalarial activity against P. falciparum strains 3D7. For formulation/extract screening, SYBR green I-based fluorescence assay was carried out as described. Sorbitol synchronized parasites were incubated under normal culture conditions at 2% hematocrit and 1% parasitemia in the absence or presence of increasing concentrations of formulations. CQ was used as positive control, while 0.4% DMSO was used as the negative control. After 48 h of incubation, 100 μl of SYBR Green I solution (0.2 μl of 10,000 X SYBR Green I (Invitrogen/ml) in lysis buffer {Tris (20 mM; pH 7.5), EDTA (5 mM), saponin (0.008%; w/v), and Triton X-100 (0.08%; v/v)} was added to each well and mixed twice gently with multi-channel pipette and incubated in dark at 37 °C for 1 h. Fluorescence was measured with a victor fluorescence multi-well plate reader (Perkin Elmer) with excitation and emission wavelength bands centered at 485 and 530 nm, respectively. The fluorescence counts were plotted against the drug concentration and the 50% inhibitory concentration (IC50) was determined by analysis of the dose–response curves. Results were validated microscopically by examination of Giemsa-stained smears of extract-treated parasite cultures:
Parasitemia = Number of parasitized RBC/Total number of RBC counts × 100.
Cytotoxic activity on Vero cells using MTT assay
The cytotoxic effects of extracts/formulations on host cells was assessed by functional assay as described using Vero cells cultured in MEM containing 10% fetal bovine serum, 0.21% sodium bicarbonate (Sigma), and 50 μg/ml penstrip (complete medium). Briefly, cells (104 cells/200 μl/well) were seeded into 96-well flat-bottom tissue culture plates in complete medium. Drug solutions/ formulation were added after 24 h of seeding and incubated for 48 h in a humidified atmosphere at 37 °C and 5% CO2. DMSO was added at 10%. Twenty microliters of a stock solution of MTT (5 mg/ml in 1X phosphate-buffered saline) was added to each well, gently mixed and incubated for another 4 h. After spinning the plate at 1500 rpm for 5 min, supernatant was removed and 100 μl of DMSO (stop agent) was added. Formation of formazan was read on a microtiter plate reader (Versa max tunable multi-well plate reader) at 570 nm. The 0.1 mg/ml concentration of freeze-dried extract and equivalent phytoniosomes was added. The 50% cytotoxic concentration (TC50) was determined by analysis of dose–response curves. Therapeutic index was calculated as a ratio of TC50 Vero/IC50 3D7. The values are shown in Fig. 4.
Fig. 4.
a Peak area of standard caffeic acid, b peak area of standard gallic acid, c Peak area of S. aromaticum, d peak area of O. tenuiflorum, e peak area of A. paniculata
Anti-bacterial activity against drug-resistant methicillin-resistant Staphylococcus aureus (MRSA)
It is a form of S. aureus that has become resistant to some antibiotics. Methicillin-resistant Staphylococcus aureus (MRSA) is not a danger to healthy individuals but people may become colonized. As per clinical and laboratory standards institute (CLSI) recommendations, Mueller–Hinton agar medium was used to grow the MRSA strain. Agar well plate method was carried out. The formulation and the extracts were added to the well and incubated at 37 °C. The zone of inhibition was measured using scale after incubation.
Preparation of capsule containing phytoniosome powder
The hard gelatin capsule size 0 was filled with 400-mg powder (combined extract of O. tenuiflorum and P. nigrum) which is reported to have good in vitro antimalarial activity. The freeze-dried phytoniosome powder of O. tenuiflorum 100 mg and P. nigrum 100 mg was blended with the calculated amount of 2% w/w of talcum as a glidant, 2% w/w of magnesium stearate as lubricant, and the total amount to be filled in the capsule was made up with the filler, microcrystalline cellulose. The mixed powder was packed into capsule by manual capsule filling machine. The capsules were stored at room temperature and studied for stability.
Evaluation of the phytoniosome capsule
Physical appearance
The physical appearances of capsule were evaluated in terms of capsule shells’ appearance and color. The content inside the capsule shell was also investigated for its physical appearance.
Uniformity of weight
First, the weight of 20 filled capsules were determined and recorded. Then, the inside contents were totally discarded and the empty shells were subsequently weighted. The weight difference between the filled capsule and the empty capsule shell was calculated as the weight of powder. The average weight and percentage of deviation were also calculated and compared with the reference.
Disintegration
Disintegration of capsules was tested in a disintegration tester. Phosphate buffer (pH 7.4) was used as a testing medium; the temperature was set at 37 °C. Six capsules were evaluated for their time of disintegration by placing each capsule into a disintegrating tube and covered using the disc. The test was carried out until the phytoniosome-loaded capsule totally disintegrated without any residues in the basket. The disintegration times were recorded and the average time was consequently calculated. The disintegration time should be less than 30 min for hard gelatin capsules as per Indian pharmacopoeia limit.
Stability studies of the prepared capsules
The prepared phytoniosome capsules were stored at room temperature (30 ± 1 °C) for stability studies. The capsules were evaluated for physical appearance, weight uniformity, flowability of the contents, and disintegration at the end of 7, 15, 30, 45, and 60 days.
Statistical analyses
All data presented as Mean ± SD of at least three samples. Significant difference among groups was determined by ANOVA, using graph pad prism version 5 for windows.
Results and discussion
Around 100 plant species belonging to 40 families have been used as antimalarial agents (Choubey and Dubey 2015). Traditional remedies are widely used to treat malaria, as they are more available and affordable. The developments of new antimalarial natural products are the need of the hour, as there is an increasing resistance of plasmodium to the available antimalarial drugs (Kayano et al. 2011). The selected plant materials of the study A. paniculata, S. aromaticum, C. verum, P. nigrum, and O. tenuiflorum were significantly determined as not toxic in previously reported studies (Worasuttayangkurn et al. 2019; Valente et al. 2009; Muhammad et al. 2017; Chunlaratthanaphorn et al. 2007; Uma et al. 2013). Hence, the investigation with O. tenuiflorum leaves, P. nigrum seeds, S. aromaticum buds, C. verum bark, and A. paniculata whole plant powder were taken to explore antimalarial activity. Through the powder microscopy, vascular fibers and stone cells of A. paniculata, prismatic crystals and stone cells of O. tenuiflorum, fibers and resin cells of C. verum, calcium oxalate crystals and starch grains of S. aromaticum, oil glands and stone cells of P. nigrum were identified and compared with standards.
Phytochemical analysis
O. tenuiflorum, A. paniculata, C. verum, S. aromaticum, and P. nigrum plant powder were extracted by soxhlet extractor using various solvents (hexane, ethanol, and water) based on the literature reports. O. tenuiflorum leaves were found to contain alkaloids, steroids, and tannins in hexane extract, but it was absent in ethanolic extract as per the reported literature (Tunsirikongkon et al. 2013; Sachan and Gupta 2014). A. paniculata whole plant powder aqueous extract was found to contain tannins, flavonoids, and cardiac glycosides but it was absent in ethanol and methanol extract as per the literature reports (Aronsson et al. 1981). C. verum powder of hexane extract was found to contain steroids, alkaloids, saponins, tannins, and phenols but it was absent in ethanolic and aqueous extract as per the literature (Gunes et al. 2017). S. aromaticum bud powder aqueous extract was found to contain tannins, flavonoids, and cardiac glycosides. However, it was absent in ethanol and hexane extracts as per the literature review (Bagavan et al. 2011). P. nigrum fruit ethanolic extract was found to contain tannins, cardiac glycosides, and alkaloids in ethanolic extracts but other relevant literature states that it is absent in petroleum ether, chloroform, and n-butanol extract (Brewer et al. 1994). The phytochemical screening results of crude extracts are shown in Table 1.
Table 1.
Phytochemical screening of extracts
| Plant part | Solvent | Alkaloids | Tannins | Cardiac glycosides | Flavonoids | Steroids | Phenols | Saponins |
|---|---|---|---|---|---|---|---|---|
| A. paniculata | Water | − | + | + | + | − | − | − |
| O. tenuiflorum | Hexane | + | + | − | − | + | − | − |
| P. nigrum | Ethanol | − | + | + | + | − | − | − |
| C. verum | Hexane | + | + | − | + | + | + | + |
| S. aromaticum | Water | − | + | + | + | − | − | − |
Present (+) negative (−)
Previous literature study reports state that water is the best extracting solvent for flavonoids in S. aromaticum (Ghallab et al. 2020) when compared to organic solvents. Ethanol extract of P. nigrum was more active than the hexane, dichloromethane, ethyl acetate, or aqueous extraction (Simas et al. 2007). Similarly, hexane extract of O. tenuiflorum and C. verum was the best extracting solvent for phytochemical compounds extraction compared to chloroform, ethyl acetate, and methanol (Simas et al. 2007; Kumar et al. 2012). The choice of solvents in this study was thus based on the above reports.
Ash value
Total ash value of O. tenuiflorum, A. paniculata, C. verum, S. aromaticum, and P. nigrum was found to be 2, 1, 1, 1, and 2%. Water insoluble ash value was found to be 2, 1, 3, 3, and 2.5%. Acid insoluble ash value was found to be 0.23, 1.5, 2, 3, and 1.5%. The ash values of the extracts are within the limit cited in ayurvedic pharmacopoeia-II (Carvalho and Krettli 1991) (Table 2).
Table 2.
Ash value for extracts
| Parameters | A. paniculata (%) | O. tenuiflorum (%) | C. verum (%) | S. aromaticum (%) | P. nigrum (%) |
|---|---|---|---|---|---|
| Total ash | 2 | 1 | 1 | 1 | 2 |
| Water-soluble ash | 2 | 1 | 3 | 3 | 2.5 |
| Acid insoluble ash | 0.23 | 1.5 | 2 | 3 | 1.5 |
Percentage yield
The percent yield of aqueous soxhlet extract of A. paniculata and S. aromaticum was found to be 8.1 and 7.6%, respectively. Ethanolic extract of P. nigrum was found to be 16.48%. Hexane extract of O. tenuiflorum and C. verum was found to be 3.88 and 9.2%, respectively. A high percentage yield was found in ethanol extract of P. nigrum (Table 3).
Table 3.
Percentage yield of extracts
| Type of extract | % yield (W/W) | Color of extract |
|---|---|---|
| Aqueous soxhlet extract of A. paniculata | 8.1 | Dark green |
| Aqueous soxhlet extract of S. aromaticum | 7.6 | Dark brown |
| Ethanolic soxhlet extract of P. nigrum | 16.48 | Light-greenish brown |
| Hexane soxhlet extract of O. tenuiflorum | 3.88 | Light green |
| Hexane extract of C. verum | 9.2 | Light brown |
Total flavonoids and phenolic content
The total amount of flavonoids content was determined. The flavonoid content of A. paniculata, S. aromaticum, C. verum, and P. nigrum was found to be 0.152 ± 0.002, 0.092 ± 0.009, 0.243 ± 0.002, and 0.214 ± 0.009 mg/g, respectively, of Gallic acid equivalent of dry extract. A significant difference (P < 0.002) in the flavonoid contents of different extracts was determined by one-way ANOVA test. Total amount of phenolic content in C. verum was found to be 0.328 ± 0.019 mg/g of Gallic acid equivalent of dry extract (Dondorp et al. 2009).
Vesicle size determination
The vesicle size of the sonicated phytoniosome was in the size range of 319.7–621.2 nm and the unsonicated phytoniosomes were slightly bigger in vesicles size when compared with sonicated phytoniosomes (Table 4). The zeta potential of the formulations ranged from − 1.39 to − 16.8 mV, which shows that all the particles are negatively charged. Extract-loaded phytoniosomes show a population with relatively homogenous sizes. The Atomic Force Microscopy (AFM) report reveals that the shape of herbal loaded phytoniosome was found to be semi-spherical (Fig. 1). All the phytoniosome-combined extract-sonicated formulations show less size and high zeta potential when compared to non-sonicated formulation, except C. verum and P. nigrum formulations (Table 4). The selected best formulation based on vesicle size was used for further evaluations.
Table 4.
Particle size and zeta potential of the prepared phytoniosomes
| Formulation code | Combined formulation | PDI | Zeta potential | Average size |
|---|---|---|---|---|
| PT1 | O. tenuiflorum and P. nigrum (phytoniosome unsonicated) | 0.103 | − 1.39 | 791.7 |
| PT2 | O. tenuiflorum and P. nigrum (phytoniosome sonicated) | 0.467 | − 14 | 621.2 |
| PN1 | A. paniculata, S. aromaticum and C. verum (phytoniosome unsonicated) | 1.000 | − 16.8 | 676.5 |
| PN2 | A. paniculata, S. aromaticum and C. verum (phytoniosome sonicated) | 1.000 | − 11.6 | 319.7 |
| PC1 | C. verum and P. nigrum (phytoniosome unsonicated) | 1.000 | − 22.3 | 613.4 |
| PC2 | C. verum and P. nigrum (phytoniosome sonicated) | 0.385 | − 15.4 | 757.2 |
Fig. 1.
2D display of atomic force microscopic image of A. paniculata, S. aromaticum and C. verum loaded phytoniosome
HPTLC method
Extracts of A. paniculata, C. verum, and S. aromaticum showed the presence of flavonoid (quercetin) in whole plant powder of A. paniculata, bark of C. verum, and buds of S. aromaticum when analyzed by HPTLC method. The Rf value of the standard Quercetin was 0.45. The Rf value of A. paniculata, C. verum, and S. aromaticum extracts was 0.45, 0.47 and 0.45 (Fig. 2). The Rf value of the formulation PN1, PC1, PC2 was 0.46, 0.45, and 0.44. The Rf value of the standard caffeic acid was 0.19 (Fig. 3). The Rf value of O. tenuiflorum extract was also found to be 0.19, which confirms the presence of phenolic acid. The Rf value of the standard gallic acid was 0.07. The Rf value of A. paniculata extract shows the presence of phenols and the value was also found to be 0.07 (Fig. 4).
Fig. 2.
a Peak area of standard quercetin, b peak area of A. paniculata, c peak area of C. verum, and d peak area of S. aromaticum
Fig. 3.
a Peak area of the formulation 1 (PN1), b peak area of the formulation 2 (PC1), and c peak area of the formulation 3 (PN1)
Antimalarial activity
The antimalarial testing for the formulations was tested in Chloroquine-sensitive 3D7 strain of P. falciparum. The phytoniosome-containing extracts of C. verum and P. nigrum show antimalarial activity at (IC50 5.25 μg/ml) against P. falciparum 3D7. Even though the sizes of the vesicles are larger after sonication, this combination has shown three times more activity in killing P. falciparum when compared to the standard drug Chloroquine (IC50 3.50 μg/ml). The A. paniculata, C. verum, and S. aromaticum combination showed good antimalarial activity (IC50 6.75 μg/ml). On the other hand, combination of O. tenuiflorum with P. nigrum shows antimalarial activity at (IC50 12 μg/ml) and A. paniculata shows antimalarial activity at (IC50 8.25 μg/ml).
A previous study reported that O. sanctum has excellent antimalarial properties. Eugenol the main constituent of this plant which is responsible for its antimalarial property and the extracts of both parts of plant, i.e., roots and leaves of O. sanctum had eliminated the propagation of parasite to a greater extent as compared to B. variegata plant used (Banyal et al. 2015).
Cytotoxicity studies
The phytoniosomes containing extracts of C. verum and P. nigrum show cytotoxicity at TC50 > 100 μg/ml against Vero cell line. The A. paniculata, C. verum, and S. aromaticum combination also showed cytotoxicity at TC50 > 100 μg/ml. On the other hand, the combination of O. tenuiflorum with P. nigrum and A. paniculata alone also shows cytotoxicity at TC50 > 100 μg/ml. Andrographolide is the major active compound of A. paniculata responsible for its bioactive properties (Karuppiah et al. 2020). The standard drug Chloroquine showed cytotoxicity at TC50 > 65 μg/ml when compared to above mentioned formulations. It confirms that the formulation was non-toxic to Vero cells but at the same time, it inhibits P. falciparum strains 3D7. These extensive abilities of herbal extracts make them important for development of new products. The reports are shown in (Fig. 5) and in (Table 5).
Fig. 5.
In vitro antimalarial activity with phytoniosomes. a Negative control (Standard), b C. verum with P. nigrum (PC1), c A. paniculata, C. verum with S. aromaticum (PN1), d O. tenuiflorum with P. nigrum (PT1), e A. paniculata, and f positive control (Chloroquine)
Table 5.
Therapeutic indices of formulations harboring potent antimalarial activity
| Sample | Antimalarial potency (IC50 μg/ml) against Plasmodium falciparum 3D7 | Cytotoxicity (TC50 μg/ml) against Vero cell line | Therapeutic index TC50/IC50 |
|---|---|---|---|
| (Sonicated C. verum and P. nigrum) | 5.25* | > 100 | > 32 |
| (Sonicated A. paniculata, C. verum and S. aromaticum) | 6.75* | > 100 | > 26 |
| (Sonicated O. tenuiflorum with P. nigrum) | 12* | > 100 | > 12.5 |
| (Sonicated A. paniculata) | 8.25* | > 100 | > 8.25 |
| Std (Chloroquine) | 3.50 | > 56 | > 11.5 |
*Significance was observed for the samples against the standard in the Student t test p < 0.001
Anti-bacterial activity against drug-resistant methicillin-resistant Staphylococcus aureus (MRSA)
The average zone of inhibition of O. tenuiflorum, and P. nigrum was found to be 15 and 13 mm, respectively. The stock solutions of O. tenuiflorum and P. nigrum (1 mg/ml) were serially diluted at various concentrations of 50, 100, 150, and 200 µl. The 200-µl concentration showed the best antibacterial activity against drug-resistant MRSA when compared to other concentrations. As concentration increases, the zone of inhibition also increases in the O. tenuiflorum and P. nigrum extracts and not in formulation (Fig. 6). A previous study reported that the essential oil extracted from O. tenuiflorum showed antimicrobial activity against S. aureus (including MRSA) and E. coli, but was less active against P. aeruginosa (Yamani et al. 2016).
Fig. 6.
Anti-bacterial activity against MRSA. a Extraction of O. tenuiflorum (15 mm) and b extraction of P. nigrum (13 mm)
Formulation of capsules and stability studies
The phytoniosomes containing the extracts of O. tenuiflorum, and P. nigrum were converted into a conventional oral dosage form-like capsule after lyophilization based on the reports of anti-bacterial activity. The capsules filled with phytoniosomes were stable till the end of 60 days. The results of the flow property study indicated a fair flow for the powder. Addition of 2% talc, 1% mannitol, and 2% magnesium stearate along with microcrystalline cellulose increased the flow properties of the lyophilized powder. The disintegration time of the prepared capsule was found to be 13 min, which was within the standard limits. The weight variation range of the prepared capsules was found to be 1.89%, which was within the standard limits. During storage, the capsules filled with the powder did not show any change in the color and appearance. Stability studies show that there were no changes in weight variation and disintegration time of the capsules till 60 days. The phytoniosomal capsules of O. tenuiflorum and P. nigrum have to be subjected for longer time stability studies.
Conclusion
To enhance therapeutic effects and outcomes especially in existing drug-resistant infections, an enormous number of attempts have been made with regard to development of drug delivery systems based on herbs. This is the first study to report with scientific evidence for the combined phytoniosome herbal formulation of C. verum and P. nigrum for antimalarial activity and the formulation with combined extract of O. tenuiflorum and P. nigrum for MRSA activity. The flavonoids present in C. verum- and P. nigrum-loaded formulations may be responsible for antimalarial activity. The phenolic acid and flavonoid in O. tenuiflorum and P. nigrum may be responsible for MRSA activity. Preclinical studies have to be carried out to confirm these antimalarial and antibacterial activities.
Acknowledgements
We would like to thank the Principal, PSG College of Pharmacy and PSG Institute of Medical Sciences and Research for providing necessary facilities to do the work.
Declarations
Conflict of interest
There is no conflict of interest pertaining to this article.
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