Polyherbal effect between Phyllanthus urinaria and Curcuma longa as an Anticancer and Antioxidant

 

Sapti Puspitarini¹, Nashi Widodo², Sri Widyarti², Yoga Dwi Jatmiko², Muhaimin Rifa’i²*

¹Doctoral Program, Biology Department, Science and Mathematics Faculty,

Brawijaya University, Malang, Indonesia.

²Biology Department, Science and Mathematics Faculty, Brawijaya University, Malang, Indonesia.

 

ABSTRACT:

A combination of herbals medicines is an alternative treatment choice for developing anticancer therapy because of its benefits, active compounds, and non-toxic side effect. This study investigates the anticancer and antioxidant activity of Phyllanthus urinaria and Curcuma longa water extract combination. The analysis of their bio-active components was done using LC-HRMS. The biological activity prediction was made using PassOnline and Cytoscape. Anticancer activity, apoptosis, and cell cycle assay were tested on treated T47D breast cancer cells line with the combination herbal extract and individual herbal extract compare to the untreated and cisplatin-treated control cells. This study also determined the antioxidant activity and phenolic and flavonoid total assay. The result showed that these herbs' compounds were predicted to have a biological function in cancer treatment. The herbal combination has efficiency inducing apoptosis with more than 50% compared to the individual herbal treatment and untreated control cells. Cell cycle analysis shows these herbals affect cell cycle arrest at G2/M phase compared to the control cells. The antioxidant activity and total phenol and flavonoid of P. urinaria and C. longa combination positively correlate with the anticancer activity result. Compusyn test results on cell toxicity parameters showed that the combination of both herbs had an additive effect (C = 1). Although this combination has no synergism effect, the water extract of P. urinaria and C. longa combination can be an anticancer agent.

 

 

 

INTRODUCTION:

Indonesia's biodiversity is the second largest in the world after the Amazon Rainforest in Brazil that causes Indonesia to have many indigenous medicinal plants¹. Indonesian herbal medicine usage has been applied in Indonesian society to promote health, prevent and cure diseases². Their benefits go hand in hand with their active compounds. Their active compounds have several pharmacology functions, such as antioxidant, anti-inflammatory, anticancer, cardioprotection, and antimicrobial effects^3,4. Therefore, herbal medicine is a potential source for new drug design^5,6.

 

 

Phyllanthus urinaria is an herbal plant widely distributed in tropical regions in Asia, America, and India. In Southeast Asia, China, India, and Brazil, P. urinaria has long been used as traditional medicine for digestive disease, liver disease, malaria, and jaundice^7,8. P. urinaria has pharmacological potency due to its active compounds. P. urinaria has pharmacological activities, such as hepatoprotective, antidiabetic, antimicrobial, antioxidant, and cardioprotective effects. P. urinaria contains 93 active compounds, including 12 flavonoids, 21 phenolics, 13 terpenoids, 22 lignans, 16 tannins, and other active compounds⁷.

 

Curcuma longa L. belongs to the Zingiberaceae family, which is extensively cultivated for its rhizomes. It is a perennial herb distributed throughout tropical and subtropical regions of the world. A great variety of pharmacological activities of turmeric have been reported. It exhibits anti-parasitic, antispasmodic, anti-inflammatory, anticarcinogenic, and gastrointestinal effects. The aqueous extracts of C. longa contain total phenol content ranging from 4.52% to 7.68%. The flavonoid content ranged from 0.29% to 0.67%^9,10.

 

P. urinaria and C. longa are different herbal extracts that have been consumed daily as Indonesian preventive medicine. People believe these herbals could prevent and cure any kind of disease. The majority of herbal medicines have a function on pharmaceutics due to their active compounds. When herbs are used in combination, the effects can be complicated because various interactions can occur among the individual components. Because of multiple elements in the mix of herbal products, the consequences of herb–herb or herb-drug interactions are often unpredictable and complicated¹¹. Combining drugs and herbs may have synergistic, additive, or antagonistic effects^12,13.

 

Antioxidant is a molecule that capable of acting as an oxidation inhibitor¹⁴. In addition, antioxidants can neutralize free radicals, such as peroxide, hydroperoxide, or lipid peroxyl, thus inhibiting the oxidative mechanisms before caused damage to cells¹⁵.  Medicinal plants contain antioxidants, such as phenolic acids, polyphenols, and flavonoids^4,16, that can increase the antioxidant capacity and reduce the risk of certain diseases such as cancer and diabetes, and heart disease¹⁷. Herbs' antioxidant activity can be determined by several assays, including 2,2–Diphenyl-1-picrylhydrazyl (DPPH) scavenging activity assay¹⁸.

 

Cancer has characteristic uncontrolled growth, invasion, and sometimes metastasis^19,20. Therefore, the invention of an anticancer agent is a challenging task for the researcher. Furthermore, cancer therapy agents should have the ability to induce apoptosis in cancer cells²¹. Chemotherapeutic treatments, including chemotherapy, surgery, and radiation, lead to several side effects, including hair loss, immunosuppression, and mucositis^15–17. So, the search for new anticancer drugs is needed to obtain new insight into anticancer agents, mainly from natural products. Therefore, this study investigates the combination of P. urinaria and C. longa as an anticancer candidate using in silico and in vitro approach.

 

MATERIALS AND METHODS:

Extraction preparation:

P. urinaria and C. longa powder was obtained from UPT. Material Medika Batu, Batu, Indonesia. The sample powder was extracted in boiling demineralized water in a ratio of 1:20 (w/v) for four h. After that, the solution was filter with a fine cloth and centrifuged using Hermle centrifuge type z446. Then the solution was filtered with a rough filter, continued with a 2,5µm Whatman filter. The water of extract was removed using freeze dryer become to a powder and frozen until usage. The extracts were called P. urinaria water extract (PUWE) and C. longa water extract (CLWE). PUWE, CLWE, and their combination (i.e., PUWE + CLWE with 1:1 ratio) were prepared in a water solvent.

 

LC-HRMS analysis:

LC-HRMS was conducted at Life Science Central Laboratory, Brawijaya University. The extract was injected into the LC-HRMS (Thermo Scientific Dionex Ultimate 3000 RSLCnano with microflow meter). The mobile phase consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile. The chromatographic separation was performed by analytical column Hypersil GOLD aQ 50 × 1mm × 1.9μm particle size with an analytical flow rate of 40μL/min. The column temperature was set at 40°C, and the run time was 30 min. The polarity was adjusted in both positive and negative. The full scan was at 70,000 resolution, and data-dependent MS2 was at 17,500 resolution. Processing data using Compound Discoverer (Thermo Scientific) with McCloud MS/MS Library.

 

Biological function prediction:

The biological function prediction of the active compound was retrieved from PassOnline Way2drug prediction (http://www.pharmaexpert.ru/passonline/). Prediction of Activity Spectra for Substances is a computer program that is used for biological activity predictions. The Pa values were utilized for PASSOnline analysis, which means the activity prediction of the active compound to carry out their functions²². Pa values have a range of 0.1 > 0.9. When the value is higher, their probability activity is more accurate. The active compounds which are used are based on the LC-HRMS result.

 

Compound–protein interaction network:

Compound–protein interaction networks were investigated using Cytoscape integrated with StringApps. Cytoscape software is an available network tool designed to analyze and visualize networks, whereas StringApps was designed to serve protein-protein association from STRING network and protein–chemical interaction from STITCH networks²³.

 

Cytotoxicity assay:

The cells were tested using WST-1 (Sigma) for cytotoxicity assay. P. urinaria and C. longa's cytotoxicity activity was tested against human breast cancer (T47D) cells. The T47D cells were obtained from the Laboratory of Structure, Development, and Physiology of Animals, Biology Department, Science and Mathematics Faculty, Brawijaya University. The T47D cells were cultured as in the previous study²². The treatments used were PUWE, CLWE, and their combination (i.e., PUWE + CLWE with 1:1 ratio). The data were analyzed using the CompuSyn program.

 

Cell cycle and apoptosis assay:

The cells were seeded to approximately 75,000 cells/well into 24 well plates. The doses used in the cell apoptosis assay were obtained from the IC₅₀ value, and this study used cisplatin three µg/mL (KALBE Farma). After incubation for 24 h, the treated T47D cells were harvested and incubated in the dyes for 30 min and then analyzed using the flow cytometer (BD FACS Calibur) equipped with CellQuest software for flow cytometry analysis. For the cell cycle assay, the cells were dyed using propidium iodide (BioLegend). For the cell apoptosis assay, the cells were tested using annexin-V/propidium iodide dyes (BioLegend).

 

DPPH scavenging activity assay:

DPPH scavenging activity assay was used to determine the antioxidant activity of the sample. Extract of 100µL (range of the sample 31.25–500µg/mL) was added to 100-µL DPPH (Sigma) in ethanol solution (SmartLab) (0.4mM) in 96 well plates. The mixture was incubated for 30 min, and its absorbance was then measured using an ELISA reader (BioTek ELx808) with λ = 490nm. The inhibition percentage was adjusted using Equation 1. The IC₅₀ value was calculated from linear regression of the cytotoxicity chart using Equation 2.

 

(Equation 1)

 

 (Equation 2)

 

Determination of total flavonoids:

Total flavonoids were estimated using aluminum chloride colorimetric assay²³. Quercetin (MarkHerb) was used as a standard in 96% ethanol solution with a serial dilution range of 1.5625–100µg/mL. Sample of 50µL (1 mg/mL) or standard was added to 10µL of AlCl₃ (Sigma) (10%, w/v) followed by 150-µL 96% ethanol solution (SmartLab). Lastly, added 10µL of CH₃COONa (Sigma) (1M) to the mixture in 96 well plates. As a blank, 96% ethanol solution was used. The mixture was incubated at room temperature for 40 min and protected from light. The absorbance was measured using an ELISA reader (BioTek ELx808) in λ = 405nm. Total flavonoid contents were expressed in Quercetin equivalent (standard curve equation of Quercetin; )mg QE/g of dry extract.

 

Determination of total phenolic content:

The total phenolic content was estimated using the Folin–Ciocalteu assay with modification²⁴. Sample or standard of 100 µL was added to 1.0 mL of the Folin–Ciocalteu reagent (Merck) (a ready-to-use reagent diluted 10-fold with distilled water). After 5 min, 1.0 mL of Na₂CO₃ (Sigma) (7,5%, w/v) was added to the mixture and incubated at room temperature for 90 min in dark conditions. Total phenolic content was measured using spectrophotometry in λ = 725nm. Gallic acid (MarkHerb) was used as standard. Gallic acid was expressed in gallic acid equivalent (standard curve equation of gallic acid) mg GAE/g of dry extract.

 

RESULTS:

LC-HRMS analysis of PUWE and CLWE:

The LC-HRMS shows the qualitative analysis of the bio-active compounds present in the PUWE and CLWE. These herbals contain bioactive compounds such as; phenolics, terpenoids, and alkaloids. Some of these compounds have been reported to have anticancer activities in several cell lines (Table 1).

 

Table 1. Compounds obtained from the LC-MS analysis

Herbal	Active Compound	Activity anticancer
P. urinaria	Rutin25	Inhibitor cell cycle; Inducer apoptosis26,27
	Quercetin28	Inhibitor cell cycle; Inducer apoptosis29–31
	Corilagin32	Inducer apoptosis33
	Isoquercitrin32	Anti-proliferative34
C. longa	ar-turmerone35,36	Inducer apoptosis37
	ferulic acid35	Inducer cell cycle arrest and autophagy38
	Vanillin35	Anti- proliferative39
	Curcumin35,36	Inducer apoptosis and anti-proliferative35

 

Active compounds of function prediction:

The prediction of functional bioactive components present in both herbal (PUWE and CLWE) shown in Table 2. The results show that most bio-active components work as anticancer through apoptosis (i.e., P53 enhancer, caspase-3 stimulant, and apoptosis agonist) and metastasis inhibitor (i.e., MMP9 and JAK2 expression inhibitor). Comparing the bio-active components in P. urinaria seems to be more potent as an anticancer than the C. longa.

 

Table 2. Function prediction as anticancer from PassOnline

Biological function prediction as anticancer (PassOnline)	Compound (Pa score)
	Rutin (CID: 5280805)	Quercetin (CID: 5280343)	Corilagin (CID: 73568)	Isoquercetin (CID: 5280804)	ar-turmerone (CID: 160512)	ferulic acid (CID: 445858)	Curcumin (CID: 969516)	Isoferulic acid (CID: 736186)
Anticarcinogenic	√ (0.983)	√ (0.757)		√ (0.965)
Chemopreventive	√ (0.968)	√ (0.717)	√ (0.794)	√ (0.956)
TP53 expression enhancer	√ (0.893)	√ (0.844)	√ (0.827)	√ (0.959)		√ (0.778)		√ (0.788)
Proliferative diseases treatment	√ (0.952)			√ (0.921)
Antineoplastic	√ (0.849)	√ (0.797)
Caspase 3 stimulant	√ (0.839)			√ (0.801)		√ (0.749)	√ (0747)	√ (0.749)
Apoptosis agonist	√ (0.747)	√ (0.887)	√ (0.794)	√ (0.792)	√ (0.714)	√ (0.702)	√ (0.803)
JAK2 expression inhibitor		√ (0.787)				√ (0.915)		√ (0.915)
MMP9 expression inhibitor		√ (0.734)				√ (0.865)	√ (0.805)	√ (0.865)

Note: √ represents the compounds that have function prediction as an anticancer agent.

 

 

Figure 1. Protein–active compound network. A. Network of PUWE; B. network of CLWE; C. PUWE–CLWE collaborative network. The blue circle represents proteins involved in the cancer disease mechanism. The green rectangle represents active compounds from herbal plants. The yellow square represents the proteins connecting two herbs. The green line represents active compound–protein interaction. The red line represents active compound–active compound interaction. The gray line represents protein-protein interaction

 

 

Compound–protein interaction network:

Compound-protein interaction networks were used to investigate the interaction of bioactive components from the herbal with the proteins. The data presented in Figure1 shows all the proteins that interact with the bio-active components play a role in cancer disease mechanism. As shown in Figure1, when all the bio-active components from both herbals were combined, EGFR is the only protein that forms interaction between two herbals. However, there is no interaction were formed between bio-active components of the two herbals.

 

Antioxidant activity and total phenol and flavonoid content:

In a comparison of the antioxidant activity, total phenol and flavonoid content of the individual herbal and their combination are shown in Table 3. The combination of herbal (PUWE+CLWE) showed intense antioxidant activity against DPPH free radical (88.06±2.16ppm), whereas the PUWE alone showed better against DPPH free radical (33.32±3.09ppm) than the combination or CLWE alone. In addition, total phenol and flavonoid content have a positive correlation with DPPH free radical scavenging activity.

 

Table 3. Antioxidant activity and total phenol and flavonoid content of the samples

Sample	IC50 of DPPH scavenging (ppm)	Total Phenol (mgGAE/g)	Total flavonoid (mgQE/g)
PUWE	33.32 ± 3.09	26.87 ± 0.25	5.14 ± 0.35
CLWE	2272.21 ± 5.87	3.95 ± 0.08	3.15 ± 0.18
PUWE+CLWE	88.06 ± 2.16	15.21 ± 0.06	4.80 ± 0.17

 

Anticancer activity:

The anticancer activity of herbals was assessed by treating breast cancer cell line T47D with single herbal and two herbal combinations. The herbal of PUWE can control cell growth better than the combination or CLWE alone. Based on IC₅₀ results indicate that PUWE (163.942µg/mL) has a better inhibitory ability against cancer cell growth followed by the herbal combination (PUWE+CLWE) (301.290) then CLWE (1343.76 µg/mL). Furthermore, the number of combination indexes shows that PUWE+CLWE is classified as nearly additive (Table 4).

 

Table 4. Anticancer assay result of the samples

Compounds concentration		Fractional inhibition (fa)	IC50	Combination index value
PUWE (µg/mL)	CLWE (µg/mL)
100	-	0.162	163.942 µg/mL (PUWE)
200	-	0.74
400	-	0.908
-	100	0.025	1343.76 µg/mL (CLWE)
-	200	0.118
-	400	0.137
50	50	0.192	301.290 µg/mL (Combination)	1.03 (nearly additive)
100	100	0.244
200	200	0.665

 

The ability of herbs to induce apoptosis and cell cycle arrest in T47D cells:

The herbal ability to induce apoptosis and cell cycle arrest was assessed by treating T47D cell line with the individual and combination herbal in different concentrations: 0 (control), ½.IC₅₀, IC₅₀, and 2.IC₅₀ (Table 5). As determined by Annexin-V/propidium iodine staining, the herbals can induce apoptosis in the T47D cell line compared to the control. The individual herbal extract of PUWE induced apoptosis in T47D cells in a dose-dependent manner. The effect is different when the T47D cells were treated with individual herbal extract of CLWE as the apoptosis was very low and was not dose-dependent. The effect of combination herbal extract (PUWE+CLWE) was significantly higher to induce apoptosis in T47D cells in a dose-dependent manner. The extent of apoptosis percentage was greater (28.08±6.9 to 75.21±2.22% for 105.5 to 602µg/mL doses) when the cells were treated with the combination herbal extract (PUWE+CLWE). The apoptosis cell percentage is shown in Figure 2A.

 

The data presented in Figure 2B shows the percentage of T47D cells being arrested in different cell cycle stages after being treated with different concentrations of herbal extract for 24 hours. The individual herbal extract of PUWE induced cell cycle arrest in the S phase, but the effect is different when the cells are treated in a higher concentration of PUWE (2.IC50) as accumulated of cell cycle arrest G2/M phase. The effect of CLWE shows the opposite result with PUWE as showing the accumulation of cell cycle arrest in the G2/M phase at low concentrations (½.IC50 and IC50) then arrest in S phase at higher concentration (2.IC50). The effect of combination herbal extract (PUWE+CLWE) was significantly higher to induce cell cycle arrest in the G2/M phase at the highest and lowest concentrations, but the accumulation of cell cycle arrest changes in the middle concentration accumulated in the G0/G1 phase.

 

Table 5. Concentration conversion of apoptosis and cell cycle assay

Concentration Label	Concentration Conversion
	PUWE (µg/mL)	CLWE (µg/mL)	PUWE+CLWE (µg/mL)
½.IC50	81,5	671,5	150,5
IC50	163	1343	301
2.IC50	326	2686	602

 

 

Figure 2. The ability of herbs to induce apoptosis and cell cycle arrest in T47D cells after 24 h incubation. A: cell apoptosis percentage. B: Cell cycle arrest percentage

 

DISCUSSION:

P. urinaria and C. longa are different herbal extracts that have been consumed daily as Indonesian preventive medicine. People believe these herbals could prevent and cure any kind of disease. These species have excellent antioxidants and contain benefits bio-active components such as; curcumin from C. longa L. and Quercetin, rutin from P. urinaria. The extract of P. urinaria and C. longa contains an active compound shown to induce apoptosis and anti-proliferative (Tabel 1.). However, because this research uses crude extract, future research needs to conduct active compounds validation (e.g., single compound isolation) to get novel compounds.

 

STITCH conducts its work by collecting information about interactions from metabolic pathways, crystal structures, binding experiments, and drug–target relationships⁴⁰. Based on this in-silico analysis of the compound-protein network, expected that the combination of this herbal extract could disturb the proteins that play a role in cancer cells progression. Our present study reported that curcumin, rutin, and Quercetin are bio-active components of herbal combination (PUWE+CLWE) that interact together, targeting EGFR protein (Figure 1.), as reported that increasing of this protein can trigger cancer cells progression⁴¹. Curcumin, routine, and Quercetin are known to have the ability to downregulate EGFR in cancer cases, thereby inducing apoptosis and inhibiting the growth of cancer cells^42–44. First, though, the mechanisms need to be investigated. Naturally, the body has a balanced system that regulates the amount of expressed protein as needed. However, in cancer cases, the body loses its balanced system, so there is an excess and a deficiency in proteins. New alternatives are required to maintain the body's balance, one of which is herbs' use. Increasing several proteins can trigger the progression of cancer cells. These proteins include HMOX1⁴⁵, CCND1⁴⁶, SERPINE1⁴⁷, EGFR and PTGS2⁴¹, AKT1, MAPK14 and MAPK8⁴⁸, STAT3⁴⁹, GSR⁵⁰, P4HB⁵¹, CYP1B1⁵², AKR1C3⁵³, HCK⁵⁴, and MCL1⁵⁵. In addition, decreasing several proteins can trigger the progression of cancer cells. These proteins include TP53 and CASP3⁵⁶. Although the resulting direct effect is unknown, the protein–compound interaction is expected to work appropriately to balance protein in the body system.

 

This study found that the combination of these aqueous herbal extracts (PUWE+CLWE) has intense antioxidant activity against DPPH free radicals (Table 3). Treatment of T47D breast cancer cells with IC50 to 2.IC50 concentrations of herbal combination has the efficiency to induce apoptosis (Figure 2A.) with more than 50% compared to the individual herbal treatment and control. The treatment also increases the number of inhibition cell growth in dose-dependent (Table 4.). Cell cycle analysis shows that T47D cells treated with these herbals increase the percentage of cells in G2/M phase compare to the control cells (Figure 2B.). These results indicated that the cells are getting arrest at G2/M phase. The antioxidant activity and total phenol and flavonoid content of PUWE, CLWE, and their combination positively correlate with anticancer activity. PUWE has strong antioxidant and anticancer activities. It also has the highest total phenol and total flavonoid content than other samples. Herbal medicine has the potential as a new drug against cancer due to its active compounds⁸. Polyphenol compounds from plant sources have health benefits in various diseases, such as antioxidant and anticancer activities.

 

The individual herbal extract of PUWE and CLWE induced T47D cell death through apoptosis and cell cycle arrest. T47D cells treated with PUWE show a higher percentage of apoptosis than CLWE; this may be due to the higher amount of phenol and flavonoid contained in PUWE (Tabel 3.). Several studies reported that herbal medicine has potential as a new drug against cancer due to its active compounds⁵. Polyphenol compounds from plant sources have health benefits in various diseases, such as antioxidant and anticancer activities⁵⁷. The research used cisplatin for control because cisplatin establishes a chemotherapy drug that can induce apoptosis through multiple mechanisms, such as activate signal transduction pathways, death receptor signaling, and mitochondrial pathways. In addition, cisplatin can cause cell cycle arrest at the G1, S, or G2-M phase by inhibiting the DNA synthesis and repair⁵⁸.

 

The cells treated with PUWE had more cell arrest in the S phase than the untreated control cells, whereas higher accumulation in the G2/M phase was shown in CLWE treatment cells at the same concentration (½.IC50). When treated with ½. IC50 combination extract (PUWE+CLWE) accumulation of cells in the G2/M phase was higher than individual treatment. This result similar to Hu et al.'s report that curcumin could induce cell cycle arrest at G2/M phase⁵⁹. This result indicated that these herbals might affect the cell cycle by regulating the expression of regulatory proteins in the G2/M cell phase.

 

In this study, the combination of P. urinaria and C. longa has an additive effect (Table 4). An additive effect is an add-up of individual effects where each agent does not affect the other (no-interactions)¹². Besides the combination herbal extract (PUWE+CLWE), PUWE may show the best anticancer effect than CLWE. PUWE showed a higher percentage of apoptosis than CLWE because the active compound content in PUWE has a better anticancer activity role than the active compound in CLWE (Table 2). This is also evidenced by the IC50 value obtained. Furthermore, PUWE is lower than CLWE (Table 4).

 

Meanwhile, it cannot be confirmed that CLWE has an anticancer effect because CLWE has a high IC₅₀ value (Table 2). The combination of natural extract observed in this study seems to be free of side effects and proven anticancer preventive potential. However, the anticancer potential needs further investigations to realize the benefit function of this combination extract. The result of the combination depends on the individual dose-effect curves and enables unequivocal definitions of synergy, additive effect, and antagonism. This effect may be influenced by the extraction process and the content and concentration of compounds in the herbs⁶⁰.

 

CONCLUSIONS:

This study concludes that the combination of P. urinaria and C. longa has potential as anticancer agents. The herbal combination extract can inhibit cancer cell growth and induce apoptosis and cell cycle arrest in T47D cells inappropriate dose of IC50. Combining both herbs has not shown a synergism interaction, but the combination of herbal compounds shown targeting the same protein (EGFR) can eliminate cancer cells. Furthermore, further study is needed to determine the effective anticancer doses of combination herbs. In addition, a study on protein expression related to cancer mechanisms needs to be conducted to clarify the effects of herbal treatment.

 

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of the Ministry of Research and Technology of the Republic of Indonesia for funding this research. The Laboratory of Structure, Development and Physiology of Animals, Biology Department, Science and Mathematics Faculty, Brawijaya University for facilitating this research is also acknowledged.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Accepted on 05.05.2021          © RJPT All right reserved

Research J. Pharm.and Tech 2022; 15(2):671-678.