Comparative Evaluation of Phytochemical Constituents by GC-MS and Antitubercular & Antimicrobial Potential of Ceiba pentandra and Parmotrema perlatum against Resistant Strains

Ceiba pentandra and Parmotrema perlatum lichen are two illustrious spices. Apart from their benefit as aromas, These are known for various therapeutic activities. Both are well known as appetizers. Decoctions of theses powders are used to relieve cough, anorexia and helminthiasis etc. Though several studies on antimicrobial activities of these two plants are available, studies on resistant microorganisms and anti tubercular activity are very limited. Hence, the author made an attempt to identify the phytochemical constituents present in methanolic and n-hexane extracts of Ceiba pentandra and Parmotrema perlatum using phytochemical tests and GC-MS method then to evaluate antibacterial activity against 5 resistant microorganisms by microtitre broth dilution method using ciprofloxacillin & streptomycin as reference standards. Methanolic extracts of Parmotrema perlatum and Ceiba pentandra had shown a considerable antibacterial activity than nOriginal Research Article Devi et al.; JPRI, 33(35A): 197-203, 2021; Article no.JPRI.67959 198 hexane extracts and exhibited 90% growth inhibition against H37Rv resistant strain of mycobacterium at 80 & 320 micrograms/ml respectively. N-hexane extracts of both species were found to be poorly effective against H37 Rv.


INTRODUCTION
Medicinal plants serve as a rich source for various therapeutically active molecules. The ayurvedic system being a traditional practice generally utilizes Phyto molecules to cure various diseases and ailments. Indian spices are wellknown remedies to treat gastrointestinal problems including helminthiasis, GIT infections etc. These spices are not only known to contribute flavor to food but also dispense therapeutically active molecules to the body. Secondary metabolites like saponins, tannins and alkaloids being bitter and acrid principles generally able to kill or sustain the growth of localized microorganisms hence many medicinal herbs are popularized as anti-infectives. Though several synthetic anti-bacterial drugs are available, antimicrobial resistance is the serious and most concern problem for today's global health. Hence it has become an emerging field of research and a challenge to the medicinal chemist to discover new molecules with a safe therapeutic index.
In the present study, author had selected the following 5 resistant microorganisms and reported the most common infections caused by them and the extent of resistance as follows.
Pneumonia, septicaemia, Urinary tract infections and intra abdominal infections are the most serious infections caused by Gram negative bacteria called Pseudomonas aeruginos [1]. However recent drugs of carbapenam category were also found to be ineffective due to drug resistance developed by pseudomonas pathogens. Including Pneumonia, blood stream infections, meningitis and pyogenic lever abscess are the life threatening conditions caused by a gram negative Klebsiella pneumoni [2] bacteria and known for its resistant [3] against carbapenams and other antibiotics. Streptococcus pyogenes [4] is a gram-positive Bacterium by which causes scarlet fever, severe pharyngitis and tonsillitis. Generally, macrolide antibiotics are used to treat the infections successfully. But recent reports have revealed that this pathogen had developed resistance to newer macrolides. Clostridium difficile is a spore forming gram positive bacteria. It causes severe inflammation in colon. Though cephalosporins are the effective antibiotics to treat various forms of colon infections, recent studies reported the resistance [5] of Clostridium pathogens against newer cephalosporins. H37Rv is the most virulent strain of tuberculosis and also a usual biomarker to study antitubercular potential of new antitubercular leads with respect to dose and other invitro pharmacodynamic effects.
Based on the above illustrations, the researcher had viewed to identify phytochemical constituents present in methanolic and n-hexane extracts of two Indian spices namely seeds of Ceiba pentandra [6] and lichen of Parmotrema perlatum [7] and to demonstrate their antibacterial and antimycobacterial activities. Ceiba pentandra is commonly known as Kapok belongs to the family Malvaceae. It is a very tall deciduous tree grows in rainfall forest areas. Parmotrema perlatum is lichen which is commonly known as Black stone flower belongs to the family Parmeliaeceae. It occurs throughout northern and southern hemispheres. Both of these spices are commonly used in India and China. Ceiba pentandra is known for antioxidant [6], anticancer, anti-inflammatory and other activities [8]. Parmotrema perlatum was reported for antibacterial activity where as evidences for phytoconstituents [9] and other biological activities [10] are very limited. So the author had focussed the present study on phytoconstituents and biological activities of Parmotrema perlatum.

Collection of Plant Material
Fruits of Kapok and black stone flower samples were purchased in local market of Trivendrum (City), Kerala (state), India then herbariums were authenticated by Dr. K. Madhava Setty, Associate professor, Taxonomist, Department of Botany, Sri Venkateswara University, Tirupati, Andhra pradesh, India as their Voucher specimen Numbers: 0429 & 0776 for Ceiba pentandra and Parmotrema perlatum respectively at S.V. university Herbarium. All the chemicals used for extraction and estimations were purchased from Sigma Aldrich, Bengaluru.

Methods
Extracts of n-hexane and methanol were prepared separately by cold maceration [11] as specified below. Fruits of kapok were broken to collect seeds. Seeds were sieved to remove debris and fibrous. Then cleaned seeds were crushed in to a coarse powder. Two samples each containing 150 grams of powder were weighed and transferred into two separate round bottom flasks and numbered the flasks as (1) & (2). 450 ml of Methanol was transferred to flask (1) and 450 ml of n-hexane was transferred to flask (2). Methanol extract was named as CP(M)E and n-hexane extract was named as CP(N)E.
Black stone flower material was cleaned to remove fibres, sand etc. Then it was cleaned under running tap water to remove adhered dust after that immediately it was dried under sunshade. Then it was crushed to produce coarse material for the preparation of extracts of methanol and n-hexane extracts as mentioned in the extraction procedure for kapok. The methanol extract was named as PP(M)E and n-hexane extract was named as PP(N)E. Then all the flasks were kept aside for 7days. At 8 th day solvents were recovered under reduced pressure using a rotary vacuum evaporator. The extracts were then used for further studies.
Phytochemical tests [12] were performed on methanolic and n-hexane extracts of Ceiba pentandra and Parmotrema perlatum extracts to identify the presence of secondary metabolites. The results obtained are represented in Table 1.

GC-MS analysis
The Gas Chromatography-Mass spectroscopy (GC-MS) analysis [13] of the components were carried by equipping Mass Selective Detector (MSD) (HP6890 GC and HP7673) autosampler, operated at 65eV using acquisition scan mode with HP-5MS at 100˚ C oven temperature held initially for 1 min and then increased to 280˚C by 20˚C/min and held for 10 min. 250˚C was the injector temperature, and Helium was used as the carrier gas at a constant column flow rate of 1.5 ml/min. By split, less mode technique 2µl aliquot of the sample extract was injected. From 10 chromatograms, target components were selected, and Comp Extractor software was used for the registration of chromatograms. After pouring all of the above into a well, the microplate was incubated at 37 o C for 18 h. The development of pink colouration due to TTC in a well indicated bacterial growth, and the absence of the colour was taken as mean inhibition of bacterial growth. The first well of the microplate was the control without any additional molecules and second well contains Ciprofloxacin as (+)ve control. The MIC value was noted at the well, where no colour was manifested. 90% of growth inhibition was considered as MIC which was calculated based on Non-linear regression method of the test sample as per standard protocol [14,15].

Determination of anti tubercular activity using Microplate Alamar Blue Assay (MABA)
The o C for five days. After 5 days of incubation, 25μl of freshly prepared 1:1 mixture of alamar blue reagent and 10% tween 80 was added to the plate and incubated for 24 hours. A blue colour in the well was interpreted as no bacterial growth and pink colour was scored as growth. The Minimum Inhibitory Concentration (MIC) at 90% growth inhibition was defined as the lowest extract concentration, which prevented the colour change from blue to pink. Streptomycin was used as standard anti-TB drug. MIC 90 was calculated based on Non-linear regression method using Win-analyst software [16,17].

RESULTS AND DISCUSSION
Alkaloids, terpenoids, flavonoids, saponins, tannins, steroids, phosphates and sulphates were identified as major constituents in methanolic extracts of Parmotrema perlatum whereas carbohydrates, and proteins were found to be absent in both extracts of Parmotrema perlatum. Methanolic extract of Ceiba pentandra contains alkaloids, tannins, saponins and flavonoids, carbohydrates, phosphates and sulphates as the constituents whereas n-hexane extract has given the positive result for alkaloids, saponins, tannins and carbohydrates. Steroids were absent in both Ceiba pentandra extracts (Table 1).

GC-MS Study
GC-MS study had revealed the presence of some of the following important constituents on comparison of the compound libraries with Retention time and peak areas. Parmotrema perlatum contains a notable amount of terpenes and sesquiterpenes like Carvone, Copaene, Cubebene, Viridifloral and Rishitin. Sambucol is an immune stimulant anthocyanin. Longidione is a tricyclic hydrocarbon, Platambin is a naphthalene derivative. Stigmastan 3,5-diene is a phytosterol. Ceiba pentandra extracts contain saturated dicarboxylic acids like azelaic and Suberic acid, limonene a liquid phenol and Limonene-6-ol, a monocyclic monoterpene ( Table 2).

Anti Tubercular Activity
Methanolic extract of Parmotrena perlatum had shown 90% growth inhibition against H37 Rv at 80 micrograms/ml and intermediate activity was observed with methanolic extract of

Fig. 1. Antitubercular activity
Ceiba pentandra at 320 micrograms/ml. nhexane extracts of both samples had shown only about 30% growth inhibition even at 320 micrograms/ml. Parmotrema perlatum has terpenes and steroids as chief chemical constituents as per the GC-MS study hence contributed the remarkable antibacterial and antitubercular activities. Isolation of active constituents and exploration of structures of principle constituents in the future study may lead to discover natural antitubercular and antimicrobial leads (Fig. 1).

CONCLUSION
It is concluded that, the study has made an attempt to identify the phytochemical constituents present in methanolic and n-hexane extracts of Ceiba pentandra and Parmotrema perlatum using phytochemical tests. These spices are not only known to contribute flavor to food but also dispense therapeutically active molecules to the body. Methanolic extracts of both spices were proven as effective antimicrobial activity against resistant microorganisms than n-hexane extract.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.