A Review Over the effect of Heavy Metal in Metabolism of Brassica juncea (L.) and Myristica fragrans

 

Vanktesh Kumar*, Navjot Kaur, Pankaj Wadhwa

Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab-144411, India.

*Corresponding Author E-mail: Vankteshkumar555@hotmail.com

 

ABSTRACT:

Brassica juncea (L.) and Myristica fragrans are common plants found in India where the concentration of heavy metals in water and soil is observed more frequently and in sufficient amounts to cause bad effects to the living entity. In plants various kinds of bad effects such as dwarfness, yellowish leaves, dead leaves, brownish and dead stem. These are kinds of symptoms that one can observe in plants suffering from the bad effects of heavy metal absorption. At a specific time, they should be identified and treated for the same otherwise this may cause an increase in dead material in the plant, day by day. This review summarizes the bad effects of heavy metals in plants like these two.

 

KEYWORDS: Brassica juncea, Myristica fragrans, Platinum, Calcium, Nut, Heavy metal.

 

 


INTRODUCTION:

Brassica juncea: Brassica juncea (L.) commonly known as Indian mustard is a plant that belongs to the mustard family i.e., Cruciferae (Brassicaceae). In India, it is being used as oilseed, edible oil, and spices [1]. Morphologically this plant has flowers on top, which is the main body of the plant including soft, straighten, and easily bendable stems [2]. Flowers of mustard commonly bear 4-5 diagonally flattered shaped petals along with the sepals in the same number supporting from the lower side of the flower [3]. Flowers are mainly in pale yellow but ranges from light green to greenish-yellow. The stems of the plant are less bulky comparing to the wild type of mustard plant [4]. The stem is found to be less or negligible hairy with a smooth surface.

 

The stem can grow up to a maximum of 75cm long at its fastest rate annually. Pods are elongated narrow, smooth surfaces and filled with small round-shaped seeds from which oil is removed using various techniques [5]. The pods are mainly positioned at the right angle to the stem but sometimes found to be at the lower side. The length of seed pods is commonly ranging from 2.0 to 2.5cm. The pods are beak-shaped from the lower-end [6]. The seeds are round-shaped and maybe black and yellowish-white depending upon the species. Almost all parts of this plant are edible and have some specific use which is either medicinal or household [7]. The edible parts include flowers, stem, seeds, stem, shoot, and roots [8]. This plant has its flavor which is why it is being used as mustard flavor to various eatables. It is found in mainly North America, various provinces of Canada including various regions of Asia, South America, Western and Eastern Siberia. After world war II Canada became the primary producer of the mustard seeds and imported to various regions of the world including Central Asia where it was being used as a spice and as cooking oil also [9]. Before the 2nd world war, western and eastern Africa was the primary producer but due to the less developed economy and infrastructure they lagged, and Canada became the king. It requires loamy, clay, and sandy soil for growth including other conditions such as optimum rain and pH during the growing season [10]. It can grow in all neutral, acidic, and alkaline soil but the soil must be containing moisture. A semi-shaded or negligible shaded area is preferred for good growth [11]. This is an annual growing plant with self-pollination. The primary pollens are heavy sticky and that is why wind cannot carry them to a great extent. The physical contact due to bending stem due to wind can result in self-pollination [12]. Heavy rains can devastate the stems and hence resulting in a reduction in the final production. The cultivation can be done only by the seeds of mustard and is an annual plant 70% of the seed are viable when sprouted for cultivation purpose [13]. The seeds are the only way to cultivate and propagate but nowadays botanists have developed the techniques to propagate it through its stem, shoots, roots, and leaves also which is a great revolution because this can reduce the cultivation cost to the farmers [14].

 

Myristica fragrans:

A tree accompanying its greenery to ecology known as Indian nutmeg. Biologically it is known to be Myristica fragrans which belong to the Myristicaceae family and has its medicinal as well as ecological importance [15]. Usually, its morphological characters involved an average length of 4-15m and exceptionally reach 20m. In further addition, leaves are dark greenish and have a dimension of 4-12cm length and 2-7cm width [16]. It is a dioecious plant having male and female both at the same site which produces roughly round or oval-shaped fruits having hard shells and rough surface [17]. The fruit size ranges from 2-5cm and the whole fruit along with the shell is known as the nutmeg apple fruit which is firstly dried and then nutmeg is obtained [18]. The shell color ranges from light brown to greyish brown and after ripening of the fruit, the breaking shell will give a flesh that will be irregular in structure and whitish brown in color is the actual eatable substance. The eatable substance has a pungent strong smell due to fixed oil in it [19]. Talking about chemical constituents 20-45% of total biomass consist of the fixed oils which include and tridecanoic, glycerides of lauric, palmitic acids, stearic, and myristic acid present in combined form and known as the nutmeg butter [20]. There is a presence of essential and volatile oil was seen (6-10%). Starch saponins, flavanols, and proteins in the minute form are also found in nutmeg. Eugenol and Myristicin are the chief constituents responsible for their medicinal use [21]. These all constituents can be isolated using simple chromatography or isolation can be done using HPLC and identification can be done using hyphenated techniques. Mainly it is applicable to comply as a flavoring agent and carminative agent but in India, it is used in designing cuisines. It is also being used as a larvicidal agent and in soap industries as a soap base. It has some antibacterial properties which make this prominent among all the evergreen trees [22]. It is also reported that extract of Myristica fragrans can inhibit the corrosion in some metals which is known as green inhibition or green protection [23]. Haldar et al reported the green protection of mild steel using the extract of the Myristica fragrans. They took mild steel of different composition which consist of C (0.083%), Fe (97.60%), Ni (0.27%), Si (0.39%), Cr (0.45), Mn (0.43%), P (0.12%) and Cu (0.43%) with a working area of 1 cm2 and before the experiment the mild steel was kept in a solution of water and acetone but just before the experiment it was taken out and dried with warm air [24]. To prepare the test inhibitory solution normal fruits with shells were taken and washed using the distilled water and dried for at least 72 hours. Then fruits flesh was taken out and ground then this ground form was refluxed at a heat of 65°C using 100 ml of ethanol for 24 h [25]. Then the resultant solution was filtered and concentrated which results in a small dark brown gummy substance (4.5g). Then different test solutions of 100mg, 200mg, 300mg, 400mg, and 500mg per Litre of 0.5 M of H2SO4 dilution were done with all the test solutions. Then the mild steel specimens were kept in the solution with a surface coverage of 20mL per cm2 at a temperature of 25°C and a weight-determining setup was added to the system [26]. Then the results of which was that the extract was successfully able to inhibit the corrosion but up to a certain limit depending upon the concentration. The protective nature of the natural product was adsorptive, and the highest concentrated solution (500mg/L) was able to protect the surface and weight of the specimen up to 87.81% compared to others. As the concentration increases there is a change in the protective ability was seen but up to a certain limit. The adsorptive nature was confirmed using Langmuir adsorption isotherm [27].

 

LITERATURE REVIEW:

Ismail et al have conducted research that proves that growing Indian mustard under the greenhouse effect can increase the product yield and improves the quality of seeds and ultimately the oil quality also [28]. Zhang et al concluded their gas chromatographic analysis of Indian mustard with 22 constituents constituting 98.4% of the total weight of the seed oil. These constituents were 3-butenylisothiocyanate (4.8 – 6.0%), Diallyl trisulfide (7.8 – 9.0%), sulphides (14.2- 20.9%) and phenethyl isothiocyanate (2.4 – 4.0%). But some chemical compounds that are not found in any other country except china are diallyl sulfide, methyl allyl disulfide, 4-(4,5-dihydro-4,4-dimethyl-2-oxazolyl)-isoxazole, diallyl tetrasulfide, 2-propen-1-thiol, diallyl trisulfide, 6-undecanol, N, N’-diallyl thiourea, diallyl disulfide, methyl allyl trisulfide, anti-9-methyl-1,6-methano-fluorene, ben-zenepropanenitrile and 2,3-dimethyl 2-butenoic acid [29]. The amount of essential oil present in the plant seeds is depending upon the factor that where the plant has been grown. Golubkina et al have shown that plants that were grown in India had 86.8% w/v, Pakistan had 84.6% w/v, China had 89.5% w/v and Bangladesh had 88.4% w/v of oil quantity present in the seeds. But when it comes to the countries outside the Asia region the quantities get reduced that Asia region such as Vietnam (70.4% w/v), Australia (68.45% w/v), Poland (68.12% w/v), Japan (68.11% w/v), Australia (68.07%w/v), Belgium (67.96% w/v), Denmark (67.76% w/v) and Mexico (66.54% w/v) [30]. But when it comes to Canada, Eastern and Western Africa the quality becomes higher than the non-Asia region and becomes nearly to the oil found in the Asia region which is 84.6% w/v and 85.32% w/v respectively. When it comes to consumption then countries like China, India, Pakistan, the US, and Mexico consume at high rates than other countries due to its medicinal as well as usual use [31]. Ahmad et al have demonstrated the effect of the heavy metals in the Brassica juncea (L.). They targeted Cadmium (Cd) which is a heavy metal and toxic at high levels. The anthropogenic release of Cd directly into the soil leads to an increase in toxicity and devastates the fertile property, which may lead to the compression of the shoot, root, and stem growth. Cd acts as a pollutant in soil when levels become so high (4,000–13,000 tons per year) [32]. But this study is about the effects of Calcium (Ca) against the Cd-stress and growth of the plant. Ca has its crucial role in plant growth and maintaining the structure of the plant as a macro-nutrient. This work consists of the effect of the Ca on the uptake of the Cd in Indian mustard. A total number of 6 plants were firstly grown under controlled conditions using nutrient media using seeds. After 26 days of controlled growth, they were treated with different levels of Cd and Causing f Cd (CdSO4.8H2O) and Ca (CaCl2) respectively dissolved n nutrient media which is as follow in Tab. 1.

 

Tab. 1: Plants treated with the concentration of Ca and Cd [33].

Sr. No

Plant name

Levels of Cd (mg L-1)

Levels of Ca (mM)

1

P0

0

0

2

P1

0

50

3

P2

200

0

4

P3

200

50

5

P4

300

0

6

P5

300

50

 

As a result of which Cd stressed plants faced a lot of reduction in growth such as height, oil content, and other compounds. Comparing with the P1, the height P4 had reduced to 17%, 46%, and 63% approx. after 30, 60, and 90 consecutive days. A lower reduction in height of the P5 plant was seen compared with P4. The reduced heights were 3%, 19%, and 21% approx. after 30, 60, and 90 consecutive days when compared with the P1 plant (lacks Cd and Ca levels), which proves that high Cd-stress can cause high reductions in the growth of the plant. While P3 and P5 faced a height reduction of 16% and 56% respectively after 90 consecutive days, which evince the statement that Ca inhibit or reduce the Cd uptake and contribute to reliving the Cd-stress of plants. Just like the heights, the root lengths were also affected by the stress. Comparing the P0, with the root length of P4 had reduced to 15%, 23%, and 24% approx. after 30, 60, and 90 days. But less reduction in root length was seen in the case of P5 compared with the P4 using P0 as a standard untreated growing plant. In the case of P5 having Ca to protect the plant from stress has root length reduction up to 36% approx. after 90 days. There was a great effect seen in the pigmentation of the flower of the plant such as in plant P2 and P4, chlorophyll A content was reduced but has more content in case of plant P3 and P5. But the highest content of chlorophyll A was seen in the untreated P0 plant. The oil quantity is reduced in the case of Cd-stressed plants (P2 and P4) as compared to the P0. The oil quantities improve when Ca is used to release the Cd-stress in plants like P3 and P5 comparing to those of P2 and P4. To determine the oil quantities 3g of seeds were taken and crushed in Na2So4 and used then to examine the oil contents. The enzymes such as Glutathione S-transferases (GST), guaiacol peroxidase (GPX), Monodehydroascorbate reductase (MDHAR) Ascorbate peroxidase, and other chemical constituents were determined using the GC-MS. To determine the effects of Ca on Cd-induced stress the accumulation data of Ca and Cd in shoots and tips were used and determined using atomic absorption spectrophotometer. These data prove that Cd is a soil pollutant when releasing beyond the limits and cause the inhibition and reduction in the plant growth and its content as well. But Ca is a rescuing macro-nutrient that can decrease the Cd uptake by the plant from the soil and lead to reduce the Cd led to stress and enhance the growth [34]. The same study was carried out to check the effect of Selenium (Se) instead of Ca over the Cd-stress. The series of experiments possess almost the same setup but there is a difference in the treatment levels of the Se which shows nearly the same effects as compared to the Ca. Although Se in various biochemical activities or reactions acts as a cofactor so the body is quite familiar with the Selenium as compare to the others. Hasanuzzaman et al demonstrated the inhibitory effect of Selenium in the case of DNA oxidation by hydrogen peroxide [35].

 

Tab. 2: Plants treated with the concentration of Cd and Se [36].

Sr. No

Plant name

Levels of Cd (mg L-1)

Levels of Se (mM)

1

Q0

0

0

2

Q1

0

50

3

Q2

200

0

4

Q3

200

50

5

Q4

300

0

6

Q5

300

50

 

Treatment of Cd and Se using different concentrations in nutrient media is as given in Tab. 2. In this case, the total number of 6 plants were taken and grown from seeds using nutrient media.

 

It was observed that a plant that has alone Cd suffered a lot of stress which reduced the height of the plant to 25% and 33% approx. in Q2 and Q4 respectively. In the case of root length, it was reduced to 35% and 56% respectively. But in the case of plants that had Cd and Se both suffered less comparing with plants having Cd alone. So, plans Q3 and Q5 have reduced height which reduced to 23% and 41% respectively. In the case of root length, it was reduced to 28% and 41.2% respectively. When it comes to dry weight Se has a positive effect on all the plants in which it was used against the Cd. It was noted that the concentration of the oil was also improved compared with plants having Cd alone with those of nutrient media having untreated plants (Q0). The stress-releasing Se was able to improve the quality and quantity of the oil present and even able to inhibit the devastation of the several enzymes which are found to be present in Indian mustard. Ahmed et al experimented to check the effects of the Trichoderma harzianum (TH) on the NaCl-induced stress in the Brassica juncea. Salinity stress has some reductive effects on plant biomass. It is used to decrease the quality and quantity of the plant and requires a lot of water for irrigation. Salinity may decrease the properties such as antioxidant, enzymatic activity, and the concentration of the chemical constituents. The increase in osmotic and ionic stress will lead to a reduction in plant growth and may also affect the fertility of the soil. In the experimental part, Trichoderma harzianumi was collected from the infected maize and cultured under controlled conditions, and provided an agar-based nutrient medium. The activity of fungal strain was confirmed using weight sapling growth. Plant seeds were made surface sterile using sodium hypochlorite and then washed with distilled water and then kept of nutrient media (Agar based) till the germination. After proper growth plants were transferred to the experimental pots. A total of 6 Plants treated with NaCl and fungal strains are as follow in Tab. 3.

 

Tab. 3: Represents the plants treated with the TH and NaCl and its concentration given [37].

Sr. No

Plant name

Concentration of NaCl (mM)

Presence/Absence of TH

1

S0

0

0

2

S1

0

TH

3

S2

100

0

4

S3

100

TH

5

S4

200

0

6

S5

200

TH

 

 

After a specific time, the plants were compared taking specific characters such as plant height, root length, and dry weight of the plant. In the case of the height of the plant, a major reduction was seen in plant S4 having high concentrations of NaCl which are 33.7%. But in the case of the S5 and S3, the values are 13.5 and 14.1% approx. respectively. Approximately the same effects were seen in the case of the root length. The results were found to be of great significance which is given in Tab. 4. But in the case of the dry weight of the plant major reduction was seen in plant S5 which is 36% but using TH against NaCl stress reduced this value to 13.12% of plant S5 and 16.11% of plant S3. Beyond all these this experiment revealed that there was a reduction seen in the plants treated with 100 and 200 mM of NaCl (25.5% and 19.8% respectively) comparing with the reference untreated plant S0. But there was improved oil concentration seen in plant S3 and S5 which was around 30.5% and 23.5% respectively which clearly shows the positive effect of TH. TH also improved the concentration reduction and activity abolishment of various enzymes present in the Indian mustard. A positive effect was seen over the production of chlorophyll A when compared with NaCl stressed and normal plants [38].

 

Tab. 4: Represents the respective height, root length, and dry weights of the plant after treatment.

Sr. No.

Treated plants

Heights (cm)

Root length (cm)

Dry Weight (g per plant)

1

S0

51.72 ± 1.57

21.11 ± 1.00

15.39 ± 0.88

2

S1

53.19 ± 1.59

23.72 ± 1.04

16.61 ± 0.95

3

S2

40.19 ± 1.31

15.29 ± 0.94

12.87 ± 0.73

4

S3

47.22 ± 1.42

20.04 ± 0.98

15.73 ± 0.90

5

S4

34.24 ± 1.11

11.81 ± 0.86

10.08 ± 0.64

6

S5

39.73 ± 1.22

14.81 ± 0.90

12.11 ± 0.70

 

So the statement can be made based on all these data that the quality of compounds and oil of Brassica juncea (L.) can be improved using some other factors against the heavy metal stress and soil pollution can be inhibited [39].

 

When it comes to the isolation of the chemical constituents then it is very difficult to obtain all the chemical constituents in sufficient proportion using conventional isolation and extraction method. So Morsy et al reported the comparison of extraction of chemical constituent using conventional and green extraction technique. In this experiment, the aim was purely to compare the presence of different chemical compounds, but it would be vast to compare each constituent, so they performed the comparison of extraction of oleoresins from nutmeg. In the conventional method, they used the dried powder powdered form of nutmeg weighing around 40 g and extracted it using absolute ethanol in a ratio (1:4 w/v) using conventional maceration for continuous 3 days. After that, it was filtered and concentrated at 40°C. In the green extraction technique, the Ultrasound-assisted extraction (UAE) was preferred in which 10g of powdered nutmeg with 40mL of absolute ethanol kept under the influence of the ultrasonic waves in ultra-sonicator for the different time (10 and 25 minutes) at 40°C temperature [40]. Besides, for qualitative and quantitative analysis the Shimadzu GC-MS was used. As a result of which different compounds were found with different concentrations. But it was very clear that there was a lot of difference between the extracted products of both techniques as in green techniques some of the constituents that were not persisted in the conventional one. But some common compounds were found in both the techniques which are Camphene, Myrcene, α-Terpinene, (Z)-trans Sabinene hydrate, Safrole, Methyl eugenol, Eugenol, Methyl isoeugenol, Linalool, Terpinene-4-ol, Piperitol isomer, Geranyl linalool isomer, Monoterpene Alcohols, Myristicin, Methoxyeugenol, Chavibetol (trans-isoeugenol), Elemicin, Cis-2- menthen-1-ol, α-Terpineol, myristicin, myristic acid, limonene, safrole, and sabinene. The concentration difference was observed with a standard deviation value using the ANOVA technique and a significant difference of >>0.05 was observed [41]. Another experiment on the chemical composition of the nutmeg is done by Rodianawati et al in which they evaluated the effect of heating on the chemical makeup and composition of the nutmeg and the ultimate effect on the antifungal property of the plant. Further in preparation of the sample which was taken and washed with distilled water and dried to maintain moisture content up to 10-15%. The dried sample was then kept in a solution containing 96% of ethanol along with and PDA (Potato Dextrose Agar) and Dichloran Rose Bengal Chloramphenicol (DRBC) which acted as growth medium of fungi. Three different strains of the fungi which are Fungi Mucor Racemosus, Aspergillus Niger, and Penicillium glabrum along with other chemicals were used. Further preparation of oleoresins from the nutmeg was done using distillation and extraction. In the first step crushed powder of fruit around 200 g were taken and distilled using water and steam distillation for up to 5 hours. Then it was filtered, and the remaining residue was dried and passed through the sieve (size 20). The particles which were passed through it were kept as it is and those which did not pass taken again for extraction using traditional techniques maceration. The extraction was done using 96% of ethanol (1:5 w/v) at 25°C for 2.5 hours. Then it was filtered using a vacuum filter and mixed with another distilled portion. The distilled water portion was separated using the separating funnel. Then the ethanol was evaporated using the rotatory evaporator and then the obtained residue is known to be nutmeg’s oleoresins when mixed with the essential oil. Then this sample was weighed 5g and taken in three different test tubes and kept at three different temperatures 100°C, 120°C, and 180°C where each test tube was heated in the closed test tube with aluminum foil using the oven. These heat phenomena were repeated three times with each of the samples and further analyzed using GC-MS (gas chromatography-mass spectroscopy). The analysis of the antifungal activity was done using 10ppm of nutmeg’s oleoresins in a sterile solution of Tween 80 and the growth of mycelium was measured using the diameter measurement of colonies grown. In the resultant analysis, it was observed that the total number of 24 compounds were found in the heat-treated samples and the most dominant compound was sabinene having around 41% of concentration and then essential oil was having a total of 18 compounds with 6 compounds in the nutmeg’s butter which were fatty acids. Myristate acid was the only acid with the highest concentration of 84.23%. The heat treatment had a versatile effect on the compounds that some new compounds were detected such as endo Bornyl acetate, α-and α-terpenic cubebene acetate. Some compounds were missing may be due to the fusion between two to form new compounds such as trans-caryophyllene, γ-cadinene, cis-sabinene hydrate, β-ocimene, and α-humulene and trans-caryophyllene, γ-cadinene, cis-sabinene hydrate, β-ocimene, and α-humulene which were undetectable. When it comes to the antifungal activity which was evaluated between the unheated sample and three heated samples (100°C, 120°C, and 180°C) has shown no significant difference in their inhibitory action. This inhibitory action was measured using the diameters of the colonies grown on Petri plate in which not even a 1 mm difference was found. This all-data states that the heating at food processing temperature has an effect on the chemical constituents but has no effect on antifungal activity. Talking about the essential oil of the nutmeg it has several medicinal activities [42]. Adiani et al have reported that essential oil obtained from the fruit of the nutmeg has antioxidant activity against numerous agents. All the materials used in this experiment were procured from well-established standard industries and research labs. Firstly, the extraction of essential oil was done using 200g of crushed fruits of nutmeg with diethyl ether as a solvent for 2 hours at a temperature of 40°C. All extractions were done using distillation assembly. After the extraction, the component was filtered using a vacuum filter and the extract was dried using the sodium sulfate and nitrogen stream to remove the diethyl ether. The obtained essential oil was stored at 20-25°C for 24 hours. The essential oil was analyzed using the GC-MS of Shimadzu having quadrupole as an analyzer. The H+ donation and radical scavenging property of the essential oil were analyzed using the assay of DPPH radical scavenging. In this 0.1ml of methanolic solution of essential oil was taken in test tubes along with 1.5ml of methanolic solution of DPPH. The total number of 5 solutions (test samples) were prepared with different concentrations of essential oil ranging from 5-50mg ml-1 Then incubated for 1 hour and the absorbance was measured at 517nm. The antioxidant activity was measured following the absorbance and expressed in μM using Trolox equivalent antioxidant capacity (TEAC). Where TEAC is having the capacity of inhibition of oxidation in μM equivalent to that of 1g of essential oil which possesses the same antioxidant capacity. All the chemical components of the essential oil were analyzed using TLC and Gas chromatography. In the DPPH assay, the antioxidant activity was measured concerning the ability of essential oil which can discolor the DPPH solution and IC50 was calculated as that amount of essential oil which can discolor 50% of the DPPH solution. The antioxidant activity was also verified using β-carotene bleaching assay. As a result of both the assay and GC-MS analysis, the 12-15% w/w of essential oil was obtained in which various ethers and mono-hydroxy terpenes were extracted. The DPPH scavenging and β-carotene bleaching required concentration against the required activity of inhibition is given in Tab. 5.

 

Table 5: The optimum concentration of essential oil required for inhibitory action corresponding to the Trolox [43].

Sample

DPPH (Trolox equivalents as μM g−1)

β-carotene/linoleic acid

(Trolox equivalents as μM g−1)

Sample 1

3.29±0.05

1.51±0.12

Sample 2

NA

2.33±0.15

Sample 3

18.19±0.5

2.89±0.01

Sample 4

3.82±0.1

1.54±0.02

Sample 5

NA

0.81±0.17

Essential oil

2.94±0.09

25.11±1.50

 

CONCLUSION:

By the literature hunt, it was found that there some serious and hazardous effects in each part of the plant. Some of which are like yellowish eaves and dad leaves either partially or fully. The roots were found to be brownish-yellow and showed very less conduction of nutrients from root to other parts. Various symptoms may be visible as the effects of heavy metal is not a sudden process. So, as a conclusion, we can say that in a specific period, the effects and symptoms must be identified and treated as well otherwise it may cause devastation and degradation of the plant body.

 

ACKNOWLEDGEMENT:

We would like to thank our parents and our teachers who provided us with opportunities to work.

 

CONFLICT OF INTREST:

The authors declared no conflict of interest in any way.

 

AUTHORS CONTRIBUTION:

All the authors were involved in the processing and preparation of the manuscript.

 

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Received on 08.12.2020            Modified on  25.01.2021

Accepted on 23.02.2021      ©Asian Pharma Press All Right Reserved

Asian Journal of Pharmaceutical Research. 2021; 11(2):97-103.

DOI: 10.52711/2231-5691.2021.00019