doi.org/10.36721/PJPS.2022.35.1.REG.059-076.1 Pharmacognostic and phytochemical study of the flowers of Cordia sebestena L. Syed Waleed Ahmed Bokhari1,2*, Hina Sharif2, Syed Muhammad Umer Gilani1, Syed Tahir Ali2, Salman Ahmed1, Maaz Uddin Ahmed Siddiqui1 and Muhammad Mohtasheemul Hasan1 1 2 Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, University of Karachi, Karachi, Pakistan Department of Pharmacognosy, Faculty of Pharmacy, Hamdard University, Karachi, Pakistan Abstract: The present study shows the pharmacognostic and phytochemical studies on the flowers of Cordia sebestena L. belongs to the family Boraginaceae. C. sebestena L. is found primarily in tropical and subtropical regions of the American, Asian and African continents. Though it is an important plant, until date no pharmacognostic work is found on its parts such as flowers. Various organoleptic characters were recorded by macroscopic study. Microscopic study of the flowers were also conducted which shows the presence of fibers, calcium oxalate crystals and multiple types of trichomes, along with fluorescence analysis. The present study also deals with the Fourier transform infrared spectroscopic analysis of C. sebestena L. FT-IR spectra revealed the presence of C-H, C=C, C-N, C-O and aromatic groups. Chemical composition of the hexane extract of the flowers of C. sebestena L. was detected through GC-MS and spectrum achieved through GC-MS were correlated with the database of National Institute of Standards and Technology (NIST) which comprise of beyond 62000 outlines of the mass spectrum. GC-MS analysis of n-hexane extract shown the existence of Retinoic acid, lupeol, β-sitosterol, stigmasterol, hexadecanoic acid along with fatty acids, esters, alkaloids and alcohols. These pharmacognostic and phytochemical studies can be valuable towards giving reliable proof of the quality of the plant which can benefit health professionals and herbal medicine manufacturers. Keywords: Cordia sebestena L, GC-MS, standardization, microscopy. INTRODUCTION Plant signifies considerable extent of the worldwide medication market. In this regard globally recognized rules are fundamental for their quality evaluation. It has been evaluated that 80% of individuals living in developing nations are totally reliant on conventional herbal medicines. Thus it gets to be greatly vital to document the standardization of these plant materials that will be use as future medications (Khan et al., 2015). Phytochemicals, also called phytoconstituents are, bioactive components extensively found in foodstuffs like fruits, whole-grain, leaves, roots, vegetables, nuts, seeds and legumes. Though ten of thousand phytochemicals have existed but only small numbers of them have been secluded from plants (Cao et al., 2017). The foremost usual phytochemicals in food incorporate polyphenols, carotenoids, flavonoids, coumarins, indoles, isoflavones, lignans, catechins, phenolic acids, stilbenoids, isothiocyanates, saponins, procyanidins, phenylpropanoids, anthraquinones, ginsenosides, alkaoloids and others (Zhao et al., 2018; Xiao, 2017). Herbal medications are safer than synthetic drugs since the phytochemicals inside the plant extract target the biochemical pathway (Nisar et al., 2018). C. sebestena L. belongs to the family Boraginaceae commonly known as Geiger tree is evergreen thick, deciduous tree. The genus Cordia generally includes ornamental plant species. The plants which belong to the family Boraginaceae are found in the tropical, subtropical and hotter regions around the world. Cordia sebestena L. is perennial plant but flowers abundantly found in June and July (Adeosun et al., 2015; Prakash et al., 2020). It grows upto a height of 25-30 feet and spreads upto 2025 feet, having green or white coloured fruit, orange-red 2-5 cm long flowers and leaves are ovate 4.5-10 cm. Geiger tree is local to Cuba, Northern West Indies, along with a few parts of Tropical North, Central and South America. Bloom are orangish red and gaudy and are appeared in bunches basically in spring and summer (Hanani et al., 2019). Bioassay of fractions of the ethyl acetate extract of C. sebestena L. have lead to the separation of sebestinoids which has restraint ability on aspartic protease (Dai et al., 2010). Seed oil of C. sebestena L. contains palmitic acid and oleic acid (Agunbiade et al., 2013). AgCuO biometallic nanomaterial from C. sebestena L. leaf extract synthesized through green synthesis (Ravi et al., 2020). Dyeing potential of flowers of C. sebestena L. also reported (Kumaresan et al., 2012). We herewith report the entire chemical arrangement of hexane extract and pharmacognostic features of the flowers of C. sebestena L. *Corresponding author: e-mail: waleed.ahmed@hamdard.edu.pk Pak. J. Pharm. Sci., Vol.35, No.1, January 2022, pp.069-076 69 Pharmacognostic and phytochemical study of the flowers of Cordia sebestena L. MATERIALS AND METHODS Plant material Flowers of C. sebestena L. were collected from University of Karachi, Karachi during August 2019. The plant specimen was identified by Taxonomist Dr. Muneeba Khan in the Center for Plant Conservation, University of Karachi with (GH No. 95282). Extract preparation of flowers of Cordia sebestena L. 500g of flowers of Cordia sebestena L. were soaked in one liter of hexane in extraction flask and kept at room temperature for 3 days and had shaken three times daily. The extract was strained through Whatman filter paper No.1. The filtered extract was dried at 40C under vacuum pressure in rotary evaporator. The resultant extract was kept in dark in amber glass bottle at ambient temperature. Pharmacognostic study Macroscopic evaluation Macroscopic evaluation of the flowers of C. sebestena L. like color, size, shape, texture and fracture was performed (Ahmed and Hasan, 2015). Microscopic evaluation Powdered microscopy of the flowers of C. sebestena L. was conducted through light microscope. Dried flowers were pulverized to mechanical grinding and passed through sieve No. 40 (Bharthi et al., 2017). Fine powder was taken on a glass slide and treated separately with water, glycerine, chloral hydrate and iodine reagents. Microscopic observations were fulfilled utilizing 4, 40 and 10 objective lenses and photomicrographs were captured (WHO, 2011; Evans, 2009). by Agilent Technologies 7000A. Triple Quadrupole Acquisition Method was applied throughout the method. The instrument was packed with a non polar column stuffed with film prepared of 95% Dimethylpolysiloxane and 5% phenyl (Agilent HP-5MS30m length × 250μm diameter × 0.25μm film thickness). For the discovery of the compounds an electron ionization source with 70eV energy was utilized. Ultra immaculate Helium gas (99.99%) was utilized as a carrier gas for mobile phase with split mode at septum purge flow rate of 3ml/min. The injection volume was 2.5L with a split ratio of 10:1. The temperature of the injector was 250C. The pressure was 9.05 psi and constant flow was 1.129 ml/min by average velocity of 38.724 cm/sec. Total runtime was 82.286 min. The solution was ready by taking 1gm of extract and making it dissolvable in 20ml of corresponding solvent. The arrangements were sifted through Whatman No.1 filter paper to evacuate any thick particles. All the chemicals utilized were of analytical grade. Nist spectral library was used to analyze spectra. Calculation of mole percent peak area was done according to the following formula (Ullah et al., 2019). Mole % component (Peak area) = area under peak/total area of all peaks x 100 RESULTS Macroscopic evaluation Fresh flowers of C. sebestena L. were orange in color present in bunches consisted of epipetalous stamen with in a throat, salveform in shape. Flowers starts with long tube and widens into polypetalous flower, actinomorphic, gamosepalous, calyx 1.2-1.5cm, crenulate corolla, involucre bract having smooth texture, bland taste, soft fracture upon breaking and measuring 2-5cm size. Fluorescence analysis Fluorescence analysis of the powder of the flowers of C. sebestena L. was also performed with different chemicals to check the existence of diverse fluorescent chemical compounds under visible and UV light of short (254nm) and long (365nm) wave length (Tang et al., 2018; Kadam et al., 2012). Microscopic evaluation Orangish brown powder of the flowers of C. sebestena showed some significant microscopic features which are shown in fig.1. Fourier transform infrared spectroscopy (FTIR) analysis To identify the distinguishing functional groups found in the phytochemicals FTIR was used. Fine powder of the flowers of C. sebestena was taken for identification of functional groups. Powder was then taken on FTIR spectroscope (Nicolet avatar 330 FTIR, Thermo Electron Co. USA.) having range of wave numbers 500-4000 cm1. Data interpretation of FT-IR spectra was carried out using correlation chart (Pavia et al., 2008) Fourier transform infrared spectroscopy (FTIR) analysis FTIR analysis of the flowers of C. sebestena L. has shown in table 2 and fig. 2. Phytochemical study GC-MS analysis The Gas chromatography mass spectroscopy of hexane extract of the flowers of C. sebestena L. was carried out 70 Fluorescence analysis Result of the fluorescence analysis of the flowers of C. sebestena L. has shown in table 1. GC-MS analysis GCMS analysis of hexane extract of the flowers of C. sebestena L. are presented in table 3 and fig. 3 and structures of compounds are in table 4 which shows more compounds of fatty acid, ester groups, hydrocarbons, triterpene, alkaloids, alcohols, sterols and vitamin class. Pak. J. Pharm. Sci., Vol.35, No.1, January 2022, pp.069-076 Syed Waleed Ahmed Bokhari et al Table 1: Fluorescence characters of the flowers of C. sebestena L. Reagent Chloroform Ethanol Ferric chloride Glacial Acetic acid Hydrochloric acid Methanol Sulphuric acid Day light Orangish brown Orangish brown Blackish brown Dark Yellow Orangish brown Brown Orangish brown UV 254nm Orangish brown Red Blackish brown Pink Orangish brown Brown Orangish brown UV 365nm Grey Bluish grey Blackish brown Bluish green Orangish brown Yellow Orangish brown Table 2: FT-IR analysis of flowers of of C. sebestena L. Absorption Frequency (cm-1) 2900 1600 1400 1300 1025 Type of Vibration Assigned C-H Aldehyde C=C Alkene Aromatic C-N Amines C-O Alcohols, ethers, esters, carboxylic acids, anhydrides Intensity w m-w m-w m-s s (Abbreviation: S; strong W; weak M; medium) Table 3: Phytochemical constituents of hexane extract of C. sebestena L. Compound Name Retinoic acid, 5,6-epoxy-5,6 -dihydro Lupeol 4,4,6a,6b,8a,11,11,14b-Octamethyl1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14boctadecahydro-2H-picen-3-one β-sitosterol Stigmasterol Octadecanoic acid,2-propenyl ester 1H-Purin-2-amine, 6-methoxy-N-methylNonacosane Heptacosane Dodecanoic acid, phenyl methyl ester 1,2-Benzenedicarboxylic acid , diisooctyl ester 9,12-Octadecadienoic acid, ethyl Ester 9-12-Octadecadienoyl chloride Hexadecanoic acid, ethyl ester n-Hexadecanoic acid Hexadecanoic acid, methyl ester Rheadan-8-ol,2,2,10,11-tetramethoxy-16-methyl 9,10-Dimethyltricyclo (2,5) decane-9,10-diol Blumenol C 3-Furanacetic acid, 4-hexyl-2,5-dihydro-2,5-dioxo Tetradecane Tridecane Dodecane Undecane Hexylene glycol Molecular Weight Retention Time Nist Number ID Number C20H28O3 C30H50O 316 426 69.650 64.160 51852 124852 5918 7814 Peak Area % 1.37 1.82 C30H48O 424 63.668 194624 153809 2.16 C29H50O C29H48O C21H40O2 C 7H 9N 5O C29H60 C27H56 C19H30O2 C24H38O4 C20H36O2 C18H31CIO C18H36O2 C16H32O2 C17H34O2 C22H27NO6 C12H20O2 C13H22O2 C12H16O5 C14H30 C13H28 C12H26 C11H24 C6H14O2 414 412 324 179 408 380 290 390 308 298 284 256 270 401 196 210 240 198 184 170 156 118 62.620 61.612 60.411 59.074 56.858 54.605 52.984 52.602 42.599 37.820 31.491 30.122 28.540 26.505 23.505 22.717 18.958 17.332 15.426 13.391 11.207 6.955 287034 352610 36559 34043 197624 79427 232922 113206 249157 76312 233204 335494 42975 64919 187529 108740 26532 113925 229227 291499 227975 234996 1913 18876 5707 132450 5478 5508 11551 20061 28827 4450 49485 6723 9049 155496 8345 5952 89100 5511 5468 21869 22005 26166 5.7 1.82 2.25 9.77 2.93 1.23 2.08 14.86 2.17 7.66 1.84 6.16 5.08 0.76 3.86 0.86 0.88 1.76 2.73 2.64 1.54 0.16 Molecular Formula Pak. J. Pharm. Sci., Vol.35, No.1, January 2022, pp.069-076 71 Pharmacognostic and phytochemical study of the flowers of Cordia sebestena L. Table 4: Chemical structures of phytoconstituents reported in n-hexane extract of flowers of C. sebestena L. Retinoic acid, 5,6-epoxy-5-6-dihydroβ-sitosterol Stigmasterol 4,4,6a,6b,8a,11,11,14b-Octamethyl, 1,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14boctadecahydro-2H-picen-3-one Lupeol 6-methoxy-N-methyl-1H-purin-2-amine Rheadan-8-ol, 2,3,10,11-tetramethoxy-16-methylHexylene glycol Blumenol C Octadecanoic acid, 2-propenyl ester Dodecanoic acid, phenylmethyl ester 1,2-Benzenedicarboxylic acid , diisooctyl ester 9,12- Octadecadienoic acid, ethyl ester 9-12- Octadecadienoyl chloride Hexadecanoic acid, methyl ester Hexadecanoic acid, ethyl ester Hexadecanoic acid Heptacosane Nonacosane Tetradecane Tridecane Dodecane Undecane 9,10-Dimethyltricyclo (2,5) decane-9,10-diol 2-Carboxymethyl-3-n-hexylmaleic acid anhydride 72 Pak. J. Pharm. Sci., Vol.35, No.1, January 2022, pp.069-076 Syed Waleed Ahmed Bokhari et al Fig. 1: Powder Microscopic features of flowers of Cordia sebestena L. (A) Trichome and oil globule; (B) Calcium oxalate; (C) Glandular trichomes with cicatrix and radiating pollen grains; (D) Unicellular trichomes; (E) Fibers with spiral thickening; (F) Fibers; (G) Pollen grains; (H) Lignified fibers (I) Dendritic calcium oxalate; (J) Multicellular trichomes; (K) Fragments of xylem with spiral thickening; (L) Unicellular trichomes with pollen grains; (M) Prismatic calcium oxalate crystals; (N) Pericyclic fibers Pak. J. Pharm. Sci., Vol.35, No.1, January 2022, pp.069-076 73 Pharmacognostic and phytochemical study of the flowers of Cordia sebestena L. Fig. 2: FTIR spectra of flowers of C. Sebestena L. Fig. 3: Chromatograms of n-hexane extract of flowers of C. sebestena L. Fatty acid esters that are present in hexane extract among them 1,2-Benzenedicarboxylic acid, diisooctyl ester was abundant in plant with a peak area percentage of 14.86 also 9-12-Octadecadienoyl chloride is present having peak area 7.66. Among alkaloids 1H-Purin-2-amine, 6methoxy-N-methyl was abundant with a peak area percentage 9.77. β-sitosterol was present with a peak area percentage of 5.7 which is high in sterols. nHexadecanoic acid was present in plant extract with a peak area % age of 6.16. Similarly in fatty acid methyl esters group Hexadecanoic acid, methyl ester was present in hexane extract with a peak area percentage of 5.08. 4,4,6a,6b,8a,11,11,14b-Octamethyl-1,4,4a,5,6,6a,6b,7,8, 8a,9,10,11,12,12a,14,14a,14b-octadecahydro-2H-picen-3one which is triterpene is present with a peak area percentage of 2.16. Among hydroxy hydrocarbons 9,10Dimethyltricyclo (2,5) decane-9,10-diol was present with peak area percentage of 3.86. DISCUSSION Plants with medicinal values are regarded as the source of new chemical entities which can be converted into drugs 74 with considerable research. A considerable number of present day drugs are synthesized either specifically or in other way from the medicinal plants. Therefore the standardization of herbal medications is of great significance in setting up its legitimate character to play a basic part in understanding its structure, science, botanical value and clinical suitability because of frequently finding of substitute or bogus herbal supplies (Zhang et al., 2021). Examination of histological characters along with macroscopic evaluation are the essential tests for standardization. In view of these practicalities authors have prepared an endeavor to fig. out histological characters and macroscopic study that can be utilize for the identification and standardization of this plant, as no standard specifications for standardization has been reported so far. The projecting microscopic features of the flowers of C. sebestena L. are fibers, calcium oxalate crystals, multiple types of trichomes shown in fig. 1. (Bijeshmon and George, 2014; Bharthi et al., 2017) reported somewhat similar types of fibers in the flowers of Tabernaemontana divaricata R. and Vitex negundo L. (Reddy et al.,2015) reported similar multicellular trichomes in the flowers of Justicia adhatoda L. (Das et al., 2021) also reported similar unicellular trichomes. (Baravalia et al., 2011; Bijeshmon and George, 2014) investigated similar fragments of xylem with spiral thickening in the flowers of Woodfordia fruticosa Kurz. and Tabernaemontana divarcata R. Fluorescence examination may be a vital parameter which demonstrates the symbol of chromophore within the drug, which is essential to perform standardization (Prasanth et al., 2017). Few constituents appeared fluorescent within the ultra-violet or visible light since they may regularly be changed over into fluorescent subsidiaries by using diverse chemicals as shown in table 1 which is useful to recognize them. Fourier-transform Infrared spectroscopy serves as a notable means for providing robust insight of various functional groups within the plant material (Selvaraju et al., 2021) It also provides major information of organic and inorganic components. In our study FT-IR analysis showed types of vibrations as shown in table 2 and fig. 2 like C-H group which shows the presence of several aliphatic, aldehyde containing compounds, C=C group present confirm the presence of alkenes, presence of carboxylic acids, ethers, esters, alcohol, anhydride confirmed by the strong peak of C-O, C-N groups indicate the presence of aliphatic amines. All these functional groups present in the plant have numerous medicinal characteristics and these functional groups construct phytochemicals present in the natural product. The identification of the chemical constituents in a plant is a vital stage because the pharmacological and biological activities of plants are dependent on these Pak. J. Pharm. Sci., Vol.35, No.1, January 2022, pp.069-076 Syed Waleed Ahmed Bokhari et al bioactive chemical constituents. Hence, GCMS analysis was used to detect the bioactive phytochemicals present in hexane extract of flowers of C. sebestena L. shown in table 3, fig. 3 and table 4 which shows fatty acid, esters, sterols and alkaloids have high peak area percentage and literature shows that these phytoconstituents possess pharmacological activities like sterols have antiatherosclerotic effects (Salehi et al., 2021), fatty acids are medicinally important and have antibacterial and antifungal activity (Casillas et al., 2021; Pohl et al., 2011). Similarly anti-inflammatory activity of alkaloids was also reported (Souza et al., 2020). GC-MS, analysis have shown the existence of n-hexadecanoic acid which is well known to have antioxidant property (Gopu et al., 2021), Hexadecanoic acid, ethyl ester have antiandrogenic activity, stigmasterol and lupeol possess anticancer, antiiflammatory, antiarthritic and diuretic activity (Rajeswari et al., 2012). Β-sitosterol alleviates inflammatory response (Sun et al., 2020). 4,4, 6a,6b, 8a,11,11,14bOctamethyl-1,4, 4a, 5, 6, 6a,6b,7,8,8a,9,10,11,12,12a,14, 14a, 14b-octadecahydro-2H-picen-3-one which is triterpene possess antibacterial, antioxidant, antitumour and cancer preventive activity (Duan et al., 2011)Hexadecanoic acid, methyl ester may have antioxidant, hypocholesterolemic, anti androgenic, hemolytic, Alpha reductase inhibitor (Pavani and Naika, 2021). Heptacosane possess antioxidant activity (Dandekar et al., 2015). Nonacosane has antibacterial activity (Ryu et al., 2020). 1,2-Benzenedicarboxylic acid, diisooctyl ester known to have antifouling and antioxidant activity (Parthipan et al., 2015) Hexane extract of C. sebestena L. also shows the presence of several hydrocarbons, these hydrocarbons contribute in chemotaxonomy of C. sebestena L (Adeosun et al., 2013). CONCLUSION Standardization of herbal drugs is a topic of great concern. The pharmacognostic parameters of the flowers of C. sebestena L. are laid down for the first time and these findings could be helpful in the identification and authentication of these plant materials in future for further research and utilization. Phytochemical studies of hexane extract also shows different findings and therefore useful in quality control of the plant drug. 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